U.S. patent application number 10/849979 was filed with the patent office on 2007-07-26 for antibodies to hhpen62 polypeptide.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Charles E. Birse, Laurie A. Brewer, Kenneth C. Carter, Reinhard Ebner, Gregory A. Endress, Kimberly A. Florence, David W. LaFleur, Paul A. Moore, Jian Ni, Henrik S. Olsen, Craig A. Rosen, Steven M. Ruben, Yanggu Shi, Daniel R. Soppet, Ying-Fei Wei, Paul E. Young.
Application Number | 20070172830 10/849979 |
Document ID | / |
Family ID | 38266832 |
Filed Date | 2007-07-26 |
United States Patent
Application |
20070172830 |
Kind Code |
A1 |
Ruben; Steven M. ; et
al. |
July 26, 2007 |
ANTIBODIES TO HHPEN62 POLYPEPTIDE
Abstract
The present invention relates to novel human secreted proteins
and isolated nucleic acids containing the coding regions of the
genes encoding such proteins. Also provided are vectors, host
cells, antibodies, and recombinant methods for producing human
secreted proteins. The invention further relates to diagnostic and
therapeutic methods useful for diagnosing and treating diseases,
disorders, and/or conditions related to these novel human secreted
proteins.
Inventors: |
Ruben; Steven M.;
(Brookeville, MD) ; Florence; Kimberly A.;
(Rockville, MD) ; Ni; Jian; (Germantown, MD)
; Rosen; Craig A.; (Laytonsville, MD) ; Carter;
Kenneth C.; (North Potomac, MD) ; Moore; Paul A.;
(North Bethesda, MD) ; Olsen; Henrik S.;
(Gaithersburg, MD) ; Shi; Yanggu; (Gaithersburg,
MD) ; Young; Paul E.; (Gaithersburg, MD) ;
Wei; Ying-Fei; (Berkeley, CA) ; Brewer; Laurie
A.; (St. Paul, MN) ; Soppet; Daniel R.;
(Centreville, VA) ; LaFleur; David W.;
(Washington, DC) ; Endress; Gregory A.; (Florence,
MA) ; Ebner; Reinhard; (Gaithersburg, MD) ;
Birse; Charles E.; (North Potomac, MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC.;INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
38266832 |
Appl. No.: |
10/849979 |
Filed: |
May 21, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09948783 |
Sep 10, 2001 |
|
|
|
10849979 |
May 21, 2004 |
|
|
|
09892877 |
Jun 28, 2001 |
|
|
|
10849979 |
May 21, 2004 |
|
|
|
09437658 |
Nov 10, 1999 |
|
|
|
09892877 |
Jun 28, 2001 |
|
|
|
PCT/US99/09847 |
May 6, 1999 |
|
|
|
09437658 |
Nov 10, 1999 |
|
|
|
60231846 |
Sep 11, 2000 |
|
|
|
60085093 |
May 12, 1998 |
|
|
|
60085094 |
May 12, 1998 |
|
|
|
60085105 |
May 12, 1998 |
|
|
|
60085180 |
May 12, 1998 |
|
|
|
60085927 |
May 18, 1998 |
|
|
|
60085906 |
May 18, 1998 |
|
|
|
60085920 |
May 18, 1998 |
|
|
|
60085924 |
May 18, 1998 |
|
|
|
60085922 |
May 18, 1998 |
|
|
|
60085923 |
May 18, 1998 |
|
|
|
60085921 |
May 18, 1998 |
|
|
|
60085925 |
May 18, 1998 |
|
|
|
60085928 |
May 18, 1998 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/320.1; 435/325; 435/6.16; 435/69.1; 514/18.9; 514/19.3;
514/20.9; 514/44R; 530/350; 530/388.1; 536/23.1 |
Current CPC
Class: |
C07K 14/47 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 514/012; 514/044; 530/350;
530/388.1; 536/023.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; A61K 48/00 20060101 A61K048/00; A61K 38/17 20060101
A61K038/17; C07K 14/47 20060101 C07K014/47; C12P 21/06 20060101
C12P021/06 |
Claims
1-24. (canceled)
25. An isolated antibody or fragment thereof that specifically
binds to a polypeptide selected from the group consisting of: (a) a
polypeptide whose amino acid sequence consists of amino acid
residues 1 to 508 of SEQ ID NO:139; (b) a polypeptide whose amino
acid sequence consists of a portion of SEQ ID NO:139, wherein said
portion is at least 30 contiguous amino acid residues of SEQ ID
NO:139; and (c) a polypeptide whose amino acid sequence consists of
a portion of SEQ ID NO:139, wherein said portion is at least 50
contiguous amino acid residues of SEQ ID NO:139.
26. The antibody or fragment thereof of claim 25 that specifically
binds polypeptide (a).
27. The antibody or fragment thereof of claim 25 that specifically
binds polypeptide (b).
28. The antibody or fragment thereof of claim 25 that specifically
binds polypeptide (c).
29. The antibody or fragment thereof of claim 27 that specifically
binds a polypeptide whose amino acid sequence consists of amino
acid residues 1 to 508 of SEQ ID NO:139.
30. The antibody or fragment thereof of claim 25, which is a
polyclonal antibody.
31. The antibody or fragment thereof of claim 25, which is a
monoclonal antibody.
32. The antibody or fragment thereof of claim 25, which is selected
from the group consisting of: (a) a chimeric antibody; (b) a human
antibody; (c) a humanized antibody; (d) a single chain antibody;
and (e) a Fab fragment.
33. The antibody or fragment thereof of claim 25, which is
labeled.
34. The antibody or fragment thereof of claim 25 wherein said
polypeptide bound by said antibody or fragment thereof is
glycosylated
35. The antibody or fragment thereof of claim 25 wherein said
antibody or fragment thereof specifically binds to said polypeptide
in a Western blot.
36. The antibody or fragment thereof of claim 25 wherein said
antibody or fragment thereof specifically binds to said polypeptide
in an ELISA.
37. An isolated cell that produces the antibody or fragment thereof
of claim 25.
38. A hybridoma that produces the antibody or fragment thereof of
claim 25.
39. The antibody or fragment thereof of claim 26, which is a
polyclonal antibody.
40. The antibody or fragment thereof of claim 26, which is a
monoclonal antibody.
41. The antibody or fragment thereof of claim 26 which is selected
from the group consisting of: (a) a chimeric antibody; (b) a human
antibody; (c) a humanized antibody; (d) a single chain antibody;
and (e) a Fab fragment.
42. The antibody or fragment thereof of claim 26, which is
labeled.
43. The antibody or fragment thereof of claim 26 wherein said
polypeptide bound by said antibody or fragment thereof is
glycosylated
44. The antibody or fragment thereof of claim 26 wherein said
antibody or fragment thereof specifically binds to said polypeptide
in a Western blot.
45. The antibody or fragment thereof of claim 26 wherein said
antibody or fragment thereof specifically binds to said polypeptide
in an ELISA.
46. An isolated cell that produces the antibody or fragment thereof
of claim 26.
47. A hybridoma that produces the antibody or fragment thereof of
claim 26.
48. A method of detecting a polypeptide comprising amino acid
residues 1 to 508 of SEQ ID NO:139 in a biological sample
comprising: (a) contacting the biological sample with the antibody
or fragment thereof of claim 25; (b) allowing a complex to form
between said polypeptide comprising amino acid residues 1 to 508 of
SEQ ID NO:139 and said antibody of claim 25; and, (c) detecting
said complex.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/948,783, filed Sep. 10, 2001, which claims
benefit under 35 U.S.C. .sctn. 119(e) of U.S. Provisional Patent
Application No. 60/231,846 filed Sep. 11, 2000; this application is
also a continuation-in-part of U.S. patent applicaton Ser. No.
09/892,877 filed Jun. 28, 2001, which is a continuation application
of U.S. patent applicaton Ser. No. 09/437,658 filed Nov. 10, 1999,
which is a continuation-in-part of International Patent Application
No: PCT/US99/09847, filed May 6, 1999, which claims benefit under
35 U.S.C. .sctn. 119(e) based on the following U.S. Provisional
Applications: No. 60/085,093, filed on May 12, 1998; No.
60/085,094, filed on May 12, 1998; No. 60/085,105, filed on May 12,
1998; No. 60/085,180, filed on May 12, 1998; No. 60/085,927, filed
on May 18, 1998; No. 60/085,906, filed on May 18, 1998; No.
60/985,920, filed on May 18, 1998; No. 60/085,924, filed on May 18,
1998; No. 60/085,922, filed on May 18, 1998; No. 60/085,923, filed
on May 18, 1998; No. 60/085,921, filed on May 18, 1998; No.
60/085,925, filed on May 18, 1998; and, No. 60/085,928, filed on
May 18, 1998. Each of the above referenced patents and/or patent
applications is hereby incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0002] This invention relates to newly identified polynucleotides,
polypeptides encoded by these polynucleotides, antibodies that bind
these polypeptides, uses of such polynucleotides, polypeptides, and
antibodies, and their production.
BACKGROUND OF THE INVENTION
[0003] Unlike bacterium, which exist as a single compartment
surrounded by a membrane, human cells and other eucaryotes are
subdivided by membranes into many functionally distinct
compartments. Each membrane-bounded compartment, or organelle,
contains different proteins essential for the function of the
organelle. The cell uses "sorting signals," which are amino acid
motifs located within the protein, to target proteins to particular
cellular organelles.
[0004] One type of sorting signal, called a signal sequence, a
signal peptide, or a leader sequence, directs a class of proteins
to an organelle called the endoplasmic reticulum (ER). The ER
separates the membrane-bounded proteins from all other types of
proteins. Once localized to the ER, both groups of proteins can be
further directed to another organelle called the Golgi apparatus.
Here, the Golgi distributes the proteins to vesicles, including
secretory vesicles, the cell membrane, lysosomes, and the other
organelles.
[0005] Proteins targeted to the ER by a signal sequence can be
released into the extracellular space as a secreted protein. For
example, vesicles containing secreted proteins can fuse with the
cell membrane and release their contents into the extracellular
space--a process called exocytosis. Exocytosis can occur
constitutively or after receipt of a triggering signal. In the
latter case, the proteins are stored in secretory vesicles (or
secretory granules) until exocytosis is triggered. Similarly,
proteins residing on the cell membrane can also be secreted into
the extracellular space by proteolytic cleavage of a "linker"
holding the protein to the membrane.
[0006] Despite the great progress made in recent years, only a
small number of genes encoding human secreted proteins have been
identified. These secreted proteins include the commercially
valuable human insulin, interferon, Factor VIII, human growth
hormone, tissue plasminogen activator, and erythropoeitin. Thus, in
light of the pervasive role of secreted proteins in human
physiology, a need exists for identifying and characterizing novel
human secreted proteins and the genes that encode them. This
knowledge will allow one to detect, to treat, and to prevent
medical diseases, disorders, and/or conditions by using secreted
proteins or the genes that encode them.
SUMMARY OF THE INVENTION
[0007] The present invention relates to novel polynucleotides and
the encoded polypeptides. Moreover, the present invention relates
to vectors, host cells, antibodies, and recombinant and synthetic
methods for producing the polypeptides and polynucleotides. Also
provided are diagnostic methods for detecting diseases, disorders,
and/or conditions related to the polypeptides and polynucleotides,
and therapeutic methods for treating such diseases, disorders,
and/or conditions. The invention further relates to screening
methods for identifying binding partners of the polypeptides.
DETAILED DESCRIPTION
Definitions
[0008] The following definitions are provided to facilitate
understanding of certain terms used throughout this
specification.
[0009] In the present invention, "isolated" refers to material
removed from its original environment (e.g., the natural
environment if it is naturally occurring), and thus is altered "by
the hand of man" from its natural state. For example, an isolated
polynucleotide could be part of a vector or a composition of
matter, or could be contained within a cell, and still be
"isolated" because that vector, composition of matter, or
particular cell is not the original environment of the
polynucleotide. The term "isolated" does not refer to genomic or
cDNA libraries, whole cell total or MRNA preparations, genomic DNA
preparations (including those separated by electrophoresis and
transferred onto blots), sheared whole cell genomic DNA
preparations or other compositions where the art demonstrates no
distinguishing features of the polynucleotide/sequences of the
present invention.
[0010] In the present invention, a "secreted" protein refers to
those proteins capable of being directed to the ER, secretory
vesicles, or the extracellular space as a result of a signal
sequence, as well as those proteins released into the extracellular
space without necessarily containing a signal sequence. If the
secreted protein is released into the extracellular space, the
secreted protein can undergo extracellular processing to produce a
"mature" protein. Release into the extracellular space can occur by
many mechanisms, including exocytosis and proteolytic cleavage.
[0011] In specific embodiments, the polynucleotides of the
invention are at least 15, at least 30, at least 50, at least 100,
at least 125, at least 500, or at least 1000 continuous nucleotides
but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb,
10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a
further embodiment, polynucleotides of the invention comprise a
portion of the coding sequences, as disclosed herein, but do not
comprise all or a portion of any intron. In another embodiment, the
polynucleotides comprising coding sequences do not contain coding
sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of
interest in the genome). In other embodiments, the polynucleotides
of the invention do not contain the coding sequence of more than
1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic
flanking gene(s).
[0012] As used herein, a "polynucleotide" refers to a molecule
having a nucleic acid sequence contained in SEQ ID NO:X or the cDNA
contained within the clone deposited with the ATCC. For example,
the polynucleotide can contain the nucleotide sequence of the full
length cDNA sequence, including the 5' and 3' untranslated
sequences, the coding region, with or without the signal sequence,
the secreted protein coding region, as well as fragments, epitopes,
domains, and variants of the nucleic acid sequence. Moreover, as
used herein, a "polypeptide" refers to a molecule having the
translated amino acid sequence generated from the polynucleotide as
broadly defined.
[0013] In the present invention, the full length sequence
identified as SEQ ID NO:X was often generated by overlapping
sequences contained in multiple clones (contig analysis). A
representative clone containing all or most of the sequence for SEQ
ID NO:X was deposited with the American Type Culture Collection
("ATCC"). As shown in Table 1, each clone is identified by a cDNA
Clone ID (Identifier) and the ATCC Deposit Number. The ATCC is
located at 10801 University Boulevard, Manassas, Va. 20110-2209,
USA. The ATCC deposit was made pursuant to the terms of the
Budapest Treaty on the international recognition of the deposit of
microorganisms for purposes of patent procedure.
[0014] A "polynucleotide" of the present invention also includes
those polynucleotides capable of hybridizing, under stringent
hybridization conditions, to sequences contained in SEQ ID NO:X,
the complement thereof, or the cDNA within the clone deposited with
the ATCC. "Stringent hybridization conditions" refers to an
overnight incubation at 42 degree C. in a solution comprising 50%
formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm
DNA, followed by washing the filters in 0.1.times.SSC at about 65
degree C.
[0015] Also contemplated are nucleic acid molecules that hybridize
to the polynucleotides of the present invention at lower stringency
hybridization conditions. Changes in the stringency of
hybridization and signal detection are primarily accomplished
through the manipulation of formamide concentration (lower
percentages of formamide result in lowered stringency); salt
conditions, or temperature. For example, lower stringency
conditions include an overnight incubation at 37 degree C. in a
solution comprising 6.times.SSPE (20.times.SSPE=3M NaCl; 0.2M
NaH.sub.2PO.sub.4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide,
100 ug/ml salmon sperm blocking DNA; followed by washes at 50
degree C. with 1.times.SSPE, 0.1% SDS. In addition, to achieve even
lower stringency, washes performed following stringent
hybridization can be done at higher salt concentrations (e.g.
5.times.SSC).
[0016] Note that variations in the above conditions may be
accomplished through the inclusion and/or substitution of alternate
blocking reagents used to suppress background in hybridization
experiments. Typical blocking reagents include Denhardt's reagent,
BLOTTO, heparin, denatured salmon sperm DNA, and commercially
available proprietary formulations. The inclusion of specific
blocking reagents may require modification of the hybridization
conditions described above, due to problems with compatibility.
[0017] Of course, a polynucleotide which hybridizes only to polyA+
sequences (such as any 3' terminal polyA+ tract of a cDNA shown in
the sequence listing), or to a complementary stretch of T (or U)
residues, would not be included in the definition of
"polynucleotide," since such a polynucleotide would hybridize to
any nucleic acid molecule containing a poly (A) stretch or the
complement thereof (e.g., practically any double-stranded cDNA
clone generated using oligo dT as a primer).
[0018] The polynucleotide of the present invention can be composed
of any polyribonucleotide or polydeoxribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. For example,
polynucleotides can be composed of single- and double-stranded DNA,
DNA that is a mixture of single- and double-stranded regions,
single- and double-stranded RNA, and RNA that is mixture of single-
and double-stranded regions, hybrid molecules comprising DNA and
RNA that may be single-stranded or, more typically, double-stranded
or a mixture of single- and double-stranded regions. In addition,
the polynucleotide can be composed of triple-stranded regions
comprising RNA or DNA or both RNA and DNA. A polynucleotide may
also contain one or more modified bases or DNA or RNA backbones
modified for stability or for other reasons. "Modified" bases
include, for example, tritylated bases and unusual bases such as
inosine. A variety of modifications can be made to DNA and RNA;
thus, "polynucleotide" embraces chemically, enzymatically, or
metabolically modified forms.
[0019] The polypeptide of the present invention can be composed of
amino acids joined to each other by peptide bonds or modified
peptide bonds, i.e., peptide isosteres, and may contain amino acids
other than the 20 gene-encoded amino acids. The polypeptides may be
modified by either natural processes, such as posttranslational
processing, or by chemical modification techniques which are well
known in the art. Such modifications are well described in basic
texts and in more detailed monographs, as well as in a voluminous
research literature. Modifications can occur anywhere in a
polypeptide, including the peptide backbone, the amino acid
side-chains and the amino or carboxyl termini. It will be
appreciated that the same type of modification may be present in
the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched, for example, as a
result of ubiquitination, and they may be cyclic, with or without
branching. Cyclic, branched, and branched cyclic polypeptides may
result from posttranslation natural processes or may be made by
synthetic methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination. (See, for instance, PROTEINS--STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION
OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs.
1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990);
Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
[0020] "SEQ ID NO:X" refers to a polynucleotide sequence while "SEQ
ID NO:Y" refers to a polypeptide sequence, both sequences
identified by an integer specified in Table 1.
[0021] "A polypeptide having biological activity" refers to
polypeptides exhibiting activity similar, but not necessarily
identical to, an activity of a polypeptide of the present
invention, including mature forms, as measured in a particular
biological assay, with or without dose dependency. In the case
where dose dependency does exist, it need not be identical to that
of the polypeptide, but rather substantially similar to the
dose-dependence in a given activity as compared to the polypeptide
of the present invention (i.e., the candidate polypeptide will
exhibit greater activity or not more than about 25-fold less and,
preferably, not more than about tenfold less activity, and most
preferably, not more than about three-fold less activity relative
to the polypeptide of the present invention.)
[0022] Polynucleotides and Polypeptides of the Invention
[0023] Features of Protein Encoded By Gene No: 1
[0024] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: WAGTQEPTGLPSTLSRSESWDH (SEQ ID NO: 225). Moreover,
fragments and variants of these polypeptides (such as, for example,
fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides,
or polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0025] The translation product of this gene shares sequence
homology with tag-7 which is thought to be important in tumor
metastasis and is itself a secretory protein (see, Kiselev S L, et
al., J Biol Chem. 273:18633 (1998) and Genetika. 1996 May; 32(5):
621-628. (Russian)), and a family of peptidoglycan recognition
proteins involved in the innate immune response to peptidoglycan in
species as diverse as insects and humans (see, Kang, D. et.al.,
PNAS 95:10078 (1998)).
[0026] This gene is expressed primarily in keratinocytes.
[0027] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
dermatological disorders, especially skin cancers such as melanoma.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the integumentary system, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., skin, cancerous and wounded tissues) or bodily
fluids (e.g., sweat, lymph, serum, plasma, urine, synovial fluid
and spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of one, two, or all three of the immunogenic epitopes shown
in SEQ ID NO: 118 as residues: Ser-25 to Ala-31, Gln-146 to
Ser-151, His-231 to Asn-236. Polynucleotides encoding said
polypeptides are encompassed by the invention, as are antibodies
that bind one or more of these peptides.
[0028] The tissue distribution in keratinocytes and homology to
tag-7 indicates that polynucleotides and polypeptides corresponding
to this gene would be useful for detection, treatment, and/or
prevention of dermatological disorders, especially skin cancers
like melanoma, and integumentary tumors (e.g., keratoses, Bowen's
disease, basal cell carcinoma, squamous cell carcinoma, malignant
melanoma, Paget's disease, mycosis fungoides, and Kaposi's
sarcoma). Tag-7 was dicovered when gene expression was compared in
a metastatic (VMR-Liv) neoplastic cell line and a related
nonmetastatic (VMR-O) neoplastic cell line by means of the
differential display method. A fragment of cDNA corresponding to
the tag-7 gene, differentially expressed in the metastatic cell
line, was isolated. The full-length tag-7 cDNA was gened and its
nucleotide sequence was determined. The gene sequence claimed in
this patent application has significant homology to tag-7 and on
that basis is expected to share significant biological activities
with tag-7. Such activities can be assayed as set forth herein and
by assays known in the art. Additionally, the homology to a
conserved peptidoglycan recognition protein family involved in
innate immunity, indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the treatment,
diagnosis, and/or prevention of various skin disorders including
congenital disorders (e.g., nevi, moles, freckles, Mongolian spots,
hemangiomas, port-wine syndrome), injuries and inflammation of the
skin (e.g., wounds, rashes, prickly heat disorder, psoriasis,
dermatitis), atherosclerosis, uticaria, eczema, photosensitivity,
autoimmune disorders (e.g., lupus erythematosus, vitiligo,
dermatomyositis, morphea, scleroderma, pemphigoid, and pemphigus),
keloids, striae, erythema, petechiae, purpura, and xanthelasma.
Moreover, such disorders may predispose increased susceptibility to
viral and bacterial infections of the skin (e.g., cold sores,
warts, chickenpox, molluscum contagiosum, herpes zoster, boils,
cellulitis, erysipelas, impetigo, tinea, althlete's foot, and
ringworm). Furthermore, the protein may also be used to determine
biological activity, to raise antibodies, as tissue markers, to
isolate cognate ligands or receptors, to identify agents that
modulate their interactions, in addition to its use as a
nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0029] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:11 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1177 of SEQ ID NO:11, b is an integer
of 15 to 1191, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:11, and where b is greater
than or equal to a+14.
[0030] Features of Protein Encoded By Gene No: 2
[0031] The translation product of this gene shares weak sequence
homology with FGF Receptor Ligand-2 which is thought to be
important in activating FGF receptor in mediating cell
proliferative functions.
[0032] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
EIIHNLPTSRMAARTKKKNDIINIKVPADCNTRMSYYYKGSGKRGEMESWLV
MSSWSILDFEFLEARPQLFNLVYTEHSTYSGRHYTRERGGFMVFKNSYSQLLL
KRKDSLCAFIQPMALNIHVPMSSKCIFPAQSGPSTFRSLWWCPHPISKCQLGL YSSQIRDIPYLA
(SEQ ID NO: 226), EIIHNLPTSRMAARTKKKNDIINIKVPADCNTRMS (SEQ ID NO:
227), YYYKGSGKRGEMESWLVMSSWSILDFEFLEARPQLF (SEQ ID NO: 228),
NLVYTEHSTYSGRHYTRERGGFMVFKNSYSQLLLKR (SEQ ID NO: 229),
KDSLCAFIQPMALNIIHVPMSSKCIFPAQSGPSTF (SEQ ID NO: 230), and/or
RSLWWCPHPISKCQLGLYSSQIRDIPYLA (SEQ ID NO: 231). Moreover, fragments
and variants of these polypeptides (such as, for example, fragments
as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention. This gene
is expressed primarily in neutrophils.
[0033] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited
to,abnormal immune reactions or disorders. Similarly, polypeptides
and antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune system tissue
and connective tissues, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., immune, cancerous and wounded tissues) or
bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of the immunogenic epitopes shown in SEQ ID NO: 119 as
residues: Met-1 to Met-6. Polynucleotides encoding said
polypeptides are encompassed by the invention, as are antibodies
that bind one or more of these peptides.
[0034] The tissue distribution and homology to FGF Receptor
Ligand-2 indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for detection,
treatment, and/or prevention of immune disorders, especially those
that are mediated by neutrophil functions. They can be utilized in
the treatment of neural and immune disorders, or to stimulate
proliferation of vertebrate cells, raise antibodies, and to screen
for antagonists useful for inhibiting tumor growth. Moreover, the
expression of this gene product indicates a role in regulating the
proliferation, survival, differentiation, and/or activation of
hematopoietic cell lineages, including blood stem cells. This gene
product may be involved in the regulation of cytokine production,
antigen presentation, or other processes that may also suggest a
usefulness in the treatment of cancer (e.g., by boosting immune
responses). Representative uses are described in the "Immune
Activity" and "Infectious Disease" sections below, in Example 11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Since the
gene is expressed in cells of lymphoid origin, the natural gene
product may be involved in immune functions. Therefore it may be
also used as an agent for immunological disorders including
arthritis, asthma, immunodeficiency diseases such as AIDS,
leukemia, rheumatoid arthritis, granulomatous disease, inflammatory
bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and
tissues. In addition, this gene product may have commercial utility
in the expansion of stem cells and committed progenitors of various
blood lineages, and in the differentiation and/or proliferation of
various cell types. Furthermore, the protein may also be used to
determine biological activity, raise antibodies, as tissue markers,
to isolate cognate ligands or receptors, to identify agents that
modulate their interactions, in addition to its use as a
nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0035] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:12 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1237 of SEQ ID NO:12, b is an integer
of 15 to 1251, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:12, and where b is greater
than or equal to a+14.
[0036] Features of Protein Encoded By Gene No: 3
[0037] The translation product of this gene shares sequence
homology with glycosyl transferase, which is thought to be
important in glycosylation of proteins (see, e.g., Genbank
Accession No. g2996578). Based on the sequence similarity, the
translation product of this clone is expected to share at least
some biological activities with glycosyltransferase proteins. Such
activities are known in the art.
[0038] The polypeptide of this gene has been determined to have
transmembrane domains at about amino acid positions 238-254,
338-354, 143-159, 13-29, 429-445, 384-400, 489-505, 462-478,
102-118, and 189-205 of the amino acid sequence referenced in Table
1 for this gene. Based upon these characteristics, it is believed
that the protein product of this gene shares structural features to
type IIIa membrane proteins.
[0039] The gene encoding the disclosed cDNA is believed to reside
on chromosome 11. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 11.
[0040] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
EACGAAAMAALTIATGTGNWFSALALGVTLLKCLLIPTYHSTDFEVHRNWL
AITHSLPISQWYYEATSEWTLDYPPFFAWFEYILSHVAKYFDQEMLNVHNLN
YSSSRTLLFQRFSVIFMDVLFVYAVRECCKCIDGKKVGKELTEKPKFILSVLLL
WNFGLLIVDHIHFQYNGFLFGLMLLSIARLFQKRHMEGAFLFAVLLHFKHIYL
YVAPAYGVYLLRSYCFTANKPDGSIRWKSFSFVRVISLGLVVFLVSALSLGPF
LALNQLPQVFSRLFPFKRGLCHAYWAPNFWALYNALDKVLSVIGLKLKFLDP
NNIPKASMTSGLVQQFQHTVLPSVTPLATLICTLIAILPSIFCLWFKPQGPRGFL
RCLTLCALSSFMFGWHVHEKAILLAILPMSLLSVGKAGDASIFLILTTTGHYSL
FPLLFTAPELPIKILLMLLFTIYSISSLKTLFRKEKPLFNWMETFYLLXLGPLEVC CEFVFPFTSW
KVKYPFIPLLLTSVYCAVGITYAWFKLYVSVLIDSAIGKTKKQ (SEQ ID NO: 232).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0041] This gene is expressed primarily in osteoclastoma cells,
B-cells, macrophage, tonsils, ovarian cancer tissue, melanocytes,
haemopoietic cells and colon tissue, and, to a lesser extent, in
several other tissues and organs.
[0042] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
disorders of the skin, blood, skeletal system and cancer.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the haemopoietic system, epithelium and skeletal system, expression
of this gene at significantly higher or lower levels may be
routinely detected in certain tissues or cell types (e.g., immune,
musculo-skeletal, cancerous and wounded tissues) or bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder. Preferred polypeptides
of the present invention comprise, or alternatively consist of one,
two, three, four or all five of the immunogenic epitopes shown in
SEQ ID NO: 120 as residues: Glu-136 to Pro-141, Ala-221 to Ser-227,
Asp-307 to Pro-312, Lys-355 to Gly-361, Phe-449 to Pro-454.
Polynucleotides encoding said polypeptides are encompassed by the
invention, as are antibodies that bind one or more of these
peptides.
[0043] The tissue distribution in musculo-skeletal and immune
tissues, and the homology to glycosyl transferase protein,
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the treatment, prevention, detection
and/or diagnosis of disorders of the haemopoietic, skeletal and
epithelial systems, and cancers thereof, as well as disorders
associated with incorrect post-translational modification of
proteins (i.e. glycosylation). The tissue distribution in immune
cells (e.g., B-cells and macrophage) indicates polynucleotides and
polypeptides corresponding to this gene would be useful for the
diagnosis detection, prevention and/or treatment of a variety of
immune system disorders. Representative uses are described in the
"Inmune Activity" and "Infectious Disease" sections below, in
Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.
Briefly, the expression indicates a role in regulating the
proliferation; survival; differentiation; and/or activation of
hematopoietic cell lineages, including blood stem cells.
Involvement in the regulation of cytokine production, antigen
presentation, or other processes indicates a usefulness for
treatment of cancer (e.g. by boosting immune responses). Expression
in cells of lymphoid origin, indicates the natural gene product
would be involved in immune functions. Therefore it would also be
useful as an agent for immunological disorders including arthritis,
asthma, immunodeficiency diseases such as AIDS, leukemia,
rheumatoid arthritis, granulomatous disease, inflammatory bowel
disease, sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus, this
gene product is thought to be useful in the expansion of stem cells
and committed progenitors of various blood lineages, and in the
differentiation and/or proliferation of various cell types.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0044] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:13 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1720 of SEQ ID NO:13, b is an integer
of 15 to 1734, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:13, and where b is greater
than or equal to a+14.
[0045] Features of Protein Encoded By Gene No: 4
[0046] The translation product of this gene shares sequence
homology with human pleckstrin protein which is thought to be
important in platelet formation or activity (see, e.g., Genbank
Accession No. g35518 and Tyers, M., et al., Nature 333 (6172),
470-473 (1988); all references available through this accession are
hereby incorporated herein by reference). Therefore, it is likely
that this gene also has activity in platelets.
[0047] This gene is expressed primarily in keratinocytes, and, to a
lesser extent, in spleen and bone marrow.
[0048] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
the following diseases and conditions which include, but are not
limited to, immune and clotting disorders. Similarly, polypeptides
and antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune and blood
clotting systems, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues or
cell types (e.g., immune, blood clotting, cancerous and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial fluid and spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder. Preferred polypeptides of the present invention comprise,
or alternatively consist of one or both of the immunogenic epitopes
shown in SEQ ID NO: 121 as residues: Leu-38 to Gly-49, Lys-75 to
Thr-80. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0049] The tissue distribution in keratinocytes, spleen and bone
marrow, and the homology to pleckstrin indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the study, diagnosis, detection, prevention and/or
treatment of immune system and clotting disorders. Furthermore,
since this protein is 50% identical to the Pleckstrin protein, it
is an excellent candidate for a protein kinase C substrate.
Identification of this protein as a target of protein kinase C, and
the exploration of its role in protein kinase C mediated responses,
such as inflammation, may lead to a better understanding of the
inflammatory response. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0050] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:14 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1526 of SEQ ID NO:14, b is an integer
of 15 to 1540, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:14, and where b is greater
than or equal to a+14.
[0051] Features of Protein Encoded By Gene No: 5
[0052] The gene encoding the disclosed cDNA is thought to reside on
chromosome 17. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 17.
[0053] This gene is expressed primarily in infant liver/spleen
tissues, T cells, bone marrow stromal cells, and thymus tissue,
and, to a lesser extent, in brain and tonsils tissues.
[0054] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
various immune system disorders and/or diseases. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues or cell types
(e.g., immune, cancerous and wounded tissues) or bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder. Preferred polypeptides
of the present invention comprise, or alternatively consist of the
immunogenic epitopes shown in SEQ ID NO: 122 as residues: Ser-46 to
Arg-54. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0055] The tissue distribution in liver/spleen tissues, T-cells,
bone marrow stromal cells, and thymus tissue indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the diagnosis, detection, prevention and/or treatment
of a variety of cancers, most notably cancers of the immune system.
Representative uses are described in the Immune Activity and
Infectious Disease sections below, in Example 11, 13, 14, 16, 18,
19, 20, and 27, and elsewhere herein. Briefly, the expression of
this gene product in a variety of cells of the immune system
indicates that polynucleotides and polypeptides corresponding to
this gene may be players in the progression of these diseases, and
may be a beneficial target for inhibitors as therapeutics.
Furthermore, the tissue distribution indicates that polynucleotides
and polypeptides corresponding to this gene would be useful for the
treatment and/or diagnosis of hematopoietic related disorders such
as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia,
since stromal cells are important in the production of cells of
hematopoietic lineages. The uses include bone marrow cell ex vivo
culture, bone marrow transplantation, bone marrow reconstitution,
radiotherapy or chemotherapy of neoplasia. The gene product may
also be involved in lymphopoiesis, therefore, it can be used in
immune disorders such as infection, inflammation, allergy,
immunodeficiency etc. In addition, this gene product may have
commercial utility in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Furthermore, the
protein may also be used to determine biological activity, to raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0056] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:15 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1544 of SEQ ID NO:15, b is an integer
of 15 to 1558, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:15, and where b is greater
than or equal to a+14.
[0057] Features of Protein Encoded By Gene No: 6
[0058] The translation product of this gene shares sequence
homology with angiopoietin-2, an anti-angiogenic factor. See, for
example, Maisonpierre, et al., Angiopoietin-2, a natural antagonist
for Tie2 that disrupts in vivo angiogenesis. Science. (1997)
277(5322): 55-60, incorporated herein by reference in its entirety.
Based on the sequence similarity, the translation product of this
gene is expected to share certain biological activities with
Angiopoietin-2 as may be assessed by assays known in the art and
described herein.
[0059] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: TABLE-US-00001 (SEQ ID NO: 233)
MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGL
LQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELR
RTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQ
NQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIE
NQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIY
NRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWE
NYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSG
GWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKML IHPTDSESFE, (SEQ
ID NO: 234) MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRF, (SEQ ID NO: 235)
AMLDDVKILANGLLQLGHGLKDFVHKTKGQINDI, (SEQ ID NO: 236)
FQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKL, (SEQ ID NO: 237)
QVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLE, (SEQ ID NO: 238)
EQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDL, (SEQ ID NO: 239)
LQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTE, (SEQ ID NO: 240)
ISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTT, (SEQ ID NO: 241)
IYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTL, (SEQ ID NO: 242)
IQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKI, (SEQ ID NO: 243)
YSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHE, (SEQ ID NO: 244)
TNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKG, (SEQ ID NO: 245)
HFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKLP, and/or (SEQ ID NO: 246)
ERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE.
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0060] The gene encoding the disclosed cDNA is believed to reside
on chromosome 1. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 1.
[0061] This gene is expressed primarily in liver.
[0062] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
angiogenesis and neovascularisation associated with tumour
development. Similarly, polypeptides and antibodies directed to
these polypeptides would be useful in providing immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the vascular system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., vascular, liver, cancerous and
wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder. Preferred polypeptides of the present invention comprise,
or alternatively consist of one, two, three or all four of the
immunogenic epitopes shown in SEQ ID NO: 123 as residues: Arg-18 to
Asp-27, Leu-29 to Arg-36, Ser-90 to Tyr-104, Val-108 to Lys-114.
Polynucleotides encoding said polypeptides are encompassed by the
invention, as are antibodies that bind one or more of these
peptides.
[0063] The tissue distribution primarily in liver and homology to
angiopoietin-2 indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the treatment,
prevention, diagnosis and/or detection of disorders associated with
angiogenesis including the inhibition of angiogenesis and
neovascularisation associated with tumour development; the
promotion of neovascularisation and wound healing; the treatment of
ischaemia; thromboembolytic disease; atherosclerosis; inflammation;
and diabetes. Moreover, polynucleotides and polypeptides
corresponding to this gene may be useful for treating disorders
and/or disease states that include, but are not limited to, solid
tumors, blood born tumors such as leukemias, tumor metastasis,
Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic
neuromas, neurofibromas, trachomas, and pyogenic granulomas,
rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for
example, diabetic retinopathy, retinopathy of prematurity, macular
degeneration, corneal graft rejection, neovascular glaucoma,
retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis,
delayed wound healing, endometriosis, vascluogenesis, granulations,
hypertrophic scars (keloids), nonunion fractures, scleroderma,
trachoma, vascular adhesions, myocardial angiogenesis, coronary
collaterals, cerebral collaterals, arteriovenous malformations,
ischemic limb angiogenesis, Osler-Webber Syndrome, plaque
neovascularization, telangiectasia, hemophiliac joints,
angiofibroma fibromuscular dysplasia, wound granulation, Crohn's
disease, atherosclerosis, birth control agent by preventing
vascularization required for embryo implantation controlling
menstruation, diseases that have angiogenesis as a pathologic
consequence such as cat scratch disease (Rochele minalia quintosa),
ulcers (Helicobacter pylori), Bartonellosis and bacillary
angiomatosis. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0064] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:16 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1622 of SEQ ID NO:16, b is an integer
of 15 to 1636, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:16, and where b is greater
than or equal to a+14.
[0065] Features of Protein Encoded By Gene No: 7
[0066] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: TABLE-US-00002 (SEQ ID NO: 247)
LPPRGPATFGSPGCPPANSPPSAPATPE PARAPERV.
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0067] When tested against fibroblast cell lines, supernatants
removed from cells containing this gene activated the EGR1 assay.
Thus, it is likely that this gene activates fibroblast cells
through a signal transduction pathway. Early growth response 1
(EGR1) is a promoter associated with certain genes that induces
various tissues and cell types upon activation, leading the cells
to undergo differentiation and proliferation. The translation
product of this gene shares sequence homology with murine claudin-1
and other murine and human members of the claudin family of
integral membrane proteins which are structurally similar and
contain four transmembrane domains (see, e.g., Genbank Acc. Nos.
gi|3335182 (AF072127) and/or gi|4128015|gnl|PID|e1363658; all
references available through these accessions are hereby
incorporated in their entirety by reference herein). Three integral
membrane proteins, claudin-1, -2, and occludin, are known to be
components of tight junction (TJ) strands. FLAG-tagged claudin-1
and -2 protein have been demonstrated using immunofluorescence
microscopy to be highly concentrated at cell contact sites as
planes through a homophilic interaction.It is believed that
claudin-1 and -2 are mainly responsible for TJ strand formation,
and occludin is an accessory protein in some function of TJ strands
(see, e.g., J. Cell Biol 143:391-401 (1998), which is hereby
incorporated by reference herein).
[0068] This gene is expressed primarily in wound healing tissues,
and various carcinoma tissues, and, to a lesser extent, in some
other tissues.
[0069] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
tumorigenesis. Similarly, polypeptides and antibodies directed to
these polypeptides would be useful in providing immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of wounded tissues, and cancerous tissues, expression
of this gene at significantly higher or lower levels may be
routinely detected in certain tissues or cell types (e.g.,
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0070] The tissue distribution in healing wound tissue and various
carcinomas indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for detection,
diagnosis, treatment, and/or prevention of wounds and tumors.
Representative uses are described elsewhere herein. Additionally,
the homology of the translation product of this gene to claudin-1,
a integral membrane protein involved in tight junction formation,
and the biological activity of supernatants from cells expressing
this gene on fibroblast cells in EGR assays indicate that
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection, diagnosis, treatment, and/or
prevention of cancer and other proliferative disorders. Expression
within cellular sources marked by proliferating cells (e.g.,
healing wound and various carcinomas) and the homology of the
translation product of this gene to a family of claudin proteins
indicates that this protein may play a role in the regulation of
cellular division and tight junction formation. Furthermore, the
protein may also be used to determine biological activity, to raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0071] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:17 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1242 of SEQ ID NO:17, b is an integer
of 15 to 1256, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:17, and where b is greater
than or equal to a+14.
[0072] Features of Protein Encoded By Gene No: 8
[0073] The translation product of this gene shares sequence
homology with fibulin which is thought to be important in cellular
adhesion and extracellular matrix organization.
[0074] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
GTRAGVSKYTGGRGVTWAPSSAAVPRISSATMRMGLTSFSTTGA (SEQ ID NO: 248),
WQSGHRLWQLEWPPPPLSADEHPWEGPLPGTSPSPKFSMPSPVPHGHHRPTL
TMTRSWRIFFNNIAYRSSSANRLFRVIRREHGDPLIEELNPGDALEPEGRGTGG
VVTDFDGDGMLDLILSHGESMAQPLSVFRGNQGFNNNWLRVVPRTRFGAFA
RGAKVVLYTKKSGAHLRIIDGGSGYLCEMEPVAHFGLGKDEASSVEVTWPD
GKMVSRNVASGEMNSVLEILYPRDEDTLQDPAPLECGQGFSQQENGHCMDT
NECIQFPFVCPRDKPVCVNTYGSYRCRTNKKCSXGLRVPTRMAHTGL (SEQ ID NO: 249),
WQSGHRLWQLEWPPPPLSADEHPWEGPLPGTSPSPK (SEQ ID NO: 250),
FSMPSPVPHGHHRPTLTMTRSWRIFFNNIAYRSSS (SEQ ID NO: 251),
ANRLFRVIRREHGDPLIEELNPGDALEPEGRGTGGVV (SEQ ID NO: 252),
TDFDGDGMLDLILSHGESMAQPLSVFRGNQGFNN (SEQ ID NO: 253),
NWLRVVPRTRFGAFARGAKVVLYTKKSGAHLRIID (SEQ ID NO: 254),
GGSGYLCEMEPVAHFGLGKDEASSVEVTWPDGKMVS (SEQ ID NO: 255),
RNVASGEMNSVLEILYPRDEDTLQDPAPLECGQGF (SEQ ID NO: 256),
SQQENGHCMDTNECIQFPFVCPRDKPVCVNTYGSYR (SEQ ID NO: 257), and/or
CRTNKKCSXGLRVPTRMAHTGL (SEQ ID NO: 258). Moreover, fragments and
variants of these polypeptides (such as, for example, fragments as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0075] The gene encoding the disclosed cDNA is believed to reside
on chromosome 10. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 10.
[0076] This gene is expressed primarily in brain, kidney, Gessler
Wilms tumor, and synovial sarcoma.
[0077] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
thrombosis, atherosclerosis, neoplasia, schizophrenia, Alzheimer's
disease, Parkinson's disease, Huntington's disease, transmissible
spongiform encephalopathies (TSE), Creutzfeldt-Jakob disease (CJD),
specific brain tumors, aphasia, mania, depression and dementia.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the central nervous and cardiovascular systems, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., brain, cancerous
and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine, synovial fluid or cerebrospinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0078] Based on the sequence similarity, the translation product of
this clone is expected to share at least some biological activities
with fibulin proteins. Such activities are known in the art, some
of which are described elsewhere herein. Fibulin itself, can be
used to manipulate adhesion of cells to fibronectin, collagen,
laminin, and possibly also other proteins. The tissue distribution
in brain and the homology to fibulin indicates that polynucleotides
and polypeptides corresponding to this gene would be useful for the
treatment, prevention, detection and/or diagnosis of developmental,
degenerative and/or neoplastic conditions (such as cancer) with
mechanisms contingent on the regulation of cellular adhesion and
extracellular matrix organization. Thrombosis, atherosclerosis and
restenosis may be potential cardiovascular targets for application.
In addition, polynucleotides and polypeptides corresponding to this
gene would be useful for the detection, treatment, and/or
prevention of neurodegenerative disease states, behavioral
disorders, or inflammatory conditions. Representative uses are
described in the "Regeneration" and "Hyperproliferative Disorders"
sections below, in Example 11, 15, and 18, and elsewhere herein.
Briefly, the uses include, but are not limited to the detection,
treatment, and/or prevention of Alzheimer's Disease, Parkinson's
Disease, Huntington's Disease, Tourette Syndrome, meningitis,
encephalitis, demyelinating diseases, peripheral neuropathies,
neoplasia, trauma, congenital malformations, spinal cord injuries,
ischemia and infarction, aneurysms, hemorrhages, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder,
depression, panic disorder, learning disabilities, ALS, psychoses,
autism, and altered behaviors, including disorders in feeding,
sleep patterns, balance, and perception. In addition, elevated
expression of this gene product in regions of the brain indicates
it plays a role in normal neural function. Furthermore, the protein
may also be used to determine biological activity, to raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0079] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:18 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1129 of SEQ ID NO:18, b is an integer
of 15 to 1143, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:18, and where b is greater
than or equal to a+14.
[0080] Features of Protein Encoded By Gene No: 9
[0081] The translation product of this gene shares sequence
homology with carbonic anhydrase VI, which is thought to be
important in protein degradation and pH regulation (see, e.g.,
GenBank Accession No.: BAA78709.1 and Mori K, et al., J Biol Chem.
274:15701-5 (1999); EMBL locus BTCARANVI (accession X96503); and
Jiang et al., Biochem. J. 318:291-296 (1996) which are hereby
incorporated herein in their entireties, by reference). Based on
this homology, it is likely that this gene would have activity
similar to carbonic anhydrase.
[0082] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: QSPIDIQTD (SEQ ID NO: 259),
LHNNGHTVQLSLPSTLYL (SEQ ID NO: 260), YVAAQLHLHWG (SEQ ID NO: 261),
AELHIVHYDSD (SEQ ID NO: 262), GQHWTYEGPHGQDHWP (SEQ ID NO: 263),
QSPIDIQTDSVTFD (SEQ ID NO: 264), LHNNGHTVQLSLPST (SEQ ID NO: 265),
KYVAAQLHLHWG (SEQ ID NO: 266), and/or AELHIVHYDSDSY (SEQ ID NO:
267). Moreover, fragments and variants of these polypeptides (such
as, for example, fragments as described herein, polypeptides at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
these polypeptides, or polypeptides encoded by a polynucleotide
which hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0083] The gene encoding the disclosed cDNA is thought to reside on
chromosome 1. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 1.
[0084] This gene is expressed primarily in fetal tissues and brain
tissue, and, to a lesser extent, in melanocytes, wilms tumor and
retinal tissues.
[0085] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
glaucoma and alkalosis resulting from disease of the kidney.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the systems regulating ionic balance and pH in the fluids of the
body, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues or cell types
(e.g., metabolic, regulatory, renal, cancerous and wounded tissues)
or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of one, two, three, four, five, six or all seven of the
immunogenic epitopes shown in SEQ ID NO: 126 as residues: Tyr-24 to
His-32, Pro-38 to Ala-44, Pro-66 to Glu-75, His-111 to Gly-116,
Tyr-139 to Ser-146, Thr-176 to Ser-181, Lys-239 to Lys-249.
Polynucleotides encoding said polypeptides are encompassed by the
invention, as are antibodies that bind one or more of these
peptides.
[0086] The tissue distribution and homology to secreted carbonic
anhydrase indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for developing drugs
that modulate ionic balance in the serum and in the retina, and may
be used for treating diseases such as glaucoma or alkalosis
secondary to renal disease. Representative uses are described
elsewhere herein. Furthermore, this protein may play a role in the
regulation of cellular division, and may show utility in the
diagnosis, treatment, and/or prevention of developmental diseases
and disorders, including cancer, and other proliferative
conditions. Representative uses are described in the
"Hyperproliferative Disorders" and "Regeneration" sections below
and elsewhere herein. Briefly, developmental tissues rely on
decisions involving cell differentiation and/or apoptosis in
pattern formation. Dysregulation of apoptosis can result in
inappropriate suppression of cell death, as occurs in the
development of some cancers, or in failure to control the extent of
cell death, as is believed to occur in acquired immunodeficiency
and certain neurodegenerative disorders, such as spinal muscular
atrophy (SMA). Alternatively, this gene product may be involved in
the pattern of cellular proliferation that accompanies early
embryogenesis. Thus, aberrant expression of this gene product in
tissues--particularly adult tissues--may correlate with patterns of
abnormal cellular proliferation, such as found in various cancers.
Because of potential roles in proliferation and differentiation,
polynucleotides and polypeptides corresponding to this gene may
have applications in the adult for tissue regeneration and the
treatment of cancers. It may also act as a morphogen to control
cell and tissue type specification. Therefore, the polynucleotides
and polypeptides of the present invention would be useful in
treating, detecting, and/or preventing said disorders and
conditions, in addition to other types of degenerative conditions.
Thus this protein may modulate apoptosis or tissue differentiation
and would be useful in the detection, treatment, and/or prevention
of degenerative or proliferative conditions and diseases.
Polynucleotides and polypeptides corresponding to this gene would
be useful in modulating the immune response to aberrant
polypeptides, as may exist in proliferating and cancerous cells and
tissues. Polynucleotides and polypeptides of the invention can also
be used to gain new insight into the regulation of cellular growth
and proliferation. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues. The protein may
also be used to determine biological activity, to raise antibodies,
as tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0087] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:19 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 toI 523 of SEQ ID NO:19, b is an integer
of 15 to 1537, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:19, and where b is greater
than or equal to a+14.
[0088] Features of Protein Encoded By Gene No: 10
[0089] The translation product of this gene shares sequence
homology with murine CD63/ME491 which is thought to be important in
activation of macrophage and platelet population (marker of); CD37
(Genbank Acc. No. gi|29794, all references available through this
accession are hereby incorporated in their entirety by reference
herein), a human leukocyte marker; and several members of the
tetraspanin protein family (see, e.g., Genbank Acc. No. gi|3152703
(AF065389) and gi|2995865 (AF053455), all references available
through these accessions are hereby incorporated in their entirety
by reference herein), which are expressed in a wide variety of
species and regulate cell adhesion, migration, proliferation and
differentiation.
[0090] This translation product of this gene appears to contain
four transmembrane domains starting from about amino acid positions
24 to about 40, from about 98 to about 114, from about position 62
to about 78, from about position 235 to about 251. Further, this
polypeptide is likely to be a Type IIIa membrane protein (Ncyt
Cexo) as identified using the PSORT analysis tool. The
transmembrane 4 superfamily (TM4SF) which has at least 16 members
is the second biggest subfamily among CD antigen superfamilies and
activation antigens of T-cells. All TM4SF members contain four
putative transmembrane domains, two extracellular loops, and two
short cytoplasmic tails. They are variously expressed on immature,
early, mature, activated lymphocytes, monocytes, macrophages,
granulocytes, platelets, eosinophils, basophils, certain leukemic
and lymphoma cells, and a variety of other cells and tissues. CD9
cell surface protein is expressed by both hematopoietic and neural
cells, and may play a role in intercellular signaling in the immune
and nervous system. CD63 is a 53-Kd lysosomal membrane glycoprotein
that has been identified as a platelet activation molecule; it
plays an important role in cell adhesion of platelets and
endothelial cells. Increased MRNA for CD63 antigen was found in
atherosclerotic lesions of Watanabe heritable hyperlipidemic
rabbits, suggesting a potential role of CD63 in progression of
atherosclerosis. CD63 is also a mast cell marker. This gene also
shares close homology with C33 antigen (CD82); CD82 was originally
identified as the target of several mAbs inhibitory to syncytium
formation induced by human T-cell leukemia virus type I (HTLV-I),
the etiological agent of adult T-cell leukemia. Therefore, this
gene could be a target for the development of a drug for this
leukemia. CD81 is the target of an antiproliferative antibody. A
diverse group of human cell lines, including hematolymphoid,
neuroectodermal, and mesenchymal cells, express the CD81 protein.
Many of the lymphoid cell lines, in particular those derived from
large cell lymphomas, were susceptible to the antiproliferative
effects of the antibody. CD81 may therefore play an important role
in the regulation of lymphoma cell growth. CD9, CD20, CD37, CD63,
CD81 and CD82 have been implicated in the regulation of cell
growth, adhesion, and signal transduction of B, T lymphocytes and
some other non-lymphoid cells. They associate with CD2, CD21, CD4,
CD8, MHC Class II molecules, integrins, and function as co-receptor
for T, B and other lymphoid cells. Some TM4SF are leukocyte
antigens, highly expressed in activated leukocytes, lymphocytes,
and are highly specific surface markers for lymphoblastic leukemia,
lymphoma, melanoma, and neuroblastoma. CD9 has been show to be
involved in cell motility and tumor metastasis. These antigen could
be a valuable immunogen or target to implement active and passive
immunotherapy in patients with cancer. Others have been shown to be
involved in inhibition of prostate cancer metastasis.
[0091] In specific embodiments, polynucleotides of the invention
comprise, or alternatively consist of, the following nucleotide
sequence: GGCCGCGCCGCCGCTGCCGCCGCCGCGCGCGATTCTGCTTCTCAGAAGAT
GCACTATTATAGATACTCTAACGCCAAGGTCAGCTGCTGGTACAAGTACC
TCCTTTTCAGCTACAACATCATCTTCTGATTGGCTGGAGTTGTCTTCCTTGG
AGTCGGGCTGTGGGCATGGAGCGAAAAGGGTGTGCTGTCCGACCTCACCA
AAGTGACCCGGATGCATGGAATCGACCCTGTGGTGCTGGTCCTGATGGTG
GGCGTGGTGATGTTCACCCTGGGGTTCGCCGGCTGCGTGGGGGCTCTGCG
GGAGAATATCTGCTTGCTCAACTTTTTCTGTGGCACCATCGTGCTCATCTT
CTTCCTGGAGCTGGCTGTGGCCGTGCTGGCCTTCCTGTTCCAGGACTGGGT
GAGGGACCGGTTCCGGGAGTTCTTCGAGAGCAACATCAAGTCCTACCGGG
ACGATATCGATCTGCAAAACCTCATCGACTCCCTTCAGAAAGCTAACCAG
TGCTGTGGCGCATATGGCCCTGAAAGACTGGGACCTCAGACGTCTACTTC
AATTGCAGCGGTGCCAGCTACAGCCGAGAGAATGCGGGGTCCCCTTCTCC
TGCTGCGTGCCAGATCCTGCGCAAAAAGTTGTGAACACACAGTGTGGATA
TGATGTCAGGATTCAGCTGAAGAGCAAGTGGGATGAGTCCATCTTCACGA
AAGGCTGCATCCAGGCGCTGGAAAGCTGGCTCCCGCGGAACATTTACATT
GTGGCTGGCGTCTTCATCGCCATCTCGCTGTTGCAGATATTTGGCATCTTC
CTGGCAAGGACGCTGATCTCAGACATCGAGGCAGTGAAGGCCGGCCATCA
CTTCTGAGGAGCAGAGTTGAGGGAGCCGAGCTGAGCCACGCTGGGAGGC
CAGAGCCTTTCTCTGCCATCAGCCCTACGTCCAGAGGGAGAGGAGCCGAC
ACCCCCAGAGCCAGTGCCCCATCTTAAGCATCAGCGTGACGTGACCTCTC
TGTTTCTGCTTGCTGGTGCTGAAGACCAAGGGTCCCCCTTGTTACCTGCCC
AAACTTGTGACTGCATCCCTCTGGAGTCTACCCAGAGACAGAGAATGTGT
CTTTATGTGGGAGTGGTGACTCTGAAAGACAGAGAGGGCTCCTGTGGCTG
CCAGGAGGGCTTGACTCAGACCCCCTGCAGCTCAAGCATGTCTGCAGGAC
ACCTGGTCCCCCTCTCCCAGTGGCATCCCAAACATCTGCTTTGGGTCCATC
CCACATCTGTGGGTGGGCCCGTGGGTAAGAAGGGAACCCCACAGGCGTG
GAACAGGGCATCCTCTCTCCCATCCAAGCAAAGCCAGCATGGGGGCCTGC
CCGTAACGGGAGGCGGACGTGGCCCCGCTGGGCCTCTGAGTGCCAGCGCA
GTCTGCTGGGACATGCACATATCAGGGGTTGTTTGCAGGATCCTCAGCCA
TGTTCAAGTGAAGTAAGCCTGAGCCAGTGCGTGGACTGGTGCCACGGGAG
TGCCTTGTCCACTGTCCCCCTGTGTCCACCAGCTATTCTCCTGGCGCCGGA
ACTGCCTCTGGTCTTGATAGCATTAAGCCCTGATTGGCCGGTGGCGCGGTG
GGCATGGTTCTTCACTGAGAGCCGGCTCTCCTTTTCTTAAAGTGTGTAAAT AGTTTATTT (SEQ
ID NO:268). In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: MHYYRYSNAKVSCWYKYLLFSYNIIFWLAGVVFLGVGLWAWSEKGVLSDL
TKVTRMHGIDPVVLVLMVGVVMFTLGFAGCVGALRENICLLNFFCGTIVLIFF
LELAVAVLAFLFQDWVRDRFREFFESNIKSYRDDIDLQNLIDSLQKANQCCGA
YGPEDWDLNVYFNCSGASYSREKCGVPFSCCVPDPAQKVVNTQCGYDVRIQ
LKSKWDESIFTKGCIQALESWLPRNIYIVAGVFIAISLLQIFGIFLARTLISDIEAV KAGHHF
(SEQ ID NO: 269) Moreover, fragments and variants of these
polypeptides (such as, for example, fragments as described herein,
polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% identical to these polypeptides, or polypeptides encoded by a
polynucleotide which hybridizes, under stringent conditions, to the
polynucleotide encoding these polypeptides) are encompassed by the
invention. Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0092] This gene maps to chromosome 10, and therefore would be
useful in linkage analysis as a marker for chromosome 10.
[0093] This gene is expressed primarily in infant and human brain
and, to a lesser extent, in pancreas islet cell tumor, Wilm's
tumor, uterine cancer, and B cell lymphomas.
[0094] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions: cancers and central nervous system
disorders. Similarly, polypeptides and antibodies directed to those
polypeptides would be useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the, immune, metabolic and central nervous system, expression of
this gene at significantly higher or lower levels may be detected
in certain tissues or cell types (e.g., CNS, cancerous and wounded
tissues) or bodily fluids (e.g., lymph, bile, serum, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue from an individual not
having the disorder. Preferred polypeptides of the present
invention comprise, or alternatively consist of the immunogenic
epitopes shown in SEQ ID NO: 127 as residues: Met-1 to Ala-9.
Polynucleotides encoding said polypeptides are encompassed by the
invention, as are antibodies that bind one or more of these
peptides.
[0095] The tissue distribution in infant and human brain, and
various tumors, and homology to murine CD63/ME491, human CD37, and
tetraspanins indicates that polynucleotides and/or polypeptides
corresponding to this gene would be useful for the study,
detection, treatment, and/or prevention of central nervous system
diseases and cancers. Moreover, the expression within embryonic
tissue and other cellular sources marked by proliferating cells,
and its homology indicates that polynucleotides and/or polypeptides
of the invention may play a role in the regulation of cellular
division, and may show utility in the diagnosis, treatment, and/or
prevention of developmental diseases and disorders, cancer, and
other proliferative conditions. Representative uses are described
in the "Hyperproliferative Disorders" and "Regeneration" sections
below and elsewhere herein. Briefly, developmental tissues rely on
decisions involving cell differentiation and/or apoptosis in
pattern formation. Dysregulation of apoptosis can result in
inappropriate suppression of cell death, as occurs in the
development of some cancers, or in failure to control the extent of
cell death, as is believed to occur in acquired immunodeficiency
and certain neurodegenerative disorders, such as spinal muscular
atrophy (SMA). Because of potential roles in proliferation and
differentiation, this gene product may have applications in the
adult for tissue regeneration and the treatment of cancers. It may
also act as a morphogen to control cell and tissue type
specification. Therefore, the polynucleotides and polypeptides of
the present invention would be useful in treating, detecting,
and/or preventing said disorders and conditions, in addition to
other types of degenerative conditions. Thus this protein may
modulate apoptosis or tissue differentiation and would be useful in
the detection, treatment, and/or prevention of degenerative or
proliferative conditions and diseases. The polynucleotides and/or
polypeptides of the invention would be useful in modulating the
immune response to aberrant polypeptides, as may exist in
proliferating and cancerous cells and tissues. The protein can also
be used to gain new insight into the regulation of cellular growth
and proliferation. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0096] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:20 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2658 of SEQ ID NO:20, b is an integer
of 15 to 2672, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:20, and where b is greater
than or equal to a+14.
[0097] Features of Protein Encoded By Gene No: 11
[0098] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
SQLLPGSVPGWAAHPLRRTVLSPSQHTHNSSHRMKANCEVSASQRLTGRIRH
PRGLLQNSPRSRKLWMRLGLRSRYSGTQARSAPAGGHIVDTAEQRQVQARV
PWAAAVARQLLRYEKAKASAGTPPAHKPCCHYRCCGYSQAQQKPTASAPQ
HLYRPTRPHFRGCRSISV (SEQ ID NO: 279),
SGNLGSADGWAYIDVEVRRPWAFVGPGCSRSSGNGSTAYGLVGSPRWLSPF
HTGGAVSLPRRPRGPGPVLGVARPCLRCVLRPEHYEPGSHYSGFAGRDASRA
FVTGDCSEAGLVDDVSDLSAAEMLTLHNWLSFYEKNYVCVGRVTGRFYGED
GLPTPALTQVEAAITRGLEANKLQLQEKQTFPPCNAEWSSARGSRLWCSQKS
GGVSRDWIGVPRKLYKPGAKEPRCVCVRTTGPPSGQMPDNPPHRNRGDLDH
PNLAEYTGCPPLAITCSFPL (SEQ ID NO: 270),
SGNLGSADGWAYIDVEVRRPWAFVGPGCSRSSGNGS (SEQ ID NO: 271),
TAYGLVGSPRWLSPFHTGGAVSLPRRPRGPGPVLGV (SEQ ID NO: 272),
ARPCLRCVLRPEHYEPGSHYSGFAGRDASRAFVTGD (SEQ ID NO: 273),
CSEAGLVDDVSDLSAAEMLTLHNWLSFYEKNYVCVG (SEQ ID NO: 274),
RVTGRFYGEDGLPTPALTQVEAAITRGLEANKLQLQ (SEQ ID NO: 275),
EKQTFPPCNAEWSSARGSRLWCSQKSGGVSRDWIGV (SEQ ID NO: 276),
PRKLYKPGAKEPRCVCVRTTGPPSGQMPD (SEQ ID NO: 277), and/or
NPPHRNRGDLDHPNLAEYTGCPPLAITCSFPL (SEQ ID NO: 278). Moreover,
fragments and variants of these polypeptides (such as, for example,
fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides,
or polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0099] The translation product of this gene shares sequence
homology to several steroid receptor proteins (see, e.g., Genbank
Acc. Nos. gnl|PID|e314174, gnl|PID|e1154367 (AJ002030), and/or
gnl|PID|e257707); all references available through these accessions
are hereby incorporated by reference herein). Based on the sequence
similarity, the translation product of this clone is expected to
share at least some biological activities with steroid receptor
binding proteins. Such activities are known in the art, some of
which are described elsewhere herein.
[0100] This gene is expressed primarily in brain, fetal tissue,
immune cells (e.g., T-cells), breasts and, to a lesser extent, in
variety of other tissues and cell types.
[0101] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
developmental, degenerative and behavioral diseases of the brain
such as schizophrenia, Alzheimer's disease, Parkinson's disease,
Huntington's disease, transmissible spongiform encephalopathies
(TSE), Creutzfeldt-Jakob disease (CJD), specific brain tumors,
aphasia, mania, depression, dementia, paranoia, addictive behavior
and sleep disorders. Similarly, polypeptides and antibodies
directed to these polypeptides would be useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the brain, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., immune, cancerous
and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder. Preferred polypeptides of the present invention comprise,
or alternatively consist of one, two or all three of the
immunogenic epitopes shown in SEQ ID NO: 128 as residues: Glu-42 to
Pro-53, Ser-67 to Thr-73, Ala-84 to Leu-90. Polynucleotides
encoding said polypeptides are encompassed by the invention, as are
antibodies that bind one or more of these peptides.
[0102] The tissue distribution in brain and the homology to steroid
receptor proteins indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection,
treatment, and/or prevention of neurodegenerative disease states,
behavioral disorders, or inflammatory conditions. Representative
uses are described in the "Regeneration" and "Hyperproliferative
Disorders" sections below, in Example 11, 15, and 18, and elsewhere
herein. Briefly, the uses include, but are not limited to the
detection, treatment, and/or prevention of Alzheimer's Disease,
Parkinson's Disease, Huntington's Disease, Tourette Syndrome,
meningitis, transmissible spongiform encephalopathy (TSE),
Creutzfeldt-Jakob disease (CJD), aphasia, specific brain tumors,
encephalitis, demyelinating diseases, peripheral neuropathies,
neoplasia, trauma, congenital malformations, spinal cord injuries,
ischemia and infarction, aneurysms, hemorrhages, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder,
depression, panic disorder, learning disabilities, ALS, psychoses,
autism, and altered behaviors, including disorders in feeding,
sleep patterns, balance, and perception. In addition, elevated
expression of this gene product in regions of the brain indicates
it plays a role in normal neural function. Potentially, this gene
product is involved in synapse formation, neurotransmission,
learning, cognition, homeostasis, or neuronal differentiation or
survival. The tissue distribution in T-cells indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the diagnosis, detection, prevention, and/or
treatment of a variety of immune system disorders. Representative
uses are described in the "Immune Activity" and "Infectious
Disease" sections below, in Example 11, 13, 14, 16, 18, 19, 20, and
27, and elsewhere herein. Briefly, the expression indicates a role
in regulating the proliferation; survival; differentiation; and/or
activation of hematopoietic cell lineages, including blood stem
cells. Involvement in the regulation of cytokine production,
antigen presentation, or other processes indicates a usefulness for
treatment of cancer (e.g., by boosting immune responses).
Expression in cells of lymphoid origin, indicates the natural gene
product would be involved in immune functions. Therefore it would
also be useful as an agent for immunological disorders including
arthritis, asthma, immunodeficiency diseases such as AIDS,
leukemia, rheumatoid arthritis, granulomatous disease, inflammatory
bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus, this
gene product is thought to be useful in the expansion of stem cells
and committed progenitors of various blood lineages, and in the
differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0103] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:21 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1494 of SEQ ID NO:21, b is an integer
of 15 to 1508, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:21, and where b is greater
than or equal to a+14.
[0104] Features of Protein Encoded By Gene No: 12
[0105] The polypeptide of this gene has been determined to have a
transmembrane domain at about amino acid position 144-160 of the
amino acid sequence referenced in Table 1 for this gene. Moreover,
a cytoplasmic tail encompassing amino acids 161-222 of this protein
has also been determined. Based upon these characteristics, it is
believed that the protein product of this gene shares structural
features to type Ia membrane proteins.
[0106] This gene is expressed primarily in kidney and gall bladder
tissues, fetal tissue, and testes tissue.
[0107] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
renal disorders, metabolic diseases, and disorders of the
reproductive and developing organs. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the renal, metabolic,
developing, and reproductive systems, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., renal, metabolic,
reproductive, cancerous and wounded tissues) or bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder. Preferred polypeptides
of the present invention comprise, or alternatively consist of the
immunogenic epitopes shown in SEQ ID NO: 129 as residues: Lys-60 to
Ala-66. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0108] The tissue distribution in kidney and gall bladder tissues,
testicular tissue, and fetal tissues, indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for treatment, prevention, detection and/or diagnosis of
disorders of the renal system, reproductive system, metabolic
system and developing systems. Furthermore, the tissue distribution
in kidney indicates that polynucleotides and polypeptides
corresponding to this gene would be useful in the treatment,
prevention, diagnosis and/or detection of kidney diseases including
renal failure, nephritus, renal tubular acidosis, proteinuria,
pyuria, edema, pyelonephritis, hydronephritis, nephrotic syndrome,
crush syndrome, glomerulonephritis, hematuria, renal colic and
kidney stones, in addition to Wilm's Tumor Disease, and congenital
kidney abnormalities such as horseshoe kidney, polycystic kidney,
and Falconi's syndrome. Alternatively, the tissue distribution
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the treatment and diagnosis of
conditions concerning proper testicular fimction (e.g., endocrine
function, sperm maturation), as well as cancer. Therefore, this
gene product would be useful in the treatment of male infertility
and/or impotence. This gene product is also useful in assays
designed to identify binding agents, as such agents (antagonists)
would be useful as male contraceptive agents. Similarly, the
protein is believed to be useful in the treatment and/or diagnosis
of testicular cancer. The testes are also a site of active gene
expression of transcripts that may be expressed, particularly at
low levels, in other tissues of the body. Therefore, this gene
product may be expressed in other specific tissues or organs where
it may play related functional roles in other processes, such as
hematopoiesis, inflammation, bone formation, and kidney function,
to name a few possible target indications. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0109] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:22 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1433 of SEQ ID NO:22, b is an integer
of 15 to 1447, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:22, and where b is greater
than or equal to a+14.
[0110] Features of Protein Encoded By Gene No: 13
[0111] The translation product of this gene shares weak homology
with O-linked GlcNAc transferases (see, e.g., Genbank Acc. No.
gi|2266994) which are important for a variety of cellular
functions, including, but not limited to, stability of secreted
proteins and proper function. Based on the sequence similarity, the
translation product of this clone is expected to share at least
some biological activities with glycosylation enzyme proteins. Such
activities are known in the art, (see, e.g., G Lubas W A, et al., J
Biol Chem. 272:9316-24 (1997); all references available through
this citation are hereby incorporated herein by reference) and some
of which are described elsewhere herein.
[0112] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: LLLCPWWLCFDWS (SEQ ID NO: 280),
MGCIPLIKSISDWRVIALAALWFCLIGLICQALCSEDGHKRRILTLGLGFLVIPF
LPASNLFFRVGFVVAECVLYLPSIGYCVLLTFGFGALSKHTKKKKLIAAVVLG
ILFINTLRCVLRTAKWRSEEQLFRSALSVCPLNAKVHYNIGKNLADKGNQTA
AIRYYREAVRLNPKYVHAMNNLGNILKERNELQEAEELLSLAVQIQPDFAAA
WMNLGIVQNSLKRFETAEQNYRTAIKHRRKYPDCYYNLGRLVRTGCPVPVE GKMGYFS (SEQ ID
NO: 281), MGCIPLIKSISDWRVIALAALWFCLIGLICQALCSEDG (SEQ ID NO: 282),
HKRRILTLGLGFLVIPFLPASNLFFRVGFVVAECVLYL (SEQ ID NO: 283),
PSIGYCVLLTFGFGALSKHTKKKKLIAAVVLGILFINT (SEQ ID NO: 284),
LRCVLRTAKWRSEEQLFRSALSVCPLNAKVHYNIGKNL (SEQ ID NO: 285),
ADKGNQTAAIRYYREAVRLNPKYVHAMNNLGNILKERN (SEQ ID NO: 286),
ELQEAEELLSLAVQIQPDFAAAWMNLGIVQNSLKRFET (SEQ ID NO: 287), and/or
AEQNYRTAIKHRRKYPDCYYNLGRLVRTGCPVPVEGKMGYFS (SEQ ID NO: 288).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0113] The polypeptide encoded by this gene has been determined to
have transmembrane domains at about amino acid position 38 to about
54, at about 136 to about 152, at about 161 to about 177, at about
192 to about 208, at about 223 to about 239, at about 243 to about
259, at about 374 to about 390, at about 402 to about 418, at about
432 to about 448, and at about 461 to about 477 of the amino acid
sequence referenced in Table 7 for this gene. Based upon these
characteristics, it is believed that the protein product of this
gene shares structural features to type IIIa membrane proteins.
[0114] Included in this invention as preferred domains are
Aldolketo reductase family putative active site signatures, which
were identified using the ProSite analysis tool (Swiss Institute of
Bioinformatics). The aldo-keto reductase family groups together a
number of structurally and functionally related NADPH-dependent
oxidoreductases as well as some other proteins. Three consensus
patterns specific to this family of proteins were developed. The
third pattern, located in the C-terminal, is centered on a lysine
residue whose chemical modification, in aldose and aldehyde
reductases, affect the catalytic efficiency. The consensus pattern
is as follows:
[LIVM]-[PAIV]-[KR]-[ST]-x(4)-R-x(2)-[GSTAEQK]-[NSL]-x(2)-[LIVMFA]
[K is a putative active site residue]. In specific embodiments,
polypeptides of the invention comprise, or alternatively consist
of, the following amino acid sequence: LIKSISDWRVIALAAL (SEQ ID NO:
289). Moreover, fragments and variants of these polypeptides (such
as, for example, fragments as described herein, polypeptides at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
these polypeptides, or polypeptides encoded by a polynucleotide
which hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention. Further preferred are polypeptides comprising the
Aldo/keto reductase family putative active site signature above,
and at least 5, 10, 15, 20, 25, 30, 50, or 75 additional contiguous
amino acid residues of the amino acid sequence referenced in Table
7 for this gene. The additional contiguous amino acid residues may
be N-terminal or C-terminal to the Aldo/keto reductase family
putative active site signatures. Alternatively, the additional
contiguous amino acid residues may be both N-terminal and
C-terminal to the Aldo/keto reductase family putative active site
signatures, wherein the total N- and C-terminal contiguous amino
acid residues equal the specified number.
[0115] FIGS. 1A-G show the nucleotide (SEQ ID NO:23) and deduced
amino acid sequence (SEQ ID NO: 130) corresponding to this
gene.
[0116] FIG. 2 shows an analysis of the amino acid sequence (SEQ ID
NO: 130). Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic
index and surface probability are shown, and all were generated
using the default settings of the recited computer algorithyms. In
the "Antigenic Index or Jameson-Wolf" graph, the positive peaks
indicate locations of the highly antigenic regions of the protein,
i.e., regions from which epitope-bearing peptides of the invention
can be obtained. Polypeptides comprising, or alternatively
consisting of, domains defined by these graphs are contemplated by
the present invention, as are polynucleotides encoding these
polypeptides.
[0117] The data presented in FIG. 2 are also represented in tabular
form in Table 3. The columns are labeled with the headings "Res",
"Position", and Roman Numerals I-XIV. The column headings refer to
the following features of the amino acid sequence presented in FIG.
2, and Table 3: "Res": amino acid residue of SEQ ID NO: 130 and
FIGS. 1A-G; "Position": position of the corresponding residue
within SEQ ID NO: 130 and FIGS. 1A-G; I: Alpha,
Regions--Garnier-Robson; II: Alpha, Regions--Chou-Fasman; III:
Beta, Regions--Garnier-Robson; IV: Beta, Regions--Chou-Fasman; V:
Turn, Regions--Gamier-Robson; VI: Turn, Regions--Chou-Fasman; VII:
Coil, Regions--Garnier-Robson; VIII: Hydrophilicity
Plot--Kyte-Doolittle; IX: Hydrophobicity Plot--Hopp-Woods; X:
Alpha, Amphipathic Regions--Eisenberg; XI: Beta, Amphipathic
Regions--Eisenberg; XII: Flexible Regions--Karplus-Schulz; XIII:
Antigenic Index--Jameson-Wolf; and XIV: Surface Probability
Plot--Emini.
[0118] Preferred embodiments of the invention in this regard
include fragments that comprise, or alternatively consisting of,
one or more of the following regions: alpha-helix and alpha-helix
forming regions ("alpha-regions"), beta-sheet and beta-sheet
forming regions ("beta-regions"), turn and turn-forming regions
("turn-regions"), coil and coil-forming regions ("coil-regions"),
hydrophilic regions, hydrophobic regions, alpha amphipathic
regions, beta amphipathic regions, flexible regions,
surface-forming regions and high antigenic index regions. The data
representing the structural or functional attributes of the protein
set forth in FIG. 2 and/or Table 3, as described above, was
generated using the various modules and algorithms of the DNA*STAR
set on default parameters. In a preferred embodiment, the data
presented in columns VIII, IX, XIII, and XIV of Table 3 can be used
to determine regions of the protein which exhibit a high degree of
potential for antigenicity. Regions of high antigenicity are
determined from the data presented in columns VIII, IX, XIII,
and/or XIV by choosing values which represent regions of the
polypeptide which are likely to be exposed on the surface of the
polypeptide in an environment in which antigen recognition may
occur in the process of initiation of an immune response.
[0119] Certain preferred regions in these regards are set out in
FIG. 2, but may, as shown in Table 3, be represented or identified
by using tabular representations of the data presented in FIG. 2.
The DNA*STAR computer algorithm used to generate FIG. 2 (set on the
original default parameters) was used to present the data in FIG. 2
in a tabular format (See Table 3). The tabular format of the data
in FIG. 2 is used to easily determine specific boundaries of a
preferred region.
[0120] The present invention is further directed to fragments of
the polynucleotide sequences described herein. By a fragment of,
for example, the polynucleotide sequence of a deposited cDNA or the
nucleotide sequence shown in SEQ ID NO:23, is intended
polynucleotide fragments at least about 15 nt, and more preferably
at least about 20 nt, at least about 25 nt, still more preferably
at least about 30 nt, at least about 35 nt, and even more
preferably, at least about 40 nt in length, at least about 45 nt in
length, at least about 50 nt in length, at least about 60 nt in
length, at least about 70 nt in length, at least about 80 nt in
length, at least about 90 nt in length, at least about 100 nt in
length, at least about 125 nt in length, at least about 150 nt in
length, at least about 175 nt in length, which are useful as
diagnostic probes and primers as discussed herein. Of course,
larger fragments 200-1500 nt in length are also useful according to
the present invention, as are fragments corresponding to most, if
not all, of the nucleotide sequence of a deposited cDNA or as shown
in SEQ ID NO:23. By a fragment at least 20 nt in length, for
example, is intended fragments which include 20 or more contiguous
bases from the nucleotide sequence of a deposited cDNA or the
nucleotide sequence as shown in SEQ ID NO:23. In this context
"about" includes the particularly recited size, an sizes larger or
smaller by several (5, 4, 3, 2, or 1) nucleotides, at either
terminus or at both termini. Representative examples of
polynucleotide fragments of the invention include, for example,
fragments that comprise, or alternatively, consist of, a sequence
from about nucleotide 1 to about 50, from about 51 to about 100,
from about 101 to about 150, from about 151 to about 200, from
about 201 to about 250, from about 251 to about 300, from about 301
to about 350, from about 351 to about 400, from about 401 to about
450, from about 451 to about 500, and from about 501 to about 550,
and from about 551 to about 600, from about 601 to about 650, from
about 651 to about 700, from about 701 to about 750, from about 751
to about 800, from about 801 to about 850, from about 851 to about
900, from about 901 to about 950, from about 951 to about 1000,
from about 1001 to about 1050, from about 1051 to about 1100, from
about 1101 to about 1150 from about 1151 to about 1200, from about
1201 to about 1250, from about 1251 to about 1300, from about 1301
to about 1350, from about 1351 to about 1400, from about 1401 to
about 1450, from about 1451 to about 1500, from about 1501 to about
1550, from about 1551 to about 1600, from about 1601 to about 1650,
from about 1651 to about 1700, from about 1701 to about 1750, from
about 1751 to about 1800, from about 1801 to about 1850, from about
1851 to about 1900, from about 1901 to about 1950, from about 1951
to about 2000, from about 2001 to about 2050, from about 2051 to
about 2100, from about 2101 to about 2150 from about 2151 to about
2200, from about 2201 to about 2250, from about 2251 to about 2300,
from about 2301 to about 2350, from about 2351 to about 2400, from
about 2401 to about 2450, from about 2451 to about 2500, 2501 to
about 2550, from about 2551 to about 2600, from about 2601 to about
2650, from about 2651 to about 2700, from about 2701 to about 2750,
from about 2751 to about 2800, from about 2801 to about 2850, from
about 2851 to about 2900, from about 2901 to about 2950, from about
2951 to about 3000, from about 3001 to about 3050, from about 3051
to about 3100, from about 3101 to about 3150 from about 3151 to
about 3200, from about 3201 to about 3250, from about 3251 to about
3300, from about 3301 to about 3350, from about 3351 to about 3400,
from about 3401 to about 3450, from about 3451 to about 3500, 3501
to about 3550, from about 3551 to about 3600, from about 3601 to
about 3650, from about 3651 to about 3700, from about 3701 to about
3750, from about 3751 to about 3800, from about 3801 to about 3850,
and from about 3851 to 3886 of SEQ ID NO:23, or the complementary
strand thereto, or the cDNA contained in a deposited clone. In this
context "about" includes the particularly recited ranges, and
ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides,
at either terminus or at both termini. In additional embodiments,
the polynucleotides of the invention encode functional attributes
of the corresponding protein.
[0121] Preferred polypeptide fragments of the invention comprise,
or alternatively consist of, the secreted protein having a
continuous series of deleted residues from the amino or the carboxy
terminus, or both. Particularly, N-terminal deletions of the
polypeptide can be described by the general formula m-760 where m
is an integer from 2 to 755, where m corresponds to the position of
the amino acid residue identified in SEQ ID NO:130. More in
particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, an amino
acid sequence selected from the group: I-2 to V-760; P-3 to V-760;
N-4 to V-760; Q-5 to V-760; H-6 to V-760; N-7 to V-760; A-8 to
V-760; G-9 to V-760; A-10 to V-760; G-11 to V-760; S-12 to V-760;
H-13 to V-760; Q-14 to V-760; P-15 to V-760; A-16 to V-760; V-17 to
V-760; F-18 to V-760; R-19 to V-760; M-20 to V-760; A-21 to V-760;
V-22 to V-760; L-23 to V-760; D-24 to V-760; T-25 to V-760; D-26 to
V-760; L-27 to V-760; D-28 to V-760; H-29 to V-760; I-30 to V-760;
L-31 to V-760; P-32 to V-760; S-33 to V-760; S-34 to V-760; V-35 to
V-760; L-36 to V-760; P-37 to V-760; P-38 to V-760; F-39 to V-760;
W-40 to V-760; A-41 to V-760; K-42 to V-760; L-43 to V-760; V-44 to
V-760; V-45 to V-760; G-46 to V-760; S-47 to V-760; V-48 to V-760;
A-49 to V-760; I-50 to V-760; V-51 to V-760; C-52 to V-760; F-53 to
V-760; A-54 to V-760; R-55 to V-760; S-56 to V-760; Y-57 to V-760;
D-58 to V-760; G-59 to V-760; D-60 to V-760; F-61 to V-760; V-62 to
V-760; F-63 to V-760; D-64 to V-760; D-65 to V-760; S-66 to V-760;
E-67 to V-760; A-68 to V-760; I-69 to V-760; V-70 to V-760; N-71 to
V-760; N-72 to V-760; K-73 to V-760; D-74 to V-760; L-75 to V-760;
Q-76 to V-760; A-77 to V-760; E-78 to V-760; T-79 to V-760; P-80 to
V-760; L-81 to V-760; G-82 to V-760; D-83 to V-760; L-84 to V-760;
W-85 to V-760; H-86 to V-760; H-87 to V-760; D-88 to V-760; F-89 to
V-760; W-90 to V-760; G-91 to V-760; S-92 to V-760; R-93 to V-760;
L-94 to V-760; S-95 to V-760; S-96 to V-760; N-97 to V-760; T-98 to
V-760; S-99 to V-760; H-100 to V-760; K-101 to V-760; S-102 to
V-760; Y-103 to V-760; R-104 to V-760; P-105 to V-760; L-106 to
V-760; T-107 to V-760; V-108 to V-760; L-109 to V-760; T-110 to
V-760; F-111 to V-760; R-112 to V-760; I-113 to V-760; N-114 to
V-760; Y-105 to V-760; Y-116 to V-760; L-117 to V-760; S-118 to
V-760; G-119 to V-760; G-120 to V-760; F-121 to V-760; H-122 to
V-760; P-123 to V-760; V-124 to V-760; G-125 to V-760; F-126 to
V-760; H-127 to V-760; V-128 to V-760; V-129 to V-760; N-130 to
V-760; I-131 to V-760; L-132 to V-760; L-133 to V-760; H-134 to
V-760; S-135 to V-760; G-136 to V-760; I-137 to V-760; S-138 to
V-760; V-139 to V-760; L-140 to V-760; M-141 to V-760; V-142 to
V-760; D-143 to V-760; V-144 to V-760; F-145 to V-760; S-146 to
V-760; V-147 to V-760; L-148 to V-760; F-149 to V-760; G-150 to
V-760; G-151 to V-760; L-152 to V-760; Q-153 to V-760; Y-154 to
V-760; T-155 to V-760; S-156 to V-760; K-157 to V-760; G-158 to
V-760; R-159 to V-760; R-160 to V-760; L-161 to V-760; H-162 to
V-760; L-163 to V-760; A-164 to V-760; P-165 to V-760; R-166 to
V-760; A-167 to V-760; S-168 to V-760; L-169 to V-760; L-170 to
V-760; A-171 to V-760; A-172 to V-760; L-173 to V-760; L-174 to
V-760; F-175 to V-760; A-176 to V-760; V-177 to V-760; H-178 to
V-760; P-179 to V-760; V-180 to V-760; H-181 to V-760; T-182 to
V-760; E-183 to V-760; C-184 to V-760; V-185 to V-760; A-186 to
V-760; G-187 to V-760; V-188 to V-760; V-189 to V-760; G-190 to
V-760; R-191 to V-760; A-192 to V-760; D-193 to V-760; L-194 to
V-760; L-195 to V-760; C-196 to V-760; A-197 to V-760; L-198 to
V-760; F-199 to V-760; F-200 to V-760; L-201 to V-760; L-202 to
V-760; S-203 to V-760; F-204 to V-760; L-205 to V-760; G-206 to
V-760; Y-207 to V-760; C-208 to V-760; K-209 to V-760; A-210 to
V-760; F-211 to V-760; R-212 to V-760; E-213 to V-760; S-214 to
V-760; N-215 to V-760; K-216 to V-760; E-217 to V-760; G-218 to
V-760; A-219 to V-760; H-220 to V-760; S-221 to V-760; S-222 to
V-760; T-223 to V-760; F-224 to V-760; W-225 to V-760; V-226 to
V-760; L-227 to V-760; L-228 to V-760; S-229 to V-760; I-230 to
V-760; F-231 to V-760; L-232 to V-760; G-233 to V-760; A-234 to
V-760; V-235 to V-760; A-236 to V-760; M-237 to V-760; L-238 to
V-760; C-239 to V-760; K-240 to V-760; E-241 to V-760; Q-242 to
V-760; G-243 to V-760; I-244 to V-760; T-245 to V-760; V-246 to
V-760; L-247 to V-760; G-248 to V-760; L-249 to V-760; N-250 to
V-760; A-251 to V-760; V-252 to V-760; F-253 to V-760; D-254 to
V-760; I-255 to V-760; L-256 to V-760; V-257 to V-760; I-258 to
V-760; G-259 to V-760; K-260 to V-760; F-261 to V-760; N-262 to
V-760; V-263 to V-760; L-264 to V-760; E-265 to V-760; I-266 to
V-760; X-267 to V-760; Q-268 to V-760; K-269 to V-760; V-270 to
V-760; L-271 to V-760; H-272 to V-760; K-273 to V-760; D-274 to
V-760; K-275 to V-760; S-276 to V-760; L-277 to V-760; E-278 to
V-760; N-279 to V-760; L-280 to V-760; G-281 to V-760; M-282 to
V-760; L-283 to V-760; R-284 to V-760; N-285 to V-760; G-286 to
V-760; G-287 to V-760; L-288 to V-760; L-289 to V-760; F-290 to
V-760; R-291 to V-760; M-292 to V-760; T-293 to V-760; L-294 to
V-760; L-295 to V-760; T-296 to V-760; S-297 to V-760; G-298 to
V-760; G-299 to V-760; A-300 to V-760; G-301 to V-760; M-302 to
V-760; L-303 to V-760; Y-304 to V-760; V-305 to V-760; R-306 to
V-760; W-307 to V-760; R-308 to V-760; I-309 to V-760; M-310 to
V-760; G-311 to V-760; T-312 to V-760; G-313 to V-760; P-314 to
V-760; X-315 to V-760; A-316 to V-760; F-317 to V-760; T-318 to
V-760; E-319 to V-760; V-320 to V-760; D-321 to V-760; N-322 to
V-760; P-323 to V-760; A-324 to V-760; S-325 to V-760; F-326 to
V-760; A-327 to V-760; D-328 to V-760; S-329 to V-760; M-330 to
V-760; L-331 to V-760; V-332 to V-760; R-333 to V-760; A-334 to
V-760; V-335 to V-760; N-336 to V-760; Y-337 to V-760; N-338 to
V-760; Y-339 to V-760; Y-340 to V-760; Y-341 to V-760; S-342 to
V-760; L-343 to V-760; N-344 to V-760; A-345 to V-760; W-346 to
V-760; L-347 to V-760; L-348 to V-760; L-349 to V-760; C-350 to
V-760; P-351 to V-760; W-352 to V-760; W-353 to V-760; L-354 to
V-760; C-355 to V-760; F-356 to V-760; D-357 to V-760; W-358 to
V-760; S-359 to V-760; M-360 to V-760; G-361 to V-760; C-362 to
V-760; I-363 to V-760; P-364 to V-760; L-365 to V-760; I-366 to
V-760; K-367 to V-760; S-368 to V-760; I-369 to V-760; S-370 to
V-760; D-371 to V-760; W-372 to V-760; R-373 to V-760; V-374 to
V-760; I-375 to V-760; A-376 to V-760; L-377 to V-760; A-378 to
V-760; A-379 to V-760; L-380 to V-760; W-381 to V-760; F-382 to
V-760; C-383 to V-760; L-384 to V-760; I-385 to V-760; G-386 to
V-760; L-387 to V-760; I-388 to V-760; C-389 to V-760; Q-390 to
V-760; A-391 to V-760; L-392 to V-760; C-393 to V-760; S-394 to
V-760; E-395 to V-760; D-396 to V-760; G-397 to V-760; H-398 to
V-760; K-399 to V-760; R-400 to V-760; R-401 to V-760; I-402 to
V-760; L-403 to V-760; T-404 to V-760; L-405 to V-760; G-406 to
V-760; L-407 to V-760; G-408 to V-760; F-409 to V-760; L-410 to
V-760; V-411 to V-760; I-412 to V-760; P-413 to V-760; F-414 to
V-760; L-415 to V-760; P-416 to V-760; A-417 to V-760; S-418 to
V-760; N-419 to V-760; L-420 to V-760; F-421 to V-760; F-422 to
V-760; R-423 to V-760; V-424 to V-760; G-425 to V-760; F-426 to
V-760; V-427 to V-760; V-428 to V-760; A-429 to V-760; E-430 to
V-760; R-431 to V-760; V-432 to V-760; L-433 to V-760; Y-434 to
V-760; L-435 to V-760; P-436 to V-760; S-437 to V-760; X-438 to
V-760; G-439 to V-760; Y-440 to V-760; C-441 to V-760; V-442 to
V-760; L-443 to V-760; L-444 to V-760; T-445 to V-760; F-446 to
V-760; G-447 to V-760; F-448 to V-760; G-449 to V-760; A-450 to
V-760; L-451 to V-760; S-452 to V-760; K-453 to V-760; H-454 to
V-760; T-455 to V-760; K-456 to V-760; K-457 to V-760; K-458 to
V-760; K-459 to V-760; L-460 to V-760; I-461 to V-760; A-462 to
V-760; A-463 to V-760; V-464 to V-760; V-465 to V-760; L-466 to
V-760; G-467 to V-760; I-468 to V-760; L-469 to V-760; F-470 to
V-760; I-471 to V-760; N-472 to V-760; T-473 to V-760; L-474 to
V-760; R-475 to V-760; C-476 to V-760; V-477 to V-760; L-478 to
V-760; R-479 to V-760; S-480 to V-760; G-481 to V-760; E-482 to
V-760; W-483 to V-760; R-484 to V-760; S-485 to V-760; E-486 to
V-760; E-487 to V-760; Q-488 to V-760; L-489 to V-760; F-490 to
V-760; R-491 to V-760; S-492 to V-760; A-493 to V-760; L-494 to
V-760; S-495 to V-760; V-496 to V-760; C-497 to V-760; P-498 to
V-760; L-499 to V-760; N-500 to V-760; A-501 to V-760; K-502 to
V-760; V-503 to V-760; H-504 to V-760; Y-505 to V-760; N-506 to
V-760; I-507 to V-760; G-508 to V-760; K-509 to V-760; N-510 to
V-760; L-511 to V-760; A-512 to V-760; D-513 to V-760; K-514 to
V-760; G-515 to V-760; N-516 to V-760; Q-517 to V-760; T-518 to
V-760; A-519 to V-760; A-520 to V-760; I-521 to V-760; R-522 to
V-760; Y-523 to V-760; Y-524 to V-760; R-525 to V-760; E-526 to
V-760; A-527 to V-760; V-528 to V-760; R-529 to V-760; L-530 to
V-760; N-531 to V-760; P-532 to V-760; K-533 to V-760; Y-534 to
V-760; V-535 to V-760; H-536 to V-760; A-537 to V-760; M-538 to
V-760; N-539 to V-760; N-540 to V-760; L-541 to V-760; G-542 to
V-760; N-543 to V-760; I-544 to V-760; L-545 to V-760; K-546 to
V-760; E-547 to V-760; R-548 to V-760; N-549 to V-760; E-550 to
V-760; L-551 to V-760; Q-552 to V-760; E-553 to V-760; A-554 to
V-760; E-555 to V-760; E-556 to V-760; L-557 to V-760; L-558 to
V-760; S-559 to V-760; L-560 to V-760; A-561 to V-760; V-562 to
V-760; Q-563 to V-760; S-564 to V-760; Q-565 to V-760; P-566 to
V-760; D-567 to V-760; F-568 to V-760; A-569 to V-760; A-570 to
V-760; A-571 to V-760; W-572 to V-760; M-573 to V-760; N-574 to
V-760; L-575 to V-760; G-576 to V-760; W-577 to V-760; V-578 to
V-760; Q-579 to V-760; N-580 to V-760; S-581 to V-760; L-582 to
V-760; K-583 to V-760; R-584 to V-760; F-585 to V-760; E-586 to
V-760; A-587 to V-760; A-588 to V-760; E-589 to V-760; Q-590 to
V-760; S-591 to V-760; Y-592 to V-760; R-593 to V-760; T-594 to
V-760; A-595 to V-760; S-596 to V-760; K-597 to V-760; H-598 to
V-760; R-599 to V-760; R-600 to V-760; K-601 to V-760; Y-602 to
V-760; P-603 to V-760; D-604 to V-760; C-605 to V-760; Y-606 to
V-760; Y-607 to V-760; N-608 to V-760; L-609 to V-760; G-610 to
V-760; R-611 to V-760; L-612 to V-760; Y-613 to V-760; A-614 to
V-760; D-615 to V-760; L-616 to V-760; N-617 to V-760; R-618 to
V-760; H-619 to V-760; V-620 to V-760; D-621 to V-760; A-622 to
V-760; L-623 to V-760; N-624 to V-760; A-625 to V-760; W-626 to
V-760; R-627 to V-760; N-628 to V-760; A-629 to V-760; T-630 to
V-760; V-631 to V-760; L-632 to V-760; K-633 to V-760; P-634 to
V-760; E-635 to V-760; H-636 to V-760; S-637 to V-760; L-638 to
V-760; A-639 to V-760; W-640 to V-760; N-641 to V-760; N-642 to
V-760; M-643 to V-760; A-644 to V-760; W-645 to V-760; L-646 to
V-760; L-647 to V-760; D-648 to V-760; N-649 to V-760; T-650 to
V-760; G-651 to V-760; N-652 to V-760; L-653 to V-760; A-654 to
V-760; Q-655 to V-760; A-656 to V-760; E-657 to V-760; A-658 to
V-760; V-659 to V-760; G-660 to V-760; R-661 to V-760; E-662 to
V-760; A-663 to V-760; L-664 to V-760; E-665 to V-760; L-666 to
V-760; E-667 to V-760; P-668 to V-760; N-669 to V-760; D-670 to
V-760; H-671 to V-760; S-672 to V-760; L-673 to V-760; M-674 to
V-760; F-675 to V-760; S-676 to V-760; L-677 to V-760; A-678 to
V-760; N-679 to V-760; V-680 to V-760; L-681 to V-760; G-682 to
V-760; K-683 to V-760; S-684 to V-760; Q-685 to V-760; K-686 to
V-760; Y-687 to V-760; K-688 to V-760; E-689 to V-760; S-690 to
V-760; E-691 to V-760; A-692 to V-760; L-693 to V-760; F-694 to
V-760; L-695 to V-760; K-696 to V-760; A-697 to V-760; L-698 to
V-760; K-699 to V-760; A-700 to V-760; N-701 to V-760; P-702 to
V-760; N-703 to V-760; A-704 to V-760; A-705 to V-760; S-706 to
V-760; Y-707 to V-760; H-708 to V-760; G-709 to V-760; N-710 to
V-760; L-711 to V-760; A-712 to V-760; V-713 to V-760; L-714 to
V-760; Y-715 to V-760; H-716 to V-760; R-717 to V-760; W-718 to
V-760; G-719 to V-760; H-720 to V-760; L-721 to V-760; D-722 to
V-760; L-723 to V-760; A-724 to V-760; K-725 to V-760; K-726 to
V-760; H-727 to V-760; Y-728 to V-760; E-729 to V-760; I-730 to
V-760; S-731 to V-760; L-732 to V-760; Q-733 to V-760; L-734 to
V-760; D-735 to V-760; P-736 to V-760; T-737 to V-760; A-738 to
V-760; S-739 to V-760; G-740 to V-760; T-741 to V-760; K-742 to
V-760; E-743 to V-760; N-744 to V-760; Y-745 to V-760; G-746 to
V-760; L-747 to V-760; L-748 to V-760; R-749 to V-760; R-750 to
V-760; K-751 to V-760; L-752 to V-760; E-753 to V-760; L-754 to
V-760; and M-755 to V-760 of SEQ ID NO:130. Polypeptides encoded by
these polynucleotides are also encompassed by the invention.
[0122] Also as mentioned above, even if deletion of one or more
amino acids from the C-terminus of a protein results in
modification of loss of one or more biological functions of the
protein, other functional activities (e.g., biological activities,
ability to multimerize, ability to bind ligand, ability to generate
antibodies, ability to bind antibodies) may still be retained. For
example the ability of the shortened polypeptide to induce and/or
bind to antibodies which recognize the complete or mature forms of
the polypeptide generally will be retained when less than the
majority of the residues of the complete or mature polypeptide are
removed from the C-terminus. Whether a particular polypeptide
lacking C-terminal residues of a complete polypeptide retains such
immunologic activities can readily be determined by routine methods
described herein and otherwise known in the art. It is not unlikely
that a polypeptide with a large number of deleted C-terminal amino
acid residues may retain some biological or immunogenic activities.
In fact, peptides composed of as few as six amino acid residues may
often evoke an immune response.
[0123] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the carboxy
terminus of the amino acid sequence of the polypeptide shown in
FIGS. 1A-G (SEQ ID NO:130), as described by the general formula
1-n, where n is an integer from 6 to 759, where n corresponds to
the position of the amino acid residue identified in SEQ ID NO:130.
More in particular, the inventioriprovides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, an amino
acid sequence selected from the group: M-1 to A-759; M-1 to K-758;
M-1 to K-757; M-1 to Q-756; M-1 to M-755; M-1 to L-754; M-1 to
E-753; M-1 to L-752; M-1 to K-751; M-1 to R-750; M-1 to R-749; M-1
to L-748; M-1 to L-747; M-1 to G-746; M-1 to Y-745; M-1 to N-744;
M-1 to E-743; M-1 to K-742; M-1 to T-741; M-1 to G-740; M-1 to
S-739; M-1 to A-738; M-1 to T-737; M-1 to P-736; M-1 to D-735; M-1
to L-734; M-1 to Q-733; M-1 to L-732; M-1 to S-731; M-1 to I-730;
M-1 to E-729; M-1 to Y-728; M-1 to H-727; M-1 to K-726; M-1 to
K-725; M-1 to A-724; M-1 to L-723; M-1 to D-722; M-1 to L-721; M-1
to H-720; M-1 to G-719; M-1 to W-718; M-1 to R-717; M-1 to H-716;
M-1 to Y-715; M-1 to L-714; M-1 to V-713; M-1 to A-712; M-1 to
L-711; M-1 to N-710; M-1 to G-709; M-1 to H-708; M-1 to Y-707; M-1
to S-706; M-1 to A-705; M-1 to A-704; M-1 to N-703; M-1 to P-702;
M-1 to N-701; M-1 to A-700; M-1 to K-699; M-1 to I-698; M-1 to
A-697; M-1 to K-696; M-1 to L-695; M-1 to F-694; M-1 to L-693; M-1
to A-692; M-1 to E-691; M-1 to S-690; M-1 to E-689; M-1 to K-688;
M-1 to Y-687; M-1 to K-686; M-1 to Q-685; M-1 to S-684; M-1 to
K-683; M-1 to G-682; M-1 to L-681; M-1 to V-680; M-1 to N-679; M-1
to A-678; M-1 to L-677; M-1 to S-676; M-1 to F-675; M-1 to M-674;
M-1 to L-673; M-1 to S-672; M-1 to H-671; M-1 to D-670; M-1 to
N-669; M-1 to P-668; M-1 to I-667; M-1 to L-666; M-1 to E-665; M-1
to L-664; M-1 to A-663; M-1 to E-662; M-1 to R-661; M-1 to G-660;
M-1 to V-659; M-1 to A-658; M-1 to E-657; M-1 to A-656; M-1 to
Q-655; M-1 to A-654; M-1 to L-653; M-1 to N-652; M-1 to G-651; M-1
to T-650; M-1 to N-649; M-1 to D-648; M-1 to L-647; M-1 to L-646;
M-1 to I-645; M-1 to I-644; M-1 to M-643; M-1 to N-642; M-1 to
N-641; M-1 to W-640; M-1 to A-639; M-1 to L-638; M-1 to S-637; M-1
to H-636; M-1 to E-635; M-1 to P-634; M-1 to K-633; M-1 to L-632;
M-1 to V-631; M-1 to T-630; M-1 to A-629; M-1 to N-628; M-1 to
R-627; M-1 to W-626; M-1 to A-625; M-1 to N-624; M-1 to L-623; M-1
to A-622; M-1 to D-621; M-1 to V-620; M-1 to H-619; M-1 to R-618;
M-1 to N-617; M-1 to L-616; M-1 to D-615; M-1 to A-614; M-1 to
Y-613; M-1 to L-612; M-1 to R-611; M-1 to G-610; M-1 to L-609; M-1
to N-608; M-1 to Y-607; M-1 to Y-606; M-1 to C-605; M-1 to D-604;
M-1 to P-603; M-1 to Y-602; M-1 to K-601; M-1 to R-600; M-1 to
R-599; M-1 to H-598; M-1 to K-597; M-1 to I-596; M-1 to A-595; M-1
to T-594; M-1 to R-593; M-1 to Y-592; M-1 to S-591; M-1 to Q-590;
M-1 to E-589; M-1 to A-588; M-1 to A-587; M-1 to E-586; M-1 to
F-585; M-1 to R-584; M-1 to K-583; M-1 to L-582; M-1 to S-581; M-1
to N-580; M-1 to Q-579; M-1 to V-578; M-1 to I-577; M-1 to G-576;
M-1 to L-575; M-1 to N-574; M-1 to M-573; M-1 to W-572; M-1 to
A-571; M-1 to A-570; M-1 to A-569; M-1 to F-568; M-1 to D-567; M-1
to P-566; M-1 to Q-565; M-1 to I-564; M-1 to Q-563; M-1 to V-562;
M-1 to A-561; M-1 to L-560; M-1 to S-559; M-1 to L-558; M-1 to
L-557; M-1 to E-556; M-1 to E-555; M-1 to A-554; M-1 to E-553; M-1
to Q-552; M-1 to L-551; M-1 to E-550; M-1 to N-549; M-1 to R-548;
M-1 to E-547; M-1 to K-546; M-1 to L-545; M-1 to I-544; M-1 to
N-543; M-1 to G-542; M-1 to L-541; M-1 to N-540; M-1 to N-539; M-1
to M-538; M-1 to A-537; M-1 to H-536; M-1 to V-535; M-1 to Y-534;
M-1 to K-533; M-1 to P-532; M-1 to N-531; M-1 to L-530; M-1 to
R-529; M-1 to V-528; M-1 to A-527; M-1 to E-526; M-1 to R-525; M-1
to Y-524; M-1 to Y-523; M-1 to R-522; M-1 to I-521; M-1 to A-520;
M-1 to A-519; M-1 to T-518; M-1 to Q-517; M-1 to N-516; M-1 to
G-515; M-1 to K-514; M-1 to D-513; M-1 to A-512; M-1 to L-511; M-1
to N-510; M-1 to K-509; M-1 to G-508; M-1 to I-507; M-1 to N-506;
M-1 to Y-505; M-1 to H-504; M-1 to V-503; M-1 to K-502; M-1 to
A-501; M-1 to N-500; M-1 to L-499; M-1 to P-498; M-1 to C-497; M-1
to V-496; M-1 to S-495; M-1 to L-494; M-1 to A-493; M-1 to S-492;
M-1 to R-491; M-1 to F-490; M-1 to L-489; M-1 to Q-488; M-1 to
E-487; M-1 to E-486; M-1 to S-485; M-1 to R-484; M-1 to W-483; M-1
to E-482; M-1 to G-481; M-1 to S-480; M-1 to R-479; M-1 to L-478;
M-1 to V-477; M-1 to C-476; M-1 to R-475; M-1 to L-474; M-1 to
T-473; M-1 to N-472; M-1 to I-471; M-1 to F-470; M-1 to L-469; M-1
to I-468; M-1 to G-467; M-1 to L-466; M-1 to V-465; M-1 to V-464;
M-1 to A-463; M-1 to A-462; M-1 to I-461; M-1 to L-460; M-1 to
K-459; M-1 to K-458; M-1 to K-457; M-1 to K-456; M-1 to T-455; M-1
to H-454; M-1 to K-453; M-1 to S-452; M-1 to L-451; M-1 to A-450;
M-1 to G-449; M-1 to F-448; M-1 to G-447; M-1 to F-446; M-1 to
T-445; M-1 to L-444; M-1 to L-443; M-1 to V-442; M-1 to C-441; M-1
to Y-440; M-1 to G-439; M-1 to X-438; M-1 to S-437; M-1 to P-436;
M-1 to L-435; M-1 to Y-434; M-1 to L-433; M-1 to V-432; M-1 to
R-431; M-1 to E-430; M-1 to A-429; M-1 to V-428; M-1 to V-427; M-1
to F-426; M-1 to G-425; M-1 to V-424; M-1 to R-423; M-1 to F-422;
M-1 to F-421; M-1 to L-420; M-1 to N-419; M-1 to S-418; M-1 to
A-417; M-1 to P-416; M-1 to L-415; M-1 to F-414; M-1 to P-413; M-1
to I-412; M-1 to V-411; M-1 to L-410; M-1 to F-409; M-1 to G-408;
M-1 to L-407; M-1 to G-406; M-1 to L-405; M-1 to T-404; M-1 to
L-403; M-1 to I-402; M-1 to R-401; M-1 to R-400; M-1 to K-399; M-1
to H-398; M-1 to G-397; M-1 to D-396; M-1 to E-395; M-1 to S-394;
M-1 to C-393; M-1 to L-392; M-1 to A-391; M-1 to Q-390; M-1 to
C-389; M-1 to I-388; M-1 to L-387; M-1 to G-386; M-1 to I-385; M-1
to L-384; M-1 to C-383; M-1 to F-382; M-1 to W-381; M-1 to L-380;
M-1 to A-379; M-1 to A-378; M-1 to L-377; M-1 to A-376; M-1 to
I-375; M-1 to V-374; M-1 to R-373; M-1 to W-372; M-1 to D-371; M-1
to S-370; M-1 to I-369; M-1 to S-368; M-1 to K-367; M-1 to I-366;
M-1 to L-365; M-1 to P-364; M-1 to I-363; M-1 to C-362; M-1 to
G-361; M-1 to M-360; M-1 to S-359; M-1 to W-358; M-1 to D-357; M-1
to F-356; M-1 to C-355; M-1 to L-354; M-1 to W-353; M-1 to W-352;
M-1 to P-351; M-1 to C-350; M-1 to L-349; M-1 to L-348; M-1 to
L-347; M-1 to W-346; M-1 to A-345; M-1 to N-344; M-1 to L-343; M-1
to S-342; M-1 to Y-341; M-1 to Y-340; M-1 to Y-339; M-1 to N-338;
M-1 to Y-337; M-1 to N-336; M-1 to V-335; M-1 to A-334; M-1 to
R-333; M-1 to V-332; M-1 to L-331; M-1 to M-330; M-1 to S-329; M-1
to D-328; M-1 to A-327; M-1 to F-326; M-1 to S-325; M-1 to A-324;
M-1 to P-323; M-1 to N-322; M-1 to D-321; M-1 to V-320; M-1 to
E-319; M-1 to T-318; M-1 to F-317; M-1 to A-316; M-1 to X-315; M-1
to P-314; M-1 to G-313; M-1 to T-312; M-1 to G-311; M-1 to M-310;
M-1 to I-309; M-1 to R-308; M-1 to W-307; M-1 to R-306; M-1 to
V-305; M-1 to Y-304; M-1 to L-303; M-1 to M-302; M-1 to G-301; M-1
to A-300; M-1 to G-299; M-1 to G-298; M-1 to S-297; M-1 to T-296;
M-1 to L-295; M-1 to L-294; M-1 to T-293; M-1 to M-292; M-1 to
R-291; M-1 to F-290; M-1 to L-289; M-1 to L-288; M-1 to G-287; M-1
to G-286; M-1 to N-285; M-1 to R-284; M-1 to L-283; M-1 to M-282;
M-1 to G-281; M-1 to L-280; M-1 to N-279; M-1 to E-278; M-1 to
L-277; M-1 to S-276; M-1 to K-275; M-1 to D-274; M-1 to K-273; M-1
to H-272; M-1 to L-271; M-1 to V-270; M-1 to K-269; M-1 to Q-268;
M-1 to X-267; M-1 to I-266; M-1 to E-265; M-1 to L-264; M-1 to
V-263; M-1 to N-262; M-1 to F-261; M-1 to K-260; M-1 to G-259; M-1
to I-258; M-1 to V-257; M-1 to L-256; M-1 to I-255; M-1 to D-254;
M-1 to F-253; M-1 to V-252; M-1 to A-251; M-1 to N-250; M-1 to
L-249; M-1 to G-248; M-1 to L-247; M-1 to V-246; M-1 to T-245; M-1
to I-244; M-1 to G-243; M-1 to Q-242; M-1 to E-241; M-1 to K-240;
M-1 to C-239; M-1 to L-238; M-1 to M-237; M-1 to A-236; M-1 to
V-235; M-1 to A-234; M-1 to G-233; M-1 to L-232; M-1 to F-231; M-1
to I-230; M-1 to S-229; M-1 to L-228; M-1 to L-227; M-1 to V-226;
M-1 to W-225; M-1 to F-224; M-1 to T-223; M-1 to S-222; M-1 to
S-221; M-1 to H-220; M-1 to A-219; M-1 to G-218; M-1 to E-217; M-1
to K-216; M-1 to N-215; M-1 to S-214; M-1 to E-213; M-1 to R-212;
M-1 to F-211; M-1 to A-210; M-1 to K-209; M-1 to C-208; M-1 to
Y-207; M-1 to G-206; M-1 to L-205; M-1 to F-204; M-1 to S-203; M-1
to L-202; M-1 to L-201; M-1 to F-200; M-1 to F-199; M-1 to L-198;
M-1 to A-197; M-1 to C-196; M-1 to L-195; M-1 to L-194; M-1 to
D-193; M-1 to A-192; M-1 to R-191; M-1 to G-190; M-1 to V-189; M-1
to V-188; M-1 to G-187; M-1 to A-186; M-1 to V-185; M-1 to C-184;
M-1 to E-183; M-1 to T-182; M-1 to H-181; M-1 to V-180; M-1 to
P-179; M-1 to H-178; M-1 to V-177; M-1 to A-176; M-1 to F-175; M-1
to L-174; M-1 to L-173; M-1 to A-172; M-1 to A-171; M-1 to L-170;
M-1 to L-169; M-1 to S-168; M-1 to A-167; M-1 to R-166; M-1 to
P-165; M-1 to A-164; M-1 to L-163; M-1 to H-162; M-1 to L-161; M-1
to R-160; M-1 to R-159; M-1 to G-158; M-1 to K-157; M-1 to S-156;
M-1 to T-155; M-1 to Y-154; M-1 to Q-153; M-1 to L-152; M-1 to
G-151; M-1 to G-150; M-1 to F-149; M-1 to L-148; M-1 to V-147; M-1
to S-146; M-1 to F-145; M-1 to V-144; M-1 to D-143; M-1 to V-142;
M-1 to M-141; M-1 to L-140; M-1 to V-139; M-1 to S-138; M-1 to
I-137; M-1 to G-136; M-1 to S-135; M-1 to H-134; M-1 to L-133; M-1
to L-132; M-1 to I-131; M-1 to N-130; M-1 to V-129; M-1 to V-128;
M-1 to H-127; M-1 to F-126; M-1 to G-125; M-1 to V-124; M-1 to
P-123; M-1 to H-122; M-1 to F-121; M-1 to G-120; M-1 to G-119; M-1
to S-118; M-1 to L-117; M-1 to Y-116; M-1 to Y-115; M-1 to N-114;
M-1 to I-113; M-1 to R-112; M-1 to F-111; M-1 to T-110; M-1 to
L-109; M-1 to V-108; M-1 to T-107; M-1 to L-106; M-1 to P-105; M-1
to R-104; M-1 to Y-103; M-1 to S-102; M-1 to K-101; M-1 to H-100;
M-1 to S-99; M-1 to T-98; M-1 to N-97; M-1 to S-96; M-1 to S-95;
M-1 to L-94; M-1 to R-93; M-1 to S-92; M-1 to G-91; M-1 to W-90;
M-1 to F-89; M-1 to D-88; M-1 to H-87; M-1 to H-86; M-1 to W-85;
M-1 to L-84; M-1 to D-83; M-1 to G-82; M-1 to L-81; M-1 to P-80;
M-1 to T-79; M-1 to E-78; M-1 to A-77; M-1 to Q-76; M-1 to L-75;
M-1 to D-74; M-1 to K-73; M-1 to N-72; M-1 to N-71; M-1 to V-70;
M-1 to I-69; M-1 to A-68; M-1 to E-67; M-1 to S-66; M-1 to D-65;
M-1 to D-64; M-1 to F-63; M-1 to V-62; M-1 to F-61; M-1 to D-60;
M-1 to G-59; M-1 to D-58; M-1 to Y-57; M-1 to S-56; M-1 to R-55;
M-1 to A-54; M-1 to F-53; M-1 to C-52; M-1 to V-51; M-1 to I-50;
M-1 to A-49; M-1 to V-48; M-1 to S-47; M-1 to G-46; M-1 to V-45;
M-1 to V-44; M-1 to L-43; M-1 to K-42; M-1 to A-41; M-1 to W-40;
M-1 to F-39; M-1 to P-38; M-1 to P-37; M-1 to L-36; M-1 to V-35;
M-1 to S-34; M-1 to S-33; M-1 to P-32; M-1 to L-31; M-1 to I-30;
M-1 to H-29; M-1 to D-28; M-1 to L-27; M-1 to D-26; M-1 to T-25;
M-1 to D-24; M-1 to L-23; M-1 to V-22; M-1 to A-21; M-1 to M-20;
M-1 to R-19; M-1 to F-18; M-1 to V-17; M-1 to A-16; M-1 to P-15;
M-1 to Q-14; M-1 to H-13; M-1 to S-12; M-1 to G-11; M-1 to A-10;
M-1 to G-9; M-1 to A-8; M-1 to N-7; and M-1 to H-6 of SEQ ID NO:
130. Polypeptides encoded by these polynucleotides are also
encompassed by the invention.
[0124] In addition, any of the above listed N- or C-terminal
deletions can be combined to produce a N- and C-terminal deleted
polypeptide. The invention also provides polypeptides comprising,
or alternatively consisting of, one or more amino acids deleted
from both the amino and the carboxyl termini, which may be
described generally as having residues m-n of SEQ ID NO:130, where
n and m are integers as described above. Polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0125] The present invention is also directed to proteins
containing polypeptides at least 80%, 85%, 90%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to a polypeptide sequence set forth
herein as m-n. In preferred embodiments, the application is
directed to proteins containing polypeptides at least 80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the
amino acid sequence of the specific N- and C-terminal deletions
recited herein. Polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0126] Also included are polynucleotide sequences encoding a
polypeptide consisting of a portion of the complete amino acid
sequence encoded by a cDNA clone contained in ATCC Deposit No.
209745, where this portion excludes any integer of amino acid
residues from 1 to about 755 amino acids from the amino terminus of
the complete amino acid sequence encoded by a cDNA clone contained
in ATCC Deposit No. 209745, or any integer of amino acid residues
from 6 to about 759 amino acids from the carboxy terminus, or any
combination of the above amino terminal and carboxy terminal
deletions, of the complete amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 209745. Polypeptides encoded by
these polynucleotides also are encompassed by the invention.
[0127] As described herein or otherwise known in the art, the
polynucleotides of the invention have uses that include, but are
not limited to, serving as probes or primers in chromosome
identification, chromosome mapping, and linkage analysis.
[0128] This gene is expressed primarily in ovarian cancer tissues
and substantia nigra and, to a lesser extent, in amygdala and
brain, striatum.
[0129] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
neurodegenerative disorders and/or disorders of the reproductive
system, including, but not limited to ovarian cancer. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
central nervous system and brain and/or reproductive system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g., CNS,
neural, nervous, neuronal, reproductive, ovarian, cancerous and
wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine, vaginal pool, synovial fluid and spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of one, two or
all three of the immunogenic epitopes shown in SEQ ID NO: 130 as
residues: Arg-93 to Arg-104, Tyr-154 to Arg-159, Arg-212 to
His-220. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0130] The tissue distribution in substantia nigra and, to a lesser
extent, in amygdala and brain, striatum, indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection, treatment, and/or prevention of
neurodegenerative disease states, behavioral disorders, or
inflammatory conditions. Representative uses are described in the
"Regeneration" and "Hyperproliferative Disorders" sections below,
in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses
include, but are not limited to the detection, treatment, and/or
prevention of Alzheimer's Disease, Parkinson's Disease,
Huntington's Disease, Tourette Syndrome, meningitis, encephalitis,
demyelinating diseases, peripheral neuropathies, neoplasia, trauma,
congenital malformations, spinal cord injuries, ischemia and
infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia,
paranoia, obsessive compulsive disorder, depression, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, elevated expression of this
gene product in regions of the brain indicates it plays a role in
normal neural function. Potentially, this gene product is involved
in synapse formation, neurotransmission, learning, cognition,
homeostasis, or neuronal differentiation or survival.
[0131] The tissue distribution in reproductive and developing
tissues indicates that polynucleotides and/or polypeptides
corresponding to this gene would be useful for the treatment,
prevention, detection, and/or diagnosis of disorders of
reproductive system organs, including cancers, disorders affecting
fertility, and/or developmental disorders. Specifically, expression
in ovarian cancer tissue, indicates that polynucleotides and/or
polypeptides corresponding to this gene, agonists, and/or
antagonists thereof (including, but not limited to antibodies or
fragments thereof, that bind polypeptides of the invention) would
be useful for the treatment, prevention, detection and diagnosis of
conditions concerning proper ovarian function (e.g., egg
maturation, endocrine function), as well as cancer. The expression
in ovarian tissue may indicate that polynucleotides and/or
polypeptides corresponding to this gene, agonists, and/or
antagonists thereof (including, but not limited to antibodies or
fragments thereof, that bind polypeptides of the invention) can be
used to treat, prevent, detect and/or diagnose disorders of the
ovary, including inflammatory disorders, such as oophoritis (e.g.,
caused by viral or bacterial infection), ovarian cysts, amenorrhea,
infertility, hirsutism, and ovarian cancer (including, but not
limited to, primary and secondary cancerous growth, endometrioid
carcinoma of the ovary, ovarian papillary serous adenocarcinoma,
ovarian mucinous adenocarcinoma, Ovarian Krukenberg tumor).
[0132] Moreover, the predicted membrane localization indicates that
polynucleotides and/or polypeptides corresponding to this gene
would be a good target for antagonists, particularly small
molecules or antibodies, which block functional activity (such as,
for example, binding of the receptor by its cognate ligand(s);
transport function; signalling function). Accordingly, preferred
are antibodies and or small molecules which specifically bind an
extracellular portion of the translation product of this gene. The
extracellular regions can be ascertained from the information
regarding the transmembrane domains as set out above. Also provided
is a kit for detecting cancer. In one embodiment, the kit would be
useful for detecting ovarian cancer. Such a kit comprises in one
embodiment an antibody specific for the translation product of this
gene bound to a solid support. Also provided is a method of
detecting cancer (for example, ovarian cancer) in an individual
which comprises a step of contacting an antibody specific for the
translation product of this gene to a bodily fluid from the
individual, preferably serum, and ascertaining whether antibody
binds to an antigen found in the bodily fluid. Preferably the
antibody is bound to a solid support and the bodily fluid is serum.
The above embodiments, as well as other treatments and diagnostic
tests (kits and methods), are more particularly described elsewhere
herein. Furthermore, the protein may also be used to determine
biological activity, to raise antibodies, as tissue markers, to
isolate cognate ligands or receptors, to identify agents that
modulate their interactions, in addition to its use as a
nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0133] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:23 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 3872 of SEQ ID NO:23, b is an integer
of 15 to 3886, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:23, and where b is greater
than or equal to a+14.
[0134] Features of Protein Encoded By Gene No: 14
[0135] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: RDNDYLLHGHRPPMF (SEQ ID NO:290),
SFRACFKSIFRIHTETGNIWTHLL (SEQ ID NO:291), and/or
GFVLFLFLGILTMLRPNMYFMAPLQEKVV (SEQ ID NO:292). Moreover, fragments
and variants of these polypeptides (such as, for example, fragments
as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0136] The gene encoding the disclosed cDNA is thought to reside on
chromosome 1. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 1.
[0137] This gene is expressed primarily in bone marrow, fetal liver
and spleen tissues, several types of leukocytes including
neutophils, and T-cells, placental tissue, and brain tissue.
[0138] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
diseases and/or disorders of the immune system and central nervous
system including AIDS, Lupus, hemotological cancers, mood
disorders, and dementia. Similarly, polypeptides and antibodies
directed to these polypeptides would be useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune system and central
nervous sytem, expression of this gene at significantly higher or
lower levels may be routinely detected in certain tissues or cell
types (e.g., immune, neural, cancerous and wounded tissues) or
bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of one, two or all three of the immunogenic epitopes shown
in SEQ ID NO: 131 as residues: Glu-24 to Tyr-35, Arg-83 to Thr-92,
Pro-148 to Gly-154. Polynucleotides encoding said polypeptides are
encompassed by the invention, as are antibodies that bind one or
more of these peptides.
[0139] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, treatment, and/or prevention of a variety of immune
system disorders. Representative uses are described in the `Immune
Activity` and `Infectious Disease` sections below, in Example 11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the
expression of this gene product in fetal liver and spleen tissues,
and several types of leukocytes, indicates a role in the regulation
of the proliferation; survival; differentiation; and/or activation
of potentially all hematopoietic cell lineages, including blood
stem cells. Polynucleotides and polypeptides of the invention may
be involved in the regulation of cytokine production, antigen
presentation, or other processes that may also suggest a usefulness
in the treatment of cancer (e.g., by boosting immune responses).
Since the gene is expressed in cells of lymphoid origin,
polynucleotides and polypeptides of the invention, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed tissues.
Therefore it may be also used as an agent for immunological
disorders including arthritis, asthma, immune deficiency diseases
such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel
disease, sepsis, acne, and psoriasis. In addition, polynucleotides
and polypeptides of the invention may have commercial utility in
the expansion of stem cells and committed progenitors of various
blood lineages, and in the differentiation and/or proliferation of
various cell types. Alternatively, the tissue distribution
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the detection, diagnosis, prevention
and/or treatment of neurodegenerative disease states and
behavioural disorders such as Alzheimer's Disease, Parkinson's
Disease, Huntington's Disease, Tourette Syndrome, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, the gene or gene product may
also play a role in the treatment and/or detection of developmental
disorders associated with the developing embryo, or sexually-linked
disorders. Furthermore, the protein may also be used to determine
biological activity, to raise antibodies, as tissue markers, to
isolate cognate ligands or receptors, to identify agents that
modulate their interactions, in addition to its use as a
nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0140] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:24 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1569 of SEQ ID NO:24, b is an integer
of 15 to 1583, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:24, and where b is greater
than or equal to a+14.
[0141] Features of Protein Encoded By Gene No: 15
[0142] The translation product of this gene shares sequence
homology with gp25L, which is thought to be important in protein
processing.
[0143] This gene is expressed primarily in stimulated synovium,
cerebellum, immune cells (e.g., T-cells), and placental tissues,
and, to a lesser extent, in several other tissues and organs.
[0144] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
inflammation, disorders of developing systems, central nervous
system, and musculo-skeletal system. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune, central nervous
system, musculo-skeletal, and developing systems, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., immune, neural,
musculo-skeletal, cancerous and wounded tissues) or bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0145] The tissue distribution and homology to gp25L indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for treatment, prevention, detection and/or diagnosis of
disorders of immune, central nervous system, musculo-skeletal, and
developing systems. In addition, the expression of this gene
product in synovium indicates a role in the detection and treatment
of disorders and conditions affecting the skeletal system, in
particular osteoporosis as well as disorders afflicting connective
tissues (e.g., arthritis, trauma, tendonitis, chrondomalacia and
inflammation), such as in the diagnosis or treatment of various
autoimmune disorders such as rheumatoid arthritis, lupus,
scleroderma, and dermatomyositis as well as dwarfism, spinal
deformation, and specific joint abnormalities as well as
chondrodysplasias (i.e., spondyloepiphyseal dysplasia congenita,
familial arthritis, Atelosteogenesis type II, metaphyseal
chondrodysplasia type Schmid). The tissue distribution and homology
to gp25L indicates that the polynucleotides and polypeptides of the
invention would be useful for treatment, prevention, detection
and/or diagnosis of disorders associated with expression of
Gp25L-H, e.g. Cushing's disease, cystic fibrosis, diabetes
mellitus, diabetes insipidus, glucose-galactose malabsorption
syndrome, hypercholesterolemia, hyper and hypoglycemia, Grave's
disease, goiter, inflammation and autoimmune disorders including
Addison's disease, adult respiratory distress syndrome, allergies
(including hay fever and hives), anemia, asthma, atherosclerosis,
bronchitis, cholecystitis, Crohn's disease, ulcerative colitis,
atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
atrophic gastritis, glomerulonephritis, gout, hypereosinophilia,
irritable bowel syndrome, lupus erythematosus, multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis, osteoporosis, pancreatitis, polymyositis,
rheumatoid arthritis, scleroderma, Sjogren's syndrome and
autoimmune thyroiditis, complications of cancer, hemodialysis,
extracorporeal circulation; viral, bacterial, fungal, parasitic,
protozoal and helminthic infections and trauma. The tissue
distribution in T-cells indicates that polynucleotides and
polypeptides of the invention would be useful for the diagnosis,
detection, prevention and/or treatment of a variety of immune
system disorders. Representative uses are described in the "Immune
Activity" and "Infectious Disease" sections below, in Example 11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the
expression indicates a role in regulating the proliferation;
survival; differentiation; and/or activation of hematopoietic cell
lineages, including blood stem cells. Involvement in the regulation
of cytokine production, antigen presentation, or other processes
indicates a usefulness for treatment of cancer (e.g. by boosting
immune responses). Expression in cells of lymphoid origin,
indicates the natural gene product would be involved in immune
functions. Therefore it would also be useful as an agent for
immunological disorders including arthritis, asthma,
immunodeficiency diseases such as AIDS, leukemia, rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease,
sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus, this
gene product is thought to be useful in the expansion of stem cells
and committed progenitors of various blood lineages, and in the
differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, raise antibodies, as tissue markers, to isolate cognate
ligands or receptors, to identify agents that modulate their
interactions, in addition to its use as a nutritional supplement.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0146] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:25 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1655 of SEQ ID NO:25, b is an integer
of 15 to 1669, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:25, and where b is greater
than or equal to a+14.
[0147] Features of Protein Encoded By Gene No: 16
[0148] The translation product of this gene shares sequence
homology with ribosomal proteins (see, e.g., Genbank accession
number gi|437926 and PID|d1011606; all references available through
these accessions are hereby incorporated in their entirety by
reference herein). Based on the sequence similarity, the
translation product of this clone is expected to share at least
some biological activities with ribosomal proteins.
[0149] This gene is expressed primarily in immune and hematopoietic
cells, fetal tissue, adipose tissue, uterine cancer tissue, ovary
tumor, breast and brain tissues, and, to a lesser extent, in
several other tissues.
[0150] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
immune and hematopoietic disorders, disorders of the central
nervous system and reproductive organs. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune system,
hematopoietic, central nervous system and reproductive system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
immune, reproductive, neural, cancerous and wounded tissues) or
bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0151] The tissue distribution in breast, brain, and immune tissues
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the treatment, prevention, detection
and/or diagnosis of disorders of the immune, hematopoietic, central
nervous and reproductive systems. Moreover, the expression within
fetal tissues and other cellular sources marked by proliferating
cells indicates that polynucleotides and polypeptides of the
invention may play a role in the regulation of cellular division,
and may show utility in the diagnosis, treatment, and/or prevention
of developmental diseases and disorders, including cancer, and
other proliferative conditions. Representative uses are described
in the "Hyperproliferative Disorders" and "Regeneration" sections
below and elsewhere herein. Briefly, developmental tissues rely on
decisions involving cell differentiation and/or apoptosis in
pattern formation. Dysregulation of apoptosis can result in
inappropriate suppression of cell death, as occurs in the
development of some cancers, or in failure to control the extent of
cell death, as is believed to occur in acquired immunodeficiency
and certain degenerative disorders, such as spinal muscular atrophy
(SMA). Alternatively, this gene product may be involved in the
pattern of cellular proliferation that accompanies early
embryogenesis. Thus, aberrant expression of this gene product in
tissues--particularly adult tissues--may correlate with patterns of
abnormal cellular proliferation, such as found in various cancers.
Because of potential roles in proliferation and differentiation,
polynucleotides and polypeptides of the invention may have
applications in the adult for tissue regeneration and the treatment
of cancers. It may also act as a morphogen to control cell and
tissue type specification. Therefore, the polynucleotides and
polypeptides of the present invention would be useful in treating,
detecting, and/or preventing said disorders and conditions, in
addition to other types of degenerative conditions. Thus this
protein may modulate apoptosis or tissue differentiation and would
be useful in the detection, treatment, and/or prevention of
degenerative or proliferative conditions and diseases. The protein
would be useful in modulating the immune response to aberrant
polypeptides, as may exist in proliferating and cancerous cells and
tissues. The protein can also be used to gain new insight into the
regulation of cellular growth and proliferation. Furthermore, the
protein may also be used to determine biological activity, raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0152] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:26 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1039 of SEQ ID NO:26, b is an integer
of 15 to 1053, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:26, and where b is greater
than or equal to a+14.
[0153] Features of Protein Encoded By Gene No: 17
[0154] The gene encoding the disclosed cDNA is believed to reside
on chromosome 11. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 11.
[0155] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
TGPEFPGSNSTVARRIKDLAADIEEELVCRLKICDGFSLQLDESADVSGLAVLL
VFVRYRFNKSIEEDLLLCESLQSNATGEEIFNCINSFMQKHEIEWEKCVDVCSD
ASRAVDGKIAEAVTLIKYVAPESTSSHCLLYRHALAVKIMPTSLKNVLDQAV
QIINYIKARPHQSRLLKILCEEMGAQHTALLLNTEVRWLSRGKVLVRLFELRR
ELLVFMDSAFRLSDCLTNSSWLLRLAYLADIFTKLNEVNLSMQGKNVTVFTV
FDKMSSLLRKLEFWASSVEEENFDCFPTLSDFLTEINSTVDKDICSAIVQHLRG
LRATLLKYFPVTNDNNAWVRNPFTVTVKPASLVARDYESLIDLTSDSQVKQN
FSELSLNDFWSSLIQEYPSIARRAVRVLLPFATMHLCETGFSYYAATKTKYRK
RLDAAPHMRIRLSNITPNIKICDKKTQKHCSH (SEQ ID NO:293),
DIEEELVCRLKICDGFSLQLDESADVSGLAV (SEQ ID NO:294),
NSFMQKHEIEWEKCVDVCSDASRAVDGKIAEAVTLI (SEQ ID NO:295),
LDQAVQIINYIKARPHQSRLLKILCEEMGAQHTALL (SEQ ID NO:296),
SAFRLSDCLTNSSWLLRLAYLADIFTKLNEVNLSMQGKNVTVFTVFDKM (SEQ ID NO:297),
SDFLTEINSTVDKDICSAIVQHLRGLRATLLK (SEQ ID NO:298), and/or
SDSQVKQNFSELSLNDFWSSLIQEYPSIARRAVRVLLP (SEQ ID NO:299). Moreover,
fragments and variants of these polypeptides (such as, for example,
fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides,
or polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0156] This gene is expressed primarily in spleen from a chronic
lymphocytic leukemia patient, and hodgkin's lymphoma, and, to a
lesser extent, in pancreatic islet cell tumors and activated T
cells.
[0157] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
chronic lymphocytic leukemia; hodgkin's lymphoma; pancreatic islet
cell cancer; cancer in general; hematopoietic disorders; immune
dysfunction. Similarly, polypeptides and antibodies directed to
these polypeptides would be useful in providing immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the immune system and pancreas, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., hematopoietic,
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0158] The tissue distribution in spleen from a chronic lymphocytic
leukemia patient, and hodgkin's lymphoma, pancreatic islet cell
tumors, and activated T-cells indicates that polynucleotides and/or
polypeptides corresponding to this gene would be useful in the
treatment, prevention, detection and/or diagnosis of a variety of
immune system disorders. Representative uses are described in the
"Immune Activity" and "Infectious Disease" sections below, in
Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.
Briefly, the protein product of this gene would be useful for the
diagnosis and/or treatment of a variety of cancers, including CLL;
Hodgkin's lymphoma; and pancreatic cancer. Expression of this gene
product in a variety of cancers indicates that it may be a bad
player and may likely be a target for inhibitors as therapeutics.
Alternately, this gene product may be expressed in both normal and
abnormal hematopoietic tissues, where it may play necessary roles
in the proliferation; survival; differentiation; or activation of
hematopoietic cell lineages. Likewise, expression in pancreatic
islet cell tumors may simply reflect a necessary role that this
protein plays in normal pancreatic function. Furthermore, the
protein may also be used to determine biological activity, to raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0159] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:27 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1463 of SEQ ID NO:27, b is an integer
of 15 to 1477, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:27, and where b is greater
than or equal to a+14.
[0160] Features of Protein Encoded By Gene No: 18
[0161] When tested against U937 Myeloid cell lines, supernatants
removed from cells containing this gene activated the GAS assay.
Thus, it is likely that this gene activates myeloid cells, and to a
lesser extent other cells, through the Jak-STAT signal transduction
pathway. The gamma activating sequence (GAS) is a promoter element
found upstream of many genes which are involved in the Jak-STAT
pathway. The Jak-STAT pathway is a large, signal transduction
pathway involved in the differentiation and proliferation of cells.
Therefore, activation of the Jak-STAT pathway, reflected by the
binding of the GAS element, can be used to indicate proteins
involved in the proliferation and differentiation of cells.
[0162] The polypeptide of this gene has been determined to have
transmembrane domains at about amino acid positions 219 to about
235, at about 114 to about 130, at about 86 to about 102, and at
about 43 to about 59 of the amino acid sequence referenced in Table
1 for this gene. Based upon these characteristics, it is believed
that the protein product of this gene shares structural features to
type IIIa membrane proteins.
[0163] The gene encoding the disclosed cDNA is believed to reside
on chromosome 17. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 17.
[0164] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: DPRVRECLQDWASFLRLAIPSMLMLCMEWWAYEVGSFLSGILGMVELGAQS
IVYELAIIVYMVPAGFSVAASVRVGNALGAGDMEQARKSSTVSLLITVLFAV
AFSVLLLSCKDHVGYIFTTDRDIINLVAQVVPIYAVSHLFEALACTSGGVLRGS
GNQKVGAIVNTIGXYVVGLPIGIALMFATTLGVMGLWSGIIICTVFQAVCFLG
FIIQLNWKKACXQAQVHANLKVNNVPRSGNSALPQDPLHPGCPENLEGILTN
DVGKTGEPQSDQQMRQEEPLPEHPQDGAKLSRKQLVLRRGLLLLGVFLILLV GILVRFYVRIQ
(SEQ ID NO:300). Moreover, fragments and variants of these
polypeptides (such as, for example, fragments as described herein,
polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% identical to these polypeptides, or polypeptides encoded by a
polynucleotide which hybridizes, under stringent conditions, to the
polynucleotide encoding these polypeptides) are encompassed by the
invention. Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0165] This gene is expressed primarily in endometrial tumor
tissue, cartilage tissue, fetal tissue, immune tissue (B-cells and
macrophages), and to a lesser extent in several other tissues and
organs.
[0166] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited
to,tumors and disorders of the musculo-skeletal system. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
musculo-skeletal system, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., musculo-skeletal, immune, cancerous and
wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder. Preferred polypeptides of the present invention comprise,
or alternatively consist of the immunogenic epitopes shown in SEQ
ID NO: 135 as residues: Met-1 to Ser-8. Polynucleotides encoding
said polypeptides are encompassed by the invention, as are
antibodies that bind one or more of these peptides.
[0167] The tissue distribution in musculo-skeletal tissues and
biological activity in the GAS assay, indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the treatment, prevention, detection and/or diagnosis
of disorders of the musculo-skeletal system, and cancers thereof.
The tissue distribution in immune cells (e.g., B-cells and
macrophages) and biological activity in the GAS assay indicates
that polynucleotides and polypeptides corresponding to this gene
would be useful for the diagnosis and treatment of a variety of
immune system disorders. Representative uses are described in the
"Immune Activity" and "Infectious Disease" sections below, in
Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.
Briefly, the expression indicates a role in regulating the
proliferation; survival; differentiation; and/or activation of
hematopoietic cell lineages, including blood stem cells.
Involvement in the regulation of cytokine production, antigen
presentation, or other processes indicates a usefulness for
treatment of cancer (e.g., by boosting immune responses).
Expression in cells of lymphoid origin, indicates the natural gene
product would be involved in immune functions. Therefore it would
also be useful as an agent for immunological disorders including
arthritis, asthma, immunodeficiency diseases such as AIDS,
leukemia, rheumatoid arthritis, granulomatous disease, inflammatory
bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus, this
gene product is thought to be useful in the expansion of stem cells
and committed progenitors of various blood lineages, and in the
differentiation and/or proliferation of various cell types. In
addition, the expression of this gene product in cartilage tissue
indicates a role in the detection and treatment of disorders and
conditions affecting the skeletal system, in particular
osteoporosis as well as disorders afflicting connective tissues
(e.g., arthritis, trauma, tendonitis, chrondomalacia and
inflammation), such as in the diagnosis or treatment of various
autoimmune disorders such as rheumatoid arthritis, lupus,
scleroderma, and dermatomyositis as well as dwarfism, spinal
deformation, and specific joint abnormalities as well as
chondrodysplasias (i.e., spondyloepiphyseal dysplasia congenita,
familial arthritis, Atelosteogenesis type II, metaphyseal
chondrodysplasia type Schmid). Furthermore, the protein may also be
used to determine biological activity, to raise antibodies, as
tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0168] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:28 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2490 of SEQ ID NO:28, b is an integer
of 15 to 2504, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:28, and where b is greater
than or equal to a+14.
[0169] Features of Protein Encoded By Gene No: 19
[0170] The gene encoding the disclosed cDNA is thought to reside on
chromosome 17. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 17.
[0171] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: GTRIHTILVYQESNRKMDSVDPASSQAMELSDVTLIEGVGNEVMVVAGVVVL
ILALVLAWLSTYVADSGSNQLLGAIVSAGDTSVLHLGHVDHLVAGQGNPEPT
ELPHPSEGNDEKAEEAGEGRGDSTGEAGAGGGVEPSLEHLLDIQGLPKRQAG
AGSSSPEAPLRSEDSTCLPPSPGLITVRLKFLNDTEELAVARPEDTVGALKSKY
FPGQESQMKLIYQGRLLQDPARTLRSLNITDNCVIHCHRSPPGSAVPGPSASLA
PSATEPPSLGVNVGSLMVPVFVVLLGVVWYFRINYRQFFTAPATVSLVGVTV FFSFLVFGMYGR
(SEQ ID NO: 301). Moreover, fragments and variants of these
polypeptides (such as, for example, fragments as described herein,
polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% identical to these polypeptides, or polypeptides encoded by a
polynucleotide which hybridizes, under stringent conditions, to the
polynucleotide encoding these polypeptides) are encompassed by the
invention. Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0172] The polypeptide of this gene has been determined to have
transmembrane domains at about amino acid positions 234 to about
250 and at about 266 to about 282 of the amino acid sequence
referenced in Table 1 for this gene. Based upon these
characteristics, it is believed that the protein product of this
gene shares structural features to type IIIa membrane proteins.
[0173] This gene is expressed primarily in breast and cerebellum
tissues, ovary cancer tissue, B-cells, tonsils, as well as in cells
of the hematopoietic system, and, to a lesser extent, in several
other organs and tissues.
[0174] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited
to,disorders of the brain, reproductive system and hematopoietic
system. Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune and hematopoietic system, central nervous system and
reproductive system, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., immune, neural, reproductive, cancerous and
wounded tissues) or bodily fluids (e.g., lymph, serun, plasma,
urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder. Preferred polypeptides of the present invention comprise,
or alternatively consist of one, two, three or all four of the
immunogenic epitopes shown in SEQ ID NO: 136 as residues: Gly-56 to
Gly-86, Leu-107 to Ala-112, Ala-121 to Thr-129, Lys-164 to Gln-174.
Polynucleotides encoding said polypeptides are encompassed by the
invention, as are antibodies that bind one or more of these
peptides.
[0175] The tissue distribution in immune, reproductive, and neural
tissues indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the treatment,
prevention, detection and/or diagnosis of disorders of the immune
and haemopoietic system, the central nervous system, and the
reproductive system. Furthermore, the expression in the breast
tissue may indicate its uses in breast neoplasia and breast
cancers, such as fibroadenoma, pipillary carcinoma, ductal
carcinoma, Paget's disease, medullary carcinoma, mucinous
carcinoma, tubular carcinoma, secretory carcinoma and apocrine
carcinoma, as well as juvenile hypertrophy and gynecomastia,
mastitis and abscess, duct ectasia, fat necrosis and fibrocystic
diseases. Alternatively, the tissue distribution in cerebellum
tissue indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the detection,
treatment, prevention and/or diagnosis of neurodegenerative disease
states and behavioural disorders such as Alzheimer's Disease,
Parkinson's Disease, Huntington's Disease, Tourette Syndrome,
schizophrenia, mania, dementia, paranoia, obsessive compulsive
disorder, panic disorder, learning disabilities, ALS, psychoses,
autism, and altered behaviors, including disorders in feeding,
sleep patterns, balance, and perception. In addition, the gene or
gene product may also play a role in the treatment and/or detection
of developmental disorders associated with the developing embryo,
or sexually-linked disorders. In addition, the tissue distribution
in immune system cells and tissues indicates that polynucleotides
and polypeptides corresponding to this gene would be useful for the
detection, diagnosis, prevention and/or treatment of immune system
disorders. Representative uses are described in the "Immune
Activity" and "Infectious Disease" sections below, in Example 11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. This gene
product may be involved in the regulation of cytokine production,
antigen presentation, or other processes that may also suggest a
usefulness in the treatment of cancer (e.g., by boosting immune
responses). Since the gene is expressed in cells of lymphoid
origin, the gene or protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues. Therefore it
may be also used as an agent for immunological disorders including
arthritis, asthma, immune deficiency diseases such as AIDS,
leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,
acne, and psoriasis. In addition, this gene product may have
commercial utility in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0176] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:29 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1852 of SEQ ID NO:29, b is an integer
of 15 to 1866, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:29, and where b is greater
than or equal to a+14.
[0177] Features of Protein Encoded By Gene No: 20
[0178] The translation product of this gene shares weak sequence
homology with dehydrogenase enzymes (see, e.g., gnl|PID|e1316908,
all references available through this accession are hereby
incorporated in their entirety by reference herein) which are
thought to be important in a variety of enzymatic conversions,
including the biosynthesis of clavulanic acid from a precursor
clavulanic acid aldehyde. The obtained clavulanic acid is in turn a
key ingredient in antibiotics.
[0179] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: DSRISLLVNNAGVGATASLLESDADK (SEQ ID NO:302)
and/or MDAMILLNVLALTRLAKAAATNFVAQGRGTIINIGSIVALAPKVLNGVYGGT
KAFVQAFSESLQHELSDKGVVVQVVLPGATATEFWDIAGLPVNNLPEAMVM
TTENLVXAALAGLAQGEAVTIPSLPDSADWDTYERARLALGPNLSHREPAAR YGLK (SEQ ID
NO:303). Moreover, fragments and variants of these polypeptides
(such as, for example, fragments as described herein, polypeptides
at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical
to these polypeptides, or polypeptides encoded by a polynucleotide
which hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0180] This gene is expressed primarily in CD34 positive
hematopoietic cells.
[0181] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
hematopoietic diseases and/or disorders; impaired immune function;
lymphomas and leukemias. Similarly, polypeptides and antibodies
directed to these polypeptides would be useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune system, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., hematopoietic,
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of, the
immunogenic epitopes shown in SEQ ID NO: 137 as residues: Pro-97 to
Pro-113. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0182] The tissue distribution in CD34 positive hematopoietic cells
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the diagnosis, detection, prevention
and/or treatment of a variety of hematopoietic disorders.
Expression of this gene product specifically in CD34 positive cells
indicates that it plays a role in early events of hematopoiesis,
including proliferation; survival; differentiation; and activation
of early stem and committed progenitor cells. Polynucleotides and
polypeptides corresponding to this gene would be useful for the
treatment and diagnosis of hematopoietic related disorders such as
anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia
since stromal cells are important in the production of cells of
hematopoietic lineages. Representative uses are described in the
"Immune Activity" and "Infectious Disease" sections below, in
Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.
Briefly, the uses include bone marrow cell ex-vivo culture, bone
marrow transplantation, bone marrow reconstitution, radiotherapy or
chemotherapy of neoplasia. The gene product may also be involved in
lymphopoiesis, therefore, it can be used in immune disorders such
as infection, inflammation, allergy, immunodeficiency etc. In
addition, this gene product may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0183] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:30 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1487 of SEQ ID NO:30, b is an integer
of 15 to 1501, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:30, and where b is greater
than or equal to a+14.
[0184] Features of Protein Encoded By Gene No: 21
[0185] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
GTPAGTGPEFPGRPTRPSRTESAQTTQHSPLRPLWRLKRDSSPCHPQTRADWG
VCPPWGGAAQGLRPGCHLAPRRCLCPGSCCPWHWAEAQWSFLWRGLWGLR
TLPTALRASPAASGTVTYSACLGTSCLLRAPCWRLRT CRQSWC (SEQ ID NO:304),
GTPAGTGPEFPGRPTRPSRTESAQTTQH (SEQ ID NO:305),
SPLRPLWRLKRDSSPCHPQTRADWGVCPPW (SEQ ID NO:306),
GGAAQGLRPGCHLAPRRCLCPGSCCPWHWA (SEQ ID NO:307),
EAQWSFLWRGLWGLRTLPTALRASPAASGT (SEQ ID NO:308),
VTYSACLGTSCLLRAPCWRLRTCRQSWC (SEQ ID NO:309), and/or
MPVPWFLLSLALGRSPVVLSLERLVGPQDATHCSPGLSCRLWDSDILCLPGDI
VPAPGPVLAPTHLQTELVLRCQKETDCDLCLRVAVHLAVHGHWEEPEDEEK
FGGAADLGVEEPRNASLQAQVVLSFQAYPTARCVLLEVQVPAALVQFGQSV
GSVVYDCFEAALGSEVRIWSYTQPRYEKELNHTQQLPDCRGLEVWNSIPSCW
ALPWLNVSADGDNVHLVLNVSEEQHFGLSLYWNQVQGPPKPRWHKNLTGP
QIITLNHTDLVPCLCIQVWPLEPDSVRTNICPFREDPRAHQNLWQAARLRLLT
LQSWLLDAPCSLPAEAALCWRAPGGDPCQPLVPPLSWENVTVDKVLEFPLLK
GHPNLCVQVNSSEKLQLQECLWADSLGPLKDDVLLLETRGPQDNRSLCALEP
SGCTSLPSKASTRAARLGEYLLQDLQSGQCLQLWDDDLGALWACPMDKYIH
KRWALVWLACLLFRRALSLILLLKKDHAKGWLRLLKQDVRSG (SEQ ID NO:310).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0186] The gene encoding the disclosed cDNA is believed to reside
on chromosome 3. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 3.
[0187] This gene is expressed primarily in osteoarthritis, breast
cancer, and uterine cancer, and, to a lesser extent, in brain.
[0188] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
cancer, particularly breast and uterine cancer; and neurological
diseases and/or disorders. Similarly, polypeptides and antibodies
directed to these polypeptides would be useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the breast, lymph node, and CNS,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
reproductive, breast, skeletal, joint, neural, and cancerous and
wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
amniotic fluid, urine, synovial fluid and spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of the
immunogenic epitopes shown in SEQ ID NO: 138 as residues: Gln-75 to
Cys-80. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0189] The tissue distribution in breast and uterine cancer
indicates that polynucleotides and/or polypeptides corresponding to
this gene would be useful for the diagnosis, detection, prevention
and/or treatment of a variety of cancers, particularly breast
cancer and uterine cancer. Expression of this gene in brain also
indicates that it may play a role in neurological function, and
that its absence may lead to disorders such as Alzheimer's and/or
Parkinson's disease. Expression of this gene product at elevated
levels within cancerous tissue indicates that it may be a player in
the progression of the disease, perhaps by driving proliferation or
blocking differentiation or apoptosis. Therefore, beneficial
therapeutics may be developed based upon attempts to block this
gene product. Representative uses are described in the
"Hyperproliferative Disorders" and "Regeneration" sections below
and elsewhere herein. Briefly, developmental tissues rely on
decisions involving cell differentiation and/or apoptosis in
pattern formation. Dysregulation of apoptosis can result in
inappropriate suppression of cell death, as occurs in the
development of some cancers, or in failure to control the extent of
cell death, as is believed to occur in acquired immunodeficiency
and certain neurodegenerative disorders, such as spinal muscular
atrophy (SMA). Because of potential roles in proliferation and
differentiation, polynucleotides and/or polypeptides corresponding
to this gene may have applications in the adult for tissue
regeneration and the treatment of cancers. It may also act as a
morphogen to control cell and tissue type specification. Therefore,
the polynucleotides and polypeptides of the present invention would
be useful in treating, detecting, and/or preventing said disorders
and conditions, in addition to other types of degenerative
conditions. Thus this protein may modulate apoptosis or tissue
differentiation and would be useful in the detection, treatment,
and/or prevention of degenerative or proliferative conditions and
diseases. The protein would be useful in modulating the immune
response to aberrant polypeptides, as may exist in proliferating
and cancerous cells and tissues. The protein can also be used to
gain new insight into the regulation of cellular growth and
proliferation. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0190] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:31 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1738 of SEQ ID NO:31, b is an integer
of 15 to 1752, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:31, and where b is greater
than or equal to a+14.
[0191] Features of Protein Encoded By Gene No: 22
[0192] This gene shares sequence homology with a yeast hypothetical
52.9 KD protein CDC26-YMR31 intergenic region (see, e.g. Genbank
Accession No. gp|D50617|YSCCHRVI.sub.--14; all references available
through this accession are hereby incorporated in their entirety by
reference herein).
[0193] This gene has been mapped to chromosome 18q22-23, and
therefore can be used in linkage analysis as a marker for
18q22-23.
[0194] This gene is expressed primarily in whole brain tissue, as
well as brain specific tissues such as hypothalamus, frontal
cortex, cerebellum, amygdala, and hippocampus tissues, as well as
other brain specific tissues.
[0195] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
schizophrenia, developmental disorders, and abnormal mental states.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the central nervous system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., neural, brain, cancerous and
wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder. Preferred polypeptides of the present invention comprise,
or alternatively consist of one, two, three, four, five, six,
seven, eight, nine or all ten of the immunogenic epitopes shown in
SEQ ID NO: 139 as residues: Met-98 to Gln-107, Gly-120 to Gly-126,
Pro-138 to Trp-145, Leu-159 to Gly-169, Val-211 to Arg-217, Cys-256
to His-262, Glu-320 to Val-327, Phe-399 to Asn-406, Asp-444 to
Ser-450, Asp-475 to Trp-488. Polynucleotides encoding said
polypeptides are encompassed by the invention, as are antibodies
that bind one or more of these peptides.
[0196] The tissue distribution in whole brain tissue and brain
specific tissues indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for treating,
preventing, detecting and/or diagnosing neural and
neurodegenerative disorders. Furthermore, the tissue distribution
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the detection, diagnosis, prevention
and/or treatment of neurodegenerative disease states and
behavioural disorders such as Alzheimer's Disease, Parkinson's
Disease, Huntington's Disease, Tourette Syndrome, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, the gene or gene product may
also play a role in the treatment and/or detection of developmental
disorders associated with the developing embryo, or sexually-linked
disorders. Elevated expression of this gene product within the
frontal cortex of the brain indicates that it may be involved in
neuronal survival; synapse formation; conductance; neural
differentiation, etc. Such involvement may impact many processes,
such as learning and cognition. Additionally, the amygdala
processes sensory information and relays this to other areas of the
brain including the endocrine and autonomic domains of the
hypothalamus and the brain stem. Thus, polynucleotides and
polypeptides corresponding to this gene may also be useful for the
detection and/or treatment of neural disorders that impact
processes mediated by the amygdala. Protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
[0197] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:32 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2138 of SEQ ID NO:32, b is an integer
of 15 to 2152, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:32, and where b is greater
than or equal to a+14.
[0198] Features of Protein Encoded By Gene No: 23
[0199] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: TABLE-US-00003 PPRPSTSGQWG (SEQ ID NO:
311) and/or RRSPFTSAQTG. (SEQ ID NO: 312)
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0200] The gene encoding the disclosed cDNA is thought to reside on
chromosome 1. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 1.
[0201] When tested against SKNMC cell lines, supernatants removed
from cells containing this gene activated the NFkB promoter
element. Thus, it is likely that this gene activates neuroblastoma
cells through the NFkB signal transduction pathway. NF-kB (Nuclear
Factor kB) is a transcription factor activated by a wide variety of
agents, leading to cell activation, differentiation, or apoptosis.
Reporter constructs utilizing the NF-kB promoter element are used
to screen supernatants for such activity.
[0202] This gene is expressed primarily in breast and soleus
tissues, and, to a lesser extent, in several cell types, including
T-cells.
[0203] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
breast cancer, and musculo-skeletal diseases and/or disorders.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the lactation system and breast, as well as the musculo-skeletal
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues or cell types
(e.g., musculo-skeletal, breast, cancerous and wounded tissues) or
bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of one or both of the immunogenic epitopes shown in SEQ ID
NO: 140 as residues: Thr-35 to Lys-43, Pro-59 to Arg-64.
Polynucleotides encoding said polypeptides are encompassed by the
invention, as are antibodies that bind one or more of these
peptides.
[0204] The tissue distribution in soleus tissue indicates that the
protein product of this gene would be useful for the detection,
treatment, and/or prevention of conditions and pathologies of the
cardiovascular system, such as heart disease, restenosis,
atherosclerosis, stoke, angina, thrombosis, and wound healing.
Representative uses are described elsewhere herein. Likewise,
expression in breast tissue indicates that polynucleotides and/or
polypeptides of the invention would be useful for diagnosis,
treatment and/or prevention of breast neoplasia and breast cancers,
such as fibroadenoma, pipillary carcinoma, ductal carcinoma,
Paget's disease, medullary carcinoma, mucinous carcinoma, tubular
carcinoma, secretory carcinoma and apocrine carcinoma, as well as
juvenile hypertrophy and gynecomastia, mastitis and abscess, duct
ectasia, fat necrosis and fibrocystic diseases. Furthermore, the
protein may also be used to determine biological activity, to raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0205] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:33 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1743 of SEQ ID NO:33, b is an integer
of 15 to 1757, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:33, and where b is greater
than or equal to a+14.
[0206] Features of Protein Encoded By Gene No: 24
[0207] The gene encoding the disclosed cDNA is believed to reside
on chromosome 3. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 3.
[0208] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: GTGWDFGLAAVCLRAAEVAGSFK (SEQ ID NO:313),
GYRRVFEEYMRVISQRYPDRIEGENYLPQPIYRHIASFLSVFKLVLIGLIIVGK
DPFAFFGMQAPSIWQWGQENKVYACMMVFFLSNMIENQCMSTGAFEITLND
VPVWSKLESGHLPSMQQLVQILDNEMKLNVHMDSIPHHRS (SEQ ID NO:314),
GYRRVFEEYMRVISQRYPDIRIEGENYLPQPIYR (SEQ ID NO:315),
HIASFLSVFKLVLIGLIIVGKDPFAFFGMQAPSI (SEQ ID NO:316),
WQWGQENKVYACMMVFFLSNMIENQCMSTGAFEI (SEQ ID NO:317),
TLNDVPVWSKLESGHLPSMQQLVQILDNEMKLNVHM (SEQ ID NO:318), and/or
DSIPHHRS (SEQ ID NO: 298). Moreover, fragments and variants of
these polypeptides (such as, for example, fragments as described
herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%, or 100% identical to these polypeptides, or polypeptides
encoded by a polynucleotide which hybridizes, under stringent
conditions, to the polynucleotide encoding these polypeptides) are
encompassed by the invention. Antibodies that bind polypeptides of
the invention and polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0209] This gene is expressed primarily in fast-growing tissues
such as early development stage tissues, cancerous tissues, and
hematopoietic tissues, and, to a lesser extent, in some other
tissues.
[0210] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
growth disorders, tumorigenesis, and immune and inflammatory
disorders. Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the fast-growing tissues such as early development stage tissues,
cancer tissues, and hematopoietic tissues, expression of this gene
at significantly higher or lower levels may be routinely detected
in certain tissues or cell types (e.g., cancerous and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial fluid and spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0211] The tissue distribution in fast-growing tissues such as
early development stage tissues, cancerous tissues, and
hematopoietic tissues, indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for
detection, treatment, and/or prevention of growth disorders,
tumorigenesis, and immune and inflammatory disorders. Similarly,
the tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, treatment, and/or prevention of cancer and other
proliferative disorders. Expression in cellular sources marked by
proliferating cells indicates that this protein may play a role in
the regulation of cellular division. Additionally, the expression
in hematopoietic cells and tissues indicates that polynucleotides
and polypeptides corresponding to this gene may play a role in the
proliferation, differentiation, and/or survival of hematopoietic
cell lineages. In such an event, polynucleotides and polypeptides
corresponding to this gene may be useful in the treatment of
lymphoproliferative disorders, and in the maintenance and
differentiation of various hematopoietic lineages from early
hematopoietic stem and committed progenitor cells. Moreover, the
expression within embryonic tissue and other cellular sources
marked by proliferating cells indicates that polynucleotides and
polypeptides corresponding to this gene may play a role in the
regulation of cellular division, and may show utility in the
diagnosis, treatment, and/or prevention of developmental diseases
and disorders, including cancer, and other proliferative
conditions. Representative uses are described in the
"Hyperproliferative Disorders" and "Regeneration" sections below
and elsewhere herein. Briefly, developmental tissues rely on
decisions involving cell differentiation and/or apoptosis in
pattern formation. Dysregulation of apoptosis can result in
inappropriate suppression of cell death, as occurs in the
development of some cancers, or in failure to control the extent of
cell death, as is believed to occur in acquired immunodeficiency
and certain degenerative disorders, such as spinal muscular atrophy
(SMA). Alternatively, this gene product may be involved in the
pattern of cellular proliferation that accompanies early
embryogenesis. Thus, aberrant expression of this gene product in
tissues--particularly adult tissues--may correlate with patterns of
abnormal cellular proliferation, such as found in various cancers.
Because of potential roles in proliferation and differentiation,
this gene product may have applications in the adult for tissue
regeneration and the treatment of cancers. It may also act as a
morphogen to control cell and tissue type specification. Therefore,
the polynucleotides and polypeptides of the present invention would
be useful in treating, detecting, and/or preventing said disorders
and conditions, in addition to other types of degenerative
conditions. Thus this protein may modulate apoptosis or tissue
differentiation and would be useful in the detection, treatment,
and/or prevention of degenerative or proliferative conditions and
diseases. The protein would be useful in modulating the immune
response to aberrant polypeptides, as may exist in proliferating
and cancerous cells and tissues. The protein can also be used to
gain new insight into the regulation of cellular growth and
proliferation. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0212] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:34 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1452 of SEQ ID NO:34, b is an integer
of 15 to 1466, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:34, and where b is greater
than or equal to a+14.
[0213] Features of Protein Encoded By Gene No: 25
[0214] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: GRARGRPPGPEAAPASLSVSLRREVHSRGE (SEQ ID NO: 320).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0215] The polypeptide of this gene has been determined to have a
transmembrane domain at about amino acid position 2 to about 18 of
the amino acid sequence referenced in Table 1 for this gene.
Moreover, a cytoplasmic tail encompassing amino acids 19 to 130 of
this protein has also been determined. Based upon these
characteristics, it is believed that the protein product of this
gene shares structural features to type Ib membrane proteins.
[0216] This gene is expressed primarily in olfactory epithelium and
prostate.
[0217] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
olfactory and prostate disorders and prostate cancer. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
olfactory system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues or
cell types (e.g., olfactory, cancerous and wounded tissues) or
bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of one or both of the immunogenic epitopes shown in SEQ ID
NO: 142 as residues: His-24 to Ala-29, Glu-42 to Glu-49.
Polynucleotides encoding said polypeptides are encompassed by the
invention, as are antibodies that bind one or more of these
peptides.
[0218] The tissue distribution primarily in the olfactory
epithelium indicates a role for polynucleotides and polypeptides
corresponding to this gene in the treatment, prevention, detection
and/or diagnosis of olfactory and sensory disorders, including loss
of the sense of smell. The expression in the prostate tissue
indicates that polynucleotides and/or polypeptides of the invention
would be useful for diagnosis, treatment and/or prevention of the
disorders of the prostate, including inflammatory disorders, such
as chronic prostatitis, granulomatous prostatitis and malacoplakia,
prostatic hyperplasia and prostate neoplastic disorders, including
adenocarcinoma, transitional cell carcinomas, ductal carcinomas,
squamous cell carcinomas, or as hormones or factors with systemic
or reproductive functions. Furthermore, the protein may also be
used to determine biological activity, raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0219] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:35 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 512 of SEQ ID NO:35, b is an integer
of 15 to 526, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:35, and where b is greater
than or equal to a+14.
[0220] Features of Protein Encoded By Gene No: 26
[0221] The gene encoding the disclosed cDNA is believed to reside
on chromosome 14. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 14.
[0222] This gene is expressed primarily in 8 week embryo.
[0223] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
developmental disorders. Similarly, polypeptides and antibodies
directed to these polypeptides would be useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly during fetal development, expression
of this gene at significantly higher or lower levels may be
routinely detected in certain tissues or cell types (e.g.,
embryonic, cancerous and wounded tissues) or bodily fluids (e.g.,
lymph, amniotic fluid, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0224] The expression of this gene primarily in the embryo,
indicates a key role for polynucleotides and polypeptides
corresponding to this gene in embryo development and further
indicates its usefulness in the treatment and/or detection of
embryonic developmental defects. Moreover, the expression within
embryonic tissue and other cellular sources marked by proliferating
cells indicates that polynucleotides and polypeptides corresponding
to this gene may play a role in the regulation of cellular
division, and may show utility in the diagnosis, treatment, and/or
prevention of developmental diseases and disorders,
including.cancer, and other proliferative conditions.
Representative uses are described in the "Hyperproliferative
Disorders" and "Regeneration" sections below and elsewhere herein.
Briefly, developmental tissues rely on decisions involving cell
differentiation and/or apoptosis in pattern formation.
Dysregulation of apoptosis can result in inappropriate suppression
of cell death, as occurs in the development of some cancers, or in
failure to control the extent of cell death, as is believed to
occur in acquired immunodeficiency and certain degenerative
disorders, such as spinal muscular atrophy (SMA). Alternatively,
polynucleotides and polypeptides corresponding to this gene may be
involved in the pattern of cellular proliferation that accompanies
early embryogenesis. Thus, aberrant expression of this gene product
in tissues--particularly adult tissues--may correlate with patterns
of abnormal cellular proliferation, such as found in various
cancers. Because of potential roles in proliferation and
differentiation, polynucleotides and polypeptides corresponding to
this gene may have applications in the adult for tissue
regeneration and the treatment of cancers. It may also act as a
morphogen to control cell and tissue type specification. Therefore,
the polynucleotides and polypeptides of the present invention would
be useful in treating, detecting, and/or preventing said disorders
and conditions, in addition to other types of degenerative
conditions. Thus this protein may modulate apoptosis or tissue
differentiation and would be useful in the detection, treatment,
and/or prevention of degenerative or proliferative conditions and
diseases. The polynucleotides and polypeptides corresponding to
this gene would be useful in modulating the immune response to
aberrant polypeptides, as may exist in proliferating and cancerous
cells and tissues. The protein can also be used to gain new insight
into the regulation of cellular growth and proliferation.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0225] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:36 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2398 of SEQ ID NO:36, b is an integer
of 15 to 2412, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:36, and where b is greater
than or equal to a+14.
[0226] Features of Protein Encoded By Gene No: 27
[0227] This gene is expressed primarily in neutrophils.
[0228] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
disorders affecting the immune system. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
immune, cancerous and wounded tissues) or bodily fluids (e.g.,
lymph, serum, plasma, urine, synovial fluid and spinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of one or both
of the immunogenic epitopes shown in SEQ ID NO: 144 as residues:
Trp-25 to Thr-38, Pro-83 to Ala-88. Polynucleotides encoding said
polypeptides are encompassed by the invention, as are antibodies
that bind one or more of these peptides.
[0229] The tissue distribution in neutrophils indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the diagnosis, detection, prevention and/or treatment
of immune system disorders, especially those affecting neutrophils.
Furthermore, polynucleotides and polypeptides corresponding to this
gene may be involved in the regulation of cytokine production,
antigen presentation, or other processes that may also suggest a
usefulness in the treatment of cancer (e.g. by boosting immune
responses). Since the gene is expressed in cells of lymphoid
origin, the gene or protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues. Therefore it
may be also used as an agent for immunological disorders including
arthritis, asthma, immune deficiency diseases such as AIDS,
leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,
acne, and psoriasis. In addition, polynucleotides and polypeptides
corresponding to this gene may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0230] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:37 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1260 of SEQ ID NO:37, b is an integer
of 15 to 1274, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:37, and where b is greater
than or equal to a+14.
[0231] Features of Protein Encoded By Gene No: 28
[0232] The translation product of this gene shares sequence
homology with protein complexes related to clathrin adaptors (see,
e.g., AAD43327 (AF155157) which are thought to play a role in
signal-mediated trafficking of integral membrane proteins in
mammalian cells (see, e.g., Le Borgne and Hoflack, Curr Opin Cell
Biol 10:499-503 (1998); all references available through this
accession and reference are hereby incorporated by reference
herein.) Based on the sequence similarity, the translation product
of this clone is expected to share at least some biological
activities with protein complexes related to clathrin adaptors.
Such activities are known in the art, some of which are described
elsewhere herein.
[0233] The gene encoding the disclosed cDNA is thought to reside on
chromosome 1. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 1.
[0234] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: QTPFTCTLIIHRACXXPVRXSRVDPRVRGKQALIWLLGVHGERIPNAPYVLE
DFVENVKSETFPAVKMELLTALLRLFLSRPAECQDMLGRLLYYCIEEEKDMA
VRDRGLFYYRLLLVGIDEVKRILCSPKSDPTLGLLEDPAERPVNSWASDFNTL
VPVYGKAHWATISKCQGAERCDPELPKTSSFAASGPLIPEENKERVQELPDSG
ALMLVPNRQLTADYFEKTWLSLKVAHQQVLPWRGEFHPDTLQMALQVVNI
QTIAMSRAGSRPWKAYLSAQDDTGCLFLTELLLEPGNSEMQISVKQNEARTE
TLNSFISVLETVIGTIEEIKS (SEQ ID NO: 321) Moreover, fragments and
variants of these polypeptides (such as, for example, fragments as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0235] This gene is expressed primarily in fetal liver, immune
cells (e.g., eosinophils and T-cells), colon tumor, and brain
tissue, and, to a lesser extent, in various other fetal and
transformed cell types.
[0236] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited
to,immune, developmental and neurological conditions. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
developing, immune and central nervous systems, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., immune,
developing, neural, cancerous and wounded tissues) or bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder. Preferred polypeptides
of the present invention comprise, or alternatively consist of one,
two, three, four, five or all six of the immunogenic epitopes shown
in SEQ ID NO: 145 as residues: Pro-75 to Asn-81, Gln-106 to
Cys-111, Glu-130 to Asp-141, Arg-176 to Asp-182, Ala-201 to
Trp-206, Lys-238 to Thr-246. Polynucleotides encoding said
polypeptides are encompassed by the invention, as are antibodies
that bind one or more of these peptides.
[0237] The tissue distribution in fetal liver and brain tissues
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the study, detection, diagnosis,
prevention and/or treatment of growth disorders and neoplasias of
the immune and central nervous systems. The tissue distribution
indicates polynucleotides and polypeptides corresponding to this
gene would be useful for the detection, treatment, and/or
prevention of neurodegenerative disease states, behavioral
disorders, or inflammatory conditions. Representative uses are
described in the "Regeneration" and "Hyperproliferative Disorders"
sections below, in Example 11, 15, and 18, and elsewhere herein.
Briefly, the uses include, but are not limited to the detection,
treatment, and/or prevention of Alzheimer's Disease, Parkinson's
Disease, Huntington's Disease, Tourette Syndrome, meningitis,
encephalitis, demyelinating diseases, peripheral neuropathies,
neoplasia, trauma, congenital malformations, spinal cord injuries,
ischemia and infarction, aneurysms, hemorrhages, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder,
depression, panic disorder, learning disabilities, ALS, psychoses,
autism, and altered behaviors, including disorders in feeding,
sleep patterns, balance, and perception. In addition, elevated
expression of this gene product in regions of the brain indicates
it plays a role in normal neural function. Potentially, this gene
product is involved in synapse formation, neurotransmission,
learning, cognition, homeostasis, or neuronal differentiation or
survival. In addition, the gene or gene product may also play a
role in the treatment and/or detection of developmental disorders
associated with the developing embryo, or sexually-linked
disorders. Alternatively, expression of this gene product in fetal
liver/spleen tissue indicates a role in the regulation of the
proliferation; survival; differentiation; and/or activation of
potentially all hematopoietic cell lineages, including blood stem
cells. Polynucleotides and polypeptides of the invention may be
involved in the regulation of cytokine production, antigen
presentation, or other processes that may also suggest a usefulness
in the treatment of cancer (e.g., by boosting immune responses).
Since the gene is expressed in cells of lymphoid origin, the gene
or protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma, immune
deficiency diseases such as AIDS, leukemia, rheumatoid arthritis,
inflammatory bowel disease, sepsis, acne, and psoriasis. In
addition, this gene product may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0238] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:38 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1022 of SEQ ID NO:38, b is an integer
of 15 to 1036, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:38, and where b is greater
than or equal to a+14.
[0239] Features of Protein Encoded By Gene No: 29
[0240] This gene shares sequence homology to fibulin (see, e.g.,
GeneSeq Accession No. R11148 and R11149; all references available
through these accessions are hereby incorporated in their entirety
by reference herein). Fibulin binds to the cytoplasmic domain of
the beta-1 subunit of integrin adhesion receptors in a
cation-dependent, EDTA-reversible manner. Thus, polynucleotides and
polypeptides of the invention may be used to manipulate adhesion of
cells to fibronectin, collagen, laminin, and possibly also other
proteins.
[0241] When tested against both U937 Myeloid cell lines and Jurkat
T-cell cell lines, supernatants removed from cells containing this
gene activated the GAS assay. Thus, it is likely that this gene
activates both T-cells and myeloid cells, and to a lesser extent
other tissues and cell types, through the Jak-STAT signal
transduction pathway. The gamma activating sequence (GAS) is a
promoter element found upstream of many genes which are involved in
the Jak-STAT pathway. The Jak-STAT pathway is a large, signal
transduction pathway involved in the differentiation and
proliferation of cells. Therefore, activation of the Jak-STAT
pathway, reflected by the binding of the GAS element, can be used
to indicate proteins involved in the proliferation and
differentiation of cells.
[0242] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: CENTEGGYRCIC (SEQ ID NO:322). Moreover, fragments and
variants of these polypeptides (such as, for example, fragments as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention. This
sequence contains an aspartic acid and asparagine hydroxylation
site of the consensus sequence: C.[DN].{4}[FY].C.C (D or N is the
hydroxylation site). Post-translational hydroxylation of aspartic
acid or asparagine to form erythro-beta-hydroxyaspartic acid or
erythro-beta-hydroxyasparagine has been identified in a number of
proteins with domains homologous to epidermal growth factor (EGF)
(see, e.g., Stenflo J., et al., J. Biol. Chem. 263:21-24 (1988)).
Examples of such proteins are the blood coagulation protein factors
VII, IX and X, proteins C, S, and Z, the LDL receptor,
thrombomodulin, etc. Based on sequence comparisons of the
EGF-homology region that contains hydroxylated Asp or Asn, a
consensus sequence has been identified that seems to be required by
the hydroxylase(s). All references are hereby incorporated in their
entirety herein by reference.
[0243] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: CDCQAGYGGEAC (SEQ ID NO: 323) and/or
CICAEGYKQMEGIC (SEQ ID NO: 324). Moreover, fragments and variants
of these polypeptides (such as, for example, fragments as described
herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%, or 100% identical to these polypeptides, or polypeptides
encoded by a polynucleotide which hybridizes, under stringent
conditions, to the polynucleotide encoding these polypeptides) are
encompassed by the invention. Antibodies that bind polypeptides of
the invention and polynucleotides encoding these polypeptides are
also encompassed by the invention. These sequences contain EGF-like
domain signatures (consensus sequence: C.C.{5}G.{2}C or
C.C.{2}[GP][FYW].{4,8}C). A sequence of about thirty to forty
amino-acid residues long found in the sequence of epidermal growth
factor (EGF) has been shown to be present, in a more or less
conserved form, in a large number of other, mostly animal proteins.
The functional significance of EGF domains in what appear to be
unrelated proteins is not yet clear. However, a common feature is
that these repeats are found in the extracellular domain of
membrane-bound proteins or in proteins known to be secreted. For
further information see, e.g., Davis C. G., New Biol. 2:410-419
(1990), Blomquist M. C., et al., Proc. Natl. Acad. Sci. U.S.A.
81:7363-7367 (1984), Barker W. C., et al., Protein Nucl. Acid Enz.
29:54-68 (1986), Doolittle R. F., et al., Nature 307:558-560
(1984), Appella E., et al., FEBS Lett. 231:1-4 (1988), Campbell I.
D., et al., Curr. Opin. Struct. Biol. 3:385-392 (1993), and/or
Tamkun J. W., et al., Cell 46:271-282 (1986). All references are
hereby incorporated in their entirety herein by reference.
[0244] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: DIDECGTEGANCGADQFCVNTEGSYEC (SEQ ID NO:
325) and/or DVDECETEVCPGENKQCENTEGGYRC (SEQ ID NO: 326). Moreover,
fragments and variants of these polypeptides (such as, for example,
fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides,
or polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention. These
sequences contain Calcium-binding EGF-like domain pattern
signatures (consensus sequence:
[DEQN].[DEQN]{2}C.{3,14}C.{3,7}C.[DN].{4}[FY].C). A sequence of
about forty amino-acid residues long found in the sequence of
epidermal growth factor (EGF) has been shown to be present in a
large number of membrane-bound and extracellular, mostly animal
proteins. Many of these proteins require calcium for their
biological function and a calcium-binding site has been found to be
located at the N-terminus of some EGF-like domains. Calcium-binding
may be crucial for numerous protein-protein interactions. Some
proteins that are known or that are predicted to contain
calcium-binding EGF-like domains include: Bone morphogenic protein
1 (BMP-1), Calcium-dependent serine proteinase (CASP), Cartilage
oligomeric matrix protein COMP, Coagulation factors VII, IX, and X,
Fibrillin 1 and fibrillin 2, and Leucocyte antigen. For references
see: New Biol. 2:410-419 (1990), Blomquist M. C., et al., Proc.
Natl. Acad. Sci. U.S.A. 81:7363-7367 (1984), Barker W. C., et al.,
Protein Nucl. Acid Enz. 29:54-68 (1986), Doolittle R. F., et al.,
Nature 307:558-560 (1984), Appella E., et al., FEBS Lett. 231:1-4
(1988) Campbell I. D., et al., Curr. Opin. Struct. Biol. 3:385-392
(1993), Rao Z., et al., Cell 82:131-141 (1995), et al., J. Biol.
Chem. 267:19642-19649 (1992). All references are hereby
incorporated in their entirety herein by reference.
[0245] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: TABLE-US-00004 (SEQ ID NO: 327)
CDCQAGYGGEACGQCGLGYFEAERNASHLVCSAC.
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention. This sequence contains a Laminin-type EGF-like (LE)
domain signature (consensus sequence:
C-x(1,2)-C-x(5)-G-x(2)-C-x(2)-C-x(3,4)-[FYW]-x(3,15)-C). Laminins
(see, e.g., Beck K., et al., FASEB J. 4:148-160(1990)) are the
major noncollagenous components of basement membranes that mediate
cell adhesion, growth migration, and differentiation. They are
composed of distinct but related alpha, beta and gamma chains. The
three chains form a cross-shaped molecule that consist of a long
arm and three short globular arms. The long arm consist of a coiled
coil structure contributed by all three chains and cross-linked by
interchain disulfide bonds. Beside different types of globular
domains each subunit contains, in its first half, consecutive
repeats of about 60 amino acids in length that include eight
conserved cysteines (see, e.g., Engel J., FEBS Lett.
251:1-7(1989)). The tertiary structure (see, e.g, Stetefeld J., et
al., J. Mol. Biol. 257:644-657(1996) Baumgartner R., et al., J.
Mol. Biol. 257:658-668(1996)) of this domain is remotely similar in
its N-terminal to that of the EGF-like module. It is known as a
`LE` or `laminin-type EGF-like` domain. The number of copies of the
LE domain in the different forms of laminins is highly variable;
from 3 up to 22 copies have been found. All references are hereby
incorporated in their entirety herein by reference.
[0246] The gene encoding the disclosed cDNA is thought to reside on
chromosome 3. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 3.
[0247] This gene is expressed primarily in cerebellum tissue, and,
to a lesser extent, in multiple tissues and cell types including
prostate, liver, T-cells, kidney, and lung tissues, as well as
musculo-skeletal tissues such as endothelial tissue, healing groin
wound tissue, fetal heart tissue, and osteosarcoma tissue.
[0248] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
diseases and/or disorders of the central nervous system, including
dementia, mood disorders, both unipolar and bipolar deppression,
and Alzheimer's disease, as well as disorders of the
musculo-skeletal, renal, and pulmonary systems. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
central nervous system, renal, pulmonary system, and
musculo-skeletal system, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., neural, musculo-skeletal, cancerous and
wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder. Preferred polypeptides of the present invention comprise,
or alternatively consist of one, two, three, four, five, six,
seven, eight, nine ten, eleven, twelve, thirteen, fourteen, or all
fifteen of the immunogenic epitopes shown in SEQ ID NO: 146 as
residues: Pro-28 to Thr-45, Arg-59 to Gly-67, Ala-71 to Glu-84,
Lys-120 to Asp-126, Pro-159 to Gly-164, Glu-167 to Gly-186, Arg-217
to Asn-225, Glu-245 to Ala-255, Gly-282 to Gly-297, Pro-312 to
Gly-324, Thr-356 to Lys-364, Gly-366 to Thr-372, Lys-377 to
Ala-383, Gly-397 to Thr-407, Thr-419 to Gly-433. Polynucleotides
encoding said polypeptides are encompassed by the invention, as are
antibodies that bind one or more of these peptides.
[0249] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
diagnosis, detection, prevention and/or treatment of a variety of
cancers, most notably cancers of the central nervous system,
pulmonary, and renal systems, as well as the disorders of the
central nervous system listed above. Representative uses are
described in the "Hyperproliferative Diseases", "Chemotaxis" and
"Binding Activity" sections below, in Examples 11, 12, 13, 14, 15,
16, 18, 19, and 20, and elsewhere herein. Briefly, the expression
of this gene product in a variety of systems indicates that
polynucleotides and polypeptides corresponding to this gene may be
a player in the progression of these diseases, and may be a
beneficial target for inhibitors as therapeutics. Alternatively,
the tissue distribution in musculo-skeletal tissues, as the
homology to fibulin, indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, diagnosis, prevention and/or treatment of disorders
involving the vasculature. Elevated expression of this gene product
by endothelial cells indicates that it may play vital roles in the
regulation of endothelial cell function; secretion; proliferation;
or angiogenesis. Alternately, this may represent a gene product
expressed by the endothelium and transported to distant sites of
action on a variety of target organs. Furthermore, the protein may
also be used to determine biological activity, to raise antibodies,
as tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0250] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:39 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1365 of SEQ ID NO:39, b is an integer
of 15 to 1379, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:39, and where b is greater
than or equal to a+14.
[0251] Features of Protein Encoded By Gene No: 30
[0252] The translation product of this gene shares sequence
homology with coxsackie and adenovirus receptor in mouse.
Particularly, this gene shares sequence homology with a human A33
antigen, which is a transmembrane protein and a novel member of the
immunoglobulin superfamily. (see, e.g., Proc. Natl. Acad. Sci.
U.S.A. 94, 469-474 (1997); see also, Accession No. 1814277; all
references available through the accession and reference are hereby
incorporateed herein by reference.) Therefore, this gene likely has
activity similar to the human A33 antigen.
[0253] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: TABLE-US-00005 (SEQ ID NO: 328)
MISLPGPLVTNLLRFLFLGLSALAPPSRAQLQLHLPANRLQAVEGGEVVL
PAWYTLHGEVSSSQPWEVPFVMWFFKQKEKEDQVLSYINGVTTSKPGVSL
VYSMPSRNLSLRLEGLQEKDSGPYSCSVNVQNKQGKSRGHSIKTLELNVL
VPPAPPSCRLQGVPHVGANVTLSCQSPRSKPAVQYQWDRQLPSFQTFFAP
ALDVIRGSLSLTNLSSSMAGVYVCKAHNEVGTAQCNVTLEVSTGPGAAVV
AGAVVGTLVGLGLLAGLVLLYHRRGKALEEPANDIKEDAIAPRTLPWPKS
SDTISKNGTLSSVTSARALRPPHGPPRPGALTPTPSLSSQALPSPRLPTT
DGAHPQPISPIPGGVSSSGLSRMGAVPVMVPAQSQAGSL, (SEQ ID NO: 329)
MISLPGPLVTNLLRFLFLGLSALAPPSRAQLQLHL, (SEQ ID NO: 330)
PANRLQAVEGGEVVLPAWYTLHGEVSSSQPWEVPF, (SEQ ID NO: 331)
VMWFFKQKEKEDQVLSYINGVTTSKPGVSLVYSMP, (SEQ ID NO: 332)
SRNLSLRLEGLQEKDSGPYSCSVNVQNKQGKSRGH, (SEQ ID NO: 333)
SIKTLELNVLVPPAPPSCRLQGVPHVGANVTLSCQ (SEQ ID NO: 334)
SPRSKPAVQYQWDRQLPSFQTFFAPALDVIRGSLS, (SEQ ID NO: 335)
LTNLSSSMAGVYVCKAHNEVGTAQCNVTLEVSTGP, (SEQ ID NO: 336)
GAAVVAGAVVGTLVGLGLLAGLVLLYHRRGKALEE, (SEQ ID NO: 337)
PANDIKEDAIAPRTLPWPKSSDTISKNGTLSSVTS, (SEQ ID NO: 338)
ARALRPPHGPPRPGALTPTPSLSSQALPSPRLPTT, and/or (SEQ ID NO: 339)
DGAHPQPISPIPGGVSSSGLSRMGAVPVMVPAQSQAGSL.
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0254] The translated product of this gene also shares some
homology with a mouse basement membrane proteoglycan (see, e.g.,
GenBank Accession AAA39911.1 and Noonan, D. M., et al., J. Biol.
Chem. 266, 22939-22947 (1991); all references available through
this citation are hereby incorporated herein by reference). Based
on the sequence similarity, the translation product of this clone
is expected to share at least some biological activities with
extracellular basement membrane proteoglcans. Such activities are
known in the art, some of which are described elsewhere herein.
[0255] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: TABLE-US-00006 (SEQ ID NO: 340)
LSLTNLSSSMAGVYVCKAHNEVGTAQCNVTLEVSTG.
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0256] Contact of cells with supernatant expressing the product of
this gene has been shown to increase the permeability of the plasma
membrane of THP-1 cell lines to calcium. Thus it is likely that the
product of this gene is involved in a signal transduction pathway
that is initiated when the product binds a receptor on the surface
of the plasma membrane of both monocytes, and to a lesser extent,
other immune and hematopoietic cells. Thus, polynucleotides and
polypeptides have uses which include, but are not limited to,
activating monocytes. Binding of a ligand to a receptor is known to
alter intracellular levels of small molecules, such as calcium,
potassium and sodium, as well as alter pH and membrane potential.
Alterations in small molecule concentration can be measured to
identify supernatants which bind to receptors of a particular
cell.
[0257] This gene is expressed in various tissues including
placenta, brain, heart, muscle, adipocytes, and liver.
[0258] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions: viral diseases, and immune diseases and/or
disorders. Similarly, polypeptides and antibodies directed to those
polypeptides would be useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system and central nervous system, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., CNS, reproductive,
vascular, cancerous and wounded tissues) or bodily fluids (e.g.,
lymph, amniotic fluid, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0259] The tissue distribution in various tissues including
placenta, brain, heart, muscle, adipocytes, and liver, and the
homology to A33 antigen indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
diagnosis, detection, prevention and/or treatment of a variety of
cancers, most notably cancers of the immune system, as well as
viral infections. Expression of this gene product indicates that
polynucleotides and polypeptides corresponding to this gene may be
a player in the progression of these diseases, and may be a
beneficial target for inhibitors as therapeutics. Representative
uses are described in the "Chemotaxis" and "Binding Activity"
sections below, in Examples 11, 12, 13, 14, 15, 16, 18, 19, and 20,
and elsewhere herein. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0260] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:40 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1918 of SEQ ID NO:40, b is an integer
of 15 to 1932, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:40, and where b is greater
than or equal to a+14.
[0261] Features of Protein Encoded By Gene No: 31
[0262] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:GSSFVVSEGSYLDISDWLNPAKLSLYY (SEQ ID
NO:341), LDISDWLNPAKL (SEQ ID NO:342), SDWLNPAKLSL (SEQ ID NO:343),
and/or DACEQLCDPETGE (SEQ ID NO:344). Moreover, fragments and
variants of these polypeptides (such as, for example, fragments as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0263] This gene is expressed primarily in human ovary and adrenal
gland tissues.
[0264] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
reproductive diseases and/or disorders, particularly ovarian
cancer. Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the reproductive system, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., reproductive, and cancerous and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial fluid and spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0265] The tissue distribution in ovary tissue indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for diagnosing and/or treating reproductive system
disorders including ovarian cancer, as well as cancers of other
tissues where expression has been observed. Representative uses are
described in the "Hyperproliferative Disorders" and "Regeneration"
sections below and elsewhere herein. Expression in ovarian tissue,
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the treatment, prevention, detection
and diagnosis of conditions concerning proper ovarian function
(e.g., egg maturation, endocrine function), as well as cancer. The
expression in ovarian tissue may indicate the gene or its products
can be used to treat, prevent, detect and/or diagnose disorders of
the ovary, including inflammatory disorders, such as oophoritis
(e.g., caused by viral or bacterial infection), ovarian cysts,
amenorrhea, infertility, hirsutism, and ovarian cancer (including,
but not limited to, primary and secondary cancerous growth,
endometrioid carcinoma of the ovary, ovarian papillary serous
adenocarcinoma, ovarian mucinous adenocarcinoma, Ovarian Krukenberg
tumor). Furthermore, the protein may also be used to determine
biological activity, to raise antibodies, as tissue markers, to
isolate cognate ligands or receptors, to identify agents that
modulate their interactions, in addition to its use as a
nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0266] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:41 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1416 of SEQ ID NO:41, b is an integer
of 15 to 1430, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:41, and where b is greater
than or equal to a+14.
[0267] Features of Protein Encoded By Gene No: 32
[0268] This gene is expressed primarily in thymus and stromal
cells.
[0269] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
aberrant immune responses, such as either chronic or acute
inflammation. Similarly, polypeptides and antibodies directed to
these polypeptides would be useful in providing immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the immune system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., immune, cancerous and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial fluid and spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0270] The tissue distribution in thymus stromal cells indicates
that polynucleotides and polypeptides corresponding to this gene
would be useful for diagnosing, detecting, preventing and/or
treating disorders of the immune system, particularly those
involving a pathological inflammatory reponse. Representative uses
are described in the "Immune Activity" and "Infectious Disease"
sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and
elsewhere herein. Furthermore, the gene product may also be
involved in lymphopoiesis, therefore, it can be used in immune
disorders such as infection, inflammation, allergy,
immunodeficiency etc. In addition, polynucleotides and polypeptides
corresponding to this gene may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0271] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:42 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1393 of SEQ ID NO:42, b is an integer
of 15 to 1407, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:42, and where b is greater
than or equal to a+14.
[0272] Features of Protein Encoded By Gene No: 33
[0273] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: EGKIKICEKKAIKVILHTCNS (SEQ ID NO: 345). Moreover,
fragments and variants of these polypeptides (such as, for example,
fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides,
or polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0274] This gene is expressed primarily in frontal cortex.
[0275] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
central nervous system (CNS) diseases and/or disorders. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the CNS,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
brain, cancerous and wounded tissues) or bodily fluids (e.g.,
lymph, serum, plasma, urine, synovial fluid or cerebrospinal fluid)
or another tissue or cell sample taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue or bodily fluid from
an individual not having the disorder. Preferred polypeptides of
the present invention comprise, or alternatively consist of the
immunogenic epitopes shown in SEQ ID NO: 150 as residues: Pro-41 to
Asp-47. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0276] The tissue distribution in frontal cortex indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for detection, treatment, and/or prevention of CNS
disorders including disorders of the brain and nervous system.
Representative uses are described in the "Regeneration" and
"Hyperproliferative Disorders" sections below, in Example 11, 15,
and 18, and elsewhere herein. Elevated expression of this gene
product within the frontal cortex of the brain indicates that it
may be involved in neuronal survival, synapse formation,
conductance, neural differentiation, etc. Such involvement may
impact many processes, such as learning and cognition. It may also
be useful in the treatment of such neurodegenerative disorders as
schizophrenia, ALS, or Alzheimer's. Furthermore, the protein may
also be used to determine biological activity, to raise antibodies,
as tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0277] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:43 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 936 of SEQ ID NO:43, b is an integer
of 15 to 950, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:43, and where b is greater
than or equal to a+14.
[0278] Features of Protein Encoded By Gene No: 34
[0279] This gene is expressed primarily in adipose tissue, human
embryo, and neutrophils.
[0280] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
obesity, Nasu-Hakola disease, cardiovascular disease,
non-insulin-dependent diabetes mellitus. Similarly, polypeptides
and antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the adipose, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., adipose, cancerous
and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0281] The tissue distribution in adipose indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the treatment, prevention, detection and diagnosis of
metabolic disorders related to lipids and adipose tissue, such as
obesity, Nasu-Hakola disease (membranous lipodystrophy),
cardiovascular disease, lipidemia, non-insulin-dependent diabetes
mellitus, stroke and carcinoma. The tissue distribution in
neutrophils indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the diagnosis and
treatment of a variety of immune system disorders. Representative
uses are described in the "Immune Activity" and "Infectious
Disease" sections below, in Example 11, 13, 14, 16, 18, 19, 20, and
27, and elsewhere herein. Briefly, the expression indicates a role
in regulating the proliferation; survival; differentiation; and/or
activation of hematopoietic cell lineages, including blood stem
cells. Involvement in the regulation of cytokine production,
antigen presentation, or other processes indicates a usefulness for
treatment of cancer (e.g., by boosting immune responses).
Expression in cells of lymphoid origin, indicates the natural gene
product would be involved in immune functions. Therefore it would
also be useful as an agent for immunological disorders including
arthritis, asthma, immunodeficiency diseases such as AIDS,
leukemia, rheumatoid arthritis, granulomatous disease, inflammatory
bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus,
polynucleotides and polypeptides corresponding to this gene are
thought to be useful in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Moreover, the
expression within embryonic tissue and other cellular sources
marked by proliferating cells indicates that polynucleotides and
polypeptides corresponding to this gene may play a role in the
regulation of cellular division, and may show utility in the
diagnosis, treatment, and/or prevention of developmental diseases
and disorders, including cancer, and other proliferative
conditions. Representative uses are described in the
"Hyperproliferative Disorders" and "Regeneration" sections below
and elsewhere herein. Briefly, developmental tissues rely on
decisions involving cell differentiation and/or apoptosis in
pattern formation. Dysregulation of apoptosis can result in
inappropriate suppression of cell death, as occurs in the
development of some cancers, or in failure to control the extent of
cell death, as is believed to occur in acquired immunodeficiency
and certain degenerative disorders, such as spinal muscular atrophy
(SMA). Alternatively, this gene product may be involved in the
pattern of cellular proliferation that accompanies early
embryogenesis. Thus, aberrant expression of this gene product in
tissues--particularly adult tissues--may correlate with patterns of
abnormal cellular proliferation, such as found in various cancers.
Because of potential roles in proliferation and differentiation,
this gene product may have applications in the adult for tissue
regeneration and the treatment of cancers. It may also act as a
morphogen to control cell and tissue type specification. Therefore,
the polynucleotides and polypeptides of the present invention would
be useful in treating, detecting, and/or preventing said disorders
and conditions, in addition to other types of degenerative
conditions. Thus polynucleotides and polypeptides corresponding to
this gene may modulate apoptosis or tissue differentiation and
would be useful in the detection, treatment, and/or prevention of
degenerative or proliferative conditions and diseases. The
polynucleotides and polypeptides corresponding to this gene would
be useful in modulating the immune response to aberrant
polypeptides, as may exist in proliferating and cancerous cells and
tissues. The protein can also be used to gain new insight into the
regulation of cellular growth and proliferation. Furthermore, the
protein may also be used to determine biological activity, to raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0282] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:44 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 990 of SEQ ID NO:44, b is an integer
of 15 to 1004, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:44, and where b is greater
than or equal to a+14.
[0283] Features of Protein Encoded By Gene No: 35
[0284] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: NSARVEFFIPPLRITQKVRSTKS (SEQ ID NO:346). Moreover,
fragments and variants of these polypeptides (such as, for example,
fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides,
or polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0285] This gene is apparently expressed primarily in IL-1- and
LPS-induced neutrophils.
[0286] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
abnormal immune reactions or disorders including, but not limited
to, chronic or cyclic neutropenia, neutrophilia, and neutrocytosis.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues or
cell types (e.g., immune, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0287] The tissue distribution in neutrophils indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for detection, treatment, and/or prevention of immune
disorders or abnormal reactions mediated by neutrophils, including
infection, inflammation, allergy, immunodeficiency, chronic or
cyclic neutropenia, neutrophilia, and neutrocytosis, and the like.
Representative uses are described in the "Immune Activity" and
"Infectious Disease" sections below, in Example 11, 13, 14, 16, 18,
19, 20, and 27, and elsewhere herein. Moreover, the expression of
this gene product indicates a role in regulating the proliferation,
survival, differentiation, and/or activation of hematopoietic cell
lineages, including blood stem cells. Polynucleotides and
polypeptides corresponding to this gene may be involved in the
regulation of cytokine production, antigen presentation, or other
processes that may also suggest a usefulness in the treatment of
cancer (e.g., by boosting immune responses). Since the gene is
expressed in cells of lymphoid origin, the natural gene product may
be involved in immune functions. Therefore it may be also used as
an agent for immunological disorders including arthritis, asthma,
immunodeficiency diseases such as AIDS, leukemia, rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease,
sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity, immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and
tissues. In addition, polynucleotides and polypeptides
corresponding to this gene may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Furthermore, the protein may also be used to
determine biological activity, raise antibodies, as tissue markers,
to isolate cognate ligands or receptors, to identify agents that
modulate their interactions, in addition to its use as a
nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0288] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:45 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1667 of SEQ ID NO:45, b is an integer
of 15 to 1681, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:45, and where b is greater
than or equal to a+14.
[0289] Features of Protein Encoded By Gene No: 36
[0290] The translated ORF of the contig has homology with the
human, porcine, and bovine INS10 double-chain insulin precursor,
especially around a region containing multiple cysteine
residues.
[0291] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
MMVWNLFPCFPPLLLLQFIDCQQSSEIEEQGFTRSLLGHPIFFCPDPCWQSCMN
CVILSVLSFFFLIRWISKIVAVQKLESSSRRKPILFLIISCEIASFIHLFLSQMSAEC
CCFYLVILICKY (SEQ ID NO:347), MMVWNLFPCFPPLLLLQFIDCQQSSEIE (SEQ ID
NO:348), QGFTRSLLGHPIFFCPDPCWQSCMNCVI (SEQ ID NO:349),
LSVLSFFFLIRWISKIVAVQKLESSSRRKPILFLI (SEQ ID NO:350), and/or
ISCEIASFIHLFLSQMSAECCCFYLVILICKY (SEQ ID NO:351). Moreover,
fragments and variants of these polypeptides (such as, for example,
fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides,
or polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0292] The polypeptide of this gene has been determined to have a
transmembrane domain at about amino acid position 50 to about 66 of
the amino acid sequence referenced in Table 1 for this gene.
Moreover, a cytoplasmic tail encompassing amino acids 67 to 90 of
this protein has also been determined. Based upon these
characteristics, it is believed that the protein product of this
gene shares structural features to type Ia membrane proteins.
[0293] The gene encoding the disclosed cDNA is believed to reside
on chromosome 21. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 21.
[0294] This gene is expressed primarily in cells and tissues
isolated from a 15 days post-incision healing abdomen wound and, to
a lesser extent, in many immune tissues (e.g., T-cells and
B-cells)and connective tissues/cells with proliferative capacity,
such as osteoclastoma, ovarian cancer, B-cell lymphoma and
hepatocellular tumor.
[0295] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,wound
healing, diabetes mellitus, and cancers of the bone and connective
tissues, lymphomas, and cancers of the liver. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly those of the
cells and tissues involved in healing tissue damages and
regeneration, diabetes mellitis, and many cancers including, but
not limited to ovarian cancer, breast cancer, colon cancer, cardiac
tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma,
lung cancer, intestinal cancer, testicular cancer, stomach cancer,
neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma,
adenoma, and the like, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of one, or both of the immunogenic epitopes shown in SEQ ID
NO: 153 as residues: Gln-22 to Phe-31, Leu-78 to Lys-85.
Polynucleotides encoding said polypeptides are encompassed by the
invention, as are antibodies that bind one or more of these
peptides.
[0296] The tissue distribution in healing wound and regenerating
tissues/cells indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for detection,
treatment, and/or prevention of tissue damages, trauma, necrosis,
and tissue regeneration. In addition, since this gene exhibits
homology with an insulin precursor, polynucleotides and
polypeptides corresponding to this gene can be used to regulate the
metabolism of glucose or other sugars, the synthesis of proteins,
and the formation and storage of neutral lipids. The tissue
distribution in immune tissues (e.g., T-cells and B-cells)
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the diagnosis and treatment of a
variety of immune system disorders. Representative uses are
described in the "Immune Activity" and "Infectious Disease"
sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and
elsewhere herein. Briefly, the expression indicates a role in
regulating the proliferation; survival; differentiation; and/or
activation of hematopoietic cell lineages, including blood stem
cells. Involvement in the regulation of cytokine production,
antigen presentation, or other processes indicates a usefulness for
treatment of cancer (e.g., by boosting immune responses).
Expression in cells of lymphoid origin, indicates the natural gene
product would be involved in immune functions. Therefore
polynucleotides and polypeptides corresponding to this gene would
also be useful as an agent for immunological disorders including
arthritis, asthma, immunodeficiency diseases such as AIDS,
leukemia, rheumatoid arthritis, granulomatous disease, inflammatory
bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus,
polynucleotides and polypeptides corresponding to this gene are
thought to be useful in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Furthermore, the
protein may also be used to determine biological activity, raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0297] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:46 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1347 of SEQ ID NO:46, b is an integer
of 15 to 1361, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:46, and where b is greater
than or equal to a+14.
[0298] Features of Protein Encoded By Gene No: 37
[0299] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: KVDTPRRHFCPEISFFLTPLPQSARNSTVRNALSGLKNLTPAMISTVSKQDTSK
LGEEE (SEQ ID NO:352). Moreover, fragments and variants of these
polypeptides (such as, for example, fragments as described herein,
polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% identical to these polypeptides, or polypeptides encoded by a
polynucleotide which hybridizes, under stringent conditions, to the
polynucleotide encoding these polypeptides) are encompassed by the
invention. Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0300] When tested against U937 Myeloid cell lines, supernatants
removed from cells containing this gene activated the GAS assay.
Thus, it is likely that this gene activates myeloid cells through
the Jak-STAT signal transduction pathway. The gamma activating
sequence (GAS) is a promoter element found upstream of many genes
which are involved in the Jak-STAT pathway. The Jak-STAT pathway is
a large, signal transduction pathway involved in the
differentiation and proliferation of cells. Therefore, activation
of the Jak-STAT pathway, reflected by the binding of the GAS
element, can be used to indicate proteins involved in the
proliferation and differentiation of cells.
[0301] The polypeptide of this gene has been determined to have a
transmembrane domain at about amino acid position 7 to about 23 of
the amino acid sequence referenced in Table 1 for this gene.
Moreover, a cytoplasmic tail encompassing amino acids 24 to 105 of
this protein has also been determined. Based upon these
characteristics, it is believed that the protein product of this
gene shares structural features to type Ia membrane proteins.
[0302] This gene is expressed primarily in B-cell lymphoma.
[0303] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
B-cell lymphoma, immunodeficient or auto-immune conditions.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues or
cell types (e.g., immune, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0304] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, treatment, and/or prevention of B-cell lymphomas, as
well as other immune disorders including: leukemias,
auto-immunities, immunodeficiencies (e.g., AIDS), immuno-supressive
conditions (transplantation) and hematopoietic disorders, such as
anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia,
since stromal cells are important in the production of cells of
hematopoietic lineages. In addition, polynucleotides and
polypeptides corresponding to this gene may be applicable in
conditions of general microbial infection, inflammation or cancer.
Representative uses are described in the "Immune Activity" and
"Infectious Disease" sections below, in Example 11, 13, 14, 16, 18,
19, 20, and 27, and elsewhere herein. The uses include bone marrow
cell ex vivo culture, bone marrow transplantation, bone marrow
reconstitution, radiotherapy or chemotherapy of neoplasia. The
polynucleotides and polypeptides corresponding to this gene may
also be involved in lymphopoiesis, therefore, it can be used in
immune disorders such as infection, inflammation, allergy,
immunodeficiency etc. In addition, the biological activity of
supernatants from cells expressing this gene in the GAS assay
indicates that this gene product may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0305] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:47 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1123 of SEQ ID NO:47, b is an integer
of 15 to 1137, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:47, and where b is greater
than or equal to a+14.
[0306] Features of Protein Encoded By Gene No: 38
[0307] The polypeptide of this gene has been determined to have a
transmembrane domain at about amino acid position 8 to about 24 of
the amino acid sequence referenced in Table 1 for this gene.
Moreover, a cytoplasmic tail encompassing amino acids 1 to 7 of
this protein has also been determined. Based upon these
characteristics, it is believed that the protein product of this
gene shares structural features to type II membrane proteins.
[0308] The gene encoding the disclosed cDNA is thought to reside on
chromosome 10. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 10.
[0309] This gene is expressed primarily in infant brain, testes,
brain, osteoblasts, and caudate nucleus tissues, and, to a lesser
extent, in various other normal and transformed cell types,
including smooth muscle and adult heart tissues, and T-cell
lymphoma.
[0310] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
neurological and growth defects. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the developing nervous
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues or cell types
(e.g., immune, neural, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0311] The tissue distribution in infant brain tissue indicates
that polynucleotides and polypeptides corresponding to this gene
would be useful for the study, detection and/or treatment of infant
and general nervous system disorders and neoplasias. The tissue
distribution indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection,
treatment, and/or prevention of neurodegenerative disease states,
behavioral disorders, or inflammatory conditions. Representative
uses are described in the "Regeneration" and "Hyperproliferative
Disorders" sections below, in Example 11, 15, and 18, and elsewhere
herein. Briefly, the uses include, but are not limited to the
detection, treatment, and/or prevention of Alzheimer's Disease,
Parkinson's Disease, Huntington's Disease, Tourette Syndrome,
meningitis, encephalitis, demyelinating diseases, peripheral
neuropathies, neoplasia, trauma, congenital malformations, spinal
cord injuries, ischemia and infarction, aneurysms, hemorrhages,
schizophrenia, mania, dementia, paranoia, obsessive compulsive
disorder, depression, panic disorder, learning disabilities, ALS,
psychoses, autism, and altered behaviors, including disorders in
feeding, sleep patterns, balance, and perception. In addition,
elevated expression of this gene product in regions of the brain
indicates it plays a role in normal neural function. Potentially,
this gene product is involved in synapse formation,
neurotransmission, learning, cognition, homeostasis, or neuronal
differentiation or survival. In addition, the gene or gene product
may also play a role in the treatment and/or detection of
developmental disorders associated with the developing embryo, or
sexually-linked disorders. Moreover, the tissue distribution in
immune cells (e.g., T-cells) indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
diagnosis, detection, prevention and/or treatment of a variety of
immune system disorders. Representative uses are described in the
"Immune Activity" and "Infectious Disease" sections below, in
Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.
Briefly, the expression indicates a role in regulating the
proliferation; survival; differentiation; and/or activation of
hematopoietic cell lineages, including blood stem cells.
Involvement in the regulation of cytokine production, antigen
presentation, or other processes indicates a usefulness for
treatment of cancer (e.g., by boosting immune responses).
Expression in cells of lymphoid origin, indicates the natural gene
product would be involved in immune functions. Therefore it would
also be useful as an agent for immunological disorders including
arthritis, asthma, immunodeficiency diseases such as AIDS,
leukemia, rheumatoid arthritis, granulomatous disease, inflammatory
bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus, this
gene product is thought to be useful in the expansion of stem cells
and committed progenitors of various blood lineages, and in the
differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0312] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:48 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2749 of SEQ ID NO:48, b is an integer
of 15 to 2763, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:48, and where b is greater
than or equal to a+14.
[0313] Features of Protein Encoded By Gene No: 39
[0314] The translated product of this gene shares some homology
with a Caenorhabditis elegans gene product containing zinc
finger-like motifs (see, e.g., Genbank Accession No.: AAA91223 and
Wilson, R., et al., Nature 368, 32-38 (1994)). Similarly, the
translated product of this gene also shares some homology with
transcriptional regulatory proteins from Saccharomyces cerevisiae
(see, e.g., GenBank Accessions Nos.: CAA92346.1, BAA04890.1, and
AAA34471.1). All references available through the above listed
accessions and citations are hereby incorporated herein by
reference. Based on the sequence similarity, the translation
product of this clone is expected to share at least some biological
activities with transcriptional regulatory proteins. Such
activities are known in the art, some of which are described
elsewhere herein.
[0315] This gene is expressed primarily in epithelial-TNFalpha and
INF induced cells and brain frontal cortex.
[0316] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
neurodegenerative diseases and/or disorders. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
central nervous system, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., CNS, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of one or both of the immunogenic epitopes shown in SEQ ID
NO: 156 as residues: Lys-35 to Asp-41, Glu-49 to Leu-63.
Polynucleotides encoding said polypeptides are encompassed by the
invention, as are antibodies that bind one or more of these
peptides.
[0317] The tissue distribution in the brain indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for detection, treatment, and/or prevention of
neurodegenerative disorders, especially those involving the frontal
cortex. Representative uses are described in the "Regeneration" and
"Hyperproliferative Disorders" sections below, in Example 11, 15,
and 18, and elsewhere herein. Briefly, the elevated expression of
this gene product within the frontal cortex of the brain indicates
that it may be involved in neuronal survival; synapse formation;
conductance; neural differentiation, etc. Such involvement may
impact many processes, such as learning and cognition.
Polynucleotides and polypeptides corresponding to this gene may
also be useful in the treatment of such neurodegenerative disorders
as schizophrenia; ALS; or Alzheimer's. Furthermore, the protein may
also be used to determine biological activity, to raise antibodies,
as tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0318] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:49 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1334 of SEQ ID NO:49, b is an integer
of 15 to 1348, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:49, and where b is greater
than or equal to a+14.
[0319] Features of Protein Encoded By Gene No: 40
[0320] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: PTRPPTRPLSFTFTKQTSSTCLSLHF (SEQ ID NO:353). Moreover,
fragments and variants of these polypeptides (such as, for example,
fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides,
or polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0321] The gene encoding the disclosed cDNA is believed to reside
on chromosome 18. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 18.
[0322] This gene is expressed primarily in infant brain, frontal
cortex, and, to a lesser extent, in melanocytes.
[0323] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
neurodegenerative diseases and/or disorders. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
central nervous system, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., CNS, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of one or both of the immunogenic epitopes shown in SEQ ID
NO: 157 as residues: Val-40 to Cys-47, Lys-49 to Gly-54.
Polynucleotides encoding said polypeptides are encompassed by the
invention, as are antibodies that bind one or more of these
peptides.
[0324] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, treatment, and/or prevention of neurodegenerative
disorders especially those involving the frontal cortex. Moreover,
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection, treatment, and/or prevention of
neurodegenerative disease states, behavioral disorders, or
inflammatory conditions. Representative uses are described in the
"Regeneration" and "Hyperproliferative Disorders" sections below,
in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses
include, but are not limited to the detection, treatment, and/or
prevention of Alzheimer's Disease, Parkinson's Disease,
Huntington's Disease, Tourette Syndrome, meningitis, encephalitis,
demyelinating diseases, peripheral neuropathies, neoplasia, trauma,
congenital malformations, spinal cord injuries, ischemia and
infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia,
paranoia, obsessive compulsive disorder, depression, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, elevated expression of this
gene product in regions of the brain indicates it plays a role in
normal neural function. Potentially, polynucleotides and
polypeptides corresponding to this gene are involved in synapse
formation, neurotransmission, learning, cognition, homeostasis, or
neuronal differentiation or survival. Furthermore, the protein may
also be used to determine biological activity, to raise antibodies,
as tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0325] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:50 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1250 of SEQ ID NO:50, b is an integer
of 15 to 1264, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:50, and where b is greater
than or equal to a+14.
[0326] Features of Protein Encoded By Gene No: 41
[0327] This gene shows structural homology with the duck insulin
precursor which is thought to be important in metabolic
homeostasis. (see, e.g., Genbank Accession No. pir|A01600|IPDK
insulin precursor; all references available through this accession
number are hereby incorporated in their entirety by reference
herein).
[0328] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: LECVLLICFRAMSAIYTHTSIGNAQKLFTDGSAFRRVREPLPKEGKSWPQ (SEQ
ID NO: 354). Moreover, fragments and variants of these polypeptides
(such as, for example, fragments as described herein, polypeptides
at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical
to these polypeptides, or polypeptides encoded by a polynucleotide
which hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0329] This gene is expressed primarily in eosinophil-IL5 induced
cells, and, to a lesser extent, in B cell lymphoma, breast lymph
node, and CD34 depleted buffy coat (cord blood).
[0330] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
immune diseases and/or disorders. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
immune, hematopoietic, and cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise immunogenic epitopes
shown in SEQ ID NO: 158 as residues: Arg-39 to Glu-56.
Polynucleotides encoding said polypeptides are encompassed by the
invention, as are antibodies that bind one or more of these
peptides.
[0331] The tissue distribution in hematopoietic tissues indicates
that polynucleotides and polypeptides corresponding to this gene
would be useful for detection, treatment, and/or prevention of
immune disorders especially those involving eosinophils and
B-cells. Polynucleotides and polypeptides corresponding to this
gene would be useful for the detection, treatment, and/or
prevention of a variety of immune system disorders. Representative
uses are described in the "Immune Activity" and "Infectious
Disease" sections below, in Example 11, 13, 14, 16, 18, 19, 20, and
27, and elsewhere herein. Briefly, the expression of this gene
product indicates a role in regulating the proliferation; survival;
differentiation; and/or activation of hematopoietic cell lineages,
including blood stem cells. Polynucleotides and polypeptides
corresponding to this gene may be involved in the regulation of
cytokine production, antigen presentation, or other processes
suggesting a usefulness in the treatment of cancer (e.g., by
boosting immune responses). Since the gene is expressed in cells of
lymphoid origin, the natural gene product may be involved in immune
functions. Therefore polynucleotides and polypeptides of the
invention may be also used as an agent for immunological disorders
including arthritis, asthma, immunodeficiency diseases such as
AIDS, leukemia, rheumatoid arthritis, granulomatous disease,
inflammatory bowel disease, sepsis, acne, neutropenia,
neutrophilia, psoriasis, hypersensitivities, such as T-cell
mediated cytotoxicity; immune reactions to transplanted organs and
tissues, such as host-versus-graft and graft-versus-host diseases,
or autoimmunity disorders, such as autoimmune infertility, lense
tissue injury, demyelination, systemic lupus erythematosis, drug
induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease,
scleroderma and tissues. Moreover, the protein may represent a
secreted factor that influences the differentiation or behavior of
other blood cells, or that recruits hematopoietic cells to sites of
injury. In addition, polynucleotides and polypeptides corresponding
to this gene may have commercial utility in the expansion of stem
cells and committed progenitors of various blood lineages, and in
the differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, raise antibodies, as tissue markers, to isolate cognate
ligands or receptors, to identify agents that modulate their
interactions, in addition to its use as a nutritional supplement.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0332] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:51 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1646 of SEQ ID NO:51, b is an integer
of 15 to 1660, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:51, and where b is greater
than or equal to a+14.
[0333] Features of Protein Encoded By Gene No: 42
[0334] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: KQNLTNLDVPVQYHVALSDKVK (SEQ ID NO: 355). Moreover,
fragments and variants of these polypeptides (such as, for example,
fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides,
or polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0335] This gene is expressed primarily in pineal gland and, to a
lesser extent, in multiple sclerosis cells.
[0336] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited
to,insomnia, multiple sclerosis, and other neurodegenerative
diseases and/or disorders. Similarly, polypeptides and antibodies
directed to these polypeptides would be useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the central nervous system and
endocrine system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues or
cell types (e.g., CNS, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of the immunogenic epitopes shown in SEQ ID NO: 159 as
residues: Pro-7 to Gly-12. Polynucleotides encoding said
polypeptides are encompassed by the invention, as are antibodies
that bind one or more of these peptides.
[0337] The tissue distribution primarily in pineal gland and, to a
lesser extent, in multiple sclerosis cells indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for treatment of insomia and jet lag through agonist or
antagonist interaction with pineal gland receptors to allow
regulation of melatonin production. Representative uses are
described elsewhere herein. This gene may also be useful in the
treatment of multiple sclerosis. Furthermore, the protein may also
be used to determine biological activity, to raise antibodies, as
tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0338] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:52 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1664 of SEQ ID NO:52, b is an integer
of 15 to 1678, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:52, and where b is greater
than or equal to a+14.
[0339] Features of Protein Encoded By Gene No: 43
[0340] The gene encoding the disclosed cDNA is believed to reside
on chromosome 2. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 2.
[0341] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: PSCPPEMKKELPVDSCLPRSLELHPQKMDPKRQHIQLLSSLTECLTVDPLSASV
WRQLYPKHLSQSSLLLXHLLSSWEQIPKKVQKSLQETIQSLKLTNQELLRKGS SNNQDVVTCD
(SEQ ID NO: 356). Moreover, fragments and variants of these
polypeptides (such as, for example, fragments as described herein,
polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% identical to these polypeptides, or polypeptides encoded by a
polynucleotide which hybridizes, under stringent conditions, to the
polynucleotide encoding these polypeptides) are encompassed by the
invention. Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0342] When tested against Jurket and U937 cell lines, supernatants
removed from cells containing this gene activated the NFkB promoter
element. Thus, it is likely that this gene activates T-cells and
myeloid cells through the NFkB signal transduction pathway. NF-kB
(Nuclear Factor kB) is a transcription factor activated by a wide
variety of agents, leading to cell activation, differentiation, or
apoptosis. Reporter constructs utilizing the NF-kB promoter element
are used to screen supernatants for such activity.
[0343] This gene is expressed primarily in ovary tumors and breast
cancer and, to a lesser extent, in normal lung and colon
tumors.
[0344] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
cancer, particularly of the ovary and breast; and colon. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the colon,
breast, or female reproductive system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., reproductive,
gastrointestinal, and cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0345] The tissue distribution primarily in ovary tumors and breast
cancer and, to a lesser extent, in normal lung and colon tumors
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the diagnosis and/or treatment of a
variety of cancers, most notably cancers of the ovary, breast, or
colon. Representative uses are described in the "Hyperproliferative
Disorders" and "Regeneration" sections below and elsewhere herein.
Briefly, the expression of polynucleotides and polypeptides
corresponding to this gene in a variety of cancers indicates that
it may be a player in the progression of the disease, and may be a
beneficial target for inhibitors as therapeutics. Similarly,
expression in ovarian tissue, indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
treatment, prevention, detection and diagnosis of conditions
concerning proper ovarian function (e.g., egg maturation, endocrine
function), as well as cancer. The expression in ovarian tissue may
indicate the gene or its products can be used to treat, prevent,
detect and/or diagnose disorders of the ovary, including
inflammatory disorders, such as oophoritis (e.g., caused by viral
or bacterial infection), ovarian cysts, amenorrhea, infertility,
hirsutism, and ovarian cancer (including, but not limited to,
primary and secondary cancerous growth, endometrioid carcinoma of
the ovary, ovarian papillary serous adenocarcinoma, ovarian
mucinous adenocarcinoma, Ovarian Krukenberg tumor). Likewise,
expression in breast tissue indicates that polynucleotides and/or
polypeptides of the invention would be useful for diagnosis,
treatment and/or prevention of breast neoplasia and breast cancers,
such as fibroadenoma, pipillary carcinoma, ductal carcinoma,
Paget's disease, medullary carcinoma, mucinous carcinoma, tubular
carcinoma, secretory carcinoma and apocrine carcinoma, as well as
juvenile hypertrophy and gynecomastia, mastitis and abscess, duct
ectasia, fat necrosis and fibrocystic diseases. The tissue
distribution in colon and colon cancer indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for diagnosis, treatment, prevention and/or detection of
tumors, especially of the intestine, such as, carcinoid tumors,
lymphomas, non-neoplastic polyps, adenomas, familial syndromes,
colorectal carcinogenesis, colorectal carcinoma, cancer of the
colon, cancer of the rectum and carcinoid tumors, as well as
cancers in other tissues where expression has been indicated. The
expression in the colon tissue may indicate that polynucleotides
and polypeptides of the invention can be used to treat, detect,
prevent and/or diagnose disorders of the colon, including
inflammatory disorders such as, congenital abnormalities, such as
atresia and stenosis, Meckel diverticulum, congenital aganglionic
megacolon-Hirschsprung disease; enterocolitis, such as diarrhea and
dysentary, infectious enterocolitis, including viral
gastroenteritis, bacterial enterocolitis, necrotizing
enterocolitis, antiboitic-associated colitis (pseudomembranous
colitis), and collagenous and lymphocytic colitis, miscellaneous
intestinal inflammatory disorders, including parasites and
protozoa, amoebic colitis, acquired immunodeficiency syndrome,
transplantation, drug-induced intestinal injury, radiation
enterocolitis, neutropenic colitis, diverticular colon disease
(DCD), inflammatory colonic disease, idiopathic inflammatory bowel
disease, such as Crohn's disease (CD), non-inflammatory bowel
disease (non-IBD) colonic inflammation; ulcerative disorders such
as, ulcerative colitis (UC); eosinophilic colitis; noncancerous
tumors, such as, polyps in the colon, adenomas, leiomyomas,
lipomas, and angiomas. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0346] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:53 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1846 of SEQ ID NO:53, b is an integer
of 15 to 1860, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:53, and where b is greater
than or equal to a+14.
[0347] Features of Protein Encoded By Gene No: 44
[0348] In an alternative reading frame, this gene shares sequence
homology with a murine testosterone induced transcript (see, e.g.,
Geneseq Accession No. 758299; all references available through this
accession are hereby incorporated by reference herein.). This same
region also shares sequence homology with a human cancer suppressor
transfer factor protein (see, e.g., Geneseq Accession No. R86875;
all references available through this accession are hereby
incorporated by reference herein.).
[0349] The gene encoding the disclosed cDNA is thought to reside on
chromosome 11. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 11.
[0350] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: KAPYSWLADSWPHPSRSPSAQEPRGSCCPSNPDPDDRYYNEAGISLYLAQTA
RGTAAPGEGPVYSTIDPAGEELQTFHGGFPQHPSGDLGPWSQYAPPEWSQG (SEQ ID NO:
357). Moreover, fragments and variants of these polypeptides (such
as, for example, fragments as described herein, polypeptides at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
these polypeptides, or polypeptides encoded by a polynucleotide
which hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0351] This gene is expressed primarily in various embryonic/fetal
tissues, particularly fetal brain tissue.
[0352] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
congenital birth defects, particularly of the central nervous
system, and cancers, such as MEN. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the central nervous system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
neural, developing, cancerous and wounded tissues) or bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0353] The tissue distribution in fetal and embryonic tissues
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the diagnosis, detection, prevention
and/or treatment of a variety of cancers, most notably cancers of
the central nervous system, such as MEN, as well as the disorders
of the central nervous system listed above. Representative uses are
described in the "Hyperproliferative Disorders" and "Regeneration"
sections below and elsewhere herein. Briefly, the expression within
embryonic tissue and other cellular sources marked by proliferating
cells indicates that polynucleotides and polypeptides of the
invention may play a role in the regulation of cellular division,
and may show utility in the detection, treatment, and/or prevention
of cancer and other proliferative disorders. Similarly, embryonic
development also involves decisions involving cell differentiation
and/or apoptosis in pattern formation. Thus, polynucleotides and
polypeptides of the invention may also be involved in apoptosis or
tissue differentiation and could again be useful in cancer therapy.
Expression of polynucleotides and polypeptides corresponding to
this gene in a variety of systems indicates that this gene may be a
player in the progression of these diseases, and may be a
beneficial target for inhibitors as therapeutics. Furthermore, the
protein may also be used to determine biological activity, to raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0354] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:54 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1649 of SEQ ID NO:54, b is an integer
of 15 to 1663, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:54, and where b is greater
than or equal to a+14.
[0355] Features of Protein Encoded By Gene No: 45
[0356] The gene encoding the disclosed cDNA is thought to reside on
chromosome 1. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 1.
[0357] This gene is highly homologous to bovine cytochrome b-5
reductase (see e.g., GENBANK: locus BOVCYB5R, accession M83104;
Strittmatter et al., J. Biol. Chem. 267:2519-2523 (1992); the
references available through the accession number and the captioned
reference are hereby incorporated herein by reference). Based on
this homology, it is likely that this gene would have activity
similar to NADH-cytochrome b5 reductase.
[0358] This gene is expressed primarily in liver and lung
tissues.
[0359] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
diseases and/or disorders of the liver and lung including chronic
liver failure, bronchitis, emphasema, and chronic lung failure.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the hepatic and pulmonary systems, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., hepatic, pulmonary, cancerous
and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder. Preferred polypeptides of the present invention comprise,
or alternatively consist of one, two, three, four, five or all six
of the immunogenic epitopes shown in SEQ ID NO: 162 as residues:
Arg-31 to Gln-37, Val-88 to Gly-95, Pro-110 to Gln-120, Gln-151 to
Ala-163, Asp-231 to Trp-237, Pro-277 to Lys-287. Polynucleotides
encoding said polypeptides are encompassed by the invention, as are
antibodies that bind one or more of these peptides.
[0360] The tissue distribution in liver tissue indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection and treatment of liver disorders and
cancers (e.g., hepatoblastoma, jaundice, hepatitis, liver metabolic
diseases and conditions that are attributable to the
differentiation of hepatocyte progenitor cells). Representative
uses are described in the "Hyperproliferative Disorders",
"Infectious Disease", and "Binding Activity" sections below, in
Example 11, and 27, and elsewhere herein. Alternatively, the tissue
distribution indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the detection and
treatment of disorders associated with developing lungs,
particularly in premature infants where the lungs are the last
tissues to develop. The tissue distribution indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the diagnosis and intervention of lung tumors, since
the gene may be involved in the regulation of cell division,
particularly since it is expressed in fetal tissue. Furthermore,
the protein may also be used to determine biological activity, to
raise antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0361] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:55 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1618 of SEQ ID NO:55, b is an integer
of 15 to 1632, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:55, and where b is greater
than or equal to a+14.
[0362] Features of Protein Encoded By Gene No: 46
[0363] This gene is expressed primarily in tonsil tissue and
neutrophils, and, to a lesser extent, in testes tissue, brain and
cerebellum tissues.
[0364] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
diseases and/or disorders of the tonsils, immune system disorders,
reproductive disorders, and neural disorders. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
tonsils, and the immune, reproductive, and neural systems,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
immune, neural, reproductive, tonsils, cancerous and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial fluid and spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder. Preferred polypeptides of the present invention comprise,
or alternatively consist of one or both of the immunogenic epitopes
shown in SEQ ID NO: 163 as residues: Pro-17 to Glu-26, Asp-60 to
Val-72. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0365] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, treatment, and/or prevention of a variety of immune
system disorders. Representative uses are described in the "Immune
Activity" and "Infectious Disease" sections below, in Example 11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the
expression ofpolynucleotides and polypeptides corresponding to this
gene in tonsils as well as neutrophils indicates a role in the
regulation of the proliferation; survival; differentiation; and/or
activation of potentially all hematopoietic cell lineages,
including blood stem cells. Polynucleotides and polypeptides
corresponding to this gene may be involved in the regulation of
cytokine production, antigen presentation, or other processes that
may also suggest a usefulness in the treatment of cancer (e.g., by
boosting immune responses). Since the gene is expressed in cells of
lymphoid origin, the gene or protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
Therefore it may be also used as an agent for immunological
disorders including arthritis, asthma, immune deficiency diseases
such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel
disease, sepsis, acne, and psoriasis. In addition, polynucleotides
and polypeptides corresponding to this gene may have commercial
utility in the expansion of stem cells and committed progenitors of
various blood lineages, and in the differentiation and/or
proliferation of various cell types. Alternatively, the tissue
distribution indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the treatment and/or
diagnosis of conditions concerning proper testicular function (e.g.
endocrine function, sperm maturation), as well as cancer.
Therefore, polynucleotides and polypeptides corresponding to this
gene would be useful in the treatment of male infertility and/or
impotence. Polynucleotides and polypeptides corresponding to this
gene is also useful in assays designed to identify binding agents,
as such agents (antagonists) would be useful as male contraceptive
agents. Similarly, the protein is believed to be useful in the
treatment and/or diagnosis of testicular cancer. The testes are
also a site of active gene expression of transcripts that may be
expressed, particularly at low levels, in other tissues of the
body. Therefore, polynucleotides and polypeptides corresponding to
this gene may be expressed in other specific tissues or organs
where it may play related functional roles in other processes, such
as hematopoiesis, inflammation, bone formation, and kidney
function, to name a few possible target indications. The tissue
distribution in brain and cerebellum tissues indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection/treatment of neurodegenerative disease
states and behavioural disorders such as Alzheimers Disease,
Parkinsons Disease, Huntingtons Disease, Tourette Syndrome,
schizophrenia, mania, dementia, paranoia, obsessive compulsive
disorder, panic disorder, learning disabilities, ALS, psychoses,
autism, and altered behaviors, including disorders in feeding,
sleep patterns, balance, and perception. In addition, the gene or
gene product may also play a role in the treatment and/or detection
of developmental disorders associated with the developing embryo,
or sexually-linked disorders. Furthermore, the protein may also be
used to determine biological activity, to raise antibodies, as
tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0366] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:56 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2219 of SEQ ID NO:56, b is an integer
of 15 to 2233, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:56, and where b is greater
than or equal to a+14.
[0367] Features of Protein Encoded By Gene No: 47
[0368] The translation product of this gene shares sequence
homology with seven trans-membrane receptors and plectin, which is
thought to be important in muscular dystrophy and multiple other
diseases.
[0369] The gene encoding the disclosed cDNA is thought to reside on
chromosome 16. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 16.
[0370] This gene is expressed primarily in brain, fetal organs and
placental tissue, and, to a lesser extent, in several other organs
and tissues.
[0371] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
diseases and/or disorders of the central nervous system, fetal and
developing organs. Similarly, polypeptides and antibodies directed
to these polypeptides would be useful in providing immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the central nervous system, developing and fetal
systems, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues or cell types
(e.g., neural, developing, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of one, two or all three of the immunogenic epitopes shown
in SEQ ID NO: 164 as residues: Arg-13 to Trp-19, Leu-76 to Ala-92,
Ser-100 to Arg-105. Polynucleotides encoding said polypeptides are
encompassed by the invention, as are antibodies that bind one or
more of these peptides.
[0372] The tissue distribution and homology to plectin and seven
transmembrane receptors indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
treatment and/or diagnosis of disorders of the central nervous
system, as well as developing and fetal systems. Moreover, the
expression within fetal tissue indicates this protein may play a
role in the regulation of cellular division, and may show utility
in the diagnosis, treatment, and/or prevention of developmental
diseases and disorders, cancer, and other proliferative conditions.
Representative uses are described in the "Hyperproliferative
Disorders" and "Regeneration" sections below and elsewhere herein.
Briefly, developmental tissues rely on decisions involving cell
differentiation and/or apoptosis in pattern formation.
Dysregulation of apoptosis can result in inappropriate suppression
of cell death, as occurs in the development of some cancers, or in
failure to control the extent of cell death, as is believed to
occur in acquired immunodeficiency and certain neurodegenerative
disorders, such as spinal muscular atrophy (SMA). Because of
potential roles in proliferation and differentiation,
polynucleotides and polypeptides corresponding to this gene may
have applications in the adult for tissue regeneration and the
treatment of cancers. It may also act as a morphogen to control
cell and tissue type specification. Therefore, the polynucleotides
and polypeptides of the present invention would be useful in
treating, detecting, and/or preventing said disorders and
conditions, in addition to other types of degenerative conditions.
Thus, polynucleotides and polypeptides corresponding to this gene
may modulate apoptosis or tissue differentiation and would be
useful in the detection, treatment, and/or prevention of
degenerative or proliferative conditions and diseases. The
polynucleotides and polypeptides corresponding to this gene would
be useful in modulating the immune response to aberrant
polypeptides, as may exist in proliferating and cancerous cells and
tissues. The protein can also be used to gain new insight into the
regulation of cellular growth and proliferation. Furthermore, the
protein may also be used to determine biological activity, to raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0373] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:57 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1949 of SEQ ID NO:57, b is an integer
of 15 to 1963, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:57, and where b is greater
than or equal to a+14.
[0374] Features of Protein Encoded By Gene No: 48
[0375] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
LQQTMQAMLHFGGRLAQSLRGTSKEAASDPSDSPNLPTPGSWW (SEQ ID NO: 358),
EQLTQASRVYASGGTEGFPLSRWAPGRHGTAAEEGAQERPLPTDE (SEQ ID NO: 359),
MAPGRGLWLGRLFGVPGGPAENENGALKSRRPSSWLPPTVSVLAL (SEQ ID NO: 360),
VKRGAPPEMPSPQELEASAPRMVQTHRAVRALCDHTAARPDQLS (SEQ ID NO: 361),
FRRGEVLRVITTVDEDWLRCGRDGMEGLVPVGYTSLVL (SEQ ID NO: 362), and/or
LQQTMQAMLHFGGRLAQSLRGTSKEAASDPSDSPNLPTPGSWWEQLTQASR
VYASGGTEGFPLSRWAPGRHGTAAEEGAQERPLPTDEMAPGRGLWLGRLFG
VPGGPAENENGALKSRRPSSWLPPTVSVLALVKRGAPPEMPSPQELEASAPR
MVQTHRAVRALCDHTAARPDQLSFRRGEVLRVITTVDEDWLRCGRDGMEG LVPVGYTSLVL (SEQ
ID NO: 363). Moreover, fragments and variants of these polypeptides
(such as, for example, fragments as described herein, polypeptides
at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical
to these polypeptides, or polypeptides encoded by a polynucleotide
which hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0376] A portion of the translation product of this gene shares
sequence homology with SH3 domain of human SH3P17 protein (see,
e.g., Genseq accession number W34234; all references available
through this accession are hereby incorporated by reference herein)
which is thought to be important in cell growth, malignancy, and/or
signal transduction processes. Therefore, it is likely that the
translation product of this gene shares at least some biological
activity with polypeptides/proteins possessing SH domains.
[0377] This gene is expressed primarily in synovium, synovial
sarcoma, and chondrosarcoma tissues, and, to a lesser extent, in
endometrial stromal cells.
[0378] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
skeletal and reproductive disorders. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the skeletal and
reproductive systems, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., skeletal, reproductive, cancerous and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial fluid and spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0379] The tissue distribution in skeletal tissues indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection, diagnosis, prevention and/or treatment
of disorders and conditions affecting the skeletal system, in
particular osteoporosis as well as disorders afflicting connective
tissues (e.g., arthritis, trauma, tendonitis, chrondomalacia and
inflammation). The polynucleotides and polypeptides of the
invention would be useful in the diagnosis or treatment of various
autoimmune disorders such as rheumatoid arthritis, lupus,
scleroderma, and dermatomyositis as well as dwarfism, spinal
deformation, and specific joint abnormalities as well as
chondrodysplasias (i.e., spondyloepiphyseal dysplasia congenita,
familial arthritis, Atelosteogenesis type II, metaphyseal
chondrodysplasia type Schmid). Alternatively, the tissue
distribution in endometrium indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for
treating female infertility. The polynucleotides and polypeptides
of the invention are likely involved in preparation of the
endometrium of implantation and could be administered either
topically or orally. Alternatively, this gene could be transfected
in gene-replacement treatments into the cells of the endometrium
and the protein products could be produced. Similarly, these
treatments could be performed during artificial insemination for
the purpose of increasing the likelyhood of implantation and
development of a healthy embryo. In both cases this gene or its
gene product could be administered at later stages of pregnancy to
promote heathy development of the endometrium. Furthermore, the
protein may also be used to determine biological activity, to raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0380] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:58 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1253 of SEQ ID NO:58, b is an integer
of 15 to 1267, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:58, and where b is greater
than or equal to a+14.
[0381] Features of Protein Encoded By Gene No: 49
[0382] The gene encoding the disclosed cDNA is believed to reside
on chromosome 7. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 7.
[0383] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
ARACPRXGAAVEKLGGKPVQPDSKPTCCSQVKAEGLIFAGLTGLKLLPSSLQ
RAVFVRQCLGFWNDGSRALQ (SEQ ID NO:364) and
MSPNLNATHTSAQTPGFMERKTTHTVAQALSHAVRTIRGARSPLRPDASRTP
TSCQMSTQSLLICKARLPSFQNPRHCLTKTALCKELGSNLSPVRPAKISPSALT
CEQHVGLESGWTGFPPSFSTAAPXLGQARA (SEQ ID NO: 365). Moreover,
fragments and variants of these polypeptides (such as, for example,
fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides,
or polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0384] This gene is expressed primarily in hypothalamus,
hepatocellular tumor, ovarian cancer reexcision and, to a lesser
extent, in other tissues.
[0385] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
obesity, metabolic disorders, and hepatocellular tumors. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the,
endocrine system, hypothalamus and hepatocellular tumor, expression
of this gene at significantly higher or lower levels may be
routinely detected in certain tissues or cell types (e.g.,
hypothalamus, cancerous and wounded tissues) or bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0386] The tissue distribution in hypothalamus and hepatocellular
tumors indicates that the protein products of this gene would be
useful for detection, treatment, and/or prevention of obesity,
metabolic disorders, and hepatocellular tumors. Similarly, the
tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the detection,
treatment, and/or prevention of various endocrine disorders and
cancers, particularly Addison's disease, Cushing's Syndrome, and
disorders and/or cancers of the pancreas (e.g., diabetes mellitus),
adrenal cortex, ovaries, pituitary (e.g., hyper-, hypopituitarism),
thyroid (e.g., hyper-, hypothyroidism), parathyroid (e.g., hyper-,
hypoparathyroidism), hypothallamus, and testes. Furthermore, the
protein may also be used to determine biological activity, to raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0387] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:59 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1281 of SEQ ID NO:59, b is an integer
of 15 to 1295, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:59, and where b is greater
than or equal to a+14.
[0388] Features of Protein Encoded By Gene No: 50
[0389] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: FQSVYHMKLQSSNLPASVYGNNLNCINSSSS (SEQ ID NO: 366).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0390] This gene is expressed primarily in brain, placenta, immune
cells (e.g., B-cells and macrophage), fetal tissue and breast.
[0391] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
reproductive, neurological and behavioural disorders. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the CNS,
immune and female reproductive systems, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., immune, reproductive, CNS,
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
breast milk, amniotic fluid, serum, plasma, urine, synovial fluid
or cerebrospinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0392] The tissue distribution in brain indicates the protein
product of this clone would be useful for the detection, treatment,
and/or prevention of neurodegenerative disease states, behavioral
disorders, or inflammatory conditions. Representative uses are
described in the "Regeneration" and "Hyperproliferative Disorders"
sections below, in Example 11, 15, and 18, and elsewhere herein.
Briefly, the uses include, but are not limited to the detection,
treatment, and/or prevention of Alzheimer's Disease, Parkinson's
Disease, Huntington's Disease, Tourette Syndrome, meningitis,
encephalitis, demyelinating diseases, peripheral neuropathies,
neoplasia, trauma, congenital malformations, spinal cord injuries,
ischemia and infarction, aneurysms, hemorrhages, schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder,
depression, panic disorder, learning disabilities, ALS, psychoses,
autism, and altered behaviors, including disorders in feeding,
sleep patterns, balance, and perception. In addition, elevated
expression of Polynucleotides and polypeptides corresponding to
this gene in regions of the brain indicates it plays a role in
normal neural function. Potentially, polynucleotides and
polypeptides corresponding to this gene would be involved in
synapse formation, neurotransmission, learning, cognition,
homeostasis, or neuronal differentiation or survival. The tissue
distribution in B-cells and macrophage indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the diagnosis and treatment of a variety of immune
system disorders. Representative uses are described in the "Immune
Activity" and "Infectious Disease" sections below, in Example 11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the
expression indicates a role in regulating the proliferation;
survival; differentiation; and/or activation of hematopoietic cell
lineages, including blood stem cells. Involvement in the regulation
of cytokine production, antigen presentation, or other processes
indicates a usefulness for treatment of cancer (e.g., by boosting
immune responses). Expression in cells of lymphoid origin,
indicates the natural gene product would be involved in immune
functions. Therefore it would also be useful as an agent for
immunological disorders including arthritis, asthma,
immunodeficiency diseases such as AIDS, leukemia, rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease,
sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus,
polynucleotides and polypeptides corresponding to this gene is
thought to be useful in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. In addition, expression
in breast and placenta indicates a role in the detection and/or
treatment of female infertility and/or pregnancy disorders. In
addition, the gene or gene product may also play a role in the
treatment and/or detection of developmental disorders associated
with the developing embryo, or sexually-linked disorders. Moreover,
the expression within fetal tissue and other cellular sources
marked by proliferating cells indicates this protein may play a
role in the regulation of cellular division, and may show utility
in the diagnosis, treatment, and/or prevention of developmental
diseases and disorders, including cancer, and other proliferative
conditions. Representative uses are described in the
"Hyperproliferative Disorders" and "Regeneration" sections below
and elsewhere herein. Briefly, developmental tissues rely on
decisions involving cell differentiation and/or apoptosis in
pattern formation. Dysregulation of apoptosis can result in
inappropriate suppression of cell death, as occurs in the
development of some cancers, or in failure to control the extent of
cell death, as is believed to occur in acquired immunodeficiency
and certain degenerative disorders, such as spinal muscular atrophy
(SMA). Alternatively, polynucleotides and polypeptides
corresponding to this gene may be involved in the pattern of
cellular proliferation that accompanies early embryogenesis. Thus,
aberrant expression of polynucleotides and polypeptides
corresponding to this gene in tissues--particularly adult
tissues--may correlate with patterns of abnormal cellular
proliferation, such as found in various cancers. Because of
potential roles in proliferation and differentiation,
polynucleotides and polypeptides corresponding to this gene may
have applications in the adult for tissue regeneration and the
treatment of cancers. It may also act as a morphogen to control
cell and tissue type specification. Therefore, the polynucleotides
and polypeptides of the present invention would be useful in
treating, detecting, and/or preventing said disorders and
conditions, in addition to other types of degenerative conditions.
Thus this protein may modulate apoptosis or tissue differentiation
and would be useful in the detection, treatment, and/or prevention
of degenerative or proliferative conditions and diseases. The
protein would be useful in modulating the immune response to
aberrant polypeptides, as may exist in proliferating and cancerous
cells and tissues. The protein can also be used to gain new insight
into the regulation of cellular growth and proliferation.
Furthermore, the protein may also be used to determine biological
activity, raise antibodies, as tissue markers, to isolate cognate
ligands or receptors, to identify agents that modulate their
interactions, in addition to its use as a nutritional supplement.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0393] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:60 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 901 of SEQ ID NO:60, b is an integer
of 15 to 915, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:60, and where b is greater
than or equal to a+14.
[0394] Features of Protein Encoded By Gene No: 51
[0395] This gene is expressed primarily in adipocytes.
[0396] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
obesity, Nasu-Hakola disease, cardiovascular disease,
non-insulin-dependent diabetes mellitus. Similarly, polypeptides
and antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the adipose, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., endocrine,
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of one or both
of the immunogenic epitopes shown in SEQ ID NO: 168 as residues:
Asp-6 to Arg-12, Lys-31 to Leu-41. Polynucleotides encoding said
polypeptides are encompassed by the invention, as are antibodies
that bind one or more of these peptides.
[0397] The tissue distribution in adipose tissue indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the treatment and diagnosis of endocrine and
metabolic disorders related to lipids and adipose tissue, such as
obesity, Nasu-Hakola disease (membranous lipodystrophy),
cardiovascular disease, lipidemia, non-insulin-dependent diabetes
mellitus, stroke and carcinoma. Furthermore, polynucleotides and
polypeptides corresponding to this gene may show utility in
ameliorating conditions which occur secondary to aberrant
fatty-acid metabolism (e.g., aberrant myelin sheath development),
either directly or indirectly. Furthermore, the protein may also be
used to determine biological activity, raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0398] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:61 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1431 of SEQ ID NO:61, b is an integer
of 15 to 1445, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:61, and where b is greater
than or equal to a+14.
[0399] Features of Protein Encoded By Gene No: 52
[0400] The gene encoding the disclosed cDNA is thought to reside on
chromosome 1. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 1.
[0401] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: GLSIHDGTWKSAI
YGFGDQSNLRKLRNVSNLKPVPLIGPKLKRRWPISYCRELKGYSIPFMGSDVS
VVRRTQRYLYENLEESPVQYAAYVTVGGITSVIKLMFAGLFFLFFVRFGIGRQ
LLIKFPWFFSFGYFSKQGPTQKQIDAASFTLTFFGQGYSQGTGTDKNKPNIKIC
TQVKGPEAGYVATPIAMVQAAMTLLSDASHLPKAGGVFTPGAAFSKTKLI
DRLNKHGIEFSVISSSEV (SEQ ID NO: 367) Moreover, fragments and
variants of these polypeptides (such as, for example, fragments as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0402] This gene is expressed primarily in testes, endometrial
tumor tissue, prostate cancer tissue, immune tissue (e.g., bone
marrow and T-cells) and placenta tissue, and, to a lesser extent,
in several other tissues and organs.
[0403] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
reproductive diseases and disorders, cancers and hematopoietic
disorders. Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the hematopoietic and reproductive system, expression of this gene
at significantly higher or lower levels may be routinely detected
in certain tissues or cell types (e.g., immune, reproductive,
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of one or both
of the immunogenic epitopes shown in SEQ ID NO: 169 as residues:
Phe-32 to Gln-41, Gln-54 to Asn-68. Polynucleotides encoding said
polypeptides are encompassed by the invention, as are antibodies
that bind one or more of these peptides.
[0404] The tissue distribution in testes tissue and bone marrow
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the treatment and/or diagnosis of
disorders of the hematopoietic and reproductive systems, and
cancers thereof. The tissue distribution in bone marrow and T-cells
indicates the protein product of this clone would be useful for the
diagnosis and treatment of a variety of immune system disorders.
Representative uses are described in the "Immune Activity" and
"Infectious Disease" sections below, in Example 11, 13, 14, 16, 18,
19, 20, and 27, and elsewhere herein. Briefly, the expression
indicates a role in regulating the proliferation; survival;
differentiation; and/or activation of hematopoietic cell lineages,
including blood stem cells. Involvement in the regulation of
cytokine production, antigen presentation, or other processes
indicates a usefulness for treatment of cancer (e.g. by boosting
immune responses). Expression in cells of lymphoid origin,
indicates the natural gene product would be involved in immune
functions. Therefore it would also be useful as an agent for
immunological disorders including arthritis, asthma,
immunodeficiency diseases such as AIDS, leukemia, rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease,
sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus,
polynucleotides and polypeptides corresponding to this gene is
thought to be useful in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. The tissue distribution
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the treatment and diagnosis of
conditions concerning proper testicular function (e.g., endocrine
function, sperm maturation), as well as cancer. Therefore,
polynucleotides and polypeptides corresponding to this gene would
be useful in the treatment of male infertility and/or impotence.
Polynucleotides and polypeptides corresponding to this gene is also
useful in assays designed to identify binding agents, as such
agents (antagonists) would be useful as male contraceptive agents.
Similarly, the protein is believed to be useful in the treatment
and/or diagnosis of testicular cancer. The testes are also a site
of active gene expression of transcripts that may be expressed,
particularly at low levels, in other tissues of the body.
Therefore, polynucleotides and polypeptides corresponding to this
gene may be expressed in other specific tissues or organs where it
may play related functional roles in other processes, such as
hematopoiesis, inflammation, bone formation, and kidney function,
to name a few possible target indications. Furthermore, the protein
may also be used to determine biological activity, to raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0405] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:62 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1086 of SEQ ID NO:62, b is an integer
of 15 to 1100, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:62, and where b is greater
than or equal to a+14.
[0406] Features of Protein Encoded By Gene No: 53
[0407] The translation product of this gene has homology with
metallothionine proteins from several organisms.
[0408] This gene is expressed primarily in ovarian cancer, fetal
tissue (e.g., liver, spleen, and heart), testes, embryo, colon,
T-cells, neutrophils, tonsils, B-cell lymphoma, and to a lesser
extent in many other tissues.
[0409] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
reproductive defects, and lymphoid and ovarian cancers. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
and female reproductive systems, and of lymphoid and ovarian
cancers, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues or cell types
(e.g., immune, reproductive, cancerous and wounded tissues) or
bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of the immunogenic epitopes shown in SEQ ID NO: 170 as
residues: Leu-39 to Ser-47. Polynucleotides encoding said
polypeptides are encompassed by the invention, as are antibodies
that bind one or more of these peptides.
[0410] The tissue distribution in ovarian cancer, tonsils, and
B-cell lymphoma indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the study, detection
and/or treatment of female reproductive disorders, gonadal and
general lymphoid neoplasias, and cancers thereof. The tissue
distribution in immune cells (e.g., neutrophils and T-cells)
indicates the protein product of this clone would be useful for the
diagnosis and treatment of a variety of immune system disorders.
Representative uses are described in the "Immune Activity" and
"Infectious Disease" sections below, in Example 11, 13, 14, 16, 18,
19, 20, and 27, and elsewhere herein. Briefly, the expression
indicates a role in regulating the proliferation; survival;
differentiation; and/or activation of hematopoietic cell lineages,
including blood stem cells. Involvement in the regulation of
cytokine production, antigen presentation, or other processes
indicates a usefulness for treatment of cancer (e.g. by boosting
immune responses). Expression in cells of lymphoid origin,
indicates the natural gene product would be involved in immune
functions. Therefore it would also be useful as an agent for
immunological disorders including arthritis, asthma,
immunodeficiency diseases such as AIDS, leukemia, rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease,
sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus,
polynucleotides and polypeptides corresponding to this gene is
thought to be useful in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Expression of
polynucleotides and polypeptides corresponding to this gene in
tonsils indicates a role in the regulation of the proliferation;
survival; differentiation; and/or activation of potentially all
hematopoietic cell lineages, including blood stem cells.
Polynucleotides and polypeptides corresponding to this gene may be
involved in the regulation of cytokine production, antigen
presentation, or other processes that may also suggest a usefulness
in the treatment of cancer (e.g., by boosting immune responses).
Since the gene is expressed in cells of lymphoid origin, the gene
or protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma, immune
deficiency diseases such as AIDS, leukemia, rheumatoid arthritis,
inflammatory bowel disease, sepsis, acne, and psoriasis. In
addition, polynucleotides and polypeptides corresponding to this
gene may have commercial utility in the expansion of stem cells and
committed progenitors of various blood lineages, and in the
differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, raise antibodies, as tissue markers, to isolate cognate
ligands or receptors, to identify agents that modulate their
interactions, in addition to its use as a nutritional supplement.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0411] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:63 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1485 of SEQ ID NO:63, b is an integer
of 15 to 1499, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:63, and where b is greater
than or equal to a+14.
[0412] Features of Protein Encoded By Gene No: 54
[0413] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
MDPDRAFICGESRQFAQCLIFGFLFLTSGMLISVLGIWVPGCGSNWAQEPLNE
TDTGDSEPRMCGFLSLQIMGPLIVLVGLCFFVVAHVKKRNTLNAGQDASERE
EGQIQIMEPVQVTVGDSVIIFPPPPPPYFPESSASAVAESPGTNSLLPNENPPSY
YSIFNYGTPTSEGAASERDCESIYTISGTNSSSEASHTPHLPSELPPRYEEKENA
AATFLPLSSEPSPP (SEQ ID NO: 369), and/or
MDPDRAFICGESRQFAQCLIFGFLFLTSGMLISVLGIWVPGCGSNWAQEPLNE TDTGDSEPR
(SEQ ID NO: 368). Moreover, fragments and variants of these
polypeptides (such as, for example, fragments as described herein,
polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% identical to these polypeptides, or polypeptides encoded by a
polynucleotide which hybridizes, under stringent conditions, to the
polynucleotide encoding these polypeptides) are encompassed by the
invention. Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0414] This gene is expressed primarily in adult kidney and
pulmonary tissues, as well as in osteoblasts.
[0415] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
metabolic, endocrine and skeletal disorders. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
endocrine, skeletal, metabolic and developmental systems,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
endocrine, skeletal, cancerous and wounded tissues) or bodily
fluids (e.g., sputum, lymph, serum, plasma, urine, synovial fluid
and spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of one, two, three, four, five or all six of the
immunogenic epitopes shown in SEQ ID NO: 171 as residues: Ala-35 to
Gly-45, Pro-67 to Pro-73, Pro-91 to Ser-97, Thr-127 to Leu-139,
Leu-143 to Asn-152, Ser-162 to Pro-167. Polynucleotides encoding
said polypeptides are encompassed by the invention, as are
antibodies that bind one or more of these peptides.
[0416] The tissue distribution in kidney tissue and osteoblasts
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the study, diagnosis and/or treatment
of various endocrine and skeletal disorders. Furthermore, elevated
levels of expression of polynucleotides and polypeptides
corresponding to this gene in osteoblasts indicates that it may
play a role in the survival, proliferation, and/or growth of
osteoblasts. Therefore, it may be useful in influencing bone mass
in such conditions as osteoporosis. Alternatively, the tissue
distribution in kidney indicates that this gene or gene product
would be useful in the treatment and/or detection of kidney
diseases including renal failure, nephritus, renal tubular
acidosis, proteinuria, pyuria, edema, pyelonephritis,
hydronephritis, nephrotic syndrome, crush syndrome,
glomerulonephritis, hematuria, renal colic and kidney stones, in
addition to Wilm's Tumor Disease, and congenital kidney
abnormalities such as horseshoe kidney, polycystic kidney, and
Falconi's syndrome. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0417] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:64 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 641 of SEQ ID NO:64, b is an integer
of 15 to 655, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:64, and where b is greater
than or equal to a+14.
[0418] Features of Protein Encoded By Gene No: 55
[0419] This gene is expressed primarily in neutrophils and
embryonic tissues.
[0420] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
immune system disorders and cancers, and developmental disorders.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune and developing systems, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., immune, developing, cancerous
and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid,
serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of one, two,
three, four, five, six, seven or all eight of the immunogenic
epitopes shown in SEQ ID NO: 172 as residues: Gln-21 to Ala-33,
Lys-48 to Leu-54, His-91 to Arg-97, Ala-143 to Gln-148, Glu-173 to
Thr-179, Ser-215 to Lys-254, Arg-262 to Glu-269, Ala-309 to
Gly-314. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0421] The tissue distribution in neutrophils and embryonic tissues
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the diagnosis, study and/or treatment
of various developmental and immune system disorders and cancers
thereof, as well as cancers of other tissues where expression of
this gene has been observed. Furthermore, expression within
embryonic tissue and other cellular sources marked by proliferating
cells indicates that this protein may play a role in the regulation
of cellular division, and may show utility in the detection,
treatment, and/or prevention of cancer and other proliferative
disorders. Similarly, embryonic development also involves decisions
involving cell differentiation and/or apoptosis in pattern
formation. Thus, this protein may also be involved in apoptosis or
tissue differentiation and could again be useful in cancer therapy.
Alternatively, expression of polynucleotides and polypeptides
corresponding to this gene in neutrophils also strongly indicates a
role for this protein in immune function and immune surveillance.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0422] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:65 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1436 of SEQ ID NO:65, b is an integer
of 15 to 1450, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:65, and where b is greater
than or equal to a+14.
[0423] Features of Protein Encoded By Gene No: 56
[0424] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: TABLE-US-00007 (SEQ ID NO: 370)
FDFIASLLKANRLSLQTCELLLAAALLPSERYKALSI.
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0425] This gene is expressed primarily in fetal liver, spleen and,
to a lesser extent, in breast.
[0426] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
immune and haemopoietic diseases and/ordisorders, in addition to,
fetal development. Similarly, polypeptides and antibodies directed
to these polypeptides would be useful in providing immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the circulatory system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., hematopoietic, developmental,
and cancerous and wounded tissues) or bodily fluids (e.g., lymph,
amniotic fluid, serum, plasma, urine, synovial fluid and spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder. Preferred polypeptides
of the present invention comprise, or alternatively consist of one
or both of the immunogenic epitopes shown in SEQ ID NO: 173 as
residues: Ile-50 to Ser-61, Pro-75 to Ser-104. Polynucleotides
encoding said polypeptides are encompassed by the invention, as are
antibodies that bind one or more of these peptides.
[0427] The tissue distribution in fetal liver and spleen indicates
that polynucleotides and polypeptides corresponding to this gene
would be useful for detection, treatment, and/or prevention of
haemopoietic disorders involving stem cell production and
maturation. Similarly, polynucleotides and polypeptides
corresponding to this gene would be useful for the treatment and
diagnosis of hematopoietic related disorders such as anemia,
pancytopenia, leukopenia, thrombocytopenia or leukemia since
stromal cells are important in the production of cells of
hematopoietic lineages. Representative uses are described in the
"Immune Activity" and "Infectious Disease" sections below, in
Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.
Briefly, the uses include bone marrow cell ex-vivo culture, bone
marrow transplantation, bone marrow reconstitution, radiotherapy or
chemotherapy of neoplasia. The gene product may also be involved in
lymphopoiesis, therefore, it can be used in immune disorders such
as infection, inflammation, allergy, immunodeficiency etc. In
addition, polynucleotides and polypeptides corresponding to this
gene may have commercial utility in the expansion of stem cells and
committed progenitors of various blood lineages, and in the
differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0428] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:66 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 656 of SEQ ID NO:66, b is an integer
of 15 to 670, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:66, and where b is greater
than or equal to a+14.
[0429] Features of Protein Encoded By Gene No: 57
[0430] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: MNKKAELKPSALPGWANVWKLMCLVTVCASLIITSDSVVSTVRLKGSCEDY
LGLSCGNTSHAY (SEQ ID NO: 371). Moreover, fragments and variants of
these polypeptides (such as, for example, fragments as described
herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%, or 100% identical to these polypeptides, or polypeptides
encoded by a polynucleotide which hybridizes, under stringent
conditions, to the polynucleotide encoding these polypeptides) are
encompassed by the invention. Antibodies that bind polypeptides of
the invention and polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0431] This gene is expressed primarily in adult pulmonary
cells.
[0432] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
emphysema and other pulmonary diseases and/or disorders. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
pulmonary system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues or
cell types (e.g., lung, cardiovascular, and cancerous and wounded
tissues) or bodily fluids (e.g., lymph, sputum, pulmonary
surfactant, serum, plasma, urine, synovial fluid and spinal fluid)
or another tissue or cell sample taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue or bodily fluid from
an individual not having the disorder.
[0433] The tissue distribution in adult pulmonary cells indicates
that polynucleotides and polypeptides corresponding to this gene
would be useful for detection, treatment, and/or prevention of
disorders of the pulmonary systems, especially emphysema, asthma,
and other similar dysfunctions. Representative uses are described
elsewhere herein. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0434] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:67 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1678 of SEQ ID NO:67, b is an integer
of 15 to 1692, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:67, and where b is greater
than or equal to a+14.
[0435] Features of Protein Encoded By Gene No: 58
[0436] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: MSADGAEADGSTQVTVEEPVQQPSVVDRVASMPLISSTCDMVSAAYASTKE
SYPHVKTVCDAAEKGVRTLTAAAVSGAQPILSKLEPQIASASEYAHRGLDKL
EENLPILQQPTEKVLADTKELVSSKVSGAQEMVSSAKDTVATQLSEAVDATR
GAVQSGVDKTKSVVTGGVQSVMGSRLGQMVLSGVDTVLGKSEEWADNHLP
LTDAELARIATSLDGFDVASVQQQRQEQSYFVRLGSLSERLRQHAYEHSLGK
LRATKQRAQEALLQLSQALSLMETVKQGVDQKLVEGQEKLHQMWLSWNQ
KQLQGPEKEPPKPEQVESRALTMFRDIAQQLQATCTSLGSSIQGLPTNVKDQV
QQARRQVEDLQATFSSIHSFQDLSSSILAQSRERVASAREALDHMVEYVAQN
TPVTWLVGPFAPGITEKAPEEKK (SEQ ID NO: 372) which shares homology with
a human adipocyte differentiation-related protein (see GenBank
Accession CAA65989 and Heid, H. W., et al., Biochem. J. 320,
1025-1030 (1996); all references available through this accession
and citation are hereby incorporated herein by reference).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention. This gene is expressed primarily in hypothalmus
(schizophrenic), and, to a lesser extent, in cerebellum.
[0437] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
schizophenia and hypothalic diseases and/or diseases. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
central nervous system, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., CNS, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0438] The tissue distribution in hypothalmus (schizophrenic) and,
to a lesser extent, in cerebellum indicates that polynucleotides
and polypeptides corresponding to this gene would be useful for
detection, treatment, and/or prevention of neurological disorders,
especially schizophenia, neurodegenerative disease states,
behavioral disorders, or inflammatory conditions. Representative
uses are described in the "Regeneration" and "Hyperproliferative
Disorders" sections below, in Example 11, 15, and 18, and elsewhere
herein. Briefly, the uses include, but are not limited to the
detection, treatment, and/or prevention of Alzheimer's Disease,
Parkinson's Disease, Huntington's Disease, Tourette Syndrome,
meningitis, encephalitis, demyelinating diseases, peripheral
neuropathies, neoplasia, trauma, congenital malformations, spinal
cord injuries, ischemia and infarction, aneurysms, hemorrhages,
schizophrenia, mania, dementia, paranoia, obsessive compulsive
disorder, depression, panic disorder, learning disabilities, ALS,
psychoses, autism, and altered behaviors, including disorders in
feeding, sleep patterns, balance, and perception. In addition,
elevated expression of this gene product in regions of the brain
indicates it plays a role in normal neural function. Potentially,
polynucleotides and polypeptides corresponding to this gene are
involved in synapse formation, neurotransmission, learning,
cognition, homeostasis, or neuronal differentiation or survival.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0439] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:68 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 641 of SEQ ID NO:68, b is an integer
of 15 to 655, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:68, and where b is greater
than or equal to a+14.
[0440] Features of Protein Encoded By Gene No: 5
[0441] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: MLCKSLLYCVVSYLYYFVFIYFFPVFLICSWLELQMWNLQIGRADCFQNTLV
YVLSLCLQYKNHPA (SEQ ID NO: 373). Moreover, fragments and variants
of these polypeptides (such as, for example, fragments as described
herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%, or 100% identical to these polypeptides, or polypeptides
encoded by a polynucleotide which hybridizes, under stringent
conditions, to the polynucleotide encoding these polypeptides) are
encompassed by the invention. Antibodies that bind polypeptides of
the invention and polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0442] This gene is expressed primarily in CD34 positive
hematopoietic cells.
[0443] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
hematopoietic diseases and/or disorders; impaired immune function;
susceptibility to infections; lymphomas and leukemias. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues or cell types
(e.g., hematopoitic, immune, cancerous and wounded tissues) or
bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0444] The tissue distribution in CD34 positive cells indicates
that polynucleotides and polypeptides corresponding to this gene
would be useful for the diagnosis, detection, prevention and/or
treatment of a variety of hematopoietic disorders. Expression of
this gene product particularly in CD34 positive cells indicates
that polynucleotides and polypeptides of the invention may play a
role in the proliferation; survival; differentiation; and/or
activation of early stem and committed progenitor cells within the
hematopoietic system. Thus, polynucleotides and polypeptides of the
invention may be useful in determining the numbers and proportions
of different hematopoietic cell lineages both in vitro and in vivo.
Additionally, the tissue distribution indicates polynucleotides and
polypeptides corresponding to this gene would be useful for the
treatment and diagnosis of hematopoietic related disorders such as
anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia
since stromal cells are important in the production of cells of
hematopoietic lineages. Representative uses are described in the
"Immune Activity" and "Infectious Disease" sections below, in
Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.
Briefly, the uses include bone marrow cell ex-vivo culture, bone
marrow transplantation, bone marrow reconstitution, radiotherapy or
chemotherapy of neoplasia. The gene product may also be involved in
lymphopoiesis, therefore, it can be used in immune disorders such
as infection, inflammation, allergy, immunodeficiency etc. In
addition, polynucleotides and polypeptides of the invention may
have commercial utility in the expansion of stem cells and
committed progenitors of various blood lineages, and in the
differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0445] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:69 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1604 of SEQ ID NO:69, b is an integer
of 15 to 1618, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:69, and where b is greater
than or equal to a+14.
[0446] Features of Protein Encoded By Gene No: 60
[0447] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: IDLSFPSTNVSLEDRNTTKPSVNVG (SEQ ID NO:
374), VAHACNPSTLGG (SEQ ID NO: 375), GGQITRSGDQDQPDQHG (SEQ ID NO:
376), GFTMLVRLVLIS (SEQ ID NO: 377), and
PRDLPTSASQSAGITGMSHPARPKLLFN (SEQ ID NO: 378). Moreover, fragments
and variants of these polypeptides (such as, for example, fragments
as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0448] This gene is expressed primarily in dermatofibrosarcoma
protuberance and 12 week old early human embryos.
[0449] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
dermatofibrosarcoma; cancer; abnormal cell proliferation;
embryological/developmental defects; inhibition of apoptosis; and
hematopoietic diseases and/or disorders. Similarly, polypeptides
and antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the skin and epithelium,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
integumentary, reproductive, developmental, and cancerous and
wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
amniotic fluid, urine, synovial fluid and spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0450] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
diagnosis and/or treatment of abnormal cellular proliferation, such
as cancer. Expression of this gene in dermatofibrosarcoma and 12
week early stage embryos indicates that polynucleotides and
polypeptides of the invention are involved in cellular
proliferation and/or a block in differentiation. Polynucleotides
and polypeptides of the invention may drive cellular proliferation
directly, or may play a role in inhibiting apoptosis or interfering
with differentiation events. Similarly, polynucleotides and
polypeptides of the invention would be useful for the treatment,
diagnosis, and/or prevention of various skin disorders.
Representative uses are described in the "Biological Activity",
"Hyperproliferative Disorders", "Infectious Disease", and
"Regeneration" sections below, in Example 11, 19, and 20, and
elsewhere herein. Briefly, the protein would be useful in
detecting, treating, and/or preventing congenital disorders (i.e.
nevi, moles, freckles, Mongolian spots, hemangiomas, port-wine
syndrome), integumentary tumors (i.e., keratoses, Bowen's disease,
basal cell carcinoma, squamous cell carcinoma, malignant melanoma,
Paget's disease, mycosis flngoides, and Kaposi's sarcoma), injuries
and inflammation of the skin (i.e., wounds, rashes, prickly heat
disorder, psoriasis, dermatitis), atherosclerosis, uticaria,
eczema, photosensitivity, autoimmune disorders (i.e., lupus
erythematosus, vitiligo, dermatomyositis, morphea, scleroderma,
pemphigoid, and pemphigus), keloids, striae, erythema, petechiae,
purpura, and xanthelasma. In addition, such disorders may
predispose increased susceptibility to viral and bacterial
infections of the skin (i.e., cold sores, warts, chickenpox,
molluscum contagiosum, herpes zoster, boils, cellulitis,
erysipelas, impetigo, tinea, althlete's foot, and ringworm).
Moreover, polynucleotides and polypeptides corresponding to this
gene may also be useful for the treatment or diagnosis of various
connective tissue disorders (i.e., arthritis, trauma, tendonitis,
chrondomalacia and inflammation, etc.), autoimmune disorders (i.e.,
rheumatoid arthritis, lupus, scleroderma, dermatomyositis, etc.),
dwarfism, spinal deformation, joint abnormalities, amd
chondrodysplasias (i.e., spondyloepiphyseal dysplasia congenita,
familial osteoarthritis, Atelosteogenesis type II, metaphyseal
chondrodysplasia type Schmid). Furthermore, the protein may also be
used to determine biological activity, to raise antibodies, as
tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0451] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:70 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1788 of SEQ ID NO:70, b is an integer
of 15 to 1802, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:70, and where b is greater
than or equal to a+14.
[0452] Features of Protein Encoded By Gene No: 61
[0453] This gene is expressed primarily in neutrophils.
[0454] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
disorders affecting the immune system. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune system
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
immune, cancerous and wounded tissues) or bodily fluids (e.g.,
lymph, serum, plasma, urine, synovial fluid and spinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue from an individual not
having the disorder.
[0455] The tissue distribution in neutrophils indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the diagnosis, detection, prevention and/or treatment
of immune system disorders, especially those affecting neutrophils.
Representative uses are described in the "Immune Activity" and
"Infectious Disease" sections below, in Example 11, 13, 14, 16, 18,
19, 20, and 27, and elsewhere herein. Briefly, this gene product
may be involved in the regulation of cytokine production, antigen
presentation, or other processes that may also suggest a usefulness
in the treatment of cancer (e.g., by boosting immune responses).
Since the gene is expressed in cells of lymphoid origin, the gene
or protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues. Therefore it may be also used as an agent for
immunological disorders including arthritis, asthma, immune
deficiency diseases such as AIDS, leukemia, rheumatoid arthritis,
inflammatory bowel disease, sepsis, acne, and psoriasis. In
addition, this gene product may have commercial utility in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0456] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:71 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1278 of SEQ ID NO:71, b is an integer
of 15 to 1292, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:71, and where b is greater
than or equal to a+14.
[0457] Features of Protein Encoded By Gene No: 62
[0458] The translation product of this gene shares sequence
homology with angiotensin II receptor which is thought to be
important in ligand binding for blood pressure regulation. (see,
e.g., GenBank Accession No. gi|387891, gi|1763532, and/or
gi|349736; all references available through these accessions are
hereby incorporated herein by reference). In specific embodiments,
polypeptides of the invention comprise, or alternatively consist
of, the following amino acid sequence (portion of extracellular
domain): PFWAAESALDFHWPFGGALCKMVLTATVLNVYASIFLITALSVARY (SEQ ID NO:
379). Moreover, fragments and variants of these polypeptides (such
as, for example, fragments as described herein, polypeptides at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
these polypeptides, or polypeptides encoded by a polynucleotide
which hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0459] This gene is expressed primarily in 7TM-pbfd and PCMIX
libraries (tissue types unknown).
[0460] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
blood pressure regulatory diseases and/or disorders. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
vascular system, expression of this gene at significantly higher or
lower levels may be routinely detected in certain tissues or cell
types (e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
lymph, serum, plasma, urine, synovial fluid and spinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of the
immunogenic epitopes shown in SEQ ID NO: 179 as residues: Gln-117
to Ser-126. Polynucleotides encoding said polypeptides are
encompassed by the invention, as are antibodies that bind one or
more of these peptides.
[0461] The tissue distribution and homology to angiotensin II
receptor indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the study,
detection, treatment, and/or prevention of vascular diseases such
as blood pressure regulatory disorders. Representative uses are
described elsewhere herein. In particular, the extracellular region
of the receptor can be used as a soluble antagonist. Moreover,
polynucleotides and polypeptides of the invention would be useful
in the detection, treatment, and/or prevention of a variety of
vascular disorders and conditions, which include, but are not
limited to miscrovascular disease, vascular leak syndrome,
aneurysm, stroke, embolism, thrombosis, coronary artery disease,
arteriosclerosis, and/or atherosclerosis. Furthermore, the protein
may also be used to determine biological activity, to raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0462] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:72 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 869 of SEQ ID NO:72, b is an integer
of 15 to 883, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:72, and where b is greater
than or equal to a+14.
[0463] Features of Protein Encoded By Gene No: 63
[0464] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: THADKNQVRNSN (SEQ ID NO: 380),
QFLSWEQCTGNTESQ (SEQ ID NO: 381), VRRPKAKGXQTSN (SEQ ID NO: 382),
PTQLNKHKPTTKERRRKGL (SEQ ID NO: 383), and/or LISKHENIY (SEQ ID NO:
384). Moreover, fragments and variants of these polypeptides (such
as, for example, fragments as described herein, polypeptides at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
these polypeptides, or polypeptides encoded by a polynucleotide
which hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0465] This gene is expressed primarily in neutrophils.
[0466] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
diseases and/or disorders affecting the immune system. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues or cell types
(e.g., immune, cancerous and wounded tissues) or bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0467] The tissue distribution in neutrophils indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the diagnosis and/or treatment of immune system
disorders, especially those affecting neutrophils. Representative
uses are described in the "Immune Activity" and "Infectious
Disease" sections below, in Example 11, 13, 14, 16, 18, 19, 20, and
27, and elsewhere herein. Briefly, polynucleotides and polypeptides
of the invention may be involved in the regulation of cytokine
production, antigen presentation, or other processes that may also
suggest a usefulness in the treatment of cancer (e.g., by boosting
immune responses). Since the gene is expressed in cells of lymphoid
origin, the gene or protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues. Therefore
polynucleotides and polypeptides of the invention may be also used
as an agent for immunological disorders including arthritis,
asthma, immune deficiency diseases such as AIDS, leukemia,
rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and
psoriasis. In addition, polynucleotides and polypeptides of the
invention may have commercial utility in the expansion of stem
cells and committed progenitors of various blood lineages, and in
the differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0468] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:73 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 771 of SEQ ID NO:73, b is an integer
of 15 to 785, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:73, and where b is greater
than or equal to a+14.
[0469] Features of Protein Encoded By Gene No: 64
[0470] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: TLYIXXMXTQTWRDQGRCGRDXINCIV (SEQ ID NO: 385). Moreover,
fragments and variants of these polypeptides (such as, for example,
fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides,
or polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0471] This gene is expressed primarily in brain tissue from a
manic depressive, in some cancer tissues such as ovarian cancer,
and in spleen from a patient with chronic lymphocytic leukemia and,
to a lesser extent, in other tissues.
[0472] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
brain disorders (e.g., manic depression), and tumorigenesis.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the central nervous system (CNS), reproductive system, and immune
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues or cell types
(e.g., brain, reproductive, immune, cancerous and wounded tissues)
or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of one or both of the immunogenic epitopes shown inSEQ ID
NO: 181 as residues: Thr-29 to Ala-37, Arg-41 to Lys-46.
Polynucleotides encoding said polypeptides are encompassed by the
invention, as are antibodies that bind one or more of these
peptides.
[0473] The tissue distribution primarily in brain tissue from a
manic depressive indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for diagnosing and
treating manic depression and tumorigenesis. The tissue
distribution in brain also indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, treatment, and/or prevention of neurodegenerative
disease states, behavioral disorders, or inflammatory conditions.
Representative uses are described in the "Regeneration" and
"Hyperproliferative Disorders" sections below, in Example 11, 15,
and 18, and elsewhere herein. Briefly, the uses include, but are
not limited to the detection, treatment, and/or prevention of
Alzheimer's Disease, Parkinson's Disease, Huntington's Disease,
Tourette Syndrome, meningitis, encephalitis, demyelinating
diseases, peripheral neuropathies, neoplasia, trauma, congenital
malformations, spinal cord injuries, ischemia and infarction,
aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia,
obsessive compulsive disorder, depression, panic disorder, learning
disabilities, ALS, psychoses, autism, and altered behaviors,
including disorders in feeding, sleep patterns, balance, and
perception. In addition, elevated expression of this gene product
in regions of the brain indicates that polynucleotides and
polypeptides corresponding to this gene may play a role in normal
neural function. Potentially, polynucleotides and polypeptides
corresponding to this gene are involved in synapse formation,
neurotransmission, learning, cognition, homeostasis, or neuronal
differentiation or survival. Furthermore, the protein may also be
used to determine biological activity, to raise antibodies, as
tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0474] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:74 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2327 of SEQ ID NO:74, b is an integer
of 15 to 2341, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:74, and where b is greater
than or equal to a+14.
[0475] Features of Protein Encoded By Gene No: 65
[0476] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: SLCTPGRGWEESWGSSLPNLTGWSVSSLDNNDV (SEQ ID NO: 386).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0477] This gene is expressed primarily in metastic melanoma
spleen, rhabdomyosarcoma, and IL-1 induced neutrophils and, to a
lesser extent, in other tissues.
[0478] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
tumorigenesis, metastasis and inflammatory disorders. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the skin,
connective tissue and immune system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., skin, cancerous and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial fluid and spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0479] The tissue distribution in metastic melanoma spleen,
rhabdomyosarcoma, and IL-1 induced neutrophils indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for detection, treatment, and/or prevention of certain
tumors such as melanoma, rhabdomyosarcoma and inflammatory
disorders. Similarly, the tissue distribution indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the treatment, diagnosis, and/or prevention of
various skin disorders including congenital disorders (e.g., nevi,
moles, freckles, Mongolian spots, hemangiomas, port-wine syndrome),
integumentary tumors (e.g., keratoses, Bowen's disease, basal cell
carcinoma, squamous cell carcinoma, malignant melanoma, Paget's
disease, mycosis fungoides, and Kaposi's sarcoma), injuries and
inflammation of the skin (e.g., wounds, rashes, prickly heat
disorder, psoriasis, dermatitis), atherosclerosis, uticaria,
eczema, photosensitivity, autoimmune disorders (e.g., lupus
erythematosus, vitiligo, dermatomyositis, morphea, scleroderna,
pemphigoid, and pemphigus), keloids, striae, erythema, petechiae,
purpura, and xanthelasma. Moreover, such disorders may predispose
increased susceptibility to viral and bacterial infections of the
skin (e.g., cold sores, warts, chickenpox, molluscum contagiosum,
herpes zoster, boils, cellulitis, erysipelas, impetigo, tinea,
althlete's foot, and ringworm). The tissue distribution in
neutrophils indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the diagnosis and
treatment of a variety of immune system disorders. Representative
uses are described in the "Immune Activity" and "Infectious
Disease" sections below, in Example 11, 13, 14, 16, 18, 19, 20, and
27, and elsewhere herein. Briefly, the expression indicates a role
in regulating the proliferation; survival; differentiation; and/or
activation of hematopoietic cell lineages, including blood stem
cells. Involvement in the regulation of cytokine production,
antigen presentation, or other processes indicates a usefulness for
treatment of cancer (e.g., by boosting immune responses).
Expression in cells of lymphoid origin, indicates the natural gene
product would be involved in immune functions. Therefore
polynucleotides and polypeptides of the invention would also be
useful as an agent for immunological disorders including arthritis,
asthma, immunodeficiency diseases such as AIDS, leukemia,
rheumatoid arthritis, granulomatous disease, inflammatory bowel
disease, sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus, this
gene product is thought to be usefil in the expansion of stem cells
and committed progenitors of various blood lineages, and in the
differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0480] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:75 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1868 of SEQ ID NO:75, b is an integer
of 15 to 1882, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:75, and where b is greater
than or equal to a+14.
[0481] Features of Protein Encoded By Gene No: 66
[0482] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
DSESSSEEEEEFGVVGNRSRFAKGDYLRCCKICYPLCGFVILAACVVACVGLV
WMQVALKEDLDALKEKFRTMESNQKSSFQEIPKLNEELLSKQKQLEKIESGE
MGLNKVWINITEMNKQISLLTSAVNHLKANVKSAADLISLPTTVEGLQKSVA
SIGXTLNSVHLAVEALQKTVDEHKKTMELLQSDMNQHFLKETPGSNQIIPSPS
ATSELDNKTHSENLKQMGDRSATLKRQSLDQVTNRTDTVKIQSIKKEG (SEQ ID NO:393),
MQVALKEDLDALKEKFRTMESNQKSSFQEIPKLNEELLSKQKQLEKIESGEM
GLNKVWINITEMNKQISLLTSAVNHLKANVKSAADLISLPTTVEGLQKSVASI
GXTLNSVHLAVEALQKTVDEHKKTMELLQSDMNQHFLKETPGSNQIIPSPSA
TSELDNKTHSENLKQMGDRSATLKRQSLDQVTNRTDTVKIQSIKKEG (SEQ ID NO:387),
MQVALKEDLDALKEKFRTMESNQKSSFQEIPKLNEELLSKQKQ (SEQ ID NO:388),
LEKIESGEMGLNKVWINITEMNKQISLLTSAVNHLKANVKSAA (SEQ ID NO:389),
DLISLPTTVEGLQKSVASIGXTLNSVHLAVEALQKTVDEHKKT (SEQ ID NO:390),
MELLQSDMNQHFLKETPGSNQIIPSPSATSELDNKTHSENLKQ (SEQ ID NO:391), and/or
MGDRSATLKRQSLDQVTNRTDTVKIQSIKKEG (SEQ ID NO: 392). Moreover,
fragments and variants of these polypeptides (such as, for example,
fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides,
or polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0483] The gene encoding the disclosed cDNA is believed to reside
on chromosome 1. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 1.
[0484] This gene is expressed primarily in fetal, placental and
infant brain tissues, and, to a lesser extent, in many normal and
neoplastic cell types.
[0485] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
developmental disorders, cancer and general growth disorders.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the reproductive, developing, and nervous systems, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., reproductive,
developmental, neural, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of the immunogenic epitopes shown in SEQ ID NO: 183 as
residues: Cys-30 to Asn-44. Polynucleotides encoding said
polypeptides are encompassed by the invention, as are antibodies
that bind one or more of these peptides.
[0486] The tissue distribution in infant brain and embryonic
tissues indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the study, detection
and/or treatment of growth and neoplastic disorders. Furthermore,
the tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, treatment, and/or prevention of cancer and other
proliferative disorders. Expression within embryonic tissue and
other cellular sources marked by proliferating cells indicates that
polynucleotides and polypeptides of the invention may play a role
in the regulation of cellular division. Embryonic development also
involves decisions involving cell differentiation and/or apoptosis
in pattern formation. Thus polynucleotides and polypeptides of the
invention may also be involved in apoptosis or tissue
differentiation and could again be useful in cancer therapy.
Alternatively, the tissue distribution in brain indicates
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection, treatment, and/or prevention of
neurodegenerative disease states, behavioral disorders, or
inflammatory conditions. Representative uses are described in the
"Regeneration" and "Hyperproliferative Disorders" sections below,
in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses
include, but are not limited to the detection, treatment, and/or
prevention of Alzheimer's Disease, Parkinson's Disease,
Huntington's Disease, Tourette Syndrome, meningitis, encephalitis,
demyelinating diseases, peripheral neuropathies, neoplasia, trauma,
congenital malformations, spinal cord injuries, ischemia and
infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia,
paranoia, obsessive compulsive disorder, depression, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, elevated expression of this
gene product in regions of the brain indicates it plays a role in
normal neural function. Potentially, polynucleotides and
polypeptides of the invention are involved in synapse formation,
neurotransmission, learning, cognition, homeostasis, or neuronal
differentiation or survival. In addition, the gene or gene product
may also play a role in the treatment and/or detection of
developmental disorders associated with the developing embryo, or
sexually-linked disorders. Furthermore, the protein may also be
used to determine biological activity, to raise antibodies, as
tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0487] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:76 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2878 of SEQ ID NO:76, b is an integer
of 15 to 2892, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:76, and where b is greater
than or equal to a+14.
[0488] Features of Protein Encoded By Gene No: 67
[0489] This gene is apparently exclusively in fetal heart
tissue.
[0490] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
cardiovascular and growth defects. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the developing
cardiovascular system, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., cardiovascular, heart, cancerous and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial fluid and spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0491] The tissue distribution in fetal heart tissue indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the study, detection and/or treatment of disorders
and growth defects of heart development and function. Furthermore,
the tissue distribution in fetal heart tissue indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection, treatment, and/or prevention of
conditions and pathologies of the cardiovascular system, such as
heart disease, restenosis, atherosclerosis, stroke, angina,
thrombosis, and wound healing. Furthermore, the protein may also be
used to determine biological activity, to raise antibodies, as
tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0492] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:77 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1659 of SEQ ID NO:77, b is an integer
of 15 to 1673, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:77, and where b is greater
than or equal to a+14.
[0493] Features of Protein Encoded By Gene No: 68
[0494] This gene is expressed primarily in pancreas islet cell
tumor tissue.
[0495] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
digestive and metabolic defects and tumors, particularly tumors of
the pancreas. Similarly, polypeptides and antibodies directed to
these polypeptides would be useful in providing immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the endocrine system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., endocrine, pancreas, cancerous
and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0496] The tissue distribution in pancreas islet cell tumor tissue
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the study, detection and/or treatment
of hormonal and neoplastic disorders of endocrine organs and
metabolism. Additionally, the tissue distribution indicates
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection, treatment, and/or prevention of
various endocrine disorders and cancers. Representative uses are
described in the "Biological Activity", "Hyperproliferative
Disorders", and "Binding Activity" sections below, in Example 11,
17, 18, 19, 20 and 27, and elsewhere herein. Briefly, the protein
can be used for the detection, treatment, and/or prevention of the
Addison's disease, Cushing's Syndrome, and disorders and/or cancers
of the pancreas (e.g., diabetes mellitus), adrenal cortex, ovaries,
pituitary (e.g., hyper-, hypopituitarism), thyroid (e.g., hyper-,
hypothyroidism), parathyroid (e.g., hyper-,hypoparathyroidism),
hypothallamus, and testes. Furthermore, the protein may also be
used to determine biological activity, to raise antibodies, as
tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0497] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:78 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1447 of SEQ ID NO:78, b is an integer
of 15 to 1461, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:78, and where b is greater
than or equal to a+14.
[0498] Features of Protein Encoded By Gene No: 69
[0499] This gene is expressed primarily in tonsils.
[0500] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
diseases and/or disorders of the tonsils, and disorders of the
immune system. Similarly, polypeptides and antibodies directed to
these polypeptides would be useful in providing immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the tonsils, and the immune system, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., tonsils, immune,
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0501] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, treatment, and/or prevention of a variety of immune
system disorders. Expression of this gene product in tonsils
indicates a role in the regulation of the proliferation; survival;
differentiation; and/or activation of potentially all hematopoietic
cell lineages, including blood stem cells. Representative uses are
described in the "Immune Activity" and "Infectious Disease"
sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and
elsewhere herein. Briefly, polynucleotides and polypeptides of the
invention may be involved in the regulation of cytokine production,
antigen presentation, or other processes that may also suggest a
usefulness in the treatment of cancer (e.g., by boosting immune
responses). Since the gene is expressed in cells of lymphoid
origin, the gene or protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues. Therefore
polynucleotides and polypeptides of the invention may be also used
as an agent for immunological disorders including arthritis,
asthma, immune deficiency diseases such as AIDS, leukemia,
rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and
psoriasis. In addition, polynucleotides and polypeptides of the
invention may have commercial utility in the expansion of stem
cells and committed progenitors of various blood lineages, and in
the differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0502] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:79 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1503 of SEQ ID NO:79, b is an integer
of 15 to 1517, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:79, and where b is greater
than or equal to a+14.
[0503] Features of Protein Encoded By Gene No: 70
[0504] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: SPQFLSSKSLPT (SEQ ID NO:394). Moreover, fragments and
variants of these polypeptides (such as, for example, fragments as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0505] This gene is expressed primarily in infant brain and spinal
cord.
[0506] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
congenital brain disorders, including various forms of mental
retardation, spina bifida, epilepsy, and various mood disorders,
including bipolar and unipolar depression. Similarly, polypeptides
and antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the central nervous system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
brain, CNS, cancerous and wounded tissues) or bodily fluids (e.g.,
lymph, amniotic fluid, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of one or both of the immunogenic epitopes shown in SEQ ID
NO: 187 as residues: Pro-42 to Lys-49, Lys-56 to Lys-71.
Polynucleotides encoding said polypeptides are encompassed by the
invention, as are antibodies that bind one or more of these
peptides.
[0507] The tissue distribution in infant brain and spinal cord
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the diagnosis, detection, prevention
and/or treatment of disorders of the brain and nervous system,
including congenital brain disorders, including various forms of
mental retardation, spina bifida, epilepsy, and various mood
disorders, including bipolar and unipolar depression. Additionally,
polynucleotides and polypeptides corresponding to this gene may
have cytostatic, thrombotic and/or osteopathic activity. It may
also be useful in the treatment of such neurodegenerative disorders
as schizophrenia; ALS; or Alzheimer's. The tissue distribution in
brain further indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the detection,
treatment, and/or prevention of neurodegenerative disease states,
behavioral disorders, or inflammatory conditions. Representative
uses are described in the "Regeneration" and "Hyperproliferative
Disorders" sections below, in Example 11, 15, and 18, and elsewhere
herein. Briefly, the uses include, but are not limited to the
detection, treatment, and/or prevention of Alzheimer's Disease,
Parkinson's Disease, Huntington's Disease, Tourette Syndrome,
meningitis, encephalitis, demyelinating diseases, peripheral
neuropathies, neoplasia, trauma, congenital malformations, spinal
cord injuries, ischemia and infarction, aneurysms, hemorrhages,
schizophrenia, mania, dementia, paranoia, obsessive compulsive
disorder, depression, panic disorder, learning disabilities, ALS,
psychoses, autism, and altered behaviors, including disorders in
feeding, sleep patterns, balance, and perception. In addition,
elevated expression of this gene product in regions of the brain
indicates that polynucleotides and polypeptides corresponding to
this gene may play a role in normal neural function. Potentially,
polynucleotides and polypeptides corresponding to this gene are
involved in synapse formation, neurotransmission, learning,
cognition, homeostasis, or neuronal differentiation or survival.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0508] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:80 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 560 of SEQ ID NO:80, b is an integer
of 15 to 574, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:80, and where b is greater
than or equal to a+14.
[0509] Features of Protein Encoded By Gene No: 71
[0510] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: TABLE-US-00008 (SEQ ID NO: 395)
GPPSPRGLPSLPLHLPAPRRYLQSRYACSQSSVSAAARRWGSGWMAWDPW
NQASGRYARITLLSVQACHQPTVWPRAGHSLPERYSLHPHNGDSTHLSGL LTVKCGA, (SEQ ID
NO: 396) GPPSPRGLPSLPLHLPAPRRYLQSRYACSQSSVSAAA, (SEQ ID NO: 397)
RRWGSGWMAWDPWNQASGRYARITLLSVQACHQ, (SEQ ID NO: 399)
GPPSPRGLPSLPLHLPAPRRYLQSRYACSQSSVSAAARRWGSGWMAWDPW
NQASGRYARITLLSVQACHQPTVWPRAGHSLPERYSLHPHNGDSTHLSGL
LTVKKCGAMAGFASYPWSDFPWCWVVCFSFXFFFLRQSESLSQKKRQVAD
ELXFGQSKRDSDGGWMLRSSAGNS, (SEQ ID NO: 400)
MESCSVVQAGVKWCDLGSLQPPPRFKQFSWEVEVAVSRDHTIALQXGGQS
KXLSQKKEKKYVLNATFLNFYFCRDKVLLCCPGWSHIVGLKQSSHLGLRK
CWDYRHGPLXLALCHFVCK, and/or (SEQ ID NO: 392)
PTVWPRAGHSLPERYSLHPHNGDSTHLSGLLTVKCGA.
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0511] This gene is expressed primarily in neutrophils.
[0512] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
infection, inflammation and other immune reactions or disorders.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues or
cell types (e.g., immune, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0513] The tissue distribution in neutrophils indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for detection, treatment, and/or prevention of immune
disorders, such as infection, inflammation, allergy and
immunodeficiency. Therefore, polynucleotides and polypeptides
corresponding to this gene may have clinical relevance in the
treatment of impaired immunity, in the correction of autoimmunity,
in immune modulation, in the treatment of allergy, and in the
regulation of inflammation. It may also play a role in influencing
differentiation of specific hematopoietic lineages, and may even
affect the hematopoietic stem cell. The tissue distribution in
neutophils also indicates that polynucleotides and polypeptides
corresponding to this gene may be useful for the diagnosis and
treatment of a variety of immune system disorders. Representative
uses are described in the "Immune Activity" and "Infectious
Disease" sections below, in Example 11, 13, 14, 16, 18, 19, 20, and
27, and elsewhere herein. Briefly, the expression indicates a role
in regulating the proliferation; survival; differentiation; and/or
activation of hematopoietic cell lineages, including blood stem
cells. Involvement in the regulation of cytokine production,
antigen presentation, or other processes indicates a usefulness for
treatment of cancer (e.g., by boosting immune responses).
Expression in cells of lymphoid origin, indicates the natural gene
product would be involved in immune functions. Therefore
polynucleotides and polypeptides corresponding to this gene would
also be useful as an agent for immunological disorders including
arthritis, asthma, immunodeficiency diseases such as AIDS,
leukemia, rheumatoid arthritis, granulomatous disease, inflammatory
bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus, this
gene product is thought to be useful in the expansion of stem cells
and committed progenitors of various blood lineages, and in the
differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, raise antibodies, as tissue markers, to isolate cognate
ligands or receptors, to identify agents that modulate their
interactions, in addition to its use as a nutritional supplement.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0514] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:81 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1441 of SEQ ID NO:81, b is an integer
of 15 to 1455, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:81, and where b is greater
than or equal to a+14.
[0515] Features of Protein Encoded By Gene No: 72
[0516] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: NQENSLQTN SYLDSTESK (SEQ ID NO: 401). Moreover, fragments
and variants of these polypeptides (such as, for example, fragments
as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0517] The polypeptide of this gene has been determined to have a
transmembrane domain at about amino acid position 12 to about 28 of
the amino acid sequence referenced in Table 1 for this gene.
Moreover, a cytoplasmic tail encompassing about amino acids 29 to
about 70 of this protein has also been determined. Based upon these
characteristics, it is believed that polynucleotides and
polypeptides corresponding to this gene shares structural features
to type Ib membrane proteins.
[0518] This gene is expressed primarily in neutrophils, activated
T-cells, tonsils, and fetal heart.
[0519] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
immune system disorders. Similarly, polypeptides and antibodies
directed to these polypeptides would be useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune system, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., immune,
cardiovascular, cancerous and wounded tissues) or bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0520] The tissue distribution neutrophils and T-cells indicates
that polynucleotides and polypeptides corresponding to this gene
would be useful for disgnosis and treatment of immune related
disorders including, infection, inflammation, allergy, tissue/organ
transplantation, immunodeficiency, etc. Representative uses are
described in the "Immune Activity" and "Infectious Disease"
sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and
elsewhere herein. Briefly, the expression indicates a role in
regulating the proliferation; survival; differentiation; and/or
activation of hematopoietic cell lineages, including blood stem
cells. Involvement in the regulation of cytokine production,
antigen presentation, or other processes indicates a usefulness for
treatment of cancer (e.g., by boosting immune responses).
Polynucleotides and polypeptides corresponding to this gene may
have clinical relevance in the treatment of impaired immunity, in
the correction of autoimmunity, in immune modulation, in the
treatment of allergy, and in the regulation of inflammation. It may
also play a role in influencing differentiation of specific
hematopoietic lineages, and may even affect the hematopoietic stem
cell. Furthermore, the protein may also be used to determine
biological activity, raise antibodies, as tissue markers, to
isolate cognate ligands or receptors, to identify agents that
modulate their interactions, in addition to its use as a
nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0521] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:82 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1626 of SEQ ID NO:82, b is an integer
of 15 to 1640, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:82, and where b is greater
than or equal to a+14.
[0522] Features of Protein Encoded By Gene No: 73
[0523] This gene is expressed primarily in hemangioperiocytoma,
placental tissue, and breast and endometrial tumor tissues, and, to
a lesser extent, in various other normal and transformed cell
types.
[0524] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
defects and tumors of female reproductive organs. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
reproductive system, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., reproductive, cancerous and wounded tissues)
or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0525] The tissue distribution in endometrial tumor tissue and
placental tissue indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the study, detection
and/or treatment of reproductive system disorders and neoplasias,
as well as cancers of other tissues where expression of this gene
has been observed. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0526] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:83 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 511 of SEQ ID NO:83, b is an integer
of 15 to 525, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:83, and where b is greater
than or equal to a+14.
[0527] Features of Protein Encoded By Gene No: 74
[0528] In an alternativie reading frame, this gene shares homology
with a DNA mismatch repair proteins, including PMS 4, and PMS1 (See
Accession No. R95251, gnl|PID|d1008095 and pir|JC2399|JC2399).
[0529] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: QKRACFPFAFCRDCQFXEXSPAMLPVQPAXL (SEQ ID
NO: 402); VSAHGIWLFRS (SEQ ID NO: 403); and/or
KHAAPPASLSLSLLLHHGQKRACFPFAFCRDCQFXEXSPAMLPVQPAXL (SEQ ID NO: 404).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention. This gene is expressed primarily in hematopoietic
cells and tissues, such as monocytes, primary dendritic cells, and
thymus; and, to a lesser extent, in brain
[0530] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
hematopoietic diseases and/or disorders; immune dysfunction;
susceptibility to infection; impaired immune surveillance;
neurological disorders, and cancers which may result from increased
genetic instability. Similarly, polypeptides and antibodies
directed to these polypeptides would be useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the immune system, CNS, and solid
tissues, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues or cell types
(e.g., hematopoietic, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0531] The tissue distribution primarily in hematopoietic cells and
tissues and the homology to DNA mismatch repair proteins indicates
that polynucleotides and polypeptides corresponding to this gene
would be useful for the diagnosis and/or treatment of a variety of
disorders, especially cancer. Representative uses are described in
the "Immune Activity" and "Infectious Disease" sections below, in
Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.
Briefly, the expression of this gene product in a number of
hematopoietic cells and tissues indicates that polynucleotides and
polypeptides of the invention may play a role in the proliferation;
differentiation; survival; and/or activation of a variety of
hematopoietic lineages, particularly the monocyte/macrophage
pathway. Expression of this gene product in a variety of brain
tissues also indicates that polynucleotides and polypeptides of the
invention may play a role in normal neuronal function or in
establishment of neural connectivity. Therefore, polynucleotides
and polypeptides of the invention may be useful in the treatment of
neurological disorders, such as Alzheimer's or Parkinson's.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0532] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:84 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 823 of SEQ ID NO:84, b is an integer
of 15 to 837, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:84, and where b is greater
than or equal to a+14.
[0533] Features of Protein Encoded By Gene No: 75
[0534] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: MCDNLIMLRTLMRYIVFLSLQCLWGQGTHSSCYPPSPLRLPLFFFLDIKLGISN
WPVVMQSCFALYLAGLICLTRSHEAIGRSSLSPSSSAPKVVARGVPS (SEQ ID NO: 405).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0535] This gene is expressed primarily in T-cell lymphoma,
endometrial tumors, and infant brain cells.
[0536] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
T-cell lymphoma, endometrial tumor, and neurodegenerative or
developmental diseases and/or disorders. Similarly, polypeptides
and antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune, central nervous
system, and reproductive systems, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., neural, immune, reproductive,
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of one or both
of the immunogenic epitopes shown in SEQ ID NO: 192 as residues:
Glu-28 to Tyr-33, Gly-50 to Tyr-57. Polynucleotides encoding said
polypeptides are encompassed by the invention, as are antibodies
that bind one or more of these peptides.
[0537] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for
detecting, diagnosing, preventing and/or treating T-cell lymphoma,
endometrial tumors, neurodegenerative or developmental disorders.
The tissue distribution in infant brain cells indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection/treatment of neurodegenerative disease
states and behavioural disorders. Representative uses are described
in the "Regeneration" and "Hyperproliferative Disorders" sections
below, in Example 11, 15, and 18, and elsewhere herein. Briefly,
the uses include, but are not limited to the detection, treatment,
and/or prevention of Alzheimer's Disease, Parkinson's Disease,
Huntington's Disease, Tourette Syndrome, schizophrenia, mania,
dementia, paranoia, obsessive compulsive disorder, panic disorder,
learning disabilities, ALS, psychoses, autism, and altered
behaviors, including disorders in feeding, sleep patterns, balance,
and perception. In addition, polynucleotides and polypeptides of
the invention may also play a role in the treatment and/or
detection of developmental disorders associated with the developing
embryo, or sexually-linked disorders. Furthermore, the protein may
also be used to determine biological activity, to raise antibodies,
as tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0538] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:85 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1560 of SEQ ID NO:85, b is an integer
of 15 to 1574, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:85, and where b is greater
than or equal to a+14.
[0539] Features of Protein Encoded By Gene No: 76
[0540] A translated product of this gene shares some homology with
C. elegans UNC-53 protein variant 7A and 8A which would be useful
to promote neuronal regeneration, revascularisation or wound
healing (see, e.g., GenSeq Accession W20057 and W20056; all
references available through these accessions are hereby
incorporated herein by reference).
[0541] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
MLVLMTLFLLLYYRYVYGFGVCVYVHIYAHIYTHTHIYNQLSIAYSSLIIYILY
SNFSNTPTKSFSPPYQYYNVPDNNITNPALTPTDFFENKQLLHAISFLYSPTGFL
QPPAHPVQLRTSTTLYGNHRGQTGCSQLD (SEQ ID NO:406), and
SNTPTKSFSPPYQYYNVPDNNITNPALTPTDFFENKQLLHAISFLYSPTGFLQPP
AHPVQLRTSTTL (SEQ ID NO: 407). Moreover, fragments and variants of
these polypeptides (such as, for example, fragments as described
herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%, or 100% identical to these polypeptides, or polypeptides
encoded by a polynucleotide which hybridizes, under stringent
conditions, to the polynucleotide encoding these polypeptides) are
encompassed by the invention. Antibodies that bind polypeptides of
the invention and polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0542] This gene is expressed primarily in cancer cells, particular
from hepatocellular carcinoma.
[0543] Homology to proteins that promote wound healing and
revascularization indicate that polynucleotides and polypeptides
corresponding to this gene would be useful in the detection,
treatment, and/or prevention of a variety of vascular disorders and
conditions, which include, but are not limited to miscrovascular
disease, vascular leak syndrome, aneurysm, stroke, embolism,
thrombosis, coronary artery disease, arteriosclerosis, and/or
atherosclerosis. Moreover, homology to proteins involved in
neuronal regeneration indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, treatment, and/or prevention of neurodegenerative
disease states, behavioral disorders, or inflammatory conditions.
Representative uses are described in the "Regeneration" and
"Hyperproliferative Disorders" sections below, in Example 11, 15,
and 18, and elsewhere herein. Briefly, the uses include, but are
not limited to the detection, treatment, and/or prevention of
Alzheimer's Disease, Parkinson's Disease, Huntington's Disease,
Tourette Syndrome, meningitis, encephalitis, demyelinating
diseases, peripheral neuropathies, neoplasia, trauma, congenital
malformations, spinal cord injuries, ischemia and infarction,
aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia,
obsessive compulsive disorder, depression, panic disorder, learning
disabilities, ALS, psychoses, autism, and altered behaviors,
including disorders in feeding, sleep patterns, balance, and
perception. In addition, elevated expression of this gene product
in regions of the brain indicates it plays a role in normal neural
function. Potentially, polynucleotides and polypeptides
corresponding to this gene are involved in synapse formation,
neurotransmission, learning, cognition, homeostasis, or neuronal
differentiation or survival. Polynucleotides and polypeptides of
the invention would be useful as reagents for differential
identification of the tissue(s) or cell type(s) present in a
biological sample and for diagnosis of diseases and conditions
which include, but are not limited to, central and peripheral
nervous system tissues, wounded and healing tissues, cardiovascular
system tissues, ocular tissues (particularly retina),
hepatocellular carcinoma and other similar cancer, particularly of
the liver. Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the hepatic system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues or
cell types (e.g., hepatic, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0544] The tissue distribution in tissues of cancerous origins,
such as hepatocellular carcinoma tissue, indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the diagnosis and/or treatment of a variety of
cancers, most notably cancers of the liver, such as hepatocellular
carcinoma. Expression of this gene product in a variety of cancers
indicates that polynucleotides and polypeptides corresponding to
this gene may be a player in the progression of these diseases, and
may be a beneficial target for inhibitors as therapeutics. Protein,
as well as, antibodies directed against the protein may show
utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0545] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:86 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1614 of SEQ ID NO:86, b is an integer
of 15 to 1628, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:86, and where b is greater
than or equal to a+14.
[0546] Features of Protein Encoded By Gene No: 77
[0547] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: MEMNYCGSRVLY (SEQ ID NO: 408) and/or
MEMNYCGSRVLYMSLILLGSPIIPLWSYTSATQAAALVTSHVWKPSLEAHQIN
ISPEPSIHYDRWHTQSNCSLINSLQ (SEQ ID NO:409). Moreover, fragments and
variants of these polypeptides (such as, for example, fragments as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0548] This gene is expressed primarily in T-cell lymphoma, and, to
a lesser extent, in hepatocellular tumor tissue.
[0549] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
T-cell lymphoma, hepatocellular tumors, and cancers. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
and hepatic systems, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
or cell types (e.g., immune, hepatic, cancerous and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial fluid and spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder. Preferred polypeptides of the present invention comprise,
or alternatively consist of the immunogenic epitopes shown in SEQ
ID NO: 194 as residues: Pro-46 to Asn-58. Polynucleotides encoding
said polypeptides are encompassed by the invention, as are
antibodies that bind one or more of these peptides.
[0550] The tissue distribution in T-cell lymphoma and
hepatocellular tumor tissue indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, diagnosis, prevention and/or treatment of T-cell
lymphomas and hepatocellular tumors, as well as cancers of other
tissues where expression of this gene has been observed.
Representative uses are described in the "Immune Activity" and
"Infectious Disease" sections below, in Example 11, 13, 14, 16, 18,
19, 20, and 27, and elsewhere herein. Furthermore, the protein may
also be used to determine biological activity, to raise antibodies,
as tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0551] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:87 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1781 of SEQ ID NO:87, b is an integer
of 15 to 1795, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:87, and where b is greater
than or equal to a+14.
[0552] Features of Protein Encoded By Gene No: 78
[0553] This gene is expressed primarily in brain tissue, and, to a
lesser extent, in ntera2 cell lines, melanocytes, normal colon, and
T-helper cells.
[0554] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
neurodegenerative diseases and/or conditions. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
nervous system, expression of this gene at significantly higher or
lower levels may be routinely detected in certain tissues or cell
types (e.g., neural, immune, hematopoietic, gastrointestinal, and
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of the
immunogenic epitopes shown in SEQ ID NO: 195 as residues: Met-1 to
Trp-6. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0555] The tissue distribution in brain tissue indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for detecting and/or treating neurodegenerative diseases
of the central nervous system. Representative uses are described in
the "Regeneration" and "Hyperproliferative Disorders" sections
below, in Example 11, 15, and 18, and elsewhere herein. Briefly,
the tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, diagnosis, prevention, and/or treatment of
neurodegenerative disease states and behavioural disorders such as
Alzheimer's Disease, Parkinson's Disease, Huntington's Disease,
Tourette Syndrome, schizophrenia, mania, dementia, paranoia,
obsessive compulsive disorder, panic disorder, learning
disabilities, ALS, psychoses, autism, and altered behaviors,
including disorders in feeding, sleep patterns, balance, and
perception. In addition, the gene or gene product may also play a
role in the treatment and/or detection of developmental disorders
associated with the developing embryo, or sexually-linked
disorders. Furthermore, the protein may also be used to determine
biological activity, to raise antibodies, as tissue markers, to
isolate cognate ligands or receptors, to identify agents that
modulate their interactions, in addition to its use as a
nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0556] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:88 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1850 of SEQ ID NO:88, b is an integer
of 15 to 1864, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:88, and where b is greater
than or equal to a+14.
[0557] Features of Protein Encoded By Gene No: 79
[0558] The gene encoding the disclosed cDNA is thought to reside on
chromosome 1. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 1.
[0559] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: IPEEASCFPSAV (SEQ ID NO: 410),
EILFGKLKSKAALCTQG (SEQ ID NO: 411), HADRYTCCRCLSPFSLAGL (SEQ ID NO:
412), LSDPLLLPDCSFSFN (SEQ ID NO: 413), KAVAYANVSCRRFKHKTTKLGPIQW
(SEQ ID NO: 414), PSSQSPEPPQPLSLFVTRLPNLYDFP (SEQ ID NO: 415),
and/or SRQIICTNLCKCTPICFLF (SEQ ID NO: 416). Moreover, fragments
and variants of these polypeptides (such as, for example, fragments
as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0560] Translated products of this gene share some homology with a
Factor VIIa protein (see, e.g., GenSeq Accession No. R13788; all
references available through this accession are hereby incorporated
herein by reference). In specific embodiments, polypeptides of the
invention comprise, or alternatively consist of, an amino acid
sequence selected from the group: KGSLPWRLLLPLNGP (SEQ ID NO: 417)
and LCRLVFESSAGHVSVCHSF (SEQ ID NO: 418). Moreover, fragments and
variants of these polypeptides (such as, for example, fragments as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed,by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0561] This gene is expressed primarily in breast tissue, fetal
liver and adult hepatoma tissues, and, to a lesser extent, in
merkel cells and osteoblasts.
[0562] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
circulatory disorders (particularly coagulatory disorders), cancers
of the liver or breast. Similarly, polypeptides and antibodies
directed to these polypeptides would be useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the circulatory system or
glandular systems, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues or
cell types (e.g., breast, liver, cancerous and wounded tissues) or
bodily fluids (e.g., lymph, breast milk, serum, plasma, urine,
synovial fluid and spinal fluid) or another tissue or cell sample
taken from an individual having such a disorder, relative to the
standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder. Preferred polypeptides of the present invention comprise,
or alternatively consist of the immunogenic epitopes shown in SEQ
ID NO: 196 as residues: Asn-25 to Gln-50. Polynucleotides encoding
said polypeptides are encompassed by the invention, as are
antibodies that bind one or more of these peptides.
[0563] The tissue distribution in breast and hepatoma tissues
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for diagnosing and/or treating tumors of
the breast or liver. Furthermore, the expression in the breast
tissue may indicate its uses in breast neoplasia and breast
cancers, such as fibroadenoma, pipillary carcinoma, ductal
carcinoma, Paget's disease, medullary carcinoma, mucinous
carcinoma, tubular carcinoma, secretory carcinoma and apocrine
carcinoma, as well as juvenile hypertrophy and gynecomastia,
mastitis and abscess, duct ectasia, fat necrosis and fibrocystic
diseases. Alternatively, the tissue distribution indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection and treatment of liver disorders and
cancers (e.g., hepatoblastoma, jaundice, hepatitis, liver metabolic
diseases and conditions that are attributable to the
differentiation of hepatocyte progenitor cells). Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and immunotherapy targets for the above listed tumors
and tissues.
[0564] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:89 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1969 of SEQ ID NO:89, b is an integer
of 15 to 1983, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:89, and where b is greater
than or equal to a+14.
[0565] Features of Protein Encoded By Gene No: 80
[0566] This gene is expressed primarily in thymus and brain
tissues.
[0567] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
diseases and/or disorders of the immune system and diseases of the
brain, including various types of mood disorders. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system and central nervous system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., immune, neural, cancerous and
wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken from an individual having such a disorder, relative to
the standard gene expression level, i.e., the expression level in
healthy tissue or bodily fluid from an individual not having the
disorder.
[0568] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, treatment, and/or prevention of a variety of immune
system disorders. Representative uses are described in the "Immune
Activity" and "Infectious Disease" sections below, in Example 11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the
expression of this gene product in thymus indicates a role in the
regulation of the proliferation; survival; differentiation; and/or
activation of potentially all hematopoietic cell lineages,
including blood stem cells. Polynucleotides and polypeptides
corresponding to this gene may be involved in the regulation of
cytokine production, antigen presentation, or other processes that
may also suggest a usefulness in the treatment of cancer (e.g., by
boosting immune responses). Since the gene is expressed in cells of
lymphoid origin, the gene or protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
Therefore polynucleotides and polypeptides of the invention may be
also used as an agent for immunological disorders including
arthritis, asthma, immune deficiency diseases such as AIDS,
leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,
acne, and psoriasis. In addition, polynucleotides and polypeptides
of the invention may have commercial utility in the expansion of
stem cells and committed progenitors of various blood lineages, and
in the differentiation and/or proliferation of various cell types.
Alternatively, the tissue distribution in brain tissue indicates
that polynucleotides and polypeptides corresponding to this gene
would be useful for the detection, diagnosis, prevention and/or
treatment of neurodegenerative disease states and behavioural
disorders such as Alzheimer's Disease, Parkinson's Disease,
Huntington's Disease, Tourette Syndrome, schizophrenia, mania,
dementia, paranoia, obsessive compulsive disorder, panic disorder,
learning disabilities, ALS, psychoses, autism, and altered
behaviors, including disorders in feeding, sleep patterns, balance,
and perception. In addition, the gene or gene product may also play
a role in the treatment and/or detection of developmental disorders
associated with the developing embryo, or sexually-linked
disorders. Furthermore, the protein may also be used to determine
biological activity, to raise antibodies, as tissue markers, to
isolate cognate ligands or receptors, to identify agents that
modulate their interactions, in addition to its use as a
nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0569] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:90 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1943 of SEQ ID NO:90, b is an integer
of 15 to 1957, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:90, and where b is greater
than or equal to a+14.
[0570] Features of Protein Encoded By Gene No: 81
[0571] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: MLLPVNTLLYI (SEQ ID NO: 419),
LLTPLCFFYGTSRP (SEQ ID NO: 420), PYLELVT (SEQ ID NO: 421),
LLKKKKQSVGFSV (SEQ ID NO: 422), CILEAGR (SEQ ID NO: 423),
MGFSAPTPGPL (SEQ ID NO: 424), FDLRRLILSIV (SEQ ID NO: 425),
AFCPHVTPCKYAVIHTV (SEQ ID NO: 426), NTPLLFLWDLQ (SEQ ID NO: 427),
ATIFRTSYLIKKEKTVC (SEQ ID NO: 428), WLLSLHLGGREVRAGAP (SEQ ID NO:
429), QTLQEGSLHSI (SEQ ID NO: 430), and/or
MGFSAPTPGPLFDLRRLILSIVAFCPHVTPCKYAVIHTVNTPLLFLWDLQATIF
RTSYLIKKEKTVCWLLSLHLGGREVRAGAPQTLQEGSLHSI (SEQ ID NO: 431).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0572] This gene is expressed primarily in brain and breast
tissues, and, to a lesser extent, in several other cell and tissue
types including colon and liver tissues.
[0573] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
breast and brain cancers, mood disorders, dementia, and Alzhiemer's
disease. Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the central nervous and lactations systems, expression of this gene
at significantly higher or lower levels may be routinely detected
in certain tissues or cell types (e.g., neural, reproductive,
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
breast milk, serum, plasma, urine, synovial fluid and spinal fluid)
or another tissue or cell sample taken from an individual having
such a disorder, relative to the standard gene expression level,
i.e., the expression level in healthy tissue or bodily fluid from
an individual not having the disorder. Preferred polypeptides of
the present invention comprise, or alternatively consist of the
immunogenic epitopes shown in SEQ ID NO: 198 as residues: Gly-21 to
Tyr-27. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0574] The expression in breast tissue indicates that
polynucleotides and/or polypeptides of the invention would be
useful for diagnosis, treatment and/or prevention of breast
neoplasia and breast cancers, such as fibroadenoma, pipillary
carcinoma, ductal carcinoma, Paget's disease, medullary carcinoma,
mucinous carcinoma, tubular carcinoma, secretory carcinoma and
apocrine carcinoma, as well as juvenile hypertrophy and
gynecomastia, mastitis and abscess, duct ectasia, fat necrosis and
fibrocystic diseases. Representative uses are described in the
"Regeneration" and "Hyperproliferative Disorders" sections below,
in Example 11, 15, and 18, and elsewhere herein. Alternatively, the
tissue distribution of this gene in brain tissue indicates that
polynucleotides and polypeptides of the invention would be useful
for the detection and/or treatment of brain cancers and neural
disorders, such as Alzheimer's Disease, Parkinson's Disease,
Huntington's Disease, Tourette Syndrome, schizophrenia, mania,
dementia, paranoia, obsessive compulsive disorder, panic disorder,
learning disabilities, ALS, psychoses, autism, and altered
behaviors, including disorders in feeding, sleep patterns, balance,
and perception. In addition, the gene or gene product may also play
a role in the treatment and/or detection of developmental disorders
associated with the developing embryo, or sexually-linked
disorders. Furthermore, the protein may also be used to determine
biological activity, to raise antibodies, as tissue markers, to
isolate cognate ligands or receptors, to identify agents that
modulate their interactions, in addition to its use as a
nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0575] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:91 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 559 of SEQ ID NO:91, b is an integer
of 15 to 573, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:91, and where b is greater
than or equal to a+14.
[0576] Features of Protein Encoded By Gene No: 82
[0577] The gene encoding the disclosed cDNA is believed to reside
on chromosome 1. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 1.
[0578] This gene is expressed primarily in liver and, to a lesser
extent, in other tissues.
[0579] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
liver/hepatocyte disorders. Similarly, polypeptides and antibodies
directed to these polypeptides would be useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the liver, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., liver, cancerous
and wounded tissues) or bodily fluids (e.g., lymph, bile, serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue
or cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0580] The tissue distribution in liver indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for detection, treatment, and/or prevention of liver
(hepatocyte) disorders and cancers (e.g., hepatoblastoma, jaundice,
hepatitis, liver metabolic diseases and conditions that are
attributable to the differentiation of hepatocyte progenitor
cells). Furthermore, the protein may also be used to determine
biological activity, to raise antibodies, as tissue markers, to
isolate cognate ligands or receptors, to identify agents that
modulate their interactions, in addition to its use as a
nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0581] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:92 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1198 of SEQ ID NO:92, b is an integer
of 15 to 1212, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:92, and where b is greater
than or equal to a+14.
[0582] Features of Protein Encoded By Gene No: 83
[0583] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: YWVSISQRSVCQQARTSIFFKDGLSREKYSNNG (SEQ ID NO: 432).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0584] This gene is expressed primarily in T cells.
[0585] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
immune disorders, including AIDS and various other diseases in
which the immune system is suppressed. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providingimmunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
immune, cancerous and wounded tissues) or bodily fluids (e.g.,
lymph, serum, plasma, urine, synovial fluid and spinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0586] The tissue distribution in T cells indicates that the
polypeptides or polynucleotides would be useful for treatment,
prophylaxis, and diagnosis of immune and autoimmune diseases, such
as lupus, transplant rejection, allergic reactions, arthritis,
asthma, immunodeficiency diseases, leukemia, and AIDS.
Representative uses are described in the "hnmune Activity" and
"Infectious Disease" sections below, in Example 11, 13, 14, 16, 18,
19, 20, and 27, and elsewhere herein. Briefly, the expression
indicates a role in regulating the proliferation; survival;
differentiation; and/or activation of hematopoietic cell lineages,
including blood stem cells. Involvement in the regulation of
cytokine production, antigen presentation, or other processes
indicates a usefulness for treatment of cancer (e.g., by boosting
immune responses). Expression in cells of lymphoid origin,
indicates the natural gene product would be involved in immune
functions. Therefore polynucleotides and polypeptides of the
invention would also be useful as an agent for immunological
disorders including arthritis, asthma, immunodeficiency diseases
such as AIDS, leukemia, rheumatoid arthritis, granulomatous
disease, inflammatory bowel disease, sepsis, acne, neutropenia,
neutrophilia, psoriasis, hypersensitivities, such as T-cell
mediated cytotoxicity; immune reactions to transplanted organs and
tissues, such as host-versus-graft and graft-versus-host diseases,
or autoimmunity disorders, such as autoimmune infertility, lense
tissue injury, demyelination, systemic lupus erythematosis, drug
induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease,
and scleroderrna. Moreover, the protein may represent a secreted
factor that influences the differentiation or behavior of other
blood cells, or that recruits hematopoietic cells to sites of
injury. Thus, polynucleotides and polypeptides of the invention are
thought to be useful in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. The polypeptides or
polynucleotides of the present invention would also be useful in
the treatment, prophlaxis, and detection of thymus disorders, such
as Grave's Disease, lymphocytic thyroiditis, hyperthyroidism, and
hypothyroidism. Similarly, elevated levels of expression of this
gene product in T cell lineages indicates that it may play an
active role in normal T cell function and in the regulation of the
immune response. For example, this gene product may be involved in
T cell activation, in the activation or control of differentiation
of other hematopoietic cell lineages, in antigen recognition, or in
T cell proliferation. Furthermore, the protein may also be used to
determine biological activity, raise antibodies, as tissue markers,
to isolate cognate ligands or receptors, to identify agents that
modulate their interactions, in addition to its use as a
nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0587] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:93 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1130 of SEQ ID NO:93, b is an integer
of 15 to 1144, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:93, and where b is greater
than or equal to a+14.
[0588] Features of Protein Encoded By Gene No: 84
[0589] The translation product of this gene shares sequence
homology with a protein which was found to accumulate during
growth-factor-induced proliferation and transformation of normal
rat fibroblasts (see, e.g., Glaichenhaus, N., and Cuzin, F., Cell
50:1081 (1987); and Genbank Acc. No. gi|207250; all references
available through this accession and reference are hereby
incorporated by reference herein.)
[0590] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
LSVRAPGVPAARPRLSSARQAGAGRGELRGQRLWLGPECGCGAGQAGSMLR
AVGSLLRLGRGLTVRCGPGAPLEATRRPAPALPPRGLPCYSSGGAPSNSGPQG
HGEIHRVPTQRRPSQFDKKILLWTGRFKSMEEIPPRIPPEMIDTARNKARVKAC YI (SEQ ID
NO:433), LSVRAPGVPAARPRLSSARQAGAGRGELRGQRLWLG (SEQ ID NO:434),
PECGCGAGQAGSMLRAVGSLLRLGRGLTVRCGPG (SEQ ID NO:435),
APLEATRRPAPALPPRGLPCYSSGGAPSNSGPQG (SEQ ID NO:436),
HGEIHRVPTQRRPSQFDKKILLWTGRF (SEQ ID NO:437), and/or
KSMEEIPPRIPPEMIDTARNKARVKACYI (SEQ ID NO:438). Moreover, fragments
and variants of these polypeptides (such as, for example, fragments
as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0591] The polypeptide of this gene has been determined to have a
transmembrane domain at about amino acid position 4 to about 20 of
the amino acid sequence referenced in Table 1 for this gene.
Moreover, a cytoplasmic tail encompassing amino acids 1 to 3 of
this protein has also been determined. Based upon these
characteristics, it is believed that polynucleotides and
polypeptides corresponding to this gene shares structural features
to type II membrane proteins.
[0592] This gene is expressed primarily in placenta.
[0593] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
developmental anomalies or fetal deficiencies, cancers or
neoplastic conditions. Similarly, polypeptides and antibodies
directed to these polypeptides would be useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the developing embryo, expression
of this gene at significantly higher or lower levels may be
routinely detected in certain tissues or cell types (e.g.,
embryonic, placental, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, amniotic fluid, serum, plasma, urine, synovial
fluid and spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0594] The tissue distribution and homology to a protein which was
found to accumulate during proliferation and transformation of
normal fibroblasts indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the treatment and
diagnosis of developmental anomalies or fetal deficiencies,
neoplasms and cancers. Additionally, the tissue distribution in
placenta indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the diagnosis and/or
treatment of disorders of the placenta. Specific expression within
the placenta indicates that polynucleotides and polypeptides of the
invention may play a role in the proper establishment and
maintenance of placental function. Alternately, polynucleotides and
polypeptides of the invention may be produced by the placenta and
then transported to the embryo, where it may play a crucial role in
the development and/or survival of the developing embryo or fetus.
Expression of this gene product in a vascular-rich tissue such as
the placenta also indicates that polynucleotides and polypeptides
of the invention may be produced more generally in endothelial
cells or within the circulation. In such instances, it may play
more generalized roles in vascular function, such as in
angiogenesis. Polynucleotides and polypeptides of the invention may
also be produced in the vasculature and have effects on other cells
within the circulation, such as hematopoietic cells. It may serve
to promote the proliferation, survival, activation, and/or
differentiation of hematopoietic cells, as well as other cells
throughout the body. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues. Furthermore,
the protein may also be used to determine biological activity, to
raise antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0595] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:94 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1260 of SEQ ID NO:94, b is an integer
of 15 to 1274, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:94, and where b is greater
than or equal to a+14.
[0596] Features of Protein Encoded By Gene No: 85
[0597] The translated product of this gene shares some homology
with a novel alpha-neurotoxin from the king cobra (Ophiophagus
hannah) venom (see, e.g., Genbank Accession No. JC1474 and P80965;
all references available through these accessions are hereby
incorporated herein by reference). Based on the sequence
similarity, the translation product of this clone is expected to
share at least some biological activities with neurotransmitter
proteins.
[0598] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group:
CSPGQDEMQDETWCSGQSETVNEAKQLRTTHSRVPNQQVCVCGWLPVNISP HSPLKK (SEQ ID
NO: 439) and/or MSGDVCVFGYAHLHSQTKHSGSQGWVLIYLFAMQKISCTKLPLLRNLKLNL
VWLSQGWVFFKGLWGEMLTGSHPQTHTCWLGTRLWVVLSCLASLTVSDCP
EHQVSSCISSWPGEHSVSFQPFPPFPHSLGGTEVGVEESQMAGVGI (SEQ ID NO: 440).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0599] The gene encoding the disclosed cDNA is thought to reside on
chromosome 3. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 3.
[0600] This gene is expressed primarily in T-cell lymphoma and
synovial sarcoma tissues, and, to a lesser extent, in fetal
liver/spleen tissue and synovial fibroblasts.
[0601] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
T-Cell lymphoma and synovial sarcoma. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
immune, hematopoietic, and cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid or spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder. Preferred polypeptides
of the present invention comprise, or alternatively consist of one
or both of the immunogenic epitopes shown in SEQ ID NO: 202 as
residues: Gly-4 to His-10, Asp-32 to Val-38. Polynucleotides
encoding said polypeptides are encompassed by the invention, as are
antibodies that bind one or more of these peptides.
[0602] The tissue distribution in T-cell lymphoma and synovial
sarcoma tissues indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the detection,
diagnosis, prevention and/or treatment of T-cell lymphomas and
synovial sarcomas, as well as cancers of other tissues where
expression of this gene has been observed. Representative uses are
described in the "Immune Activity" and "Infectious Disease"
sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and
elsewhere herein. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0603] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:95 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1766 of SEQ ID NO:95, b is an integer
of 15 to 1780, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:95, and where b is greater
than or equal to a+14.
[0604] Features of Protein Encoded By Gene No: 86
[0605] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: LNILISLTVSSHCKL (SEQ ID NO: 441),
INYHSGFIHQFLA (SEQ ID NO: 442), and/or MANNSLSSQFI (SEQ ID NO:
443). Moreover, fragments and variants of these polypeptides (such
as, for example, fragments as described herein, polypeptides at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
these polypeptides, or polypeptides encoded by a polynucleotide
which hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0606] The translated product of this gene shares some homology
with Integrin Beta 5 subunit protein (see, e.g., GenBank Accession
No. Q64657; all references available through this accession are
hereby incorporated herein by reference).
[0607] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: ISGVLIFNLIASSWVLCFPLCDLSCQKTLRIFFASFFHAVCVHVSCTSWQPLVLF
IKWWVVGCSP (SEQ ID NO: 444). Moreover, fragments and variants of
these polypeptides (such as, for example, fragments as described
herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%, or 100% identical to these polypeptides, or polypeptides
encoded by a polynucleotide which hybridizes, under stringent
conditions, to the polynucleotide encoding these polypeptides) are
encompassed by the invention. Antibodies that bind polypeptides of
the invention and polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0608] The translated product of this gene also contains a Zinc
finger (C2H2 type) domain consistent with the consensus pattern:
C.{2,4}C.{3 }[LIVMFYWC].{8}H.{3,5}H (identified using the ProSite
analysis tool (Swiss Institute of Bioinformatics)). Accordingly, in
specific embodiments, polypeptides of the invention comprise, or
alternatively consist of, the following amino acid sequence:
CDLSCQKTLRIFFASFFHAVCVH (SEQ ID NO: 445). Moreover, fragments and
variants of these polypeptides (such as, for example, fragments as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0609] This gene is expressed primarily in thymus tissue.
[0610] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
diseases and/or disorders of the immune system. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the immune
system, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues or cell types
(e.g., immune, cancerous and wounded tissues) or bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder.
[0611] The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, treatment, and/or prevention of a variety of immune
system disorders. Representative uses are described in the "Immune
Activity" and "Infectious Disease" sections below, in Example 11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the
expression of this gene product in thymus indicates a role in the
regulation of the proliferation; survival; differentiation; and/or
activation of potentially all hematopoietic cell lineages,
including blood stem cells. Polynucleotides and polypeptides
corresponding to this gene may be involved in the regulation of
cytokine production, antigen presentation, or other processes that
may also suggest a usefulness in the treatment of cancer (e.g., by
boosting immune responses). Since the gene is expressed in cells of
lymphoid origin, the gene or protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
Therefore polynucleotides and polypeptides of the invention may be
also used as an agent for immunological disorders including
arthritis, asthma, immune deficiency diseases such as AIDS,
leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,
acne, and psoriasis. In addition, polynucleotides and polypeptides
of the invention may have commercial utility in the expansion of
stem cells and committed progenitors of various blood lineages, and
in the differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0612] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:96 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1780 of SEQ ID NO:96, b is an integer
of 15 to 1794, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:96, and where b is greater
than or equal to a+14.
[0613] Features of Protein Encoded By Gene No: 87
[0614] The gene encoding the disclosed cDNA is believed to reside
on chromosome 10. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 10.
[0615] This gene is expressed primarily in brain, kidney, testes,
colon cancer, parathyroid tumor, immune cells (e.g., T-cells) and
to a lesser extent, in many other tissues.
[0616] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
kidney diseases and various diseases of the brain including mood
disorders. Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the brain and renal systems, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., kidney, CNS, immune, cancerous
and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine, synovial fluid or cerebrospinal fluid) or another tissue or
cell sample taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of the
immunogenic epitopes shown in SEQ ID NO: 204 as residues: Arg-68 to
Lys-78. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0617] The tissue distribution in kidney indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful in the treatment, prevention, diagnosis and/or detection
of kidney diseases including renal failure, nephritis, renal
tubular acidosis, proteinuria, pyuria, edema, pyelonephritis,
hydronephritis, nephrotic syndrome, crush syndrome,
glomerulonephritis, hematuria, renal colic and kidney stones, in
addition to Wilm's Tumor Disease, and congenital kidney
abnormalities such as horseshoe kidney, polycystic kidney, and
Falconi's syndrome. The tissue distribution in brain indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection, treatment, and/or prevention of
neurodegenerative disease states, behavioral disorders, or
inflammatory conditions. Representative uses are described in the
"Regeneration" and "Hyperproliferative Disorders" sections below,
in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses
include, but are not limited to the detection, treatment, and/or
prevention of Alzheimer's Disease, Parkinson's Disease,
Huntington's Disease, Tourette Syndrome, meningitis, encephalitis,
demyelinating diseases, peripheral neuropathies, neoplasia, trauma,
congenital malformations, spinal cord injuries, ischemia and
infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia,
paranoia, obsessive compulsive disorder, depression, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, elevated expression of this
gene product in regions of the brain indicates it plays a role in
normal neural function. Potentially, this gene product is involved
in synapse formation, neurotransmission, learning, cognition,
homeostasis, or neuronal differentiation or survival. The tissue
distribution in testes, kidney, and other tissues associates with
the endocrine system indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
detection, treatment, and/or prevention of various endocrine
disorders and cancers. Representative uses are described in the
"Biological Activity", "Hyperproliferative Disorders", and "Binding
Activity" sections below, in Example 11, 17, 18, 19, 20 and 27, and
elsewhere herein. Briefly, the protein can be used for the
detection, treatment, and/or prevention of Addison's disease,
Cushing's Syndrome, and disorders and/or cancers of the pancrease
(e.g., diabetes mellitus), adrenal cortex, ovaries, pituitary
(e.g., hyper-, hypopituitarism), thyroid (e.g., hyper-,
hypothyroidism), parathyroid (e.g., hyper-,hypoparathyroidism),
hypothallamus, and testes. The tissue distribution in immune cells
(e.g., T-cells) indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the diagnosis and
treatment of a variety of immune system disorders. Representative
uses are described in the "Immune Activity" and "Infectious
Disease" sections below, in Example 11, 13, 14, 16, 18, 19, 20, and
27, and elsewhere herein. Briefly, the expression indicates a role
in regulating the proliferation; survival; differentiation; and/or
activation of hematopoietic cell lineages, including blood stem
cells. Involvement in the regulation of cytokine production,
antigen presentation, or other processes indicates a usefulness for
treatment of cancer (e.g. by boosting immune responses). Expression
in cells of lymphoid origin, indicates the natural gene product
would be involved in immune functions. Therefore it would also be
useful as an agent for immunological disorders including arthritis,
asthma, immunodeficiency diseases such as AIDS, leukemia,
rheumatoid arthritis, granulomatous disease, inflammatory bowel
disease, sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus, this
gene product is thought to be usefil in the expansion of stem cells
and committed progenitors of various blood lineages, and in the
differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0618] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:97 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 2051 of SEQ ID NO:97, b is an integer
of 15 to 2065, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:97, and where b is greater
than or equal to a+14.
[0619] Features of Protein Encoded By Gene No: 88
[0620] It has been discovered that this gene is expressed primarily
in neutrophils.
[0621] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
immune and inflammatory disorders. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune and inflammatory
systems, expression of this gene at significantly higher or lower
levels may be routinely detected in certain tissues or cell types
(e.g., immune, cancerous and wounded tissues) or bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder. Preferred polypeptides
of the present invention comprise, or alternatively consist of the
immunogenic epitopes shown in SEQ ID NO: 205 as residues: Pro-41 to
Gln-48. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0622] The tissue distribution in neutrophils indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the study, diagnosis, detection prevention and/or
treatment of immune and inflammatory diseases. Representative uses
are described in the "Immune Activity" and "Infectious Disease"
sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and
elsewhere herein. Briefly, the expression of this gene product
indicates a role in regulating the proliferation; survival;
differentiation; and/or activation of hematopoietic cell lineages,
including blood stem cells. Furthermore, polynucleotides and
polypeptides of the invention may be involved in the regulation of
cytokine production, antigen presentation, or other processes that
may also suggest a usefulness in the treatment of cancer (e.g., by
boosting immune responses). Since the gene is expressed in cells of
lymphoid origin, the gene or protein, as well as, antibodies
directed against the protein may show utility as a tumor marker
and/or immunotherapy targets for the above listed tissues.
Therefore polynucleotides and polypeptides of the invention may be
also used as an agent for immunological disorders including
arthritis, asthma, immune deficiency diseases such as AIDS,
leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,
acne, and psoriasis. In addition, this gene product may have
commercial utility in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation
and/or proliferation of various cell types. Furthermore, the
protein may also be used to determine biological activity, to raise
antibodies, as tissue markers, to isolate cognate ligands or
receptors, to identify agents that modulate their interactions, in
addition to its use as a nutritional supplement. Protein, as well
as, antibodies directed against the protein may show utility as a
tumor marker and/or immunotherapy targets for the above listed
tissues.
[0623] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:98 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1140 of SEQ ID NO:98, b is an integer
of 15 to 1154, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:98, and where b is greater
than or equal to a+14.
[0624] Features of Protein Encoded By Gene No: 89
[0625] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: ELAIGESCS (SEQ ID NO: 446). Moreover, fragments and
variants of these polypeptides (such as, for example, fragments as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0626] The translation product of this gene shares sequence
homology with NY-REN-8 antigen (see, e.g., Genbank accession number
AF155098 (AD42864); all references available through this accession
are hereby incorporated by reference herein) which is an antigen
recognized by autologous antibody in patients with renal-cell
carcinoma and may be important in cancer diagnosis, therapy, and/or
prevention. Based on the sequence similarity, the translation
product of this clone is expected to share at least some biological
activities with NY-REN-8 antigen and other related antigens.
[0627] This gene is expressed primarily in brain, testes, and fetal
tissue.
[0628] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
developmental, degenerative and behavioral diseases of the brain
such as schizophrenia, Alzheimer's disease, Parkinson's disease,
Huntington's disease, transmissible spongiform encephalopathies
(TSE), Creutzfeldt-Jakob disease (CJD), specific brain tumors,
aphasia, mania, depression, dementia, paranoia, addictive behavior
and sleep disorders. Similarly, polypeptides and antibodies
directed to these polypeptides would be useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the brain, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., CNS, endocrine,
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, synovial fluid or cerebrospinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of the
immunogenic epitopes shown in SEQ ID NO: 206 as residues: Gly-45 to
Thr-50. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0629] The tissue distribution in brain indicates polynucleotides
and polypeptides corresponding to this gene would be useful for the
detection, treatment, and/or prevention of neurodegenerative
disease states, behavioral disorders, or inflammatory conditions.
Representative uses are described in the "Regeneration" and
"Hyperproliferative Disorders" sections below, in Example 11, 15,
and 18, and elsewhere herein. Briefly, the uses include, but are
not limited to the detection, treatment, and/or prevention of
Alzheimer's Disease, Parkinson's Disease, Huntington's Disease,
Tourette Syndrome, meningitis, encephalitis, demyelinating
diseases, peripheral neuropathies, neoplasia, trauma, congenital
malformations, spinal cord injuries, ischemia and infarction,
aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia,
obsessive compulsive disorder, depression, panic disorder, learning
disabilities, ALS, psychoses, autism, and altered behaviors,
including disorders in feeding, sleep patterns, balance, and
perception. In addition, elevated expression of this gene product
in regions of the brain indicates that polynucleotides and
polypeptides of the invention may play a role in normal neural
function. Potentially, polynucleotides and polypeptides of the
invention are involved in synapse formation, neurotransmission,
learning, cognition, homeostasis, or neuronal differentiation or
survival. Moreover, the expression within fetal tissue and other
cellular sources marked by proliferating cells indicates that
polynucleotides and polypeptides of the invention may play a role
in the regulation of cellular division, and may show utility in the
diagnosis, treatment, and/or prevention of developmental diseases
and disorders, including cancer, and other proliferative
conditions. Representative uses are described in the
"Hyperproliferative Disorders" and "Regeneration" sections below
and elsewhere herein. Briefly, developmental tissues rely on
decisions involving cell differentiation and/or apoptosis in
pattern formation. Dysregulation of apoptosis can result in
inappropriate suppression of cell death, as occurs in the
development of some cancers, or in failure to control the extent of
cell death, as is believed to occur in acquired immunodeficiency
and certain degenerative disorders, such as spinal muscular atrophy
(SMA). Alternatively, polynucleotides and polypeptides of the
invention may be involved in the pattern of cellular proliferation
that accompanies early embryogenesis. Thus, aberrant expression of
this gene product in tissues--particularly adult tissues--may
correlate with patterns of abnormal cellular proliferation, such as
found in various cancers. Because of potential roles in
proliferation and differentiation, polynucleotides and polypeptides
of the invention may have applications in the adult for tissue
regeneration and the treatment of cancers. It may also act as a
morphogen to control cell and tissue type specification. Therefore,
the polynucleotides and polypeptides of the present invention would
be useful in treating, detecting, and/or preventing said disorders
and conditions, in addition to other types of degenerative
conditions. Thus polynucleotides and polypeptides of the invention
may modulate apoptosis or tissue differentiation and would be
useful in the detection, treatment, and/or prevention of
degenerative or proliferative conditions and diseases.
Polynucleotides and polypeptides of the invention would be useful
in modulating the immune response to aberrant polypeptides, as may
exist in proliferating and cancerous cells and tissues. The protein
can also be used to gain new insight into the regulation of
cellular growth and proliferation. Furthermore, the protein may
also be used to determine biological activity, to raise antibodies,
as tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0630] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:99 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 601 of SEQ ID NO:99, b is an integer
of 15 to 615, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:99, and where b is greater
than or equal to a+14.
[0631] Features of Protein Encoded By Gene No: 90
[0632] The gene encoding the disclosed cDNA is believed to reside
on chromosome 3. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 3.
[0633] This gene is expressed primarily in brain tissue, kidney,
tonsils, bone marow, colon, testes, ovary tumor, and to a lesser
extent many other tissues.
[0634] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
neurological and behavioural disorders. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the central nervous system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g., CNS,
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, synovial fluid or cerebrospinal fluid) or
another tissue or cell sample taken from an individual having such
a disorder, relative to the standard gene expression level, i.e.,
the expression level in healthy tissue or bodily fluid from an
individual not having the disorder.
[0635] The tissue distribution in brain indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection, treatment, and/or prevention of
neurodegenerative disease states, behavioral disorders, or
inflammatory conditions. Representative uses are described in the
"Regeneration" and "Hyperproliferative Disorders" sections below,
in Example 11, 15, and 18, and elsewhere herein. Briefly, the uses
include, but are not limited to the detection, treatment, and/or
prevention of Alzheimer's Disease, Parkinson's Disease,
Huntington's Disease, Tourette Syndrome, meningitis, encephalitis,
demyelinating diseases, peripheral neuropathies, neoplasia, trauma,
congenital malformations, spinal cord injuries, ischemia and
infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia,
paranoia, obsessive compulsive disorder, depression, panic
disorder, learning disabilities, ALS, psychoses, autism, and
altered behaviors, including disorders in feeding, sleep patterns,
balance, and perception. In addition, elevated expression of this
gene product in regions of the brain indicates it plays a role in
normal neural function. Potentially, polynucleotides and
polypeptides of the invention are involved in synapse formation,
neurotransmission, learning, cognition, homeostasis, or neuronal
differentiation or survival. The tissue distribution in bone marrow
and other immune tissues indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
diagnosis and treatment of a variety of immune system disorders.
Representative uses are described in the "Immune Activity" and
"Infectious Disease" sections below, in Example 11, 13, 14, 16, 18,
19, 20, and 27, and elsewhere herein. Briefly, the expression
indicates a role in regulating the proliferation; survival;
differentiation; and/or activation of hematopoietic cell lineages,
including blood stem cells. Involvement in the regulation of
cytokine production, antigen presentation, or other processes
indicates a usefulness for treatment of cancer (e.g., by boosting
immune responses). Expression in cells of lymphoid origin,
indicates the natural gene product would be involved in immune
functions. Therefore polynucleotides and polypeptides of the
invention would also be useful as an agent for immunological
disorders including arthritis, asthma, immunodeficiency diseases
such as AIDS, leukemia, rheumatoid arthritis, granulomatous
disease, inflammatory bowel disease, sepsis, acne, neutropenia,
neutrophilia, psoriasis, hypersensitivities, such as T-cell
mediated cytotoxicity; immune reactions to transplanted organs and
tissues, such as host-versus-graft and graft-versus-host diseases,
or autoimmunity disorders, such as autoimmune infertility, lense
tissue injury, demyelination, systemic lupus erythematosis, drug
induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease,
and scleroderma. Moreover, the protein may represent a secreted
factor that influences the differentiation or behavior of other
blood cells, or that recruits hematopoietic cells to sites of
injury. Thus, this gene product is thought to be useful in the
expansion of stem cells and committed progenitors of various blood
lineages, and in the differentiation and/or proliferation of
various cell types. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0636] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:100 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1610 of SEQ ID NO:100, b is an
integer of 15 to 1624, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:100, and where
b is greater than or equal to a+14.
[0637] Features of Protein Encoded By Gene No: 91
[0638] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: PVIWPDGKRIVLLAEVS (SEQ ID NO: 447). Moreover, fragments
and variants of these polypeptides (such as, for example, fragments
as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0639] This gene is expressed primarily in adrenal gland tumor.
[0640] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
adrenal gland cancer. Similarly, polypeptides and antibodies
directed to these polypeptides would be useful in providing
immunological probes for differential identification of the
tissue(s) or cell type(s). For a number of disorders of the above
tissues or cells, particularly of the adrenal system, expression of
this gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., adrenal gland,
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of the
immunogenic epitopes shown in SEQ ID NO: 208 as residues: Arg-49 to
Gln-56. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0641] The tissue distribution in adrenal gland indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the diagnosis, detection, prevention and/or treatment
of disorders involving the adrenal gland. Expression of this gene
product in adrenal gland tumor indicates that polynucleotides and
polypeptides of the invention may play a role in the proliferation
of cells of the adrenal gland, or potentially in the proliferation
of cells in general. In such an event, it may play a role in
determining the course and severity of cancer. Alternatively,
polynucleotides and polypeptides of the invention may play a role
in the normal function of adrenal glands, such as in the production
of corticosteroids, androgens, or epinephrines. Thus
polynucleotides and polypeptides of the invention may play a role
in general homeostasis, as well as in disorders involving the
androgen hormones. Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify
agents that modulate their interactions, in addition to its use as
a nutritional supplement. Protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
[0642] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:101 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1742 of SEQ ID NO: 101, b is an
integer of 15 to 1756, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:101, and where
b is greater than or equal to a+14.
[0643] Features of Protein Encoded By Gene No: 92
[0644] The gene encoding the disclosed cDNA is thought to reside on
chromosome 2. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 2.
[0645] This gene is expressed in multiple tissues, including the
thymus, and cell types, including B cells and monocytes.
[0646] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
disorders and/or disorders afflicting the immune system, such as
AIDS and autoimmune diseases. Similarly, polypeptides and
antibodies directed to these polypeptides would be useful in
providing immunological probes for differential identification of
the tissue(s) or cell type(s). For a number of disorders of the
above tissues or cells, particularly of the immune system,
expression of this gene at significantly higher or lower levels may
be routinely detected in certain tissues or cell types (e.g.,
immune, cancerous and wounded tissues) or bodily fluids (e.g.,
lymph, serum, plasma, urine, synovial fluid and spinal fluid) taken
from an individual having such a disorder, relative to the standard
gene expression level, i.e., the expression level in healthy tissue
or bodily fluid from an individual not having the disorder.
[0647] The tissue distribution in immune system tissues and cells
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the diagnosis, detection, prevention
and/or treatment of disorders affecting the immune system,
especially autoimmune diseases and AIDS. Representative uses are
described in the "Immune Activity" and "Infectious Disease"
sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and
elsewhere herein. Briefly, polynucleotides and polypeptides of the
invention may be involved in the regulation of cytokine production,
antigen presentation, or other processes that may also suggest a
usefulness in the treatment of cancer (e.g., by boosting immune
responses). Since the gene is expressed in cells of lymphoid
origin, the gene or protein, as well as, antibodies directed
against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues. Therefore
polynucleotides and polypeptides of the invention may be also used
as an agent for immunological disorders including arthritis,
asthma, immune deficiency diseases such as AIDS, leukemia,
rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and
psoriasis. In addition, this gene product may have commercial
utility in the expansion of stem cells and committed progenitors of
various blood lineages, and in the differentiation and/or
proliferation of various cell types. Furthermore, the protein may
also be used to determine biological activity, to raise antibodies,
as tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0648] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:102 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1402 of SEQ ID NO:102, b is an
integer of 15 to 1416, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:102, and where
b is greater than or equal to a+14.
[0649] Features of Protein Encoded By Gene No: 93
[0650] The translated product of this gene shares some homology
with an X-linked retinopathy protein (see, e.g., Genbank Accession
No. AAB26149.1 and Wong, P., et al., Genomics 15(3):467-71 (1993);
all references available through this accession and citation are
hereby incorporated herein by reference).
[0651] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: FYYFWRQGGSCFVQTGVQWCDHGSLQL (SEQ ID NO:
448) and TPGRQSKTPS (SEQ ID NO: 449). Moreover, fragments and
variants of these polypeptides (such as, for example, fragments as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0652] The translated product of this gene also shares some
homology with a Human histiocyte-secreted factor (HSF) protein
(see, e.g., GenSeq Accession No. R96800; all references available
through this accession are hereby incorporated herein by
reference).
[0653] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: YFIIFGDREGLALFRLECSGVIMAHCNFELLGDR (SEQ ID NO: 450).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0654] This gene is expressed primarily in fetal lung tissue.
[0655] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to
ocular, immune, and lung diseases and/or disorders. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the eye
(especially retina), immune system, and lung, expression of this
gene at significantly higher or lower levels may be routinely
detected in certain tissues or cell types (e.g., retina, blood,
pulmonary, cancerous and wounded tissues) or bodily fluids (e.g.,
lymph, serum, plasma, urine, sputum, pulmonary surfactant, synovial
fluid and spinal fluid) or another tissue or cell sample taken from
an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively
consist of the immunogenic epitopes shown in SEQ ID NO: 210 as
residues: Leu-32 to His-38. Polynucleotides encoding said
polypeptides are encompassed by the invention, as are antibodies
that bind one or more of these peptides.
[0656] The tissue distribution in fetal lung tissue indicates that
polynucleotides and polypeptides corresponding to this gene would
be useful for the detection, diagnosis, prevention and/or treatment
of lung diseases and/or disorders. Representative uses are
described elsewhere herein. Furthermore, the tissue distribution
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the detection and treatment of
disorders associated with developing lungs, particularly in
premature infants where the lungs are the last tissues to develop.
The tissue distribution indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
diagnosis and intervention of lung tumors, since the gene may be
involved in the regulation of cell division, particularly since it
is expressed in fetal tissue. Furthermore, the protein may also be
used to determine biological activity, to raise antibodies, as
tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0657] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:103 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 690 of SEQ ID NO:103, b is an integer
of 15 to 704, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:103, and where b is greater
than or equal to a+14.
[0658] Features of Protein Encoded By Gene No: 94
[0659] The translated product of this gene shares some homology
with peripheral benzodiazepine receptor interacting protein (see
Genbank Accession No. AAD11957.1; all references available through
this accession are hereby incorporated herein by reference).
[0660] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence
selected from the group: TABLE-US-00009 (SEQ ID NO: 451)
CFLSVSFQWN, (SEQ ID NO: 452) VTIAQVGIFVCFVHCCT, (SEQ ID NO: 453)
PGQVPSKHLGSNASVRA, (SEQ ID NO: 454) DEGAKVQRRPWGSQTHSPVLFL, (SEQ ID
NO: 455) LTRPGLWGSLLPVQQQRG, (SEQ ID NO: 456) CASLGVLRANRSPCV, (SEQ
ID NO: 457) SWLEVTTLSAPGPVITTY, (SEQ ID NO: 458)
PGQWVREIXLVGRAVARV, (SEQ ID NO: 459) LTWPPXGPMGTVWPGF, (SEQ ID NO:
460) MADIPGTFLALGCHGQR, (SEQ ID NO: 461) VGRGSWASGWTNQSA, (SEQ ID
NO: 462) PDHPLPVGLLEAWRVE and/or (SEQ ID NO: 463)
WGSQTHSPVLFLLTRPGLWGSLLPVQQQRGCASLGVLRANRSPCVSWLEV
TTLSAPGPVITTYPGQWVREIXLVGRAVARVLTWPPXGPMGTVWPGFMAD
IPGTFLALGCHGQRVGRGSWASGWTNQ-SAFPAGPPDHPLPV.
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0661] This gene is expressed primarily neutrophils and
eosinophils, and, to a lesser extent, in bone marrow and fetal
liver/spleen tissue.
[0662] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
asthma and diseases and/or disorders afflicting the immune system.
Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the immune system, expression of this gene at significantly higher
or lower levels may be routinely detected in certain tissues or
cell types (e.g., immune, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) taken from an individual having such a disorder,
relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of the
immunogenic epitopes shown in SEQ ID NO: 211 as residues: Ser-2 to
Trp-7. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0663] The tissue distribution in immune system cells and tissues
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the diagnosis, detection, prevention
and/or treatment of asthma or other disorders affecting the immune
system. Representative uses are described in the "Immune Activity"
and "Infectious Disease" sections below, in Example 11, 13, 14, 16,
18, 19, 20, and 27, and elsewhere herein. Briefly, polynucleotides
and polypeptides of the invention may be involved in the regulation
of cytokine production, antigen presentation, or other processes
that may also suggest a usefulness in the treatment of cancer
(e.g., by boosting immune responses). Since the gene is expressed
in cells of lymphoid origin, the gene or protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed tissues.
Therefore polynucleotides and polypeptides of the invention may be
also used as an agent for immunological disorders including
arthritis, asthma, immune deficiency diseases such as AIDS,
leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,
acne, and psoriasis. In addition, polynucleotides and polypeptides
of the invention may have commercial utility in the expansion of
stem cells and committed progenitors of various blood lineages, and
in the differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0664] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:104 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1245 of SEQ ID NO:104, b is an
integer of 15 to 1259, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:104, and where
b is greater than or equal to a+14.
[0665] Features of Protein Encoded By Gene No: 95
[0666] This gene shares sequence homology to the rat cornichon-like
protein (see, e.g., Genbank Accession No. 2317276), the murine
cornichon protein (see, e.g., Genbank Accession No. gi|2460430),
and the human comichon protein (see, e.g., Genbank Accession No.
gi|4063709). All references available through these accessions are
hereby incorporated by reference herein.The Drosophila cornichon
gene is thought to be involved in signaling processes necessary for
both anterior-posterior and dorsal-ventral pattern formation in
Drosophila. Thus, it is likely that this gene plays a similar role
in human development.
[0667] The gene encoding the disclosed cDNA is thought to reside on
chromosome 1. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 1.
[0668] This gene is expressed primarily in endometrial tumor tissue
and infant brain tissue, and, to a lesser extent, in frontal cortex
tissue.
[0669] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
endometrial tumor, and neural and developmental diseases and/or
disorders. Similarly, polypeptides and antibodies directed to these
polypeptides would be useful in providing immunological probes for
differential identification of the tissue(s) or cell type(s). For a
number of disorders of the above tissues or cells, particularly of
the neural and reproductive organs, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., neural, reproductive,
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, amniotic fluid, synovial fluid and spinal
fluid) or another tissue or cell sample taken from an individual
having such a disorder, relative to the standard gene expression
level, i.e., the expression level in healthy tissue or bodily fluid
from an individual not having the disorder. Preferred polypeptides
of the present invention comprise, or alternatively consist of the
immunogenic epitopes shown in SEQ ID NO: 212 as residues: Glu-33 to
Phe-38. Polynucleotides encoding said polypeptides are encompassed
by the invention, as are antibodies that bind one or more of these
peptides.
[0670] The tissue distribution in infant brain tissue and frontal
cortex tissue, and the homology to cornichon proteins, indicates
that polynucleotides and polypeptides corresponding to this gene
would be useful for detecting, diagnosing, preventing and/or
treating neural and developmental disorders. The tissue
distribution indicates that polynucleotides and polypeptides
corresponding to this gene would be useful for the detection,
diagnosis, prevention and/or treatment of neurodegenerative disease
states and behavioural disorders such as Alzheimer's Disease,
Parkinson's Disease, Huntington's Disease, Tourette Syndrome,
schizophrenia, mania, dementia, paranoia, obsessive compulsive
disorder, panic disorder, learning disabilities, ALS, psychoses,
autism, and altered behaviors, including disorders in feeding,
sleep patterns, balance, and perception. In addition,
polynucleotides and polypeptides of the invention may also play a
role in the treatment and/or detection of developmental disorders
associated with the developing embryo, or sexually-linked
disorders. Representative uses are described in the "Regeneration"
and "Hyperproliferative Disorders" sections below, in Example 11,
15, and 18, and elsewhere herein. Briefly, the elevated expression
of this gene product within the frontal cortex of the brain
indicates that polynucleotides and polypeptides of the invention
may be involved in neuronal survival; synapse formation;
conductance; neural differentiation, etc. Such involvement may
impact many processes, such as learning and cognition.
Alternatively, the tissue distribution in endometrial tumor tissue
indicates that polynucleotides and polypeptides of the invention
would be useful for the detection and/or treatment of endometrial
tumors and/or reproductive disorders, as well as tumors of other
tissues where expression of this gene has been observed.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0671] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:105 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 1790 of SEQ ID NO:105, b is an
integer of 15 to 1804, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:105, and where
b is greater than or equal to a+14.
[0672] Features of Protein Encoded By Gene No: 96
[0673] The translation product of this gene shares significant
sequence homology with a protein which was recently sequenced by
another group, which was named paraplegin by this group (see, e.g.,
Genbank Accession No. g3273089).
[0674] The gene encoding the disclosed cDNA is thought to reside on
chromosome 16. Accordingly, polynucleotides related to this
invention would be useful as a marker in linkage analysis for
chromosome 16.
[0675] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: LARADPPGCRRRGWRPSSAELQLRLLTPTFEGINGLLLKQHLVQNPVRLWQL
LGGTFYFNTSRLKQKNKE KDKSKGKAPEEDEXERRRRERDDQ (SEQ ID NO: 464).
Moreover, fragments and variants of these polypeptides (such as,
for example, fragments as described herein, polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these
polypeptides, or polypeptides encoded by a polynucleotide which
hybridizes, under stringent conditions, to the polynucleotide
encoding these polypeptides) are encompassed by the invention.
Antibodies that bind polypeptides of the invention and
polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0676] When tested against Jurkat T-cell cell lines, supernatants
removed from cells containing this gene activated the GAS assay.
Thus, it is likely that this gene activates T-cells, and to a
lesser extent other immune cells, through the Jak-STAT signal
transduction pathway. The gamma activating sequence (GAS) is a
promoter element found upstream of many genes which are involved in
the Jak-STAT pathway. The Jak-STAT pathway is a large, signal
transduction pathway involved in the differentiation and
proliferation of cells. Therefore, activation of the Jak-STAT
pathway, reflected by the binding of the GAS element, can be used
to indicate proteins involved in the proliferation and
differentiation of cells.
[0677] This gene is expressed primarily in Jurkat T-cells,
Macrophage, T-Cell Lymphoma, tonsils, and salivary glands.
[0678] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
T-Cell lymphomas. Similarly, polypeptides and antibodies directed
to these polypeptides would be useful in providing immunological
probes for differential identification of the tissue(s) or cell
type(s). For a number of disorders of the above tissues or cells,
particularly of the immune system, expression of this gene at
significantly higher or lower levels may be routinely detected in
certain tissues or cell types (e.g., immune, hematopoietic, and
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or cell sample taken from an individual having such a
disorder, relative to the standard gene expression level, i.e., the
expression level in healthy tissue or bodily fluid from an
individual not having the disorder. Preferred polypeptides of the
present invention comprise, or alternatively consist of one, two,
three, four, five, six or all seven of the immunogenic epitopes
shown in SEQ ID NO: 213 as residues: Met-1 to Leu-6, Asp-84 to
Lys-89, Asp-124 to Gly-130, Ser-138 to Trp-143, His-145 to Ser-153,
Thr-170 to Pro-183, Trp-191 to Pro-198. Polynucleotides encoding
said polypeptides are encompassed by the invention, as are
antibodies that bind one or more of these peptides.
[0679] The tissue distribution in immune tissues and T-cells, in
conjunction with the detected GAS biological activity data,
indicates that polynucleotides and polypeptides corresponding to
this gene would be useful for the detection and/or treatment of
T-cell lymphomas. Representative uses are described in the "Immune
Activity" and "Infectious Disease" sections below, in Example 11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the
expression of this gene product in T cell lymphoma indicates that
polynucleotides and polypeptides of the invention may play a role
in the proliferation of the lymphoid cell lineages, and may be
involved in normal antigen recognition and activation of T cells
during the immune process. Furthermore, the protein may also be
used to determine biological activity, to raise antibodies, as
tissue markers, to isolate cognate ligands or receptors, to
identify agents that modulate their interactions, in addition to
its use as a nutritional supplement. Protein, as well as,
antibodies directed against the protein may show utility as a tumor
marker and/or immunotherapy targets for the above listed
tissues.
[0680] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:106 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 957 of SEQ ID NO:106, b is an integer
of 15 to 971, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:106, and where b is greater
than or equal to a+14.
[0681] Features of Protein Encoded By Gene No: 97
[0682] In specific embodiments, polypeptides of the invention
comprise, or alternatively consist of, the following amino acid
sequence: FLRFWCTCHVSS (SEQ ID NO: 465). Moreover, fragments and
variants of these polypeptides (such as, for example, fragments as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% identical to these polypeptides, or
polypeptides encoded by a polynucleotide which hybridizes, under
stringent conditions, to the polynucleotide encoding these
polypeptides) are encompassed by the invention. Antibodies that
bind polypeptides of the invention and polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0683] This gene is expressed primarily in bladder, dermal
endothelial cells, retina, and dendritic cells.
[0684] Polynucleotides and polypeptides of the invention would be
useful as reagents for differential identification of the tissue(s)
or cell type(s) present in a biological sample and for diagnosis of
diseases and conditions which include, but are not limited to,
diseases of the bladder, including bladder cancer. Similarly,
polypeptides and antibodies directed to these polypeptides would be
useful in providing immunological probes for differential
identification of the tissue(s) or cell type(s). For a number of
disorders of the above tissues or cells, particularly of the
urinary system, expression of this gene at significantly higher or
lower levels may be routinely detected in certain tissues or cell
types (e.g., bladder, cancerous and wounded tissues) or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and
spinal fluid) or another tissue or cell sample taken from an
individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or
bodily fluid from an individual not having the disorder.
[0685] The tissue distribution in bladder indicates that the
polynucleotides and polypeptides corresponding to this gene would
be useful for treatment, prevention, detection and/or diagnosis of
urinary tract disorders (e.g., cystitis, urinary tract calcui,
incontinance) and bladder tumors or cancers. The tissue
distribution in endothelial cells indicates that polynucleotides
and polypeptides corresponding to this gene would be useful for the
diagnosis, detection, prevention and/or treatment of disorders
involving the vasculature and/or dermal tissue. Elevated expression
of this gene product by endothelial cells indicates that it may
play vital roles in the regulation of endothelial cell function;
secretion; proliferation; or angiogenesis. Alternately, this may
represent a gene product expressed by the endothelium and
transported to distant sites of action on a variety of target
organs. Expression of this gene product by hematopoietic cells also
indicates involvement in the proliferation; survival; activation;
or differentiation of all blood cell lineages. The tissue
distribution in retina indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the
treatment, prevention, diagnosis and/or detection of eye disorders
including blindness, color blindness, impaired vision, short and
long sightedness, retinitis pigmentosa, retinitis proliferans, and
retinoblastoma, retinochoroiditis, retinopathy and retinoschisis.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0686] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:107 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where
a is any integer between 1 to 807 of SEQ ID NO:107, b is an integer
of 15 to 821, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:107, and where b is greater
than or equal to a+1. TABLE-US-00010 5'NT NT of ATCC SEQ 5'NT 3'NT
5'NT First SEQ AA AA First Last Deposit ID Total of of of AA of ID
of of AA of AA Gene cDNA Nr and NO: NT Clone Clone Start Signal NO:
Sig Sig Secreted of No. Clone ID Date Vector X Seq. Seq. Seq. Codon
Pep Y Pep Pep Portion ORF 1 HKABZ65 209683 pCMVSport 11 1191 1 1191
69 69 118 1 17 18 243 Mar. 20, 1998 2.0 2 KNGIC80 209683 Uni-ZAP XR
12 1251 1 1251 24 24 119 1 24 25 41 Mar. 20, 1998 3 HDPUG50 209745
pCMVSport 13 1734 1 1734 22 22 120 1 34 35 526 Apr. 7, 1998 3.0 4
HAEAB66 209745 pBluescript 14 1540 914 1537 105 105 121 1 30 31 354
Apr. 7, 1998 SK 5 HHEPF59 209746 pCMVSport 15 1558 1 1558 38 38 122
1 21 22 63 Apr. 7, 1998 3.0 6 HE9BK23 209683 Uni-ZAP XR 16 1636 1
1636 39 39 123 1 21 22 309 Mar. 20, 1998 7 HCYBI36 209683
pBluescript 17 1256 148 1256 235 235 124 1 23 24 211 Mar. 20, 1998
SK- 8 HSSDXS1 209683 Uni-ZAP XR 18 1143 1 1143 133 133 125 1 20 21
50 Mar. 20, 1998 9 HSDAJ46 209746 Uni-ZAP XR 19 1537 92 1537 299
299 126 1 18 19 262 Apr. 7, 1998 10 HRACG45 209745 pCMVSport 20
2672 222 2672 178 178 127 1 42 43 270 Apr. 7, 1998 3.0 11 HAPPW30
209683 Uni-ZAP XR 21 1508 14 1501 54 54 128 1 22 23 91 Mar. 20,
1998 12 HE2ESS1 209745 Uni-ZAP XR 22 1447 1 1447 77 77 129 1 14 15
222 Apr. 7, 1998 13 HAGGJ80 209745 Uni-ZAP XR 23 3886 1289 3886 251
251 130 1 56 57 760 Apr. 7, 1998 13 HAGGJ80 209745 Uni-ZAP XR 108
1576 1 1576 40 40 215 1 34 35 84 Apr. 7, 1998 14 HTXDW56 209746
Uni-ZAP XR 2 4 1583 1 1583 217 217 131 1 22 23 201 Apr. 7, 1998 15
HEEAG23 209745 Uni-ZAP XR 25 1669 25 1280 57 57 132 1 18 19 46 Apr.
7, 1998 16 HDPKI93 209745 pCMVSport 26 1053 1 1053 46 46 133 1 21
22 305 Apr. 7, 1998 3.0 17 HDLAC10 209745 pCMVSport 27 1477 1 1477
132 132 134 1 29 30 81 Apr. 7, 1998 2.0 18 HDPOH06 209745 pCMVSport
28 2504 1 2504 252 252 135 1 29 30 242 Apr. 7, 1998 3.0 19 HCE4G61
209745 Uni-ZAP XR 29 1866 1 1866 130 130 136 1 23 24 285 Apr. 7,
1998 19 HCE4G61 209745 Uni-ZAP XR 109 1779 1 1720 125 125 216 1 20
21 81 Apr. 7, 1998 20 HCWUI13 209745 ZAP Express 30 1501 1 1501 80
80 137 1 18 19 157 Apr. 7, 1998 21 HDPSP01 209745 pCMVSport 31 1752
1 1752 227 227 138 1 20 21 308 Apr. 7, 1998 3.0 22 HHPEN62 209746
Uni-ZAP XR 32 2152 141 2152 183 183 139 1 27 28 508 Apr. 7, 1998 23
HUKBT29 209746 Lambda ZAP 33 1757 56 1757 74 74 140 1 19 20 506
Apr. 7, 1998 II 24 HMAJR50 209683 Uni-ZAP XR 34 1466 32 1466 70 70
141 1 21 22 48 Mar. 20, 1998 25 HBIMB51 209683 pCMVSport 35 526 1
526 93 93 142 1 21 22 130 Mar. 20, 1998 3.0 26 HE8DX88 209683
Uni-ZAP XR 36 2412 1 2412 256 256 143 1 29 30 43 Mar. 20, 1998 27
HNGHT03 209746 Uni-ZAP XR 37 1274 65 1274 305 305 144 1 24 25 91
Apr. 7, 1998 28 HWABU17 209745 pCMVSport 38 1036 1 1036 202 202 145
1 18 19 266 Apr. 7, 1998 3.0 29 HDTAT90 209746 pCMVSport 39 1379 8
1379 78 78 146 1 26 27 434 Apr. 7, 1998 2.0 30 HHFGR93 209746
Uni-ZAP XR 40 1932 1 1836 130 130 147 1 29 30 236 Apr. 7, 1998 31
HOVCB25 209746 pSport1 41 1430 1 1430 150 150 148 1 18 19 99 Apr.
7, 1998 32 HSYAV66 209746 pCMVSport 42 1407 1 1407 186 186 149 1 28
29 69 Apr. 7, 1998 3.0 33 HFPCT29 209683 Uni-ZAP XR 43 950 1 950
268 268 150 1 26 27 61 Mar. 20, 1998 34 HAWAT25 209683 pBluescript
44 1004 56 1004 149 149 151 1 32 33 88 Mar. 20, 1998 SK- 35 HNHFR04
209683 Uni-ZAP XR 45 1681 1 1681 71 71 152 1 21 22 78 Mar. 20, 1998
36 HOSFT61 209683 Uni-ZAPXIR 46 1361 1 1361 210 210 153 1 21 22 123
Mar. 20, 1998 36 HOSFT61 209683 Uni-ZAP XR 110 1365 1 1365 211 211
217 1 21 22 90 Mar. 20, 1998 37 HBJIO81 209683 Uni-ZAP XR 47 1137 1
1137 220 220 154 1 23 24 68 Mar. 20, 1998 38 HADCLSS 209745 pSport1
48 2763 15 2763 60 60 155 1 29 30 43 Apr. 7, 1998 39 HAIBO81 209745
Uni-ZAP XR 49 1348 1 1348 250 250 156 1 18 19 63 Apr. 7, 1998 40
HBBBC37 209745 pCMVSport1 50 1264 1 1264 81 81 157 1 17 18 61 Apr.
7, 1998 41 HBIMX85 209745 Uni-ZAP XR 51 1660 39 1660 45 45 158 1 18
19 82 Apr. 7, 1998 42 HCEES66 209745 Uni-ZAP XR 52 1678 1 1678 178
178 159 1 39 40 46 Apr. 7, 1998 43 HCEMP62 209745 Uni-ZAP XR 53
1860 269 1726 352 352 160 1 30 31 187 Apr. 7, 1998 43 HCEMP62
209745 Uni-ZAP XR 111 1957 582 1823 19 19 218 1 33 34 335 Apr. 7,
1998 44 HE2FB90 209746 Uni-ZAP XR 54 1663 1 1663 205 205 161 1 27
28 113 Apr. 7, 1998 45 HTHDJ94 209746 Uni-ZAP XR 55 1632 20 1632 66
66 162 1 26 27 292 Apr. 7, 1998 46 HTOHJ89 209746 Uni-ZAP XR 56
2233 1 2233 42 42 163 1 17 18 86 Apr. 7, 1998 47 HUSHB62 209745
LambdaZAP 57 1963 1 1760 130 130 164 1 49 50 106 Apr. 7, 1998 II 48
HSXAG02 209683 Uni-ZAP XR 58 1267 411 1243 600 600 165 1 22 23 58
Mar. 20, 1998 49 HHTLH52 209683 ZAP Express 59 1295 1 1295 218 218
166 1 22 23 40 Mar. 20, 1998 50 HCFMS9S 209683 pSport1 60 915 1 915
123 123 167 1 22 23 65 Mar. 20, 1998 51 HOUCT90 209683 Uni-ZAP XR
61 1445 1 1445 74 74 168 1 30 31 46 Mar. 20, 1998 52 HCFLR78 209745
pSport1 62 1100 224 1100 475 475 169 1 16 17 140 Apr. 7, 1998 53
HTOHT18 209745 Uni-ZAP XR 63 1499 267 1499 433 433 170 1 24 25 53
Apr. 7, 1998 54 HKPMB11 209745 pBluescript 64 655 1 655 55 55 171 1
25 26 167 Apr. 7, 1998 54 HKPMB11 209745 pBluescript 112 1135 490
1135 350 350 219 1 30 31 229 Apr. 7, 1998 55 HNFHS38 209745 Uni-ZAP
XR 65 1450 1 1450 172 172 172 1 18 19 325 Apr. 7, 1998 55 HNFHS38
209745 Uni-ZAP XR 113 1446 1 1446 171 171 220 1 18 19 62 Apr. 7,
1998 56 HAIBU10 209745 Uni-ZAP XR 66 670 1 669 201 201 173 1 20 21
113 Apr. 7, 1998 57 HAPOK30 209745 Uni-ZAP XR 67 1692 1 1692 300
300 174 1 19 20 61 Apr. 7, 1998 58 HCEEM18 209745 Uni-ZAP XR 68 655
18 655 157 157 175 1 30 31 41 Apr. 7, 1998 59 HCWUA22 209745 ZAP
Express 69 1618 48 1618 233 233 176 1 33 34 42 Apr. 7, 1998 60
HDSAG91 209745 Uni-ZAP XR 70 1802 1 1802 156 156 177 1 23 24 47
Apr. 7, 1998 61 HNEDJ35 209746 Uni-ZAP XR 71 1292 1 1292 71 71 178
1 36 37 50 Apr. 7, 1998 62 H7TBA62 209745 PCRII 72 883 1 807 199
199 179 1 65 66 227 Apr. 7, 1998 62 H7TBA62 209745 PCRII 114 733 9
718 224 224 221 1 36 37 170 Apr. 7, 1998 63 HNGIO50 209746 Uni-ZAP
XR 73 785 1 785 132 132 180 1 27 28 44 Apr. 7, 1998 64 HMIAW81
209683 Uni-ZAP XR 74 2341 1 2215 229 229 181 1 17 18 46 Mar. 20,
1998 65 HMMCJ60 209683 pSport1 75 1882 1 1882 132 132 182 1 16 17
41 Mar. 20, 1998 66 HDPIO09 209745 pCMVSport 76 2892 17 2892 85 85
183 1 36 37 47 Apr. 7, 1998 3.0 67 HHFHH34 209745 Uni-ZAP XR 77
1673 1 1673 16 16 184 1 22 23 70 Apr. 7, 1998 68 HISCL83 209745
pSport1 78 1461 1 1461 259 259 185 1 21 22 41 Apr. 7, 1998 69
HTOAI70 209746 Uni-ZAP XR 79 1517 1 1517 190 190 186 1 19 20 92
Apr. 7, 1998 69 HTOAI70 209746 Uni-ZAP XR 115 1518 1 1518 190 190
222 1 19 20 42
Apr. 7, 1998 70 HSDER95 209683 Uni-ZAP XR 80 574 1 574 72 72 187 1
25 26 71 Mar. 20, 1998 71 HNECL25 209683 Uni-ZAP XR 81 1455 1 1455
322 322 188 1 32 33 66 Mar. 20, 1998 72 HNFGZ45 209683 Uni-ZAP XR
82 1640 1 1640 450 450 189 1 38 39 70 Mar. 20, 1998 73 HHGCU49
209745 Lambda ZAP 83 525 1 525 173 173 190 1 23 24 40 Apr. 7, 1998
II 74 HDPND68 209745 pCMVSport 84 837 1 837 154 154 191 1 17 18 66
Apr. 7, 1998 3.0 75 HETDT81 209746 Uni-ZAP XR 85 1574 1 1574 189
189 192 1 25 26 66 Apr. 7, 1998 76 HHLBA14 209746 pBluescript 86
1628 353 1627 546 546 193 1 24 25 48 Apr. 7, 1998 SK- 77 HLTBU43
209746 Uni-ZAP XR 87 1795 1 1795 198 198 194 1 19 20 66 Apr. 7,
1998 78 HNTSJ84 209746 pSport1 88 1864 239 1864 336 336 195 1 22 23
57 Apr. 7, 1998 79 HOHCG16 209746 pCMVSport 89 1983 1 1983 257 257
196 1 18 19 52 Apr. 7, 1998 2.0 80 HTHCB31 209746 Uni-ZAP XR 90
1957 1 1957 46 46 197 1 17 18 43 Apr. 7, 1998 81 HUKAM16 209746
Lambda ZAP 91 573 1 573 178 178 198 1 23 24 52 Apr. 7, 1998 II 82
HLDOJ66 209683 pCMVSport 92 1212 1 1212 313 313 199 1 20 21 40 Mar.
20, 1998 3.0 83 HTXKF10 209683 Uni-ZAP XR 93 1144 1 1144 334 334
200 1 32 33 71 Mar. 20, 1998 84 HPMAI22 209683 Uni-ZAP XR 94 1274
334 1274 483 483 201 1 16 17 59 Mar. 20, 1998 85 HL2AG57 209746
Uni-ZAP XR 95 1780 349 1780 560 560 202 1 31 32 80 Apr. 7, 1998 86
HTHBH29 209746 Uni-ZAP XR 96 1794 1223 1431 93 93 203 1 30 31 70
Apr. 7, 1998 86 HTHBH29 209746 Uni-ZAP XR 116 1054 1 1054 52 52 223
1 24 25 56 Apr. 7, 1998 87 HUSAM59 209683 Lambda ZAP 97 2065 1 2065
475 475 204 1 17 18 78 Mar. 20, 1998 II 88 HNGGR26 209745 Uni-ZAP
XR 98 1154 1 1154 50 50 205 1 27 28 115 Apr. 7, 1998 89 HTLCX30
209683 Uni-ZAP XR 99 615 1 459 60 60 206 1 28 29 50 Mar. 20, 1998
90 HCEBC87 209683 Uni-ZAP XR 100 1624 243 1624 517 517 207 1 23 24
57 Mar. 20, 1998 91 HATCB92 209683 Uni-ZAP XR 101 1756 1 1756 247
247 208 1 40 41 56 Mar. 20, 1998 92 HMSCX69 209746 Uni-ZAP XR 102
1416 207 1416 246 246 209 1 16 17 49 Apr. 7, 1998 93 HLHAL68 209746
Uni-ZAP XR 103 704 1 704 30 30 210 1 21 22 44 Apr. 7, 1998 94
HEOMR73 209746 pSport1 104 1259 644 1259 354 354 211 1 24 25 44
Apr. 7, 1998 95 HETIB83 209746 Uni-ZAP XR 105 1804 1 1804 104 104
212 1 30 31 160 Apr. 7, 1998 96 HJPDD28 209746 Uni-ZAP XR 106 971
260 971 283 283 213 1 21 22 198 Apr. 7, 1998 96 HJPDD28 209746
Uni-ZAP XR 117 921 1 921 31 31 224 1 21 22 96 Apr. 7, 1998 97
HBAMB15 209683 pSport1 107 821 330 821 390 390 214 1 19 20 59 Mar.
20, 1998
Table 1 summarizes the information corresponding to each "Gene No."
described above. The nucleotide sequence identified as "NT SEQ ID
NO:X" was assembled from partially homologous ("overlapping")
sequences obtained from the "cDNA clone ID" identified in Table 1
and, in some cases, from additional related DNA clones. The
overlapping sequences were assembled into a single contiguous
sequence of high redundancy (usually three to five overlapping
sequences at each nucleotide position), resulting in a final
sequence identified as SEQ ID NO:X.
[0687] The cDNA Clone ID was deposited on the date and given the
corresponding deposit number listed in "ATCC Deposit No:Z and
Date." Some of the deposits contain multiple different clones
corresponding to the same gene. "Vector" refers to the type of
vector contained in the cDNA Clone ID.
[0688] "Total NT Seq." refers to the total number of nucleotides in
the contig identified by "Gene No." The deposited clone may contain
all or most of these sequences, reflected by the nucleotide
position indicated as "5' NT of Clone Seq." and the "3' NT of Clone
Seq." of SEQ ID NO:X. The nucleotide position of SEQ ID NO:X of the
putative start codon (methionine) is identified as "5' NT of Start
Codon." Similarly, the nucleotide position of SEQ ID NO:X of the
predicted signal sequence is identified as "5' NT of First AA of
Signal Pep."
[0689] The translated amino acid sequence, beginning with the
methionine, is identified as "AA SEQ ID NO:Y," although other
reading frames can also be easily translated using known molecular
biology techniques. The polypeptides produced by these alternative
open reading frames are specifically contemplated by the present
invention.
[0690] The first and last amino acid position of SEQ ID NO:Y of the
predicted signal peptide is identified as "First AA of Sig Pep" and
"Last AA of Sig Pep." The predicted first amino acid position of
SEQ ID NO:Y of the secreted portion is identified as "Predicted
First AA of Secreted Portion." Finally, the amino acid position of
SEQ ID NO:Y of the last amino acid in the open reading frame is
identified as "Last AA of ORF."
[0691] SEQ ID NO:X (where X may be any of the polynucleotide
sequences disclosed in the sequence listing) and the translated SEQ
ID NO:Y (where Y may be any of the polypeptide sequences disclosed
in the sequence listing) are sufficiently accurate and otherwise
suitable for a variety of uses well known in the art and described
further below. For instance, SEQ ID NO:X is useful for designing
nucleic acid hybridization probes that will detect nucleic acid
sequences contained in SEQ ID NO:X or the cDNA contained in the
deposited clone. These probes will also hybridize to nucleic acid
molecules in biological samples, thereby enabling a variety of
forensic and diagnostic methods of the invention. Similarly,
polypeptides identified from SEQ ID NO:Y may be used, for example,
to generate antibodies which bind specifically to proteins
containing the polypeptides and the secreted proteins encoded by
the cDNA clones identified in Table 1.
[0692] Nevertheless, DNA sequences generated by sequencing
reactions can contain sequencing errors. The errors exist as
misidentified nucleotides, or as insertions or deletions of
nucleotides in the generated DNA sequence. The erroneously inserted
or deleted nucleotides cause frame shifts in the reading frames of
the predicted amino acid sequence. In these cases, the predicted
amino acid sequence diverges from the actual amino acid sequence,
even though the generated DNA sequence may be greater than 99.9%
identical to the actual DNA sequence (for example, one base
insertion or deletion in an open reading frame of over 1000
bases).
[0693] Accordingly, for those applications requiring precision in
the nucleotide sequence or the amino acid sequence, the present
invention provides not only the generated nucleotide sequence
identified as SEQ ID NO:X and the predicted translated amino acid
sequence identified as SEQ ID NO:Y, but also a sample of plasmid
DNA containing a human cDNA of the invention deposited with the
ATCC, as set forth in Table 1. The nucleotide sequence of each
deposited clone can readily be determined by sequencing the
deposited clone in accordance with known methods. The predicted
amino acid sequence can then be verified from such deposits.
Moreover, the amino acid sequence of the protein encoded by a
particular clone can also be directly determined by peptide
sequencing or by expressing the protein in a suitable host cell
containing the deposited human cDNA, collecting the protein, and
determining its sequence.
[0694] The present invention also relates to the genes
corresponding to SEQ ID NO:X, SEQ ID NO:Y, or the deposited clone.
The corresponding gene can be isolated in accordance with known
methods using the sequence information disclosed herein. Such
methods include preparing probes or primers from the disclosed
sequence and identifying or amplifying the corresponding gene from
appropriate sources of genomic material.
[0695] Also provided in the present invention are allelic variants,
orthologs, and/or species homologs. Procedures known in the art can
be used to obtain full-length genes, allelic variants, splice
variants, full-length coding portions, orthologs, and/or species
homologs of genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or a
deposited clone, using information from the sequences disclosed
herein or the clones deposited with the ATCC. For example, allelic
variants and/or species homologs may be isolated and identified by
making suitable probes or primers from the sequences provided
herein and screening a suitable nucleic acid source for allelic
variants and/or the desired homologue.
[0696] Table 2 summarizes the expression profile of polynucleotides
corresponding to the clones disclosed in Table 1. The first column
provides a unique clone identifier, "Clone ID", for a cDNA clone
related to each contig sequence disclosed in Table 1. Column 2,
"Library Code" shows the expression profile of tissue and/or cell
line libraries which express the polynucleotides of the invention.
Each Library Code in column 2 represents a tissue/cell source
identifier code corresponding to the Library Code and Library
description provided in Table 4. Expression of these
polynucleotides was not observed in the other tissues and/or cell
libraries tested. One of skill in the art could routinely use this
information to identify tissues which show a predominant expression
pattern of the corresponding polynucleotide of the invention or to
identify polynucleotides which show predominant and/or specific
tissue expression.
[0697] Table 3, column 1, provides a nucleotide sequence
identifier, "SEQ ID NO:X," that matches a nucleotide SEQ ID NO:X
disclosed in Table 1, column 5. Table 3, column 2, provides the
chromosomal location, "Cytologic Band or Chromosome," of
polynucleotides corresponding to SEQ ID NO:X. Chromosomal location
was determined by finding exact matches to EST and cDNA sequences
contained in the NCBI (National Center for Biotechnology
Information) UniGene database. Given a presumptive chromosomal
location, disease locus association was determined by comparison
with the Morbid Map, derived from Online Mendelian Inheritance in
Man (Online Mendelian Inheritance in Man, OMIM.TM..
McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins
University (Baltimore, Md.) and National Center for Biotechnology
Information, National Library of Medicine (Bethesda, Md.) 2000.
World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/). If the
putative chromosomal location of the Query overlapped with the
chromosomal location of a Morbid Map entry, the OMIM reference
identification number of the morbid map entry is provided in Table
3, column 3, labelled "OMIM ID." A key to the OMIM reference
identification numbers is provided in Table 5.
[0698] Table 4 provides a key to the Library Code disclosed in
Table 2. Column 1 provides the Library Code disclosed in Table 2,
column 2. Column 2 provides a description of the tissue or cell
source from which the corresponding library was derived.
[0699] Table 5 provides a key to the OMIM reference identification
numbers disclosed in Table 3, column 3. OMIM reference
identification numbers (Column 1) were derived from Online
Mendelian Inheritance in Man (Online Mendelian Inheritance in Man,
OMIM. McKusick-Nathans Institute for Genetic Medicine, Johns
Hopkins University (Baltimore, Md.) and National Center for
Biotechnology Information, National Library of Medicine, (Bethesda,
Md.) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/).
Column 2 provides diseases associated with the cytologic band
disclosed in Table 3, column 2, as determined using the Morbid Map
database. TABLE-US-00011 TABLE 2 Clone ID Library Codes HKABZ65
H0494 HNGIC80 S0052 HDPUG50 H0013 H0038 H0046 H0083 H0144 H0212
H0438 H0457 H0488 H0494 H0497 H0521 H0543 H0545 H0580 H0581 H0583
H0591 H0597 H0599 H0616 H0627 H0659 H0661 H0665 H0672 H0673 H0674
H0682 H0685 L0055 L0163 L0362 L0517 L0545 L0659 L0662 L0740 L0747
L0748 L0758 L0759 L0763 L0766 L0767 L0770 L0771 L0776 L0777 L0779
L0782 S0010 S0026 S0142 S0214 S0344 S0360 S0390 S0420 S0434 HAEAB66
H0266 H0494 H0646 H0676 L0383 L0517 L0596 L0659 L0662 L0747 L0748
L0749 L0750 L0752 L0755 L0758 L0761 L0764 L0771 L0774 L0777 L0783
L0789 L0792 L0800 L0803 L0804 L0806 L0809 S0116 S0356 S0358 S0402
T0048 T0109 HHEPF59 H0038 H0063 H0254 H0255 H0264 H0318 H0333 H0389
H0392 H0413 H0422 H0428 H0445 H0449 H0483 H0521 H0542 H0543 H0556
H0583 H0606 H0615 H0648 H0664 H0673 H0702 L0157 L0382 L0439 L0447
L0471 L0595 L0646 L0650 L0655 L0659 L0662 L0665 L0666 L0748 L0756
L0761 L0764 L0766 L0768 L0769 L0779 L0782 L0789 L0791 L0803 L0809
S0027 S0028 S0049 S0212 S0418 HE9BK23 H0014 H0098 H0144 H0355 H0393
H0509 H0510 H0574 H0632 L0581 L0748 L0775 L0790 L0803 L0804 HCYBI36
H0014 H0031 H0123 H0156 H0170 H0171 H0188 H0264 H0295 H0341 H0428
H0431 H0435 H0445 H0479 H0494 H0509 H0520 H0521 H0529 H0530 H0543
H0547 H0551 H0574 H0575 H0586 H0587 H0592 H0596 H0620 H0633 H0638
H0648 H0658 H0661 H0670 H0672 H0674 H0684 H0690 L0021 L0157 L0362
L0448 L0451 L0483 L0525 L0589 L0602 L0637 L0646 L0648 L0649 L0653
L0655 L0657 L0662 L0664 L0665 L0717 L0731 L0740 L0747 L0748 L0749
L0752 L0754 L0755 L0758 L0759 L0761 L0763 L0764 L0766 L0770 L0774
L0775 L0776 L0777 L0779 L0780 L0803 L0804 L0806 L0809 S0003 S0014
S0052 S0122 S0132 S0194 S0212 S0242 S0352 S0358 S0374 S0378 S0388
S0422 S0450 S3014 T0002 T0010 T0023 T0040 T0114 HSSDX51 H0050 H0052
H0069 H0135 H0391 H0575 H0652 H0690 L0021 L0438 L0439 L0554 L0599
L0653 L0665 L0717 L0774 L0775 S0038 S0049 S0222 S0312 S0334 S0338
T0006 T0082 HSDAJ46 H0009 H0052 H0144 H0352 H0392 L0593 L0595 L0598
L0608 L0740 L0741 L0745 L0746 L0748 L0749 L0759 L0769 L0770 L0777
L0783 L0809 S0031 HRACG45 H0009 H0030 H0036 H0059 H0555 L0599 S0358
HAPPW30 H0009 H0012 H0038 H0052 H0103 H0135 H0169 H0188 H0208 H0213
H0266 H0292 H0388 H0412 H0424 H0521 H0538 H0539 H0545 H0547 H0575
H0616 H0653 H0663 H0672 L0163 L0591 L0599 L0638 L0665 L0731 L0742
L0747 L0748 L0752 L0753 L0755 L0757 L0758 L0759 L0764 L0767 L0769
L0770 L0772 L0774 L0775 L0776 L0777 L0779 L0786 L0809 S0010 S0027
S0045 S0049 S0392 S0474 T0040 T0041 T0042 HE2ES51 H0015 H0038 H0170
H0356 H0622 L0774 L0803 S0015 S0438 HAGGJ80 H0040 H0144 H0327 H0422
H0427 H0539 H0542 H0547 H0551 H0561 H0581 H0648 H0658 H0659 H0672
H0684 L0157 L0352 L0362 L0438 L0471 L0519 L0591 L0659 L0662 L0663
L0665 L0731 L0756 L0758 L0759 L0764 L0766 L0774 L0775 L0777 L0779
L0783 S0003 S0028 S0036 S0051 S0150 S0152 S0342 S0346 S0358 S0360
S0374 HTXDW56 H0009 H0024 H0031 H0038 H0039 H0040 H0042 H0046 H0051
H0061 H0069 H0083 H0100 H0123 H0144 H0156 H0208 H0251 H0264 H0265
H0266 H0271 H0295 H0327 H0351 H0370 H0393 H0427 H0431 H0435 H0436
H0457 H0484 H0485 H0494 H0519 H0521 H0522 H0529 H0542 H0543 H0545
H0547 H0551 H0556 H0561 H0580 H0581 H0586 H0616 H0617 H0622 H0624
H0635 H0642 H0644 H0656 H0658 H0660 H0661 H0667 H0687 H0688 H0696
L0021 L0040 L0373 L0439 L0515 L0565 L0591 L0595 L0596 L0598 L0605
L0626 L0636 L0637 L0638 L0653 L0655 L0659 L0662 L0663 L0664 L0665
L0666 L0731 L0740 L0742 L0744 L0745 L0747 L0748 L0749 L0750 L0751
L0752 L0754 L0755 L0756 L0757 L0758 L0759 L0761 L0763 L0764 L0766
L0770 L0771 L0776 L0789 L0794 L0803 L0804 L0805 L0806 L0809 S0002
S0003 S0010 S0026 S0027 S0040 S0042 S0044 S0045 S0114 S0116 S0132
S0134 S0192 S0212 S0278 S0316 S0328 S0330 S0356 S0358 S0360 S0374
S0376 S0378 S0380 S0412 S0414 S0426 S0462 S0474 T0082 HEEAG23 H0038
H0052 H0123 H0144 H0194 H0255 H0286 H0328 H0375 H0436 H0484 H0521
H0542 H0549 H0556 H0624 L0748 L0789 S0027 S0030 S0126 S0196 S0222
S0278 S0300 S0358 S0420 HDPKI93 H0024 H0039 H0052 H0059 H0087 H0135
H0144 H0255 H0264 H0265 H0295 H0341 H0393 H0478 H0494 H0510 H0521
H0522 H0539 H0543 H0549 H0574 H0597 H0598 H0616 H0677 L0565 L0588
L0596 L0665 L0738 L0743 L0747 L0749 L0751 L0769 S0126 S0146 S0206
S0210 S0356 S0360 HDLAC10 H0031 H0170 H0320 H0373 H0422 H0445 H0485
H0494 H0519 H0539 H0543 H0550 H0555 H0581 H0586 H0650 H0657 H0658
H0672 H0690 L0374 L0438 L0599 L0606 L0635 L0638 L0655 L0665 L0666
L0667 L0743 L0745 L0759 L0761 L0764 L0766 L0777 L0779 L0803 L0804
S0134 S0212 S0218 S0358 S0360 T0067 HDPOH06 H0046 H0087 H0318 H0431
H0521 H0522 L0599 L0608 L0662 L0663 L0666 L0731 L0748 L0749 L0774
L0775 L0777 L0783 L0803 S0318 S0344 HCWUI13 H0589 HDPSP01 H0052
H0059 H0100 H0123 H0135 H0370 H0392 H0427 H0478 H0494 H0521 H0545
H0550 H0551 H0555 H0586 H0617 H0618 H0620 H0684 L0665 L0666 L0731
L0743 L0745 L0747 L0750 L0751 L0752 L0755 L0759 L0764 L0769 L0771
L0774 L0775 L0777 L0780 L0783 L0792 L0804 L0805 L0806 L0809 S0051
S0132 S0314 S0328 S0418 S3014 HHPEN62 H0046 H0051 H0052 H0100 H0261
H0305 H0327 H0438 L0635 L0741 L0769 L0770 L0803 S0010 S0036 S0051
S0112 S0260 S0282 S0346 HUKBT29 H0002 H0051 H0059 H0116 H0149 H0255
H0522 H0543 H0555 H0599 L0366 L0460 L0485 L0604 L0747 L0777 L0803
S0330 S0364 S0366 S0428 S0430 S0446 HMAJR50 H0013 H0014 H0031 H0032
H0038 H0040 H0046 H0051 H0052 H0056 H0059 H0069 H0090 H0123 H0130
H0134 H0144 H0170 H0250 H0267 H0316 H0327 H0328 H0341 H0357 H0402
H0412 H0416 H0421 H0423 H0436 H0441 H0445 H0497 H0519 H0520 H0521
H0529 H0542 H0543 H0546 H0547 H0549 H0551 H0553 H0556 H0560 H0574
H0587 H0598 H0615 H0619 H0623 H0625 H0632 H0638 H0640 H0641 H0644
H0650 H0657 H0695 L0387 L0438 L0439 L0471 L0586 L0588 L0593 L0598
L0607 L0637 L0642 L0646 L0648 L0655 L0659 L0662 L0663 L0664 L0665
L0666 L0667 L0731 L0738 L0740 L0747 L0748 L0750 L0752 L0754 L0755
L0756 L0757 L0758 L0759 L0767 L0770 L0771 L0775 L0776 L0779 L0806
S0003 S0013 S0026 S0028 S0036 S0053 S0116 S0126 S0132 S0144 S0152
S0196 S0210 S0222 S0260 S0278 S0348 S0352 S0354 S0356 S0358 S0360
S0374 S0378 S0380 S0418 S0452 S0474 T0006 T0082 HBIMB51 H0593 S0152
HE8DX88 H0013 HNGHT03 S0052 HWABU17 H0024 H0052 H0208 H0422 H0457
H0581 H0624 S0002 S0031 S0344 S0360 S0364 T0041 HDTAT90 H0052 H0224
H0252 H0280 H0486 H0539 H0592 H0616 S0045 S0150 HHFGR93 H0024 H0030
H0040 H0042 H0046 H0050 H0051 H0056 H0124 H0144 H0265 H0305 H0328
H0361 H0413 H0422 H0427 H0441 H0485 H0506 H0519 H0543 H0553 H0555
H0556 H0569 H0575 H0586 H0599 H0616 H0619 H0644 L0363 L0471 L0599
L0603 L0605 L0644 L0659 L0662 L0665 L0666 L0731 L0747 L0748 L0749
L0750 L0751 L0754 L0755 L0764 L0769 L0770 L0775 L0779 L0783 L0794
L0800 L0803 L0804 L0806 S0038 S0045 S0046 S0146 S0280 S0358 S3012
HOVCB25 H0428 HSYAV66 H0036 H0551 HFPCT29 S0222 HAWAT25 H0100 H0135
H0171 H0263 H0670 L0774 L0803 S0216 T0060 HNHFR04 S0053 S0428
HOSFT61 H0170 H0328 H0331 H0428 H0519 H0521 H0529 H0542 H0546 H0576
H0583 H0587 H0601 H0615 H0624 H0658 H0660 H0683 L0367 L0438 L0439
L0471 L0731 L0754 L0759 L0791 S0003 S0026 S0114 S0194 S0212 S0214
S0222 S0420 T0041 HBJIO81 H0318 L0766 HADCL55 H0013 H0031 H0038
H0144 H0253 H0266 H0310 H0424 H0427 H0497 H0519 H0521 H0522 H0539
H0545 H0549 H0553 H0555 H0581 H0591 H0599 H0618 H0633 H0661 H0664
L0021 L0142 L0438 L0439 L0649 L0659 L0662 L0664 L0665 L0740 L0745
L0747 L0748 L0758 L0759 L0769 L0779 L0790 S0010 S0126 S0144 S0218
S0360 S0390 S0418 S0422 S0426 S0452 S3014 HAIBO81 S0001 S0132
HBBBC37 H0013 H0014 H0038 H0069 H0096 H0100 H0201 H0264 H0374 H0486
H0494 H0543 H0551 H0587 H0687 L0021 L0105 L0369 L0438 L0439 L0471
L0485 L0591 L0598 L0599 L0659 L0717 L0740 L0743 L0748 L0749 L0751
L0752 L0755 L0756 L0758 L0768 L0769 L0770 L0771 L0774 L0775 L0776
L0777 L0779 L0792 L0794 L0803 L0804 L0805 L0806 S0001 S0003 S0122
S0222 S0260 S0330 S0346 S0388 S0468 T0023 T0039 T0042 HBJMX85 H0254
H0255 H0306 H0318 H0327 H0402 H0421 H0436 H0445 H0457 H0486 H0506
H0543 H0555 H0556 H0583 S0007 S0114 S0140 S0218 S0348 S0358 HCEES66
H0052 L0753 L0756 HCEMP62 H0024 H0030 H0040 H0041 H0046 H0052 H0063
H0123 H0135 H0165 H0179 H0181 H0188 H0208 H0264 H0266 H0286 H0290
H0318 H0370 H0402 H0411 H0428 H0436 H0445 H0484 H0489 H0506 H0509
H0521 H0522 H0543 H0547 H0551 H0553 H0556 H0561 H0575 H0581 H0583
H0586 H0587 H0593 H0596 H0600 H0617 H0620 H0622 H0667 H0668 H0672
H0702 H0707 L0372 L0517 L0521 L0565 L0599 L0637 L0657 L0662 L0663
L0664 L0665 L0666 L0717 L0731 L0744 L0747 L0748 L0749 L0751 L0754
L0757 L0759 L0761 L0763 L0764 L0766 L0768 L0769 L0770 L0776 L0777
L0803 S0001 S0002 S0037 S0044 S0049 S0150 S0212 S0216 S0250 S0278
S0354 S0358 S0360 S0364 S0380 S0426 S0446 S0458 S3012 T0039 HE2FB90
H0012 H0050 H0130 H0171 H0318 H0333 H0428 H0539 H0549 H0571 H0624
H0662 L0439 L0639 L0665 L0750 L0755 L0756 L0764 L0769 L0772 L0792
L0794 S0046 HTHDJ94 H0009 H0013 H0039 H0042 H0046 H0052 H0063 H0123
H0124 H0135 H0144 H0150 H0156 H0163 H0170 H0200 H0264 H0295 H0423
H0445 H0486 H0494 H0519 H0520 H0521 H0543 H0544 H0545 H0553 H0556
H0561 H0575 H0581 H0593 H0599 H0600 H0606 H0644 H0645 H0652 H0658
H0662 H0673 H0674 L0005 L0055 L0143 L0369 L0438 L0439 L0485 L0519
L0520 L0526 L0536 L0549 L0637 L0659 L0731 L0740 L0748 L0750 L0752
L0753 L0755 L0757 L0758 L0759 L0763 L0764 L0766 L0768 L0770 L0774
L0776 L0777 L0779 L0783 L0803 L0806 L0809 S0002 S0010 S0027 S0032
S0132 S0358 S0364 S0434 S0466 S0474 S3012 HTOHJ89 H0264 HUSHB62
H0012 H0013 H0030 H0031 H0032 H0038 H0039 H0044 H0046 H0052 H0056
H0059 H0070 H0083 H0090 H0100 H0122 H0123 H0124 H0134 H0135 H0144
H0150 H0156 H0194 H0201 H0220 H0231 H0253 H0255 H0261 H0264 H0266
H0271 H0306 H0351 H0352 H0356 H0370 H0375 H0393 H0402 H0412 H0413
H0422 H0423 H0424 H0427 H0429 H0431 H0435 H0436 H0437 H0438 H0441
H0445 H0478 H0483 H0484 H0486 H0494 H0506 H0510 H0518 H0521 H0529
H0539 H0542 H0543 H0545 H0549 H0551 H0553 H0555 H0556 H0561 H0575
H0580 H0581 H0583 H0586 H0587 H0591 H0593 H0599 H0615 H0616 H0617
H0618 H0620 H0622 H0623 H0626 H0634 H0635 H0641 H0644 H0646 H0650
H0656 H0657 H0661 H0662 H0664 H0665 H0670 H0672 H0673 H0679 H0682
H0685 H0687 H0691 H0696 H0702 H0707 L0041 L0142 L0143 L0157 L0352
L0362 L0372 L0375 L0378 L0388 L0438 L0439 L0493 L0498 L0511 L0515
L0517 L0518 L0529 L0540 L0553 L0560 L0564 L0596 L0599 L0600 L0603
L0608 L0612 L0635 L0638 L0641 L0644 L0645 L0646 L0650 L0651 L0656
L0657 L0658 L0659 L0662 L0663 L0664 L0665 L0666 L0667 L0697 L0731
L0740 L0741 L0742 L0743 L0744 L0745 L0747 L0748 L0749 L0750 L0751
L0752 L0754 L0758 L0759 L0761 L0762 L0763 L0766 L0767 L0768 L0769
L0770 L0771 L0774 L0775 L0777 L0779 L0783 L0786 L0789 L0791 L0794
L0796 L0803 L0804 L0806 L0809 S0007 S0010 S0011 S0027 S0028 S0031
S0032 S0037 S0038 S0040 S0045 S0046 S0049 S0053 S0116 S0126 S0132
S0140 S0144 S0192 S0194 S0212 S0222 S0260 S0278 S0280 S0282 S0350
S0354 S0356 S0358 S0360 S0376 S0378 S0384 S0390 S0418 S0426 S0428
S3012 S3014 S6024 T0002 T0049 T0067 HSXAG02 H0013 H0014 H0024 H0031
H0032 H0038 H0039 H0040 H0046 H0050 H0051 H0052 H0056 H0057 H0081
H0085 H0086 H0087 H0100 H0105 H0116 H0123 H0124 H0135 H0144 H0150
H0163 H0171 H0178 H0181 H0188 H0196 H0208 H0242 H0251 H0252 H0253
H0264 H0266 H0268 H0269 H0274 H0284 H0286 H0290 H0292 H0294 H0309
H0316 H0318 H0333 H0343 H0352 H0381 H0392 H0411 H0412 H0413 H0427
H0428 H0437 H0484 H0485 H0486 H0506 H0519 H0520 H0539 H0544 H0545
H0546 H0547 H0549 H0550 H0551 H0553 H0575 H0586 H0587 H0590 H0592
H0594 H0597 H0598 H0599 H0600 H0602 H0617 H0619 H0620 H0622 H0623
H0624 H0626 H0628 H0631 H0647 H0648 H0653 H0659 H0660 H0664 H0665
H0667 H0673 H0677 H0684 H0687 H0688 H0689 H0690 H0691 H0696 L0005
L0021 L0053 L0361 L0364 L0372 L0375 L0378 L0384 L0426 L0438 L0439
L0471 L0493 L0517 L0521 L0523 L0542 L0565 L0588 L0592 L0596 L0597
L0598 L0629 L0637 L0645 L0646 L0648 L0649 L0651 L0653 L0654 L0656
L0657 L0659 L0662 L0663 L0664 L0665 L0666 L0717 L0731 L0740 L0742
L0743 L0744 L0747 L0748 L0749 L0750 L0751 L0754 L0755 L0757 L0758
L0759 L0762 L0763 L0764 L0768 L0769 L0770 L0771 L0772 L0774 L0775
L0776 L0777 L0779 L0780 L0783 L0796 L0800 L0803 L0806 L0807 S0001
S0011 S0022 S0026 S0027 S0028 S0036 S0037 S0038 S0040 S0044 S0045
S0046 S0116 S0126 S0192 S0194 S0196 S0208 S0210 S0212 S0242 S0250
S0294 S0328 S0330 S0332 S0342 S0352 S0354 S0356 S0358 S0360 S0364
S0374 S0376 S0388 S0418 S0420 S0432 S0446 S3012 S3014 T0003 T0004
T0040 T0049 HHTLH52 H0615 S6014 HCFMS95 H0061 H0068 H0090 H0170
H0255 H0265 H0266 H0309 H0413 H0423 H0457 H0486 H0494 H0521 H0539
H0549 H0551 H0575 H0581 H0618 H0637 H0638 H0648 H0657 H0658 H0659
H0670 H0682 H0689 L0055 L0363 L0369 L0438 L0439 L0593 L0601 L0638
L0645 L0651 L0655 L0657 L0659 L0663 L0664 L0666 L0731 L0740 L0743
L0744 L0746 L0748 L0749 L0751 L0752 L0754 L0758 L0761 L0764 L0767
L0768 L0769 L0771 L0774 L0775 L0776 L0779 L0803 L0806 L0809 S0002
S0045 S0134 S0142 S0196 S0250 S0354 S0358 S0360 S0376 S0378 S0426
T0008 T0049 T0060 HOUCT90 S0040 HCFLR78 H0009 H0013 H0032 H0038
H0039 H0040 H0042 H0046 H0050 H0051 H0068 H0083 H0090 H0100 H0144
H0169 H0170 H0196 H0250 H0252 H0265 H0284 H0286 H0294 H0327 H0331
H0333 H0341 H0351 H0355
H0400 H0403 H0423 H0428 H0458 H0509 H0510 H0521 H0539 H0542 H0543
H0547 H0556 H0560 H0561 H0574 H0575 H0581 H0593 H0596 H0616 H0617
H0619 H0622 H0623 H0624 H0644 H0645 H0656 H0658 H0690 L0362 L0366
L0369 L0411 L0438 L0439 L0530 L0595 L0599 L0606 L0651 L0654 L0657
L0659 L0662 L0663 L0666 L0731 L0740 L0747 L0748 L0749 L0750 L0752
L0754 L0755 L0757 L0758 L0759 L0763 L0764 L0766 L0769 L0770 L0771
L0772 L0774 L0775 L0776 L0777 L0791 L0792 L0796 L0809 S0003 S0007
S0010 S0013 S0026 S0027 S0028 S0038 S0040 S0044 S0046 S0051 S0126
S0134 S0142 S0144 S0152 S0192 S0194 S0212 S0222 S0242 S0250 S0278
S0330 S0342 S0344 S0354 S0358 S0360 S0366 S0374 S0376 S0380 S0392
S0420 S0462 T0006 T0048 T0110 HTOHT18 H0013 H0014 H0038 H0081 H0090
H0144 H0251 H0252 H0264 H0265 H0290 H0318 H0328 H0352 H0370 H0413
H0435 H0484 H0494 H0497 H0521 H0522 H0543 H0545 H0574 H0581 H0597
H0616 H0619 H0624 H0657 H0665 H0667 H0668 L0363 L0364 L0375 L0439
L0588 L0601 L0664 L0666 L0717 L0747 L0748 L0749 L0750 L0758 L0762
L0764 L0766 L0769 L0771 L0776 L0777 L0779 L0794 L0800 L0804 L0805
L0806 S0045 S0050 S0140 S0210 S0354 S0358 S0420 T0002 T0042 T0049
HKPMB11 H0453 H0575 L0803 S0126 S0210 HNFHS38 H0013 H0271 S0152
S0342 HAIBU10 H0087 H0135 H0166 H0171 H0188 H0213 H0252 H0263 H0333
H0343 H0427 H0457 H0545 H0556 H0580 H0587 H0594 H0624 H0634 H0660
H0666 H0674 H0689 L0021 L0471 L0615 L0637 L0644 L0653 L0659 L0663
L0665 L0717 L0731 L0743 L0748 L0750 L0753 L0754 L0757 L0758 L0759
L0761 L0762 L0763 L0764 L0766 L0769 L0770 L0775 L0776 L0779 L0790
L0791 L0794 L0800 L0803 L0804 L0805 L0809 S0013 S0116 S0132 S0134
S0144 S0354 S0358 S0450 HAPOK30 H0575 H0592 H0670 L0352 L0439 L0517
L0600 L0608 L0663 L0740 L0747 L0752 L0755 L0756 L0759 L0763 L0764
L0766 L0768 L0770 L0777 L0785 L0794 L0803 L0809 S0010 S0222 S0328
HCEEM18 H0012 H0014 H0023 H0024 H0031 H0036 H0051 H0052 H0069 H0081
H0111 H0123 H0124 H0179 H0253 H0266 H0271 H0294 H0305 H0309 H0327
H0333 H0341 H0370 H0429 H0449 H0486 H0494 H0506 H0510 H0521 H0539
H0543 H0544 H0550 H0551 H0575 H0581 H0586 H0599 H0616 H0620 H0623
H0628 H0635 H0644 H0653 H0657 H0665 H0683 L0382 L0471 L0565 L0601
L0604 L0651 L0664 L0745 L0750 L0752 L0754 L0757 L0758 L0759 L0766
L0769 L0779 L0789 L0794 L0800 L0803 S0002 S0022 S0027 S0028 S0037
S0040 S0044 S0045 S0046 S0051 S0126 S0142 S0144 S0152 S0212 S0220
S0278 S0344 S0356 S0358 S0360 S0420 S0424 S3014 T0010 T0040 T0041
T0042 T0049 HCWUA22 H0305 H0589 HDSAG91 H0329 H0635 L0766 HNEDJ35
H0179 H0435 H7TBA62 S0198 S0228 S0252 S0264 S0268 S0270 S0274
HNGIO50 S0052 HMIAW81 H0046 H0328 H0445 L0519 S6028 HMMCJ60 H0124
H0444 S0053 HDPIO09 H0006 H0013 H0014 H0031 H0032 H0039 H0040 H0051
H0052 H0059 H0090 H0196 H0252 H0265 H0266 H0294 H0309 H0328 H0373
H0375 H0421 H0422 H0423 H0427 H0428 H0431 H0445 H0486 H0488 H0497
H0510 H0521 H0529 H0542 H0547 H0550 H0553 H0556 H0561 H0574 H0580
H0591 H0596 H0622 H0623 H0624 H0628 H0634 H0637 H0641 H0644 H0648
H0658 H0659 H0661 H0676 H0684 H0687 L0439 L0481 L0485 L0512 L0517
L0563 L0638 L0646 L0651 L0659 L0661 L0662 L0663 L0664 L0665 L0666
L0682 L0697 L0731 L0740 L0745 L0747 L0748 L0749 L0750 L0751 L0752
L0754 L0755 L0756 L0757 L0758 L0759 L0763 L0764 L0766 L0768 L0769
L0770 L0774 L0775 L0776 L0777 L0779 L0780 L0783 L0789 L0809 S0001
S0002 S0003 S0010 S0027 S0028 S0038 S0046 S0051 S0114 S0116 S0142
S0218 S0222 S0276 S0294 S0328 S0330 S0346 S0354 S0356 S0374 T0042
HHFHH34 H0050 H0520 HISCL83 H0539 HTOAI70 H0264 HSDER95 H0009 H0321
H0362 H0427 H0547 H0658 H0690 L0438 L0588 L0592 L0598 L0740 L0749
L0756 L0759 L0766 L0769 L0773 L0775 L0776 L0791 L0803 L0804 S0031
S0136 S0176 S0328 S0374 HNECL25 H0179 HNFGZ45 H0179 H0264 H0271
H0422 H0619 S0358 HHGCU49 H0013 H0086 H0087 H0100 H0123 H0124 H0150
H0163 H0181 H0288 H0333 H0422 H0544 H0545 H0546 H0547 H0550 H0553
H0619 H0628 H0644 H0658 H0665 L0384 L0521 L0565 L0603 L0605 L0623
L0655 L0656 L0659 L0743 L0744 L0751 L0754 L0757 L0771 L0777 L0794
L0803 L0809 S0027 S0028 S0037 S0052 S0206 S0212 S0360 HDPND68 H0063
H0144 H0264 H0305 H0316 H0402 H0427 H0431 H0517 H0522 H0690 L0021
L0378 L0381 L0527 L0534 L0539 L0562 L0589 L0665 L0745 L0748 L0751
L0766 L0770 S0001 S0002 S0038 S0052 HETDT81 H0038 H0046 H0090 H0253
H0539 H0617 L0439 L0455 L0646 L0649 L0658 L0659 L0662 L0750 L0754
L0764 L0766 L0771 L0777 L0780 L0789 L0803 S0142 S0344 S0358 HHLBA14
H0013 H0264 H0427 H0547 L0438 L0439 S0010 S0222 T0041 T0091 HLTBU43
H0090 HNTSJ84 H0013 H0428 H0542 H0547 H0622 L0636 L0662 L0717 L0740
L0749 L0766 L0769 L0779 L0789 S0007 S0242 S0282 S0354 HOHCG16 H0411
H0509 H0538 L0439 L0532 L0743 L0744 L0748 L0749 S0250 HTHCB31 H0063
H0170 L0589 S0001 HUKAM16 H0028 H0059 H0081 H0135 H0194 H0231 H0255
H0264 H0352 H0423 H0483 H0521 H0529 H0542 H0547 H0553 H0587 H0616
H0628 H0662 H0663 H0687 L0439 L0471 L0526 L0605 L0639 L0664 L0665
L0743 L0744 L0745 L0747 L0748 L0759 L0769 L0774 L0776 L0777 L0809
S0002 S0007 S0036 S0212 S0330 S0360 S0378 S0418 S0428 T0010 HLDOJ66
H0510 HTXKF10 H0556 HPMAI22 H0031 H0662 L0600 L0657 L0755 L0756
L0767 L0768 L0779 L0794 HL2AG57 H0013 H0090 H0131 H0135 H0264 H0341
H0359 H0519 H0689 L0439 L0637 L0640 L0647 L0659 L0665 L0764 L0768
L0779 S0212 HTHBH29 H0063 H0100 H0520 HUSAM59 H0032 H0052 H0068
H0083 H0090 H0156 H0170 H0171 H0212 H0266 H0268 H0309 H0392 H0411
H0422 H0423 H0435 H0441 H0445 H0494 H0519 H0529 H0543 H0547 H0561
H0574 H0591 H0596 H0628 H0633 H0656 H0657 H0658 H0667 H0686 H0696
L0438 L0439 L0471 L0519 L0521 L0581 L0598 L0601 L0649 L0653 L0659
L0662 L0664 L0665 L0666 L0717 L0740 L0742 L0745 L0747 L0750 L0752
L0753 L0754 L0755 L0756 L0758 L0764 L0766 L0768 L0770 L0773 L0775
L0777 L0779 L0780 L0782 L0783 L0789 L0794 L0803 L0804 L0809 S0011
S0022 S0042 S0051 S0192 S0242 S0358 S0360 S0374 S0380 S0402 S0424
S0474 S6028 T0069 T0114 HNGGR26 S0052 HTLCX30 H0253 L0758 L0794
HCEBC87 H0052 H0163 H0171 H0351 H0411 H0415 H0592 H0694 L0439 L0465
L0520 L0592 L0650 L0657 L0666 L0745 L0748 L0751 L0752 L0755 L0756
L0758 L0766 L0777 L0779 L0783 L0788 L0803 L0805 S0010 S0136 S0358
HATCB92 H0156 HMSCX69 H0063 H0100 H0139 H0144 H0264 H0318 H0327
H0331 H0538 H0650 H0656 L0381 L0438 L0606 L0638 L0740 L0749 L0750
L0754 L0756 L0759 L0761 L0766 L0769 L0770 L0774 L0777 L0779 L0792
S0002 S0053 T0010 HLHAL68 H0024 HEOMR73 H0179 H0271 H0457 H0695
L0748 HETIB83 H0046 H0134 H0306 H0318 H0396 H0402 H0429 H0445 H0560
H0581 H0638 H0650 H0656 H0657 H0689 L0438 L0439 L0655 L0740 L0761
L0766 L0777 L0789 L0794 S0002 S0038 S0050 S0278 S0344 HJPDD28 H0002
H0014 H0015 H0024 H0031 H0036 H0040 H0046 H0052 H0083 H0090 H0169
H0204 H0214 H0264 H0265 H0266 H0352 H0370 H0393 H0421 H0431 H0435
H0448 H0494 H0583 H0620 H0635 H0642 H0653 H0656 H0658 L0021 L0364
L0372 L0374 L0462 L0588 L0596 L0599 L0622 L0644 L0647 L0659 L0663
L0665 L0666 L0731 L0740 L0747 L0750 L0751 L0752 L0753 L0754 L0758
L0759 L0765 L0766 L0769 L0771 L0772 L0773 L0783 L0806 S0038 S0040
S0142 S0280 S0356 S0358 S0366 S0442 S3014 S6028 HBAMB15 H0328 H0410
H0530 L0455 L0740
[0700] TABLE-US-00012 TABLE 3 Cytologic SEQ ID Band or NO: X
Chromosome: OMIM Reference(s): 19 1q21 104770 107670 110700 135940
145001 146790 152445 159001 174000 179755 182860 191315 230800
266200 600897 601105 601412 601652 602491 21 9q33-q34.1 103000
114350 120900 131195 146150 185000 189980 223900 253800 268900
600184 602575 57 16q13 114835 132700 172490 600968 66 12
[0701] TABLE-US-00013 TABLE 4 Library Code Library Description BL29
Burkitt's lymphoma, Pascalis Sideras H0002 Human Adult Heart H0006
Human Frontal Lobe of Brain H0009 Human Fetal Brain H0012 Human
Fetal Kidney H0013 Human 8 Week Whole Embryo H0014 Human Gall
Bladder H0015 Human Gall Bladder, fraction II H0023 Human Fetal
Lung H0024 Human Fetal Lung III H0028 Human Old Ovary H0030 Human
Placenta H0031 Human Placenta H0032 Human Prostate H0036 Human
Adult Small Intestine H0038 Human Testes H0039 Human Pancreas Tumor
H0040 Human Testes Tumor H0041 Human Fetal Bone H0042 Human Adult
Pulmonary H0044 Human Cornea H0046 Human Endometrial Tumor H0050
Human Fetal Heart H0051 Human Hippocampus H0052 Human Cerebellum
H0056 Human Umbilical Vein, Endo. remake H0057 Human Fetal Spleen
H0059 Human Uterine Cancer H0061 Human Macrophage H0063 Human
Thymus H0068 Human Skin Tumor H0069 Human Activated T-Cells H0070
Human Pancreas H0081 Human Fetal Epithelium (Skin) H0083 HUMAN
JURKAT MEMBRANE BOUND POLYSOMES H0085 Human Colon H0086 Human
epithelioid sarcoma H0087 Human Thymus H0090 Human T-Cell Lymphoma
H0096 Human Parotid Cancer H0098 Human Adult Liver, subtracted
H0100 Human Whole Six Week Old Embryo H0103 Human Fetal Brain,
subtracted H0105 Human Fetal Heart, subtracted H0111 Human
Placenta, subtracted H0116 Human Thymus Tumor, subtracted H0122
Human Adult Skeletal Muscle H0123 Human Fetal Dura Mater H0124
Human Rhabdomyosarcoma H0130 LNCAP untreated H0131 LNCAP + o.3 nM
R1881 H0134 Raji Cells, cyclohexamide treated H0135 Human Synovial
Sarcoma H0139 Activated T-Cells, 4 hrs. H0144 Nine Week Old Early
Stage Human H0149 7 Week Old Early Stage Human, subtracted H0150
Human Epididymus H0156 Human Adrenal Gland Tumor H0163 Human
Synovium H0165 Human Prostate Cancer, Stage B2 H0166 Human Prostate
Cancer, Stage B2 fraction H0169 Human Prostate Cancer, Stage C
fraction H0170 12 Week Old Early Stage Human H0171 12 Week Old
Early Stage Human, II H0178 Human Fetal Brain H0179 Human
Neutrophil H0181 Human Primary Breast Cancer H0188 Human Normal
Breast H0194 Human Cerebellum, subtracted H0196 Human
Cardiomyopathy, subtracted H0200 Human Greater Omentum, fract II
remake, H0201 Human Hippocampus, subtracted H0204 Human Colon
Cancer, subtracted H0208 Early Stage Human Lung, subtracted H0212
Human Prostate, subtracted H0213 Human Pituitary, subtracted H0214
Raji cells, cyclohexamide treated, subtracted H0220 Activated
T-Cells, 4 hrs, subtracted H0224 Activated T-Cells, 12 hrs,
subtracted H0231 Human Colon, subtraction H0242 Human Fetal Heart,
Differential (Fetal-Specific) H0250 Human Activated Monocytes H0251
Human Chondrosarcoma H0252 Human Osteosarcoma H0253 Human adult
testis, large inserts H0254 breast lymph node CDNA library H0255
breast lymph node CDNA library H0261 H. cerebellum, Enzyme
subtracted H0263 human colon cancer H0264 human tonsils H0265
Activated T-Cell (12 hs)/Thiouridine labelledEco H0266 Human
Microvascular Endothelial Cells, fract. A H0267 Human Microvascular
Endothelial Cells, fract. B H0268 Human Umbilical Vein Endothelial
Cells, fract. A H0269 Human Umbilical Vein Endothelial Cells,
fract. B H0271 Human Neutrophil, Activated H0274 Human Adult
Spleen, fractionII H0280 K562 + PMA (36 hrs) H0284 Human OB MG63
control fraction I H0286 Human OB MG63 treated (10 nM E2) fraction
I H0288 Human OB HOS control fraction I H0290 Human OB HOS treated
(1 nM E2) fraction I H0292 Human OB HOS treated (10 nM E2) fraction
I H0294 Amniotic Cells - TNF induced H0295 Amniotic Cells - Primary
Culture H0305 CD34 positive cells (Cord Blood) H0306 CD34 depleted
Buffy Coat (Cord Blood) H0309 Human Chronic Synovitis H0310 human
caudate nucleus H0316 HUMAN STOMACH H0318 HUMAN B CELL LYMPHOMA
H0320 Human frontal cortex H0321 HUMAN SCHWANOMA H0327 human corpus
colosum H0328 human ovarian cancer H0329 Dermatofibrosarcoma
Protuberance H0331 Hepatocellular Tumor H0333 Hemangiopericytoma
H0341 Bone Marrow Cell Line (RS4,11) H0343 stomach cancer (human)
H0351 Glioblastoma H0352 wilm's tumor H0355 Human Liver H0356 Human
Kidney H0357 H. Normalized Fetal Liver, II H0359 KMH2 cell line
H0361 Human rejected kidney H0362 HeLa cell line H0366 L428 cell
line H0370 H. Lymph node breast Cancer H0373 Human Heart H0374
Human Brain H0375 Human Lung H0381 Bone Cancer H0388 Human Rejected
Kidney, 704 re-excision H0389 H. Brain, X-Chromosome hybridization
H0391 H. Meniingima, M6 H0392 H. Meningima, M1 H0393 Fetal Liver,
subtraction II H0396 L1 Cell line H0400 Human Striatum Depression,
re-rescue H0402 CD34 depleted Buffy Coat (Cord Blood), re-excision
H0403 H. Umbilical Vein Endothelial Cells, IL4 induced H0410 H.
Male bladder, adult H0411 H Female Bladder, Adult H0412 Human
umbilical vein endothelial cells, IL-4 induced H0413 Human
Umbilical Vein Endothelial Cells, uninduced H0415 H. Ovarian Tumor,
II, OV5232 H0416 Human Neutrophils, Activated, re-excision H0421
Human Bone Marrow, re-excision H0422 T-Cell PHA 16 hrs H0423 T-Cell
PHA 24 hrs H0424 Human Pituitary, subt IX H0427 Human Adipose H0428
Human Ovary H0429 K562 + PMA (36 hrs), re-excision H0431 H. Kidney
Medulla, re-excision H0435 Ovarian Tumor 10-3-95 H0436 Resting
T-Cell Library, II H0437 H Umbilical Vein Endothelial Cells, frac
A, re-excision H0438 H. Whole Brain #2, re-excision H0441 H. Kidney
Cortex, subtracted H0444 Spleen metastic melanoma H0445 Spleen,
Chronic lymphocytic leukemia H0448 Salivary gland, subtracted H0449
CD34+ cell, I H0453 H. Kidney Pyramid, subtracted H0457 Human
Eosinophils H0458 CD34+ cell, I, frac II H0478 Salivary Gland, Lib
2 H0479 Salivary Gland, Lib 3 H0483 Breast Cancer cell line, MDA 36
H0484 Breast Cancer Cell line, angiogenic H0485 Hodgkin's Lymphoma
I H0486 Hodgkin's Lymphoma II H0488 Human Tonsils, Lib 2 H0489
Crohn's Disease H0494 Keratinocyte H0497 HEL cell line H0506
Ulcerative Colitis H0509 Liver, Hepatoma H0510 Human Liver, normal
H0517 Nasal polyps H0518 pBMC stimulated w/ poly I/C H0519 NTERA2,
control H0520 NTERA2 + retinoic acid, 14 days H0521 Primary
Dendritic Cells, lib 1 H0522 Primary Dendritic cells, frac 2 H0529
Myoloid Progenitor Cell Line H0530 Human Dermal Endothelial Cells,
untreated H0538 Merkel Cells H0539 Pancreas Islet Cell Tumor H0542
T Cell helper I H0543 T cell helper II H0544 Human endometrial
stromal cells H0545 Human endometrial stromal cells-treated with
progesterone H0546 Human endometrial stromal cells-treated with
estradiol H0547 NTERA2 teratocarcinoma cell line + retinoic acid
(14 days) H0549 H. Epididiymus, caput & corpus H0550 H.
Epididiymus, cauda H0551 Human Thymus Stromal Cells H0553 Human
Placenta H0555 Rejected Kidney, lib 4 H0556 Activated T-cell(12
h)/Thiouridine-re-excision H0560 KMH2 H0561 L428 H0569 Human Fetal
Brain, normalized CO H0571 Human Fetal Brain, normalized C500HE
H0574 Hepatocellular Tumor, re-excision H0575 Human Adult
Pulmonary, re-excision H0576 Resting T-Cell, re-excision H0580
Dendritic cells, pooled H0581 Human Bone Marrow, treated H0583 B
Cell lymphoma H0586 Healing groin wound, 6.5 hours post incision
H0587 Healing groin wound, 7.5 hours post incision H0589 CD34
positive cells (cord blood), re-ex H0590 Human adult small
intestine, re-excision H0591 Human T-cell lymphoma, re-excision
H0592 Healing groin wound - zero hr post-incision (control) H0593
Olfactory epithelium, nasalcavity H0594 Human Lung Cancer,
re-excision H0596 Human Colon Cancer, re-excision H0597 Human
Colon, re-excision H0598 Human Stomach, re-excision H0599 Human
Adult Heart, re-excision H0600 Healing Abdomen wound, 70&90 min
post incision H0601 Healing Abdomen Wound, 15 days post incision
H0602 Healing Abdomen Wound, 21&29 days post incision H0606
Human Primary Breast Cancer, re-excision H0615 Human Ovarian Cancer
Reexcision H0616 Human Testes, Reexcision H0617 Human Primary
Breast Cancer Reexcision H0618 Human Adult Testes, Large Inserts,
Reexcision H0619 Fetal Heart H0620 Human Fetal Kidney, Reexcision
H0622 Human Pancreas Tumor, Reexcision H0623 Human Umbilical Vein,
Reexcision H0624 12 Week Early Stage Human II, Reexcision H0625 Ku
812F Basophils Line H0626 Saos2 Cells, Untreated H0627 Saos2 Cells,
Vitamin D3 Treated H0628 Human Pre-Differentiated Adipocytes H0631
Saos2, Dexamethosome Treated
H0632 Hepatocellular Tumor, re-excision H0633 Lung Carcinoma A549
TNFalpha activated H0634 Human Testes Tumor, re-excision H0635
Human Activated T-Cells, re-excision H0637 Dendritic Cells From
CD34 Cells H0638 CD40 activated monocyte dendridic cells H0640
Ficolled Human Stromal Cells, Untreated H0641 LPS activated derived
dendritic cells H0642 Hep G2 Cells, lambda library H0644 Human
Placenta (re-excision) H0645 Fetal Heart, re-excision H0646 Lung,
Cancer (4005313 A3): Invasive Poorly Differentiated Lung
Adenocarcinoma, H0647 Lung, Cancer (4005163 B7): Invasive, Poorly
Diff. Adenocarcinoma, Metastatic H0648 Ovary, Cancer: (4004562 B6)
Papillary Serous Cystic Neoplasm, Low Malignant Pot H0650 B-Cells
H0652 Lung, Normal: (4005313 B1) H0653 Stromal Cells H0656 B-cells
(unstimulated) H0657 B-cells (stimulated) H0658 Ovary, Cancer
(9809C332): Poorly differentiated adenocarcinoma H0659 Ovary,
Cancer (15395A1F): Grade II Papillary Carcinoma H0660 Ovary,
Cancer: (15799A1F) Poorly differentiated carcinoma H0661 Breast,
Cancer: (4004943 A5) H0662 Breast, Normal: (4005522B2) H0663
Breast, Cancer: (4005522 A2) H0664 Breast, Cancer: (9806C012R)
H0665 Stromal cells 3.88 H0666 Ovary, Cancer: (4004332 A2) H0667
Stromal cells(HBM3.18) H0668 stromal cell clone 2.5 H0670 Ovary,
Cancer(4004650 A3): Well-Differentiated Micropapillary Serous
Carcinoma H0672 Ovary, Cancer: (4004576 A8) H0673 Human Prostate
Cancer, Stage B2, re-excision H0674 Human Prostate Cancer, Stage C,
re-excission H0676 Colon, Cancer: (9808C064R)-total RNA H0677 TNFR
degenerate oligo H0679 screened clones from Tonsil library H0682
Ovarian cancer, Serous Papillary Adenocarcinoma H0683 Ovarian
cancer, Serous Papillary Adenocarcinoma H0684 Ovarian cancer,
Serous Papillary Adenocarcinoma H0685 Adenocarcinoma of Ovary,
Human Cell Line, # OVCAR-3 H0686 Adenocarcinoma of Ovary, Human
Cell Line H0687 Human normal ovary(#9610G215) H0688 Human Ovarian
Cancer(#9807G017) H0689 Ovarian Cancer H0690 Ovarian Cancer, #
9702G001 H0691 Normal Ovary, #9710G208 H0694 Prostate cancer
(adenocarcinoma) H0695 mononucleocytes from patient H0696 Prostate
Adenocarcinoma H0702 NK15(IL2 treated for 48 hours) H0707 Stomach
Cancer(S007635) L0005 Clontech human aorta polyA+ mRNA (#6572)
L0017 Human (J. Swensen) L0021 Human adult (K. Okubo) L0040 Human
colon mucosa L0041 Human epidermal keratinocyte L0053 Human
pancreatic tumor L0055 Human promyelocyte L0103 DKFZphamy1 L0105
Human aorta polyA+ (TFujiwara) L0142 Human placenta cDNA
(TFujiwara) L0143 Human placenta polyA+ (TFujiwara) L0157 Human
fetal brain (TFujiwara) L0163 Human heart cDNA (YNakamura) L0352
Normalized infant brain, Bento Soares L0361 Stratagene ovary
(#937217) L0362 Stratagene ovarian cancer (#937219) L0363
NCI_CGAP_GC2 L0364 NCI_CGAP_GC5 L0366 Stratagene schizo brain S11
L0367 NCI_CGAP_Sch1 L0369 NCI_CGAP_AA1 L0372 NCI_CGAP_Co12 L0373
NCI_CGAP_Co11 L0374 NCI_CGAP_Co2 L0375 NCI_CGAP_Kid6 L0378
NCI_CGAP_Lu1 L0381 NCI_CGAP_HN4 L0382 NCI_CGAP_Pr25 L0383
NCI_CGAP_Pr24 L0384 NCI_CGAP_Pr23 L0387 NCI_CGAP_GCB0 L0388
NCI_CGAP_HN6 L0411 1-NIB L0426 b4HB3MA-Cot51.5-HAP-Ft L0438
normalized infant brain cDNA L0439 Soares infant brain 1NIB L0447
NHB3MK L0448 3HFLSK20 L0451 N3HFLSK20 L0455 Human retina cDNA
randomly primed sublibrary L0460 Adult heart, Lambda gt11 L0462
WATM1 L0465 TEST1, Human adult Testis tissue L0471 Human fetal
heart, Lambda ZAP Express L0481 CD34+DIRECTIONAL L0483 Human
pancreatic islet L0485 STRATAGENE Human skeletal muscle cDNA
library, cat. #936215. L0493 NCI_CGAP_Ov26 L0498 NCI_CGAP_HSC3
L0511 NCI_CGAP_Ov34 L0512 NCI_CGAP_Ov36 L0515 NCI_CGAP_Ov32 L0517
NCI_CGAP_Pr1 L0518 NCI_CGAP_Pr2 L0519 NCI_CGAP_Pr3 L0520
NCI_CGAP_Alv1 L0521 NCI_CGAP_Ew1 L0523 NCI_CGAP_Lip2 L0525
NCI_CGAP_Li2 L0526 NCI_CGAP_Pr12 L0527 NCI_CGAP_Ov2 L0529
NCI_CGAP_Pr6 L0530 NCI_CGAP_Pr8 L0532 NCI_CGAP_Thy1 L0534
Chromosome 7 Fetal Brain cDNA Library L0536 NCI_CGAP_Br4 L0539
Chromosome 7 Placental cDNA Library L0540 NCI_CGAP_Pr10 L0542
NCI_CGAP_Pr11 L0545 NCI_CGAP_Pr4.1 L0549 NCI_CGAP_HN10 L0553
NCI_CGAP_Co22 L0554 NCI_CGAP_Li8 L0560 NCI_CGAP_HN12 L0562
Chromosome 7 HeLa cDNA Library L0563 Human Bone Marrow Stromal
Fibroblast L0564 Jia bone marrow stroma L0565 Normal Human
Trabecular Bone Cells L0581 Stratagene liver (#937224) L0586 HTCDL1
L0588 Stratagene endothelial cell 937223 L0589 Stratagene fetal
retina 937202 L0591 Stratagene HeLa cell s3 937216 L0592 Stratagene
hNT neuron (#937233) L0593 Stratagene neuroepithelium (#937231)
L0595 Stratagene NT2 neuronal precursor 937230 L0596 Stratagene
colon (#937204) L0597 Stratagene corneal stroma (#937222) L0598
Morton Fetal Cochlea L0599 Stratagene lung (#937210) L0600 Weizmann
Olfactory Epithelium L0601 Stratagene pancreas (#937208) L0602
Pancreatic Islet L0603 Stratagene placenta (#937225) L0604
Stratagene muscle 937209 L0605 Stratagene fetal spleen (#937205)
L0606 NCI_CGAP_Lym5 L0607 NCI_CGAP_Lym6 L0608 Stratagene lung
carcinoma 937218 L0612 Schiller oligodendroglioma L0615 22 week old
human fetal liver cDNA library L0622 HM1 L0623 HM3 L0626
NCI_CGAP_GC1 L0629 NCI_CGAP_Mel3 L0635 NCI_CGAP_PNS1 L0636
NCI_CGAP_Pit1 L0637 NCI_CGAP_Brn53 L0638 NCI_CGAP_Brn35 L0639
NCI_CGAP_Brn52 L0640 NCI_CGAP_Br18 L0641 NCI_CGAP_Co17 L0642
NCI_CGAP_Co18 L0644 NCI_CGAP_Co20 L0645 NCI_CGAP_Co21 L0646
NCI_CGAP_Co14 L0647 NCI_CGAP_Sar4 L0648 NCI_CGAP_Eso2 L0649
NCI_CGAP_GU1 L0650 NCI_CGAP_Kid13 L0651 NCI_CGAP_Kid8 L0653
NCI_CGAP_Lu28 L0654 NCI_CGAP_Lu31 L0655 NCI_CGAP_Lym12 L0656
NCI_CGAP_Ov38 L0657 NCI_CGAP_Ov23 L0658 NCI_CGAP_Ov35 L0659
NCI_CGAP_Pan1 L0661 NCI_CGAP_Mel15 L0662 NCI_CGAP_Gas4 L0663
NCI_CGAP_Ut2 L0664 NCI_CGAP_Ut3 L0665 NCI_CGAP_Ut4 L0666
NCI_CGAP_Ut1 L0667 NCI_CGAP_CML1 L0682 Stanley Frontal NB pool 2
L0697 Testis 1 L0717 Gessler Wilms tumor L0731
Soares_pregnant_uterus_NbHPU L0738 Human colorectal cancer L0740
Soares melanocyte 2NbHM L0741 Soares adult brain N2b4HB55Y L0742
Soares adult brain N2b5HB55Y L0743 Soares breast 2NbHBst L0744
Soares breast 3NbHBst L0745 Soares retina N2b4HR L0746 Soares
retina N2b5HR L0747 Soares_fetal_heart_NbHH19W L0748 Soares fetal
liver spleen 1NFLS L0749 Soares_fetal_liver_spleen_1NFLS_S1 L0750
Soares_fetal_lung_NbHL19W L0751 Soares ovary tumor NbHOT L0752
Soares_parathyroid_tumor_NbHPA L0753 Soares_pineal_gland_N3HPG
L0754 Soares placenta Nb2HP L0755
Soares_placenta_8to9weeks_2NbHP8to9W L0756
Soares_multiple_sclerosis_2NbHMSP L0757
Soares_senescent_fibroblasts_NbHSF L0758 Soares_testis_NHT L0759
Soares_total_fetus_Nb2HF8_9w L0761 NCI_CGAP_CLL1 L0762
NCI_CGAP_Br1.1 L0763 NCI_CGAP_Br2 L0764 NCI_CGAP_Co3 L0765
NCI_CGAP_Co4 L0766 NCI_CGAP_GCB1 L0767 NCI_CGAP_GC3 L0768
NCI_CGAP_GC4 L0769 NCI_CGAP_Brn25 L0770 NCI_CGAP_Brn23 L0771
NCI_CGAP_Co8 L0772 NCI_CGAP_Co10 L0773 NCI_CGAP_Co9 L0774
NCI_CGAP_Kid3 L0775 NCI_CGAP_Kid5 L0776 NCI_CGAP_Lu5 L0777
Soares_NhHMPu_S1 L0779 Soares_NFL_T_GBC_S1 L0780
Soares_NSF_F8_9W_OT_PA_P_S1 L0782 NCI_CGAP_Pr21 L0783 NCI_CGAP_Pr22
L0785 Barstead spleen HPLRB2 L0786 Soares_NbHFB L0788 NCI_CGAP_Sub2
L0789 NCI_CGAP_Sub3 L0790 NCI_CGAP_Sub4 L0791 NCI_CGAP_Sub5 L0792
NCI_CGAP_Sub6 L0794 NCI_CGAP_GC6 L0796 NCI_CGAP_Brn50 L0800
NCI_CGAP_Co16 L0803 NCI_CGAP_Kid11 L0804 NCI_CGAP_Kid12
L0805 NCI_CGAP_Lu24 L0806 NCI_CGAP_Lu19 L0807 NCI_CGAP_Ov18 L0809
NCI_CGAP_Pr28 S0001 Brain frontal cortex S0002 Monocyte activated
S0003 Human Osteoclastoma S0007 Early Stage Human Brain S0010 Human
Amygdala S0011 STROMAL - OSTEOCLASTOMA S0013 Prostate S0014 Kidney
Cortex S0015 Kidney medulla S0022 Human Osteoclastoma Stromal Cells
- unamplified S0026 Stromal cell TF274 S0027 Smooth muscle, serum
treated S0028 Smooth muscle, control S0029 brain stem S0030 Brain
pons S0031 Spinal cord S0032 Smooth muscle-ILb induced S0036 Human
Substantia Nigra S0037 Smooth muscle, IL1b induced S0038 Human
Whole Brain #2 - Oligo dT >1.5 Kb S0040 Adipocytes S0042 Testes
S0044 Prostate BPH S0045 Endothelial cells-control S0046
Endothelial-induced S0049 Human Brain, Striatum S0050 Human Frontal
Cortex, Schizophrenia S0051 Human Hypothalmus, Schizophrenia S0052
neutrophils control S0053 Neutrophils IL-1 and LPS induced S0112
Hypothalamus S0114 Anergic T-cell S0116 Bone marrow S0122
Osteoclastoma-normalized A S0126 Osteoblasts S0132 Epithelial-TNFa
and INF induced S0134 Apoptotic T-cell S0136 PERM TF274 S0140
eosinophil-IL5 induced S0142 Macrophage-oxLDL S0144 Macrophage
(GM-CSF treated) S0146 prostate-edited S0150 LNCAP prostate cell
line S0152 PC3 Prostate cell line S0176 Prostate, normal,
subtraction I S0192 Synovial Fibroblasts (control) S0194 Synovial
hypoxia S0196 Synovial IL-1/TNF stimulated S0198 7TM-pbfd S0206
Smooth Muscle- HASTE normalized S0208 Messangial cell, frac 1 S0210
Messangial cell, frac 2 S0212 Bone Marrow Stromal Cell, untreated
S0214 Human Osteoclastoma, re-excision S0216 Neutrophils IL-1 and
LPS induced S0218 Apoptotic T-cell, re-excision S0220 H.
hypothalamus, frac A, re-excision S0222 H. Frontal cortex,
epileptic, re-excision S0228 PSMIX S0242 Synovial Fibroblasts
(Il1/TNF), subt S0250 Human Osteoblasts II S0252 7TM-PIMIX S0260
Spinal Cord, re-excision S0264 PPMIX S0268 PRMIX S0270 PTMIX S0274
PCMIX S0276 Synovial hypoxia-RSF subtracted S0278 H Macrophage
(GM-CSF treated), re-excision S0280 Human Adipose Tissue,
re-excision S0282 Brain Frontal Cortex, re-excision S0294 Larynx
tumor S0300 Frontal lobe, dementia, re-excision S0312 Human
osteoarthritic, fraction II S0314 Human osteoarthritis, fraction I
S0316 Human Normal Cartilage, Fraction I S0318 Human Normal
Cartilage Fraction II S0328 Palate carcinoma S0330 Palate normal
S0332 Pharynx carcinoma S0334 Human Normal Cartilage Fraction III
S0338 Human Osteoarthritic Cartilage Fraction III S0342 Adipocytes,
re-excision S0344 Macrophage-oxLDL, re-excision S0346 Human
Amygdala, re-excision S0348 Cheek Carcinoma S0350 Pharynx Carcinoma
S0352 Larynx Carcinoma S0354 Colon Normal II S0356 Colon Carcinoma
S0358 Colon Normal III S0360 Colon Tumor II S0364 Human Quadriceps
S0366 Human Soleus S0374 Normal colon S0376 Colon Tumor S0378
Pancreas normal PCA4 No S0380 Pancreas Tumor PCA4 Tu S0384 Tongue
carcinoma S0388 Human Hypothalamus, schizophrenia, re-excision
S0390 Smooth muscle, control, re-excision S0392 Salivary Gland
S0402 Adrenal Gland, normal S0412 Temporal cortex-Alzheizmer,
subtracted S0414 Hippocampus, Alzheimer Subtracted S0418 CHME Cell
Line, treated 5 hrs S0420 CHME Cell Line, untreated S0422 Mo7e Cell
Line GM-CSF treated (1 ng/ml) S0424 TF-1 Cell Line GM-CSF Treated
S0426 Monocyte activated, re-excision S0428 Neutrophils control,
re-excision S0430 Aryepiglottis Normal S0432 Sinus piniformis
Tumour S0434 Stomach Normal S0438 Liver Normal Met5No S0442 Colon
Normal S0446 Tongue Tumour S0450 Larynx Tumour S0452 Thymus S0456
Tongue Normal S0458 Thyroid Normal (SDCA2 No) S0462 Thyroid
Thyroiditis S0466 Larynx Tumor S0468 Ea.hy.926 cell line S0474
Human blood platelets S3012 Smooth Muscle Serum Treated, Norm S3014
Smooth muscle, serum induced, re-exc S6014 H. hypothalamus, frac A
S6024 Alzheimers, spongy change S6028 Human Manic Depression Tissue
T0002 Activated T-cells T0003 Human Fetal Lung T0004 Human White
Fat T0006 Human Pineal Gland T0008 Colorectal Tumor T0010 Human
Infant Brain T0023 Human Pancreatic Carcinoma T0039 HSA 172 Cells
T0040 HSC172 cells T0041 Jurkat T-cell G1 phase T0042 Jurkat
T-Cell, S phase T0048 Human Aortic Endothelium T0049 Aorta
endothelial cells + TNF-a T0060 Human White Adipose T0067 Human
Thyroid T0069 Human Uterus, normal T0082 Human Adult Retina T0091
Liver, hepatocellular carcinoma T0109 Human (HCC) cell line liver
(mouse) metastasis, remake T0110 Human colon carcinoma (HCC) cell
line, remake T0114 Human (Caco-2) cell line, adenocarcinoma, colon,
remake
[0702] TABLE-US-00014 TABLE 5 OMIM ID OMIM Description 103000
Hemolytic anemia due to adenylate kinase deficiency (3) 104770
?Amyloidosis, secondary, susceptibility to (1) 107670
Apolipoprotein A-II deficiency (3) 110700 Vivax malaria,
susceptibility to (1) 114350 Leukemia, acute myeloid (2) 114835
Monocyte carboxyesterase deficiency (1) (?) 120900 C5 deficiency
(1) 131195 Hereditary hemorrhagic telangiectasia-1, 187300 (3)
132700 Cylindromatosis (2) 135940 Ichthyosis vulgaris, 146700 (1)
(?) 145001 Hyperparathyroidism-jaw tumor syndrome (2) 146150
Hypomelanosis of Ito (2) (?) 146790 Lupus nephritis, susceptibility
to (3) 152445 Erythrokeratoderma, progressive symmetric, 602036 (3)
Vohwinkel syndrome, 124500 (3) 159001 Muscular dystrophy,
limb-girdle, type 1B (2) 172490 Phosphorylase kinase deficiency of
liver and muscle, 261750 (2) (?) 174000 Medullary cystic kidney
disease, AD (2) 179755 Renal cell carcinoma, papillary, 1 (2)
182860 Elliptocytosis-2 (3) Pyropoikilocytosis (3) Spherocytosis,
recessive (3) 185000 Stomatocytosis I (1) (?) 189980 Leukemia,
chronic myeloid (3) 191315 Insensitivity to pain, congenital, with
anhidrosis, 256800 (3) 223900 Dysautonomia, familial (2) 230800
Gaucher disease (3) Gaucher disease with cardiovascular
calcification (3) 253800 Fukuyama type congenital muscular
dystrophy (2) Walker-Warburg syndrome, 236670 (2) (?) 266200
Anemia, hemolytic, due to PK deficiency (3) 268900 [Sarcosinemia]
(2) 600184 Carnitine acetyltransferase deficiency (1) (?) 600897
Cataract, zonular pulverulent-1, 116200 (3) 600968 Gitelman
syndrome, 263800 (3) 601105 Pycnodysostosis, 265800 (3) 601412
Deafness, autosomal dominant 7 (2) 601652 Glaucoma 1A, primary open
angle, juvenile-onset, 137750 (3) 602491 Hyperlipidemia, familial
combined, 1 (2) 602575 Nail-patella syndrome with open-angle
glaucoma, 137750 (3) Nail-patella syndrome, 161200 (3)
[0703] The polypeptides of the invention can be prepared in any
suitable manner. Such polypeptides include isolated naturally
occurring polypeptides, recombinantly produced polypeptides,
synthetically produced polypeptides, or polypeptides produced by a
combination of these methods. Means for preparing such polypeptides
are well understood in the art.
[0704] The polypeptides may be in the form of the secreted protein,
including the mature form, or may be a part of a larger protein,
such as a fusion protein (see below). It is often advantageous to
include an additional amino acid sequence which contains secretory
or leader sequences, pro-sequences, sequences which aid in
purification, such as multiple histidine residues, or an additional
sequence for stability during recombinant production.
[0705] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. A recombinantly produced version of a polypeptide,
including the secreted polypeptide, can be substantially purified
using techniques described herein or otherwise known in the art,
such as, for example, by the one-step method described in Smith and
Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also
can be purified from natural, synthetic or recombinant sources
using techniques described herein or otherwise known in the art,
such as, for example, antibodies of the invention raised against
the secreted protein.
[0706] The present invention provides a polynucleotide comprising,
or alternatively consisting of, the nucleic acid sequence of SEQ ID
NO:X, and/or a cDNA contained in ATCC deposit Z. The present
invention also provides a polypeptide comprising, or alternatively,
consisting of, the polypeptide sequence of SEQ ID NO:Y and/or a
polypeptide encoded by the cDNA contained in ATCC deposit Z.
Polynucleotides encoding a polypeptide comprising, or alternatively
consisting of the polypeptide sequence of SEQ ID NO:Y and/or a
polypeptide sequence encoded by the cDNA contained in ATCC deposit
Z are also encompassed by the invention.
[0707] Signal Sequences
[0708] The present invention also encompasses mature forms of the
polypeptide having the polypeptide sequence of SEQ ID NO:Y and/or
the polypeptide sequence encoded by the cDNA in a deposited clone.
Polynucleotides encoding the mature forms (such as, for example,
the polynucleotide sequence in SEQ ID NO:X and/or the
polynucleotide sequence contained in the cDNA of a deposited clone)
are also encompassed by the invention. According to the signal
hypothesis, proteins secreted by mammalian cells have a signal or
secretary leader sequence which is cleaved from the mature protein
once export of the growing protein chain across the rough
endoplasmic reticulum has been initiated. Most mammalian cells and
even insect cells cleave secreted proteins with the same
specificity. However, in some cases, cleavage of a secreted protein
is not entirely uniform, which results in two or more mature
species of the protein. Further, it has long been known that
cleavage specificity of a secreted protein is ultimately determined
by the primary structure of the complete protein, that is, it is
inherent in the amino acid sequence of the polypeptide.
[0709] Methods for predicting whether a protein has a signal
sequence, as well as the cleavage point for that sequence, are
available. For instance, the method of McGeoch, Virus Res.
3:271-286 (1985), uses the information from a short N-terminal
charged region and a subsequent uncharged region of the complete
(uncleaved) protein. The method of von Heinje, Nucleic Acids Res.
14:4683-4690 (1986) uses the information from the residues
surrounding the cleavage site, typically residues -13 to +2, where
+1 indicates the amino terminus of the secreted protein. The
accuracy of predicting the cleavage points of known mammalian
secretory proteins for each of these methods is in the range of
75-80%. (von Heinje, supra.) However, the two methods do not always
produce the same predicted cleavage point(s) for a given
protein.
[0710] In the present case, the deduced amino acid sequence of the
secreted polypeptide was analyzed by a computer program called
SignalP (Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)),
which predicts the cellular location of a protein based on the
amino acid sequence. As part of this computational prediction of
localization, the methods of McGeoch and von Heinje are
incorporated. The analysis of the amino acid sequences of the
secreted proteins described herein by this program provided the
results shown in Table 1.
[0711] As one of ordinary skill would appreciate, however, cleavage
sites sometimes vary from organism to organism and cannot be
predicted with absolute certainty. Accordingly, the present
invention provides secreted polypeptides having a sequence shown in
SEQ ID NO:Y which have an N-terminus beginning within 5 residues
(i.e., + or -5 residues) of the predicted cleavage point.
Similarly, it is also recognized that in some cases, cleavage of
the signal sequence from a secreted protein is not entirely
uniform, resulting in more than one secreted species. These
polypeptides, and the polynucleotides encoding such polypeptides,
are contemplated by the present invention.
[0712] Moreover, the signal sequence identified by the above
analysis may not necessarily predict the naturally occurring signal
sequence. For example, the naturally occurring signal sequence may
be further upstream from the predicted signal sequence. However, it
is likely that the predicted signal sequence will be capable of
directing the secreted protein to the ER. Nonetheless, the present
invention provides the mature protein produced by expression of the
polynucleotide sequence of SEQ ID NO:X and/or the polynucleotide
sequence contained in the cDNA of a deposited clone, in a mammalian
cell (e.g., COS cells, as desribed below). These polypeptides, and
the polynucleotides encoding such polypeptides, are contemplated by
the present invention.
[0713] Polynucleotide and Polypeptide Variants
[0714] The present invention is directed to variants of the
polynucleotide sequence disclosed in SEQ ID NO:X, the complementary
strand thereto, and/or the cDNA sequence contained in a deposited
clone.
[0715] The present invention also encompasses variants of the
polypeptide sequence disclosed in SEQ ID NO:Y and/or encoded by a
deposited clone.
[0716] "Variant" refers to a polynucleotide or polypeptide
differing from the polynucleotide or polypeptide of the present
invention, but retaining essential properties thereof. Generally,
variants are overall closely similar, and, in many regions,
identical to the polynucleotide or polypeptide of the present
invention.
[0717] The present invention is also directed to nucleic acid
molecules which comprise, or alternatively consist of, a nucleotide
sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
identical to, for example, the nucleotide coding sequence in SEQ ID
NO:X or the complementary strand thereto, the nucleotide coding
sequence contained in a deposited cDNA clone or the complementary
strand thereto, a nucleotide sequence encoding the polypeptide of
SEQ ID NO:Y, a nucleotide sequence encoding the polypeptide encoded
by the cDNA contained in a deposited clone, and/or polynucleotide
fragments of any of these nucleic acid molecules (e.g., those
fragments described herein). Polynucleotides which hybridize to
these nucleic acid molecules under stringent hybridization
conditions or lower stringency conditions are also encompassed by
the invention, as are polypeptides encoded by these
polynucleotides.
[0718] The present invention is also directed to polypeptides which
comprise, or alternatively consist of, an amino acid sequence which
is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to,
for example, the polypeptide sequence shown in SEQ ID NO:Y, the
polypeptide sequence encoded by the cDNA contained in a deposited
clone, and/or polypeptide fragments of any of these polypeptides
(e.g., those fragments described herein).
[0719] By a nucleic acid having a nucleotide sequence at least, for
example, 95% "identical" to a reference nucleotide sequence of the
present invention, it is intended that the nucleotide sequence of
the nucleic acid is identical to the reference sequence except that
the nucleotide sequence may include up to five point mutations per
each 100 nucleotides of the reference nucleotide sequence encoding
the polypeptide. In other words, to obtain a nucleic acid having a
nucleotide sequence at least 95% identical to a reference
nucleotide sequence, up to 5% of the nucleotides in the reference
sequence may be deleted or substituted with another nucleotide, or
a number of nucleotides up to 5% of the total nucleotides in the
reference sequence may be inserted into the reference sequence. The
query sequence may be an entire sequence shown in Table 1, the ORF
(open reading frame), or any fragment specified as described
herein.
[0720] As a practical matter, whether any particular nucleic acid
molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% identical to a nucleotide sequence of the presence
invention can be determined conventionally using known computer
programs. A preferred method for determining the best overall match
between a query sequence (a sequence of the present invention) and
a subject sequence, also referred to as a global sequence
alignment, can be determined using the FASTDB computer program
based on the algorithm of Brutlag et al. (Comp. App. Biosci.
6:237-245(1990)). In a sequence alignment the query and subject
sequences are both DNA sequences. An RNA sequence can be compared
by converting U's to T's. The result of said global sequence
alignment is in percent identity. Preferred parameters used in a
FASTDB alignment of DNA sequences to calculate percent identiy are:
Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,
Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap
Size Penalty 0.05, Window Size=500 or the lenght of the subject
nucleotide sequence, whichever is shorter.
[0721] If the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions, a
manual correction must be made to the results. This is because the
FASTDB program does not account for 5' and 3' truncations of the
subject sequence when calculating percent identity. For subject
sequences truncated at the 5' or 3' ends, relative to the query
sequence, the percent identity is corrected by calculating the
number of bases of the query sequence that are 5' and 3' of the
subject sequence, which are not matched/aligned, as a percent of
the total bases of the query sequence. Whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the
present invention. Only bases outside the 5' and 3' bases of the
subject sequence, as displayed by the FASTDB alignment, which are
not matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score.
[0722] For example, a 90 base subject sequence is aligned to a 100
base query sequence to determine percent identity. The deletions
occur at the 5' end of the subject sequence and therefore, the
FASTDB alignment does not show a matched/alignment of the first 10
bases at 5' end. The 10 unpaired bases represent 10% of the
sequence (number of bases at the 5' and 3' ends not matched/total
number of bases in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 bases were perfectly matched the final percent
identity would be 90%. In another example, a 90 base subject
sequence is compared with a 100 base query sequence. This time the
deletions are internal deletions so that there are no bases on the
5' or 3' of the subject sequence which are not matched/aligned with
the query. In this case the percent identity calculated by FASTDB
is not manually corrected. Once again, only bases 5' and 3' of the
subject sequence which are not matched/aligned with the query
sequence are manually corrected for. No other manual corrections
are to made for the purposes of the present invention.
[0723] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence of the
present invention, it is intended that the amino acid sequence of
the subject polypeptide is identical to the query sequence except
that the subject polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the query amino acid
sequence. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a query amino acid
sequence, up to 5% of the amino acid residues in the subject
sequence may be inserted, deleted, (indels) or substituted with
another amino acid. These alterations of the reference sequence may
occur at the amino or carboxy terminal positions of the reference
amino acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0724] As a practical matter, whether any particular polypeptide is
at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for
instance, an amino acid sequences shown in Table 1 (SEQ ID NO:Y) or
to the amino acid sequence encoded by cDNA contained in a deposited
clone can be determined conventionally using known computer
programs. A preferred method for determing the best overall match
between a query sequence (a sequence of the present invention) and
a subject sequence, also referred to as a global sequence
alignment, can be determined using the FASTDB computer program
based on the algorithm of Brutlag et al. (Comp. App. Biosci.
6:237-245(1990)). In a sequence alignment the query and subject
sequences are either both nucleotide sequences or both amino acid
sequences. The result of said global sequence alignment is in
percent identity. Preferred parameters used in a FASTDB amino acid
alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining
Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window
Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
Size=500 or the length of the subject amino acid sequence,
whichever is shorter.
[0725] If the subject sequence is shorter than the query sequence
due to N- or C-terminal deletions, not because of internal
deletions, a manual correction must be made to the results. This is
because the FASTDB program does not account for N- and C-terminal
truncations of the subject sequence when calculating global percent
identity. For subject sequences truncated at the N- and C-termini,
relative to the query sequence, the percent identity is corrected
by calculating the number of residues of the query sequence that
are N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. Whether a residue is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of the present invention. Only residues to the N- and C-termini of
the subject sequence, which are not matched/aligned with the query
sequence, are considered for the purposes of manually adjusting the
percent identity score. That is, only query residue positions
outside the farthest N- and C-terminal residues of the subject
sequence.
[0726] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The
10 unpaired residues represent 10% of the sequence (number of
residues at the N- and C-termini not matched/total number of
residues in the query sequence) so 10% is subtracted from the
percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequnce are manually corrected for.
No other manual corrections are to made for the purposes of the
present invention.
[0727] The variants may contain alterations in the coding regions,
non-coding regions, or both. Especially preferred are
polynucleotide variants containing alterations which produce silent
substitutions, additions, or deletions, but do not alter the
properties or activities of the encoded polypeptide. Nucleotide
variants produced by silent substitutions due to the degeneracy of
the genetic code are preferred. Moreover, variants in which 5-10,
1-5, or 1-2 amino acids are substituted, deleted, or added in any
combination are also preferred. Polynucleotide variants can be
produced for a variety of reasons, e.g., to optimize codon
expression for a particular host (change codons in the human MRNA
to those preferred by a bacterial host such as E. coli).
[0728] Naturally occurring variants are called "allelic variants,"
and refer to one of several alternate forms of a gene occupying a
given locus on a chromosome of an organism. (Genes II, Lewin, B.,
ed., John Wiley & Sons, New York (1985).) These allelic
variants can vary at either the polynucleotide and/or polypeptide
level and are included in the present invention. Alternatively,
non-naturally occurring variants may be produced by mutagenesis
techniques or by direct synthesis.
[0729] Using known methods of protein engineering and recombinant
DNA technology, variants may be generated to improve or alter the
characteristics of the polypeptides of the present invention. For
instance, one or more amino acids can be deleted from the
N-terminus or C-terminus of the secreted protein without
substantial loss of biological function. The authors of Ron et al.,
J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGF proteins
having heparin binding activity even after deleting 3, 8, or 27
amino-terminal amino acid residues. Similarly, Interferon gamma
exhibited up to ten times higher activity after deleting 8-10 amino
acid residues from the carboxy terminus of this protein. (Dobeli et
al., J. Biotechnology 7:199-216 (1988).)
[0730] Moreover, ample evidence demonstrates that variants often
retain a biological activity similar to that of the naturally
occurring protein. For example, Gayle and coworkers (J. Biol. Chem
268:22105-22111 (1993)) conducted extensive mutational analysis of
human cytokine IL-1a. They used random mutagenesis to generate over
3,500 individual IL-1a mutants that averaged 2.5 amino acid changes
per variant over the entire length of the molecule. Multiple
mutations were examined at every possible amino acid position. The
investigators found that "[m]ost of the molecule could be altered
with little effect on either [binding or biological activity]."
(See, Abstract.) In fact, only 23 unique amino acid sequences, out
of more than 3,500 nucleotide sequences examined, produced a
protein that significantly differed in activity from wild-type.
[0731] Furthermore, even if deleting one or more amino acids from
the N-terminus or C-terminus of a polypeptide results in
modification or loss of one or more biological functions, other
biological activities may still be retained. For example, the
ability of a deletion variant to induce and/or to bind antibodies
which recognize the secreted form will likely be retained when less
than the majority of the residues of the secreted form are removed
from the N-terminus or C-terminus. Whether a particular polypeptide
lacking N- or C-terminal residues of a protein retains such
immunogenic activities can readily be determined by routine methods
described herein and otherwise known in the art.
[0732] Thus, the invention further includes polypeptide variants
which show substantial biological activity. Such variants include
deletions, insertions, inversions, repeats, and substitutions
selected according to general rules known in the art so as have
little effect on activity. For example, guidance concerning how to
make phenotypically silent amino acid substitutions is provided in
Bowie et al., Science 247:1306-1310 (1990), wherein the authors
indicate that there are two main strategies for studying the
tolerance of an amino acid sequence to change.
[0733] The first strategy exploits the tolerance of amino acid
substitutions by natural selection during the process of evolution.
By comparing amino acid sequences in different species, conserved
amino acids can be identified. These conserved amino acids are
likely important for protein function. In contrast, the amino acid
positions where substitutions have been tolerated by natural
selection indicates that these positions are not critical for
protein function. Thus, positions tolerating amino acid
substitution could be modified while still maintaining biological
activity of the protein.
[0734] The second strategy uses genetic engineering to introduce
amino acid changes at specific positions of a cloned gene to
identify regions critical for protein function. For example, site
directed mutagenesis or alanine-scanning mutagenesis (introduction
of single alanine mutations at every residue in the molecule) can
be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The
resulting mutant molecules can then be tested for biological
activity.
[0735] As the authors state, these two strategies have revealed
that proteins are surprisingly tolerant of amino acid
substitutions. The authors further indicate which amino acid
changes are likely to be permissive at certain amino acid positions
in the protein. For example, most buried (within the tertiary
structure of the protein) amino acid residues require nonpolar side
chains, whereas few features of surface side chains are generally
conserved. Moreover, tolerated conservative amino acid
substitutions involve replacement of the aliphatic or hydrophobic
amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl
residues Ser and Thr; replacement of the acidic residues Asp and
Glu; replacement of the amide residues Asn and Gln, replacement of
the basic residues Lys, Arg, and His; replacement of the aromatic
residues Phe, Tyr, and Trp, and replacement of the small-sized
amino acids Ala, Ser, Thr, Met, and Gly.
[0736] Besides conservative amino acid substitution, variants of
the present invention include (i) substitutions with one or more of
the non-conserved amino acid residues, where the substituted amino
acid residues may or may not be one encoded by the genetic code, or
(ii) substitution with one or more of amino acid residues having a
substituent group, or (iii) fusion of the mature polypeptide with
another compound, such as a compound to increase the stability
and/or solubility of the polypeptide (for example, polyethylene
glycol), or (iv) fusion of the polypeptide with additional amino
acids, such as, for example, an IgG Fc fusion region peptide, or
leader or secretory sequence, or a sequence facilitating
purification. Such variant polypeptides are deemed to be within the
scope of those skilled in the art from the teachings herein.
[0737] For example, polypeptide variants containing amino acid
substitutions of charged amino acids with other charged or neutral
amino acids may produce proteins with improved characteristics,
such as less aggregation. Aggregation of pharmaceutical
formulations both reduces activity and increases clearance due to
the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp.
Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845
(1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993).)
[0738] A further embodiment of the invention relates to a
polypeptide which comprises the amino acid sequence of the present
invention having an amino acid sequence which contains at least one
amino acid substitution, but not more than 50 amino acid
substitutions, even more preferably, not more than 40 amino acid
substitutions, still more preferably, not more than 30 amino acid
substitutions, and still even more preferably, not more than 20
amino acid substitutions. Of course, in order of ever-increasing
preference, it is highly preferable for a peptide or polypeptide to
have an amino acid sequence which comprises the amino acid sequence
of the present invention, which contains at least one, but not more
than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In
specific embodiments, the number of additions, substitutions,
and/or deletions in the amino acid sequence of the present
invention or fragments thereof (e.g., the mature form and/or other
fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or
50-150, conservative amino acid substitutions are preferable.
[0739] Polynucleotide and Polypeptide Fragments
[0740] The present invention is also directed to polynucleotide
fragments of the polynucleotides of the invention.
[0741] In the present invention, a "polynucleotide fragment" refers
to a short polynucleotide having a nucleic acid sequence which: is
a portion of that contained in a deposited clone, or encoding the
polypeptide encoded by the cDNA in a deposited clone; is a portion
of that shown in SEQ ID NO:X or the complementary strand thereto,
or is a portion of a polynucleotide sequence encoding the
polypeptide of SEQ ID NO:Y. The nucleotide fragments of the
invention are preferably at least about 15 nt, and more preferably
at least about 20 nt, still more preferably at least about 30 nt,
and even more preferably, at least about 40 nt, at least about 50
nt, at least about 75 nt, or at least about 150 nt in length. A
fragment "at least 20 nt in length," for example, is intended to
include 20 or more contiguous bases from the cDNA sequence
contained in a deposited clone or the nucleotide sequence shown in
SEQ ID NO:X. In this context "about" includes the particularly
recited value, a value larger or smaller by several (5, 4, 3, 2, or
1) nucleotides, at either terminus or at both termini. These
nucleotide fragments have uses that include, but are not limited
to, as diagnostic probes and primers as discussed herein. Of
course, larger fragments (e.g., 50, 150, 500, 600, 2000
nucleotides) are preferred.
[0742] Moreover, representative examples of polynucleotide
fragments of the invention, include, for example, fragments
comprising, or alternatively consisting of, a sequence from about
nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300,
301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700,
701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050,
1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350,
1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650,
1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950,
1951-2000, or 2001 to the end of SEQ ID NO:X, or the complementary
strand thereto, or the cDNA contained in a deposited clone. In this
context "about" includes the particularly recited ranges, and
ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides,
at either terminus or at both termini. Preferably, these fragments
encode a polypeptide which has biological activity. More
preferably, these polynucleotides can be used as probes or primers
as discussed herein. Polynucleotides which hybridize to these
nucleic acid molecules under stringent hybridization conditions or
lower stringency conditions are also encompassed by the invention,
as are polypeptides encoded by these polynucleotides.
[0743] In the present invention, a "polypeptide fragment" refers to
an amino acid sequence which is a portion of that contained in SEQ
ID NO:Y or encoded by the cDNA contained in a deposited clone.
Protein (polypeptide) fragments may be "free-standing," or
comprised within a larger polypeptide of which the fragment forms a
part or region, most preferably as a single continuous region.
Representative examples of polypeptide fragments of the invention,
include, for example, fragments comprising, or alternatively
consisting of, from about amino acid number 1-20, 21-40, 41-60,
61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the
coding region. Moreover, polypeptide fragments can be about 20, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids
in length. In this context "about" includes the particularly
recited ranges or values, and ranges or values larger or smaller by
several (5, 4, 3, 2, or 1) amino acids, at either extreme or at
both extremes. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0744] Preferred polypeptide fragments include the secreted protein
as well as the mature form. Further preferred polypeptide fragments
include the secreted protein or the mature form having a continuous
series of deleted residues from the amino or the carboxy terminus,
or both. For example, any number of amino acids, ranging from 1-60,
can be deleted from the amino terminus of either the secreted
polypeptide or the mature form. Similarly, any number of amino
acids, ranging from 1-30, can be deleted from the carboxy terminus
of the secreted protein or mature form. Furthermore, any
combination of the above amino and carboxy terminus deletions are
preferred. Similarly, polynucleotides encoding these polypeptide
fragments are also preferred.
[0745] Also preferred are polypeptide and polynucleotide fragments
characterized by structural or functional domains, such as
fragments that comprise alpha-helix and alpha-helix forming
regions, beta-sheet and beta-sheet-forming regions, turn and
turn-forming regions, coil and coil-forming regions, hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions,
substrate binding region, and high antigenic index regions.
Polypeptide fragments of SEQ ID NO:Y falling within conserved
domains are specifically contemplated by the present invention.
Moreover, polynucleotides encoding these domains are also
contemplated.
[0746] Other preferred polypeptide fragments are biologically
active fragments. Biologically active fragments are those
exhibiting activity similar, but not necessarily identical, to an
activity of the polypeptide of the present invention. The
biological activity of the fragments may include an improved
desired activity, or a decreased undesirable activity.
Polynucleotides encoding these polypeptide fragments are also
encompassed by the invention.
[0747] Preferably, the polynucleotide fragments of the invention
encode a polypeptide which demonstrates a functional activity. By a
polypeptide demonstrating a "functional activity" is meant, a
polypeptide capable of displaying one or more known functional
activities associated with a full-length (complete) polypeptide of
invention protein. Such functional activities include, but are not
limited to, biological activity, antigenicity [ability to bind (or
compete with a polypeptide of the invention for binding) to an
antibody to the polypeptide of the invention], immunogenicity
(ability to generate antibody which binds to a polypeptide of the
invention), ability to form multimers with polypeptides of the
invention, and ability to bind to a receptor or ligand for a
polypeptide of the invention.
[0748] The functional activity of polypeptides of the invention,
and fragments, variants derivatives, and analogs thereof, can be
assayed by various methods.
[0749] For example, in one embodiment where one is assaying for the
ability to bind or compete with full-length polypeptide of the
invention for binding to an antibody of the polypeptide of the
invention, various immunoassays known in the art can be used,
including but not limited to, competitive and non-competitive assay
systems using techniques such as radioimmunoassays, ELISA (enzyme
linked immunosorbent assay), "sandwich" immunoassays,
immunoradiometric assays, gel diffusion precipitation reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold,
enzyme or radioisotope labels, for example), western blots,
precipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays), complement fixation
assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody
binding is detected by detecting a label on the primary antibody.
In another embodiment, the primary antibody is detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In a further embodiment, the secondary antibody is
labeled. Many means are known in the art for detecting binding in
an immunoassay and are within the scope of the present
invention.
[0750] In another embodiment, where a ligand for a polypeptide of
the invention identified, or the ability of a polypeptide fragment,
variant or derivative of the invention to multimerize is being
evaluated, binding can be assayed, e.g., by means well-known in the
art, such as, for example, reducing and non-reducing gel
chromatography, protein affinity chromatography, and affinity
blotting. See generally, Phizicky, E., et al., 1995, Microbiol.
Rev. 59:94-123. In another embodiment, physiological correlates of
binding of a polypeptide of the invention to its substrates (signal
transduction) can be assayed.
[0751] In addition, assays described herein (see Examples) and
otherwise known in the art may routinely be applied to measure the
ability of polypeptides of the invention and fragments, variants
derivatives and analogs thereof to elicit related biological
activity related to that of the polypeptide of the invention
(either in vitro or in vivo). Other methods will be known to the
skilled artisan and are within the scope of the invention.
[0752] Epitopes and Antibodies
[0753] The present invention encompasses polypeptides comprising,
or alternatively consisting of, an epitope of the polypeptide
having an amino acid sequence of SEQ ID NO:Y, or an epitope of the
polypeptide sequence encoded by a polynucleotide sequence contained
in ATCC deposit No. Z or encoded by a polynucleotide that
hybridizes to the complement of the sequence of SEQ ID NO:X or
contained in ATCC deposit No. Z under stringent hybridization
conditions or lower stringency hybridization conditions as defined
supra. The present invention further encompasses polynucleotide
sequences encoding an epitope of a polypeptide sequence of the
invention (such as, for example, the sequence disclosed in SEQ ID
NO:X), polynucleotide sequences of the complementary strand of a
polynucleotide sequence encoding an epitope of the invention, and
polynucleotide sequences which hybridize to the complementary
strand under stringent hybridization conditions or lower stringency
hybridization conditions defined supra.
[0754] The term "epitopes," as used herein, refers to portions of a
polypeptide having antigenic or immunogenic activity in an animal,
preferably a mammal, and most preferably in a human. In a preferred
embodiment, the present invention encompasses a polypeptide
comprising an epitope, as well as the polynucleotide encoding this
polypeptide. An "immunogenic epitope," as used herein, is defined
as a portion of a protein that elicits an antibody response in an
animal, as determined by any method known in the art, for example,
by the methods for generating antibodies described infra. (See, for
example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002
(1983)). The term "antigenic epitope," as used herein, is defined
as a portion of a protein to which an antibody can
immunospecifically bind its antigen as determined by any method
well known in the art, for example, by the immunoassays described
herein. Immunospecific binding excludes non-specific binding but
does not necessarily exclude cross-reactivity with other antigens.
Antigenic epitopes need not necessarily be immunogenic.
[0755] Fragments which function as epitopes may be produced by any
conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci.
USA 82:5131-5135 (1985), further described in U.S. Pat. No.
4,631,211).
[0756] In the present invention, antigenic epitopes preferably
contain a sequence of at least 4, at least 5, at least 6, at least
7, more preferably at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least
20, at least 25, at least 30, at least 40, at least 50, and, most
preferably, between about 15 to about 30 amino acids. Preferred
polypeptides comprising immunogenic or antigenic epitopes are at
least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, or 100 amino acid residues in length. Additional
non-exclusive preferred antigenic epitopes include the antigenic
epitopes disclosed herein, as well as portions thereof. Antigenic
epitopes are useful, for example, to raise antibodies, including
monoclonal antibodies, that specifically bind the epitope.
Preferred antigenic epitopes include the antigenic epitopes
disclosed herein, as well as any combination of two, three, four,
five or more of these antigenic epitopes. Antigenic epitopes can be
used as the target molecules in immunoassays. (See, for instance,
Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science
219:660-666 (1983)).
[0757] Similarly, immunogenic epitopes can be used, for example, to
induce antibodies according to methods well known in the art. (See,
for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow
et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al.,
J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes
include the immunogenic epitopes disclosed herein, as well as any
combination of two, three, four, five or more of these immunogenic
epitopes. The polypeptides comprising one or more immunogenic
epitopes may be presented for eliciting an antibody response
together with a carrier protein, such as an albumin, to an animal
system (such as rabbit or mouse), or, if the polypeptide is of
sufficient length (at least about 25 amino acids), the polypeptide
may be presented without a carrier. However, immunogenic epitopes
comprising as few as 8 to 10 amino acids have been shown to be
sufficient to raise antibodies capable of binding to, at the very
least, linear epitopes in a denatured polypeptide (e.g., in Western
blotting).
[0758] Epitope-bearing polypeptides of the present invention may be
used to induce antibodies according to methods well known in the
art including, but not limited to, in vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe et
al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-2354 (1985). If in vivo immunization is used,
animals may be immunized with free peptide; however, anti-peptide
antibody titer may be boosted by coupling the peptide to a
macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or
tetanus toxoid. For instance, peptides containing cysteine residues
may be coupled to a carrier using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.g of peptide or carrier protein
and Freund's adjuvant or any other adjuvant known for stimulating
an immune response. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0759] As one of skill in the art will appreciate, and as discussed
above, the polypeptides of the present invention comprising an
immunogenic or antigenic epitope can be fused to other polypeptide
sequences. For example, the polypeptides of the present invention
may be fused with the constant domain of immunoglobulins (IgA, IgE,
IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination
thereof and portions thereof) resulting in chimeric polypeptides.
Such fusion proteins may facilitate purification and may increase
half-life in vivo. This has been shown for chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins. See, e.g., EP 394,827;
Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of
an antigen across the epithelial barrier to the immune system has
been demonstrated for antigens (e.g., insulin) conjugated to an
FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT
Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that
have a disulfide-linked dimeric structure due to the IgG portion
desulfide bonds have also been found to be more efficient in
binding and neutralizing other molecules than monomeric
polypeptides or fragments thereof alone. See, e.g., Fountoulakis et
al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the
above epitopes can also be recombined with a gene of interest as an
epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid
in detection and purification of the expressed polypeptide. For
example, a system described by Janknecht et al. allows for the
ready purification of non-denatured fusion proteins expressed in
human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci.
USA 88:8972-897). In this system, the gene of interest is subcloned
into a vaccinia recombination plasmid such that the open reading
frame of the gene is translationally fused to an amino-terminal tag
consisting of six histidine residues. The tag serves as a matrix
binding domain for the fusion protein. Extracts from cells infected
with the recombinant vaccinia virus are loaded onto Ni2+
nitriloacetic acid-agarose column and histidine-tagged proteins can
be selectively eluted with imidazole-containing buffers.
[0760] Additional fusion proteins of the invention may be generated
through the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling"). DNA shuffling may be employed to modulate the
activities of polypeptides of the invention, such methods can be
used to generate polypeptides with altered activity, as well as
agonists and antagonists of the polypeptides. See, generally, U.S.
Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and
5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33
(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson,
et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco,
Biotechniques 24(2):308-13 (1998) (each of these patents and
publications are hereby incorporated by reference in its entirety).
In one embodiment, alteration of polynucleotides corresponding to
SEQ ID NO:X and the polypeptides encoded by these polynucleotides
may be achieved by DNA shuffling. DNA shuffling involves the
assembly of two or more DNA segments by homologous or site-specific
recombination to generate variation in the polynucleotide sequence.
In another embodiment, polynucleotides of the invention, or the
encoded polypeptides, may be altered by being subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or
other methods prior to recombination. In another embodiment, one or
more components, motifs, sections, parts, domains, fragments, etc.,
of a polynucleotide encoding a polypeptide of the invention may be
recombined with one or more components, motifs, sections, parts,
domains, fragments, etc. of one or more heterologous molecules.
[0761] Antibodies
[0762] Further polypeptides of the invention relate to antibodies
and T-cell antigen receptors (TCR) which immunospecifically bind a
polypeptide, polypeptide fragment, or variant of SEQ ID NO:Y,
and/or an epitope, of the present invention (as determined by
immunoassays well known in the art for assaying specific
antibody-antigen binding). Antibodies of the invention include, but
are not limited to, polyclonal, monoclonal, multispecific, human,
humanized or chimeric antibodies, single chain antibodies, Fab
fragments, F(ab') fragments, fragments produced by a Fab expression
library, anti-idiotypic (anti-Id) antibodies (including, e.g.,
anti-Id antibodies to antibodies of the invention), and
epitope-binding fragments of any of the above. The term "antibody,"
as used herein, refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e.,
molecules that contain an antigen binding site that
immunospecifically binds an antigen. The immunoglobulin molecules
of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA
and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or
subclass of immunoglobulin molecule.
[0763] Most preferably the antibodies are human antigen-binding
antibody fragments of the present invention and include, but are
not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv),
single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments
comprising either a VL or VH domain. Antigen-binding antibody
fragments, including single-chain antibodies, may comprise the
variable region(s) alone or in combination with the entirety or a
portion of the following: hinge region, CH1, CH2, and CH3 domains.
Also included in the invention are antigen-binding fragments also
comprising any combination of variable region(s) with a hinge
region, CH1, CH2, and CH3 domains. The antibodies of the invention
may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine (e.g., mouse and rat),
donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As
used herein, "human" antibodies include antibodies having the amino
acid sequence of a human immunoglobulin and include antibodies
isolated from human immunoglobulin libraries or from animals
transgenic for one or more human immunoglobulin and that do not
express endogenous immunoglobulins, as described infra and, for
example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
[0764] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for a
heterologous epitope, such as a heterologous polypeptide or solid
support material. See, e.g., PCT publications WO 93/17715; WO
92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;
5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553
(1992).
[0765] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention which they recognize or specifically bind.
The epitope(s) or polypeptide portion(s) may be specified as
described herein, e.g., by N-terminal and C-terminal positions, by
size in contiguous amino acid residues, or listed in the Tables and
Figures. Antibodies which specifically bind any epitope or
polypeptide of the present invention may also be excluded.
Therefore, the present invention includes antibodies that
specifically bind polypeptides of the present invention, and allows
for the exclusion of the same.
[0766] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homolog of a polypeptide of
the present invention are included. Antibodies that bind
polypeptides with at least 95%, at least 90%, at least 85%, at
least 80%, at least 75%, at least 70%, at least 65%, at least 60%,
at least 55%, and at least 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
In specific embodiments, antibodies of the present invention
cross-react with murine, rat and/or rabbit homologs of human
proteins and the corresponding epitopes thereof. Antibodies that do
not bind polypeptides with less than 95%, less than 90%, less than
85%, less than 80%, less than 75%, less than 70%, less than 65%,
less than 60%, less than 55%, and less than 50% identity (as
calculated using methods known in the art and described herein) to
a polypeptide of the present invention are also included in the
present invention. In a specific embodiment, the above-described
cross-reactivity is with respect to any single specific antigenic
or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or
more of the specific antigenic and/or immunogenic polypeptides
disclosed herein. Further included in the present invention are
antibodies which bind polypeptides encoded by polynucleotides which
hybridize to a polynucleotide of the present invention under
stringent hybridization conditions (as described herein).
Antibodies of the present invention may also be described or
specified in terms of their binding affinity to a polypeptide of
the invention. Preferred binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-2 M,
10.sup.-2 M, 5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M,
10.sup.-4 M, 5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M,
10.sup.-6M, 5.times.10.sup.-7 M, 10.sup.7 M, 5.times.10.sup.-8 M,
10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10
M, 10.sup.10 M, 5.times.10.sup.-11 M, 10.sup.-11 M,
5.times.10.sup.-12 M, .sup.10-12 M, 5.times.10.sup.13 M, 10.sup.13
M, 5.times.10.sup.14 M, 10.sup.14 M, 5.times.10.sup.-15 M, or
10.sup.-15 M.
[0767] The invention also provides antibodies that competitively
inhibit binding of an antibody to an epitope of the invention as
determined by any method known in the art for determining
competitive binding, for example, the immunoassays described
herein. In preferred embodiments, the antibody competitively
inhibits binding to the epitope by at least 95%, at least 90%, at
least 85%, at least 80%, at least 75%, at least 70%, at least 60%,
or at least 50%.
[0768] Antibodies of the present invention may act as agonists or
antagonists of the polypeptides of the present invention. For
example, the present invention includes antibodies which disrupt
the receptor/ligand interactions with the polypeptides of the
invention either partially or fully. Preferrably, antibodies of the
present invention bind an antigenic epitope disclosed herein, or a
portion thereof. The invention features both receptor-specific
antibodies and ligand-specific antibodies. The invention also
features receptor-specific antibodies which do not prevent ligand
binding but prevent receptor activation. Receptor activation (i.e.,
signaling) may be determined by techniques described herein or
otherwise known in the art. For example, receptor activation can be
determined by detecting the phosphorylation (e.g., tyrosine or
serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example,
as described supra). In specific embodiments, antibodies are
provided that inhibit ligand activity or receptor activity by at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%,
at least 70%, at least 60%, or at least 50% of the activity in
absence of the antibody.
[0769] The invention also features receptor-specific antibodies
which both prevent ligand binding and receptor activation as well
as antibodies that recognize the receptor-ligand complex, and,
preferably, do not specifically recognize the unbound receptor or
the unbound ligand. Likewise, included in the invention are
neutralizing antibodies which bind the ligand and prevent binding
of the ligand to the receptor, as well as antibodies which bind the
ligand, thereby preventing receptor activation, but do not prevent
the ligand from binding the receptor. Further included in the
invention are antibodies which activate the receptor. These
antibodies may act as receptor agonists, i.e., potentiate or
activate either all or a subset of the biological activities of the
ligand-mediated receptor activation, for example, by inducing
dimerization of the receptor. The antibodies may be specified as
agonists, antagonists or inverse agonists for biological activities
comprising the specific biological activities of the peptides of
the invention disclosed herein. The above antibody agonists can be
made using methods known in the art. See, e.g., PCT publication WO
96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood
92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678
(1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et
al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol.
160(7):3170-3179 (1998); Prat et al., J. Cell. Sci.
111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods
205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241
(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);
Taryman et al., Neuron 14(4):755-762 (1995); Muller et al.,
Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine
8(1):14-20 (1996) (which are all incorporated by reference herein
in their entireties).
[0770] Antibodies of the present invention may be used, for
example, but not limited to, to purify, detect, and target the
polypeptides of the present invention, including both in vitro and
in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in immunoassays for qualitatively and
quantitatively measuring levels of the polypeptides of the present
invention in biological samples. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its
entirety).
[0771] As discussed in more detail below, the antibodies of the
present invention may be used either alone or in combination with
other compositions. The antibodies may further be recombinantly
fused to a heterologous polypeptide at the N- or C-terminus or
chemically conjugated (including covalently and non-covalently
conjugations) to polypeptides or other compositions. For example,
antibodies of the present invention may be recombinantly fused or
conjugated to molecules useful as labels in detection assays and
effector molecules such as heterologous polypeptides, drugs,
radionuclides, or toxins. See, e.g., PCT publications WO 92/08495;
WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP
396,387.
[0772] The antibodies of the invention include derivatives that are
modified, i.e, by the covalent attachment of any type of molecule
to the antibody such that covalent attachment does not prevent the
antibody from generating an anti-idiotypic response. For example,
but not by way of limitation, the antibody derivatives include
antibodies that have been modified, e.g., by glycosylation,
acetylation, pegylation, phosphylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical
amino acids.
[0773] The antibodies of the present invention may be generated by
any suitable method known in the art. Polyclonal antibodies to an
antigen-of-interest can be produced by various procedures well
known in the art. For example, a polypeptide of the invention can
be administered to various host animals including, but not limited
to, rabbits, mice, rats, etc. to induce the production of sera
containing polyclonal antibodies specific for the antigen. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and include but are not limited to,
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
Such adjuvants are also well known in the art.
[0774] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0775] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art
and are discussed in detail in the Examples (e.g., Example 16). In
a non-limiting example, mice can be immunized with a polypeptide of
the invention or a cell expressing such peptide. Once an immune
response is detected, e.g., antibodies specific for the antigen are
detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well known
techniques to any suitable myeloma cells, for example cells from
cell line SP20 available from the ATCC. Hybridomas are selected and
cloned by limited dilution. The hybridoma clones are then assayed
by methods known in the art for cells that secrete antibodies
capable of binding a polypeptide of the invention. Ascites fluid,
which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0776] Accordingly, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention.
[0777] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
[0778] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular embodiment,
such phage can be utilized to display antigen binding domains
expressed from a repertoire or combinatorial antibody library
(e.g., human or murine). Phage expressing an antigen binding domain
that binds the antigen of interest can be selected or identified
with antigen, e.g., using labeled antigen or antigen bound or
captured to a solid surface or bead. Phage used in these methods
are typically filamentous phage including fd and M13 binding
domains expressed from phage with Fab, Fv or disulfide stabilized
Fv antibody domains recombinantly fused to either the phage gene
III or gene VIII protein. Examples of phage display methods that
can be used to make the antibodies of the present invention include
those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50
(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);
Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et
al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology
57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT
publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO
93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743
and 5,969,108; each of which is incorporated herein by reference in
its entirety.
[0779] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988) (said
references incorporated by reference in their entireties).
[0780] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. A chimeric antibody is a molecule in which
different portions of the antibody are derived from different
animal species, such as antibodies having a variable region derived
from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567;
and 4,816397, which are incorporated herein by reference in their
entirety. Humanized antibodies are antibody molecules from
non-human species antibody that binds the desired antigen having
one or more complementarity determining regions (CDRs) from the
non-human species and a framework regions from a human
immunoglobulin molecule. Often, framework residues in the human
framework regions will be substituted with the corresponding
residue from the CDR donor antibody to alter, preferably improve,
antigen binding. These framework substitutions are identified by
methods well known in the art, e.g., by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089;
Riechmann et al., Nature 332:323 (1988), which are incorporated
herein by reference in their entireties.) Antibodies can be
humanized using a variety of techniques known in the art including,
for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967;
U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or
resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology
28(4/5):489-498 (1991); Studnicka et al., Protein Engineering
7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and
chain shuffling (U.S. Pat. No. 5,565,332).
[0781] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See also, U.S. Pat. Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO
98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and
WO 91/10741; each of which is incorporated herein by reference in
its entirety.
[0782] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring which express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar,
Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO
96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; and 5,939,598, which are incorporated by
reference herein in their entirety. In addition, companies such as
Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.)
can be engaged to provide human antibodies directed against a
selected antigen using technology similar to that described
above.
[0783] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., Bio/technology 12:899-903 (1988)).
[0784] Further, antibodies to the polypeptides of the invention
can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic" polypeptides of the invention using techniques well known
to those skilled in the art. (See, e.g., Greenspan & Bona,
FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol.
147(8):2429-2438 (1991)). For example, antibodies which bind to and
competitively inhibit polypeptide multimerization and/or binding of
a polypeptide of the invention to a ligand can be used to generate
anti-idiotypes that "mimic" the polypeptide multimerization and/or
binding domain and, as a consequence, bind to and neutralize
polypeptide and/or its ligand. Such neutralizing anti-idiotypes or
Fab fragments of such anti-idiotypes can be used in therapeutic
regimens to neutralize polypeptide ligand. For example, such
anti-idiotypic antibodies can be used to bind a polypeptide of the
invention and/or to bind its ligands/receptors, and thereby block
its biological activity.
[0785] Polynucleotides Encoding Antibodies
[0786] The invention further provides polynucleotides comprising a
nucleotide sequence encoding an antibody of the invention and
fragments thereof. The invention also encompasses polynucleotides
that hybridize under stringent or lower stringency hybridization
conditions, e.g., as defined supra, to polynucleotides that encode
an antibody, preferably, that specifically binds to a polypeptide
of the invention, preferably, an antibody that binds to a
polypeptide having the amino acid sequence of SEQ ID NO:Y.
[0787] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. For example, if the nucleotide sequence of the antibody is
known, a polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligating of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[0788] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library, or a cDNA library generated from, or nucleic acid,
preferably poly A+ RNA, isolated from, any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody of the invention) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR may then be cloned into replicable cloning vectors
using any method well known in the art.
[0789] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody is determined, the nucleotide sequence of
the antibody may be manipulated using methods well known in the art
for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998,
Current Protocols in Molecular Biology, John Wiley & Sons, NY,
which are both incorporated by reference herein in their entireties
), to generate antibodies having a different amino acid sequence,
for example to create amino acid substitutions, deletions, and/or
insertions.
[0790] In a specific embodiment, the amino acid sequence of the
heavy and/or light chain variable domains may be inspected to
identify the sequences of the complementarity determining regions
(CDRs) by methods that are well know in the art, e.g., by
comparison to known amino acid sequences of other heavy and light
chain variable regions to determine the regions of sequence
hypervariability. Using routine recombinant DNA techniques, one or
more of the CDRs may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody, as
described supra. The framework regions may be naturally occurring
or consensus framework regions, and preferably human framework
regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479
(1998) for a listing of human framework regions). Preferably, the
polynucleotide generated by the combination of the framework
regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one
or more amino acid substitutions may be made within the framework
regions, and, preferably, the amino acid substitutions improve
binding of the antibody to its antigen. Additionally, such methods
may be used to make amino acid substitutions or deletions of one or
more variable region cysteine residues participating in an
intrachain disulfide bond to generate antibody molecules lacking
one or more intrachain disulfide bonds. Other alterations to the
polynucleotide are encompassed by the present invention and within
the skill of the art.
[0791] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci.
81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984);
Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[0792] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science
242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can
be adapted to produce single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide. Techniques for the assembly of functional
Fv fragments in E. coli may also be used (Skerra et al., Science
242:1038-1041 (1988)).
[0793] Methods of Producing Antibodies
[0794] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombinant
expression techniques.
[0795] Recombinant expression of an antibody of the invention, or
fragment, derivative or analog thereof, (e.g., a heavy or light
chain of an antibody of the invention or a single chain antibody of
the invention), requires construction of an expression vector
containing a polynucleotide that encodes the antibody. Once a
polynucleotide encoding an antibody molecule or a heavy or light
chain of an antibody, or portion thereof (preferably containing the
heavy or light chain variable domain), of the invention has been
obtained, the vector for the production of the antibody molecule
may be produced by recombinant DNA technology using techniques well
known in the art. Thus, methods for preparing a protein by
expressing a polynucleotide containing an antibody encoding
nucleotide sequence are described herein. Methods which are well
known to those skilled in the art can be used to construct
expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule of
the invention, or a heavy or light chain thereof, or a heavy or
light chain variable domain, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464)
and the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy or light chain.
[0796] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the invention.
Thus, the invention includes host cells containing a polynucleotide
encoding an antibody of the invention, or a heavy or light chain
thereof, or a single chain antibody of the invention, operably
linked to a heterologous promoter. In preferred embodiments for the
expression of double-chained antibodies, vectors encoding both the
heavy and light chains may be co-expressed in the host cell for
expression of the entire immunoglobulin molecule, as detailed
below.
[0797] A variety of host-expression vector systems may be utilized
to express the antibody molecules of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody molecule, are used for the expression of
a recombinant antibody molecule. For example, mammalian cells such
as Chinese hamster ovary cells (CHO), in conjunction with a vector
such as the major intermediate early gene promoter element from
human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,
Bio/Technology 8:2 (1990)).
[0798] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited, to the E. coli expression vector pUR278
(Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody
coding sequence may be ligated individually into the vector in
frame with the lac Z coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
24:5503-5509 (1989)); and the like. pGEX vectors may also be used
to express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption and
binding to matrix glutathione-agarose beads followed by elution in
the presence of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the
cloned target gene product can be released from the GST moiety.
[0799] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter).
[0800] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non- essential
region of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts. (e.g., see Logan & Shenk,
Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544 (1987)).
[0801] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell
lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal mammary gland cell line such as, for example, CRL7030 and
Hs578Bst.
[0802] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compounds that interact directly or indirectly
with the antibody molecule.
[0803] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:202 (1992)), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to
the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
1993, TIB TECH 11(5):155-215); and hygro, which confers resistance
to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods
commonly known in the art of recombinant DNA technology may be
routinely applied to select the desired recombinant clone, and such
methods are described, for example, in Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,
Dracopoli et al. (eds), Current Protocols in Human Genetics, John
Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981), which are incorporated by reference herein in their
entireties.
[0804] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning,
Vol.3. (Academic Press, New York, 1987)). When a marker in the
vector system expressing antibody is amplifiable, increase in the
level of inhibitor present in culture of host cell will increase
the number of copies of the marker gene. Since the amplified region
is associated with the antibody gene, production of the antibody
will also increase (Crouse et al., Mol. Cell. Biol. 3:257
(1983)).
[0805] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes, and is capable of expressing, both heavy and light
chain polypeptides. In such situations, the light chain should be
placed before the heavy chain to avoid an excess of toxic free
heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl.
Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy
and light chains may comprise cDNA or genomic DNA.
[0806] Once an antibody molecule of the invention has been produced
by an animal, chemically synthesized, or recombinantly expressed,
it may be purified by any method known in the art for purification
of an immunoglobulin molecule, for example, by chromatography
(e.g., ion exchange, affinity, particularly by affinity for the
specific antigen after Protein A, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins. In
addition, the antibodies of the present invention or fragments
thereof can be fused to heterologous polypeptide sequences
described herein or otherwise known in the art, to facilitate
purification.
[0807] The present invention encompasses antibodies recombinantly
fused or chemically conjugated (including both covalently and
non-covalently conjugations) to a polypeptide (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention to generate
fusion proteins. The fusion does not necessarily need to be direct,
but may occur through linker sequences. The antibodies may be
specific for antigens other than polypeptides (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention. For example,
antibodies may be used to target the polypeptides of the present
invention to particular cell types, either in vitro or in vivo, by
fusing or conjugating the polypeptides of the present invention to
antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to the polypeptides of the present
invention may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095;
Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No.
5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al.,
J. Immunol. 146:2446-2452(1991), which are incorporated by
reference in their entireties.
[0808] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region, or portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the constant region, hinge region, CH1 domain, CH2
domain, and CH3 domain or any combination of whole domains or
portions thereof. The polypeptides may also be fused or conjugated
to the above antibody portions to form multimers. For example, Fc
portions fused to the polypeptides of the present invention can
form dimers through disulfide bonding between the Fc portions.
Higher multimeric forms can be made by fusing the polypeptides to
portions of IgA and IgM. Methods for fusing or conjugating the
polypeptides of the present invention to antibody portions are
known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929;
5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166;
PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc.
Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J.
Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad.
Sci. USA 89:11337-11341(1992) (said references incorporated by
reference in their entireties).
[0809] As discussed, supra, the polypeptides corresponding to a
polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may
be fused or conjugated to the above antibody portions to increase
the in vivo half life of the polypeptides or for use in
immunoassays using methods known in the art. Further, the
polypeptides corresponding to SEQ ID NO:Y may be fused or
conjugated to the above antibody portions to facilitate
purification. One reported example describes chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins. (EP 394,827; Traunecker et
al., Nature 331:84-86 (1988). The polypeptides of the present
invention fused or conjugated to an antibody having
disulfide-linked dimeric structures (due to the IgG) may also be
more efficient in binding and neutralizing other molecules, than
the monomeric secreted protein or protein fragment alone.
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many
cases, the Fc part in a fusion protein is beneficial in therapy and
diagnosis, and thus can result in, for example, improved
pharmacokinetic properties. (EP A 232,262). Alternatively, deleting
the Fc part after the fusion protein has been expressed, detected,
and purified, would be desired. For example, the Fc portion may
hinder therapy and diagnosis if the fusion protein is used as an
antigen for immunizations. In drug discovery, for example, human
proteins, such as hIL-5, have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58
(1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[0810] Moreover, the antibodies or fragments thereof of the present
invention can be fused to marker sequences, such as a peptide to
facilitate purification. In preferred embodiments, the marker amino
acid sequence is a hexa-histidine peptide, such as the tag provided
in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
Calif., 91311), among others, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA
86:821-824 (1989), for instance, hexa-histidine provides for
convenient purification of the fusion protein. Other peptide tags
useful for purification include, but are not limited to, the "HA"
tag, which corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the
"flag" tag.
[0811] The present invention further encompasses antibodies or
fragments thereof conjugated to a diagnostic or therapeutic agent.
The antibodies can be used diagnostically to, for example, monitor
the development or progression of a tumor as part of a clinical
testing procedure to, e.g., determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials,
radioactive materials, positron emitting metals using various
positron emission tomographies, and nonradioactive paramagnetic
metal ions. The detectable substance may be coupled or conjugated
either directly to the antibody (or fragment thereof) or
indirectly, through an intermediate (such as, for example, a linker
known in the art) using techniques known in the art. See, for
example, U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics according to the
present invention. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include 125I, 131I, 111In or 99Tc.
[0812] Further, an antibody or fragment thereof may be conjugated
to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or
cytotoxic agent includes any agent that is detrimental to cells.
Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0813] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, a-interferon, .beta.-interferon, nerve growth
factor, platelet derived growth factor, tissue plasminogen
activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM II (See,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See,
International Publication No. WO 99/23105), a thrombotic agent or
an anti-angiogenic agent, e.g., angiostatin or endostatin; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophage colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0814] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0815] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62:119-58 (1982).
[0816] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980, which is incorporated herein by
reference in its entirety.
[0817] An antibody, with or without a therapeutic moiety conjugated
to it, administered alone or in combination with cytotoxic
factor(s) and/or cytokine(s) can be used as a therapeutic.
[0818] Immunophenotyping
[0819] The antibodies of the invention may be utilized for
immunophenotyping of cell lines and biological samples. The
translation product of the gene of the present invention may be
useful as a cell specific marker, or more specifically as a
cellular marker that is differentially expressed at various stages
of differentiation and/or maturation of particular cell types.
Monoclonal antibodies directed against a specific epitope, or
combination of epitopes, will allow for the screening of cellular
populations expressing the marker. Various techniques can be
utilized using monoclonal antibodies to screen for cellular
populations expressing the marker(s), and include magnetic
separation using antibody-coated magnetic beads, "panning" with
antibody attached to a solid matrix (i.e., plate), and flow
cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al.,
Cell, 96:737-49 (1999)).
[0820] These techniques allow for the screening of particular
populations of cells, such as might be found with hematological
malignancies (i.e. minimal residual disease (MRD) in acute leukemic
patients) and "non-self" cells in transplantations to prevent
Graft-versus-Host Disease (GVHD). Alternatively, these techniques
allow for the screening of hematopoietic stem and progenitor cells
capable of undergoing proliferation and/or differentiation, as
might be found in human umbilical cord blood.
[0821] Assays For Antibody Binding
[0822] The antibodies of the invention may be assayed for
immunospecific binding by any method known in the art. The
immunoassays which can be used include but are not limited to
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety). Exemplary immunoassays are described briefly below (but
are not intended by way of limitation).
[0823] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1-4 hours) at
4.degree. C., adding protein A and/or protein G sepharose beads to
the cell lysate, incubating for about an hour or more at 4.degree.
C., washing the beads in lysis buffer and resuspending the beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a particular antigen can be assessed by, e.g.,
western blot analysis. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase
the binding of the antibody to an antigen and decrease the
background (e.g., pre-clearing the cell lysate with sepharose
beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.16.1.
[0824] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer, blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
32P or 125I) diluted in blocking buffer, washing the membrane in
wash buffer, and detecting the presence of the antigen. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected and to reduce the
background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0825] ELISAs comprise preparing antigen, coating the well of a 96
well microtiter plate with the antigen, adding the antibody of
interest conjugated to a detectable compound such as an enzymatic
substrate (e.g., horseradish peroxidase or alkaline phosphatase) to
the well and incubating for a period of time, and detecting the
presence of the antigen. In ELISAs the antibody of interest does
not have to be conjugated to a detectable compound; instead, a
second antibody (which recognizes the antibody of interest)
conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antigen, the antibody
may be coated to the well. In this case, a second antibody
conjugated to a detectable compound may be added following the
addition of the antigen of interest to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York at 11.2.1.
[0826] The binding affinity of an antibody to an antigen and the
off-rate of an antibody-antigen interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., 3H or 125I) with the antibody of interest in the
presence of increasing amounts of unlabeled antigen, and the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody of interest for a particular antigen and
the binding off-rates can be determined from the data by scatchard
plot analysis. Competition with a second antibody can also be
determined using radioimmunoassays. In this case, the antigen is
incubated with antibody of interest conjugated to a labeled
compound (e.g., 3H or 125I) in the presence of increasing amounts
of an unlabeled second antibody.
[0827] Therapeutic Uses
[0828] The present invention is further directed to antibody-based
therapies which involve administering antibodies of the invention
to an animal, preferably a mammal, and most preferably a human,
patient for treating one or more of the disclosed diseases,
disorders, or conditions. Therapeutic compounds of the invention
include, but are not limited to, antibodies of the invention
(including fragments, analogs and derivatives thereof as described
herein) and nucleic acids encoding antibodies of the invention
(including fragments, analogs and derivatives thereof and
anti-idiotypic antibodies as described herein). The antibodies of
the invention can be used to treat, inhibit or prevent diseases,
disorders or conditions associated with aberrant expression and/or
activity of a polypeptide of the invention, including, but not
limited to, any one or more of the diseases, disorders, or
conditions described herein. The treatment and/or prevention of
diseases, disorders, or conditions associated with aberrant
expression and/or activity of a polypeptide of the invention
includes, but is not limited to, alleviating symptoms associated
with those diseases, disorders or conditions. Antibodies of the
invention may be provided in pharmaceutically acceptable
compositions as known in the art or as described herein.
[0829] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding
polynucleotides or polypeptides of the present invention locally or
systemically in the body or by direct cytotoxicity of the antibody,
e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the
art will know how to use the antibodies of the present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
[0830] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors
(such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to
increase the number or activity of effector cells which interact
with the antibodies.
[0831] The antibodies of the invention may be administered alone or
in combination with other types of treatments (e.g., radiation
therapy, chemotherapy, hormonal therapy, immunotherapy and
anti-tumor agents). Generally, administration of products of a
species origin or species reactivity (in the case of antibodies)
that is the same species as that of the patient is preferred. Thus,
in a preferred embodiment, human antibodies, fragments derivatives,
analogs, or nucleic acids, are administered to a human patient for
therapy or prophylaxis.
[0832] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against polypeptides or
polynucleotides of the present invention, fragments or regions
thereof, for both immunoassays directed to and therapy of disorders
related to polynucleotides or polypeptides, including fragments
thereof, of the present invention. Such antibodies, fragments, or
regions, will preferably have an affinity for polynucleotides or
polypeptides of the invention, including fragments thereof.
Preferred binding affinities include those with a dissociation
constant or Kd less than 5.times.10.sup.-2 M, 10.sup.-2 M,
5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M,
5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M,
5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M,
5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10
M, 5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
10.sup.-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M, and
10.sup.-15 M.
[0833] Gene Therapy
[0834] In a specific embodiment, nucleic acids comprising sequences
encoding antibodies or functional derivatives thereof, are
administered to treat, inhibit or prevent a disease or disorder
associated with aberrant expression and/or activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded protein that
mediates a therapeutic effect.
[0835] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0836] For general reviews of the methods of gene therapy, see
Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990).
[0837] In a preferred aspect, the compound comprises nucleic acid
sequences encoding an antibody, said nucleic acid sequences being
part of expression vectors that express the antibody or fragments
or chimeric proteins or heavy or light chains thereof in a suitable
host. In particular, such nucleic acid sequences have promoters
operably linked to the antibody coding region, said promoter being
inducible or constitutive, and, optionally, tissue-specific. In
another particular embodiment, nucleic acid molecules are used in
which the antibody coding sequences and any other desired sequences
are flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the antibody encoding nucleic acids (Koller and
Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra
et al., Nature 342:435-438 (1989). In specific embodiments, the
expressed antibody molecule is a single chain antibody;
alternatively, the nucleic acid sequences include sequences
encoding both the heavy and light chains, or fragments thereof, of
the antibody.
[0838] Delivery of the nucleic acids into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0839] In a specific embodiment, the nucleic acid sequences are
directly administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing them as part of an
appropriate nucleic acid expression vector and administering it so
that they become intracellular, e.g., by infection using defective
or attenuated retrovirals or other viral vectors (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180; WO 92/22635; W092/20316; W093/14188, WO 93/20221).
Alternatively, the nucleic acid can be introduced intracellularly
and incorporated within host cell DNA for expression, by homologous
recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438
(1989)).
[0840] In a specific embodiment, viral vectors that contains
nucleic acid sequences encoding an antibody of the invention are
used. For example, a retroviral vector can be used (see Miller et
al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors
contain the components necessary for the correct packaging of the
viral genome and integration into the host cell DNA. The nucleic
acid sequences encoding the antibody to be used in gene therapy are
cloned into one or more vectors, which facilitates delivery of the
gene into a patient. More detail about retroviral vectors can be
found in Boesen et al., Biotherapy 6:291-302 (1994), which
describes the use of a retroviral vector to deliver the mdrl gene
to hematopoietic stem cells in order to make the stem cells more
resistant to chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are: Clowes et al., J. Clin.
Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994);
Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and
Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114
(1993).
[0841] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, Current Opinion in Genetics and
Development 3:499-503 (1993) present a review of adenovirus-based
gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory epithelia of rhesus monkeys. Other instances of the use
of adenoviruses in gene therapy can be found in Rosenfeld et al.,
Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155
(1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT
Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783
(1995). In a preferred embodiment, adenovirus vectors are used.
[0842] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med.
204:289-300 (1993); U.S. Pat. No. 5,436,146).
[0843] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0844] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen
et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther.
29:69-92m (1985) and may be used in accordance with the present
invention, provided that the necessary developmental and
physiological functions of the recipient cells are not disrupted.
The technique should provide for the stable transfer of the nucleic
acid to the cell, so that the nucleic acid is expressible by the
cell and preferably heritable and expressible by its cell
progeny.
[0845] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[0846] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as Tlymphocytes, Blymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0847] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0848] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding an antibody are introduced
into the cells such that they are expressible by the cells or their
progeny, and the recombinant cells are then administered in vivo
for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which
can be isolated and maintained in vitro can potentially be used in
accordance with this embodiment of the present invention (see e.g.
PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985
(1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow
and Scott, Mayo Clinic Proc. 61:771 (1986)).
[0849] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription. Demonstration of
Therapeutic or Prophylactic Activity
[0850] The compounds or pharmaceutical compositions of the
invention are preferably tested in vitro, and then in vivo for the
desired therapeutic or prophylactic activity, prior to use in
humans. For example, in vitro assays to demonstrate the therapeutic
or prophylactic utility of a compound or pharmaceutical composition
include, the effect of a compound on a cell line or a patient
tissue sample. The effect of the compound or composition on the
cell line and/or tissue sample can be determined utilizing
techniques known to those of skill in the art including, but not
limited to, rosette formation assays and cell lysis assays. In
accordance with the invention, in vitro assays which can be used to
determine whether administration of a specific compound is
indicated, include in vitro cell culture assays in which a patient
tissue sample is grown in culture, and exposed to or otherwise
administered a compound, and the effect of such compound upon the
tissue sample is observed.
[0851] Therapeutic/Prophylactic Administration and Composition
[0852] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of a compound or pharmaceutical composition of the invention,
preferably an antibody of the invention. In a preferred aspect, the
compound is substantially purified (e.g., substantially free from
substances that limit its effect or produce undesired
side-effects). The subject is preferably an animal, including but
not limited to animals such as cows, pigs, horses, chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably
human.
[0853] Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid or an
immunoglobulin are described above; additional appropriate
formulations and routes of administration can be selected from
among those described herein below.
[0854] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The compounds or
compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0855] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention,
care must be taken to use materials to which the protein does not
absorb.
[0856] In another embodiment, the compound or composition can be
delivered in a vesicle, in particular a liposome (see Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein,
ibid., pp. 317-327; see generally ibid.)
[0857] In yet another embodiment, the compound or composition can
be delivered in a controlled release system. In one embodiment, a
pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed.
Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton,
Fla. (1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger
and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983);
see also Levy et al., Science 228:190 (1985); During et al., Ann.
Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)).
In yet another embodiment, a controlled release system can be
placed in proximity of the therapeutic target, i.e., the brain,
thus requiring only a fraction of the systemic dose (see, e.g.,
Goodson, in Medical Applications of Controlled Release, supra, vol.
2, pp. 115-138 (1984)).
[0858] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0859] In a specific embodiment where the compound of the invention
is a nucleic acid encoding a protein, the nucleic acid can be
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl.
Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination.
[0860] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0861] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0862] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0863] The amount of the compound of the invention which will be
effective in the treatment, inhibition and prevention of a disease
or disorder associated with aberrant expression and/or activity of
a polypeptide of the invention can be determined by standard
clinical techniques. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0864] For antibodies, the dosage administered to a patient is
typically 0. 1 mg/kg to 100 mg/kg of the patient's body weight.
Preferably, the dosage administered to a patient is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Further, the dosage
and frequency of administration of antibodies of the invention may
be reduced by enhancing uptake and tissue penetration (e.g., into
the brain) of the antibodies by modifications such as, for example,
lipidation.
[0865] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
[0866] Diagnosis and Imaging
[0867] Labeled antibodies, and derivatives and analogs thereof,
which specifically bind to a polypeptide of interest can be used
for diagnostic purposes to detect, diagnose, or monitor diseases,
disorders, and/or conditions associated with the aberrant
expression and/or activity of a polypeptide of the invention. The
invention provides for the detection of aberrant expression of a
polypeptide of interest, comprising (a) assaying the expression of
the polypeptide of interest in cells or body fluid of an individual
using one or more antibodies specific to the polypeptide interest
and (b) comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of aberrant expression.
[0868] The invention provides a diagnostic assay for diagnosing a
disorder, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of a particular disorder. With
respect to cancer, the presence of a relatively high amount of
transcript in biopsied tissue from an individual may indicate a
predisposition for the development of the disease, or may provide a
means for detecting the disease prior to the appearance of actual
clinical symptoms. A more definitive diagnosis of this type may
allow health professionals to employ preventative measures or
aggressive treatment earlier thereby preventing the development or
further progression of the cancer.
[0869] Antibodies of the invention can be used to assay protein
levels in a biological sample using classical immunohistological
methods known to those of skill in the art (e.g., see Jalkanen, et
al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell.
Biol. 105:3087-3096 (1987)). Other antibody-based methods useful
for detecting protein gene expression include immunoassays, such as
the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase;
radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur
(35S), tritium (3H), indium (112In), and technetium (99Tc);
luminescent labels, such as luminol; and fluorescent labels, such
as fluorescein and rhodamine, and biotin.
[0870] One aspect of the invention is the detection and diagnosis
of a disease or disorder associated with aberrant expression of a
polypeptide of interest in an animal, preferably a mammal and most
preferably a human. In one embodiment, diagnosis comprises: a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled
molecule which specifically binds to the polypeptide of interest;
b) waiting for a time interval following the administering for
permitting the labeled molecule to preferentially concentrate at
sites in the subject where the polypeptide is expressed (and for
unbound labeled molecule to be cleared to background level); c)
determining background level; and d) detecting the labeled molecule
in the subject, such that detection of labeled molecule above the
background level indicates that the subject has a particular
disease or disorder associated with aberrant expression of the
polypeptide of interest. Background level can be determined by
various methods including, comparing the amount of labeled molecule
detected to a standard value previously determined for a particular
system.
[0871] It will be understood in the art that the size of the
subject and the imaging system used will determine the quantity of
imaging moiety needed to produce diagnostic images. In the case of
a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of 99mTc. The labeled antibody or antibody fragment
will then preferentially accumulate at the location of cells which
contain the specific protein. In vivo tumor imaging is described in
S. W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled
Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The
Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes,
eds., Masson Publishing Inc. (1982).
[0872] Depending on several variables, including the type of label
used and the mode of administration, the time interval following
the administration for permitting the labeled molecule to
preferentially concentrate at sites in the subject and for unbound
labeled molecule to be cleared to background level is 6 to 48 hours
or 6 to 24 hours or 6 to 12 hours. In another embodiment the time
interval following administration is 5 to 20 days or 5 to 10
days.
[0873] In an embodiment, monitoring of the disease or disorder is
carried out by repeating the method for diagnosing the disease or
disease, for example, one month after initial diagnosis, six months
after initial diagnosis, one year after initial diagnosis, etc.
[0874] Presence of the labeled molecule can be detected in the
patient using methods known in the art for in vivo scanning. These
methods depend upon the type of label used. Skilled artisans will
be able to determine the appropriate method for detecting a
particular label. Methods and devices that may be used in the
diagnostic methods of the invention include, but are not limited
to, computed tomography (CT), whole body scan such as position
emission tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[0875] In a specific embodiment, the molecule is labeled with a
radioisotope and is detected in the patient using a radiation
responsive surgical instrument (Thurston et al., U.S. Pat. No.
5,441,050). In another embodiment, the molecule is labeled with a
fluorescent compound and is detected in the patient using a
fluorescence responsive scanning instrument. In another embodiment,
the molecule is labeled with a positron emitting metal and is
detected in the patent using positron emission-tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic
label and is detected in a patient using magnetic resonance imaging
(MRI).
[0876] Kits
[0877] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises an antibody of
the invention, preferably a purified antibody, in one or more
containers. In a specific embodiment, the kits of the present
invention contain a substantially isolated polypeptide comprising
an epitope which is specifically immunoreactive with an antibody
included in the kit. Preferably, the kits of the present invention
further comprise a control antibody which does not react with the
polypeptide of interest. In another specific embodiment, the kits
of the present invention contain a means for detecting the binding
of an antibody to a polypeptide of interest (e.g., the antibody may
be conjugated to a detectable substrate such as a fluorescent
compound, an enzymatic substrate, a radioactive compound or a
luminescent compound, or a second antibody which recognizes the
first antibody may be conjugated to a detectable substrate).
[0878] In another specific embodiment of the present invention, the
kit is a diagnostic kit for use in screening serum containing
antibodies specific against proliferative and/or cancerous
polynucleotides and polypeptides. Such a kit may include a control
antibody that does not react with the polypeptide of interest. Such
a kit may include a substantially isolated polypeptide antigen
comprising an epitope which is specifically immunoreactive with at
least one anti-polypeptide antigen antibody. Further, such a kit
includes means for detecting the binding of said antibody to the
antigen (e.g., the antibody may be conjugated to a fluorescent
compound such as fluorescein or rhodamine which can be detected by
flow cytometry). In specific embodiments, the kit may include a
recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to
a solid support.
[0879] In a more specific embodiment the detecting means of the
above-described kit includes a solid support to which said
polypeptide antigen is attached. Such a kit may also include a
non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can
be detected by binding of the said reporter-labeled antibody.
[0880] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing antigens of
the polypeptide of the invention. The diagnostic kit includes a
substantially isolated antibody specifically immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the
binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal
antibody. The detecting means of the kit may include a second,
labeled monoclonal antibody. Alternatively, or in addition, the
detecting means may include a labeled, competing antigen.
[0881] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound antigen obtained by
the methods of the present invention. After binding with specific
antigen antibody to the reagent and removing unbound serum
components by washing, the reagent is reacted with reporter-labeled
anti-human antibody to bind reporter to the reagent in proportion
to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and
the amount of reporter associated with the reagent is determined.
Typically, the reporter is an enzyme which is detected by
incubating the solid phase in the presence of a suitable
fluorometric, luminescent or calorimetric substrate (Sigma, St.
Louis, Mo.).
[0882] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[0883] Thus, the invention provides an assay system or kit for
carrying out this diagnostic method. The kit generally includes a
support with surface-bound recombinant antigens, and a
reporter-labeled anti-human antibody for detecting surface-bound
anti-antigen antibody.
[0884] Fusion Proteins
[0885] Any polypeptide of the present invention can be used to
generate fusion proteins. For example, the polypeptide of the
present invention, when fused to a second protein, can be used as
an antigenic tag. Antibodies raised against the polypeptide of the
present invention can be used to indirectly detect the second
protein by binding to the polypeptide. Moreover, because secreted
proteins target cellular locations based on trafficking signals,
the polypeptides of the present invention can be used as targeting
molecules once fused to other proteins.
[0886] Examples of domains that can be fused to polypeptides of the
present invention include not only heterologous signal sequences,
but also other heterologous functional regions. The fusion does not
necessarily need to be direct, but may occur through linker
sequences.
[0887] Moreover, fusion proteins may also be engineered to improve
characteristics of the polypeptide of the present invention. For
instance, a region of additional amino acids, particularly charged
amino acids, may be added to the N-terminus of the polypeptide to
improve stability and persistence during purification from the host
cell or subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to facilitate
handling of polypeptides are familiar and routine techniques in the
art.
[0888] Moreover, polypeptides of the present invention, including
fragments, and specifically epitopes, can be combined with parts of
the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or
portions thereof (CH1, combination thereof, including both entire
domains and portions thereof), resulting in chimeric polypeptides.
These fusion proteins facilitate purification and show an increased
half-life in vivo. One reported example describes chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et
al., Nature 331:84-86 (1988).) Fusion proteins having
disulfide-linked dimeric structures (due to the IgG) can also be
more efficient in binding and neutralizing other molecules, than
the monomeric secreted protein or protein fragment alone.
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)
[0889] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)
discloses fusion proteins comprising various portions of constant
region of immunoglobulin molecules together with another human
protein or part thereof. In many cases, the Fc part in a fusion
protein is beneficial in therapy and diagnosis, and thus can result
in, for example, improved pharmacokinetic properties. (EP-A 0232
262. ) Alternatively, deleting the Fc part after the fusion protein
has been expressed, detected, and purified, would be desired. For
example, the Fc portion may hinder therapy and diagnosis if the
fusion protein is used as an antigen for immunizations. In drug
discovery, for example, human proteins, such as hIL-5, have been
fused with Fc portions for the purpose of high-throughput screening
assays to identify antagonists of hIL-5. (See, D. Bennett et al.,
J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J.
Biol. Chem. 270:9459-9471 (1995).)
[0890] Moreover, the polypeptides of the present invention can be
fused to marker sequences, such as a peptide which facilitates
purification of the fused polypeptide. In preferred embodiments,
the marker amino acid sequence is a hexa-histidine peptide, such as
the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. As described in Gentz et al., Proc. Natl.
Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine
provides for convenient purification of the fusion protein. Another
peptide tag useful for purification, the "HA" tag, corresponds to
an epitope derived from the influenza hemagglutinin protein.
(Wilson et al., Cell 37:767 (1984).)
[0891] Thus, any of these above fusions can be engineered using the
polynucleotides or the polypeptides of the present invention.
[0892] Vectors, Host Cells, and Protein Production
[0893] The present invention also relates to vectors containing the
polynucleotide of the present invention, host cells, and the
production of polypeptides by recombinant techniques. The vector
may be, for example, a phage, plasmid, viral, or retroviral vector.
Retroviral vectors may be replication competent or replication
defective. In the latter case, viral propagation generally will
occur only in complementing host cells.
[0894] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0895] The polynucleotide insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp, phoA and tac promoters, the SV40 early and late
promoters and promoters of retroviral LTRs, to name a few. Other
suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription
initiation, termination, and, in the transcribed region, a ribosome
binding site for translation. The coding portion of the transcripts
expressed by the constructs will preferably include a translation
initiating codon at the beginning and a termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to
be translated.
[0896] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase, G418 or neomycin resistance for eukaryotic cell culture
and tetracycline, kanamycin or ampicillin resistance genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells,
such as E. coli, Streptomyces and Salmonella typhimurium cells;
fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae
or Pichia pastoris (ATCC Accession No. 201178)); insect cells such
as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as
CHO, COS, 293, and Bowes melanoma cells; and plant cells.
Appropriate culture mediums and conditions for the above-described
host cells are known in the art.
[0897] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors,
Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from
Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among
preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and
pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL
available from Pharmacia. Preferred expression vectors for use in
yeast systems include, but are not limited to pYES2, pYD1,
pTEF1/Zeo, pYES2/GS, pPICZ,pGAPZ, pGAPZalph, pPIC9, pPIC3.5,
pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PA0815 (all available from
Invitrogen, Carlbad, Calif.). Other suitable vectors will be
readily apparent to the skilled artisan.
[0898] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection, or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986). It is
specifically contemplated that the polypeptides of the present
invention may in fact be expressed by a host cell lacking a
recombinant vector.
[0899] A polypeptide of this invention can be recovered and
purified from recombinant cell cultures by well-known methods
including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Most preferably, high
performance liquid chromatography ("HPLC") is employed for
purification.
[0900] Polypeptides of the present invention, and preferably the
secreted form, can also be recovered from: products purified from
natural sources, including bodily fluids, tissues and cells,
whether directly isolated or cultured; products of chemical
synthetic procedures; and products produced by recombinant
techniques from a prokaryotic or eukaryotic host, including, for
example, bacterial, yeast, higher plant, insect, and mammalian
cells. Depending upon the host employed in a recombinant production
procedure, the polypeptides of the present invention may be
glycosylated or may be non-glycosylated. In addition, polypeptides
of the invention may also include an initial modified methionine
residue, in some cases as a result of host-mediated processes.
Thus, it is well known in the art that the N-terminal methionine
encoded by the translation initiation codon generally is removed
with high efficiency from any protein after translation in all
eukaryotic cells. While the N-terminal methionine on most proteins
also is efficiently removed in most prokaryotes, for some proteins,
this prokaryotic removal process is inefficient, depending on the
nature of the amino acid to which the N-terminal methionine is
covalently linked.
[0901] In one embodiment, the yeast Pichia pastoris is used to
express the polypeptide of the present invention in a eukaryotic
system. Pichia pastoris is a methylotrophic yeast which can
metabolize methanol as its sole carbon source. A main step in the
methanol metabolization pathway is the oxidation of methanol to
formaldehyde using O.sub.2. This reaction is catalyzed by the
enzyme alcohol oxidase. In order to metabolize methanol as its sole
carbon source, Pichia pastoris must generate high levels of alcohol
oxidase due, in part, to the relatively low affinity of alcohol
oxidase for O.sub.2. Consequently, in a growth medium depending on
methanol as a main carbon source, the promoter region of one of the
two alcohol oxidase genes (AOX1) is highly active. In the presence
of methanol, alcohol oxidase produced from the AOX1 gene comprises
up to approximately 30% of the total soluble protein in Pichia
pastoris. See, Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21
(1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F.,
et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous
coding sequence, such as, for example, a polynucleotide of the
present invention, under the transcriptional regulation of all or
part of the AOX1 regulatory sequence is expressed at exceptionally
high levels in Pichia yeast grown in the presence of methanol.
[0902] In one example, the plasmid vector pPIC9K is used to express
DNA encoding a polypeptide of the invention, as set forth herein,
in a Pichea yeast system essentially as described in "Pichia
Protocols: Methods in Molecular Biology," D. R. Higgins and J.
Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression
vector allows expression and secretion of a protein of the
invention by virtue of the strong AOX1 promoter linked to the
Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide
(i.e., leader) located upstream of a multiple cloning site.
[0903] Many other yeast vectors could be used in place of pPIC9K,
such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,
pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815,
as one skilled in the art would readily appreciate, as long as the
proposed expression construct provides appropriately located
signals for transcription, translation, secretion (if desired), and
the like, including an in-frame AUG as required.
[0904] In another embodiment, high-level expression of a
heterologous coding sequence, such as, for example, a
polynucleotide of the present invention, may be achieved by cloning
the heterologous polynucleotide of the invention into an expression
vector such as, for example, pGAPZ or pGAPZalpha, and growing the
yeast culture in the absence of methanol.
[0905] In addition to encompassing host cells containing the vector
constructs discussed herein, the invention also encompasses
primary, secondary, and immortalized host cells of vertebrate
origin, particularly mammalian origin, that have been engineered to
delete or replace endogenous genetic material (e.g., coding
sequence), and/or to include genetic material (e.g., heterologous
polynucleotide sequences) that is operably associated with the
polynucleotides of the invention, and which activates, alters,
and/or amplifies endogenous polynucleotides. For example,
techniques known in the art may be used to operably associate
heterologous control regions (e.g., promoter and/or enhancer) and
endogenous polynucleotide sequences via homologous recombination,
resulting in the formation of a new transcription unit (see, e.g.,
U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No.
5,733,761, issued Mar. 31, 1998; International Publication No. WO
96/29411, published Sep. 26, 1996; International Publication No. WO
94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad.
Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature
342:435-438 (1989), the disclosures of each of which are
incorporated by reference in their entireties).
[0906] In addition, polypeptides of the invention can be chemically
synthesized using techniques known in the art (e.g., see Creighton,
1983, Proteins: Structures and Molecular Principles, W. H. Freeman
& Co., N.Y., and Hunkapiller et al., Nature, 310:105-111
(1984)). For example, a polypeptide corresponding to a fragment of
a polypeptide sequence of the invention can be synthesized by use
of a peptide synthesizer. Furthermore, if desired, nonclassical
amino acids or chemical amino acid analogs can be introduced as a
substitution or addition into the polypeptide sequence.
Non-classical amino acids include, but are not limited to, to the
D-isomers of the common amino acids, 2,4-diaminobutyric acid,
a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric
acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric
acid, 3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic
acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino
acids such as b-methyl amino acids, Ca-methyl amino acids,
Na-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[0907] The invention encompasses polypeptides which are
differentially modified during or after translation, e.g., by
glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand,
etc. Any of numerous chemical modifications may be carried out by
known techniques, including but not limited, to specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8
protease, NaBH.sub.4; acetylation, formylation, oxidation,
reduction; metabolic synthesis in the presence of tunicamycin;
etc.
[0908] Additional post-translational modifications encompassed by
the invention include, for example, e.g., N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends),
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of procaryotic host cell expression. The polypeptides may
also be modified with a detectable label, such as an enzymatic,
fluorescent, isotopic or affinity label to allow for detection and
isolation of the protein.
[0909] Also provided by the invention are chemically modified
derivatives of the polypeptides of the invention which may provide
additional advantages such as increased solubility, stability and
circulating time of the polypeptide, or decreased immunogenicity
(see U.S. Pat. No.: 4,179,337). The chemical moieties for
derivitization may be selected from water soluble polymers such as
polyethylene glycol, ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at random positions within the
molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
[0910] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about 1 kDa and about 100 kDa (the term
"about" indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and manufacturing. Other
sizes may be used, depending on the desired therapeutic profile
(e.g., the duration of sustained release desired, the effects, if
any on biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the polyethylene
glycol to a therapeutic protein or analog).
[0911] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the protein with consideration of
effects on functional or antigenic domains of the protein. There
are a number of attachment methods available to those skilled in
the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol.
20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl
chloride). For example, polyethylene glycol may be covalently bound
through amino acid residues via a reactive group, such as, a free
amino or carboxyl group. Reactive groups are those to which an
activated polyethylene glycol molecule may be bound. The amino acid
residues having a free amino group may include lysine residues and
the N-terminal amino acid residues; those having a free carboxyl
group may include aspartic acid residues glutamic acid residues and
the C-terminal amino acid residue. Sulfhydryl groups may also be
used as a reactive group for attaching the polyethylene glycol
molecules. Preferred for therapeutic purposes is attachment at an
amino group, such as attachment at the N-terminus or lysine
group.
[0912] One may specifically desire proteins chemically modified at
the N-terminus. Using polyethylene glycol as an illustration of the
present composition, one may select from a variety of polyethylene
glycol molecules (by molecular weight, branching, etc.), the
proportion of polyethylene glycol molecules to protein
(polypeptide) molecules in the reaction mix, the type of pegylation
reaction to be performed, and the method of obtaining the selected
N-terminally pegylated protein. The method of obtaining the
N-terminally pegylated preparation (i.e., separating this moiety
from other monopegylated moieties if necessary) may be by
purification of the N-terminally pegylated material from a
population of pegylated protein molecules. Selective proteins
chemically modified at the N-terminus modification may be
accomplished by reductive alkylation which exploits differential
reactivity of different types of primary amino groups (lysine
versus the N-terminal) available for derivatization in a particular
protein. Under the appropriate reaction conditions, substantially
selective derivatization of the protein at the N-terminus with a
carbonyl group containing polymer is achieved.
[0913] The polypeptides of the invention may be in monomers or
multimers (i.e., dimers, trimers, tetramers and higher multimers).
Accordingly, the present invention relates to monomers and
multimers of the polypeptides of the invention, their preparation,
and compositions (preferably, Therapeutics) containing them. In
specific embodiments, the polypeptides of the invention are
monomers, dimers, trimers or tetramers. In additional embodiments,
the multimers of the invention are at least dimers, at least
trimers, or at least tetramers.
[0914] Multimers encompassed by the invention may be homomers or
heteromers. As used herein, the term homomer, refers to a multimer
containing only polypeptides corresponding to the amino acid
sequence of SEQ ID NO:Y or encoded by the cDNA contained in a
deposited clone (including fragments, variants, splice variants,
and fusion proteins, corresponding to these polypeptides as
described herein). These homomers may contain polypeptides having
identical or different amino acid sequences. In a specific
embodiment, a homomer of the invention is a multimer containing
only polypeptides having an identical amino acid sequence. In
another specific embodiment, a homomer of the invention is a
multimer containing polypeptides having different amino acid
sequences. In specific embodiments, the multimer of the invention
is a homodimer (e.g., containing polypeptides having identical or
different amino acid sequences) or a homotrimer (e.g., containing
polypeptides having identical and/or different amino acid
sequences). In additional embodiments, the homomeric multimer of
the invention is at least a homodimer, at least a homotrimer, or at
least a homotetramer.
[0915] As used herein, the term heteromer refers to a multimer
containing one or more heterologous polypeptides (i.e.,
polypeptides of different proteins) in addition to the polypeptides
of the invention. In a specific embodiment, the multimer of the
invention is a heterodimer, a heterotrimer, or a heterotetramer. In
additional embodiments, the heteromeric multimer of the invention
is at least a heterodimer, at least a heterotrimer, or at least a
heterotetramer.
[0916] Multimers of the invention may be the result of hydrophobic,
hydrophilic, ionic and/or covalent associations and/or may be
indirectly linked, by for example, liposome formation. Thus, in one
embodiment, multimers of the invention, such as, for example,
homodimers or homotrimers, are formed when polypeptides of the
invention contact one another in solution. In another embodiment,
heteromultimers of the invention, such as, for example,
heterotrimers or heterotetramers, are formed when polypeptides of
the invention contact antibodies to the polypeptides of the
invention (including antibodies to the heterologous polypeptide
sequence in a fusion protein of the invention) in solution. In
other embodiments, multimers of the invention are formed by
covalent associations with and/or between the polypeptides of the
invention. Such covalent associations may involve one or more amino
acid residues contained in the polypeptide sequence (e.g., that
recited in the sequence listing, or contained in the polypeptide
encoded by a deposited clone). In one instance, the covalent
associations are cross-linking between cysteine residues located
within the polypeptide sequences which interact in the native
(i.e., naturally occurring) polypeptide. In another instance, the
covalent associations are the consequence of chemical or
recombinant manipulation. Alternatively, such covalent associations
may involve one or more amino acid residues contained in the
heterologous polypeptide sequence in a fusion protein of the
invention.
[0917] In one example, covalent associations are between the
heterologous sequence contained in a fusion protein of the
invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific
example, the covalent associations are between the heterologous
sequence contained in an Fc fusion protein of the invention (as
described herein). In another specific example, covalent
associations of fusion proteins of the invention are between
heterologous polypeptide sequence from another protein that is
capable of forming covalently associated multimers, such as for
example, oseteoprotegerin (see, e.g., International Publication NO:
WO 98/49305, the contents of which are herein incorporated by
reference in its entirety). In another embodiment, two or more
polypeptides of the invention are joined through peptide linkers.
Examples include those peptide linkers described in U.S. Pat. No.
5,073,627 (hereby incorporated by reference). Proteins comprising
multiple polypeptides of the invention separated by peptide linkers
may be produced using conventional recombinant DNA technology.
[0918] Another method for preparing multimer polypeptides of the
invention involves use of polypeptides of the invention fused to a
leucine zipper or isoleucine zipper polypeptide sequence. Leucine
zipper and isoleucine zipper domains are polypeptides that promote
multimerization of the proteins in which they are found. Leucine
zippers were originally identified in several DNA-binding proteins
(Landschulz et al., Science 240:1759, (1988)), and have since been
found in a variety of different proteins. Among the known leucine
zippers are naturally occurring peptides and derivatives thereof
that dimerize or trimerize. Examples of leucine zipper domains
suitable for producing soluble multimeric proteins of the invention
are those described in PCT application WO 94/10308, hereby
incorporated by reference. Recombinant fusion proteins comprising a
polypeptide of the invention fused to a polypeptide sequence that
dimerizes or trimerizes in solution are expressed in suitable host
cells, and the resulting soluble multimeric fusion protein is
recovered from the culture supernatant using techniques known in
the art.
[0919] Trimeric polypeptides of the invention may offer the
advantage of enhanced biological activity. Preferred leucine zipper
moieties and isoleucine moieties are those that preferentially form
trimers. One example is a leucine zipper derived from lung
surfactant protein D (SPD), as described in Hoppe et al. (FEBS
Letters 344:191, (1994)) and in U.S. patent application Ser. No.
08/446,922, hereby incorporated by reference. Other peptides
derived from naturally occurring trimeric proteins may be employed
in preparing trimeric polypeptides of the invention.
[0920] In another example, proteins of the invention are associated
by interactions between Flag.RTM. polypeptide sequence contained in
fusion proteins of the invention containing Flag.RTM. polypeptide
seuqence. In a further embodiment, associations proteins of the
invention are associated by interactions between heterologous
polypeptide sequence contained in Flag.RTM. fusion proteins of the
invention and anti-Flag.RTM. antibody.
[0921] The multimers of the invention may be generated using
chemical techniques known in the art. For example, polypeptides
desired to be contained in the multimers of the invention may be
chemically cross-linked using linker molecules and linker molecule
length optimization techniques known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). Additionally, multimers of the invention may be
generated using techniques known in the art to form one or more
inter-molecule cross-links between the cysteine residues located
within the sequence of the polypeptides desired to be contained in
the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Further, polypeptides
of the invention may be routinely modified by the addition of
cysteine or biotin to the C terminus or N-terminus of the
polypeptide and techniques known in the art may be applied to
generate multimers containing one or more of these modified
polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Additionally,
techniques known in the art may be applied to generate liposomes
containing the polypeptide components desired to be contained in
the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925,
which is herein incorporated by reference in its entirety).
[0922] Alternatively, multimers of the invention may be generated
using genetic engineering techniques known in the art. In one
embodiment, polypeptides contained in multimers of the invention
are produced recombinantly using fusion protein technology
described herein or otherwise known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In a specific embodiment, polynucleotides coding for
a homodimer of the invention are generated by ligating a
polynucleotide sequence encoding a polypeptide of the invention to
a sequence encoding a linker polypeptide and then further to a
synthetic polynucleotide encoding the translated product of the
polypeptide in the reverse orientation from the original C-terminus
to the N-terminus (lacking the leader sequence) (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In another embodiment, recombinant techniques
described herein or otherwise known in the art are applied to
generate recombinant polypeptides of the invention which contain a
transmembrane domain (or hyrophobic or signal peptide) and which
can be incorporated by membrane reconstitution techniques into
liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety).
[0923] Uses of the Polynucleotides
[0924] Each of the polynucleotides identified herein can be used in
numerous ways as reagents. The following description should be
considered exemplary and utilizes known techniques.
[0925] The polynucleotides of the present invention are useful for
chromosome identification. There exists an ongoing need to identify
new chromosome markers, since few chromosome marking reagents,
based on actual sequence data (repeat polymorphisms), are presently
available. Each polynucleotide of the present invention can be used
as a chromosome marker.
[0926] Briefly, sequences can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 bp) from the sequences shown in SEQ
ID NO:X. Primers can be selected using computer analysis so that
primers do not span more than one predicted exon in the genomic
DNA. These primers are then used for PCR screening of somatic cell
hybrids containing individual human chromosomes. Only those hybrids
containing the human gene corresponding to the SEQ ID NO:X will
yield an amplified fragment.
[0927] Similarly, somatic hybrids provide a rapid method of PCR
mapping the polynucleotides to particular chromosomes. Three or
more clones can be assigned per day using a single thermal cycler.
Moreover, sublocalization of the polynucleotides can be achieved
with panels of specific chromosome fragments. Other gene mapping
strategies that can be used include in situ hybridization,
prescreening with labeled flow-sorted chromosomes, and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0928] Precise chromosomal location of the polynucleotides can also
be achieved using fluorescence in situ hybridization (FISH) of a
metaphase chromosomal spread. This technique uses polynucleotides
as short as 500 or 600 bases; however, polynucleotides 2,000-4,000
bp are preferred. For a review of this technique, see Verma et al.,
"Human Chromosomes: a Manual of Basic Techniques," Pergamon Press,
New York (1988).
[0929] For chromosome mapping, the polynucleotides can be used
individually (to mark a single chromosome or a single site on that
chromosome) or in panels (for marking multiple sites and/or
multiple chromosomes). Preferred polynucleotides correspond to the
noncoding regions of the cDNAs because the coding sequences are
more likely conserved within gene families, thus increasing the
chance of cross hybridization during chromosomal mapping.
[0930] Once a polynucleotide has been mapped to a precise
chromosomal location, the physical position of the polynucleotide
can be used in linkage analysis. Linkage analysis establishes
coinheritance between a chromosomal location and presentation of a
particular disease. (Disease mapping data are found, for example,
in V. McKusick, Mendelian Inheritance in Man (available on line
through Johns Hopkins University Welch Medical Library).) Assuming
1 megabase mapping resolution and one gene per 20 kb, a cDNA
precisely localized to a chromosomal region associated with the
disease could be one of 50-500 potential causative genes.
[0931] Thus, once coinheritance is established, differences in the
polynucleotide and the corresponding gene between affected and
unaffected individuals can be examined. First, visible structural
alterations in the chromosomes, such as deletions or
translocations, are examined in chromosome spreads or by PCR. If no
structural alterations exist, the presence of point mutations are
ascertained. Mutations observed in some or all affected
individuals, but not in normal individuals, indicates that the
mutation may cause the disease. However, complete sequencing of the
polypeptide and the corresponding gene from several normal
individuals is required to distinguish the mutation from a
polymorphism. If a new polymorphism is identified, this polymorphic
polypeptide can be used for further linkage analysis.
[0932] Furthermore, increased or decreased expression of the gene
in affected individuals as compared to unaffected individuals can
be assessed using polynucleotides of the present invention. Any of
these alterations (altered expression, chromosomal rearrangement,
or mutation) can be used as a diagnostic or prognostic marker.
[0933] Thus, the invention also provides a diagnostic method useful
during diagnosis of a disorder, involving measuring the expression
level of polynucleotides of the present invention in cells or body
fluid from an individual and comparing the measured gene expression
level with a standard level of polynucleotide expression level,
whereby an increase or decrease in the gene expression level
compared to the standard is indicative of a disorder.
[0934] In still another embodiment, the invention includes a kit
for analyzing samples for the presence of proliferative and/or
cancerous polynucleotides derived from a test subject. In a general
embodiment, the kit includes at least one polynucleotide probe
containing a nucleotide sequence that will specifically hybridize
with a polynucleotide of the present invention and a suitable
container. In a specific embodiment, the kit includes two
polynucleotide probes defining an internal region of the
polynucleotide of the present invention, where each probe has one
strand containing a 31'mer-end internal to the region. In a further
embodiment, the probes may be useful as primers for polymerase
chain reaction amplification.
[0935] Where a diagnosis of a disorder, has already been made
according to conventional methods, the present invention is useful
as a prognostic indicator, whereby patients exhibiting enhanced or
depressed polynucleotide of the present invention expression will
experience a worse clinical outcome relative to patients expressing
the gene at a level nearer the standard level.
[0936] By "measuring the expression level of polynucleotide of the
present invention" is intended qualitatively or quantitatively
measuring or estimating the level of the polypeptide of the present
invention or the level of the mRNA encoding the polypeptide in a
first biological sample either directly (e.g., by determining or
estimating absolute protein level or mRNA level) or relatively
(e.g., by comparing to the polypeptide level or mRNA level in a
second biological sample). Preferably, the polypeptide level or
mRNA level in the first biological sample is measured or estimated
and compared to a standard polypeptide level or mRNA level, the
standard being taken from a second biological sample obtained from
an individual not having the disorder or being determined by
averaging levels from a population of individuals not having a
disorder. As will be appreciated in the art, once a standard
polypeptide level or mRNA level is known, it can be used repeatedly
as a standard for comparison.
[0937] By "biological sample" is intended any biological sample
obtained from an individual, body fluid, cell line, tissue culture,
or other source which contains the polypeptide of the present
invention or mRNA. As indicated, biological samples include body
fluids (such as semen, lymph, sera, plasma, urine, synovial fluid
and spinal fluid) which contain the polypeptide of the present
invention, and other tissue sources found to express the
polypeptide of the present invention. Methods for obtaining tissue
biopsies and body fluids from mammals are well known in the art.
Where the biological sample is to include mRNA, a tissue biopsy is
the preferred source.
[0938] The method(s) provided above may preferrably be applied in a
diagnostic method and/or kits in which polynucleotides and/or
polypeptides are attached to a solid support. In one exemplary
method, the support may be a "gene chip" or a "biological chip" as
described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174.
Further, such a gene chip with polynucleotides of the present
invention attached may be used to identify polymorphisms between
the polynucleotide sequences, with polynucleotides isolated from a
test subject. The knowledge of such polymorphisms (i.e. their
location, as well as, their existence) would be beneficial in
identifying disease loci for many disorders, including cancerous
diseases and conditions. Such a method is described in U.S. Pat.
Nos. 5,858,659 and 5,856,104. The U.S. Patents referenced supra are
hereby incorporated by reference in their entirety herein.
[0939] The present invention encompasses polynucleotides of the
present invention that are chemically synthesized, or reproduced as
peptide nucleic acids (PNA), or according to other methods known in
the art. The use of PNAs would serve as the preferred form if the
polynucleotides are incorporated onto a solid support, or gene
chip. For the purposes of the present invention, a peptide nucleic
acid (PNA) is a polyamide type of DNA analog and the monomeric
units for adenine, guanine, thymine and cytosine are available
commercially (Perceptive Biosystems). Certain components of DNA,
such as phosphorus, phosphorus oxides, or deoxyribose derivatives,
are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm,
R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.
Egholm, O. Buchardt, L.Christensen, C. Behrens, S. M. Freier, D. A.
Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature
365, 666 (1993), PNAs bind specifically and tightly to
complementary DNA strands and are not degraded by nucleases. In
fact, PNA binds more strongly to DNA than DNA itself does. This is
probably because there is no electrostatic repulsion between the
two strands, and also the polyamide backbone is more flexible.
Because of this, PNA/DNA duplexes bind under a wider range of
stringency conditions than DNA/DNA duplexes, making it easier to
perform multiplex hybridization. Smaller probes can be used than
with DNA due to the strong binding. In addition, it is more likely
that single base mismatches can be determined with PNA/DNA
hybridization because a single mismatch in a PNA/DNA 15-mer lowers
the melting point (T.sub.m) by 8.degree.-20.degree. C., vs.
4.degree.-16.degree. C. for the DNA/DNA 15-mer duplex. Also, the
absence of charge groups in PNA means that hybridization can be
done at low ionic strengths and reduce possible interference by
salt during the analysis.
[0940] The present invention is useful for detecting cancer in
mammals. In particular the invention is useful during diagnosis of
pathological cell proliferative neoplasias which include, but are
not limited to: acute myelogenous leukemias including acute
monocytic leukemia, acute myeloblastic leukemia, acute
promyelocytic leukemia, acute myelomonocytic leukemia, acute
erythroleukemia, acute megakaryocytic leukemia, and acute
undifferentiated leukemia, etc.; and chronic myelogenous leukemias
including chronic myelomonocytic leukemia, chronic granulocytic
leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs,
cows, pigs, horses, rabbits and humans. Particularly preferred are
humans.
[0941] Pathological cell proliferative diseases, disorders, and/or
conditions are often associated with inappropriate activation of
proto-oncogenes. (Gelmann, E. P. et al., "The Etiology of Acute
Leukemia: Molecular Genetics and Viral Oncology," in Neoplastic
Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds., 161-182
(1985)). Neoplasias are now believed to result from the qualitative
alteration of a normal cellular gene product, or from the
quantitative modification of gene expression by insertion into the
chromosome of a viral sequence, by chromosomal translocation of a
gene to a more actively transcribed region, or by some other
mechanism. (Gelmann et al., supra) It is likely that mutated or
altered expression of specific genes is involved in the
pathogenesis of some leukemias, among other tissues and cell types.
(Gelmann et al., supra) Indeed, the human counterparts of the
oncogenes involved in some animal neoplasias have been amplified or
translocated in some cases of human leukemia and carcinoma.
(Gelmann et al., supra)
[0942] For example, c-myc expression is highly amplified in the
non-lymphocytic leukemia cell line HL-60. When HL-60 cells are
chemically induced to stop proliferation, the level of c-myc is
found to be downregulated. (International Publication Number WO
91/15580) However, it has been shown that exposure of HL-60 cells
to a DNA construct that is complementary to the 5' end of c-myc or
c-myb blocks translation of the corresponding mRNAs which
downregulates expression of the c-myc or c-myb proteins and causes
arrest of cell proliferation and differentiation of the treated
cells. (International Publication Number WO 91/15580; Wickstrom et
al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al., Proc.
Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisan
would appreciate the present invention's usefulness would not be
limited to treatment of proliferative diseases, disorders, and/or
conditions of hematopoietic cells and tissues, in light of the
numerous cells and cell types of varying origins which are known to
exhibit proliferative phenotypes.
[0943] In addition to the foregoing, a polynucleotide can be used
to control gene expression through triple helix formation or
antisense DNA or RNA. Antisense techniques are discussed, for
example, in Okano, J. Neurochem. 56: 560 (1991);
"Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRCPress, Boca Raton, Fla. (1988). Triple helix formation is
discussed in, for instance Lee et al., Nucleic Acids Research 6:
3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et
al., Science 251: 1360 (1991). Both methods rely on binding of the
polynucleotide to a complementary DNA or RNA. For these techniques,
preferred polynucleotides are usually oligonucleotides 20 to 40
bases in length and complementary to either the region of the gene
involved in transcription (triple helix--see Lee et al., Nucl.
Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988);
and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself
(antisense--Okano, J. Neurochem. 56:560 (1991);
Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988).) Triple helix formation
optimally results in a shut-off of RNA transcription from DNA,
while antisense RNA hybridization blocks translation of an mRNA
molecule into polypeptide. Both techniques are effective in model
systems, and the information disclosed herein can be used to design
antisense or triple helix polynucleotides in an effort to treat or
prevent disease.
[0944] Polynucleotides of the present invention are also useful in
gene therapy. One goal of gene therapy is to insert a normal gene
into an organism having a defective gene, in an effort to correct
the genetic defect. The polynucleotides disclosed in the present
invention offer a means of targeting such genetic defects in a
highly accurate manner. Another goal is to insert a new gene that
was not present in the host genome, thereby producing a new trait
in the host cell.
[0945] The polynucleotides are also useful for identifying
individuals from minute biological samples. The United States
military, for example, is considering the use of restriction
fragment length polymorphism (RFLP) for identification of its
personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed on a
Southern blot to yield unique bands for identifying personnel. This
method does not suffer from the current limitations of "Dog Tags"
which can be lost, switched, or stolen, making positive
identification difficult. The polynucleotides of the present
invention can be used as additional DNA markers for RFLP.
[0946] The polynucleotides of the present invention can also be
used as an alternative to RFLP, by determining the actual
base-by-base DNA sequence of selected portions of an individual's
genome. These sequences can be used to prepare PCR primers for
amplifying and isolating such selected DNA, which can then be
sequenced. Using this technique, individuals can be identified
because each individual will have a unique set of DNA sequences.
Once an unique ID database is established for an individual,
positive identification of that individual, living or dead, can be
made from extremely small tissue samples.
[0947] Forensic biology also benefits from using DNA-based
identification techniques as disclosed herein. DNA sequences taken
from very small biological samples such as tissues, e.g., hair or
skin, or body fluids, e.g., blood, saliva, semen, synovial fluid,
amniotic fluid, breast milk, lymph, pulmonary sputum or
surfactant,urine,fecal matter, etc., can be amplified using PCR. In
one prior art technique, gene sequences amplified from polymorphic
loci, such as DQa class II HLA gene, are used in forensic biology
to identify individuals. (Erlich, H., PCR Technology, Freeman and
Co. (1992).) Once these specific polymorphic loci are amplified,
they are digested with one or more restriction enzymes, yielding an
identifying set of bands on a Southern blot probed with DNA
corresponding to the DQa class II HLA gene. Similarly,
polynucleotides of the present invention can be used as polymorphic
markers for forensic purposes.
[0948] There is also a need for reagents capable of identifying the
source of a particular tissue. Such need arises, for example, in
forensics when presented with tissue of unknown origin. Appropriate
reagents can comprise, for example, DNA probes or primers specific
to particular tissue prepared from the sequences of the present
invention. Panels of such reagents can identify tissue by species
and/or by organ type. In a similar fashion, these reagents can be
used to screen tissue cultures for contamination.
[0949] In the very least, the polynucleotides of the present
invention can be used as molecular weight markers on Southern gels,
as diagnostic probes for the presence of a specific mRNA in a
particular cell type, as a probe to "subtract-out" known sequences
in the process of discovering novel polynucleotides, for selecting
and making oligomers for attachment to a "gene chip" or other
support, to raise anti-DNA antibodies using DNA immunization
techniques, and as an antigen to elicit an immune response.
[0950] Uses of the Polypeptides
[0951] Each of the polypeptides identified herein can be used in
numerous ways. The following description should be considered
exemplary and utilizes known techniques.
[0952] A polypeptide of the present invention can be used to assay
protein levels in a biological sample using antibody-based
techniques. For example, protein expression in tissues can be
studied with classical immunohistological methods. (Jalkanen, M.,
et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J.
Cell. Biol. 105:3087-3096 (1987).) Other antibody-based methods
useful for detecting protein gene expression include immunoassays,
such as the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur
(35S), tritium (3H), indium (112In), and technetium (99mTc), and
fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0953] In addition to assaying secreted protein levels in a
biological sample, proteins can also be detected in vivo by
imaging. Antibody labels or markers for in vivo imaging of protein
include those detectable by X-radiography, NMR or ESR. For
X-radiography, suitable labels include radioisotopes such as barium
or cesium, which emit detectable radiation but are not overtly
harmful to the subject. Suitable markers for NMR and ESR include
those with a detectable characteristic spin, such as deuterium,
which may be incorporated into the antibody by labeling of
nutrients for the relevant hybridoma.
[0954] A protein-specific antibody or antibody fragment which has
been labeled with an appropriate detectable imaging moiety, such as
a radioisotope (for example, 131I, 112In, 99mTc), a radio-opaque
substance, or a material detectable by nuclear magnetic resonance,
is introduced (for example, parenterally, subcutaneously, or
intraperitoneally) into the mammal. It will be understood in the
art that the size of the subject and the imaging system used will
determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a
human subject, the quantity of radioactivity injected will normally
range from about 5 to 20 millicuries of 99mTc. The labeled antibody
or antibody fragment will then preferentially accumulate at the
location of cells which contain the specific protein. In vivo tumor
imaging is described in S. W. Burchiel et al.,
"Immunopharmacokinetics of Radiolabeled Antibodies and Their
Fragments." (Chapter 13 in Tumor Imaging: The Radiochemical
Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson
Publishing Inc. (1982).)
[0955] Thus, the invention provides a diagnostic method of a
disorder, which involves (a) assaying the expression of a
polypeptide of the present invention in cells or body fluid of an
individual; (b) comparing the level of gene expression with a
standard gene expression level, whereby an increase or decrease in
the assayed polypeptide gene expression level compared to the
standard expression level is indicative of a disorder. With respect
to cancer, the presence of a relatively high amount of transcript
in biopsied tissue from an individual may indicate a predisposition
for the development of the disease, or may provide a means for
detecting the disease prior to the appearance of actual clinical
symptoms. A more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further
progression of the cancer.
[0956] Moreover, polypeptides of the present invention can be used
to treat, prevent, and/or diagnose disease. For example, patients
can be administered a polypeptide of the present invention in an
effort to replace absent or decreased levels of the polypeptide
(e.g., insulin), to supplement absent or decreased levels of a
different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD,
catalase, DNA repair proteins), to inhibit the activity of a
polypeptide (e.g., an oncogene or tumor supressor), to activate the
activity of a polypeptide (e.g., by binding to a receptor), to
reduce the activity of a membrane bound receptor by competing with
it for free ligand (e.g., soluble TNF receptors used in reducing
inflammation), or to bring about a desired response (e.g., blood
vessel growth inhibition, enhancement of the immune response to
proliferative cells or tissues).
[0957] Similarly, antibodies directed to a polypeptide of the
present invention can also be used to treat, prevent, and/or
diagnose disease. For example, administration of an antibody
directed to a polypeptide of the present invention can bind and
reduce overproduction of the polypeptide. Similarly, administration
of an antibody can activate the polypeptide, such as by binding to
a polypeptide bound to a membrane (receptor).
[0958] At the very least, the polypeptides of the present invention
can be used as molecular weight markers on SDS-PAGE gels or on
molecular sieve gel filtration columns using methods well known to
those of skill in the art. Polypeptides can also be used to raise
antibodies, which in turn are used to measure protein expression
from a recombinant cell, as a way of assessing transformation of
the host cell. Moreover, the polypeptides of the present invention
can be used to test the following biological activities.
[0959] Gene Therapy Methods
[0960] Another aspect of the present invention is to gene therapy
methods for treatingor preventing disorders, diseases and
conditions. The gene therapy methods relate to the introduction of
nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an
animal to achieve expression of a polypeptide of the present
invention. This method requires a polynucleotide which codes for a
polypeptide of the invention that operatively linked to a promoter
and any other genetic elements necessary for the expression of the
polypeptide by the target tissue. Such gene therapy and delivery
techniques are known in the art, see, for example, WO90/11092,
which is herein incorporated by reference.
[0961] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) comprising a promoter operably
linked to a polynucleotide of the invention ex vivo, with the
engineered cells then being provided to a patient to be treated
with the polypeptide. Such methods are well-known in the art. For
example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216
(1993); Ferrantini et al., Cancer Research, 53:107-1112 (1993);
Ferrantini et al., J. Immunology 153: 4604-4615 (1994); Kaido, T.,
et al., Int. J. Cancer 60: 221-229 (1995); Ogura et al., Cancer
Research 50: 5102-5106 (1990); Santodonato, et al., Human Gene
Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy
4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38
(1996)), which are herein incorporated by reference. In one
embodiment, the cells which are engineered are arterial cells. The
arterial cells may be reintroduced into the patient through direct
injection to the artery, the tissues surrounding the artery, or
through catheter injection.
[0962] As discussed in more detail below, the polynucleotide
constructs can be delivered by any method that delivers injectable
materials to the cells of an animal, such as, injection into the
interstitial space of tissues (heart, muscle, skin, lung, liver,
and the like). The polynucleotide constructs may be delivered in a
pharmaceutically acceptable liquid or aqueous carrier.
[0963] In one embodiment, the polynucleotide of the invention is
delivered as a naked polynucleotide. The term "naked"
polynucleotide, DNA or RNA refers to sequences that are free from
any delivery vehicle that acts to assist, promote or facilitate
entry into the cell, including viral sequences, viral particles,
liposome formulations, lipofectin or precipitating agents and the
like. However, the polynucleotides of the invention can also be
delivered in liposome formulations and lipofectin formulations and
the like can be prepared by methods well known to those skilled in
the art. Such methods are described, for example, in U.S. Pat. Nos.
5,593,972, 5,589,466, and 5,580,859, which are herein incorporated
by reference.
[0964] The polynucleotide vector constructs of the invention used
in the gene therapy method are preferably constructs that will not
integrate into the host genome nor will they contain sequences that
allow for replication. Appropriate vectors include pWLNEO, pSV2CAT,
pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG
and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and
pRc/CMV2 available from Invitrogen. Other suitable vectors will be
readily apparent to the skilled artisan.
[0965] Any strong promoter known to those skilled in the art can be
used for driving the expression of polynucleotide sequence of the
invention. Suitable promoters include adenoviral promoters, such as
the adenoviral major late promoter; or heterologous promoters, such
as the cytomegalovirus (CMV) promoter; the respiratory syncytial
virus (RSV) promoter; inducible promoters, such as the MMT
promoter, the metallothionein promoter; heat shock promoters; the
albumin promoter; the ApoAI promoter; human globin promoters; viral
thymidine kinase promoters, such as the Herpes Simplex thymidine
kinase promoter; retroviral LTRs; the b-actin promoter; and human
growth hormone promoters. The promoter also may be the native
promoter for the polynucleotides of the invention.
[0966] Unlike other gene therapy techniques, one major advantage of
introducing naked nucleic acid sequences into target cells is the
transitory nature of the polynucleotide synthesis in the cells.
Studies have shown that non-replicating DNA sequences can be
introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
[0967] The polynucleotide construct of the invention can be
delivered to the interstitial space of tissues within the an
animal, including of muscle, skin, brain, lung, liver, spleen, bone
marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas,
kidney, gall bladder, stomach, intestine, testis, ovary, uterus,
rectum, nervous system, eye, gland, and connective tissue.
Interstitial space of the tissues comprises the intercellular,
fluid, mucopolysaccharide matrix among the reticular fibers of
organ tissues, elastic fibers in the walls of vessels or chambers,
collagen fibers of fibrous tissues, or that same matrix within
connective tissue ensheathing muscle cells or in the lacunae of
bone. It is similarly the space occupied by the plasma of the
circulation and the lymph fluid of the lymphatic channels. Delivery
to the interstitial space of muscle tissue is preferred for the
reasons discussed below. They may be conveniently delivered by
injection into the tissues comprising these cells. They are
preferably delivered to and expressed in persistent, non-dividing
cells which are differentiated, although delivery and expression
may be achieved in non-differentiated or less completely
differentiated cells, such as, for example, stem cells of blood or
skin fibroblasts. In vivo muscle cells are particularly competent
in their ability to take up and express polynucleotides.
[0968] For the nakednucleic acid sequence injection, an effective
dosage amount of DNA or RNA will be in the range of from about 0.05
mg/kg body weight to about 50 mg/kg body weight. Preferably the
dosage will be from about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration.
[0969] The preferred route of administration is by the parenteral
route of injection into the interstitial space of tissues. However,
other parenteral routes may also be used, such as, inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial
tissues, throat or mucous membranes of the nose. In addition, naked
DNA constructs can be delivered to arteries during angioplasty by
the catheter used in the procedure.
[0970] The naked polynucleotides are delivered by any method known
in the art, including, but not limited to, direct needle injection
at the delivery site, intravenous injection, topical
administration, catheter infusion, and so-called "gene guns". These
delivery methods are known in the art.
[0971] The constructs may also be delivered with delivery vehicles
such as viral sequences, viral particles, liposome formulations,
lipofectin, precipitating agents, etc. Such methods of delivery are
known in the art.
[0972] In certain embodiments, the polynucleotide constructs of the
invention are complexed in a liposome preparation. Liposomal
preparations for use in the instant invention include cationic
(positively charged), anionic (negatively charged) and neutral
preparations. However, cationic liposomes are particularly
preferred because a tight charge complex can be formed between the
cationic liposome and the polyanionic nucleic acid. Cationic
liposomes have been shown to mediate intracellular delivery of
plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA,
84:7413-7416 (1987), which is herein incorporated by reference);
mRNA (Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077-6081
(1989), which is herein incorporated by reference); and purified
transcription factors (Debs et al., J. Biol. Chem., 265:10189-10192
(1990), which is herein incorporated by reference), in functional
form.
[0973] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are particularly useful and are available under the trademark
Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner
et al., Proc. Natl Acad. Sci. USA, 84:7413-7416 (1987), which is
herein incorporated by reference). Other commercially available
liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
[0974] Other cationic liposomes can be prepared from readily
available materials using techniques well known in the art. See,
e.g. PCT Publication NO: WO 90/11092 (which is herein incorporated
by reference) for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
Preparation of DOTMA liposomes is explained in the literature, see,
e.g., Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417,
which is herein incorporated by reference. Similar methods can be
used to prepare liposomes from other cationic lipid materials.
[0975] Similarly, anionic and neutral liposomes are readily
available, such as from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl, choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[0976] For example, commercially dioleoylphosphatidyl choline
(DOPC), dioleoylphosphatidyl glycerol (DOPG), and
dioleoylphosphatidyl ethanolamine (DOPE) can be used in various
combinations to make conventional liposomes, with or without the
addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can
be prepared by drying 50 mg each of DOPG and DOPC under a stream of
nitrogen gas into a sonication vial. The sample is placed under a
vacuum pump overnight and is hydrated the following day with
deionized water. The sample is then sonicated for 2 hours in a
capped vial, using a Heat Systems model 350 sonicator equipped with
an inverted cup (bath type) probe at the maximum setting while the
bath is circulated at 15EC. Alternatively, negatively charged
vesicles can be prepared without sonication to produce
multilamellar vesicles or by extrusion through nucleopore membranes
to produce unilamellar vesicles of discrete size. Other methods are
known and available to those of skill in the art.
[0977] The liposomes can comprise multilamellar vesicles (MLVs),
small unilamellar vesicles (SUVs), or large unilamellar vesicles
(LUVs), with SUVs being preferred. The various liposome-nucleic
acid complexes are prepared using methods well known in the art.
See, e.g., Straubinger et al., Methods of Immunology, 101:512-527
(1983), which is herein incorporated by reference. For example,
MLVs containing nucleic acid can be prepared by depositing a thin
film of phospholipid on the walls of a glass tube and subsequently
hydrating with a solution of the material to be encapsulated. SUVs
are prepared by extended sonication of MLVs to produce a
homogeneous population of unilamellar liposomes. The material to be
entrapped is added to a suspension of preformed MLVs and then
sonicated. When using liposomes containing cationic lipids, the
dried lipid film is resuspended in an appropriate solution such as
sterile water or an isotonic buffer solution such as 10 mM
Tris/NaCl, sonicated, and then the preformed liposomes are mixed
directly with the DNA. The liposome and DNA form a very stable
complex due to binding of the positively charged liposomes to the
cationic DNA. SUVs find use with small nucleic acid fragments. LUVs
are prepared by a number of methods, well known in the art.
Commonly used methods include Ca2.sup.+-EDTA chelation
(Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975);
Wilson et al., Cell, 17:77 (1979)); ether injection (Deamer et al.,
Biochim. Biophys. Acta, 443:629 (1976); Ostro et al., Biochem.
Biophys. Res. Commun., 76:836 (1977); Fraley et al., Proc. Natl.
Acad. Sci. USA, 76:3348 (1979)); detergent dialysis (Enoch et al.,
Proc. Natl. Acad. Sci. USA, 76:145 (1979)); and reverse-phase
evaporation (REV) (Fraley et al., J. Biol. Chem., 255:10431 (1980);
Szoka et al., Proc. Natl. Acad. Sci. USA, 75:145 (1978);
Schaefer-Ridder et al., Science, 215:166 (1982)), which are herein
incorporated by reference.
[0978] Generally, the ratio of DNA to liposomes will be from about
10:1 to about 1:10. Preferably, the ration will be from about 5:1
to about 1:5. More preferably, the ration will be about 3:1 to
about 1:3. Still more preferably, the ratio will be about 1:1.
[0979] U.S. Pat. No.: 5,676,954 (which is herein incorporated by
reference) reports on the injection of genetic material, complexed
with cationic liposomes carriers, into mice. U.S. Pat. Nos.
4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622,
5,580,859, 5,703,055, and international publication NO: WO 94/9469
(which are herein incorporated by reference) provide cationic
lipids for use in transfecting DNA into cells and mammals. U.S.
Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and
international publication NO: WO 94/9469 (which are herein
incorporated by reference) provide methods for delivering
DNA-cationic lipid complexes to mammals.
[0980] In certain embodiments, cells are engineered, ex vivo or in
vivo, using a retroviral particle containing RNA which comprises a
sequence encoding polypeptides of the invention. Retroviruses from
which the retroviral plasmid vectors may be derived include, but
are not limited to, Moloney Murine Leukemia Virus, spleen necrosis
virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis
virus, gibbon ape leukemia virus, human immunodeficiency virus,
Myeloproliferative Sarcoma Virus, and mammary tumor virus.
[0981] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X,
VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines
as described in Miller, Human Gene Therapy, 1:5-14 (1990), which is
incorporated herein by reference in its entirety. The vector may
transduce the packaging cells through any means known in the art.
Such means include, but are not limited to, electroporation, the
use of liposomes, and CaPO.sub.4 precipitation. In one alternative,
the retroviral plasmid vector may be encapsulated into a liposome,
or coupled to a lipid, and then administered to a host.
[0982] The producer cell line generates infectious retroviral
vector particles which include polynucleotide encoding polypeptides
of the invention. Such retroviral vector particles then may be
employed, to transduce eukaryotic cells, either in vitro or in
vivo. The transduced eukaryotic cells will express polypeptides of
the invention.
[0983] In certain other embodiments, cells are engineered, ex vivo
or in vivo, with polynucleotides of the invention contained in an
adenovirus vector. Adenovirus can be manipulated such that it
encodes and expresses polypeptides of the invention, and at the
same time is inactivated in terms of its ability to replicate in a
normal lytic viral life cycle. Adenovirus expression is achieved
without integration of the viral DNA into the host cell chromosome,
thereby alleviating concerns about insertional mutagenesis.
Furthermore, adenoviruses have been used as live enteric vaccines
for many years with an excellent safety profile (Schwartzet al.,
Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally, adenovirus
mediated gene transfer has been demonstrated in a number of
instances including transfer of alpha-1-antitrypsin and CFTR to the
lungs of cotton rats (Rosenfeld et al.,Science , 252:431-434
(1991); Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore,
extensive studies to attempt to establish adenovirus as a causative
agent in human cancer were uniformly negative (Green et al. Proc.
Natl. Acad. Sci. USA, 76:6606 (1979)).
[0984] Suitable adenoviral vectors useful in the present invention
are described, for example, in Kozarsky and Wilson, Curr. Opin.
Genet. Devel., 3:499-503 (1993); Rosenfeld et al., Cell, 68:143-155
(1992); Engelhardt et al., Human Genet. Ther., 4:759-769 (1993);
Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al.,
Nature, 365:691-692 (1993); and U.S. Pat. No.: 5,652,224, which are
herein incorporated by reference. For example, the adenovirus
vector Ad2 is useful and can be grown in human 293 cells. These
cells contain the E1 region of adenovirus and constitutively
express E1a and E1b, which complement the defective adenoviruses by
providing the products of the genes deleted from the vector. In
addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and
Ad7) are also useful in the present invention.
[0985] Preferably, the adenoviruses used in the present invention
are replication deficient. Replication deficient adenoviruses
require the aid of a helper virus and/or packaging cell line to
form infectious particles. The resulting virus is capable of
infecting cells and can express a polynucleotide of interest which
is operably linked to a promoter, but cannot replicate in most
cells. Replication deficient adenoviruses may be deleted in one or
more of all or a portion of the following genes: E1a, E1b, E3, E4,
E2a, or L1 through L5.
[0986] In certain other embodiments, the cells are engineered, ex
vivo or in vivo, using an adeno-associated virus (AAV). AAVs are
naturally occurring defective viruses that require helper viruses
to produce infectious particles (Muzyczka, Curr. Topics in
Microbiol. Immunol., 158:97 (1992)). It is also one of the few
viruses that may integrate its DNA into non-dividing cells. Vectors
containing as little as 300 base pairs of AAV can be packaged and
can integrate, but space for exogenous DNA is limited to about 4.5
kb. Methods for producing and using such AAVs are known in the art.
See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377.
[0987] For example, an appropriate AAV vector for use in the
present invention will include all the sequences necessary for DNA
replication, encapsidation, and host-cell integration. The
polynucleotide construct containing polynucleotides of the
invention is inserted into the AAV vector using standard cloning
methods, such as those found in Sambrook et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Press (1989). The
recombinant AAV vector is then transfected into packaging cells
which are infected with a helper virus, using any standard
technique, including lipofection, electroporation, calcium
phosphate precipitation, etc. Appropriate helper viruses include
adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes
viruses. Once the packaging cells are transfected and infected,
they will produce infectious AAV viral particles which contain the
polynucleotide construct of the invention. These viral particles
are then used to transduce eukaryotic cells, either ex vivo or in
vivo. The transduced cells will contain the polynucleotide
construct integrated into its genome, and will express the desired
gene product.
[0988] Another method of gene therapy involves operably associating
heterologous control regions and endogenous polynucleotide
sequences (e.g. encoding the polypeptide sequence of interest) via
homologous recombination (see, e.g., U.S. Pat. No.: 5,641,670,
issued Jun. 24, 1997; International Publication NO: WO 96/29411,
published Sep. 26, 1996; International Publication NO: WO 94/12650,
published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA,
86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438
(1989). This method involves the activation of a gene which is
present in the target cells, but which is not normally expressed in
the cells, or is expressed at a lower level than desired.
[0989] Polynucleotide constructs are made, using standard
techniques known in the art, which contain the promoter with
targeting sequences flanking the promoter. Suitable promoters are
described herein. The targeting sequence is sufficiently
complementary to an endogenous sequence to permit homologous
recombination of the promoter-targeting sequence with the
endogenous sequence. The targeting sequence will be sufficiently
near the 5' end of the desired endogenous polynucleotide sequence
so the promoter will be operably linked to the endogenous sequence
upon homologous recombination.
[0990] The promoter and the targeting sequences can be amplified
using PCR. Preferably, the amplified promoter contains distinct
restriction enzyme sites on the 5' and 3' ends. Preferably, the 3'
end of the first targeting sequence contains the same restriction
enzyme site as the 5' end of the amplified promoter and the 5' end
of the second targeting sequence contains the same restriction site
as the 3' end of the amplified promoter. The amplified promoter and
targeting sequences are digested and ligated together.
[0991] The promoter-targeting sequence construct is delivered to
the cells, either as naked polynucleotide, or in conjunction with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, whole viruses, lipofection,
precipitating agents, etc., described in more detail above. The P
promoter-targeting sequence can be delivered by any method,
included direct needle injection, intravenous injection, topical
administration, catheter infusion, particle accelerators, etc. The
methods are described in more detail below.
[0992] The promoter-targeting sequence construct is taken up by
cells. Homologous recombination between the construct and the
endogenous sequence takes place, such that an endogenous sequence
is placed under the control of the promoter. The promoter then
drives the expression of the endogenous sequence.
[0993] The polynucleotides encoding polypeptides of the present
invention may be administered along with other polynucleotides
encoding other angiongenic proteins. Angiogenic proteins include,
but are not limited to, acidic and basic fibroblast growth factors,
VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor
alpha and beta, platelet-derived endothelial cell growth factor,
platelet-derived growth factor, tumor necrosis factor alpha,
hepatocyte growth factor, insulin like growth factor, colony
stimulating factor, macrophage colony stimulating factor,
granulocyte/macrophage colony stimulating factor, and nitric oxide
synthase.
[0994] Preferably, the polynucleotide encoding a polypeptide of the
invention contains a secretory signal sequence that facilitates
secretion of the protein. Typically, the signal sequence is
positioned in the coding region of the polynucleotide to be
expressed towards or at the 5' end of the coding region. The signal
sequence may be homologous or heterologous to the polynucleotide of
interest and may be homologous or heterologous to the cells to be
transfected. Additionally, the signal sequence may be chemically
synthesized using methods known in the art.
[0995] Any mode of administration of any of the above-described
polynucleotides constructs can be used so long as the mode results
in the expression of one or more molecules in an amount sufficient
to provide a therapeutic effect. This includes direct needle
injection, systemic injection, catheter infusion, biolistic
injectors, particle accelerators (i.e., "gene guns"), gelfoam
sponge depots, other commercially available depot materials,
osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid
(tablet or pill) pharmaceutical formulations, and decanting or
topical applications during surgery. For example, direct injection
of naked calcium phosphate-precipitated plasmid into rat liver and
rat spleen or a protein-coated plasmid into the portal vein has
resulted in gene expression of the foreign gene in the rat livers.
(Kaneda et al., Science, 243:375 (1989)).
[0996] A preferred method of local administration is by direct
injection. Preferably, a recombinant molecule of the present
invention complexed with a delivery vehicle is administered by
direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries
refers to injecting the composition centimeters and preferably,
millimeters within arteries.
[0997] Another method of local administration is to contact a
polynucleotide construct of the present invention in or around a
surgical wound. For example, a patient can undergo surgery and the
polynucleotide construct can be coated on the surface of tissue
inside the wound or the construct can be injected into areas of
tissue inside the wound.
[0998] Therapeutic compositions useful in systemic administration,
include recombinant molecules of the present invention complexed to
a targeted delivery vehicle of the present invention. Suitable
delivery vehicles for use with systemic administration comprise
liposomes comprising ligands for targeting the vehicle to a
particular site.
[0999] Preferred methods of systemic administration, include
intravenous injection, aerosol, oral and percutaneous (topical)
delivery. Intravenous injections can be performed using methods
standard in the art. Aerosol delivery can also be performed using
methods standard in the art (see, for example, Stribling et al.,
Proc. Natl. Acad. Sci. USA, 189:11277-11281 (1992), which is
incorporated herein by reference). Oral delivery can be performed
by complexing a polynucleotide construct of the present invention
to a carrier capable of withstanding degradation by digestive
enzymes in the gut of an animal. Examples of such carriers, include
plastic capsules or tablets, such as those known in the art.
Topical delivery can be performed by mixing a polynucleotide
construct of the present invention with a lipophilic reagent (e.g.,
DMSO) that is capable of passing into the skin.
[1000] Determining an effective amount of substance to be delivered
can depend upon a number of factors including, for example, the
chemical structure and biological activity of the substance, the
age and weight of the animal, the precise condition requiring
treatment and its severity, and the route of administration. The
frequency of treatments depends upon a number of factors, such as
the amount of polynucleotide constructs administered per dose, as
well as the health and history of the subject. The precise amount,
number of doses, and timing of doses will be determined by the
attending physician or veterinarian. Therapeutic compositions of
the present invention can be administered to any animal, preferably
to mammals and birds. Preferred mammals include humans, dogs, cats,
mice, rats, rabbits sheep, cattle, horses and pigs, with humans
being particularly
[1001] Biological Activities
[1002] The polynucleotides or polypeptides, or agonists or
antagonists of the present invention can be used in assays to test
for one or more biological activities. If these polynucleotides and
polypeptides do exhibit activity in a particular assay, it is
likely that these molecules may be involved in the diseases
associated with the biological activity. Thus, the polynucleotides
or polypeptides, or agonists or antagonists could be used to treat
the associated disease.
[1003] Immune Activity
[1004] Polynucleotides, polypeptides, antibodies, and/or agonists
or antagonists of the present invention may be useful in treating,
preventing, and/or diagnosing diseases, disorders, and/or
conditions of the immune system, by, for example, activating or
inhibiting the proliferation, differentiation, or mobilization
(chemotaxis) of immune cells. Immune cells develop through a
process called hematopoiesis, producing myeloid (platelets, red
blood cells, neutrophils, and macrophages) and lymphoid (B and T
lymphocytes) cells from pluripotent stem cells. The etiology of
these immune diseases, disorders, and/or conditions may be genetic,
somatic, such as cancer and some autoimmune diseases, acquired
(e.g., by chemotherapy or toxins), or infectious. Moreover,
polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention can be used as a marker or
detector of a particular immune system disease or disorder.
[1005] Polynucleotides, polypeptides, antibodies, and/or agonists
or antagonists of the present invention may be useful in treating,
preventing, and/or diagnosing diseases, disorders, and/or
conditions of hematopoietic cells. Polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
could be used to increase differentiation and proliferation of
hematopoietic cells, including the pluripotent stem cells, in an
effort to treat or prevent those diseases, disorders, and/or
conditions associated with a decrease in certain (or many) types
hematopoietic cells. Examples of immunologic deficiency syndromes
include, but are not limited to: blood protein diseases, disorders,
and/or conditions (e.g., agammaglobulinemia, dysgammaglobulinemia),
ataxia telangiectasia, common variable immunodeficiency, Digeorge
Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion
deficiency syndrome, lymphopenia, phagocyte bactericidal
dysfunction, severe combined immunodeficiency (SCIDs),
Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or
hemoglobinuria.
[1006] Moreover, polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention could also be used
to modulate hemostatic (the stopping of bleeding) or thrombolytic
activity (clot formation). For example, by increasing hemostatic or
thrombolytic activity, polynucleotides or polypeptides, and/or
agonists or antagonists of the present invention could be used to
treat or prevent blood coagulation diseases, disorders, and/or
conditions (e.g., afibrinogenemia, factor deficiencies), blood
platelet diseases, disorders, and/or conditions (e.g.,
thrombocytopenia), or wounds resulting from trauma, surgery, or
other causes. Alternatively, polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
that can decrease hemostatic or thrombolytic activity could be used
to inhibit or dissolve clotting. These molecules could be important
in the treatment or prevention of heart attacks (infarction),
strokes, or scarring.
[1007] The polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be useful in
treating, preventing, and/or diagnosing autoimmune disorders. Many
autoimmune disorders result from inappropriate recognition of self
as foreign material by immune cells. This inappropriate recognition
results in an immune response leading to the destruction of the
host tissue. Therefore, the administration of polynucleotides and
polypeptides of the invention that can inhibit an immune response,
particularly the proliferation, differentiation, or chemotaxis of
T-cells, may be an effective therapy in preventing autoimmune
disorders.
[1008] Autoimmune diseases or disorders that may be treated,
prevented, and/or diagnosed by polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
include, but are not limited to, one or more of the following:
autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia,
idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolytic
anemia, antiphospholipid syndrome, dermatitis, allergic
encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic
heart disease, glomerulonephritis (e.g, IgA nephropathy), Multiple
Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies,
Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease,
Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, Autism,
Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and
autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism
(i.e., Hashimoto's thyroiditis, systemic lupus erhythematosus,
Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as,
for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c)
insulin resistance, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura, rheumatoid arthritis, schleroderma with
anti-collagen antibodies, mixed connective tissue disease,
polymyositis/dermatomyositis, pernicious anemia, idiopathic
Addison's disease, infertility, glomerulonephritis such as primary
glomerulonephritis and IgA nephropathy, bullous pemphigoid,
Sjogren's syndrome, diabetes millitus, and adrenergic drug
resistance (including adrenergic drug resistance with asthma or
cystic fibrosis), chronic active hepatitis, primary biliary
cirrhosis, other endocrine gland failure, vitiligo, vasculitis,
post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma,
inflammatory myopathies, and other inflammatory, granulamatous,
degenerative, and atrophic disorders.
[1009] Additional autoimmune disorders (that are probable) that may
be treated, prevented, and/or diagnosed with the compositions of
the invention include, but are not limited to, rheumatoid arthritis
(often characterized, e.g., by immune complexes in joints),
scleroderma with anti-collagen antibodies (often characterized,
e.g., by nucleolar and other nuclear antibodies), mixed connective
tissue disease (often characterized, e.g., by antibodies to
extractable nuclear antigens (e.g., ribonucleoprotein)),
polymyositis (often characterized, e.g., by nonhistone ANA),
pernicious anemia (often characterized, e.g., by antiparietal cell,
microsomes, and intrinsic factor antibodies), idiopathic Addison's
disease (often characterized, e.g., by humoral and cell-mediated
adrenal cytotoxicity, infertility (often characterized, e.g., by
antispermatozoal antibodies), glomerulonephritis (often
characterized, e.g., by glomerular basement membrane antibodies or
immune complexes), bullous pemphigoid (often characterized, e.g.,
by IgG and complement in basement membrane), Sjogren's syndrome
(often characterized, e.g., by multiple tissue antibodies, and/or a
specific nonhistone ANA (SS-B)), diabetes millitus (often
characterized, e.g., by cell-mediated and humoral islet cell
antibodies), and adrenergic drug resistance (including adrenergic
drug resistance with asthma or cystic fibrosis) (often
characterized, e.g., by beta-adrenergic receptor antibodies).
[1010] Additional autoimmune disorders (that are possible) that may
be treated, prevented, and/or diagnosed with the compositions of
the invention include, but are not limited to, chronic active
hepatitis (often characterized, e.g., by smooth muscle antibodies),
primary biliary cirrhosis (often characterized, e.g., by
mitchondrial antibodies), other endocrine gland failure (often
characterized, e.g., by specific tissue antibodies in some cases),
vitiligo (often characterized, e.g., by melanocyte antibodies),
vasculitis (often characterized, e.g., by Ig and complement in
vessel walls and/or low serum complement), post-MI (often
characterized, e.g., by myocardial antibodies), cardiotomy syndrome
(often characterized, e.g., by myocardial antibodies), urticaria
(often characterized, e.g., by IgG and IgM antibodies to IgE),
atopic dermatitis (often characterized, e.g., by IgG and IgM
antibodies to IgE), asthma (often characterized, e.g., by IgG and
IgM antibodies to IgE), and many other inflammatory, granulamatous,
degenerative, and atrophic disorders.
[1011] In a preferred embodiment, the autoimmune diseases and
disorders and/or conditions associated with the diseases and
disorders recited above are treated, prevented, and/or diagnosed
using for example, antagonists or agonists, polypeptides or
polynucleotides, or antibodies of the present invention.
[1012] In a preferred embodiment polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
could be used as an agent to boost immunoresponsiveness among B
cell and/or T cell immunodeficient individuals.
[1013] B cell immunodeficiencies that may be ameliorated or treated
by administering the polypeptides or polynucleotides of the
invention, and/or agonists thereof, include, but are not limited
to, severe combined immunodeficiency (SCID)-X linked,
SCID-autosomal, adenosine deaminase deficiency (ADA deficiency),
X-linked agammaglobulinemia (XLA), Bruton's disease, congenital
agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired
agammaglobulinemia, adult onset agammaglobulinemia, late-onset
agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,
transient hypogammaglobulinemia of infancy, unspecified
hypogammaglobulinemia, agammaglobulinemia, common variable
immunodeficiency (CVI) (acquired), Wiskott-Aldrich Syndrome (WAS),
X-linked immunodeficiency with hyper IgM, non X-linked
immunodeficiency with hyper IgM, selective IgA deficiency, IgG
subclass deficiency (with or without IgA deficiency), antibody
deficiency with normal or elevated Igs, immunodeficiency with
thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell
lymphoproliferative disorder (BLPD), selective IgM
immunodeficiency, recessive agammaglobulinemia (Swiss type),
reticular dysgenesis, neonatal neutropenia, severe congenital
leukopenia, thymic alymophoplasia-aplasia or dysplasia with
immunodeficiency, ataxia-telangiectasia, short limbed dwarfism,
X-linked lymphoproliferative syndrome (XLP), Nezelof
syndrome-combined immunodeficiency with Igs, purine nucleoside
phosphorylase deficiency (PNP), MHC Class II deficiency (Bare
Lymphocyte Syndrome) and severe combined immunodeficiency.
[1014] T cell deficiencies that may be ameliorated or treated by
administering the polypeptides or polynucleotides of the invention,
and/or agonists thereof include, but are not limited to, for
example, DiGeorge anomaly, thymic hypoplasia, third and fourth
pharyngeal pouch syndrome, 22q11.2 deletion, chronic mucocutaneous
candidiasis, natural killer cell deficiency (NK), idiopathic
CD4+T-lymphocytopenia, immunodeficiency with predominant T cell
defect (unspecified), and unspecified immunodeficiency of cell
mediated immunity. In specific embodiments, DiGeorge anomaly or
conditions associated with DiGeorge anomaly are ameliorated or
treated by, for example, administering the polypeptides or
polynucleotides of the invention, or antagonists or agonists
thereof.
[1015] Other immunodeficiencies that may be ameliorated or treated
by administering polypeptides or polynucleotides of the invention,
and/or agonists thereof, include, but are not limited to, severe
combined immunodeficiency (SCID; e.g., X-linked SCID, autosomal
SCID, and adenosine deaminase deficiency), ataxia-telangiectasia,
Wiskott-Aldrich syndrome, short-limber dwarfism, X-linked
lymphoproliferative syndrome (XLP), Nezelof syndrome (e.g., purine
nucleoside phosphorylase deficiency), MHC Class II deficiency. In
specific embodiments, ataxia-telangiectasia or conditions
associated with ataxia-telangiectasia are ameliorated or treated by
administering the polypeptides or polynucleotides of the invention,
and/or agonists thereof.
[1016] In a specific preferred embodiment, rheumatoid arthritis is
treated, prevented, and/or diagnosed using polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention. In another specific preferred embodiment,
systemic lupus erythemosus is treated, prevented, and/or diagnosed
using polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention. In another specific preferred
embodiment, idiopathic thrombocytopenia purpura is treated,
prevented, and/or diagnosed using polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present
invention. In another specific preferred embodiment IgA nephropathy
is treated, prevented, and/or diagnosed using polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention. In a preferred embodiment, the autoimmune
diseases and disorders and/or conditions associated with the
diseases and disorders recited above are treated, prevented, and/or
diagnosed using antibodies against the protein of the
invention.
[1017] Similarly, allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated, prevented, and/or diagnosed using polypeptides,
antibodies, or polynucleotides of the invention, and/or agonists or
antagonists thereof. Moreover, these molecules can be used to
treat, prevent, and/or diagnose anaphylaxis, hypersensitivity to an
antigenic molecule, or blood group incompatibility.
[1018] Moreover, inflammatory conditions may also be treated,
diagnosed, and/or prevented with polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present
invention. Such inflammatory conditions include, but are not
limited to, for example, respiratory disorders (such as, e.g.,
asthma and allergy); gastrointestinal disorders (such as, e.g.,
inflammatory bowel disease); cancers (such as, e.g., gastric,
ovarian, lung, bladder, liver, and breast); CNS disorders (such as,
e.g., multiple sclerosis, blood-brain barrier permeability,
ischemic brain injury and/or stroke, traumatic brain injury,
neurodegenerative disorders (such as, e.g., Parkinson's disease and
Alzheimer's disease), AIDS-related dementia, and prion disease);
cardiovascular disorders (such as, e.g., atherosclerosis,
myocarditis, cardiovascular disease, and cardiopulmonary bypass
complications); as well as many additional diseases, conditions,
and disorders that are characterized by inflammation (such as,
e.g., chronic hepatitis (B and C), rheumatoid arthritis, gout,
trauma, septic shock, pancreatitis, sarcoidosis, dermatitis, renal
ischemia-reperfusion injury, Grave's disease, systemic lupus
erythematosis, diabetes mellitus (i.e., type 1 diabetes), and
allogenic transplant rejection).
[1019] In specific embodiments, polypeptides, antibodies, or
polynucleotides of the invention, and/or agonists or antagonists
thereof, are useful to treat, diagnose, and/or prevent
transplantation rejections, graft-versus-host disease, autoimmune
and inflammatory diseases (e.g., immune complex-induced vasculitis,
glomerulonephritis, hemolytic anemia, myasthenia gravis, type II
collagen-induced arthritis, experimental allergic and hyperacute
xenograft rejection, rheumatoid arthritis, and systemic lupus
erythematosus (SLE). Organ rejection occurs by host immune cell
destruction of the transplanted tissue through an immune response.
Similarly, an immune response is also involved in GVHD, but, in
this case, the foreign transplanted immune cells destroy the host
tissues. Polypeptides, antibodies, or polynucleotides of the
invention, and/or agonists or antagonists thereof, that inhibit an
immune response, particularly the activation, proliferation,
differentiation, or chemotaxis of T-cells, may be an effective
therapy in preventing organ rejection or GVHD.
[1020] Similarly, polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may also be used
to modulate and/or diagnose inflammation. For example, since
polypeptides, antibodies, or polynucleotides of the invention,
and/or agonists or antagonists of the invention may inhibit the
activation, proliferation and/or differentiation of cells involved
in an inflammatory response, these molecules can be used to treat,
diagnose, or prognose, inflammatory conditions, both chronic and
acute conditions, including, but not limited to, inflammation
associated with infection (e.g., septic shock, sepsis, or systemic
inflammatory response syndrome (SIRS)), ischemia-reperfusion
injury, endotoxin lethality, arthritis, complement-mediated
hyperacute rejection, nephritis, cytokine or chemokine induced lung
injury, inflammatory bowel disease, Crohn's disease, and resulting
from over production of cytokines (e.g., TNF or IL-1. ).
[1021] Polypeptides, antibodies, polynucleotides and/or agonists or
antagonists of the invention can be used to treat, detect, and/or
prevent infectious agents. For example, by increasing the immune
response, particularly increasing the proliferation activation
and/or differentiation of B and/or T cells, infectious diseases may
be treated, detected, and/or prevented. The immune response may be
increased by either enhancing an existing immune response, or by
initiating a new immune response. Alternatively, polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention may also directly inhibit the infectious agent
(refer to section of application listing infectious agents, etc),
without necessarily eliciting an immune response.
[1022] Additional preferred embodiments of the invention include,
but are not limited to, the use of polypeptides, antibodies,
polynucleotides and/or agonists or antagonists in the following
applications:
[1023] Administration to an animal (e.g., mouse, rat, rabbit,
hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse,
cow, sheep, dog, cat, non-human primate, and human, most preferably
human) to boost the immune system to produce increased quantities
of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce
higher affinity antibody production (e.g., IgG, IgA, IgM, and IgE),
and/or to increase an immune response.
[1024] Administration to an animal (including, but not limited to,
those listed above, and also including transgenic animals)
incapable of producing functional endogenous antibody molecules or
having an otherwise compromised endogenous immune system, but which
is capable of producing human immunoglobulin molecules by means of
a reconstituted or partially reconstituted immune system from
another animal (see, e.g., published PCT Application Nos.
WO98/24893, WO/9634096, WO/9633735, and WO/9110741.
[1025] A vaccine adjuvant that enhances immune responsiveness to
specific antigen.
[1026] An adjuvant to enhance tumor-specific immune responses.
[1027] An adjuvant to enhance anti-viral immune responses.
Anti-viral immune responses that may be enhanced using the
compositions of the invention as an adjuvant, include virus and
virus associated diseases or symptoms described herein or otherwise
known in the art. In specific embodiments, the compositions of the
invention are used as an adjuvant to enhance an immune response to
a virus, disease, or symptom selected from the group consisting of:
AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B).
In another specific embodiment, the compositions of the invention
are used as an adjuvant to enhance an immune response to a virus,
disease, or symptom selected from the group consisting of:
HIV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus, Japanese
B encephalitis, Influenza A and B, Parainfluenza, Measles,
Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever,
Herpes simplex, and yellow fever.
[1028] An adjuvant to enhance anti-bacterial or anti-fingal immune
responses. Anti-bacterial or anti-fungal immune responses that may
be enhanced using the compositions of the invention as an adjuvant,
include bacteria or fungus and bacteria or fungus associated
diseases or symptoms described herein or otherwise known in the
art. In specific embodiments, the compositions of the invention are
used as an adjuvant to enhance an immune response to a bacteria or
fungus, disease, or symptom selected from the group consisting of:
tetanus, Diphtheria, botulism, and meningitis type B. In another
specific embodiment, the compositions of the invention are used as
an adjuvant to enhance an immune response to a bacteria or fungus,
disease, or symptom selected from the group consisting of: Vibrio
cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella
paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group
B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,
Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium
(malaria).
[1029] An adjuvant to enhance anti-parasitic immune responses.
Anti-parasitic immune responses that may be enhanced using the
compositions of the invention as an adjuvant, include parasite and
parasite associated diseases or symptoms described herein or
otherwise known in the art. In specific embodiments, the
compositions of the invention are used as an adjuvant to enhance an
immune response to a parasite. In another specific embodiment, the
compositions of the invention are used as an adjuvant to enhance an
immune response to Plasmodium (malaria).
[1030] As a stimulator of B cell responsiveness to pathogens.
[1031] As an activator of T cells.
[1032] As an agent that elevates the immune status of an individual
prior to their receipt of immunosuppressive therapies.
[1033] As an agent to induce higher affinity antibodies.
[1034] As an agent to increase serum immunoglobulin
concentrations.
[1035] As an agent to accelerate recovery of immunocompromised
individuals.
[1036] As an agent to boost immunoresponsiveness among aged
populations.
[1037] As an immune system enhancer prior to, during, or after bone
marrow transplant and/or other transplants (e.g., allogeneic or
xenogeneic organ transplantation). With respect to transplantation,
compositions of the invention may be administered prior to,
concomitant with, and/or after transplantation. In a specific
embodiment, compositions of the invention are administered after
transplantation, prior to the beginning of recovery of T-cell
populations. In another specific embodiment, compositions of the
invention are first administered after transplantation after the
beginning of recovery of T cell populations, but prior to full
recovery of B cell populations.
[1038] As an agent to boost immunoresponsiveness among individuals
having an acquired loss of B cell function. Conditions resulting in
an acquired loss of B cell function that may be ameliorated or
treated by administering the polypeptides, antibodies,
polynucleotides and/or agonists or antagonists thereof, include,
but are not limited to, HIV Infection, AIDS, bone marrow
transplant, and B cell chronic lymphocytic leukemia (CLL).
[1039] As an agent to boost immunoresponsiveness among individuals
having a temporary immune deficiency. Conditions resulting in a
temporary immune deficiency that may be ameliorated or treated by
administering the polypeptides, antibodies, polynucleotides and/or
agonists or antagonists thereof, include, but are not limited to,
recovery from viral infections (e.g., influenza), conditions
associated with malnutrition, recovery from infectious
mononucleosis, or conditions associated with stress, recovery from
measles, recovery from blood transfusion, recovery from
surgery.
[1040] As a regulator of antigen presentation by monocytes,
dendritic cells, and/or B-cells. In one embodiment,
polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention enhance antigen presentation
or antagonizes antigen presentation in vitro or in vivo. Moreover,
in related embodiments, said enhancement or antagonization of
antigen presentation may be useful as an anti-tumor treatment or to
modulate the immune system.
[1041] As an agent to direct an individuals immune system towards
development of a humoral response (i.e. TH2) as opposed to a THI
cellular response.
[1042] As a means to induce tumor proliferation and thus make it
more susceptible to anti-neoplastic agents. For example, multiple
myeloma is a slowly dividing disease and is thus refractory to
virtually all anti-neoplastic regimens. If these cells were forced
to proliferate more rapidly their susceptibility profile would
likely change.
[1043] As a stimulator of B cell production in pathologies such as
AIDS, chronic lymphocyte disorder and/or Common Variable
Immunodificiency.
[1044] As a therapy for generation and/or regeneration of lymphoid
tissues following surgery, trauma or genetic defect.
[1045] As a gene-based therapy for genetically inherited disorders
resulting in immuno-incompetence such as observed among SCID
patients.
[1046] As an antigen for the generation of antibodies to inhibit or
enhance immune mediated responses against polypeptides of the
invention.
[1047] As a means of activating T cells.
[1048] As a means of activating monocytes/macrophages to defend
against parasitic diseases that effect monocytes such as
Leshmania.
[1049] As pretreatment of bone marrow samples prior to transplant.
Such treatment would increase B cell representation and thus
accelerate recover.
[1050] As a means of regulating secreted cytokines that are
elicited by polypeptides of the invention.
[1051] Additionally, polypeptides or polynucleotides of the
invention, and/or agonists thereof, may be used to treat or prevent
IgE-mediated allergic reactions. Such allergic reactions include,
but are not limited to, asthma, rhinitis, and eczema.
[1052] All of the above described applications as they may apply to
veterinary medicine.
[1053] Antagonists of the invention include, for example, binding
and/or inhibitory antibodies, antisense nucleic acids, or
ribozymes. These would be expected to reverse many of the
activities of the ligand described above as well as find clinical
or practical application as:
[1054] A means of blocking various aspects of immune responses to
foreign agents or self. Examples include autoimmune disorders such
as lupus, and arthritis, as well as immunoresponsiveness to skin
allergies, inflammation, bowel disease, injury and pathogens.
[1055] A therapy for preventing the B cell proliferation and Ig
secretion associated with autoimmune diseases such as idiopathic
thrombocytopenic purpura, systemic lupus erythramatosus and MS.
[1056] An inhibitor of B and/or T cell migration in endothelial
cells. This activity disrupts tissue architecture or cognate
responses and is useful, for example in disrupting immune
responses, and blocking sepsis.
[1057] An inhibitor of graft versus host disease or transplant
rejection.
[1058] A therapy for B cell and/or T cell malignancies such as ALL,
Hodgkins disease, non-Hodgkins lymphoma, Chronic lymphocyte
leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, and
EBV-transformed diseases.
[1059] A therapy for chronic hypergammaglobulinemeia evident in
such diseases as monoclonalgammopathy of undetermined significance
(MGUS), Waldenstrom's disease, related idiopathic
monoclonalgammopathies, and plasmacytomas.
[1060] A therapy for decreasing cellular proliferation of Large
B-cell Lymphomas.
[1061] A means of decreasing the involvement of B cells and Ig
associated with Chronic Myelogenous Leukemia.
[1062] An immunosuppressive agent(s).
[1063] Polynucleotides, polypeptides, antibodies, and/or agonists
or antagonists of the present invention may be used to modulate IgE
concentrations in vitro or in vivo.
[1064] In another embodiment, administration of polypeptides,
antibodies, polynucleotides and/or agonists or antagonists of the
invention, may be used to treat or prevent IgE-mediated allergic
reactions including, but not limited to, asthma, rhinitis, and
eczema.
[1065] The agonists and antagonists may be employed in a
composition with a pharmaceutically acceptable carrier, e.g., as
described herein.
[1066] The agonists or antagonists may be employed for instance to
inhibit polypeptide chemotaxis and activation of macrophages and
their precursors, and of neutrophils, basophils, B lymphocytes and
some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and
natural killer cells, in certain auto-immune and chronic
inflammatory and infective diseases. Examples of autoimmune
diseases are described herein and include multiple sclerosis, and
insulin-dependent diabetes. The antagonists or agonists may also be
employed to treat infectious diseases including silicosis,
sarcoidosis, idiopathic pulmonary fibrosis by, for example,
preventing the recruitment and activation of mononuclear
phagocytes. They may also be employed to treat idiopathic
hyper-eosinophilic syndrome by, for example, preventing eosinophil
production and migration. The antagonists or agonists or may also
be employed for treating atherosclerosis, for example, by
preventing monocyte infiltration in the artery wall.
[1067] Antibodies against polypeptides of the invention may be
employed to treat ARDS.
[1068] Agonists and/or antagonists of the invention also have uses
in stimulating wound and tissue repair, stimulating angiogenesis,
stimulating the repair of vascular or lymphatic diseases or
disorders. Additionally, agonists and antagonists of the invention
may be used to stimulate the regeneration of mucosal surfaces.
[1069] In a specific embodiment, polynucleotides or polypeptides,
and/or agonists thereof are used to treat or prevent a disorder
characterized by primary or acquired immunodeficiency, deficient
serum immunoglobulin production, recurrent infections, and/or
immune system dysfunction. Moreover, polynucleotides or
polypeptides, and/or agonists thereof may be used to treat or
prevent infections of the joints, bones, skin, and/or parotid
glands, blood-borne infections (e.g., sepsis, meningitis, septic
arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those
disclosed herein), inflammatory disorders, and malignancies, and/or
any disease or disorder or condition associated with these
infections, diseases, disorders and/or malignancies) including, but
not limited to, CVID, other primary immune deficiencies, HIV
disease, CLL, recurrent bronchitis, sinusitis, otitis media,
conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster
(e.g., severe herpes zoster), and/or pneumocystis carnii.
[1070] In another embodiment, polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
are used to treat, and/or diagnose an individual having common
variable immunodeficiency disease ("CVID"; also known as "acquired
agammaglobulinemia" and "acquired hypogammaglobulinemia") or a
subset of this disease.
[1071] In a specific embodiment, polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
may be used to treat, diagnose, and/or prevent (1) cancers or
neoplasms and (2) autoimmune cell or tissue-related cancers or
neoplasms. In a preferred embodiment, polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the
present invention conjugated to a toxin or a radioactive isotope,
as described herein, may be used to treat, diagnose, and/or prevent
acute myelogeneous leukemia. In a further preferred embodiment,
polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention conjugated to a toxin or a
radioactive isotope, as described herein, may be used to treat,
diagnose, and/or prevent, chronic myelogeneous leukemia, multiple
myeloma, non-Hodgkins lymphoma, and/or Hodgkins disease.
[1072] In another specific embodiment, polynucleotides or
polypeptides, and/or agonists or antagonists of the invention may
be used to treat, diagnose, prognose, and/or prevent selective IgA
deficiency, myeloperoxidase deficiency, C2 deficiency,
ataxia-telangiectasia, DiGeorge anomaly, common variable
immunodeficiency (CVI), X-linked agammaglobulinemia, severe
combined immunodeficiency (SCID), chronic granulomatous disease
(CGD), and Wiskott-Aldrich syndrome.
[1073] Examples of autoimmune disorders that can be treated or
detected are described above and also include, but are not limited
to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome,
rheumatoid arthritis, dermatitis, allergic encephalomyelitis,
glomerulonephritis, Goodpasture's Syndrome, Grave' Disease,
Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia,
Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura,
Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis,
Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,
Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and
autoimmune inflammatory eye disease.
[1074] In a preferred embodiment, the autoimmune diseases and
disorders and/or conditions associated with the diseases and
disorders recited above are treated, prognosed, prevented, and/or
diagnosed using antibodies against the polypeptide of the
invention.
[1075] As an agent to boost immunoresponsiveness among B cell
immunodeficient individuals, such as, for example, an individual
who has undergone a partial or complete splenectomy.
[1076] Additionally, polynucleotides, polypeptides, and/or
antagonists of the invention may affect apoptosis, and therefore,
would be useful in treating a number of diseases associated with
increased cell survival or the inhibition of apoptosis. For
example, diseases associated with increased cell survival or the
inhibition of apoptosis that could be treated or detected by
polynucleotides, polypeptides, and/or antagonists of the invention,
include cancers (such as follicular lymphomas, carcinomas with p53
mutations, and hormone-dependent tumors, including, but not limited
to colon cancer, cardiac tumors, pancreatic cancer, melanoma,
retinoblastoma, glioblastoma, lung cancer, intestinal cancer,
testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,
lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,
chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's
sarcoma and ovarian cancer); autoimmune disorders (such as,
multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis,
biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis,
systemic lupus erythematosus and immune-related glomerulonephritis
and rheumatoid arthritis) and viral infections (such as herpes
viruses, pox viruses and adenoviruses), inflammation, graft v. host
disease, acute graft rejection, and chronic graft rejection. In
preferred embodiments, polynucleotides, polypeptides, and/or
antagonists of the invention are used to inhibit growth,
progression, and/or metastisis of cancers, in particular those
listed above.
[1077] Additional diseases or conditions associated with increased
cell survival that could be treated or detected by polynucleotides,
polypeptides, and/or antagonists of the invention, include, but are
not limited to, progression, and/or metastases of malignancies and
related disorders such as leukemia (including acute leukemias
(e.g., acute lymphocytic leukemia, acute myelocytic leukemia
(including myeloblastic, promyelocytic, myelomonocytic, monocytic,
and erythroleukemia)) and chronic leukemias (e.g., chronic
myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease
and non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors including,
but not limited to, sarcomas and carcinomas such as fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[1078] Diseases associated with increased apoptosis that could be
treated or detected by polynucleotides, polypeptides, and/or
antagonists of the invention, include AIDS; neurodegenerative
disorders (such as Alzheimer's disease, Parkinson's disease,
Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar
degeneration and brain tumor or prior associated disease);
autoimmune disorders (such as, multiple sclerosis, Sjogren's
syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's
disease, Crohn's disease, polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis and rheumatoid
arthritis) myelodysplastic syndromes (such as aplastic anemia),
graft v. host disease, ischemic injury (such as that caused by
myocardial infarction, stroke and reperfusion injury), liver injury
(e.g., hepatitis related liver injury, ischemia/reperfusion injury,
cholestosis (bile duct injury) and liver cancer); toxin-induced
liver disease (such as that caused by alcohol), septic shock,
cachexia and anorexia.
[1079] Hyperproliferative diseases and/or disorders that could be
detected and/or treated by polynucleotides, polypeptides, and/or
antagonists of the invention, include, but are not limited to
neoplasms located in the: liver, abdomen, bone, breast, digestive
system, pancreas, peritoneum, endocrine glands (adrenal,
parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye,
head and neck, nervous (central and peripheral), lymphatic system,
pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
[1080] Similarly, other hyperproliferative disorders can also be
treated or detected by polynucleotides, polypeptides, and/or
antagonists of the invention. Examples of such hyperproliferative
disorders include, but are not limited to: hypergammaglobulinemia,
lymphoproliferative disorders, paraproteinemias, purpura,
sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia,
Gaucher's Disease, histiocytosis, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
[1081] Hyperproliferative Disorders
[1082] A polynucleotides or polypeptides, or agonists or
antagonists of the invention can be used to treat, prevent, and/or
diagnose hyperproliferative diseases, disorders, and/or conditions,
including neoplasms. A polynucleotides or polypeptides, or agonists
or antagonists of the present invention may inhibit the
proliferation of the disorder through direct or indirect
interactions. Alternatively, a polynucleotides or polypeptides, or
agonists or antagonists of the present invention may proliferate
other cells which can inhibit the hyperproliferative disorder.
[1083] For example, by increasing an immune response, particularly
increasing antigenic qualities of the hyperproliferative disorder
or by proliferating, differentiating, or mobilizing T-cells,
hyperproliferative diseases, disorders, and/or conditions can be
treated, prevented, and/or diagnosed. This immune response may be
increased by either enhancing an existing immune response, or by
initiating a new immune response. Alternatively, decreasing an
immune response may also be a method of treating, preventing,
and/or diagnosing hyperproliferative diseases, disorders, and/or
conditions, such as a chemotherapeutic agent.
[1084] Examples of hyperproliferative diseases, disorders, and/or
conditions that can be treated, prevented, and/or diagnosed by
polynucleotides or polypeptides, or agonists or antagonists of the
present invention include, but are not limited to neoplasms located
in the: colon, abdomen, bone, breast, digestive system, liver,
pancreas, peritoneum, endocrine glands (adrenal, parathyroid,
pituitary, testicles, ovary, thymus, thyroid), eye, head and neck,
nervous (central and peripheral), lymphatic system, pelvic, skin,
soft tissue, spleen, thoracic, and urogenital.
[1085] Similarly, other hyperproliferative diseases, disorders,
and/or conditions can also be treated, prevented, and/or diagnosed
by a polynucleotides or polypeptides, or agonists or antagonists of
the present invention. Examples of such hyperproliferative
diseases, disorders, and/or conditions include, but are not limited
to: hypergammaglobulinemia, lymphoproliferative diseases,
disorders, and/or conditions, paraproteinemias, purpura,
sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia,
Gaucher's Disease, histiocytosis, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
[1086] One preferred embodiment utilizes polynucleotides of the
present invention to inhibit aberrant cellular division, by gene
therapy using the present invention, and/or protein fusions or
fragments thereof.
[1087] Thus, the present invention provides a method for treating
or preventing cell proliferative diseases, disorders, and/or
conditions by inserting into an abnormally proliferating cell a
polynucleotide of the present invention, wherein said
polynucleotide represses said expression.
[1088] Another embodiment of the present invention provides a
method of treating or preventing cell-proliferative diseases,
disorders, and/or conditions in individuals comprising
administration of one or more active gene copies of the present
invention to an abnormally proliferating cell or cells. In a
preferred embodiment, polynucleotides of the present invention is a
DNA construct comprising a recombinant expression vector effective
in expressing a DNA sequence encoding said polynucleotides. In
another preferred embodiment of the present invention, the DNA
construct encoding the poynucleotides of the present invention is
inserted into cells to be treated utilizing a retrovirus, or more
preferrably an adenoviral vector (See G J. Nabel, et. al., PNAS
1999 96: 324-326, which is hereby incorporated by reference). In a
most preferred embodiment, the viral vector is defective and will
not transform non-proliferating cells, only proliferating cells.
Moreover, in a preferred embodiment, the polynucleotides of the
present invention inserted into proliferating cells either alone,
or in combination with or fused to other polynucleotides, can then
be modulated via an external stimulus (i.e. magnetic, specific
small molecule, chemical, or drug administration, etc.), which acts
upon the promoter upstream of said polynucleotides to induce
expression of the encoded protein product. As such the beneficial
therapeutic affect of the present invention may be expressly
modulated (i.e. to increase, decrease, or inhibit expression of the
present invention) based upon said external stimulus.
[1089] Polynucleotides of the present invention may be useful in
repressing expression of oncogenic genes or antigens. By
"repressing expression of the oncogenic genes "is intended the
suppression of the transcription of the gene, the degradation of
the gene transcript (pre-message RNA), the inhibition of splicing,
the destruction of the messenger RNA, the prevention of the
post-translational modifications of the protein, the destruction of
the protein, or the inhibition of the normal function of the
protein.
[1090] For local administration to abnormally proliferating cells,
polynucleotides of the present invention may be administered by any
method known to those of skill in the art including, but not
limited to transfection, electroporation, microinjection of cells,
or in vehicles such as liposomes, lipofectin, or as naked
polynucleotides, or any other method described throughout the
specification. The polynucleotide of the present invention may be
delivered by known gene delivery systems such as, but not limited
to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke,
Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci.
U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.
Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems
(Yates et al., Nature 313:812 (1985)) known to those skilled in the
art. These references are exemplary only and are hereby
incorporated by reference. In order to specifically deliver or
transfect cells which are abnormally proliferating and spare
non-dividing cells, it is preferable to utilize a retrovirus, or
adenoviral (as described in the art and elsewhere herein) delivery
system known to those of skill in the art. Since host DNA
replication is required for retroviral DNA to integrate and the
retrovirus will be unable to self replicate due to the lack of the
retrovirus genes needed for its life cycle. Utilizing such a
retroviral delivery system for polynucleotides of the present
invention will target said gene and constructs to abnormally
proliferating cells and will spare the non-dividing normal
cells.
[1091] The polynucleotides of the present invention may be
delivered directly to cell proliferative disorder/disease sites in
internal organs, body cavities and the like by use of imaging
devices used to guide an injecting needle directly to the disease
site. The polynucleotides of the present invention may also be
administered to disease sites at the time of surgical
intervention.
[1092] By "cell proliferative disease" is meant any human or animal
disease or disorder, affecting any one or any combination of
organs, cavities, or body parts, which is characterized by single
or multiple local abnormal proliferations of cells, groups of
cells, or tissues, whether benign or malignant.
[1093] Any amount of the polynucleotides of the present invention
may be administered as long as it has a biologically inhibiting
effect on the proliferation of the treated cells. Moreover, it is
possible to administer more than one of the polynucleotide of the
present invention simultaneously to the same site. By "biologically
inhibiting" is meant partial or total growth inhibition as well as
decreases in the rate of proliferation or growth of the cells. The
biologically inhibitory dose may be determined by assessing the
effects of the polynucleotides of the present invention on target
malignant or abnormally proliferating cell growth in tissue
culture, tumor growth in animals and cell cultures, or any other
method known to one of ordinary skill in the art.
[1094] The present invention is further directed to antibody-based
therapies which involve administering of anti-polypeptides and
anti-polynucleotide antibodies to a mammalian, preferably human,
patient for treating, preventing, and/or diagnosing one or more of
the described diseases, disorders, and/or conditions. Methods for
producing anti-polypeptides and anti-polynucleotide antibodies
polyclonal and monoclonal antibodies are described in detail
elsewhere herein. Such antibodies may be provided in
pharmaceutically acceptable compositions as known in the art or as
described herein.
[1095] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding
polynucleotides or polypeptides of the present invention locally or
systemically in the body or by direct cytotoxicity of the antibody,
e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the
art will know how to use the antibodies of the present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
[1096] In particular, the antibodies, fragments and derivatives of
the present invention are useful for treating, preventing, and/or
diagnosing a subject having or developing cell proliferative and/or
differentiation diseases, disorders, and/or conditions as described
herein. Such treatment comprises administering a single or multiple
doses of the antibody, or a fragment, derivative, or a conjugate
thereof.
[1097] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors,
for example, which serve to increase the number or activity of
effector cells which interact with the antibodies.
[1098] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against polypeptides or
polynucleotides of the present invention, fragments or regions
thereof, for both immunoassays directed to and therapy of diseases,
disorders, and/or conditions related to polynucleotides or
polypeptides, including fragements thereof, of the present
invention. Such antibodies, fragments, or regions, will preferably
have an affinity for polynucleotides or polypeptides, including
fragements thereof. Preferred binding affinities include those with
a dissociation constant or Kd less than 5.times.10.sup.-6 M,
10.sup.-6 M, 5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M,
10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10
M, 10.sup.-10 M, 5.times.10.sup.-11 M, 10.sup.-11 M,
5.times.10.sup.-12 M, 10.sup.-12 M, 5.times.10.sup.-13 M,
10.sup.-13 M, 5.times.10.sup.-14 M, 10.sup.-14 M,
5.times.10.sup.-15 M, and 10.sup.-15 M.
[1099] Moreover, polypeptides of the present invention are useful
in inhibiting the angiogenesis of proliferative cells or tissues,
either alone, as a protein fusion, or in combination with other
polypeptides directly or indirectly, as described elsewhere herein.
In a most preferred embodiment, said anti-angiogenesis effect may
be achieved indirectly, for example, through the inhibition of
hematopoietic, tumor-specific cells, such as tumor-associated
macrophages (See Joseph I B, et al. J Natl Cancer Inst,
90(21):1648-53 (1998), which is hereby incorporated by reference).
Antibodies directed to polypeptides or polynucleotides of the
present invention may also result in inhibition of angiogenesis
directly, or indirectly (See Witte L, et al., Cancer Metastasis
Rev. 17(2):155-61 (1998), which is hereby incorporated by
reference)).
[1100] Polypeptides, including protein fusions, of the present
invention, or fragments thereof may be useful in inhibiting
proliferative cells or tissues through the induction of apoptosis.
Said polypeptides may act either directly, or indirectly to induce
apoptosis of proliferative cells and tissues, for example in the
activation of a death-domain receptor, such as tumor necrosis
factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related
apoptosis-mediated protein (TRAMP) and TNF-related
apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See
Schulze-Osthoff K, et.al., Eur J Biochem 254(3):439-59 (1998),
which is hereby incorporated by reference). Moreover, in another
preferred embodiment of the present invention, said polypeptides
may induce apoptosis through other mechanisms, such as in the
activation of other proteins which will activate apoptosis, or
through stimulating the expression of said proteins, either alone
or in combination with small molecule drugs or adjuviants, such as
apoptonin, galectins, thioredoxins, antiinflammatory proteins (See
for example, Mutat Res 400(1-2):447-55 (1998), Med
Hypotheses.50(5):423-33 (1998), Chem Biol Interact. April
24;111-112:23-34 (1998), J Mol Med.76(6):402-12 (1998), int J
Tissue React;20(1):3-15 (1998), which are all hereby incorporated
by reference).
[1101] Polypeptides, including protein fusions to, or fragments
thereof, of the present invention are useful in inhibiting the
metastasis of proliferative cells or tissues. Inhibition may occur
as a direct result of administering polypeptides, or antibodies
directed to said polypeptides as described elsewere herein, or
indirectly, such as activating the expression of proteins known to
inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr
Top Microbiol Immunol 1998;231:125-41, which is hereby incorporated
by reference). Such thereapeutic affects of the present invention
may be achieved either alone, or in combination with small molecule
drugs or adjuvants.
[1102] In another embodiment, the invention provides a method of
delivering compositions containing the polypeptides of the
invention (e.g., compositions containing polypeptides or
polypeptide antibodes associated with heterologous polypeptides,
heterologous nucleic acids, toxins, or prodrugs) to targeted cells
expressing the polypeptide of the present invention. Polypeptides
or polypeptide antibodes of the invention may be associated with
with heterologous polypeptides, heterologous nucleic acids, toxins,
or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent
interactions.
[1103] Polypeptides, protein fusions to, or fragments thereof, of
the present invention are useful in enhancing the immunogenicity
and/or antigenicity of proliferating cells or tissues, either
directly, such as would occur if the polypeptides of the present
invention `vaccinated` the immune response to respond to
proliferative antigens and immunogens, or indirectly, such as in
activating the expression of proteins known to enhance the immune
response (e.g. chemokines), to said antigens and immunogens.
[1104] Cardiovascular Disorders
[1105] Polynucleotides or polypeptides, or agonists or antagonists
of the invention may be used to treat, prevent, and/or diagnose
cardiovascular diseases, disorders, and/or conditions, including
peripheral artery disease, such as limb ischemia.
[1106] Cardiovascular diseases, disorders, and/or conditions
include cardiovascular abnormalities, such as arterio-arterial
fistula, arteriovenous fistula, cerebral arteriovenous
malformations, congenital heart defects, pulmonary atresia, and
Scimitar Syndrome. Congenital heart defects include aortic
coarctation, cor triatriatum, coronary vessel anomalies, crisscross
heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,
Eisenmenger complex, hypoplastic left heart syndrome, levocardia,
tetralogy of fallot, transposition of great vessels, double outlet
right ventricle, tricuspid atresia, persistent truncus arteriosus,
and heart septal defects, such as aortopulmonary septal defect,
endocardial cushion defects, Lutembacher's Syndrome, trilogy of
Fallot, ventricular heart septal defects.
[1107] Cardiovascular diseases, disorders, and/or conditions also
include heart disease, such as arrhythmias, carcinoid heart
disease, high cardiac output, low cardiac output, cardiac
tamponade, endocarditis (including bacterial), heart aneurysm,
cardiac arrest, congestive heart failure, congestive
cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart
hypertrophy, congestive cardiomyopathy, left ventricular
hypertrophy, right ventricular hypertrophy, post-infarction heart
rupture, ventricular septal rupture, heart valve diseases,
myocardial diseases, myocardial ischemia, pericardial effusion,
pericarditis (including constrictive and tuberculous),
pneumopericardium, postpericardiotomy syndrome, pulmonary heart
disease, rheumatic heart disease, ventricular dysfunction,
hyperemia, cardiovascular pregnancy complications, Scimitar
Syndrome, cardiovascular syphilis, and cardiovascular
tuberculosis.
[1108] Arrhythmias include sinus arrhythmia, atrial fibrillation,
atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome,
bundle-branch block, sinoatrial block, long QT syndrome,
parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type
pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus
syndrome, tachycardias, and ventricular fibrillation. Tachycardias
include paroxysmal tachycardia, supraventricular tachycardia,
accelerated idioventricular rhythm, atrioventricular nodal reentry
tachycardia, ectopic atrial tachycardia, ectopic junctional
tachycardia, sinoatrial nodal reentry tachycardia, sinus
tachycardia, Torsades de Pointes, and ventricular tachycardia.
[1109] Heart valve disease include aortic valve insufficiency,
aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral
valve prolapse, tricuspid valve prolapse, mitral valve
insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary
valve insufficiency, pulmonary valve stenosis, tricuspid atresia,
tricuspid valve insufficiency, and tricuspid valve stenosis.
[1110] Myocardial diseases include alcoholic cardiomyopathy,
congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic
subvalvular stenosis, pulmonary subvalvular stenosis, restrictive
cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis,
endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion
injury, and myocarditis.
[1111] Myocardial ischemias include coronary disease, such as
angina pectoris, coronary aneurysm, coronary arteriosclerosis,
coronary thrombosis, coronary vasospasm, myocardial infarction and
myocardial stunning.
[1112] Cardiovascular diseases also include vascular diseases such
as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,
Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome,
Sturge-Weber Syndrome, angioneurotic edema, aortic diseases,
Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive diseases, arteritis, enarteritis, polyarteritis nodosa,
cerebrovascular diseases, disorders, and/or conditions, diabetic
angiopathies, diabetic retinopathy, embolisms, thrombosis,
erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,
hypertension, hypotension, ischemia, peripheral vascular diseases,
phlebitis, pulmonary venoocclusive disease, Raynaud's disease,
CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior
vena cava syndrome, telangiectasia, atacia telangiectasia,
hereditary hemorrhagic telangiectasia, varicocele, varicose veins,
varicose ulcer, vasculitis, and venous insufficiency.
[1113] Aneurysms include dissecting aneurysms, false aneurysms,
infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral
aneurysms, coronary aneurysms, heart aneurysms, and iliac
aneurysms.
[1114] Arterial occlusive diseases include arteriosclerosis,
intermittent claudication, carotid stenosis, fibromuscular
dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal
artery obstruction, retinal artery occlusion, and thromboangiitis
obliterans.
[1115] Cerebrovascular diseases, disorders, and/or conditions
include carotid artery diseases, cerebral amyloid angiopathy,
cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis,
cerebral arteriovenous malformation, cerebral artery diseases,
cerebral embolism and thrombosis, carotid artery thrombosis, sinus
thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural
hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral
infarction, cerebral ischemia (including transient), subclavian
steal syndrome, periventricular leukomalacia, vascular headache,
cluster headache, migraine, and vertebrobasilar insufficiency.
[1116] Embolisms include air embolisms, amniotic fluid embolisms,
cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary
embolisms, and thromoboembolisms. Thrombosis include coronary
thrombosis, hepatic vein thrombosis, retinal vein occlusion,
carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome,
and thrombophlebitis.
[1117] Ischemia includes cerebral ischemia, ischemic colitis,
compartment syndromes, anterior compartment syndrome, myocardial
ischemia, reperfusion injuries, and peripheral limb ischemia.
Vasculitis includes aortitis, arteritis, Behcet's Syndrome,
Churg-Strauss Syndrome, mucocutaneous lymph node syndrome,
thromboangiitis obliterans, hypersensitivity vasculitis,
Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and
Wegener's granulomatosis.
[1118] Polynucleotides or polypeptides, or agonists or antagonists
of the invention, are especially effective for the treatment of
critical limb ischemia and coronary disease.
[1119] Polypeptides may be administered using any method known in
the art, including, but not limited to, direct needle injection at
the delivery site, intravenous injection, topical administration,
catheter infusion, biolistic injectors, particle accelerators,
gelfoam sponge depots, other commercially available depot
materials, osmotic pumps, oral or suppositorial solid
pharmaceutical formulations, decanting or topical applications
during surgery, aerosol delivery. Such methods are known in the
art. Polypeptides of the invention may be administered as part of a
Therapeutic, described in more detail below. Methods of delivering
polynucleotides of the invention are described in more detail
herein.
[1120] Anti-Angiogenesis Activity
[1121] The naturally occurring balance between endogenous
stimulators and inhibitors of angiogenesis is one in which
inhibitory influences predominate. Rastinejad et al., Cell
56:345-355 (1989). In those rare instances in which
neovascularization occurs under normal physiological conditions,
such as wound healing, organ regeneration, embryonic development,
and female reproductive processes, angiogenesis is stringently
regulated and spatially and temporally delimited. Under conditions
of pathological angiogenesis such as that characterizing solid
tumor growth, these regulatory controls fail. Unregulated
angiogenesis becomes pathologic and sustains progression of many
neoplastic and non-neoplastic diseases. A number of serious
diseases are dominated by abnormal neovascularization including
solid tumor growth and metastases, arthritis, some types of eye
diseases, disorders, and/or conditions, and psoriasis. See, e.g.,
reviews by Moses et al., Biotech. 9:630-634 (1991); Folkman et al.,
N. Engl. J Med., 333:1757-1763 (1995); Auerbach et al., J
Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer
Research, eds. Klein and Weinhouse, Academic Press, New York, pp.
175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and
Folkman et al., Science 221:719-725 (1983). In a number of
pathological conditions, the process of angiogenesis contributes to
the disease state. For example, significant data have accumulated
which suggest that the growth of solid tumors is dependent on
angiogenesis. Folkman and Klagsbrun, Science 235:442-447
(1987).
[1122] The present invention provides for treatment of diseases,
disorders, and/or conditions associated with neovascularization by
administration of the polynucleotides and/or polypeptides of the
invention, as well as agonists or antagonists of the present
invention. Malignant and metastatic conditions which can be treated
with the polynucleotides and polypeptides, or agonists or
antagonists of the invention include, but are not limited to,
malignancies, solid tumors, and cancers described herein and
otherwise known in the art (for a review of such disorders, see
Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co.,
Philadelphia (1985)). Thus, the present invention provides a method
of treating, preventing, and/or diagnosing an angiogenesis-related
disease and/or disorder, comprising administering to an individual
in need thereof a therapeutically effective amount of a
polynucleotide, polypeptide, antagonist and/or agonist of the
invention. For example, polynucleotides, polypeptides, antagonists
and/or agonists may be utilized in a variety of additional methods
in order to therapeutically treator prevent a cancer or tumor.
Cancers which may be treated, prevented, and/or diagnosed with
polynucleotides, polypeptides, antagonists and/or agonists include,
but are not limited to solid tumors, including prostate, lung,
breast, ovarian, stomach, pancreas, larynx, esophagus, testes,
liver, parotid, biliary tract, colon, rectum, cervix, uterus,
endometrium, kidney, bladder, thyroid cancer; primary tumors and
metastases; melanomas; glioblastoma; Kaposi's sarcoma;
leiomyosarcoma; non-small cell lung cancer; colorectal cancer;
advanced malignancies; and blood born tumors such as leukemias. For
example, polynucleotides, polypeptides, antagonists and/or agonists
may be delivered topically, in order to treat or prevent cancers
such as skin cancer, head and neck tumors, breast tumors, and
Kaposi's sarcoma.
[1123] Within yet other aspects, polynucleotides, polypeptides,
antagonists and/or agonists may be utilized to treat superficial
forms of bladder cancer by, for example, intravesical
administration. Polynucleotides, polypeptides, antagonists and/or
agonists may be delivered directly into the tumor, or near the
tumor site, via injection or a catheter. Of course, as the artisan
of ordinary skill will appreciate, the appropriate mode of
administration will vary according to the cancer to be treated.
Other modes of delivery are discussed herein.
[1124] Polynucleotides, polypeptides, antagonists and/or agonists
may be useful in treating, preventing, and/or diagnosing other
diseases, disorders, and/or conditions, besides cancers, which
involve angiogenesis. These diseases, disorders, and/or conditions
include, but are not limited to: benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic granulomas; artheroscleric plaques; ocular angiogenic
diseases, for example, diabetic retinopathy, retinopathy of
prematurity, macular degeneration, corneal graft rejection,
neovascular glaucoma, retrolental fibroplasia, rubeosis,
retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth)
of the eye; rheumatoid arthritis; psoriasis; delayed wound healing;
endometriosis; vasculogenesis; granulations; hypertrophic scars
(keloids); nonunion fractures; scleroderma; trachoma; vascular
adhesions; myocardial angiogenesis; coronary collaterals; cerebral
collaterals; arteriovenous malformations; ischemic limb
angiogenesis; Osler-Webber Syndrome; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; fibromuscular
dysplasia; wound granulation; Crohn's disease; and
atherosclerosis.
[1125] For example, within one aspect of the present invention
methods are provided for treating, preventing, and/or diagnosing
hypertrophic scars and keloids, comprising the step of
administering a polynucleotide, polypeptide, antagonist and/or
agonist of the invention to a hypertrophic scar or keloid.
[1126] Within one embodiment of the present invention
polynucleotides, polypeptides, antagonists and/or agonists are
directly injected into a hypertrophic scar or keloid, in order to
prevent the progression of these lesions. This therapy is of
particular value in the prophylactic treatment of conditions which
are known to result in the development of hypertrophic scars and
keloids (e.g., burns), and is preferably initiated after the
proliferative phase has had time to progress (approximately 14 days
after the initial injury), but before hypertrophic scar or keloid
development. As noted above, the present invention also provides
methods for treating, preventing, and/or diagnosing neovascular
diseases of the eye, including for example, corneal
neovascularization, neovascular glaucoma, proliferative diabetic
retinopathy, retrolental fibroplasia and macular degeneration.
[1127] Moreover, Ocular diseases, disorders, and/or conditions
associated with neovascularization which can be treated, prevented,
and/or diagnosed with the polynucleotides and polypeptides of the
present invention (including agonists and/or antagonists) include,
but are not limited to: neovascular glaucoma, diabetic retinopathy,
retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of
prematurity macular degeneration, corneal graft neovascularization,
as well as other eye inflammatory diseases, ocular tumors and
diseases associated with choroidal or iris neovascularization. See,
e.g., reviews by Waltman et al., Am. J Ophthal. 85:704-710 (1978)
and Gartner et al., Surv. Ophthal. 22:291-312 (1978).
[1128] Thus, within one aspect of the present invention methods are
provided for treating or preventing neovascular diseases of the eye
such as corneal neovascularization (including corneal graft
neovascularization), comprising the step of administering to a
patient a therapeutically effective amount of a compound (as
described above) to the cornea, such that the formation of blood
vessels is inhibited. Briefly, the cornea is a tissue which
normally lacks blood vessels. In certain pathological conditions
however, capillaries may extend into the cornea from the
pericorneal vascular plexus of the limbus. When the cornea becomes
vascularized, it also becomes clouded, resulting in a decline in
the patient's visual acuity. Visual loss may become complete if the
cornea completely opacitates. A wide variety of diseases,
disorders, and/or conditions can result in corneal
neovascularization, including for example, corneal infections
(e.g., trachoma, herpes simplex keratitis, leishmaniasis and
onchocerciasis), immunological processes (e.g., graft rejection and
Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of
any cause), toxic and nutritional deficiency states, and as a
complication of wearing contact lenses.
[1129] Within particularly preferred embodiments of the invention,
may be prepared for topical administration in saline (combined with
any of the preservatives and antimicrobial agents commonly used in
ocular preparations), and administered in eyedrop form. The
solution or suspension may be prepared in its pure form and
administered several times daily. Alternatively, anti-angiogenic
compositions, prepared as described above, may also be administered
directly to the cornea. Within preferred embodiments, the
anti-angiogenic composition is prepared with a mucoadhesive polymer
which binds to cornea. Within further embodiments, the
anti-angiogenic factors or anti-angiogenic compositions may be
utilized as an adjunct to conventional steroid therapy. Topical
therapy may also be useful prophylactically in corneal lesions
which are known to have a high probability of inducing an
angiogenic response (such as chemical burns). In these instances
the treatment, likely in combination with steroids, may be
instituted immediately to help prevent subsequent
complications.
[1130] Within other embodiments, the compounds described above may
be injected directly into the corneal stroma by an ophthalmologist
under microscopic guidance. The preferred site of injection may
vary with the morphology of the individual lesion, but the goal of
the administration would be to place the composition at the
advancing front of the vasculature (i.e., interspersed between the
blood vessels and the normal cornea). In most cases this would
involve perilimbic corneal injection to "protect" the cornea from
the advancing blood vessels. This method may also be utilized
shortly after a corneal insult in order to prophylactically prevent
corneal neovascularization. In this situation the material could be
injected in the perilimbic cornea interspersed between the corneal
lesion and its undesired potential limbic blood supply. Such
methods may also be utilized in a similar fashion to prevent
capillary invasion of transplanted corneas. In a sustained-release
form injections might only be required 2-3 times per year. A
steroid could also be added to the injection solution to reduce
inflammation resulting from the injection itself.
[1131] Within another aspect of the present invention, methods are
provided for treating or preventing neovascular glaucoma,
comprising the step of administering to a patient a therapeutically
effective amount of a polynucleotide, polypeptide, antagonist
and/or agonist to the eye, such that the formation of blood vessels
is inhibited. In one embodiment, the compound may be administered
topically to the eye in order to treat or prevent early forms of
neovascular glaucoma. Within other embodiments, the compound may be
implanted by injection into the region of the anterior chamber
angle. Within other embodiments, the compound may also be placed in
any location such that the compound is continuously released into
the aqueous humor. Within another aspect of the present invention,
methods are provided for treating or preventing proliferative
diabetic retinopathy, comprising the step of administering to a
patient a therapeutically effective amount of a polynucleotide,
polypeptide, antagonist and/or agonist to the eyes, such that the
formation of blood vessels is inhibited.
[1132] Within particularly preferred embodiments of the invention,
proliferative diabetic retinopathy may be treated by injection into
the aqueous humor or the vitreous, in order to increase the local
concentration of the polynucleotide, polypeptide, antagonist and/or
agonist in the retina. Preferably, this treatment should be
initiated prior to the acquisition of severe disease requiring
photocoagulation.
[1133] Within another aspect of the present invention, methods are
provided for treating or preventing retrolental fibroplasia,
comprising the step of administering to a patient a therapeutically
effective amount of a polynucleotide, polypeptide, antagonist
and/or agonist to the eye, such that the formation of blood vessels
is inhibited. The compound may be administered topically, via
intravitreous injection and/or via intraocular implants.
[1134] Additionally, diseases, disorders, and/or conditions which
can be treated, prevented, and/or diagnosed with the
polynucleotides, polypeptides, agonists and/or agonists include,
but are not limited to, hemangioma, arthritis, psoriasis,
angiofibroma, atherosclerotic plaques, delayed wound healing,
granulations, hemophilic joints, hypertrophic scars, nonunion
fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma,
trachoma, and vascular adhesions.
[1135] Moreover, diseases, disorders, and/or conditions and/or
states, which can be treated, prevented, and/or diagnosed with the
the polynucleotides, polypeptides, agonists and/or agonists
include, but are not limited to, solid tumors, blood born tumors
such as leukemias, tumor metastasis, Kaposi's sarcoma, benign
tumors, for example hemangiomas, acoustic neuromas, neurofibromas,
trachomas, and pyogenic granulomas, rheumatoid arthritis,
psoriasis, ocular angiogenic diseases, for example, diabetic
retinopathy, retinopathy of prematurity, macular degeneration,
corneal graft rejection, neovascular glaucoma, retrolental
fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound
healing, endometriosis, vascluogenesis, granulations, hypertrophic
scars (keloids), nonunion fractures, scleroderma, trachoma,
vascular adhesions, myocardial angiogenesis, coronary collaterals,
cerebral collaterals, arteriovenous malformations, ischemic limb
angiogenesis, Osler-Webber Syndrome, plaque neovascularization,
telangiectasia, hemophiliac joints, angiofibroma fibromuscular
dysplasia, wound granulation, Crohn's disease, atherosclerosis,
birth control agent by preventing vascularization required for
embryo implantation controlling menstruation, diseases that have
angiogenesis as a pathologic consequence such as cat scratch
disease (Rochele minalia quintosa), ulcers (Helicobacter pylori),
Bartonellosis and bacillary angiomatosis.
[1136] In one aspect of the birth control method, an amount of the
compound sufficient to block embryo implantation is administered
before or after intercourse and fertilization have occurred, thus
providing an effective method of birth control, possibly a "morning
after" method. Polynucleotides, polypeptides, agonists and/or
agonists may also be used in controlling menstruation or
administered as either a peritoneal lavage fluid or for peritoneal
implantation in the treatment of endometriosis.
[1137] Polynucleotides, polypeptides, agonists and/or agonists of
the present invention may be incorporated into surgical sutures in
order to prevent stitch granulomas.
[1138] Polynucleotides, polypeptides, agonists and/or agonists may
be utilized in a wide variety of surgical procedures. For example,
within one aspect of the present invention a compositions (in the
form of, for example, a spray or film) may be utilized to coat or
spray an area prior to removal of a tumor, in order to isolate
normal surrounding tissues from malignant tissue, and/or to prevent
the spread of disease to surrounding tissues. Within other aspects
of the present invention, compositions (e.g., in the form of a
spray) may be delivered via endoscopic procedures in order to coat
tumors, or inhibit angiogenesis in a desired locale. Within yet
other aspects of the present invention, surgical meshes which have
been coated with anti-angiogenic compositions of the present
invention may be utilized in any procedure wherein a surgical mesh
might be utilized. For example, within one embodiment of the
invention a surgical mesh laden with an anti-angiogenic composition
may be utilized during abdominal cancer resection surgery (e.g.,
subsequent to colon resection) in order to provide support to the
structure, and to release an amount of the anti-angiogenic
factor.
[1139] Within further aspects of the present invention, methods are
provided for treating tumor excision sites, comprising
administering a polynucleotide, polypeptide, agonist and/or agonist
to the resection margins of a tumor subsequent to excision, such
that the local recurrence of cancer and the formation of new blood
vessels at the site is inhibited. Within one embodiment of the
invention, the anti-angiogenic compound is administered directly to
the tumor excision site (e.g., applied by swabbing, brushing or
otherwise coating the resection margins of the tumor with the
anti-angiogenic compound). Alternatively, the anti-angiogenic
compounds may be incorporated into known surgical pastes prior to
administration. Within particularly preferred embodiments of the
invention, the anti-angiogenic compounds are applied after hepatic
resections for malignancy, and after neurosurgical operations.
[1140] Within one aspect of the present invention, polynucleotides,
polypeptides, agonists and/or agonists may be administered to the
resection margin of a wide variety of tumors, including for
example, breast, colon, brain and hepatic tumors. For example,
within one embodiment of the invention, anti-angiogenic compounds
may be administered to the site of a neurological tumor subsequent
to excision, such that the formation of new blood vessels at the
site are inhibited.
[1141] The polynucleotides, polypeptides, agonists and/or agonists
of the present invention may also be administered along with other
anti-angiogenic factors. Representative examples of other
anti-angiogenic factors include: Anti-Invasive Factor, retinoic
acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor
of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,
Plasminogen Activator Inhibitor-1, Plasminogen Activator
Inhibitor-2, and various forms of the lighter "d group" transition
metals.
[1142] Lighter "d group" transition metals include, for example,
vanadium, molybdenum, tungsten, titanium, niobium, and tantalum
species. Such transition metal species may form transition metal
complexes. Suitable complexes of the above-mentioned transition
metal species include oxo transition metal complexes.
[1143] Representative examples of vanadium complexes include oxo
vanadium complexes such as vanadate and vanadyl complexes. Suitable
vanadate complexes include metavanadate and orthovanadate complexes
such as, for example, ammonium metavanadate, sodium metavanadate,
and sodium orthovanadate. Suitable vanadyl complexes include, for
example, vanadyl acetylacetonate and vanadyl sulfate including
vanadyl sulfate hydrates such as vanadyl sulfate mono- and
trihydrates.
[1144] Representative examples of tungsten and molybdenum complexes
also include oxo complexes. Suitable oxo tungsten complexes include
tungstate and tungsten oxide complexes. Suitable tungstate
complexes include ammonium tungstate, calcium tungstate, sodium
tungstate dihydrate, and tungstic acid. Suitable tungsten oxides
include tungsten(IV) oxide and tungsten(VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and
molybdenyl complexes. Suitable molybdate complexes include ammonium
molybdate and its hydrates, sodium molybdate and its hydrates, and
potassium molybdate and its hydrates. Suitable molybdenum oxides
include molybdenum(VI) oxide, molybdenum(VI) oxide, and molybdic
acid. Suitable molybdenyl complexes include, for example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum
complexes include hydroxo derivatives derived from, for example,
glycerol, tartaric acid, and sugars.
[1145] A wide variety of other anti-angiogenic factors may also be
utilized within the context of the present invention.
Representative examples include platelet factor 4; protamine
sulphate; sulphated chitin derivatives (prepared from queen crab
shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated
Polysaccharide Peptidoglycan Complex (SP-PG) (the fimction of this
compound may be enhanced by the presence of steroids such as
estrogen, and tamoxifen citrate); Staurosporine; modulators of
matrix metabolism, including for example, proline analogs,
cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,
alpha,alpha-dipyridyl, aminopropionitrile fumarate;
4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate;
Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3
(Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin
(Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin
Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et
al., Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST";
Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987);
anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol.
Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer
Institute); Lobenzarit disodium
(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or "CCA";
Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;
Angostatic steroid; AGM-1470; carboxynaminolmidazole; and
metalloproteinase inhibitors such as BB94.
[1146] Diseases at the Cellular Level
[1147] Diseases associated with increased cell survival or the
inhibition of apoptosis that could be treated, prevented, and/or
diagnosed by the polynucleotides or polypeptides and/or antagonists
or agonists of the invention, include cancers (such as follicular
lymphomas, carcinomas with p53 mutations, and hormone-dependent
tumors, including, but not limited to colon cancer, cardiac tumors,
pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung
cancer, intestinal cancer, testicular cancer, stomach cancer,
neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma,
adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and
ovarian cancer); autoimmune diseases, disorders, and/or conditions
(such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's
thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,
polymyositis, systemic lupus erythematosus and immune-related
glomerulonephritis and rheumatoid arthritis) and viral infections
(such as herpes viruses, pox viruses and adenoviruses),
inflammation, graft v. host disease, acute graft rejection, and
chronic graft rejection. In preferred embodiments, the
polynucleotides or polypeptides, and/or agonists or antagonists of
the invention are used to inhibit growth, progression, and/or
metasis of cancers, in particular those listed above.
[1148] Additional diseases or conditions associated with increased
cell survival that could be treated, prevented or diagnosed by the
polynucleotides or polypeptides, or agonists or antagonists of the
invention, include, but are not limited to, progression, and/or
metastases of malignancies and related disorders such as leukemia
(including acute leukemias (e.g., acute lymphocytic leukemia, acute
myelocytic leukemia (including myeloblastic, promyelocytic,
myelomonocytic, monocytic, and erythroleukemia)) and chronic
leukemias (e.g., chronic myelocytic (granulocytic) leukemia and
chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,
Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease, and solid
tumors including, but not limited to, sarcomas and carcinomas such
as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[1149] Diseases associated with increased apoptosis that could be
treated, prevented, and/or diagnosed by the polynucleotides or
polypeptides, and/or agonists or antagonists of the invention,
include AIDS; neurodegenerative diseases, disorders, and/or
conditions (such as Alzheimer's disease, Parkinson's disease,
Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar
degeneration and brain tumor or prior associated disease);
autoimmune diseases, disorders, and/or conditions (such as,
multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis,
biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis,
systemic lupus erythematosus and immune-related glomerulonephritis
and rheumatoid arthritis) myelodysplastic syndromes (such as
aplastic anemia), graft v. host disease, ischemic injury (such as
that caused by myocardial infarction, stroke and reperfusion
injury), liver injury (e.g., hepatitis related liver injury,
ischemia/reperfusion injury, cholestosis (bile duct injury) and
liver cancer); toxin-induced liver disease (such as that caused by
alcohol), septic shock, cachexia and anorexia.
[1150] Wound Healing and Epithelial Cell Proliferation
[1151] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing the
polynucleotides or polypeptides, and/or agonists or antagonists of
the invention, for therapeutic purposes, for example, to stimulate
epithelial cell proliferation and basal keratinocytes for the
purpose of wound healing, and to stimulate hair follicle production
and healing of dermal wounds. Polynucleotides or polypeptides, as
well as agonists or antagonists of the invention, may be clinically
useful in stimulating wound healing including surgical wounds,
excisional wounds, deep wounds involving damage of the dermis and
epidermis, eye tissue wounds, dental tissue wounds, oral cavity
wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial
ulcers, venous stasis ulcers, bums resulting from heat exposure or
chemicals, and other abnormal wound healing conditions such as
uremia, malnutrition, vitamin deficiencies and complications
associted with systemic treatment with steroids, radiation therapy
and antineoplastic drugs and antimetabolites. Polynucleotides or
polypeptides, and/or agonists or antagonists of the invention,
could be used to promote dermal reestablishment subsequent to
dermal loss.
[1152] The polynucleotides or polypeptides, and/or agonists or
antagonists of the invention, could be used to increase the
adherence of skin grafts to a wound bed and to stimulate
re-epithelialization from the wound bed. The following are a
non-exhaustive list of grafts that polynucleotides or polypeptides,
agonists or antagonists of the invention, could be used to increase
adherence to a wound bed: autografts, artificial skin, allografts,
autodermic graft, autoepdermic grafts, avacular grafts, Blair-Brown
grafts, bone graft, brephoplastic grafts, cutis graft, delayed
graft, dermic graft, epidermic graft, fascia graft, full thickness
graft, heterologous graft, xenograft, homologous graft,
hyperplastic graft, lamellar graft, mesh graft, mucosal graft,
Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,
penetrating graft, split skin graft, thick split graft. The
polynucleotides or polypeptides, and/or agonists or antagonists of
the invention, can be used to promote skin strength and to improve
the appearance of aged skin.
[1153] It is believed that the polynucleotides or polypeptides,
and/or agonists or antagonists of the invention, will also produce
changes in hepatocyte proliferation, and epithelial cell
proliferation in the lung, breast, pancreas, stomach, small
intesting, and large intestine. The polynucleotides or
polypeptides, and/or agonists or antagonists of the invention,
could promote proliferation of epithelial cells such as sebocytes,
hair follicles, hepatocytes, type II pneumocytes, mucin-producing
goblet cells, and other epithelial cells and their progenitors
contained within the skin, lung, liver, and gastrointestinal tract.
The polynucleotides or polypeptides, and/or agonists or antagonists
of the invention, may promote proliferation of endothelial cells,
keratinocytes, and basal keratinocytes.
[1154] The polynucleotides or polypeptides, and/or agonists or
antagonists of the invention, could also be used to reduce the side
effects of gut toxicity that result from radiation, chemotherapy
treatments or viral infections. The polynucleotides or
polypeptides, and/or agonists or antagonists of the invention, may
have a cytoprotective effect on the small intestine mucosa. The
polynucleotides or polypeptides, and/or agonists or antagonists of
the invention, may also stimulate healing of mucositis (mouth
ulcers) that result from chemotherapy and viral infections.
[1155] The polynucleotides or polypeptides, and/or agonists or
antagonists of the invention, could further be used in full
regeneration of skin in full and partial thickness skin defects,
including burns, (i.e., repopulation of hair follicles, sweat
glands, and sebaceous glands), treatment of other skin defects such
as psoriasis. The polynucleotides or polypeptides, and/or agonists
or antagonists of the invention, could be used to treat
epidermolysis bullosa, a defect in adherence of the epidermis to
the underlying dermis which results in frequent, open and painful
blisters by accelerating reepithelialization of these lesions. The
polynucleotides or polypeptides, and/or agonists or antagonists of
the invention, could also be used to treat gastric and doudenal
ulcers and help heal by scar formation of the mucosal lining and
regeneration of glandular mucosa and duodenal mucosal lining more
rapidly. Inflamamatory bowel diseases, such as Crohn's disease and
ulcerative colitis, are diseases which result in destruction of the
mucosal surface of the small or large intestine, respectively.
Thus, the polynucleotides or polypeptides, and/or agonists or
antagonists of the invention, could be used to promote the
resurfacing of the mucosal surface to aid more rapid healing and to
prevent progression of inflammatory bowel disease. Treatment with
the polynucleotides or polypeptides, and/or agonists or antagonists
of the invention, is expected to have a significant effect on the
production of mucus throughout the gastrointestinal tract and could
be used to protect the intestinal mucosa from injurious substances
that are ingested or following surgery. The polynucleotides or
polypeptides, and/or agonists or antagonists of the invention,
could be used to treat diseases associate with the under expression
of the polynucleotides of the invention.
[1156] Moreover, the polynucleotides or polypeptides, and/or
agonists or antagonists of the invention, could be used to prevent
and heal damage to the lungs due to various pathological states. A
growth factor such as the polynucleotides or polypeptides, and/or
agonists or antagonists of the invention, which could stimulate
proliferation and differentiation and promote the repair of alveoli
and brochiolar epithelium to prevent or treat acute or chronic lung
damage. For example, emphysema, which results in the progressive
loss of aveoli, and inhalation injuries, i.e., resulting from smoke
inhalation and burns, that cause necrosis of the bronchiolar
epithelium and alveoli could be effectively treated, prevented,
and/or diagnosed using the polynucleotides or polypeptides, and/or
agonists or antagonists of the invention. Also, the polynucleotides
or polypeptides, and/or agonists or antagonists of the invention,
could be used to stimulate the proliferation of and differentiation
of type II pneumocytes, which may help treat or prevent disease
such as hyaline membrane diseases, such as infant respiratory
distress syndrome and bronchopulmonary displasia, in premature
infants.
[1157] The polynucleotides or polypeptides, and/or agonists or
antagonists of the invention, could stimulate the proliferation and
differentiation of hepatocytes and, thus, could be used to
alleviate or treat liver diseases and pathologies such as fulminant
liver failure caused by cirrhosis, liver damage caused by viral
hepatitis and toxic substances (i.e., acetaminophen, carbon
tetraholoride and other hepatotoxins known in the art).
[1158] In addition, the polynucleotides or polypeptides, and/or
agonists or antagonists of the invention, could be used treat or
prevent the onset of diabetes mellitus. In patients with newly
diagnosed Types I and II diabetes, where some islet cell function
remains, the polynucleotides or polypeptides, and/or agonists or
antagonists of the invention, could be used to maintain the islet
function so as to alleviate, delay or prevent permanent
manifestation of the disease. Also, the polynucleotides or
polypeptides, and/or agonists or antagonists of the invention,
could be used as an auxiliary in islet cell transplantation to
improve or promote islet cell function.
[1159] Neurological Diseases
[1160] Nervous system diseases, disorders, and/or conditions, which
can be treated, prevented, and/or diagnosed with the compositions
of the invention (e.g., polypeptides, polynucleotides, and/or
agonists or antagonists), include, but are not limited to, nervous
system injuries, and diseases, disorders, and/or conditions which
result in either a disconnection of axons, a diminution or
degeneration of neurons, or demyelination. Nervous system lesions
which may be treated, prevented, and/or diagnosed in a patient
(including human and non-human mammalian patients) according to the
invention, include but are not limited to, the following lesions of
either the central (including spinal cord, brain) or peripheral
nervous systems: (1) ischemic lesions, in which a lack of oxygen in
a portion of the nervous system results in neuronal injury or
death, including cerebral infarction or ischemia, or spinal cord
infarction or ischemia; (2) traumatic lesions, including lesions
caused by physical injury or associated with surgery, for example,
lesions which sever a portion of the nervous system, or compression
injuries; (3) malignant lesions, in which a portion of the nervous
system is destroyed or injured by malignant tissue which is either
a nervous system associated malignancy or a malignancy derived from
non-nervous system tissue; (4) infectious lesions, in which a
portion of the nervous system is destroyed or injured as a result
of infection, for example, by an abscess or associated with
infection by human immunodeficiency virus, herpes zoster, or herpes
simplex virus or with Lyme disease, tuberculosis, syphilis; (5)
degenerative lesions, in which a portion of the nervous system is
destroyed or injured as a result of a degenerative process
including but not limited to degeneration associated with
Parkinson's disease, Alzheimer's disease, Huntington's chorea, or
amyotrophic lateral sclerosis (ALS); (6) lesions associated with
nutritional diseases, disorders, and/or conditions, in which a
portion of the nervous system is destroyed or injured by a
nutritional disorder or disorder of metabolism including but not
limited to, vitamin B 12 deficiency, folic acid deficiency,
Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami
disease (primary degeneration of the corpus callosum), and
alcoholic cerebellar degeneration; (7) neurological lesions
associated with systemic diseases including, but not limited to,
diabetes (diabetic neuropathy, Bell's palsy), systemic lupus
erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by
toxic substances including alcohol, lead, or particular
neurotoxins; and (9) demyelinated lesions in which a portion of the
nervous system is destroyed or injured by a demyelinating disease
including, but not limited to, multiple sclerosis, human
immunodeficiency virus-associated myelopathy, transverse myelopathy
or various etiologies, progressive multifocal leukoencephalopathy,
and central pontine myelinolysis.
[1161] In a preferred embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to protect neural cells from the damaging effects of cerebral
hypoxia. According to this embodiment, the compositions of the
invention are used to treat, prevent, and/or diagnose neural cell
injury associated with cerebral hypoxia. In one aspect of this
embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the invention are used to treat, prevent, and/or
diagnose neural cell injury associated with cerebral ischemia. In
another aspect of this embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to treat, prevent, and/or diagnose neural cell injury
associated with cerebral infarction. In another aspect of this
embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the invention are used to treat, prevent, and/or
diagnose or prevent neural cell injury associated with a stroke. In
a further aspect of this embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to treat, prevent, and/or diagnose neural cell injury
associated with a heart attack.
[1162] The compositions of the invention which are useful for
treating or preventing a nervous system disorder may be selected by
testing for biological activity in promoting the survival or
differentiation of neurons. For example, and not by way of
limitation, compositions of the invention which elicit any of the
following effects may be useful according to the invention: (1)
increased survival time of neurons in culture; (2) increased
sprouting of neurons in culture or in vivo; (3) increased
production of a neuron-associated molecule in culture or in vivo,
e.g., choline acetyltransferase or acetylcholinesterase with
respect to motor neurons; or (4) decreased symptoms of neuron
dysfunction in vivo. Such effects may be measured by any method
known in the art. In preferred, non-limiting embodiments, increased
survival of neurons may routinely be measured using a method set
forth herein or otherwise known in the art, such as, for example,
the method set forth in Arakawa et al. (J. Neurosci. 10:3507-3515
(1990)); increased sprouting of neurons may be detected by methods
known in the art, such as, for example, the methods set forth in
Pestronk et al. (Exp. Neurol. 70:65-82 (1980)) or Brown et al.
(Ann. Rev. Neurosci. 4:17-42 (1981)); increased production of
neuron-associated molecules may be measured by bioassay, enzymatic
assay, antibody binding, Northern blot assay, etc., using
techniques known in the art and depending on the molecule to be
measured; and motor neuron dysfunction may be measured by assessing
the physical manifestation of motor neuron disorder, e.g.,
weakness, motor neuron conduction velocity, or functional
disability.
[1163] In specific embodiments, motor neuron diseases, disorders,
and/or conditions that may be treated, prevented, and/or diagnosed
according to the invention include, but are not limited to,
diseases, disorders, and/or conditions such as infarction,
infection, exposure to toxin, trauma, surgical damage, degenerative
disease or malignancy that may affect motor neurons as well as
other components of the nervous system, as well as diseases,
disorders, and/or conditions that selectively affect neurons such
as amyotrophic lateral sclerosis, and including, but not limited
to, progressive spinal muscular atrophy, progressive bulbar palsy,
primary lateral sclerosis, infantile and juvenile muscular atrophy,
progressive bulbar paralysis of childhood (Fazio-Londe syndrome),
poliomyelitis and the post polio syndrome, and Hereditary
Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
[1164] Infectious Disease
[1165] A polypeptide or polynucleotide and/or agonist or antagonist
of the present invention can be used to treat, prevent, and/or
diagnose infectious agents. For example, by increasing the immune
response, particularly increasing the proliferation and
differentiation of B and/or T cells, infectious diseases may be
treated, prevented, and/or diagnosed. The immune response may be
increased by either enhancing an existing immune response, or by
initiating a new immune response. Alternatively, polypeptide or
polynucleotide and/or agonist or antagonist of the present
invention may also directly inhibit the infectious agent, without
necessarily eliciting an immune response.
[1166] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated, prevented, and/or
diagnosed by a polynucleotide or polypeptide and/or agonist or
antagonist of the present invention. Examples of viruses, include,
but are not limited to Examples of viruses, include, but are not
limited to the following DNA and RNA viruses and viral families:
Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Bimaviridae,
Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue,
EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae
(such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster),
Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,
Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B,
and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae,
Picomaviridae, Poxviridae (such as Smallpox or Vaccinia),
Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,
Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling
within these families can cause a variety of diseases or symptoms,
including, but not limited to: arthritis, bronchiollitis,
respiratory syncytial virus, encephalitis, eye infections (e.g.,
conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A,
B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin,
Chikungunya, Rift Valley fever, yellow fever, meningitis,
opportunistic infections (e.g., AIDS), pneumonia, Burkitt's
Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,
sexually transmitted diseases, skin diseases (e.g., Kaposi's,
warts), and viremia. polynucleotides or polypeptides, or agonists
or antagonists of the invention, can be used to treat, prevent,
and/or diagnose any of these symptoms or diseases. In specific
embodiments, polynucleotides, polypeptides, or agonists or
antagonists of the invention are used to treat, prevent, and/or
diagnose: meningitis, Dengue, EBV, and/or hepatitis (e.g.,
hepatitis B). In an additional specific embodiment polynucleotides,
polypeptides, or agonists or antagonists of the invention are used
to treat patients nonresponsive to one or more other commercially
available hepatitis vaccines. In a further specific embodiment
polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat, prevent, and/or diagnose AIDS.
[1167] Similarly, bacterial or fungal agents that can cause disease
or symptoms and that can be treated, prevented, and/or diagnosed by
a polynucleotide or polypeptide and/or agonist or antagonist of the
present invention include, but not limited to, include, but not
limited to, the following Gram-Negative and Gram-positive bacteria
and bacterial families and fungi: Actinomycetales (e.g.,
Corynebacterium, Mycobacterium, Norcardia), Cryptococcus
neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax,
Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia
(e.g., Borrelia burgdorferi), Brucellosis, Candidiasis,
Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses,
E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E.
coli), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella
typhi, and Salmonella paratyphi), Serratia, Yersinia),
Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis,
Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae,
Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal),
Meisseria meningitidis, Pasteurellacea Infections (e.g.,
Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B),
Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis,
Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal and
Streptococcal (e.g., Streptococcus pneumoniae and Group B
Streptococcus). These bacterial or fungal families can cause the
following diseases or symptoms, including, but not limited to:
bacteremia, endocarditis, eye infections (conjunctivitis,
tuberculosis, uveitis), gingivitis, opportunistic infections (e.g.,
AIDS related infections), paronychia, prosthesis-related
infections, Reiter's Disease, respiratory tract infections, such as
Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch
Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid,
pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B),
Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,
Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo,
Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin
diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract
infections, wound infections. Polynucleotides or polypeptides,
agonists or antagonists of the invention, can be used to treat,
prevent, and/or diagnose any of these symptoms or diseases. In
specific embodiments, polynucleotides, polypeptides, agonists or
antagonists of the invention are used to treat, prevent, and/or
diagnose: tetanus, Diptheria, botulism, and/or meningitis type
B.
[1168] Moreover, parasitic agents causing disease or symptoms that
can be treated, prevented, and/or diagnosed by a polynucleotide or
polypeptide and/or agonist or antagonist of the present invention
include, but not limited to, the following families or class:
Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis,
Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Hehninthiasis,
Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and
Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium
falciparium, Plasmodium malariae and Plasmodium ovale). These
parasites can cause a variety of diseases or symptoms, including,
but not limited to: Scabies, Trombiculiasis, eye infections,
intestinal disease (e.g., dysentery, giardiasis), liver disease,
lung disease, opportunistic infections (e.g., AIDS related),
malaria, pregnancy complications, and toxoplasmosis.
polynucleotides or polypeptides, or agonists or antagonists of the
invention, can be used totreat, prevent, and/or diagnose any of
these symptoms or diseases. In specific embodiments,
polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat, prevent, and/or diagnose malaria.
[1169] Preferably, treatment or prevention using a polypeptide or
polynucleotide and/or agonist or antagonist of the present
invention could either be by administering an effective amount of a
polypeptide to the patient, or by removing cells from the patient,
supplying the cells with a polynucleotide of the present invention,
and returning the engineered cells to the patient (ex vivo
therapy). Moreover, the polypeptide or polynucleotide of the
present invention can be used as an antigen in a vaccine to raise
an immune response against infectious disease.
[1170] Regeneration
[1171] A polynucleotide or polypeptide and/or agonist or antagonist
of the present invention can be used to differentiate, proliferate,
and attract cells, leading to the regeneration of tissues. (See,
Science 276:59-87 (1997).) The regeneration of tissues could be
used to repair, replace, or protect tissue damaged by congenital
defects, trauma (wounds, bums, incisions, or ulcers), age, disease
(e.g. osteoporosis, osteocarthritis, periodontal disease, liver
failure), surgery, including cosmetic plastic surgery, fibrosis,
reperfusion injury, or systemic cytokine damage.
[1172] Tissues that could be regenerated using the present
invention include organs (e.g., pancreas, liver, intestine, kidney,
skin, endothelium), muscle (smooth, skeletal or cardiac),
vasculature (including vascular and lymphatics), nervous,
hematopoietic, and skeletal (bone, cartilage, tendon, and ligament)
tissue. Preferably, regeneration occurs without or decreased
scarring. Regeneration also may include angiogenesis.
[1173] Moreover, a polynucleotide or polypeptide and/or agonist or
antagonist of the present invention may increase regeneration of
tissues difficult to heal. For example, increased tendon/ligament
regeneration would quicken recovery time after damage. A
polynucleotide or polypeptide and/or agonist or antagonist of the
present invention could also be used prophylactically in an effort
to avoid damage. Specific diseases that could be treated,
prevented, and/or diagnosed include of tendinitis, carpal tunnel
syndrome, and other tendon or ligament defects. A further example
of tissue regeneration of non-healing wounds includes pressure
ulcers, ulcers associated with vascular insufficiency, surgical,
and traumatic wounds.
[1174] Similarly, nerve and brain tissue could also be regenerated
by using a polynucleotide or polypeptide and/or agonist or
antagonist of the present invention to proliferate and
differentiate nerve cells. Diseases that could be treated,
prevented, and/or diagnosed using this method include central and
peripheral nervous system diseases, neuropathies, or mechanical and
traumatic diseases, disorders, and/or conditions (e.g., spinal cord
disorders, head trauma, cerebrovascular disease, and stoke).
Specifically, diseases associated with peripheral nerve injuries,
peripheral neuropathy (e.g., resulting from chemotherapy or other
medical therapies), localized neuropathies, and central nervous
system diseases (e.g., Alzheimer's disease, Parkinson's disease,
Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager
syndrome), could all be treated, prevented, and/or diagnosed using
the polynucleotide or polypeptide and/or agonist or antagonist of
the present invention.
[1175] Chemotaxis
[1176] A polynucleotide or polypeptide and/or agonist or antagonist
of the present invention may have chemotaxis activity. A chemotaxic
molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts,
neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells) to a particular site in the body, such as
inflammation, infection, or site of hyperproliferation. The
mobilized cells can then fight off and/or heal the particular
trauma or abnormality.
[1177] A polynucleotide or polypeptide and/or agonist or antagonist
of the present invention may increase chemotaxic activity of
particular cells. These chemotactic molecules can then be used to
treat, prevent, and/or diagnose inflammation, infection,
hyperproliferative diseases, disorders, and/or conditions, or any
immune system disorder by increasing the number of cells targeted
to a particular location in the body. For example, chemotaxic
molecules can be used to treat, prevent, and/or diagnose wounds and
other trauma to tissues by attracting immune cells to the injured
location. Chemotactic molecules of the present invention can also
attract fibroblasts, which can be used to treat, prevent, and/or
diagnose wounds.
[1178] It is also contemplated that a polynucleotide or polypeptide
and/or agonist or antagonist of the present invention may inhibit
chemotactic activity. These molecules could also be used totreat,
prevent, and/or diagnose diseases, disorders, and/or conditions.
Thus, a polynucleotide or polypeptide and/or agonist or antagonist
of the present invention could be used as an inhibitor of
chemotaxis.
[1179] Binding Activity
[1180] A polypeptide of the present invention may be used to screen
for molecules that bind to the polypeptide or for molecules to
which the polypeptide binds. The binding of the polypeptide and the
molecule may activate (agonist), increase, inhibit (antagonist), or
decrease activity of the polypeptide or the molecule bound.
Examples of such molecules include antibodies, oligonucleotides,
proteins (e.g., receptors),or small molecules.
[1181] Preferably, the molecule is closely related to the natural
ligand of the polypeptide, e.g., a fragment of the ligand, or a
natural substrate, a ligand, a structural or functional mimetic.
(See, Coligan et al., Current Protocols in Immunology 1(2):Chapter
5 (1991).) Similarly, the molecule can be closely related to the
natural receptor to which the polypeptide binds, or at least, a
fragment of the receptor capable of being bound by the polypeptide
(e.g., active site). In either case, the molecule can be rationally
designed using known techniques.
[1182] Preferably, the screening for these molecules involves
producing appropriate cells which express the polypeptide, either
as a secreted protein or on the cell membrane. Preferred cells
include cells from mammals, yeast, Drosophila, or E. coli. Cells
expressing the polypeptide (or cell membrane containing the
expressed polypeptide) are then preferably contacted with a test
compound potentially containing the molecule to observe binding,
stimulation, or inhibition of activity of either the polypeptide or
the molecule.
[1183] The assay may simply test binding of a candidate compound to
the polypeptide, wherein binding is detected by a label, or in an
assay involving competition with a labeled competitor. Further, the
assay may test whether the candidate compound results in a signal
generated by binding to the polypeptide.
[1184] Alternatively, the assay can be carried out using cell-free
preparations, polypeptide/molecule affixed to a solid support,
chemical libraries, or natural product mixtures. The assay may also
simply comprise the steps of mixing a candidate compound with a
solution containing a polypeptide, measuring polypeptide/molecule
activity or binding, and comparing the polypeptide/molecule
activity or binding to a standard.
[1185] Preferably, an ELISA assay can measure polypeptide level or
activity in a sample (e.g., biological sample) using a monoclonal
or polyclonal antibody. The antibody can measure polypeptide level
or activity by either binding, directly or indirectly, to the
polypeptide or by competing with the polypeptide for a
substrate.
[1186] Additionally, the receptor to which a polypeptide of the
invention binds can be identified by numerous methods known to
those of skill in the art, for example, ligand panning and FACS
sorting (Coligan, et al., Current Protocols in Immun., 1(2),
Chapter 5, (1991)). For example, expression cloning is employed
wherein polyadenylated RNA is prepared from a cell responsive to
the polypeptides, for example, NIH3T3 cells which are known to
contain multiple receptors for the FGF family proteins, and SC-3
cells, and a cDNA library created from this RNA is divided into
pools and used to transfect COS cells or other cells that are not
responsive to the polypeptides. Transfected cells which are grown
on glass slides are exposed to the polypeptide of the present
invention, after they have been labelled. The polypeptides can be
labeled by a variety of means including iodination or inclusion of
a recognition site for a site-specific protein kinase.
[1187] Following fixation and incubation, the slides are subjected
to auto-radiographic analysis. Positive pools are identified and
sub-pools are prepared and re-transfected using an iterative
sub-pooling and re-screening process, eventually yielding a single
clones that encodes the putative receptor.
[1188] As an alternative approach for receptor identification, the
labeled polypeptides can be photoaffinity linked with cell membrane
or extract preparations that express the receptor molecule.
Cross-linked material is resolved by PAGE analysis and exposed to
X-ray film. The labeled complex containing the receptors of the
polypeptides can be excised, resolved into peptide fragments, and
subjected to protein microsequencing. The amino acid sequence
obtained from microsequencing would be used to design a set of
degenerate oligonucleotide probes to screen a cDNA library to
identify the genes encoding the putative receptors.
[1189] Moreover, the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling") may be employed to modulate the activities of
polypeptides of the invention thereby effectively generating
agonists and antagonists of polypeptides of the invention. See
generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721,
5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion
Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol.
16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol.
287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques
24(2):308-13 (1998) (each of these patents and publications are
hereby incorporated by reference). In one embodiment, alteration of
polynucleotides and corresponding polypeptides of the invention may
be achieved by DNA shuffling. DNA shuffling involves the assembly
of two or more DNA segments into a desired polynucleotide sequence
of the invention molecule by homologous, or site-specific,
recombination. In another embodiment, polynucleotides and
corresponding polypeptides of the invention may be alterred by
being subjected to random mutagenesis by error-prone PCR, random
nucleotide insertion or other methods prior to recombination. In
another embodiment, one or more components, motifs, sections,
parts, domains, fragments, etc., of the polypeptides of the
invention may be recombined with one or more components, motifs,
sections, parts, domains, fragments, etc. of one or more
heterologous molecules. In preferred embodiments, the heterologous
molecules are family members. In further preferred embodiments, the
heterologous molecule is a growth factor such as, for example,
platelet-derived growth factor (PDGF), insulin-like growth factor
(IGF-D), transforming growth factor (TGF)-alpha, epidermal growth
factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone
morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins
A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth
differentiation factors (GDFs), nodal, MIS, inhibin-alpha,
TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived
neurotrophic factor (GDNF).
[1190] Other preferred fragments are biologically active fragments
of the polypeptides of the invention. Biologically active fragments
are those exhibiting activity similar, but not necessarily
identical, to an activity of the polypeptide. The biological
activity of the fragments may include an improved desired activity,
or a decreased undesirable activity.
[1191] Additionally, this invention provides a method of screening
compounds to identify those which modulate the action of the
polypeptide of the present invention. An example of such an assay
comprises combining a mammalian fibroblast cell, a the polypeptide
of the present invention, the compound to be screened and 3[H]
thymidine under cell culture conditions where the fibroblast cell
would normally proliferate. A control assay may be performed in the
absence of the compound to be screened and compared to the amount
of fibroblast proliferation in the presence of the compound to
determine if the compound stimulates proliferation by determining
the uptake of 3[H] thymidine in each case. The amount of fibroblast
cell proliferation is measured by liquid scintillation
chromatography which measures the incorporation of 3[H] thymidine.
Both agonist and antagonist compounds may be identified by this
procedure.
[1192] In another method, a mammalian cell or membrane preparation
expressing a receptor for a polypeptide of the present invention is
incubated with a labeled polypeptide of the present invention in
the presence of the compound. The ability of the compound to
enhance or block this interaction could then be measured.
Alternatively, the response of a known second messenger system
following interaction of a compound to be screened and the receptor
is measured and the ability of the compound to bind to the receptor
and elicit a second messenger response is measured to determine if
the compound is a potential agonist or antagonist. Such second
messenger systems include but are not limited to, cAMP guanylate
cyclase, ion channels or phosphoinositide hydrolysis.
[1193] All of these above assays can be used as diagnostic or
prognostic markers. The molecules discovered using these assays can
be used to treat, prevent, and/or diagnose disease or to bring
about a particular result in a patient (e.g., blood vessel growth)
by activating or inhibiting the polypeptide/molecule. Moreover, the
assays can discover agents which may inhibit or enhance the
production of the polypeptides of the invention from suitably
manipulated cells or tissues. Therefore, the invention includes a
method of identifying compounds which bind to the polypeptides of
the invention comprising the steps of: (a) incubating a candidate
binding compound with the polypeptide; and (b) determining if
binding has occurred. Moreover, the invention includes a method of
identifying agonists/antagonists comprising the steps of: (a)
incubating a candidate compound with the polypeptide, (b) assaying
a biological activity, and (b) determining if a biological activity
of the polypeptide has been altered.
[1194] Also, one could identify molecules bind a polypeptide of the
invention experimentally by using the beta-pleated sheet regions
contained in the polypeptide sequence of the protein. Accordingly,
specific embodiments of the invention are directed to
polynucleotides encoding polypeptides which comprise, or
alternatively consist of, the amino acid sequence of each beta
pleated sheet regions in a disclosed polypeptide sequence.
Additional embodiments of the invention are directed to
polynucleotides encoding polypeptides which comprise, or
alternatively consist of, any combination or all of contained in
the polypeptide sequences of the invention. Additional preferred
embodiments of the invention are directed to polypeptides which
comprise, or alternatively consist of, the amino acid sequence of
each of the beta pleated sheet regions in one of the polypeptide
sequences of the invention. Additional embodiments of the invention
are directed to polypeptides which comprise, or alternatively
consist of, any combination or all of the beta pleated sheet
regions in one of the polypeptide sequences of the invention.
[1195] Targeted Delivery
[1196] In another embodiment, the invention provides a method of
delivering compositions to targeted cells expressing a receptor for
a polypeptide of the invention, or cells expressing a cell bound
form of a polypeptide of the invention.
[1197] As discussed herein, polypeptides or antibodies of the
invention may be associated with heterologous polypeptides,
heterologous nucleic acids, toxins, or prodrugs via hydrophobic,
hydrophilic, ionic and/or covalent interactions. In one embodiment,
the invention provides a method for the specific delivery of
compositions of the invention to cells by administering
polypeptides of the invention (including antibodies) that are
associated with heterologous polypeptides or nucleic acids. In one
example, the invention provides a method for delivering a
therapeutic protein into the targeted cell. In another example, the
invention provides a method for delivering a single stranded
nucleic acid (e.g., antisense or ribozymes) or double stranded
nucleic acid (e.g., DNA that can integrate into the cell's genome
or replicate episomally and that can be transcribed) into the
targeted cell.
[1198] In another embodiment, the invention provides a method for
the specific destruction of cells (e.g., the destruction of tumor
cells) by administering polypeptides of the invention (e.g.,
polypeptides of the invention or antibodies of the invention) in
association with toxins or cytotoxic prodrugs.
[1199] By "toxin" is meant compounds that bind and activate
endogenous cytotoxic effector systems, radioisotopes, holotoxins,
modified toxins, catalytic subunits of toxins, or any molecules or
enzymes not normally present in or on the surface of a cell that
under defined conditions cause the cell's death. Toxins that may be
used according to the methods of the invention include, but are not
limited to, radioisotopes known in the art, compounds such as, for
example, antibodies (or complement fixing containing portions
thereof) that bind an inherent or induced endogenous cytotoxic
effector system, thymidine kinase, endonuclease, RNAse, alpha
toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin,
saporin, momordin, gelonin, pokeweed antiviral protein,
alpha-sarcin and cholera toxin. By "cytotoxic prodrug" is meant a
non-toxic compound that is converted by an enzyme, normally present
in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may
be used according to the methods of the invention include, but are
not limited to, glutamyl derivatives of benzoic acid mustard
alkylating agent, phosphate derivatives of etoposide or mitomycin
C, cytosine arabinoside, daunorubisin, and phenoxyacetamide
derivatives of doxorubicin.
[1200] Drug Screening
[1201] Further contemplated is the use of the polypeptides of the
present invention, or the polynucleotides encoding these
polypeptides, to screen for molecules which modify the activities
of the polypeptides of the present invention. Such a method would
include contacting the polypeptide of the present invention with a
selected compound(s) suspected of having antagonist or agonist
activity, and assaying the activity of these polypeptides following
binding.
[1202] This invention is particularly useful for screening
therapeutic compounds by using the polypeptides of the present
invention, or binding fragments thereof, in any of a variety of
drug screening techniques. The polypeptide or fragment employed in
such a test may be affixed to a solid support, expressed on a cell
surface, free in solution, or located intracellularly. One method
of drug screening utilizes eukaryotic or prokaryotic host cells
which are stably transformed with recombinant nucleic acids
expressing the polypeptide or fragment. Drugs are screened against
such transformed cells in competitive binding assays. One may
measure, for example, the formulation of complexes between the
agent being tested and a polypeptide of the present invention.
[1203] Thus, the present invention provides methods of screening
for drugs or any other agents which affect activities mediated by
the polypeptides of the present invention. These methods comprise
contacting such an agent with a polypeptide of the present
invention or a fragment thereof and assaying for the presence of a
complex between the agent and the polypeptide or a fragment
thereof, by methods well known in the art. In such a competitive
binding assay, the agents to screen are typically labeled.
Following incubation, free agent is separated from that present in
bound form, and the amount of free or uncomplexed label is a
measure of the ability of a particular agent to bind to the
polypeptides of the present invention.
[1204] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to the polypeptides of the present invention, and is described in
great detail in European Patent Application 84/03564, published on
Sep. 13, 1984, which is incorporated herein by reference herein.
Briefly stated, large numbers of different small peptide test
compounds are synthesized on a solid substrate, such as plastic
pins or some other surface. The peptide test compounds are reacted
with polypeptides of the present invention and washed. Bound
polypeptides are then detected by methods well known in the art.
Purified polypeptides are coated directly onto plates for use in
the aforementioned drug screening techniques. In addition,
non-neutralizing antibodies may be used to capture the peptide and
immobilize it on the solid support.
[1205] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding polypeptides of the present invention specifically compete
with a test compound for binding to the polypeptides or fragments
thereof. In this manner, the antibodies are used to detect the
presence of any peptide which shares one or more antigenic epitopes
with a polypeptide of the invention.
[1206] Antisense And Ribozyme (Antagonists)
[1207] In specific embodiments, antagonists according to the
present invention are nucleic acids corresponding to the sequences
contained in SEQ ID NO:X, or the complementary strand thereof,
and/or to nucleotide sequences contained a deposited clone. In one
embodiment, antisense sequence is generated internally by the
organism, in another embodiment, the antisense sequence is
separately administered (see, for example, O'Connor, Neurochem.,
56:560 (1991). Oligodeoxynucleotides as Anitsense Inhibitors of
Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense
technology can be used to control gene expression through antisense
DNA or RNA, or through triple-helix formation. Antisense techniques
are discussed for example, in Okano, Neurochem., 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988). Triple helix formation is
discussed in, for instance, Lee et al., Nucleic Acids Research,
6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan
et al., Science, 251:1300 (1991). The methods are based on binding
of a polynucleotide to a complementary DNA or RNA.
[1208] For example, the use of c-myc and c-myb antisense RNA
constructs to inhibit the growth of the non-lymphocytic leukemia
cell line HL-60 and other cell lines was previously described.
(Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments
were performed in vitro by incubating cells with the
oligoribonucleotide. A similar procedure for in vivo use is
described in WO 91/15580. Briefly, a pair of oligonucleotides for a
given antisense RNA is produced as follows: A sequence
complimentary to the first 15 bases of the open reading frame is
flanked by an EcoR1 site on the 5 end and a HindIII site on the 3
end. Next, the pair of oligonucleotides is heated at 90.degree. C.
for one minute and then annealed in 2.times.ligation buffer (20 mM
TRIS HCl pH 7.5, 110 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2
mM ATP) and then ligated to the EcoR1/HindIII site of the
retroviral vector PMV7 (WO 91/15580).
[1209] For example, the 5' coding portion of a polynucleotide that
encodes the mature polypeptide of the present invention may be used
to design an antisense RNA oligonucleotide of from about 10 to 40
base pairs in length. A DNA oligonucleotide is designed to be
complementary to a region of the gene involved in transcription
thereby preventing transcription and the production of the
receptor. The antisense RNA oligonucleotide hybridizes to the mRNA
in vivo and blocks translation of the mRNA molecule into receptor
polypeptide.
[1210] In one embodiment, the antisense nucleic acid of the
invention is produced intracellularly by transcription from an
exogenous sequence. For example, a vector or a portion thereof, is
transcribed, producing an antisense nucleic acid (RNA) of the
invention. Such a vector would contain a sequence encoding the
antisense nucleic acid of the invention. Such a vector can remain
episomal or become chromosomally integrated, as long as it can be
transcribed to produce the desired antisense RNA. Such vectors can
be constructed by recombinant DNA technology methods standard in
the art. Vectors can be plasmid, viral, or others known in the art,
used for replication and expression in vertebrate cells. Expression
of the sequence encoding a polypeptide of the invention, or
fragments thereof, can be by any promoter known in the art to act
in vertebrate, preferably human cells. Such promoters can be
inducible or constitutive. Such promoters include, but are not
limited to, the SV40 early promoter region (Bemoist and Chambon,
Nature, 29:304-310 (1981), the promoter contained in the 3' long
terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell,
22:787-797 (1980), the herpes thymidine promoter (Wagner et al.,
Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445 (1981), the regulatory
sequences of the metallothionein gene (Brinster et al., Nature,
296:39-42 (1982)), etc.
[1211] The antisense nucleic acids of the invention comprise a
sequence complementary to at least a portion of an RNA transcript
of a gene of interest. However, absolute complementarity, although
preferred, is not required. A sequence "complementary to at least a
portion of an RNA," referred to herein, means a sequence having
sufficient complementarity to be able to hybridize with the RNA,
forming a stable duplex; in the case of double stranded antisense
nucleic acids of the invention, a single strand of the duplex DNA
may thus be tested, or triplex formation may be assayed. The
ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid
Generally, the larger the hybridizing nucleic acid, the more base
mismatches with a RNA sequence of the invention it may contain and
still form a stable duplex (or triplex as the case may be). One
skilled in the art can ascertain a tolerable degree of mismatch by
use of standard procedures to determine the melting point of the
hybridized complex.
[1212] Oligonucleotides that are complementary to the 5' end of the
message, e.g., the 5' untranslated sequence up to and including the
AUG initiation codon, should work most efficiently at inhibiting
translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs have been shown to be effective at
inhibiting translation of mRNAs as well. See generally, Wagner, R.,
Nature, 372:333-335 (1994). Thus, oligonucleotides complementary to
either the 5'- or 3'- non-translated, non-coding regions of a
polynucleotide sequence of the invention could be used in an
antisense approach to inhibit translation of endogenous mRNA.
Oligonucleotides complementary to the 5' untranslated region of the
mRNA should include the complement of the AUG start codon.
Antisense oligonucleotides complementary to mRNA coding regions are
less efficient inhibitors of translation but could be used in
accordance with the invention. Whether designed to hybridize to the
5'-, 3'- or coding region of mRNA, antisense nucleic acids should
be at least six nucleotides in length, and are preferably
oligonucleotides ranging from 6 to about 50 nucleotides in length.
In specific aspects the oligonucleotide is at least 10 nucleotides,
at least 17 nucleotides, at least 25 nucleotides or at least 50
nucleotides.
[1213] The polynucleotides of the invention can be DNA or RNA or
chimeric mixtures or derivatives or modified versions thereof,
single-stranded or double-stranded. The oligonucleotide can be
modified at the base moiety, sugar moiety, or phosphate backbone,
for example, to improve stability of the molecule, hybridization,
etc. The oligonucleotide may include other appended groups such as
peptides (e.g., for targeting host cell receptors in vivo), or
agents facilitating transport across the cell membrane (see, e.g.,
Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556
(1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652 (1987);
PCT Publication NO: WO88/09810, published Dec. 15, 1988) or the
blood-brain barrier (see, e.g., PCT Publication NO: WO89/10134,
published April 25, 1988), hybridization-triggered cleavage agents.
(See, e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or
intercalating agents. (See, e.g., Zon, Pharm. Res., 5:539-549
(1988)). To this end, the oligonucleotide may be conjugated to
another molecule, e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
[1214] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including,
but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[1215] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including, but not
limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
[1216] In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group including, but not limited to, a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[1217] In yet another embodiment, the antisense oligonucleotide is
an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms
specific double-stranded hybrids with complementary RNA in which,
contrary to the usual b-units, the strands run parallel to each
other (Gautier et al., Nucl. Acids Res., 15:6625-6641 (1987)). The
oligonucleotide is a 2-0-methylribonucleotide (Inoue et al., Nucl.
Acids Res., 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue
(Inoue et al., FEBS Lett. 215:327-330 (1987)).
[1218] Polynucleotides of the invention may be synthesized by
standard methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
(Nucl. Acids Res., 16:3209 (1988)), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.,
85:7448-7451 (1988)), etc.
[1219] While antisense nucleotides complementary to the coding
region sequence of the invention could be used, those complementary
to the transcribed untranslated region are most preferred.
[1220] Potential antagonists according to the invention also
include catalytic RNA, or a ribozyme (See, e.g., PCT International
Publication WO 90/11364, published Oct. 4, 1990; Sarver et al,
Science, 247:1222-1225 (1990). While ribozymes that cleave mRNA at
site specific recognition sequences can be used to destroy mRNAs
corresponding to the polynucleotides of the invention, the use of
hammerhead ribozymes is preferred. Hammerhead ribozymes cleave
mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. The sole requirement
is that the target mRNA have the following sequence of two bases:
5'-UG-3'. The construction and production of hammerhead ribozymes
is well known in the art and is described more filly in Haseloff
and Gerlach, Nature, 334:585-591 (1988). There are numerous
potential hammerhead ribozyme cleavage sites within each nucleotide
sequence disclosed in the sequence listing. Preferably, the
ribozyme is engineered so that the cleavage recognition site is
located near the 5' end of the mRNA corresponding to the
polynucleotides of the invention; i.e., to increase efficiency and
minimize the intracellular accumulation of non-functional mRNA
transcripts.
[1221] As in the antisense approach, the ribozymes of the invention
can be composed of modified oligonucleotides (e.g. for improved
stability, targeting, etc.) and should be delivered to cells which
express the polynucleotides of the invention in vivo. DNA
constructs encoding the ribozyme may be introduced into the cell in
the same manner as described above for the introduction of
antisense encoding DNA. A preferred method of delivery involves
using a DNA construct "encoding" the ribozyme under the control of
a strong constitutive promoter, such as, for example, pol III or
pol II promoter, so that transfected cells will produce sufficient
quantities of the ribozyme to destroy endogenous messages and
inhibit translation. Since ribozymes unlike antisense molecules,
are catalytic, a lower intracellular concentration is required for
efficiency.
[1222] Antagonist/agonist compounds may be employed to inhibit the
cell growth and proliferation effects of the polypeptides of the
present invention on neoplastic cells and tissues, i.e. stimulation
of angiogenesis of tumors, and, therefore, retard or prevent
abnormal cellular growth and proliferation, for example, in tumor
formation or growth.
[1223] The antagonist/agonist may also be employed to prevent
hyper-vascular diseases, and prevent the proliferation of
epithelial lens cells after extracapsular cataract surgery.
Prevention of the mitogenic activity of the polypeptides of the
present invention may also be desirous in cases such as restenosis
after balloon angioplasty.
[1224] The antagonist/agonist may also be employed to prevent the
growth of scar tissue during wound healing.
[1225] The antagonist/agonist may also be employed to treat,
prevent, and/or diagnose the diseases described herein.
[1226] Thus, the invention provides a method of treating or
preventing diseases, disorders, and/or conditions, including but
not limited to the diseases, disorders, and/or conditions listed
throughout this application, associated with overexpression of a
polynucleotide of the present invention by administering to a
patient (a) an antisense molecule directed to the polynucleotide of
the present invention, and/or (b) a ribozyme directed to the
polynucleotide of the present invention. invention, and/or (b) a
ribozyme directed to the polynucleotide of the present
invention
[1227] Other Activities
[1228] The polypeptide of the present invention, as a result of the
ability to stimulate vascular endothelial cell growth, may be
employed in treatment for stimulating re-vascularization of
ischemic tissues due to various disease conditions such as
thrombosis, arteriosclerosis, and other cardiovascular conditions.
These polypeptide may also be employed to stimulate angiogenesis
and limb regeneration, as discussed above.
[1229] The polypeptide may also be employed for treating wounds due
to injuries, bums, post-operative tissue repair, and ulcers since
they are mitogenic to various cells of different origins, such as
fibroblast cells and skeletal muscle cells, and therefore,
facilitate the repair or replacement of damaged or diseased
tissue.
[1230] The polypeptide of the present invention may also be
employed stimulate neuronal growth and to treat, prevent, and/or
diagnose neuronal damage which occurs in certain neuronal disorders
or neuro-degenerative conditions such as Alzheimer's disease,
Parkinson's disease, and AIDS-related complex. The polypeptide of
the invention may have the ability to stimulate chondrocyte growth,
therefore, they may be employed to enhance bone and periodontal
regeneration and aid in tissue transplants or bone grafts.
[1231] The polypeptide of the present invention may be also be
employed to prevent skin aging due to sunburn by stimulating
keratinocyte growth.
[1232] The polypeptide of the invention may also be employed for
preventing hair loss, since FGF family members activate
hair-forming cells and promotes melanocyte growth. Along the same
lines, the polypeptides of the present invention may be employed to
stimulate growth and differentiation of hematopoietic cells and
bone marrow cells when used in combination with other
cytokines.
[1233] The polypeptide of the invention may also be employed to
maintain organs before transplantation or for supporting cell
culture of primary tissues.
[1234] The polypeptide of the present invention may also be
employed for inducing tissue of mesodermal origin to differentiate
in early embryos.
[1235] The polypeptide or polynucleotides and/or agonist or
antagonists of the present invention may also increase or decrease
the differentiation or proliferation of embryonic stem cells,
besides, as discussed above, hematopoietic lineage.
[1236] The polypeptide or polynucleotides and/or agonist or
antagonists of the present invention may also be used to modulate
mammalian characteristics, such as body height, weight, hair color,
eye color, skin, percentage of adipose tissue, pigmentation, size,
and shape (e.g., cosmetic surgery). Similarly, polypeptides or
polynucleotides and/or agonist or antagonists of the present
invention may be used to modulate mammalian metabolism affecting
catabolism, anabolism, processing, utilization, and storage of
energy.
[1237] Polypeptide or polynucleotides and/or agonist or antagonists
of the present invention may be used to change a mammal's mental
state or physical state by influencing biorhythms, caricadic
rhythms, depression (including depressive diseases, disorders,
and/or conditions), tendency for violence, tolerance for pain,
reproductive capabilities (preferably by Activin or Inhibin-like
activity), hormonal or endocrine levels, appetite, libido, memory,
stress, or other cognitive qualities.
[1238] Polypeptide or polynucleotides and/or agonist or antagonists
of the present invention may also be used as a food additive or
preservative, such as to increase or decrease storage capabilities,
fat content, lipid, protein, carbohydrate, vitamins, minerals,
cofactors or other nutritional components.
[1239] Other Preferred Embodiments
[1240] Other preferred embodiments of the claimed invention include
an isolated nucleic acid molecule comprising a nucleotide sequence
which is at least 95% identical to a sequence of at least about 50
contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X
wherein X is any integer as defined in Table 1.
[1241] Also preferred is a nucleic acid molecule wherein said
sequence of contiguous nucleotides is included in the nucleotide
sequence of SEQ ID NO:X in the range of positions beginning with
the nucleotide at about the position of the 5' Nucleotide of the
Clone Sequence and ending with the nucleotide at about the position
of the 3' Nucleotide of the Clone Sequence as defined for SEQ ID
NO:X in Table 1.
[1242] Also preferred is a nucleic acid molecule wherein said
sequence of contiguous nucleotides is included in the nucleotide
sequence of SEQ ID NO:X in the range of positions beginning with
the nucleotide at about the position of the 5' Nucleotide of the
Start Codon and ending with the nucleotide at about the position of
the 3' Nucleotide of the Clone Sequence as defined for SEQ ID NO:X
in Table 1.
[1243] Similarly preferred is a nucleic acid molecule wherein said
sequence of contiguous nucleotides is included in the nucleotide
sequence of SEQ ID NO:X in the range of positions beginning with
the nucleotide at about the position of the 5' Nucleotide of the
First Amino Acid of the Signal Peptide and ending with the
nucleotide at about the position of the 3' Nucleotide of the Clone
Sequence as defined for SEQ ID NO:X in Table 1.
[1244] Also preferred is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
a sequence of at least about 150 contiguous nucleotides in the
nucleotide sequence of SEQ ID NO:X.
[1245] Further preferred is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
a sequence of at least about 500 contiguous nucleotides in the
nucleotide sequence of SEQ ID NO:X.
[1246] A further preferred embodiment is a nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
the nucleotide sequence of SEQ ID NO:X beginning with the
nucleotide at about the position of the 5' Nucleotide of the First
Amino Acid of the Signal Peptide and ending with the nucleotide at
about the position of the 3' Nucleotide of the Clone Sequence as
defined for SEQ ID NO:X in Table 1.
[1247] A further preferred embodiment is an isolated nucleic acid
molecule comprising a nucleotide sequence which is at least 95%
identical to the complete nucleotide sequence of SEQ ID NO:X.
[1248] Also preferred is an isolated nucleic acid molecule which
hybridizes under stringent hybridization conditions to a nucleic
acid molecule, wherein said nucleic acid molecule which hybridizes
does not hybridize under stringent hybridization conditions to a
nucleic acid molecule having a nucleotide sequence consisting of
only A residues or of only T residues.
[1249] Also preferred is a composition of matter comprising a DNA
molecule which comprises a human cDNA clone identified by a cDNA
Clone Identifier in Table 1, which DNA molecule is contained in the
material deposited with the American Type Culture Collection and
given the ATCC Deposit Number shown in Table 1 for said cDNA Clone
Identifier.
[1250] Also preferred is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
a sequence of at least 50 contiguous nucleotides in the nucleotide
sequence of a human cDNA clone identified by a cDNA Clone
Identifier in Table 1, which DNA molecule is contained in the
deposit given the ATCC Deposit Number shown in Table 1.
[1251] Also preferred is an isolated nucleic acid molecule, wherein
said sequence of at least 50 contiguous nucleotides is included in
the nucleotide sequence of the complete open reading frame sequence
encoded by said human cDNA clone.
[1252] Also preferred is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
sequence of at least 150 contiguous nucleotides in the nucleotide
sequence encoded by said human cDNA clone.
[1253] A further preferred embodiment is an isolated nucleic acid
molecule comprising a nucleotide sequence which is at least 95%
identical to sequence of at least 500 contiguous nucleotides in the
nucleotide sequence encoded by said human cDNA clone.
[1254] A further preferred embodiment is an isolated nucleic acid
molecule comprising a nucleotide sequence which is at least 95%
identical to the complete nucleotide sequence encoded by said human
cDNA clone.
[1255] A further preferred embodiment is a method for detecting in
a biological sample a nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least
50 contiguous nucleotides in a sequence selected from the group
consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is
any integer as defined in Table 1; and a nucleotide sequence
encoded by a human cDNA clone identified by a cDNA Clone Identifier
in Table 1 and contained in the deposit with the ATCC Deposit
Number shown for said cDNA clone in Table 1; which method comprises
a step of comparing a nucleotide sequence of at least one nucleic
acid molecule in said sample with a sequence selected from said
group and determining whether the sequence of said nucleic acid
molecule in said sample is at least 95% identical to said selected
sequence.
[1256] Also preferred is the above method wherein said step of
comparing sequences comprises determining the extent of nucleic
acid hybridization between nucleic acid molecules in said sample
and a nucleic acid molecule comprising said sequence selected from
said group. Similarly, also preferred is the above method wherein
said step of comparing sequences is performed by comparing the
nucleotide sequence determined from a nucleic acid molecule in said
sample with said sequence selected from said group. The nucleic
acid molecules can comprise DNA molecules or RNA molecules.
[1257] A further preferred embodiment is a method for identifying
the species, tissue or cell type of a biological sample which
method comprises a step of detecting nucleic acid molecules in said
sample, if any, comprising a nucleotide sequence that is at least
95% identical to a sequence of at least 50 contiguous nucleotides
in a sequence selected from the group consisting of: a nucleotide
sequence of SEQ ID NO:X wherein X is any integer as defined in
Table 1; and a nucleotide sequence encoded by a human cDNA clone
identified by a cDNA Clone Identifier in Table 1 and contained in
the deposit with the ATCC Deposit Number shown for said cDNA clone
in Table 1.
[1258] The method for identifying the species, tissue or cell type
of a biological sample can comprise a step of detecting nucleic
acid molecules comprising a nucleotide sequence in a panel of at
least two nucleotide sequences, wherein at least one sequence in
said panel is at least 95% identical to a sequence of at least 50
contiguous nucleotides in a sequence selected from said group.
[1259] Also preferred is a method for diagnosing in a subject a
pathological condition associated with abnormal structure or
expression of a gene encoding a secreted protein identified in
Table 1, which method comprises a step of detecting in a biological
sample obtained from said subject nucleic acid molecules, if any,
comprising a nucleotide sequence that is at least 95% identical to
a sequence of at least 50 contiguous nucleotides in a sequence
selected from the group consisting of: a nucleotide sequence of SEQ
ID NO:X wherein X is any integer as defined in Table 1; and a
nucleotide sequence encoded by a human cDNA clone identified by a
cDNA Clone Identifier in Table 1 and contained in the deposit with
the ATCC Deposit Number shown for said cDNA clone in Table 1.
[1260] The method for diagnosing a pathological condition can
comprise a step of detecting nucleic acid molecules comprising a
nucleotide sequence in a panel of at least two nucleotide
sequences, wherein at least one sequence in said panel is at least
95% identical to a sequence of at least 50 contiguous nucleotides
in a sequence selected from said group.
[1261] Also preferred is a composition of matter comprising
isolated nucleic acid molecules wherein the nucleotide sequences of
said nucleic acid molecules comprise a panel of at least two
nucleotide sequences, wherein at least one sequence in said panel
is at least 95% identical to a sequence of at least 50 contiguous
nucleotides in a sequence selected from the group consisting of: a
nucleotide sequence of SEQ ID NO:X wherein X is any integer as
defined in Table 1; and a nucleotide sequence encoded by a human
cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained in the deposit with the ATCC Deposit Number shown for
said cDNA clone in Table 1. The nucleic acid molecules can comprise
DNA molecules or RNA molecules.
[1262] Also preferred is an isolated polypeptide comprising an
amino acid sequence at least 90% identical to a sequence of at
least about 10 contiguous amino acids in the amino acid sequence of
SEQ ID NO:Y wherein Y is any integer as defined in Table 1.
[1263] Also preferred is a polypeptide, wherein said sequence of
contiguous amino acids is included in the amino acid sequence of
SEQ ID NO:Y in the range of positions beginning with the residue at
about the position of the First Amino Acid of the Secreted Portion
and ending with the residue at about the Last Amino Acid of the
Open Reading Frame as set forth for SEQ ID NO:Y in Table 1.
[1264] Also preferred is an isolated polypeptide comprising an
amino acid sequence at least 95% identical to a sequence of at
least about 30 contiguous amino acids in the amino acid sequence of
SEQ ID NO:Y.
[1265] Further preferred is an isolated polypeptide comprising an
amino acid sequence at least 95% identical to a sequence of at
least about 100 contiguous amino acids in the amino acid sequence
of SEQ ID NO:Y.
[1266] Further preferred is an isolated polypeptide comprising an
amino acid sequence at least 95% identical to the complete amino
acid sequence of SEQ ID NO:Y.
[1267] Further preferred is an isolated polypeptide comprising an
amino acid sequence at least 90% identical to a sequence of at
least about 10 contiguous amino acids in the complete amino acid
sequence of a secreted protein encoded by a human cDNA clone
identified by a cDNA Clone Identifier in Table 1 and contained in
the deposit with the ATCC Deposit Number shown for said cDNA clone
in Table 1.
[1268] Also preferred is a polypeptide wherein said sequence of
contiguous amino acids is included in the amino acid sequence of a
secreted portion of the secreted protein encoded by a human cDNA
clone identified by a cDNA Clone Identifier in Table 1 and
contained in the deposit with the ATCC Deposit Number shown for
said cDNA clone in Table 1.
[1269] Also preferred is an isolated polypeptide comprising an
amino acid sequence at least 95% identical to a sequence of at
least about 30 contiguous amino acids in the amino acid sequence of
the secreted portion of the protein encoded by a human cDNA clone
identified by a cDNA Clone Identifier in Table 1 and contained in
the deposit with the ATCC Deposit Number shown for said cDNA clone
in Table 1.
[1270] Also preferred is an isolated polypeptide comprising an
amino acid sequence at least 95% identical to a sequence of at
least about 100 contiguous amino acids in the amino acid sequence
of the secreted portion of the protein encoded by a human cDNA
clone identified by a cDNA Clone Identifier in Table 1 and
contained in the deposit with the ATCC Deposit Number shown for
said cDNA clone in Table 1.
[1271] Also preferred is an isolated polypeptide comprising an
amino acid sequence at least 95% identical to the amino acid
sequence of the secreted portion of the protein encoded by a human
cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained in the deposit with the ATCC Deposit Number shown for
said cDNA clone in Table 1.
[1272] Further preferred is an isolated antibody which binds
specifically to a polypeptide comprising an amino acid sequence
that is at least 90% identical to a sequence of at least 10
contiguous amino acids in a sequence selected from the group
consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is
any integer as defined in Table 1; and a complete amino acid
sequence of a protein encoded by a human cDNA clone identified by a
cDNA Clone Identifier in Table 1 and contained in the deposit with
the ATCC Deposit Number shown for said cDNA clone in Table 1.
[1273] Further preferred is a method for detecting in a biological
sample a polypeptide comprising an amino acid sequence which is at
least 90% identical to a sequence of at least 10 contiguous amino
acids in a sequence selected from the group consisting of: an amino
acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1; and a complete amino acid sequence of a protein encoded by
a human cDNA clone identified by a CDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC Deposit Number shown for
said cDNA clone in Table 1; which method comprises a step of
comparing an amino acid sequence of at least one polypeptide
molecule in said sample with a sequence selected from said group
and determining whether the sequence of said polypeptide molecule
in said sample is at least 90% identical to said sequence of at
least 10 contiguous amino acids.
[1274] Also preferred is the above method wherein said step of
comparing an amino acid sequence of at least one polypeptide
molecule in said sample with a sequence selected from said group
comprises determining the extent of specific binding of
polypeptides in said sample to an antibody which binds specifically
to a polypeptide comprising an amino acid sequence that is at least
90% identical to a sequence of at least 10 contiguous amino acids
in a sequence selected from the group consisting of: an amino acid
sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1; and a complete amino acid sequence of a protein encoded by
a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC Deposit Number shown for
said cDNA clone in Table 1.
[1275] Also preferred is the above method wherein said step of
comparing sequences is performed by comparing the amino acid
sequence determined from a polypeptide molecule in said sample with
said sequence selected from said group.
[1276] Also preferred is a method for identifying the species,
tissue or cell type of a biological sample which method comprises a
step of detecting polypeptide molecules in said sample, if any,
comprising an amino acid sequence that is at least 90% identical to
a sequence of at least 10 contiguous amino acids in a sequence
selected from the group consisting of: an amino acid sequence of
SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a
complete amino acid sequence of a secreted protein encoded by a
human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC Deposit Number shown for
said cDNA clone in Table 1.
[1277] Also preferred is the above method for identifying the
species, tissue or cell type of a biological sample, which method
comprises a step of detecting polypeptide molecules comprising an
amino acid sequence in a panel of at least two amino acid
sequences, wherein at least one sequence in said panel is at least
90% identical to a sequence of at least 10 contiguous amino acids
in a sequence selected from the above group.
[1278] Also preferred is a method for diagnosing in a subject a
pathological condition associated with abnormal structure or
expression of a gene encoding a secreted protein identified in
Table 1, which method comprises a step of detecting in a biological
sample obtained from said subject polypeptide molecules comprising
an amino acid sequence in a panel of at least two amino acid
sequences, wherein at least one sequence in said panel is at least
90% identical to a sequence of at least 10 contiguous amino acids
in a sequence selected from the group consisting of: an amino acid
sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1; and a complete amino acid sequence of a secreted protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier
in Table 1 and contained in the deposit with the ATCC Deposit
Number shown for said cDNA clone in Table 1.
[1279] In any of these methods, the step of detecting said
polypeptide molecules includes using an antibody.
[1280] Also preferred is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
a nucleotide sequence encoding a polypeptide wherein said
polypeptide comprises an amino acid sequence that is at least 90%
identical to a sequence of at least 10 contiguous amino acids in a
sequence selected from the group consisting of: an amino acid
sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1; and a complete amino acid sequence of a secreted protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier
in Table 1 and contained in the deposit with the ATCC Deposit
Number shown for said cDNA clone in Table 1.
[1281] Also preferred is an isolated nucleic acid molecule, wherein
said nucleotide sequence encoding a polypeptide has been optimized
for expression of said polypeptide in a prokaryotic host.
[1282] Also preferred is an isolated nucleic acid molecule, wherein
said polypeptide comprises an amino acid sequence selected from the
group consisting of: an amino acid sequence of SEQ ID NO:Y wherein
Y is any integer as defined in Table 1; and a complete amino acid
sequence of a secreted protein encoded by a human cDNA clone
identified by a cDNA Clone Identifier in Table 1 and contained in
the deposit with the ATCC Deposit Number shown for said cDNA clone
in Table 1.
[1283] Further preferred is a method of making a recombinant vector
comprising inserting any of the above isolated nucleic acid
molecule into a vector. Also preferred is the recombinant vector
produced by this method. Also preferred is a method of making a
recombinant host cell comprising introducing the vector into a host
cell, as well as the recombinant host cell produced by this
method.
[1284] Also preferred is a method of making an isolated polypeptide
comprising culturing this recombinant host cell under conditions
such that said polypeptide is expressed and recovering said
polypeptide. Also preferred is this method of making an isolated
polypeptide, wherein said recombinant host cell is a eukaryotic
cell and said polypeptide is a secreted portion of a human secreted
protein comprising an amino acid sequence selected from the group
consisting of: an amino acid sequence of SEQ ID NO:Y beginning with
the residue at the position of the First Amino Acid of the Secreted
Portion of SEQ ID NO:Y wherein Y is an integer set forth in Table 1
and said position of the First Amino Acid of the Secreted Portion
of SEQ ID NO:Y is defined in Table 1; and an amino acid sequence of
a secreted portion of a protein encoded by a human cDNA clone
identified by a cDNA Clone Identifier in Table 1 and contained in
the deposit with the ATCC Deposit Number shown for said cDNA clone
in Table 1. The isolated polypeptide produced by this method is
also preferred.
[1285] Also preferred is a method of treatment of an individual in
need of an increased level of a secreted protein activity, which
method comprises administering to such an individual a
pharmaceutical composition comprising an amount of an isolated
polypeptide, polynucleotide, or antibody of the claimed invention
effective to increase the level of said protein activity in said
individual.
[1286] The above-recited applications have uses in a wide variety
of hosts. Such hosts include, but are not limited to, human,
murine, rabbit, goat, guinea pig, camel, horse, mouse, rat,
hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat,
non-human primate, and human. In specific embodiments, the host is
a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig,
sheep, dog or cat. In preferred embodiments, the host is a mammal.
In most preferred embodiments, the host is a human.
[1287] In specific embodiments of the invention, for each "Contig
ID" listed in the fourth column of Table 6, preferably excluded are
one or more polynucleotides comprising, or alternatively consisting
of, a nucleotide sequence referenced in the fifth column of Table 6
and described by the general formula of a-b, whereas a and b are
uniquely determined for the corresponding SEQ ID NO:X referred to
in column 3 of Table 6. Further specific embodiments are directed
to polynucleotide sequences excluding one, two, three, four, or
more of the specific polynucleotide sequences referred to in the
fifth column of Table 6. In no way is this listing meant to
encompass all of the sequences which may be excluded by the general
formula, it is just a representative example. All references
available through these accessions are hereby incorporated by
reference in their entirety. TABLE-US-00015 TABLE 6 Clone ID NO: Z
SEQ ID NO.: X Contig ID: Accession #'s HKABZ65 11 665424 AA715814,
AA503019, AW338860, AL044701, AA715173, AA715075, AI568659,
AA525144, AF109907, AC005071, AC004878, AC005081, AC002549,
AC020663, AC006064, AC004858, AC007666, AL022318, AL035086,
AC004656, AC004067, AC004477, AC006023, Z98884, AC007637, AL080243,
AC002369, Z84487, AL031311, AL049776, AC004686, AL080317, AC002310,
AL050318, AL132712, D87675, AC007546, AC004675, AL035683, AC002288,
AF030453, Z95331, AC006077, AC008101, AF088219, AC005175, AL021391,
AC005670, AL133163, AL031123, AC004770, AC004659, AL078463,
AC002492, AC006084, AC005089, AL031670, AC005088, AC004491,
AC005887, AP000008, AC002457, AC009946, AC005200, AC006581,
AL022316, AC005180, AC005015, AP000553, Z98742, AC007283, AC005920,
AC004832, AL035462, AC002352, AC005037, AF111169, AC004854,
AF067844, U95090, AL109623, AF053356, U78027, Z85987, AP000704,
AC004263, AC004232, AC005661, AC005409, AP000563, AC005005,
AL031848, AC004881, AC004685, AC005480, AC003950, AC000026,
AC007563, AL021155, AC002470, AL031767, AP000557, Z73358, AL023553,
AC002375, U52112, AC002395, AC002425, AC005280, AC006101, AL034418,
AC005538, AC002476, AL049569, AL109801, AL035422, AC005102,
AC002059, Z85996, AC004813, Z98304, AC003663, AC005562, AL034402,
AC004804, AC002477, AC007225, AC005399, AL121825, AL034421,
AL008718, Z84486, AC007051, AC005366, Z98950, AL049795, AC002073,
AC006536, AC004905, AL035405, AC004587, U91327, AL096791, AC005393,
AP000513, AL109753, AC003029, AL021918, AC005703, AL035659, Z93020,
and AL031279. HNGIC80 12 637909 AL118503 HDPUG50 13 684120
AI217895, AI983150, AW385698, AW374106, AI660124, AI339010,
AW374124, AA166971, AA542906, AA689356, AI285269, AI346870, N27706,
AW236815, AI821227, AI821074, AL134542, AA166818, AA836112, D20721,
AI221030, AA627350, AW027663, N35710, AI221246, AW372396, AI285231,
T95430, AW372395, AI699709, AL134543, AA055338, AA449417, AW197834,
R83129, AI418208, AA375954, AA450383, AA961046, N20259, AA336834,
AA226636, AI911109, AA225691, N20865, AA825421, AI932769, AA938413,
AW197872, AA370379, N29162, C03633, AI620095, AA055337, AI932771,
AA976076, AI821821, AA173926, AA173884, AA569611, AI821883,
AA772955, AW383971, AI432644, AI431307, AI431316, AI432666,
AI431238, AI623302, AI432653, AI431323, AI921241, AI431347,
AI431350, AI432655, AW081103, AI431321, AL042853, AL042729,
AI431243, AI431230, AI431328, AI432654, AI431310, AI431312,
AI432650, AI432677, AI431247, AI432657, AI492519, AI431231,
AI791349, AI431257, AI431235, AI431315, AI431354, AI431318,
AI431353, AI432661, AI431246, AI432649, AI432643, AI432675,
AI431337, AI432651, AI432647, AI432674, AI431330, AW129223,
AL045327, AI431248, AL042931, AI432665, AL042519, AJ224875, Y17793,
AF019249, AL133082, and AF064854. HAEAB66 14 580083 AI659421,
AI632698, AI969812, AI394313, AW139577, AI739006, AW271206,
AW293868, AI805043, AI799897, AI923666, AA640596, AA308562, H80192,
AA833662, AA910928, AI275400, AW377553, AW377527, AI191675,
AI041565, AW138256, AI693984, AI392758, AI597816, AA776304,
AI956051, AI085021, AI288918, AI076685, AA725434, AI824191,
AI471844, AA524228, N70113, AA143492, AA143493, AA226122, AA226045,
AI123234, AA858158, AA532806, W01829, N70775, AI183697, AI693773,
AA757995, AA304772, H78816, AI276951, AA613815, AA152444, AI076680,
AI283120, AA152445, AF228603, AF157600, and AF170564. HHEPF59 15
695722 AL120852, AI922659, AA932542, AA262051, AA526382, AW205846,
N39596, AI459931, AW406797, AI866992, AI373687, AI475825, AA582869,
AI862875, AA223668, R96889, T90824, AA642941, R43602, D60935,
R44585, AA812110, AI669230, AI928028, AI199166, AI369241, AI799999,
AI963565, N52647, D80065, N68066, R96890, AI083867, T85729, R19318,
N80503, AW451196, N72375, AI571518, AI797299, AI685620, AW002004,
AW194849, AW197067, AI498711, N46743, AW243761, AA974737, AW189464,
AI383927, T23990, AI373614, AI633402, AA247241, AI961589, AL120853,
AI587156, AI702073, AI537261, AI862139, AI500714, AI627988,
AI921248, AI433157, AI633125, AW084425, AI659585, AI677796,
AL121564, AW152182, AI277008, AI677797, AW167448, AI670009,
AI280637, AI873923, AI620003, AI570989, AW029638, AI590630,
AI812107, AW129271, AI620089, AI963193, AI281772, AI624293,
AW105383, AI683173, AI682971, AI745656, AW080327, AI874166,
AI868740, AI241744, AI612852, AI886181, AI637584, AI241923,
AW090550, AW029329, AW170635, AI610770, AI587114, AI500061,
AI583085, AI564719, AI469532, AI538564, AI432030, AI499285,
AI827154, AI633000, AI538829, AI445025, AI434223, AW148536,
AI002285, AI541056, AW129722, AI884318, AI554186, AI357940,
AI569637, AI568138, AI445992, AI582932, AW151714, AI521560,
AI569975, AA641818, AI287233, AI473536, AW104724, AI866469,
AA502794, AW162194, AW104827, AI932949, AI288285, AW192652,
AW026087, AI148272, AI591387, AI631095, AI800155, AI610690,
AI275640, AI669459, AW149925, AI915291, AI963346, AW148408,
AI499393, AI469112, AW193530, AW073270, AI632408, AW090071,
AI866801, AI690426, AW058243, AL048656, AW087207, AI499986,
AI368868, AI635067, AW081653, AI801152, AI540382, AI890507,
AI921464, AI690748, AI521103, AW148363, AW130068, AW051088,
AI309244, AI290154, AI362347, AW105431, AI698391, AI142101,
AI471909, AI355779, AW190194, AW090736, AI476478, AI922561,
AI889189, AA805434, AW008353, AI362248, AI687362, AL037454,
AI889376, AI609375, AL039086, AI619426, AW151893, AI744988,
AA983883, AI796743, AW162118, AI538716, AI499947, AI648567, W74529,
AI280732, AI797538, AI242248, AW075667, AI978703, AI696829,
AI249877, AI648508, AW167021, AL046595, AI254731, AI540674,
AW132056, AI624084, AI287793, AI569583, AI522052, AI539800,
AI579901, AI760435, AW087160, AW193231, AW129916, AI269205,
AI933589, AI963458, AW129230, AI925502, AW169604, AI340982,
AL037030, AI520862, AW264727, AL040241, AW150453, AW262552,
AI568855, AI286256, AW169653, AI261344, AW263823, AW168788,
AC004596, AF090901, E05822, I89947, AL137429, AL133112, AR038854,
AL137523, AL137539, AF061981, AL050116, I48978, AL137459, AC004883,
AL133072, AL137480, AL050149, AL078630, AL110280, A08910, A08909,
A08908, Z82022, A08916, A77033, A77035, AF106657, E12747, AF113019,
Y10936, AJ000937, A08913, U35846, AL049938, AL049452, AF185576,
A18788, AF026816, AL049283, I33392, AF111849, AC004093, U80742,
AL117435, AJ006417, A21103, S36676, AL080159, AF183393, AL122100,
AL137529, AF106862, L19437, AL117460, AF000301, AL137488, AF061573,
AC006840, AC005968, I89931, AB022159, AL117416, Y14314, I49625,
I48979, A08912, AL137463, AB016226, X82434, AF118094, AL137560,
U53505, A49139, AF026124, AL080148, A58524, A58523, A18777,
AL050366, AL117648, AF067790, AF153205, AF109906, AF139986,
AL050092, AF008439, AL137557, A07647, L31396, X52034, L31397,
X81464, AF182215, AR020905, X87582, Y11254, AL133080, AL049466,
X94372, AL137550, S77771, AL122093, AL133113, AL133031, AL050277,
AF067728, AF087943, AL137271, E02349, A93350, U87620, AL137533,
AL050138, AL133606, AF032666, AJ005690, I89934, AF119337, AL133067,
AL133640, I03321, AL133075, M27260, AP000020, AL137476, Y10655,
A08907, AC004227, AF090903, I68732, AF177401, D83032, Y10080,
AL122110, X79812, AL049430, AF031147, AL117457, AL096744, A03736,
AF100931, A65341, AF111851, U58996, AL110197, AF159615, AL110221,
U66274, I09499, AL137547, S76508, U88966, E01573, E02319, AL080124,
AL050146, AF091084, AF113677, U67958, AL080154, AF079763, I17544,
E01314, A45787, X57961, AL117440, AL110225, AL137292, Z97214,
AL137276, AF054599, AF126247, AF175903, AF097996, AL133558,
AL050024, AF113699, Y09972, AL023657, AF125948, AF162270, Y07905,
U42766, X96540, AL110218, X72889, Y09885, E03348, AL049300,
AL110296, AL117583, AJ238278, E08631, AF090896, A23630, AL110222,
A08911, AI5345, AC002467, AL137283, Y11587, AF118070, AL049382,
AL049314, AF106827, I17767, AF142672, Z37987, E07108, AF061795,
AF151685, AL050108, AL133665, AL133010, AL137521, X89102, AL137479,
X53587, AR011880, I89944, AF090934, L13297, AL137478, AL110196,
AL133081, AF210052, A08915, AF158248, L04504, X98834, AL049465,
AL133560, L04849, AF003737, AF113690, AF031903, AF017437, AF113689,
AL080074, I26207, E04233, AR038969, AL122050, and AF051325. HE9BK23
16 675382 AW299658, AW058550, AI796131, AW299514, AI767984,
AI634858, AW235128, AI498692, AI373251, AI796532, R86161, AW295829,
T73510, T73442, N71226, C15737, and AF152562. HCYBI36 17 666358
AI801638, AW089881, AA484795, AI700113, AI452474, AI220875, H03348,
T79392, AA305424, H04030, AI392810, AA375432, AW366425, H95362,
AI950113, AA375883, AF101051, AF115546, and AF072127. HSSDX51 18
566879 AI792073, AI791928, AW206230, AA317765, R60584, H22857,
AI694498, F03192, R05669, AA365484, and U78304. HSDAJ46 19 692358
AI800075, AI686505, AW023374, AA418208, H97489, AA620395, AA418073,
AW027850, AA401879, N67776, AI168759, N36146, AA700811, N28007,
AA012999, H99095, AI015805, H82563, H60753, R87427, H59689,
AA688368, W03403, H83666, R85022, AI582759, AA205528, H83667,
N35665, AA322820, AW072108, H60754, R07653, AA339201, H59688,
R07706, N26551, N69337, H84836, N88280, AA640177, AA221012,
AA094140, and AB025904. HRACG45 20 671767 AL043880, AW005102,
AA137033, AA523117, AA810411, AI671452, AI339682, AA437080, W90768,
AI091057, AI288535, AA528033, AA137115, AW130160, AI338926,
AI683304, AI199890, AI625514, AW083986, AW194157, AI859189,
AI199896, AI990810, AI269181, AA114896, AA114897, AA922395,
AI349372, AI168791, AI812097, AI274942, AA128536, AI266219, W90701,
AI636417, AI743815, AI862000, AW204921, R51136, H48319, N78924,
AI023431, AA128362, AI305176, AI873659, AI091111, AI056132,
AI087400, H48227, R52200, AW339362, AI886641, H79689, R62399,
R62400, AI680503, F11141, AA423981, AA938005, AA330985, W05294,
M78260, Z46207, AI350674, AI915189, R12869, AA523876, R38443,
F08308, R51028, F05074, AA339020, H79690, F08811, AI982566,
AA343945, F04534, R43758, AA368664, AW089229, D78779, AA465535,
AA368064, AI751158, AW062626, and T05279. HAPPW30 21 684272
AW341517, AA868588, AA479992, AA305964, AA758865, AI276502,
AA846842, AI183515, N41325, AW273135, AA775255, H57026, AA954695,
AI337591, AI685296, N95033, AA969117, AI147710, AA962530, AA150989,
AA758255, AI675402, AI167695, AI151098, AI798973, AA383301,
AW172620, AI359078, AI688288, AI911606, H83172, AI078598, AI188832,
H58146, AA446238, AA310796, AA724109, AA864698, AI240610, AA953573,
AA421572, H41807, W15373, H48433, AA977855, AA757910, H87382,
H46522, AI216014, AA098821, AA877407, W38885, AI739312, R11443,
H46521, R19191, H82944, W72627, C04986, AI479980, H56935, AI216655,
AA339733, R99133, AA975974, AA922234, AA375160, AW183259, AA421590,
AI459843, T61945, AI216656, AI191499, AI902298, H70309, AI902295,
T71506, AA150942, AA383302, N57057, AA568552, T62175, R94393,
AW021717, AI811212, AI924051, AW411235, AW411351, AW411265,
AW410902, AI923989, AW166742, AI284509, AA742505, AA100772,
AI804524, AW162189, AI654329, AI244704, AI049850, AI628333,
AI343379, AI567204, AI457113, AA585298, T29005, AI889191, AI289436,
R42275, AW411298, N63128, AW409775, AI954425, AR037084, AR054173,
AL137258, AR068753, S71381, M19658, AR068751, AL133015, Y11254,
AF065135, A76337, A76335, I92592, A91160, S68736, A93016, Y10936,
AR015970, AF093542, AL137254, AL133560, Y16645, AF118094, AJ132433,
E03671, AF139986, U88966, AL096751, AL031903, AL133024, L04504,
E08517, AF130470, AL050024, AF081366, S69385, AL050172, AF132341,
AC006203, and AC006112. HE2ES51 22 684278 AI792241, AI793025,
AW242855, AI767568, AA999850, AI911520, AI765078, AI373739,
AI793193, AI985237, AI433883, AI478325, AI671437, AI613056,
AI253234, AI524824, AI650909, AW299600, AI431850, AA131483,
AI470468, AI473091, AA345162, AA065156, AA076448, AA282824,
AL134259, AI862043, AI801088, AI583966, AI473471, AL120446,
AI520946, W45039, AL035890, AW020619, AI239701, AI741637, AI468959,
AA159625, AW410316, AC003669, U83112, AR030544, AF113677, I06996,
AL137550, S71381, AR064250, X15132, AF091084, X67813, AL080234,
AF113691, AF010191, E15324, AR054173, A23327, A49723, A49722,
AB029755, E16086, E13052, U53505, X66417, I29004, AF113690, and
A03736. HAGGJ80 23 1158546 HTXDW56 24 695765 AI765620, AA725071,
AW271710, AI916562, AI634990, AI654165, AI991405, AI983985,
AW299864, AI670830, AI570128, AW168930, AW009948, AA704525,
AI749744, AW000916, AA861614, AI955276, AI492455, AI676055,
AI276897, AI681128, AI796805, AW275120, AA430567, AI659635,
AW419101, AA890343, AW167370, AI635116, AI361022, AI700668,
AI968287, AW276391, AI935478, AI089414, AI955265, AI912091,
AI718821, AI935972, AI216100, AA699534, AA030011, AA587495,
AI025329, AI300305, AI342565, AI308169, N38817, AI298732, AA704532,
AI628899, AI018477, AA115429, AI334626, AA774557, AA045856,
AI160398, AA628480, AA555219, AA189132, AA780575, AI983713,
AI686341, AI350088, AI917769, AI263986, AI131166, AI207172,
AI347097, AI094833, W31093, AA216738, AA922079, AI095199, AA911845,
AI356898, R24001, AI827291, AI251444, AI828631, AA190469, AA774568,
N66175, W95063, T51687, R74559, H83131, AW341133, H83132, AI356017,
AA028993, H44274, AW086147, N98698, R51880, AI431971, AI203034,
AA766045, AI823509, AW071576, AW295813, R62584, AA765554, AI752881,
AA017568, N49971, H48233, AI277213, R79503, H78111, R62585, R22076,
AI206462, N54046, AI220633, AA580842, H05682, H67771, AI949890,
AI675025, C21303, AW384997, AI583730, AA317754, AA191411, AI540820,
H41654, H78112, AA359438, R74460, AW022361, AI434155, AA404726,
D25572, R43514, N79216, AI468840, H53574, R22023, R20396, H53895,
AA095942, R24167, AA362292, AA853334, N66781, N67446, H67770,
H48324, N62548, N45446, N77843, AA045973, AI910254, W91944,
AA115428, AF151803, and AL049839. HEEAG23 25 684254 AI279852,
H57654, AI472339, H85172, AA383569, H96534, AA903404, AA719530,
AI084916, AW135894, AA993772, AA890589,
W61170, AA807443, AA814409, AI828884, R76166, AA470533, AI829062,
AA737653, H96878, R62923, AA714658, AA705115, AI964064, AA569749,
AI343340, AA769402, AW080830, AA494452, AI871834, AW193760,
AL045053, D58349, AW275510, AI445674, AW168618, AA635739, AI039584,
AI656744, AA158461, AA551552, AA490183, AA169263, AC004938,
AR052481, AL031777, AL035079, AC004882, AL031680, AL109938,
AC006312, AP000223, AL121578, AC005033, AC005082, U63313, AC006511,
AC004477, AC006315, Z84572, AL035462, Z83823, AC002350, AC010206,
D83253, AC004151, AC005291, AC005225, AC004652, AL021940, AP000962,
AL031721, AL031666, AC004167, AL009183, U78027, AL021393, AC004223,
AC002400, AF141309, AC006101, AL136297, AL035422, AL022100,
AL049643, AC005694, AC003006, AL035659, AL022165, AC007066,
AL133448, AL080243, AL137100, AC007014, AC004752, AL031427, Y18000,
AC004832, AC003065, AC005800, AC006515, AC006011, AL035587,
AC006017, AC004913, AC005387, AP000045, AC002039, AL117258,
AC004076, AL031228, AD000092, AC006049, AL121934, AL022316,
AC006205, Z82244, AC005393, AC006057, AC007324, AP000228, AC007919,
AC007051, AC011718, AC006127, AC007227, AC005696, AC004754,
AC004534, Z86090, AF107885, AP000547, AC005488, AP000689, AL035409,
AC005037, AL049793, Z97196, AP000140, AC009743, AP000359, AP000088,
Z69705, Z98051, Z84816, AF178030, AC002492, AC006211, Z92540,
AC005725, AC000387, AC006125, AL033392, Z95116, AC008079, AC000115,
AL008725, AC006581, AL133289, AC005776, AL139054, AF141308,
AC002347, Z97632, Z93241, Z98752, AC005529, AL022311, AL096707,
AL078593, AC010722, AC005323, AL031650, AC006544, AC002476,
AC004883, AC004814, AF037338, Z98950, AC006543, AC007358, AL035420,
AL109839, AL022238, AC005531, AF196969, AC002299, AL034420,
AC004525, AP000104, AF200465, AC004833, AC005913, AL024507, Z93016,
AL031577, AC007262, AL049766, AC004130, AC005486, AC002312,
AL050308, AL009181, AC006285, AL078474, AL035252, AC006253,
AC005632, AF190465, AC004854, AF035396, AL049776, U95740, AF109907,
AC005702, AL022163, AC009263, AC007688, AC005829, AC005779,
AC005088, AC005060, AC004808, AC004686, AC004057, AC016025,
AC005527, AC005228, AC006974, AC007226, AC005512, Z81369, AC005378,
AC004690, AC003102, AC005618, Z73359, AL079295, AL022395, U91327,
AC018769, AC004673, AF111169, Z95400, AC002300, AC005859, AL035423,
AC004675, AF053356, AC003684, AF205588, AL049830, AC004019,
AC004463, AL035455, AC008033, AC005736, AC004890, AC002990,
AP000552, AL031602, AF106656, AC004933, AD000812, AF064861,
AL049757, AC005933, AA252707, and AA252834. HDPKI93 26 683964
AW026665, AI038157, AW160610, AA291566, AI313184, AA513729,
AI028306, R72376, AI167786, AI744406, AI185677, AI660416, AA856738,
AA397659, AA479875, AI918286, AA479739, W68014, AA628725, AA216390,
R62410, AA399031, AW162156, AI032922, W67956, AA229418, AI628273,
AA759314, AI564387, AI874106, AA100111, AA852988, AA852989,
AA677565, AA627551, AW269334, AI885854, AA100172, AI886985,
AI369442, H40744, AI927077, AA188450, N94366, AA291402, AA187325,
AI342982, AW136745, AW136249, AI499025, T24752, AI241302, AI990643,
AI971492, AI074558, AW166318, AA428682, T55849, AA837459, T18597,
AI525852, AI557312, AI557262, Z33559, Z32887, D59751, D50992,
AI525661, AI535660, AI541205, AI525556, N71206, AI536138, AI535639,
AI525500, AI557533, AA058620, AI557474, AI526078, AI536150,
AI557084, AI525302, AI557809, AI541321, AI541075, AI541365,
AI557602, AI546829, AI557082, AI557697, AI541353, AI541034, R29657,
AI541450, AI540974, AI525856, AI541069, AI557039, AI547177,
AI535994, AI536070, AI557543, AF132956, AF086160, U94592, AR050070,
A62298, A82595, A82593, Z30183, and A62300. HDLAC10 27 692299
AL049012, AW161772, AI963569, AI627938, AA430167, AW150904,
AA811288, AW148833, AA016001, AA534493, AI580793, AI473859,
AA552599, AI762820, AA630256, AI249503, AI289630, AI093700,
AI683179, AA902142, R50658, AA994326, Z43651, AI796343, Z39714,
T36201, R50558, D60811, AI796404, AW023060, AI560541, AA445981,
Z45738, F04619, AF205600, and AF205601. HDPOH06 28 683371 AI378660,
AA669141, AI985796, AA688220, AI042515, AI372881, AI014423,
AW025175, AI335099, AW263024, AI491990, AW128917, AI570270,
AI128127, R91019, W85883, N59550, AW305279, AA679558, AI635705,
AI559984, F00878, AW340645, R08677, W85967, AI262108, T98198,
AA670170, T53837, AA337112, AA583164, R88760, AA902605, N78291,
T98199, AI540509, AC003108, AC003684, Z95152, AC004694, AC002365,
AL121603, AL049872, AC005071, AC007425, and R08585. HCE4G61 29
846836 AF039237, AL041798, AI831480, AW340563, AW025258, AI127613,
AI818249, AA523520, AI750911, AA315462, AI216595, AA564125,
AA058690, N24213, AW005271, AI735048, AA677314, AI278958, AA416726,
R72506, AA071513, H39933, AI202794, AI871329, AA287126, AI269956,
AA045523, C05686, AA040646, AA296927, AW386990, D80945, D61149,
R37394, AI261950, Z44618, D60504, AI342287, AA297041, AA323642,
AI289604, R85079, AI867501, T35835, R37967, AI021950, R72507,
AA082290, AI350532, AI925788, AI369584, AA889809, AA297013,
AW105104, AW391339, Z40486, R08860, R13486, R72016, AI188886,
AI784381, AI186121, F01836, AA977138, AA534946, C15524, AA327396,
AA057306, AA058536, Z42306, AI801558, AA416805, AI631998, AI750912,
D31575, R72047, R72058, and AC004596. HCWUI13 30 695679 HDPSP01 31
689129 AI871101, AI560217, AA047000, AW190726, AA419038, AI479404,
AA035467, AI361637, AI198435, AA725194, AI093316, AA442664,
AI078128, AI274339, AA915909, AI677732, AI283200, AI769275,
AI857306, AI275083, AA423792, AA046943, AI291474, AI291805,
AI983969, AA427407, AA661657, AI141350, AA031475, N92812, W24931,
AA035466, AA031617, AA961077, AA250784, AW070742, AA411122,
AA378564, AW051192, AW452102, AW293787, H91665, AA514348, AW149476,
H91759, T86488, AW392670, Z99396, AL119363, U46347, AL119457,
AW363220, AW384394, AL119497, AL119319, AL119444, AW372827,
AL119443, U46350, U46351, U46349, AL119324, AL119484, AL119391,
AL119355, AL119483, AL119439, U46346, U46341, AL134525, AL134533,
AL119341, AL119335, AL119522, AL037205, AL119418, AL134528,
AL119399, AL134531, AL134527, AL134538, U46345, AL119396, AL119496,
AL042614, AL043003, AI142134, AL042542, AL042544, AL042450,
AL043019, AL042984, AL042965, AL042975, AL043029, AL119304,
AL042551, AL119464, AB026436, AR054110, AR060234, A81671, AR066494,
and AR069079. HHPEN62 32 695134 AI939620, AI480056, AW300615,
AW300620, AI589129, AI911546, AI361251, AI498527, H41544, AA326679,
AA348503, AI422476, AA912288, and AI423129. HUKBT29 33 694590
AI889172, AI080136, AA211445, AA211523, F24617, AA211502, F27978,
AI862904, F28119, F30666, F29048, AI972919, AA211549, AI128717,
Z24989, AW302460, F28086, F26294, Z28706, AA413432, and R45814.
HMAJR50 34 654004 AW408305, AW403731, AW117933, AA947938, AW268857,
AI983988, AI566347, AW129984, AW051493, AI741765, AI803337,
AW081302, AI811384, AI828939, AL037800, AI146996, AA720675,
AI819563, N21132, AI419827, AI460230, AI080555, AW195872, AI348121,
AA833715, AW193550, AI285275, N31147, H97793, AW189406, AI240056,
AI806449, AA928209, AW277257, AI225247, N50918, AA907019, AI597972,
AW302355, AI050898, H82455, AA364171, AI610240, AI376029, AI989465,
AA746601, R68913, AA157064, N21022, N47537, AW087619, Z44334,
AA156969, H10287, AW197890, AA047176, AI074218, H59319, H13601,
AI613266, T35974, R19227, AI338148, AI281563, AA992483, T35248,
N42759, T34340, R59345, R62736, T30641, D56630, H10230, W02661,
N31923, R38953, R19536, AA995898, T80302, AA248245, N67312, R68809,
AI355476, R21115, AA694574, AA904354, Z40284, R59344, R45755,
D56961, T34357, T35247, AA313414, T30099, AA301358, AI277161,
T31945, T32601, AI865075, R44491, R43889, AA906085, AI560586,
T36242, AA585150, N83938, AW197781, AI824759, W25731, Z28514,
H59272, R78516, R29225, AI798671, N47536, AW378845, T24535,
AA057047, AI583065, AI619777, AI312428, AI923989, AL045266,
AI288305, AI866573, AI590043, AI538885, AI335426, AI348777,
AI500061, AI432969, AI287326, AI866465, AI468872, AI682798,
AI433157, AA572758, AI702073, AL119836, AI887308, AI539771,
AI500523, AI582932, AI249877, AL040241, AI633125, AI698391,
AI815232, AI915291, AI874261, AI207656, AA420722, AI819326,
AI889189, AL079963, AI608936, AI799273, AI340603, AA420758,
AW163834, AL038605, AW129230, AI675052, AI637584, AI798404,
AL041150, AI625464, AI872910, AW161579, AI539153, AI632408,
AW118496, AI570989, AI499986, AW102924, AI570861, AL110306,
AW410969, AI929108, AW190042, AW087462, AI866770, AL037521,
AI633419, AI345347, AA176980, AI446373, AI340533, AL045500,
AW088903, AA635382, AW151136, AW148536, AI538085, AI801325,
AW022682, AI345608, AW051056, AL036403, AI284517, AW409914,
AI697045, AI783504, AI538342, AI572021, AI863082, AW151485,
AI288285, AL121014, AL036802, AI635067, AW411235, AI580435,
AL048656, AI473799, AL036396, AL039086, AL038565, AI890214,
AL119791, AW166903, AW059713, AI611738, AI963194, AI270099,
AI251221, AI801793, AI340519, N33175, AI471909, AI352497, AI631273,
AI571439, AI619748, AI500706, AI537677, AI934035, AI521560,
AI500662, AI345745, AI623396, AI927233, AL036541, AA579232,
AI888661, AI539687, AI475430, AI445992, AL135022, AF131856,
AF195141, AC005915, E07108, Y11587, I48978, AF090901, AF090903,
I89947, AF067728, A08916, A08913, I00734, A08910, A08909, AL117460,
AF158248, AF106862, E03348, AF177401, AF113677, Z82022, I48979,
AL133067, AL049283, E00617, E00717, E00778, E02349, AF017152,
A65341, Y11254, S68736, X65873, A93016, I89931, AL049382, AF078844,
I49625, AL133080, AL137271, AL133075, AL137550, AL137557, I61429,
AL110228, AF090934, AL080159, Y14314, AL122093, U35846, U80742,
AF113019, AL133014, AL133072, S78214, AL133560, Y16645, AL110196,
A77033, A77035, AF125949, X93495, AF079765, AL122050, AF087943,
AL117457, AL050116, I17767, AL137533, AL117585, AF090896, AF106657,
E05822, I42402, AJ238278, AF039138, AF039137, AL137283, AL080074,
AF118090, AF111851, AL137459, AL050149, AL096744, AL122121,
AL080060, AF091084, AL050024, AF183393, AL117435, AF104032,
AF061943, AF113013, AL050393, S61953, AL122110, AL136884, AJ000937,
AL049430, AF107847, AL137281, AF125948, AL080124, AB019565, X82434,
AL049452, AF118094, AL117463, AL117583, AF153205, A93350, AL137656,
A03736, AL049938, I26207, I33392, AF113699, AF090900, AL117394,
X98834, AF113691, A65340, AR038969, AL110221, A08912, AL050108,
AL110225, AL133113, AL050138, AL133606, E15569, A58524, A58523,
E01573, E02319, AF119337, AF008439, AF118070, I03321, AF146568,
AL050092, U42766, AL122123, AL049466, AL050146, AR020905, AL133104,
AF113690, AF113689, U67958, AF113676, U73682, AJ012755, U72620,
X72889, X53587, AL049314, AR011880, A08911, AL050277, AR000496,
U39656, AF141289, AL133077, AL137521, X63574, AF026816, A18777,
I09360, AF106827, AL122098, AL137527, AL133565, AR059958, E08263,
E08264, U00763, AF114168, AF113694, AF017437, AF097996, AL137560,
AL133640, X84990, AF026124, AL133016, I09499, AL137548, S76508,
X96540, AR038854, AL137463, Y10655, AF145233, AL137538, S83440,
AL110280, AF111112, AL049464, AF069506, AJ242859, AF111849,
AL137554, AL133557, AF126488, Y09972, E08631, L31396, AL117440,
AF185576, A12297, AL133031, L31397, AL110159, AF090943, AF118064,
AL133093, L40363, X70685, Z72491, AL137648, AL080127, AL080137,
AR068753, E07361, AF000145, AL137556, AL122049, X79812, U58996,
U87620, U89295, A08907, AF207750, A08908, AL050172, AF079763,
AB007812, I92592, Z37987, I66342, U72621, Y07905, AR068751, A76335,
AF120268, S53987, D16301, AL137480, and AA075938. HBIMB51 35 672711
AW293249. HE8DX88 36 663511 AI352035, and AL049871. HNGHT03 37
692430 HWABU17 38 678671 N58127, AI970999, AA543049, AA805508,
AA481100, AW086144, AI224173, N52797, N49240, AW439223, AA480173,
W87476, AA481045, AI702077, AA968423, AI208249, AA676568, AI339421,
AA551673, H61729, H90630, AA354107, H18441, H90534, AA203228,
Z41571, AA948533, AI990383, H24029, H18549, H22748, H61939,
AW075792, W87571, AI270746, AA002111, AA002112, AW072594, N57619,
AA977512, AL043010, AF092094, AF155157, and AF004231. HDTAT90 39
692291 AL041807, AA315553, AA578538, R60726, AA578520, W25198,
N34727, AW160746, R90863, R84524, AW246146, AA081697, T52130,
AW177731, AI525011, AW177733, AW068182, C04045, R51326, AA251576,
AA081290, and AL050275. HHFGR93 40 691402 AW190823, W52782,
AI921717, AA707399, AA780017, AI809901, AI656071, AI870870,
AI633244, AA046658, AA913618, AA428298, AI014541, AW300019,
AW173046, H12307, AA428713, H12782, AI141481, AI092488, W58612,
AW172540, AI184646, W58613, AI359381, AW361707, AI126255, R77354,
AI970137, AI949837, AW081182, AI923177, AI187105, AI624748, R69232,
AA514466, AI521359, R69114, AI347221, R76149, AA664044, R73827,
R79810, H12841, R78260, H12629, R76098, R63063, R32862, R78261,
T47327, AI189377, R73853, R62315, R68433, AI828342, H12360,
AA618505, H12680, T50332, R79923, R79910, AI216465, AA733001,
R35438, AA683601, AW009057, R81664, T98690, H00855, R33685, H02334,
AI189455, R73852, AW365832, AI873711, R67936, H02440, AI569353,
H02804, R68432, R66838, H38189, R76065, R64387, R33581, R35749,
AW235425, T98640, R27675, AA991630, AI189443, R75889, R81467,
R31360, AA367816, R27576, R63218, AA359117, R31889, R34252,
AI762218, AW002259, W52486, H01235, AI199859, R62314, AA046788,
AA249358, R64386, AW407088, N55686, R67441, AI002022, D45691,
AA446485, AA430177, AI432644, AI492519, AI623302, AI432655,
AI432661, AI432653, AI431354, AI431312, AI431347, AI431230,
AI431328, AI432654, AI431310, AW081103, AI432677, AI431337,
AI431351, AI432675, AI431353, AW128900, AI432674, AI432651,
AI432647, AI431330, AI431243, AI432650, AI431248, AI431255,
AI431307, AI431316, AI432649, AI432672, AI432665, AI431254,
AI431357, AI432662, AI431241, AI791349, AI432676, AI431345,
AI431346, AI432673, AI432658, AI432666, AI431340, AI431238,
AW128846, AI432664, AI431308, AI432657, AI432645, AI431321,
AI431247, AW128897, AI431231, AI432643, AI431257, AI431323,
AI431350, AI431318, AW128884, AI431235, AI431315, AI492520,
AI492510, AI431246, AI431751, AI492509, AW129223, AI431314,
AL042729, AL042931, Y17793, AF064854, and AF019249. HOVCB25 41
691357 AA318972, AB014534, AF116574, AF116573, AC000029, and
AC003678. HSYAV66 42 686437 AF126372. HFPCT29 43 668239 HAWAT25 44
677480 AI992139, AW173625, AI802924, AI263005, AI286190, AA694076,
AW168835, AA699535, AA625080, AI912832, AA854042, AA320461,
AA704943, AI762162, AA740929, AI700148, AI241269, AA330308,
AI640185, AC006359, AL021920, and AC004455. HNHFR04 45 646709
HOSFT61 46 862050 AI768188, AI935495, AI819745, AI422744,
AI423415,
AI140447, AI969550, AI332649, AI942442, AA127755, AI075724,
AI199841, AI422431, AI129261, AI140453, AI050878, AI419482,
AI766108, AI080121, AI675245, AI280479, AI809228, AI372882,
AI335707, AI423608, AA678475, AA807943, AI221599, N79574, AA449772,
AI375330, AI094106, AA987838, R44044, AW274423, AI914896, R41865,
R55755, AA831552, N51677, H23272, AI014757, AW444813, R26737,
AW089977, Z38205, AI540756, AA029258, T24879, R26969, AP000118,
AP000165, AP000315, and AC016831. HBJIO81 47 625977 AW301022,
AA748554, and AA761415. HADCL55 48 686761 AI891111, AW273154,
AI421861, AI937106, AA844641, AI435050, AW080343, AI903718,
AA010290, AW363110, AI963329, AA460436, AA460435, C18387, AA010291,
W26232, N20813, H09922, AI862319, AW363122, AI131459, AI422844,
AA926645, AI671988, C01597, H22803, AI147703, AA017133, Z42698,
H23009, H58948, AW295951, F05928, H09826, R92329, AA156440, M78768,
AA824261, AA995248, Z38858, AA812976, AA092371, AL136827, and
AB023199. HAIBO81 49 695698 AI061313, AL046519, AI733856, AA805848,
AI609972, AA469327, AI753113, AI291439, AI537995, AI130709,
AI687343, AW021154, AI814682, AW302659, AW302705, AI536858,
AA829036, AL041375, AA829044, AW148775, T71936, AI815210, AL020997,
AC002425, AC006011, AC004975, Z82214, AP000510, AC005919, AC005225,
AC008033, AC004020, AL021707, AC005209, AC003110, AL133163,
AC000353, AC005288, AC005529, AC006530, Z98751, AL035407, AC007226,
AC005484, AL049759, AC007666, AL024474, AC007055, Z97876, AP000552,
AC002470, AC007036, AC008372, AL031295, AC005261, AC006312,
AC005514, AC009247, AP000514, AC007686, AC003080, AC006139,
AC004655, AC006130, AC005952, AC006241, AC002369, AL109627,
AF038458, U91323, AC002544, AC005231, AL049229, Z83840, AC005015,
AC004408, AL078463, M89651, M30688, AL035071, AL031767, AC005088,
AL118516, AC006023, AC004491, AC006511, AP000557, AC005696,
AL049766, AC005363, AL035413, AC002996, AF050154, AC007536,
AC005726, Z96074, AC005280, AL049569, AC004156, AL031283, Z83826,
AC005060, X54486, AC005324, AD000092, AC006942, AC005251, AC007371,
AL031293, AL008725, AC005049, AL031428, Z84466, Z93023, AC003669,
AC004024, AC004707, AL021453, AL031577, AF030453, AP000692,
AL021546, AP000350, AP000260, AB014078, AC005067, AL031670,
AP000116, AC006050, AC004099, AC007207, AL133353, AC007685,
AL031311, AC004878, AB023049, AC005069, Z82976, AC007637, AP000049,
AC005534, AC003101, AC005519, U78027, AF001548, AC002504, AC004771,
AC004477, AL035587, AC005520, AC009516, AC006487, AF165926,
AL031584, AC002039, AC003982, AC005759, AC005736, AC007690,
AL049699, AP000555, AC004000, L78810, AC005837, AC005409, AP000563,
AB001523, AC007938, AC005488, AC005578, AP000311, AP000036,
AF196779, AL050318, AC005355, AC006480, AC005954, AC004228,
AC005089, AP000215, AC005694, AC007227, AP000117, AL022311,
AP000558, AC002314, AC005940, AL024498, AL035249, AC005207,
AL109984, AL022323, AP000210, AC003043, AL109801, AL109758,
AC007193, Z95331, AC006101, AC005912, AJ011930, AC005046, AC005971,
AF129077, AC002404, AL049539, AF165147, AL022302, AC004770,
AC004796, AC007731, U63721, AL049780, AC006441, AL031846, AC002400,
AC005500, AL049538, AC009731, AL049839, AL023575, AC004216,
AP000344, AL031659, AL022316, AL031985, AC002429, AB014079,
AL121658, AC005200, U52112, AL022336, AC004150, AL035422, AL049694,
U91326, AC006597, AL121653, AP000337, AF053356, AL132718, AL031597,
and AC002549. HBBBC37 50 695702 AI953024, AI570581, AI052251,
AW072845, AI283137, AW418961, AI276972, AI765673, AA443232,
AI218363, H98529, AI819979, AA284497, AI187773, W31829, AA971941,
H19433, AI674860, AI359631, AA443194, AA857996, AA975354, AW022944,
AI032489, R59463, C02118, H23263, AA776510, R60979, Z38831,
AA102625, N28938, D51172, T34946, R59403, AA953086, N81166,
AI823922, T90503, R45520, AA872986, R45152, W04580, AI277164,
R20541, H97160, AI560504, N22268, Z19348, AA287201, M62100, R44572,
AA094604, R11284, Z42669, and AB032961. HBJMX85 51 692971 AI673085,
AA716494, AW151554, AW445050, AA807345, AA926684, AI989351,
AW071081, AI687590, AI523580, AW451331, AW075954, AI131215,
AI333008, AA974138, AW291257, AA769392, T84096, H91806, AA765936,
AW451758, C04782, AW402336, AI473525, AW028312, AA814453, AI568709,
T78707, AI801411, AW085753, AW085750, AA471314, AA831522, and
AL096808. HCEES66 52 694592 AI650353, AW129672, AI564414, AI805921,
N51082, AI239923, H52585, H22566, AA007234, AI241833, R42536,
H52176, H24419, and N54208. HCEMP62 53 684780 AI688113, AI554392,
AA911109, AW173438, AW382483, AA486370, AA778384, AI382028,
AA776265, AA563686, AI493765, AI523553, AA484857, AI362311,
AA811238, AA906681, AA838288, AA460659, AI276177, AW404956,
AA479791, AA259052, AI097482, AI082243, AA488079, AA088205,
AI609703, AI093069, AW438882, AW366250, AA477188, AI350871,
AI953839, AI033274, AA285058, AA648139, AI087234, AA226399,
AA594766, H53631, H04050, AI298774, H03363, T86181, AI687929,
AI270613, H48473, AA496296, H53672, H70534, AI433271, R99170,
AW188898, AA359247, AA374856, R23345, H28080, R70772, R33920,
AI500391, AA852639, R81465, AA297085, T83919, AA428830, R33033,
AA297469, AW088943, AA621048, AI400220, AA853069, R81663, AI963710,
R23264, AA290677, AI687795, AW027045, AI289188, AA808274, AW074305,
AA290975, AA461006, AA297403, R26089, AA226370, AA297468, R23497,
AA359017, AA258974, AW392388, AI683668, T86180, AI653763, AA291083,
H26077, T83747, AW058461, AW016612, AW023590, AL079963, AI241923,
AI611738, AW163834, AW051088, AL039086, AI282679, AW054964,
AI868931, AI917252, AW118518, AL040241, AI570807, AI582932,
AI802542, AI174394, AL037030, AW198075, AI446373, AI912510,
AI499890, AI890907, AW026882, AI474146, AW074869, AI280732,
AI619502, AI677796, AI680162, AI352497, AI863382, AI886753,
AI824576, AI923370, AW083778, AI624293, AI280607, AI433157,
AI702073, AL037649, AI310575, AL037582, AL037602, AI457369,
AI762739, AI932794, AI340533, AI520809, AI633125, AI698391,
AI270706, AI915291, AW152182, AW166903, AL036638, AL047675,
AW131294, AI889189, AI473536, AI270183, AW088899, W74529, AI627988,
AI537837, AI288305, AL046990, AI572096, AI783997, AI288050,
AW169234, AI675052, AW088903, AW079572, AW168373, AI699865,
AI917963, AI926878, AI284131, AL036150, AW071417, AI620284,
AI340603, AL036673, AI500061, AI493248, AI886181, AI632408,
AI866770, AI620089, AA449768, AI886123, AI554821, AI269862,
AI933589, AI635067, AI434468, AW149925, AI537261, AI863191,
AL119863, AI445992, AA806720, AI623941, AI538085, AI564749,
AW150794, R32821, AI288285, AW079409, AW078818, AW029611, AW151136,
AL046944, AW130930, AI866469, AW161156, AW051258, AI590686,
AI468872, AI886415, AI349645, AI473451, AI926367, AI537677,
AA916133, AL042440, AI540821, AI569583, AW072719, AA641818,
AI499285, AI690748, AI587606, AI340519, AI872423, AL048482,
AI670009, AI521560, AI348847, AW268122, AW268302, AI559586,
AI343091, AI610402, AI345677, AI310582, AI340627, N33175, AW162189,
AI859991, AL110280, I89947, I48978, AL050092, AF113689, AF111849,
AF026816, AF113691, Y14314, AR038854, A45787, AL137271, Y16645,
A08916, U80742, AL117435, A08910, A08909, A08913, X84990, A03736,
I33392, A93350, I48979, AF183393, I89931, AL110221, AF177401,
I49625, AL080159, AF111851, AL133075, AL050149, AF113013, AL050277,
AF087943, A08912, AJ000937, A77033, A77035, AR000496, U39656,
AL137533, AF090901, AL137476, AR020905, X82434, X87582, AF153205,
S78214, AF090900, U78525, AL050138, U35846, AL137480, A58524,
A58523, AF162270, AF100931, AF113677, AL050024, AL137648, AL050146,
E02221, AL137463, AR011880, AF113019, L19437, AL137478, Z82022,
E07108, AF078844, AR038969, AF090934, AF017437, Y11587, AL137538,
AL117460, AL137488, AL137292, X83508, X72889, AF091084, AL137550,
AL080074, AF061981, AL080060, E03348, A18777, E06743, AL137560,
AL049382, M30514, AL137294, AL133560, AF008439, AF111112, AL122123,
S61953, A65341, E05822, E04233, AL133640, AF090903, I09499,
AL110225, S68736, Y07905, AL050393, AL122121, AJ012755, AF081197,
AF081195, X53587, AF113694, U67958, AF210052, L30117, E15569,
AF061795, AF151685, AL133016, X65873, AL133565, AF104032, AL133606,
AF119337, AL110196, I42402, AL133665, AL117457, AF158248, U49434,
AF139986, AF079765, AF067790, I03321, Z37987, AL023657, AF125949,
AF061573, AF097996, A08908, E02349, AL050116, L31396, I66342,
AL133113, L31397, U91329, X96540, AF118064, AR059958, AF026124,
AF125948, AL117440, AF185576, E12747, AF106862, X81464, AL122110,
AL137521, AF113690, AL133067, AL137556, AL049466, AF067728,
AF132676, AF061836, AL117585, AL122100, U96683, AF146568, AL122118,
X93495, AL117432, U72620, AL080124, AL049300, AF090943, AL137557,
AL049283, AL133080, AL049430, AF113699, U00763, U58996, AL117583,
AF079763, AL133557, Y09972, E08631, AL050108, AF090896, AL080148,
I00734, A12297, AL133072, AB019565, Y11254, AL122050, AL080154,
M86826, AB007812, AF113676, E00617, E00717, E00778, X63574, A07647,
AF126247, AL137526, AF118094, AL049452, AL133081, AL137459,
AL096744, AJ003118, AL080137, AF057300, AF057299, AL080086, I26207,
AF003737, AL122049, AL049938, AL049314, AJ238278, U68233, AL117649,
I92592, AL122093, AL049464, U42766, AF032666, U88966, A21103,
AL133104, I09360, AL133093, AF118070, X62580, A90832, Z72491,
AL122111, AL117416, AL133098, AL122098, X92070, and AF017152.
HE2FB90 54 691077 AI857437, AI857436, AI278048, AA507045, AW273440,
AW297803, AA493364, R47896, AI292326, AI364487, N66632, N58844,
AI361304, AA347485, AA357233, N80769, AI374919, H08044, R47895,
AW189621, AW439143, AA887910, AI394536, AI591191, AI279880,
AI280275, W65494, AI797532, AA357422, H07938, W65483, and AI123607.
HTHDJ94 55 693652 AI290720, AI741602, D79185, AW024422, AA401528,
AA417131, AI333681, W47348, AA280813, AA905310, AA569922, AA573334,
AA902128, AW027880, AA570689, AI312759, AA976250, AI092605,
AA558902, AA151226, AI041784, AW262597, AA280806, N36166, W32108,
AA151227, AA406299, AI090180, AA781961, AA115004, AI623995,
AW239455, AI027447, AA065116, AI377228, N59607, AA451762, AI804317,
AA724950, AA449952, AA450034, AA115005, AI186329, H10448, AA482977,
AI242335, AA453022, AI032607, AI804465, AA640751, C16610, AI149260,
AA987598, AA781332, AI804069, AA973798, AA452663, AA127134,
AA872873, H82385, T86790, T82258, H10449, F30722, T78950, W32213,
AI424359, AA338139, AA296988, T78898, AI285049, AI278719, AA451764,
W47452, AA541483, F06459, Z28571, Z39388, AA297494, T86695,
AI318411, F01234, AA808781, AA297421, AI991656, AA661544, D31389,
AA280856, AA280942, AA064799, Z24822, AA031579, AA298704, AI670708,
AW238447, AA494107, AA296942, AA031458, AA297411, AA297354,
AA099261, AA098866, T83540, AA297420, AI675090, AA194682, AA368017,
AA297201, D20890, AI908416, AA897425, AA530981, AA411374, H70649,
AA449811, F24096, AF125533, AF169481, and AF091084. HTOHJ89 56
695763 AA101269, AI792578, AI054419, AA847499, AW023111, AI612070,
AA477503, AI611533, AI379719, AI440117, AW162288, AC002310, Z97196,
AL022165, AC002470, AF129756, AC004139, AC006064, AF031078,
AC006509, AL022320, Y14768, AF030876, AC005088, AL022336, AF024533,
AL109801, AC007308, AL132712, AL022326, AL022316, AL031255,
AL031577, AL034549, AC004221, AL009181, AL022327, AC006241,
AP000045, AP000113, AP000501, AP000356, AC004622, AF111168,
AC006965, AC005280, AC004216, AC005702, AC005969, AC004812,
AL121754, AP000302, AL032821, AC004408, AC004382, I34294, AL121655,
AC009946, AD000092, U91326, AL031677, AC007227, AC002350, AC005694,
AB023048, AC009516, AL031767, AC005837, AC008372, AC005730,
AC003006, AC003042, AC006011, AL024498, AL035249, AP000114,
AP000046, AC007226, AC004476, AC007151, AC005829, AL031775,
AC007182, Z95116, AC005911, Z95115, AF196779, AC006130, Z83826,
AL050341, AC007766, AC005370, Z85986, AC002133, AC009542, AC002364,
U85195, AC005231, AC006511, AC007685, AC005379, AL050318, U95742,
AE000658, AC009247, AC004967, AP000555, AC005484, AP000252,
AC004890, AF106918, AC007686, AP000143, AC007240, AC007216,
AF165926, AL080243, AP000500, AC005726, AL049758, AC005914,
AC002365, AC009330, AL031650, AC005071, AL021546, AC004381,
AL008735, AC020663, AF196969, AC005067, AL049692, AL049748,
AC007546, U91322, AL109798, AC007371, AC006449, Z94044, AP000354,
AC004805, AC000134, AC007774, AC005212, AL035659, AC002544,
AC005261, AC007367, AP000115, Z98946, AC005545, Z98742, AL031602,
Z85987, Z86090, L44140, AC005933, AL049776, Z93017, AC005562,
AC006101, Z98051, AL022311, AC004195, AC006538, AL022476, Z84487,
AL035587, AC006958, AP000090, AC005527, and AC004780. HUSHB62 57
680495 AI096616, AI937128, AA478989, AW148649, AI635678, AA580461,
AI871452, D61293, AA701343, AA947641, AI077699, AA587392, N92014,
N22807, AW090032, AI913164, AI566329, AI304741, AI832816, AI920824,
AI688989, AA505810, AI474080, AI591133, AW024245, AI423395,
AA918351, AI285282, AA834943, AI870350, AI559195, AA838061,
AA775249, AI435008, AA703175, R97195, AI421979, AA723388, N63869,
AI339068, AA478599, T16357, AA868966, AI272181, AL039420, AA769928,
H28566, AA363734, AA916277, AW337191, AI262344, D81371, H19664,
AI239934, R56668, H03426, AI282295, H13067, AW276930, AI197924,
AW363835, R21517, AI863591, AA299531, AI001889, AI420251, R53825,
AA384624, AA962591, AL121415, C17798, AI276296, AA534355, AA013482,
AA507743, Z45873, AI193397, R76057, AA947445, D78950, H53668,
AI803908, R56831, AA327530, R21620, AA304103, AA327544, AI631817,
F09191, AI695253, H11336, T16635, H53629, T35087, AI610263,
AW275794, R12800, AI601236, R97196, AI287902, AW128986, H04135,
AA574194, AW118490, Z41505, AA865671, AW192504, AW151452, AI205173,
AI244106, AA477926, AW316864, AA322273, AA363735, R39499, R39500,
F11529, T35056, AL039419, AA960860, R75882, T19229, AW376282,
AI934081, AA557576, T06706, H28565, R19084, AW384792, AJ011001,
AL137591, AF106858, and AF166382. HSXAG02 58 667848 AW370368,
AW137077, AI601240, AI803696, AI168184, AA121075, AA527028,
AI334348, AA603723, AI432655, AW296548, AA420755, AI371852, N28275,
AI268286, H93203, AW300705, AW453008, AA420796, T09453, AI761383,
AA555003, AA122417, AI864017, AI698470, T09015, F11705, AA987938,
AW083439, AA826633, AA972661, AW149398, C00601, AW183138, AI828119,
D51113, R49527, AI251099, AA405926, AA936580, F09363, AW449144,
C16721, R24145, H66763, W30922, Z39944, AW136862, H08616, T30425,
AA405803, H46486, T30746, T51043, T50980, AA666060, AW131331,
AW020419, H50168, AI469290, AI918408, AA720850, AI360816, AI434731,
AA765198, AI334893, H95782, R37188, AI828795, AI699175, AL134598,
AI432110, AA883351, AW195253, R75918, AI358271, AI263331, AI887775,
AI619820, AA862606, AA806605, AA824513, AI783808, AI335235,
AL042365,
AI274811, AI683585, AI804505, AI500659, AI147877, AW151929,
AI446046, AI815232, AI801325, AI866691, AI500523, AI538850,
AI582932, AI923989, AI590043, AI284517, AI872423, AI440260,
AI500706, AI289791, AI926593, AI889189, AI500662, AW151974,
AI284509, AI538885, AI927233, AI866573, AI401697, AI866469,
AI888661, AI500714, AI285439, AI859991, AI355779, AI889147,
AI581033, AI491710, AI611728, AI584118, AI440238, AI860003,
AI539260, H03560, AW151979, AI571699, AI285419, AI494201, AI866581,
AW074057, AI567953, AI690930, AL047152, N23647, AA928539, AI251221,
AW104746, AI932970, AI174819, AI799179, AW087915, AI621341,
AW058275, AI921379, AI066497, AI261815, AL079910, AI538637,
AW000738, AI800473, AI499325, AI250646, AI868180, AA587120,
AL047422, AI537943, AI307569, R25628, AI521596, AA643038, AW130785,
AI800159, AI349186, AW021662, AB026894, AL080083, A65890, AF140224,
E01614, E13364, AL137479, AF161493, E12580, X73361, AJ001388,
AF061795, AF151685, AL133053, AL133015, X56530, AL137292, AR005195,
AF114168, AL133054, X87582, AL137716, AF161699, AL110218, AL137298,
AL117438, U42766, AR038854, AR050959, Y10080, A08912, I32738,
A08911, L24896, A18777, U75932, AF141315, AF097996, AL049423,
AL133084, U30290, AL133608, AL133049, S76508, AL110159, AF100781,
AF017437, S63521, AL109672, AF093119, A44314, AL117457, AF113694,
A08913, AL133557, I28326, A08910, AF054831, A08909, U78525,
AJ004832, I77092, L04859, AF002985, A08908, AF035321, AF090900,
AR034821, L13297, AF111845, AF043345, AL050277, A91160, AF177401,
S68736, AF112208, E08443, A93016, AF028823, A20553, Y09972, U62807,
A60092, A60094, AF031572, AC006288, I13140, S36676, X60786,
AF131826, AF039202, AR068753, S82852, AL137537, AF026816, AF090943,
U58996, M85165, AF199509, AL080140, AF004162, AF151109, I08319,
AL133560, Y10655, M19658, AF131814, A07588, AF098484, AR068751,
X83544, AL117583, AF076633, AF200464, X52128, AL080162, AF090886,
AL133016, I09499, AL137533, AF199027, AF158248, AF090903, X99971,
AL080126, AL050092, X72889, AL049276, I89947, AF161418, S71381,
AL136884, AF090923, Y11254, AF065135, A76337, A76335, I08608,
AF017790, X55446, I92592, S73498, A90832, X06146, X57961, AL049557,
I48978, AF054289, A26498, AF118094, AL050024, AR029490, A49139,
AJ006039, S83440, AF106657, Y07915, AF124435, U75604, AF107018,
AL022165, I29004, X66417, X00861, AF167995, X15132, AF091084,
X75295, AL117460, AL080234, AF125948, AL136842, AL110224, AL137521,
M79462, AL110280, AL137267, E06743, A23630, X97332, AL110171,
AF105427, AF113019, AL049347, AL133623, and AF056194. HHTLH52 59
665722 AI805189, AW136027, AI634613, AA292087, AI201246, AI693706,
AI675765, AW390785, AA868564, AI654869, AW390814, AI077669,
AW082330, AI695580, AA995665, AA758454, AW373785, AA757902,
AC004893, AL133396, AF165142, AL034374, AC004837, AC002416,
AP000045, AP000113, L44140, AC006501, AC003071, AC006255, AC005058,
AL096776, AC008014, AC002326, AC005663, AC004797, AL049795, U95739,
AC004878, AL031721, AC005488, AC005291, AP000116, AC007193,
AC004084, AL009031, AF199364, AL022165, AL031390, AF030453,
AL035450, AC002301, AL022163, AC005088, AC005224, AC004966,
AC006210, AL049553, AC005520, AC011311, AC005216, AC002527,
AP000013, AC004477, AF001549, AF045555, AC007425, AC004613,
AL022315, AC016027, AC004213, U95742, AL121603, AC007216, AC002558,
AC007358, AC005971, AC006312, AC003666, AL033521, AC005562,
AC005519, L78753, AC005081, AL021329, AC002394, AC007773, AC005028,
Z84486, AC007245, AP000226, Z83838, AP000250, Z98036, AL031282,
AL031283, AP000087, AC005962, AC002565, AC004884, AL022723,
AC003089, AC000119, AC005157, AC002086, AC005250, AC002432, U51560,
AL079342, AC005102, AC007055, AL109865, Z84480, AP000211, AP000133,
AP000030, AC004139, AP000344, AC002377, Z80896, AC003108, AC005242,
AC002365, AC006120, Z93930, AC006344, AL035072, AC002430, AF064865,
AC009113, AF111168, AL133445, AC003111, AC005074, AC002289,
AC007384, AC004134, AL021937, AC006203, AC004895, AC006112, Z82214,
U91323, U62317, AC005479, Z99297, Z93244, AF067844, AC006139,
AC008372, AC004465, AL031774, AL021393, AC007151, AC005993,
AC003029, AF088219, AP000359, AC009320, AC007237, AL022721,
AC002504, and AC000025. HCFMS95 60 674464 AW276842, AL036382,
AI963720, AI637587, AI954260, AI708009, AA610491, AA581903,
AW439558, AI633390, AI859284, AW193265, AW085780, AI345654,
AW082108, AA491814, AI956144, AI831819, AA569471, AL046409,
AA493471, AA623002, AW304584, AA526979, AI344844, AI564454,
AL042420, AI061334, AW088846, AA177120, AI284640, AI064952,
AI814735, AI890570, AI890928, AI561060, AI568678, AL119691,
AW265393, AI860020, AL121385, AI273968, AI783494, AA714453,
AA828042, AW303196, AW274349, AI355206, AA551552, AI431303,
AI670124, AI345681, AW301350, AI345675, AA578861, AI801600,
AI956131, R66997, AI350211, AW070892, AI305766, AW238278, AA947364,
AW088202, AW083402, AI613280, AI270117, AA601492, AW276827,
AA598824, AA371434, AL041690, AA806796, AI368745, AW303876,
AW419262, AI141675, AA594725, AA683258, AW083364, AW250970,
AI446464, AA226153, AW438643, AW302013, AI807650, AA535661,
AA225246, AI239488, AI085719, AW189303, AI619997, AI064864,
AI281881, AA559290, AI334443, AA136338, AI634384, F36273, AW265009,
AA469451, AA483223, AA219129, AL040130, AI873916, AW102955,
AA781975, AI669453, AA525898, AL037683, AW082492, AA507824,
AA468131, AI368256, AA643962, AA678671, AA775230, AI471481,
AI431240, AA525790, AA632994, AA079831, AI623898, AA809029,
AI110688, N54894, AI625244, AI561255, AW249224, AI754658, AL048925,
AI345157, AI053790, AA526787, AW270619, AW104748, AI374809,
AA503475, AI375710, AA649642, H71429, AI133164, AA526191, AW151855,
AI889923, AA513141, AW276435, AW193432, D82290, AI688846, AW088718,
AI160117, W79504, AI969436, AI538433, AA513181, AW029038, AL044940,
AA570227, AI908381, AI240168, AA468022, AA584881, AI537955, T53128,
AI801591, AA523837, AI798473, AW085794, AA552856, N53150, AW410400,
AI251436, AA583955, W47183, AA877817, AA531372, AA720702, AW162049,
AA652057, AI962050, AI929531, AA280632, AL138265, AA493206,
AA857486, AW261871, AI859742, AI610159, AI689222, AA665021,
AI636627, AI370094, AI345518, AW162246, AA908422, AI635818,
AW088224, AW270382, AI370074, AW265385, AI798266, AI312309,
AL022316, AC005031, U80017, AC004999, AF084195, AC005773, AL031055,
AL079340, Z82217, AC007993, U85195, AE000658, AP000082, AC019014,
U57004, AL031429, AL034412, AC005480, AC005264, AC008078, AL117471,
U57007, AC004234, U73479, AC006392, AJ251973, AC006059, AF015155,
AC004087, M94634, Z93017, U01102, S75201, AL033375, AL021938,
AC007382, AC004941, AC004010, AF015153, AF015148, AL009029,
AL049844, AC006238, Z98043, AF184110, X51956, AL022156, Z97987,
U47924, AC006292, AC006276, U57009, AC006512, AF181449, AL031577,
AB028893, AC004098, AC004009, AC002449, X74558, AC004870, D83989,
AC005221, AL022476, AP000305, AC006449, AC005250, M19364, AP000047,
AC005815, AL031293, AL049761, U18391, U18392, U57005, X55925,
AL118497, Z83844, X69951, U38672, AC007151, U18394, AC006077,
AC006126, AC006057, AP000115, X53550, AF015157, AC005323, AF077058,
Z82198, AL035659, AC007068, AL031255, AL133353, X54181, AC005391,
AC005548, AL022302, AC005162, X54180, U18398, U57006, U18395,
U18393, U57008, AL034562, AL023553, X54178, AC007671, U18387,
U40369, X75335, AF076952, AF156539, X55926, U18396, Z95124,
AC003695, AC004853, AL136295, X54175, X54176, AC006536, I51997,
U18400, AF050154, AF144630, AL078581, AL121603, AF064863, AC003081,
X55931, AC004745, AP000348, AL022322, AL096678, U18390, AL109865,
AL078474, X55924, AC004594, AL121934, AL133396, AC005599, AC006539,
AF015151, AL117344, AC005696, AC005919, AC005261, AC007537,
AC005076, U18399, AC005154, AC006041, AC005210, AF045448, AL023807,
AC004047, AC004501, AL022328, AC006367, AC005518, AP000359,
AF121781, AF088219, AC006130, U62317, AC007065, AC007681, AC007564,
AF015147, AF015156, AC005682, AC007103, AC002508, AC004019,
AL031313, AC006312, AC003050, X54179, AL023803, AP001137, AC006989,
AF196779, AL035681, AC006597, Z98751, AC004894, AL031315, X55932,
L81583, AF085442, X55922, AL049631, AC002470, AF015152, AC008249,
AC006537, AC004895, Z98200, AC004672, AC005690, AC007676, AF015154,
AP001172, Z98744, AC002564, AF190465, AL031585, AC006120, AC005701,
M37551, U67801, Z82245, AC000115, AC007324, AL008732, AL035695,
AC008101, AC007043, AC006213, AF031077, AL049709, AF135028,
AC005084, AF015167, AC005324, AF117829, AL078604, Z97632, AL050308,
AL031446, AL136504, AC008079, AP000567, AC005304, AF015158,
AC003957, AC005102, AL031668, AL022323, AP000356, AF074708,
AC002368, AC007371, AC007656, and L48038. HOUCT90 61 646817
AW023515, AA715814, AA659232, AA513851, AA704393, AW237905,
AI753672, T05118, AA602906, AI683131, AA535216, AI307201, AA019973,
AW327422, AI076228, AA559241, AW023975, AI267269, AI635440,
AA410788, AI249365, AI380617, AW023111, AI523316, AA644090,
AA683069, AA654778, AA668291, AI144081, AA569591, AW062682,
AI923052, AI792521, AA169245, AW302711, AW304580, AI056177,
AA633892, AI365625, AI929410, AI792499, AA640430, AA630535,
AA492015, AA515048, AA182731, AI887235, AA115863, AI912401,
AA470567, AA455483, AA484267, AI246796, AL079734, AA659832,
AA825827, AI053793, AI049709, AW316599, AA747757, AI669421,
AA572813, AA084609, AA487272, AI754170, AI192440, AA669238,
AL038842, AA583394, N35306, AA515728, W96277, AW270619, AA640410,
H78898, AI053934, AW407632, N23913, AA780515, R95840, AL037714,
AI874201, AI538491, AI628859, AI675615, AW410354, N34477, AA502532,
AA313025, N72170, AA503298, AA282820, AI224619, AA622801, AA714011,
AI362442, AC004876, Y07848, AC000026, AB023050, AC002059, AP000511,
AL022238, U89335, AC006992, AC007021, AC006549, Z85987, AC002511,
AL049776, AC006441, AC007663, AC006430, AL050332, Z85996, AL031281,
AC003950, AC004686, AF031078, AC005104, AL109798, AF030876, U95742,
AP000558, AC009516, AL031685, AC018633, AC006130, U07562, AC005058,
AD001527, AC007688, Z95116, AP000047, AL021394, Z82176, AC000070,
AC004707, AC000082, AC004408, AC006064, AP000115, AL121655,
AL022336, AC004794, AJ246003, AC006312, AC006547, AC005390,
AP000553, AL049872, AF134726, AC007684, AC005482, AP000304,
AC004851, AC005081, AC002504, AL049745, AP000302, AC007216, L78833,
AC002302, AC004382, AC000097, AC004814, AC004067, AC004032,
AC004991, AC006468, AC005071, AL034548, AL049757, Y10196, Z73359,
AF222686, AL049538, AC005180, L44140, AC005908, AP000141, AP000143,
AC007151, AL031666, AL031588, AC006077, Z84469, AC004079, Z93024,
AC004212, AC000393, AC004106, Z97184, AP001060, AC005284, AP000694,
AC004638, AJ009610, AF042090, Z82215, AC003042, AC005829, AC005138,
X58050, AC004671, AC006543, AC005041, AC005800, U78027, AC002404,
AC005412, AC004057, AL031664, AC003109, AC005324, AC005529,
AC005183, AC007731, AC004694, AL031282, AL035413, AF111168,
AC006080, AC004655, AL035422, AC002350, AL109758, AL023284,
AC004882, AC005212, AC007899, AC004531, AC006211, AL035417,
AF196969, AC005005, AC004783, Z98044, AC004019, AF109907, AP000114,
AP000046, AC007546, AL021940, AC007540, AL021707, AP000152, Z92542,
AC005695, AC005031, AL022165, AC004878, AC005043, AL022237,
AC006011, AP000557, AL079342, AB004907, AL022316, AC005046,
AL133163, AF050154, AC002310, AC002394, AC006544, U80017, AF031076,
AC000025, AP000505, AL020997, AP000279, AL109801, AL035658,
AC002365, AF001549, AC004945, AC005952, AL031005, AC008038,
AC009247, AC007386, X55922, AL035659, Z98036, AL031427, AC004834,
AC007537, AC008040, AL008715, AC004678, AP000512, AL021391,
AC007868, D87675, AL021546, AC003065, AC006449, AC005500, AC005527,
AL031589, AC005033, AL031659, AC005765, AL133448, AC004955, Y14768,
AP000501, AC002476, AC002984, AC002425, Z73360, AL023807, Z81365,
AC004887, AL133500, AC006930, AL022302, AC010205, AC003972,
AF129756, AC007156, AC005409, AL035461, AC005667, AC004033,
AC000090, Z93241, AC000159, AC005015, AC007051, AL109984, and
AC003983. HCFLR78 62 679532 AA573144, AA005018, AI978717, AI983151,
AA007460, AW129961, AI023529, AI023528, AI097101, AI138990,
AA429301, AI307122, AI281472, AA724365, AA074611, W47513, AI126957,
AA843528, AA621024, N92125, AI151489, AA005019, AI023888, AI963099,
W74039, AW296547, W47514, AI052664, AI369723, AI470114, AA203390,
AA634442, AI289000, H75708, AI288995, AI131387, AI093686, H77802,
AA470883, H09852, AI092232, AA758859, AA873611, AA463447, AA663901,
AA365043, AI080292, H12975, AA937147, H17224, AI014968, R74259,
AA612948, AA682858, AA609107, AA358497, AW009774, AW383643,
AA443488, AI076595, AJ243243, AA426125, AA862640, T99925, AI636974,
AA336306, AI246182, AI050921, AA508211, D55400, AW445069, W19308,
AA865885, H66722, N77454, AI266464, AI018497, R02421, AA889290,
AI247759, H75637, H00584, H00583, R28482, D53939, AI798308,
AI909657, R09589, AA449327, W72360, W90696, R32666, AA873850,
AA074610, AW264053, AA524719, N63685, AA515478, AI742730, AA425024,
F29136, AA429478, AA843783, AA635518, AI263801, D52605, AW008958,
and AF151807. HTOHT18 63 628300 and AC004928. HKPMB11 64 688048
AI767027, AI985304, AA552150, AW058459, AI762127, and AW363648.
HNFHS38 65 872798 AA576409, R33161, AL036490, AF064782, I89937, and
I89938. HAIBU10 66 695699 AI767136, AW003744, AA553744, AI360184,
AI565814, AW051486, AA505513, AW301029, AA679066, AI376801,
AI341735, AI268928, AI761796, AI090327, AI767627, AI700593,
AI538258, AA987212, AW003752, AI185049, AI682919, AA460766,
AI343947, AI174548, R72610, AW027615, AA460166, C01651, R68273,
R72274, AW207668, W46139, AA329290, AI393145, AI624837, AA810925,
R11148, R94606, T84462, AI749190, AI659411, AA757401, R11149,
AI424444, AI653008, AI337023, AI539289, AW173060, AA961062,
AI150522, AA535727, AI797658, AW304422, AI620080, AA609486,
AI911307, AW269588, AI357832, AI762382, AA723777, AW449936,
AI937537, AI699787, AW235819, AI806859, AW182752, T96516, AA039581,
AA993113, AW196492, AA931719, AA749267, AW003753, AA938372, N87192,
AI750035, AW074390, AI239833, AI979278, AA722791, R68308, N95006,
R94246, R97183, AA385572, AA811736, AA090761, AW294034, AI675733,
R29264, AI628714, AW418794, AI700162, AI418602, AA905467, AW362416,
and AW362436. HAPOK30 67 685705 AI350913, AA456130, AA459754,
AA922659, AA729889, AA897044, AW207589, R60787, AA373089, AA514352,
AI797424, AA737686, AI434406, AI025403, AW295994, AI492263,
AA811057, AA629548, AI823834, AW027718, AI741138, AI637804,
AI521795, AI094328, AI420179, AI277236, N26754, T16381, AW271660,
AW337972, N66648, AI242353, AI094233, AI042022, AI690072, AI273673,
AL036251, AA487475, AC004659, AF126403, AC005184, AC001231,
AE000658,
AC007564, U85195, AC005829, AC007227, AC007934, AL049775, AL109938,
AL035079, AC005377, AC005250, AC002070, AC011504, Z83821, AL020991,
AC006313, AP000514, AB014080, and Z99128. HCEEM18 68 694615
AI433694, AI287242, AA016140, AA769504, AA248824, AI174876, and
AL031230. HCWUA22 69 695683 HDSAG91 70 692361 AL048773, AA837369,
AA056206, AI200051, AW265393, F32668, AW023111, AA657918, AI307201,
AA484148, AI815425, AL046746, AI267349, AW269488, R96401, H98660,
AI349874, AW438542, AA664521, AA218851, AI264743, AI267450,
AI267847, AW021116, AI623720, R90740, AI267356, AI499503, AI287832,
AW028429, AA664604, H73070, AW270768, AW277174, AL045848, AA693370,
AA713570, AA604843, AA167744, AI538852, AA713569, AA826144,
AL118947, AA317170, AA381762, AA651864, AA584581, AI921188,
AA577852, AI801482, AI446205, AA714595, AA877817, AL133243,
AC008015, AP000046, AC006960, AC005215, AL031276, AL023284,
AC005228, AL033527, AL022162, AC005091, AC002504, AC007191, U82695,
U76377, AL035458, AC007539, AC002364, AC007130, AL049872, AL117351,
AC004549, AC006061, AL034345, Z97205, AC006487, AC002543, AL078581,
AP000338, AC007172, AP000216, AC006455, AC004531, AL031228,
AC008282, AL031652, AC012384, AL109628, Z82243, AF107045, AC006344,
AC004381, AC004087, AL117355, AL023694, AC005516, AC007216,
AP000090, Z95118, Z84487, AL117258, AC004712, AC005161, AC004982,
AL049569, AC002302, AC007030, Z74739, AC007057, AC005829, AC007347,
AC004217, AF015416, AC004975, AC002105, AL049761, AC003957,
AF196971, AL139165, AB022537, AL024508, AL049563, D87675, Z93931,
Z98751, AC006088, AC004900, AL049552, AC004858, AL035361, Z82184,
AC000118, AC005669, AL021328, AL021937, AC004894, AC002454,
AC007102, AL121694, AF109907, AC005249, AL122003, AC007551,
AC007314, AC002457, AR036572, AL078462, AC006101, U91328, AC007970,
AL122023, AL022726, Z73358, AL049697, AC005075, AL031668, AJ010770,
AC005005, AL109613, AC016025, AL022401, AC004848, AL050308, X54175,
AC004702, AC007656, AF067844, AC007114, AC004013, Z95113, AL035454,
AC006285, AC008040, AF165926, AF114156, AP000965, AC005154,
AC013417, AL121769, AC018633, AL031255, AC002476, AC006084,
AC004703, AL078474, AC005902, AP000011, AC004638, AP000030,
AF104455, AL121595, AL021453, AC002531, Z93016, AC005722, AC004128,
AP000211, AP000133, AP000884, AC005011, AL049835, Z93244, AC005158,
AC005288, AC007676, AL031283, AC006115, AC002331, AL022723,
AL121658, AC002377, AJ246003, AF205588, AL033521, AL023655,
AC004522, U73634, U11309, AC007617, AL031132, AC006430, AC004458,
AC004242, AC006241, Z95115, AL139054, AC007488, AC005094, AC007666,
AC006530, AC004802, AL121852, AC005048, AC005191, Z85986, AP000153,
AC008071, AC002472, AC002981, AC003007, AC005007, AC005785,
AP000472, AC006160, AJ250235, AC008033, AC004806, AL137100,
AC004987, AC004111, AC005907, AC006333, AL050318, AC005519,
AC004002, U96629, AC005058, Z49258, AB020867, AL031428, AP000964,
AC002430, AL133485, U85195, Z83841, AP000025, AL022345, AE000658,
AC002429, AL035693, and AL133500. HNEDJ35 71 695744 AI613459,
AI090377, N68677, AW275432, AI310670, AI224583, AA573067, AI016704,
AW085751, AW410844, AW151102, AI124798, AA489390, AA595661,
AW021917, AW080811, AI547110, AI654285, AA809125, AI334435,
AA013168, AL121039, AI433952, AI702049, AI284583, AL037910,
AW272815, AW075979, AA365586, R23873, AA324108, AA019973, AW117740,
AA831426, AA425283, AA350886, AI174703, AA693484, AW148821,
AW151247, AA904275, AI753131, AI251696, AI523205, AI355572,
AW265468, AW327868, AI349130, AI049999, AI690379, AI935827,
AA904137, AI926728, AA631915, AL037067, AW327624, AI078409,
AI473671, AI061313, AW270652, AA484143, AL041375, AA629992,
AW270258, H07953, AW302659, AA743996, AW302705, AA584360, AL045709,
AA828045, AW265385, AI921161, T74524, AA602906, AW302315, AI801563,
AA525423, AL079734, AA311535, AL039117, AA640305, AA720732,
AI439525, AA679946, AA280886, AI754037, AA720582, AI887468, H62123,
AI452836, N99245, AA507623, AI445815, AW327852, AA642809, AI754421,
AW167799, AA878149, Z49154, AC007207, AC005386, AL080243, AC007541,
AC005516, AC002558, AL022328, AC006132, AL117340, AL049869, Z83826,
AC005993, AC006271, AC016027, AC002302, AL132641, U95742, AC005004,
AC004253, AF038458, AC007216, AC007421, AC002314, AC002430,
AL035072, AC007384, AC005488, AC000052, AC005081, AL021707,
AC016830, AL022320, AC006251, AC004150, AC006543, AC004000,
AP000111, AP000043, AC008044, AC002563, AL021978, AC006285, Z98200,
AC003101, AC005632, AC003035, AL031848, AC004820, AC005668,
AC005531, AL049776, AF205588, AC006544, AL021453, AL133246,
AC004019, AL034420, AC006449, AF045555, AP000326, AC007676,
AL050343, AC006236, AL133243, AC005940, AP000169, AP000122,
AP000054, AP000359, AL078581, AC012331, AL049760, AL096791,
AC005288, AC005527, U80460, AC005060, AC007395, AC008082, AC005247,
AP000688, AL049694, AL031587, AL050348, AC003029, AC002347, Z83856,
AC008101, AL121754, AL133448, AC005971, AC004560, AC005234,
AC004796, AC005696, AL049589, AC000085, AC005529, AP000557,
AC002425, AL117258, AL121653, AJ003147, AL117337, AB023050, Z95152,
Z92543, AC005703, AL035414, AL049570, AL008582, AC005057, AL022316,
AC004771, AL136295, U85195, AC004893, AC005694, AC002044, AF207550,
Z98256, AC003042, AE000658, AL049794, AC005502, AC004050, AC000025,
AF109907, Y18000, AL031427, AC006137, AL121658, AL117694, AC005899,
AC006126, AC005291, AC005225, AL049766, AC005218, AC008079,
AL121603, AC007204, AC007308, AC006347, AC004079, AC005837,
AC007151, AC004087, AC004590, AF134726, AC006241, AC005841,
AC006120, AC006013, AP000509, AP000009, AJ011930, AC003982,
AL049758, Z84480, Z94056, AC004678, AC004212, AC002395, U91324,
AC005856, AC007731, AC004686, AC007666, AP000248, AC005500,
AC005553, AL022326, AC005921, U91321, AP000692, AC007298, AP000552,
AC008009, AC005800, D84394, AP000556, AL031255, AP000512, AP000354,
Z75887, AL035696, AC005519, AL109985, AP000301, AC002428, AC003043,
AC005399, AC002091, AC004878, AC002470, AC004895, AL034549,
AC005832, AC004821, AC005046, AL049576, AL096701, AC005257,
AL121655, AL109963, Z83840, AC005091, AC007845, U73023, AC006373,
AC007537, AC005255, AF196779, AC003665, AC006160, AC003070,
AL049780, AD000812, AC005300, AC007055, AL049757, AL031650, U62293,
AC002300, AC004975, AC002312, AP000080, AL021391, AC007041,
AL049778, AE000659, AL031588, AC005284, AL035415, AC005003,
AC002301, AC006480, AC006441, AC004020, AF196970, AC007225,
AC005231, AL033527, AC005220, and AD000092. H7TBA62 72 861995
R85215, AA514191, AA514190, AF121051, AF095853, AF033115, AF018071,
D44443, L39119, AR038762, AJ005168, Y11107, AF046029, U67221,
AJ245869, AC005501, I58669, I15353, AR018866, U89924, A58521,
AB029348, D88984, AF054142, A49700, U85943, AF033196, AF095855,
I65436, AR062871, D49729, AJ001044, AJ006789, AF013625, AF044960,
AJ250192, U92795, AF019721, and AF045229. HNGIO50 73 691288 HMIAW81
74 667504 AA614239, and AC006518. HMMCJ60 75 663467 AL118912,
U91321, and AC003982. HDPIO09 76 686765 AI804463, N32803, AW373516,
N32577, AW182870, AA676790, W87853, AI038081, AA478157, AI334288,
AA776795, AA306403, AI351376, AI203592, AA280706, AI089377,
AW205251, AW391127, AI675872, AW391220, N30775, AI123770, AI148454,
AI222245, AA133659, W90305, AI220222, W87683, AA418887, AI472099,
AA133660, R13704, AA127057, AW367495, AA932329, AA287753, AA948045,
H04073, AA826530, AI266054, AA742688, W90615, AI024654, AW408660,
AI184331, AA255740, AA025466, AA418888, AI094116, AA431370,
AW391079, AA343934, AA456495, AA545796, R81593, AA419218, AI191834,
AI275548, AA001310, H69110, AA432367, AA255539, AA680159, AI218857,
AW166628, AL120413, AI468727, AW102705, AA203443, AA384514,
AI740920, AA846384, AA001680, AA478158, AI417822, AI863025,
AW383212, AW205723, AW374902, T98259, AA361259, AA001638, AW166981,
AL121099, T98314, AA360747, AI148591, R27882, R18829, AA476304,
AA125934, AW390975, AA361382, AA013465, AW391073, AA767404,
AA262028, T25723, AA832312, C21543, AA373327, AA419219, R81340,
H03380, AA993124, AW139299, AA628282, and AB007963. HHFHH34 77
688045 AL109984, AC007216, AC004228, U95742, AL049759, AC004770,
AC005694, U95090, AB023048, AP000694, AC004883, AC007238, AL022238,
AC006536, AL031053, and AC006947. HISCL83 78 688047 HTOAI70 79
840223 AI002744, AA636025, AA838190, AI223700, AC004049, AC004408,
AC006511, AC006212, AC005747, AC002565, AC007066, AC005736,
AL031311, AC004032, AL133448, AL096703, AL034417, AC004967,
AC005071, AC006013, AC003101, Z85987, AC002492, AC007546, AC005191,
AC002554, AL035423, AF001550, AC000029, AL022323, AC006101,
AL035400, AC000353, AC005914, AL022336, AL121653, AF111169,
AC008134, AC002351, AC004832, AP000696, AC005682, AC006449,
AC004067, AL050307, AF038458, Z82194, U91321, AC006946, AC008072,
AC007842, AL132985, AL031985, AC005280, U52112, AC005189, AC007227,
AC005384, AC004976, AL008718, AP000248, AC004216, AC004526,
AC016026, AC005486, AC005512, AF176915, AL035249, AC006139,
AP000514, AC004531, AC005181, AL031283, AC004703, Z98304, AC007021,
AL035420, AC003661, AF001549, AC004491, AC006960, AC007461,
AL020997, AC000105, AC010582, AC004112, AC002551, AC010197,
AC006006, AC005015, Z98950, AC005229, AC009263, AC005562, AC006973,
AC004815, AC005880, AC005043, Z97054, AC005207, AP000100, AL096791,
AP000688, AL034548, AC006568, AC005666, AL121655, AC004770,
AF003529, AC005722, AC005608, AC007327, AC009464, AL022165,
AC006991, U96629, AC005031, AC003663, Z85996, AC002543, AC005255,
AC005081, U91327, AC004099, U95626, AC004887, AC004520, AC004905,
AC005300, AC004707, AC006084, AL035417, AC004381, AP000359,
AC004841, AC002369, AC007488, AC005046, AF111168, AL121756,
AC002070, AP000117, AC004876, AL122021, Z77249, AC000025, AL031721,
and AC001226. HSDER95 80 664502 AW005333, AA631227, AA143192,
AA181022, AI301959, H98648, AA594850, AI478582, AA287457, AI393857,
N75788, AA211849, F06608, N22567, AW450628, AA563681, AW195766,
AI915322, AA186657, AA992992, AA143136, AI302352, AA631048,
AI341927, AI870902, N75929, AA973384, AA160641, and AA338837.
HNECL25 81 618777 AW243793, AW022608, AW270258, AA287872, AA490908,
AW304580, F00564, AA487475, AL048275, AI050070, AL042756, AA631396,
AW117829, AA601278, AC000159, Z95152, AC004832, AC005089, AC005527,
AC007546, AC005529, Z97054, AC005899, AC005081, AL049540, AC007227,
AC006211, AL049776, AF111168, AP000030, AC004263, AL109865, Z93016,
U52112, U85195, AL031602, AC004966, AE000658, Z93017, AC005519,
AC007225, AL109801, AC004531, AL133448, AL049779, AC005736,
AC005071, AC002126, AL022323, AF134726, AC005844, AC007666,
AC004228, AL121653, AC005088, AC003665, AL133163, AC005874,
AF134471, AP000553, AC005225, AC006530, AC005049, AL035587,
AC016027, AC006480, AL022313, AL020997, AL034429, AL049795,
AC005944, AC005197, Y14768, AL049758, AC008072, AL022311, Z86090,
AC002115, AC005484, AL096791, AC016830, AC004019, AL080243,
AC002425, AP000505, AC007114, AC007934, AC007308, AC005291,
AC003029, AL035659, AC005200, AF196971, AP000251, U95740, AC010197,
Z98884, AL009183, AL034549, AC004821, AP000152, Z98036, AL031848,
AL096701, AC006312, AC004659, AC005913, AC006241, AL035458,
AC004217, AC004686, AC020663, AC005821, AF001548, AL031680,
AP000503, U91323, AL022316, AC005104, AC004841, AC005668, AL021154,
AL049757, AC007057, AC007371, AF030453, AC002316, AP000116,
AC005670, AC007842, AF118808, AC002400, AL049713, AC005231,
AC004674, AL022165, AP000350, AC004963, AL049764, AC005839,
AL022320, AC009247, AL121652, Z99128, Z83846, AC015853, AC004106,
AD000812, AC005632, AL121603, AC005781, AL049636, U96629, AF111169,
AC002365, AL050321, AC006449, AP000351, AC005488, AC000052,
AP000269, AL139054, AL031311, AC004876, AP000555, AL023284, U91318,
AC003678, AC002350, AC007688, AL133445, U62317, AC005209, AC007676,
AC004703, AC004922, AC004084, AF067844, AP000557, AC007637,
AL031589, U62293, AL035089, AC004813, AC006441, Z93023, AC006111,
AC004678, AC002483, AC002470, AP000500, AC004967, AL031846, Z85987,
AC004087, AC002312, AC005778, AC005015, AC005740, AC007450,
AC006057, AC007686, U47924, AC005531, AC005046, AC004962, AC002551,
AC009516, AP000103, and AL022721. HNFGZ45 82 618786 AI887235,
AI192440, AW410784, AA282951, AA579130, AA904211, AW023111,
AA579152, AA568747, T50676, AW270258, AW303098, AI250552, AI362442,
AI278972, AI251284, AI251203, AI284543, AA613630, AA084609,
AA482776, AI251034, AW020088, AI431513, AI275982, AI366555,
AI590906, AA602951, R83708, AI446452, AW029515, AI561210, AI872216,
AA115863, AI821714, AI792133, AA595499, AI791913, AW189113,
AI254770, AI799421, AA912287, AA225406, AA613624, AI792464,
AI888468, AW303196, AW274349, AI693979, AI821785, AW272294,
AA550850, AI284092, AA947265, T06518, AW079761, AW301350, AI249853,
AI859946, AI272052, AI732789, AI380617, AI669421, AI635028, N22058,
AC005189, AP000346, AP000347, AC004125, AL049779, AC007993, Z95115,
AC006160, AC002073, Z94056, AC006088, AC003957, AC006537, AC004084,
AC007151, AL023096, AL121578, AF196969, AL020997, AC007842,
AC007637, AC005902, AC006064, AL117258, AC003070, AL031311,
AC005703, AC002347, Z85996, AP000359, AL031597, AL034548, AL079342,
AC006006, AF001552, AC005920, AC004408, AC002395, AC006312,
AL132712, Z82244, AC004834, AC006965, AL022726, AC005736, AC005914,
L78810, AL078463, AC006948, Z85987, AL031589, AF165926, AC004000,
AC005081, AC005514, AC004982, AC004097, U89336, AC005031, AC006450,
AP000302, AB023049, AF129756, AC004837, AP000115, AL132800,
AL096701, AC005192, AC007425, AC007688, AL035683, AL049778, U91322,
AC008170, D87011, AC010206, AC002375, AL021808, AF037338, AC008085,
AL132641, AC007221, AL133355, AC002302, AL009181, AL034420,
AC002554, AL035249, AL049569, AC005971, AC004520, U91319, AL009172,
Z82215, AC005005, U95739, AL035587, AB003151, AC002563, AP000513,
AP000114, AP000046, AL031729, AC004466, Z83844, Z93017, AC004477,
AL121603, AL021707, AC006449, AL022323, Z68869, AC006050, AC005082,
AC002551, AL035462, AC002352, Y10196, AC004929, AC006111, AL022163,
Z82188, AF196972, Z99570, AC007242, U91323, AC002492, AC008044,
AC000392, AP001049, AC004492, AC004659, AF017104, AC006487,
AC005316, AC005488, AL031228, AC004525, AL031291, AL021918,
AC004815, AC005231, AC005291, AL049872, Z85986, AL031659, Z86090,
AC008080, AC005225, AC005837, Z97183, AC006505, AC005066, AL035361,
U91318, AC002303, AC006398, AC004927, AC005988, AC004526, AP000172,
AL050307, AC008249, AC004448, AC009044, AC002300, AC006040,
AC004812,
AL122023, AP000057, AC004778, AC005071, AL031680, AP000085,
AP000694, U91327, AC005520, AC005317, AL049540, AP000125, Z98257,
AC004531, AL022336, AC005666, AL049712, AL109802, L77570, U95090,
AL031276, AL020995, AC005778, AL021397, AL022316, AL080278,
AC005562, AC002301, AL021391, AL034412, AL031670, AC003029,
AL022721, U47924, AC004876, AL034554, AF207550, AF111167, AL021155,
AP000493, AF196779, AC002565, AL049759, AP000558, Z83838, AF006501,
AC008018, AL031678, AP000550, AC005245, AL031984, AC004230,
AL021878, AC004099, AC007160, AC004816, AC005874, AF134471,
AL022476, AP000338, AC007292, AL049539, Z97195, AF099810, AL096712,
D86992, AL031846, AC000075, AC005003, AC004797, AL021407, AP000689,
AP000696, AL078638, and AC007386. HHGCU49 83 688046 AA610125, and
AA679911. HDPND68 84 693214 AA663075, AA047850, N58606, AA077458,
AA856937, AA828871, AA807210, AA078031, AA077602, W58609, AW129389,
AI186819, T87361, AI089362, AI287723, AI874364, AA450298, AA323098,
AA411245, AA437334, AW105275, R50539, AI123195, AI566985, AA928290,
AI418162, AA411170, AA731141, AA810894, AA812128, AA419599,
AA463970, AA427583, AA862081, W58608, AA306683, AI866126, AI500451,
C01071, AW058069, AW058057, AA291009, AA447967, AA001158, AA078563,
AA290621, AI805526, AA015835, AI805341, AA610550, AA078595,
AA393321, AA077098, R27266, R66759, AI520978, AA291720, N26141,
AI571067, AI096500, AI027709, AI885893, AI225016, AI434401,
AI889278, AA291823, H82512, AA635813, AA583169, AA503752, AA780801,
AW089311, AI206412, AI888819, T16137, AI141818, AA035214, AA161026,
AI282157, AI375694, AA282042, AA437275, AI761298, AA908680,
AA411808, AA770647, AI985940, AA098984, AI500026, AI355393,
AI039000, AI039733, AA917605, AA621085, AA037468, AI282159,
AA077481, AC006014, AC004878, AC005488, AC005071, AC004084,
AC006480, AC005088, AF030453, AC002045, AL049760, AC004656,
AL049694, AC002492, AC006210, AC004998, AL023283, AC004865,
AL034408, AL121576, AC005090, AL022308, AC009411, AL049828,
AC005066, AP000702, AP000701, and Z95889. HETDT81 85 684320
AI823398, W27116, AI581128, AL119465, AA897785, AW004741, AI808377,
AI280964, W28405, R60290, AW006936, AI872266, AW137140, AA383051,
AI167420, AA215764, AI362366, AI184026, R28463, R26454, W07404,
AI701315, AI871468, and AF110799. HHLBA14 86 690808 AL046937,
H08012, H08129, AA309286, Z43050, R22834, and AC006924. HLTBU43 87
695735 AI133350, AI739016, AW300169, N36266, AI972422, AI826155,
AI678721, N28853, AA769899, T41222, T40368, AI086442, AL050309,
Z69733, AC004038, AC010349, AC005062, AL121654, AC002524, AC005389,
AC008109, AC009411, AC007527, AC003035, AC005250, AC006600,
AL050334, AC005969, AC004025, AL031114, AL023876, AC000110,
AL121578, AC004020, AC004865, AC004029, AC011422, AL049843,
AL022575, AL030995, AC005145, AC003971, AC004015, AL022401, Z92846,
AC002980, AC010196, AC006971, AL035552, AL136297, AC002075,
AC005014, AL008708, AP000233, AP000147, AC003080, AP000152,
AP000011, Z93341, AP000360, AL049834, Z99128, AC000053, Z93403,
Z74696, AC002479, AL121782, AL034350, AL132800, AC004629, AC003013,
AC002060, AC000004, AJ006997, AL031387, AL049875, AB014088,
AC005518, L11910, AL133233, AL049830, Z95325, AC004384, AP001137,
AL109620, AL031054, AP000516, AC004835, AP000526, AC007363,
AC005537, Z98746, Z82203, AL031885, AL035423, AC006155, AC006367,
AC004103, Z95124, AC005002, AP000069, AL122023, AC002476, and
AC002448. HNTSJ84 88 689474 AI827770, AI962616, AA805764, AA824592,
AW136273, AI952021, AI991996, AW182593, D61365, AI362069, AI934672,
AI126330, AW169950, AA252349, AI362077, AW006061, AA598594, H79076,
AI167818, AI040236, N35889, AA972128, N20949, AI928628, AA252198,
AL134524, AL038878, AL045327, AL134110, AL045328, U46344, AL135012,
AL042898, AL047163, AL045494, AL042523, AL038024, AI318479,
AL042420, AL037295, AL038838, AL038761, AL037343, AL038041,
AL045891, AI547295, AL038983, AI142134, D29033, AL037436, AL048657,
AL037335, AL042655, AL037323, AL048677, AL038651, AL042468,
AL042741, AL038040, AL042519, AL038745, AL037727, AL037443,
AL038532, AL038822, AW363350, AL045326, AL043089, AL043321,
AL046356, AL042488, AL037341, AL041955, AL039432, AI547258,
AL039360, AL039643, AB033037, AL133028, AR066494, A93916, A93931,
AL133053, A85203, AL122101, A93923, D17247, AL133074, AL133049, and
AR023813. HOHCG16 89 679018 W03235, N69989, N58737, R01251, R72832,
R46310, R73289, AA493321, AA002182, R46782, N72648, H43991, H43992,
R46517, R50293, R37273, R46214, R72018, R72019, R45144, R01365,
AI473522, R46877, R07516, R46516, R07468, T78715, R93643, AA382228,
and AB028980. HTHCB31 90 693201 AA490084, AA694325, AC007541,
AC007686, AC005225, and Z93023. HUKAM16 91 695767 AI720817,
AA625436, AI279619, AI686480, AI080242, W76270, AA778254, W72455,
W72270, W76514, AA894476, H28670, H77984, AA886785, AA299801,
AI589802, H24539, R54675, AI538603, AA441799, AI476560, R15815,
AI969936, AI866817, AA363431, AI560023, H39513, AL048656, AI889168,
AI819545, AL121328, AI890057, AI801167, AI679550, AI886055,
AI446405, AW409775, AA908294, AI698437, AI929108, AW152182,
AI250871, AA514684, AW161202, AA769318, AI365256, AI934147,
AW084247, AI679916, AI857296, AA830406, AL036705, AA464646,
AW162194, AW151034, AA128660, AI277008, AI559863, AW026630,
AI918554, AI677797, AI804505, AW089932, AI628214, AA835966,
AL110306, AA830821, AI250627, AL046463, AW162189, AW082532,
AW074374, AW084772, AI570966, AI436644, AW166612, AI500061,
AW263804, W38553, AI089766, AI950664, AI799244, AI652028, AI499920,
AL138386, AL038575, AA693347, AI859654, AI696378, AI683634,
AI669459, AL120736, AW105601, AI921746, AI634626, AW161892,
AI524179, AW172723, AI244380, AI280661, AW149876, AW083573,
AI687568, AW085786, AI612732, AW050725, AI679622, AI829495, W46513,
AI540606, AI915295, AW026905, AI919345, AI539153, AI251205,
AI689420, AI050666, AI567582, AI624529, AI089970, AI924971, N80094,
AI491852, AI628344, AI336634, AW151652, AW265004, AL042384,
AL042787, AW087199, AI636619, AI583578, AI249946, AW301410,
AI699788, AW088162, AW081343, AI924686, AI874243, AW193467,
AI802372, AI800411, AI623736, AI371228, AA827630, AI690813, N27632,
AW163823, F37364, AI582912, AI824360, AW068845, AW161098, AI921386,
AI419374, AW194185, AW193530, AI349622, AI270039, AI798373,
AI431424, AI784028, AI889147, AL038445, AI255071, AI683465,
AW088944, AI857241, AW051048, AI801605, AI590575, AI620093,
AW268261, AW084097, AI358701, AI679800, AW078680, N71180, AI799234,
AI961403, AI934000, AL038541, AI633125, AI684145, AI621171,
AI873605, AI590035, AW188382, N75771, AI591407, AW263569, AI802826,
AL036652, AI864857, AW073898, AI952862, AI634219, AW087203,
AW073697, AW083804, AI282903, AI827058, AI096771, AI824557, H42825,
AW020480, AI373914, AA911767, AI521538, AI141288, AL079794,
AL039086, AI635013, AI783861, AI345415, AI689614, AI360560,
AW088560, AI418254, AI951222, AI355277, AI624668, AI521095,
AW078729, AW026730, AI497733, AW079334, AA808175, AI886206,
AA627473, AI553669, AW102816, W33163, AI434242, AI251221, AW075084,
AI310925, AI633477, AW190725, AI537617, AI696969, AI312399,
AI952217, AI349937, AI469112, AI745713, AI918408, AW089572,
AI334884, AI307543, U42031, AF036941, X06146, AL110197, AL137529,
Z72491, AF158248, AF126247, AF067420, AB019565, AF113690, AF017152,
AF118094, AJ010277, AL133568, AF031147, M86826, AF012536, AL137526,
L13297, AL122098, U87620, I48978, AL117460, AL117585, E04233,
AL110221, U92992, AF114170, AL137557, AF141289, AL050172, AL117435,
I89947, E03348, U92068, AF065135, E03349, S69510, X59414, A08913,
AF102578, E15569, AL133104, A18777, I89931, AL049452, AJ003118,
S68736, S77771, A08912, A08910, A08911, Y11435, I49625, A08909,
AB026995, AB025103, L31396, L31397, AR038854, A08907, A08908,
E01963, U96683, S76508, I00734, I89934, X79812, X62580, X70685,
M27260, X54971, E00617, E00717, E00778, AL133077, AL133645, X80340,
I26207, U90884, I42402, E02221, AB029065, AL050310, AL137556,
AL109672, Y11587, E01812, U75370, AJ006417, X62773, AF207750,
AL133558, AL050024, AL133081, AL137536, I17544, AF090886, AF036268,
AF081571, X67813, AL133072, AL137665, AL137300, AR029580, U00763,
AF030165, U68233, I92592, I66342, AF182215, AL035458, Z22828,
AF114818, AF113676, A12297, X72387, U77594, A57389, X76228, X56039,
AL137478, U35146, AF125949, AF015958, AL137548, E01614, E13364,
I89944, X60786, AF038847, AR019470, AL137530, AL137641, X84990,
AL117578, AL122045, S69407, AL137660, AL137555, AF118070, L40363,
AL049460, AL122106, AL122111, AL137459, AF017790, U66274, AF106934,
AF139986, AF119358, AL133049, AF113013, A08916, S61953, AF078844,
AF067790, AF113694, AF051325, AL050280, AF159148, AL137538,
AF151109, AL137656, AL133014, A27171, U80742, A03736, U91329,
AF008439, AF113691, D55641, AF026816, E01573, E02319, AL110171,
AL122110, S75997, AR020905, AJ001838, AL137283, AL049464, S63521,
AL133636, X99257, AL110196, AL137560, AF132676, AL117626, AF061836,
A94751, AL117649, AL137547, AF016271, AL137658, A90832, Z97214,
AF167995, AL137276, E02914, Y10655, AF119337, L10353, X66871,
AF090943, AF069506, M30514, I52013, AJ242859, I17767, U95114,
E15324, AF185614, J05032, Z37987, AF000301, AL117457, AL137558,
AF199027, U68387, U57352, AF177401, AL110225, AL117394, A52563,
AL080126, AL137294, AF137367, AF057300, AF057299, A93016, M85164,
AF089818, AF107847, AF210052, I30339, I30334, AL050116, AL137256,
AL122118, AL080137, S79832, AL110222, AF022363, AL117629, X72889,
AF113699, and AF179633. HLDOJ66 92 665402 AA805014, AA728939,
AA736485, AI754286, AI610651, AW192331, AA582746, AL042221,
AI654655, AA664879, AA971711, AW083934, AA551582, AA070899, H05449,
AA507282, AI499954, AI922217, H67234, AC004859, AF001549, AL035415,
AL022330, AC004032, AC007792, AL031311, AC002300, AC005225,
AC002477, AC004967, AC007425, AL050307, AL121754, AL079342,
AC005015, AC006449, AL031680, AC004832, AL031276, AC006211, U15422,
AL031685, AL022336, AC006966, AB015355, AC007225, AC004791,
AC005486, AC004383, AL121652, AC005071, AC004491, AL022165,
AL049591, AL049843, AL031594, U07561, AL021917, AL049780, AL008723,
AC007388, AC005520, AC002302, AL031003, AC007676, AF111169,
AL031657, AC006530, AC002115, AC005803, AL049776, AC005081,
AC005878, AL049709, AC005736, AC005620, AC007563, AC005796,
AL049749, AC005412, AL121603, AF064860, AC004813, AP000704,
AF196971, AB023048, Z82188, AC005295, AC006480, AP000100, AC006441,
AL109984, AC003663, AL022399, AL035400, AC002308, AC004797,
AC005082, U91321, AL133353, Z98941, U96629, AC005041, Z93023,
AL024498, AC002347, AC006581, AC007151, AC007030, AC007546,
AC007993, AC005844, Z93930, AL008718, AF196969, AC016025, AL049697,
AC008126, AL049631, AP000350, AC002994, AC006537, U52112, AP000008,
AC005291, AC005821, AC000025, AL049794, AL023807, AC005060,
AC004805, AC010206, Z98752, Z83819, AC004000, AP000104, AC005529,
AC005332, AC006205, AL049869, AC002425, AF111167, AC006013,
AL021327, AC005874, AP000511, AF134471, AP000509, Z98036, AF093117,
AC016830, Z82203, AC007021, AC002404, AC004477, AL133245, AC004844,
AC004638, AL049712, AC005632, AF088219, U95090, AC005531, Z83844,
AC005881, AC016027, AL035447, AC007263, AC002301, AL031123, D84394,
AC003684, AP001053, AC004540, Z77249, AC002351, AL139054, AL008627,
AL035086, AC004216, AC004878, and AC007227. HTXKF10 93 663473
AC006484, AC007878, AC005226, Z94056, AC012330, AC006055, AC005480,
AC007981, AC004943, AP000550, AC007325, AC008018, AL022163,
AC002073, AL049843, AC004216, AC005736, AL022476, AC002301,
AC007308, AC004933, AC005274, AL121748, AL024498, AL020993,
AC004797, AC002375, Z99716, AC004859, AC005305, AL035423, AC005800,
AC002477, AL022328, AP000563, AC005225, AJ010770, AC006088,
AC005156, and AL121603. HPMAI22 94 635491 AI540210, AW173208,
AW006589, AW104434, AI148598, AI656207, AI350808, AW297121,
AW237250, AA918535, AA918200, AI357673, AW235193, AW083055,
AI350807, AI200477, AI991567, AA953496, AI825590, AA738163, N59298,
AA369466, H71562, AA369367, AA369366, H71045, T48746, and H71557.
HL2AG57 95 695733 AI829139, AW264273, AW070588, AA872984, H06954,
AI369038, AW134647, AA974445, AA902284, H14753, AI904699, AW006498,
AA970510, H06955, AW243991, AA306732, and AA333155. HTHBH29 96
882405 AI284640, AW276435, AI610159, AI613280, AI580652, AL046409,
AW276817, AA623002, AI821271, AI249997, AA488395, AA469451,
AI888518, AI653636, AI963720, AA569471, AW303196, AI334443,
AL041690, AI246119, AW023672, AA856954, AI814735, AI887483,
AI744995, AW301350, AA610491, AA613345, AI358343, AI053790,
AW270270, AW408717, AW406162, AL048925, AW406447, AA877817,
AI434695, AW169537, AI357288, AW148792, AI149478, AI053672, C75026,
AI589230, AI133164, AA394271, AW274349, AW338086, AI085719,
AI267818, AI537955, AI499938, AA583955, AW238278, AI890348,
AI431303, AA908357, AA653618, AI374809, AA244357, AI133102,
AL042420, AI564185, AA493471, AL119691, AL138455, AA488746,
AA577906, AA808877, AA665021, AA653975, AL037683, AI336660,
AA490183, AA503475, AI830390, AA587256, AA581903, AI521679,
AI270559, AI286356, AW008952, AI148277, AI246796, AW071196,
AL138265, AW265385, AI349850, AL044940, AA468131, AA649642,
AI951889, AA758934, AI005388, AW301809, AI749559, AW265393,
AL044858, AW162489, AI471481, AI951928, AA669840, AW327868,
AW303876, AA708678, AL046898, AI434706, AI368256, AI564454,
AI871722, AI270117, AI281903, AL046205, M77974, AW083402, AI565581,
AW088202, AI817516, AA503258, AI446601, AI307201, AA491814,
AA126051, AA126035, AA491284, AI298710, AA514737, AA908422,
AI266576, AI669453, AI951863, AI457397, AI801591, AI471543,
AA515905, AA633936, AA079462, AI061334, AW088846, H94870, AA572713,
AA829225, AI744188, AI635818, AI341664, AW304584, AW157651,
AI634384, AI928898, AI859251, AI633390, AA838333, AI561255,
AI282310, AL135405, AA828856, H88666, AA486559, AW261871, AA503154,
AI510838, AI224093, AA610271, AI744827, AW084252, T07287, AI654525,
AA100599, AI734872, AI865905, AL119984, N48230, AW407578, AI305547,
AI471534, AI801600, AW089322, AI287651, AA613232, AL040130,
AI128353, AI207401, H71429, H09313, AI683577, AW243960, X75335,
AF015150, X74558, U67221, AL021579, AF015153, D83989, AL049869,
AF015151, AF015156, X55926, AC004664, AC000003, AC006946, AL096701,
U18391, U18394, AF015148, AC005399, U18393, AC005756, M37551,
Z81369, U18395, U85195, AF015147, Z81308, U47924, AE000658,
AF015149, U57009, AF109907, X55925, AL079295, AL050312, U18387,
Z82244, X54175, U91326, AC003101, I51997, AC004760, U18396,
AC005411, AC002432, AC002465, AC002553, U57007, U38672, AC006111,
AC002544, U18392, AC005291, AC007012, X54181, X54176, U38673,
U91323, U57005, AF077058,
U02532, Z22650, AL022336, AL031683, AF141976, AC005304, AF015155,
AC005545, AC005181, X54178, X55924, X55931, AP000047, AP000049,
AC004686, U38675, AL132641, X55927, AL035458, AL078621, AF015154,
AC003086, AC005206, AP000116, AC006277, AP000115, AC005667,
AP000311, AC005844, AP000305, AC007787, U67211, AC005225, AC004195,
AB004907, AC005488, AC006548, X54179, AC002056, AF037338, AF015157,
AL021707, Z95329, X53550, U02531, AP000556, AC006239, AF129756,
AC002375, AC006312, AP000504, AF111168, AC003029, AC004862,
AB023060, AC004777, M87916, AF001548, X54180, U67827, AC006047,
U67801, AC004219, AC005697, AL023803, AC007685, AP000512, AC005771,
AC007666, AC005666, U11309, AB023051, AC005274, AL050341, U18399,
Z46936, U57008, AL117356, U95740, AC007216, AL080243, AL023553,
U95742, Z99916, AC007283, AL031594, AP000509, AC000085, X55922,
AL122020, AC008008, AL133448, AC006014, AL049776, AP001054,
AC004019, AC004699, AP000967, AC009516, AC000090, AF169121,
AC006359, AC007390, Z97053, Z93017, AC004957, AC004895, AJ006995,
AL117258, AP000477, U34879, AC007664, D84394, AF015160, AL049594,
AC004617, AL031311, AC008372, AC006130, AP000503, AL035423,
AL024498, AC006166, AP000510, AC007011, AL133396, AL121754,
AC009405, AC004447, AC004547, AC002457, AL096791, AC004825, X62025,
AL031295, AL031277, AC005412, AC006116, AC002395, AP000020,
AC005786, Z97200, AL009028, AC004216, AC004991, AL121603, AC006084,
AP000355, AF039590, AC005529, AC000070, AC004253, Z99716, AC006020,
AC004854, AC006538, AC005944, AC005231, AC006333, M87917, AC004525,
X54177, AC006241, AC007199, AC002091, AL049764, AC006088, AL121655,
AC000083, AC006947, AC005057, AP000345, AP000207, AP000129,
AL132777, U18398, AP000356, Z84814, AP000567, AC005159, X55448,
U72787, AJ010598, AL034420, AC006275, AL049697, AP000351, and
AL031283. HUSAM59 97 664505 AI097107, AW070331, AA885479, AI808635,
AI808760, AI806642, AI359136, AA526850, AI743373, AA830249,
AI097104, AI418914, AI263892, AI091575, AI742264, AI660637,
AI218017, N25567, AI687290, AI149468, AI423504, AA056058, AI425096,
AW080003, AA662766, AI699928, AW136639, AI674380, AA278628,
AA789184, AW172424, H09719, AI950490, AI435913, AI092275, F34735,
W31769, AI192998, AA749063, AA659932, AW003176, AL134147, AA907350,
AA768824, AI968635, AA253111, W32476, AA576431, AI470700, AW303675,
AW196635, H14752, AI216029, AI624069, AI005582, AA021513, AI659546,
N68031, R63310, R12287, AA280617, AA158531, H13396, AA843425,
AI474557, AA319692, AI087824, AA912809, R18232, R84736, AA770430,
T73345, R25515, R36860, AA988334, H96095, AA868055, AA768638,
AA299057, AA369839, AI762252, N52758, AI758489, AA809759, R20615,
AI184905, H46838, AA029481, AA778585, R16041, AA253055, Z39839,
H40110, AA483843, AA278627, R37936, H06060, R46123, AA282001,
W04668, AA730323, H40174, AA215402, AA903630, AW304327, AI091241,
AA885041, AA813856, and AC006263. HNGGR26 98 688054 HTLCX30 99
675636 AI656951, AA470111, AA897538, AW452331, AI970293, AA469941,
AI890868, AW014651, R44057, AA812843, AW071964, AI916916, AI480054,
AW002112, AI248788, AI248426, AI675608, AI926984, W88679, AA768893,
AA709247, AW006299, T18862, T52506, AA779510, AI014337, AA992522,
AI964071, D29261, AW103701, AA404555, AF155098, AF135016, AP000547,
D87003, AC004527, AC002041, and AA132101. HCEBC87 100 646713
AL046649, AA446161, AI936225, AW015005, AW150185, AI382086,
AI768700, AI207383, AA723583, AI221375, AA454867, AI025876,
AW078908, AI373629, AA282528, AA747091, AI041381, AA160990,
AA480040, N46327, AA455209, AI240230, AI024163, AA321984, N46331,
D61581, N63661, T31481, AW088780, AI025261, AA948537, Z20134,
H10717, AI764971, T89167, Z21302, Z20133, R40286, Z21303, AI702127,
N40996, AA018284, AA452557, AA479066, T85209, and AA452740. HATCB92
101 603948 AI253043, AA621792, N84222, AA094505, and AB020635.
HMSCX69 102 692125 AI123531, W07173, AA480028, AI803282, AI708223,
N62829, AI203954, AI094156, AA872061, N49953, AW005810, AA433846,
AA411225, AA761754, AA745807, AI708531, N50810, AA243455, AA422108,
AA824620, AI281390, R26124, AI246265, AA257995, AI766585, W01365,
N78578, AA243448, AA761087, AA883328, AA257994, AA766741, AI361501,
Z41792, AA479057, AA740668, AI868726, AA837149, Z46162, AA282715,
N52735, N31703, N48176, and AB020653. HLHAL68 103 684216 AA359084,
AC008149, AC006057, AC007308, AF064861, AL021329, AC005180, and
AC005197. HEOMR73 104 691244 AI341807, AI651516, AI337376, W86648,
N46466, AA522544, AI279721, W86523, AI245138, and AI262580. HETIB83
105 690863 AL042250, R38741, F06975, R24942, T80007, R45205,
Z40078, Z44674, F03694, AF070524, and D13896. HJPDD28 106 842041
AI620815, AL049064, AL042003, AA781401, AW248711, AI568876, R01265,
AL044223, AA682242, AA887723, AW238884, AW372959, Y16610, AF090912,
AF080514, AF080515, AF080512, AF080513, and AF080516. HBAMB15 107
671835 W27833, AI860764, AA809619, AA768248, AI370876, AI291737,
H96013, AW051697, AI633038, and AI784315.
[1288] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1
Isolation of a Selected CDNA Clone From the Deposited Sample
[1289] Each cDNA clone in a cited ATCC deposit is contained in a
plasmid vector. Table 1 identifies the vectors used to construct
the cDNA library from which each clone was isolated. In many cases,
the vector used to construct the library is a phage vector from
which a plasmid has been excised. The table immediately below
correlates the related plasmid for each phage vector used in
constructing the cDNA library. For example, where a particular
clone is identified in Table 1 as being isolated in the vector
"Lambda Zap," the corresponding deposited clone is in
"pBluescript." TABLE-US-00016 Corresponding Deposited Vector Used
to Construct Library Plasmid Lambda Zap pBluescript (pBS) Uni-Zap
XR pBluescript (pBS) Zap Express pBK lafmid BA plafmid BA pSport1
pSport1 pCMVSport 2.0 pCMVSport 2.0 pCMVSport 3.0 pCMVSport 3.0 pCR
.RTM. 2.1 pCR .RTM. 2.1
[1290] Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636),
Uni-Zap XR (U.S. Pat. Nos. 5,128, 256 and 5,286,636), Zap Express
(U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short,
J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees,
M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK
(Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are
commercially available from Stratagene Cloning Systems, Inc., 11011
N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an
ampicillin resistance gene and pBK contains a neomycin resistance
gene. Both can be transformed into E. coli strain XL-1 Blue, also
available from Stratagene. pBS comes in 4 forms SK+, SK-, KS+and
KS. The S and K refers to the orientation of the polylinker to the
T7 and T3 primer sequences which flank the polylinker region ("S"
is for SacI and "K" is for KpnI which are the first sites on each
respective end of the linker). "+" or "-" refer to the orientation
of the f1 origin of replication ("ori"), such that in one
orientation, single stranded rescue initiated from the f1 ori
generates sense strand DNA and in the other, antisense.
[1291] Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were
obtained from Life Technologies, Inc., P.O. Box 6009, Gaithersburg,
Md. 20897. All Sport vectors contain an ampicillin resistance gene
and may be transformed into E. coli strain DHiOB, also available
from Life Technologies. (See, for instance, Gruber, C. E., et al.,
Focus 15:59 (1993).) Vector lafmid BA (Bento Soares, Columbia
University, NY) contains an ampicillin resistance gene and can be
transformed into E. coli strain XL-1 Blue. Vector pCR.RTM.2.1,
which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad,
Calif. 92008, contains an ampicillin resistance gene and may be
transformed into E. coli strain DH10B, available from Life
Technologies. (See, for instance, Clark, J. M., Nuc. Acids Res.
16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).)
Preferably, a polynucleotide of the present invention does not
comprise the phage vector sequences identified for the particular
clone in Table 1, as well as the corresponding plasmid vector
sequences designated above.
[1292] The deposited material in the sample assigned the ATCC
Deposit Number cited in Table 1 for any given cDNA clone also may
contain one or more additional plasmids, each comprising a cDNA
clone different from that given clone. Thus, deposits sharing the
same ATCC Deposit Number contain at least a plasmid for each cDNA
clone identified in Table 1. Typically, each ATCC deposit sample
cited in Table 1 comprises a mixture of approximately equal amounts
(by weight) of about 50 plasmid DNAs, each containing a different
cDNA clone; but such a deposit sample may include plasmids for more
or less than 50 cDNA clones, up to about 500 cDNA clones.
[1293] Two approaches can be used to isolate a particular clone
from the deposited sample of plasmid DNAs cited for that clone in
Table 1. First, a plasmid is directly isolated by screening the
clones using a polynucleotide probe corresponding to SEQ ID
NO:X.
[1294] Particularly, a specific polynucleotide with 30-40
nucleotides is synthesized using an Applied Biosystems DNA
synthesizer according to the sequence reported. The oligonucleotide
is labeled, for instance, with P.sup.32P-.gamma.-ATP using T4
polynucleotide kinase and purified according to routine methods.
(E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Press, Cold Spring, NY (1982).) The plasmid
mixture is transformed into a suitable host, as indicated above
(such as XL-1 Blue (Stratagene)) using techniques known to those of
skill in the art, such as those provided by the vector supplier or
in related publications or patents cited above. The transformants
are plated on 1.5% agar plates (containing the appropriate
selection agent, e.g., ampicillin) to a density of about 150
transformants (colonies) per plate. These plates are screened using
Nylon membranes according to routine methods for bacterial colony
screening (e.g., Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press,
pages 1.93 to 1.104), or other techniques known to those of skill
in the art.
[1295] Alternatively, two primers of 17-20 nucleotides derived from
both ends of the SEQ ID NO:X (i.e., within the region of SEQ ID
NO:X bounded by the 5' NT and the 3' NT of the clone defined in
Table 1) are synthesized and used to amplify the desired cDNA using
the deposited cDNA plasmid as a template. The polymerase chain
reaction is carried out under routine conditions, for instance, in
25 ul of reaction mixture with 0.5 ug of the above cDNA template. A
convenient reaction mixture is 1.5-5 mM MgCl.sub.2, 0.01% (w/v)
gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each
primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR
(denaturation at 94 degree C. for 1 min; annealing at 55 degree C.
for 1 min; elongation at 72 degree C. for 1 min) are performed with
a Perkin-Elmer Cetus automated thermal cycler. The amplified
product is analyzed by agarose gel electrophoresis and the DNA band
with expected molecular weight is excised and purified. The PCR
product is verified to be the selected sequence by subcloning and
sequencing the DNA product.
[1296] Several methods are available for the identification of the
5' or 3' non-coding portions of a gene which may not be present in
the deposited clone. These methods include but are not limited to,
filter probing, clone enrichment using specific probes, and
protocols similar or identical to 5' and 3' "RACE" protocols which
are well known in the art. For instance, a method similar to 5'
RACE is available for generating the missing 5' end of a desired
full-length transcript. (Fromont-Racine et al., Nucleic Acids Res.
21(7):1683-1684 (1993).)
[1297] Briefly, a specific RNA oligonucleotide is ligated to the 5'
ends of a population of RNA presumably containing full-length gene
RNA transcripts. A primer set containing a primer specific to the
ligated RNA oligonucleotide and a primer specific to a known
sequence of the gene of interest is used to PCR amplify the 5'
portion of the desired full-length gene. This amplified product may
then be sequenced and used to generate the full length gene.
[1298] This above method starts with total RNA isolated from the
desired source, although poly-A+ RNA can be used. The RNA
preparation can then be treated with phosphatase if necessary to
eliminate 5' phosphate groups on degraded or damaged RNA which may
interfere with the later RNA ligase step. The phosphatase should
then be inactivated and the RNA treated with tobacco acid
pyrophosphatase in order to remove the cap structure present at the
5' ends of messenger RNAs. This reaction leaves a 5' phosphate
group at the 5' end of the cap cleaved RNA which can then be
ligated to an RNA oligonucleotide using T4 RNA ligase.
[1299] This modified RNA preparation is used as a template for
first strand cDNA synthesis using a gene specific oligonucleotide.
The first strand synthesis reaction is used as a template for PCR
amplification of the desired 5' end using a primer specific to the
ligated RNA oligonucleotide and a primer specific to the known
sequence of the gene of interest. The resultant product is then
sequenced and analyzed to confirm that the 5' end sequence belongs
to the desired gene.
Example 2
Isolation of Genomic Clones Corresponding to a Polynucleotide
[1300] A human genomic P1 library (Genomic Systems, Inc.) is
screened by PCR using primers selected for the cDNA sequence
corresponding to SEQ ID NO:X., according to the method described in
Example 1. (See also, Sambrook.)
Example 3
Tissue Distribution of Polypeptide
[1301] Tissue distribution of mRNA expression of polynucleotides of
the present invention is determined using protocols for Northern
blot analysis, described by, among others, Sambrook et al. For
example, a cDNA probe produced by the method described in Example 1
is labeled with P.sup.32 using the rediprime.TM. DNA labeling
system (Amersham Life Science), according to manufacturer's
instructions. After labeling, the probe is purified using CHROMA
SPIN-100.TM. column (Clontech Laboratories, Inc.), according to
manufacturer's protocol number PT1200-1. The purified labeled probe
is then used to examine various human tissues for mRNA
expression.
[1302] Multiple Tissue Northern (MTN) blots containing various
human tissues (H) or human immune system tissues (IM) (Clontech)
are examined with the labeled probe using ExpressHybTm
hybridization solution (Clontech) according to manufacturer's
protocol number PT1190-1. Following hybridization and washing, the
blots are mounted and exposed to film at -70 degree C. overnight,
and the films developed according to standard procedures.
Example 4
Chromosomal Mapping of the Polynucleotides
[1303] An oligonucleotide primer set is designed according to the
sequence at the 5' end of SEQ ID NO:X. This primer preferably spans
about 100 nucleotides. This primer set is then used in a polymerase
chain reaction under the following set of conditions: 30 seconds,95
degree C.; 1 minute, 56 degree C.; 1 minute, 70 degree C. This
cycle is repeated 32 times followed by one 5 minute cycle at 70
degree C. Human, mouse, and hamster DNA is used as template in
addition to a somatic cell hybrid panel containing individual
chromosomes or chromosome fragments (Bios, Inc). The reactions is
analyzed on either 8% polyacrylamide gels or 3.5% agarose gels.
Chromosome mapping is determined by the presence of an
approximately 100 bp PCR fragment in the particular somatic cell
hybrid.
Example 5
Bacterial Expression of a Polypeptide
[1304] A polynucleotide encoding a polypeptide of the present
invention is amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' ends of the DNA sequence, as
outlined in Example 1, to synthesize insertion fragments. The
primers used to amplify the cDNA insert should preferably contain
restriction sites, such as BamHI and XbaI, at the 5' end of the
primers in order to clone the amplified product into the expression
vector. For example, BamHI and XbaI correspond to the restriction
enzyme sites on the bacterial expression vector pQE-9. (Qiagen,
Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic
resistance (Amp.sup.r), a bacterial origin of replication (ori), an
IPTG-regulatable promoter/operator (P/O), a ribosome binding site
(RBS), a 6-histidine tag (6-His), and restriction enzyme cloning
sites.
[1305] The pQE-9 vector is digested with BamHI and XbaI and the
amplified fragment is ligated into the pQE-9 vector maintaining the
reading frame initiated at the bacterial RBS. The ligation mixture
is then used to transform the E. coli strain M15/rep4 (Qiagen,
Inc.) which contains multiple copies of the plasmid pREP4, which
expresses the lacI repressor and also confers kanamycin resistance
(Kan.sup.r). Transformants are identified by their ability to grow
on LB plates and ampicillin/kanamycin resistant colonies are
selected. Plasmid DNA is isolated and confirmed by restriction
analysis.
[1306] Clones containing the desired constructs are grown overnight
(O/N) in liquid culture in LB media supplemented with both Amp (100
ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a
large culture at a ratio of 1:00 to 1:250. The cells are grown to
an optical density 600 (O.D..sup.600) of between 0.4 and 0.6. IPTG
(Isopropyl-B-D-thiogalacto pyranoside) is then added to a final
concentration of 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/O leading to increased gene
expression.
[1307] Cells are grown for an extra 3 to 4 hours. Cells are then
harvested by centrifugation (20 mins at 6000.times.g). The cell
pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl
by stirring for 3-4 hours at 4 degree C. The cell debris is removed
by centrifugation, and the supernatant containing the polypeptide
is loaded onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity
resin column (available from QIAGEN, Inc., supra). Proteins with a
6 x His tag bind to the Ni-NTA resin with high affinity and can be
purified in a simple one-step procedure (for details see: The
QIAexpressionist (1995) QIAGEN, Inc., supra).
[1308] Briefly, the supernatant is loaded onto the column in 6 M
guanidine-HCl, pH 8, the column is first washed with 10 volumes of
6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M
guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M
guanidine-HCl, pH 5.
[1309] The purified protein is then renatured by dialyzing it
against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6
buffer plus 200 mM NaCl. Alternatively, the protein can be
successfully refolded while immobilized on the Ni-NTA column. The
recommended conditions are as follows: renature using a linear 6
M-1 M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH
7.4, containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation
the proteins are eluted by the addition of 250 mM immidazole.
Immidazole is removed by a final dialyzing step against PBS or 50
mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified
protein is stored at 4 degree C. or frozen at -80 degree C.
[1310] In addition to the above expression vector, the present
invention further includes an expression vector comprising phage
operator and promoter elements operatively linked to a
polynucleotide of the present invention, called pHE4a. (ATCC
Accession Number 209645, deposited on Feb. 25, 1998. ) This vector
contains: 1) a neomycinphosphotransferase gene as a selection
marker, 2) an E. coli origin of replication, 3) a T5 phage promoter
sequence, 4) two lac operator sequences, 5) a Shine-Delgarno
sequence, and 6) the lactose operon repressor gene (laclq). The
origin of replication (oriC) is derived from pUC19 (LTI,
Gaithersburg, Md.). The promoter sequence and operator sequences
are made synthetically.
[1311] DNA can be inserted into the pHEa by restricting the vector
with NdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted
product on a gel, and isolating the larger fragment (the stuffer
fragment should be about 310 base pairs). The DNA insert is
generated according to the PCR protocol described in Example 1,
using PCR primers having restriction sites for NdeI (5' primer) and
XbaI, BamHI, XhoI, or Asp718 (3' primer). The PCR insert is gel
purified and restricted with compatible enzymes. The insert and
vector are ligated according to standard protocols.
[1312] The engineered vector could easily be substituted in the
above protocol to express protein in a bacterial system.
Example 6
Purification of a Polypeptide from an Inclusion Body
[1313] The following alternative method can be used to purify a
polypeptide expressed in E coli when it is present in the form of
inclusion bodies. Unless otherwise specified, all of the following
steps are conducted at 4-10 degree C.
[1314] Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10 degree C. and the
cells harvested by continuous centrifugation at 15,000 rpm (Heraeus
Sepatech). On the basis of the expected yield of protein per unit
weight of cell paste and the amount of purified protein required,
an appropriate amount of cell paste, by weight, is suspended in a
buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The
cells are dispersed to a homogeneous suspension using a high shear
mixer.
[1315] The cells are then lysed by passing the solution through a
microfluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at
4000-6000 psi. The homogenate is then mixed with NaCl solution to a
final concentration of 0.5 M NaCl, followed by centrifugation at
7000.times.g for 15 min. The resultant pellet is washed again using
0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
[1316] The resulting washed inclusion bodies are solubilized with
1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After
7000.times.g centrifugation for 15 min., the pellet is discarded
and the polypeptide containing supernatant is incubated at 4 degree
C. overnight to allow further GuHCl extraction.
[1317] Following high speed centrifugation (30,000.times.g) to
remove insoluble particles, the GuHCl solubilized protein is
refolded by quickly mixing the GuHCl extract with 20 volumes of
buffer containing 50 mM sodium, pH 4.5,150 mM NaCl, 2 mM EDTA by
vigorous stirring. The refolded diluted protein solution is kept at
4 degree C. without mixing for 12 hours prior to further
purification steps.
[1318] To clarify the refolded polypeptide solution, a previously
prepared tangential filtration unit equipped with 0.16 um membrane
filter with appropriate surface area (e.g., Filtron), equilibrated
with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample
is loaded onto a cation exchange resin (e.g., Poros HS-50,
Perseptive Biosystems). The column is washed with 40 mM sodium
acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500
mM NaCl in the same buffer, in a stepwise manner. The absorbance at
280 nm of the effluent is continuously monitored. Fractions are
collected and further analyzed by SDS-PAGE.
[1319] Fractions containing the polypeptide are then pooled and
mixed with 4 volumes of water. The diluted sample is then loaded
onto a previously prepared set of tandem columns of strong anion
(Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-20,
Perseptive Biosystems) exchange resins. The columns are
equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are
washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20
column is then eluted using a 10 column volume linear gradient
ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M
NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under
constant A.sub.280 monitoring of the effluent. Fractions containing
the polypeptide (determined, for instance, by 16% SDS-PAGE) are
then pooled.
[1320] The resultant polypeptide should exhibit greater than 95%
purity after the above refolding and purification steps. No major
contaminant bands should be observed from Commassie blue stained
16% SDS-PAGE gel when 5 ug of purified protein is loaded. The
purified protein can also be tested for endotoxin/LPS
contamination, and typically the LPS content is less than 0.1 ng/ml
according to LAL assays.
Example 7
Cloning and Expression of a Polypeptide in a Baculovirus Expression
System
[1321] In this example, the plasmid shuttle vector pA2 is used to
insert a polynucleotide into a baculovirus to express a
polypeptide. This expression vector contains the strong polyhedrin
promoter of the Autographa californica nuclear polyhedrosis virus
(AcMNPV) followed by convenient restriction sites such as BamHI,
XbaI and Asp718. The polyadenylation site of the simian virus 40
("SV40") is used for efficient polyadenylation. For easy selection
of recombinant virus, the plasmid contains the beta-galactosidase
gene from E. coli under control of a weak Drosophila promoter in
the same orientation, followed by the polyadenylation signal of the
polyhedrin gene. The inserted genes are flanked on both sides by
viral sequences for cell-mediated homologous recombination with
wild-type viral DNA to generate a viable virus that express the
cloned polynucleotide.
[1322] Many other baculovirus vectors can be used in place of the
vector above, such as pAc373, pVL941, and pAcIM1, as one skilled in
the art would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation,
secretion and the like, including a signal peptide and an in-frame
AUG as required. Such vectors are described, for instance, in
Luckow et al., Virology 170:31-39 (1989).
[1323] Specifically, the cDNA sequence contained in the deposited
clone, including the AUG initiation codon and the naturally
associated leader sequence identified in Table 1, is amplified
using the PCR protocol described in Example 1. If the naturally
occurring signal sequence is used to produce the secreted protein,
the pA2 vector does not need a second signal peptide.
Alternatively, the vector can be modified (pA2 GP) to include a
baculovirus leader sequence, using the standard methods described
in Summers et al., "A Manual of Methods for Baculovirus Vectors and
Insect Cell Culture Procedures," Texas Agricultural Experimental
Station Bulletin No. 1555 (1987).
[1324] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Calif.). The fragment then is digested with appropriate
restriction enzymes and again purified on a 1% agarose gel.
[1325] The plasmid is digested with the corresponding restriction
enzymes and optionally, can be dephosphorylated using calf
intestinal phosphatase, using routine procedures known in the art.
The DNA is then isolated from a 1% agarose gel using a commercially
available kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.).
[1326] The fragment and the dephosphorylated plasmid are ligated
together with T4 DNA ligase. E. coli HB101 or other suitable E.
coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla,
Calif.) cells are transformed with the ligation mixture and spread
on culture plates. Bacteria containing the plasmid are identified
by digesting DNA from individual colonies and analyzing the
digestion product by gel electrophoresis. The sequence of the
cloned fragment is confirmed by DNA sequencing.
[1327] Five ug of a plasmid containing the polynucleotide is
co-transfected with 1.0 ug of a commercially available linearized
baculovirus DNA ("BaculoGold.TM. baculovirus DNA", Pharmingen, San
Diego, Calif.), using the lipofection method described by Felgner
et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of
BaculoGold.TM. virus DNA and 5 ug of the plasmid are mixed in a
sterile well of a microtiter plate containing 50 ul of serum-free
Grace's medium (Life Technologies Inc., Gaithersburg, Md.).
Afterwards, 10 ul Lipofectin plus 90 ul Grace's medium are added,
mixed and incubated for 15 minutes at room temperature. Then the
transfection mixture is added drop-wise to Sf9 insect cells (ATCC
CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's
medium without serum. The plate is then incubated for 5 hours at 27
degrees C. The transfection solution is then removed from the plate
and 1 ml of Grace's insect medium supplemented with 10% fetal calf
serum is added. Cultivation is then continued at 27 degrees C. for
four days.
[1328] After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, supra. An
agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg)
is used to allow easy identification and isolation of
gal-expressing clones, which produce blue-stained plaques. (A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10. ) After appropriate incubation, blue stained plaques are
picked with the tip of a micropipettor (e.g., Eppendorf). The agar
containing the recombinant viruses is then resuspended in a
microcentrifuge tube containing 200 ul of Grace's medium and the
suspension containing the recombinant baculovirus is used to infect
Sf9 cells seeded in 35 mm dishes. Four days later the supernatants
of these culture dishes are harvested and then they are stored at 4
degree C.
[1329] To verify the expression of the polypeptide, Sf9 cells are
grown in Grace's medium supplemented with 10% heat-inactivated FBS.
The cells are infected with the recombinant baculovirus containing
the polynucleotide at a multiplicity of infection ("MOI") of about
2. If radiolabeled proteins are desired, 6 hours later the medium
is removed and is replaced with SF900 II medium minus methionine
and cysteine (available from Life Technologies Inc., Rockville,
Md.). After 42 hours, 5 uCi of .sup.35S-methionine and 5 uCi
.sup.35S-cysteine (available from Amersham) are added. The cells
are further incubated for 16 hours and then are harvested by
centrifugation. The proteins in the supernatant as well as the
intracellular proteins are analyzed by SDS-PAGE followed by
autoradiography (if radiolabeled).
[1330] Microsequencing of the amino acid sequence of the amino
terminus of purified protein may be used to determine the amino
terminal sequence of the produced protein.
Example 8
Expression of a Polypeptide in Mammalian Cells
[1331] The polypeptide of the present invention can be expressed in
a mammalian cell. A typical mammalian expression vector contains a
promoter element, which mediates the initiation of transcription of
mRNA, a protein coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription is achieved with the early
and late promoters from SV40, the long terminal repeats (LTRs) from
Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the
cytomegalovirus (CMV). However, cellular elements can also be used
(e.g., the human actin promoter).
[1332] Suitable expression vectors for use in practicing the
present invention include, for example, vectors such as pSVL and
pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr
(ATCC 37146), pBCl2MI (ATCC 67109), pCMVSport 2.0, and pCMVSport
3.0. Mammalian host cells that could be used include, human Hela,
293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7
and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary
(CHO) cells.
[1333] Alternatively, the polypeptide can be expressed in stable
cell lines containing the polynucleotide integrated into a
chromosome. The co-transfection with a selectable marker such as
dhfr, gpt, neomycin, hygromycin allows the identification and
isolation of the transfected cells.
[1334] The transfected gene can also be amplified to express large
amounts of the encoded protein. The DHFR (dihydrofolate reductase)
marker is useful in developing cell lines that carry several
hundred or even several thousand copies of the gene of interest.
(See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370
(1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta,
1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology
9:64-68 (1991).) Another useful selection marker is the enzyme
glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279
(1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using
these markers, the mammalian cells are grown in selective medium
and the cells with the highest resistance are selected. These cell
lines contain the amplified gene(s) integrated into a chromosome.
Chinese hamster ovary (CHO) and NSO cells are often used for the
production of proteins.
[1335] Derivatives of the plasmid pSV2-dhfr (ATCC Accession No.
37146), the expression vectors pC4 (ATCC Accession No. 209646) and
pC6 (ATCC Accession No.209647) contain the strong promoter (LTR) of
the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular
Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer
(Boshart et al., Cell 41:521-530 (1985).) Multiple cloning sites,
e.g., with the restriction enzyme cleavage sites BamHI, XbaI and
Asp718, facilitate the cloning of the gene of interest. The vectors
also contain the 3' intron, the polyadenylation and termination
signal of the rat preproinsulin gene, and the mouse DHFR gene under
control of the SV40 early promoter.
[1336] Specifically, the plasmid pC6, for example, is digested with
appropriate restriction enzymes and then dephosphorylated using
calf intestinal phosphates by procedures known in the art. The
vector is then isolated from a 1% agarose gel.
[1337] A polynucleotide of the present invention is amplified
according to the protocol outlined in Example 1. If the naturally
occurring signal sequence is used to produce the secreted protein,
the vector does not need a second signal peptide. Alternatively, if
the naturally occurring signal sequence is not used, the vector can
be modified to include a heterologous signal sequence. (See, e.g.,
WO 96/34891. )
[1338] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Calif.). The fragment then is digested with appropriate
restriction enzymes and again purified on a 1% agarose gel.
[1339] The amplified fragment is then digested with the same
restriction enzyme and purified on a 1% agarose gel. The isolated
fragment and the dephosphorylated vector are then ligated with T4
DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed
and bacteria are identified that contain the fragment inserted into
plasmid pC6 using, for instance, restriction enzyme analysis.
[1340] Chinese hamster ovary cells lacking an active DHFR gene is
used for transfection. Five .mu.g of the expression plasmid pC6 a
pC4 is cotransfected with 0.5 ug of the plasmid pSVneo using
lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a
dominant selectable marker, the neo gene from Tn5 encoding an
enzyme that confers resistance to a group of antibiotics including
G418. The cells are seeded in alpha minus MEM supplemented with 1
mg/ml G418. After 2 days, the cells are trypsinized and seeded in
hybridoma cloning plates (Greiner, Germany) in alpha minus MEM
supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml
G418. After about 10-14 days single clones are trypsinized and then
seeded in 6-well petri dishes or 10 ml flasks using different
concentrations of methotrexate (50 riM, 100 nM, 200 nM, 400 nM, 800
nM). Clones growing at the highest concentrations of methotrexate
are then transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM).
The same procedure is repeated until clones are obtained which grow
at a concentration of 100-200 uM. Expression of the desired gene
product is analyzed, for instance, by SDS-PAGE and Western blot or
by reversed phase HPLC analysis.
Example 9
Protein Fusions
[1341] The polypeptides of the present invention are preferably
fused to other proteins. These fusion proteins can be used for a
variety of applications. For example, fusion of the present
polypeptides to His-tag, HA-tag, protein A, IgG domains, and
maltose binding protein facilitates purification. (See Example 5;
see also EP A 394,827; Traunecker, et al., Nature 331:84-86
(1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases
the halflife time in vivo. Nuclear localization signals fused to
the polypeptides of the present invention can target the protein to
a specific subcellular localization, while covalent heterodimer or
homodimers can increase or decrease the activity of a fusion
protein. Fusion proteins can also create chimeric molecules having
more than one function. Finally, fusion proteins can increase
solubility and/or stability of the fused protein compared to the
non-fused protein. All of the types of fusion proteins described
above can be made by modifying the following protocol, which
outlines the fusion of a polypeptide to an IgG molecule, or the
protocol described in Example 5.
[1342] Briefly, the human Fc portion of the IgG molecule can be PCR
amplified, using primers that span the 5' and 3' ends of the
sequence described below. These primers also should have convenient
restriction enzyme sites that will facilitate cloning into an
expression vector, preferably a mammalian expression vector.
[1343] For example, if pC4 (Accession No. 209646) is used, the
human Fc portion can be ligated into the BamHI cloning site. Note
that the 3' BamHI site should be destroyed. Next, the vector
containing the human Fc portion is re-restricted with BamHI,
linearizing the vector, and a polynucleotide of the present
invention, isolated by the PCR protocol described in Example 1, is
ligated into this BamHI site. Note that the polynucleotide is
cloned without a stop codon, otherwise a fusion protein will not be
produced.
[1344] If the naturally occurring signal sequence is used to
produce the secreted protein, pC4 does not need a second signal
peptide. Alternatively, if the naturally occurring signal sequence
is not used, the vector can be modified to include a heterologous
signal sequence. (See, e.g., WO 96/34891.)
[1345] Human IgG Fc region: TABLE-US-00017 (SEQ ID NO:1)
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGG
TGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG
TAAATGAGTGCGACGGCCGCGACTCTAGAGGAT
Example 10
Production of an Antibody from a Polypeptide
[1346] The antibodies of the present invention can be prepared by a
variety of methods. (See, Current Protocols, Chapter 2.) As one
example of such methods, cells expressing a polypeptide of the
present invention is administered to an animal to induce the
production of sera containing polyclonal antibodies. In a preferred
method, a preparation of the secreted protein is prepared and
purified to render it substantially free of natural contaminants.
Such a preparation is then introduced into an animal in order to
produce polyclonal antisera of greater specific activity.
[1347] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or protein binding fragments
thereof). Such monoclonal antibodies can be prepared using
hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler
et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J.
Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies
and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In
general, such procedures involve immunizing an animal (preferably a
mouse) with polypeptide or, more preferably, with a secreted
polypeptide-expressing cell. Such cells may be cultured in any
suitable tissue culture medium; however, it is preferable to
culture cells in Earle's modified Eagle's medium supplemented with
10% fetal bovine serum (inactivated at about 56 degrees C.), and
supplemented with about 10 g/l of nonessential amino acids, about
1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.
[1348] The splenocytes of such mice are extracted and fused with a
suitable myeloma cell line. Any suitable myeloma cell line may be
employed in accordance with the present invention; however, it is
preferable to employ the parent myeloma cell line (SP20), available
from the ATCC. After fusion, the resulting hybridoma cells are
selectively maintained in HAT medium, and then cloned by limiting
dilution as described by Wands et al. (Gastroenterology 80:225-232
(1981).) The hybridoma cells obtained through such a selection are
then assayed to identify clones which secrete antibodies capable of
binding the polypeptide.
[1349] Alternatively, additional antibodies capable of binding to
the polypeptide can be produced in a two-step procedure using
anti-idiotypic antibodies. Such a method makes use of the fact that
antibodies are themselves antigens, and therefore, it is possible
to obtain an antibody which binds to a second antibody. In
accordance with this method, protein specific antibodies are used
to immunize an animal, preferably a mouse. The splenocytes of such
an animal are then used to produce hybridoma cells, and the
hybridoma cells are screened to identify clones which produce an
antibody whose ability to bind to the protein-specific antibody can
be blocked by the polypeptide. Such antibodies comprise
anti-idiotypic antibodies to the protein-specific antibody and can
be used to immunize an animal to induce formation of further
protein-specific antibodies.
[1350] It will be appreciated that Fab and F(ab')2 and other
fragments of the antibodies of the present invention may be used
according to the methods disclosed herein. Such fragments are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab fragments) or pepsin (to produce F(ab')2
fragments). Alternatively, secreted protein-binding fragments can
be produced through the application of recombinant DNA technology
or through synthetic chemistry.
[1351] For in vivo use of antibodies in humans, it may be
preferable to use "humanized" chimeric monoclonal antibodies. Such
antibodies can be produced using genetic constructs derived from
hybridoma cells producing the monoclonal antibodies described
above. Methods for producing chimeric antibodies are known in the
art. (See, for review, Morrison, Science 229:1202 (1985); Oi et
al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;
Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature
314:268 (1985).)
Example 11
Production Of Secreted Protein For High-Throughput Screening
Assays
[1352] The following protocol produces a supernatant containing a
polypeptide to be tested. This supernatant can then be used in the
Screening Assays described herein.
[1353] First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim)
stock solution (1 mg/ml in PBS) 1:20 in PBS (w/o calcium or
magnesium 17-516F Biowhittaker) for a working solution of 50 ug/ml.
Add 200 ul of this solution to each well (24 well plates) and
incubate at RT for 20 minutes. Be sure to distribute the solution
over each well (note: a 12-channel pipetter may be used with tips
on every other channel). Aspirate off the Poly-D-Lysine solution
and rinse with lml PBS (Phosphate Buffered Saline). The PBS should
remain in the well until just prior to plating the cells and plates
may be poly-lysine coated in advance for up to two weeks.
[1354] Plate 293T cells (do not carry cells past P+20) at
2.times.10.sup.5 cells/well in 0.5 ml DMEM(Dulbecco's Modified
Eagle Medium)(with 4.5 G/L glucose and L-glutamine (12-604F
Biowhittaker))/10% heat inactivated FBS(14-503F
Biowhittaker)/1.times.Penstrep(17-602E Biowhittaker). Let the cells
grow overnight.
[1355] The next day, mix together in a sterile solution basin: 300
ul Lipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070
Gibco/BRL)/96-well plate. With a small volume multi-channel
pipetter, aliquot approximately 2 ug of an expression vector
containing a polynucleotide insert, produced by the methods
described in Examples 8 or 9, into an appropriately labeled 96-well
round bottom plate. With a multi-channel pipetter, add 50 ul of the
Lipofectamine/Optimem I mixture to each well. Pipette up and down
gently to mix. Incubate at RT 15-45 minutes. After about 20
minutes, use a multi-channel pipetter to add 150 ul Optimem I to
each well. As a control, one plate of vector DNA lacking an insert
should be transfected with each set of transfections.
[1356] Preferably, the transfection should be performed by
tag-teaming the following tasks. By tag-teaming, hands on time is
cut in half, and the cells do not spend too much time on PBS.
First, person A aspirates off the media from four 24-well plates of
cells, and then person B rinses each well with 0.5-1 ml PBS. Person
A then aspirates off PBS rinse, and person B, using a 12-channel
pipetter with tips on every other channel, adds the 200 ul of
DNA/Lipofectamine/Optimem I complex to the odd wells first, then to
the even wells, to each row on the 24-well plates. Incubate at 37
degrees C. for 6 hours.
[1357] While cells are incubating, prepare appropriate media,
either 1% BSA in DMEM with 1.times.penstrep, or CHO-5 media (116.6
mg/L of CaCl2 (anhyd); 0.00130 mg/L CuSO.sub.4-5H.sub.2O; 0.050
mg/L of Fe(NO.sub.3).sub.3-9H.sub.2O; 0.417 mg/L of
FeSO.sub.4-7H.sub.2O; 311.80 mg/L of Kc1; 28.64 mg/L of MgCl.sub.2;
48.84 mg/L of MgSO.sub.4; 6995.50 mg/L of NaCl; 2400.0 mg/L of
NaHCO.sub.3; 62.50 mg/L of NaH.sub.2PO.sub.4-H.sub.2O; 71.02 mg/L
of Na.sub.2HPO4; .4320 mg/L of ZnSO.sub.4-7H.sub.2O; 0.002 mg/L of
Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L of
DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010
mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of
Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic
Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20
mg/L of Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of
L-Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml of
L-Asparagine-H.sub.2O; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml
of L-Cystine-2HCL-H.sub.2O; 31.29 mg/ml of L-Cystine-2HCL; 7.35
mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml
of Glycine; 52.48 mg/ml of L-Histidine-HCL-H.sub.2O; 106.97 mg/ml
of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of
L-Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of
L-Phenylalainine; 40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine;
101.05 mg/ml of L-Threonine; 19.22 mg/ml of L-Tryptophan; 91.79
mg/ml of L-Tryrosine-2Na-2H.sub.2O; 99.65 mg/ml of L-Valine; 0.0035
mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of
Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of
i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L of Pyridoxal HCL;
0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3.17 mg/L
of Thiamine HCL; 0.365 mg/L of Thymidine; and 0.680 mg/L of Vitamin
B.sub.12; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine;
0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL;
55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM
of Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of
Methyl-B-Cyclodextrin complexed with Linoleic Acid; 33.33 mg/L of
Methyl-B-Cyclodextrin complexed with Oleic Acid; and 10 mg/L of
Methyl-B-Cyclodextrin complexed with Retinal) with 2 mm glutamine
and 1.times.penstrep. (BSA (81-068-3 Bayer) 100 gm dissolved in 1L
DMEM for a 10% BSA stock solution). Filter the media and collect 50
ul for endotoxin assay in 15 ml polystyrene conical.
[1358] The transfection reaction is terminated, preferably by
tag-teaming, at the end of the incubation period. Person A
aspirates off the transfection media, while person B adds 1.5 ml
appropriate media to each well. Incubate at 37 degrees C. for 45 or
72 hours depending on the media used: 1% BSA for 45 hours or CHO-5
for 72 hours.
[1359] On day four, using a 300 ul multichannel pipetter, aliquot
600 ul in one 1 ml deep well plate and the remaining supernatant
into a 2 ml deep well. The supernatants from each well can then be
used in the assays described in Examples 13-20.
[1360] It is specifically understood that when activity is obtained
in any of the assays described below using a supernatant, the
activity originates from either the polypeptide directly (e.g., as
a secreted protein) or by the polypeptide inducing expression of
other proteins, which are then secreted into the supernatant. Thus,
the invention further provides a method of identifying the protein
in the supernatant characterized by an activity in a particular
assay.
Example 12
Construction of GAS Reporter Construct
[1361] One signal transduction pathway involved in the
differentiation and proliferation of cells is called the Jaks-STATs
pathway. Activated proteins in the Jaks-STATs pathway bind to gamma
activation site "GAS" elements or interferon-sensitive responsive
element ("ISRE"), located in the promoter of many genes. The
binding of a protein to these elements alter the expression of the
associated gene.
[1362] GAS and ISRE elements are recognized by a class of
transcription factors called Signal Transducers and Activators of
Transcription, or "STATs." There are six members of the STATs
family. Stat1 and Stat3 are present in many cell types, as is Stat2
(as response to IFN-alpha is widespread). Stat4 is more restricted
and is not in many cell types though it has been found in T helper
class I, cells after treatment with IL-12. Stat5 was originally
called mammary growth factor, but has been found at higher
concentrations in other cells including myeloid cells. It can be
activated in tissue culture cells by many cytokines.
[1363] The STATs are activated to translocate from the cytoplasm to
the nucleus upon tyrosine phosphorylation by a set of kinases known
as the Janus Kinase ("Jaks") family. Jaks represent a distinct
family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2,
and Jak3. These kinases display significant sequence similarity and
are generally catalytically inactive in resting cells.
[1364] The Jaks are activated by a wide range of receptors
summarized in the Table below. (Adapted from review by Schidler and
Darnell, Ann. Rev. Biochem. 64:621-51 (1995).) A cytokine receptor
family, capable of activating Jaks, is divided into two groups: (a)
Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9,
IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and
thrombopoietin; and (b) Class 2 includes IFN-a, IFN-g, and IL-10.
The Class 1 receptors share a conserved cysteine motif (a set of
four conserved cysteines and one tryptophan) and a WSXWS motif (a
membrane proximal region encoding Trp-Ser-Xxx-Trp-Ser (SEQ ID
NO:2)).
[1365] Thus, on binding of a ligand to a receptor, Jaks are
activated, which in turn activate STATs, which then translocate and
bind to GAS elements. This entire process is encompassed in the
Jaks-STATs signal transduction pathway.
[1366] Therefore, activation of the Jaks-STATs pathway, reflected
by the binding of the GAS or the ISRE element, can be used to
indicate proteins involved in the proliferation and differentiation
of cells. For example, growth factors and cytokines are known to
activate the Jaks-STATs pathway. (See Table below.) Thus, by using
GAS elements linked to reporter molecules, activators of the
Jaks-STATs pathway can be identified. TABLE-US-00018 JAKs Ligand
tyk2 Jak1 Jak2 Jak3 STATS GAS(elements) or ISRE IFN family IFN-a/B
+ + - - 1, 2, 3 ISRE IFN-g + + - 1 GAS (IRF1 > Lys6 > IFP)
Il-10 + ? ? - 1, 3 gp130 family IL-6 (Pleiotrophic) + + + ? 1, 3
GAS (IRF1 > Lys6 > IFP) Il-11 (Pleiotrophic) ? + ? ? 1, 3 OnM
(Pleiotrophic) ? + + ? 1, 3 LIF (Pleiotrophic) ? + + ? 1, 3 CNTF
(Pleiotrophic) -/+ + + ? 1, 3 G-CSF (Pleiotrophic) ? + ? ? 1, 3
IL-12 (Pleiotrophic) + - + + 1, 3 g-C family IL-2 (lymphocytes) - +
- + 1, 3, 5 GAS IL-4 (lymph/myeloid) - + - + 6 GAS (IRF1 = IFP
>> Ly6)(IgH) IL-7 (lymphocytes) - + - + 5 GAS IL-9
(lymphocytes) - + - + 5 GAS IL-13 (lymphocyte) - + ? ? 6 GAS IL-15
? + ? + 5 GAS gp140 family IL-3 (myeloid) - - + - 5 GAS (IRF1 >
IFP >> Ly6) IL-5 (myeloid) - - + - 5 GAS GM-CSF (myeloid) - -
+ - 5 GAS Growth hormone family GH ? - + - 5 PRL ? +/- + - 1, 3, 5
EPO ? - + - 5 GAS(B-CAS > IRF1 = IFP >> Ly6) Receptor
Tyrosine Kinases EGF ? + + - 1, 3 GAS (IRF1) PDGF ? + + - 1, 3
CSF-1 ? + + - 1, 3 GAS (not IRF1)
[1367] To construct a synthetic GAS containing promoter element,
which is used in the Biological Assays described in Examples 13-14,
a PCR based strategy is employed to generate a GAS-SV40 promoter
sequence. The 5' primer contains four tandem copies of the GAS
binding site found in the IRF1 promoter and previously demonstrated
to bind STATs upon induction with a range of cytokines (Rothman et
al., Immunity 1:457-468 (1994).), although other GAS or ISRE
elements can be used instead. The 5' primer also contains 18 bp of
sequence complementary to the SV40 early promoter sequence and is
flanked with an XhoI site. The sequence of the 5' primer is:
TABLE-US-00019 (SEQ ID NO:3)
5':GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCC
CCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3'
[1368] The downstream primer is complementary to the SV40 promoter
and is flanked with a HindIII site:
5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NO:4).
[1369] PCR amplification is performed using the SV40 promoter
template present in the B-gal:promoter plasmid obtained from
Clontech. The resulting PCR fragment is digested with XhoI/HindIII
and subcloned into BLSK2-. (Stratagene.) Sequencing with forward
and reverse primers confirms that the insert contains the following
sequence: TABLE-US-00020 (SEQ ID NO:5)
5':CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGA
AATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTC
CCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCA
TTCTCCGCCCCATGGCTGACTAATTTTTTTTTATTTATGCAGAGGCCGAG
GCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGG
AGGCCTAGGCTTTTGCAAAAAGCTT:3'.
[1370] With this GAS promoter element linked to the SV40 promoter,
a GAS:SEAP2 reporter construct is next engineered. Here, the
reporter molecule is a secreted alkaline phosphatase, or "SEAP."
Clearly, however, any reporter molecule can be instead of SEAP, in
this or in any of the other Examples. Well known reporter molecules
that can be used instead of SEAP include chloramphenicol
acetyltransferase (CAT), luciferase, alkaline phosphatase,
B-galactosidase, green fluorescent protein (GFP), or any protein
detectable by an antibody.
[1371] The above sequence confirmed synthetic GAS-SV40 promoter
element is subcloned into the pSEAP-Promoter vector obtained from
Clontech using HindIII and XhoI, effectively replacing the SV40
promoter with the amplified GAS:SV40 promoter element, to create
the GAS-SEAP vector. However, this vector does not contain a
neomycin resistance gene, and therefore, is not preferred for
mammalian expression systems.
[1372] Thus, in order to generate mammalian stable cell lines
expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed
from the GAS-SEAP vector using SalI and NotI, and inserted into a
backbone vector containing the neomycin resistance gene, such as
pGFP-1 (Clontech), using these restriction sites in the multiple
cloning site, to create the GAS-SEAP/Neo vector. Once this vector
is transfected into mammalian cells, this vector can then be used
as a reporter molecule for GAS binding as described in Examples
13-14.
[1373] Other constructs can be made using the above description and
replacing GAS with a different promoter sequence. For example,
construction of reporter molecules containing NFK-B and EGR
promoter sequences are described in Examples 15 and 16. However,
many other promoters can be substituted using the protocols
described in these Examples. For instance, SRE, IL-2, NFAT, or
Osteocalcin promoters can be substituted, alone or in combination
(e.g., GAS/NF-KB/EGR, GAS/NF-KB, Il-2/NFAT, or NF-KB/GAS).
Similarly, other cell lines can be used to test reporter construct
activity, such as HELA (epithelial), HUVEC (endothelial), Reh
(B-cell), Saos-2 (osteoblast), HUVAC (aortic), or
Cardiomyocyte.
Example 13
High-Throughput Screening Assay for T-cell Activity.
[1374] The following protocol is used to assess T-cell activity by
identifying factors, and determining whether sup emate containing a
polypeptide of the invention proliferates and/or differentiates
T-cells. T-cell activity is assessed using the GAS/SEAP/Neo
construct produced in Example 12. Thus, factors that increase SEAP
activity indicate the ability to activate the Jaks-STATS signal
transduction pathway. The T-cell used in this assay is Jurkat
T-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCC
Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No.
CRL-1582) cells can also be used.
[1375] Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In
order to generate stable cell lines, approximately 2 million Jurkat
cells are transfected with the GAS-SEAP/neo vector using DMRIE-C
(Life Technologies)(transfection procedure described below). The
transfected cells are seeded to a density of approximately 20,000
cells per well and transfectants resistant to 1 mg/ml genticin
selected. Resistant colonies are expanded and then tested for their
response to increasing concentrations of interferon gamma. The dose
response of a selected clone is demonstrated.
[1376] Specifically, the following protocol will yield sufficient
cells for 75 wells containing 200 ul of cells. Thus, it is either
scaled up, or performed in multiple to generate sufficient cells
for multiple 96 well plates. Jurkat cells are maintained in
RPMI+10% serum with 1% Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life
Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml
OPTI-MEM containing 50 ul of DMRIE-C and incubate at room
temperature for 15-45 mins.
[1377] During the incubation period, count cell concentration, spin
down the required number of cells (10.sup.7 per transfection), and
resuspend in OPTI-MEM to a final concentration of 10.sup.7
cells/ml. Then add 1 ml of 1.times.10.sup.7 cells in OPTI-MEM to
T25 flask and incubate at 37 degrees C. for 6 hrs. After the
incubation, add 10 ml of RPMI+15% serum.
[1378] The Jurkat:GAS-SEAP stable reporter lines are maintained in
RPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are
treated with supernatants containing polypeptides of the invention
and/or induced polypeptides of the invention as produced by the
protocol described in Example 11.
[1379] On the day of treatment with the supernatant, the cells
should be washed and resuspended in fresh RPMI+10% serum to a
density of 500,000 cells per ml. The exact number of cells required
will depend on the number of supernatants being screened. For one
96 well plate, approximately 10 million cells (for 10 plates, 100
million cells) are required.
[1380] Transfer the cells to a triangular reservoir boat, in order
to dispense the cells into a 96 well dish, using a 12 channel
pipette. Using a 12 channel pipette, transfer 200 ul of cells into
each well (therefore adding 100,000 cells per well).
[1381] After all the plates have been seeded, 50 ul of the
supernatants are transferred directly from the 96 well plate
containing the supernatants into each well using a 12 channel
pipette. In addition, a dose of exogenous interferon gamma (0.1,
1.0, 10 ng) is added to wells H9, H10, and H11 to serve as
additional positive controls for the assay.
[1382] The 96 well dishes containing Jurkat cells treated with
supernatants are placed in an incubator for 48 hrs (note: this time
is variable between 48-72 hrs). 35 ul samples from each well are
then transferred to an opaque 96 well plate using a 12 channel
pipette. The opaque plates should be covered (using sellophene
covers) and stored at -20 degrees C. until SEAP assays are
performed according to Example 17. The plates containing the
remaining treated cells are placed at 4 degrees C. and serve as a
source of material for repeating the assay on a specific well if
desired.
[1383] As a positive control, 100 Unit/ml interferon gamma can be
used which is known to activate Jurkat T cells. Over 30 fold
induction is typically observed in the positive control wells.
[1384] The above protocol may be used in the generation of both
transient, as well as, stable transfected cells, which would be
apparent to those of skill in the art.
Example 14
High-Throughput Screening Assay Identifying Myeloid Activity
[1385] The following protocol is used to assess myeloid activity by
determining whether polypeptides of the invention proliferates
and/or differentiates myeloid cells. Myeloid cell activity is
assessed using the GAS/SEAP/Neo construct produced in Example 12.
Thus, factors that increase SEAP activity indicate the ability to
activate the Jaks-STATS signal transduction pathway. The myeloid
cell used in this assay is U937, a pre-monocyte cell line, although
TF-1, HL60, or KG1 can be used.
[1386] To transiently transfect U937 cells with the GAS/SEAP/Neo
construct produced in Example 12, a DEAE-Dextran method (Kharbanda
et. al., 1994, Cell Growth & Differentiation, 5:259-265) is
used. First, harvest 2.times.10e.sup.7 U937 cells and wash with
PBS. The U937 cells are usually grown in RPMI 1640 medium
containing 10% heat-inactivated fetal bovine serum (FBS)
supplemented with 100 units/ml penicillin and 100 mg/ml
streptomycin.
[1387] Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4)
buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid
DNA, 140 mM NaCl, 5 mM KCl, 375 uM Na.sub.2HPO.sub.4.7H.sub.2O, 1
mM MgCl.sub.2, and 675 uM CaCl.sub.2. Incubate at 37 degrees C. for
45 min.
[1388] Wash the cells with RPMI 1640 medium containing 10% FBS and
then resuspend in 10 ml complete medium and incubate at 37 degrees
C. for 36 hr.
[1389] The GAS-SEAP/U937 stable cells are obtained by growing the
cells in 400 ug/ml G418. The G418-free medium is used for routine
growth but every one to two months, the cells should be re-grown in
400 ug/ml G418 for couple of passages.
[1390] These cells are tested by harvesting 1.times.10.sup.8 cells
(this is enough for ten 96-well plates assay) and wash with PBS.
Suspend the cells in 200 ml above described growth medium, with a
final density of 5.times.10.sup.5 cells/ml. Plate 200 ul cells per
well in the 96-well plate (or 1.times.10.sup.5 cells/well).
[1391] Add 50 ul of the supernatant prepared by the protocol
described in Example 11. Incubate at 37 degrees C. for 48 to 72 hr.
As a positive control, 100 Unit/ml interferon gamma can be used
which is known to activate U937 cells. Over 30 fold induction is
typically observed in the positive control wells. SEAP assay the
supernatant according to the protocol described in Example 17.
Example 15
High-Throughput Screening Assay Identifying Neuronal Activity
[1392] When cells undergo differentiation and proliferation, a
group of genes are activated through many different signal
transduction pathways. One of these genes, EGR1 (early growth
response gene 1), is induced in various tissues and cell types upon
activation. The promoter of EGR1 is responsible for such induction.
Using the EGR1 promoter linked to reporter molecules, activation of
cells can be assessed.
[1393] Particularly, the following protocol is used to assess
neuronal activity in PC12 cell lines. PC12 cells (rat
phenochromocytoma cells) are known to proliferate and/or
differentiate by activation with a number of mitogens, such as TPA
(tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF
(epidermal growth factor). The EGR1 gene expression is activated
during this treatment. Thus, by stably transfecting PC12 cells with
a construct containing an EGR promoter linked to SEAP reporter,
activation of PC12 cells can be assessed.
[1394] The EGR/SEAP reporter construct can be assembled by the
following protocol. The EGR-1 promoter sequence (-633 to
+1)(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR
amplified from human genomic DNA using the following primers:
TABLE-US-00021 (SEQ ID NO:6) 5' GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3'
(SEQ ID NO:7) 5' GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3'.
[1395] Using the GAS:SEAP/Neo vector produced in Example 12, EGR1
amplified product can then be inserted into this vector. Linearize
the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII,
removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product
with these same enzymes. Ligate the vector and the EGR1
promoter.
[1396] To prepare 96 well-plates for cell culture, two mls of a
coating solution (1:30 dilution of collagen type I (Upstate Biotech
Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per
one 10 cm plate or 50 ml per well of the 96-well plate, and allowed
to air dry for 2 hr.
[1397] PC12 cells are routinely grown in RPMI-1640 medium (Bio
Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. #
12449-78P), 5% heat-inactivated fetal bovine serum (FBS)
supplemented with 100 units/ml penicillin and 100 ug/ml
streptomycin on a precoated 10 cm tissue culture dish. One to four
split is done every three to four days. Cells are removed from the
plates by scraping and resuspended with pipetting up and down for
more than 15 times.
[1398] Transfect the EGR/SEAP/Neo construct into PC12 using the
Lipofectamine protocol described in Example 11. EGR-SEAP/PC12
stable cells are obtained by growing the cells in 300 ug/ml G418.
The G418-free medium is used for routine growth but every one to
two months, the cells should be re-grown in 300 ug/ml G418 for
couple of passages.
[1399] To assay for neuronal activity, a 10 cm plate with cells
around 70 to 80% confluent is screened by removing the old medium.
Wash the cells once with PBS (Phosphate buffered saline). Then
starve the cells in low serum medium (RPMI-1640 containing 1% horse
serum and 0.5% FBS with antibiotics) overnight.
[1400] The next morning, remove the medium and wash the cells with
PBS. Scrape off the cells from the plate, suspend the cells well in
2 ml low serum medium. Count the cell number and add more low serum
medium to reach final cell density as 5.times.10.sup.5
cells/ml.
[1401] Add 200 ul of the cell suspension to each well of 96-well
plate (equivalent to 1.times.10.sup.5 cells/well). Add 50 ul
supernatant produced by Example 11, 37.degree. C. for 48 to 72 hr.
As a positive control, a growth factor known to activate PC12 cells
through EGR can be used, such as 50 ng/ul of Neuronal Growth Factor
(NGF). Over fifty-fold induction of SEAP is typically seen in the
positive control wells. SEAP assay the supernatant according to
Example 17.
Example 16
High-Throughput Screening Assay for T-cell Activity
[1402] NF-KB (Nuclear Factor KB) is a transcription factor
activated by a wide variety of agents including the inflammatory
cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and
lymphotoxin-beta, by exposure to LPS or thrombin, and by expression
of certain viral gene products. As a transcription factor, NF-KB
regulates the expression of genes involved in immune cell
activation, control of apoptosis (NF-KB appears to shield cells
from apoptosis), B and T-cell development, anti-viral and
antimicrobial responses, and multiple stress responses.
[1403] In non-stimulated conditions, NF-KB is retained in the
cytoplasm with I-KB (Inhibitor KB). However, upon stimulation, I-KB
is phosphorylated and degraded, causing NF-KB to shuttle to the
nucleus, thereby activating transcription of target genes. Target
genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and
class 1 MHC.
[1404] Due to its central role and ability to respond to a range of
stimuli, reporter constructs utilizing the NF-KB promoter element
are used to screen the supernatants produced in Example 11.
Activators or inhibitors of NF-KB would be useful in treating
diseases. For example, inhibitors of NF-KB could be used to treat
those diseases related to the acute or chronic activation of NF-KB,
such as rheumatoid arthritis.
[1405] To construct a vector containing the NF-KB promoter element,
a PCR based strategy is employed. The upstream primer contains four
tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID
NO:8), 18 bp of sequence complementary to the 5' end of the SV40
early promoter sequence, and is flanked with an XhoI site:
TABLE-US-00022 (SEQ ID NO:9)
5':GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGG
ACTTTCCATCCTGCCATCTCAATTAG:3'
[1406] The downstream primer is complementary to the 3' end of the
SV40 promoter and is flanked with a HindIII site: TABLE-US-00023
(SEQ ID NO:4) 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3'
[1407] PCR amplification is performed using the SV40 promoter
template present in the pB-gal:promoter plasmid obtained from
Clontech. The resulting PCR fragment is digested with XhoI and
HindIII and subcloned into BLSK2-. (Stratagene) Sequencing with the
T7 and T3 primers confirms the insert contains the following
sequence: TABLE-US-00024 (SEQ ID NO:10)
5':CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTT
CCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCG
CCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGG
CTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTG
AGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGC AAAAAGCTT:3'
[1408] Next, replace the SV40 minimal promoter element present in
the pSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40
fragment using XhoI and HindIII. However, this vector does not
contain a neomycin resistance gene, and therefore, is not preferred
for mammalian expression systems.
[1409] In order to generate stable mammalian cell lines, the
NF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP
vector using restriction enzymes SalI and NotI, and inserted into a
vector containing neomycin resistance. Particularly, the
NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech),
replacing the GFP gene, after restricting pGFP-1 with SalI and
NotI.
[1410] Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat
T-cells are created and maintained according to the protocol
described in Example 13. Similarly, the method for assaying
supernatants with these stable Jurkat T-cells is also described in
Example 13. As a positive control, exogenous TNF alpha (0.1,1, 10
ng) is added to wells H9, H10, and H11, with a 5-10 fold activation
typically observed.
Example 17
Assay for SEAP Activity
[1411] As a reporter molecule for the assays described in Examples
13-16, SEAP activity is assayed using the Tropix Phospho-light Kit
(Cat. BP-400) according to the following general procedure. The
Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction
Buffers used below.
[1412] Prime a dispenser with the 2.5.times.Dilution Buffer and
dispense 15 ul of 2.5.times.dilution buffer into Optiplates
containing 35 ul of a supernatant. Seal the plates with a plastic
sealer and incubate at 65 degree C. for 30 min. Separate the
Optiplates to avoid uneven heating.
[1413] Cool the samples to room temperature for 15 minutes. Empty
the dispenser and prime with the Assay Buffer. Add 50 ml Assay
Buffer and incubate at room temperature 5 min. Empty the dispenser
and prime with the Reaction Buffer (see the table below). Add 50 ul
Reaction Buffer and incubate at room temperature for 20 minutes.
Since the intensity of the chemiluminescent signal is time
dependent, and it takes about 10 minutes to read 5 plates on
luminometer, one should treat 5 plates at each time and start the
second set 10 minutes later.
[1414] Read the relative light unit in the luminometer. Set H12 as
blank, and print the results. An increase in chemiluminescence
indicates reporter activity. TABLE-US-00025 Reaction Buffer
Formulation: # of plates Rxn buffer diluent (ml) CSPD (ml) 10 60 3
11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 16 90 4.5 17 95
4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22 120 6 23 125
6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.5 29 155
7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 185
9.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41
215 10.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12
47 245 12.25 48 250 12.5 49 255 12.75 50 260 13
Example 18
High-Throughput Screening Assay Identifying Changes in Small
Molecule Concentration and Membrane Permeability
[1415] Binding of a ligand to a receptor is known to alter
intracellular levels of small molecules, such as calcium,
potassium, sodium, and pH, as well as alter membrane potential.
These alterations can be measured in an assay to identify
supernatants which bind to receptors of a particular cell. Although
the following protocol describes an assay for calcium, this
protocol can easily be modified to detect changes in potassium,
sodium, pH, membrane potential, or any other small molecule which
is detectable by a fluorescent probe.
[1416] The following assay uses Fluorometric Imaging Plate Reader
("FLIPR") to measure changes in fluorescent molecules (Molecular
Probes) that bind small molecules. Clearly, any fluorescent
molecule detecting a small molecule can be used instead of the
calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.;
catalog no. F-14202), used here.
[1417] For adherent cells, seed the cells at 10,000-20,000
cells/well in a Co-star black 96-well plate with clear bottom. The
plate is incubated in a CO.sub.2 incubator for 20 hours. The
adherent cells are washed two times in Biotek washer with 200 ul of
HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after
the final wash.
[1418] A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic
acid DMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4
is added to each well. The plate is incubated at 37 degrees C. in a
CO.sub.2 incubator for 60 min. The plate is washed four times in
the Biotek washer with HBSS leaving 100 ul of buffer.
[1419] For non-adherent cells, the cells are spun down from culture
media. Cells are re-suspended to 2-5.times.10.sup.6 cells/ml with
HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-4 solution in
10% pluronic acid DMSO is added to each ml of cell suspension. The
tube is then placed in a 37 degrees C. water bath for 30-60 min.
The cells are washed twice with HBSS, resuspended to
1.times.10.sup.6 cells/ml, and dispensed into a microplate, 100
ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate
is then washed once in Denley CellWash with 200 ul, followed by an
aspiration step to 100 ul final volume.
[1420] For a non-cell based assay, each well contains a fluorescent
molecule, such as fluo-4. The supernatant is added to the well, and
a change in fluorescence is detected.
[1421] To measure the fluorescence of intracellular calcium, the
FLIPR is set for the following parameters: (1) System gain is
300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is
F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6)
Sample addition is 50 ul. Increased emission at 530 nm indicates an
extracellular signaling event which has resulted in an increase in
the intracellular Ca.sup.++ concentration.
Example 19
High-Throughput Screening Assay Identifying Tyrosine Kinase
Activity
[1422] The Protein Tyrosine Kinases (PTK) represent a diverse group
of transmembrane and cytoplasmic kinases. Within the Receptor
Protein Tyrosine Kinase RPTK) group are receptors for a range of
mitogenic and metabolic growth factors including the PDGF, FGF,
EGF, NGF, HGF and Insulin receptor subfamilies. In addition there
are a large family of RPTKs for which the corresponding ligand is
unknown. Ligands for RPTKs include mainly secreted small proteins,
but also membrane-bound and extracellular matrix proteins.
[1423] Activation of RPTK by ligands involves ligand-mediated
receptor dimerization, resulting in transphosphorylation of the
receptor subunits and activation of the cytoplasmic tyrosine
kinases. The cytoplasmic tyrosine kinases include receptor
associated tyrosine kinases of the src-family (e.g., src, yes, Ick,
lyn, fyn) and non-receptor linked and cytosolic protein tyrosine
kinases, such as the Jak family, members of which mediate signal
transduction triggered by the cytokine superfamily of receptors
(e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
[1424] Because of the wide range of known factors capable of
stimulating tyrosine kinase activity, the identification of novel
human secreted proteins capable of activating tyrosine kinase
signal transduction pathways are of interest. Therefore, the
following protocol is designed to identify those novel human
secreted proteins capable of activating the tyrosine kinase signal
transduction pathways.
[1425] Seed target cells (e.g., primary keratinocytes) at a density
of approximately 25,000 cells per well in a 96 well Loprodyne
Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.).
The plates are sterilized with two 30 minute rinses with 100%
ethanol, rinsed with water and dried overnight. Some plates are
coated for 2 hr with 100 ml of cell culture grade type I collagen
(50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can
be purchased from Sigma Chemicals (St. Louis, Mo.) or 10% Matrigel
purchased from Becton Dickinson (Bedford, Mass.), or calf serum,
rinsed with PBS and stored at 4 degree C. Cell growth on these
plates is assayed by seeding 5,000 cells/well in growth medium and
indirect quantitation of cell number through use of alamarBlue as
described by the manufacturer Alamar Biosciences, Inc. (Sacramento,
Calif.) after 48 hr. Falcon plate covers #3071 from Becton
Dickinson (Bedford, Mass.) are used to cover the Loprodyne Silent
Screen Plates. Falcon Microtest III cell culture plates can also be
used in some proliferation experiments.
[1426] To prepare extracts, A431 cells are seeded onto the nylon
membranes of Loprodyne plates (20,000/200 ml/well) and cultured
overnight in complete medium. Cells are quiesced by incubation in
serum-free basal medium for 24 hr. After 5-20 minutes treatment
with EGF (60 ng/ml) or 50 ul of the supernatant produced in Example
11, the medium was removed and 100 ml of extraction buffer ((20 mM
HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4,
2 mM Na4P2O7 and a cocktail of protease inhibitors (# 1836170)
obtained from Boeheringer Mannheim (Indianapolis, Ind.) is added to
each well and the plate is shaken on a rotating shaker for 5
minutes at 4 degrees C. The plate is then placed in a vacuum
transfer manifold and the extract filtered through the 0.45 mm
membrane bottoms of each well using house vacuum. Extracts are
collected in a 96-well catch/assay plate in the bottom of the
vacuum manifold and immediately placed on ice. To obtain extracts
clarified by centrifugation, the content of each well, after
detergent solubilization for 5 minutes, is removed and centrifuged
for 15 minutes at 4 degrees C. at 16,000.times.g.
[1427] Test the filtered extracts for levels of tyrosine kinase
activity. Although many methods of detecting tyrosine kinase
activity are known, one method is described here.
[1428] Generally, the tyrosine kinase activity of a supernatant is
evaluated by determining its ability to phosphorylate a tyrosine
residue on a specific substrate (a biotinylated peptide).
Biotinylated peptides that can be used for this purpose include
PSK1 (corresponding to amino acids 6-20 of the cell division kinase
cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin).
Both peptides are substrates for a range of tyrosine kinases and
are available from Boehringer Mannheim.
[1429] The tyrosine kinase reaction is set up by adding the
following components in order. First, add 10 ul of 5 uM
Biotinylated Peptide, then 10 ul ATP/Mg.sub.2+ (5 mM ATP/50 mM
MgCl.sub.2), then 10 ul of 5.times.Assay Buffer (40 mM imidazole
hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100
MM MgCl.sub.2, 5 mM MnCl.sub.2, 0.5 mg/ml BSA), then 5 ul of Sodium
Vanadate(1 mM), and then 5 ul of water. Mix the components gently
and preincubate the reaction mix at 30 degrees C. for 2 min.
Initial the reaction by adding 10 ul of the control enzyme or the
filtered supernatant.
[1430] The tyrosine kinase assay reaction is then terminated by
adding 10 ul of 120 mm EDTA and place the reactions on ice.
[1431] Tyrosine kinase activity is determined by transferring 50 ul
aliquot of reaction mixture to a microtiter plate (MTP) module and
incubating at 37 degrees C. for 20 min. This allows the
streptavadin coated 96 well plate to associate with the
biotinylated peptide. Wash the MTP module with 300 ul/well of PBS
four times. Next add 75 ul of anti-phospotyrosine antibody
conjugated to horse radish peroxidase(anti-P-Tyr-POD(0.5 u/ml)) to
each well and incubate at 37 degrees C. for one hour. Wash the well
as above.
[1432] Next add 100 ul of peroxidase substrate solution (Boehringer
Mannheim) and incubate at room temperature for at least 5 mins (up
to 30 min). Measure the absorbance of the sample at 405 nm by using
ELISA reader. The level of bound peroxidase activity is quantitated
using an ELISA reader and reflects the level of tyrosine kinase
activity.
Example 20
High-Throughput Screening Assay Identifying Phosphorylation
Activity
[1433] As a potential alternative and/or compliment to the assay of
protein tyrosine kinase activity described in Example 19, an assay
which detects activation (phosphorylation) of major intracellular
signal transduction intermediates can also be used. For example, as
described below one particular assay can detect tyrosine
phosphorylation of the Erk-1 and Erk-2 kinases. However,
phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map
kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase
(MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine,
phosphotyrosine, or phosphothreonine molecule, can be detected by
substituting these molecules for Erk-1 or Erk-2 in the following
assay.
[1434] Specifically, assay plates are made by coating the wells of
a 96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr
at room temp, (RT). The plates are then rinsed with PBS and blocked
with 3% BSA/PBS for 1 hr at RT. The protein G plates are then
treated with 2 commercial monoclonal antibodies (100 ng/well)
against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology).
(To detect other molecules, this step can easily be modified by
substituting a monoclonal antibody detecting any of the above
described molecules.) After 3-5 rinses with PBS, the plates are
stored at 4 degrees C. until use.
[1435] A431 cells are seeded at 20,000/well in a 96-well Loprodyne
filterplate and cultured overnight in growth medium. The cells are
then starved for 48 hr in basal medium (DMEM) and then treated with
EGF (6 ng/well) or 50 ul of the supernatants obtained in Example 11
for 5-20 minutes. The cells are then solubilized and extracts
filtered directly into the assay plate.
[1436] After incubation with the extract for 1 hr at RT, the wells
are again rinsed. As a positive control, a commercial preparation
of MAP kinase (10 ng/well) is used in place of A431 extract. Plates
are then treated with a commercial polyclonal (rabbit) antibody (1
ug/ml) which specifically recognizes the phosphorylated epitope of
the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is
biotinylated by standard procedures. The bound polyclonal antibody
is then quantitated by successive incubations with
Europium-streptavidin and Europium fluorescence enhancing reagent
in the Wallac DELFIA instrument (time-resolved fluorescence). An
increased fluorescent signal over background indicates a
phosphorylation.
Example 21
Method of Determining Alterations in a Gene Corresponding to a
Polynucleotide
[1437] RNA isolated from entire families or individual patients
presenting with a phenotype of interest (such as a disease) is be
isolated. cDNA is then generated from these RNA samples using
protocols known in the art. (See, Sambrook.) The cDNA is then used
as a template for PCR, employing primers surrounding regions of
interest in SEQ ID NO:X. Suggested PCR conditions consist of 35
cycles at 95 degrees C. for 30 seconds; 60-120 seconds at 52-58
degrees C.; and 60-120 seconds at 70 degrees C., using buffer
solutions described in Sidransky et al., Science 252:706
(1991).
[1438] PCR products are then sequenced using primers labeled at
their 5' end with T4 polynucleotide kinase, employing SequiTherm
Polymerase. (Epicentre Technologies). The intron-exon borders of
selected exons is also determined and genomic PCR products analyzed
to confirm the results. PCR products harboring suspected mutations
is then cloned and sequenced to validate the results of the direct
sequencing.
[1439] PCR products is cloned into T-tailed vectors as described in
Holton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced
with T7 polymerase (United States Biochemical). Affected
individuals are identified by mutations not present in unaffected
individuals.
[1440] Genomic rearrangements are also observed as a method of
determining alterations in a gene corresponding to a
polynucleotide. Genomic clones isolated according to Example 2 are
nick-translated with digoxigenindeoxy-uridine 5'-triphosphate
(Boehringer Manheim), and FISH performed as described in Johnson et
al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the
labeled probe is carried out using a vast excess of human cot-1 DNA
for specific hybridization to the corresponding genomic locus.
[1441] Chromosomes are counterstained with
4,6-diamino-2-phenylidole and propidium iodide, producing a
combination of C- and R-bands. Aligned images for precise mapping
are obtained using a triple-band filter set (Chroma Technology,
Brattleboro, Vt.) in combination with a cooled charge-coupled
device camera (Photometrics, Tucson, Ariz.) and variable excitation
wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75
(1991).) Image collection, analysis and chromosomal fractional
length measurements are performed using the ISee Graphical Program
System. (Inovision Corporation, Durham, N.C.) Chromosome
alterations of the genomic region hybridized by the probe are
identified as insertions, deletions, and translocations. These
alterations are used as a diagnostic marker for an associated
disease.
Example 22
Method of Detecting Abnormal Levels of a Polypeptide in a
Biological Sample
[1442] A polypeptide of the present invention can be detected in a
biological sample, and if an increased or decreased level of the
polypeptide is detected, this polypeptide is a marker for a
particular phenotype. Methods of detection are numerous, and thus,
it is understood that one skilled in the art can modify the
following assay to fit their particular needs.
[1443] For example, antibody-sandwich ELISAs are used to detect
polypeptides in a sample, preferably a biological sample. Wells of
a microtiter plate are coated with specific antibodies, at a final
concentration of 0.2 to 10 ug/ml. The antibodies are either
monoclonal or polyclonal and are produced by the method described
in Example 10. The wells are blocked so that non-specific binding
of the polypeptide to the well is reduced.
[1444] The coated wells are then incubated for >2 hours at RT
with a sample containing the polypeptide. Preferably, serial
dilutions of the sample should be used to validate results. The
plates are then washed three times with deionized or distilled
water to remove unbounded polypeptide.
[1445] Next, 50 ul of specific antibody-alkaline phosphatase
conjugate, at a concentration of 25-400 ng, is added and incubated
for 2 hours at room temperature. The plates are again washed three
times with deionized or distilled water to remove unbounded
conjugate.
[1446] Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or
p-nitrophenyl phosphate (NPP) substrate solution to each well and
incubate 1 hour at room temperature. Measure the reaction by a
microtiter plate reader. Prepare a standard curve, using serial
dilutions of a control sample, and plot polypeptide concentration
on the X-axis (log scale) and fluorescence or absorbance of the
Y-axis (linear scale). Interpolate the concentration of the
polypeptide in the sample using the standard curve.
Example 23
Formulation
[1447] The invention also provides methods of treatment and/or
prevention diseases, disorders, and/or conditions (such as, for
example, any one or more of the diseases or disorders disclosed
herein) by administration to a subject of an effective amount of a
Therapeutic. By therapeutic is meant a polynucleotides or
polypeptides of the invention (including fragments and variants),
agonists or antagonists thereof, and/or antibodies thereto, in
combination with a pharmaceutically acceptable carrier type (e.g.,
a sterile carrier).
[1448] The Therapeutic will be formulated and dosed in a fashion
consistent with good medical practice, taking into account the
clinical condition of the individual patient (especially the side
effects of treatment with the Therapeutic alone), the site of
delivery, the method of administration, the scheduling of
administration, and other factors known to practitioners. The
"effective amount" for purposes herein is thus determined by such
considerations.
[1449] As a general proposition, the total pharmaceutically
effective amount of the Therapeutic administered parenterally per
dose will be in the range of about 1 ug/kg/day to 10 mg/kg/day of
patient body weight, although, as noted above, this will be subject
to therapeutic discretion. More preferably, this dose is at least
0.01 mg/kg/day, and most preferably for humans between about 0.01
and 1 mg/kg/day for the hormone. If given continuously, the
Therapeutic is typically administered at a dose rate of about 1
ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections per day
or by continuous subcutaneous infusions, for example, using a
mini-pump. An intravenous bag solution may also be employed. The
length of treatment needed to observe changes and the interval
following treatment for responses to occur appears to vary
depending on the desired effect.
[1450] Therapeutics can be are administered orally, rectally,
parenterally, intracistemally, intravaginally, intraperitoneally,
topically (as by powders, ointments, gels, drops or transdermal
patch), bucally, or as an oral or nasal spray. "Pharmaceutically
acceptable carrier" refers to a non-toxic solid, semisolid or
liquid filler, diluent, encapsulating material or formulation
auxiliary of any. The term "parenteral" as used herein refers to
modes of administration which include intravenous, intramuscular,
intraperitoneal, intrastemal, subcutaneous and intraarticular
injection and infusion.
[1451] Therapeutics of the invention are also suitably administered
by sustained-release systems. Suitable examples of
sustained-release Therapeutics are administered orally, rectally,
parenterally, intracistemally, intravaginally, intraperitoneally,
topically (as by powders, ointments, gels, drops or transdermal
patch), bucally, or as an oral or nasal spray. "Pharmaceutically
acceptable carrier" refers to a non-toxic solid, semisolid or
liquid filler, diluent, encapsulating material or formulation
auxiliary of any type. The term "parenteral" as used herein refers
to modes of administration which include intravenous,
intramuscular, intraperitoneal, intrastemal, subcutaneous and
intraarticular injection and infusion.
[1452] Therapeutics of the invention are also suitably administered
by sustained-release systems. Suitable examples of
sustained-release Therapeutics include suitable polymeric materials
(such as, for example, semi-permeable polymer matrices in the form
of shaped articles, e.g., films, or mirocapsules), suitable
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, and sparingly soluble derivatives
(such as, for example, a sparingly soluble salt).
[1453] Sustained-release matrices include polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and
gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556
(1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J.
Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech.
12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[1454] Sustained-release Therapeutics also include liposomally
entrapped Therapeutics of the invention (see generally, Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)).
Liposomes containing the Therapeutic are prepared by methods known
per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA)
82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.(USA)
77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949;
EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045
and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the
small (about 200-800 Angstroms) unilamellar type in which the lipid
content is greater than about 30 mol. percent cholesterol, the
selected proportion being adjusted for the optimal Therapeutic.
[1455] In yet an additional embodiment, the Therapeutics of the
invention are delivered by way of a pump (see Langer, supra;
Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,
Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574
(1989)).
[1456] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[1457] For parenteral administration, in one embodiment, the
Therapeutic is formulated generally by mixing it at the desired
degree of purity, in a unit dosage injectable form (solution,
suspension, or emulsion), with a pharmaceutically acceptable
carrier, i.e., one that is non-toxic to recipients at the dosages
and concentrations employed and is compatible with other
ingredients of the formulation. For example, the formulation
preferably does not include oxidizing agents and other compounds
that are known to be deleterious to the Therapeutic.
[1458] Generally, the formulations are prepared by contacting the
Therapeutic uniformly and intimately with liquid carriers or finely
divided solid carriers or both. Then, if necessary, the product is
shaped into the desired formulation. Preferably the carrier is a
parenteral carrier, more preferably a solution that is isotonic
with the blood of the recipient. Examples of such carrier vehicles
include water, saline, Ringer's solution, and dextrose solution.
Non-aqueous vehicles such as fixed oils and ethyl oleate are also
useful herein, as well as liposomes.
[1459] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, manose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
[1460] The Therapeutic is typically formulated in such vehicles at
a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10
mg/ml, at a pH of about 3 to 8. It will be understood that the use
of certain of the foregoing excipients, carriers, or stabilizers
will result in the formation of polypeptide salts.
[1461] Any pharmaceutical used for therapeutic administration can
be sterile. Sterility is readily accomplished by filtration through
sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutics generally are placed into a container having a sterile
access port, for example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle.
[1462] Therapeutics ordinarily will be stored in unit or multi-dose
containers, for example, sealed ampoules or vials, as an aqueous
solution or as a lyophilized formulation for reconstitution. As an
example of a lyophilized formulation, 10-ml vials are filled with 5
ml of sterile-filtered 1% (w/v) aqueous Therapeutic solution, and
the resulting mixture is lyophilized. The infusion solution is
prepared by reconstituting the lyophilized Therapeutic using
bacteriostatic Water-for-Injection.
[1463] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the Therapeutics of the invention. Associated with
such container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration. In addition, the Therapeutics may be employed in
conjunction with other therapeutic compounds.
[1464] The Therapeutics of the invention may be administered alone
or in combination with adjuvants. Adjuvants that may be
administered with the Therapeutics of the invention include, but
are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE
(Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL. In a
specific embodiment, Therapeutics of the invention are administered
in combination with alum. In another specific embodiment,
Therapeutics of the invention are administered in combination with
QS-21. Further adjuvants that may be administered with the
Therapeutics of the invention include, but are not limited to,
Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,
CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.
Vaccines that may be administered with the Therapeutics of the
invention include, but are not limited to, vaccines directed toward
protection against MMR (measles, mumps, rubella), polio, varicella,
tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae
B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,
cholera, yellow fever, Japanese encephalitis, poliomyelitis,
rabies, typhoid fever, and pertussis. Combinations may be
administered either concomitantly, e.g., as an admixture,
separately but simultaneously or concurrently; or sequentially.
This includes presentations in which the combined agents are
administered together as a therapeutic mixture, and also procedures
in which the combined agents are administered separately but
simultaneously, e.g., as through separate intravenous lines into
the same individual. Administration "in combination" further
includes the separate administration of one of the compounds or
agents given first, followed by the second.
[1465] The Therapeutics of the invention may be administered alone
or in combination with other therapeutic agents. Therapeutic agents
that may be administered in combination with the Therapeutics of
the invention, include but not limited to, other members of the TNF
family, chemotherapeutic agents, antibiotics, steroidal and
non-steroidal anti-inflammatories, conventional immunotherapeutic
agents, cytokines and/or growth factors. Combinations may be
administered either concomitantly, e.g., as an admixture,
separately but simultaneously or concurrently; or sequentially.
This includes presentations in which the combined agents are
administered together as a therapeutic mixture, and also procedures
in which the combined agents are administered separately but
simultaneously, e.g., as through separate intravenous lines into
the same individual. Administration "in combination" further
includes the separate administration of one of the compounds or
agents given first, followed by the second.
[1466] In one embodiment, the Therapeutics of the invention are
administered in combination with members of the TNF family. TNF,
TNF-related or TNF-like molecules that may be administered with the
Therapeutics of the invention include, but are not limited to,
soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known
as TNF-beta), LT-beta (found in complex heterotrimer
LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3,
OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I
(International Publication No. WO 97/33899), endokine-alpha
(International Publication No. WO 98/07880), TR6 (International
Publication No. WO 98/30694), OPG, and neutrokine-alpha
(International Publication No. WO 98/18921, OX40, and nerve growth
factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB,
TR2 (International Publication No. WO 96/34095), DR3 (International
Publication No. WO 97/33904), DR4 (International Publication No. WO
98/32856), TR5 (International Publication No. WO 98/30693), TR6
(International Publication No. WO 98/30694), TR7 (International
Publication No. WO 98/41629), TRANK, TR9 (International Publication
No. WO 98/56892),TR10 (International Publication No. WO 98/54202),
312C2 (International Publication No. WO 98/06842), and TR12, and
soluble forms CD154, CD70, and CD153.
[1467] In certain embodiments, Therapeutics of the invention are
administered in combination with antiretroviral agents, nucleoside
reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors, and/or protease inhibitors. Nucleoside
reverse transcriptase inhibitors that may be administered in
combination with the Therapeutics of the invention, include, but
are not limited to, RETROVIR.TM. (zidovudine/AZT), VIDEX.TM.
(didanosine/ddI), HIVID.TM. (zalcitabine/ddC), ZERIT.TM.
(stavudine/d4T), EPIVIR.TM. (lamivudine/3TC), and COMBIVIR.TM.
(zidovudine/lamivudine). Non-nucleoside reverse transcriptase
inhibitors that may be administered in combination with the
Therapeutics of the invention, include, but are not limited to,
VIRAMUNE.TM. (nevirapine), RESCRIPTOR.TM. (delavirdine), and
SUSTIVA.TM. (efavirenz). Protease inhibitors that may be
administered in combination with the Therapeutics of the invention,
include, but are not limited to, CRIXVAN.TM. (indinavir),
NORVIR.TM. (ritonavir), INVIRASE.TM. (saquinavir), and VIRACEPT.TM.
(nelfinavir). In a specific embodiment, antiretroviral agents,
nucleoside reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors, and/or protease inhibitors may be used in
any combination with Therapeutics of the invention to treat AIDS
and/or to prevent or treat HIV infection.
[1468] In other embodiments, Therapeutics of the invention may be
administered in combination with anti-opportunistic infection
agents. Anti-opportunistic agents that may be administered in
combination with the Therapeutics of the invention, include, but
are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM.,
PENTAMIDINE.TM., ATOVAQUONE.TM., ISONIAZID.TM., RIFAMPIN.TM.,
PYRAZINAMIDE.TM., ETHAMBUTOL.TM., RIFABUTIN.TM.,
CLARITHROMYCIN.TM., AZITHROMYCIN.TM., GANCICLOVIR.TM.,
FOSCARNET.TM., CIDOFOVIR.TM., FLUCONAZOLE.TM., ITRACONAZOLE.TM.,
KETOCONAZOLE.TM., ACYCLOVIR.TM., FAMCICOLVIR.TM.,
PYRIMETHAMINE.TM., LEUCOVORIN.TM., NEUPOGEN.TM. (filgrastim/G-CSF),
and LEUKINE.TM. (sargramostim/GM-CSF). In a specific embodiment,
Therapeutics of the invention are used in any combination with
TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM., PENTAMIDINE.TM.,
and/or ATOVAQUONE.TM. to prophylactically treat or prevent an
opportunistic Pneumocystis carinii pneumonia infection. In another
specific embodiment, Therapeutics of the invention are used in any
combination with ISONIAZID.TM., RIFAMPIN.TM., PYRAZINAMIDE.TM.,
and/or ETHAMBUTOL.TM. to prophylactically treat or prevent an
opportunistic Mycobacterium avium complex infection. In another
specific embodiment, Therapeutics of the invention are used in any
combination with RIFABUTIN.TM., CLARITHROMYCIN.TM., and/or
AZITHROMYCIN.TM. to prophylactically treat or prevent an
opportunistic Mycobacterium tuberculosis infection. In another
specific embodiment, Therapeutics of the invention are used in any
combination with GANCICLOVIR.TM., FOSCARNET.TM., and/or
CIDOFOVIR.TM. to prophylactically treat or prevent an opportunistic
cytomegalovirus infection. In another specific embodiment,
Therapeutics of the invention are used in any combination with
FLUCONAZOLE.TM., ITRACONAZOLE.TM., and/or KETOCONAZOLE.TM. to
prophylactically treat or prevent an opportunistic fingal
infection. In another specific embodiment, Therapeutics of the
invention are used in any combination with ACYCLOVIR.TM. and/or
FAMCICOLVIR.TM. to prophylactically treat or prevent an
opportunistic herpes simplex virus type I and/or type II infection.
In another specific embodiment, Therapeutics of the invention are
used in any combination with PYRIMETHAMINE.TM. and/or
LEUCOVORIN.TM. to prophylactically treat or prevent an
opportunistic Toxoplasma gondii infection. In another specific
embodiment, Therapeutics of the invention are used in any
combination with LEUCOVORIN.TM. and/or NEUPOGEN.TM. to
prophylactically treat or prevent an opportunistic bacterial
infection.
[1469] In a further embodiment, the Therapeutics of the invention
are administered in combination with an antiviral agent. Antiviral
agents that may be administered with the Therapeutics of the
invention include, but are not limited to, acyclovir, ribavirin,
amantadine, and remantidine.
[1470] In a further embodiment, the Therapeutics of the invention
are administered in combination with an antibiotic agent.
Antibiotic agents that may be administered with the Therapeutics of
the invention include, but are not limited to, amoxicillin,
beta-lactamases, aminoglycosides, beta-lactam (glycopeptide),
beta-lactamases, Clindamycin, chloramphenicol, cephalosporins,
ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones,
macrolides, metronidazole, penicillins, quinolones, rifampin,
streptomycin, sulfonamide, tetracyclines, trimethoprim,
trimethoprim-sulfamthoxazole, and vancomycin.
[1471] Conventional nonspecific immunosuppressive agents, that may
be administered in combination with the Therapeutics of the
invention include, but are not limited to, steroids, cyclosporine,
cyclosporine analogs, cyclophosphamide methylprednisone,
prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other
immunosuppressive agents that act by suppressing the function of
responding T cells.
[1472] In specific embodiments, Therapeutics of the invention are
administered in combination with immunosuppressants.
Immunosuppressants preparations that may be administered with the
Therapeutics of the invention include, but are not limited to,
ORTHOCLONE.TM. (OKT3), SANDIMMUNE.TM./NEORAL.TM./SANGDYA.TM.
(cyclosporin), PROGRAF.TM. (tacrolimus), CELLCEPT.TM.
(mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE.TM.
(sirolimus). In a specific embodiment, immunosuppressants may be
used to prevent rejection of organ or bone marrow
transplantation.
[1473] In an additional embodiment, Therapeutics of the invention
are administered alone or in combination with one or more
intravenous immune globulin preparations. Intravenous immune
globulin preparations that may be administered with the
Therapeutics of the invention include, but not limited to,
GAMMAR.TM., IVEEGAM.TM., SANDOGLOBULIN.TM., GAMMAGARD S/D.TM., and
GAMIMUNE.TM.. In a specific embodiment, Therapeutics of the
invention are administered in combination with intravenous immune
globulin preparations in transplantation therapy (e.g., bone marrow
transplant).
[1474] In an additional embodiment, the Therapeutics of the
invention are administered alone or in combination with an
anti-inflammatory agent. Anti-inflammatory agents that may be
administered with the Therapeutics of the invention include, but
are not limited to, glucocorticoids and the nonsteroidal
anti-inflammatories, aminoarylcarboxylic acid derivatives,
arylacetic acid derivatives, arylbutyric acid derivatives,
arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,
pyrazolones, salicylic acid derivatives, thiazinecarboxamides,
e-acetamidocaproic acid, S-adenosylmethionine,
3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,
bucolome, difenpiramide, ditazol, emorfazone, guaiazulene,
nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal,
pifoxime, proquazone, proxazole, and tenidap.
[1475] In another embodiment, compostions of the invention are
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with the
Therapeutics of the invention include, but are not limited to,
antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin,
and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites
(e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon
alpha-2b, glutamic acid, plicamycin, mercaptopurine, and
6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU,
lomustine, CCNU, cytosine arabinoside, cyclophosphamide,
estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,
cis-platin, and vincristine sulfate); hormones (e.g.,
medroxyprogesterone, estramustine phosphate sodium, ethinyl
estradiol, estradiol, megestrol acetate, methyltestosterone,
diethylstilbestrol diphosphate, chlorotrianisene, and
testolactone); nitrogen mustard derivatives (e.g., mephalen,
chorambucil, mechlorethamine (nitrogen mustard) and thiotepa);
steroids and combinations (e.g., bethamethasone sodium phosphate);
and others (e.g., dicarbazine, asparaginase, mitotane, vincristine
sulfate, vinblastine sulfate, and etoposide).
[1476] In a specific embodiment, Therapeutics of the invention are
administered in combination with CHOP (cyclophosphamide,
doxorubicin, vincristine, and prednisone) or any combination of the
components of CHOP. In another embodiment, Therapeutics of the
invention are administered in combination with Rituximab. In a
further embodiment, Therapeutics of the invention are administered
with Rituxmab and CHOP, or Rituxmab and any combination of the
components of CHOP.
[1477] In an additional embodiment, the Therapeutics of the
invention are administered in combination with cytokines. Cytokines
that may be administered with the Therapeutics of the invention
include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7,
IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha.
In another embodiment, Therapeutics of the invention may be
administered with any interleukin, including, but not limited to,
IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,
IL-18, IL-19, IL-20, and IL-21.
[1478] In an additional embodiment, the Therapeutics of the
invention are administered in combination with angiogenic proteins.
Angiogenic proteins that may be administered with the Therapeutics
of the invention include, but are not limited to, Glioma Derived
Growth Factor (GDGF), as disclosed in European Patent Number
EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed
in European Patent Number EP-6821 10; Platelet Derived Growth
Factor-B (PDGF-B), as disclosed in European Patent Number
EP-282317; Placental Growth Factor (PlGF), as disclosed in
International Publication Number WO 92/06194; Placental Growth
Factor-2 (PIGF-2), as disclosed in Hauser et al., Gorwth Factors,
4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as
disclosed in International Publication Number WO 90/13649; Vascular
Endothelial Growth Factor-A (VEGF-A), as disclosed in European
Patent Number EP-506477; Vascular Endothelial Growth Factor-2
(VEGF-2), as disclosed in International Publication Number WO
96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular
Endothelial Growth Factor B-186 (VEGF-B 186), as disclosed in
International Publication Number WO 96/26736; Vascular Endothelial
Growth Factor-D (VEGF-D), as disclosed in International Publication
Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D),
as disclosed in International Publication Number WO 98/07832; and
Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in
German Patent Number DE19639601. The above mentioned references are
incorporated herein by reference herein.
[1479] In an additional embodiment, the Therapeutics of the
invention are administered in combination with hematopoietic growth
factors. Hematopoietic growth factors that may be administered with
the Therapeutics of the invention include, but are not limited to,
LEUKINE.TM. (SARGRAMOSTIM.TM.) and NEUPOGEN.TM.
(FILGRASTIM.TM.).
[1480] In an additional embodiment, the Therapeutics of the
invention are administered in combination with Fibroblast Growth
Factors. Fibroblast Growth Factors that may be administered with
the Therapeutics of the invention include, but are not limited to,
FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9,
FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
[1481] In additional embodiments, the Therapeutics of the invention
are administered in combination with other therapeutic or
prophylactic regimens, such as, for example, radiation therapy.
Example 24
Method of Treating Decreased Levels of the Polypeptide
[1482] The present invention relates to a method for treating an
individual in need of an increased level of a polypeptide of the
invention in the body comprising administering to such an
individual a composition comprising a therapeutically effective
amount of an agonist of the invention (including polypeptides of
the invention). Moreover, it will be appreciated that conditions
caused by a decrease in the standard or normal expression level of
a secreted protein in an individual can be treated by administering
the polypeptide of the present invention, preferably in the
secreted form. Thus, the invention also provides a method of
treatment of an individual in need of an increased level of the
polypeptide comprising administering to such an individual a
Therapeutic comprising an amount of the polypeptide to increase the
activity level of the polypeptide in such an individual.
[1483] For example, a patient with decreased levels of a
polypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide
for six consecutive days. Preferably, the polypeptide is in the
secreted form. The exact details of the dosing scheme, based on
administration and formulation, are provided in Example 23.
Example 25
Method of Treating Increased Levels of the Polypeptide
[1484] The present invention also relates to a method of treating
an individual in need of a decreased level of a polypeptide of the
invention in the body comprising administering to such an
individual a composition comprising a therapeutically effective
amount of an antagonist of the invention (including polypeptides
and antibodies of the invention).
[1485] In one example, antisense technology is used to inhibit
production of a polypeptide of the present invention. This
technology is one example of a method of decreasing levels of a
polypeptide, preferably a secreted form, due to a variety of
etiologies, such as cancer. For example, a patient diagnosed with
abnormally increased levels of a polypeptide is administered
intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and
3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day
rest period if the treatment was well tolerated. The formulation of
the antisense polynucleotide is provided in Example 23.
Example 26
Method of Treatment Using Gene Therapy-Ex Vivo
[1486] One method of gene therapy transplants fibroblasts, which
are capable of expressing a polypeptide, onto a patient. Generally,
fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in tissue-culture medium and separated
into small pieces. Small chunks of the tissue are placed on a wet
surface of a tissue culture flask, approximately ten pieces are
placed in each flask. The flask is turned upside down, closed tight
and left at room temperature over night. After 24 hours at room
temperature, the flask is inverted and the chunks of tissue remain
fixed to the bottom of the flask and fresh media (e.g., Ham's F12
media, with 10% FBS, penicillin and streptomycin) is added. The
flasks are then incubated at 37 degree C. for approximately one
week.
[1487] At this time, fresh media is added and subsequently changed
every several days. After an additional two weeks in culture, a
monolayer of fibroblasts emerge. The monolayer is trypsinized and
scaled into larger flasks.
[1488] pMV-7 (Kirschmeier, P.T. et al., DNA, 7:219-25 (1988)),
flanked by the long terminal repeats of the Moloney murine sarcoma
virus, is digested with EcoRI and HindIII and subsequently treated
with calf intestinal phosphatase. The linear vector is fractionated
on agarose gel and purified, using glass beads.
[1489] The cDNA encoding a polypeptide of the present invention can
be amplified using PCR primers which correspond to the 5' and 3'
end sequences respectively as set forth in Example 1 using primers
and having appropriate restriction sites and initiation/stop
codons, if necessary. Preferably, the 5' primer contains an EcoRI
site and the 3' primer includes a HindIII site. Equal quantities of
the Moloney murine sarcoma virus linear backbone and the amplified
EcoRI and HindIII fragment are added together, in the presence of
T4 DNA ligase. The resulting mixture is maintained under conditions
appropriate for ligation of the two fragments. The ligation mixture
is then used to transform bacteria HB101, which are then plated
onto agar containing kanamycin for the purpose of confirming that
the vector has the gene of interest properly inserted.
[1490] The amphotropic pA317 or GP+am12 packaging cells are grown
in tissue culture to confluent density in Dulbecco's Modified
Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and
streptomycin. The MSV vector containing the gene is then added to
the media and the packaging cells transduced with the vector. The
packaging cells now produce infectious viral particles containing
the gene (the packaging cells are now referred to as producer
cells).
[1491] Fresh media is added to the transduced producer cells, and
subsequently, the media is harvested from a 10 cm plate of
confluent producer cells. The spent media, containing the
infectious viral particles, is filtered through a millipore filter
to remove detached producer cells and this media is then used to
infect fibroblast cells. Media is removed from a sub-confluent
plate of fibroblasts and quickly replaced with the media from the
producer cells. This media is removed and replaced with fresh
media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is very low, then it is necessary to use a retroviral vector
that has a selectable marker, such as neo or his. Once the
fibroblasts have been efficiently infected, the fibroblasts are
analyzed to determine whether protein is produced.
[1492] The engineered fibroblasts are then transplanted onto the
host, either alone or after having been grown to confluence on
cytodex 3 microcarrier beads.
Example 27
Gene Therapy Using Endogenous Genes Corresponding To
Polynucleotides of the Invention
[1493] Another method of gene therapy according to the present
invention involves operably associating the endogenous
polynucleotide sequence of the invention with a promoter via
homologous recombination as described, for example, in U.S. Pat.
No.: 5,641,670, issued Jun. 24, 1997; International Publication NO:
WO 96/29411, published Sep. 26, 1996; International Publication NO:
WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl.
Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature,
342:435-438 (1989). This method involves the activation of a gene
which is present in the target cells, but which is not expressed in
the cells, or is expressed at a lower level than desired.
[1494] Polynucleotide constructs are made which contain a promoter
and targeting sequences, which are homologous to the 5' non-coding
sequence of endogenous polynucleotide sequence, flanking the
promoter. The targeting sequence will be sufficiently near the 5'
end of the polynucleotide sequence so the promoter will be operably
linked to the endogenous sequence upon homologous recombination.
The promoter and the targeting sequences can be amplified using
PCR. Preferably, the amplified promoter contains distinct
restriction enzyme sites on the 5' and 3' ends. Preferably, the 3'
end of the first targeting sequence contains the same restriction
enzyme site as the 5' end of the amplified promoter and the 5' end
of the second targeting sequence contains the same restriction site
as the 3' end of the amplified promoter.
[1495] The amplified promoter and the amplified targeting sequences
are digested with the appropriate restriction enzymes and
subsequently treated with calf intestinal phosphatase. The digested
promoter and digested targeting sequences are added together in the
presence of T4 DNA ligase. The resulting mixture is maintained
under conditions appropriate for ligation of the two fragments. The
construct is size fractionated on an agarose gel then purified by
phenol extraction and ethanol precipitation.
[1496] In this Example, the polynucleotide constructs are
administered as naked polynucleotides via electroporation. However,
the polynucleotide constructs may also be administered with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, precipitating agents, etc. Such methods
of delivery are known in the art.
[1497] Once the cells are transfected, homologous recombination
will take place which results in the promoter being operably linked
to the endogenous polynucleotide sequence. This results in the
expression of polynucleotide corresponding to the polynucleotide in
the cell. Expression may be detected by immunological staining, or
any other method known in the art.
[1498] Fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in DMEM+ 10% fetal calf serum.
Exponentially growing or early stationary phase fibroblasts are
trypsinized and rinsed from the plastic surface with nutrient
medium. An aliquot of the cell suspension is removed for counting,
and the remaining cells are subjected to centrifugation. The
supernatant is aspirated and the pellet is resuspended in 5 ml of
electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl,
0.7 mM Na.sub.2 HPO.sub.4, 6 mM dextrose). The cells are
recentrifuged, the supernatant aspirated, and the cells resuspended
in electroporation buffer containing 1 mg/ml acetylated bovine
serum albumin. The final cell suspension contains approximately
3.times.10.sup.6 cells/ml. Electroporation should be performed
immediately following resuspension.
[1499] Plasmid DNA is prepared according to standard techniques.
For example, to construct a plasmid for targeting to the locus
corresponding to the polynucleotide of the invention, plasmid pUC
18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMV
promoter is amplified by PCR with an XbaI site on the 5' end and a
BamHI site on the 3'end. Two non-coding sequences are amplified via
PCR: one non-coding sequence (fragment 1) is amplified with a
HindIII site at the 5' end and an Xba site at the 3'end; the other
non-coding sequence (fragment 2) is amplified with a BamHI site at
the 5'end and a HindIII site at the 3'end. The CMV promoter and the
fragments (1 and 2) are digested with the appropriate enzymes (CMV
promoter--XbaI and BamHI; fragment 1--XbaI; fragment 2--BamHI) and
ligated together. The resulting ligation product is digested with
HindIII, and ligated with the HindIII-digested pUC18 plasmid.
[1500] Plasmid DNA is added to a sterile cuvette with a 0.4 cm
electrode gap (Bio-Rad). The final DNA concentration is generally
at least 120 .mu.g/ml. 0.5 ml of the cell suspension (containing
approximately 1.5..times.10.sup.6 cells) is then added to the
cuvette, and the cell suspension and DNA solutions are gently
mixed. Electroporation is performed with a Gene-Pulser apparatus
(Bio-Rad). Capacitance and voltage are set at 960 .mu.F and 250-300
V, respectively. As voltage increases, cell survival decreases, but
the percentage of surviving cells that stably incorporate the
introduced DNA into their genome increases dramatically. Given
these parameters, a pulse time of approximately 14-20 mSec should
be observed.
[1501] Electroporated cells are maintained at room temperature for
approximately 5 min, and the contents of the cuvette are then
gently removed with a sterile transfer pipette. The cells are added
directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf
serum) in a 10 cm dish and incubated at 37 degree C. The following
day, the media is aspirated and replaced with 10 ml of fresh media
and incubated for a further 16-24 hours.
[1502] The engineered fibroblasts are then injected into the host,
either alone or after having been grown to confluence on cytodex 3
microcarrier beads. The fibroblasts now produce the protein
product. The fibroblasts can then be introduced into a patient as
described above.
Example 28
Method of Treatment Using Gene Therapv--In Vivo
[1503] Another aspect of the present invention is using in vivo
gene therapy methods to treat disorders, diseases and conditions.
The gene therapy method relates to the introduction of naked
nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an
animal to increase or decrease the expression of the polypeptide.
The polynucleotide of the present invention may be operatively
linked to a promoter or any other genetic elements necessary for
the expression of the polypeptide by the target tissue. Such gene
therapy and delivery techniques and methods are known in the art,
see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos. 5,693,622,
5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479
(1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff,
Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene
Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation
94(12):3281-3290 (1996) (incorporated herein by reference).
[1504] The polynucleotide constructs may be delivered by any method
that delivers injectable materials to the cells of an animal, such
as, injection into the interstitial space of tissues (heart,
muscle, skin, lung, liver, intestine and the like). The
polynucleotide constructs can be delivered in a pharmaceutically
acceptable liquid or aqueous carrier.
[1505] The term "naked" polynucleotide, DNA or RNA, refers to
sequences that are free from any delivery vehicle that acts to
assist, promote, or facilitate entry into the cell, including viral
sequences, viral particles, liposome formulations, lipofectin or
precipitating agents and the like. However, the polynucleotides of
the present invention may also be delivered in liposome
formulations (such as those taught in Felgner P.L. et al. (1995)
Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol.
Cell 85(1):1-7) which can be prepared by methods well known to
those skilled in the art.
[1506] The polynucleotide vector constructs used in the gene
therapy method are preferably constructs that will not integrate
into the host genome nor will they contain sequences that allow for
replication. Any strong promoter known to those skilled in the art
can be used for driving the expression of DNA. Unlike other gene
therapies techniques, one major advantage of introducing naked
nucleic acid sequences into target cells is the transitory nature
of the polynucleotide synthesis in the cells. Studies have shown
that non-replicating DNA sequences can be introduced into cells to
provide production of the desired polypeptide for periods of up to
six months.
[1507] The polynucleotide construct can be delivered to the
interstitial space of tissues within the an animal, including of
muscle, skin, brain, lung, liver, spleen, bone marrow, thymus,
heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous
system, eye, gland, and connective tissue. Interstitial space of
the tissues comprises the intercellular fluid, mucopolysaccharide
matrix among the reticular fibers of organ tissues, elastic fibers
in the walls of vessels or chambers, collagen fibers of fibrous
tissues, or that same matrix within connective tissue ensheathing
muscle cells or in the lacunae of bone. It is similarly the space
occupied by the plasma of the circulation and the lymph fluid of
the lymphatic channels. Delivery to the interstitial space of
muscle tissue is preferred for the reasons discussed below. They
may be conveniently delivered by injection into the tissues
comprising these cells. They are preferably delivered to and
expressed in persistent, non-dividing cells which are
differentiated, although delivery and expression may be achieved in
non-differentiated or less completely differentiated cells, such
as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle cells are particularly competent in their ability to take up
and express polynucleotides.
[1508] For the naked polynucleotide injection, an effective dosage
amount of DNA or RNA will be in the range of from about 0.05 g/kg
body weight to about 50 mg/kg body weight. Preferably the dosage
will be from about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration. The
preferred route of administration is by the parenteral route of
injection into the interstitial space of tissues. However, other
parenteral routes may also be used, such as, inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial
tissues, throat or mucous membranes of the nose. In addition, naked
polynucleotide constructs can be delivered to arteries during
angioplasty by the catheter used in the procedure.
[1509] The dose response effects of injected polynucleotide in
muscle in vivo is determined as follows. Suitable template DNA for
production of mRNA coding for polypeptide of the present invention
is prepared in accordance with a standard recombinant DNA
methodology. The template DNA, which may be either circular or
linear, is either used as naked DNA or complexed with liposomes.
The quadriceps muscles of mice are then injected with various
amounts of the template DNA.
[1510] Five to six week old female and male Balb/C mice are
anesthetized by intraperitoneal injection with 0.3 ml of 2.5%
Avertin. A 1.5 cm incision is made on the anterior thigh, and the
quadriceps muscle is directly visualized. The template DNA is
injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge
needle over one minute, approximately 0.5 cm from the distal
insertion site of the muscle into the knee and about 0.2 cm deep. A
suture is placed over the injection site for future localization,
and the skin is closed with stainless steel clips.
[1511] After an appropriate incubation time (e.g., 7 days) muscle
extracts are prepared by excising the entire quadriceps. Every
fifth 15 um cross-section of the individual quadriceps muscles is
histochemically stained for protein expression. A time course for
protein expression may be done in a similar fashion except that
quadriceps from different mice are harvested at different times.
Persistence of DNA in muscle following injection may be determined
by Southern blot analysis after preparing total cellular DNA and
HIRT supernatants from injected and control mice. The results of
the above experimentation in mice can be use to extrapolate proper
dosages and other treatment parameters in humans and other animals
using naked DNA.
Example 29
Transgenic Animals
[1512] The polypeptides of the invention can also be expressed in
transgenic animals. Animals of any species, including, but not
limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs,
micro-pigs, goats, sheep, cows and non-human primates, e.g.,
baboons, monkeys, and chimpanzees may be used to generate
transgenic animals. In a specific embodiment, techniques described
herein or otherwise known in the art, are used to express
polypeptides of the invention in humans, as part of a gene therapy
protocol.
[1513] Any technique known in the art may be used to introduce the
transgene (i.e., polynucleotides of the invention) into animals to
produce the founder lines of transgenic animals. Such techniques
include, but are not limited to, pronuclear microinjection
(Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994);
Carver et al., Biotechnology (NY) 11: 1263-1270 (1993); Wright et
al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S.
Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into
germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA
82:6148-6152 (1985)), blastocysts or embryos; gene targeting in
embryonic stem cells (Thompson et al., Cell 56:313-321 (1989));
electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.
3:1803-1814 (1983)); introduction of the polynucleotides of the
invention using a gene gun (see, e.g., Ulmer et al., Science
259:1745 (1993); introducing nucleic acid constructs into embryonic
pleuripotent stem cells and transferring the stem cells back into
the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,
Cell 57:717-723 (1989); etc. For a review of such techniques, see
Gordon, "Transgenic Animals," Intl. Rev. Cytol. 115:171-229 (1989),
which is incorporated by reference herein in its entirety.
[1514] Any technique known in the art may be used to produce
transgenic clones containing polynucleotides of the invention, for
example, nuclear transfer into enucleated oocytes of nuclei from
cultured embryonic, fetal, or adult cells induced to quiescence
(Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature
385:810-813 (1997)).
[1515] The present invention provides for transgenic animals that
carry the transgene in all their cells, as well as animals which
carry the transgene in some, but not all their cells, i.e., mosaic
animals or chimeric. The transgene may be integrated as a single
transgene or as multiple copies such as in concatamers, e.g.,
head-to-head tandems or head-to-tail tandems. The transgene may
also be selectively introduced into and activated in a particular
cell type by following, for example, the teaching of Lasko et al.
(Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The
regulatory sequences required for such a cell-type specific
activation will depend upon the particular cell type of interest,
and will be apparent to those of skill in the art. When it is
desired that the polynucleotide transgene be integrated into the
chromosomal site of the endogenous gene, gene targeting is
preferred. Briefly, when such a technique is to be utilized,
vectors containing some nucleotide sequences homologous to the
endogenous gene are designed for the purpose of integrating, via
homologous recombination with chromosomal sequences, into and
disrupting the function of the nucleotide sequence of the
endogenous gene. The transgene may also be selectively introduced
into a particular cell type, thus inactivating the endogenous gene
in only that cell type, by following, for example, the teaching of
Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory
sequences required for such a cell-type specific inactivation will
depend upon the particular cell type of interest, and will be
apparent to those of skill in the art.
[1516] Once transgenic animals have been generated, the expression
of the recombinant gene may be assayed utilizing standard
techniques. Initial screening may be accomplished by Southern blot
analysis or PCR techniques to analyze animal tissues to verify that
integration of the transgene has taken place. The level of mRNA
expression of the transgene in the tissues of the transgenic
animals may also be assessed using techniques which include, but
are not limited to, Northern blot analysis of tissue samples
obtained from the animal, in situ hybridization analysis, and
reverse transcriptase-PCR (rt-PCR). Samples of transgenic
gene-expressing tissue may also be evaluated immunocytochemically
or immunohistochemically using antibodies specific for the
transgene product.
[1517] Once the founder animals are produced, they may be bred,
inbred, outbred, or crossbred to produce colonies of the particular
animal. Examples of such breeding strategies include, but are not
limited to: outbreeding of founder animals with more than one
integration site in order to establish separate lines; inbreeding
of separate lines in order to produce compound transgenics that
express the transgene at higher levels because of the effects of
additive expression of each transgene; crossing of heterozygous
transgenic animals to produce animals homozygous for a given
integration site in order to both augment expression and eliminate
the need for screening of animals by DNA analysis; crossing of
separate homozygous lines to produce compound heterozygous or
homozygous lines; and breeding to place the transgene on a distinct
background that is appropriate for an experimental model of
interest.
[1518] Transgenic animals of the invention have uses which include,
but are not limited to, animal model systems useful in elaborating
the biological function of polypeptides of the present invention,
studying diseases, disorders, and/or conditions associated with
aberrant expression, and in screening for compounds effective in
ameliorating such diseases, disorders, and/or conditions.
Example 30
Knock-Out Animals
[1519] Endogenous gene expression can also be reduced by
inactivating or "knocking out" the gene and/or its promoter using
targeted homologous recombination. (E.g., see Smithies et al.,
Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512
(1987); Thompson et al., Cell 5:313-321 (1989); each of which is
incorporated by reference herein in its entirety). For example, a
mutant, non-functional polynucleotide of the invention (or a
completely unrelated DNA sequence) flanked by DNA homologous to the
endogenous polynucleotide sequence (either the coding regions or
regulatory regions of the gene) can be used, with or without a
selectable marker and/or a negative selectable marker, to transfect
cells that express polypeptides of the invention in vivo. In
another embodiment, techniques known in the art are used to
generate knockouts in cells that contain, but do not express the
gene of interest. Insertion of the DNA construct, via targeted
homologous recombination, results in inactivation of the targeted
gene. Such approaches are particularly suited in research and
agricultural fields where modifications to embryonic stem cells can
be used to generate animal offspring with an inactive targeted gene
(e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra).
However this approach can be routinely adapted for use in humans
provided the recombinant DNA constructs are directly administered
or targeted to the required site in vivo using appropriate viral
vectors that will be apparent to those of skill in the art.
[1520] In further embodiments of the invention, cells that are
genetically engineered to express the polypeptides of the
invention, or alternatively, that are genetically engineered not to
express the polypeptides of the invention (e.g., knockouts) are
administered to a patient in vivo. Such cells may be obtained from
the patient (i.e., animal, including human) or an MHC compatible
donor and can include, but are not limited to fibroblasts, bone
marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle
cells, endothelial cells etc. The cells are genetically engineered
in vitro using recombinant DNA techniques to introduce the coding
sequence of polypeptides of the invention into the cells, or
alternatively, to disrupt the coding sequence and/or endogenous
regulatory sequence associated with the polypeptides of the
invention, e.g., by transduction (using viral vectors, and
preferably vectors that integrate the transgene into the cell
genome) or transfection procedures, including, but not limited to,
the use of plasmids, cosmids, YACs, naked DNA, electroporation,
liposomes, etc. The coding sequence of the polypeptides of the
invention can be placed under the control of a strong constitutive
or inducible promoter or promoter/enhancer to achieve expression,
and preferably secretion, of the polypeptides of the invention. The
engineered cells which express and preferably secrete the
polypeptides of the invention can be introduced into the patient
systemically, e.g., in the circulation, or intraperitoneally.
[1521] Alternatively, the cells can be incorporated into a matrix
and implanted in the body, eg., genetically engineered fibroblasts
can be implanted as part of a skin graft; genetically engineered
endothelial cells can be implanted as part of a lymphatic or
vascular graft. (See, for example, Anderson et al. U.S. Pat. No.
5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each
of which is incorporated by reference herein in its entirety).
[1522] When the cells to be administered are non-autologous or
non-MHC compatible cells, they can be administered using well known
techniques which prevent the development of a host immune response
against the introduced cells. For example, the cells may be
introduced in an encapsulated form which, while allowing for an
exchange of components with the immediate extracellular
environment, does not allow the introduced cells to be recognized
by the host immune system.
[1523] Transgenic and "knock-out" animals of the invention have
uses which include, but are not limited to, animal model systems
useful in elaborating the biological function of polypeptides of
the present invention, studying diseases, disorders, and/or
conditions associated with aberrant expression, and in screening
for compounds effective in ameliorating such diseases, disorders,
and/or conditions.
Example 31
Production of an Antibody
[1524] Hybridoma Technology
[1525] The antibodies of the present invention can be prepared by a
variety of methods. (See, Current Protocols, Chapter 2. ) As one
example of such methods, cells expressing polypeptide(s) of the
invention are administered to an animal to induce the production of
sera containing polyclonal antibodies. In a preferred method, a
preparation of polypeptide(s) of the invention is prepared and
purified to render it substantially free of natural contaminants.
Such a preparation is then introduced into an animal in order to
produce polyclonal antisera of greater specific activity.
[1526] Monoclonal antibodies specific for polypeptide(s) of the
invention are prepared using hybridoma technology. (Kohler et al.,
Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511
(1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et
al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier,
N.Y., pp. 563-681 (1981)). In general, an animal (preferably a
mouse) is immunized with polypeptide(s) of the invention, or, more
preferably, with a secreted polypeptide-expressing cell. Such
polypeptide-expressing cells are cultured in any suitable tissue
culture medium, preferably in Earle's modified Eagle's medium
supplemented with 10% fetal bovine serum (inactivated at about
56.degree. C.), and supplemented with about 10 g/l of nonessential
amino acids, about 1,000 U/ml of penicillin, and about 100 .mu.g/ml
of streptomycin.
[1527] The splenocytes of such mice are extracted and fused with a
suitable myeloma cell line. Any suitable myeloma cell line may be
employed in accordance with the present invention; however, it is
preferable to employ the parent myeloma cell line (SP20), available
from the ATCC. After fusion, the resulting hybridoma cells are
selectively maintained in HAT medium, and then cloned by limiting
dilution as described by Wands et al. (Gastroenterology 80:225-232
(1981)). The hybridoma cells obtained through such a selection are
then assayed to identify clones which secrete antibodies capable of
binding the polypeptide(s) of the invention.
[1528] Alternatively, additional antibodies capable of binding
polypeptide(s) of the invention can be produced in a two-step
procedure using anti-idiotypic antibodies. Such a method makes use
of the fact that antibodies are themselves antigens, and therefore,
it is possible to obtain an antibody which binds to a second
antibody. In accordance with this method, protein specific
antibodies are used to immunize an animal, preferably a mouse. The
splenocytes of such an animal are then used to produce hybridoma
cells, and the hybridoma cells are screened to identify clones
which produce an antibody whose ability to bind to the
polypeptide(s) of the invention protein-specific antibody can be
blocked by polypeptide(s) of the invention. Such antibodies
comprise anti-idiotypic antibodies to the polypeptide(s) of the
invention protein-specific antibody and are used to immunize an
animal to induce formation of further polypeptide(s) of the
invention protein-specific antibodies.
[1529] For in vivo use of antibodies in humans, an antibody is
"humanized". Such antibodies can be produced using genetic
constructs derived from hybridoma cells producing the monoclonal
antibodies described above. Methods for producing chimeric and
humanized antibodies are known in the art and are discussed herein.
(See, for review, Morrison, Science 229:1202 (1985); Oi et al.,
BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;
Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature
314:268 (1985).)
[1530] Isolation Of Antibody Fragments Directed
[1531] Polypeptide(s) of the Invention From A Library Of scFvs
[1532] Naturally occurring V-genes isolated from human PBLs are
constructed into a library of antibody fragments which contain
reactivities against polypeptide(s) of the invention to which the
donor may or may not have been exposed (see e.g., U.S. Pat. No.
5,885,793 incorporated herein by reference in its entirety).
[1533] Rescue of the Library. A library of scFvs is constructed
from the RNA of human PBLs as described in PCT publication WO
92/01047. To rescue phage displaying antibody fragments,
approximately 109 E. coli harboring the phagemid are used to
inoculate 50 ml of 2.times.TY containing 1% glucose and 100
.mu.g/ml of ampicillin (2.times.TY-AMP-GLU) and grown to an O.D. of
0.8 with shaking. Five ml of this culture is used to innoculate 50
ml of 2.times.TY-AMP-GLU, 2.times.108 TU of delta gene 3 helper
(M13 delta gene III, see PCT publication WO 92/01047) are added and
the culture incubated at 37.degree. C. for 45 minutes without
shaking and then at 37.degree. C. for 45 minutes with shaking. The
culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet
resuspended in 2 liters of 2.times.TY containing 100 .mu.g/ml
ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are
prepared as described in PCT publication WO 92/01047.
[1534] M13 delta gene III is prepared as follows: M13 delta gene
III helper phage does not encode gene III protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious Ml 3 delta gene III particles are
made by growing the helper phage in cells harboring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),
resuspended in 300 ml 2.times.TY broth containing 100 .mu.g
ampicillin/ml and 25 .mu.g kanamycin/ml (2.times.TY-AMP-KAN) and
grown overnight, shaking at 37.degree. C. Phage particles are
purified and concentrated from the culture medium by two
PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS
and passed through a 0.45 .mu.m filter (Minisart NML; Sartorius) to
give a final concentration of approximately 1013 transducing
units/ml (ampicillin-resistant clones).
[1535] Panning of the Library. Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100 .mu.g/ml or 10 .mu.g/ml of
a polypeptide of the present invention. Tubes are blocked with 2%
Marvel-PBS for 2 hours at 37.degree. C. and then washed 3 times in
PBS. Approximately 1013 TU of phage is applied to the tube and
incubated for 30 minutes at room temperature tumbling on an over
and under turntable and then left to stand for another 1.5 hours.
Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with
PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and
rotating 15 minutes on an under and over turntable after which the
solution is immediately neutralized with 0.5 ml of 1.0 M Tris-HCl,
pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1
by incubating eluted phage with bacteria for 30 minutes at
37.degree. C. The E. coli are then plated on TYE plates containing
1% glucose and 100 .mu.g/ml ampicillin. The resulting bacterial
library is then rescued with delta gene 3 helper phage as described
above to prepare phage for a subsequent round of selection. This
process is then repeated for a total of 4 rounds of affinity
purification with tube-washing increased to 20 times with PBS, 0.1%
Tween-20 and 20 times with PBS for rounds 3 and 4.
[1536] Characterization of Binders. Eluted phage from the 3rd and
4th rounds of selection are used to infect E. coli HB 2151 and
soluble scFv is produced (Marks, et al., 1991) from single colonies
for assay. ELISAs are performed with microtitre plates coated with
either 10 pg/ml of the polypeptide of the present invention in 50
mM bicarbonate pH 9.6. Clones positive in ELISA are further
characterized by PCR fingerprinting (see, e.g., PCT publication WO
92/01047) and then by sequencing. These ELISA positive clones may
also be further characterized by techniques known in the art, such
as, for example, epitope mapping, binding affinity, receptor signal
transduction, ability to block or competitively inhibit
antibody/antigen binding, and competitive agonistic or antagonistic
activity.
Example 32
Assays Detecting Stimulation or Inhibition of B cell Proliferation
and Differentiation
[1537] Generation of functional humoral immune responses requires
both soluble and cognate signaling between B-lineage cells and
their microenvironment. Signals may impart a positive stimulus that
allows a B-lineage cell to continue its programmed development, or
a negative stimulus that instructs the cell to arrest its current
developmental pathway. To date, numerous stimulatory and inhibitory
signals have been found to influence B cell responsiveness
including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and
IL-15. Interestingly, these signals are by themselves weak
effectors but can, in combination with various co-stimulatory
proteins, induce activation, proliferation, differentiation,
homing, tolerance and death among B cell populations.
[1538] One of the best studied classes of B-cell co-stimulatory
proteins is the TNF-superfamily. Within this family CD40, CD27, and
CD30 along with their respective ligands CD154, CD70, and CD153
have been found to regulate a variety of immune responses. Assays
which allow for the detection and/or observation of the
proliferation and differentiation of these B-cell populations and
their precursors are valuable tools in determining the effects
various proteins may have on these B-cell populations in terms of
proliferation and differentiation. Listed below are two assays
designed to allow for the detection of the differentiation,
proliferation, or inhibition of B-cell populations and their
precursors.
[1539] In Vitro Assay-Purified polypeptides of the invention, or
truncated forms thereof, is assessed for its ability to induce
activation, proliferation, differentiation or inhibition and/or
death in B-cell populations and their precursors. The activity of
the polypeptides of the invention on purified human tonsillar B
cells, measured qualitatively over the dose range from 0.1 to
10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation
assay in which purified tonsillar B cells are cultured in the
presence of either formalin-fixed Staphylococcus aureus Cowan I
(SAC) or immobilized anti-human IgM antibody as the priming agent.
Second signals such as IL-2 and IL-15 synergize with SAC and IgM
crosslinking to elicit B cell proliferation as measured by
tritiated-thymidine incorporation. Novel synergizing agents can be
readily identified using this assay. The assay involves isolating
human tonsillar B cells by magnetic bead (MACS) depletion of
CD3-positive cells. The resulting cell population is greater than
95% B cells as assessed by expression of CD45R(B220).
[1540] Various dilutions of each sample are placed into individual
wells of a 96-well plate to which are added 10.sup.5 B-cells
suspended in culture medium (RPMI 1640 containing 10% FBS,
5.times.10.sup.-5 M 2ME, 100 U/ml penicillin, 10 ug/ml
streptomycin, and 10.sup.-5 dilution of SAC) in a total volume of
150 ul. Proliferation or inhibition is quantitated by a 20 h pulse
(1 uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post
factor addition. The positive and negative controls are IL2 and
medium respectively.
[1541] In Vivo Assay--BALB/c mice are injected (i.p.) twice per day
with buffer only, or 2 mg/Kg of a polypeptide of the invention, or
truncated forms thereof. Mice receive this treatment for 4
consecutive days, at which time they are sacrificed and various
tissues and serum collected for analyses. Comparison of H&E
sections from normal spleens and spleens treated with polypeptides
of the invention identify the results of the activity of the
polypeptides on spleen cells, such as the diffusion of
peri-arterial lymphatic sheaths, and/or significant increases in
the nucleated cellularity of the red pulp regions, which may
indicate the activation of the differentiation and proliferation of
B-cell populations. Immunohistochemical studies using a B cell
marker, anti-CD45R(B220), are used to determine whether any
physiological changes to splenic cells, such as splenic
disorganization, are due to increased B-cell representation within
loosely defined B-cell zones that infiltrate established T-cell
regions.
[1542] Flow cytometric analyses of the spleens from mice treated
with polypeptide is used to indicate whether the polypeptide
specifically increases the proportion of ThB+, CD45R(B220)dull B
cells over that which is observed in control mice.
[1543] Likewise, a predicted consequence of increased mature B-cell
representation in vivo is a relative increase in serum Ig titers.
Accordingly, serum IgM and IgA levels are compared between buffer
and polypeptide-treated mice.
[1544] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides of the invention (e.g., gene therapy), agonists,
and/or antagonists of polynucleotides or polypeptides of the
invention.
Example 33
T Cell Proliferation Assay
[1545] Proliferation assay for Resting PBLs.
[1546] A CD3-induced proliferation assay is performed on PBMCs and
is measured by the uptake of .sup.3H-thymidine. The assay is
performed as follows. Ninety-six well plates are coated with 100
microliters per well of mAb to CD3 (HIT3a, Pharmingen) or
isotype-matched control mAb (B33.1) overnight at 4.degree. C. (1
microgram/ml in 0.05 M bicarbonate buffer, pH 9.5), then washed
three times with PBS. PBMC are isolated by F/H gradient
centrifugation from human peripheral blood and added to
quadruplicate wells (5.times.10.sup.4/well) of mAb coated plates in
RPMI containing 10% FCS and P/S in the presence of varying
concentrations of TNF Delta and/or TNF Epsilon protein (total
volume 200 microliters). Relevant protein buffer and medium alone
are controls. After 48 hr. culture at 37.degree. C., plates are
spun for 2 min. at 1000 rpm and 100 microliters of supernatant is
removed and stored -20.degree. C. for measurement of IL-2 (or other
cytokines) if effect on proliferation is observed. Wells are
supplemented with 100 microliters of medium containing 0.5
microcuries of .sup.3H-thymidine and cultured at 37.degree. C. for
18-24 hr. Wells are harvested and incorporation of
.sup.3H-thymidine used as a measure of proliferation. Anti-CD3
alone is the positive control for proliferation. IL-2 (100 U/ml) is
also used as a control which enhances proliferation. Control
antibody which does not induce proliferation of T cells is used as
the negative controls for the effects of TNF Delta and/or TNF
Epsilon proteins.
[1547] Alternatively, a proliferation assay on resting PBL
(peripheral blood lymphocytes) is measured by the up-take of
.sup.3H-thymidine. The assay is performed as follows. PBMC are
isolated by Ficoll (LSM, ICN Biotechnologies, Aurora, Ohio.)
gradient centrifugation from human peripheral blood, and are
cultured overnight in 10% (Fetal Calf Serum, Biofluids, Rockville,
Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). This overnight incubation
period allows the adherent cells to attach to the plastic, which
results in a lower background in the assay as there are fewer cells
that can act as antigen presenting cells or that might be producing
growth factors. The following day the non-adherent cells are
collected, washed and used in the proliferation assay. The assay is
performed in a 96 well plate using 2.times.10.sup.4 cells/well in a
final volume of 200 microliters. The supernatants (e.g., CHO or
293T supernatants) expressing the protein of interest are tested at
a 30% final dilution, therefore 60 ul are added to 140 ul of 10%
FCS/RPMI containing the cells. Control supernatants are used at the
same final dilution and express the following proteins: vector
(negative control), IL-2 (*), IFN.gamma., TNF.alpha., IL-10 and
TR2. In addition to the control supernatants, recombinant human
IL-2 (R & D Systems, Minneapolois, Minn.) at a final
concentration of 100 ng/ml is also used. After 24 hours of culture,
each well is pulsed with 1 uCi of .sup.3H-thymidine (Nen, Boston,
Mass.). Cells are then harvested 20 hours following pulsing and
incorporation of .sup.3H-thymidine is used as a measure of
proliferation. Results are expressed as an average of triplicate
samples plus or minus standard error. [1548] (*) The amount of the
control cytokines IL-2, IFN.gamma., TNF.alpha. and IL-10 produced
in each transfection varies between 300 pg to 5 ng/ml.
[1549] Costimulation Assay.
[1550] A costimulation assay on resting PBL (peripheral blood
lymphocytes) is performed in the presence of immobilized antibodies
to CD3 and CD28. The use of antibodies specific for the invariant
regions of CD3 mimic the induction of T cell activation that would
occur through stimulation of the T cell receptor by an antigen.
Cross-linking of the TCR (first signal) in the absence of a
costimulatory signal (second signal) causes very low induction of
proliferation and will eventually result in a state of "anergy",
which is characterized by the absence of growth and inability to
produce cytokines. The addition of a costimulatory signal such as
an antibody to CD28, which mimics the action of the costimulatory
molecule. B7-1 expressed on activated APCs, results in enhancement
of T cell responses including cell survival and production of IL-2.
Therefore this type of assay allows to detect both positive and
negative effects caused by addition of supernatants expressing the
proteins of interest on T cell proliferation.
[1551] The assay is performed as follows. Ninety-six well plates
are coated with 100 ng/ml anti-CD3 and 5 ug/ml anti-CD28
(Pharmingen, San Diego, Calif.) in a final volume of 100 ul and
incubated overnight at 4C. Plates are washed twice with PBS before
use. PBMC are isolated by Ficoll (LSM, ICN Biotechnologies, Aurora,
Ohio.) gradient centrifugation from human peripheral blood, and are
cultured overnight in 10% FCS(Fetal Calf Serum, Biofluids,
Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). This overnight
incubation period allows the adherent cells to attach to the
plastic, which results in a lower background in the assay as there
are fewer cells that can act as antigen presenting cells or that
might be producing growth factors. The following day the non
adherent cells are collected, washed and used in the proliferation
assay. The assay is performed in a 96 well plate using
2.times.10.sup.4 cells/well in a final volume of 200 ul. The
supernatants (e.g., CHO or 293T supernatants) expressing the
protein of interest are tested at a 30% final dilution, therefore
60 ul are added to 140 ul of 10% FCS/RPMI containing the cells.
Control supernatants are used at the same final dilution and
express the following proteins: vector only (negative control),
IL-2, IFN.gamma., TNF.alpha., IL-10 and TR2. In addition to the
control supernatants recombinant human IL-2 (R & D Systems,
Minneapolis, Minn.) at a final concentration of 10 ng/ml is also
used. After 24 hours of culture, each well is pulsed with 1 uCi of
.sup.3H-thymidine (Nen, Boston, Mass.). Cells are then harvested 20
hours following pulsing and incorporation of .sup.3H-thymidine is
used as a measure of proliferation. Results are expressed as an
average of triplicate samples plus or minus standard error.
[1552] Proliferation Assay for Preactivated-resting T Cells.
[1553] A proliferation assay on preactivated-resting T cells is
performed on cells that are previously activated with the lectin
phytohemagglutinin (PHA). Lectins are polymeric plant proteins that
can bind to residues on T cell surface glycoproteins including the
TCR and act as polyclonal activators. PBLs treated with PHA and
then cultured in the presence of low doses of IL-2 resemble
effector T cells. These cells are generally more sensitive to
further activation induced by growth factors such as IL-2. This is
due to the expression of high affinity IL-2 receptors that allows
this population to respond to amounts of IL-2 that are 100 fold
lower than what would have an effect on a naive T cell. Therefore
the use of this type of cells might enable to detect the effect of
very low doses of an unknown growth factor, that would not be
sufficient to induce proliferation on resting (naive) T cells.
[1554] The assay is performed as follows. PBMC are isolated by F/H
gradient centrifugation from human peripheral blood, and are
cultured in 10% FCS(Fetal Calf Serum, Biofluids, Rockville,
Md.)/RPMI (Gibco BRL, Gaithersburg, Md.) in the presence of 2 ug/ml
PHA (Sigma, Saint Louis, Mo.) for three days. The cells are then
washed in PBS and cultured in 10% FCS/RPMI in the presence of 5
ng/ml of human recombinant IL-2 (R & D Systems, Minneapolis,
Minn.) for 3 days. The cells are washed and rested in starvation
medium (1% FCS/RPMI) for 16 hours prior to the beginning of the
proliferation assay. An aliquot of the cells is analyzed by FACS to
determine the percentage of T cells (CD3 positive cells) present;
this usually ranges between 93-97% depending on the donor. The
assay is performed in a 96 well plate using 2.times.10.sup.4
cells/well in a final volume of 200 ul. The supernatants (e.g., CHO
or 293T supernatants) expressing the protein of interest are tested
at a 30% final dilution, therefore 60 ul are added to 140 ul of in
10% FCS/RPMI containing the cells. Control supernatants are used at
the same final dilution and express the following proteins: vector
(negative control), IL-2, IFN.gamma., TNF.alpha., IL-10 and TR2. In
addition to the control supernatants recombinant human IL-2 at a
final concentration of 10 ng/ml is also used. After 24 hours of
culture, each well is pulsed with 1 uCi of .sup.3H-thymidine(Nen,
Boston, Mass.). Cells are then harvested 20 hours following pulsing
and incorporation of .sup.3H-thymidine is used as a measure of
proliferation. Results are expressed as an average of triplicate
samples plus or minus standard error.
[1555] The studies described in this example test activity of
polypeptides of the invention. However, one skilled in the art
could easily modify the exemplified studies to test the activity of
polynucleotides of the invention (e.g., gene therapy), agonists,
and/or antagonists of polynucleotides or polypeptides of the
invention.
Example 34
Effect of Polypeptides of the Invention on the Expression of MHC
Class II Costimulatory and Adhesion Molecules and Cell
Differentiation of Monocytes and Monocyte-Derived Human Dendritic
Cells
[1556] Dendritic cells are generated by the expansion of
proliferating precursors found in the peripheral blood: adherent
PBMC or elutriated monocytic fractions are cultured for 7-10 days
with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells
have the characteristic phenotype of immature cells (expression of
CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with
activating factors, such as TNF-.alpha., causes a rapid change in
surface phenotype (increased expression of MHC class I and II,
costimulatory and adhesion molecules, downregulation of
FC.gamma.RII, upregulation of CD83). These changes correlate with
increased antigen-presenting capacity and with functional
maturation of the dendritic cells.
[1557] FACS analysis of surface antigens is performed as follows.
Cells are treated 1-3 days with increasing concentrations of
polypeptides of the invention or LPS (positive control), washed
with PBS containing 1% BSA and 0.02 mM sodium azide, and then
incubated with 1:20 dilution of appropriate FITC- or PE-labeled
monoclonal antibodies for 30 minutes at 4 degrees C. After an
additional wash, the labeled cells are analyzed by flow cytometry
on a FACScan (Becton Dickinson).
[1558] Effect on the production of cyctokines. Cytokines generated
by dendritic cells, in particular IL-12, are important in the
initiation of T-cell dependent immune responses. IL-12 strongly
influences the development of Th1 helper T-cell immune response,
and induces cytotoxic T and NK cell function. An ELISA is used to
measure the IL-12 release as follows. Dendritic cells (10.sup.6/ml)
are treated with increasing concentrations of polypeptides of the
invention for 24 hours. LPS (100 ng/ml) is added to the cell
culture as positive control. Supernatants from the cell cultures
are then collected and analyzed for IL-12 content using commercial
ELISA kit (e.g, R & D Systems (Minneapolis, Minn.)). The
standard protocols provided with the kits are used.
[1559] Effect on the expression of MHC Class II, costimulatory and
adhesion molecules. Three major families of cell surface antigens
can be identified on monocytes: adhesion molecules, molecules
involved in antigen presentation, and Fc receptor. Modulation of
the expression of MHC class II antigens and other costimulatory
molecules, such as B7 and ICAM-1, may result in changes in the
antigen presenting capacity of monocytes and ability to induce T
cell activation. Increase expression of Fc receptors may correlate
with improved monocyte cytotoxic activity, cytokine release and
phagocytosis.
[1560] FACS analysis is used to examine the surface antigens as
follows. Monocytes are treated 1-5 days with increasing
concentrations of polypeptides of the invention or LPS (positive
control), washed with PBS containing 1% BSA and 0.02 mM sodium
azide, and then incubated with 1:20 dilution of appropriate FITC-
or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C.
After an additional wash, the labeled cells are analyzed by flow
cytometry on a FACScan (Becton Dickinson).
[1561] Monocyte activation and/or increased survival. Assays for
molecules that activate (or alternatively, inactivate) monocytes
and/or increase monocyte survival (or alternatively, decrease
monocyte survival) are known in the art and may routinely be
applied to determine whether a molecule of the invention functions
as an inhibitor or activator of monocytes. Polypeptides, agonists,
or antagonists of the invention can be screened using the three
assays described below. For each of these assays, Peripheral blood
mononuclear cells (PBMC) are purified from single donor leukopacks
(American Red Cross, Baltimore, Md.) by centrifugation through a
Histopaque gradient (Sigma). Monocytes are isolated from PBMC by
counterflow centrifugal elutriation.
[1562] Monocyte Survival Assay. Human peripheral blood monocytes
progressively lose viability when cultured in absence of serum or
other stimuli. Their death results from internally regulated
process (apoptosis). Addition to the culture of activating factors,
such as TNF-alpha dramatically improves cell survival and prevents
DNA fragmentation. Propidium iodide (PI) staining is used to
measure apoptosis as follows. Monocytes are cultured for 48 hours
in polypropylene tubes in serum-free medium (positive control), in
the presence of 100 ng/ml TNF-alpha (negative control), and in the
presence of varying concentrations of the compound to be tested.
Cells are suspended at a concentration of 2.times.10.sup.6/ml in
PBS containing PI at a final concentration of 5 .mu.g/ml, and then
incubaed at room temperature for 5 minutes before FACScan analysis.
PI uptake has been demonstrated to correlate with DNA fragmentation
in this experimental paradigm.
[1563] Effect on cytokine release. An important function of
monocytes/macrophages is their regulatory activity on other
cellular populations of the immune system through the release of
cytokines after stimulation. An ELISA to measure cytokine release
is performed as follows. Human monocytes are incubated at a density
of 5.times.15 cells/ml with increasing concentrations of the a
polypeptide of the invention and under the same conditions, but in
the absence of the polypeptide. For IL-12 production, the cells are
primed overnight with IFN (100 U/ml) in presence of a polypeptide
of the invention. LPS (10 ng/ml) is then added. Conditioned media
are collected after 24 h and kept frozen until use. Measurement of
TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a
commercially available ELISA kit (e.g, R & D Systems
(Minneapolis, Minn.)) and applying the standard protocols provided
with the kit.
[1564] Oxidative burst. Purified monocytes are plated in 96-w plate
at 2-1.times.10.sup.5 cell/well. Increasing concentrations of
polypeptides of the invention are added to the wells in a total
volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine and
antibiotics). After 3 days incubation, the plates are centrifuged
and the medium is removed from the wells. To the macrophage
monolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10
mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM
phenol red and 19 U/ml of HRPO) is added, together with the
stimulant (200 nM PMA). The plates are incubated at 37.degree. C.
for 2 hours and the reaction is stopped by adding 20 .mu.l 1N NaOH
per well. The absorbance is read at 610 nm. To calculate the amount
of H.sub.2O.sub.2 produced by the macrophages, a standard curve of
a H.sub.2O.sub.2 solution of known molarity is performed for each
experiment.
[1565] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polypeptides, polynucleotides (e.g., gene therapy), agonists,
and/or antagonists of the invention.
Example 35
Biological Effects of Polypeptides of the Invention
[1566] Astrocyte and Neuronal Assays
[1567] Recombinant polypeptides of the invention, expressed in
Escherichia coli and purified as described above, can be tested for
activity in promoting the survival, neurite outgrowth, or
phenotypic differentiation of cortical neuronal cells and for
inducing the proliferation of glial fibrillary acidic protein
immunopositive cells, astrocytes. The selection of cortical cells
for the bioassay is based on the prevalent expression of FGF-1 and
FGF-2 in cortical structures and on the previously reported
enhancement of cortical neuronal survival resulting from FGF-2
treatment. A thymidine incorporation assay, for example, can be
used to elucidate a polypeptide of the invention's activity on
these cells.
[1568] Moreover, previous reports describing the biological effects
of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro
have demonstrated increases in both neuron survival and neurite
outgrowth (Walicke et al., "Fibroblast growth factor promotes
survival of dissociated hippocampal neurons and enhances neurite
extension." Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay
herein incorporated by reference in its entirety). However, reports
from experiments done on PC-12 cells suggest that these two
responses are not necessarily synonymous and may depend on not only
which FGF is being tested but also on which receptor(s) are
expressed on the target cells. Using the primary cortical neuronal
culture paradigm, the ability of a polypeptide of the invention to
induce neurite outgrowth can be compared to the response achieved
with FGF-2 using, for example, a thymidine incorporation assay.
[1569] Fibroblast and Endothelial Cell Assays
[1570] Human lung fibroblasts are obtained from Clonetics (San
Diego, Calif.) and maintained in growth media from Clonetics.
Dermal microvascular endothelial cells are obtained from Cell
Applications (San Diego, Calif.). For proliferation assays, the
human lung fibroblasts and dermal microvascular endothelial cells
can be cultured at 5,000 cells/well in a 96-well plate for one day
in growth medium. The cells are then incubated for one day in 0.1%
BSA basal medium. After replacing the medium with fresh 0.1% BSA
medium, the cells are incubated with the test proteins for 3 days.
Alamar Blue (Alamar Biosciences, Sacramento, Calif.) is added to
each well to a final concentration of 10%. The cells are incubated
for 4 hr. Cell viability is measured by reading in a CytoFluor
fluorescence reader. For the PGE.sub.2 assays, the human lung
fibroblasts are cultured at 5,000 cells/well in a 96-well plate for
one day. After a medium change to 0.1% BSA basal medium, the cells
are incubated with FGF-2 or polypeptides of the invention with or
without IL-1.alpha. for 24 hours. The supernatants are collected
and assayed for PGE.sub.2 by EIA kit (Cayman, Ann Arbor, Mich.).
For the IL-6 assays, the human lung fibroblasts are cultured at
5,000 cells/well in a 96-well plate for one day. After a medium
change to 0.1% BSA basal medium, the cells are incubated with FGF-2
or with or without polypeptides of the invention IL-1.alpha. for 24
hours. The supernatants are collected and assayed for IL-6 by ELISA
kit (Endogen, Cambridge, Mass.).
[1571] Human lung fibroblasts are cultured with FGF-2 or
polypeptides of the invention for 3 days in basal medium before the
addition of Alamar Blue to assess effects on growth of the
fibroblasts. FGF-2 should show a stimulation at 10-2500 ng/ml which
can be used to compare stimulation with polypeptides of the
invention.
[1572] Parkinson Models.
[1573] The loss of motor function in Parkinson's disease is
attributed to a deficiency of striatal dopamine resulting from the
degeneration of the nigrostriatal dopaminergic projection neurons.
An animal model for Parkinson's that has been extensively
characterized involves the systemic administration of 1-methyl-4
phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is
taken-up by astrocytes and catabolized by monoamine oxidase B to
1-methyl-4-phenyl pyridine (MPP.sup.+) and released. Subsequently,
MPP.sup.+ is actively accumulated in dopaminergic neurons by the
high-affinity reuptake transporter for dopamine. MPP.sup.+ is then
concentrated in mitochondria by the electrochemical gradient and
selectively inhibits nicotidamide adenine disphosphate: ubiquinone
oxidoreductionase (complex I), thereby interfering with electron
transport and eventually generating oxygen radicals.
[1574] It has been demonstrated in tissue culture paradigms that
FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic
neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's
group has demonstrated that administering FGF-2 in gel foam
implants in the striatum results in the near complete protection of
nigral dopaminergic neurons from the toxicity associated with MPTP
exposure (Otto and Unsicker, J. Neuroscience, 1990).
[1575] Based on the data with FGF-2, polypeptides of the invention
can be evaluated to determine whether it has an action similar to
that of FGF-2 in enhancing dopaminergic neuronal survival in vitro
and it can also be tested in vivo for protection of dopaminergic
neurons in the striatum from the damage associated with MPTP
treatment. The potential effect of a polypeptide of the invention
is first examined in vitro in a dopaminergic neuronal cell culture
paradigm. The cultures are prepared by dissecting the midbrain
floor plate from gestation day 14 Wistar rat embryos. The tissue is
dissociated with trypsin and seeded at a density of 200,000
cells/cm.sup.2 on polyorthinine-laminin coated glass coverslips.
The cells are maintained in Dulbecco's Modified Eagle's medium and
F12 medium containing hormonal supplements (N1). The cultures are
fixed with paraformaldehyde after 8 days in vitro and are processed
for tyrosine hydroxylase, a specific marker for dopminergic
neurons, immunohistochemical staining. Dissociated cell cultures
are prepared from embryonic rats. The culture medium is changed
every third day and the factors are also added at that time.
[1576] Since the dopaminergic neurons are isolated from animals at
gestation day 14, a developmental time which is past the stage when
the dopaminergic precursor cells are proliferating, an increase in
the number of tyrosine hydroxylase immunopositive neurons would
represent an increase in the number of dopaminergic neurons
surviving in vitro. Therefore, if a polypeptide of the invention
acts to prolong the survival of dopaminergic neurons, it would
suggest that the polypeptide may be involved in Parkinson's
Disease.
[1577] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 36
The Effect of Polypeptides of the Invention on the Growth of
Vascular Endothelial Cells
[1578] On day 1, human umbilical vein endothelial cells (HUVEC) are
seeded at 2-5.times.10.sup.4 cells/35 mm dish density in M199
medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin,
and 50 units/ml endothelial cell growth supplements (ECGS,
Biotechnique, Inc.). On day 2, the medium is replaced with M199
containing 10% FBS, 8 units/ml heparin. A polypeptide having the
amino acid sequence of SEQ ID NO:Y, and positive controls, such as
VEGF and basic FGF (bFGF) are added, at varying concentrations. On
days 4 and 6, the medium is replaced. On day 8, cell number is
determined with a Coulter Counter.
[1579] An increase in the number of HUVEC cells indicates that the
polypeptide of the invention may proliferate vascular endothelial
cells.
[1580] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 37
Stimulatory Effect of Polypeptides of the Invention on the
Proliferation of Vascular Endothelial Cells
[1581] For evaluation of mitogenic activity of growth factors, the
colorimetric MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)2H-tetrazolium) assay with the electron coupling reagent PMS
(phenazine methosulfate) was performed (CellTiter 96 AQ, Promega).
Cells are seeded in a 96-well plate (5,000 cells/well) in 0.1 mL
serum-supplemented medium and are allowed to attach overnight.
After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF,
VEGF.sub.165 or a polypeptide of the invention in 0.5% FBS) with or
without Heparin (8 U/ml) are added to wells for 48 hours. 20 mg of
MTS/PMS mixture (1:0.05) are added per well and allowed to incubate
for 1 hour at 37.degree. C. before measuring the absorbance at 490
nm in an ELISA plate reader. Background absorbance from control
wells (some media, no cells) is subtracted, and seven wells are
performed in parallel for each condition. See, Leak et. al. In
Vitro Cell. Dev. Biol. 30A:512-518 (1994).
[1582] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 38
Inhibition of PDGF-induced Vascular Smooth Muscle Cell
Proliferation Stimulatory Effect
[1583] HAoSMC proliferation can be measured, for example, by BrdUrd
incorporation. Briefly, subconfluent, quiescent cells grown on the
4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP.
Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd.
After 24 h, immunocytochemistry is performed by using BrdUrd
Staining Kit (Zymed Laboratories). In brief, the cells are
incubated with the biotinylated mouse anti-BrdUrd antibody at 4
degrees C. for 2 h after being exposed to denaturing solution and
then incubated with the streptavidin-peroxidase and
diaminobenzidine. After counterstaining with hematoxylin, the cells
are mounted for microscopic examination, and the BrdUrd-positive
cells are counted. The BrdUrd index is calculated as a percent of
the BrdUrd-positive cells to the total cell number. In addition,
the simultaneous detection of the BrdUrd staining (nucleus) and the
FITC uptake (cytoplasm) is performed for individual cells by the
concomitant use of bright field illumination and dark field-UV
fluorescent illumination. See, Hayashida et al., J. Biol. Chem.
6:271(36):21985-21992 (1996).
[1584] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 39
Stimulation of Endothelial Migration
[1585] This example will be used to explore the possibility that a
polypeptide of the invention may stimulate lymphatic endothelial
cell migration.
[1586] Endothelial cell migration assays are performed using a 48
well microchemotaxis chamber (Neuroprobe Inc., Cabin John, M D;
Falk, W., et al., J. Immunological Methods 1980;33:239-247).
Polyvinylpyrrolidone-free polycarbonate filters with a pore size of
8 urn (Nucleopore Corp. Cambridge, Mass.) are coated with 0.1%
gelatin for at least 6 hours at room temperature and dried under
sterile air. Test substances are diluted to appropriate
concentrations in M199 supplemented with 0.25% bovine serum albumin
(BSA), and 25 ul of the final dilution is placed in the lower
chamber of the modified Boyden apparatus. Subconfluent, early
passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for
the minimum time required to achieve cell detachment. After placing
the filter between lower and upper chamber, 2.5.times.10.sup.5
cells suspended in 50 ul M199 containing 1% FBS are seeded in the
upper compartment. The apparatus is then incubated for 5 hours at
37.degree. C. in a humidified chamber with 5% CO2 to allow cell
migration. After the incubation period, the filter is removed and
the upper side of the filter with the non-migrated cells is scraped
with a rubber policeman. The filters are fixed with methanol and
stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park,
Ill.). Migration is quantified by counting cells of three random
high-power fields (40.times.) in each well, and all groups are
performed in quadruplicate.
[1587] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 40
Stimulation of Nitric Oxide Production by Endothelial Cells
[1588] Nitric oxide released by the vascular endothelium is
believed to be a mediator of vascular endothelium relaxation. Thus,
activity of a polypeptide of the invention can be assayed by
determining nitric oxide production by endothelial cells in
response to the polypeptide.
[1589] Nitric oxide is measured in 96-well plates of confluent
microvascular endothelial cells after 24 hours starvation and a
subsequent 4 hr exposure to various levels of a positive control
(such as VEGF-1) and the polypeptide of the invention. Nitric oxide
in the medium is determined by use of the Griess reagent to measure
total nitrite after reduction of nitric oxide-derived nitrate by
nitrate reductase. The effect of the polypeptide of the invention
on nitric oxide release is examined on HUVEC.
[1590] Briefly, NO release from cultured HUVEC monolayer is
measured with a NO-specific polarographic electrode connected to a
NO meter (Iso-NO, World Precision Instruments Inc.) (1049).
Calibration of the NO elements is performed according to the
following equation:
2KNO.sub.2+2KI+2H.sub.2SO.sub.462NO+I.sub.2+2H.sub.2O+2K.sub.2SO.sub.4
[1591] The standard calibration curve is obtained by adding graded
concentrations of KNO.sub.2 (0, 5, 10, 25, 50, 100, 250, and 500
nmol/L) into the calibration solution containing KI and
H.sub.2SO.sub.4. The specificity of the Iso-NO electrode to NO is
previously determined by measurement of NO from authentic NO gas
(1050). The culture medium is removed and HUVECs are washed twice
with Dulbecco's phosphate buffered saline. The cells are then
bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well
plates, and the cell plates are kept on a slide warmer (Lab Line
Instruments Inc.) To maintain the temperature at 37.degree. C. The
NO sensor probe is inserted vertically into the wells, keeping the
tip of the electrode 2 mm under the surface of the solution, before
addition of the different conditions. S-nitroso acetyl penicillamin
(SNAP) is used as a positive control. The amount of released NO is
expressed as picomoles per 1.times.10.sup.6 endothelial cells. All
values reported are means of four to six measurements in each group
(number of cell culture wells). See, Leak et al. Biochem. and
Biophys. Res. Comm. 217:96-105 (1995).
[1592] The studies described in this example tested activity of
polypeptides of the invention. However, one skilled in the art
could easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 41
Effect of Polypepides of the Invention on Cord Formation in
Angiogenesis
[1593] Another step in angiogenesis is cord formation, marked by
differentiation of endothelial cells. This bioassay measures the
ability of microvascular endothelial cells to form capillary-like
structures (hollow structures) when cultured in vitro.
[1594] CADMEC (microvascular endothelial cells) are purchased from
Cell Applications, Inc. as proliferating (passage 2) cells and are
cultured in Cell Applications' CADMEC Growth Medium and used at
passage 5. For the in vitro angiogenesis assay, the wells of a
48-well cell culture plate are coated with Cell Applications'
Attachment Factor Medium (200 ml/well) for 30 min. at 37.degree. C.
CADMEC are seeded onto the coated wells at 7,500 cells/well and
cultured overnight in Growth Medium. The Growth Medium is then
replaced with 300 mg Cell Applications' Chord Formation Medium
containing control buffer or a polypeptide of the invention (0.1 to
100 ng/ml) and the cells are cultured for an additional 48 hr. The
numbers and lengths of the capillary-like chords are quantitated
through use of the Boeckeler VIA-170 video image analyzer. All
assays are done in triplicate.
[1595] Commercial (R&D) VEGF (50 ng/ml) is used as a positive
control. b-esteradiol (1 ng/ml) is used as a negative control. The
appropriate buffer (without protein) is also utilized as a
control.
[1596] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 42
Angiogenic Effect on Chick Chorioallantoic Membrane
[1597] Chick chorioallantoic membrane (CAM) is a well-established
system to examine angiogenesis. Blood vessel formation on CAM is
easily visible and quantifiable. The ability of polypeptides of the
invention to stimulate angiogenesis in CAM can be examined.
[1598] Fertilized eggs of the White Leghorn chick (Gallus gallus)
and the Japanese qual (Coturnix coturnix) are incubated at
37.8.degree. C. and 80% humidity. Differentiated CAM of 16-day-old
chick and 13-day-old qual embryos is studied with the following
methods.
[1599] On Day 4 of development, a window is made into the egg shell
of chick eggs. The embryos are checked for normal development and
the eggs sealed with cellotape. They are further incubated until
Day 13. Thermanox coverslips (Nunc, Naperville, Ill.) are cut into
disks of about 5 mm in diameter. Sterile and salt-free growth
factors are dissolved in distilled water and about 3.3 mg/5 ml are
pipetted on the disks. After air-drying, the inverted disks are
applied on CAM. After 3 days, the specimens are fixed in 3%
glutaraldehyde and 2% formaldehyde and rinsed in 0.12 M sodium
cacodylate buffer. They are photographed with a stereo microscope
[Wild M8] and embedded for semi- and ultrathin sectioning as
described above. Controls are performed with carrier disks
alone.
[1600] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 43
Angiogenesis Assay Using a Matrigel Implant in Mouse
[1601] In vivo angiogenesis assay of a polypeptide of the invention
measures the ability of an existing capillary network to form new
vessels in an implanted capsule of murine extracellular matrix
material (Matrigel). The protein is mixed with the liquid Matrigel
at 4 degree C. and the mixture is then injected subcutaneously in
mice where it solidifies. After 7 days, the solid "plug" of
Matrigel is removed and examined for the presence of new blood
vessels. Matrigel is purchased from Becton Dickinson
Labware/Collaborative Biomedical Products.
[1602] When thawed at 4 degree C. the Matrigel material is a
liquid. The Matrigel is mixed with a polypeptide of the invention
at 150 ng/ml at 4 degrees C. and drawn into cold 3 ml syringes.
Female C57B1/6 mice approximately 8 weeks old are injected with the
mixture of Matrigel and experimental protein at 2 sites at the
midventral aspect of the abdomen (0.5 ml/site). After 7 days, the
mice are sacrificed by cervical dislocation, the Matrigel plugs are
removed and cleaned (i.e., all clinging membranes and fibrous
tissue is removed). Replicate whole plugs are fixed in neutral
buffered 10% formaldehyde, embedded in paraffin and used to produce
sections for histological examination after staining with Masson's
Trichrome. Cross sections from 3 different regions of each plug are
processed. Selected sections are stained for the presence of vWF.
The positive control for this assay is bovine basic FGF (150
ng/ml). Matrigel alone is used to determine basal levels of
angiogenesis.
[1603] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 44
Rescue of Ischemia in Rabbit Lower Limb Model
[1604] To study the in vivo effects of polynucleotides and
polypeptides of the invention on ischemia, a rabbit hindlimb
ischemia model is created by surgical removal of one femoral
arteries as described previously (Takeshita et al., Am J. Pathol
147:1649-1660 (1995)). The excision of the femoral artery results
in retrograde propagation of thrombus and occlusion of the external
iliac artery. Consequently, blood flow to the ischemic limb is
dependent upon collateral vessels originating from the internal
iliac artery (Takeshitaet al. Am J. Pathol 147:1649-1660 (1995)).
An interval of 10 days is allowed for post-operative recovery of
rabbits and development of endogenous collateral vessels. At 10 day
post-operatively (day 0), after performing a baseline angiogram,
the internal iliac artery of the ischemic limb is transfected with
500 mg naked expression plasmid containing a polynucleotide of the
invention by arterial gene transfer technology using a
hydrogel-coated balloon catheter as described (Riessen et al. Hum
Gene Ther. 4:749-758 (1993); Leclerc et al. J. Clin. Invest. 90:
936-944 (1992)). When a polypeptide of the invention is used in the
treatment, a single bolus of 500 mg polypeptide of the invention or
control is delivered into the internal iliac artery of the ischemic
limb over a period of 1 min. through an infusion catheter. On day
30, various parameters are measured in these rabbits: (a) BP
ratio--The blood pressure ratio of systolic pressure of the
ischemic limb to that of normal limb; (b) Blood Flow and Flow
Reserve--Resting FL: the blood flow during undilated condition and
Max FL: the blood flow during fully dilated condition (also an
indirect measure of the blood vessel amount) and Flow Reserve is
reflected by the ratio of max FL: resting FL; (c) Angiographic
Score--This is measured by the angiogram of collateral vessels. A
score is determined by the percentage of circles in an overlaying
grid that with crossing opacified arteries divided by the total
number m the rabbit thigh; (d) Capillary density--The number of
collateral capillaries determined in light microscopic sections
taken from hindlimbs.
[1605] The studies described in this example tested activity of
polynucleotides and polypeptides of the invention. However, one
skilled in the art could easily modify the exemplified studies to
test the agonists, and/or antagonists of the invention.
Example 45
Effect of Polypeptides of the Invention on Vasodilation
[1606] Since dilation of vascular endothelium is important in
reducing blood pressure, the ability of polypeptides of the
invention to affect the blood pressure in spontaneously
hypertensive rats (SHR) is examined. Increasing doses (0, 10, 30,
100, 300, and 900 mg/kg) of the polypeptides of the invention are
administered to 13-14 week old spontaneously hypertensive rats
(SHR). Data are expressed as the mean +/- SEM. Statistical analysis
are performed with a paired t-test and statistical significance is
defined as p<0.05 vs. the response to buffer alone.
[1607] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 46
Rat Ischemic Skin Flap Model
[1608] The evaluation parameters include skin blood flow, skin
temperature, and factor VIII immunohistochemistry or endothelial
alkaline phosphatase reaction. Expression of polypeptides of the
invention, during the skin ischemia, is studied using in situ
hybridization.
[1609] The study in this model is divided into three parts as
follows:
[1610] a) Ischemic skin
[1611] b) Ischemic skin wounds
[1612] c) Normal wounds
[1613] The experimental protocol includes:
[1614] a) Raising a 3.times.4 cm, single pedicle full-thickness
random skin flap (myocutaneous flap over the lower back of the
animal).
[1615] b) An excisional wounding (4-6 mm in diameter) in the
ischemic skin (skin-flap).
[1616] c) Topical treatment with a polypeptide of the invention of
the excisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the
following various dosage ranges: 1 mg to 100 mg.
[1617] d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and
21 post-wounding for histological, immunohistochemical, and in situ
studies.
[1618] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 47
Peripheral Arterial Disease Model
[1619] Angiogenic therapy using a polypeptide of the invention is a
novel therapeutic strategy to obtain restoration of blood flow
around the ischemia in case of peripheral arterial diseases. The
experimental protocol includes:
[1620] a) One side of the femoral artery is ligated to create
ischemic muscle of the hindlimb, the other side of hindlimb serves
as a control.
[1621] b) a polypeptide of the invention, in a dosage range of 20
mg-500 mg, is delivered intravenously and/or intramuscularly 3
times (perhaps more) per week for 2-3 weeks.
[1622] c) The ischemic muscle tissue is collected after ligation of
the femoral artery at 1, 2, and 3 weeks for the analysis of
expression of a polypeptide of the invention and histology. Biopsy
is also performed on the other side of normal muscle of the
contralateral hindlimb.
[1623] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 48
Ischemic Myocardial Disease Model
[1624] A polypeptide of the invention is evaluated as a potent
mitogen capable of stimulating the development of collateral
vessels, and restructuring new vessels after coronary artery
occlusion. Alteration of expression of the polypeptide is
investigated in situ. The experimental protocol includes:
[1625] a) The heart is exposed through a left-side thoracotomy in
the rat. Immediately, the left coronary artery is occluded with a
thin suture (6-0) and the thorax is closed.
[1626] b) a polypeptide of the invention, in a dosage range of 20
mg-500 mg, is delivered intravenously and/or intramuscularly 3
times (perhaps more) per week for 2-4 weeks.
[1627] c) Thirty days after the surgery, the heart is removed and
cross-sectioned for morphometric and in situ analyzes.
[1628] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 49
Rat Corneal Wound Healing Model
[1629] This animal model shows the effect of a polypeptide of the
invention on neovascularization. The experimental protocol
includes:
[1630] a) Making a 1-1.5 mm long incision from the center of cornea
into the stromal layer.
[1631] b) Inserting a spatula below the lip of the incision facing
the outer corner of the eye.
[1632] c) Making a pocket (its base is 1-1.5 mm form the edge of
the eye).
[1633] d) Positioning a pellet, containing 50 ng-5 ug of a
polypeptide of the invention, within the pocket.
[1634] e) Treatment with a polypeptide of the invention can also be
applied topically to the comeal wounds in a dosage range of 20
mg-500 mg (daily treatment for five days).
[1635] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 50
Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models
[1636] A. Diabetic db+/db+ Mouse Model.
[1637] To demonstrate that a polypeptide of the invention
accelerates the healing process, the genetically diabetic mouse
model of wound healing is used. The full thickness wound healing
model in the db+/db+ mouse is a well characterized, clinically
relevant and reproducible model of impaired wound healing. Healing
of the diabetic wound is dependent on formation of granulation
tissue and re-epithelialization rather than contraction (Gartner,
M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G. et
al., Am. J Pathol. 136:1235 (1990)).
[1638] The diabetic animals have many of the characteristic
features observed in Type II diabetes mellitus. Homozygous
(db+/db+) mice are obese in comparison to their normal heterozygous
(db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single
autosomal recessive mutation on chromosome 4 (db+) (Coleman et al.
Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show
polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+)
have elevated blood glucose, increased or normal insulin levels,
and suppressed cell-mediated immunity (Mandel et al., J. Immunol.
120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.
51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55
(1985)). Peripheral neuropathy, myocardial complications, and
microvascular lesions, basement membrane thickening and glomerular
filtration abnormalities have been described in these animals
(Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et
al., Diabetes 29(1):60-67 (1980); Giacomelli et Lab Invest.
40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl): 1-6
(1982)). These homozygous diabetic mice develop hyperglycemia that
is resistant to insulin analogous to human type II diabetes (Mandel
et al., J. Immunol. 120:1375-1377 (1978)).
[1639] The characteristics observed in these animals suggests that
healing in this model may be similar to the healing observed in
human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246
(1990)).
[1640] Genetically diabetic female C57BL/KsJ (db+/db+) mice and
their non-diabetic (db+/+m) heterozygous littermates are used in
this study (Jackson Laboratories). The animals are purchased at 6
weeks of age and are 8 weeks old at the beginning of the study.
Animals are individually housed and received food and water ad
libitum. All manipulations are performed using aseptic techniques.
The experiments are conducted according to the rules and guidelines
of Human Genome Sciences, Inc. Institutional Animal Care and Use
Committee and the Guidelines for the Care and Use of Laboratory
Animals.
[1641] Wounding protocol is performed according to previously
reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med.
172:245-251 (1990)). Briefly, on the day of wounding, animals are
anesthetized with an intraperitoneal injection of Avertin (0.01
mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in
deionized water. The dorsal region of the animal is shaved and the
skin washed with 70% ethanol solution and iodine. The surgical area
is dried with sterile gauze prior to wounding. An 8 mm
full-thickness wound is then created using a Keyes tissue punch.
Immediately following wounding, the surrounding skin is gently
stretched to eliminate wound expansion. The wounds are left open
for the duration of the experiment. Application of the treatment is
given topically for 5 consecutive days commencing on the day of
wounding. Prior to treatment, wounds are gently cleansed with
sterile saline and gauze sponges.
[1642] Wounds are visually examined and photographed at a fixed
distance at the day of surgery and at two day intervals thereafter.
Wound closure is determined by daily measurement on days 1-5 and on
day 8. Wounds are measured horizontally and vertically using a
calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is no longer visible and the wound is covered by
a continuous epithelium.
[1643] A polypeptide of the invention is administered using at a
range different doses, from 4 mg to 500 mg per wound per day for 8
days in vehicle. Vehicle control groups received 50 mL of vehicle
solution.
[1644] Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology and
immunohistochemistry. Tissue specimens are placed in 10% neutral
buffered formalin in tissue cassettes between biopsy sponges for
further processing.
[1645] Three groups of 10 animals each (5 diabetic and 5
non-diabetic controls) are evaluated: 1) Vehicle placebo control,
2) untreated group, and 3) treated group.
[1646] Wound closure is analyzed by measuring the area in the
vertical and horizontal axis and obtaining the total square area of
the wound. Contraction is then estimated by establishing the
differences between the initial wound area (day 0) and that of post
treatment (day 8). The wound area on day 1 is 64 mm2, the
corresponding size of the dermal punch. Calculations are made using
the following formula: [Open area on day 8]-[Open area on day
1]/[Open area on day 1]
[1647] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) and cut using a Reichert-Jung microtome. Routine
hematoxylin-eosin (H&E) staining is performed on cross-sections
of bisected wounds. Histologic examination of the wounds are used
to assess whether the healing process and the morphologic
appearance of the repaired skin is altered by treatment with a
polypeptide of the invention. This assessment included verification
of the presence of cell accumulation, inflammatory cells,
capillaries, fibroblasts, re-epithelialization and epidermal
maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235
(1990)). A calibrated lens micrometer is used by a blinded
observer.
[1648] Tissue sections are also stained immunohistochemically with
a polyclonal rabbit anti-human keratin antibody using ABC Elite
detection system. Human skin is used as a positive tissue control
while non-immune IgG is used as a negative control. Keratinocyte
growth is determined by evaluating the extent of
reepithelialization of the wound using a calibrated lens
micrometer.
[1649] Proliferating cell nuclear antigen/cyclin (PCNA) in skin
specimens is demonstrated by using anti-PCNA antibody (1:50) with
an ABC Elite detection system. Human colon cancer can serve as a
positive tissue control and human brain tissue can be used as a
negative tissue control. Each specimen includes a section with
omission of the primary antibody and substitution with non-immune
mouse IgG. Ranking of these sections is based on the extent of
proliferation on a scale of 0-8, the lower side of the scale
reflecting slight proliferation to the higher side reflecting
intense proliferation.
[1650] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
[1651] B. Steroid Impaired Rat Model
[1652] The inhibition of wound healing by steroids has been well
documented in various in vitro and in vivo systems (Wahl,
Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid
Action: Basic and Clinical Aspects. 280-302 (1989); Wahlet al., J.
Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med.
147:1684-1694 (1978)). Glucocorticoids retard wound healing by
inhibiting angiogenesis, decreasing vascular permeability (Ebert et
al., An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation,
and collagen synthesis (Beck et al., Growth Factors. 5: 295-304
(1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)) and
producing a transient reduction of circulating monocytes (Haynes et
al., J. Clin. Invest. 61: 703-797 (1978); Wahl, "Glucocorticoids
and wound healing", In: Antiinflammatory Steroid Action: Basic and
Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)).
The systemic administration of steroids to impaired wound healing
is a well establish phenomenon in rats (Beck et al., Growth
Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61:
703-797 (1978); Wahl, " Glucocorticoids and wound healing", In:
Antiinflammatory Steroid Action: Basic and Clinical Aspects,
Academic Press, New York, pp. 280-302 (1989); Pierce et al., Proc.
Natl. Acad. Sci. USA 86: 2229-2233 (1989)).
[1653] To demonstrate that a polypeptide of the invention can
accelerate the healing process, the effects of multiple topical
applications of the polypeptide on full thickness excisional skin
wounds in rats in which healing has been impaired by the systemic
administration of methylprednisolone is assessed.
[1654] Young adult male Sprague Dawley rats weighing 250-300 g
(Charles River Laboratories) are used in this example. The animals
are purchased at 8 weeks of age and are 9 weeks old at the
beginning of the study. The healing response of rats is impaired by
the systemic administration of methylprednisolone (17 mg/kg/rat
intramuscularly) at the time of wounding. Animals are individually
housed and received food and water ad libitum. All manipulations
are performed using aseptic techniques. This study is conducted
according to the rules and guidelines of Human Genome Sciences,
Inc. Institutional Animal Care and Use Committee and the Guidelines
for the Care and Use of Laboratory Animals.
[1655] The wounding protocol is followed according to section A,
above. On the day of wounding, animals are anesthetized with an
intramuscular injection of ketamine (50 mg/kg) and xylazine (5
mg/kg). The dorsal region of the animal is shaved and the skin
washed with 70% ethanol and iodine solutions. The surgical area is
dried with sterile gauze prior to wounding. An 8 mm full-thickness
wound is created using a Keyes tissue punch. The wounds are left
open for the duration of the experiment. Applications of the
testing materials are given topically once a day for 7 consecutive
days commencing on the day of wounding and subsequent to
methylprednisolone administration. Prior to treatment, wounds are
gently cleansed with sterile saline and gauze sponges.
[1656] Wounds are visually examined and photographed at a fixed
distance at the day of wounding and at the end of treatment. Wound
closure is determined by daily measurement on days 1-5 and on day
8. Wounds are measured horizontally and vertically using a
calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is no longer visible and the wound is covered by
a continuous epithelium.
[1657] The polypeptide of the invention is administered using at a
range different doses, from 4 mg to 500 mg per wound per day for 8
days in vehicle. Vehicle control groups received 50 mL of vehicle
solution.
[1658] Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology. Tissue specimens
are placed in 10% neutral buffered formalin in tissue cassettes
between biopsy sponges for further processing.
[1659] Four groups of 10 animals each (5 with methylprednisolone
and 5 without glucocorticoid) are evaluated: 1) Untreated group 2)
Vehicle placebo control 3) treated groups.
[1660] Wound closure is analyzed by measuring the area in the
vertical and horizontal axis and obtaining the total area of the
wound. Closure is then estimated by establishing the differences
between the initial wound area (day 0) and that of post treatment
(day 8). The wound area on day 1 is 64 mm.sup.2, the corresponding
size of the dermal punch. Calculations are made using the following
formula: [Open area on day 8]--[Open area on day 1]/[Open area on
day 1]
[1661] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) and cut using an Olympus. microtome. Routine hematoxylin-eosin
(H&E) staining is performed on cross-sections of bisected
wounds. Histologic examination of the wounds allows assessment of
whether the healing process and the morphologic appearance of the
repaired skin is improved by treatment with a polypeptide of the
invention. A calibrated lens micrometer is used by a blinded
observer to determine the distance of the wound gap.
[1662] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
[1663] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 51
Lymphadema Animal Model
[1664] The purpose of this experimental approach is to create an
appropriate and consistent lymphedema model for testing the
therapeutic effects of a polypeptide of the invention in
lymphangiogenesis and re-establishment of the lymphatic circulatory
system in the rat hind limb. Effectiveness is measured by swelling
volume of the affected limb, quantification of the amount of
lymphatic vasculature, total blood plasma protein, and
histopathology. Acute lymphedema is observed for 7-10 days. Perhaps
more importantly, the chronic progress of the edema is followed for
up to 3-4 weeks.
[1665] Prior to beginning surgery, blood sample is drawn for
protein concentration analysis. Male rats weighing approximately
.about.350 g are dosed with Pentobarbital. Subsequently, the right
legs are shaved from knee to hip. The shaved area is swabbed with
gauze soaked in 70% EtOH. Blood is drawn for serum total protein
testing. Circumference and volumetric measurements are made prior
to injecting dye into paws after marking 2 measurement levels (0.5
cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of
both right and left paws are injected with 0.05 ml of 1% Evan's
Blue. Circumference and volumetric measurements are then made
following injection of dye into paws.
[1666] Using the knee joint as a landmark, a mid-leg inguinal
incision is made circumferentially allowing the femoral vessels to
be located. Forceps and hemostats are used to dissect and separate
the skin flaps. After locating the femoral vessels, the lymphatic
vessel that runs along side and underneath the vessel(s) is
located. The main lymphatic vessels in this area are then
electrically coagulated suture ligated.
[1667] Using a microscope, muscles in back of the leg (near the
semitendinosis and adductors) are bluntly dissected. The popliteal
lymph node is then located. The 2 proximal and 2 distal lymphatic
vessels and distal blood supply of the popliteal node are then and
ligated by suturing. The popliteal lymph node, and any accompanying
adipose tissue, is then removed by cutting connective tissues.
[1668] Care is taken to control any mild bleeding resulting from
this procedure. After lymphatics are occluded, the skin flaps are
sealed by using liquid skin (Vetbond) (AJ Buck). The separated skin
edges are sealed to the underlying muscle tissue while leaving a
gap of .about.0.5 cm around the leg. Skin also may be anchored by
suturing to underlying muscle when necessary.
[1669] To avoid infection, animals are housed individually with
mesh (no bedding). Recovering animals are checked daily through the
optimal edematous peak, which typically occurred by day 5-7. The
plateau edematous peak are then observed. To evaluate the intensity
of the lymphedema, the circumference and volumes of 2 designated
places on each paw before operation and daily for 7 days are
measured. The effect plasma proteins on lymphedema is determined
and whether protein analysis is a useful testing perimeter is also
investigated. The weights of both control and edematous limbs are
evaluated at 2 places. Analysis is performed in a blind manner.
[1670] Circumference Measurements: Under brief gas anesthetic to
prevent limb movement, a cloth tape is used to measure limb
circumference. Measurements are done at the ankle bone and dorsal
paw by 2 different people then those 2 readings are averaged.
Readings are taken from both control and edematous limbs.
[1671] Volumetric Measurements: On the day of surgery, animals are
anesthetized with Pentobarbital and are tested prior to surgery.
For daily volumetrics animals are under brief halothane anesthetic
(rapid immobilization and quick recovery), both legs are shaved and
equally marked using waterproof marker on legs. Legs are first
dipped in water, then dipped into instrument to each marked level
then measured by Buxco edema software(Chen/Victor). Data is
recorded by one person, while the other is dipping the limb to
marked area.
[1672] Blood-plasma protein measurements: Blood is drawn, spun, and
serum separated prior to surgery and then at conclusion for total
protein and Ca2+ comparison.
[1673] Limb Weight Comparison: After drawing blood, the animal is
prepared for tissue collection. The limbs are amputated using a
quillitine, then both experimental and control legs are cut at the
ligature and weighed. A second weighing is done as the
tibio-cacaneal joint is disarticulated and the foot is weighed.
[1674] Histological Preparations: The transverse muscle located
behind the knee (popliteal) area is dissected and arranged in a
metal mold, filled with freezeGel, dipped into cold methylbutane,
placed into labeled sample bags at -80EC until sectioning. Upon
sectioning, the muscle is observed under fluorescent microscopy for
lymphatics.
[1675] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 52
Suppression of TNF Alpha-induced Adhesion Molecule Expression by a
Polypeptide of the Invention
[1676] The recruitment of lymphocytes to areas of inflammation and
angiogenesis involves specific receptor-ligand interactions between
cell surface adhesion molecules (CAMs) on lymphocytes and the
vascular endothelium. The adhesion process, in both normal and
pathological settings, follows a multi-step cascade that involves
intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion
molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1
(E-selectin) expression on endothelial cells (EC). The expression
of these molecules and others on the vascular endothelium
determines the efficiency with which leukocytes may adhere to the
local vasculature and extravasate into the local tissue during the
development of an inflammatory response. The local concentration of
cytokines and growth factor participate in the modulation of the
expression of these CAMs.
[1677] Tumor necrosis factor alpha (TNF-a), a potent
proinflammatory cytokine, is a stimulator of all three CAMs on
endothelial cells and may be involved in a wide variety of
inflammatory responses, often resulting in a pathological
outcome.
[1678] The potential of a polypeptide of the invention to mediate a
suppression of TNF-a induced CAM expression can be examined. A
modified ELISA assay which uses ECs as a solid phase absorbent is
employed to measure the amount of CAM expression on TNF-a treated
ECs when co-stimulated with a member of the FGF family of
proteins.
[1679] To perform the experiment, human umbilical vein endothelial
cell (HUVEC) cultures are obtained from pooled cord harvests and
maintained in growth medium (EGM-2; Clonetics, San Diego, Calif.)
supplemented with 10% FCS and 1% penicillin/streptomycin in a 37
degree C. humidified incubator containing 5% CO.sub.2. HUVECs are
seeded in 96-well plates at concentrations of 1.times.10.sup.4
cells/well in EGM medium at 37 degree C. for 18-24 hrs or until
confluent. The monolayers are subsequently washed 3 times with a
serum-free solution of RPMI-1640 supplemented with 100 U/ml
penicillin and 100 mg/ml streptomycin, and treated with a given
cytokine and/or growth factor(s) for 24 h at 37 degree C. Following
incubation, the cells are then evaluated for CAM expression.
[1680] Human Umbilical Vein Endothelial cells (HUVECS) are grown in
a standard 96 well plate to confluence. Growth medium is removed
from the cells and replaced with 90 ul of 199 Medium (10% FBS).
Samples for testing and positive or negative controls are added to
the plate in triplicate (in 10 ul volumes). Plates are incubated at
37 degree C. for either 5 h (selectin and integrin expression) or
24 h (integrin expression only). Plates are aspirated to remove
medium and 100 .mu.l of 0.1% paraformaldehyde-PBS(with Ca++ and
Mg++) is added to each well. Plates are held at 4.degree. C. for 30
min.
[1681] Fixative is then removed from the wells and wells are washed
1.times. with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the
wells to dry. Add 10 .mu.l of diluted primary antibody to the test
and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and
Anti-E-selectin-Biotin are used at a concentration of 10 .mu.g/ml
(1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at
37.degree. C. for 30 min. in a humidified environment. Wells are
washed X3 with PBS(+Ca,Mg)+0.5% BSA.
[1682] Then add 20 .mu.l of diluted ExtrAvidin-Alkaline Phosphotase
(1:5,000 dilution) to each well and incubated at 37.degree. C. for
30 min. Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of
p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of glycine buffer
(pH 10.4). 100 .mu.l of pNPP substrate in glycine buffer is added
to each test well. Standard wells in triplicate are prepared from
the working dilution of the ExtrAvidin-Alkaline Phosphotase in
glycine buffer: 1:5,000
(10.sup.0)>10.sup.-0.5>10.sup.-1>10.sup.-1.50.5 .mu.l of
each dilution is added to triplicate wells and the resulting AP
content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100
.mu.l of pNNP reagent must then be added to each of the standard
wells. The plate must be incubated at 37.degree. C. for 4 h. A
volume of 50 .mu.l of 3 M NaOH is added to all wells. The results
are quantified on a plate reader at 405 nm. The background
subtraction option is used on blank wells filled with glycine
buffer only. The template is set up to indicate the concentration
of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng;
0.18 ng]. Results are indicated as amount of bound AP-conjugate in
each sample.
[1683] The studies described in this example tested activity of a
polypeptide of the invention. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of the invention.
Example 53
Assay for the Stimulation of Bone Marrow CD34+ Cell
Proliferation
[1684] This assay is based on the ability of human CD34+ to
proliferate in the presence of hematopoietic growth factors and
evaluates the ability of isolated polypeptides expressed in
mammalian cells to stimulate proliferation of CD34+ cells.
[1685] It has been previously shown that most mature precursors
will respond to only a single signal. More immature precursors
require at least two signals to respond. Therefore, to test the
effect of polypeptides on hematopoietic activity of a wide range of
progenitor cells, the assay contains a given polypeptide in the
presence or absence of other hematopoietic growth factors. Isolated
cells are cultured for 5 days in the presence of Stem Cell Factor
(SCF) in combination with tested sample. SCF alone has a very
limited effect on the proliferation of bone marrow (BM) cells,
acting in such conditions only as a "survival" factor. However,
combined with any factor exhibiting stimulatory effect on these
cells (e.g., IL-3), SCF will cause a synergistic effect. Therefore,
if the tested polypeptide has a stimulatory effect on a
hematopoietic progenitors, such activity can be easily detected.
Since normal BM cells have a low level of cycling cells, it is
likely that any inhibitory effect of a given polypeptide, or
agonists or antagonists thereof, might not be detected.
Accordingly, assays for an inhibitory effect on progenitors is
preferably tested in cells that are first subjected to in vitro
stimulation with SCF+IL+3, and then contacted with the compound
that is being evaluated for inhibition of such induced
proliferation.
[1686] Briefly, CD34+ cells are isolated using methods known in the
art. The cells are thawed and resuspended in medium (QBSF 60
serum-free medium with 1% L-glutamine (500 ml) Quality Biological,
Inc., Gaithersburg, Md. Cat# 160-204-101). After several gentle
centrifugation steps at 200.times.g, cells are allowed to rest for
one hour. The cell count is adjusted to 2.5.times.10.sup.5
cells/ml. During this time, 100 .mu.l of sterile water is added to
the peripheral wells of a 96-well plate. The cytokines that can be
tested with a given polypeptide in this assay is rhSCF (R&D
Systems, Minneapolis, Minn., Cat# 255-SC) at 50 ng/ml alone and in
combination with rhSCF and rhIL-3 (R&D Systems, Minneapolis,
Minn., Cat# 203-ML) at 30 ng/ml. After one hour, 10 .mu.l of
prepared cytokines, 50 .mu.l SID (supernatants at 1:2 dilution=50
.mu.l) and 20 .mu.l of diluted cells are added to the media which
is already present in the wells to allow for a final total volume
of 100 .mu.l. The plates are then placed in a 37.degree. C./5%
CO.sub.2 incubator for five days.
[1687] Eighteen hours before the assay is harvested, 0.5
.mu.Ci/well of [3H] Thymidine is added in a 10 .mu.l volume to each
well to determine the proliferation rate. The experiment is
terminated by harvesting the cells from each 96-well plate to a
filtermat using the Tomtec Harvester 96. After harvesting, the
filtermats are dried, trimmed and placed into OmniFilter assemblies
consisting of one OmniFilter plate and one OmniFilter Tray. 60
.mu.l Microscint is added to each well and the plate sealed with
TopSeal-A press-on sealing film A bar code 15 sticker is affixed to
the first plate for counting. The sealed plates is then loaded and
the level of radioactivity determined via the Packard Top Count and
the printed data collected for analysis. The level of radioactivity
reflects the amount of cell proliferation.
[1688] The studies described in this example test the activity of a
given polypeptide to stimulate bone marrow CD34+ cell
proliferation. One skilled in the art could easily modify the
exemplified studies to test the activity of polynucleotides (e.g.,
gene therapy), antibodies, agonists, and/or antagonists and
fragments and variants thereof. As a nonlimiting example, potential
antagonists tested in this assay would be expected to inhibit cell
proliferation in the presence of cytokines and/or to increase the
inhibition of cell proliferation in the presence of cytokines and a
given polypeptide. In contrast, potential agonists tested in this
assay would be expected to enhance cell proliferation and/or to
decrease the inhibition of cell proliferation in the presence of
cytokines and a given polypeptide.
[1689] The ability of a gene to stimulate the proliferation of bone
marrow CD34+ cells indicates that polynucleotides and polypeptides
corresponding to the gene are useful for the diagnosis and
treatment of disorders affecting the immune system and
hematopoiesis. Representative uses are described in the "Immune
Activity" and "Infectious Disease" sections above, and elsewhere
herein.
Example 54
Assay for Extracellular Matrix Enhanced Cell Response (EMECR)
[1690] The objective of the Extracellular Matrix Enhanced Cell
Response (EMECR) assay is to identify gene products (e.g., isolated
polypeptides) that act on the hematopoietic stem cells in the
context of the extracellular matrix (ECM) induced signal.
[1691] Cells respond to the regulatory factors in the context of
signal(s) received from the surrounding microenvironment. For
example, fibroblasts, and endothelial and epithelial stem cells
fail to replicate in the absence of signals from the ECM.
Hematopoietic stem cells can undergo self-renewal in the bone
marrow, but not in in vitro suspension culture. The ability of stem
cells to undergo self-renewal in vitro is dependent upon their
interaction with the stromal cells and the ECM protein fibronectin
(fn). Adhesion of cells to fn is mediated by the
.alpha..sub.5..beta..sub.l and .alpha..sub.4..beta..sub.l integrin
receptors, which are expressed by human and mouse hematopoietic
stem cells. The factor(s) which integrate with the ECM environment
and responsible for stimulating stem cell self-renewal has not yet
been identified. Discovery of such factors should be of great
interest in gene therapy and bone marrow transplant
applications
[1692] Briefly, polystyrene, non tissue culture treated, 96-well
plates are coated with fn fragment at a coating concentration of
0.2 .mu.g/cm.sup.2. Mouse bone marrow cells are plated (1,000
cells/well ) in 0.2 ml of serum-free medium. Cells cultured in the
presence of IL-3 (5 ng/ml )+SCF (50 ng/ml ) would serve as the
positive control, conditions under which little self-renewal but
pronounced differentiation of the stem cells is to be expected.
Gene products are tested with appropriate negative controls in the
presence and absence of SCF(5.0 ng/ml), where test factor sup
emates represent 10% of the total assay volume. The plated cells
are then allowed to grow by incubating in a low oxygen environment
(5% CO.sub.2, 7% O.sub.2, and 88% N.sub.2) tissue culture incubator
for 7 days. The number of proliferating cells within the wells is
then quantitated by measuring thymidine incorporation into cellular
DNA.
Verification of the positive hits in the assay will require
phenotypic characterization of the cells, which can be accomplished
by scaling up of the culture system and using appropriate antibody
reagents against cell surface antigens and FACScan.
[1693] One skilled in the art could easily modify the exemplified
studies to test the activity of polynucleotides (e.g., gene
therapy), antibodies, agonists, and/or antagonists and fragments
and variants thereof.
[1694] If a particular gene product is found to be a stimulator of
hematopoietic progenitors, polynucleotides and polypeptides
corresponding to the gene may be useful for the diagnosis and
treatment of disorders affecting the immune system and
hematopoiesis. Representative uses are described in the "Immune
Activity" and "Infectious Disease" sections above, and elsewhere
herein. The gene product may also be useful in the expansion of
stem cells and committed progenitors of various blood lineages, and
in the differentiation and/or proliferation of various cell
types.
[1695] Additionally, the polynucleotides and/or polypeptides of the
gene of interest and/or agonists and/or antagonists thereof, may
also be employed to inhibit the proliferation and differentiation
of hematopoietic cells and therefore may be employed to protect
bone marrow stem cells from chemotherapeutic agents during
chemotherapy. This antiproliferative effect may allow
administration of higher doses of chemotherapeutic agents and,
therefore, more effective chemotherapeutic treatment.
[1696] Moreover, polynucleotides and polypeptides corresponding to
the gene of interest may also be useful for the treatment and
diagnosis of hematopoietic related disorders such as, for example,
anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia
since stromal cells are important in the production of cells of
hematopoietic lineages. The uses include bone marrow cell ex-vivo
culture, bone marrow transplantation, bone marrow reconstitution,
radiotherapy or chemotherapy of neoplasia.
Example 55
Human Dermal Fibroblast and Aortic Smooth Muscle Cell
Proliferation
[1697] The polypeptide of interest is added to cultures of normal
human dermal fibroblasts (NHDF) and human aortic smooth muscle
cells (AoSMC) and two co-assays are performed with each sample. The
first assay examines the effect of the polypeptide of interest on
the proliferation of normal human dermal fibroblasts (NHDF) or
aortic smooth muscle cells (AoSMC). Aberrant growth of fibroblasts
or smooth muscle cells is a part of several pathological processes,
including fibrosis, and restenosis. The second assay examines IL6
production by both NHDF and SMC. IL6 production is an indication of
functional activation. Activated cells will have increased
production of a number of cytokines and other factors, which can
result in a proinflammatory or immunomodulatory outcome. Assays are
run with and without co-TNFa stimulation, in order to check for
costimulatory or inhibitory activity.
[1698] Briefly, on day 1, 96-well black plates are set up with 1000
cells/well (NHDF) or 2000 cells/well (AoSMC) in 100 .mu.l culture
media. NHDF culture media contains: Clonetics FB basal media, 1
mg/ml hFGF, 5 mg/ml insulin, 50 mg/ml gentamycin, 2% FBS, while
AoSMC culture media contains Clonetics SM basal media, 0.5 .mu.g/ml
hEGF, 5 mg/ml insulin, 1 .mu.g/ml hFGF, 50 mg/ml gentamycin, 50
.mu.g/ml Amphotericin B, 5% FBS. After incubation @ 37.degree. C.
for at least 4-5 hours culture media is aspirated and replaced with
growth arrest media. Growth arrest media for NHDF contains
fibroblast basal media, 50 mg/ml gentamycin, 2% FBS, while growth
arrest media for AoSMC contains SM basal media, 50 mg/ml
gentamycin, 50 .mu.g/ml Amphotericin B, 0.4% FBS. Incubate at 37 C
until day 2.
[1699] On day 2, serial dilutions and templates of the polypeptide
of interest are designed which should always include media controls
and known-protein controls. For both stimulation and inhibition
experiments, proteins are diluted in growth arrest media. For
inhibition experiments, TNFa is added to a final concentration of 2
ng/ml (NHDF) or 5 ng/ml (AoSMC). Then add 1/3 vol media containing
controls or supernatants and incubate at 37 C/5% CO.sub.2 until day
5.
[1700] Transfer 60 .mu.l from each well to another labeled 96-well
plate, cover with a plate-sealer, and store at 4 C until Day 6 (for
IL6 ELISA). To the remaining 100 .mu.l in the cell culture plate,
aseptically add Alamar Blue in an amount equal to 10% of the
culture volume (10 .mu.l). Return plates to incubator for 3 to 4
hours. Then measure fluorescence with excitation at 530 nm and
emission at 590 nm using the CytoFluor. This yields the growth
stimulation/inhibition data.
[1701] On day 5, the IL6 ELISA is performed by coating a 96 well
plate with 50-100 ul/well of Anti-Human IL6 Monoclonal antibody
diluted in PBS, pH 7.4, incubate ON at room temperature.
[1702] On day 6, empty the plates into the sink and blot on paper
towels. Prepare Assay Buffer containing PBS with 4% BSA. Block the
plates with 200 .mu.l/well of Pierce Super Block blocking buffer in
PBS for 1-2 hr and then wash plates with wash buffer (PBS, 0.05%
Tween-20). Blot plates on paper towels. Then add 50 .mu.l/well of
diluted Anti-Human IL-6 Monoclonal, Biotin-labeled antibody at 0.50
mg/ml. Make dilutions of IL-6 stock in media (30, 10, 3, 1, 0.3, 0
ng/ml). Add duplicate samples to top row of plate. Cover the plates
and incubate for 2 hours at RT on shaker.
[1703] Wash plates with wash buffer and blot on paper towels.
Dilute EU-labeled Streptavidin 1:1000 in Assay buffer, and add 100
.mu.l/well. Cover the plate and incubate 1 h at RT. Wash plates
with wash buffer. Blot on paper towels.
[1704] Add 100 .mu.l/well of Enhancement Solution. Shake for 5
minutes. Read the plate on the Wallac DELFIA Fluorometer. Readings
from triplicate samples in each assay were tabulated and
averaged.
[1705] A positive result in this assay suggests AoSMC cell
proliferation and that the gene product of interest may be involved
in dermal fibroblast proliferation and/or smooth muscle cell
proliferation. A positive result also suggests many potential uses
of polypeptides, polynucleotides, agonists and/or antagonists of
the gene/gene product of interest. For example, inflammation and
immune responses, wound healing, and angiogenesis, as detailed
throughout this specification. Particularly, polypeptides of the
gene product and polynucleotides of the gene may be used in wound
healing and dermal regeneration, as well as the promotion of
vasculargenesis, both of the blood vessels and lymphatics. The
growth of vessels can be used in the treatment of, for example,
cardiovascular diseases. Additionally, antagonists of polypeptides
of the gene product and polynucleotides of the gene may be useful
in treating diseases, disorders, and/or conditions which involve
angiogenesis by acting as an anti-vascular (e.g.,
anti-angiogenesis). These diseases, disorders, and/or conditions
are known in the art and/or are described herein, such as, for
example, malignancies, solid tumors, benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic granulomas; artheroscleric plaques; ocular angiogenic
diseases, for example, diabetic retinopathy, retinopathy of
prematurity, macular degeneration, corneal graft rejection,
neovascular glaucoma, retrolental fibroplasia, rubeosis,
retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth)
of the eye; rheumatoid arthritis; psoriasis; delayed wound healing;
endometriosis; vasculogenesis; granulations; hypertrophic scars
(keloids); nonunion fractures; scleroderma; trachoma; vascular
adhesions; myocardial angiogenesis; coronary collaterals; cerebral
collaterals; arteriovenous malformations; ischemic limb
angiogenesis; Osler-Webber Syndrome; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; fibromuscular
dysplasia; wound granulation; Crohn's disease; and atherosclerosis.
Moreover, antagonists of polypeptides of the gene product and
polynucleotides of the gene may be useful in treating
anti-hyperproliferative diseases and/or anti-inflammatory known in
the art and/or described herein.
[1706] One skilled in the art could easily modify the exemplified
studies to test the activity of polynucleotides (e.g., gene
therapy), antibodies, agonists, and/or antagonists and fragments
and variants thereof.
Example 56
Cellular Adhesion Molecule (CAM) Expression on Endothelial
Cells
[1707] The recruitment of lymphocytes to areas of inflammation and
angiogenesis involves specific receptor-ligand interactions between
cell surface adhesion molecules (CAMs) on lymphocytes and the
vascular endothelium. The adhesion process, in both normal and
pathological settings, follows a multi-step cascade that involves
intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion
molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1
(E-selectin) expression on endothelial cells (EC). The expression
of these molecules and others on the vascular endothelium
determines the efficiency with which leukocytes may adhere to the
local vasculature and extravasate into the local tissue during the
development of an inflammatory response. The local concentration of
cytokines and growth factor participate in the modulation of the
expression of these CAMs.
[1708] Briefly, endothelial cells (e.g., Human Umbilical Vein
Endothelial cells (HUVECs)) are grown in a standard 96 well plate
to confluence, growth medium is removed from the cells and replaced
with 100 .mu.l of 199 Medium (10% fetal bovine serum (FBS)).
Samples for testing and positive or negative controls are added to
the plate in triplicate (in 10 .mu.l volumes). Plates are then
incubated at 37.degree. C. for either 5 h (selectin and integrin
expression) or 24 h (integrin expression only). Plates are
aspirated to remove medium and 100 .mu.l of 0.1%
paraformaldehyde-PBS(with Ca++ and Mg++) is added to each well.
Plates are held at 4.degree. C. for 30 min. Fixative is removed
from the wells and wells are washed 1.times. with PBS(+Ca,Mg)+0.5%
BSA and drained. 10 .mu.l of diluted primary antibody is added to
the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin
and Anti-E-selectin-Biotin are used at a concentration of 10
.mu.g/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are
incubated at 37.degree. C. for 30 min. in a humidified environment.
Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA. 20 .mu.l of
diluted ExtrAvidin-Alkaline Phosphotase (1:5,000 dilution, refered
to herein as the working dilution) are added to each well and
incubated at 37.degree. C. for 30 min. Wells are washed three times
with PBS(+Ca,Mg)+0.5% BSA. Dissolve 1 tablet of p-Nitrophenol
Phosphate pNPP per 5 ml of glycine buffer (pH 10.4). 100 .mu.l of
pNPP substrate in glycine buffer is added to each test well.
Standard wells in triplicate are prepared from the working dilution
of the ExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000
(10.sup.0)>10.sup.-0.5>10.sup.-1>10.sup.-1.50.5 .mu.l of
each dilution is added to triplicate wells and the resulting AP
content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100
.mu.l of pNNP reagent is then added to each of the standard wells.
The plate is incubated at 37.degree. C. for 4 h. A volume of 50
.mu.l of 3 M NaOH is added to all wells. The plate is read on a
plate reader at 405 nm using the background subtraction option on
blank wells filled with glycine buffer only. Additionally, the
template is set up to indicate the concentration of AP-conjugate in
each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results
are indicated as amount of bound AP-conjugate in each sample.
Example 57
Alamar Blue Endothelial Cells Proliferation Assay
[1709] This assay may be used to quantitatively determine protein
mediated inhibition of bFGF-induced proliferation of Bovine
Lymphatic Endothelial Cells (LECs), Bovine Aortic Endothelial Cells
(BAECs) or Human Microvascular Uterine Myometrial Cells (UTMECs).
This assay incorporates a fluorometric growth indicator based on
detection of metabolic activity. A standard Alamar Blue
Proliferation Assay is prepared in EGM-2MV with 10 ng/ml of bFGF
added as a source of endothelial cell stimulation. This assay may
be used with a variety of endothelial cells with slight changes in
growth medium and cell concentration. Dilutions of the protein
batches to be tested are diluted as appropriate. Serum-free medium
(GIBCO SFM) without bFGF is used as a non-stimulated control and
Angiostatin or TSP-1 are included as a known inhibitory
controls.
[1710] Briefly, LEC, BAECs or UTMECs are seeded in growth media at
a density of 5000 to 2000 cells/well in a 96 well plate and placed
at 37-C overnight. After the overnight incubation of the cells, the
growth media is removed and replaced with GIBCO EC-SFM. The cells
are treated with the appropriate dilutions of the protein of
interest or control protein sample(s) (prepared in SFM ) in
triplicate wells with additional bFGF to a concentration of 10
ng/ml. Once the cells have been treated with the samples, the
plate(s) is/are placed back in the 37.degree. C. incubator for
three days. After three days 10 ml of stock alamar blue (Biosource
Cat# DAL1100) is added to each well and the plate(s) is/are placed
back in the 37.degree. C. incubator for four hours. The plate(s)
are then read at 530 nm excitation and 590 nm emission using the
CytoFluor fluorescence reader. Direct output is recorded in
relative fluorescence units.
[1711] Alamar blue is an oxidation-reduction indicator that both
fluoresces and changes color in response to chemical reduction of
growth medium resulting from cell growth. As cells grow in culture,
innate metabolic activity results in a chemical reduction of the
immediate surrounding environment. Reduction related to growth
causes the indicator to change from oxidized (non-fluorescent blue)
form to reduced (fluorescent red) form. i.e. stimulated
proliferation will produce a stronger signal and inhibited
proliferation will produce a weaker signal and the total signal is
proportional to the total number of cells as well as their
metabolic activity. The background level of activity is observed
with the starvation medium alone. This is compared to the output
observed from the positive control samples (bFGF in growth medium)
and protein dilutions.
Example 58
Detection of Inhibition of a Mixed Lymphocyte Reaction
[1712] This assay can be used to detect and evaluate inhibition of
a Mixed Lymphocyte Reaction (MLR) by gene products (e.g., isolated
polypeptides). Inhibition of a MLR may be due to a direct effect on
cell proliferation and viability, modulation of costimulatory
molecules on interacting cells, modulation of adhesiveness between
lymphocytes and accessory cells, or modulation of cytokine
production by accessory cells. Multiple cells may be targeted by
these polypeptides since the peripheral blood mononuclear fraction
used in this assay includes T, B and natural killer lymphocytes, as
well as monocytes and dendritic cells.
[1713] Polypeptides of interest found to inhibit the MLR may find
application in diseases associated with lymphocyte and monocyte
activation or proliferation. These include, but are not limited to,
diseases such as asthma, arthritis, diabetes, inflammatory skin
conditions, psoriasis, eczema, systemic lupus erythematosus,
multiple sclerosis, glomerulonephritis, inflammatory bowel disease,
crohn's disease, ulcerative colitis, arteriosclerosis, cirrhosis,
graft vs. host disease, host vs. graft disease, hepatitis, leukemia
and lymphoma.
[1714] Briefly, PBMCs from human donors are purified by density
gradient centrifugation using Lymphocyte Separation Medium
(LSM.RTM., density 1.0770 g/ml, Organon Teknika Corporation, West
Chester, Pa.). PBMCs from two donors are adjusted to
2.times.10.sup.6 cells/ml in RPMI-1640 (Life Technologies, Grand
Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCs
from a third donor is adjusted to 2.times.10.sup.5 cells/ml. Fifty
microliters of PBMCs from each donor is added to wells of a 96-well
round bottom microtiter plate. Dilutions of test materials (50
.mu.l) is added in triplicate to microtiter wells. Test samples (of
the protein of interest) are added for final dilution of 1:4;
rhuIL-2 (R&D Systems, Minneapolis, Minn, catalog number 202-IL)
is added to a final concentration of 1 .mu.g/ml; anti-CD4 mAb
(R&D Systems, clone 34930.11, catalog number MAB379) is added
to a final concentration of 10 .mu.g/ml. Cells are cultured for 7-8
days at 37.degree. C. in 5% CO.sub.2, and 1 .mu.C of [.sup.3H]
thymidine is added to wells for the last 16 hrs of culture. Cells
are harvested and thymidine incorporation determined using a
Packard TopCount. Data is expressed as the mean and standard
deviation of triplicate determinations.
[1715] Samples of the protein of interest are screened in separate
experiments and compared to the negative control treatment,
anti-CD4 mAb, which inhibits proliferation of lymphocytes and the
positive control treatment, IL-2 (either as recombinant material or
supernatant), which enhances proliferation of lymphocytes.
[1716] One skilled in the art could easily modify the exemplified
studies to test the activity of polynucleotides (e.g., gene
therapy), antibodies, agonists, and/or antagonists and fragments
and variants thereof. TABLE-US-00026 TABLE 7 Res Position I II III
IV V VI VII VIII IX X XI XII XIII XIV Met 1 . . B . . . . 0.24 0.41
. . . -0.40 0.65 Ile 2 . . B . . T . 0.60 0.39 . . . 0.10 0.88 Pro
3 . . B . . T . 0.99 0.46 . . . -0.20 0.93 Asn 4 . . . . T T . 0.79
0.43 . . . 0.35 1.51 Gln 5 . . B . . T . 0.83 0.31 . . F 0.40 2.18
His 6 . . . . . . C 0.84 0.06 . . F 0.40 1.40 Asn 7 . . . . . . C
1.39 0.13 . . . 0.10 0.88 Ala 8 . . . . . . C 1.30 0.16 . . . 0.10
0.50 Gly 9 . . . . T T . 1.27 0.14 . . . 0.50 0.49 Ala 10 . . . . T
T . 1.27 0.14 . . F 0.65 0.42 Gly 11 . . . . . T C 1.09 0.14 . . F
0.45 0.72 Ser 12 . . . . . T C 0.50 0.07 . . F 0.60 1.12 His 13 . A
. . . . C 0.23 0.14 . . F 0.20 1.12 Gln 14 . A B . . . . -0.12 0.29
* * F -0.15 0.84 Pro 15 . A B . . . . 0.58 0.64 * * F -0.45 0.54
Ala 16 . A B . . . . 0.32 0.26 * * . -0.30 0.78 Val 17 A A . . . .
. 0.03 0.37 . * . -0.30 0.45 Phe 18 . A B . . . . -0.79 0.47 . * .
-0.60 0.29 Arg 19 . A B . . . . -1.60 0.69 . . . -0.60 0.21 Met 20
. A B . . . . -1.39 0.87 . * . -0.60 0.24 Ala 21 A A . . . . .
-1.11 0.23 . * . -0.30 0.46 Val 22 A A . . . . . -0.26 -0.07 . * .
0.30 0.34 Leu 23 A A . . . . . -0.37 -0.07 . * . 0.30 0.57 Asp 24 A
. . . . T . -0.48 -0.00 . * F 0.85 0.47 Thr 25 A . . . . T . 0.09
-0.50 * . F 1.30 1.05 Asp 26 A . . . . T . -0.21 -0.64 * * F 1.30
1.73 Leu 27 A . . . . T . -0.17 -0.64 * * . 1.00 0.73 Asp 28 A . .
. . . . 0.43 0.04 * * . -0.10 0.41 His 29 . . B . . . . 0.13 -0.01
. * . 0.50 0.38 Ile 30 . . B . . . . 0.14 0.37 * . . -0.10 0.62 Leu
31 . . B . . T . -0.71 0.07 . . . 0.10 0.50 Pro 32 . . B . . T .
-0.71 0.71 . . F -0.05 0.27 Ser 33 . . . . T T . -0.92 0.90 . . F
0.35 0.32 Ser 34 . . B . . T . -1.10 0.64 . . F -0.05 0.60 Val 35 .
. B . . . . -0.91 0.39 . . F 0.05 0.60 Leu 36 . . B . . . . -0.39
0.74 . . F -0.25 0.39 Pro 37 . . B . . T . -0.77 1.27 . . F -0.05
0.31 Pro 38 . . B . . T . -0.42 1.39 * * . -0.20 0.42 Phe 39 A . .
. . T . -0.93 0.74 * . . -0.05 1.01 Trp 40 A . . . . T . -0.93 0.74
. * . -0.20 0.54 Ala 41 A A . B . . . -0.98 0.96 * . . -0.60 0.26
Lys 42 . A B B . . . -1.11 1.17 . * . -0.60 0.22 Leu 43 . A B B . .
. -1.20 0.81 . . . -0.60 0.21 Val 44 . A B B . . . -1.36 0.29 . . .
-0.30 0.28 Val 45 . . B B . . . -1.66 0.43 . . . -0.60 0.10 Gly 46
. . B B . . . -1.96 0.93 . . . -0.60 0.13 Ser 47 . A B B . . .
-2.86 0.93 . . . -0.60 0.12 Val 48 . A B B . . . -2.71 0.93 . . .
-0.60 0.12 Ala 49 . A B B . . . -2.56 0.86 . . . -0.60 0.06 Ile 50
. A B B . . . -2.29 1.21 * . . -0.60 0.04 Val 51 . A B B . . .
-1.83 1.33 * . . -0.60 0.06 Cys 52 . A B B . . . -1.83 0.69 * . .
-0.60 0.11 Phe 53 . A B B . . . -1.22 0.57 * . . -0.26 0.21 Ala 54
. A B B . . . -0.63 0.64 * . . 0.08 0.44 Arg 55 . A B B . . . -0.09
-0.00 * . . 1.47 1.38 Ser 56 . . . . T T . 0.77 -0.14 * . F 2.76
1.57 Tyr 57 . . . . T T . 0.73 -0.93 . . F 3.40 2.60 Asp 58 . . . .
T T . 0.58 -0.64 . . F 3.06 1.15 Gly 59 . . . . T T . 0.47 -0.00 .
* F 2.27 0.64 Asp 60 . . B . . . . 0.36 0.40 . * F 0.73 0.35 Phe 61
. . B . . . . 0.66 -0.36 . * . 0.84 0.35 Val 62 . . B . . . . 0.60
-0.36 . * . 0.50 0.59 Phe 63 . . B . . . . 0.60 -0.40 . * . 0.50
0.48 Asp 64 A . . . . T . 0.36 -0.40 * . F 0.85 0.95 Asp 65 A . . .
. T . -0.53 -0.69 * . F 1.30 1.30 Ser 66 A . . . . T . -0.69 -0.64
* . F 1.30 1.05 Glu 67 A . . . . T . 0.17 -0.79 * . F 1.15 0.47 Ala
68 A . . . . . . 0.87 -0.39 . . . 0.50 0.45 Ile 69 A . . . . . .
0.91 0.01 . . . -0.10 0.54 Val 70 A . . . . . . 0.91 -0.37 . . .
0.50 0.62 Asn 71 A . . . . . . 0.40 -0.37 . . F 0.80 1.03 Asn 72 A
. . . . T . 0.40 -0.19 . . F 1.00 1.21 Lys 73 A . . . . T . 0.40
-0.47 . * F 1.00 2.83 Asp 74 A . . . . T C 1.29 -0.61 . * F 1.50
1.78 Leu 75 A . . . . T . 1.83 -1.01 . . F 1.30 1.91 Gln 76 . . B .
. . . 1.62 -0.93 . * . 0.95 1.38 Ala 77 . . B . . . . 0.81 -0.50 .
* F 1.35 1.28 Glu 78 . . B . . . . 0.42 0.19 . * F 0.70 1.28 Thr 79
. . B . . T . 0.42 -0.07 . * F 1.60 0.73 Pro 80 A . . . . T . 0.42
-0.47 * * F 2.00 1.21 Leu 81 . . . . T T . 0.13 -0.29 * . F 2.50
0.57 Gly 82 A . . . . T . 0.69 0.63 * . F 0.95 0.42 Asp 83 A A . .
. . . 0.66 0.64 . . . 0.15 0.37 Leu 84 A A . . . . . 0.97 0.71 * .
. -0.10 0.61 Trp 85 A A . . . . . 0.48 0.03 . . . 0.10 1.03 His 86
. A B . . . . 1.00 0.39 . . . -0.30 0.53 His 87 . A B . . . . 1.00
1.30 . . . -0.60 0.68 Asp 88 . A . . T . . 0.70 1.04 . . . -0.20
0.64 Phe 89 . A . . T . . 1.62 0.51 . . . -0.20 0.63 Trp 90 . A . .
T . . 1.10 0.01 . * . 0.10 0.91 Gly 91 . . . . . T C 0.83 0.20 . .
F 0.45 0.45 Ser 92 . . . . . T C 0.57 0.59 . * F 0.15 0.69 Arg 93 .
. . . . T C 0.57 0.19 . * F 0.45 0.88 Leu 94 . . . . . T C 0.96
-0.33 . * F 1.20 1.43 Ser 95 . . . . . T C 0.94 -0.27 * * F 1.20
1.54 Ser 96 . . . . . T C 1.26 -0.27 * * F 1.20 1.06 Asn 97 . . . .
. T C 1.60 0.23 * * F 0.60 1.74 Thr 98 . . . . T T . 1.19 -0.46 * *
F 1.74 2.60 Ser 99 . . . . T . . 1.76 -0.46 * * F 1.88 2.60 His 100
. . . . T T . 2.17 -0.09 * . F 2.42 2.53 Lys 101 . . . . T T . 2.26
-0.49 * . F 2.76 3.44 Ser 102 . . . . T T . 1.44 -0.54 * . F 3.40
3.97 Tyr 103 . . B . . T . 1.44 -0.24 * . F 2.36 2.40 Arg 104 . . B
B . . . 0.89 -0.26 * . F 1.62 1.74 Pro 105 . . B B . . . 0.11 0.39
* . F 0.53 0.96 Leu 106 . . B B . . . -0.24 0.69 * . . -0.26 0.51
Thr 107 . . B B . . . -0.64 0.41 * * . -0.60 0.37 Val 108 . . B B .
. . -0.29 1.20 * * . -0.60 0.21 Leu 109 . . B B . . . -1.29 0.77 *
* . -0.60 0.50 Thr 110 . . B B . . . -1.08 0.77 . * . -0.60 0.24
Phe 111 . . B B . . . -0.51 0.69 . * . -0.60 0.52 Arg 112 . . B B .
. . -0.44 0.80 . * . -0.60 0.99 Ile 113 . . B B . . . -0.40 0.87 .
* . -0.45 1.08 Asn 114 . . B B . . . 0.11 1.07 . * . -0.45 1.02 Tyr
115 . . B B . . . 0.08 0.67 . * . -0.60 0.70 Tyr 116 . . . B T . .
0.43 1.10 * * . -0.20 0.99 Leu 117 . . . . T T . -0.38 0.84 * * .
0.20 0.61 Ser 118 . . . . T T . 0.48 1.23 * * F 0.35 0.34 Gly 119 .
. . . T T . 0.27 0.97 . * F 0.35 0.29 Gly 120 . . . . T T . -0.34
0.64 . . F 0.35 0.55 Phe 121 . . B . . . . -0.44 0.60 . . . -0.40
0.30 His 122 . . B . . T . -0.33 0.64 . . . -0.20 0.30 Pro 123 . .
B . . T . -0.07 1.00 . . . -0.20 0.27 Val 124 . . B . . T . -0.58
1.07 . . . -0.20 0.42 Gly 125 . . B . . T . -1.09 0.93 * . . -0.20
0.23 Phe 126 . . B B . . . -0.39 1.07 * . . -0.60 0.11 His 127 . .
B B . . . -1.24 1.04 * . . -0.60 0.24 Val 128 . . B B . . . -1.84
1.09 * . . -0.60 0.17 Val 129 . . B B . . . -1.80 1.34 * . . -0.60
0.16 Asn 130 . . B B . . . -1.49 1.24 * . . -0.60 0.10 Ile 131 . .
B B . . . -1.09 1.24 * . . -0.60 0.18 Leu 132 . . B B . . . -1.40
0.99 * * . -0.60 0.32 Leu 133 A . . B . . . -1.43 0.77 . * . -0.60
0.20 His 134 . . . . . T C -0.88 1.06 * * . 0.00 0.20 Ser 135 . . .
. . T C -1.73 0.76 . * . 0.00 0.32 Gly 136 . . B . . T . -1.66 0.71
. * . -0.20 0.29 Ile 137 . . B . . T . -1.44 0.71 . . . -0.20 0.17
Ser 138 . . B B . . . -1.49 0.83 . . . -0.60 0.13 Val 139 . . B B .
. . -1.46 1.09 . . . -0.60 0.10 Leu 140 . . B B . . . -2.01 0.66 .
. . -0.60 0.23 Met 141 . . B B . . . -2.37 0.61 . . . -0.60 0.13
Val 142 . . B B . . . -1.78 1.01 . . . -0.60 0.15 Asp 143 . . B B .
. . -2.33 0.76 . . . -0.60 0.24 Val 144 . . B B . . . -2.29 0.71 .
. . -0.60 0.18 Phe 145 . . B B . . . -2.18 0.79 . . . -0.60 0.20
Ser 146 . . B B . . . -1.92 0.93 * . . -0.60 0.10 Val 147 . . B B .
. . -1.41 1.36 * . . -0.60 0.14 Leu 148 . . B B . . . -2.22 1.14 *
. . -0.60 0.16 Phe 149 . . B B . . . -1.37 1.04 * . . -0.60 0.10
Gly 150 . . . B T . . -0.91 1.06 . . . -0.20 0.23 Gly 151 . . . B .
. C -0.92 1.17 . . . -0.40 0.44 Leu 152 . . B B . . . -0.37 0.97 *
* . -0.60 0.73 Gln 153 . . B B . . . 0.49 0.57 * * . -0.26 0.98 Tyr
154 . . B B . . . 0.84 0.14 . * F 0.68 1.98 Thr 155 . . . B T . .
1.30 0.14 * * F 1.42 2.38 Ser 156 . . . . T T . 1.76 -0.54 * * F
3.06 2.69 Lys 157 . . . . T T . 1.76 -0.94 * * F 3.40 3.37 Gly 158
. . . . T T . 1.72 -1.01 * * F 3.06 1.92 Arg 159 . . B . . T . 1.16
-1.00 * . F 2.32 1.95 Arg 160 . A B . . . . 0.88 -0.70 * . F 1.43
0.81 Leu 161 . A B . . . . 0.97 -0.20 * * . 0.64 0.82 His 162 . A B
. . . . 1.03 -0.20 * * . 0.30 0.65 Leu 163 . A B . . . . 0.79 -0.20
* * . 0.30 0.65 Ala 164 . A B . . . . 0.38 0.30 * * . -0.30 0.80
Pro 165 A A . . . . . -0.54 0.00 . * F -0.15 0.78 Arg 166 A A . . .
. . -0.54 0.19 * * F -0.15 0.78 Ala 167 A A . . . . . -1.10 0.19 .
* . -0.30 0.64 Ser 168 A A . . . . . -0.88 0.19 . * . -0.30 0.42
Leu 169 A A . . . . . -1.10 0.26 . * . -0.30 0.22 Leu 170 A A . . .
. . -1.70 0.94 . * . -0.60 0.18 Ala 171 A A . . . . . -2.51 1.13 .
* . -0.60 0.11 Ala 172 A A . B . . . -2.51 1.53 . . . -0.60 0.11
Leu 173 A A . B . . . -3.07 1.34 . . . -0.60 0.14 Leu 174 A A . B .
. . -2.29 1.30 . . . -0.60 0.10 Phe 175 A A . B . . . -1.69 1.30 .
. . -0.60 0.14 Ala 176 A A . B . . . -1.96 1.23 . . . -0.60 0.26
Val 177 A A . B . . . -1.40 1.19 . . . -0.60 0.23 His 178 A A . B .
. . -0.90 1.00 . . . -0.60 0.37 Pro 179 A A . B . . . -0.09 0.70 .
. . -0.60 0.52 Val 180 A A . B . . . -0.06 0.20 . . . -0.15 1.22
His 181 A A . B . . . -0.32 0.13 . . . -0.30 0.48 Thr 182 A A . B .
. . -0.06 0.27 . . . -0.30 0.23 Glu 183 A A B B . . . -0.37 0.34 .
. . -0.30 0.31 Cys 184 . A B B . . . -1.01 0.13 . . . -0.30 0.23
Val 185 . . B B . . . -1.01 0.27 . . . -0.30 0.12 Ala 186 . . B B .
. . -1.32 0.43 * . . -0.60 0.05 Gly 187 . . B B . . . -0.90 0.86 *
* . -0.60 0.09 Val 188 . . B B . . . -1.49 0.29 * * . -0.30 0.24
Val 189 . . B B . . . -0.82 0.14 . * . -0.30 0.24 Gly 190 A A . . .
. . -0.78 -0.36 . * . 0.30 0.41 Arg 191 A A . . . . . -1.00 -0.10 .
* . 0.30 0.46 Ala 192 A A . . . . . -1.32 -0.06 . * . 0.30 0.51 Asp
193 A A . . . . . -1.06 -0.13 . * . 0.30 0.28 Leu 194 A A . . . . .
-1.01 -0.06 . * . 0.30 0.14 Leu 195 A A . . . . . -1.37 0.63 . * .
-0.60 0.12 Cys 196 A A . . . . . -2.18 0.91 . * . -0.60 0.06 Ala
197 A A . . . . . -2.40 1.70 . . . -0.60 0.06 Leu 198 A A . . . . .
-3.21 1.70 * . . -0.60 0.06 Phe 199 A A . . . . . -2.70 1.70 . . .
-0.60 0.10 Phe 200 A A . . . . . -2.59 1.51 . . . -0.60 0.13 Leu
201 A A . . . . . -2.73 1.80 . . . -0.60 0.14 Leu 202 A A . . . . .
-2.49 1.80 . . . -0.60 0.13 Ser 203 A . . . . . . -1.92 1.44 . . .
-0.40 0.15 Phe 204 A . . . . . . -1.89 1.41 . . . -0.40 0.28 Leu
205 A . . . . T . -1.14 1.30 . * . -0.20 0.18 Gly 206 . . . . T T .
-0.92 0.61 . . . 0.20 0.27 Tyr 207 . . . . T T . -0.81 0.73 * * .
0.20 0.32 Cys 208 A . . . . T . -0.40 0.73 * . . -0.20 0.33 Lys 209
A A . . . . . 0.30 0.04 * . . -0.30 0.66 Ala 210 A A . . . . . 0.81
-0.39 * . . 0.30 0.73 Phe 211 A A . . . . . 1.16 -0.76 * . . 0.75
1.82 Arg 212 A A . . . . . 1.44 -0.93 * . F 0.90 1.47 Glu 213 A A .
. . . . 2.11 -0.93 * . F 0.90 2.90 Ser 214 A A . . . . . 1.72 -1.43
* . F 0.90 5.80 Asn 215 A . . . . T . 1.72 -1.79 * . F 1.30 2.93
Lys 216 A . . . . T . 2.39 -1.29 * * F 1.30 1.71 Glu 217 A . . . .
T . 1.98 -0.79 * . F 1.30 1.74 Gly 218 A . . . . T . 1.68 -0.79 . .
F 1.30 1.45 Ala 219 A . . . . . . 1.67 -0.80 . . F 0.95 0.97 His
220 . . . . . T C 0.97 -0.31 . . F 1.05 0.81 Ser 221 . . . . . T C
0.63 0.47 . . F 0.15 0.71 Ser 222 . . . . . T C -0.22 0.96 . . F
0.15 0.74 Thr 223 . . B . . T . -0.69 1.10 . . F -0.05 0.40 Phe 224
. . B B . . . -0.91 1.29 . . . -0.60 0.25 Trp 225 . . B B . . .
-1.18 1.59 . . . -0.60 0.15 Val 226 . . B B . . . -1.77 1.59 . . .
-0.60 0.14 Leu 227 . . B B . . . -2.17 1.79 . . . -0.60 0.11 Leu
228 . . B B . . . -2.67 1.79 . . . -0.60 0.09 Ser 229 . . B B . . .
-2.31 1.56 . . . -0.60 0.10 Ile 230 A . . B . . . -2.61 1.34 . . .
-0.60 0.13 Phe 231 A . . B . . . -2.61 1.16 . . . -0.60 0.15 Leu
232 A . . B . . . -2.39 1.11 . . . -0.60 0.09 Gly 233 A . . B . . .
-2.18 1.23 . . . -0.60 0.12 Ala 234 A . . B . . . -2.69 1.16 . . .
-0.60 0.14 Val 235 A . . B . . . -2.47 1.06 * . . -0.60 0.14 Ala
236 A . . B . . . -1.72 0.94 * . . -0.60 0.08 Met 237 A . . B . . .
-0.91 0.51 . . . -0.60 0.15 Leu 238 A . . B . . . -0.57 0.01 . . .
-0.30 0.35 Cys 239 A . . B . . . -0.32 -0.23 . . . 0.30 0.60 Lys
240 A . . B . . . -0.36 -0.30 . . F 0.45 0.60 Glu 241 A . . . . . .
-0.08 -0.23 . . F 0.65 0.51
Gln 242 A . . B . . . -0.33 -0.43 . . F 0.60 1.38 Gly 243 . . B B .
. . -0.33 -0.36 . . F 0.45 0.51 Ile 244 . . B B . . . -0.01 0.33 .
. F -0.15 0.24 Thr 245 . . B B . . . -0.87 0.76 . . . -0.60 0.14
Val 246 . . B B . . . -0.87 1.04 . . . -0.60 0.12 Leu 247 . . B B .
. . -1.46 1.01 . . . -0.60 0.27 Gly 248 . . B B . . . -1.97 0.83 .
. . -0.60 0.19 Leu 249 . . B B . . . -1.78 0.99 . * . -0.60 0.19
Asn 250 . . B B . . . -1.47 1.13 . * . -0.60 0.20 Ala 251 A . . B .
. . -1.50 0.44 * * . -0.60 0.33 Val 252 A . . B . . . -1.50 0.70 *
* . -0.60 0.28 Phe 253 . . B B . . . -2.01 0.70 * * . -0.60 0.14
Asp 254 . . B B . . . -2.09 0.94 * * . -0.60 0.11 Ile 255 . . B B .
. . -2.43 1.13 * * . -0.60 0.10 Leu 256 . . B B . . . -1.80 0.91 .
* . -0.60 0.11 Val 257 . . B B . . . -1.64 0.13 . * . -0.30 0.14
Ile 258 A . . B . . . -0.94 0.91 . * . -0.60 0.17 Gly 259 A . . B .
. . -1.80 0.63 . * . -0.60 0.33 Lys 260 A . . B . . . -1.72 0.59 .
* . -0.60 0.33 Phe 261 A . . B . . . -0.91 0.63 . . . -0.60 0.39
Asn 262 A . . B . . . -0.94 -0.06 . * . 0.30 0.68 Val 263 A . . B .
. . -0.44 0.20 . . . -0.30 0.24 Leu 264 A . . B . . . -0.10 0.63 .
* . -0.60 0.35 Glu 265 A . . B . . . -0.10 0.24 * * . -0.30 0.38
Ile 266 A . . B . . . -0.26 -0.16 * . F 0.60 1.02 Xxx 267 A . . B .
. . -1.07 -0.16 * . F 0.45 0.91 Gln 268 A . . B . . . -0.24 -0.16 *
* F 0.45 0.44 Lys 269 A . . B . . . 0.61 0.34 * * F -0.15 0.85 Val
270 A . . B . . . 0.61 -0.34 * * F 0.60 1.32 Leu 271 A . . B . . .
1.54 -0.77 * * . 0.75 1.28 His 272 A . . B . . . 1.63 -1.17 * . .
0.75 1.28 Lys 273 A . . B . . . 0.82 -0.79 * . F 0.90 2.30 Asp 274
A . . . . T . 0.78 -0.74 * . F 1.30 2.30 Lys 275 A . . . . T . 1.63
-1.43 * . F 1.30 2.93 Ser 276 A . . . . T . 1.63 -1.53 . . F 1.30
2.36 Leu 277 A . . . . T . 1.32 -0.84 . . F 1.30 1.16 Glu 278 A A .
. . . . 0.68 -0.41 . . F 0.45 0.58 Asn 279 A A . . . . . -0.13 0.20
. . . -0.30 0.43 Leu 280 A A . . . . . -0.07 0.50 . . . -0.60 0.43
Gly 281 A A . . . . . 0.23 -0.19 . . . 0.30 0.48 Met 282 . A B . .
. . 0.70 0.21 . . . -0.30 0.48 Leu 283 . . B . . T . 0.36 0.24 . .
. 0.10 0.58 Arg 284 . . B . . T . -0.46 -0.01 . . F 0.85 0.58 Asn
285 . . . . T T . -0.46 0.24 * . F 0.65 0.48 Gly 286 . . . . T T .
-0.81 0.31 * * F 0.65 0.48 Gly 287 . . . B . . C -0.10 0.41 * * F
-0.25 0.21 Leu 288 . . B B . . . 0.11 0.41 * * . -0.60 0.26 Leu 289
. . B B . . . -0.31 0.63 * * . -0.60 0.26 Phe 290 . . B B . . .
-1.12 0.69 . * . -0.60 0.38 Arg 291 . . B B . . . -1.59 0.94 . * .
-0.60 0.38 Met 292 . . B B . . . -1.56 0.94 . * . -0.60 0.38 Thr
293 . . B B . . . -1.04 0.74 . * . -0.60 0.63 Leu 294 . . B B . . .
-0.58 0.34 . * . -0.30 0.43 Leu 295 . . B B . . . -0.22 0.77 . * F
-0.45 0.43 Thr 296 . . . . . T C -0.92 0.59 . * F 0.15 0.30 Ser 297
. . . . . T C -0.67 0.60 . . F 0.15 0.36 Gly 298 . . . . . T C
-0.96 0.34 . . F 0.45 0.43 Gly 299 . . . . . T C -0.96 0.27 . . F
0.45 0.30 Ala 300 . . . . . . C -0.39 0.47 . . F -0.05 0.18 Gly 301
. . B B . . . -0.93 0.84 . * . -0.60 0.29 Met 302 . . B B . . .
-0.52 1.06 . * . -0.60 0.22 Leu 303 . . B B . . . -0.47 0.63 . * .
-0.60 0.42 Tyr 304 . . B B . . . -0.01 1.04 . * . -0.60 0.45 Val
305 . . B B . . . -0.31 0.61 . * . -0.60 0.89 Arg 306 . . B B . . .
-0.57 0.69 . * . -0.60 0.75 Trp 307 . . B B . . . -0.31 0.61 . * .
-0.60 0.48 Arg 308 . . B B . . . 0.19 0.29 . * . -0.30 0.63 Ile 309
. . B B . . . 0.09 0.13 . * . -0.30 0.47 Met 310 . . B B . . . 0.73
0.56 . * . -0.60 0.44 Gly 311 . . . . T . . 0.23 0.07 . * . 0.30
0.35 Thr 312 . . . . . . C -0.07 0.50 * * F -0.05 0.63 Gly 313 . .
. . . T C -0.88 0.31 * * F 0.45 0.65 Pro 314 . . . . . T C -0.30
0.49 * . F 0.15 0.57 Xxx 315 . . . . . T C 0.30 0.54 * . F 0.15
0.57 Ala 316 . . B . . T . -0.21 0.06 . . . 0.10 0.99 Phe 317 . . B
. . . . 0.10 0.27 * . . -0.10 0.48 Thr 318 . . B . . . . 0.44 -0.16
* . . 0.77 0.62 Glu 319 . . B . . . . 0.44 -0.19 * . F 1.19 0.99
Val 320 . . B . . . . 0.24 -0.26 * . F 1.61 1.77 Asp 321 . . . . .
. C 0.53 -0.54 * * F 2.38 1.24 Asn 322 . . . . . T C 0.53 -0.64 * .
F 2.70 0.96 Pro 323 A . . . . T . 0.26 0.14 * . F 1.48 1.12 Ala 324
A . . . . T . 0.26 -0.00 * . F 1.66 0.68 Ser 325 A . . . . T . 0.81
-0.00 * . . 1.24 0.70 Phe 326 A . . . . . . 0.21 -0.01 * . . 0.77
0.61 Ala 327 A . . . . . . -0.60 0.17 . . . -0.10 0.60 Asp 328 A .
. . . . . -1.24 0.36 * * . -0.10 0.37 Ser 329 A . . B . . . -0.54
0.61 * * . -0.60 0.31 Met 330 A . . B . . . -0.83 -0.17 * . . 0.30
0.61 Leu 331 A . . B . . . -0.99 -0.17 * . . 0.30 0.37 Val 332 . .
B B . . . -0.40 0.47 * . . -0.60 0.20 Arg 333 . . B B . . . -0.64
0.49 * . . -0.60 0.33 Ala 334 . . B B . . . -0.34 0.63 * . . -0.60
0.63 Val 335 . . B B . . . 0.01 0.34 * . . -0.15 1.36 Asn 336 . . B
. . T . 0.58 0.46 * . . -0.05 1.09 Tyr 337 . . B . . T . 1.19 1.21
* . . -0.05 1.69 Asn 338 . . B . . T . 0.78 1.47 . * . -0.05 3.57
Tyr 339 . . B . . T . 0.56 1.21 . * . -0.05 2.98 Tyr 340 . . B . .
. . 1.41 1.50 . * . -0.25 1.57 Tyr 341 . . B . . . . 0.82 1.14 . *
. -0.25 1.57 Ser 342 . A B . . . . 0.78 1.24 . * . -0.45 1.01 Leu
343 . A B . . . . -0.03 1.40 . * . -0.60 0.68 Asn 344 . A B . . . .
-0.60 1.33 . * . -0.60 0.36 Ala 345 . A B . . . . -1.17 1.26 . . .
-0.60 0.22 Trp 346 . A B . . . . -1.59 1.56 . * . -0.60 0.22 Leu
347 . A B . . . . -1.50 1.44 . . . -0.60 0.07 Leu 348 . A B . . . .
-0.98 1.47 . . . -0.60 0.11 Leu 349 . A B . . . . -1.27 1.89 . . .
-0.60 0.11 Cys 350 . . B . . T . -1.49 1.89 . . . -0.20 0.14 Pro
351 . . . . T T . -1.87 1.89 . . . 0.20 0.14 Trp 352 . . . . T T .
-1.76 1.77 . . . 0.20 0.09 Trp 353 . . B . . T . -0.94 1.87 . * .
-0.20 0.15 Leu 354 . . B . . . . -0.42 1.30 . * . -0.40 0.16 Cys
355 . . . . T T . -0.06 1.79 . * . 0.20 0.16 Phe 356 . . . . T T .
-0.44 1.26 . * . 0.20 0.21 Asp 357 . . . . T T . -0.50 0.96 . * .
0.20 0.25 Trp 358 . . . . T T . -0.88 0.70 . * . 0.20 0.46 Ser 359
. . . . T T . -0.96 0.70 . * . 0.20 0.28 Met 360 . . . . T T .
-0.50 0.60 . * . 0.20 0.12 Gly 361 . . . . T T . -0.61 1.03 . . .
0.20 0.17 Cys 362 . . B . . T . -1.50 0.80 * . . -0.20 0.11 Ile 363
. . B B . . . -1.17 1.10 . . . -0.60 0.08 Pro 364 . . B B . . .
-1.17 0.49 * . . -0.60 0.15 Leu 365 . . B B . . . -1.46 0.44 * . .
-0.60 0.38 Ile 366 . . B B . . . -1.41 0.56 * . . -0.60 0.38 Lys
367 . . B B . . . -0.74 0.26 * . F -0.15 0.33 Ser 368 . . B B . . .
-0.14 -0.17 * * F 0.45 0.68 Ile 369 . . . B T . . 0.18 0.06 * * F
0.40 1.01 Ser 370 . . B B . . . 0.13 -0.63 * * F 0.75 0.99 Asp 371
. . . B T . . 0.13 0.01 . * F 0.25 0.55 Trp 372 . . B B . . . -0.50
0.31 . * . -0.30 0.55 Arg 373 . A B . . . . -1.01 0.13 . . . -0.30
0.41 Val 374 . A B . . . . -0.71 0.43 . . . -0.60 0.20 Ile 375 . A
B . . . . -1.00 0.93 . * . -0.60 0.20 Ala 376 A A . . . . . -1.81
0.51 . * . -0.60 0.10 Leu 377 A A . . . . . -1.81 1.20 * * . -0.60
0.11 Ala 378 A A . . . . . -2.62 1.47 * . . -0.60 0.17 Ala 379 A A
. . . . . -2.43 1.57 . . . -0.60 0.14 Leu 380 A A . . . . . -2.36
1.64 . . . -0.60 0.09 Trp 381 A A . . . . . -2.66 1.64 . . . -0.60
0.08 Phe 382 A A . . . . . -2.19 1.83 . . . -0.60 0.05 Cys 383 A A
. . . . . -2.41 1.76 . . . -0.60 0.06 Leu 384 A A . . . . . -2.71
1.76 . . . -0.60 0.05 Ile 385 . A B . . . . -2.57 1.53 * . . -0.60
0.04 Gly 386 . A . . T . . -2.28 1.31 * . . -0.20 0.04 Leu 387 . A
. . T . . -2.17 1.14 * . . -0.20 0.09 Ile 388 . A B . . . . -2.31
0.96 * . . -0.60 0.12 Cys 389 . A B . . . . -2.17 0.96 * . . -0.60
0.10 Gln 390 . A B . . . . -1.58 1.10 . . . -0.60 0.07 Ala 391 . A
B . . . . -1.23 0.80 . . . -0.60 0.13 Leu 392 . A B . . . . -0.42
0.11 . . . -0.30 0.41 Cys 393 A A . . . . . 0.12 -0.46 . . . 0.30
0.40 Ser 394 A . . . . T . 0.76 -0.43 . . F 0.85 0.39 Glu 395 A . .
. . T . 0.80 -0.43 . . F 0.85 0.64 Asp 396 A . . . . T . 1.50 -1.11
. . F 1.30 2.39 Gly 397 A . . . . T . 2.42 -1.69 . . F 1.30 3.50
His 398 A . . . . . . 2.20 -2.07 . . F 1.10 3.95 Lys 399 A . . B .
. . 1.69 -1.39 . . F 0.90 1.66 Arg 400 . . B B . . . 1.38 -0.70 . .
F 0.90 1.38 Arg 401 . . B B . . . 0.57 -0.64 * . . 0.75 1.47 Ile
402 . . B B . . . 0.57 -0.46 * . . 0.30 0.61 Leu 403 . . B B . . .
-0.21 -0.03 * . . 0.30 0.31 Thr 404 . . B B . . . -0.60 0.66 * . .
-0.60 0.13 Leu 405 . . B B . . . -1.41 1.09 * . . -0.60 0.18 Gly
406 . . B B . . . -2.33 1.19 * * . -0.60 0.19 Leu 407 . . B B . . .
-2.30 1.19 . . . -0.60 0.11 Gly 408 . . B B . . . -2.38 1.34 . . .
-0.60 0.10 Phe 409 . . B B . . . -2.28 1.34 . . . -0.60 0.07 Leu
410 . . B B . . . -2.17 1.34 . . . -0.60 0.13 Val 411 . . B B . . .
-2.63 1.44 . . . -0.60 0.11 Ile 412 . . B B . . . -2.03 1.70 . . .
-0.60 0.11 Pro 413 . . B . . . . -2.28 1.34 . . . -0.40 0.20 Phe
414 . . B . . . . -1.88 1.16 . . . -0.40 0.28 Leu 415 . . B . . . .
-1.07 0.90 . . . -0.40 0.53 Pro 416 . . . . . . C -1.02 0.61 . . .
-0.20 0.55 Ala 417 . . . . T T . -0.83 0.87 . . . 0.20 0.52 Ser 418
. . . . . T C -1.32 0.87 * * . 0.00 0.55 Asn 419 A . . . . T .
-0.51 0.97 * * . -0.20 0.31 Leu 420 . . B . . T . -0.56 0.54 * * .
-0.20 0.60 Phe 421 . . B B . . . -0.69 0.69 * * . -0.60 0.33 Phe
422 . . B B . . . -0.80 0.73 * * . -0.60 0.20 Arg 423 . . B B . . .
-1.36 1.11 * * . -0.60 0.21 Val 424 . . B B . . . -2.21 1.07 * * .
-0.60 0.18 Gly 425 . . B B . . . -1.99 0.93 * * . -0.60 0.16 Phe
426 . . B B . . . -1.29 0.64 * * . -0.60 0.08 Val 427 A . . B . . .
-0.48 0.64 * * . -0.60 0.19 Val 428 A . . B . . . -1.44 -0.00 * * .
0.30 0.37 Ala 429 A . . B . . . -1.40 0.21 * . . -0.30 0.32 Glu 430
A . . B . . . -1.30 0.11 * . . -0.30 0.36 Arg 431 . . B B . . .
-1.41 0.23 * . . -0.30 0.75 Val 432 . . B B . . . -0.77 0.27 * . .
-0.30 0.62 Leu 433 . . B B . . . -0.21 0.20 * . . -0.30 0.55 Tyr
434 . . B B . . . -0.01 0.59 * . . -0.60 0.38 Leu 435 . . B B . . .
-0.36 1.01 * . . -0.60 0.65 Pro 436 . . . B T . . -0.71 0.80 * . F
-0.05 0.78 Ser 437 . . . . T T . -0.52 0.87 . . F 0.35 0.78 Xxx 438
. . . . T T . -0.57 0.69 . . F 0.35 0.50 Gly 439 . . B . . T .
-1.13 0.64 . . F -0.05 0.24 Tyr 440 . . B . . T . -1.13 0.90 . . .
-0.20 0.15 Cys 441 . . B B . . . -1.23 1.20 . . . -0.60 0.10 Val
442 . . B B . . . -1.63 1.26 . . . -0.60 0.14 Leu 443 . . B B . . .
-1.59 1.61 . . . -0.60 0.08 Leu 444 . . B B . . . -1.94 1.29 . . .
-0.60 0.14 Thr 445 . . B B . . . -2.04 1.50 . * . -0.60 0.17 Phe
446 . . B B . . . -1.97 1.29 . . . -0.60 0.20 Gly 447 A . . B . . .
-1.92 1.10 . . . -0.60 0.25 Phe 448 A . . B . . . -1.41 1.10 * . .
-0.60 0.14 Gly 449 A . . . . . . -0.56 1.00 . . . -0.40 0.22 Ala
450 A . . . . . . -0.28 0.21 . . . -0.10 0.44 Leu 451 A . . . . . .
0.11 0.29 * . . -0.10 0.69 Ser 452 A . . . . T . 0.50 -0.01 * . F
1.00 1.01 Lys 453 A . . . . T . 1.24 -0.44 * . F 1.00 1.99 His 454
A . . . . T . 1.63 -0.94 . . F 1.30 4.83 Thr 455 A . . . . T . 2.27
-1.63 . . F 1.30 7.20 Lys 456 A A . . . . . 2.27 -2.01 . . F 0.90
7.20 Lys 457 A A . . . . . 1.68 -1.33 . . F 0.90 4.36 Lys 458 A A .
B . . . 1.04 -1.14 . . F 0.90 2.12 Lys 459 A A . B . . . 0.49 -1.13
. . F 0.90 1.07 Leu 460 A A . B . . . -0.06 -0.63 . . . 0.60 0.54
Ile 461 A A . B . . . -0.96 0.01 . . . -0.30 0.20 Ala 462 A A . B .
. . -1.81 0.66 . . . -0.60 0.07 Ala 463 A A . B . . . -2.20 1.34 *
. . -0.60 0.07 Val 464 A A . B . . . -3.13 1.09 * . . -0.60 0.11
Val 465 . A B B . . . -3.13 1.09 . . . -0.60 0.07 Leu 466 . A B B .
. . -2.94 1.27 . . . -0.60 0.06 Gly 467 . A B B . . . -3.24 1.56 .
. . -0.60 0.07 Ile 468 . . B B . . . -2.66 1.60 . . . -0.60 0.07
Leu 469 . . B B . . . -2.11 1.36 . . . -0.60 0.13 Phe 470 . . B B .
. . -2.07 1.16 * . . -0.60 0.19 Ile 471 . . B B . . . -1.14 1.41 *
* . -0.60 0.22 Asn 472 . . B B . . . -1.47 0.73 * . . -0.60 0.52
Thr 473 . . B B . . . -1.43 0.61 * * . -0.60 0.32 Leu 474 . . B B .
. . -1.43 0.47 * * . -0.60 0.34 Arg 475 . . B B . . . -0.62 0.47 *
* . -0.60 0.18 Cys 476 . . B B . . . -0.03 0.07 * * . -0.30 0.24
Val 477 . . B B . . . -0.38 -0.03 * * . 0.30 0.39 Leu 478 . . B B .
. . -0.07 -0.29 * * . 0.60 0.20 Arg 479 . . B B . . . 0.46 -0.29 *
* F 1.05 0.63 Ser 480 . . . . . T C 0.46 0.06 * * F 1.35 0.89 Gly
481 . . . . . T C 0.82 -0.59 . * F 2.70 2.12 Glu 482 . . . . . T C
1.68 -0.89 * * F 3.00 1.45 Trp 483 . . . . . T C 2.49 -0.89 . * F
2.70 1.88 Arg 484 A A . . . . . 2.38 -1.27 . * F 1.80 3.29 Ser 485
A A . . . . . 1.87 -1.30 * * F 1.50 3.29 Glu 486 A A . . . . . 1.51
-0.61 * * F 1.20 2.58 Glu 487 A A . . . . . 1.62 -0.74 * * F 0.90
1.14 Gln 488 A A . . . . . 1.61 -0.74 * * F 0.90 1.67 Leu 489 A A .
. . . . 0.91 -0.74 * * F 0.90 1.29 Phe 490 A A . . . . . 0.40 -0.24
* . . 0.30 0.75 Arg 491 A A . . . . . 0.10 0.44 * * . -0.60 0.36
Ser 492 A A . . . . . -0.76 0.43 * * . -0.60 0.58
Ala 493 A A . . . . . -1.42 0.39 * * . -0.30 0.50 Leu 494 . A B . .
. . -0.82 0.17 * * . -0.30 0.14 Ser 495 . A B . . . . -0.93 0.60 .
* . -0.60 0.16 Val 496 . . B . . . . -1.04 0.90 . * . -0.40 0.13
Cys 497 . . B . . T . -1.33 0.80 . * . -0.20 0.25 Pro 498 A . . . .
T . -0.70 0.61 . * . -0.20 0.19 Leu 499 A . . . . T . -0.74 0.23 .
* . 0.10 0.51 Asn 500 A . . . . T . -0.48 0.23 . * . 0.10 0.70 Ala
501 A . . . . . . 0.13 0.16 . * . -0.10 0.62 Lys 502 A . . B . . .
0.80 0.49 . * . -0.45 1.18 Val 503 . . B B . . . 0.12 0.20 . * .
-0.15 1.18 His 504 . . B B . . . 0.59 0.49 * * . -0.60 0.82 Tyr 505
. . B B . . . 0.63 0.41 * * . -0.60 0.40 Asn 506 . . B B . . . 1.22
0.41 * * . -0.45 1.09 Ile 507 . . B B . . . 0.37 0.17 * * . -0.15
1.29 Gly 508 . . B . . T . 0.63 0.36 * * F 0.25 0.68 Lys 509 . . B
. . T . 0.67 0.10 * . F 0.59 0.43 Asn 510 . . B . . T . 0.96 -0.30
* . F 1.68 1.02 Leu 511 . . B . . T . 0.61 -0.99 * . F 2.32 2.05
Ala 512 . . B . . . . 1.50 -0.99 * . F 2.46 1.02 Asp 513 . . . . T
T . 1.84 -0.59 * . F 3.40 1.02 Lys 514 . . . . T T . 1.49 -0.59 * .
F 3.06 2.13 Gly 515 . . . . T T . 0.90 -0.79 * . F 2.72 3.05 Asn
516 A . . . . T . 1.12 -0.79 . . F 1.98 1.84 Gln 517 A A . . . . .
0.82 -0.29 . * F 0.79 0.93 Thr 518 . A B . . . . 0.93 0.40 . * F
-0.15 0.66 Ala 519 . A B . . . . 0.64 -0.03 . * . 0.30 0.80 Ala 520
. A B . . . . 0.74 0.33 * * . -0.30 0.73 Ile 521 . A B . . . . 0.86
0.69 * . . -0.60 0.79 Arg 522 . . B . . . . 0.86 0.20 * . . 0.05
1.53 Tyr 523 . . B . . . . 0.58 -0.30 * . . 0.65 2.62 Tyr 524 . A B
. . . . 0.31 -0.30 * * . 0.45 3.78 Arg 525 . A B . . . . 1.01 -0.34
* * . 0.45 1.43 Glu 526 . A B . . . . 1.09 -0.34 * * . 0.45 1.79
Ala 527 . A B . . . . 0.98 -0.41 * * . 0.30 0.94 Val 528 . A B . .
. . 1.01 -0.77 * * . 0.60 0.77 Arg 529 . A B . . . . 1.30 -0.34 * *
. 0.30 0.69 Leu 530 A . . . . . . 0.94 -0.34 * * . 0.65 1.37 Asn
531 . . . . . T C 0.09 -0.09 * * . 1.05 2.89 Pro 532 A . . . . T .
0.64 -0.09 * * F 1.00 1.09 Lys 533 A . . . . T . 0.91 0.41 * * .
-0.05 1.80 Tyr 534 . . B . . T . 0.20 0.23 * * . 0.25 1.13 Val 535
. . B . . . . 1.01 0.44 * . . -0.40 0.73 His 536 . . B . . . . 1.01
0.41 * . . -0.40 0.58 Ala 537 . . B . . T . 0.41 0.81 * . . -0.20
0.60 Met 538 . . B . . T . 0.02 0.74 * . . -0.20 0.67 Asn 539 A . .
. . T . 0.27 0.53 * . . -0.20 0.48 Asn 540 A . . . . T . 0.23 0.43
* . . -0.20 0.77 Leu 541 A . . . . . . -0.54 0.61 * . . -0.40 0.55
Gly 542 A . . . . . . 0.09 0.69 * . . -0.40 0.28 Asn 543 A A . . .
. . 0.69 0.29 * * . -0.30 0.35 Ile 544 A A . . . . . 0.80 -0.11 * .
. 0.30 0.73 Leu 545 A A . . . . . 0.80 -0.80 * * F 0.90 1.45 Lys
546 A A . . . . . 1.61 -0.83 * * F 0.90 1.45 Glu 547 A A . . . . .
1.14 -1.23 . * F 0.90 3.57 Arg 548 A A . . . . . 1.14 -1.23 * * F
0.90 3.57 Asn 549 A A . . . . . 2.03 -1.51 * * F 0.90 3.09 Glu 550
A A . . . . . 2.26 -1.51 . * F 0.90 3.09 Leu 551 A A . . . . . 2.21
-1.01 * * F 0.90 1.60 Gln 552 A A . . . . . 2.21 -1.01 * * F 0.90
1.72 Glu 553 A A . . . . . 1.29 -1.41 * * F 0.90 1.72 Ala 554 A A .
. . . . 0.48 -0.73 * . F 0.90 1.72 Glu 555 A A . . . . . 0.18 -0.73
* . F 0.75 0.82 Glu 556 A A . . . . . 0.18 -0.74 * . F 0.75 0.63
Leu 557 A A . . . . . -0.41 -0.06 * . . 0.30 0.52 Leu 558 A A . . .
. . -1.27 -0.06 * * . 0.30 0.30 Ser 559 A A . . . . . -0.68 0.59 *
* . -0.60 0.13 Leu 560 A A . . . . . -1.57 0.99 * * . -0.60 0.27
Ala 561 A A . . . . . -1.57 0.99 * * . -0.60 0.23 Val 562 A A . . .
. . -0.97 0.70 * * . -0.60 0.30 Gln 563 . A B . . . . -0.16 0.74 *
* . -0.60 0.56 Ile 564 . A B . . . . -0.56 0.06 . * . -0.30 0.92
Gln 565 . . B . . T . -0.33 0.34 . * F 0.40 1.08 Pro 566 . . B . .
T . -0.33 0.20 . * F 0.25 0.63 Asp 567 A . . . . T . -0.07 0.30 . *
. 0.10 0.91 Phe 568 A . . . . T . -0.36 0.11 . * . 0.10 0.53 Ala
569 A A . . . . . -0.07 0.63 . * . -0.60 0.36 Ala 570 A A . . . . .
-0.07 0.81 . * . -0.60 0.21 Ala 571 A A . . . . . -0.67 1.21 * . .
-0.60 0.40 Trp 572 A A . . . . . -1.01 1.11 . * . -0.60 0.32 Met
573 A A . . . . . -1.20 1.04 . . . -0.60 0.32 Asn 574 A A . B . . .
-1.47 1.23 . . . -0.60 0.22 Leu 575 A A . B . . . -0.88 1.37 . . .
-0.60 0.15 Gly 576 . A B B . . . -0.29 0.86 . . . -0.60 0.27 Ile
577 . . B B . . . -0.30 0.64 . . . -0.60 0.27 Val 578 . . B . . T .
-0.51 0.63 . * . -0.20 0.44 Gln 579 . . B . . T . -0.47 0.63 * . F
-0.05 0.37 Asn 580 . . B . . T . 0.46 0.20 * . F 0.40 1.05 Ser 581
. . . . . T C 0.10 -0.49 * . F 1.20 2.76 Leu 582 . A . . . . C 0.99
-0.34 * . F 0.80 1.38 Lys 583 . A . . . . C 1.26 -0.74 * . F 1.10
1.49 Arg 584 A A . . . . . 0.67 -0.64 * . . 0.75 1.12 Phe 585 A A .
. . . . 0.67 -0.53 * . . 0.75 1.37 Glu 586 A A . . . . . 0.97 -1.21
* . . 0.75 1.19 Ala 587 A A . . . . . 1.48 -0.81 * . . 0.75 1.05
Ala 588 A A . . . . . 1.19 -0.43 * * . 0.45 1.63 Glu 589 A A . . .
. . 1.19 -0.46 . * F 0.60 1.47 Gln 590 A . . . . T . 1.58 -0.46 * .
F 1.00 2.86 Ser 591 A . . . . T . 0.99 -0.47 * * F 1.00 4.08 Tyr
592 A . . . . T . 0.69 -0.47 * * F 1.00 2.38 Arg 593 A . . . . T .
1.32 0.21 * * F 0.25 0.96 Thr 594 A A . B . . . 1.29 -0.19 * * .
0.45 1.44 Ala 595 A A . B . . . 1.40 -0.07 * * . 0.45 1.25 Ile 596
A A . B . . . 1.81 -0.83 * * . 1.05 1.25 Lys 597 . A B B . . . 2.10
-0.83 * * . 1.35 1.69 His 598 . A B . . . . 1.74 -1.31 * * F 1.80
3.35 Arg 599 . A . . T . . 1.84 -1.06 * * F 2.50 7.50 Arg 600 . . .
. T . . 2.43 -1.31 * * F 3.00 5.80 Lys 601 . . . . T . . 2.66 -1.31
* * F 2.70 7.12 Tyr 602 . . B . . T . 2.37 -1.24 * . F 2.20 1.95
Pro 603 . . . . T T . 2.16 -0.49 . . F 2.00 1.56 Asp 604 . . . . T
T . 2.04 0.27 * * . 0.95 1.22 Cys 605 . . B . . T . 1.12 0.67 * . .
-0.05 1.25 Tyr 606 . . B . . . . 0.73 0.60 * . . -0.40 0.67 Tyr 607
. . B . . . . 1.09 0.60 * . . -0.40 0.40 Asn 608 . . B . . . . 0.49
0.60 * . . -0.25 1.45 Leu 609 . . B . . . . 0.24 0.71 * * . -0.40
0.76 Gly 610 . . B . . . . 0.32 0.71 * * . -0.40 0.76 Arg 611 . A B
. . . . 0.57 0.46 * * . -0.60 0.48 Leu 612 . A B . . . . -0.00 0.06
* * . -0.30 0.97 Tyr 613 . A B . . . . -0.00 0.06 * * . -0.30 0.81
Ala 614 . A B . . . . 0.92 0.03 * * . -0.30 0.66 Asp 615 A A . . .
. . 1.23 0.03 * * . -0.15 1.57 Leu 616 . A B . . . . 0.27 -0.16 * *
. 0.45 1.37 Asn 617 . A B . . . . 1.08 -0.27 * * . 0.45 1.00 Arg
618 . A B . . . . 0.73 -0.77 * . . 0.75 1.00 His 619 A A . . . . .
0.51 -0.27 * . . 0.45 1.23 Val 620 A A . . . . . 0.51 -0.27 * . .
0.30 0.63 Asp 621 A A . . . . . 0.73 -0.27 * . . 0.30 0.52 Ala 622
A A . . . . . 0.44 0.23 * . . -0.30 0.38 Leu 623 A A . . . . . 0.44
0.64 * . . -0.60 0.54 Asn 624 A A . . . . . 0.48 -0.00 * . . 0.30
0.64 Ala 625 A A . . . . . 0.74 0.40 * . . -0.15 1.02 Trp 626 A A .
. . . . 0.43 0.40 * . . -0.15 1.25 Arg 627 A A . . . . . 0.17 0.20
* . . -0.15 1.12 Asn 628 A A . B . . . 0.17 0.44 . . . -0.60 0.82
Ala 629 A A . B . . . 0.21 0.63 * . . -0.60 0.64 Thr 630 . A B B .
. . 0.59 -0.29 . * . 0.30 0.66 Val 631 . A B B . . . 0.88 0.14 . *
. -0.30 0.63 Leu 632 . . B B . . . 0.73 -0.26 . . . 0.45 1.08 Lys
633 . . B B . . . 0.43 -0.26 . * F 0.60 1.02 Pro 634 . . B . . . .
0.21 -0.36 * . F 0.80 1.85 Glu 635 A A . . . . . -0.07 -0.31 . * F
0.60 1.85 His 636 A A . . . . . 0.50 -0.50 * * . 0.60 0.93 Ser 637
A A . . . . . 1.31 0.41 . * . -0.60 0.63 Leu 638 A A . . . . . 1.27
0.39 . * . -0.30 0.59 Ala 639 A A . . . . . 0.88 0.79 . . . -0.60
0.70 Trp 640 A A . . . . . -0.01 0.90 . . . -0.60 0.51 Asn 641 A A
. . . . . -0.87 1.20 . . . -0.60 0.44 Asn 642 A A . B . . . -1.38
1.20 . . . -0.60 0.30 Met 643 . A B B . . . -1.38 1.39 . . . -0.60
0.24 Ile 644 . . B B . . . -0.79 1.16 * . . -0.60 0.12 Ile 645 . .
B B . . . -0.50 0.76 * . . -0.60 0.13 Leu 646 . . B B . . . -0.81
0.76 . . . -0.60 0.21 Leu 647 . . B B . . . -1.16 0.63 * . . -0.60
0.42 Asp 648 . . B B . . . -0.56 0.37 . * F -0.15 0.60 Asn 649 . .
. . . T C -0.48 0.09 . . F 0.60 1.17 Thr 650 . . . . . T C -0.18
0.09 * . F 0.60 1.17 Gly 651 . . . . . T C 0.63 -0.10 * . F 1.05
0.71 Asn 652 . . . . . T C 0.86 0.30 . . F 0.45 0.76 Leu 653 A A .
. . . . 0.86 0.40 . . . -0.30 0.53 Ala 654 A A . . . . . 0.27 -0.09
. . . 0.30 0.93 Gln 655 A A . . . . . -0.28 -0.01 . . . 0.30 0.58
Ala 656 A A . . . . . -0.28 0.23 . * . -0.30 0.53 Glu 657 A A . . .
. . -0.17 -0.03 . * . 0.30 0.52 Ala 658 A A . . . . . 0.64 -0.53 *
* . 0.60 0.58 Val 659 A A . . . . . 0.64 -0.93 * * . 0.60 1.00 Gly
660 A A . . . . . -0.17 -0.93 * * . 0.60 0.58 Arg 661 A A . . . . .
0.42 -0.24 * * F 0.45 0.48 Glu 662 A A . . . . . -0.39 -0.74 * * .
0.75 1.11 Ala 663 A A . . . . . -0.69 -0.70 * * . 0.60 0.93 Leu 664
A A . . . . . -0.04 -0.44 * * . 0.30 0.33 Glu 665 A A . . . . .
0.30 -0.01 * * . 0.30 0.30 Leu 666 A A . . . . . 0.19 0.39 * * .
-0.30 0.47 Ile 667 A . . . . T . 0.16 -0.11 * . . 0.70 0.95 Pro 668
A . . . . T . 0.44 -0.30 . . F 0.85 0.75 Asn 669 A . . . . T . 0.44
0.09 . . F 0.40 1.22 Asp 670 A . . . . T . -0.16 0.09 . . F 0.40
1.43 His 671 A A . . . . . -0.04 0.01 . . F -0.15 0.92 Ser 672 A A
. . . . . 0.54 0.37 . * . -0.30 0.49 Leu 673 . A B . . . . -0.06
0.36 . . . -0.30 0.40 Met 674 . A B . . . . -0.64 1.04 . . . -0.60
0.24 Phe 675 A A . . . . . -0.64 1.04 . . . -0.60 0.18 Ser 676 A A
. . . . . -1.47 1.06 * . . -0.60 0.35 Leu 677 A A . . . . . -1.98
1.01 * . . -0.60 0.26 Ala 678 A A . . . . . -1.51 1.09 * . . -0.60
0.25 Asn 679 A A . . . . . -0.87 0.73 * . . -0.60 0.19 Val 680 A A
. . . . . -0.47 0.34 . * . -0.30 0.45 Leu 681 A A . . . . . -0.17
0.04 * . . -0.30 0.60 Gly 682 A . . . . T . 0.69 -0.06 * . F 0.85
0.64 Lys 683 A . . . . T . 1.03 -0.46 . * F 1.00 1.74 Ser 684 A . .
. . T . 1.08 -0.34 . . F 1.00 3.30 Gln 685 A . . . . T . 1.93 -1.03
. . F 1.30 6.67 Lys 686 A . . . . . . 2.44 -1.46 . . F 1.10 5.77
Tyr 687 A . . . . T . 2.79 -1.07 . . F 1.30 5.77 Lys 688 A . . . .
T . 2.16 -1.46 . . F 1.30 5.77 Glu 689 A . . . . T . 1.64 -1.36 . .
F 1.30 2.92 Ser 690 A . . . . T . 0.94 -0.67 . . F 1.30 1.54 Glu
691 A A . . . . . 0.09 -0.64 . . F 0.75 0.66 Ala 692 A A . . . . .
0.38 0.04 * . . -0.30 0.32 Leu 693 A A . . . . . -0.26 0.04 * . .
-0.30 0.47 Phe 694 A A . . . . . -1.14 0.16 * . . -0.30 0.28 Leu
695 A A . . . . . -0.80 0.84 * . . -0.60 0.19 Lys 696 A A . . . . .
-1.39 0.34 . . . -0.30 0.46 Ala 697 A A . . . . . -0.80 0.16 * . .
-0.30 0.54 Ile 698 A A . . . . . -0.20 -0.23 * * . 0.66 1.05 Lys
699 A A . . . . . 0.50 -0.49 * * F 0.87 0.82 Ala 700 A A . . . . .
0.72 -0.09 * * F 1.23 1.30 Asn 701 . . . . . T C 0.09 -0.09 * * F
2.04 1.87 Pro 702 . . . . . T C 0.38 -0.27 * * F 2.10 0.95 Asn 703
. . . . T T . 1.02 0.11 . * . 1.49 1.25 Ala 704 A . . . . T . 0.94
0.37 . * . 0.88 1.22 Ala 705 . . B . . . . 1.19 0.47 . . . 0.17
1.07 Ser 706 . . B . . . . 1.19 0.47 . * . -0.19 0.66 Tyr 707 . . B
. . . . 0.59 0.47 . . . -0.25 1.05 His 708 . . B . . T . -0.00 0.66
. * . -0.20 0.86 Gly 709 . . B . . T . -0.27 0.66 . * . -0.20 0.65
Asn 710 . . B . . T . -0.49 0.91 . * . -0.20 0.31 Leu 711 . . B . .
T . -0.43 0.84 . * . -0.20 0.19 Ala 712 . . B B . . . -0.22 1.10 .
* . -0.60 0.29 Val 713 . . B B . . . -0.08 1.17 . * . -0.60 0.25
Leu 714 . . B B . . . -0.02 0.77 * . . -0.60 0.59 Tyr 715 . . B B .
. . -0.37 1.00 * . . -0.60 0.62 His 716 . . B . . T . 0.41 0.93 * .
. -0.20 0.82 Arg 717 . . . . T T . 0.19 0.79 . . . 0.35 1.36 Trp
718 A . . . . T . 1.04 0.79 . . . -0.20 0.71 Gly 719 A . . . . T .
1.04 0.03 . . . 0.10 0.88 His 720 A A . . . . . 0.70 0.21 . . .
-0.30 0.37 Leu 721 A A . . . . . 0.78 0.71 . . . -0.60 0.35 Asp 722
A A . . . . . 0.71 -0.20 . . . 0.30 0.72 Leu 723 A A . . . . . 0.97
-0.63 . . . 0.75 1.05 Ala 724 A A . . . . . 1.07 -0.63 * * . 0.75
1.74 Lys 725 A A . . . . . 1.10 -0.56 * * F 0.90 1.63 Lys 726 A A .
. . . . 1.02 -0.56 * * F 0.90 3.42 His 727 A A . . . . . 0.72 -0.56
* * . 0.75 2.37 Tyr 728 A A . . . . . 0.72 -0.67 * * . 0.75 1.59
Glu 729 . A B . . . . 1.31 0.01 * * . -0.30 0.66 Ile 730 . A B . .
. . 0.46 0.41 . * . -0.60 0.83 Ser 731 . A B . . . . 0.41 0.60 . *
. -0.60 0.44 Leu 732 . A B . . . . 0.23 -0.16 . * . 0.30 0.42 Gln
733 . A B . . . . 0.17 0.27 . * . -0.30 0.93 Leu 734 . A B . . . .
-0.42 0.07 . * . -0.15 1.01 Asp 735 . . . . . T C 0.17 0.19 . * F
0.60 1.23 Pro 736 . . . . . T C 0.12 -0.11 . * F 1.31 0.95 Thr 737
. . . . . T C 0.62 -0.09 . * F 1.72 1.14 Ala 738 . . B . . T C 0.67
-0.29 . * F 1.83 0.99 Ser 739 . . . . . . C 1.48 -0.29 . * F 2.04
1.28 Gly 740 . . . . . . C 1.48 -0.71 . . F 2.60 1.54 Thr 741 . . .
. . . C 1.44 -0.80 . . F 2.34 2.44 Lys 742 . . B . . . . 1.41 -0.54
. . F 1.88 2.86 Glu 743 . . B . . . . 1.19 -0.50 . . F 1.62
2.86
Asn 744 . . B . . T . 0.68 -0.24 * . F 1.26 1.63 Tyr 745 . . B . .
T . 1.13 -0.04 * . . 0.70 0.67 Gly 746 A . . . . T . 1.56 -0.04 * .
. 0.70 0.76 Leu 747 A . . . . T . 1.56 -0.04 * * . 0.70 0.93 Leu
748 A A . . . . . 0.74 -0.44 * * . 0.45 1.18 Arg 749 A A . . . . .
0.74 -0.51 * * F 0.75 0.99 Arg 750 A A . . . . . 0.18 -0.94 * * F
0.90 2.07 Lys 751 A A . . . . . -0.08 -0.94 * * F 0.90 2.07 Leu 752
A A . . . . . 0.73 -1.01 * * . 0.75 1.05 Glu 753 A A . . . . . 1.59
-0.61 * * . 0.60 0.93 Leu 754 A A . . . . . 1.52 -0.61 * * . 0.60
0.93 Met 755 A A . . . . . 0.82 -0.61 * . . 0.75 2.24 Gln 756 A A .
. . . . -0.08 -0.80 . * . 0.75 1.31 Lys 757 A A . . . . . 0.34
-0.16 . . F 0.60 1.18 Lys 758 A A . . . . . -0.04 -0.41 . . . 0.45
1.52 Ala 759 A A . . . . . 0.38 -0.60 . . . 0.75 1.12 Val 760 A A .
. . . . 0.59 -0.57 . . . 0.60 0.72
[1717] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples. Numerous modifications and variations of
the present invention are possible in light of the above teachings
and, therefore, are within the scope of the appended claims.
[1718] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) in the Background
of the Invention, Detailed Description, and Examples is hereby
incorporated herein by reference. Further, the hard copy of the
sequence listing submitted herewith and the corresponding computer
readable form are both incorporated herein by reference in their
entireties.
Sequence CWU 1
1
465 1 733 DNA Homo sapiens 1 gggatccgga gcccaaatct tctgacaaaa
ctcacacatg cccaccgtgc ccagcacctg 60 aattcgaggg tgcaccgtca
gtcttcctct tccccccaaa acccaaggac accctcatga 120 tctcccggac
tcctgaggtc acatgcgtgg tggtggacgt aagccacgaa gaccctgagg 180
tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg
240 aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg
caccaggact 300 ggctgaatgg caaggagtac aagtgcaagg tctccaacaa
agccctccca acccccatcg 360 agaaaaccat ctccaaagcc aaagggcagc
cccgagaacc acaggtgtac accctgcccc 420 catcccggga tgagctgacc
aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480 atccaagcga
catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga 540
ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg
600 acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat
gaggctctgc 660 acaaccacta cacgcagaag agcctctccc tgtctccggg
taaatgagtg cgacggccgc 720 gactctagag gat 733 2 5 PRT Homo sapiens
Site (3) Xaa equals any of the twenty naturally ocurring L-amino
acids 2 Trp Ser Xaa Trp Ser 1 5 3 86 DNA Homo sapiens 3 gcgcctcgag
atttccccga aatctagatt tccccgaaat gatttccccg aaatgatttc 60
cccgaaatat ctgccatctc aattag 86 4 27 DNA Homo sapiens 4 gcggcaagct
ttttgcaaag cctaggc 27 5 271 DNA Homo sapiens 5 ctcgagattt
ccccgaaatc tagatttccc cgaaatgatt tccccgaaat gatttccccg 60
aaatatctgc catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
120 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
ttttttttat 180 ttatgcagag gccgaggccg cctcggcctc tgagctattc
cagaagtagt gaggaggctt 240 ttttggaggc ctaggctttt gcaaaaagct t 271 6
32 DNA Homo sapiens 6 gcgctcgagg gatgacagcg atagaacccc gg 32 7 31
DNA Homo sapiens 7 gcgaagcttc gcgactcccc ggatccgcct c 31 8 12 DNA
Homo sapiens 8 ggggactttc cc 12 9 73 DNA Homo sapiens 9 gcggcctcga
ggggactttc ccggggactt tccggggact ttccgggact ttccatcctg 60
ccatctcaat tag 73 10 256 DNA Homo sapiens 10 ctcgagggga ctttcccggg
gactttccgg ggactttccg ggactttcca tctgccatct 60 caattagtca
gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc 120
cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
180 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
gaggcctagg 240 cttttgcaaa aagctt 256 11 1191 DNA Homo sapiens 11
gctgggctgg aacacaagar cccacagggc tgccgtccac actctcccgg tcagagtcct
60 gggaccacat ggggacgctg ccatggcttc ttgccttctt cattctgggt
ctccaggctt 120 gggatactcc caccatcgtc tcccgcaagg agtggggggc
aagaccgctc gcctgcaggg 180 ccctgctgac cctgcctgtg gcctacatca
tcacagacca gctcccaggg atgcagtgcc 240 agcagcagag cgtttgcagc
cagatgctgc gggggttgca gtcccattcc gtctacacca 300 taggctggtg
cgacgtggcg tacaacttcc tggttgggga tgatggcagg gtgtatgaag 360
gtgttggctg gaacatccaa ggcttgcaca cccagggcta caacaacatt tccctgggca
420 tcgccttctt tggcaataag ataagcagca gtcccagccc tgctgcctta
tcagctgcag 480 agggtctgat ctcctatgcc atccagaagg gtcacctgtc
gcccaggtat attcagccac 540 ttcttctgaa agaagagacc tgcctggacc
ctcaacatcc agtgatgccc agraaggttt 600 gccccaacat catcaaacga
tctgcttggg aagccagaga gacacactgc cctaaaatga 660 acctcccagc
caaatatgtc atcatcatcc acaccgctgg cacaagctgc actgtatcca 720
cagactgcca gactgtcgtc cgaaacatac agtcctttca catggacaca cggaactttt
780 gtgacattgg atatcaataa ggccaggcgt ggcggcgatt acgtctgtaa
tcccaggact 840 ttgggaggcc aaggcgggca gatcacttca ggccaggaat
tcaagagcag cctggccaat 900 atggcgaaac tctgtctcta ctgaaaacaa
acaaacaaac aaacaaacaa acaaagaaac 960 aacaaaaatt agccgggtgt
ggtggcacac gcctgtagtc ccagctactc aggaggctga 1020 ggcataagaa
ttgcttgaac cctggaggcg gaggttgcag tgagctgaga ttgggccacc 1080
gcactccagt ctgggagaca gagtgagact gtctcaaaac aacaacaaaa aaatccctaa
1140 cataatctca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa agggcggccg c 1191
12 1251 DNA Homo sapiens 12 ggcacaggtc agccaactaa caaatgaagc
gcagggaaat gactcaattc ttattgagtc 60 tagttgctct taattgctgc
tctatttctt tgggaagatt gacatatcca ggaggttttc 120 atctaaaact
agacccctta gaactctgaa gtcagagcaa ctttccctct gtcaatccta 180
ctcactactt ttgtamcctt gaccagagaa gttgcttaat cttttggggc ctgcattctc
240 atatacctaa agtaggaata aaaatacctg cttagagact tgctcagtcc
atcaaatrag 300 agattataca caaccttccc acttcaagga tggctgcaag
gacaaaaaag aaaaatgaca 360 taataaatat aaaggtccct gcagactgta
atactaggat gagttattac tacaaaggct 420 cagggaaaag aggagagatg
gagtcttggt tggtcatgtc atcatggtct attttagatt 480 ttgagttttt
agaggcaaga ccacagttgt ttaatttagt gtatacagaa cattccactt 540
attcagggag acattatact agggaaaggg gtgggttcat ggtgttcaaa aattcatact
600 cacagttatt attaaaaaga aaggattctc tatgtgcttt tattcagccc
atggctttaa 660 atatcatcca tgtgcctatg tcttccaaat gtatttttcc
agcccagtct ggtccctcga 720 cattcagatc cttatggtgg tgccctcacc
ctatatccaa atgccaactt ggtctctact 780 ctagtcagat tagagatatc
ccatacttgg catgactaaa atggaacttt aacttgtttc 840 ttatctctat
ctcagtaaat cacaccacca cagtgcatca ttttcctaaa tcaaattcct 900
aagaatcatc cttgattttt cccttccttt tgtcccttgc catcccagat tatcctgcaa
960 aaactgtcta tgctacctac aaaagtatct gccacatgtc atactaattg
tcratatcct 1020 agagcaccmt tcatctgcct tcacctgtgg tgttgctgca
attgtctcct tcctggctgc 1080 cctgattata tccattctcc ctgtctccaa
aagcattctg cgcacagcag acacagatgt 1140 ttcataaatg taagtctggt
catgcgctcc tctacctaaa accattagat ggtttttcat 1200 tgcactcaca
actagagttt cctgaccatg acttgcaggc taagctcgta g 1251 13 1734 DNA Homo
sapiens SITE (1417) n equals a,t,g, or c SITE (1703) n equals
a,t,g, or c SITE (1714) n equals a,t,g, or c SITE (1715) n equals
a,t,g, or c SITE (1731) n equals a,t,g, or c SITE (1732) n equals
a,t,g, or c 13 gaagcgtgcg gtgccgcagc aatggcggcg ctcacaattg
ccacgggtac tggcaattgg 60 ttttcggctt tggcgctcgg ggtgactctt
ctcaaatgcc ttctcatccc cacataccat 120 tccacagatt ttgaagtaca
ccgaaactgg cttgctatca ctcacagttt gccaatatca 180 cagtggtatt
atgaggcaac ttcagagtgg acgttggatt accccccttt ctttgcatgg 240
tttgagtata tcctgtcaca tgttgccaaa tattttgatc aagaaatgct gaatgtccat
300 aatttgaatt actccagctc aaggacctta cttttccaga gattttccgt
catctttatg 360 gatgtactct ttgtgtatgc tgtccgtgag tgctgtaaat
gcattgatgg aaaaaaagtg 420 ggtaaagaac ttacagaaaa gccaaaattt
attctgtcgg tattacttct gtggaacttc 480 gggttattaa ttgtggacca
tattcatttt cagtacaatg gctttttatt tggattaatg 540 ctactctcca
ttgcacgatt atttcagaaa aggcatatgg aaggagcatt tctctttgct 600
gttctcctac atttcaagca tatctacctc tatgtagcac cagcttatgg tgtatatctg
660 ctgcgatcct actgtttcac tgcaaataaa ccagatgggt ctattcgatg
gaagagtttc 720 agctttgttc gtgttatttc cctgggactg gttgttttct
tagtttctgc tctttcattg 780 ggtcctttcc tggccttgaa tcagctgcct
caagtctttt cccgactctt tcctttcaag 840 aggggcctct gtcatgcata
ttgggctcca aacttctggg ctttgtacaa tgctttggac 900 aaagtgctgt
ctgtcatcgg tttgaaattg aaatttcttg atcccaacaa tattcccaag 960
gcctcaatga caagtggttt ggttcagcag ttccaacaca cagtccttcc ctcagtgact
1020 cccttggcaa ccctcatctg cacactgatt gccatattgc cctctatttt
ctgtctttgg 1080 tttaaacccc aagggcccag aggctttctc cgatgtctaa
ctctttgtgc cttgagctcc 1140 tttatgtttg ggtggcatgt tcatgaaaaa
gccatacttc tagcaattct cccaatgagc 1200 cttttgtctg tgggaaaagc
aggagacgct tcgatttttc tgattctgac cacaacagga 1260 cattattccc
tctttcctct gctcttcact gcaccagaac ttcccattaa aatcttactc 1320
atgttactat tcaccatata tagtatttcg tcactgaaga ctttattcag aaaagaaaaa
1380 cctcttttta attggatgga aactttctac ctgcttngcc tggggcctct
ggaagtctgc 1440 tgtgaatttg tattcccttt cacctcctgg aaggtgaagt
accccttcat ccctttgtta 1500 ctaacctcag tgtattgtgc agtaggcatc
acatatgctt ggttcaaact gtatgtttca 1560 gtattgattg actctgctat
tggcaagaca aagaaacaat gaataaagga actgcttaga 1620 aaaaaaaaaa
aaaaaaaaaa aaagggcggc cgctctagag gatccctcga gggcccaagc 1680
ttacgcgtgc atgcgagtca tantctctcc tggnntgatc gtatgaagct nngc 1734 14
1540 DNA Homo sapiens SITE (22) n equals a,t,g, or c SITE (430) n
equals a,t,g, or c 14 gcctgggcgc cgtgggcgcg gnactgcgcg ggctgcgcgg
gtgccgagga gcgcgaggcg 60 cggggaaggc gcacctgggg tggccctggc
gtgcgggcgg cgacatggag gacggcgtgc 120 tcaaggaggg cttcctggtc
aagaggggcc acattgtcca caactggaag gcgcgatggt 180 tcatccttcg
gcagaacacg ctggtgtact acaagcttga ggggggtcgg agagtgaccc 240
ctcccaaggg ccggatcctc ctggatggct gcaccatcac ctgcccctgc ctggagtatg
300 aaaaccgacc gctcctcatt aagctgaaga ctcaaacatc cacggagtac
ttcctggagg 360 cctgttctcg agaggaagcg ggatgcctgg gcctttkaag
rtyaccgggg ctattcatgc 420 agggcagccn ggggaaggtc cagcagctgc
acagcctgag aaactccttc amgctgcccc 480 cgcacatcar gctgyatcgy
attgtggaca agatgcacga tagcaacacc ggwatccgtt 540 caagccccaa
catggagcag agaagcacct ataaaaagam cttyctcggc tcctccctgg 600
tggactggyt yatctycaam agcttcamgg gcagccgtct kgaggcggtg amcctggcct
660 ccatgytcat rgaggagaac ttcctcaggt ctgtggctgt acgatgcatg
ggaggcattc 720 ggtctgggga tctggccgag cagttcctgg atgactccac
agccctgtac acttttsctg 780 agagctacam aaagawgata agccccaagg
aagaaattag cctgagcact gtggagttaa 840 gtggcacggt ggtgaaacaa
ggctacctgg ccaagcaggg acacaagagg aaaaactgga 900 aggtgcgtcg
ctttgttcta aggaaggatc cagctttcct gcattactat gacccttcca 960
aagaagagaa caggccagtg ggtgggtttt ctcttcgtgg ttcactcgtg tctgctctgg
1020 aagataatgg cgttcccact ggggttaaag ggaatgtcca gggaaacctc
ttcaaagtga 1080 ttactaagga tgacacacac tattacattc aggccagcag
caaggctgag cgagccgagt 1140 ggattgaagc tatcaaaaag ctaacatgac
aaggacctga gggaaccagg attcctccct 1200 cctaccagat gacacagaca
agagttcctg gagaatggga gtgttaagac ttttgacttc 1260 tttgtaagtt
ttgtactgct ttggagagtg aatgctgcca agagttcctc agattacaaa 1320
cagcagtggt gccatttcct tccccatctt catgttacaa acctggaaag gctagaacag
1380 ccattaggcg tcagcatctt gacttttccc cagcatcaca aacagccatt
tcctcgggca 1440 ccaaagtagg ttccctttgt tggaacaatt acactggcca
tgccataatg ttgaataaaa 1500 ctctcttctt atgaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1540 15 1558 DNA Homo sapiens 15 ccacgtcgtc cgaacctttt
aaaaatggtc ttgatgtatg tggaagagag tatgtgtatg 60 tgtgttcctg
tacatagcat gggtgcagct gtggatgtgt gcaaaagagt gtgagtgtgt 120
gtgtgtgtgt gtaaaggggt ctgtcctaga gcccacatca gtttgttgtg aatctggaaa
180 aagggtcggt gagggccggg agatgttgac cctggtggga gcaggctgag
gctgccccgt 240 tctccacatc ctctgttttg cccagtctct gattccatta
gggggagtgt gctgaagcca 300 ttctcggatg cttcccagac caggctccct
ctgccagagt cacatgcatc cgagctgctg 360 gtctccattg tccagcagga
aggcggaaag gcaggcaaga tggtgtgaag cttaaagctt 420 gtatttgatg
gaaaaggtct cccctgttca tctgagaggc caagcctggc caccccaggc 480
tcagaacctg ggcttcaaga aatgtgctgg gagctcctaa cttacacatc cctccagcct
540 tccttgaatc ctcccaccac cccctatttc ctttaatttc tcaggtctgc
tccctcctcc 600 cccaacccca cagctgggca agaagtctgc aaaagctgca
tctgcagctg tctctaactc 660 ttcccagcca tctcccgtat tttttggtac
cttgattcct tgactcttaa taagccaagc 720 caccttatct ctgtagttct
tatttttttg ttgactaaat ttggggggtt cttttttatg 780 gtcatgtcac
tgacctatta aattggggct tggtgctttt ccaccttccc cctctgaatg 840
aaagccaagg aatgggggaa gagcgggaac tctgccgcgg aggtggagca agaacggtga
900 agggccctgg tcccagagag gctggtgggt ccctctccca aaggaaggca
gacagtctct 960 gctttgcctt ggaccttggt gctgggggtg gggaggcctg
ggggggacac tccccactcc 1020 cattcccctt cctttgtcct aatcctggaa
ttaagtacag gggtttatag gttctatttc 1080 ttcccaagag ccctgcaaag
aaccccagtt tcctatttgg atgcccctac actgttgtgt 1140 ttcagtggaa
tgtattttca tttaaaaaca actttgaatg gggcactttt tctttcctgt 1200
tttaaaaatt gaaaaattct tacagtacaa acaggactgt cagggtgggg gtgttggtgc
1260 tgtaagaggt tactcttgag tgcattttgg cactgggatg ggatggctgg
ggtgggaaga 1320 cccccatccc cacccccaac ttcttttcta atatttaagg
agtgttttgt aggattcaac 1380 aaccaccaca acttgaattt gtatcatggg
aggtgggagg gagtggctta gaggtgtctg 1440 cctatgctta aagccaactg
tggaagtttt gttttccctt ttttgtataa taaagtgaaa 1500 aacaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1558 16 1636
DNA Homo sapiens SITE (424) n equals a,t,g, or c SITE (823) n
equals a,t,g, or c SITE (960) n equals a,t,g, or c 16 gaattcggca
cgagttgaaa ttgaaaatca agataaaaat gttcacaatt aagctccttc 60
tttttattgt tcctctagtt atttcctcca gaattgatca agacaattca tcatttgatt
120 ctctatctcc agagccaaaa tcaagatttg ctatgttaga cgatgtaaaa
attttagcca 180 atggcctcct tcagttggga catggtctta aagactttgt
ccataagacg aagggccaaa 240 ttaatgacat atttcaaaaa ctcaacatat
ttgatcagtc tttttatgat ctatcgctgc 300 aaaccagtga aatcaaagar
gaagaaaagg aactgagaag aactacmtat aaactacaag 360 tcaaaaatga
agaggtaaag aatatgtcac ttgaactcaa ctcaaaactt gaaagcctcc 420
tagnagaaaa aattctactt caacaaaaag tgaaatattt agaagagcaa ctaactaact
480 taattcaaaa tcaacctgaa actccagaac acccagaagt aacttcactt
aaaacttttg 540 tagaaaaaca agataatagc atcaaagacy ttctccagac
cgtggaagac caatatwaac 600 aattaaacca acagcatagt caaataaaag
aratagaaaa tcagctcaga aggactagta 660 ttcaagaacc cacagaaatt
tctctatctt ccaagccaag agcaccaaga actactccct 720 ttcttcagtt
gaatgaaata agaaatgtaa aacatgatgg cattcctgct gaatgtacca 780
ccatttataa cagaggtgaa catacaagtg gcatgtatgc atncagaccc agcaactctc
840 aagtttttca tgtctactgt gatgttatat caggtagtcc atggacatta
attcaacatc 900 gaatagatgg atcacaaaac ttcaatgaaa cgtgggagaa
ctacaaatat ggttttgggn 960 aggcttgatg gagaattttg gttgggccta
gagaagatat actccatagt gaagcaatct 1020 aattatgttt tacgaattga
gttggaagac tggaaagaca acaaacatta tattgaatat 1080 tctttttact
tgggaaatca cgaaaccaac tatacgctac atctagttgc gattactggc 1140
aatgtcccca atgcaatccc ggaaaacaaa gatttggtgt tttctacttg ggatcacaaa
1200 gcaaaaggac acttcaactg tccagagggt tattcaggag gctggtggtg
gcatgatgag 1260 tgtggagaaa acaacctaaa tggtaaatat aacaaaccaa
gagcaaaatc taagccagag 1320 aggagaagag gattatcttg gaagtctcaa
aatggaaggt tatactctat aaaatcaacc 1380 aaaatgttga tccatccaac
agattcagaa agctttgaat gaactgaggc aaatttaaaa 1440 ggcaataatt
taaacattaa cctcattcca agttaatgtg gtctaataat ctggtattaa 1500
atccttaaga gaaagcttga gaaatagatt ttttttatct taaagtcact gtctatttaa
1560 gattaaacat acaatcacat aaccttaaaa aaaaaaaaaa aaaaactcga
ggggggcccg 1620 gtacccaatt cgccgg 1636 17 1256 DNA Homo sapiens
SITE (1240) n equals a,t,g, or c 17 tcgacccacg cgtccgagca
accgcagctt ctagtatcca gactccagcg ccgccccggg 60 cgcggacccc
aaccccgacc cagagcttct ccagcggcgg cgcacgagca gggctccccg 120
ccttaacttc ctccgcgggg cccagccacc ttcgggagtc cgggttgccc acctgcaaac
180 tctccgcctt ctgcacctgc cacccctgag ccagcgcggg cgcccgagcg
agtcatggcc 240 aacgcggggc tgcagctgtt gggcttcatt ctcgccttcc
tgggatggat cggcgccatc 300 gtcagcactg ccctgcccca gtggaggatt
tactcctatg ccggcgacaa catcgtgacc 360 gcccaggcca tgtacgaggg
gctgtggatg tcctgcgtgt cgcagagcac cgggcagatc 420 cagtgcaaag
tctttgactc cttgctgaat ctgagcagca cattgcaagc aacccgtgcc 480
ttgatggtgg ttggcatcct cctgggagtg atagcaatct ttgtggccam cgttggcatg
540 aagtgtatga agtgcttgga agacgatgag gtgcagaaga tgaggatggc
tgtcattggg 600 ggcgcgatat ttcttcttgc aggtctggct attttagttg
ccacagcatg gtatggcaat 660 agaatcgttc aagaattcta tgaccctatg
accccagtca atgccaggta cgaatttggt 720 caggctctct tcactggctg
ggctgctgct tctctctgcc ttctgggagg tgccctactt 780 tgctgttcct
gtccccgaaa aacaacctct tacccaacac caaggcccta tccaaaacct 840
gcaccttcca gcgggaaaga ctacgtgtga cacagaggca aaaggagaaa atcatgttga
900 aacaaaccga aaatggacat tgagatacta tcattaacat taggacctta
gaattttggg 960 tattgtaatc tgaagtatgg tattacaaaa caaacaaaca
aacaaaaaac ccatgtgtta 1020 aaatactcag tgctaaacat ggcttaatct
tattttatct tctttcctca atataggagg 1080 gaagattttt ccatttgtat
tactgcttcc cattgagtaa tcatactcaa ctgggggaag 1140 gggtgctcct
taaatatata tagatatgta tatatacatg tttttctatt aaaaatagac 1200
agtaaaatwc taaaaaaaaa aaaaaaamcy cggggggggn ccggtaccca ttcgcc 1256
18 1143 DNA Homo sapiens SITE (1100) n equals a,t,g, or c 18
ggcacgaggg ctggggtcag caaatataca gggggccgag gcgtcacgtg ggccccatcc
60 tcagcagcag tgcctcggat atcttctgcg acaatgagaa tgggcctaac
ttccttttcc 120 acaaccgggg cgatggcacc tttgtggacg ctgcggccag
tgctggtgtg gacgaccccc 180 accagcatgg gcgaggtgtc gccctggctg
acttcaaccg tgatggcaaa gtggacatcg 240 tctatggcaa ctggaatggc
ccccaccgcc tctatctgca gatgagcacc catgggaagg 300 tccgcttccg
gggacatcgc cttcacccaa gttctccatg ccctcccctg ttccgcacgg 360
tcatcaccgg ccgactttga caatgaccag gagctggaga atcttcttca acaacattgc
420 ctaccgcagc tcctcagcca accgcctctt ccgcgtcatc cgtagagagc
acggagaccc 480 cctcatcgag gagctcaatc ccggcgacgc cttggagcct
gagggccggg gcacaggggg 540 tgtggtgacc gacttcgacg gagacgggat
gctggacctc atcttgtccc atggagagtc 600 catggctcaa ccgctgtccg
tcttccgggg caatcagggc ttcaacaaca actggctgcg 660 agtggtgcca
cgcacccggt ttggggcctt tgccagggga gctaaggtcg tgctctacac 720
caagaagagt ggggcccacc tgaggatcat cgacgggggc tcaggctacc tgtgtgagat
780 ggagcccgtg gcacactttg gcctggggaa ggatgaagcc agcagtgtgg
aggtgacgtg 840 gccagatggc aagatggtga gccggaacgt ggccagcggg
gagatgaact cagtgctgga 900 gatcctctac ccccgggatg aggacacact
tcaggaccca gccccactgg agtgtggcca 960 aggattctcc cagcaggaaa
atggccattg catggacacc aatgaatgca tccagttccc 1020 attcgtgtgc
cctcgagaca agcccgtatg tgtcaacacc tatggaagct acaggtgccg 1080
gaccaacaag aagtgcagtn cggggctacg agtcccaacg aggatggcac atacgggctt
1140 gtc 1143 19 1537 DNA Homo sapiens 19 atcatatagg aaacggtagc
ctgcagtacc ggtccggaat tcccgggtcg acccacgcgt 60 ccggagcagc
aagagatttg tcctggggat ccagaaaccc atgataccct actgaacacc 120
gaatcccctg gaagcccaca gagacagaga cagcaagaga agcagagata aatacactca
180 cgccaggagc tcgctcgctc tctctctctc tctctcactc ctccctccct
ctctctctgc 240 ctgtcctagt cctctagtcc tcaaattccc agtcccctgc
accccttcct gggacactat 300 gttgttctcc gccctcctgc tggaggtgat
ttggatcctg gctgcagatg ggggtcaaca 360 ctggacgtat gagggcccac
atggtcagga ccattggcca gcctcttacc ctgagtgtgg 420 aaacaatgcc
cagtcgccca tcgatattca gacagacagt gtgacatttg accctgattt 480
gcctgctctg cagccccacg gatatgacca gcctggcacc gagcctttgg acctgcacaa
540 caatggccac acagtgcaac tctctctgcc ctctaccctg tatctgggtg
gacttccccg 600 aaaatatgta gctgcccagc tccacctgca ctggggtcag
aaaggatccc caggggggtc 660 agaacaccag atcaacagtg aagccacatt
tgcagagctc cacattgtac attatgactc 720 tgattcctat gacagcttga
gtgaggctgc tgagaggcct cagggcctgg ctgtcctggg 780 catcctaatt
gagctggaaa agcttcaggg gacattgttc tccacagaag
aggagccctc 840 taagcttctg gtacagaact accgagccct tcagcctctc
aatcagcgca tggtctttgc 900 ttctttcatc caagcaggat cctcgtatac
cacaggtgaa atgctgagtc taggtgtagg 960 aatcttggtt ggctgtctct
gccttctcct ggctgtttat ttcattgcta gaaagattcg 1020 gaagaagagg
ctggaaaacc gaaagagtgt ggtcttcacc tcagcacaag ccacgactga 1080
ggcataaatt ccttctcaga taccatggat gtggatgact tcccttcatg cctatcagga
1140 agcctctaaa atggggtgta ggatctggcc agaaacactg taggagtagt
aagcagatgt 1200 cctccttccc ctggacatct cctagagagg aatggaccca
ggctgtcatt ccaggaagaa 1260 ctgcagagcc ttcagcctct ccaaacatgt
aggaggaaat gaggaaatcg ctgtgttgtt 1320 aatgcagaga acaaactctg
tttagttgca ggggaagttt gggatatacc ccaaagtcct 1380 ctaccccctc
acttttatgg ccctttccct agatatactg cgggatctct ccttaggata 1440
aagagttgct gttgaagttg tatatttttg atcaatatat ttggaaatta aagtttctga
1500 ctttaaaaaa aaaaaaaaaa aaaaaactcg agggggg 1537 20 2672 DNA Homo
sapiens SITE (16) n equals a,t,g, or c SITE (28) n equals a,t,g, or
c SITE (47) n equals a,t,g, or c SITE (52) n equals a,t,g, or c
SITE (93) n equals a,t,g, or c 20 cccaaagttc ggaaantaaa ccttcaanta
aagggaaaca aaaagcngga gnttcccacc 60 gcgggtgggc ggcccgttct
agaattaagt ggnatccccc cggggctgcc aggaatttcc 120 gagccggggc
cgcgccgccg ctgcccgccg ccgcgsgcgg attytgcttc tcagaagatg 180
cactattata gatactctaa cgccaaggtc agctgctggt acaagtacct ccttttcagc
240 tacaacatca tcttctgrtt ggctggagtt gtcttccttg gagtcgggct
gtgggcatgg 300 agcgaaaagg gtgtgctgtc cgacctcacc aaagtgaccc
ggatgcatgg aatcgaccct 360 gtggtgctgg tcctgatggt gggcgtggtg
atgttcaccc tggggttcgc cggctgcgtg 420 ggggctctgc gggagaatat
ctgcttgctc aactttttct gtggcaccat cgtgctcatc 480 ttcttcctgg
agctggctgt ggccgtgctg gccttcctgt tccaggactg ggtgagggac 540
cggttccggg agttcttcga gagcaacatc aagtcctacc gggacgatat cgatctgcaa
600 aacctcatcg actcccttca gaaagctaac cagtgctgtg gcgcatatgg
ccctgaagac 660 tgggacctca acgtctactt caattgcagc ggtgccagct
acagccgaga gaagtgcggg 720 gtccccttct cctgctgcgt gccagatcct
gcgcaaaaag ttgtgaacac acagtgtgga 780 tatgatgtca ggattcagct
gaagagcaag tgggatgagt ccatcttcac gaaaggctgc 840 atccaggcgc
tggaaagctg gctcccgcgg aacatttaca ttgtggctgg cgtcttcatc 900
gccatctcgc tgttgcagat atttggcatc ttcctggcaa ggacgctgat ctcagacatc
960 gaggcagtga aggccggcca tcacttctga ggagcagagt tgagggagcc
gagctgagcc 1020 acgctgggag gccagagcct ttctctgcca tcagccctac
gtccagaggg agaggagccg 1080 acacccccag agccagtgcc ccatcttaag
catcagcgtg acgtgacctc tctgtttctg 1140 cttgctggtg ctgaagacca
agggtccccc ttgttacctg cccaaacttg tgactgcatc 1200 cctctggagt
ctacccagag acagagaatg tgtctttatg tgggagtggt gactctgaaa 1260
gacagagagg gctcctgtgg ctgccaggag ggcttgactc agaccccctg cagctcaagc
1320 atgtctgcag gacaccctgg tcccctctcc actggcatcc agacatctgc
tttgggtcat 1380 ccacatctgt gggtgggccg tgggtagagg gacccacagg
cgtggacagg gcatctctct 1440 ccatcaagca aagcagcatg ggggcctgcc
cgtaacggga ggcggacgtg gccccgctgg 1500 gcctctgagt gccagcgcag
tctgctggga catgcacata tcaggggttg tttgcaggat 1560 cctcagccat
gttcaagtga agtaagcctg agccagtgcg tggactggtg ccacgggagt 1620
gccttgtcca ctgtccccct gtgtccacca gctattctcc tggcgccgga actgcctctg
1680 gtcttgatag cattaagccc tgatggcgcc ggtggcggtt gggcatggtt
cttcactgag 1740 agccggctct ccttttctta aagtgtgtaa atagtttatt
tataggggta agaatgttct 1800 cacaccattt cacttcctct tcctctcctc
cagcattctc ctctgagcag ccttagatag 1860 tgtccatggc tggagccgac
cctttgagtc cccttgagtg tcttaagaac cagcccacaa 1920 cagcctctct
ttctcctcca catactgcag cctccctcca tgcatcccac atacaagcac 1980
tcccccactc cccagcgtgg cctcactgtc ttctggtctt ggtgctactg aaattgtcac
2040 ccagaatttg aatcctgacc ctccccactg caagcccagg gagccccagc
ccaagatggc 2100 cagcctgaaa ctgttggcca gggctcctct tgtggccatg
tacccagggc tggctggcct 2160 gccatttgcc tctccccgga gacagccgtt
cttctgcaac cacaccccgt gcctagccac 2220 aaccccaggc tgcagctgct
cagaagctcc aggcattttg tttctggtga ccgcccctaa 2280 tgggatatcg
gtgatcactg gtccaccctt cctgtcaggg cttttctggg gctgctcttg 2340
gaaatgaagt cttaagtact gaataactcc cctggggata gctggggcat ttgtctagct
2400 gggctacttt ctaacacttt gccatagctc agaccacttc tcatcgttca
gggatggact 2460 gcaaccttaa tttacttgcc ggagtgtaca ttctagtgtg
gtgtatactg gtggctgttg 2520 atgatgattt tttttttttt tttacacaat
tctctgtaga ctaggagaag aatgcttgtg 2580 tttttcggaa gtgtgatgct
tctctttgac tgccaaactc ttttatggaa tatatcttta 2640 tattaaaaaa
aaaaaaaaac aaaaaaaaaa aa 2672 21 1508 DNA Homo sapiens 21
ggcacagaga tagagcggca acctcggaag tgcggacggg tgggcctata tagatgttga
60 ggtgcggagg ccgtgggctt ttgttgggcc tggctgtagc cgcagcagcg
gtaatggcag 120 cacggcttat gggctggtgg ggtccccgcg ctggctttcg
ccttttcata ccggaggagc 180 tgtctcgcta ccgcggcggc ccaggggacc
cgggcctgta cttggcgttg ctcggccgtg 240 tctacgatgt gtcctccggc
cggagcacta cgagcctggg tcccactata gcggcttcgc 300 aggccgagac
gcatccagag ctttcgtgac cggggactgt tctgaagcag gcctcgtgga 360
tgacgtatcc gacctgtcag ccgctgagat gctgacactt cacaattggc tttcattcta
420 tgagaagaat tatgtgtgtg ttgggagggt gacaggacgg ttctacggag
aggatgggct 480 gcccaccccg gcactgaccc aggtagaagc tgcgatcacc
agaggcttgg aggccaacaa 540 actacagctg caagagaagc agacattccc
gccgtgcaac gcggagtgga gctcagccag 600 gggcagccgg ctctggtgct
cccagaagag tggaggtgtg agcagagact ggattggcgt 660 ccccaggaag
ctgtataagc caggtgctaa ggagccccgc tgcgtgtgtg tgagaaccac 720
cggcccccct agtggccaga tgccggacaa ccctccacac agaaatcgtg gggacctgga
780 ccacccaaac ttggcagagt acacaggctg cccaccgcta gccatcacat
gctcctttcc 840 actctaagcc gtagcctctt ctgttaataa cacacagaga
gctctgccaa gcacctgagt 900 aggcccttga cacttgtgtg ccctgggatg
cctcctggcg cgaatcagga gggtctggaa 960 ggactctggc tatattctgc
aaatgtggct catgcccctt accgtggctc ggcgttgtgg 1020 tgcctgaggg
acagccggcc acctgcccag tactggtcag cttttcaaca ctattccctt 1080
tgacctactg gccatcttcc tcacagccct cagatatcaa cgggcacaaa taagaccaac
1140 tcaatttcca cttgaattta caaccaaaag cctgctgagt tgattacagc
tgggccaata 1200 cagtacgagg caataacaaa ttagtgtggg ttgattctgg
aattggaaaa gcttttgctt 1260 gtatggatac agcaaatcca gatgtctctg
aacaaagcaa caatttaaag caacgacatt 1320 ttctgtcctt taagcactta
aaatcaggtg tggtgtgttt tcaaaggcag aagtctgcat 1380 tttgagcaaa
aggtggcttc ccagctctaa caaggtaact ggttagcatg acattaaagc 1440
ttgggcaagg cttcaaactt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1500 aactcgag 1508 22 1447 DNA Homo sapiens 22 aattcggcac
gagagattta agtgcagcgt ggattttttt tttctcactt tgccttgtgt 60
tttccaccct gaaagaatgt tgtggctgct cttttttctg gtgactgcca ttcatgctga
120 actctgtcaa ccaggtgcag aaaatgcttt taaagtgaga cttagtatca
gaacagctct 180 gggagataaa gcatatgcct gggataccaa tgaagaatac
ctcttcaaag cgatggtagc 240 tttctccatg agaaaagttc ccaacagaga
agcaacagaa atttcccatg tcctactttg 300 caatgtaacc cagagggtat
cattctggtt tgtggttaca gacccttcaa aaaatcacac 360 ccttcctgct
gttgaggtgc aatcagccat aagaatgaac aagaaccgga tcaacaatgc 420
cttctttcta aatgmccaaa ctctggaatt tttaaaaatc ccttccacac ttgcaccacc
480 catggaccca tctgtgccca tctggattat tatatttggt gtgatatttt
gcatcatcat 540 agttgcaatt gcactactga ttttatcagg gatctggcaa
cgtagaagaa agaacaaaga 600 accatctgaa gtggatgacg ctgaagataa
gtgtgaaaac atgatcacaa ttgaaaatgg 660 catcccctct gatcccctgg
acatgaaggg agggcatatt aatgatgcct tcatgacaga 720 ggatgagagg
ctcacccctc tctgaagggc tgttgttctg cttcctcaag aaattaaaca 780
tttgtttctg tgtgactgct gagcatcctg aaataccaag agcagatcat atattttgtt
840 tcaccattct tcttttgtaa taaattttga atgtgcttga aagtgaaaag
caatcaatta 900 tacccaccaa caccactgaa atcataagct attcacgact
caaaatattc taaaatattt 960 ttctgacagt atagtgtata aatgtggtca
tgtggtattt gtagttattg atttaagcat 1020 ttttagaaat aagatcaggc
atatgtatat attttcacac ttcaaagacc taaggaaaaa 1080 taaattttcc
agtggagaat acatataata tggtgtagaa atcattgaaa atggatcctt 1140
tttgacgatc acttatatca ctctgkatat gactaagtaa acaaaagtga gaagtaatta
1200 ttgtaaatgg atggataaaa atggaattac tcatatacag ggtggaattt
tatcctgtta 1260 tcacaccaac agttgattat atattttctg aatatcagcc
cctaatagga caattctatt 1320 tgttgaccat ttctacaatt tgtaaaagtc
caatctgtgc taacttaata aagtaataat 1380 catctctttt tgattgtgaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440 actcgag 1447 23
3886 DNA Homo sapiens SITE (1050) n equals a,t,g, or c SITE (3743)
n equals a,t,g, or c SITE (3848) n equals a,t,g, or c SITE (3877) n
equals a,t,g, or c SITE (3882) n equals a,t,g, or c SITE (3885) n
equals a,t,g, or c 23 gcacccggga gggagatgcg gccggggctc aggctccttg
cagttgtaat ttagattcga 60 gaagtggttt atcctttgac tggaaaagaa
aagtagctgc agtattcccc cagcacttgc 120 tgagagcatg ccgtatgcca
ggctgtgagg ctcgagagac aagcagtgga agagttgcgg 180 cctgtttcat
ctctggattg taaatctgag cctccttctg gcccctggaa ggggacagca 240
tcaccatgga atgattccta accagcataa tgctggagcc gggagccacc aacctgcagt
300 tttcagaatg gccgtgttgg acactgattt ggatcacatt cttccatctt
ctgttcttcc 360 tccattctgg gctaagttag tagtgggatc ggttgccatt
gtgtgttttg cacgcagcta 420 tgatggagac tttgtctttg atgactcaga
agctattgtt aacaataagg acctccaagc 480 agaaacgccc ctgggggacc
tgtggcatca tgacttctgg ggcagtagac tgagcagcaa 540 caccagccac
aagtcctacc ggcctctcac cgtcctgact ttcaggatta actactacct 600
ctcgggaggc ttccaccccg tgggctttca cgtggtcaac atcctcctgc acagtggcat
660 ctctgtcctc atggtggacg tcttctcggt tctgtttggc ggcctgcagt
acaccagtaa 720 aggccggagg ctgcacctcg cccccagggc gtccctgctg
gccgcgctgc tgtttgctgt 780 ccatcctgtg cacaccgagt gtgttgctgg
tgttgtcggc cgtgcagacc tcctgtgtgc 840 cctgttcttc ttgttatctt
tccttggcta ctgtaaagca tttagagaaa gtaacaagga 900 gggagcgcat
tcttccacct tctgggtgct gctgagtatc tttctgggag cagtggccat 960
gctgtgcaaa gagcaaggga tcactgtgct gggtttaaat gcggtatttg acatcttggt
1020 gataggcaaa ttcaatgttc tggaaattgn ccagaaggta ctacataagg
acaagtcatt 1080 agagaatctc ggcatgctca ggaacggggg cctcctcttc
agaatgaccc tgctcacctc 1140 tggaggggct gggatgctct acgtgcgctg
gaggatcatg ggcacgggcc cgycggcctt 1200 caccgaggtg gacaacccgg
cctcctttgc tgacagcatg ctggtgaggg ccgtaaacta 1260 caattactac
tattcattga atgcctggct gctgctgtgt ccctggtggc tgtgttttga 1320
ttggtcaatg ggctgcatcc ccctcattaa gtccatcagc gactggaggg taattgcact
1380 tgcagcactc tggttctgcc taattggcct gatatgccaa gccctgtgct
ctgaagacgg 1440 ccacaagaga aggatcctta ctctgggcct gggatttctc
gttatcccat ttctccccgc 1500 gagtaacctg ttcttccgag tgggcttcgt
ggtcgcggag cgtgtcctct acctccccag 1560 crttgggtac tgtgtgctgc
tgacttttgg attcggagcc ctgagcaaac ataccaagaa 1620 aaagaaactc
attgccgctg tcgtgctggg aatcttattc atcaacacgc tgagatgtgt 1680
gctgcgcagc ggcgagtggc ggagtgagga acagcttttc agaagtgctc tgtctgtgtg
1740 tcccctcaat gctaaggttc actacaacat tggcaaaaac ctggctgata
aaggcaacca 1800 gacagctgcc atcagatact accgggaagc tgtaagatta
aatcccaagt atgttcatgc 1860 catgaataat cttggaaata tcttaaaaga
aaggaatgag ctacaggaag ctgaggagct 1920 gctgtctttg gctgttcaaa
tacagccaga ctttgccgct gcgtggatga atctaggcat 1980 agtgcagaat
agcctgaaac ggtttgaagc agcagagcaa agttaccgga cagcaattaa 2040
acacagaagg aaatacccag actgttacta caacctcggg cgtctgtatg cagatctcaa
2100 tcgccacgtg gatgccttga atgcgtggag aaatgccacc gtgctgaaac
cagagcacag 2160 cctggcctgg aacaacatga ttatactcct cgacaataca
ggtaatttag cccaagctga 2220 agcagttgga agagaggcac tggaattaat
acctaatgat cactctctca tgttctcgtt 2280 ggcaaacgtg ctggggaaat
cccagaaata caaggaatct gaagctttat tcctcaaggc 2340 aattaaagca
aatccaaatg ctgcaagtta ccatggtaat ttggctgtgc tttatcatcg 2400
ttggggacat ctagacttgg ccaagaaaca ctatgaaatc tccttgcagc ttgaccccac
2460 ggcatcagga actaaggaga attacggtct gctgagaaga aagctagaac
taatgcaaaa 2520 gaaagctgtc tgatcctgtt tccttcatgt tttgagtttg
agtgtgtgtg tgcatgaggc 2580 atatcattaa tagtatgtgg ttacatttaa
ccatttaaaa gtcttagaca tgttatttta 2640 ctgatttttt tctatgaaaa
caaagacatg caaaaagatt atagcaccag caatatactc 2700 ttgaatgcgt
gatatgattt ttcattgaaa ttgtattttt tcagacaact caaatgtaat 2760
tctaaaattc caaaaatgtc ttttttaatt aaacagaaaa agagaaaaaa ttatcttgag
2820 caacttttag tagaattgag cttacatttg ggatctgagc cttgtcgtgt
atggactagc 2880 actattaaac ttcaattatg accaagaaag gatacactgg
cccctacaat ttgtataaat 2940 attgaacatg tctatatatt agcattttta
tttaatgaca aagcaaatta agttttttta 3000 tctctttttt ttaaaacaac
atactgtgaa ctttgtaagg aaatatttat ttgtattttt 3060 atgttttgaa
tagggcaaat aatcgaatga ggaatggaag ttttaacata gtatatctat 3120
atgcttttcc ccataggaag aaattgactc ttgcagtttt tggatgctct gacttgtgca
3180 atttcaatac acaggagatt atgtaatgta atatttttca taagcggtta
ctatcaattg 3240 aaagttcaag ccatgcttta ggcaagagca ggcagcctca
catctttatt tttgttacat 3300 ccaaggtgaa gagggcaaca catctgtgta
agctgctttt tagtgtgttt atctgaaggc 3360 cgttttccat tttgcttaat
gtaactacag acattatcca gaaaatgcaa aattttctat 3420 caaatggagc
cacattcggg gaattcgtgg tatttttaag aattgagttg ttcctgctgt 3480
tttttatttg atccaaacaa tgttttgttt tgttcttctc tgtatgctgt tgacctaatg
3540 atttatgcaa tctctgtaat ttcttatgca gtaaaattac tacacaaact
agcaaaaaaa 3600 aaaaaaaaaa aaaaagggcg gccgctctag aggatccaag
cttacgtacg cgtgcatgcg 3660 acgtcatagc tcttctatag tgcacctaaa
ttcaattcac tgggccgtcg ttttacaacg 3720 tcgtgactgg gaaaaccctg
cgntacccaa cttaatcgcc ttgcagcaca tccccctttc 3780 gccaagctgg
cgtaatagcg aaaaggcccg gaccgacggc ctttccaaca gttgccaacc 3840
tgaaggcnaa agggaccccc cctggacggg gcataanccc gnggnt 3886 24 1583 DNA
Homo sapiens 24 ggcacgaggg acaacgacta tctgctacat ggtcatagac
ctcccatgtt ctcctttcgg 60 gcttgcttca agagcatctt ccgcattcat
acagaaactg gcaacatctg gacccatctg 120 cttggtttcg tgctgtttct
ctttttggga atcttgacca tgctcagacc aaatatgtac 180 ttcatggccc
ctctacagga gaaggtggtt tttgggatgt tctttttggg tgcagtgctc 240
tgcctcagct tctcctggct ctttcacacc gtctattgtc attcagagaa agtctctcgg
300 actttttcca aactggacta ttcagggatt gctcttctaa ttatggggag
ctttgtcccc 360 tggctctatt attccttcta ctgctcccca cagccacggc
tcatctacct ctccatcgtc 420 tgtgtcctgg gcatttctgc catcattgtg
gcgcagtggg accggtttgc cactcctaag 480 caccggcaga caagagcagg
cgtgttcctg ggacttggct tgagtggcgt cgtgcccacc 540 atgcacttta
ctatcgctga gggctttgtc aaggccacca cagtgggcca gatgggctgg 600
ttcttcctca tggctgtgat gtacatcact ggagctggcc tttatgctgc tcgaattcct
660 gagcgcttct ttcctggaaa atttgacata tggttccagt ctcatcagat
tttccatgtc 720 ctggtggtgg cagcagcctt tgtccacttc tatggagtct
ccaaccttca ggaattccgt 780 tacggcctag aaggcggctg tactgatgac
acccttctct gagccttccc acctgcgggg 840 tggaggagga acttcccaag
tgcttttaaa aataacttct ttgctgaagt gagaggaaga 900 gtctgagttg
tctgtttcta gaagaaacct cttagagaat tcagtaccaa ccaagcttca 960
gcccactttc acacccactg ggcaataaac tttccatttc cattctccta gctggggatg
1020 gggcatggtc aaacttagcc atcccctcct cagcaaggca tctaccggcc
cctcacagag 1080 acagtacttt gaaactcatg ttgagatttt accctctcct
ccaaccattt tgggaaaatt 1140 atggactggg actcttcaga aattctgtct
tttcttctgg aagaaaatgt ccctccctta 1200 cccccatcct taactttgta
tcctggctta taacaggcca tccatttttg tagcacactt 1260 ttcaaaaaca
attatatacc ctggtcccat ctttctaggg cctggatctg cttatagagc 1320
aggaagaata aagccaccaa cttttaccta gcccggctaa tcatggaagt gtgtccaggc
1380 ttcaagtaac ttgagtttta attttttttt ttttcttggc agagtaatgt
aaaatttaaa 1440 tggggaaaga tatttaatat ttaatactaa gctttaaaaa
gaaacctgct atcattgcta 1500 tgtatcttga tgcaaagact atgatgttaa
taaaagaaag tacagaagac acttggcatt 1560 caaaaaaaaa aaaaaaaaaa aaa
1583 25 1669 DNA Homo sapiens SITE (587) n equals a,t,g, or c SITE
(1634) n equals a,t,g, or c SITE (1648) n equals a,t,g, or c SITE
(1659) n equals a,t,g, or c SITE (1668) n equals a,t,g, or c 25
aggcgcttag gggctgaggc gcgatggcag gtgtcggggc tgggcctctg cgggcgatgg
60 ggcggcaggc cctgctgctt ctcgcgctgt gcgccacagg cgcccagggg
ctctacttcc 120 acatcggcga gaccgagaag cgctgtttca tcgaggaaat
ccccgacgag accatggtca 180 tcggtcaggc gggctgaggg tggggaggcc
ctttgtaccc agctcagccc tcggcggcgc 240 tccctcctcc cgagcccagc
cgggtcgctg gctcccccag tacctagcct gagggtgccc 300 cgaggacgcc
aggccccctg cctagagctc cgggccgcac gtcggagggg gccgggcgga 360
gaggcggccc actagggccg gtcgtgacta tgtgtctgcc ccgcaggcaa ctatcgtacc
420 cagatgtggg ataagcagaa ggaggtcttc ctgccctcga cccctggcct
gggcatgcac 480 gtggaagtga aggaccccga cggcaaggtg gtgctgtccc
ggcagtacgg ctcggagggc 540 cgcttcacgt tcacctccca cacgcccggt
gaccatcaaa tctgtcngca ctccaattct 600 accaggatgg ctctcttcgc
tggtggcaaa ctgcgkgtgc atctcgacat ccaggttggg 660 gagcatgcca
acaactaccc tgagattgct gcaaaagata agctgacgga gctacagctc 720
cgcgcccgcc agttgcttga tcaggtggaa cagattcaga aggagcagga ttaccaaagg
780 gcaagtgcat atctccttgt aatttgagag ggcagttgac ctttataccc
actataccta 840 ctcaagtttc tgcttgggag atcagctctg cagagaatgg
aatgagaagt attggtttag 900 ataggttgtt tgtttgttgt ttttgagacg
gagtttcact cttgttgccc atgctggagt 960 gcaatgccat gatcttggct
cactgcaacc tccgcctccc caggctgagg caggagaatg 1020 gcgtgagctc
gggaggtgga gcttgcagtg agctgagatc gtgccactgc actccagcct 1080
gggcgacaga gtgagactcc ttctaaaaaa caaaaacaaa accaaaacag tagttagggt
1140 acacacacac aaattctagt gattttcccc ccagtactac ccttgacttt
tgaaattcct 1200 gctttctcag agtttacaac atccttacca aacagccttc
tccctcctta ccacaaaaaa 1260 araaaaaaaa gttctggggt tgaggggaca
ctccattctt aacatcctct attatcccag 1320 cccaattccc cagctctcac
tgggactagt tgtacctatc ttcattcatt tggtcccagc 1380 atgactacct
gttggtgcat gagctgatct ctcctaacct aacagccaga tgctagtctc 1440
tggtactyag atgctgggct gcatcagata ggatgcacag gatcatcctg ggaagcttgt
1500 tgacatagat tcctgtgcaa cacttcagat atagtcttaa tgtagatttg
tgttggggtg 1560 gtatggtagg tagaataatg ggcctaccac tgtgtaaaca
tatggatatg tttacctaac 1620 atgacagaag aganttaagt tgctaatnag
atgactgtna aataaatna 1669 26 1053 DNA Homo sapiens SITE (1025) n
equals a,t,g, or c 26 ctaggagcac cgagcagctt ggctaaaagt aagggtgtcg
tgctgatggc cctgtgcgca 60 ctgacccgcg ctctgckctc tctgaacctg
gcgcccccga ccgtcgccgc ccctgccccg 120 agtctgttcc ccgccgccca
gatgatgaac aatggcctcc tccaacagcc ctctgccttg 180 atgttgctcc
cctgccgccc agttcttact tctgtggccc ttaatgccaa ctttgtgtcc 240
tggaagagtc gtaccaagta caccattaca ccagtgaaga tgaggaagtc tgggggccga
300 gaccacacag gccgaatccg ggtgcatggt attggcgggg gccacaagca
acgttatcga 360 atgattgact ttctgcgttt ccggcctgag gagaccaagt
caggaccctt tgaggagaag 420 gttatccaag tccgctatga tccctgtagg
tcagcagaca tagctctggt tgctgggggc 480 agccggaaac gctggatcat
cgccacagaa aacatgcagg ctggagatac aatcttgaac 540 tctaaccaca
taggccgaat ggcagttgct gctcgggaag gggatgcgca tcctcttggg 600
gctctgcctg tggggaccct catcaacaac gtggaaagtg agccaggccg gggtgcccaa
660 tatatccgag ctgcagggac gtgtggtgtg ctactgcgga aggtgaatgg
cacagccatt 720
atccagctgc cctctaagag gcagatgcag gtgctggaaa cgtgcgtagc aacagtaggc
780 cgagtatcca acgttgatca taacaaacgg gtcattggca aggcaggtcg
caaccgctgg 840 ctgggcaaga ggcctaacag tgggcggtgg caccgcaagg
ggggctgggc tggccgaaag 900 attcggccac taccccccat gaagagttac
gtgaagctgc cttctgcttc tgcccaaagc 960 tgatatccct gtactctaat
aaaatgcccc ccccccccgt taaaaaaaaa aaaaaaaaaa 1020 ctcgnggggg
ggcccggtaa ccaattcggc cta 1053 27 1477 DNA Homo sapiens SITE (7) n
equals a,t,g, or c 27 tgcaggnacc ggtccggaat tcccgggatc aaacagtact
gttgcacgtc gaattaagga 60 tctagctgct gacattgaag aagagcttgt
ttgtagactg aaaatttgcg atgggttttc 120 actgcaacta gatgaatcag
ctgatgtttc aggacttgct gtgctgcttg tgtttgttcg 180 ttataggttt
aataagtcta ttgaggaaga cctactcctg tgtgaatctt tgcaaagtaa 240
tgctaccggt gaagaaatat tcaactgtat caacagtttt atgcagaaac atgaaattga
300 atgggaaaaa tgtgttgatg tttgtagtga tgcttctagg gcagtggatg
ggaaaattgc 360 cgaagctgtc accttaataa aatatgtggc tcccgaaagc
accagtagtc actgcctatt 420 atacagacat gcactggcag ttaaaataat
gcctacatct ctaaaaaatg tgctagacca 480 ggcagtacaa atcatcaatt
atattaaagc tcgaccacat caatccagac tattaaaaat 540 tttatgtgag
gaaatgggtg ctcagcacac agcacttctt ctaaatacag aggtgaggtg 600
gctttctcga ggtaaagttc ttgtaagact ttttgaactt cgtcgtgaac ttttggtttt
660 catggattct gcttttcgac tatctgattg tttaacaaat tcatcttggc
tgctaagact 720 tgcatatctt gcagatattt ttactaaatt aaatgaagtt
aatttgtcaa tgcaaggaaa 780 aaatgtgacc gtttttacag tatttgataa
aatgtcgtca ttgttaagaa aattggaatt 840 ttgggcctca tctgtagaag
aagaaaactt tgattgtttt cctacactca gtgatttttt 900 gactgaaatt
aattctacag ttgataaaga tatttgcagt gccattgtgc agcacctaag 960
gggtttgcgc gctactctgt taaaatactt tcctgtaaca aatgacaata atgcttgggt
1020 tagaaatcca tttacagtta ctgttaaacc agcttcatta gtagcacggg
actatgagag 1080 cctgattgat ttaacatctg attctcaagt gaagcaaaat
tttagtgaac tttcactaaa 1140 tgatttttgg agtagcctaa ttcaggaata
cccaagcatt gcaaggcgtg cagtgcgtgt 1200 acttcttcct tttgctacaa
tgcacctgtg tgaaacgggg ttttcatatt acgctgcaac 1260 aaaaacaaaa
tataggaaaa gacttgatgc tgcacctcat atgcgaatcc gacttagcaa 1320
tattacacct aatattaagc ggatatgtga taaaaagaca caaaaacact gttctcatta
1380 aaattggagg agtttgcatg tctcatgata accaaatgta agatgaaaat
aaaagatgat 1440 ttacttcaaa aaaaaaaaaa aaaaaaaggg cggccgc 1477 28
2504 DNA Homo sapiens 28 tcgacccacg cgtccgcgag tgcctgcagg
actgggcctc cttcctccgc ctggccatcc 60 ccagcatgct catgctgtgc
atggagtggt gggcctatga ggtcgggagc ttcctcagtg 120 gcatcctcgg
catggtggag ctgggcgctc agtccatcgt gtatgaactg gccatcattg 180
tgtacatggt ccctgcaggc ttcagtgtgg ctgccagtgt ccgggtagga aacgctctgg
240 gtgctggaga catggagcag gcacggaagt cctctaccgt ttccctgctg
attacagtgc 300 tctttgctgt agccttcagt gtcctgctgt taagctgtaa
ggatcacgtg gggtacattt 360 ttactaccga ccgagacatc attaatctgg
tggctcaggt ggttccaatt tatgctgttt 420 cccacctctt tgaagctctt
gcttgcacga gtggtggtgt tctgaggggg agtggaaatc 480 agaaagttgg
agccattgtg aataccattg ggtamtatgt ggttggcctc cccatcggga 540
tcgcgctgat gtttgcaacc acacttggag tgatgggtct gtggtcaggg atcatcatct
600 gtacagtctt tcaagctgtg tgttttctag gctttattat tcagctaaat
tggaaaaaag 660 cctgtcmgca ggctcaggta cacgccaatt tgaaagtaaa
caacgtgcct cggagtggga 720 attctgctct ccctcaggat ccgcttcacc
cagggtgccc tgaaaacctt gaaggaattt 780 taacgaacga tgttggaaag
acaggcgagc ctcagtcaga tcagcagatg cgccaagaag 840 aacctttgcc
ggaacatcca caggacggcg ctaaattgtc caggaaacag ctggtgctgc 900
ggcgagggct tctgctcctg ggggtcttct taatcttgct ggtggggatt ttagtgagat
960 tctatgtcag aattcagtga cgtggtagga aagaaagtca ggtcaagtga
tgcttttgag 1020 cttacacaca attcacaggc ccaccagtga caatttactg
tgagttaatg tcattcaggt 1080 gtgcccatgg attttgaggg ctggaaatgc
aaagacacat ttttctataa aaagaaaaag 1140 caactaaggt taaaagctat
attgtggccc aagacactgt ctgaaagatg acatgagtag 1200 taattcacca
ctatctgaac caagcaagga tcaatgtgct gactgcattg gccaatggct 1260
ttgatacttc tgctattttt ttagacacaa acccataaac taactgctta agaattcata
1320 ctgcttgaat tatgtaaaat atattttaca gtatatcttt ccttgggcct
tagattacta 1380 ttcactgggc aaatggtatt tgtttttgtt ttaatttttt
ttttaataga cggaagtctt 1440 gctctgtcat gcaggctgga gtgcggtggt
gcgatcatag ctcactgcag cctcgaactc 1500 ttgggcttca agcaatcctc
ctgtgtcagc caccagagta gctgagacta caggggtatg 1560 ccaccatgcc
cagctggcat ttgttaatct tcatttgagg tctagatcta ggcactgtgg 1620
acactgaaaa acagttggga aatctttcga gctgtggaaa tccaaacaaa gactgataat
1680 tcctggtarg ggtgtgtgcs tgacgtactg carcctyaam ctyctgggct
yaagtgatcc 1740 tcccacctca gcctcctgag tagctgagac cacaggcgtg
tgccaccacg cctagctaat 1800 ttttwawacc rgggtcwamc ctttgtttcc
caggstggty ttgaattcct gggatcaagc 1860 aatycttcca cctkgsmctc
ccaaagtgtt gggattatag gcatgagcca ccasgactgg 1920 ccagaggaca
aaattttaat aaaggtctta gcttaagcag taatcytact tcattaagcc 1980
ttcctggggt gcggtacaca ccgttaattc agcaaccctc agtacatact aagtatgctc
2040 agtgctgtga aagtggatta caccaaatta agtcattctt atcacaccca
atcaaaagtc 2100 aagaagccag ggataaaagc acctcaggca cataacatta
atctagtaat gtaattctct 2160 gcacatccag ctggtgaaac tgcgtgctgt
aagctgggac cagctttgtc cataactgct 2220 gagagaactt gctgaagctc
taggaataat tttgcctgcc cggttgctca ccagttgtag 2280 cttgccagct
cccaacaccc ttcctggtgc caataaactt tctcaaagag caatactgac 2340
atttcttttg ataaaacctc cagccttctc tgtgttgttc cgacataccg aggaccaact
2400 ggtctacatg gatgccctga acatgcaatt ctttcttcca aaataaaaca
ttaaatagag 2460 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagggc ggcc
2504 29 1866 DNA Homo sapiens 29 ggcacgagaa tacatacgat ccttgtctac
caggagtcta atagaaagat ggacagcgtg 60 gaccctgcca gcagccaggc
catggagctc tctgatgtca ccctcattga gggtgtgggt 120 aatgaggtga
tggtggtggc aggtgtggtg gtgctgattc tagccttggt cctagcttgg 180
ctctctacct acgtagcaga cagcggtagc aaccagctcc tgggcgctat tgtgtcagca
240 ggcgacacat ccgtcctcca cctggggcat gtggaccacc tggtggcagg
ccaaggcaac 300 cccgagccaa ctgaactccc ccatccatca gagggtaatg
atgagaaggc tgaagaggcg 360 ggtgaaggtc ggggagactc cactggggag
gctggagctg ggggtggtgt tgagcccagc 420 cttgagcatc tccttgacat
ccaaggcctg cccaaaagac aagcaggtgc aggcagcagc 480 agtccagagg
cccccctgag atctgaggat agcacctgcc tccctcccag ccctggcctc 540
atcactgtgc ggctcaaatt cctcaatgat accgaggagc tggctgtggc taggccagag
600 gataccgtgg gtgccctgaa gagcaaatac ttccctggac aagaaagcca
gatgaaactg 660 atctaccagg gccgcctgct acaagaccca gcccgcacac
tgcgttctct gaacattacc 720 gacaactgtg tgattcactg ccaccgctca
cccccagggt cagctgttcc aggcccctca 780 gcctccttgg ccccctcggc
cactgagcca cccagccttg gtgtcaatgt gggcagcctc 840 atggtgcctg
tctttgtggt gctgttgggt gtggtctggt acttccgaat caattaccgc 900
caattcttca cagcacctgc cactgtctcc ctggtgggag tcaccgtctt cttcagcttc
960 ctagtatttg ggatgtatgg acgataagga cataggaaga aaatgaaagg
catggtcttt 1020 ctcctttatg gcctccccac ttttcctggc cagagctggg
cccaagggcc ggggagggag 1080 gggtggaaag gatgtgatgg aaatctcctc
cataggacac aggaggcaag tatgcggcct 1140 ccccttctca tccacaggag
tacagatgtc cctcccgtgc gagcacaact caggtagaaa 1200 tgaggatgtc
atcttccttc acttttaggg tcctctgaag gagttcaaag ctgctggcca 1260
agctcagtgg ggagcctggg ctctgagatt ccctcccacc tgtggttctg actcttccca
1320 gtgtcctgca tgtctgcccc cagcacccag ggctgcctgc aagggcagct
cagcatggcc 1380 ccagcacaac tccgtaggga gcctggagta tccttccatt
tctcagccaa atactcatct 1440 tttgagactg aaatcacact ggcgggaatg
aagattgtgc cagccttctc ttatgggcac 1500 ctagccgcct tcaccttctt
cctctacccc ttagcaggaa tagggtgtcc tcccttcttt 1560 caaagcactt
tgcttgcatt ttattttatt tttttaagag tccttcatag agctcagtca 1620
ggaaggggat ggggcaccaa gccaagcccc cagcattggg agcggccagg ccacagctgc
1680 tgctcccgta gtcctcaggc tgtaagcaag agacagcact ggcccttggc
cagcgtccta 1740 ccctgcccaa ctccaaggac tgggtatgga ttgctgggcc
ctaggctctt gcttctgggg 1800 ctattggagg gtcagtgtct gtgactgaat
aaagttccat tttgtggtaa aaaaaaaaaa 1860 aaaaaa 1866 30 1501 DNA Homo
sapiens SITE (434) n equals a,t,g, or c SITE (441) n equals a,t,g,
or c SITE (1300) n equals a,t,g, or c 30 ggacagccgt atcagcctgc
tggtgaataa cgccggtgtg ggcgccacgg cttcgctgct 60 ggagtcggat
gccgacaaaa tggacgcgat gattctgctg aacgtactgg cgctgacccg 120
cctggccaaa gccgcggcaa ccaactttgt cgcccagggc cgtggcacga tcatcaacat
180 cggctcgatt gtcgctctcg ctcccaaagt gctgaacggc gtgtatggcg
gtaccaaagc 240 gttcgtgcag gcgttcagcg aatcgctgca gcatgagctg
agtgacaagg gcgtagtggt 300 ccaggtggtg ctgccaggcg ctaccgccac
ggagttctgg gacatcgccg gcctgcctgt 360 gaaacaacct gccggaagcc
atggtgatga ccaccgaaaa cctggtggac gccgccctgs 420 caggccttgc
ccanggcraa ncgtgacgat tccgtccctg ccggacagcg cagattggga 480
cactacgaac gcgcgcggct ggccctgggt ccgaacctgt cgcaccgtga acccgccgct
540 cgttatgggt tgaagtaatc cggactagcg cagccgggtt taaacgcagg
cttcctgatt 600 gcctgggagg cctgttcata cccgtaggcg accgacagca
acgtggcttc gctcaaattt 660 ttcccataga agtgaacggc tgtcggcatc
ccttcgtcgt ccatgcccga tggtatggag 720 ataccgggat aaccggccac
cgccgagtag tagtaactgt atgagtgaaa gttggacatc 780 attgcatcaa
gcttatgctc ggccagcggc ttatcgatgg tgcttttgaa aatcgggccg 840
atggcagccc ataactcatt gcgcgcctca tcactgatat ccatcccgtt gatcatggtg
900 agcatctgtt gatccggcac acccggaccg ctgttgcgct cgttgaattc
aatcagctca 960 gccagcgact tcaccggcaa gcctgcccgt ccggccaggt
aggcttcaag ctggtgttta 1020 acgtccgata acaacgcgtc gttatattgt
tcatgggttt cgtacgggac gccctcaccc 1080 agttgaccca cgggtaccaa
tgtcgcgccc ttgcctcgca gcaacgtaat ggcatcctcg 1140 aagtgctsct
gtcggctttt ttcgccgggt cgttggcatc ttctacagat aactcaggca 1200
acggcgtata accgatgcgc ttgcccacca aggcgtcagg cttgattccc tgggtgtagc
1260 ggttggtatc cgtcatcgca tccagtgctt gcgccgcatn acgcacgtta
cgggtgaagg 1320 tgcccaccgt gtcctggcgg gaactggtca tcacccttcg
gtactcacta atccttcggt 1380 cggtttgaaa ccaataacac cgttgtaagc
cgccggcgta atgattgaac cattggtttc 1440 gacccccaat gccaagggca
caatcccttg tgcaacggct accgcagagc ccgtactcga 1500 g 1501 31 1752 DNA
Homo sapiens SITE (1099) n equals a,t,g, or c 31 aaggtacgcc
tgcaggtacc ggtccggaat tcccgggtcg acccacgcgt ccgtccagga 60
cagagagtgc acaaactacc cagcacagcc ccctccgccc cctctggagg ctgaagaggg
120 attccagccc ctgccaccca cagacacggg ctgactgggg tgtctgcccc
ccttgggggg 180 gggcagcaca gggcctcagg cctgggtgcc acctggcacc
tagaagatgc ctgtgccctg 240 gttcttgctg tccttggcac tgggccgaag
cccagtggtc ctttctctgg agaggcttgt 300 ggggcctcag gacgctaccc
actgctctcc gggcctctcc tgccgcctct gggacagtga 360 catactctgc
ctgcctgggg acatcgtgcc tgctccgggc cccgtgctgg cgcctacgca 420
cctgcagaca gagctggtgc tgaggtgcca gaaggagacc gactgtgacc tctgtctgcg
480 tgtggmtgtc cacttggccg tgcatgggca ctgggaagag cctgaagatg
aggaaaagtt 540 tggaggagca gctgacttag gggtggagga gcctaggaat
gcctctctcc aggcccaagt 600 cgtgctctcc ttccaggcct accctactgc
ccgctgcgtc ctgctggagg tgcaagtgcc 660 tgctgccctt gtgcagtttg
gtcagtctgt gggctctgtg gtatatgact gcttcgaggc 720 tgccctaggg
agtgaggtac gaatctggtc ctatactcag cccaggtacg agaaggaayt 780
caaccacaca cagcagctgc ctgactgcag ggggctcgaa gtctggaaca gcatcccgag
840 ctgctgggcc ctgccctggc tcaacgtgtc agcagatggt gacaacgtgc
atctggttct 900 gaatgtctct gaggagcagc acttcggcct ctccctgtac
tggaatcagg tccagggccc 960 cccaaaaccc cggtggcaca aaaacctgac
tggaccgcag atcattacct tgaaccacac 1020 agacctggtt ccctgcctct
gtattcaggt gtggcctctg gaacctgact ccgttagacg 1080 aacatctgcc
ccttcaggna ggacccccgc gcacaccaga acctctggca agccgcccga 1140
ctgcgactgc tgaccctgca gagctggctg ctggacgcac cgtgctcgct gcccgcagaa
1200 gcggcactgt gctggcgggc tccgggtggg gacccctgcc agccactggt
cccaccgctt 1260 tcctgggaga aygtcactgt ggacaaggtt ctcgagttcc
cattgctgaa aggccaccct 1320 aacctctgtg ttcaggtgaa cagctcggag
aagctgcagc tgcaggagtg cttgtgggct 1380 gactccctgg ggcctctcaa
agacgatgtg ctactgttgg agacacgagg cccccaggac 1440 aacagatccc
tctgtgcctt ggaacccagt ggctgtactt cactacccag caaagcctcc 1500
acgagggcag ctcgccttgg agagtactta ctacaagacc tgcagtcagg ccagtgtctg
1560 cagctatggg acgatgactt gggagcgcta tgggcctgcc ccatggacaa
atacatccac 1620 aagcgctggg ccctcgtgtg gctggcctgc ctactctttg
cctgcgcttt ccctcatcct 1680 ccttctcaaa aaggatcacg cgaaagggtg
gctgaggctc ttgaaacagg acgtccgctc 1740 gggggcggcc gc 1752 32 2152
DNA Homo sapiens 32 ccgctttgtt ctccagatgt gaatagctcc actataccag
cctcgtcttc cttccggggg 60 acaacgtggg tcagggcaca gagagatatt
taatgtcacc ctcttggggc tttcatggga 120 ctccctctgc cacatttttt
ggaggttggg aaagttgcta gaggcttcag aactccagcc 180 taatggatcc
caaactcggg agaatggctg cgtccctgct ggctgtgctg ctgctgctgc 240
tgctggagcg cggcatgttc tcctcaccct ccccgccccc ggcgctgtta gagaaagtct
300 tccagtacat tgacctccat caggatgaat ttgtgcagac gctgaaggag
tgggtggcca 360 tcgagagcga ctctgtccag cctgtgcctc gcttcagaca
agagctcttc agaatgatgg 420 ccgtggctgc ggacacgctg cagcgcctgg
gggcccgtgt ggcctcggtg gacatgggtc 480 ctcagcagct gcccgatggt
cagagtcttc caatacctcc cgtcatcctg gccgaactgg 540 ggagcgatcc
cacgaaaggc accgtgtgct tctacggcca cttggacgtg cagcctgctg 600
accggggcga tgggtggctc acggacccct atgtgctgac ggaggtagac gggaaacttt
660 atggacgagg agcgaccgac aacaaaggcc ctgtcttggc ttggatcaat
gctgtgagcg 720 ccttcagagc cctggagcaa gatcttcctg tgaatatcaa
attcatcatt gaggggatgg 780 aagaggctgg ctctgttgcc ctggaggaac
ttgtggaaaa agaaaaggac cgattcttct 840 ctggtgtgga ctacattgta
atttcagata acctgtggat cagccaaagg aagccagcaa 900 tcacttatgg
aacccggggg aacagctact tcatggtgga ggtgaaatgc agagaccagg 960
attttcactc aggaaccttt ggtggcatcc ttcatgaacc aatggctgat ctggttgctc
1020 ttctcggtag cctggtagac tcgtctggtc atatcctggt ccctggaatc
tatgatgaag 1080 tggttcctct tacagaagag gaaataaata catacaaagc
catccatcta gacctagaag 1140 aataccggaa tagcagccgg gttgagaaat
ttctgttcga tactaaggag gagattctaa 1200 tgcacctctg gaggtaccca
tctctttcta ttcatgggat cgagggcgcg tttgatgagc 1260 ctggaactaa
aacagtcata cctggccgag ttataggaaa attttcaatc cgtctagtcc 1320
ctcacatgaa tgtgtctgcg gtggaaaaac aggtgacacg acatcttgaa gatgtgttct
1380 ccaaaagaaa tagttccaac aagatggttg tttccatgac tctaggacta
cacccgtgga 1440 ttgcaaatat tgatgacacc cagtatctcg cagcaaaaag
agcgatcaga acagtgtttg 1500 gaacagaacc agatatgatc cgggatggat
ccaccattcc aattgccaaa atgttccagg 1560 agatcgtcca caagagcgtg
gtgctaattc cgctgggagc tgttgatgat ggagaacatt 1620 cgcagaatga
gaaaatcaac aggtggaact acatagaggg aaccaaatta tttgctgcct 1680
ttttcttaga gatggcccag ctccattaat cacaagaacc ttctagtctg atctgatcca
1740 ctgacagatt cacctccccc acatccctag acagggatgg aatgtaaata
tccagagaat 1800 ttgggtctag tatagtacat tttcccttcc atttaaaatg
tcttgggata tctggatcag 1860 taataaaata tttcaaaggc acagatgttg
gaaatggttt aaggtccccc actgcacacc 1920 ttcctcaagt catagctgct
tgcagcaact tgatttcccc aagtcctgtg caatagcccc 1980 aggattggat
tccttccaac cttttagcat atctccaacc ttgcaatttg attggcataa 2040
tcactccggt ttgctttcta ggtcctcaag tgctcgtgac acataatcat tccatccaat
2100 gatcgccttt gctttaccay tctttccttt tatcttatta ataaaaatgt tg 2152
33 1757 DNA Homo sapiens 33 aggctttcca cccagaccgt caacttcggg
acagtggggg agacggtcac ccttcacatc 60 tgcccagaca gggatgggga
tgaggcggca cagcctgatg ctgctgccat ggtggcttgg 120 ggcagcgggg
agaaaggagt gtcacaggga gcagctcgtg gctgcagtgg aagtcactga 180
gcaagagact aaagtcccca agaaaaccgt catcatagaa gagaccatca ccactgtggt
240 gaagagccca cgtggccaac gacggtyccc cagcaagtcc ccctcccgct
caccttcccg 300 ctgctctgcc agcccgctga ggccaggcct actggccccc
gacctgctgt acctgccagg 360 tgctggccag ccccgcaggc cggargcaga
accaggccag aagcccrtgg tgcccacact 420 gtatgtgacg gaggccgagg
cccactctcc agctctgccc ggactctcgg ggccccagcc 480 caagtgggtg
gaggtggagg agaccattga agtccgggtg aagaagatgg gcccgcaggg 540
tgtgtctccc accacagagg tgcccaggag ctcatcgggg catctcttca cactgcccgg
600 tgcgaccccc ggaggggacc ccaattccaa caactccaac aacaagctgc
tggcccagga 660 ggcctgggcc cagggcacag ccatggtcgg cgtcagagag
ccccttgtct tccgcgtgga 720 tgccagaggc agtgtggact gggctgcttc
tggcatgggc agcctggagg aggagggcac 780 catggaggag gcgggagagg
aagaggggga agacggagac gcctttgtga cggaggagtc 840 ccaggacaca
cacagccttg gggatcgtga ccccaagatc ctcacgcaca acggccgcat 900
gctgacactg gctgacctgg aagattacgt gcctggggaa ggggagacct tccactgtgg
960 tggccctggg cctggcgccc ctgatgaccc tccctgcgag gtctcggtga
tccagagaga 1020 gatcggggag cccacggtgg gcagcctgtg ctgctcagcg
tggggcatgc actgggtccc 1080 cgaggccctc tcggcctctt taggcctgag
ccccgtgggg cgtcaccacc gggaccccag 1140 gtccgtagcc ttgagggcac
ctccttcctc ttgcgggagg ccccggctcg gcctgtgggc 1200 agtgctccct
ggacgcagtc tttctgcacc cgcatccggc gttctgcgga cagtggccag 1260
agcagcttca ccacagagct ttccacccag accgtcaact tcgggacagt gggggagacg
1320 gtcacccttc acatctgtcs ctggccwcgg gccttcttac ctcactcaac
ttcagccagg 1380 aggactgggt ggtgcttgca atgttggaat gaccggctca
aagacctcag ctctgggctg 1440 tttcctgtca gcctggcagg agcctcagga
ctgtggacga aggatgtggc cttgggcatt 1500 tgtcctgttc ccacatgggc
ctggtccctc cctcctggcc ccagccacag ctgccaggcc 1560 tgacatggcc
ttgcctctcc tgcagtcttg gtgactgaga cccttgggtg gcgcttccca 1620
gctctgcagg ccctcctggc cttttctgca gggtggacac agggtctgtg tgtgggcagc
1680 agcccctgtc tctcagcaag aataaagcag cttcctgtgc aaaaaaaaaa
aaaaaaaaaa 1740 aactcgagcg gcacgag 1757 34 1466 DNA Homo sapiens 34
ggcacaggct gggactttgg gctggctgca gtctgtctga gggcggccga agtggctggc
60 tcatttaaga tgaggcttct gctgcttctc ctagtggcgg cgtctgcgat
ggtccggagc 120 gaggcctcgg ccaatctggg cggcgtgccc agcaagagat
taaagatgca gtacgccacg 180 gggccgctgc tcaagttcca gatttgtgtt
tcctgaggtt ataggcgggt gtttgaggag 240 tacatgcggg ttattagcca
gcggtaccca gacatccgca ttgaaggaga gaattacctc 300 cctcaaccaa
tatatagaca catagcatct ttcctgtcag tcttcaaact agtattaata 360
ggcttaataa ttgttggcaa ggatcctttt gctttctttg gcatgcaagc tcctagcatc
420 tggcagtggg gccaagaaaa taaggtttat gcatgtatga tggttttctt
cttgagcaac 480 atgattgaga accagtgtat gtcaacaggt gcatttgaga
taactttaaa tgatgtacct 540 gtgtggtcta agctggaatc tggtcacctt
ccatccatgc aacaacttgt tcaaattctt 600 gacaatgaaa tgaagctcaa
tgtgcatatg gattcaatcc cacaccatcg atcatagcac 660 cacctatcag
cactgaaaac tcttttgcat taagggatca ttgcaagagc agcgtgactg 720
acattatgaa ggcctgtact gaagacagca agctgttagt acagaccaga tgctttcttg
780 gcaggctcgt tgtacctctt ggaaaacctc aatgcaagat agtgtttcag
tgctggcata 840 ttttggaatt ctgcacattc atggagtgca ataatactgt
atagctttcc ccacctccca 900 caaaatcacc cagttaatgt gtgtgtgtgt
ttttttttta aggtaaacat tactacttgt 960 aacttttttt cttagtcata
tttgaaaaag tagaaaattg agttacaatt tgattttttt 1020 tccaaagatg
tctgttaaat ctgttgtgct tttatatgaa tatttgtttt ttatagttta 1080
aaattgatcc
tttgggaatc cagttgaagt tcccaaatac tttataagag tttatcagac 1140
atctctaatt tggccatgtc cagtttatac agtttacaaa atatagcaga tgcaagatta
1200 tgggggaaat cctatattca gagtactcta taaatttttg tgtatgtgtg
tatgtgcgtg 1260 tgattaccag agaactacta aaaaaaccaa ctgcttttta
aatcctattg tgtagttaaa 1320 gtgtcatgcc ttgaccaatc taatgaattg
attaattaac tgggccttta tacttaacta 1380 aataaaaaac taagcagata
tgagttaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440 aaaaaaaaaa
aaaaaaaaaa actcga 1466 35 526 DNA Homo sapiens SITE (283) n equals
a,t,g, or c 35 ggggacgtgc acggggccgc cctcctggcc ctgaagctgc
gccggcctcc ctgagcgttt 60 cgctgcggag ggaagtccac tctcggggag
agatgctgat gccggtccac ttcctgctgc 120 tcctgctgct gctcctgggg
ggccccagga caggcctccc ccacaagttc tacaaagcca 180 agcccatctt
cagctgcctc aacaccgccc tgtctgaggc tgagaagggc cagtgggagg 240
atgcatccct gctgagcaag aggagcttcc actacctgcg canaagsacg cctcttcggg
300 agaggaggag gagggcaaag agaaaaagac tttccccatc tctggggcca
ggggtggarc 360 cagaggcacc cggtacagat acgtgtccca agcacagccc
aggggaaagc cacgccagga 420 cacggccaag agtccccacc gcaccaagtt
caccctgtcc ctcgacgtcc ccaccaacat 480 catgaacctc ctcttcaaca
tcgccaaggc caagaactgc gtgccc 526 36 2412 DNA Homo sapiens SITE
(329) n equals a,t,g, or c SITE (340) n equals a,t,g, or c SITE
(977) n equals a,t,g, or c SITE (1117) n equals a,t,g, or c 36
cacgagtttt aaatcaattt tttttcaagc aatcagattc ttttctccta gaggagctgt
60 gggcaagaaa actaatgaat tctacatcct tctcatcacc tggtttaaat
tgttttctgc 120 tctgagtaaa cagtaattac tgtttaagta catctcagca
gaattttatc ccaattgcaa 180 cagttcatgt tcctcctaat gtaatctctg
cggaggaaat gatcgtcaag ggaagcaggc 240 tgacctgctc acgggatggc
gttcttacaa tctgcatctt atgtaatggt gattctgtgt 300 gcctgtgtca
taattattgg aatattatnt tatgcttttn tttttgagac tctatctcca 360
aaaaaaagaa gagacataga aatttgaaga aggatccttt aatggtctac accgtcttcc
420 aaagtcaaga agtggcagct gatatccatt tgaaagtaga atcctagctt
ttcagagcta 480 gacmaggcct cagaaactat agttgaattc ctcattgtac
caatgagaaa ctcaggccta 540 gatgggtaaa aagaggtgtg ttgtagcagt
gctgggacag atctcggttt ttctgcttcc 600 tatacaatcc tcttcaaccc
aatactacaa tgtatttatt atcacatatt aagctggaga 660 tttgtagcca
tgttattaga gttgcaactg tttatcctat agattccagc cacattttaa 720
acacataact tcatgtagtt aggccactaa aaataaagta atccatcaaa ctagtaatac
780 actagagaat ttgacctaca tactaagatg cctgaaatcc acagtatatg
gcaatttaac 840 ccccatctaa tagtggctca atcaagtagc taaacatatt
tatttcactc agatggttgg 900 ttgtttggta gaaggaatgg actccttggg
ctattttggg aacaaaaaag gactaggaca 960 caaatcaaag ccacatncac
agtaagaaat ccgggctgat ctctgccaag aaaagktaca 1020 aagaataatt
acttgatcac gtggggaaat ttcgacataa aagaagtaat ggataaaarg 1080
aaagaaaaat gaccaattgc tgragmcaat aattatngca accctaaacc agaaagcact
1140 aagccaggaa gtcaaaaact aagtcatmca catatgacaa ggtgcggggg
ttggtcctga 1200 gacttcagtg agaatatgtc cgatcaggat atgcaaagaa
ccatttggaa gatttctagt 1260 tcataaggga agtaccaaat gaagtggatg
ggaccatacg caatttgcat aggaccccca 1320 aggaggaaat agtatgacat
ggtagtaaaa aagcaatcac gactacactc acaattttag 1380 gagaaaataa
aactaaatcc agaatttgaa gccaacaaca acaaaaaaag tcattattta 1440
gggtatacgt tcctgtgggc agtaccttgc aaagtagaac atcttcaaga agaaatattt
1500 gacttgaggt aaggctttca agattgccat attacattca taaaggcaaa
ctcatccttg 1560 agaccaaagt gacagaagat tagaaattaa ggctttgttt
taaagaaatg ttgacatcat 1620 actggaaatt attatccagc tacttacaca
ttcgttttta aatccatccc tatgtttagc 1680 tgccaaaatg caaactgcgc
attgywctct cacggagagc gccacaggtc accagctatt 1740 attctcccag
gagtcattga gtaggctgcc ccaagtacca cataggaaac tcaacgaact 1800
attttcattt caaggaccat tagaaacaga aaggaaaaga gaaggtcagg gaaacttagt
1860 ttctaacaaa ggaagtgagg cactttgaaa aagaaaatat ttagagaacg
gagaggaagc 1920 taaacccaaa caaccaaaac gcacagctga caattattcc
gggaagttgg taacttctgc 1980 ctggtctcta gaagcacagg aagaaaggac
tgttagcgtg aagaacactc cagggttctg 2040 ggtatctagg cagagtcagt
caacagggct aaccatgtga taatcctggg taattccacc 2100 tcacagttca
ctaaaaaaca agcggaaccc tgggcaaagc cctttggggc tttaatagca 2160
atggaggaca tcaccctgtc actttctctg cttctacaca gcaggcaatc aaggaaaact
2220 tgccaagaaa tatgagtgaa taaatgattt tgaaagtttc attgagcagg
aacatgaaaa 2280 ggatgatttg gggatagctg gaaggatagt tacttgcatg
aataatattt attcaccgtc 2340 agtgtgatat ttctcaatag aaagattgta
tttaaaatgt acaactacaa aaaaaaaaaa 2400 aaaaaactcg ag 2412 37 1274
DNA Homo sapiens 37 ggcacgagga aagaccaact ggccggtctt ctgagcagat
ggattcctat aggaccagtg 60 gggagaggat tacacagtac ctctgaaccc
tcaacacaaa ataatatctc ttctattgtt 120 ggttagtttc actatctgct
tcattctttt aaaatgtcaa gtgttttctc ttgagggaac 180 cttctgaatt
actttgcctg cactatacct aattcttatt acaatgctgt gaactttgaa 240
ttattaccct tgttcacccc aaatggaaaa tcaaagctca aagaggtgag tgactgccca
300 gctcatgcag ctgatagaat caagatttca tttcaggtgt gtctggatcc
tgcacttact 360 tgctcttttc tcaacatggc ctcctaagga tccagaagga
agcccgccat cagcaaccag 420 cagcccactc accccccacc tcagtctcac
cttgccattc aaacaggctc cagtttcaaa 480 tgtcagttct gccattcacg
tgatgctgga caagtcagtt agcctctctg agattcaatt 540 ttctcatatg
cctaatggaa aaagagcatc taccttataa attgcatatt tactcttcct 600
tcccactgac tcaatagaac tatattcccc aytcccatag atgctggcct tagacttgtg
660 acttgcattg gccaatgaga tgagacatac accaatcaaa agagaggcat
taaatgtgca 720 tgactggttt agcttggcct cttatgstcc tgccatgcgt
rattagatca tgcctgagta 780 gccactgctc attcagcctg agttctggag
tgagaaacag gtggagcagt cctggactcc 840 atccacagcc cagagcagag
caccatggcc cacccgtagg gctgtaagtg agaaagaaat 900 gtctattgtt
ataaaccagt ggttttcaag gcatggtccc tcagtatcaa cgtcacttag 960
aaatttggag aaatgcacat tctcagtcct catccaaacc tgcttaatca gaaactctgg
1020 gggttgggcc cagcaatctg tattttaaaa aggcctctag gtaattctga
tgcaggctca 1080 ggcttggaat ccactgttat aaatcactga catttgggga
ttatttgttg ctgtgtgaaa 1140 gctgactaat acatctaccc tttgaggttg
ctatgaggac acagtaagat aagatgtgag 1200 aagctcctgg aatgaggttc
ctcctgatag tcctaagcct ggcatccaaa attcttcata 1260 atctgatcct cgag
1274 38 1036 DNA Homo sapiens SITE (43) n equals a,t,g, or c SITE
(47) n equals a,t,g, or c SITE (58) n equals a,t,g, or c 38
caaaccccat ttacgtgcac actgatacac cgtcacgcct gcnggtnacc ggtccggnat
60 tcccgggtcg acccacgcgt ccgggggaag caagcactta tttggctact
tggtgtccat 120 ggggaaagaa ttcctaatgc tccttatgtg ttagaggact
ttgttgagaa tgtgaagtcg 180 gaaacatttc cagctgttaa gatggagctg
ctcactgctt tgctgcgcct tttcctctcc 240 cgacctgctg agtgccagga
catgctagga cgtttgttgt attactgcat agaggaagaa 300 aaagatatgg
ctgtacggga ccgaggtctc ttctattatc gcctcctctt agttggcatt 360
gatgaagtta agcggattct gtgtagccct aaatctgacc ctactcttgg acttttggag
420 gatccggcag aaagacctgt gaatagctgg gcctcagact tcaacacact
ggtgccagtg 480 tatggcaaag cccactgggc aactatctct aaatgccagg
gggcagagcg ttgtgaccca 540 gagcttccta aaacttcatc ctttgccgca
tcaggaccct tgattcctga agagaacaag 600 gagagggtac aagaactccc
tgattctgga gccctcatgc tagtccccaa tcgccagctt 660 actgctgatt
attttgagaa aacttggctt agccttaaag ttgctcatca gcaagtgttg 720
ccttggcggg gagaattcca tcctgacacc ctccagatgg ctcttcaagt agtgaacatc
780 cagaccatcg caatgagtag ggctgggtct cggccatgga aagcatacct
cagtgctcag 840 gatgatactg gctgtctgtt cttaacagaa ctgctattgg
agcctggaaa ctcagaaatg 900 cagatctctg tgaaacaaaa tgaagcaaga
acggagacgc tgaatagttt tatttctgta 960 ttagaaactg tgattggaac
aattgaagaa ataaaatcat aacagagaaa aaaaaaaaaa 1020 aaaaaagggc ggccgc
1036 39 1379 DNA Homo sapiens 39 gcggcgcggg tgggggttgt gcgttttacg
caggctgtgg cagcgacgcg gtccccagcc 60 tgggtaaaga tggccccatg
gcccccgaag gcctagtccc agctgtgctc tggggcctca 120 gcctcttcct
caacctccca ggacctatct ggctccagcc ctctccacct ccccagtctt 180
ctcccccgcc tcagccccat ccgtgtcata cctgccgggg actggttgac agctttaaca
240 agggcctgga gagaaccatc cgggacaact ttggaggtgg aaacactgcc
tgggaggaag 300 agaatttgtc caaatacaaa gacagtgaga cccgcctggt
agaggtgctg gagggtgtgt 360 gcagcaagtc agacttcgag tgccaccgcc
tgctggagct gagtgaggag ctggtggaga 420 gctggtggtt tcacaagcag
caggaggccc cggacctctt ccagtggctg tgctcagatt 480 ccctgaagct
ctgctgcccc gcaggcacct tcgggccctc ctgccttccc tgtcctgggg 540
gaacagagag gccctgcggt ggctacgggc agtgtgaagg agaagggaca cgagggggca
600 gcgggcactg tgactgccaa gccggctacg ggggtgaggc ctgtggccag
tgtggccttg 660 gctactttga ggcagaacgc aacgccagcc atctggtatg
ttcggcttgt tttggcccct 720 gtgcccgatg ctcaggacct gaggaatcaa
actgtttgca atgcaagaag ggctgggccc 780 tgcatcacct caagtgtgta
gacattgatg agtgtggcac agagggagcc aactgtggag 840 ctgaccaatt
ctgcgtgaac actgagggct cctatgagtg ccgagactgt gccaaggcct 900
gcctaggctg catgggggca gggccaggtc gctgtaagaa gtgtagccct ggctatcagc
960 aggtgggctc caagtgtctc gatgtggatg agtgtgagac agaggtgtgt
ccgggagaga 1020 acaagcagtg tgaaaacacc gagggcggtt atcgctgcat
ctgtgccgag ggctacaagc 1080 agatggaagg catctgtgtg aaggagcaga
tcccaggtgc attccccatc ttaactgatt 1140 taacccctga aacaacccga
cgctggaagt tgggttctca tccccactct acatatgtaa 1200 aaatgaagat
gcagagagat gaagctactt tcccagggct atatggcaag caagtcgcaa 1260
agctgggatc ccaatccaga cagtctgacc gtggaacgag actcatacac gtaataaatg
1320 ctctgccccc aacttgtcca ccacaaaaaa aaaaaaaaaa aaaaaaaaag
ggcggccgc 1379 40 1932 DNA Homo sapiens SITE (293) n equals a,t,g,
or c 40 ggcacgaggc cgccctgggt gtcagcggct cggctcccgc gcacgctccg
gccgtcgcgc 60 asctcggcac ctgcaggtcc gtgcgtcccg cggctggcgc
ccctgactcc gtcccggcca 120 gggagggcca tgatttccct cccggggccc
ctggtgacca acttgctgcg gtttttgttc 180 ctggggctga gtgccctcgc
gcccccctcg cgggcccagc tgcaactgca cttgcccgcc 240 aaccggttgc
aggcggtgga gggaggggaa gtggtgcttc cagcgtggta cancttgcac 300
ggggaggtgt cttcatccca gccatgggag gtgccctttg tgatgtggtt cttcaaacag
360 aaagaaaagg aggatcaggt gttgtcctac atcaatgggg tcacaacaag
caaacctgga 420 gtatccttgg tctactccat gccctcccgg aacctgtccc
tgcggctgga gggtctccag 480 gagaaagact ctggccccta cagctgctcc
gtgaatgtgc aagacaaaca aggcaaatct 540 aggggccaca gcatcaaaac
cttagaactc aatgtactgg ttcctccagc tcctccatcc 600 tgccgtctcc
agggtgtgcc ccatgtgggg gcaaacgtga ccctgagctg ccagtctcca 660
aggagtaagc ccgctgtcca ataccagtgg gatcggcagc ttccatcctt ccagactttc
720 tttgcaccag cattagatgt catccgtggg tctttaagcc tcaccaacct
ttcgtcttcc 780 atggctggag tctatgtctg caaggcccac aatgaggtgg
gcactgccaa tgtaatgtga 840 cgctggaagt gagcacaggg cctggagctg
cagtggttgc tggagctgtt gtgggtaccc 900 tggttggact ggggttgctg
gctgggctgg tcctcttgta ccaccgccgg ggcaaggccc 960 tggaggagcc
agccaatgat atcaaggagg atgccattgc tccccggacc ctgccctggc 1020
ccaagagctc agacacaatc tccaagaatg ggaccctttc ctctgtcacc tccgcacgag
1080 ccctccggcc accccatggc cctcccaggc ctggtgcatt gacccccacg
cccagtctct 1140 ccagccaggc cctgccctca ccaagactgc ccacgacaga
tggggcccac cctcaaccaa 1200 tatcccccat ccctggtggg gtttcttcct
ctggcttgag ccgcatgggt gctgtgcctg 1260 tgatggtgcc tgcccagagt
caagctggct ctctggtatg atgaccccac cactcattgg 1320 ctaaaggatt
tggggtctct ccttcctata rgggtcacct ctagcacaga ggcctgagtc 1380
atgggaaaga gtcacactcc tgacccttag tactctgccc ccacctctct ttactgtggg
1440 aaaaccatct cagtaagacc taagtgtcca ggagacagaa ggagaagagg
aagtggatct 1500 ggaattggga ggagcctcca cccacccctg actcctcctt
atgaagccag ctgctgaaat 1560 tagctactca ccaagagtga ggggcagaga
cttccagtca ctgagtctcc caggccccct 1620 tgatctgtac cccaccccta
tctaacacca cccttggctc ccactccagc tccctgtatt 1680 gatataacct
gtcaggctgg cttggttagg ttttactggg gcagaggata gggaatctct 1740
tattaaaact aacatgaaat atgtgttgtt ttcatttgca aatttaaata aagatacata
1800 atgtttgtat garaaaaaaa aaaaaaaaaa aaaaagggcg gccgctctag
aggatccctc 1860 gaggggccca agcttacgcg tgcatgcgac gtcatagctc
tctccctata gtgagtcgta 1920 ttataagcta gg 1932 41 1430 DNA Homo
sapiens 41 aatttgaccc tacttccctc tcagtcctaa gggcctatct ttcatcacta
ggttgaatta 60 tctcccatgt ttgatttgcc tctatctccc tatgggcttg
caacaccatg acgggcacat 120 tgcaagtgca cttakacaaa tgagatcaga
tgacctgggg aacgtggctt gtacacacct 180 ttctgtgttc tgtagcatca
gctaagacct taaaatcagt aagaaagtat ctgtctctct 240 gttcacccat
aggaagcagc ttcgtggtga gtgaagggag ctacctggac atctccgact 300
ggttaaaccc ggccaagctt tccctgtatt accagatcaa tgccacctcc ccatgggtga
360 gggacctctg tggacaaagg wcgacagatg cctgtgagca gctctgcgac
ccagaaaccg 420 gtgagccatg ggagccggga tggggataga aggtgggaga
ggctgggttg aaagaggcat 480 tgtgctccct ctacctaaag aaccatggrk
tctgagccat tgacaagtgg ctgaataaga 540 aggcccatca atctaataaa
cactaatgta tgtgctgcca ttgcctttca aggggggaaa 600 ttcttagaga
gccacagact ctcagagtaa gaaaggacca cagagaacat ctggcctagc 660
tccagacaag caaaatgtct gcacttcaga tatccctgca ttcagagcct atcttcttgg
720 tactagctgg gtgatcttga gcaagacact gcttaccttt cagtacaaga
gaatgaaaat 780 agcaccaacc cacaaaactg tcattaggat tgaatgagaa
gctgtgtgga aatctcacag 840 catattcatt aattcactca acaggtattt
ctttagtagc caggcatatt tttaggtatt 900 gggaagacag cagtgatcaa
aatatgcaaa atctccaccc tcatgaagct tacatgctag 960 tggggtagac
actaaacatg cactgtggaa tatggtagcc actagctaca tgtggcattt 1020
tattttaaat taattgaaat taaataaaag taaacattca ttttcccagt cataccaccc
1080 agatttcaag tgttccatag ccacacacta gcagctacat tgttgcacaa
catagmtata 1140 gaatatcttc atcactctga aaatttctca tgggacagtg
ctgcagtggt caaacaagca 1200 ggtaaattat atgactctgt taggtgatga
tagatgccgg tgtaggggaa aagaatgatg 1260 tacaaagcat gtggagtgct
aagctgggag tttgggtgga gtttcattat acagaaagtg 1320 gtcaggggag
gcctctctga ggtaagagga tcacttgagc ctaggagttc gagtccagcc 1380
tggacaacat agcaagatct catctctaaa aaaaaaaaaa aagggcggcc 1430 42 1407
DNA Homo sapiens SITE (353) n equals a,t,g, or c 42 gcttaagatg
aaaagttcct tttcttgtgt taatggatgg cacaactggc ataaaaggtc 60
attaaatgct aatagaccca cttgaggtat gctcgcttaa tggaggatta gagcaaaaca
120 gacttaaaag accaacatgc cagttgtgcc atcccttaag atgaaaagtt
ccttttcttg 180 tgttaatgta caaagctttt cttttggcac tgacaactgt
gttctacctg ggaattttga 240 atagccattt tcatggctgt gtgttgtgta
acacaaatgt ttttaaatgg tattctcacc 300 cagtaggcca gctctccaaa
cgttgcttag atgcttcaaa attagcatat ttnaagttta 360 ccagtataaa
ataccaatgc aactactcta catagccaaa tgtttgtaaa tcacgtctta 420
ttttcctgak gtttttcact ccaccaaatc ttacaaatsr ttgaaagaaa tatattctaa
480 cagtacgcac tgaatagtga aaataattag acattttaag aaccagagcc
atagaattat 540 tttaaattag tagaaaagag gagctatttc cgaatctata
gaataaagta ccacctaaaa 600 ctgaatttta tcatataasc aagtaatacc
tattagtcat acctaaattt ttcagcactt 660 cattcaatta aaatmcatga
attttaaata ttttacatga tgtgaatagg catgataata 720 cttttagtat
aaaatctaaa ctttttccat ttatcagaaa tgataaaatc cagttaccac 780
atatcacgtt tataaaatcc ttaattaaat gagtaacttc taaaatataa caatactaaa
840 tatcacactg cgatggaggt cccaaatatg tggtctatca ccactgaatt
catgtaatag 900 ataagaaaaa aattagaggt ggatgtcttg ttttgtgtca
tgaattacta aaatctctta 960 gtagttgtgg tatatttttg agtaaaatta
ccatttccag atttgagttt gaagggcttt 1020 tatagtkgta ttttcctcct
cactgttaat aatcataatc ctttttcagt attttagtgg 1080 cctgaacaac
tggtttatct acaatctcaa atcctaagtg tataattatg tgcatgttca 1140
atacctcata taatacttgc tcaacagtat agtggtacca tggcattaag atggtgtttt
1200 tgttctacat atttttcaat atttattctt tctatgttga aattatatca
ggctttaccg 1260 gtttttttag ttgtttaaat aagtaatatt ttcaaaagaa
taaaataacc aatgatatct 1320 cttggaataa tctgtaaaac gtagttataa
aattctattt tctacttaga aaaaaaaaaa 1380 aaaaaaaaaa aaaaaaaaag ggcggcc
1407 43 950 DNA Homo sapiens 43 ggcacgaggt taccagcctg tttaattaca
gcagacttcc cacttttctc ccacttagta 60 tttccaattt gctgcttcct
gaaacctagg aagaattgaa aattgtctag agaataagca 120 tgccagattt
gttaaatcag cgaccttatt ttatatatat ttctaagtca tggccatggg 180
catagaagct tcttttttaa ttaagaagga aaaataaaaa tatgtgaaaa gaaagccata
240 aaggtcattt tacacacatg taactccatg cacgaatgcc agtccttccc
cttgtgtgtg 300 cacttgagac tagttctact actatccttc aaaacccaag
tgcatgaatt ccatgaagtt 360 tttccccatt attctcattt taattttcct
tctctgaaca actatgacat taatttatta 420 cttaatcatg aattatggca
tacaactccc taattgatgt ttgtgggttt ttttctcccc 480 cagctagatt
ttaatttcct tgaagacaga agccatgctc ttactgtgct agaatatctg 540
tctcccgtag ctcctgacac agtgctctgt gtatagaggg tgcttgttgg ctcaccaatt
600 tgttctttac accaaatgcc cagggaaatc ttacatagag tttataccag
gcaagaaaag 660 gatatgctag attctccagc tgccaaagac tggaatgtca
ctggtatcca gtcaccacaa 720 tctctaggtc cctcattttg ttcttggtga
gaaaggagca ctaaggagat ttcgtccttg 780 aaaaggcaga aagcaagtgt
agtatcatct tgccatctag cttggaaatt aacacttgat 840 cctaaattag
gtaatcttcc cttcacatct cagagttttc caggcaacag acactcagta 900
cgaacaacaa caacaacaac aacaaaaata ccaaaaaaaa aaaaaaaaaa 950 44 1004
DNA Homo sapiens 44 aattcggcac gagcagcatt ccacttgcaa ttggaagccg
agagaaacca ttgtttatga 60 aakkaaagag gctkctcaga tgactgcaaa
ccagccttcc ttactggttt tatcactggt 120 aatgttataa agacagttgt
ccagtttcat gaatcttgta ggtttttgtt tgtttatttg 180 tttgcttttr
atgttgttgt tgttgctgtt gttttccaaa ttcagtattg tagaaaaata 240
tgctgcccca gaagagatga ttggacactc tccagcgtgg tgttggactt tgtcatctct
300 tgcacagcca tctccagacc ttagtgttta cctcacgtta gttttttata
ttctgcaaag 360 acaaamccaa aataatccaa atttgacaca aatacctggg
atacatctta tttgagatgt 420 ttaacaaatg tctggatcat cttttcttac
attggattat aacgcaggaa acactgtgaa 480 gtaagtaaag ttggaattcc
caagtcmaag accatttgaa tatttacaag tagatttgag 540 gcaggaataa
tacagggtgg ccgcagggta acaaattcta ggcagcagat ttacatgact 600
tgaggctatg ggctgataag acgctgaaaa accagggtgt ggaccaagct ggctaagact
660 gactggaccc aatgtggtgc tagatttgag gtaggtttta cctaggccct
cattatacac 720 ttattaacat actaaatcac acacccacca gtgccatgac
agttctgaga ccaatatgtg 780 atgtaaaaat ggatggcacc acagttccga
gaaatcacct ttacccagga attttcacga 840 atattccact ccttggttaa
agaaacccat tgagatgaaa ccccagaacc cattgttctc 900 tctcgggtat
gcccgaactc ccctttcttg agtgtgtact ttctgctttg caatacatct 960
cttctttcac tatttgctga ctcatccttg acttggttct cgag 1004 45 1681 DNA
Homo sapiens 45 gaattcggca cgagtcgagt tttttattcc tccactgaga
atcacacaaa aagttagaag 60 cacaaaaagt atgatgggta atgatttgct
ccacctcgtg ttcttgcaac taagtttagg 120 tgtagcatca gggggatgga
ttttgtggcc actgaggaga ttgggtggtg cccatacgag 180 taaggatmca
aataaaaatg gmcacsytgt gcattgcttg gtcattacca atgagcctct 240
agtttccamc aagaagattg ggctctcttc tcctcacact tgtccatcaa ctctccaaca
300 gttttgatcc ccactgtaat taaactagta tcttctaaac acaaaatctt
cactctacct 360 cagtagcgct tggcagctga aatcttttct atttagaata
tcccaccttt ctatcttgaa 420 attttgtcca agctaaatgc ctcctactaa
tctctgcgta cctgcgggaa
cacaatgtgg 480 ctaccacatt ggctaccagg gctgtaggga ggattgtctc
aaaatcctct ccatttatca 540 caraaaggga ggcgggaara ggaaraaagt
aggttatgcc ctgaggctca aggctactgg 600 atggccaatc tgtgctaggt
ttgctggtca gaaagtagga tgatatgagc tgatatagsa 660 gagaaatata
gggtacagtt tctaccctga ggggctgtat tttagttggg gagatacatg 720
caatgactgg acaccaccac caaggataag gaagtcctgg gattgtgtga aagccacagc
780 agttcagaga ggagaaggaa aaagactcca tggaaatgat gggaattgaa
ccaggcctgg 840 gttttccccc tctcaggcac actggaggct gtttgcctac
cctgttgcat ctcttggctt 900 ttccaagttt ctgtcttgtt acagactctt
tcctctcttc ctcctcctag aaatattggc 960 aagcttcttt agtcatttgt
gtttctttac attacaggcc agaggtgtat cttctctgat 1020 agataatggc
cctcagttaa gactagggaa agctattttg cttgctgtat tagcgcccta 1080
ttttagaata atcctattcc cttgattctt tagtatttac aatttttcta agtaccgatt
1140 atattttcta agtcaaagtg gggtaaaatt agtgcattgt atcctgttgt
tgccgctttc 1200 tggagtagtc agtcttacat atttgaacaa taccaccctg
gtgtaatttt aaaaagtaag 1260 agcttgattc tttaaaaaac acttagccag
gcagtgtgag ctctctctga ggatcctcac 1320 attaggagtg ttttacatac
atcacacaaa aggaaaatgc gttctgaggg gatcggggct 1380 cctccgagct
gagagctgga cctgatgaat tgtgacaaat gggcctgttt ctgccagctg 1440
cacgttctca gccaggtgac gtctgaggct gcctgccagt aatggtttgt ggtttgggga
1500 gcaagaggga ggccctggac atactcactg gtggggaaca ggaaaaagtc
aggcccaatc 1560 agaaatagta actctcctca gtgttcccca gctaagtaag
actatgcatt taccatacag 1620 tccccatcct aaaactcatg aaatgaagaa
ttagtgacac actgggggag tagtggctcg 1680 a 1681 46 1361 DNA Homo
sapiens 46 ggcacgaggg agaactgctt taattagcct aggtgaaaag tagtcctagc
agtgtaaata 60 tgtataatta gagttttcta atttcactgt gagatctcta
acttttgagt ggcaaacaga 120 tcaagtcttt tgctcataga cttttctgtg
gggttattaa aatgcaaaag ctttattttt 180 ttaataatgc catactccat
tagtgtcaga tgatggtatg gaatttgttc ccttgctttc 240 ccccactgtt
actgcttcag tttatagatt gccagcagag ttcagaaata gagcagggat 300
ttacccgttc tttgcttgga catcccattt tcttttgtcc agacccatgt tggcaatcat
360 gtatgaactg tgttatactt tcagtgcttt cttttttctt tttgataaga
tggatatcaa 420 aaatagttgc tgtgcaaaag ttagagtctt cttcaagaag
aaaaccaatt ctttttctaa 480 taatatcctg tgaaattgct tcattcattc
atttattttt aagccaaatg tcagcagagt 540 gctgctgctt ttatctagta
attttgatat gtaagtatta atgcattttt aaaagatgtc 600 tacattgaaa
catgttcttc ccagtgtcct gcttatgatg ctttgttcag attttttgta 660
agagaccagt tagtacactg ggggtgtata ttgtgtacat gtgtcatttt agttaggcat
720 gtaggccaaa tgtgattata aatgaagttg atgaacatta attttgttat
tagtgagttt 780 tttgaattgt aaatggattt ccagtttacc ttctgttgtc
tacagctttt ttaattttaa 840 ggtttgacta attgtatcca tctcattgta
cagtgtttta gttgcaagca gaaagtagaa 900 tttggtataa agcaggttat
ttctatattg aaaggagtac agttgaaatt gtagatttaa 960 gattgttaaa
atcatgacaa ttctaacttg tctattctaa cctattgtgt acaatctgat 1020
tttttaaaat tgtaaacatg tatgatcttg gtttcatgtg tttttgaaag tgttattgtt
1080 taaaaaatga aaaaagcata tctgctaaag agctgtcagt tttcattact
gactctgtaa 1140 aatacactgt tctttgtgta ctgtgtgtta ttttgccagc
tgctgcatta gccttcaaaa 1200 gtatttggaa acttaagatg aactacattt
cttgcaaagt acattccttt ctgtggtatt 1260 ttgtcctgta actgaagtat
agtaattatt ttatggaaat gttagcaatt ctgtaccaac 1320 tttgaataaa
atgaaaaatt tataaaaaaa aaaaaaaaaa a 1361 47 1137 DNA Homo sapiens 47
gggctttttg tcaacctgaa gcacgttcta agtcgatggt agaaagtgga cacccccaga
60 agacactttt gcccagaaat ctctttcttc ctgacccctc ttccccagag
tgcccggaat 120 tccactgtca gaaatgcatt gtctgggtta aaaaacttaa
cacctgctat gatttcaaca 180 gtgtcaaaac aggatacgtc aaaactgggc
gaggaggaaa tgtatttggg ttctaggata 240 gtgaaagctc tattttttct
acttttctgt attttccata tttggtacaa tgagcacgta 300 cttagaacgg
ttttagattt acgaaaatat gcaaacacag tacagatagt tcttgcgtcc 360
cccatgccta gttcctctat tgctaacgtc tcaacgttag tgtggtgcgt ttgttgcaat
420 gggtgaatga atattcgtgg gctgttatta aagtcagtgc ttcaccccta
tttccccagc 480 tttcctctta catccttttc tgttccaaga tgcatccagg
atgccgcgtt acattagtct 540 tcacacttcc ttaggttcct cttggtatga
tggtttctca gatttttctt gtttttgata 600 atcttgacag ttcgaggagt
atttgtcagg cattttgtca aatgttcttc aactggggtc 660 tctggtggtt
ttctcatgat tagtctggga atgtgctttt gggaggaaga ccacagagat 720
gatgtgccag tctcagaaca tcgtactaag aaaaggttct gccaacttga cttaccactg
780 ttgctggtga ctttgagccc ccggctgagg ttactccttt gtaaagttac
tctttttttc 840 tcctttccat gctgtatgtt ttagaaggaa gtcactatgc
tgctccaagc aactcaagtt 900 tgatgaatgg ggagttccgc cccacctcct
tgagggcaga gtagctacat aaattacttg 960 gaatttctca aggagatttg
tctgtactcc cagtttatta tataaataaa tgatttattt 1020 atattacagg
gacccaggga tctttactgt atgctttggg ttataatcca atggtacttt 1080
actttgtggc tcaagtatac tacttttaaa ttggaaaaaa aaaaaaaaaa aactcga 1137
48 2763 DNA Homo sapiens 48 agagtttgac cctggaaagg tgctttgtat
atgttctttt cacatagtgc ccagcttgca 60 tgaaatgtac agagaaatgt
gtggtcgtat tttttacttt tgtcttgtat atgtatgtat 120 attgggtcct
ctgggcagta gaggcaaagc tcacctccca tgtagcacat gaaatgcttg 180
tgagttgttg acattggaca ggtgaacagt agggcattac atttgtgtga attaaatgtg
240 aacttctgta ttacgttgcg gcgtcggcag tcctgcgttc cctggagtaa
ctgtacgtat 300 ctgcctttgc tgggaagact gyggggctgc ctgtgttggc
tggcgaccag caggattgct 360 ccaggatttt gtgtttacct cgcgtgaagt
tcagcacgtg ctgtcgtgta gtcagcttct 420 actctaattt ctgttacagt
tctgcaaagg taacctggag tttagaagtt aaaaaaaagc 480 atgggatgtt
ggatttgcac catttggagt ttctttaggg aagaaaagtt ttctgctttt 540
ttatagaaaa tcatttcagt ctcccgaggt ctcatgctag caaattttga aataggattc
600 taatcactga tttcaaatat taagcaaaat gtaaagcact ttaatttata
gctatggtta 660 taaacaggtt ttagatgttt caaatgactt gtccactgaa
tgtcacttga ccttgataag 720 aggccgcctg cacacagagc ccagttaatt
ctccgcacct cggttgtgtg cttccgaatg 780 ggctcactcc cgtggtggtg
tttgagagcc aacaacacta cctcagagac gggtctttgg 840 gaaactttgg
gtctcactgt tgcctggctg gagcactttg gtttatagct ggaatactga 900
gttcagttca gaaggcagga aagacagtca caccgacgtg tcctgaaggt gtaggctctc
960 cacttaggcg cacaagctga cggctgcagc cagcaggccc cggtgacgag
acacttccag 1020 gtcttgtggt ggggacgcct ctcagtgcca gtcccgccac
tgctgagtga gcctggtgtt 1080 cttgccttct tggaaattac tgctcacctg
gtatctgtac gttaatgttt cttgctgagt 1140 tacagttttg ataaagaggc
tctcatttcc tgtgtcttgt atattcagtc ctttcaatac 1200 gtccacctgg
aggctcacca cttggagaga cacaggaagg taatatttac agctgtcatg 1260
tgacatcccc aggtctttgt gttttgccct gttttacggt gaggtaggag ggaacccatc
1320 tggggaccgg taggtgcagg tgcagtagga cgtgggactt ttggacccgt
cctttggtgc 1380 agctcgccag ggatgagagg cacctcccta cttgggtctt
caggagctgg tccaaggagc 1440 ttcgaatcta agtcatctag aatgaccctg
aaatgactga cagccccggg cccaagaaaa 1500 acccataacc acctcagatg
gatctgacgt ggctaaggga caaacagcaa atatttcagt 1560 cattttgatt
ttacaaataa aaaatgtgtt gtgtttttgt ccgacattat ttcctgactg 1620
cactgttctg agaatggagt ccacctggtc cctctggttg attagaatct caggtttcag
1680 ctcctgctgt cctgagcgaa cttgcctgat gcagggctgt gctgtgtcca
gatgttgctg 1740 gggcctcact ttttctcttg gctggaggtc caattgccag
agcctcccac actgcacata 1800 caaaggtytg agcccagggc agcttctggg
gccactgcac aggccacctg cttgggttcc 1860 tcggagttta atttgaaagt
ctgggtgtct taggatgatg gttaggaaca ttgaaaaatg 1920 gctgcaaata
gccaaatcaa acttaagaac cagatctctg ccagattaaa catttttgaa 1980
gcttttaaaa gtcaatattc ctagtggcca ctgagttcca ggcacactgg tgccctttac
2040 tgccacagct gctcaccttg tctggcaaac tggagggacc tcagaaactg
gactcctgca 2100 tgtccttggg ggcgcagccc tgtggtgctc aggcagagct
ctcaggagcc ggggcacctt 2160 gctgttcgct gctgtgtcgt cttctaatgt
gagctcatcc actgctgctg cagcgtggtg 2220 atcaggagtc acagacaaga
tcggggatgg tgtgtgtgtg tgtgtgtgtg tgtgtgcacg 2280 tgtgtgtggc
taaattaagt catactgtca accacacgtg atctcgtctg aaacagtgtt 2340
tggaagtggg aacagttttg tcctgtatgc tgatgtgtcc agaatttcat ttaatgatag
2400 acggaaaatg tgtggttact gaaaactgta tatgatacag aatttcataa
gagccatgct 2460 gttgggcaaa gcaactcttt ttcaaccact gctcatcagt
ttctgtagag acaaaaactc 2520 tgtacatatt ttggaatctg aagaatccta
tgtaaatcat ttgttactta agtctgtgaa 2580 aaacatattt ctttggagga
aaatgtatgc atttataagt gttccatgga atcagttttt 2640 attgtatcga
tataattgtc tctaagtgtt gactgtcttc attgcaatat gaaattcatt 2700
aaaatgtcca tgttccataa ttactattat aaaaaaaaaa aaaaaaaaaa aaagggcggc
2760 cgc 2763 49 1348 DNA Homo sapiens 49 gttaaaaaac atgtaaaacc
gtatttatct tcgaattaca gtgttatgtg tttgaatggt 60 tttagatgtt
aaaaagtagc aaattgaaac ttaatgttta aagtctttgt taattgaaaa 120
attgatcttc aatagtggta ctatttgcag tatgattcgt tcctttaatg tacatacgta
180 tatattagta catacgagag tgatgttaga cctgtagaaa tgaaggtgtt
gttttaattg 240 aaaacattta tgtttatttt gctgatagtg tttgtatttt
caaaaagtaa acaagttctg 300 tcaatatgtt tgaaaatttt taaagttgag
ataaatagca tctcattttg taaaaataaa 360 aaatataaag atttaccata
tgcgtttgca tcagaaaaga ctggaaggac atactcaaat 420 gtcaacaatg
attatctctg aatatgggat tatgggcaga tttttatatt ctttttactt 480
atctgtattt tcaaaaactt ctacagtaag tgaactgcat ttataatact gttttaaaag
540 attgaaccac caaagataga ggttattaaa aattatatcc ctactcacat
gattatagta 600 attggattat ttttggattt caagaaacat tagtattagt
ttaagagaat gttgctatat 660 gtaaagcatt gtactaaaaa ctatgggaga
tatacagaag gaaaagatag cttactttca 720 aggaagctgt atttcaaaaa
atgtgtgtag aaagtgccag agtggcaagg aaatttgctc 780 accagttatc
ccactcctta atacagtttc ctggcaaatc tttgtttctt tcttagacta 840
atacttggag acctatgtct ccttgtactc ttctttcaaa tctaactttg tttttttaat
900 ggatcatgaa agataaattt ctgtaattga tgttttattc atagcatgaa
gattttcctc 960 taaactgttt cttccttttc tggtaatcat ttacagtggt
ctttatgtta caatttgaaa 1020 cacagtagaa gtacaaaaat atggccaggc
gcggcggctc acgcctataa tcccagcact 1080 ttgggaggcc aacgtgggtg
gatcacttga gctggggagt tcaagaccag cttggtcaac 1140 atggtgaaac
cctgtctcta ctaaaaatac gaaaattagt cgggcgtggt ggcacatgcc 1200
tgtaatccca gctgcttggg aggctgaggc acgagaatcg cttgaacatg ggaggtggag
1260 gttgcagtga gccaaggttg caccactgca ctccagccta ggcaaccaag
cgagactttg 1320 tctcaaaaaa aaaaaaaaaa aaaaaaaa 1348 50 1264 DNA
Homo sapiens 50 gacccacgcg tccgcccacg cgtccgcttt cattcacatt
cacaaagcaa acatctagta 60 catgtctttc acttcacttt atgatagtgt
attggatgat ttgggcatta cgatcacctc 120 ttaccacagc acagaacata
cattcttcaa cagcattaac ggagtttgcc aagtgcatta 180 aagaggtcac
gtggagggta cgttcatatg aaacaatctg cagaaagtgg ggtaagaaag 240
ggcacatggc acagttaaag ttgtagaaat caaattacta tcattttttg ttgccaaaac
300 aaagtcttac atttaacccc cctttctacc acccccctcc acacttcacg
tcagctacat 360 agtttccaca gggtaattca ctaagagctt gtggagcttg
gttttaaaat ccttagcctg 420 gtctgacttt aggcatagct tcagttcttc
ttccgtgtcc tggtttcttg ttcagtttta 480 cttctaatcc accaccaaaa
gaaatgtctg gctggtctca gctagagtct atgtgtctta 540 gagcatgtgt
gcgtatctga ccatcatccc tgctctcatc tcagctccct ccaggctgag 600
caccggttcc ttttgtccca tacgtcatga agtccactat tgggaaacct gtgcttccct
660 ctccatggct taactccctg tcagtgtcgg agtgtataag aatgcttgta
aatactgtaa 720 tatatttatt aatatttgaa aggcattcat tcagtggaca
gtgggaatta actctcccaa 780 ggcaagtgaa aatgaatgat tgacgtacgt
tgatttaaca atcttactag attttaattc 840 ttaaggattt caaatgaaac
cagaaggtgg ttatgtaaga ggcttaaaat gatcttatgt 900 ttaaagagat
tctgttatta gcaccatgaa ctcgtactat gaaattttta agccttttat 960
ttttctaact atattactgt aggactggat attaggtgtc atataggaag cacaaaagtt
1020 tattgctgtt tgctaaagca aaatagcaga aaattttgta tatgcaaaac
tgttgaagga 1080 ccatagagaa tgtgtactac tgcggggctt ttactaggct
tcctgcgtgt gtaaaagtcg 1140 aggtattgct ggcattcagg gtgacatgat
ggtactaaat gttttccatt aaagtcttct 1200 attttaaaat ttagagaaaa
ataaaatggc tttccatcag aaaaaaaaaa aaaaaaaaaa 1260 aaaa 1264 51 1660
DNA Homo sapiens 51 acccacgcgt ccgtatacat atctattagt atagtatctc
ttgaatgcga ttttttctag 60 aatgtgttct gctgatttgt tttagagcca
tgagtgcaat ttatacacat acatctattg 120 ggaatgctca gaagttgttt
actgatggaa gtgccttcag aagagtccgg gaaccacttc 180 ctaaggaagg
aaagagctgg ccacagttag agcaagcctg cctggggccc tgctctgtgt 240
tccagctgca aactgcctgc atcatccctt cctgttactc ttccttcacc tgagacagtc
300 gaggccacag cgtcagccag ggccagagct gggatttgaa cccaggcact
cgggctccag 360 agccacactg ccccagtgtg gggcttagtg gcggctcctg
gccctgactg aggggctgac 420 tgaagcctgg tgagagcgtg ctgggtcagc
ctctccctgg cgggaatcct ctccgtccag 480 tcttctaacc tagcagcctc
acgtccacag agctgccttg tgaaactcag cagagccctg 540 gcttcctgca
gagccgtgtt ctcccagcct gcttcatggc tccctggttg agccaagctt 600
gcggatccgt ggggtgaagg tacccgcacc gcctgggcct tagtggtatg tacgggcctg
660 catcgtgagc agcgggcggg ggcccaggca ggtgaggcag ctggccacaa
gggcagggcc 720 cggcccccct cccaaggctg tgtctsatat tcttgagcct
gttcgagttt ccttttccaa 780 gcctcctgtt ctcccacccc camccctgcc
atgctgcagt gactaaatct gtggttctca 840 tccttggaga acacctgagt
agctgctaca agctggccac agcccagcga ctctgatgtg 900 gttgggctgg
gtgtggccag cagccagggc atcgggactt ttcgaagctc ccaggtgact 960
caccggcagc tggggatgag aactgtcagg agggaaggtc agaagtccca ggatgcactt
1020 gaaaagcctc tagctccacc agtgaccagc tcctggctgg actcctggtc
tggactcagc 1080 atcagggagg ctctggcctc tcgccctcag gctgggggct
tcttcacatg gtcatcaaag 1140 acttggccag ttccgcctct cccacggccg
tccttgtctc acccagcatc acgacgcatc 1200 agttcaccaa caaacacgat
tcagtgctgc tagtgctggg tcctgttctg ggggctggtg 1260 atgaggccaa
gagggaaaga gggagctctg tgttccatcg agggggcgac aagcctggac 1320
cagatgaaag tgactcatgt tgttaattag cggcttaggg ccaaaggtgg cccctggacc
1380 agtggcctca gcatcacctg ggaaaaggtt agaaatgaac attcccaggc
cccacctcag 1440 cctcctgaat cagagcatcc ctttggcaaa ccatactgag
aaacaaccac gtgcatcacc 1500 aagcgctgtg aagaaggcaa gctttgagac
cttgaaggaa tcatcaaact ctgggcctcg 1560 gtgtgctcac ccagggcgag
acaaagacgc catgctcctc caagtggcct ccaagattaa 1620 atgagcaatg
acttttaaaa aaaaaaaaaa aaaactcgag 1660 52 1678 DNA Homo sapiens 52
aattcggcac gagccaagct gcactattgg gaatggattg tggctgaaca gcaaatcaaa
60 acaccagaaa tatttttata tgttaacgtc atattatgtt aatgttgctg
aaaacaaaac 120 ctaacaaacc ttgatgtacc agtccaatac catgtagcgc
tgagtgataa agttaaaatg 180 tgctgtgctt cccacccttg tcagagggaa
gggtggctat gtgttatttt cactgtcttt 240 ttgaaagtta cagtatgtgt
tttcactttc gtgcagataa ctggaagtaa agcggcaaac 300 agtgctatta
catgctaaag ttaccttctc tttgtttttt gcatatctgg aattacacct 360
ttaaagactg atatgaatca gtacggtcac tatacatttt atgatttttc tgtcatctta
420 aaattgtatg atcgtaacat tatttattac cacaaaacag caaaatcttc
aatgtctaag 480 aaaactagct taaaatgttt aaatatagtt ctgattgggt
attaattact tgattaagaa 540 aaaattaaca ttatagatac tctggcatta
cgcttctata ccttttaggt cttccttgca 600 atactggaac ataattcttt
tgtgtagctc actattagcc agctaagttc atctttttaa 660 taccataaaa
aggttatatg tacagttcct attttagctt gcttacaaag ggagcattat 720
ttttatttaa agtattgcta gtaaatgatt tgtagaaact tggttttcta agcatagttc
780 ttccataacc accttttgtt gtttgagcac aagggattct tttcctagtt
ctatgtgttt 840 gtttccctat atgcagtctt taaaggatta caacacttaa
aattgaatgg acttgtgtca 900 agctttttgc atcatacatt ttttgaaaga
tttttaaaaa agcctacaac ttacatatgt 960 agtagaatca gccattgctc
tgctcctggc atagagtcac ctgttatgtg gattaaatag 1020 ttttaaaata
catatttgaa gmcctttgag aatgctttag tgtttgattt gaaataaaag 1080
gaaattttag caaggattaa agaaaaaagc tatcagctgt atgttaagag agactcttac
1140 taacatgttg taaatattac aattcatgaa atgttattgt aagtctgtaa
cttaattttt 1200 tccctgtttt agttatacag gttggtttgg aaatttgtgt
tttggcataa acaagtaaaa 1260 tgtgcccatt ttatggkttc catgcttttg
taatcctaaa aatattaatg tctagttgtt 1320 ctatattata accacatttg
cgctctatgc aagcccttgg aacagaacat actcatcttc 1380 atgtaggacc
tatgaaaatt gtctattttt atctatatat ttaaagtttt ctaaaaatga 1440
taaaaggtta ttacgaattt tgttgtacaa aatctgtaca aaaatctgtt tttacatcat
1500 aatgcaagaa ttggaaattt ttctatggta gcctagttat ttgagcctgg
tttcaatgtg 1560 agaaccacgt ttactgttat tgtatttaat tttcttttcc
ttttcaacaa tctcctaata 1620 aaactgtctg aaatctccct gtgactttaa
aaaaaaaaaa aaaaaaaaaa aactcgag 1678 53 1860 DNA Homo sapiens SITE
(912) n equals a,t,g, or c 53 cctagctgtc cccctgagat gaagaaagag
ctccctgttg acagctgcct gccccgctca 60 ctcgagcttc accctcagaa
gatggatccc aagagacagc acattcagct cctgagcagc 120 ctgactgagt
gcctgacggt ggaccccctc agtgccagcg tctggaggca gctgtaccct 180
aagcacctgt cacagtccag ccttctgctg kagcacttgc tcagctcctg ggagcagatt
240 cccaagaagg tacagaagtc tttgcaagaa accattcagt ccctcaagct
taccaaccag 300 gagctgctga ggaagggtag cagtaacaac caggatgtcg
tcacctgtga catggcctgc 360 aagggcctgt tgcagcaggt tcagggtcct
cggctgccct ggacgcggct cctcctgttg 420 ctgctggtct tcgctgtagg
cttcctgtgc catgacctgc cggtcacaca gctccttcca 480 ggctggctgg
gggagacact gccgctctgg ggctcccacc tgctcaccgt ggtgcggccc 540
agcttgcagc tggcctgggc tcacaccaat gccacagtca gcttcctttc tgcccactgt
600 gcctctcacc ttgcgtggtt tggtgacagt ctcaccagtc tctctcagag
gctacagatc 660 cagctccccg attccgtgaa tcagctactc cgctatctga
gagagctgcc cctgcttttc 720 caccagaatg tgctgctgcc actgtggcac
ctcttgcttg aggccctggc ctgggcccag 780 gagcactgcc atgaggcatg
cagaggtgag gtgacctggg actgcatgaa gacacagctc 840 agtgaggctg
tccactggac ctggctttgc tacaggacat tacagtggct ttcttggact 900
gggcacttgc cntgatatcc cagcagtagg ccctgccttc ctggccactg atttctgcat
960 gggtagacca tccaagactg cagcgggtag aaggtggcag ttcttcatgg
gagtcttttt 1020 aacttggtgc ctgagttctc tcctaggcaa gtggccagtt
gcctccacct cagttcttcc 1080 atctttggtg gggacagggc ccagcagcat
ctcagcctcc tacccacaat tccactgaac 1140 acttttctgg ccctactgca
catggccccc agcctccatc cttgtgctgg tagcctctca 1200 caactccgcc
cttgccctct gccttccact tccttccatc tcatttctaa accccaaaca 1260
gctcatctct aaaaagatag aactcccagc aggtggcttc tgtgttcttc tgacaaatga
1320 ttcctgcttc tccagacttt agcagcctcc tgttcccatt cttggtcaca
gctctagcca 1380 cagcagaagg aaaggggctt ccagaagaat atagcaccgc
attgggaaac agcagcctca 1440 cctccacctg aagcctgggt gtggctgtca
gtggacatgg ggagctggat ggaaatgcct 1500 ctcacttcaa aatgcccagc
ctgccccaaa tgcctctaag cccctccctg tcccctccct 1560 tgtagtccta
cttcttccaa ctttccattc cccatcatgc tgggggtctt ggtcacaagg 1620
ctcagcttct ctccactgtc catccctcct atcatctgta gagcagagca caggcagttg
1680 tgtgccttgg gcccagggaa ccctccatca acctgagaca ggactcagta
tatggttctt 1740 gggtatgccc taccaggtgg aataaaggac acagatttga
tttctaraaa aaaaaaaaaa 1800 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860 54 1663 DNA Homo sapiens SITE
(975) n equals a,t,g, or c 54 aattcggcag agttttctga tcagactctt
tttattgttt tgttttttat aaacaagtct 60 caggtggaaa aagaaagaaa
gggaggagct agctctctgc cttctcagcc aattgaaatc 120 gtggaaacca
atgggcttca gctagcccca ctcatcactg ctggggggga aaagacatcc 180
ctactcccct tccccgtggc
actcatgata ttctcaatgc cccaacaagg gtcatcttgg 240 ttcctctcgg
cgtttctgtc ctggcctttg gctctggctc cggctctgac tccgactccg 300
gctccggcca gggccccggg agcccctaga gctgctggag cccctggaag agttgctgcc
360 ggccgtggaa catgtgctgg tgccctggcc ccgggacagg aagcttggtc
tgctgtatgg 420 gagccaggcc tcttcatctg ggtggagcac ccgctgggct
gccaggggca cggcctggac 480 cgctttcctc tccccactgc gctcccgctc
cagggaggac atgctgcctg ctgccctcag 540 ctctagggcc cagctcgcct
cttcctctgg cggtggcaag ggtggtgggg gcaagtcccc 600 aggactgttc
tccctcctgt agggaagagc cttgggtttc ttccggaatc gagcacgggg 660
tccttgaagt gggggagtca tctccccatt cccctgccag gttctgcctg gggcactgct
720 ggctgtgcta ggggcaggac tggggctgag gtggggtgag gctgcagggc
cagcacccaa 780 gccagcaggc ctcgcttcac ggatgcccag catgggctgg
gatacactga gaggggaact 840 cggcccaagg ggcaccytcc tgggcatctg
atggagatgg ggcatgtcag ttgggggctg 900 gggagggtca ggaggtgagg
gtgtaagagt ggctgtggac tgctgtccat aggaaggtgt 960 gggagagggg
gtttnccttc gggatggggt gaccaggcac cctccactgg agctgggctc 1020
cgtcaggtga cttctctcag gcattggcgg gcaccactcc tctggctctg agctgccctc
1080 cagctcctcc tccggccctt ctaggcagct cagttcacaa gaaggaggag
gtgggggcag 1140 ggcttctggc cagttcagag agggcatctg cacaggtttc
cccagaagct tcactttgcc 1200 tcccttggct ccactgtccc cctggctcca
ctctggagga gcgtactggc tccagggacc 1260 cagatctcct gagggatgtt
gggggaagcc cccatggaag gtctgcagct cctcccccgc 1320 tgggtcaatg
gtgctataga caggaccctc gccaggggcg gccgtgcccc tggccgtctg 1380
agctagatac agggagattc ccgcttcgtt gtaatatctg tcgtccgggt caggattgct
1440 agggcagcag cttcccctgg gttcctgggc cgaggggctt cgagatgggt
ggggccacga 1500 atctgccagc catgagtagg gggctttccg tcctcgaact
tgccccttct ttatggagat 1560 ggttgcaaag cctggcctcc tcgtggcgtc
ttagaggcaa acgtcatcca gatcccgccc 1620 cgtcttggcc cgcagccctc
cctagtcctg gcagctcctc gag 1663 55 1632 DNA Homo sapiens 55
cccgccccgc ggcgcattgt gggatctgtc ggcttgtcag gtggtggagg aaaaggcgct
60 ccgtcatggg gatccagacg agccccgtcc tgctggcctc cctgggggtg
gggctggtca 120 ctctgctcgg cctggctgtg ggctcctact tggttcggag
gtcccgccgg cctcaggtca 180 ctctcctgga ccccaatgaa aagtacctgc
tacgactgct agacaagacg actgtgagcc 240 accacactct ggggctgcct
gtgggcaaac atatctacct ctccacccga attgatggca 300 gcctggtcat
caggccatac actcctgtca ccagtgatga ggatcaaggc tatgtggatc 360
ttgtcatcaa ggtctacctg aagggtgtgc accccaaatt tcctgaggga gggaagatgt
420 ctcagtacct ggatagcctg aaggttgggg atgtggtgga gtttcggggg
ccaagcgggt 480 tgctcactta cactggaaaa gggcatttta acattcagcc
caacaagaaa tctccaccag 540 aaccccgagt ggcgaagaaa ctgggaatga
ttgccggcgg gacaggaatc accccaatgc 600 tacagctgat ccgggccatc
ctgaaagtcc ctgaagatcc aacccagtgc tttctgcttt 660 ttgccaacca
gacagaaaag gatatcatct tgcgggagga cttagaggaa ctgcaggccc 720
gctatcccaa tcgctttaag ctctggttca ctctggatca tcccccaaaa gattgggcct
780 acagcaaggg ctttgtgact gccgacatga tccgggaaca cctgcccgct
ccaggggatg 840 atgtgctggt actgctttgt gggccacccc caatggtgca
gctggcctgc catcccaact 900 tggacaaact gggctactca caaaagatgc
gattcaccta ctgagcatcc tccagcttcc 960 ctggtgctgt tcgctgcagt
tgttccccat cagtactcaa gcactataag ccttagattc 1020 ctttcctcag
agtttcaggt tttttcagtt acatctagag ctgaaatctg gatagtacct 1080
gcaggaacaa tattcctgta gccatggaag agggccaagg ctcagtcact ccttggatgg
1140 cctcctaaat ctccccgtgg caacaggtcc aggagaggcc catggagcag
tctcttccat 1200 ggagtaagaa ggaagggagc atgtacgctt ggtccaagat
tggctagttc cttgatagca 1260 tcttactctc accttctttg tgtctgtgat
gaaaggaaca gtctgtgcaa tgggttttac 1320 ttaaacttca ctgttcaacc
tatgagcaaa tctgtatgtg tgagtataag ttgagcatag 1380 catacttcca
gaggtggtct tatggagatg gcaagaaagg aggaaatgat ttcttcagat 1440
ctcaaaggag tctgaaatat catatttctg tgtgtgtctc tctcagcccc tgcccaggct
1500 agagggaaac agctactgat aatcgaaaac tgctgtttgt ggcaggaacc
cctggctgtg 1560 caaataaawr kgctgaggcc cctgtgtgat attgaaaaaa
aaaaaaaaaa aaaaaaaaaa 1620 aaaaaactcg ag 1632 56 2233 DNA Homo
sapiens 56 ggcacgagct tgatttgata tggtaagcag taatatttaa aatggtgatg
gtattcttct 60 taacattttc tggctcccac ggatgtgttc cgacatctca
gccctggaag gatgctgaag 120 accaggttgg gtgtgtccat gccgtagctt
gggtgaactc agctctttac acagtcctct 180 gcccctttct gggaaagccc
aaatgttcat tctcatttga taggaacgag agtgaggatt 240 tgaataagca
ggaggttaag tgcagggcag tgcctgtctc tgtgtcgagc tcaatgttgt 300
aattgtgctg tgtaaaaggc ctgtgtggtg aacaagggtg agctcactcc agggaggaga
360 aggactgtta gaagactttt gtggcacctg acagccctgt ggggtcagct
tattctctcg 420 taccctgaac aacttggtcc taaggcctag tagagatttg
aaggaagaaa gcaacccagt 480 cctcaactct gcttttttta gaatgaagaa
cagactagca aaatagcatt gccatacatc 540 tcaaggcaga gagatgcgac
agggattgga agccaggtaa ttggtcagga aacattctgg 600 agacaaattt
ggggaccaag actcaaggat tgggaaggac aaggaaatag gatctaggtg 660
gtctaccgtc taggcctgtt ggttctccct tctccatgat agttagtggg gaaatcccac
720 gtaaggaaag cacgggtagt aagaaacttg ggaacaaata acacctagaa
actgaggcag 780 caagatgcac cttagtctag gaagccttct tgaaagaggg
gagtctctgg taagaatttg 840 aaagaaaaga aatatggctt gcttagcaag
aatataagaa aggctttgag gaagaaaaga 900 tagccagtga gtgccaagca
tctggttggg cttgagggtg agcacaaaca ggaagcaacc 960 cggccagccc
ctctgtgttt ctgccacagt caaacagtgc tcaaggaata tgaatacggc 1020
tgtcctgatt gtgaaagaag agaggggccc gaggcaaagg aagctggcag gcagctcctg
1080 ctgatcctcc agatgctagt tgataaaggc ccaatttcaa atgaaggttt
tgaaagcaaa 1140 aggacagtag gaacccggag gcagggaatg aatcacagga
cttgggagcg ggtgtggggt 1200 gaacctgaaa ttgagacagg attaaaaacg
acctgtctga gatgggacag gggctggctt 1260 gtttcacgga cttcaatgct
tctggcagca atggggaaat tgggcaggct ggctatcata 1320 ggaggctggg
cacagaccct gagcccaggg gatggtacat tgagtagcca gtggccccgg 1380
gtgaaagttc tgcagccaaa aacaactggg ggatgaggaa aaaaggaaaa attcaattct
1440 agtctctccc attaagcccc cttcccaatt tgaagactgg cccaagaggc
cttcgggaat 1500 acccctcctg tcttccaccc ttctcatcac ttccctgtcc
cttctctgtc ctttccccca 1560 actctccccc tcaagcccag tctcgttgtc
accaaggctt ctaggtgatt agagaatccc 1620 acctcatctc cacctggaac
cctccctcca cttctgcact cctagggata aaccgttgca 1680 cacccctgcc
ccacctggaa gggcctacag ggtctccagt gaaaaacctg tgaactgttg 1740
aacctcctgt ttggtggcat attattttga tttttggtga ctttttcttg gaataagtca
1800 acaaatatta accaagtgcc taccacatgc caagcgctgc tctaggtata
cagtggtgag 1860 caaagttggg ttgagttttt caatagaaaa tccatgtttg
ggtaatttaa gcttaaaata 1920 tcatgcaaac aggctggatg cattggctca
cacctgtggt cctagtactt tgggaggccg 1980 aggcagacag atcacttgag
gtcaggagtt caagactagc ctggccaaca tggcgaaaca 2040 ctgtctctac
taaaaaaata caaaaattag ccggacgtgg tggcgggcgc ctgtaatccc 2100
agctacccgg gaggctgagg gatgagaatc gcttgaaccc aggagtcgga ggttgcagtg
2160 agccgagatc ccgccactgc actccagtat gggcagcaga atgagactcc
atctcaaaaa 2220 aaaaaaaaaa aaa 2233 57 1963 DNA Homo sapiens SITE
(1540) n equals a,t,g, or c SITE (1935) n equals a,t,g, or c 57
ggcacgagct ttgaagagag agttcaagag ggcgtcatct acccttccat gtgctggatc
60 cgggactccc tggtcagcta catcaccaac ctgggcctct tcagcctggt
gtttctgttc 120 aacatggcca tgctagccac catggtggtg cagatcctgc
ggctgcgccc ccacacccaa 180 aagtggtcac atgtgctgac actgctgggc
ctcagcctgg tccttggcct gccctgggcc 240 ttgatcttct tctcctttgc
ttctggcacc ttccagcttg tcgtcctcta ccttttcagc 300 atcatcacct
ccttccaagg cttcctcatc ttcatctggt actggtccat gcggctgcag 360
gcccggggtg gcccctcccc tctgaagagc aactcagaca gcgccaggct ccccatcagc
420 tcgggcagca cctcgtccag ccgcatctag gcctccagcc cacctgccca
tgtgatgaag 480 cagagatgcg gcctcgtcgm acactgcctg tggcccccga
gccmggccca gccccaggcc 540 agtcagccgc agactttgga aagcccaacg
accatggaga gatgggccgt tgccatggtg 600 gacggaytcc cgggctgggc
ttttgaattg gscttgggga ctactcggct ctcactcagc 660 tcccacggga
ctcagaagtg cgccgccatg ctgcctaggg tactgtcccc acatctgtcc 720
caacccagct ggaggcctgg tctctcctta yaacccctgg gcccagccct cattgctggg
780 ggccaggcct tggatcttga gggtctggca catccttaat cctgtgcccc
tgcctgggac 840 agaaatgtgg ctccagttgc tctgtctctc gtggtcaccc
tgagggcact ctgcatcctc 900 tgtcatttta acctcaggtg gcacccaggg
cgaatggggc ccagggcaga ccttcagggc 960 cagagccctg gcggaggaga
ggccctttgc caggagcaca gcagcagctc gcctacctct 1020 gagcccaggc
cccctccctc cctcagcccc ccagtcctcc ctccatcttc cctggggttc 1080
tcctcctctc ccagggcctc cttgctcctt cgttcacagc tgggggtccc cgattccaat
1140 gctgtttttt ggggagtggt ttccaggagc tgcctggtgt ctgctgtaaa
tgtttgtcta 1200 ctgcacaagc ctcggcctgc ccctgagcca ggctcggtac
cgatgcgtgg gctgggctag 1260 gtccctctgt ccatctgggc ctttgtatga
gctgcattgc ccttgctcac cctgaccaag 1320 cacacgcctc agaggggccc
tcagcctctc ctgaagccct cttgtggcaa gaactgtgga 1380 ccatgccagt
cccgtctggt ttccatccca ccactccaag gactgagact gacctcctct 1440
ggtgacactg gcctagrgcc tgacactctc ctaagaggtt ctctccaagc ccccaaatag
1500 ctccaggcgc cctcggccgc ccatcatggt taattctgtn ccaacaaaca
cacacgggta 1560 gattgctggc ctgttgtagg tggtagggac acagatgacc
gacctggtca ctcctcctgc 1620 caacattcag tctggtatgt gaggcgtgcg
tgaagcaaga actcctggag ctacagggac 1680 agggagccat cattcctgcc
tgggaatcct ggaagacttc ctgcaggagt cagcgttcaa 1740 tcttgacctt
gaagatggga aggatgttct ttttacgtac caattctttt gtcttttgat 1800
attaaaaaga agtacatgtt cattgtagag aatttggaaa ctgtagaaga gaatcaagaa
1860 gaaaaataaa aatcagctgt tgtaatcacc tagcaaaaaa aaaaaaaaaa
aaaaccggca 1920 cgaggggggg cccgntaccc aattcggcct ttggaaatga gat
1963 58 1267 DNA Homo sapiens SITE (1248) n equals a,t,g, or c SITE
(1255) n equals a,t,g, or c 58 gctgcagcag actatgcaag ccatgctgca
ctttgggggc cggctggccc agagccttcg 60 ggggacttcc aaggaagctg
cttcagaccc ctctgactct ccaaaccttc ccacaccagg 120 gagctggtgg
gagcagttga cccaggcctc ccgggtctat gcctctgggg gcactgaggg 180
ctttcctctt tcccgatggg caccggggcg tcatgggact gcagctgaag aaggtgcaca
240 ggagagaccc ctgcccacag atgagatggc accaggcagg ggcctctggt
tgggaagact 300 atttggagtg cctgggggcc ccgcagaaaa tgagaatgga
gccctaaagt ccaggagacc 360 atctagctgg ctgcccccga cagtgagtgt
gttggctctt gtgaagcggg gggcacctcc 420 cgagatgcct tctcctcagg
agcttgaggc ctcagcaccc aggatggtgc aaacccatag 480 ggcagtgcgg
gctctctgtg atcacactgc tgcaagacct gaccagttga gcttccggcg 540
tggggaagtg ctgcgtgtca tcaccacagt ggatgaggac tggctccgct gtgggcggga
600 tggcatggag ggtctggtgc ctgtggggta tacctccctt gttctgtagc
cctgggaccc 660 tttcctgcgt atgtgtctcc ttcctgtcac ctgggaatgg
aatggccagt gaacaccatc 720 ccagaagcat tttccctctg caaaatgacg
tttcttccca cgtctgtttc tgctaatatt 780 taaaataaac tttccttctt
ccctcctata cccacctgta aggtgaaatc tgctcttctt 840 ccaaatatat
aaaaaaggaa ttgccctcca ggtaatccct ttcctttttc ccgtctatat 900
aagggaatgt cttccttcct atctatctgc aaaatggaaa tctagacctc cttcttcatc
960 cataagtgga ctgtgccagt acaatacatg cctcagcccc caagcctaga
aggacctcta 1020 gtctccttcc tgtgtggaat cttccccact ccatccctcc
caagttgcct gtattgataa 1080 tgtactcact catgctgtac taggtgctga
agcctggaca cccttggtgg gtgggcctgt 1140 ggtgatggtt tgcatccttc
ctcctttgtc ccaataaagt atgggagttg aaaaaaaaaa 1200 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaackc gcggccgnaa gcttntttcc 1260 ctttagt
1267 59 1295 DNA Homo sapiens 59 ggcacgagct tgtcccaggm ctggagcagc
tgtggagaaa ctgggaggga agcccgtcca 60 gcctgattcc aagcccacat
gctgctcaca ggtcaaggcc gagggactga tttttgccgg 120 tctgactgga
ctcaagttac ttcccagttc cttgcagaga gctgtctttg tgagacagtg 180
tcttgggttc tggaatgatg ggagccgtgc tttgcaaatg aggagtgatt gcgtgctcat
240 ctggcagctg gtgggtgtcc tgctggcatc aggcctgagc ggtgaccgtg
ctcctctgat 300 tgtcctcact gcgtgtgaca aggcctgggc cactgtgtga
gtcgtcttgc gctccatgaa 360 gcctggtgtc tgtgcagatg tgtgggtggc
gttaaggttg ggggacattt gtctttcaca 420 ctggagaatg ggagtctgga
gctggtgcta ctggtgagga agaggcccgg cctgctgcca 480 ggttcgccca
caccttcccc ctggttgttg ggaaaaccaa ccttggaatg gccaaggcag 540
gagatagcac ctccccggtg aagatccagg agctctcatg agctccacgt ggaaagatca
600 aggatctgga gtctggagcc cttcaggcag caactcagtg accatgaacc
tcagctctgt 660 ccacccggca cagcattgct gggagctgga cccgggaggc
tgccggctcc agagtgagga 720 gggtccagac catgcagaca atatgccctt
tttctccaaa caccatttca agcaaacccg 780 caggtctcct ccacggctgt
cagcagcttc tcgtagagct tctcatagga ctcatatggt 840 ggaatgtcga
tccggttaaa gctgaaaaag gacaaaagag agtcaccgtg tgggcagtcc 900
agccctagga ccaacctcaa ggccaaggac aggcagtgag aaagacaggg tctcgctagg
960 ttgcccaggc tgctctcaaa ctcctggcct caagtgaacc tcctgccttg
ccctctcaac 1020 gtgctgggag ccactgtgcc caatcaacac acagtaaagg
ggaagctcat ttccagtatt 1080 tgtgcaaaga aaaagacatc ctttaagaag
ctatcgtagc aaaccaaaaa atacaaaatt 1140 gtgacccaga ggatgtacag
tgacttctgg ctttctaggg tgctgtggca ggtgctgtgg 1200 cttttgagtt
ctgatgatga caaaaatatt ttggcagaga ctccatctca aaaaaaaaaa 1260
aaaaaaaaaa aaaaaaaaaa aaaaaaaaac tcgag 1295 60 915 DNA Homo sapiens
60 acccacgcgt ccgtgttcac agacagtagt ttcaaagtgt gtaccacatg
aagttgcagt 60 cttccaacct tccagccagt gtgtatggaa ataacctgaa
ttgtattaat agcagttctt 120 caatgtgggc ctgctggggg atgcttggtt
gtattccgtt gtttgttccc tgggtgcccg 180 tcttgggcaa gcatttctct
ggatgtcyct atttatgtgg caggtmaccc tgctggattg 240 ccttcatctg
tgtgcggact ccctgtggac caactacagc gcctacagct actttgaagt 300
ggtcaccatt kgygacttga taatgawcct cgccttttac ctggtccacc tcttccgctt
360 ctaccgcgtg ctcacctgta tcagctggcc cctgtcggta agagagtggt
ctggccctgt 420 cctccgcatg cacaagtcag gatgttagct agagtactga
gacctgacag agtttttccc 480 gtctgcccat ctcacctctt taaccattct
ttgctgcctc tgccctgaat ttcctattgt 540 ttggtggaca tctctgcttg
atgtcctgct ggtttttaaa actcactttc cagctacaag 600 aaggctgtgg
ctggccgggc gcggtggctc acgctggtaa tcccagcact ttgggaggct 660
gaggcgggcg gatcacgagg tcaggagttc gagaccacgg tgaaaccccg tctctactaa
720 aaaatacaaa aaatcagccg ggcgtggtgg cgggtgcctg tagtcccagc
tactcagaga 780 ggctgaggca ggagaatggc gtgaacccgg gaggcggagc
ttgcagtgag ccgagatcga 840 gccactgcac tccagcctgg gtgacagagc
gagactcctc tcaaaaaaaa aaaaaaaaaa 900 aaaaagggcg gccgc 915 61 1445
DNA Homo sapiens SITE (1047) n equals a,t,g, or c 61 aggaattcgg
cacgagcggc acgaggactc cttctcttct gcagaagcag atgggaatat 60
gctcttttaa actatgagat actggacaga catgaggagg aactaccgtg tcacgtatca
120 agtagtgttg ttatttctgt gcttctccct cctaacagaa tgtaaaacct
ttgaacccag 180 gtcagagagg tctttatttt catatcccct gtgatgtcta
atttatttgg atttacagat 240 aaatgatcgg taaactttag aaacagcact
ccagtttata gctctgtgct gtagacttac 300 tgaacaacta cagtgaaacc
aattcaaaaa gggatatttt gtattatgat ttagtctcct 360 acttccaagg
ctagttttta aggctgtgaa gggaagctga aaatgacaca gtgtttctgg 420
gatgaccaga cagacactgt atccagagat gctgtctgcg cagcggggga tagtaaaccc
480 cttagtacaa cattaattgg catggtggtt tatgagttaa tgtaatacca
aatattaaca 540 taaataaaaa tatatttaag tgataactaa gctggacata
tatcttaaaa gacaactaca 600 gcccagaaaa caatgaacat tgttgtccta
cagctatttt gtcactgtga tgatacctaa 660 ttttaatctt aaagggagct
gatgtttata acctagaagt tgattttgat aacatttgag 720 aaaacttcat
aaagctggca caggtaacat atttagtttt gtatatctgc tgtccaattt 780
gagtctctaa aaattatctt agaatgaata tgaaattcgc aggtataaag accaagtttt
840 cagaaataaa aaatgtccaa gtactttgaa acatctattt ttcactcatt
attcagccta 900 ggatattagc acttgtgtcc ttgaacagag atgagaatgt
ttgttatcca aagaccagga 960 aggtcaccag ccaagggata tacagtcgtg
cctcatcttc tgtgcctttg tattccttta 1020 tgctttgtag cttaacaaaa
ggttttncct tgtacttgtt aagtttccat atatttgtta 1080 aatatatact
tcacacttca cagttgctca tgtcagaaca gactattgaa aatgtaaacc 1140
tggccaggca cggtgctcac gcctgtaatc ccagcacatt gggaggctga ggcaggcgga
1200 tcacttgagg tcaggagttt gagaccagcc tggccaacat ggtgaaacct
tgtatctgct 1260 aaaaatgcca aaaaattagc taggcatagt ggtgcacgcc
tataacccca gctacttggg 1320 aggctgaggc aggagaattg cttgaaccca
ggaggcggag gttgcagtga accaagatca 1380 caccactgca ctccagccwa
ggtgatagag tgacactctc tcaaaaaaaa aaaaaaaaaa 1440 ctcga 1445 62 1100
DNA Homo sapiens 62 ggtgactgct ccctagctgg tcatgaaaat tctcctcaag
attattaaat cagggattat 60 gtcttgtcca aatataagtg aaatattgtt
tgtaacaatg ataagttact tggctttaca 120 ttttagtaac taccctttca
tgtttcttta actcttgaaa tattttatta ggggttgagc 180 attcatgatg
gtacctggaa gtcagcaatt tatggttttg gagatcagag taatttgaga 240
aaactaagaa atgtatcaaa tctgaaacct gtcccgctca ttggtccaaa attgaagaga
300 aggtggccaa tttcttattg tcgggaactc aaaggttatt ccattccttt
tatgggatct 360 gatgtgtctg ttgtaaggag gactcaacgt tacttgtatg
aaaatttaga ggaatcacca 420 gttcagtatg ctgcgtatgt aactgtggga
ggcatcacct ctgttattaa gctgatgttt 480 gcaggacttt tctttttgtt
ctttgtgagg tttggaattg gaaggcaact tctcataaaa 540 ttcccatggt
tcttctcctt tggctatttt tcaaaacaag gcccaacaca aaaacagatt 600
gatgctgcct cattcacgct gacattcttt ggtcaaggat acagccaagg cactggtaca
660 gataagaaca aaccaaatat caaaatttgt actcaggtga aaggaccaga
ggctggctat 720 gtggctaccc ccatagctat ggttcaggca gccatgactc
ttctaagtga tgcttctcat 780 ctgcctaagg cgggcggggt cttcacacct
ggagcagctt tttccaaaac aaagttgatt 840 gacagactca acaaacacgg
tattgagttt agtgttatta gcagctctga agtctaaaca 900 ctggaagaat
taactgaagt cataacgtgc gtgaattaac agcttctcta tttgatattt 960
gaaattcttc tgtaagcctg tctgagtgta tgtggaaacg attgtcaaat ctaaaatatc
1020 tatatattaa aaagtaggaa attgtcctag cttaccctaa atttcaaaaa
aaaaaaaaaa 1080 aaaaaaaaaa gggcggccgc 1100 63 1499 DNA Homo sapiens
SITE (52) n equals a,t,g, or c SITE (66) n equals a,t,g, or c SITE
(84) n equals a,t,g, or c 63 agcttattgc aaagacaaat gtttgaagtg
tttgttgaga tttcctgttg tncttcctga 60 ggcagncaca gcataagctc
tttnaccctc tacttctcag cacataagct ttcttaccat 120 ctatcactgg
agtcaggggt gaggggagga ccgcatgaca gttggttaat atacacttat 180
tttttggcaa aaacgttttc tctgggacca gaatgatctt gatactgaaa aaatttctag
240 tgctagatcc tctttctaag tgtgaaagga cttatctgga atgctccaga
atgatcccaa 300 gtgttgagct gagagggacc tggcagcaga atctgattat
tgaaaagtgg caattgttga 360 tttattgaag acagaataat aactcagcag
aactgttatg ttgagctgaa cccgacctcc 420 ttcagccgaa tcatgcaaga
atgcctgctg catggctgtt gctgctactt attaaggctt 480 ggtgttctgg
gcacagtgca atgcatttct acatggttga tcctcacagc aaatgaacaa 540
cacaggctta aggaaacaag caactctcaa agtcctgcag tgagtagagc ttagctgttg
600 gtagtcaaca tgccacgcga ttcggragtt gagcctgtct ccagaggtta
gagatgttca 660 gtttcctctt aaggttctta cgtagatttt tttcatgact
ttatctacat cctccttaaa 720 tttacgtttt tagtccttac tggctcttga
tatcaccagt tttgttgtta ttagtaattt 780 ctaactgccc taaatttgtc
tgttttaaga ttcaagggat gatacctcag tctgttatct 840 ggaatatggt
ttacaaatcc attttttctc ttcaaggctt tgaaaacatt gacattgtct 900
cctcctaaca tttttatttg tcttgcagac
tcctaattta tttaatttat cgttaggaag 960 acgacttttc tgtcttttga
tgattttagc tgcccttctc tagaccttgc tgattccatt 1020 atctttacca
agaattgaaa gtgaaagtgg catttgtcat agaatgccat ggtcttattc 1080
caaagtatct taggatggaa caatacaagg cataatatgg ggtcagtgag gtttgttaca
1140 cgagtgaatg accaacaaca ctactgtctg ttcaaaccca gtctgaaggg
tgaatcagac 1200 cgaccattgg ccgtgagggt ctggactgct cagtattatc
tcaaggatat caagggttat 1260 tggaaactgt gtgatcaaag gggctccatg
actttatgca gggattcagt agggagccaa 1320 gaaggttgag aatagttcag
agaccagagt ctaagaccaa tcaagaagaa tggatcaatt 1380 agagatatga
attctggtgc ttatattttt gtggagctgg ttgtgagata aaaggtcaag 1440
cctaccagac tgaaaagtgt atgtgaaagc tctttaaaaa aaaaaaaaaa aaactcgag
1499 64 655 DNA Homo sapiens 64 ggcacgaggc aggaaccgct aaacgagaca
gacactggcg actcagagcc ccggatgtgt 60 gggttccttt ctctgcagat
catggggccc ttgattgtgc ttgtgggatt gtgtttcttc 120 gtggttgccc
atgttaagaa gagaaacacg ctgaatgctg gccaggatgc ctctgagaga 180
gaagagggac agatccagat tatggagcct gtccaggtca ctgtaggtga ctcggtaata
240 atatttccac cccctccacc accttacttt cctgaatctt cagcttctgc
ggtcgctgag 300 agtcctggaa ctaacagtct gcttccgaat gaaaaccccc
cttcatatta cagtattttc 360 aactatggga ccccaacttc agagggtgca
gcctctgaaa gagactgtga atctatatat 420 accatttctg ggacgaattc
atcttctgag gcctcacaca ctccacatct tccatctgaa 480 ttgcctccta
gatatgaaga aaaagaaaat gctgcagcta cattcttgcc tctatcttct 540
gagccttccc caccgtaaac tatggactct agttcagttt tatatgcaat ggatcactac
600 tccatcaatt tcttcaaaca aaaaaacaac agcaaaaaaa aaaaaaaaaa aaaaa
655 65 1450 DNA Homo sapiens 65 ggcacgagcg gaagtgcaac tcgaacttgg
tcggggcgcg gatcccgaga gggaaagtca 60 taacaaccgc acgagggagt
tcgactggcg aactggaagg ccacgcctcc tcccgcctgc 120 cccctcagcc
ctgtggctgg gggcagagct cagactgtct tctgaagatt gatgtctatt 180
tccttgagct ctttaatttt gttgccaatt tggataaaca tggcacaaat ccagcaggga
240 ggtccagatg aaaaagaaaa gactaccgca ctgaaagatt tattatctag
gatagatttg 300 gatgaactaa tgaaaaaaga tgaaccgcct cttgattttc
ctgataccct ggaaggattt 360 gaatatgctt ttaatgaaaa gggacagtta
agacacataa aaactgggga accatttgtt 420 tttaactacc gggaagattt
acacagatgg aaccagaaaa gatacgaggc tctaggagag 480 atcatcacga
agtatgtata tgagctcctg gaaaaggatt gtaatttgaa aaaagtatct 540
attccagtag atgccactga gagtgaacca aagagtttta tctttatgag tgaggatgct
600 ttgacaaatc cacagaaact gatggtttta attcatggta gtggtgttgt
cagggcaggg 660 cagtgggcta gaagacttat tataaatgaa gatctggaca
gtggcacaca gataccgttt 720 attaaaagag ctgtggctga aggatatgga
gtaatagtac taaatcccaa tgaaaactat 780 attgaagtag aaaagccgaa
gatacacgta cagtcatcat ctgatagttc agatgaacca 840 gcagaaaaac
gggaaagaaa agataaagtt tctaaagaaa caaagaagcg acgtgatttc 900
tatgagaagt atcgtaaccc ccaaagagaa aaagaaatga tgcaattgta tatcagagaa
960 aatggttctc ctgaagaaca tgcaatctat gtttgggatc atttcatagc
tcaggctgct 1020 gctgagaatg tgtttttcgt tgctcacagc tatggaggac
ttgcttttgt tgaactgcaa 1080 ctcatgatca aacaagctaa ttcagatgct
gggaagtgct ttcgcttagc tatgtggaag 1140 aaccattgac tgtatacaac
caacaagtgt atggtgcaac aggagatcca ttgaaaaccg 1200 tttataggac
tgaacgacaa ccccaaatgc aagtgaccat gagcaactac aaataggtat 1260
acatatgcat ttgagctgaa cagactttct gacatataat ttagtcaaaa ttgctgtatt
1320 tcttcccctt aaatttatac ataatcagct tcttgtatgg acccaaattg
gagaaatgta 1380 attcagtagt tggtgagaaa taaaggattg tgacctctgt
gtaattatca ggaaaaaaaa 1440 aaaaaaaaaa 1450 66 670 DNA Homo sapiens
66 ggcacgagag gcgctaaggg gaacaccccc ttccccaggt cttttatttg
tttaagttat 60 ttttgcacaa atgactcttt tatatttaat tcgatttcat
tgcctccctt cttaaagcca 120 acaggctcag tttacaaacc tgtgagctac
tgttggctgc tgccctcctt cccagtgaaa 180 ggtacaaagc aataagcatc
atgcatcctc cccttacccc tccaacaccc ctctgcctct 240 ggctcaggtt
gctcaaagca cagatcctct cttaccccgt ccccaggttt gaaacacata 300
gcctcatttc aaggtgtagc caggttcccc cgactttcct ctgggatata aaaaaggggg
360 taagggggca aagagagccc tctgggcctc tcctcccata cacactacac
tgccccttct 420 ccccccatca aaacgctcag agacgttgtg atgatgcgac
tgaggattat gcaacgtggt 480 ccaaccggag cggccagcat gaccagctgt
ccaggggctg cctcctgcct tttcttttgt 540 aaagacaaga cccttgggag
ttttaattct gttttgtact tgccctgtgg ggcctccact 600 gcttttctat
gggagacact cttaatttaa cagatgagaa tattttgaaa aaaaaaaaaa 660
aaaaaaaaaa 670 67 1692 DNA Homo sapiens 67 tgcagtccta gctactgggg
aggtggaggc tgcagtgagc cgagatcaca ccactgcact 60 acagcctggg
cgacagagag agactctccc aaaacaaaca aacaaaaccc aaaaataaag 120
aagtcatctt gaaagaagtt tcaacatttg ccttttcatt ctgagattac agttttctat
180 aaacatctaa gagtgaagag tctgacgttt tttggtcaca gctgagccac
tgcgtgaccc 240 ccgccccgcc ccacactcac tttgctctag gcaaagctgt
actctgaaag ctggccccaa 300 tggggaggtt aggactgtgc ctgctcagaa
gtctgtgggt gcctcagaga agggcaacaa 360 ccctaggctg gaccctagcc
ttgagagtac ttcctactgc cagagcccsc agatyycttc 420 cggtggcagc
agatactgcc agaagagcct gcggtgcaca caccagaatc cgggtacttg 480
gatgagaagg acacattact gatcaccttc ctccaggcaa ccctgtcagt taaggactac
540 agtcccgccc ccattatgta gatagggaaa cagaggcaaa gaagttagga
aactcgccca 600 gaactctcag ctcatgaata aaaaagcaga actaaaaccc
agtgctctcc ctggctgggc 660 aaacgtgtgg aagttgatgt gcctggttac
tgtttgtgct tcgcttatca taaccagtga 720 cagcgtggtt agcactgttc
gcctcaaggg cagctgtgag gattacttgg gattgtcctg 780 tggaaacact
tcacatgcat attaactagg agaaaagcca ctggagaatg agctttatga 840
gctctatcaa tcaccacagc tagtctgacc taggggtaag caaaatggaa gacaggaaaa
900 agggaataca tttgctyagg acagcgtgag ggccacgtga gctgcttgat
tggtagcgat 960 ttgtacaggg gctttatgga tcactaggtt ttaatttgca
aggcctgaaa ctgtccttag 1020 cattctctga aacccacagt gccagtcgcc
cttcacgcct cggccagcag aaagctcctc 1080 atgagtggat cctcttgaga
acttcagagg ggtcaggtga cggtgactga gactgcctca 1140 gtgatcacgc
tcggtgctat gagctgaaat ctgggccaag ggcacagtaa gttcaggcag 1200
ctagtatgtt taaaataact acttttcggg agctaagcca tgaggacgta aaggcattaa
1260 gaatgataca atggactttg gggactcagg ggaaagggtt ggggtgaggg
ataaaagggt 1320 ccagtgtaca ctgcttgggt gatgggtgcc ccaaaatcct
ggaaatcacc gctaaagaac 1380 ctcacgtaac caaacaccac ctgaacccca
aaaacctact gaaactttta aaaattaaaa 1440 atacatacat aaaatagcta
cttttactgc tgtcaacagc atgttcctga aaaatgttgg 1500 aattcaaact
ttctggaggg cagctggtca agaaacttat tcacgtcagg agttttctaa 1560
aatttgtttt taatgcttat tggtacttct gcattagaag taactacaaa tgtcttatta
1620 aagtttccac tttaaatgca aaaaaaaaaa aaaaaaatga ccctcgaggg
ggggcccggt 1680 acccaattcg cc 1692 68 655 DNA Homo sapiens 68
gatgtagagc agactgagct catccatcat gatttcttcg tgatattact gccaagcaga
60 ttataaggtg aagtcaatgt gacaaaagga aattcggcta aaagcttcct
gaagcctttt 120 gatgctaagc agtccttctt ttgatattta atacccatgg
acataaactt ctgccttaga 180 ggtcgccatg gagttttgtt ttgttttgtt
ttgttttgtt tttgccatct gttaacagtc 240 ctgagtaccc atagagcctt
ttactattta tcagcatyct agagtcgtca gtatggattg 300 tcaaaacttg
cattkgtctc ttttttgttc agtgttgtgt gcatccacat ttyctttctt 360
ttttaaacaa ccctgcttat gtaacatcca cattttctga cttacctttc aaacctgcca
420 gaaagcagaa gtgatattta awacacttgg tatgttttat atatwgattc
taatgataat 480 gtttrgtcta agatggacct gacaaggcca ggcatrgtgg
ttcaacagca ctttgagagg 540 ctgaggcagg atgattgcct gagcctggga
gttcaaggtt acagtgaact gtgatcacat 600 cctgccttct agcctgggtg
acagagcaag accctgtctc aaaaaaaaaa aaaaa 655 69 1618 DNA Homo sapiens
69 taacgcgcct gcaggtcgac actagtggat ccaaagaatt sggcacagta
aaaaaaaaag 60 aaaaaaaaag aatactgcct cacatcaaat ggtctatgtt
acttagtata tatgatcaag 120 taacatgcag tcatcatcaa aactgtatta
caatgtttag aagagtttcc tattgacaaa 180 ataaataaaa tgtttctgct
ttatgattaa ataaatccat cattgtttat gcatgattaa 240 gttgcaaaaa
gtttcagagg ttataaaggt tttaaagatg cttctatatc ctttggtttt 300
gcttttatct ttgaaattgg atacaaaagc cacaatcttt gctgtgttgg aagatgtata
360 ggaatagaaa catgaaaccc acaaacataa aggtttacct tgaagtggta
gactttttaa 420 aaatgagaac acttgaatta gaaatactga aagcttacca
aaagtttgtc aaaccgggaa 480 tcaagaccta ttgtgtcgct catccttgac
cccacatcta ctcactttcc aactcctatg 540 tagcaaatcc cctaaatacc
tctcaaattt attcacttgt ctccatacct acagccatca 600 atcactctcg
tcaaagtcaa tgctgtctat taactggttc ttaaaattgc tacattcttt 660
tctgtgcctc ggcttttact ccttactatc ctaaattcta tattcaggca gggtgattct
720 tgtattggag acaaagagag agcacataga ccaaggtgtt ttggaaacag
tcggccctcc 780 ctatctgcag gttccacatc tgcagctcta accaactgca
gatcaaaaat actgggaaga 840 agtatataaa aacaaaataa tacaaataag
aaacaacaca gtataacaat gatttacata 900 gcatttacat tgtattagat
ataagtactc tagaaatgat ttgaagtatt gtttgacact 960 tgaacaacat
gagggttagg gatgccaatc tcccccgcac acagtcaaaa atctgtgttt 1020
aacttttgag ttcccaaaaa cttacctatt atccaattgt tgacaggaag ccttactgat
1080 aatacagtca attaacacat attttgcacg tcatatatat tatatactgt
attcctacaa 1140 tgaagtaagc tagagaaaat gttaacaaaa ttataaagaa
taaaacacat attttatata 1200 cttttttaga gagagagttc tcactatctt
tgcaaggctg gactcgaatt tctgggctca 1260 agcaatcctt ctgtctctgc
ctcctgagta gctgggacta caggcacttg ctaccacacc 1320 cagctcctat
atttattatt tattaagtgg aagtggatca tcttcatcct tctcatcttc 1380
aggtggagta ggctgaggag gagcagggag aagagggttg ggtgttgctg tctcaggggt
1440 ggcagaggca gaagaaagta taagtgaacc catgcagttc aaacccatat
tgttcaagta 1500 tcagctgtaa acaggagggc gtgtataggt tatatgcaaa
tattaaacca ctttatatga 1560 gggacttggg catccatgaa ttttggcatt
tagaggttcc tggaaccaat ccctcgag 1618 70 1802 DNA Homo sapiens SITE
(1790) n equals a,t,g, or c SITE (1792) n equals a,t,g, or c SITE
(1801) n equals a,t,g, or c 70 gaattcggca cgagtctctc tcacttttga
aatgcttatt attttaatga caataatgca 60 gagagagaga gtatttttga
atagacttaa gttttccttc aactaatgtc tccttggagg 120 acagaaatac
aactaaaccc tctgtcaacg tgggtatgta tttttttact ttctattttt 180
caattagttc ttttatgttt tttcttctag tcattgttaa agctaccaat ggaccaagat
240 atgttgtggg ttgtcgtcga caggtaatac tttatatttg tatagtgcct
gatgattgac 300 aaagcagttt catgtaagtk attgtctcya attcttgagg
cwagcaggtg gagcatttat 360 gcccataact cacaaggatg atttgttcag
acatagctag ttattaacaa agcctgaatt 420 caamccatgg gctttgactc
ctggcattcc gtactttcta ctgtattaca ttgtctcagt 480 cagatctgtt
aatagccact tagaaataaa agtattttag aactggaaaa cagacatttt 540
attttaatgt catttttaaa gaggacttaa aagtgttaga tatcatcagt tacctgtgtt
600 tatatttaga cattcagaac tgttacttat ggactgtacc atggcctaag
ttaattttgt 660 atgaggtcat ttagattagg gtagggcaag ttgaaataat
tctaaatttt attttacagt 720 tatcaaagat gccaacaaat gacctcaagt
cattcagtag tgtctgaaat caatttatgt 780 attattcttt aggaagtgtc
cttagataat tcttttaaat tcattggaag agttttctct 840 gtttaattgt
catttcaggt tcaggtttta aaacattcac agaacatggc tgtaagggag 900
aatttaatcc aggaactata aatctcctat taggattttg cctagtatat aagcggttga
960 cattttctaa gtcaaaatat tagataccta aactgacaag ggattttcat
gtccctttca 1020 gggctctgtg gatgccgaaa gttggcattt ctaagatatt
tcaggttgca tgaggacaag 1080 actgtatttg aagactaaaa aacattagaa
aagccgaagt atatataagt tgagtatccc 1140 ttatccaaaa tgcttgagcc
agaaatgtgt tttagatttt ggcttttttt ttttcaggtt 1200 ttagaatatt
tgtgktgkac tggttgagca tycctaatta aaaaaaatca aaagtttgaa 1260
atgctccgat gagcattttc tttgagcatc atgtcagcat tcaaaaaatt tcacattgkg
1320 gagcattttg gattttcaga ttaagaatac tcagcctgka tttcctatag
atgtaaacat 1380 tgaaatagct tcatattgat ttctcctctt attttttcaa
gtaacctcac ttcttagccg 1440 ttttttcctt aattgttata ttaatcctag
tgttttgcct atcttcctaa atttgaagct 1500 ctttgtaaaa tcctgtgaca
agtggtcagt aatttatatg attccgaaat tgtattggca 1560 cgcagttttt
taaactatta aaaagtaact tgggtcgggc ggggtggctc atgcctgtaa 1620
tcccagcact ttgggaggct gaggtgggca gatcacgagg tcaggagatc aagaccagcc
1680 tgaccaacat ggtgaaaccc cgtctttact aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1740 aaaaaaaaaa aaactcgagg gggggcccgt acccaattcg
ccctatagtn antatagtga 1800 nt 1802 71 1292 DNA Homo sapiens 71
ggatgataga tgatctgtaa atattttctc tcattccata ttcctctggc tccttttaag
60 atttttcttt atgtctggtt tcaaggaatt tgattttgtt gtgccctggt
ggagtataag 120 ctttcttttg agttttttgc tcttgttgtt aagcttttgg
agtttgtggg tttatacttt 180 tcatcagatt tggaacatct ttggctatta
tttctccaaa tagtcacaca tcgctcctcg 240 gattccagtt acatatatat
tattaggttc ttgaagttgt cccatacctt actgatgctc 300 tgctcttttt
ctttggtctt atatttgggt ttcatttgga tagtttttat ttctgtgtct 360
ttacattcac tcgtctttcc ttctgctgtg tcttgactgc tgctagttcc atccaatgta
420 tttcatttat atatctataa tttgtggttt gatagaaatg cagtgatgta
gcaggtatca 480 ataaatactg ccttaatttg ttgcgaaaat ataacagatt
cctgttctgt atgttagcta 540 aaaaggtatg caaaccaccc tgtatgtcat
attaacattt atgtcccttt gtttccatgt 600 caacttttag tttctctgcc
aaaacctaca tatgtttttt ttatatgatt attctacatt 660 ttctgctgag
agtggacatc tgcattagta gttctatgat atttgtttta taagttgcca 720
gaatggttgc tctgtttggc agactgcaga caaatattta tctatgattc gttgcatgat
780 atgaccatga ttttgctaca aaaaacttga aatagatttt aatattttct
ttactattat 840 cagagagaga gctggattac ctgcaaaagt gtacttttgc
ttattgctgt cattgataac 900 tcagtgccag ctgggcgtgg tcactggtat
tacctccatg tgatcacttt ttgttcacta 960 atgttaattt aaaaaatttt
aggctgggcg caggtggctc acacctgtaa tcccagcact 1020 ttgggaggcc
gaggcagggg gatcatgagg tcaggagatc aagaccagcc tggccaacat 1080
ggtgaaaccc agtctctact gaaaatacaa aaattagcct ggcatggtgg taagcgcctg
1140 ttatgccagc tacttgggag gatgaggcag gagaatcgct tgaacctggg
aggtggaggt 1200 tgcagtgagc caagattgca ccattgcact ccagcctggg
caacaagagc aaaactctgt 1260 ctcaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 1292
72 883 DNA Homo sapiens SITE (8) n equals a,t,g, or c SITE (28) n
equals a,t,g, or c SITE (30) n equals a,t,g, or c SITE (47) n
equals a,t,g, or c SITE (873) n equals a,t,g, or c 72 aaaatagnaa
taactaaaag gcgaattnan ccctctagat gcatgcncga cacggccgcc 60
agtgtgatgg atatctgcag aattcggctt atcgtgaacc tggctttggt ggacctggga
120 ctggcactca ctctcccctt ttgggcagcc gagtcggcac tggactttca
ctggcccttc 180 ggaggtgccc tctgcaagat ggttctgacg gccactgtcc
tcaacgtcta tgccagcatc 240 ttcctcatca cagcgctgag cgttgctcgc
tactgggtgg tggccatggc tgcggggcca 300 ggcacccacc tctcactctt
ctgggcccga atagccaccc tggcagtgtg ggcggcagct 360 gccctggtga
cggtgcccac agctgtcttc ggggtggagg gtgaggtgtg tggtgtgcgc 420
ctttgcctgc tgcgtttccc cagcaggtct tggctggggg cctaccagct gcagagggtg
480 gtgctggctt tcatggtgcc cttgggcgtc atcaccacca gctacctgct
gctgctggcc 540 ttcctgcagc ggcggcaacg gcggcggcag gacagcaggg
tcgtggcccg ctctgtccgc 600 atcctggtgg cttccttctt cctctgctgg
tttcccaacc atgtggtcac tctctggggt 660 gtcctggtgc agtttgccct
ggtgccctgg atcagtactt tctatactct ccagccgtat 720 gtcttccctg
tcactacttg cttggcacac agcaatagct gtctcaaccc tattgcctat 780
gtcttaagcc gaattccagc acactggcgg ccgttactag tggatccgag ctcggtacca
840 agcttgatgc atagcttgag tattcatagt gcncctaaat agt 883 73 785 DNA
Homo sapiens SITE (716) n equals a,t,g, or c SITE (731) n equals
a,t,g, or c SITE (772) n equals a,t,g, or c 73 ctgcaggaat
tcggcacgag gttttatcat ccaggatatg gtcactctca gtggcatatt 60
ccatgtgcat ctgataagga tgtatgttct gctcttcctg ggtaaagtgt tataaattca
120 aattgttgat aatgttcagg tcatctatat ccttaatggt tttctccctg
attcttttat 180 taactactga gagaagaata ttggcatgtc cacctataat
tttgaattcg tctatttttc 240 tttcagatct gtctgttttg ccttaaacat
tccttatctt tcagaataat taaaagtaaa 300 aaaacattgt tacttgtttt
ttccatttct gatgttctcc attttgttgc atagatccaa 360 gtttctgagc
ttttaccctg tgaatcatag tcattttaaa tttcttgtca tatgtgagag 420
tttagttctg attactgctt tgtcttttca gattgtgttt tattgtgtat tttcacattc
480 cttgtaattt tttatgttaa aaaaattgtg tatgtgcmaa gctgaacata
ggacagaaga 540 cactgaagta aatgttttca tgcttggaaa tgagcaggcc
tttcctcctc ctctctttag 600 tcgtgggytt gtgcttgttt agttgagttg
ggtttgaagt ttgktcacct ttggctttgg 660 gtctcctaac ctgactttct
gtgtttcctg tgcactgctc ccaagataga aactgnttct 720 gggctatctt
ncagttggaa ttccttactt gattcttatc agcatgggtt angaagggaa 780 acatg
785 74 2341 DNA Homo sapiens SITE (161) n equals a,t,g, or c SITE
(163) n equals a,t,g, or c SITE (170) n equals a,t,g, or c SITE
(1229) n equals a,t,g, or c SITE (2243) n equals a,t,g, or c SITE
(2309) n equals a,t,g, or c 74 gcccagttcc tcttgaaaag gcagagaatt
tagacagaaa ttcaccaact gctttcttac 60 agaaagtaaa ccaatttctg
cttccagaaa aatggagtaa atgtattttg ccctattcct 120 tctactaaga
aaaactataa accctgaaca ttatatataa nanatatgan aactcagacc 180
tggagagacc aaggcagatg tggtagggac ttmataaatt gtatagtgat gaatcctcta
240 agttttcttt tctgctttat aatttgcaga cttttagctg aaaatgccat
caacatagaa 300 atactaacag gcacatatga gaatttccca acaaaagcct
attattttag gcaaaggtca 360 aggaaatagt ctaccaaggc agaaaacatt
tcgacaataa ccactctact gtagtcaagt 420 accacagaaa acactattac
ctcaagtgaa gagcttagat ctttagayct tcataccagc 480 caggctgtga
caaggtgtcc caaccctcct ccagaatagt atctcagaat agcagaagtt 540
ggaactttca tccccaactt gtggtaataa gcccctcact ctccttccac accttgatat
600 gactggagag caaatgggga gctggatcta ctctaaaagc agcaatgaag
aagcaccctc 660 ctttccatac caggtggtgc ttgtggaggc catgtgggaa
acagtaacaa gtcacttctt 720 cctccgagac aggctatcag tggaggccca
gtggtgaccc agaatccacc ctccagccag 780 cagtaatgag gaacctccgc
tgcctaggtg tcaacagaga ttgagaggaa acctttattt 840 ctatcatcac
ctggcagtaa tgcagtgtcc ctccctcact cccttgcctt gctggagtag 900
tgtctgagga agctagctaa gacagaaaag gtaaataagt tctagagtct cataatgcct
960 aaaatgtcct ggttcattta gaaatcattt ggtatacaaa gaaccaggaa
aaatctcaac 1020 ttgaatgtaa aaggtaatta gaagattcca gaacaaaaat
gacaaagatg ttggaattat 1080 tcagaaaata ttttaaagca gtcatcataa
aaatgcttcc agtatattgs ttacaacata 1140 tatgaamcaa atttaaaaat
tatctyagcc aaaaaattaa aatatwtgaa agaactgaat 1200 ggacatttta
gaactgaaac ttacaatanc cacataaaaa attcatgaag gtaagcagga 1260
aaaaactata aacacagcct cagggacctg tagtattata actgaaggcc taatttttgt
1320 gttatcagag tcccagaagg agagaagaaa tgggcaactt tgagaaaggt
ctcaaagact 1380 gaaaacttcc ttaatttggc aataggcaaa aacccacrga
ttcctwaatt cargcaamcc 1440 caaaatctct tagcactgta tcagaatacc
atagaatggg tggtttatwa aaacaaaaat 1500 gtgttgctca caatactgga
ggctggaaga ccgtgatcag aatgccagca cagatgagtt 1560 ctgctgaaga
cattttttgg ctatagatgg acatcatctc attgtatcct cacatgttgg 1620
agaaaagaaa aagatatctc ttgtctcctt ctccctctct ctctctcttt ttttttttat
1680 aaggcctctg atctcaacrt gagggcccca mmctcatrac ktartctaac
cctaattacc 1740 tcccaaaggc ctaacctcca aataacatca cattgaattt
aggatgtcta catatgaatt 1800 ttgaggggac acaaactttc agtgcataaa
actaaccaag acaaacacaa agaatccaaa 1860 ctaaggtata ccatggtaaa
atatctgaaa attaaaagaa agaacaaatt ttgaaagcag 1920
ctagaggaaa tagctcatct ataggagaga aaacaataca aatggaagca ggaaacatca
1980 gaaatagatg aaagccatag aaaagtggca caacactgtc tatgtgatga
aataaaataa 2040 ctttcaattc tggtttttat atctggtata tttgtctttt
aggaatggaa gggctataaa 2100 gacatttgat gaaagaaagc tgagaggatt
tgtcaccaga aggtctrcct tttaaarrgg 2160 ggctcaagar rrttctctat
ccaggaaaaa aaaaagaaaa agtttaaaaa agaaacttta 2220 aaacaccaga
tttaaagaaa acncagtgga aagggaaaaa tgagtggctt catcttcctt 2280
ttcctcttca gtttggtaga tttatttgnc cagctgaagt taaaattatg ccattatcag
2340 a 2341 75 1882 DNA Homo sapiens SITE (755) n equals a,t,g, or
c SITE (1237) n equals a,t,g, or c SITE (1866) n equals a,t,g, or c
75 gcaagttttg tgtttggccc tcaataaact agtctctctg tacccctggc
agggggtggg 60 aggagtcctg ggggagctcc cttccaaatc ttacagggtg
gtctgtttct tctttggata 120 ataatgatgt aatggctagt ctcttgagaa
cttgctgtgt tccatacatt gtactaagca 180 tttatttgga ttatctcatt
aaatcttcac aatcacttta tttaacagat ggagaaatta 240 aggcacatgg
aacctaagtt gttcaaggtc atggagccag taagtgttag agccaagtcg 300
tttggctcca gagcctgtgt tcttaactac tactttgtag tgtctttctt acatattagt
360 tgggcctgtg tattgctagt tgaattcctc ttcccagtgg caggccttca
cgtgtttgac 420 catggttttc atgttctcca aacctcagtt ctctagattt
gtactttggt aggtcatcat 480 tttccacaga tcctacctct ttaggtcaga
aaatcttgcc agtttataaa gattctctgg 540 gactaactcc cacaaagcaa
ggtcacaaga gatcaatgta caaatgaagc agttcagtga 600 gtttgtctac
cattctccat aagtacatgg grgacamctg atgattggaa ggtttggttc 660
acctcatggg agctgtgata tctcactcac cacacagatc tgctcttctg agggaccatc
720 ttgccaattt ccagagagtt gcagggatat taaanttttg cacattaagc
ttcctctttc 780 caagctgsac atgggscctg ctaccgkttg tgaamagtct
tctagagtga tawaggttct 840 agctttctta gttaagatcg tattttctga
taccactccc ttgtcacttt gcctgaaatg 900 agaaactccc aacctcaact
gcttttctag tctcttccaa tgaatgcctt ccaaagggct 960 ggtgtcctcc
agggtgtatt agttgttact aatttcatcc tccaaggctg atctgatttt 1020
caagatctgt agagagacct tagtatattg ccttgcctgt accaaatmca gtcattatgg
1080 cmcaggaaaa tctcaaatmc cttattggaa acccaggcaa atatttattt
gaccttaatg 1140 aaatgaaaaa gacattggat gcatacattt aaagaaaacc
caaaactttg gaatctttac 1200 caaggagggt atcttttgaa aaggacagkc
tggaacnaag aacttgataa aatagaagta 1260 aaggttgaca cttttttttt
ttttttttga gatctatatc actctgtcgc ccgggctgga 1320 gtgtagtggc
gtgatcttgg ctcactgaaa cctcggcctc ctgggtacag gtgattctca 1380
tgcctcagcc tcctgagtag ctggcactat gggcatgtgc caccatgccc agctaatttt
1440 kgtgtttttg gtggagacag ggttttaccg tgttggctag ctggtcctga
cctcctggcc 1500 tcaagtgatc cacccgactt ggcctcccaa agtgaaagtc
ggcattacta gccctgttca 1560 gcacatgaga cagggcactg gatggtgtct
acctaatgat tttcaaccca ggggcccttg 1620 gcccaagcgt atcactggta
taaagggcct ctgccagcta atgtgagggt gagtgtggct 1680 gttgtttcca
tgagagaact cctgggagtt ctacactcag caaacgtttg ttgttggact 1740
atgaaggcgg acacagattt tatacgaatt tgtaatgcta acatctagca taagaattgg
1800 caaccataga aaatactacg tgtatatata tgtttatagt ctcaaaaaaa
aaaaaaaaaa 1860 aaaaanaaaa aagggcggcc gc 1882 76 2892 DNA Homo
sapiens SITE (858) n equals a,t,g, or c 76 agactctgag tccagctccg
aagaggaaga ggaattcggt gtggttggaa atcgctctcg 60 ctttgccaag
ggagactatt tacgatgctg caagatctgt tatccgctct gtggttttgt 120
catccttgct gcctgtgttg tggcctgtgt tggcttggtg tggatgcagg ttgctctcaa
180 ggaggatctg gatgccctca aggaaaaatt tcgaacaatg gaatctaatc
agaaaagctc 240 attccaagaa atccccaaac ttaatgaaga actactcagc
aagcaaaaac aacttgagaa 300 gattgaatct ggagagatgg gtttgaacaa
agtctggata aacatcacag aaatgaataa 360 gcagatttct ctgttgactt
ctgcagtgaa ccacctcaaa gccaatgtta agtcagctgc 420 agacttgatt
agcctgccta ccactgtaga gggacttcag aagagtgtag cttccattgg 480
cmatacttta aacagcgtcc atcttgctgt ggaagcacta cagaaaactg tggatgaaca
540 caagaaaacg atggaattac tgcagagtga tatgaatcag cacttcttga
aggagactcc 600 tggaagcaac cagatcattc cgtcaccttc agccacatca
gaacttgaca ataaaaccca 660 cagtgagaat ttgaaacaga tgggtgatag
atctgccact ctgaaaagac agtctttgga 720 tcaagtcacc aacagaacag
atacagtaaa aatccaaagc ataaagaaag aaggatagtt 780 ccaaattctc
caggtatccc aagcttaaga gagraactcc agcttgatcc agtgctctta 840
cmaacmaacc tgrgagcnac mggcctccag agaccgccga tgargagcaa gtagagagtt
900 cacatcaaag ccatcagcat tgccaaaatt ttcacagttt cttggagacc
cagttgagaa 960 agctgcccaa ctaagaccta tctccctacc aggagtttct
agcactgaag atcttcagga 1020 tttattccgc aagactggcc aggacgtgga
tgggaagctg acctaccagg aaatctggac 1080 ctccctaggt tctgctatgc
cagaaccaga gagcttgaga gcatttgatt ccgatggaga 1140 tggaagatac
tcattcctgg agctaagggt agctttaggt atctagcttc atcaggcata 1200
ttttagaaat ggactgccta atatctattt acctaacaac aaaacaaccc ttacttaccc
1260 atcagtcctc tagtcctcca aactactgta gcagatactt tgccaccttt
taacttgttt 1320 gaagaagcta tataaaagtt atttttttaa agaagaagac
cattttactt atgatgttca 1380 gaaatctatg atttcctaca accagtaaga
tcttacattt taaaattgcc agaaaaaaaa 1440 ttaaagccct ctttttttct
ctttcctttt tttgagggga ggagacctta tcttttaaag 1500 ctgggaaatg
tatatagaga gagaataagc cacttttata tttcacttaa atttgcctta 1560
aattagctgc actttataga gactcagaaa atgtcttttc tttaaaagat aggccttttc
1620 tgtttgtaaa tatttaaatg aaagaaagca ttgtgcatat tgtgtggaaa
gtaggaagaa 1680 tggttttgaa caggatatga acaaatgact tattaaaaat
tgctgatctg gtgtaggtgg 1740 cagctgaaac tacatccatg tctccataag
gyatccctca aaggcccagg cgctgccagg 1800 gggtttgtcc tggtagctgg
aggaaccgat ttcagggagt agacactgga gacaatactg 1860 actccaggca
tggctcatgg aagtaggatt ctggttcttt gttcctattc cctcagctaa 1920
tcccaacctg ggaatcagag aagtcttggg gatttttctc atttttagta ctatttcagg
1980 gtttatgagc ataaaaagtt atccattggg gagctccatt ttccctgctg
agtgagctag 2040 attgccttcc ccacccaccc acttaagtct gtcttaaagc
cgtagctggc tcccaccacc 2100 agtaccatct ccatttgaat ggcagggcta
aattccccca gccattatct cacactgacc 2160 acccagagct ttagaagaga
gctgtgcttc taattttgac ccagaaaacc ataccccttg 2220 agattttacc
tagaggctaa ccaagagcct aatatgtttc tctgggggat gactaaagcc 2280
aaaaaggctg tgagatgaaa catgtgaaat aatattcagt ttccttacca ttaccagctc
2340 agaagtagct agaggctttc tacccaaagg atgccaaagt atagcagggc
aggcctggag 2400 ctagggcctt cacatggtgg tagcaagttt ttcaaatcta
atacaatcaa gtacaatact 2460 tcctttaaat gcttctgtgg acctggcatg
aaagatccct agattgaaag gaataatacc 2520 tccatgtctc ctgtatgttg
agtctagaat tgctgtgttg ttcttagaag cagtctttgg 2580 gcaacaactt
gaaaggggaa aaaaaaacta caaaaactta actttggtat aggccaagtc 2640
agggagaaag tagagaaagc tgtcatgcca cagacttctt tagtggagat catttccttt
2700 ttaactttgt tcaggttgcc cttcaccatg gatacagtcc ggtaccctta
aacatttaag 2760 ggctgttttt tttttcttta catgatgttc agcttggtat
taaccaaact taaatttttt 2820 ttccagaagt attaaaattt agttaaagca
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2880 aagggcggcc gc 2892 77 1673
DNA Homo sapiens 77 cggcacgagc tggaaatgaa atttgcccct gtttatatgt
acctgtcttt tatttgcctc 60 tgtctttttt attgcaactc aatagacaca
caccattgct ttgtctctga ttatttggca 120 tttgaatcgt caatgaggga
agcttttaca gaacttttga tactaataaa aggtgagtca 180 aatgttttaa
aaaagatgca gaatcatcat ttatgtcaga gctactgact cacacttaaa 240
ttgcagtgtt agcactgaaa aagaaatgta tatggatggg aatatagatt gcaggccaat
300 taggacccct cttttgaagt tggaattgag ggatagctac tgttctcttc
tatctttgag 360 ggttaggaga actttattca gtgttgaata actgtattcc
tcctgtttat taatgtttgt 420 tgtgggggtc ttctattcag cacccatctc
tgcctgtcct gctcccccgc ccccagagga 480 ggatataata agaggcatgg
gacaggggct tataataata agacatggga ggggttgatt 540 acccagtgtc
ttcaagtaac ttttacgaga gatttgaaat agccagcgat caatgcaaaa 600
tagcaatggc cttggcagaa tttgcacata catactcaat gtttacagtt taaactctgg
660 tgtcagacag ggtcatagtt accccgattg gatgcatccc atctctggtg
cagaacctct 720 aaaacttggg aaatcattga aagtcatctg cttattaaaa
aagcagattc tcagactcac 780 atcagactag gagaagtcct gagaaatcta
aatttttagc acatgctttg ggggattctt 840 tacatcacgt gtgtttggga
aactgtgctg attgatgtcc atggaaagca gcctcaggca 900 tggggagggg
ctggaaaaga attatttagg tcagtttcgg gatcttagat tgtttcttgg 960
ctacactggc cactttttaa agtgtgctta gaaagagtat gacacctttt taattttcaa
1020 aaggacttgg gttcagtgta tgtccttatg ttaaagaaac agccctcttt
gtagttactc 1080 tagaaatagg tagaatggca gaaagagcgc tggctgtctg
tgtttgaggc ctgttttgta 1140 cttcatgtgg ccatgtggta tgggaacatc
ctgggatttc tgtgagcctc tgtgaactca 1200 gattccccat ctggaaaaca
ggagtaacaa cactggttgg aacctttatg gagtgtaaat 1260 aaagtgatag
ctctttgtaa gcgacgaaga gccaggtcag tgtttaattt tattttctca 1320
gaaatagtac tagttattaa ggcctttaac aaaaaaaaat ctttgaaaag gctaatgggg
1380 gcctggtata gtgtgtcatg cctgtaagcc cagcattttg gcaggctaaa
ggggggagga 1440 tcacttgagg ccaggagttt gagagcagcg tgggtaacat
ggtgacatcc tgtctgtaca 1500 aaaaataaaa acattagctg aatgtggtgg
catgcgccta tagtcccagc tactcggaag 1560 ctgaggtggg aagattgttt
gagcccagga gggtgaggga agctataatt atgccactgt 1620 actccagcct
gggcgacaga gtgagatcct gtcttaaaaa aaaaaaaaaa aaa 1673 78 1461 DNA
Homo sapiens 78 ccacgcgtcc ggagagttat ggagaatgct gattttgatt
attatgatgc cagatactga 60 gaatatctta catgtatctt ctgagcagag
cttctgttcc acaaagttaa atccatgctt 120 aatataattt ttgccaagta
aattttagtc gattgcacct cagttgttga ttagtaaccc 180 atcggcagta
gaaagatggc agtgtttttt ccaggctgtt tgttcctcta agtatctaga 240
cgaggccgag tcagccttat gggtctaaag ctgccaattt tcctgtggtt tctttatttc
300 tttatccctt tatccagctg ctacttactg ctattgccac atttgccctc
tggctcatgg 360 gatagcatgc ttagcttccc ctgaggctac tgttaatgct
tcctttttac tctgctggct 420 ggaaatgtac ttggcatcct tagtcttaaa
cctctcctcc ctcttttttc cacagacacc 480 aggcacttaa gtagcacttt
cagcctgcac cagttatcag tagtagcttt caacccctca 540 tttctggtct
ggtaactcag cacactgtcc caagagagct tgactaagcc aatttgcccc 600
ctcttccctt cttcctctgt ctgttcatct ttcttttttc tttttcctac ccatccattt
660 ccttgactct ccttttattt ttctcttact ctctttaatc tcccaaatga
tttttttctg 720 cttttagtat agcagatgcc ccagaattag gcagatactt
gtaatacaaa ataaaacaat 780 agtaaatttt aaaattaaac atttgctcaa
gattggatca actaaaaaac gagtttattt 840 tttatgactg gtctattcgc
ccctttatgg ctataatgca gattttttgt attaaaagtg 900 tataggtttg
tgtttttgtt ttttttgtgc ttttacataa agagttgtga agatcgtttt 960
tatgcaggcc tgctcattca agatgatctg tgatgtggga aaaaagtaaa atctttttct
1020 agctaatgtt ttacaaggaa aaggaaagct acttttattt ttatttattt
atttttttac 1080 atacaatgat tcgaatacac agtttgagtt atttttcaaa
ctaactttct ctgaatatgc 1140 tataaatgtt ggctgttcat ttttcaagta
atggtttgta aacaactttt aggcattctt 1200 agctaactaa tatttatgac
caatagttta ggacataaag attataccta tgaattgggg 1260 gatcaagaac
agtaacagtg ctctgcaggc ctcgatcatt aactgccaac aaaatctaca 1320
ggacaattcc aaatgtctgc aaaagaaaaa catgaaaaat tcatactgat aattatagat
1380 cagaatcatt taaagccctt atctccttcc tcctctcatt tccctaatct
taattctttc 1440 ctctggaaaa aaaaaaaaaa a 1461 79 1517 DNA Homo
sapiens SITE (1145) n equals a,t,g, or c 79 aggagaaact ctaaaaactg
cagatattat ttcatgctat atgttccatc ctctgatgag 60 aatgtgagga
aagaaaattg tatcctgcat ggctgaaaat ggtcccctac aaaaatatca 120
tgttggacaa ctaatctgag atagtggtat ctctggaaag cagtttagca ctggtgagtt
180 tggactttca tggcaggctg ccttggttca tatcttttgg taatgatact
tatcctctgt 240 raggcccatt tctttatttg tggaaatgaa gacaatagag
tgcttagata taatttagaa 300 caatgtccgt cacatagtaa acacgtaata
aacggtagct cttattgtta ttattattac 360 tattattacc ttgaagacag
gggctctgtc ttgttcatca ttccatctcc agctcttagc 420 acagtccctg
gcacaattca aacatgtatt tggatgaatg acaaatagct actgaatatt 480
tgccctgttc caagcattgt tagaggtaca tgggacaggg cagtgaacaa aacagacaaa
540 acctcctgct gtctcagagt tcacactcta atggggagac ccaggcaatg
aggaaataat 600 taaaatatac aatgtgtctt atggcaataa atgacaaaga
aaaataaagc agaggtgaga 660 aacagtggca gtgttttggt gatcatttgc
tttgcaacaa gccactcccc aaagttagtg 720 gcctaaaaca atttaatcac
agttcatgtt ctggctacaa caatacacat ccctctcatg 780 tgcaaaatac
actcactcct ccctcagagc ctcgtaccat taagggttca ggttcaaagc 840
ttaagatctt atcctctgaa gtaggtttag ggacaaacaa gtcttctcag gtacttcttc
900 tggggacaca gagacttgtg aactaaaaga caagttacct accttccaac
acaactgaca 960 tgcaatgggg atataggaaa agataatttc aataggcgct
tctgtgcaaa agcgggggaa 1020 atgagagtca ctcagcagtc acggttcata
ttaatctaaa atctagccag gcatatatcc 1080 caagtcttcc tgatgtgagg
acaagaatta tttcttgatt agggctcact twwtctcttt 1140 gaggntggtt
cgcctcagct tttggatttg tcctctgaat catccttcct tgtctataaa 1200
atgcatgtat atactcatac atacatagag agaaagagag agagagagag agagagactc
1260 tgtcacgcag gctggagtgc aatggtgtga tctcagctca ctgcaaccta
caactcctgg 1320 gttcaagcaa ttctcctgtc tcagcctccc gagcacctgt
agtccctgct actcaggagg 1380 ctgaggcagg agaattgctt gaatccgaga
ggcagaggtt gtcagtgagc agagattaca 1440 ccactgcact ccagcttggg
tgacagagca aggcttcatc tcaaaaaaag acaaaaaaaa 1500 aaaaaaaaaa ctcgtag
1517 80 574 DNA Homo sapiens 80 tagtagagcg cgtgtataga ggcagagagg
agtgaagtcc acagttcctc tcctccaaga 60 gcctgccgac catgcccgcg
ggcgtgccca tgtccaccta cctgaaaatg ttcgcagcca 120 gtctcctggc
catgtgcgca ggggcagaag tggtgcacag gtactaccga ccggacctga 180
caatacctga aattccacca aagcgtggag aactcaaaac ggagcttttg ggactgaaag
240 aaagaaaaca caaacctcaa gtttctcaac aggaggaact taaataacta
tgccaagaat 300 tctgtgaaca atataagtct taaatatgta tttcttaatt
tattgcatca aactacttgt 360 ccttaagcac ttagtctaat gctaactgca
agaggaggtg ctcagtggat gtttagccga 420 tacgttgaaa tttaattacg
gtttgattga tatttcttga aaactgccaa agcacatatc 480 atcaaaccat
ttcatgaata tggtttggaa gatgtttagt cttgaatata acgcgaaata 540
gaatatttgt aagtctacta taaaaaaaaa aaaa 574 81 1455 DNA Homo sapiens
SITE (390) n equals a,t,g, or c SITE (456) n equals a,t,g, or c
SITE (1100) n equals a,t,g, or c SITE (1293) n equals a,t,g, or c
SITE (1409) n equals a,t,g, or c 81 ggtccaccct cccccagggg
cctccccagc ctccctctcc acctccctgc cccccggaga 60 tacctccaaa
gccggtacgc ctgttcccag agttcggtga gtgctgcagc caggagatgg 120
ggctctgggt ggatggcctg ggatccctgg aatcaggcct ctggaaggta tgcaaggatc
180 acactccttt ctgtgcaagc ctgccaccag cccactgtgt ggccccgggc
aggtcacagc 240 ctccctgagc gctattctct tcatcctcac aatggagaca
gcacccacct ctctggcctc 300 ctgaccgtta agtgtggggc catggccggc
tttgccagtt acccatggtc tgattttcca 360 tggtgttggg tggtttgctt
ttctttttkn tttttttttt tgagacagag cgagagtctg 420 tctcaaaaaa
aaagacaagt tgcagatgag ctgagntttg ggcagagcaa gcgggattct 480
gatggggggt ggatgttgcg ctcgtcagca ggcaatagtt agttggttga gggttttgat
540 camggggtag ctactgcctg ccccatttta tccagctctg tagttgctat
agagttgcta 600 gaaccttggc acatcactta tcagttttgt cacctcagat
ggcttcttca ctacttgggg 660 tgtctcctgg gtgtggggct ctccttcctg
tggcctctgc tgactgcctg gcactggcac 720 acatgctctg gtgaggggag
gaccaacggt ttttcccgtt tgttttctgc ttcctcgttt 780 aaccctcctc
gtcttgtaag atgaatgtwc ttgtctctgt tcactatgca gatgaggact 840
ttgaggctca gagacgccac taacttgcct ggtccaagcc ttttgggcct ctcaggctgc
900 agccagcaat gctgcagtga agtttgcctg ggaggctgac cctaggagtc
tgcaggcgtg 960 ttaggacccc cgatctagaa gacagcagag atgtaggcca
gggaggacca ataccgagca 1020 tctgagggca ggcacacctc agactgacca
gaatacaaat gaattcgagt cacttacaaa 1080 caaagtggca taaggccagn
cacagtggcc catgcctata atcccagcac tttcggaggc 1140 cgaggtggga
ggattgcttg aggccaacga tgtgagacca gcctgggcaa catagcaaga 1200
ccttgtctct acaaaaataa aaattcaaaa aagtggcatt taacacatac tttttttctt
1260 ttttttgaga cagarttttg ctctgtcccc cangctggag tgcaatggtg
tgatctcggc 1320 tcactgcaac ctccacctcc caggagaact gcttgaacct
gggaggcggt tgcagtgagc 1380 caagatcgca ccacttcact ccagcctgna
caacggagca agactccatc taaaaaaaaa 1440 aaaaaaaaaa ctcga 1455 82 1640
DNA Homo sapiens SITE (687) n equals a,t,g, or c SITE (764) n
equals a,t,g, or c 82 gtgagcactg gtttaagcac ctcatagact ggcatttctg
cctcccacaa gataactgga 60 cctggcttag gaatctgaat agcagcatgc
atggagtgct tctatgtgtc aagcactgtt 120 ttaagcacgt ttgaaataac
tcacttcatt tgaaataact gagtctacat gatgactgta 180 aaaggttggt
tctgttgtta cctgcatttt accgatgagg aaactgaagc cttcagaagt 240
gcagtcactt gtccagggcc acatagcagg ctgagatttg aaccgccagg ccttttgact
300 ccagagctta cactcttaac tccattcatc tgctaagtcc ttccctgtcc
tcttgcaaga 360 tgccttaatc cagggattat caaacttttt cttaaaatca
ggagaactca ttgcaaacca 420 attcatacct agattccaca gaatcaaaga
tgcagccgag ttacccattg agctggagtg 480 ggggcgtara attgccctgt
ttggcctcct tsctgacatt gctgttccta ctgcagcctc 540 tgatgcttcc
ccttggaggc tcccagaccc agttgggcaa ccacagtgtt gtccgtctgc 600
ttctcccagt tcagaggctc ggctttgccg aagtccctcc actcgaagtg gcacagagtt
660 gaggtctctt ccaggcacac tggcgtnccc tcactgggct cctgtccctg
ccttggtcaa 720 catcctggtg cgcactgggt gggtgactaa caacattttt
gganttgtgg ctggagccca 780 ggtgactact ccaaatcacg gttttccatt
ctgtgtgaga tggcctcatg cctttctatg 840 cctctgacag gcagttctct
gaatttcgaa ggctcttgtc ttaagagact gtcagaagtc 900 cctttggcaa
gggactgtgg gcaaaccgcc cagcggctgt ggtcaattcc tctctctgat 960
ggcagtagtg ctacctaggg ggccgcctgg gtgaaacggg cttttttgca tacttccaaa
1020 ctggttccct gtagctaggg gaccaaacaa ttattgtctg aaccaagatg
ctcctgagag 1080 tgaagagaat gtaaagtgct cagtcctgga cagatggtat
atatgatcgc cgtaaataca 1140 gccagccctt gccagaagtg ggtctggaga
aatggtgcgg gggggcgtga aaagggctta 1200 caacccgcag tcctgtgtct
ctgctaggtg aattggtagc atcagtcctc actctgctta 1260 ttcagaccaa
aaaattgtta agttcttccc accaccacgg agcacagact tgattaagat 1320
ccagaaaggt cagccgggtg cagtgacttg cgcctgtaat cccagcactt tgggaggccg
1380 aggcgggtgg ctcacttgag gtcaggagtt tgagaccagc ctggccaacc
tggtaaaacc 1440 ctgtctctac taaaaataca aaaattakcc asgcatggtg
gcccatgcca taatcccagc 1500 tactggcggg gctgaggcag gagaattgct
tgaacccggg aggcgaaggt tgcagtgagc 1560 tgagatcgtg ccatgcactc
cagcctgggg gacagagtga gactctgtct caaaaaaaaa 1620 aaaaaaaaaa
aaaaactcga 1640 83 525 DNA Homo sapiens 83 ggcacgagga gaactgatgg
gggtggagag aagctccttg tgggaggaga gggaactacc 60 agcagagccc
ctcctaccgc agacacagga tcggagacaa cctccaaccc cacctgcctc 120
ctgaagtgct gctgacatgc aactgcctta actttgccta cctggcctcc ttatgatccc
180 cctccggcgt ggtatggttg gggggcttct tttgctgctg gccacggcaa
acaagctgct 240 tgctgcttcc ttcagagacc tcatggatgt tcttacatgc
ccccgacccc ggtagatggc 300 tccctgttgt ttggggagcc tggaaggtgg
ttatgccttt tggatgcagg agaggagcaa 360 gaaagagtgg agagggagaa
tgggggagcc ggaccctgac ctccctgggt tctggttgga 420 gatgaaaaaa
ttagaagcat caggtctaag atcagcttct cttggaagca gagcctgaga 480
caagatataa atgccagtca tttattaaaa aaaaaaaaaa aaaaa 525 84 837 DNA
Homo sapiens SITE (717) n equals a,t,g, or c 84 cactatagaa
ggtacgcctg caggtaccgg
tccggaattc ccgggtcgac ccacgcgtcc 60 gggtgggaga tgattggctc
atggcggctg acgtccccct tgctggtctc gtgatagtga 120 gtgagcgctc
atgggatctg gttgtttaga agcatgcagc acctcctgct tcactctctc 180
tgtctctcct gctccaccat ggccagaaac gtgcctgctt ccccttcgcc ttctgccgtg
240 attgtcagtt tcytgaggsc tccccagcca tgcttcctgt acagcctgca
raactgtgag 300 tcaattaaac ctcttttctt cataaattcc ccagtttcca
gtagttcttt atagcagtgt 360 gaaaacagac taatggaccc ttctggttga
aggaatgyag ccattctgct tgtttrasta 420 tktcctttct attcatctct
atttccyggg aggtgtttat ccaagtgcaa taggagrtat 480 tggtgacygc
asagtcccct cagtgttctg ctagtaaata gttgaaggtt gatcaktgat 540
ctycwgcrtt ttcagtctgg catggaaaag ccccyrtgya actggtaaag rtatcartaa
600 gcaccaggag gtatctaaat ccaccaggag ccataggcat cacgttgacg
tccatttacc 660 agtcttccct ggcaagattc ttctgaattg tgctgccttg
gccaaaagag gtatggnagg 720 ggctgggcrc agtggctyry gcctgtratc
ccagcaggag ttcgagacca ggcaggagaa 780 tcactagcag agaatatgtc
tccccaaccc ctctcaaaaa aaaaaaaggg cggccgc 837 85 1574 DNA Homo
sapiens SITE (19) n equals a,t,g, or c SITE (873) n equals a,t,g,
or c 85 gtgatctttg taatatctnc tgttgtttct atgatatagg agctagggga
agggggttgt 60 ttgccttctt caggacctga ctggacagat ggacctggct
caagcaacta ctctggatgc 120 actttgctgt gtgggatgaa ctaaaagtgt
ctgaattttg ctgataactt tataaaactc 180 actatggcat gcttccctcc
tggtgggccc taggatggat gacactcaag atactacaga 240 tgtgggtgca
ggcatgcaca cacacgatgg aatatggcca ttcctacaca ggtggggtag 300
agagtgggtc agcagcctgg cacctcacag aggtgggacc taagaggact catgattatg
360 cagagaattg gattgggtct ctgtcataga ttgagtaatc tcttccctta
cctcaattcc 420 atctccaccc atctctacat ctgggcacag caacccagag
atggccaaaa gcattcaagc 480 ctgggggaag atgtttgact attgctgctc
ttcaccagaa cctcacacct ctcctgggac 540 tggaaccctt cagtgggtgt
gtggccagtt ttggaggctg gaatgatggg ccagggtgta 600 ggattcattc
tccatgtaaa gtttcctttc atcctgccta gccatcccca aggtttattt 660
ccagaagaaa ggaatatctc tacttggatc aattctggtc atttcaagag gatggaggcc
720 tcaagtgtgg gaacttcccc tactccctgg atgtgtgtac ctagcacact
tccttctccc 780 accccttttt ccagttggat ttgtttttct gttctcttct
gtcctgtctt atactgcaac 840 tgtgtctcct aggggacaga tggccttctt
tgncatcttc actctccacc cccagagagg 900 agtcagagcc ataactcaat
cactcagccc ctccaaagat agttgatgtg tgataatctc 960 ataatgttga
gaaccctgat gagatacatt gtcttcctct ccctacaatg cctctggggc 1020
caaggcaccc attcttcttg ctatcctcca tcccccttga ggcttccact tttttttttt
1080 ttagacataa agctgggcat cagcaactgg cctgtggtga tgcaaagctg
ctttgctctg 1140 tatctggctg gactgatctg tctcacaaga agccatgagg
ccatagggag aagctccctc 1200 tccccttcat cttctgctcc aaaggtggta
gcaagaggag tacccagtta ggggttggag 1260 cccccatata acatcttcct
gtcagaagac tgatggatct ttttcattcc aaccatctcc 1320 ctttcccccg
atgaatgcaa taaaactctg tgacaccagc aaccattgct ctttagaaat 1380
gggttttctg atcatatggc tgatgtgtta tgggcagtat ggatgtcttc atttgttgct
1440 tctgtttttc atcttttttg ttttattaat aaaaatttat gtatttgctc
ctgttactat 1500 aataatacag ggaataaatt attcaatcca aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1560 aaaaaaaact cgag 1574 86 1628 DNA Homo
sapiens 86 tccctctctc ctgcctgtgc tcaaacaccc acagagaact ctggaaggag
aagaaaaaca 60 tacctctctc cttccccggc ctttccccac acacactgag
gttgagaagc tgggaataaa 120 cagcgtccaa cacttttaaa tggcggtggc
agctccagac aagggagaag ggaaggactg 180 agagaagaaa ttattagact
ttttagactg gctgaaccca gaacctttac attggttaca 240 aaatcatgcc
tggaggtgaa aaattcaata gcattatacc aagtgtttgg gcaacggtgt 300
agagacaata gtagagacaa gctacagctt tagacagtgg tagagagaag ctacaacttt
360 agcttgtaga aacagctgtt gggtgttcag cagatgcagt ctaggtgcat
gcaaacccac 420 tgtgtgcagc atgccttcac tctcacgaca agggagccag
accttgtgtc tgcggctggc 480 tgaatattgc atggaatctg tggattcaca
gaggcttctt ctcagctaag agggagtgtg 540 gctggatgca tttctctgtg
gctcattcaa tttggggtat actcatacta ctgagtctct 600 atgaaggagt
gataagctgg gtcttcaatt tccaaatgtt taccaaactc ctactatgtg 660
ccaagcacta ttcccactgc tttgagagct aacggtaaac aacaacagyg aaaaaacatg
720 taacatccca accagtgctt ctaaggattt aaaatatgct ggtacttatg
actttattct 780 tacttctgta ttatagatat gtatatggct ttggggtatg
tgtatatgta cacatatatg 840 cacacatata cacacacaca catatatata
atcagctgtc catagcctac tcatctctga 900 taatttatat tttgtactca
aatttctcga atacccctac caaatcattc tctcctcctt 960 accaatatta
taatgtccct gacaataaca taacaaaccc agctctaaca cctacagatt 1020
tctttgagaa caaacaactt ctacatgcaa tttcttttct atactcacca actggttttc
1080 ttcaacctcc tgcccaccct gtccagctca ggacatcaac aaccctttat
ggaaaccacc 1140 gaggtcagac tggatgcagt cagttggact gattcatcat
gactagttca attaagagct 1200 gatcaccttc aaacagctct gactttggaa
gcaattgatt tgactgcctc tttggtcaca 1260 tggccagatt tacccataat
tttttgcaaa cttggatgca tctttagata cagagcaatg 1320 ctttggcatc
tgggggaggg ggttgttcct ggtgctgtca cttgtaccca ctctcctgtt 1380
tcctctccaa ctatgagtca cattttccct caatatctcc tatcttactt tttgagtgat
1440 cagccctgac tttcaagtct aaatttctcc tccacgccaa aaacaaaaca
aaaacagaaa 1500 aacaaaaaaa gctttttgct gtatcacacc acctaaagtt
tggctagtga acatgagcag 1560 acctcttctg aatcccacac atcagccatg
ctcttgcagc catgtagagg agctggaggt 1620 gggtgggc 1628 87 1795 DNA
Homo sapiens 87 ggcacgagaa caaactataa actacttacc tgcatattgc
tttactggga aaaatcttag 60 cagatgatac ttccttacat ttgtagagta
gaatgtgttt tatgtctttt attagtatag 120 atgactggcc ctatatcatc
taatagatag tccttttcat catggagatg aattattgtg 180 ggtccagagt
tttgtatatg tctctaatcc tgctagggag tccaatcata cccttgtggt 240
cctatacttc agccacacag gctgcagctt tagtgacatc acacgtgtgg aaaccctctc
300 tagaggctca ccagatcaat atttctcctg aaccttcaat acattatgat
agatggcaca 360 ctcagagtaa ttgtagttta ataaattctc ttcaataaat
ggttctggaa aaacaatatc 420 tatatgcaga agaatagaag aagactgccc
acttctaaca atatacaaaa atcaaatgaa 480 aattaaagaa ttaaatctaa
gaccctgagc tatgaagcta ctacaagaaa actttgggaa 540 aaatcttcag
gacattgacc tgggcaaaga ttttttgagt aatactccat aagtacaggc 600
aaccaaagca aaaaatgaac aaatgggatc acatcaagtt aaaaagcttc cacacaacaa
660 agaaaacaat caaagtgaag agacaaccca cagaatggga gaaaatattt
gcaaactacc 720 catttgaatg ggattaataa tcagaatata tgaggagctc
aaacaactct atagaaaaaa 780 atataataat ctgatcaaaa aatgggcaaa
agatttgagt agacattcct caaaagaaga 840 catgcaaatg gtaaacaaac
atattgcgaa gtactcaaca tcactgatca tcagagaaat 900 gcagatcaaa
aactacaatg agatatcatc tcatctcaat taaaatggct tctttttcca 960
aaagagaggc aataactaat gctggtgaga atgggaagaa aaaaagaatc ctcatgcact
1020 gttggtggga atataaacta gtaaaaccac tatggagaac agtttggagt
ttcctcaaaa 1080 aactaaaaat ggagctacta tataatccag caatttcacg
cctgggtata tacccaaaag 1140 aaaataaatc catgtatcaa agaaatattt
gcactttcat atttgttgta gcaatgttca 1200 caatagtcaa gatttggaag
caacctgagt ccacaaacag ataaatgaat aaagaaaatg 1260 tactatacac
aatggagtta ctattcagcc atgaaaaaga atgagatgct atcatttgca 1320
acaacataga tggaactgga agtcattgtg ttaagtgaaa taagccagaa acagaaagac
1380 aaacatcaca tgtcctcact tatttgtggg atctaaaaat cagaacactt
gaactcatgg 1440 acatagagag tagaaggatg attaccagag gctgggaagg
gtagtgggag gaaggtgggt 1500 gttggggtga aggatgtggg gatggttaat
gggtaccaaa aattgaatga ataaggccta 1560 ctatttgata gcacaacagg
ctgactacag ccaataatag tttaactaca ttttaaaata 1620 actaagagta
taattggatt gtttgtaaca caaagataaa tgcttgaggg gatggatgtc 1680
tcattttcca ttatgtgatt attacacatt gcatgcctat ataaaacatc tcatatctca
1740 tgcaccccat caatataaac acctactgtg tatccacaaa aaaaaaaaaa aaaaa
1795 88 1864 DNA Homo sapiens SITE (1844) n equals a,t,g, or c 88
cccaagccag ccatttatta caagaagcaa caggttattg acattacatg tttgaaaatt
60 ccctttggtc tttagggaaa ataaacagga agccaagatt tggagccttt
gtaataagga 120 cttcctgcag aaagtctttt ctttactata attgagtaat
tcatatttag agtcacatgt 180 ccagtagcat ttctaatttt gagcattcac
cttgctacct ttaaaaaaca tctgagtttt 240 aagtggcctt tttatcatca
tacacatgtg catacaaaga agggacttgg cagtttaaaa 300 gccacatata
ttcactttta ttgccctaaa tttacatgaa acagacatac tggcaaactc 360
acatattgct ggtgctaacc ttatatttca tagtgttggc atattcccct tttcttagat
420 tcttactccg aaatataggt acacatcctt tgctctgtgc agagggaatt
acatcctttt 480 tcctctccta caaaaacatg ctttattaag tatccatcat
tactttcctt tatgctcgct 540 caatatgcaa tgtgctgtta ttctaccatg
taccttaaat aaaggatgat ggcaaagtta 600 tttaccatgt agaaaccatt
ttctttctag aaacaatagc tcagcctcac tgtagcagct 660 ggcatgtgtg
gtcaagtgga tagttgtact cttgcaagtt ggatttaata tcatatatac 720
tggaccttca gactgttaaa aatcaatgta accttttttt attgctatgg caagcaatta
780 gtatttcact gcacgtcttc catactaatg ttcatttcta aatcttatat
gtaggcattt 840 gttagttcca atgatttcct cactaatata acacttttta
atgggaatct ttccacctac 900 agccctggaa tgataatgct acagtaattc
ttctgaattg actttttctt tcatcctgtc 960 agctttggac aatatcccaa
ttatggcagg gaacaggtgg ggaactaaga tcagttacaa 1020 aaagttgtag
atgtgtcaac tttgtatggc tgggatcact gtgcccaaac aaaacaggcg 1080
aaatacctca gttaaaattt ttccatcaaa gtctttaaaa gaagagtata ctgaagaaag
1140 ggcagtcata atacttactt ctaacagctt ctaaagggta catgtttaac
atttcatttc 1200 aaaatcaccc caaatttgca ctaaatacca atgaagtgtt
attttgcttt agtagtcttc 1260 tgagcaacaa actatgggga attctgkaaa
amcatataaa aagtycaagm cttttttttt 1320 aaatgaatga ttactatgtt
aaatgcaaac tttttttttt ttttatttaa acaaacatac 1380 acttctcctg
gcaaggttat agatgattaa cctctgttca tagacttata tataaaacta 1440
gagggttttt tgtttacttt tttaattttt caagtgcaat tgtttcttac acagacatta
1500 ttactattaa attatcattt agccagttat ctgcaaatat atagtatgta
ttgtctcttc 1560 ttgtgacgtt tagtttaatt gcttatttta aagcagaaam
attagttaca agtgtcttac 1620 aatattttta ccaacagtaa agtagagact
taatgaaaat accttagtgt gattttaata 1680 taatttgcat attttagttg
tataaagttt taatgtaaaa tgtccattat tgaagggaaa 1740 agatctttca
ataaaaaata cccacgaaaa aaaaaaaaaa aaaaagggcg gccgctctag 1800
aggatccaag cttacgtacg cgtgcatgcg acgtcatagc tctnaaaagg ggactccaga
1860 gctt 1864 89 1983 DNA Homo sapiens 89 gacgttgaag atgagaacaa
gcagaagaaa caattggatt tctatgaaaa gaaaacagat 60 tggtgtacac
ttacacaaat ttgtgcagat tatttgtcta gaaggaaagt catacaggtt 120
gggcagtctg gtcacaaaaa gggacagggg ttgagggggt tctggtgact gtgatgaagg
180 cctcactctc aggcctccgg tcccactgaa ggtcagatga aaggtagtct
tccctggcgg 240 ttgctgctgc cactgaatgg gccctaactt tgtcgtcttg
tgtttgaatc ttctgcagga 300 cacgttagcg tatgccacag ctttgttgaa
tgaaaaagag caatcaggaa gcagtaatgg 360 gtcggagagt agtcctgcca
atgagaacgg agacaggcat ctacagcagg tataacggtc 420 agcatgtcct
tgtgtgcaaa gggcagcctt gctcttaagc tttccaaaaa gaatttccac 480
agctgaggga aaacaagatg cttcctctgg aatgtgagtc caaagagtta ccagcgctgc
540 cctctagtga tctcagctca gcatatgcac taaccgtgtg tttacagggc
tgagtagtgc 600 tgcagtgtga agtgaatgga aggcctcgag gtgtttgtgg
ctggccaccc tgatcagcct 660 gcaggtagtc ccgatgaagc cagggcacag
ggggattcgt tccagcttgt tcactttatt 720 ctgccttgcc aggttactga
aagtccctcg tttgctctca ccagccttcc tggaaatgtg 780 gactcttgaa
agaaaagctc ccgtgctctt gaagtatacc tgcttgccag gggagtccaa 840
gaaaattttg acatgtattt ttaaaaaaag aaaaaaaaac agctttaata ccaatcatta
900 tagtagaaaa agaaaataaa tatgtattga acacccactg tgtgcaaaca
ctgaactaag 960 tgtcagttaa tcattacgtc tttccaatag tctgtaactt
tccttaacag cagtctcctc 1020 tgtggtccct tcacagtact tggtacagaa
taggccccat taaatgaatg ttactgatgt 1080 agtaggtgtc attttttttt
aagtgttatc tttcggatcc tcataagcac tatgtgaggc 1140 agctgtcacc
ctgattttac agaaaggtaa ctgcagccca gcacagtgat gtgacttagc 1200
ccaaggtcac tccacacatt acctcatcac ctacttcatt tgcagagaaa ataaaagctg
1260 tcacaggaga gctcctgcgg ccactaattc ccaagcatct gcactgttct
tgtstcctct 1320 cctgtgacag tgggaagttt gcctctgtcc acccaaagcc
cctagcgctc atccccgccc 1380 accttggcag agctttgcgt tctaatgtgt
atgtaactct tcaatatcca gaacgctyca 1440 ccctgccaga cccttcccag
cgacgtctca gcacactggt ttctcttctg ccctgtcaaa 1500 gcctctcttc
tgccctgtca aagcctctct tctccctgtt gcccctgcct tcttttctct 1560
tctttgcagc caaacttcga ctaattctct aaacttaact ttccccattt tcttatctct
1620 cactcgctct tcagcctctt ccctgctaac tccctcttct ctccaactca
gcagttgggg 1680 tgacaggtgg cctgcagctt tcaggcctca tcttagccga
ctgctcggca gcatctagcg 1740 ctcctggcgc tcttcccgtt tgaaacacta
ttccagggct ttcctgacac ttctctctcg 1800 tagttttcct caaacccttc
tggctgttcc ttctctgtct ccttcctagt actgcctctt 1860 ctggaccacc
agtaaaggtt tgtggagtct ctaacctgta tcctcctgcc ctcactccat 1920
attctctccg cgtcgacgcg gccgcgaatt cccgggtcga cgagctcact agtcggcggc
1980 cgc 1983 90 1957 DNA Homo sapiens SITE (349) n equals a,t,g,
or c 90 gtttctctgc aattatttgg ttcagtttta attttatctg taacaatgat
agtaattgct 60 gtttcacttt ctctcttctg tgatgttgtt tcctctgaat
gtatgagctg ctttactcct 120 aagtttgctg acattgttgc aaatgcttat
cagaatgaat cctatatttt tatttaaaat 180 gatcgtgtca ttttcaatca
ggcagcccat ccaaacatgc ggacctatta tttctgcact 240 gatacaggaa
aggaaatgga gttgtggatg aaagccatgt tagatgctgc cctagtacag 300
acagaacctg tgaaaaggta aaggcttgta gaaaaaatga tggtgattnc cacttccatt
360 ttattccatg ccttgcaagt atttcactgt catagtmcat atcattttaa
tagtcatggt 420 atgaaatcat tttttcctca gaaagcaagg atcaattcct
gttctgaatt aaattaatac 480 acattttgtt agtttgcgat accacctaca
tttttattcc acttttcttc tttttcttct 540 ttattcattt tcacctatcg
gtgtactggg gtgaatccag aatcctaaca ttcaaactga 600 atgttctttc
ttcttacaga attaccttta atttccggtg agtacgtttt taattgttac 660
cttaaagcta cacagatttt tatcctttga gatagtgttt ttaagattct aaatcttaga
720 agagagttta tttttatgaa gttaatttgt gtttttctgt aatagtgtgt
tgatgtttct 780 aaagtgtgat gaattacagt aagaamcttt gatartttca
ttttttcaac atttctgatt 840 aatttttatt gtttttgtaa tgaatgtctc
cagaaaatag ttcgtcaagc atttagattg 900 tttccaaatc cacttcttgg
tgaattgtac cttttttata ttgaaactcc actactcaga 960 tyccttgata
atatagataa gtgctgttaa aattgaccca tgtatttttc cctgcttgaa 1020
gatacgaatc attttaatat tcttcagtat agctagttag aggaaatctg attctcagac
1080 tacataaata caagtagtat aatgtgcttt ttaaaaaata tgtattcctg
taattcgaag 1140 aaaaaattat gatgcaagtt aatttttctt ccagtcagtg
acagctgagc acatatctta 1200 tgtaagaaag atgctaatgt gcatcttttt
tccctcttct tttttttccc tcttctcagg 1260 aaattaggga ttgttmcagt
atacatctag tcctttgttt ttcttattct agtgtgcatt 1320 ttaataaagt
cttggctttt tggctaaaag acttaggttg atgctgtgta tttgtgctat 1380
ttttgtaaat atcaagtcta aatcaagtta cccaatcact agtaattaga gctggggaaa
1440 aactgaaaag aaaagagggt ctaggatata gctctaggac atctattttt
aagaaaaacc 1500 acttttgcca catgcatatt gcaggatgag agcagataga
aggaaaatct gtttttggaa 1560 ttgcatgtgt aaaaattacc tgagtagcat
aaagatgagg tggttagcac tgataacgag 1620 agaaaatgtg taggtgaaga
gaattcattt aaaatcttca ggctgagcat ggtggctcac 1680 acctgtaatt
ctagcacttt gggaggttga gggatcactt gagcccagga ttttgagatc 1740
agcctgggca acatgatgaa acaccatctg taccaaaaat acaaaaatta gctgggcgta
1800 gtaccacact cttgtagtcc ctgctactcg ggaggctgaa gcatgaggat
cacttgaatc 1860 aggaggttga ggctgctgtg agctgtgact gtgccactgc
actccagcct ggacaacgga 1920 gtgagaccct gtcaaaaaaa aaaaaaaaaa aactcga
1957 91 573 DNA Homo sapiens 91 ggcacgagtg aatattaact gtgttatttt
tatacacttt ttaagcctta actcgccatt 60 gatttaccag tttaacgttt
cctggggttt ctttgcccat ggggttctct gcccccaccc 120 ccggcccttt
gtttgacttg cgtcgtctga tactcagtat tgtagctttt tgtccgcatg 180
ttactccctg taaatacgct gttatacata ctgttaacac ccctttgctt tttctatggg
240 acctccaggc caccatattt agaactagtt accttattaa aaaagaaaaa
acagtctgtt 300 ggcttctcag tctgcatctt ggaggcaggg aggtgagggc
aggtgcccct cagacacttc 360 aggaaggtag tttgcattct atttaaaaaa
gggagtgggg agcaaatgaa aatcaaatgt 420 ggggggaaaa cactaaaggg
ggcaagaaac aaaggaatta caaaccctct gctctttgta 480 tttctctgtt
gtgaagaata aactgtacct gcacccggaa aaaaaaaaaa aaaaaaaaaa 540
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 573 92 1212 DNA Homo sapiens
92 gccctcccca gctagaatga acattctgcc aggcagggac caatatgttt
tatctctgaa 60 tctctaggac agtgctaatc aaataataga ccatcagtaa
tattcgaata catgtatgga 120 taagggattt gttccaggtc acatagctag
ttgttgctaa tagaaaggac aagtatgtag 180 ataccagcca cagttttttt
agtatctcta cgccctattg cctttccttt aactttaagc 240 ttggtcttac
ccatattttc tgtagtttaa ccttgctttt gatccctcta aggggctgtt 300
ttatataaac tcatgatcat tgttcttttt tctctctctt tccttcccct ccttccttcc
360 cttcttctct cttcctacct ctgtctcttt ttctttcctt cccagtctcc
ctcctctttc 420 ttttttcact tgtaggcttc tgttaattaa tcaatatggt
acttattaag cactgagtca 480 aatgtctaac actgtactgt atcctatgag
aaatgaaata gaagcagatt gaagacatac 540 cattacttga ggaatttaat
attttattag ccccttcttc tcaatggcct ttgtgctctt 600 ctggttctgg
ttatctgtgt tcttttctgg ccttctgcct tgaccatttc ttttggcccc 660
tgccttggaa attagtacat aatttaccct cattttggct tcacatgatc cagctacagc
720 aagacccaaa taagaaaaga tgttacagcg acattgatga agttggtcta
acacagaaac 780 tgaaagagtg agagagacag aagaaagaag catgaagtag
ggaatgagga gtagagaatg 840 tcaccaacgg ggaattacat gtgaccaaaa
aatcaaaaga ttatgactgg gtacatatga 900 aaaataggta caggccaggt
gtagtggctc acacctgtaa tcccagcact tggggaagcc 960 gargtgggtg
gattgcttga gcccaggagt ttgagaccag cctgggcaac atggtgaaac 1020
cccatctcta caaaaaatmc aaaaattagc csggcatggt ggcacacaac tgtagtctca
1080 gctactcagg aagctgaggt gggaagayca ttgagcccag raggcaragg
ttgcagtgag 1140 ctgtgatcct gccactgsac tccagcctgg gtgacagggc
aagaccctgt ytmaaaaaaa 1200 aaaaaaaaaa aa 1212 93 1144 DNA Homo
sapiens SITE (849) n equals a,t,g, or c SITE (865) n equals a,t,g,
or c SITE (1087) n equals a,t,g, or c 93 aattcggcac gagggacagt
cagctaacta ggcaagtcac aatcttatat agcataatca 60 tggaagtaac
actccatcat ctttgctgtg ttctattggt tagaagcaag tcactaggct 120
agcccatact actgggagag gattacacaa gaacatgtgg gtagaaatgg gaataacttc
180 agctgtccaa caatcttaca ggtatatcct tcatcaatca ttagctataa
gtaatattgg 240 gtttccatta gtcaaagatc tgtgtgtcag caagccagga
cttcaatatt ttttaaagat 300 ggtctttcta gagaaaaata cagtaataat
gggatgacag aaggccatgt gttttgtttt 360 gctttgtgtt gtgtcttggt
tttcctctct atgactttgc ttgttaycag cttagaaaaa 420 actaacgcag
gtggggtgat agcatggggc tgtatctcag tctctgtgca gacacaaact 480
ttttcctctc ctaccagtta ccaaacattg tttattgcct gtaagctctg gaatcccaga
540 aaactttagt tttaatcttt atcatcatca ttatcacata atttacatcc
tagtttagat 600 ttggagcttg ttttagatta atackttaca gagtagtttt
acatgaataa gcttaaacat 660 tttcccccga ttttagttct ctggcttacc
agaaaaatga aaaacaacaa caacaaaatc 720 cccaaaactg agaacccagg
aatgatagac aacaaacttg tgttttaatt ttcatgattc 780 tagttgttca
acctgttttt ttgacactct gtatctgcat tcatttattc actaaaaaga 840
tgcttagtna attgtaagta tcatnttagg cactgtgaat tcattgataa gatattctct
900 ctctctctct
ttttttcttt tgagatggag tctctgtctg ttgcccaggc tggagtacag 960
tggcatgatc tcgtcggctc actgcagcct ctgcctcccg ggttcaatcc attctcctgc
1020 ctcagctact ccagcggctg aggcagaaga attgcttgaa cctgggcagc
ggaggttgca 1080 gtgagcnaag attacgccac tgcactccag tctttctcaa
aaaaaaaaaa aaaaaaaact 1140 cgag 1144 94 1274 DNA Homo sapiens SITE
(722) n equals a,t,g, or c 94 agctgagtgt gcgagcgcca ggggttccag
ctgcacgtcc caggctctcc agcgcgcggc 60 aggccggggc gggacgagga
gagctgcggg gacaacgcct gtggctgggt ccggagtgcg 120 ggtgcggcgc
gggacaagcg ggcagcatgc tcagggcggt cgggagccta ctgcgccttg 180
gccgcgggct aacagtccgc tgcggccccg gggcgcctct cgaggccacg cgacggcccg
240 caccggctct tccgccccgg ggtctcccct gctactccag cggcggggcc
cccagcaatt 300 ctgggcccca aggtcacggg gagattcacc gagtccccac
gcagcgcagg ccttcgcagt 360 tcgacaagaa aatcctgctg tggacagggc
gtttcaaatc gatggaggag atcccgcctc 420 ggatcccgcc agaaatgata
gacaccgcaa gaaacaaagc tcgagtgaaa gcttgttaca 480 taatgattgg
actcacaatt atcgcctgct ttgctgtgat agtgtcagcc aaaagggctg 540
tagaacgaca tgaatcctta acaagttgga acttggcaaa gaaagctaag tgscgtgaag
600 aagctgcatt ggctgcacag gctaaagcta atgatattct aagtgacaaa
gtgttcacct 660 gaataccatc cctgtcatca gcaacagtag aagatgggaa
aaatagaata tttaccaaaa 720 tntctgccat ggttttattt tggtaacaag
aagcacaatg tcttttttat ttttattttt 780 tagtaaactt ttactgaagt
ataccatgca ttcaaaaagt ggacaaaact gtatacagtc 840 tgatagatat
ttatgtcgtg aacacctgtg taaccactgc caaagtgaag atgtagaata 900
ttggcaacac ttcacagcct cattcctgcc ttttctcagc cattacctcc caaacatagc
960 agtttttctg agtttcatca cctttgattc attttgcctg tttttgaact
ttatataaat 1020 ggatttatac attatgcact tgtgtgtgtg gattatttac
ctgacagtta taaggttaat 1080 ccacaaattg tgtgtaccat tagttcatcc
attgtcattg ctgtattctg ttgtataaac 1140 ataccacaat ttattttgat
atttggcaca gtttctggcc actacatata atgctaaaat 1200 gagcacattg
tatatgtcat taaaatgagg ttgaactaaa aaaaaaaaaa aaaaaaaaaa 1260
aaaaaaaact cgag 1274 95 1780 DNA Homo sapiens 95 tatttgggat
tatactgaac ctatttgtcc aataacctga gttttcaaat aattttagtt 60
ctataagtac tataattata taaatattaa tgaattcaga ttagctgaaa ggaaaaaaag
120 tagaagcctg actacttggt gctaactact aaagattttg gcagaatcaa
tgttggattt 180 ggctttcctg tcccttcccc atgccagccc cccagagtgt
tctgccttgt gctgcctccc 240 ttcacckgga gtgccacacc cctctctctg
ccagttcagc tcttcattct tcaaggcctg 300 accttgtctg acccttgtgc
ctctaaaccc gtggccccac ctctcttggg cacgagctat 360 gtcaggtgat
gtttgtgttt ttggttatgc ccatctccat agccagacca agcactctgg 420
aagccagggt tgggtgctta tttatctgtt tgccatgcag aaaatatctt gcacaaaatt
480 acctctgtta aggaatctga agctgaattt agtttggctg agtcagggtt
gggttttttt 540 taaggggctg tggggtgaaa tgttgactgg aagccaccca
caaacacaca cctgctggtt 600 aggaacccgg ctgtgggtgg ttctgagctg
tttggcttca ttgacagttt ctgattgccc 660 tgagcaccag gtctcatctt
gcatctcatc ctggcctgga gaacattcag tttccttcca 720 acccttccca
cctttccccc actcccttgg aggaactgaa gttggggttg aggagagcca 780
gatggctgga gtgggtattt gaaggtcttt ctgtcacctg ttcagtgtgg tctgccccac
840 ccctgctgac caagactgac tgaaatgtaa aataatacag accatctcaa
ctcagaaagc 900 tggcacattt ttgaaagccc aagtgtgggt aagtgcgtgg
aacaacgata attcacactg 960 ctttatgagt agaaattgtg agaaatattg
tgccaggcaa tttgcaaaat cttggaaggt 1020 tgtgtgcact taaccaccca
gcaactactc ctggatgcat cctagagaag tgccatgtga 1080 acagagaatg
attttaagac ttcactgaag tattgtttag gtagcaagat tgggaaaagc 1140
ctgcatttca tcagcagaag aatggataaa taaatgagtt gtttttggtc cttggaaagt
1200 gaatatgaaa gagttacgtc tcaacacaga tagatgaaaa attatgctga
gaaagttggt 1260 gaagctacat acaaggtacc cttagtgtaa agttaagcat
actgtgtacc tgtgggcacg 1320 ttacttcaac ttgtttttca ctttttctgt
aaaatgggat agtagtggca atctcacagg 1380 gtgattgtgg gtgggggggt
ggtcaatgaa gtaatgcatg taaaatgctt agaatagtgt 1440 ctagcatgta
agccttgtgg acatatagaa agtgttattg ttttgcacag taatctattt 1500
tctgtggatt caaataatat gaaatgagta taaaatcatg tattggaacg atgtgtgcaa
1560 gtcaccattc tgccttccta aggcaggaga cctgatggat ttgggggggg
tacatggggc 1620 cttcagttgt gttttctttg tttttttcta aaaattgatg
cagaggcatc acaatgttaa 1680 gattttaaca gggtagtgtg gtgggtactt
tttaactgtt tgcttaaagt gtttcaaagt 1740 aaaaatattt cttaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1780 96 1794 DNA Homo sapiens SITE (457) n
equals a,t,g, or c 96 gaagcattta gtaggatttt aaagaaactt gagaactgtt
acataaggtg atgaattggg 60 catagcatgt aaaattatat ttaagcaagg
aaatgatctc tggtgtttta atattcaact 120 tgattgcttc ctcttgggtt
ctgtgtttcc cactgtgtga cctgagctgt cagaaaacct 180 taagaatttt
ctttgcatca tttttccatg cagtttgtgt acatgtctca tgtacctcgt 240
ggcagccact ggttttgttc atcaaatggt gggttgtggg atgctctcct gcagtctccc
300 tctaattaaa gaggttaaat tgccgtttgc tcagccttta gttcctttcc
acagcttcct 360 aggctcttaa aaattagcac tatattcctt tcagattaaa
aaaaaaacaa aaacaaaaac 420 ctgtttgctg tctttactgc tgtggtcttg
tctagangca aatctgaaca aactgattga 480 aaggggtgtt tggtggctgg
tgttctcttt gactaaagag gcttacatgt actgtggtac 540 agtctgctta
cttaaaaggt gaggcttgaa ttaaaatmca gccagataka agccagactc 600
taatcaaatg aggtgattag atcaatgaat gaagagagga gaggagtcag gtgttgcctt
660 tccctggctg ttgaatagct gatgttccag attgccctac agtgttgtgt
tagggcatcc 720 aggagggatm cttttcaggc ttaggtacac ctcagtcttt
aaaatgagga attakgacac 780 attcatgtgt gtgtccctaa tctgctcctg
agaagagaag tgcaatcagg gtcttatttt 840 gtgaccactg acttgcacac
tgagacaaaa gggccatctg caagctgaaa atagtggatt 900 ccttaaaata
aaacactatt cacatttgat ggtgtggtag ttttrakaaa atgttcaagt 960
gtcaagttca ttttcattta taatctgaga cagttttata agtcacctcc ctgggggtaa
1020 aaatgcatgt tctgtcctca tagtgagaca catcttctgc ttagagtcta
gaaagctcta 1080 agaaagattt atgccatctg tgcagctggc atttttatag
taaaattttt tttactttgc 1140 tccaagttta agttatctca tgacaaactt
tcttgaaaga ggcattcact attattatag 1200 gaagtatact yctttattga
aaaggagata atgtatcagg taacttatta aagtattttc 1260 tcaaagttta
gtatctttag gaatacagtg cctcaataca atataaaata ttttgtaaat 1320
aatagaatga attcatttta gaatttaaat gatgctaata aaatagacca ttattctaaa
1380 agtttaacta atttagaatc aaccctggtt gaaaataagc cttaagctgt
ttttttggaa 1440 gactttaaat cctttatggc taagagatga cagacagggc
cgagtgcggt kgctcatgcc 1500 tgtaatccca gcactttggg aggccgaggc
gggcggatca cgaggtcagg aaatcaagac 1560 catcctggct aacacggtga
aaccctgtct ctactaaaaa atacaaaaaa aattagccgg 1620 gcgtggtggc
gggcgcctgt agtcccagct actcaggagg ctgaagcagg agcatggtgt 1680
gaacccagga ggcagagctt gcagtgagct gagatcacac cactgcactc cagcctgggc
1740 aacagagcga gagagtgaga ctctgtctca aaaaaaaaaa aaaaaaaaac tcga
1794 97 2065 DNA Homo sapiens 97 ggcacgagat taaaaggcct ttcaaaagaa
tgggtttgaa aaactcagta ccctttaata 60 catgtacatt tctttccttt
tttcatttaa tgtaacatgt ctgttgtaac tatgtttctt 120 aaatattatt
ttaaggttat gtgttcttta attatggtca aatataattt ggtcaccaaa 180
aatgaaataa tagtttaaaa caagtagctg ttactaagtg tgctaaaaat actcatttta
240 taattaattt tagttttctt agtatattat tataaattgt gccctaagtc
aggtacaaat 300 gtacacatca aaatgcccat attgtatcta tctgtagtcg
tttaatgtga attatatgtg 360 aatttttttc aaaattttac taaccagaat
tctgttatag gcacctaacc acgcagcatg 420 aggaaaacgg cacaacacaa
tcttgaggtg ccttctgaat catcagatta aattatgctt 480 catatgtttt
tgcttttact gtatttcttt aaaaactcta aatctttatt catgtgtcac 540
tggattaatt tatctgataa tgtgtctcac aagaatctgt tagatcgttt attcttcagt
600 tgtactttga atggtggggt ggaagtttca ggtgaacaat ggataacaaa
aagcaagtta 660 tggaagattg tgaagaggat ggaaaaactg aatacaagat
accaaaaatg aaaaaaagtg 720 tcccattttt aataactata ttctattatt
ttataaatgt gtaataaagg ggtccctctt 780 tattggttgt tatcccctta
atctttggtc tttttcagta attttaagtt ttctgggatt 840 ttttttggtt
tataaaactt gtgtttagac tttatcttgc tatggagttt tcacacttct 900
atagcacata tcctagtatc tagtcatttc tgttttaata tgaatttcag taatttaatt
960 ttaatctggt gacatattaa tcgaaaataa ggagtaatgt atacctccac
atgtcctttc 1020 tttttgtctt ctcttaaatt cacaatatcc agtaggagtg
gttattcaat ttcttcgtgg 1080 ttttaatcat caaatgaagt tagagaagta
tactaatccc agcaactatg actcatctag 1140 gcatgttaag accataaagt
aattcaggaa actattttcc tgatttttaa ataactttta 1200 gtgttatgta
acatctatcc ttctgtttta gacatgcatt tcacatatag ttgaattcta 1260
gattctaaga taattcattt tgggtaatac ttcagagtac tggatctaga atcaggcttc
1320 ctgaatttaa actcaggctc cccattaact gtgtgtctgt gagcccagtt
tctcatctgt 1380 aaaatggggc aacagtggca ctcatcttaa agggttggat
aataaaataa tgcatgtaag 1440 gccctaagca tagtgcctgg cacagaatta
ctgctcaaat gttagctgtc gtattaatat 1500 tgcacttttg cacactgatg
tacatttcct gttgaccagg ctcattcttt aagcattctc 1560 catgcttaaa
ccagttccat aatccctagg cctgtactcc agggattgag actgaaagga 1620
tcatttatgc catgtttctc taaaagcatc attgctggaa gacttttgat aagtctgatg
1680 tgtctcaagc tattctcagg ccttttttgt agagtttaga aatgaagtat
ttgaatcaat 1740 ttagtatctc ctttactatg tttctccttt taatctcagc
caacccccta cctgcaggta 1800 aacccagcat tcattaagag ctgggttggg
gtactctatt ctgtatgcat cataatagct 1860 taacattatt tagtagctgt
aacttacagg tttaatgcta gatgaggatg tctcaagccg 1920 tgagtgtgct
tgtgtaaaaa tggtggcaac atcatctcgt tggtaggaat tttttacttg 1980
aattgttatt ttgggaaaat gttaacagat ttcttctgga taaagaaaat aaattggatg
2040 atgtataaaa aaaaaaaaaa aaaaa 2065 98 1154 DNA Homo sapiens 98
ggcacgaggt gccgtgtgtg tgtgcgtgtg taagtgtgca tgtgcataca tgtgcatgtc
60 tgtaggtgca cacatctgtg tgtgtgtgtg catgtgtgtg ctgcatgtct
gtggggaggt 120 gtcctccgtg agagcgtgtg acagctggga tttgcactct
tgcgtgctgc cccagagacc 180 acagcctggg caggccctga ccttctgtgc
cccgtgcatc gagccggtct gctgcgggtg 240 cctgtggccg ccaatgggga
actcgggtga gctggcagga gggtgtgccc agagccctgg 300 ctgctgctac
tgccactcag cacagctggg ccaggctgtt gccccagagg gcgtcagacg 360
tgaactttgg gaacatcttt attctgtttt aaagtgagca caaattatta gacactttcc
420 ccaaaatcca tgtgtttggg gcgtcttccg gccatgccac acatctgtgt
ttgcctggct 480 gtttctgcac cgagttccgt ccacagcccg ggtttctgtt
gttttaagtc ttgagccctg 540 ggccgggggc cacttctcat tggtggctgg
aggctcggcc aagtgagggg ctgcttctgg 600 ttggagaggg gagtttctgg
aagggggttc cccatgtgtc tccagcgctt cctgcagtct 660 ggggaggggc
ttggcaggag caggtctggt gagaaagccc tggccggggg tggaggctca 720
gtcctgggag tgggcggggc agctgggctc ggggtgttaa cagggtcctg cggggggact
780 ctgtgctgag tcaaaggagc cggaagctgg tgtgggccgg gtggggtggg
gaaggtgggt 840 gcaggcaggg gagggggctt ggactgaagg tgagacccag
gcctgggcaa ggatgcggtg 900 tgcccagagc tggcagagtc atctgcctga
agcctgactg tggcctgggt ggggtaagga 960 aggtttggag aggctttggg
gcctgcggga aagggggctg tggagagaga ggctgaccga 1020 gggctgccga
gaggaagacc agtgttgctg gagcctgtgg tggagagggg cttggtgggt 1080
gaaccctcca gggaaggcct ggggcagggc tcagaggacc tggaaggtgt gcagagttgt
1140 gtccagcagg agct 1154 99 615 DNA Homo sapiens 99 ccagggagac
agcagcgtgg tcagagtggt aggagctggc catcggtgag agctgctcca 60
tgcctggctg ctgggtgcta gagcttgtgg accactggct tgcctcactg tggttggtgg
120 tggcggtgac agagtgtgca gcacgaccag agtggctttt ctggctttgc
ccgcccagct 180 gctccatgcc aggaggagga ggagacacct agagcctgcg
acaccatggc tcgsctcgct 240 gcagtgtagg ktctacccat gtaacagatg
aggaaaccaa ggagcacagt tatttactaa 300 ctcgcacaag gttcgaggcc
gagctcagac ctgtggagca gaagctgagt gcgctgcagt 360 ccccgctggc
ccagargccc ttcttcgagg tgccctcacc sctgggcgcc gtggacctgt 420
acgagtaygc atgcggggay gaggacctgg agccgctgtg acgccgcccg cgagaaacgc
480 cgcrcggggc cgctccccac gtgccaccac cgggccaccg cggctcgtgt
aaaaactgtt 540 gtggaaaatg agtgcgtttg tacggaatga taaactttta
tttattcaca aaaaaaaaaa 600 aaaaaaaaaa aattc 615 100 1624 DNA Homo
sapiens SITE (117) n equals a,t,g, or c 100 atatgtttct gaatagatcc
agttgaatag tctcattcaa tttgagactg ttgaacaact 60 gttgttttct
cacatacatt taaagtcagg gcacatgcgt cactgcttat ttttcgnact 120
tgacatattc cctgcatttc catgtctgcc tgtcctttag ataaacagta aaagtttccc
180 atgtgccagt atttctcaaa tggtttacat cagaatcacc tggaggactt
ttaagctaga 240 ttgtttggtc ccatcccagg gattctgaat caacaggttt
gcgatgggtc cagatagttt 300 acttttccaa gtttactaac aagtttgcca
agttccccca gtttattacc attagaccat 360 acctttttgt ccaatcattt
aaamcaaatt tttatataat aagttttatt tgtatgtaat 420 aaattttatt
atataaaaat aagttttaat atatattata taaaaagttt taataaatac 480
ctaatatatt atttaatatg ataaaactta tattaaatga aattttatgc tgtyctcttg
540 tcaatctgtc ttttgttatc ttgctggtgt gcctgtcatg tgagggactg
caatctgata 600 tgcctatttt ccacagtcaa agcaattaca agagaattgt
tacaattacc cagttatgtc 660 aagagatttt tttttaattc actaaggtag
agataaggag aatgtattaa aataggatat 720 tttaattata aatgcatgac
tggggagggg gtattgtttt tgaataaaat atgaggttat 780 ttgccatgac
aaaaaaaaaa agaagtagga aaatcccatg gaaatttatg ttccttctaa 840
cttttaaaac tacctaaaaa atataattga tttaaattat atctcaatat tccccattct
900 tttatatccc cttaaatagg tacccatgaa gagattatga actacttgaa
ggtggagact 960 gtacggtggt gtgttggagc tggcttgtaa tgtcttatga
gtgacaatcg ttagtttgag 1020 gaattttgtg agacagttgt caaattgttg
ctagcttgaa atctgcggca attggagtat 1080 ttacaccata gaaatgctat
aagtgaagrc ctacctttcc cttaagagct agttgttaaa 1140 cctttaccag
cataccactg gaccttgtct aaaatttctt tgtgttccca gtgtcttgcc 1200
cagtagatac aagataaata ttgccagaat cagatatcag gaagtagtaa gaaaaggagt
1260 taatatgcaa actaaatcac tcgctcaatt gaataattga gatcttctgt
tcatttgttc 1320 cttggacctt aatcatttgc attttggaga aaattttttc
tgctttaaaa gtctgtaatt 1380 tcagtttttg tgtcggggag agggaaaaac
tatttgtctg tagttgcttt ttgtgacaaa 1440 gtgaataccc actgggctaa
gtttcatatc taaagcttgt cactaagaat tttcattttt 1500 aggggtcaaa
aacctatttt gaaaatagtg ttgtgtgaat gctgtaagtg ttgtacatgt 1560
ctctggtttc agaattaaaa gaattcagag ttaaaaaaaa aaaaaaaaaa aaaaaaaact
1620 cgta 1624 101 1756 DNA Homo sapiens 101 ggcacgagtt ttcctctcac
atatatattg ttttgtgtcc ctggctaaag tacaagcttt 60 ttgaaggcag
aaaccatgtc tttggtttct tttgtatttc ccatagcacc ttttactgtg 120
agagtgggca cacagtatat gttgtggaat gacatcctga gtgatccctc cctggctggg
180 cctcagatta aattccctga aatggaacag tcctaaccaa cacaggacag
gtattctcca 240 tctggcatgt tggttgctcc tttcaacctg ctatttgaaa
tggctccctt caacatcttt 300 ctgttcccac agtggggctt gctatggcta
atgctgtact tgctgtatgt gttccaggcg 360 agtctgcgga caccagaact
gacctgggag cgagtgagat ctcaagttga ccagtgatat 420 ggcctgatgg
caagaggata gtactgctgg cagaggtaag ctgagactgg caaaaatact 480
cccccacaac aggagagact gcaataccca ggtcccctcc tcctcatgtt ctcgaatact
540 ttcaactcct ctgttaagca caagtttgac tactttccca atggatttta
cttctaattg 600 tgaaagatct tttcattcag caattaagaa actattttgg
ttccccactt ttcaccaatt 660 atcctgtctc tccacgtcaa tccacaggtt
gagttagata attattacta tagaaggaat 720 tcacagatag aaccagtgcc
actttgagtg atgcatacaa agagataatg tcacttgtgg 780 gatgttttaa
tcactaagca caaagtagat atgcccgact gtaaccagga ctatcttagg 840
caagttctgg gaatgtatgt ttttactgat agattccctg tttttgaagt ccattccctt
900 gaattgagcc agatgagtat aggtacctac ctagatatca attgctcaat
tgatatttcc 960 ccatcctagc tcctagctca cattgacact attgactttc
attttattgg cttccatgtc 1020 agtgtttgac cacttttcct ttcttaaaag
ctcctcttcc ctagtcctgg attcctgaca 1080 gctataatat tagatgcctt
ctattcttac cttgaagctt tctcttcttc agagaaagat 1140 accaaaatat
caaggaggat aataatactt ttctcaattt tgattttcag ttggtttttt 1200
ttcttttttt atattaaaga acctgaatat gaaaatgtaa aatatacatt gtctttatct
1260 aggggcccat aagttaggag tttttagtgt ccttactgtt tcttcacatt
ttcctcactt 1320 tatctcatct tctcagatac ttcagggcat ttgtaaaggg
actgaactat ttcttcacaa 1380 ggaaggagta tatatgagga ggagatgggc
agattgccaa atatgcatta atagctttga 1440 tgtcagtctg ctgactgatg
acttgtttct agctgcccta ggaggtccca cctggtaatt 1500 ttggtgacaa
aagcaagtac catgggtgtt tttggctaga tggttgagca aaaaggtggt 1560
caggcttcat aggaaacaaa ataggaaagg gtggcattgg gggcaatttc tagttcttct
1620 actgtctgaa tcaccaactc aaaatacaag gctgacaatg ctgtctttga
attcaggaga 1680 agcaaactga aggagaagca caaaaatcat cacagctatg
gtgaaaccct gtctctacaa 1740 aaaaaaaaaa aaaaaa 1756 102 1416 DNA Homo
sapiens 102 tacatagtta ttctttttta ttttttactc aagttacatt taatatcttt
atcacaggaa 60 ggctggcaat ataaaacttc ctatgtacga aaactcaaaa
ataaccaaag tggcaagtga 120 ataattcctt tgagaagcaa aagaacagta
caatgtttat taacacgttt cttcctgaat 180 tttcttcaat ttttttaaac
acacaaaaag cttttctgta cttagattgc tgtttgctgt 240 ttttaatgtt
gttaacatgc atattattgc atttatggat agtagtagat agtgtaatat 300
acatgaaacc aacatctagg gatggctgcc ttctgagtgc tttacagatg gcacgttctc
360 ttattatcca gcttaatcac agctcctcca actgataact tcacatcatc
tgcagtattt 420 ccaatctgta aatctggttg gcacaagttg gttttagcgt
atttggaacc gtattttaaa 480 tcactggaac tactttgcct taatgcccat
gggctgtcag ctcccaaggg ctaagaccaa 540 gtttttctta actttgtgca
tatagcgtgg gacctgccca gaacaggtac tcaacaattt 600 tgctgagcag
aactgtcctc aatggagaaa agaaaggaga aaggctttac tgaagactgc 660
cccaaataca aacaaattcc attttaaatg gaatatatac actttagccc ccaaatgcag
720 accagtgcac gtctgtgtag tttccgacta gtcacctggt aatagatcat
tcctgtcatt 780 cacaggctca gtcccagctc tatttttcag tatcttgaat
caagttctct ctcctctaat 840 catggaagaa atagacccat aactagttat
tttgggtaaa tgggagctat ttaaaaaatt 900 gatattttaa aagcttaaat
gaaatgttaa tcaaatatga tttatgatta ttttctttct 960 atgagtattc
tttaattgtg gaaggcagtt tcttaggaag ggaacaaggg ttctctttta 1020
caaccaaaag tttggtggtg gttttttttt cacaaaatta ttgagtttaa aaaaattgat
1080 ggttgttttg catttcacct agtagcttat tcaatggttt gtttttctgc
taaatgttaa 1140 ccgtcaaaac ttgaattaat ttcttaattg ctatttctac
ttcaggaatc ttaagaaaga 1200 tggcttaacc cagtcagaag ggacaagcat
aattttcttc catggctatt taagtaagta 1260 ttaggagagc tttcacgacc
atgctatagc ttcctagtga cgcagaattg gtaagacttg 1320 tgtgatatat
acatgtgtga cttgttacat atcatagcaa ctgtgtagtg ggaaggatgt 1380
aaacagttcg atatcaagct tatcgatacc gtcgac 1416 103 704 DNA Homo
sapiens SITE (287) n equals a,t,g, or c 103 actgtgtctg tcttgtctct
gatatttata tgccattatg tggcctctac tgccttagga 60 ttctaatgtt
cccactaaga tcagctaact cagttccact acagtgttta ccaccatcat 120
ctctcgcaaa caaagacagc cacttcagag ctcctaggaa atagtggtgc tcccatcatc
180 attgcattcc ttaatsacat ggtgaaaatt aacaatggct aaggagcctt
tgtgttttct 240 cctctacaat atgcccagga atttctggca ttttggccat
cttattnata ggctattact 300 gaatttmagc ctmatcctmc caaattatta
atgccaaaat attaactctt gattcttagg 360 tgagtgcacc catgccaata
aatttgccat gatctaacct taaatgtatt ctcatatatg 420 ctgtccaagt
ttctrctgat taaaatggca aggcctttag ttctcctaca taggttttct 480
ctctccagag aaggcctcaa ttctctgact aggctatgtt gggatataac tggaggcact
540 aataggtagt agggtaaatt ctttatttta ttatttttgg agacagggag
ggtcttgctt
600 tgttcagact ggagtgcagt ggtgtgatca tggctcattg caactttgaa
ctcctgggcg 660 acagagcaag actccatctc aaaaaaaaaa aaaaaaaaac tcga 704
104 1259 DNA Homo sapiens 104 gacggggacc agagcacgtt cctggctgca
gaggccacaa gtcacgctgt ctctgagagc 60 cacggtggcc tcatctctct
gccataaact tgccaattat cctgctgctg cctcattgac 120 ttcgcaccca
ctcttccctc tggaacagag gacactctcg ccagctctcc ccatggcgga 180
tccttgtcta gggtcaggcc tctgctccaa agtcacccct ggggacacct tctctgacca
240 gcccctcatt cctatggcct catgctgttt ttatttcttc ctaggactta
gcacgtatcc 300 tagaaattaa cctgctggta tatcctgttt cttgtctgtc
tctttccagt ggaatgtcac 360 catcgcccag gtggggattt ttgtgtgttt
tgttcactgc tgtacamcca gcccccagca 420 cagcgsctgt ccaggacaag
tgcccagtaa acacttggga agcaatgcaa gcgtgcgtgc 480 atggataagt
awttctttss cagatgaggg ggctaaggtt cagagaaggc cctgggggtc 540
tcagactcat agcccagtgc tctttctgct gacacgccct ggtctctggg gcagtttgtt
600 gcctgttcag caacaaagag ggtgtgcctc gttaggggtc ctgcgtgcga
atcgcagtcc 660 ctgcgtgtct tggctggagg tmacmaccct ytctgctcca
gggcctgtaa ttaccactta 720 ccctggtcaa tgggtccgag agattmccct
tgtaggcagg gctgtggcca gggtgctcac 780 ctggccccca gsaggtccca
tgggcactgt ctggccgggc ttcatggctg acattccagg 840 tacatttcta
gccctgggct gccatgggca gagggtgggg agagggtcgt gggcttcagg 900
ctggacaaac cagtcagcct tcccagctgg gccgcctgac cacccacttc ctgtggggct
960 ccttgaggcc tggagggtgg agggggtctc tgttcaaccc ccacccatgc
cctcttccct 1020 tctctccctc ggcaggtcyt cccagcagyt cctgcaaaca
gacccccgac ccaagccctt 1080 ccttctgsct ccactgccac cactgctgct
catctctgct ggcacagaag tctcttccct 1140 ggtcttccag aaatcccctc
tccacactca gccagaggga gctattaaaa ctgtgggcca 1200 gcccacatca
gtccacagca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaactcgag 1259 105 1804
DNA Homo sapiens 105 ggcagagcag acgcgkctcc ttggagggag tgcggtcctc
tagggaggca tcgggctcct 60 aggggcttct tggcgtgtgt ggtgggattg
gggtccgccg gccatggcct tcactttcgc 120 tgcgttctgc tacatgctgt
ctctggtgct gtgcgctgcg ctcatcttct tcgccatctg 180 gcacataatt
gcctttgatg agttaaggac agattttaag agccccatag accagtgcaa 240
tcctgttcat gcgagggaac ggttgaggaa catcgagcgc atctgcttcc ttctgcgaaa
300 gctggtgctg ccagaatact ccatccatag cctcttctgc attatgttcc
tgtgtgcgca 360 agagtggctc acgctggggc tgaatgtccc tctacttttc
tatcacttct ggaggtattt 420 ccactgtcca gcagatagct cagaactagc
ctacgaccca ccggtggtca tgaatgccga 480 cactttgagt tactgtcaga
aggaggcckg gtgtaagctg gccttctatc tcctctcctt 540 cttctactac
ctttactgca tgatctacac tttagtgagc tcttaacgca aagaccatgc 600
acatcatcag agactgagat gggagaggcc tgagacggag aggtgcattt ctgctggtga
660 ctggaggagg gaccagaatg aggatacgtg agaaatagac ccggcaggca
gtcagactga 720 atgggagctg gaatcacgca gcagctggga gccgagttaa
ccctgcgtgt ctgtgtcacc 780 ctgtttgtca atctttggca ttcgaattcc
acacacgggg tcctagagcc cttctgagca 840 tcagtggtgt gggggagtag
gtgacgaaac actagacctc tcctgagaga gaattgctgc 900 ttcctgaatc
cacttcattg aacagcacct tgcaagttca aatgagttcc tgggagtgga 960
ggctggaagg ccacaaggtg cttgctaagg aacagaatga cccagagtca aggccaagtc
1020 tgcagggacc tgttgaaagc ctcgagaatg kcttggctgc ccaagactct
tgktgccttt 1080 cttccaagcc atggccatgc cctttttctc aaatgggarg
ggctggargg tgtgtgggat 1140 ttgtcttcag ctgcaaccag ccttgagcct
gctgggctat tttcagctga ggaggggtga 1200 atataggaaa aatgcatttt
tgaaacrttt gcaacatgat caaggtgtta gttctccacc 1260 acacaagttg
tattcttctt ttgccacctc aaaccatcac agagtcttta aatgcaaatc 1320
aattggtcaa tgctagtcaa agctatgttc ttacaaaaac cccagacagc tcagagctca
1380 gaaaatcctg tggagtggct gctctgtacc gtgggcatcc ggcagccagg
aagtgagaca 1440 acataattat aactttgttt tatgatgctg catcatttgt
actgtttagg tcgacrtgag 1500 gacatcatct tatttagaat tttccgtttg
gcattctctt ttgggtggga gttatgctgg 1560 gggttgtaaa taatgacaag
gctgagattt ttatgatgtt taaattgggc acaatgattt 1620 tgaccttatt
ccccaaactt cttttctttt ctactgttta acatacacag gctatttata 1680
cacgtcccca gctcccatct gaaacctgtg actcaggttt atgaatggtg tttgtgtagc
1740 aacacattgt gtgctatgtt tattaaaatg cagcgacaaa aaaaaraaaa
aaaaaaaact 1800 cgag 1804 106 971 DNA Homo sapiens 106 ctagcccggg
cggatccccc gggctgcagg cgccgaggct ggaggccgag ctctgcagag 60
ttacaattga gactgctaac ccctaccttt gaagggatca acggattgtt gttgaaacaa
120 catttagttc agaatccagt cagactctgg caacttttag gtggtacttt
ctattttaac 180 acctcaaggt tgaagcagaa gaataaggag aaggataagt
cgaaggggaa ggcgcctgaa 240 gaggacgaak aggagaggag acgccgtgag
cgggacgacc agatgtaccg agagcggctg 300 cgcaccttgc tggtcatcgc
ggttgtcatg agcctcctga atgctctcag caccagcgga 360 ggcagcattt
cctggaacga ctttgtccac gagatgctgg ccaagggcga ggtgcagcgc 420
gtccaggtgg tgcctgagag cgacgtggtg gaagtctacc tgcaccctgg agccgtggtg
480 tttgggcggc ctcggctagc cttgatgtac cgaatgcagg ttgcaaatat
tgacaagttt 540 gaagagaagc ttcgagcagc tgaagatgag ctgaatatcg
aggccaagga caggatccca 600 gtttcctaca agcgaacagg attctttggg
aaatgccctg tactctgtgg ggatgacggy 660 agtgggcctg gccatcctgt
ggtatgtttt ccgtctggcc gggatgactg gaggcaccgc 720 cggcgatgga
cgtccaggtc ccggctcctg tgctggaaag cgttgatggg gagcgtcggc 780
gctgaccaca ckcgggagct gcggaagccc agcggttcac acaggcctcc cttcaacgta
840 gtcatcccct ggtggtggaa gcaagacgac ggcccctgac gtgcagccac
acacagaaaa 900 ggctgctgtg aaacatttta atgcttcgac tttttttttc
ttccagcctg gagcaacaag 960 agcaaaactc c 971 107 821 DNA Homo sapiens
107 gttttgagtg tgtgaattac atatatgaac atctgaraaa atcctataag
cagtttaatc 60 aactgttcca ctccactcca agtgagtcca taggcagaat
tgagttatgg ggagagcggc 120 ctagtaataa ttggtttgcg taatacaaag
ttctactggg tagtgatgtt gtagaagttc 180 atatagaatc agctgagctt
tcagaaatgg tgaaagggtg gtaatagtca taacttagat 240 tgtaattttt
ttcccatagg cttttaaaaa atattcatga ggttcttttt ttatttcaat 300
agtttttggg gaacaggtgg tttttggtta catgataagt tcttcagtgg tgatttctga
360 gattttggtg cacctgtcat gtgagcagta tgaactctac tttatgtgta
gtcttatccc 420 tcatgtgtat gaactccacc ttatgtgtag tcttatccct
cacccactcc tgcccttccc 480 cacaagtccc caaagtccat tatatgatct
ttatgccttt acatcttcac agtttagctc 540 tcacacaact tattataatt
tataagtaag ccagcattgg atatagttgt attccattat 600 taatttaaga
aaccttatgc aagtaattat tagtcatcat cccaaaaaaa agggagaaca 660
gggttagatt cagaatactt tgataagagc taaatactat catgagtgct gtcagtctgt
720 agtaactttc cattggtatt ctatgtcttt taggcttaca gatacttttt
acactcttac 780 aaaatgtgca caagaagaag ctgcagctca gagctcgtgc c 821
108 1576 DNA Homo sapiens SITE (252) n equals a,t,g, or c SITE
(804) n equals a,t,g, or c 108 ctgctgctgt gtccctggtg gctgtgtttt
gattggtcaa tgggctgcat ccccctcatt 60 aagtccatca gcgactggag
ggtaattgca cttgcagcac tctggttctg cctaattggc 120 ctgatatgcc
aagccctgtg ctctgaagac ggccacaaga gaaggatcct tactctgggc 180
ctgggatttc tcgttatccc atttctcccc gcgagtaacc tgttcttccg agtgggcttc
240 gtggtcgcgg antgttcctc tacctcccca gcattgggta ctgtgtgctg
ctgacttttg 300 gattcggagc cctgagcaaa cataccaaga aaaagaaact
cattgccgct gtcgtgctgg 360 gaatcttatt catcaacacg ctgagatgtg
tgctgcgcac ggcgagtggc ggagtgagga 420 acagcttttc agaagtgctc
tgtctgtgtg tcccctcaat gytaaggttc actmcamcat 480 tggcaaaaac
ctggctgata aaggcaacca gacagctgcc atcagatact accgggaagc 540
tgtaagatta aatcccaagt atgttcatgc catgaataat cttggaaata tcttaaaaga
600 aaggaatgag ctacaggaag ctgaggagct gctgtctttg gctgttcaaa
tacagccaga 660 ctttgccgct gcgtggatga atctaggcat agtgcagaat
agcctgaaaa cggtttgaag 720 cagcagagca aaakttaccg gacagcaatt
aaacacagaa gggaaatacc cmgactgtta 780 ctacaacctc gggcgtctgg
taancgcggg gtgccctgtg cctgtggaag gaaagatggg 840 ttattttyct
tatttataat aaaatgacat agtgacaccc acctagccca tacattttat 900
aaagttcytt cacatgtttc tayctcattt gaaggtagct atttgattyc cttttgagta
960 attttttaaa gctctcatta gagagcagta cagtgtgaat tagtcaagtt
taagaggtca 1020 cccacgcaaa aggttaaacc caggaataaa ttaacatgtt
aaagtcccgt ccgccctgta 1080 aaacagcact ccaatgggta acttcctgat
aaacatcagt ttctctgttt ttaaaacaag 1140 aattgagtaa gaacagagat
taaagtaaca aatycgtagt atgatttctg agctcccttg 1200 ttctccttct
tcaagggagc agagctcttc atctgcaggg agcatttccc ccaaaaaagg 1260
cagctttgga gggcacggga tttatttgaa agggctttga cattatttgg tggaaataga
1320 aaataacgtg ttctgtagta gctttatatt tttggttatt gacaggatgt
ttacgaagat 1380 ctgattgctc ttgattttct tgacaaaaat aaaatgagac
acacacatag caaaattctt 1440 taaacacgaa tggttgtctt ctccctataa
tcaatcattt aatttggttt caagaaaaca 1500 aatacatatg ttcctaatat
atttagatgt attcaataaa cattgttaat taaaaaaaaa 1560 aaaaaaaaaa ctcgag
1576 109 1779 DNA Homo sapiens 109 aggaatacat acgatccttg tctaccagga
gtctaataga aagatggaca gcgtggaccc 60 tgccagcagc caggccatgg
agctctctga tgtcaccctc attgagggtg tgggtaatga 120 ggtgatggtg
gtggcaggtg tggtggtgct gattctagcc ttggtcctag cttggctctc 180
tacctacgta gcagacagcg gtagcaacca gctcctgggc gctattgtgt cagcaggcga
240 cacatccgtc ctccacctgg ggcatgtgga ccacctggtg gcaggccaag
gcaaccccga 300 gccaactgaa ctcccccatc catcagagga caagcaggtg
caggcagcag cagtccagag 360 gcccccctga gatctgagga tagcacctgc
ctccctccca gccctggcct catcactgtg 420 cggctcaaat tcctcaatga
taccgaggag ctggctgtgg ctaggccaga ggataccgtg 480 ggtgccctga
agagcaaata cttccctgga caagaaagcc agatgaaact gatctaccag 540
ggccgcctgc tacaagaccc agcccgcaca ctgcgttctc tgaacattac cgacaactgt
600 gtgattcact gccaccgctc acccccaggg tcagctgttc caggcccctc
agcctccttg 660 gccccctcgg ccactgagcc acccagcctt ggtgtcaatg
tgggcagcct catggtgcct 720 gtctttgtgg tgctgttggg tgtggtctgg
tacttccgaa tcaattaccg ccaattcttc 780 acagcacctg ccactgtctc
cctggtggga gtcaccgtct tcttcagctt cctagtattt 840 gggatgtatg
gacgataagg acataggaag aaaatgaaag gcatggtctt tctcctttat 900
ggcctcccca cttttcctgg ccagagctgg gcccaagggc cggggaggga ggggtggaaa
960 ggatgtgatg gaaatctcct ccataggaca caggaggcaa gtatgcggcc
tccccttctc 1020 atccacagga gtacagatgt ccctcccgtg cgagcacaac
tcaggtagaa atgaggatgt 1080 catcttcctt cacttttagg gtcctctgaa
ggagttcaaa gctgctggcc aagctcagtg 1140 gggagcctgg gctctgagat
tccctcccac ctgtggttct gactcttccc agtgtcctgc 1200 atgtctgccc
ccagcaccca gggctgcctg caagggcagc tcagcatggc cccagcacaa 1260
ctccgtaggg agcctggagt atccttccat ttctcagcca aatactcatc ttttgagact
1320 gaaatcacac tggcgggaat gaagattgtg ccagccttct cttatgggca
cctagccgcc 1380 ttcaccttct tcctctaccc cttagcagga atagggtgtc
ctcccttctt tcaaagcact 1440 ttgcttgcat tttattttat ttttttaaga
gtccttcata gagctcagtc aggaagggga 1500 tggggcacca agccaagccc
ccagcattgg gagcggccag gccacagctg ctgctcccgt 1560 agtcctcagg
ctgtaagcaa gagacagcac tggcccttgg ccagcgtcct accctgccca 1620
actccaagga ctgggtatgg atygctgggc cctaggctct tgcttctggg gctattggag
1680 ggtcagtgtc tgtgactgaa taaagttcca ttttgtggta aaaaaaaaaa
aaaaaaaaaa 1740 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa agggcggcc 1779 110
1365 DNA Homo sapiens 110 ggcacgaggg agaactgctt taattagcct
aggtgaaaag tagtcctagc agtgtaaata 60 tgtataatta gagttttcta
atttcactgt gagatctcta acttttgagt ggcaaacaga 120 tcaagtcttt
tgctcataga cttttctgtg gggttattaa aatgcaaaag ctttattttt 180
tttaataatg ccatactcca ttagtgtcag atgatggtat ggaatttgtt cccttgcttt
240 cccccactgt tactgcttca gtttatagat tgccagcaga gttcagaaat
agagcaggga 300 tttacccgtt ctttgcttgg acatcccatt ttcttttgtc
cagacccatg ttggcaatca 360 tgtatgaact gtgttatact tctcagtgct
ttcttttttc tttttgataa gatggatatc 420 aaaaatagtt gctgtgcaaa
agttagtagt cttcttcaag aagaaaacca attctttttc 480 taataatatc
ctgtgaaatt gcttcattca ttcatttatt tttaagccaa atgtcagcag 540
agtgctgctg cttttatcta gtaattttga tatgtaagta ttaatgcatt tttaaaagat
600 gtctacattg aaacatgttc ttcccagtgt cctgcttatg atgctttgtt
cagatttttt 660 gtaagagacc agttagtaca ctgggggtgt atattgtgta
catgtgtcat tttagttagg 720 cattgtaggc caaatgtgat tataaatgaa
gttgatgaac attaattttg ttattagtga 780 gttttttgaa ttgtaaatgg
atttccagtt taccttctgt tgtctacagc ttttttaatt 840 ttaaggtttg
actaattgta tccatctcat tgtacagtgt tttagttgca agcagaaagt 900
agaatttggt ataaagcagg ttatttctat attgaaagga gtacagttga aattgtagat
960 ttaagattgt taaaatcatg acaattctaa cttgtctatt ctaacctatt
gtgtacaatc 1020 tgatttttta aaattgtaaa catgtatgat cttggtttca
tgtgtttttg aaagtgttat 1080 tgtttaaaaa atgaaaaaag catatctgct
aaagagctgt cagttttcat tactgactct 1140 gtaaaataca ctgttctttg
tgtactgtgt gttattttgc cagctgctgc attagccttc 1200 aaaagtattt
ggaaacttaa gatgaactac atttcttgca aagtacattc ctttctgtgg 1260
tattttgtcc tgtaactgaa gtatagtaat tattttatgg aaatgttagc aattctgtac
1320 caactttgaa taaaatgaaa aatttataaa aaaaaaaaaa aaaaa 1365 111
1957 DNA Homo sapiens 111 cctagctgtc cccctgagat gaagaaagag
ctccctgttg acagctgcct gccccgctca 60 ctcgagcttc accctcagaa
gatggatccc aagagacagc acattcagct cctgagcagc 120 ctgactgagt
gcctgacggt ggaccccctc agtgccagcg tctggaggca gctgtaccct 180
aagcacctgt cacagtccag ccttctgctg gagcacttgc tcagctcctg ggagcagatt
240 cccaagaagg tacagaagtc tttgcaagaa accattcagt ccctcaagct
taccaaccag 300 gagctgctga ggaagggtag cagtaacaac caggatgtcg
tcacctgtga catggcctgc 360 aagggcctgt tgcagcaggt tcagggtcct
cggctgccct ggacgcggct cctcctgttg 420 ctgctggtct tcgctgtagg
cttcctgtgc catgacctcc ggtcacacag ctccttccag 480 gcctccctta
ctggccggtt gcttcgatca tctggcttct tacctgctag ccaacaagcg 540
tgtgccaagc tctactccta cagtctgcaa ggctacagct ggctggggga gacactgccg
600 ctctggggct cccacctgct caccgtggtg cggcccagct tgcagctggc
ctgggctcac 660 accaatgcca cagtcagctt cctttctgcc cactgtgcct
ctcaccttgc gtggtttggt 720 gacagtctca ccagtctctc tcagaggcta
cagatccagc tccccgattc cgtgaatcag 780 ctactccgct atctgagaga
gctgcccctg cttttccacc agaatgtgct gctgccactg 840 tggcacctct
tgcttgaggc cctggcctgg gcccaggagc actgccatga ggcatgcaga 900
ggtgaggtga cctgggactg catgaagaca cagctcagtg aggctgtcca ctggacctgg
960 ctttgcctac aggacattac agtggctttc ttggactggg cacttgccct
gatatcccag 1020 cagtaggccc tgccttcctg gccactgatt tctgcatggg
tagaccatcc aagactgcag 1080 cgggtagaag gtggcagttc ttcatgggag
tctttttaac ttggtgcctg agttctctcc 1140 taggcaagtg gccagttgcc
tccacctcag ttcttccatc tttggtgggg acagggccca 1200 gcagcatctc
agcctcctac ccacaattcc actgaacact tttctggccc tactgcacat 1260
ggcccccagc ctccatcctt gtgctggtag cctctcacaa ctccgccctt gccctctgcc
1320 ttccacttcc ttccatctca tttctaaacc ccaaacagct catctctaaa
aagatagaac 1380 tcccagcagg tggcttctgt gttcttctga caaatgattc
ctgcttctcc agactttagc 1440 agcctcctgt tcccattctt ggtcacagct
ctagccacag cagaaggaaa ggggcttcca 1500 gaagaatata gcaccgcatt
gggaaacagc agcctcacct ccacctgaag cctgggtgtg 1560 gctgtcagtg
gacatgggga gctggatgga aatgcctctc acttcaaaat gcccagcctg 1620
ccccaaatgc ctctaagccc ctccctgtcc cctcccttgt agtcctactt cttccaactt
1680 tccattcccc atcatgctgg gggtcttggt cacaaggctc agcttctctc
cactgtccat 1740 ccctcctatc atctgtagag cagagcacag gcagttgtgt
gccttgggcc cagggaaccc 1800 tccatcaacc tgagacagga ctcagtatat
ggttcttggg tatgccctac caggtggaat 1860 aaaggacaca gatttgattt
ctaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaa 1957 112 1135 DNA Homo sapiens 112
acgagctgaa atcttggagg gaagaaaaca catcccaccc tgcctccggg aaggggcctc
60 tcctggacat gtctcctgca gctgctgctg agccagatgg ggaccagcag
gacagacacg 120 tcagcaaact catcttctgc ttctttgtct tcggcgccgt
cttgttgtgt gtgggagtcc 180 tgctctccat ctttgggttc caggcatgcc
aatataagcc cctcccagac tgccccatgg 240 tgctcaaggt ggcggggctg
catgtgccgt ggttgggctt ggggctgtga tcctggcccg 300 ctcccgggcg
caacttcagc tccgtgcagg gctgcagaga ggtcagcaga tggaccccga 360
ccgagccttc atctgtggag agagccgcca gtttgcccag tgccttatct ttgggtttct
420 gttcttgaca agcggcatgc tcatcagcgt cctgggcatt tgggtccctg
gatgtggctc 480 caactgggcg caggaaccgc taaacgagac agacactggc
gactcagagc cccggatgtg 540 tgggttcctt tctctgcaga tcatggggcc
cttgattgtg cttgtgggat tgtgtttctt 600 cgtggttgcc catgttaaga
agagaaacac gctgaatgct ggccaggatg cctctgagag 660 agaagaggga
cagatccaga ttatggagcc tgtccaggtc actgtaggtg actcggtaat 720
aatatttcca ccccctccac caccttactt tcctgaatct tcagcttctg cggtcgctga
780 gagtcctgga actaacagtc tgcttccgaa tgaaaacccc ccttcatatt
acagtatttt 840 caactatggg accccaactt cagagggtgc agcctctgaa
agagactgtg aatctatata 900 taccatttct gggacgaatt catcttctga
ggcctcacac actccacatc ttccatctga 960 attgcctcct agatatgaag
aaaaagaaaa tgctgcagct acattcttgc ctctatcttc 1020 tgagccttcc
ccaccgtaaa ctatggactc tagttcagtt ttatatgcaa tggatcacta 1080
ttttatttaa ttttttttaa ataaaaaata caatagcaaa aaaaaaaaaa aaaaa 1135
113 1446 DNA Homo sapiens 113 ggcacgagcg gaagtgcaac tcgaacttgg
tcggggcgcg gatcccgaga gggaaagtca 60 taacaaccgc acgagggagt
tcgactggcg aactggaagg ccacgcctcc tcccgcctgc 120 cccctcagcc
ctgtggctgg ggcagagctc agactgtctt ctgaagattg atgtctattt 180
ccttgagctc tttaattttg ttgccaattt ggataaacat ggcacaaatc cagcagggag
240 gtccagatga aaaagaaaag actaccgcac tgaaagattt attatctagg
atagatttgg 300 atgaactaat gaaaaaagat gaaccgcctc ttgatttcct
gataccctgg aaggtttgaa 360 tatgctttta atgaaaaggg acagttaaga
cacataaaaa ctggggaacc atttgttttt 420 aactaccggg aagatttaca
cagatggaac cagaaaagat acgaggctct aggagagatc 480 atcacgaata
tgtatatgag ctcctggaaa aggattgtaa tttgaaaaaa gtatctattc 540
cagtagatgc cactgagagt gaaccaaaga gttttatctt tatgagtgag gatgctttga
600 caaatccaca gaagctgatg gttttaattc atggtagtgg tgttgtcagg
gcagggcagt 660 gggctagaag acttattata aatgaagatc tggacagtgg
cacacagata ccgtttatta 720 aaagagctgt ggctgaagga tatggagtaa
tagtactaaa tcccaatgaa aactatattg 780 aagtagaaaa gccgaagata
cacgtacagt catcatctga tagttcagat gaaccagcag 840 aaaaacggga
aagaaaagat aaagtttcta aagaaacaaa gaaccgacgt gatttctatg 900
agaactatcg taacccccaa agagaaaaag aaaggatgca attgtatatc agagaaaatg
960 gttctcctga agaacatgca atctatgttt gggatcattt catagctcag
gctgctgctg 1020 agaatgtgtt tttcgttgct cacagctatg gaggacttgc
ttttgttgaa ctgcaactca 1080 tgatcaaaca agctaattca gatgctggga
agtgctttcg cttagctatg tggaagaacc 1140 attgactgta tacaaccaac
aagtgtatgg tgcaacagga gatccattga aaaccgttta 1200 taggactgaa
cgacaacccc aaatgcaagt gaccatgagc aactacaaat aggtatacat 1260
atgcatttga gctgaacaga ctttctgaca tataatttag tcaaaattgc tgtatttctt
1320 ccccttaaat ttatacataa tcagcttctt gtatggaccc aaattggaga
aatgtaattc 1380 agtagttggt gagaaataaa ggattgtgac ctctgtgtaa
ttatcaggaa aaaaaaaaaa 1440 aaaaaa 1446 114 733 DNA Homo sapiens 114
tgtgaccgat atctgcaraa ttcggcttat cgygaacctg gctttggygg acctgggact
60 ggcactcact ctcccctttt gggcagccga gtcggcactg gactttcact
ggcccttcgg 120 aggtgccctc tgcaagatgg ttctgacggc cactgtcctc
aacgtctatg
ccagcatctt 180 cctcatcaca gcgctgagcg ttgctcgcta ctgggtggtg
gccatggctg cggggccagg 240 cacccacctc tcactcttct gggcccgaat
agccaccctg gcagtgtggg cggcggctgc 300 cctggtgacg gtgcccacag
ctgtcttcgg ggtggarggt gargtgtgtg gtgtgcgcct 360 ttgcctgctg
cgtttcccca gcaggtactg gctgggggcc taccagctgc agagggtggt 420
gctggctttc atggtgccct tgggcgtcat caccaccagc tacctgctgc tgctggcctt
480 cctgcagcgg cggcaacggc ggcggcagga cagcagggtc gtggcccgct
ctgtccgcat 540 cctggtggct tccttcttcc tctgctggtt tcccaaccat
gtggtcactc tctggggtgt 600 cctggtgaag tttgacctgg tgccctggaa
cagtactttc tatactatcc agacgtatgt 660 cttccctgtc actacttgct
tggcacacag caatagctgc ctsaacccaw tagcytaygt 720 cttaagcmga att 733
115 1518 DNA Homo sapiens SITE (1146) n equals a,t,g, or c 115
aggagaaact ctaaaaactg cagatattat ttcatgctat atgttccatc ctctgatgag
60 aatgtgagga aagaaaattg tatcctgcat ggctgaaaat ggtcccctac
aaaaatatca 120 tgttggacaa ctaatctgag atagtggtat ctctggaaag
cagtttagca ctggtgagtt 180 tggactttca tggcaggctg ccttggttca
tatcttttgg taatgatact tatcctctgt 240 raggcccatt tctttatttg
tggaaatgaa gacaatagag tgcttagata taatttasca 300 acaatgtccg
tcacatagta aacacgtaat aaacggtagc tcttattgtt attattatta 360
ctattattac cttgaagaca ggggctctgt cttgttcatc attccatctc cagctcttag
420 cacagtccct ggcacaattc aaacatgtat ttggatgaat gacaaatagc
tactgaatat 480 ttgccctgtt ccaagcattg ttagaggtac atgggacagg
gcagtgaaca aaacagacaa 540 aacctcctgc tgtctcagag ttcacactct
aatggggaga cccaggcaat gaggaaataa 600 ttaaaatata caatgtgtct
tatggcaata aatgacaaag aaaaataaag cagaggtgag 660 aaacagtggc
agtgttttgg tgatcatttg ctttgcaaca agccactccc caaagttagt 720
ggcctaaaac aatttaatca cagttcatgt tctggctaca acaatacaca tccctctcat
780 gtgcaaaata cactcactcc tccctcagag cctcgtacca ttaagggttc
aggttcaaag 840 cttaagatct tatcctctga agtaggttta gggacaaaca
agtcttctca ggtacttctt 900 ctggggacac agagacttgt gaactaaaag
acaagttacc taccttccaa cacaactgac 960 atgcaatggg gatataggaa
aagataattt caataggcgc ttctgtgcaa aagcggggga 1020 aatgagagtc
actcagcagt cacggttcat attaatctaa aatctagcca ggcatatatc 1080
ccaagtcttc ctgatgtgag gacaagaatt atttcttgat tagggctcac ttwwtctctt
1140 tgaggntggt tcgcctcagc ttttggattt gtcctctgaa tcatccttcc
ttgtctataa 1200 aatgcatgta tatactcata catacataga gagaaagaga
gagagagaga gagagagact 1260 ctgtcacgca ggctggagtg caatggtgtg
atctcagctc actgcaacct acaactcctg 1320 ggttcaagca attctcctgt
ctcagcctcc cgagcacctg tagtccctgc tactcaggag 1380 gctgaggcag
gagaattgct tgaatccgag aggcagaggt tgtcagtgag cagagattac 1440
accactgcac tccagcttgg gtgacagagc aaggcttcat ctcaaaaaaa gacaaaaaaa
1500 aaaaaaaaaa actcgtag 1518 116 1054 DNA Homo sapiens 116
ggcacgagtg tgtgtgtgtg tgtgtgtgtg tgtctgtctg tgtgtctgtg tatgtgtatt
60 tctggctgtc tgttccattg ctctatatgt ctgtttttta tgctggtacc
atactgtttt 120 gattactgtt tagtaatgta ttttgaaatc aagacatgtg
ggtacctcct gctttgttct 180 ccttgtcaag attattccag gtcttttgtt
gcttcttcat agacgaatta actgctgatt 240 tatgaacttg aatattctga
tttctttgac agttagttct cattgtaaat tgataaatta 300 tcactctggt
tttatacatc agtttttagc tatggctaat aacagtcttt cctcacaatt 360
catatttagc atgttggcaa aatcatattt tggaacctgc aagacatagt ctctggtcta
420 tagtaaatca agctgctagg ttgtagtctg acaacttgtg taatatttta
gctctggatg 480 atattaattt ttaagattat taaattttat ttttcagtgt
tttacattga cagcaaaatt 540 gagtgggaag tacatactaa tttttctgta
tcttagaatt tctttgggat cattttaact 600 attttaatgt tttaaatttt
attgtgaatc tttttaagga aggctgagct gttgctacaa 660 ctgtaaaata
aatattctta aagcaggcag tgatgatcaa aatcttgcca tttgaccatt 720
aagctgctag aatatgagag tgataattta ggaatgagtt gattaaagaa aataacaaag
780 tagtttacta aggaattaat aatagcaaat aaaaggttta acaaacaaca
ataaatattc 840 tgttgatatt gcaccttaac tttccatcat catcttggga
gctgactttt ttgctgattt 900 cattccgata agataagttc atttgaccac
gtgattatta tttaatacat ctactgataa 960 ctctataata gaaagtggca
gattttagat aaagggtttg tgatttttaa ggttgatatt 1020 aacaggtagt
atcataaaaa aaaaaaaaaa aaaa 1054 117 921 DNA Homo sapiens 117
ggcacgagac gccgtgagcg ggacgaccag atgtaccgag agcggctgcg caccttgctg
60 gtcatcgcgg ttgtcatgag cctcctgaat gctctcagca ccagcggagg
cagcatttcc 120 tggaacgact ttgtccacga gatgctggcc aagggcgagg
tgcagcgcgt ccaggtggtg 180 cctgagagcg acgtggtgga agtctacctg
caccctggag ccgtggtgtt tgggcggcct 240 cggctagcct tgatgtaccg
aatgcagttg caaatattga caagtttgaa gagaagcttc 300 gagcagctga
agatgagctg aatatcgagg ccaaggacag gatcccagtt tcctacaagc 360
gaacaggatt ctttgggaaa tgccctgtac tctgtgggga tgacggtagt gggcctggcc
420 atcctgtggt atgttttccg tctggccggg atgactggag gcaccgccgg
cgatggacgt 480 ccatgtcccg gctcctgtgc tggaaagcgt tgatggggag
cgtcggcgct gaccacacgc 540 gggagctgcg gaagcccagc ggttcacaca
ggcctccctt caacgtagtc atcccctggt 600 ggtggaagca agacgacggc
ccctgacgtg cagccacaca cagaaaaggc tgctgtgaac 660 attttatgct
tcgacttttt ttttcttcag agacagggtg tcgttctgtc gcccaggctg 720
gagtgcagtg ccaccatcat agctcactgc agcctccacc tcctaggctc aagcttccta
780 agtagttggg actcaaggct tgagtcacca tgccaggctc tgttttttca
gtctgtgaaa 840 aataaagtca tcagcatgtg aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 900 aaaaaaaaaa aaaaaaaaaa a 921 118 243 PRT
Homo sapiens 118 Met Gly Thr Leu Pro Trp Leu Leu Ala Phe Phe Ile
Leu Gly Leu Gln 1 5 10 15 Ala Trp Asp Thr Pro Thr Ile Val Ser Arg
Lys Glu Trp Gly Ala Arg 20 25 30 Pro Leu Ala Cys Arg Ala Leu Leu
Thr Leu Pro Val Ala Tyr Ile Ile 35 40 45 Thr Asp Gln Leu Pro Gly
Met Gln Cys Gln Gln Gln Ser Val Cys Ser 50 55 60 Gln Met Leu Arg
Gly Leu Gln Ser His Ser Val Tyr Thr Ile Gly Trp 65 70 75 80 Cys Asp
Val Ala Tyr Asn Phe Leu Val Gly Asp Asp Gly Arg Val Tyr 85 90 95
Glu Gly Val Gly Trp Asn Ile Gln Gly Leu His Thr Gln Gly Tyr Asn 100
105 110 Asn Ile Ser Leu Gly Ile Ala Phe Phe Gly Asn Lys Ile Ser Ser
Ser 115 120 125 Pro Ser Pro Ala Ala Leu Ser Ala Ala Glu Gly Leu Ile
Ser Tyr Ala 130 135 140 Ile Gln Lys Gly His Leu Ser Pro Arg Tyr Ile
Gln Pro Leu Leu Leu 145 150 155 160 Lys Glu Glu Thr Cys Leu Asp Pro
Gln His Pro Val Met Pro Arg Lys 165 170 175 Val Cys Pro Asn Ile Ile
Lys Arg Ser Ala Trp Glu Ala Arg Glu Thr 180 185 190 His Cys Pro Lys
Met Asn Leu Pro Ala Lys Tyr Val Ile Ile Ile His 195 200 205 Thr Ala
Gly Thr Ser Cys Thr Val Ser Thr Asp Cys Gln Thr Val Val 210 215 220
Arg Asn Ile Gln Ser Phe His Met Asp Thr Arg Asn Phe Cys Asp Ile 225
230 235 240 Gly Tyr Gln 119 41 PRT Homo sapiens 119 Met Lys Arg Arg
Glu Met Thr Gln Phe Leu Leu Ser Leu Val Ala Leu 1 5 10 15 Asn Cys
Cys Ser Ile Ser Leu Gly Arg Leu Thr Tyr Pro Gly Gly Phe 20 25 30
His Leu Lys Leu Asp Pro Leu Glu Leu 35 40 120 526 PRT Homo sapiens
SITE (466) Xaa equals any of the naturally occurring L-amino acids
120 Met Ala Ala Leu Thr Ile Ala Thr Gly Thr Gly Asn Trp Phe Ser Ala
1 5 10 15 Leu Ala Leu Gly Val Thr Leu Leu Lys Cys Leu Leu Ile Pro
Thr Tyr 20 25 30 His Ser Thr Asp Phe Glu Val His Arg Asn Trp Leu
Ala Ile Thr His 35 40 45 Ser Leu Pro Ile Ser Gln Trp Tyr Tyr Glu
Ala Thr Ser Glu Trp Thr 50 55 60 Leu Asp Tyr Pro Pro Phe Phe Ala
Trp Phe Glu Tyr Ile Leu Ser His 65 70 75 80 Val Ala Lys Tyr Phe Asp
Gln Glu Met Leu Asn Val His Asn Leu Asn 85 90 95 Tyr Ser Ser Ser
Arg Thr Leu Leu Phe Gln Arg Phe Ser Val Ile Phe 100 105 110 Met Asp
Val Leu Phe Val Tyr Ala Val Arg Glu Cys Cys Lys Cys Ile 115 120 125
Asp Gly Lys Lys Val Gly Lys Glu Leu Thr Glu Lys Pro Lys Phe Ile 130
135 140 Leu Ser Val Leu Leu Leu Trp Asn Phe Gly Leu Leu Ile Val Asp
His 145 150 155 160 Ile His Phe Gln Tyr Asn Gly Phe Leu Phe Gly Leu
Met Leu Leu Ser 165 170 175 Ile Ala Arg Leu Phe Gln Lys Arg His Met
Glu Gly Ala Phe Leu Phe 180 185 190 Ala Val Leu Leu His Phe Lys His
Ile Tyr Leu Tyr Val Ala Pro Ala 195 200 205 Tyr Gly Val Tyr Leu Leu
Arg Ser Tyr Cys Phe Thr Ala Asn Lys Pro 210 215 220 Asp Gly Ser Ile
Arg Trp Lys Ser Phe Ser Phe Val Arg Val Ile Ser 225 230 235 240 Leu
Gly Leu Val Val Phe Leu Val Ser Ala Leu Ser Leu Gly Pro Phe 245 250
255 Leu Ala Leu Asn Gln Leu Pro Gln Val Phe Ser Arg Leu Phe Pro Phe
260 265 270 Lys Arg Gly Leu Cys His Ala Tyr Trp Ala Pro Asn Phe Trp
Ala Leu 275 280 285 Tyr Asn Ala Leu Asp Lys Val Leu Ser Val Ile Gly
Leu Lys Leu Lys 290 295 300 Phe Leu Asp Pro Asn Asn Ile Pro Lys Ala
Ser Met Thr Ser Gly Leu 305 310 315 320 Val Gln Gln Phe Gln His Thr
Val Leu Pro Ser Val Thr Pro Leu Ala 325 330 335 Thr Leu Ile Cys Thr
Leu Ile Ala Ile Leu Pro Ser Ile Phe Cys Leu 340 345 350 Trp Phe Lys
Pro Gln Gly Pro Arg Gly Phe Leu Arg Cys Leu Thr Leu 355 360 365 Cys
Ala Leu Ser Ser Phe Met Phe Gly Trp His Val His Glu Lys Ala 370 375
380 Ile Leu Leu Ala Ile Leu Pro Met Ser Leu Leu Ser Val Gly Lys Ala
385 390 395 400 Gly Asp Ala Ser Ile Phe Leu Ile Leu Thr Thr Thr Gly
His Tyr Ser 405 410 415 Leu Phe Pro Leu Leu Phe Thr Ala Pro Glu Leu
Pro Ile Lys Ile Leu 420 425 430 Leu Met Leu Leu Phe Thr Ile Tyr Ser
Ile Ser Ser Leu Lys Thr Leu 435 440 445 Phe Arg Lys Glu Lys Pro Leu
Phe Asn Trp Met Glu Thr Phe Tyr Leu 450 455 460 Leu Xaa Leu Gly Pro
Leu Glu Val Cys Cys Glu Phe Val Phe Pro Phe 465 470 475 480 Thr Ser
Trp Lys Val Lys Tyr Pro Phe Ile Pro Leu Leu Leu Thr Ser 485 490 495
Val Tyr Cys Ala Val Gly Ile Thr Tyr Ala Trp Phe Lys Leu Tyr Val 500
505 510 Ser Val Leu Ile Asp Ser Ala Ile Gly Lys Thr Lys Lys Gln 515
520 525 121 354 PRT Homo sapiens SITE (98) Xaa equals any of the
naturally occurring L-amino acids SITE (100) Xaa equals any of the
naturally occurring L-amino acids SITE (109) Xaa equals any of the
naturally occurring L-amino acids SITE (123) Xaa equals any of the
naturally occurring L-amino acids SITE (129) Xaa equals any of the
naturally occurring L-amino acids SITE (131) Xaa equals any of the
naturally occurring L-amino acids SITE (159) Xaa equals any of the
naturally occurring L-amino acids SITE (169) Xaa equals any of the
naturally occurring L-amino acids SITE (171) Xaa equals any of the
naturally occurring L-amino acids SITE (172) Xaa equals any of the
naturally occurring L-amino acids SITE (175) Xaa equals any of the
naturally occurring L-amino acids SITE (183) Xaa equals any of the
naturally occurring L-amino acids SITE (188) Xaa equals any of the
naturally occurring L-amino acids SITE (189) Xaa equals any of the
naturally occurring L-amino acids SITE (225) Xaa equals any of the
naturally occurring L-amino acids SITE (229) Xaa equals any of the
naturally occurring L-amino acids SITE (231) Xaa equals any of the
naturally occurring L-amino acids 121 Met Glu Asp Gly Val Leu Lys
Glu Gly Phe Leu Val Lys Arg Gly His 1 5 10 15 Ile Val His Asn Trp
Lys Ala Arg Trp Phe Ile Leu Arg Gln Asn Thr 20 25 30 Leu Val Tyr
Tyr Lys Leu Glu Gly Gly Arg Arg Val Thr Pro Pro Lys 35 40 45 Gly
Arg Ile Leu Leu Asp Gly Cys Thr Ile Thr Cys Pro Cys Leu Glu 50 55
60 Tyr Glu Asn Arg Pro Leu Leu Ile Lys Leu Lys Thr Gln Thr Ser Thr
65 70 75 80 Glu Tyr Phe Leu Glu Ala Cys Ser Arg Glu Glu Ala Gly Cys
Leu Gly 85 90 95 Leu Xaa Arg Xaa Pro Gly Leu Phe Met Gln Gly Ser
Xaa Gly Lys Val 100 105 110 Gln Gln Leu His Ser Leu Arg Asn Ser Phe
Xaa Leu Pro Pro His Ile 115 120 125 Xaa Leu Xaa Arg Ile Val Asp Lys
Met His Asp Ser Asn Thr Gly Ile 130 135 140 Arg Ser Ser Pro Asn Met
Glu Gln Arg Ser Thr Tyr Lys Lys Xaa Phe 145 150 155 160 Leu Gly Ser
Ser Leu Val Asp Trp Xaa Ile Xaa Xaa Ser Phe Xaa Gly 165 170 175 Ser
Arg Leu Glu Ala Val Xaa Leu Ala Ser Met Xaa Xaa Glu Glu Asn 180 185
190 Phe Leu Arg Ser Val Ala Val Arg Cys Met Gly Gly Ile Arg Ser Gly
195 200 205 Asp Leu Ala Glu Gln Phe Leu Asp Asp Ser Thr Ala Leu Tyr
Thr Phe 210 215 220 Xaa Glu Ser Tyr Xaa Lys Xaa Ile Ser Pro Lys Glu
Glu Ile Ser Leu 225 230 235 240 Ser Thr Val Glu Leu Ser Gly Thr Val
Val Lys Gln Gly Tyr Leu Ala 245 250 255 Lys Gln Gly His Lys Arg Lys
Asn Trp Lys Val Arg Arg Phe Val Leu 260 265 270 Arg Lys Asp Pro Ala
Phe Leu His Tyr Tyr Asp Pro Ser Lys Glu Glu 275 280 285 Asn Arg Pro
Val Gly Gly Phe Ser Leu Arg Gly Ser Leu Val Ser Ala 290 295 300 Leu
Glu Asp Asn Gly Val Pro Thr Gly Val Lys Gly Asn Val Gln Gly 305 310
315 320 Asn Leu Phe Lys Val Ile Thr Lys Asp Asp Thr His Tyr Tyr Ile
Gln 325 330 335 Ala Ser Ser Lys Ala Glu Arg Ala Glu Trp Ile Glu Ala
Ile Lys Lys 340 345 350 Leu Thr 122 63 PRT Homo sapiens 122 Met Trp
Lys Arg Val Cys Val Cys Val Phe Leu Tyr Ile Ala Trp Val 1 5 10 15
Gln Leu Trp Met Cys Ala Lys Glu Cys Glu Cys Val Cys Val Cys Val 20
25 30 Lys Gly Ser Val Leu Glu Pro Thr Ser Val Cys Cys Glu Ser Gly
Lys 35 40 45 Arg Val Gly Glu Gly Arg Glu Met Leu Thr Leu Val Gly
Ala Gly 50 55 60 123 309 PRT Homo sapiens SITE (129) Xaa equals any
of the naturally occurring L-amino acids SITE (178) Xaa equals any
of the naturally occurring L-amino acids SITE (187) Xaa equals any
of the naturally occurring L-amino acids SITE (262) Xaa equals any
of the naturally occurring L-amino acids SITE (308) Xaa equals any
of the naturally occurring L-amino acids 123 Met Phe Thr Ile Lys
Leu Leu Leu Phe Ile Val Pro Leu Val Ile Ser 1 5 10 15 Ser Arg Ile
Asp Gln Asp Asn Ser Ser Phe Asp Ser Leu Ser Pro Glu 20 25 30 Pro
Lys Ser Arg Phe Ala Met Leu Asp Asp Val Lys Ile Leu Ala Asn 35 40
45 Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp Phe Val His Lys Thr
50 55 60 Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu Asn Ile Phe
Asp Gln 65 70 75 80 Ser Phe Tyr Asp Leu Ser Leu Gln Thr Ser Glu Ile
Lys Glu Glu Glu 85 90 95 Lys Glu Leu Arg Arg Thr Thr Tyr Lys Leu
Gln Val Lys Asn Glu Glu 100 105 110 Val Lys Asn Met Ser Leu Glu Leu
Asn Ser Lys Leu Glu Ser Leu Leu 115 120 125 Xaa Glu Lys Ile Leu Leu
Gln Gln Lys Val Lys Tyr Leu Glu Glu Gln 130 135 140 Leu Thr Asn Leu
Ile Gln Asn Gln Pro Glu Thr Pro Glu His Pro Glu 145 150 155 160 Val
Thr Ser Leu Lys Thr Phe Val Glu Lys Gln Asp Asn Ser Ile Lys 165 170
175 Asp Xaa Leu Gln Thr Val Glu Asp Gln Tyr Xaa Gln Leu Asn Gln Gln
180 185 190 His Ser Gln Ile Lys Glu Ile Glu Asn Gln Leu Arg Arg Thr
Ser Ile 195 200 205 Gln Glu Pro Thr Glu Ile Ser Leu Ser Ser Lys Pro
Arg Ala Pro Arg 210 215 220 Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile
Arg Asn Val Lys His Asp 225 230 235 240 Gly Ile Pro Ala Glu Cys Thr
Thr Ile Tyr Asn Arg Gly Glu His Thr 245 250 255 Ser Gly Met Tyr Ala
Xaa Arg Pro Ser Asn Ser Gln Val Phe His Val 260 265 270 Tyr Cys
Asp
Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg 275 280 285 Ile
Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr 290 295
300 Gly Phe Gly Xaa Ala 305 124 211 PRT Homo sapiens SITE (99) Xaa
equals any of the naturally occurring L-amino acids 124 Met Ala Asn
Ala Gly Leu Gln Leu Leu Gly Phe Ile Leu Ala Phe Leu 1 5 10 15 Gly
Trp Ile Gly Ala Ile Val Ser Thr Ala Leu Pro Gln Trp Arg Ile 20 25
30 Tyr Ser Tyr Ala Gly Asp Asn Ile Val Thr Ala Gln Ala Met Tyr Glu
35 40 45 Gly Leu Trp Met Ser Cys Val Ser Gln Ser Thr Gly Gln Ile
Gln Cys 50 55 60 Lys Val Phe Asp Ser Leu Leu Asn Leu Ser Ser Thr
Leu Gln Ala Thr 65 70 75 80 Arg Ala Leu Met Val Val Gly Ile Leu Leu
Gly Val Ile Ala Ile Phe 85 90 95 Val Ala Xaa Val Gly Met Lys Cys
Met Lys Cys Leu Glu Asp Asp Glu 100 105 110 Val Gln Lys Met Arg Met
Ala Val Ile Gly Gly Ala Ile Phe Leu Leu 115 120 125 Ala Gly Leu Ala
Ile Leu Val Ala Thr Ala Trp Tyr Gly Asn Arg Ile 130 135 140 Val Gln
Glu Phe Tyr Asp Pro Met Thr Pro Val Asn Ala Arg Tyr Glu 145 150 155
160 Phe Gly Gln Ala Leu Phe Thr Gly Trp Ala Ala Ala Ser Leu Cys Leu
165 170 175 Leu Gly Gly Ala Leu Leu Cys Cys Ser Cys Pro Arg Lys Thr
Thr Ser 180 185 190 Tyr Pro Thr Pro Arg Pro Tyr Pro Lys Pro Ala Pro
Ser Ser Gly Lys 195 200 205 Asp Tyr Val 210 125 50 PRT Homo sapiens
125 Met Ala Pro Leu Trp Thr Leu Arg Pro Val Leu Val Trp Thr Thr Pro
1 5 10 15 Thr Ser Met Gly Glu Val Ser Pro Trp Leu Thr Ser Thr Val
Met Ala 20 25 30 Lys Trp Thr Ser Ser Met Ala Thr Gly Met Ala Pro
Thr Ala Ser Ile 35 40 45 Cys Arg 50 126 262 PRT Homo sapiens 126
Met Leu Phe Ser Ala Leu Leu Leu Glu Val Ile Trp Ile Leu Ala Ala 1 5
10 15 Asp Gly Gly Gln His Trp Thr Tyr Glu Gly Pro His Gly Gln Asp
His 20 25 30 Trp Pro Ala Ser Tyr Pro Glu Cys Gly Asn Asn Ala Gln
Ser Pro Ile 35 40 45 Asp Ile Gln Thr Asp Ser Val Thr Phe Asp Pro
Asp Leu Pro Ala Leu 50 55 60 Gln Pro His Gly Tyr Asp Gln Pro Gly
Thr Glu Pro Leu Asp Leu His 65 70 75 80 Asn Asn Gly His Thr Val Gln
Leu Ser Leu Pro Ser Thr Leu Tyr Leu 85 90 95 Gly Gly Leu Pro Arg
Lys Tyr Val Ala Ala Gln Leu His Leu His Trp 100 105 110 Gly Gln Lys
Gly Ser Pro Gly Gly Ser Glu His Gln Ile Asn Ser Glu 115 120 125 Ala
Thr Phe Ala Glu Leu His Ile Val His Tyr Asp Ser Asp Ser Tyr 130 135
140 Asp Ser Leu Ser Glu Ala Ala Glu Arg Pro Gln Gly Leu Ala Val Leu
145 150 155 160 Gly Ile Leu Ile Glu Leu Glu Lys Leu Gln Gly Thr Leu
Phe Ser Thr 165 170 175 Glu Glu Glu Pro Ser Lys Leu Leu Val Gln Asn
Tyr Arg Ala Leu Gln 180 185 190 Pro Leu Asn Gln Arg Met Val Phe Ala
Ser Phe Ile Gln Ala Gly Ser 195 200 205 Ser Tyr Thr Thr Gly Glu Met
Leu Ser Leu Gly Val Gly Ile Leu Val 210 215 220 Gly Cys Leu Cys Leu
Leu Leu Ala Val Tyr Phe Ile Ala Arg Lys Ile 225 230 235 240 Arg Lys
Lys Arg Leu Glu Asn Arg Lys Ser Val Val Phe Thr Ser Ala 245 250 255
Gln Ala Thr Thr Glu Ala 260 127 270 PRT Homo sapiens SITE (27) Xaa
equals any of the naturally occurring L-amino acids 127 Met His Tyr
Tyr Arg Tyr Ser Asn Ala Lys Val Ser Cys Trp Tyr Lys 1 5 10 15 Tyr
Leu Leu Phe Ser Tyr Asn Ile Ile Phe Xaa Leu Ala Gly Val Val 20 25
30 Phe Leu Gly Val Gly Leu Trp Ala Trp Ser Glu Lys Gly Val Leu Ser
35 40 45 Asp Leu Thr Lys Val Thr Arg Met His Gly Ile Asp Pro Val
Val Leu 50 55 60 Val Leu Met Val Gly Val Val Met Phe Thr Leu Gly
Phe Ala Gly Cys 65 70 75 80 Val Gly Ala Leu Arg Glu Asn Ile Cys Leu
Leu Asn Phe Phe Cys Gly 85 90 95 Thr Ile Val Leu Ile Phe Phe Leu
Glu Leu Ala Val Ala Val Leu Ala 100 105 110 Phe Leu Phe Gln Asp Trp
Val Arg Asp Arg Phe Arg Glu Phe Phe Glu 115 120 125 Ser Asn Ile Lys
Ser Tyr Arg Asp Asp Ile Asp Leu Gln Asn Leu Ile 130 135 140 Asp Ser
Leu Gln Lys Ala Asn Gln Cys Cys Gly Ala Tyr Gly Pro Glu 145 150 155
160 Asp Trp Asp Leu Asn Val Tyr Phe Asn Cys Ser Gly Ala Ser Tyr Ser
165 170 175 Arg Glu Lys Cys Gly Val Pro Phe Ser Cys Cys Val Pro Asp
Pro Ala 180 185 190 Gln Lys Val Val Asn Thr Gln Cys Gly Tyr Asp Val
Arg Ile Gln Leu 195 200 205 Lys Ser Lys Trp Asp Glu Ser Ile Phe Thr
Lys Gly Cys Ile Gln Ala 210 215 220 Leu Glu Ser Trp Leu Pro Arg Asn
Ile Tyr Ile Val Ala Gly Val Phe 225 230 235 240 Ile Ala Ile Ser Leu
Leu Gln Ile Phe Gly Ile Phe Leu Ala Arg Thr 245 250 255 Leu Ile Ser
Asp Ile Glu Ala Val Lys Ala Gly His His Phe 260 265 270 128 91 PRT
Homo sapiens 128 Met Leu Arg Cys Gly Gly Arg Gly Leu Leu Leu Gly
Leu Ala Val Ala 1 5 10 15 Ala Ala Ala Val Met Ala Ala Arg Leu Met
Gly Trp Trp Gly Pro Arg 20 25 30 Ala Gly Phe Arg Leu Phe Ile Pro
Glu Glu Leu Ser Arg Tyr Arg Gly 35 40 45 Gly Pro Gly Asp Pro Gly
Leu Tyr Leu Ala Leu Leu Gly Arg Val Tyr 50 55 60 Asp Val Ser Ser
Gly Arg Ser Thr Thr Ser Leu Gly Pro Thr Ile Ala 65 70 75 80 Ala Ser
Gln Ala Glu Thr His Pro Glu Leu Ser 85 90 129 222 PRT Homo sapiens
SITE (120) Xaa equals any of the naturally occurring L-amino acids
129 Met Leu Trp Leu Leu Phe Phe Leu Val Thr Ala Ile His Ala Glu Leu
1 5 10 15 Cys Gln Pro Gly Ala Glu Asn Ala Phe Lys Val Arg Leu Ser
Ile Arg 20 25 30 Thr Ala Leu Gly Asp Lys Ala Tyr Ala Trp Asp Thr
Asn Glu Glu Tyr 35 40 45 Leu Phe Lys Ala Met Val Ala Phe Ser Met
Arg Lys Val Pro Asn Arg 50 55 60 Glu Ala Thr Glu Ile Ser His Val
Leu Leu Cys Asn Val Thr Gln Arg 65 70 75 80 Val Ser Phe Trp Phe Val
Val Thr Asp Pro Ser Lys Asn His Thr Leu 85 90 95 Pro Ala Val Glu
Val Gln Ser Ala Ile Arg Met Asn Lys Asn Arg Ile 100 105 110 Asn Asn
Ala Phe Phe Leu Asn Xaa Gln Thr Leu Glu Phe Leu Lys Ile 115 120 125
Pro Ser Thr Leu Ala Pro Pro Met Asp Pro Ser Val Pro Ile Trp Ile 130
135 140 Ile Ile Phe Gly Val Ile Phe Cys Ile Ile Ile Val Ala Ile Ala
Leu 145 150 155 160 Leu Ile Leu Ser Gly Ile Trp Gln Arg Arg Arg Lys
Asn Lys Glu Pro 165 170 175 Ser Glu Val Asp Asp Ala Glu Asp Lys Cys
Glu Asn Met Ile Thr Ile 180 185 190 Glu Asn Gly Ile Pro Ser Asp Pro
Leu Asp Met Lys Gly Gly His Ile 195 200 205 Asn Asp Ala Phe Met Thr
Glu Asp Glu Arg Leu Thr Pro Leu 210 215 220 130 760 PRT Homo
sapiens SITE (267) Xaa equals any of the naturally occurring
L-amino acids SITE (315) Xaa equals any of the naturally occurring
L-amino acids SITE (438) Xaa equals any of the naturally occurring
L-amino acids 130 Met Ile Pro Asn Gln His Asn Ala Gly Ala Gly Ser
His Gln Pro Ala 1 5 10 15 Val Phe Arg Met Ala Val Leu Asp Thr Asp
Leu Asp His Ile Leu Pro 20 25 30 Ser Ser Val Leu Pro Pro Phe Trp
Ala Lys Leu Val Val Gly Ser Val 35 40 45 Ala Ile Val Cys Phe Ala
Arg Ser Tyr Asp Gly Asp Phe Val Phe Asp 50 55 60 Asp Ser Glu Ala
Ile Val Asn Asn Lys Asp Leu Gln Ala Glu Thr Pro 65 70 75 80 Leu Gly
Asp Leu Trp His His Asp Phe Trp Gly Ser Arg Leu Ser Ser 85 90 95
Asn Thr Ser His Lys Ser Tyr Arg Pro Leu Thr Val Leu Thr Phe Arg 100
105 110 Ile Asn Tyr Tyr Leu Ser Gly Gly Phe His Pro Val Gly Phe His
Val 115 120 125 Val Asn Ile Leu Leu His Ser Gly Ile Ser Val Leu Met
Val Asp Val 130 135 140 Phe Ser Val Leu Phe Gly Gly Leu Gln Tyr Thr
Ser Lys Gly Arg Arg 145 150 155 160 Leu His Leu Ala Pro Arg Ala Ser
Leu Leu Ala Ala Leu Leu Phe Ala 165 170 175 Val His Pro Val His Thr
Glu Cys Val Ala Gly Val Val Gly Arg Ala 180 185 190 Asp Leu Leu Cys
Ala Leu Phe Phe Leu Leu Ser Phe Leu Gly Tyr Cys 195 200 205 Lys Ala
Phe Arg Glu Ser Asn Lys Glu Gly Ala His Ser Ser Thr Phe 210 215 220
Trp Val Leu Leu Ser Ile Phe Leu Gly Ala Val Ala Met Leu Cys Lys 225
230 235 240 Glu Gln Gly Ile Thr Val Leu Gly Leu Asn Ala Val Phe Asp
Ile Leu 245 250 255 Val Ile Gly Lys Phe Asn Val Leu Glu Ile Xaa Gln
Lys Val Leu His 260 265 270 Lys Asp Lys Ser Leu Glu Asn Leu Gly Met
Leu Arg Asn Gly Gly Leu 275 280 285 Leu Phe Arg Met Thr Leu Leu Thr
Ser Gly Gly Ala Gly Met Leu Tyr 290 295 300 Val Arg Trp Arg Ile Met
Gly Thr Gly Pro Xaa Ala Phe Thr Glu Val 305 310 315 320 Asp Asn Pro
Ala Ser Phe Ala Asp Ser Met Leu Val Arg Ala Val Asn 325 330 335 Tyr
Asn Tyr Tyr Tyr Ser Leu Asn Ala Trp Leu Leu Leu Cys Pro Trp 340 345
350 Trp Leu Cys Phe Asp Trp Ser Met Gly Cys Ile Pro Leu Ile Lys Ser
355 360 365 Ile Ser Asp Trp Arg Val Ile Ala Leu Ala Ala Leu Trp Phe
Cys Leu 370 375 380 Ile Gly Leu Ile Cys Gln Ala Leu Cys Ser Glu Asp
Gly His Lys Arg 385 390 395 400 Arg Ile Leu Thr Leu Gly Leu Gly Phe
Leu Val Ile Pro Phe Leu Pro 405 410 415 Ala Ser Asn Leu Phe Phe Arg
Val Gly Phe Val Val Ala Glu Arg Val 420 425 430 Leu Tyr Leu Pro Ser
Xaa Gly Tyr Cys Val Leu Leu Thr Phe Gly Phe 435 440 445 Gly Ala Leu
Ser Lys His Thr Lys Lys Lys Lys Leu Ile Ala Ala Val 450 455 460 Val
Leu Gly Ile Leu Phe Ile Asn Thr Leu Arg Cys Val Leu Arg Ser 465 470
475 480 Gly Glu Trp Arg Ser Glu Glu Gln Leu Phe Arg Ser Ala Leu Ser
Val 485 490 495 Cys Pro Leu Asn Ala Lys Val His Tyr Asn Ile Gly Lys
Asn Leu Ala 500 505 510 Asp Lys Gly Asn Gln Thr Ala Ala Ile Arg Tyr
Tyr Arg Glu Ala Val 515 520 525 Arg Leu Asn Pro Lys Tyr Val His Ala
Met Asn Asn Leu Gly Asn Ile 530 535 540 Leu Lys Glu Arg Asn Glu Leu
Gln Glu Ala Glu Glu Leu Leu Ser Leu 545 550 555 560 Ala Val Gln Ile
Gln Pro Asp Phe Ala Ala Ala Trp Met Asn Leu Gly 565 570 575 Ile Val
Gln Asn Ser Leu Lys Arg Phe Glu Ala Ala Glu Gln Ser Tyr 580 585 590
Arg Thr Ala Ile Lys His Arg Arg Lys Tyr Pro Asp Cys Tyr Tyr Asn 595
600 605 Leu Gly Arg Leu Tyr Ala Asp Leu Asn Arg His Val Asp Ala Leu
Asn 610 615 620 Ala Trp Arg Asn Ala Thr Val Leu Lys Pro Glu His Ser
Leu Ala Trp 625 630 635 640 Asn Asn Met Ile Ile Leu Leu Asp Asn Thr
Gly Asn Leu Ala Gln Ala 645 650 655 Glu Ala Val Gly Arg Glu Ala Leu
Glu Leu Ile Pro Asn Asp His Ser 660 665 670 Leu Met Phe Ser Leu Ala
Asn Val Leu Gly Lys Ser Gln Lys Tyr Lys 675 680 685 Glu Ser Glu Ala
Leu Phe Leu Lys Ala Ile Lys Ala Asn Pro Asn Ala 690 695 700 Ala Ser
Tyr His Gly Asn Leu Ala Val Leu Tyr His Arg Trp Gly His 705 710 715
720 Leu Asp Leu Ala Lys Lys His Tyr Glu Ile Ser Leu Gln Leu Asp Pro
725 730 735 Thr Ala Ser Gly Thr Lys Glu Asn Tyr Gly Leu Leu Arg Arg
Lys Leu 740 745 750 Glu Leu Met Gln Lys Lys Ala Val 755 760 131 201
PRT Homo sapiens 131 Met Phe Phe Leu Gly Ala Val Leu Cys Leu Ser
Phe Ser Trp Leu Phe 1 5 10 15 His Thr Val Tyr Cys His Ser Glu Lys
Val Ser Arg Thr Phe Ser Lys 20 25 30 Leu Asp Tyr Ser Gly Ile Ala
Leu Leu Ile Met Gly Ser Phe Val Pro 35 40 45 Trp Leu Tyr Tyr Ser
Phe Tyr Cys Ser Pro Gln Pro Arg Leu Ile Tyr 50 55 60 Leu Ser Ile
Val Cys Val Leu Gly Ile Ser Ala Ile Ile Val Ala Gln 65 70 75 80 Trp
Asp Arg Phe Ala Thr Pro Lys His Arg Gln Thr Arg Ala Gly Val 85 90
95 Phe Leu Gly Leu Gly Leu Ser Gly Val Val Pro Thr Met His Phe Thr
100 105 110 Ile Ala Glu Gly Phe Val Lys Ala Thr Thr Val Gly Gln Met
Gly Trp 115 120 125 Phe Phe Leu Met Ala Val Met Tyr Ile Thr Gly Ala
Gly Leu Tyr Ala 130 135 140 Ala Arg Ile Pro Glu Arg Phe Phe Pro Gly
Lys Phe Asp Ile Trp Phe 145 150 155 160 Gln Ser His Gln Ile Phe His
Val Leu Val Val Ala Ala Ala Phe Val 165 170 175 His Phe Tyr Gly Val
Ser Asn Leu Gln Glu Phe Arg Tyr Gly Leu Glu 180 185 190 Gly Gly Cys
Thr Asp Asp Thr Leu Leu 195 200 132 46 PRT Homo sapiens 132 Met Gly
Arg Gln Ala Leu Leu Leu Leu Ala Leu Cys Ala Thr Gly Ala 1 5 10 15
Gln Gly Leu Tyr Phe His Ile Gly Glu Thr Glu Lys Arg Cys Phe Ile 20
25 30 Glu Glu Ile Pro Asp Glu Thr Met Val Ile Gly Gln Ala Gly 35 40
45 133 305 PRT Homo sapiens SITE (11) Xaa equals any of the
naturally occurring L-amino acids 133 Met Ala Leu Cys Ala Leu Thr
Arg Ala Leu Xaa Ser Leu Asn Leu Ala 1 5 10 15 Pro Pro Thr Val Ala
Ala Pro Ala Pro Ser Leu Phe Pro Ala Ala Gln 20 25 30 Met Met Asn
Asn Gly Leu Leu Gln Gln Pro Ser Ala Leu Met Leu Leu 35 40 45 Pro
Cys Arg Pro Val Leu Thr Ser Val Ala Leu Asn Ala Asn Phe Val 50 55
60 Ser Trp Lys Ser Arg Thr Lys Tyr Thr Ile Thr Pro Val Lys Met Arg
65 70 75 80 Lys Ser Gly Gly Arg Asp His Thr Gly Arg Ile Arg Val His
Gly Ile 85 90 95 Gly Gly Gly His Lys Gln Arg Tyr Arg Met Ile Asp
Phe Leu Arg Phe 100 105 110 Arg Pro Glu Glu Thr Lys Ser Gly Pro Phe
Glu Glu Lys Val Ile Gln 115 120 125 Val Arg Tyr Asp Pro Cys Arg Ser
Ala Asp Ile Ala Leu Val Ala Gly 130 135 140 Gly Ser Arg Lys Arg Trp
Ile Ile Ala Thr Glu Asn Met Gln Ala Gly 145 150 155 160 Asp Thr Ile
Leu Asn Ser Asn His Ile Gly Arg Met Ala Val Ala Ala 165 170 175 Arg
Glu Gly Asp Ala His Pro Leu Gly Ala Leu Pro Val Gly Thr Leu 180
185 190 Ile Asn Asn Val Glu Ser Glu Pro Gly Arg Gly Ala Gln Tyr Ile
Arg 195 200 205 Ala Ala Gly Thr Cys Gly Val Leu Leu Arg Lys Val Asn
Gly Thr Ala 210 215 220 Ile Ile Gln Leu Pro Ser Lys Arg Gln Met Gln
Val Leu Glu Thr Cys 225 230 235 240 Val Ala Thr Val Gly Arg Val Ser
Asn Val Asp His Asn Lys Arg Val 245 250 255 Ile Gly Lys Ala Gly Arg
Asn Arg Trp Leu Gly Lys Arg Pro Asn Ser 260 265 270 Gly Arg Trp His
Arg Lys Gly Gly Trp Ala Gly Arg Lys Ile Arg Pro 275 280 285 Leu Pro
Pro Met Lys Ser Tyr Val Lys Leu Pro Ser Ala Ser Ala Gln 290 295 300
Ser 305 134 81 PRT Homo sapiens 134 Met Asn Gln Leu Met Phe Gln Asp
Leu Leu Cys Cys Leu Cys Leu Phe 1 5 10 15 Val Ile Gly Leu Ile Ser
Leu Leu Arg Lys Thr Tyr Ser Cys Val Asn 20 25 30 Leu Cys Lys Val
Met Leu Pro Val Lys Lys Tyr Ser Thr Val Ser Thr 35 40 45 Val Leu
Cys Arg Asn Met Lys Leu Asn Gly Lys Asn Val Leu Met Phe 50 55 60
Val Val Met Leu Leu Gly Gln Trp Met Gly Lys Leu Pro Lys Leu Ser 65
70 75 80 Pro 135 242 PRT Homo sapiens SITE (88) Xaa equals any of
the naturally occurring L-amino acids SITE (139) Xaa equals any of
the naturally occurring L-amino acids 135 Met Glu Gln Ala Arg Lys
Ser Ser Thr Val Ser Leu Leu Ile Thr Val 1 5 10 15 Leu Phe Ala Val
Ala Phe Ser Val Leu Leu Leu Ser Cys Lys Asp His 20 25 30 Val Gly
Tyr Ile Phe Thr Thr Asp Arg Asp Ile Ile Asn Leu Val Ala 35 40 45
Gln Val Val Pro Ile Tyr Ala Val Ser His Leu Phe Glu Ala Leu Ala 50
55 60 Cys Thr Ser Gly Gly Val Leu Arg Gly Ser Gly Asn Gln Lys Val
Gly 65 70 75 80 Ala Ile Val Asn Thr Ile Gly Xaa Tyr Val Val Gly Leu
Pro Ile Gly 85 90 95 Ile Ala Leu Met Phe Ala Thr Thr Leu Gly Val
Met Gly Leu Trp Ser 100 105 110 Gly Ile Ile Ile Cys Thr Val Phe Gln
Ala Val Cys Phe Leu Gly Phe 115 120 125 Ile Ile Gln Leu Asn Trp Lys
Lys Ala Cys Xaa Gln Ala Gln Val His 130 135 140 Ala Asn Leu Lys Val
Asn Asn Val Pro Arg Ser Gly Asn Ser Ala Leu 145 150 155 160 Pro Gln
Asp Pro Leu His Pro Gly Cys Pro Glu Asn Leu Glu Gly Ile 165 170 175
Leu Thr Asn Asp Val Gly Lys Thr Gly Glu Pro Gln Ser Asp Gln Gln 180
185 190 Met Arg Gln Glu Glu Pro Leu Pro Glu His Pro Gln Asp Gly Ala
Lys 195 200 205 Leu Ser Arg Lys Gln Leu Val Leu Arg Arg Gly Leu Leu
Leu Leu Gly 210 215 220 Val Phe Leu Ile Leu Leu Val Gly Ile Leu Val
Arg Phe Tyr Val Arg 225 230 235 240 Ile Gln 136 285 PRT Homo
sapiens 136 Met Val Val Ala Gly Val Val Val Leu Ile Leu Ala Leu Val
Leu Ala 1 5 10 15 Trp Leu Ser Thr Tyr Val Ala Asp Ser Gly Ser Asn
Gln Leu Leu Gly 20 25 30 Ala Ile Val Ser Ala Gly Asp Thr Ser Val
Leu His Leu Gly His Val 35 40 45 Asp His Leu Val Ala Gly Gln Gly
Asn Pro Glu Pro Thr Glu Leu Pro 50 55 60 His Pro Ser Glu Gly Asn
Asp Glu Lys Ala Glu Glu Ala Gly Glu Gly 65 70 75 80 Arg Gly Asp Ser
Thr Gly Glu Ala Gly Ala Gly Gly Gly Val Glu Pro 85 90 95 Ser Leu
Glu His Leu Leu Asp Ile Gln Gly Leu Pro Lys Arg Gln Ala 100 105 110
Gly Ala Gly Ser Ser Ser Pro Glu Ala Pro Leu Arg Ser Glu Asp Ser 115
120 125 Thr Cys Leu Pro Pro Ser Pro Gly Leu Ile Thr Val Arg Leu Lys
Phe 130 135 140 Leu Asn Asp Thr Glu Glu Leu Ala Val Ala Arg Pro Glu
Asp Thr Val 145 150 155 160 Gly Ala Leu Lys Ser Lys Tyr Phe Pro Gly
Gln Glu Ser Gln Met Lys 165 170 175 Leu Ile Tyr Gln Gly Arg Leu Leu
Gln Asp Pro Ala Arg Thr Leu Arg 180 185 190 Ser Leu Asn Ile Thr Asp
Asn Cys Val Ile His Cys His Arg Ser Pro 195 200 205 Pro Gly Ser Ala
Val Pro Gly Pro Ser Ala Ser Leu Ala Pro Ser Ala 210 215 220 Thr Glu
Pro Pro Ser Leu Gly Val Asn Val Gly Ser Leu Met Val Pro 225 230 235
240 Val Phe Val Val Leu Leu Gly Val Val Trp Tyr Phe Arg Ile Asn Tyr
245 250 255 Arg Gln Phe Phe Thr Ala Pro Ala Thr Val Ser Leu Val Gly
Val Thr 260 265 270 Val Phe Phe Ser Phe Leu Val Phe Gly Met Tyr Gly
Arg 275 280 285 137 157 PRT Homo sapiens SITE (114) Xaa equals any
of the naturally occurring L-amino acids SITE (119) Xaa equals any
of the naturally occurring L-amino acids SITE (120) Xaa equals any
of the naturally occurring L-amino acids SITE (121) Xaa equals any
of the naturally occurring L-amino acids 137 Met Asp Ala Met Ile
Leu Leu Asn Val Leu Ala Leu Thr Arg Leu Ala 1 5 10 15 Lys Ala Ala
Ala Thr Asn Phe Val Ala Gln Gly Arg Gly Thr Ile Ile 20 25 30 Asn
Ile Gly Ser Ile Val Ala Leu Ala Pro Lys Val Leu Asn Gly Val 35 40
45 Tyr Gly Gly Thr Lys Ala Phe Val Gln Ala Phe Ser Glu Ser Leu Gln
50 55 60 His Glu Leu Ser Asp Lys Gly Val Val Val Gln Val Val Leu
Pro Gly 65 70 75 80 Ala Thr Ala Thr Glu Phe Trp Asp Ile Ala Gly Leu
Pro Val Lys Gln 85 90 95 Pro Ala Gly Ser His Gly Asp Asp His Arg
Lys Pro Gly Gly Arg Arg 100 105 110 Pro Xaa Arg Pro Cys Pro Xaa Xaa
Xaa Val Thr Ile Pro Ser Leu Pro 115 120 125 Asp Ser Ala Asp Trp Asp
Thr Thr Asn Ala Arg Gly Trp Pro Trp Val 130 135 140 Arg Thr Cys Arg
Thr Val Asn Pro Pro Leu Val Met Gly 145 150 155 138 308 PRT Homo
sapiens SITE (87) Xaa equals any of the naturally occurring L-amino
acids SITE (185) Xaa equals any of the naturally occurring L-amino
acids 138 Met Pro Val Pro Trp Phe Leu Leu Ser Leu Ala Leu Gly Arg
Ser Pro 1 5 10 15 Val Val Leu Ser Leu Glu Arg Leu Val Gly Pro Gln
Asp Ala Thr His 20 25 30 Cys Ser Pro Gly Leu Ser Cys Arg Leu Trp
Asp Ser Asp Ile Leu Cys 35 40 45 Leu Pro Gly Asp Ile Val Pro Ala
Pro Gly Pro Val Leu Ala Pro Thr 50 55 60 His Leu Gln Thr Glu Leu
Val Leu Arg Cys Gln Lys Glu Thr Asp Cys 65 70 75 80 Asp Leu Cys Leu
Arg Val Xaa Val His Leu Ala Val His Gly His Trp 85 90 95 Glu Glu
Pro Glu Asp Glu Glu Lys Phe Gly Gly Ala Ala Asp Leu Gly 100 105 110
Val Glu Glu Pro Arg Asn Ala Ser Leu Gln Ala Gln Val Val Leu Ser 115
120 125 Phe Gln Ala Tyr Pro Thr Ala Arg Cys Val Leu Leu Glu Val Gln
Val 130 135 140 Pro Ala Ala Leu Val Gln Phe Gly Gln Ser Val Gly Ser
Val Val Tyr 145 150 155 160 Asp Cys Phe Glu Ala Ala Leu Gly Ser Glu
Val Arg Ile Trp Ser Tyr 165 170 175 Thr Gln Pro Arg Tyr Glu Lys Glu
Xaa Asn His Thr Gln Gln Leu Pro 180 185 190 Asp Cys Arg Gly Leu Glu
Val Trp Asn Ser Ile Pro Ser Cys Trp Ala 195 200 205 Leu Pro Trp Leu
Asn Val Ser Ala Asp Gly Asp Asn Val His Leu Val 210 215 220 Leu Asn
Val Ser Glu Glu Gln His Phe Gly Leu Ser Leu Tyr Trp Asn 225 230 235
240 Gln Val Gln Gly Pro Pro Lys Pro Arg Trp His Lys Asn Leu Thr Gly
245 250 255 Pro Gln Ile Ile Thr Leu Asn His Thr Asp Leu Val Pro Cys
Leu Cys 260 265 270 Ile Gln Val Trp Pro Leu Glu Pro Asp Ser Val Arg
Arg Thr Ser Ala 275 280 285 Pro Ser Gly Arg Thr Pro Ala His Thr Arg
Thr Ser Gly Lys Pro Pro 290 295 300 Asp Cys Asp Cys 305 139 508 PRT
Homo sapiens 139 Met Asp Pro Lys Leu Gly Arg Met Ala Ala Ser Leu
Leu Ala Val Leu 1 5 10 15 Leu Leu Leu Leu Leu Glu Arg Gly Met Phe
Ser Ser Pro Ser Pro Pro 20 25 30 Pro Ala Leu Leu Glu Lys Val Phe
Gln Tyr Ile Asp Leu His Gln Asp 35 40 45 Glu Phe Val Gln Thr Leu
Lys Glu Trp Val Ala Ile Glu Ser Asp Ser 50 55 60 Val Gln Pro Val
Pro Arg Phe Arg Gln Glu Leu Phe Arg Met Met Ala 65 70 75 80 Val Ala
Ala Asp Thr Leu Gln Arg Leu Gly Ala Arg Val Ala Ser Val 85 90 95
Asp Met Gly Pro Gln Gln Leu Pro Asp Gly Gln Ser Leu Pro Ile Pro 100
105 110 Pro Val Ile Leu Ala Glu Leu Gly Ser Asp Pro Thr Lys Gly Thr
Val 115 120 125 Cys Phe Tyr Gly His Leu Asp Val Gln Pro Ala Asp Arg
Gly Asp Gly 130 135 140 Trp Leu Thr Asp Pro Tyr Val Leu Thr Glu Val
Asp Gly Lys Leu Tyr 145 150 155 160 Gly Arg Gly Ala Thr Asp Asn Lys
Gly Pro Val Leu Ala Trp Ile Asn 165 170 175 Ala Val Ser Ala Phe Arg
Ala Leu Glu Gln Asp Leu Pro Val Asn Ile 180 185 190 Lys Phe Ile Ile
Glu Gly Met Glu Glu Ala Gly Ser Val Ala Leu Glu 195 200 205 Glu Leu
Val Glu Lys Glu Lys Asp Arg Phe Phe Ser Gly Val Asp Tyr 210 215 220
Ile Val Ile Ser Asp Asn Leu Trp Ile Ser Gln Arg Lys Pro Ala Ile 225
230 235 240 Thr Tyr Gly Thr Arg Gly Asn Ser Tyr Phe Met Val Glu Val
Lys Cys 245 250 255 Arg Asp Gln Asp Phe His Ser Gly Thr Phe Gly Gly
Ile Leu His Glu 260 265 270 Pro Met Ala Asp Leu Val Ala Leu Leu Gly
Ser Leu Val Asp Ser Ser 275 280 285 Gly His Ile Leu Val Pro Gly Ile
Tyr Asp Glu Val Val Pro Leu Thr 290 295 300 Glu Glu Glu Ile Asn Thr
Tyr Lys Ala Ile His Leu Asp Leu Glu Glu 305 310 315 320 Tyr Arg Asn
Ser Ser Arg Val Glu Lys Phe Leu Phe Asp Thr Lys Glu 325 330 335 Glu
Ile Leu Met His Leu Trp Arg Tyr Pro Ser Leu Ser Ile His Gly 340 345
350 Ile Glu Gly Ala Phe Asp Glu Pro Gly Thr Lys Thr Val Ile Pro Gly
355 360 365 Arg Val Ile Gly Lys Phe Ser Ile Arg Leu Val Pro His Met
Asn Val 370 375 380 Ser Ala Val Glu Lys Gln Val Thr Arg His Leu Glu
Asp Val Phe Ser 385 390 395 400 Lys Arg Asn Ser Ser Asn Lys Met Val
Val Ser Met Thr Leu Gly Leu 405 410 415 His Pro Trp Ile Ala Asn Ile
Asp Asp Thr Gln Tyr Leu Ala Ala Lys 420 425 430 Arg Ala Ile Arg Thr
Val Phe Gly Thr Glu Pro Asp Met Ile Arg Asp 435 440 445 Gly Ser Thr
Ile Pro Ile Ala Lys Met Phe Gln Glu Ile Val His Lys 450 455 460 Ser
Val Val Leu Ile Pro Leu Gly Ala Val Asp Asp Gly Glu His Ser 465 470
475 480 Gln Asn Glu Lys Ile Asn Arg Trp Asn Tyr Ile Glu Gly Thr Lys
Leu 485 490 495 Phe Ala Ala Phe Phe Leu Glu Met Ala Gln Leu His 500
505 140 506 PRT Homo sapiens SITE (65) Xaa equals any of the
naturally occurring L-amino acids SITE (112) Xaa equals any of the
naturally occurring L-amino acids SITE (423) Xaa equals any of the
naturally occurring L-amino acids SITE (425) Xaa equals any of the
naturally occurring L-amino acids 140 Met Gly Met Arg Arg His Ser
Leu Met Leu Leu Pro Trp Trp Leu Gly 1 5 10 15 Ala Ala Gly Arg Lys
Glu Cys His Arg Glu Gln Leu Val Ala Ala Val 20 25 30 Glu Val Thr
Glu Gln Glu Thr Lys Val Pro Lys Lys Thr Val Ile Ile 35 40 45 Glu
Glu Thr Ile Thr Thr Val Val Lys Ser Pro Arg Gly Gln Arg Arg 50 55
60 Xaa Pro Ser Lys Ser Pro Ser Arg Ser Pro Ser Arg Cys Ser Ala Ser
65 70 75 80 Pro Leu Arg Pro Gly Leu Leu Ala Pro Asp Leu Leu Tyr Leu
Pro Gly 85 90 95 Ala Gly Gln Pro Arg Arg Pro Glu Ala Glu Pro Gly
Gln Lys Pro Xaa 100 105 110 Val Pro Thr Leu Tyr Val Thr Glu Ala Glu
Ala His Ser Pro Ala Leu 115 120 125 Pro Gly Leu Ser Gly Pro Gln Pro
Lys Trp Val Glu Val Glu Glu Thr 130 135 140 Ile Glu Val Arg Val Lys
Lys Met Gly Pro Gln Gly Val Ser Pro Thr 145 150 155 160 Thr Glu Val
Pro Arg Ser Ser Ser Gly His Leu Phe Thr Leu Pro Gly 165 170 175 Ala
Thr Pro Gly Gly Asp Pro Asn Ser Asn Asn Ser Asn Asn Lys Leu 180 185
190 Leu Ala Gln Glu Ala Trp Ala Gln Gly Thr Ala Met Val Gly Val Arg
195 200 205 Glu Pro Leu Val Phe Arg Val Asp Ala Arg Gly Ser Val Asp
Trp Ala 210 215 220 Ala Ser Gly Met Gly Ser Leu Glu Glu Glu Gly Thr
Met Glu Glu Ala 225 230 235 240 Gly Glu Glu Glu Gly Glu Asp Gly Asp
Ala Phe Val Thr Glu Glu Ser 245 250 255 Gln Asp Thr His Ser Leu Gly
Asp Arg Asp Pro Lys Ile Leu Thr His 260 265 270 Asn Gly Arg Met Leu
Thr Leu Ala Asp Leu Glu Asp Tyr Val Pro Gly 275 280 285 Glu Gly Glu
Thr Phe His Cys Gly Gly Pro Gly Pro Gly Ala Pro Asp 290 295 300 Asp
Pro Pro Cys Glu Val Ser Val Ile Gln Arg Glu Ile Gly Glu Pro 305 310
315 320 Thr Val Gly Ser Leu Cys Cys Ser Ala Trp Gly Met His Trp Val
Pro 325 330 335 Glu Ala Leu Ser Ala Ser Leu Gly Leu Ser Pro Val Gly
Arg His His 340 345 350 Arg Asp Pro Arg Ser Val Ala Leu Arg Ala Pro
Pro Ser Ser Cys Gly 355 360 365 Arg Pro Arg Leu Gly Leu Trp Ala Val
Leu Pro Gly Arg Ser Leu Ser 370 375 380 Ala Pro Ala Ser Gly Val Leu
Arg Thr Val Ala Arg Ala Ala Ser Pro 385 390 395 400 Gln Ser Phe Pro
Pro Arg Pro Ser Thr Ser Gly Gln Trp Gly Arg Arg 405 410 415 Ser Pro
Phe Thr Ser Val Xaa Gly Xaa Gly Pro Ser Tyr Leu Thr Gln 420 425 430
Leu Gln Pro Gly Gly Leu Gly Gly Ala Cys Asn Val Gly Met Thr Gly 435
440 445 Ser Lys Thr Ser Ala Leu Gly Cys Phe Leu Ser Ala Trp Gln Glu
Pro 450 455 460 Gln Asp Cys Gly Arg Arg Met Trp Pro Trp Ala Phe Val
Leu Phe Pro 465 470 475 480 His Gly Pro Gly Pro Ser Leu Leu Ala Pro
Ala Thr Ala Ala Arg Pro 485 490 495 Asp Met Ala Leu Pro Leu Leu Gln
Ser Trp 500 505 141 48 PRT Homo sapiens 141 Met Arg Leu Leu Leu Leu
Leu Leu Val Ala Ala Ser Ala Met Val Arg 1 5 10 15 Ser Glu Ala Ser
Ala Asn Leu Gly Gly Val Pro Ser Lys Arg Leu Lys 20 25 30 Met Gln
Tyr Ala Thr Gly Pro Leu Leu Lys Phe Gln Ile Cys Val Ser 35 40 45
142 130 PRT Homo sapiens SITE (64) Xaa equals any of the naturally
occurring L-amino acids SITE (65) Xaa equals any of the naturally
occurring L-amino acids 142 Met Leu Met Pro Val His Phe Leu Leu Leu
Leu Leu Leu Leu Leu Gly 1 5 10 15 Gly Pro Arg Thr Gly Leu Pro His
Lys Phe Tyr Lys Ala Lys Pro Ile 20 25
30 Phe Ser Cys Leu Asn Thr Ala Leu Ser Glu Ala Glu Lys Gly Gln Trp
35 40 45 Glu Asp Ala Ser Leu Leu Ser Lys Arg Ser Phe His Tyr Leu
Arg Xaa 50 55 60 Xaa Thr Pro Leu Arg Glu Arg Arg Arg Arg Ala Lys
Arg Lys Arg Leu 65 70 75 80 Ser Pro Ser Leu Gly Pro Gly Val Glu Pro
Glu Ala Pro Gly Thr Asp 85 90 95 Thr Cys Pro Lys His Ser Pro Gly
Glu Ser His Ala Arg Thr Arg Pro 100 105 110 Arg Val Pro Thr Ala Pro
Ser Ser Pro Cys Pro Ser Thr Ser Pro Pro 115 120 125 Thr Ser 130 143
43 PRT Homo sapiens SITE (25) Xaa equals any of the naturally
occurring L-amino acids SITE (29) Xaa equals any of the naturally
occurring L-amino acids 143 Met Ala Phe Leu Gln Ser Ala Ser Tyr Val
Met Val Ile Leu Cys Ala 1 5 10 15 Cys Val Ile Ile Ile Gly Ile Leu
Xaa Tyr Ala Phe Xaa Phe Glu Thr 20 25 30 Leu Ser Pro Lys Lys Arg
Arg Asp Ile Glu Ile 35 40 144 91 PRT Homo sapiens 144 Met Gln Leu
Ile Glu Ser Arg Phe His Phe Arg Cys Val Trp Ile Leu 1 5 10 15 His
Leu Leu Ala Leu Phe Ser Thr Trp Pro Pro Lys Asp Pro Glu Gly 20 25
30 Ser Pro Pro Ser Ala Thr Ser Ser Pro Leu Thr Pro His Leu Ser Leu
35 40 45 Thr Leu Pro Phe Lys Gln Ala Pro Val Ser Asn Val Ser Ser
Ala Ile 50 55 60 His Val Met Leu Asp Lys Ser Val Ser Leu Ser Glu
Ile Gln Phe Ser 65 70 75 80 His Met Pro Asn Gly Lys Arg Ala Ser Thr
Leu 85 90 145 266 PRT Homo sapiens 145 Met Glu Leu Leu Thr Ala Leu
Leu Arg Leu Phe Leu Ser Arg Pro Ala 1 5 10 15 Glu Cys Gln Asp Met
Leu Gly Arg Leu Leu Tyr Tyr Cys Ile Glu Glu 20 25 30 Glu Lys Asp
Met Ala Val Arg Asp Arg Gly Leu Phe Tyr Tyr Arg Leu 35 40 45 Leu
Leu Val Gly Ile Asp Glu Val Lys Arg Ile Leu Cys Ser Pro Lys 50 55
60 Ser Asp Pro Thr Leu Gly Leu Leu Glu Asp Pro Ala Glu Arg Pro Val
65 70 75 80 Asn Ser Trp Ala Ser Asp Phe Asn Thr Leu Val Pro Val Tyr
Gly Lys 85 90 95 Ala His Trp Ala Thr Ile Ser Lys Cys Gln Gly Ala
Glu Arg Cys Asp 100 105 110 Pro Glu Leu Pro Lys Thr Ser Ser Phe Ala
Ala Ser Gly Pro Leu Ile 115 120 125 Pro Glu Glu Asn Lys Glu Arg Val
Gln Glu Leu Pro Asp Ser Gly Ala 130 135 140 Leu Met Leu Val Pro Asn
Arg Gln Leu Thr Ala Asp Tyr Phe Glu Lys 145 150 155 160 Thr Trp Leu
Ser Leu Lys Val Ala His Gln Gln Val Leu Pro Trp Arg 165 170 175 Gly
Glu Phe His Pro Asp Thr Leu Gln Met Ala Leu Gln Val Val Asn 180 185
190 Ile Gln Thr Ile Ala Met Ser Arg Ala Gly Ser Arg Pro Trp Lys Ala
195 200 205 Tyr Leu Ser Ala Gln Asp Asp Thr Gly Cys Leu Phe Leu Thr
Glu Leu 210 215 220 Leu Leu Glu Pro Gly Asn Ser Glu Met Gln Ile Ser
Val Lys Gln Asn 225 230 235 240 Glu Ala Arg Thr Glu Thr Leu Asn Ser
Phe Ile Ser Val Leu Glu Thr 245 250 255 Val Ile Gly Thr Ile Glu Glu
Ile Lys Ser 260 265 146 434 PRT Homo sapiens 146 Met Ala Pro Glu
Gly Leu Val Pro Ala Val Leu Trp Gly Leu Ser Leu 1 5 10 15 Phe Leu
Asn Leu Pro Gly Pro Ile Trp Leu Gln Pro Ser Pro Pro Pro 20 25 30
Gln Ser Ser Pro Pro Pro Gln Pro His Pro Cys His Thr Cys Arg Gly 35
40 45 Leu Val Asp Ser Phe Asn Lys Gly Leu Glu Arg Thr Ile Arg Asp
Asn 50 55 60 Phe Gly Gly Gly Asn Thr Ala Trp Glu Glu Glu Asn Leu
Ser Lys Tyr 65 70 75 80 Lys Asp Ser Glu Thr Arg Leu Val Glu Val Leu
Glu Gly Val Cys Ser 85 90 95 Lys Ser Asp Phe Glu Cys His Arg Leu
Leu Glu Leu Ser Glu Glu Leu 100 105 110 Val Glu Ser Trp Trp Phe His
Lys Gln Gln Glu Ala Pro Asp Leu Phe 115 120 125 Gln Trp Leu Cys Ser
Asp Ser Leu Lys Leu Cys Cys Pro Ala Gly Thr 130 135 140 Phe Gly Pro
Ser Cys Leu Pro Cys Pro Gly Gly Thr Glu Arg Pro Cys 145 150 155 160
Gly Gly Tyr Gly Gln Cys Glu Gly Glu Gly Thr Arg Gly Gly Ser Gly 165
170 175 His Cys Asp Cys Gln Ala Gly Tyr Gly Gly Glu Ala Cys Gly Gln
Cys 180 185 190 Gly Leu Gly Tyr Phe Glu Ala Glu Arg Asn Ala Ser His
Leu Val Cys 195 200 205 Ser Ala Cys Phe Gly Pro Cys Ala Arg Cys Ser
Gly Pro Glu Glu Ser 210 215 220 Asn Cys Leu Gln Cys Lys Lys Gly Trp
Ala Leu His His Leu Lys Cys 225 230 235 240 Val Asp Ile Asp Glu Cys
Gly Thr Glu Gly Ala Asn Cys Gly Ala Asp 245 250 255 Gln Phe Cys Val
Asn Thr Glu Gly Ser Tyr Glu Cys Arg Asp Cys Ala 260 265 270 Lys Ala
Cys Leu Gly Cys Met Gly Ala Gly Pro Gly Arg Cys Lys Lys 275 280 285
Cys Ser Pro Gly Tyr Gln Gln Val Gly Ser Lys Cys Leu Asp Val Asp 290
295 300 Glu Cys Glu Thr Glu Val Cys Pro Gly Glu Asn Lys Gln Cys Glu
Asn 305 310 315 320 Thr Glu Gly Gly Tyr Arg Cys Ile Cys Ala Glu Gly
Tyr Lys Gln Met 325 330 335 Glu Gly Ile Cys Val Lys Glu Gln Ile Pro
Gly Ala Phe Pro Ile Leu 340 345 350 Thr Asp Leu Thr Pro Glu Thr Thr
Arg Arg Trp Lys Leu Gly Ser His 355 360 365 Pro His Ser Thr Tyr Val
Lys Met Lys Met Gln Arg Asp Glu Ala Thr 370 375 380 Phe Pro Gly Leu
Tyr Gly Lys Gln Val Ala Lys Leu Gly Ser Gln Ser 385 390 395 400 Arg
Gln Ser Asp Arg Gly Thr Arg Leu Ile His Val Ile Asn Ala Leu 405 410
415 Pro Pro Thr Cys Pro Pro Gln Lys Lys Lys Lys Lys Lys Lys Lys Gly
420 425 430 Gly Arg 147 236 PRT Homo sapiens SITE (55) Xaa equals
any of the naturally occurring L-amino acids 147 Met Ile Ser Leu
Pro Gly Pro Leu Val Thr Asn Leu Leu Arg Phe Leu 1 5 10 15 Phe Leu
Gly Leu Ser Ala Leu Ala Pro Pro Ser Arg Ala Gln Leu Gln 20 25 30
Leu His Leu Pro Ala Asn Arg Leu Gln Ala Val Glu Gly Gly Glu Val 35
40 45 Val Leu Pro Ala Trp Tyr Xaa Leu His Gly Glu Val Ser Ser Ser
Gln 50 55 60 Pro Trp Glu Val Pro Phe Val Met Trp Phe Phe Lys Gln
Lys Glu Lys 65 70 75 80 Glu Asp Gln Val Leu Ser Tyr Ile Asn Gly Val
Thr Thr Ser Lys Pro 85 90 95 Gly Val Ser Leu Val Tyr Ser Met Pro
Ser Arg Asn Leu Ser Leu Arg 100 105 110 Leu Glu Gly Leu Gln Glu Lys
Asp Ser Gly Pro Tyr Ser Cys Ser Val 115 120 125 Asn Val Gln Asp Lys
Gln Gly Lys Ser Arg Gly His Ser Ile Lys Thr 130 135 140 Leu Glu Leu
Asn Val Leu Val Pro Pro Ala Pro Pro Ser Cys Arg Leu 145 150 155 160
Gln Gly Val Pro His Val Gly Ala Asn Val Thr Leu Ser Cys Gln Ser 165
170 175 Pro Arg Ser Lys Pro Ala Val Gln Tyr Gln Trp Asp Arg Gln Leu
Pro 180 185 190 Ser Phe Gln Thr Phe Phe Ala Pro Ala Leu Asp Val Ile
Arg Gly Ser 195 200 205 Leu Ser Leu Thr Asn Leu Ser Ser Ser Met Ala
Gly Val Tyr Val Cys 210 215 220 Lys Ala His Asn Glu Val Gly Thr Ala
Asn Val Met 225 230 235 148 99 PRT Homo sapiens SITE (78) Xaa
equals any of the naturally occurring L-amino acids 148 Met Thr Trp
Gly Thr Trp Leu Val His Thr Phe Leu Cys Ser Val Ala 1 5 10 15 Ser
Ala Lys Thr Leu Lys Ser Val Arg Lys Tyr Leu Ser Leu Cys Ser 20 25
30 Pro Ile Gly Ser Ser Phe Val Val Ser Glu Gly Ser Tyr Leu Asp Ile
35 40 45 Ser Asp Trp Leu Asn Pro Ala Lys Leu Ser Leu Tyr Tyr Gln
Ile Asn 50 55 60 Ala Thr Ser Pro Trp Val Arg Asp Leu Cys Gly Gln
Arg Xaa Thr Asp 65 70 75 80 Ala Cys Glu Gln Leu Cys Asp Pro Glu Thr
Gly Glu Pro Trp Glu Pro 85 90 95 Gly Trp Gly 149 69 PRT Homo
sapiens SITE (56) Xaa equals any of the naturally occurring L-amino
acids 149 Met Tyr Lys Ala Phe Leu Leu Ala Leu Thr Thr Val Phe Tyr
Leu Gly 1 5 10 15 Ile Leu Asn Ser His Phe His Gly Cys Val Leu Cys
Asn Thr Asn Val 20 25 30 Phe Lys Trp Tyr Ser His Pro Val Gly Gln
Leu Ser Lys Arg Cys Leu 35 40 45 Asp Ala Ser Lys Leu Ala Tyr Xaa
Lys Phe Thr Ser Ile Lys Tyr Gln 50 55 60 Cys Asn Tyr Ser Thr 65 150
61 PRT Homo sapiens 150 Met His Glu Cys Gln Ser Phe Pro Leu Cys Val
His Leu Arg Leu Val 1 5 10 15 Leu Leu Leu Ser Phe Lys Thr Gln Val
His Glu Phe His Glu Val Phe 20 25 30 Pro His Tyr Ser His Phe Asn
Phe Pro Ser Leu Asn Asn Tyr Asp Ile 35 40 45 Asn Leu Leu Leu Asn
His Glu Leu Trp His Thr Thr Pro 50 55 60 151 88 PRT Homo sapiens
SITE (73) Xaa equals any of the naturally occurring L-amino acids
151 Met Asn Leu Val Gly Phe Cys Leu Phe Ile Cys Leu Leu Leu Met Leu
1 5 10 15 Leu Leu Leu Leu Leu Phe Ser Lys Phe Ser Ile Val Glu Lys
Tyr Ala 20 25 30 Ala Pro Glu Glu Met Ile Gly His Ser Pro Ala Trp
Cys Trp Thr Leu 35 40 45 Ser Ser Leu Ala Gln Pro Ser Pro Asp Leu
Ser Val Tyr Leu Thr Leu 50 55 60 Val Phe Tyr Ile Leu Gln Arg Gln
Xaa Gln Asn Asn Pro Asn Leu Thr 65 70 75 80 Gln Ile Pro Gly Ile His
Leu Ile 85 152 78 PRT Homo sapiens SITE (40) Xaa equals any of the
naturally occurring L-amino acids SITE (46) Xaa equals any of the
naturally occurring L-amino acids SITE (60) Xaa equals any of the
naturally occurring L-amino acids 152 Met Met Gly Asn Asp Leu Leu
His Leu Val Phe Leu Gln Leu Ser Leu 1 5 10 15 Gly Val Ala Ser Gly
Gly Trp Ile Leu Trp Pro Leu Arg Arg Leu Gly 20 25 30 Gly Ala His
Thr Ser Lys Asp Xaa Asn Lys Asn Gly His Xaa Val His 35 40 45 Cys
Leu Val Ile Thr Asn Glu Pro Leu Val Ser Xaa Lys Lys Ile Gly 50 55
60 Leu Ser Ser Pro His Thr Cys Pro Ser Thr Leu Gln Gln Phe 65 70 75
153 123 PRT Homo sapiens 153 Met Met Val Trp Asn Leu Phe Pro Cys
Phe Pro Pro Leu Leu Leu Leu 1 5 10 15 Gln Phe Ile Asp Cys Gln Gln
Ser Ser Glu Ile Glu Gln Gly Phe Thr 20 25 30 Arg Ser Leu Leu Gly
His Pro Ile Phe Phe Cys Pro Asp Pro Cys Trp 35 40 45 Gln Ser Cys
Met Asn Cys Val Ile Leu Ser Val Leu Ser Phe Phe Phe 50 55 60 Leu
Ile Arg Trp Ile Ser Lys Ile Val Ala Val Gln Lys Leu Glu Ser 65 70
75 80 Ser Ser Arg Arg Lys Pro Ile Leu Phe Leu Ile Ile Ser Cys Glu
Ile 85 90 95 Ala Ser Phe Ile His Leu Phe Leu Ser Gln Met Ser Ala
Glu Cys Cys 100 105 110 Cys Phe Tyr Leu Val Ile Leu Ile Cys Lys Tyr
115 120 154 68 PRT Homo sapiens 154 Met Tyr Leu Gly Ser Arg Ile Val
Lys Ala Leu Phe Phe Leu Leu Phe 1 5 10 15 Cys Ile Phe His Ile Trp
Tyr Asn Glu His Val Leu Arg Thr Val Leu 20 25 30 Asp Leu Arg Lys
Tyr Ala Asn Thr Val Gln Ile Val Leu Ala Ser Pro 35 40 45 Met Pro
Ser Ser Ser Ile Ala Asn Val Ser Thr Leu Val Trp Cys Val 50 55 60
Cys Cys Asn Gly 65 155 43 PRT Homo sapiens 155 Met Lys Cys Thr Glu
Lys Cys Val Val Val Phe Phe Thr Phe Val Leu 1 5 10 15 Tyr Met Tyr
Val Tyr Trp Val Leu Trp Ala Val Glu Ala Lys Leu Thr 20 25 30 Ser
His Val Ala His Glu Met Leu Val Ser Cys 35 40 156 63 PRT Homo
sapiens 156 Met Phe Ile Leu Leu Ile Val Phe Val Phe Ser Lys Ser Lys
Gln Val 1 5 10 15 Leu Ser Ile Cys Leu Lys Ile Phe Lys Val Glu Ile
Asn Ser Ile Ser 20 25 30 Phe Cys Lys Asn Lys Lys Tyr Lys Asp Leu
Pro Tyr Ala Phe Ala Ser 35 40 45 Glu Lys Thr Gly Arg Thr Tyr Ser
Asn Val Asn Asn Asp Tyr Leu 50 55 60 157 61 PRT Homo sapiens 157
Met Ile Val Tyr Trp Met Ile Trp Ala Leu Arg Ser Pro Leu Thr Thr 1 5
10 15 Ala Gln Asn Ile His Ser Ser Thr Ala Leu Thr Glu Phe Ala Lys
Cys 20 25 30 Ile Lys Glu Val Thr Trp Arg Val Arg Ser Tyr Glu Thr
Ile Cys Arg 35 40 45 Lys Trp Gly Lys Lys Gly His Met Ala Gln Leu
Lys Leu 50 55 60 158 82 PRT Homo sapiens 158 Met Arg Phe Phe Leu
Glu Cys Val Leu Leu Ile Cys Phe Arg Ala Met 1 5 10 15 Ser Ala Ile
Tyr Thr His Thr Ser Ile Gly Asn Ala Gln Lys Leu Phe 20 25 30 Thr
Asp Gly Ser Ala Phe Arg Arg Val Arg Glu Pro Leu Pro Lys Glu 35 40
45 Gly Lys Ser Trp Pro Gln Leu Glu Gln Ala Cys Leu Gly Pro Cys Ser
50 55 60 Val Phe Gln Leu Gln Thr Ala Cys Ile Ile Pro Ser Cys Tyr
Ser Ser 65 70 75 80 Phe Thr 159 46 PRT Homo sapiens 159 Met Cys Cys
Ala Ser His Pro Cys Gln Arg Glu Gly Trp Leu Cys Val 1 5 10 15 Ile
Phe Thr Val Phe Leu Lys Val Thr Val Cys Val Phe Thr Phe Val 20 25
30 Gln Ile Thr Gly Ser Lys Ala Ala Asn Ser Ala Ile Thr Cys 35 40 45
160 187 PRT Homo sapiens 160 Met Ala Cys Lys Gly Leu Leu Gln Gln
Val Gln Gly Pro Arg Leu Pro 1 5 10 15 Trp Thr Arg Leu Leu Leu Leu
Leu Leu Val Phe Ala Val Gly Phe Leu 20 25 30 Cys His Asp Leu Pro
Val Thr Gln Leu Leu Pro Gly Trp Leu Gly Glu 35 40 45 Thr Leu Pro
Leu Trp Gly Ser His Leu Leu Thr Val Val Arg Pro Ser 50 55 60 Leu
Gln Leu Ala Trp Ala His Thr Asn Ala Thr Val Ser Phe Leu Ser 65 70
75 80 Ala His Cys Ala Ser His Leu Ala Trp Phe Gly Asp Ser Leu Thr
Ser 85 90 95 Leu Ser Gln Arg Leu Gln Ile Gln Leu Pro Asp Ser Val
Asn Gln Leu 100 105 110 Leu Arg Tyr Leu Arg Glu Leu Pro Leu Leu Phe
His Gln Asn Val Leu 115 120 125 Leu Pro Leu Trp His Leu Leu Leu Glu
Ala Leu Ala Trp Ala Gln Glu 130 135 140 His Cys His Glu Ala Cys Arg
Gly Glu Val Thr Trp Asp Cys Met Lys 145 150 155 160 Thr Gln Leu Ser
Glu Ala Val His Trp Thr Trp Leu Cys Tyr Arg Thr 165 170 175 Leu Gln
Trp Leu Ser Trp Thr Gly His Leu Pro 180 185 161 113 PRT Homo
sapiens 161 Met Ile Phe Ser Met Pro Gln Gln Gly Ser Ser Trp Phe Leu
Ser Ala 1 5 10 15 Phe Leu Ser Trp Pro Leu Ala Leu Ala Pro Ala Leu
Thr Pro Thr Pro 20 25 30 Ala Pro Ala Arg Ala Pro Gly Ala Pro Arg
Ala Ala Gly Ala Pro Gly 35 40 45 Arg Val Ala Ala Gly Arg Gly Thr
Cys Ala Gly Ala Leu Ala Pro
Gly 50 55 60 Gln Glu Ala Trp Ser Ala Val Trp Glu Pro Gly Leu Phe
Ile Trp Val 65 70 75 80 Glu His Pro Leu Gly Cys Gln Gly His Gly Leu
Asp Arg Phe Pro Leu 85 90 95 Pro Thr Ala Leu Pro Leu Gln Gly Gly
His Ala Ala Cys Cys Pro Gln 100 105 110 Leu 162 292 PRT Homo
sapiens 162 Met Gly Ile Gln Thr Ser Pro Val Leu Leu Ala Ser Leu Gly
Val Gly 1 5 10 15 Leu Val Thr Leu Leu Gly Leu Ala Val Gly Ser Tyr
Leu Val Arg Arg 20 25 30 Ser Arg Arg Pro Gln Val Thr Leu Leu Asp
Pro Asn Glu Lys Tyr Leu 35 40 45 Leu Arg Leu Leu Asp Lys Thr Thr
Val Ser His His Thr Leu Gly Leu 50 55 60 Pro Val Gly Lys His Ile
Tyr Leu Ser Thr Arg Ile Asp Gly Ser Leu 65 70 75 80 Val Ile Arg Pro
Tyr Thr Pro Val Thr Ser Asp Glu Asp Gln Gly Tyr 85 90 95 Val Asp
Leu Val Ile Lys Val Tyr Leu Lys Gly Val His Pro Lys Phe 100 105 110
Pro Glu Gly Gly Lys Met Ser Gln Tyr Leu Asp Ser Leu Lys Val Gly 115
120 125 Asp Val Val Glu Phe Arg Gly Pro Ser Gly Leu Leu Thr Tyr Thr
Gly 130 135 140 Lys Gly His Phe Asn Ile Gln Pro Asn Lys Lys Ser Pro
Pro Glu Pro 145 150 155 160 Arg Val Ala Lys Lys Leu Gly Met Ile Ala
Gly Gly Thr Gly Ile Thr 165 170 175 Pro Met Leu Gln Leu Ile Arg Ala
Ile Leu Lys Val Pro Glu Asp Pro 180 185 190 Thr Gln Cys Phe Leu Leu
Phe Ala Asn Gln Thr Glu Lys Asp Ile Ile 195 200 205 Leu Arg Glu Asp
Leu Glu Glu Leu Gln Ala Arg Tyr Pro Asn Arg Phe 210 215 220 Lys Leu
Trp Phe Thr Leu Asp His Pro Pro Lys Asp Trp Ala Tyr Ser 225 230 235
240 Lys Gly Phe Val Thr Ala Asp Met Ile Arg Glu His Leu Pro Ala Pro
245 250 255 Gly Asp Asp Val Leu Val Leu Leu Cys Gly Pro Pro Pro Met
Val Gln 260 265 270 Leu Ala Cys His Pro Asn Leu Asp Lys Leu Gly Tyr
Ser Gln Lys Met 275 280 285 Arg Phe Thr Tyr 290 163 86 PRT Homo
sapiens 163 Met Val Met Val Phe Phe Leu Thr Phe Ser Gly Ser His Gly
Cys Val 1 5 10 15 Pro Thr Ser Gln Pro Trp Lys Asp Ala Glu Asp Gln
Val Gly Cys Val 20 25 30 His Ala Val Ala Trp Val Asn Ser Ala Leu
Tyr Thr Val Leu Cys Pro 35 40 45 Phe Leu Gly Lys Pro Lys Cys Ser
Phe Ser Phe Asp Arg Asn Glu Ser 50 55 60 Glu Asp Leu Asn Lys Gln
Glu Val Lys Cys Arg Ala Val Pro Val Ser 65 70 75 80 Val Ser Ser Ser
Met Leu 85 164 106 PRT Homo sapiens 164 Met Leu Ala Thr Met Val Val
Gln Ile Leu Arg Leu Arg Pro His Thr 1 5 10 15 Gln Lys Trp Ser His
Val Leu Thr Leu Leu Gly Leu Ser Leu Val Leu 20 25 30 Gly Leu Pro
Trp Ala Leu Ile Phe Phe Ser Phe Ala Ser Gly Thr Phe 35 40 45 Gln
Leu Val Val Leu Tyr Leu Phe Ser Ile Ile Thr Ser Phe Gln Gly 50 55
60 Phe Leu Ile Phe Ile Trp Tyr Trp Ser Met Arg Leu Gln Ala Arg Gly
65 70 75 80 Gly Pro Ser Pro Leu Lys Ser Asn Ser Asp Ser Ala Arg Leu
Pro Ile 85 90 95 Ser Ser Gly Ser Thr Ser Ser Ser Arg Ile 100 105
165 58 PRT Homo sapiens 165 Met Ala Trp Arg Val Trp Cys Leu Trp Gly
Ile Pro Pro Leu Phe Cys 1 5 10 15 Ser Pro Gly Thr Leu Ser Cys Val
Cys Val Ser Phe Leu Ser Pro Gly 20 25 30 Asn Gly Met Ala Ser Glu
His His Pro Arg Ser Ile Phe Pro Leu Gln 35 40 45 Asn Asp Val Ser
Ser His Val Cys Phe Cys 50 55 166 40 PRT Homo sapiens 166 Met Arg
Ser Asp Cys Val Leu Ile Trp Gln Leu Val Gly Val Leu Leu 1 5 10 15
Ala Ser Gly Leu Ser Gly Asp Arg Ala Pro Leu Ile Val Leu Thr Ala 20
25 30 Cys Asp Lys Ala Trp Ala Thr Val 35 40 167 65 PRT Homo sapiens
SITE (29) Xaa equals any of the naturally occurring L-amino acids
SITE (35) Xaa equals any of the naturally occurring L-amino acids
SITE (63) Xaa equals any of the naturally occurring L-amino acids
SITE (64) Xaa equals any of the naturally occurring L-amino acids
167 Met Trp Ala Cys Trp Gly Met Leu Gly Cys Ile Pro Leu Phe Val Pro
1 5 10 15 Trp Val Pro Val Leu Gly Lys His Phe Ser Gly Cys Xaa Tyr
Leu Cys 20 25 30 Gly Arg Xaa Pro Cys Trp Ile Ala Phe Ile Cys Val
Arg Thr Pro Cys 35 40 45 Gly Pro Thr Thr Ala Pro Thr Ala Thr Leu
Lys Trp Ser Pro Xaa Xaa 50 55 60 Thr 65 168 46 PRT Homo sapiens 168
Met Arg Tyr Trp Thr Asp Met Arg Arg Asn Tyr Arg Val Thr Tyr Gln 1 5
10 15 Val Val Leu Leu Phe Leu Cys Phe Ser Leu Leu Thr Glu Cys Lys
Thr 20 25 30 Phe Glu Pro Arg Ser Glu Arg Ser Leu Phe Ser Tyr Pro
Leu 35 40 45 169 140 PRT Homo sapiens 169 Met Phe Ala Gly Leu Phe
Phe Leu Phe Phe Val Arg Phe Gly Ile Gly 1 5 10 15 Arg Gln Leu Leu
Ile Lys Phe Pro Trp Phe Phe Ser Phe Gly Tyr Phe 20 25 30 Ser Lys
Gln Gly Pro Thr Gln Lys Gln Ile Asp Ala Ala Ser Phe Thr 35 40 45
Leu Thr Phe Phe Gly Gln Gly Tyr Ser Gln Gly Thr Gly Thr Asp Lys 50
55 60 Asn Lys Pro Asn Ile Lys Ile Cys Thr Gln Val Lys Gly Pro Glu
Ala 65 70 75 80 Gly Tyr Val Ala Thr Pro Ile Ala Met Val Gln Ala Ala
Met Thr Leu 85 90 95 Leu Ser Asp Ala Ser His Leu Pro Lys Ala Gly
Gly Val Phe Thr Pro 100 105 110 Gly Ala Ala Phe Ser Lys Thr Lys Leu
Ile Asp Arg Leu Asn Lys His 115 120 125 Gly Ile Glu Phe Ser Val Ile
Ser Ser Ser Glu Val 130 135 140 170 53 PRT Homo sapiens 170 Met Gln
Glu Cys Leu Leu His Gly Cys Cys Cys Tyr Leu Leu Arg Leu 1 5 10 15
Gly Val Leu Gly Thr Val Gln Cys Ile Ser Thr Trp Leu Ile Leu Thr 20
25 30 Ala Asn Glu Gln His Arg Leu Lys Glu Thr Ser Asn Ser Gln Ser
Pro 35 40 45 Ala Val Ser Arg Ala 50 171 167 PRT Homo sapiens 171
Met Cys Gly Phe Leu Ser Leu Gln Ile Met Gly Pro Leu Ile Val Leu 1 5
10 15 Val Gly Leu Cys Phe Phe Val Val Ala His Val Lys Lys Arg Asn
Thr 20 25 30 Leu Asn Ala Gly Gln Asp Ala Ser Glu Arg Glu Glu Gly
Gln Ile Gln 35 40 45 Ile Met Glu Pro Val Gln Val Thr Val Gly Asp
Ser Val Ile Ile Phe 50 55 60 Pro Pro Pro Pro Pro Pro Tyr Phe Pro
Glu Ser Ser Ala Ser Ala Val 65 70 75 80 Ala Glu Ser Pro Gly Thr Asn
Ser Leu Leu Pro Asn Glu Asn Pro Pro 85 90 95 Ser Tyr Tyr Ser Ile
Phe Asn Tyr Gly Thr Pro Thr Ser Glu Gly Ala 100 105 110 Ala Ser Glu
Arg Asp Cys Glu Ser Ile Tyr Thr Ile Ser Gly Thr Asn 115 120 125 Ser
Ser Ser Glu Ala Ser His Thr Pro His Leu Pro Ser Glu Leu Pro 130 135
140 Pro Arg Tyr Glu Glu Lys Glu Asn Ala Ala Ala Thr Phe Leu Pro Leu
145 150 155 160 Ser Ser Glu Pro Ser Pro Pro 165 172 325 PRT Homo
sapiens 172 Met Ser Ile Ser Leu Ser Ser Leu Ile Leu Leu Pro Ile Trp
Ile Asn 1 5 10 15 Met Ala Gln Ile Gln Gln Gly Gly Pro Asp Glu Lys
Glu Lys Thr Thr 20 25 30 Ala Leu Lys Asp Leu Leu Ser Arg Ile Asp
Leu Asp Glu Leu Met Lys 35 40 45 Lys Asp Glu Pro Pro Leu Asp Phe
Pro Asp Thr Leu Glu Gly Phe Glu 50 55 60 Tyr Ala Phe Asn Glu Lys
Gly Gln Leu Arg His Ile Lys Thr Gly Glu 65 70 75 80 Pro Phe Val Phe
Asn Tyr Arg Glu Asp Leu His Arg Trp Asn Gln Lys 85 90 95 Arg Tyr
Glu Ala Leu Gly Glu Ile Ile Thr Lys Tyr Val Tyr Glu Leu 100 105 110
Leu Glu Lys Asp Cys Asn Leu Lys Lys Val Ser Ile Pro Val Asp Ala 115
120 125 Thr Glu Ser Glu Pro Lys Ser Phe Ile Phe Met Ser Glu Asp Ala
Leu 130 135 140 Thr Asn Pro Gln Lys Leu Met Val Leu Ile His Gly Ser
Gly Val Val 145 150 155 160 Arg Ala Gly Gln Trp Ala Arg Arg Leu Ile
Ile Asn Glu Asp Leu Asp 165 170 175 Ser Gly Thr Gln Ile Pro Phe Ile
Lys Arg Ala Val Ala Glu Gly Tyr 180 185 190 Gly Val Ile Val Leu Asn
Pro Asn Glu Asn Tyr Ile Glu Val Glu Lys 195 200 205 Pro Lys Ile His
Val Gln Ser Ser Ser Asp Ser Ser Asp Glu Pro Ala 210 215 220 Glu Lys
Arg Glu Arg Lys Asp Lys Val Ser Lys Glu Thr Lys Lys Arg 225 230 235
240 Arg Asp Phe Tyr Glu Lys Tyr Arg Asn Pro Gln Arg Glu Lys Glu Met
245 250 255 Met Gln Leu Tyr Ile Arg Glu Asn Gly Ser Pro Glu Glu His
Ala Ile 260 265 270 Tyr Val Trp Asp His Phe Ile Ala Gln Ala Ala Ala
Glu Asn Val Phe 275 280 285 Phe Val Ala His Ser Tyr Gly Gly Leu Ala
Phe Val Glu Leu Gln Leu 290 295 300 Met Ile Lys Gln Ala Asn Ser Asp
Ala Gly Lys Cys Phe Arg Leu Ala 305 310 315 320 Met Trp Lys Asn His
325 173 113 PRT Homo sapiens 173 Met His Pro Pro Leu Thr Pro Pro
Thr Pro Leu Cys Leu Trp Leu Arg 1 5 10 15 Leu Leu Lys Ala Gln Ile
Leu Ser Tyr Pro Val Pro Arg Phe Glu Thr 20 25 30 His Ser Leu Ile
Ser Arg Cys Ser Gln Val Pro Pro Thr Phe Leu Trp 35 40 45 Asp Ile
Lys Lys Gly Val Arg Gly Gln Arg Glu Pro Ser Gly Pro Leu 50 55 60
Leu Pro Tyr Thr Leu His Cys Pro Phe Ser Pro His Gln Asn Ala Gln 65
70 75 80 Arg Arg Cys Asp Asp Ala Thr Glu Asp Tyr Ala Thr Trp Ser
Asn Arg 85 90 95 Ser Gly Gln His Asp Gln Leu Ser Arg Gly Cys Leu
Leu Pro Phe Leu 100 105 110 Leu 174 61 PRT Homo sapiens SITE (37)
Xaa equals any of the naturally occurring L-amino acids SITE (39)
Xaa equals any of the naturally occurring L-amino acids 174 Met Gly
Arg Leu Gly Leu Cys Leu Leu Arg Ser Leu Trp Val Pro Gln 1 5 10 15
Arg Arg Ala Thr Thr Leu Gly Trp Thr Leu Ala Leu Arg Val Leu Pro 20
25 30 Thr Ala Arg Ala Xaa Arg Xaa Leu Pro Val Ala Ala Asp Thr Ala
Arg 35 40 45 Arg Ala Cys Gly Ala His Thr Arg Ile Arg Val Leu Gly 50
55 60 175 41 PRT Homo sapiens SITE (41) Xaa equals any of the
naturally occurring L-amino acids 175 Met Asp Ile Asn Phe Cys Leu
Arg Gly Arg His Gly Val Leu Phe Cys 1 5 10 15 Phe Val Leu Phe Cys
Phe Cys His Leu Leu Thr Val Leu Ser Thr His 20 25 30 Arg Ala Phe
Tyr Tyr Leu Ser Ala Xaa 35 40 176 42 PRT Homo sapiens 176 Met Ile
Lys Leu Gln Lys Val Ser Glu Val Ile Lys Val Leu Lys Met 1 5 10 15
Leu Leu Tyr Pro Leu Val Leu Leu Leu Ser Leu Lys Leu Asp Thr Lys 20
25 30 Ala Thr Ile Phe Ala Val Leu Glu Asp Val 35 40 177 47 PRT Homo
sapiens 177 Met Tyr Phe Phe Thr Phe Tyr Phe Ser Ile Ser Ser Phe Met
Phe Phe 1 5 10 15 Leu Leu Val Ile Val Lys Ala Thr Asn Gly Pro Arg
Tyr Val Val Gly 20 25 30 Cys Arg Arg Gln Val Ile Leu Tyr Ile Cys
Ile Val Pro Asp Asp 35 40 45 178 50 PRT Homo sapiens 178 Met Ser
Gly Phe Lys Glu Phe Asp Phe Val Val Pro Trp Trp Ser Ile 1 5 10 15
Ser Phe Leu Leu Ser Phe Leu Leu Leu Leu Leu Ser Phe Trp Ser Leu 20
25 30 Trp Val Tyr Thr Phe His Gln Ile Trp Asn Ile Phe Gly Tyr Tyr
Phe 35 40 45 Ser Lys 50 179 227 PRT Homo sapiens 179 Met Val Leu
Thr Ala Thr Val Leu Asn Val Tyr Ala Ser Ile Phe Leu 1 5 10 15 Ile
Thr Ala Leu Ser Val Ala Arg Tyr Trp Val Val Ala Met Ala Ala 20 25
30 Gly Pro Gly Thr His Leu Ser Leu Phe Trp Ala Arg Ile Ala Thr Leu
35 40 45 Ala Val Trp Ala Ala Ala Ala Leu Val Thr Val Pro Thr Ala
Val Phe 50 55 60 Gly Val Glu Gly Glu Val Cys Gly Val Arg Leu Cys
Leu Leu Arg Phe 65 70 75 80 Pro Ser Arg Ser Trp Leu Gly Ala Tyr Gln
Leu Gln Arg Val Val Leu 85 90 95 Ala Phe Met Val Pro Leu Gly Val
Ile Thr Thr Ser Tyr Leu Leu Leu 100 105 110 Leu Ala Phe Leu Gln Arg
Arg Gln Arg Arg Arg Gln Asp Ser Arg Val 115 120 125 Val Ala Arg Ser
Val Arg Ile Leu Val Ala Ser Phe Phe Leu Cys Trp 130 135 140 Phe Pro
Asn His Val Val Thr Leu Trp Gly Val Leu Val Gln Phe Ala 145 150 155
160 Leu Val Pro Trp Ile Ser Thr Phe Tyr Thr Leu Gln Pro Tyr Val Phe
165 170 175 Pro Val Thr Thr Cys Leu Ala His Ser Asn Ser Cys Leu Asn
Pro Ile 180 185 190 Ala Tyr Val Leu Ser Arg Ile Pro Ala His Trp Arg
Pro Leu Leu Val 195 200 205 Asp Pro Ser Ser Val Pro Ser Leu Met His
Ser Leu Ser Ile His Ser 210 215 220 Ala Pro Lys 225 180 44 PRT Homo
sapiens 180 Met Phe Arg Ser Ser Ile Ser Leu Met Val Phe Ser Leu Ile
Leu Leu 1 5 10 15 Leu Thr Thr Glu Arg Arg Ile Leu Ala Cys Pro Pro
Ile Ile Leu Asn 20 25 30 Ser Ser Ile Phe Leu Ser Asp Leu Ser Val
Leu Pro 35 40 181 46 PRT Homo sapiens 181 Met Asn Pro Leu Ser Phe
Leu Phe Cys Phe Ile Ile Cys Arg Leu Leu 1 5 10 15 Ala Glu Asn Ala
Ile Asn Ile Glu Ile Leu Thr Gly Thr Tyr Glu Asn 20 25 30 Phe Pro
Thr Lys Ala Tyr Tyr Phe Arg Gln Arg Ser Arg Lys 35 40 45 182 41 PRT
Homo sapiens 182 Met Ala Ser Leu Leu Arg Thr Cys Cys Val Pro Tyr
Ile Val Leu Ser 1 5 10 15 Ile Tyr Leu Asp Tyr Leu Ile Lys Ser Ser
Gln Ser Leu Tyr Leu Thr 20 25 30 Asp Gly Glu Ile Lys Ala His Gly
Thr 35 40 183 47 PRT Homo sapiens 183 Met Leu Gln Asp Leu Leu Ser
Ala Leu Trp Phe Cys His Pro Cys Cys 1 5 10 15 Leu Cys Cys Gly Leu
Cys Trp Leu Gly Val Asp Ala Gly Cys Ser Gln 20 25 30 Gly Gly Ser
Gly Cys Pro Gln Gly Lys Ile Ser Asn Asn Gly Ile 35 40 45 184 70 PRT
Homo sapiens 184 Met Lys Phe Ala Pro Val Tyr Met Tyr Leu Ser Phe
Ile Cys Leu Cys 1 5 10 15 Leu Phe Tyr Cys Asn Ser Ile Asp Thr His
His Cys Phe Val Ser Asp 20 25 30 Tyr Leu Ala Phe Glu Ser Ser Met
Arg Glu Ala Phe Thr Glu Leu Leu 35 40 45 Ile Leu Ile Lys Gly Glu
Ser Asn Val Leu Lys Lys Met Gln Asn His 50 55 60 His Leu Cys Gln
Ser Tyr 65 70 185 41 PRT Homo sapiens 185 Met Gly Leu Lys Leu Pro
Ile Phe Leu Trp Phe Leu Tyr Phe Phe Ile 1 5 10 15 Pro Leu Ser Ser
Cys Tyr Leu Leu Leu Leu Pro His Leu Pro Ser Gly 20
25 30 Ser Trp Asp Ser Met Leu Ser Phe Pro 35 40 186 92 PRT Homo
sapiens SITE (18) Xaa equals any of the naturally occurring L-amino
acids 186 Met Ala Gly Cys Leu Gly Ser Tyr Leu Leu Val Met Ile Leu
Ile Leu 1 5 10 15 Cys Xaa Ala His Phe Phe Ile Cys Gly Asn Glu Asp
Asn Arg Val Leu 20 25 30 Arg Tyr Asn Leu Glu Gln Cys Pro Ser His
Ser Lys His Val Ile Asn 35 40 45 Gly Ser Ser Tyr Cys Tyr Tyr Tyr
Tyr Tyr Tyr Tyr Leu Glu Asp Arg 50 55 60 Gly Ser Val Leu Phe Ile
Ile Pro Ser Pro Ala Leu Ser Thr Val Pro 65 70 75 80 Gly Thr Ile Gln
Thr Cys Ile Trp Met Asn Asp Lys 85 90 187 71 PRT Homo sapiens 187
Met Pro Ala Gly Val Pro Met Ser Thr Tyr Leu Lys Met Phe Ala Ala 1 5
10 15 Ser Leu Leu Ala Met Cys Ala Gly Ala Glu Val Val His Arg Tyr
Tyr 20 25 30 Arg Pro Asp Leu Thr Ile Pro Glu Ile Pro Pro Lys Arg
Gly Glu Leu 35 40 45 Lys Thr Glu Leu Leu Gly Leu Lys Glu Arg Lys
His Lys Pro Gln Val 50 55 60 Ser Gln Gln Glu Glu Leu Lys 65 70 188
66 PRT Homo sapiens SITE (23) Xaa equals any of the naturally
occurring L-amino acids SITE (45) Xaa equals any of the naturally
occurring L-amino acids 188 Met Ala Gly Phe Ala Ser Tyr Pro Trp Ser
Asp Phe Pro Trp Cys Trp 1 5 10 15 Val Val Cys Phe Ser Phe Xaa Phe
Phe Phe Leu Arg Gln Ser Glu Ser 20 25 30 Leu Ser Gln Lys Lys Arg
Gln Val Ala Asp Glu Leu Xaa Phe Gly Gln 35 40 45 Ser Lys Arg Asp
Ser Asp Gly Gly Trp Met Leu Arg Ser Ser Ala Gly 50 55 60 Asn Ser 65
189 70 PRT Homo sapiens SITE (14) Xaa equals any of the naturally
occurring L-amino acids SITE (21) Xaa equals any of the naturally
occurring L-amino acids 189 Met Gln Pro Ser Tyr Pro Leu Ser Trp Ser
Gly Gly Val Xaa Leu Pro 1 5 10 15 Cys Leu Ala Ser Xaa Leu Thr Leu
Leu Phe Leu Leu Gln Pro Leu Met 20 25 30 Leu Pro Leu Gly Gly Ser
Gln Thr Gln Leu Gly Asn His Ser Val Val 35 40 45 Arg Leu Leu Leu
Pro Val Gln Arg Leu Gly Phe Ala Glu Val Pro Pro 50 55 60 Leu Glu
Val Ala Gln Ser 65 70 190 40 PRT Homo sapiens 190 Met Ile Pro Leu
Arg Arg Gly Met Val Gly Gly Leu Leu Leu Leu Leu 1 5 10 15 Ala Thr
Ala Asn Lys Leu Leu Ala Ala Ser Phe Arg Asp Leu Met Asp 20 25 30
Val Leu Thr Cys Pro Arg Pro Arg 35 40 191 66 PRT Homo sapiens SITE
(36) Xaa equals any of the naturally occurring L-amino acids 191
Met Gln His Leu Leu Leu His Ser Leu Cys Leu Ser Cys Ser Thr Met 1 5
10 15 Ala Arg Asn Val Pro Ala Ser Pro Ser Pro Ser Ala Val Ile Val
Ser 20 25 30 Phe Leu Arg Xaa Pro Gln Pro Cys Phe Leu Tyr Ser Leu
Gln Asn Cys 35 40 45 Glu Ser Ile Lys Pro Leu Phe Phe Ile Asn Ser
Pro Val Ser Ser Ser 50 55 60 Ser Leu 65 192 66 PRT Homo sapiens 192
Met Leu Pro Ser Trp Trp Ala Leu Gly Trp Met Thr Leu Lys Ile Leu 1 5
10 15 Gln Met Trp Val Gln Ala Cys Thr His Thr Met Glu Tyr Gly His
Ser 20 25 30 Tyr Thr Gly Gly Val Glu Ser Gly Ser Ala Ala Trp His
Leu Thr Glu 35 40 45 Val Gly Pro Lys Arg Thr His Asp Tyr Ala Glu
Asn Trp Ile Gly Ser 50 55 60 Leu Ser 65 193 48 PRT Homo sapiens 193
Met His Phe Ser Val Ala His Ser Ile Trp Gly Ile Leu Ile Leu Leu 1 5
10 15 Ser Leu Tyr Glu Gly Val Ile Ser Trp Val Phe Asn Phe Gln Met
Phe 20 25 30 Thr Lys Leu Leu Leu Cys Ala Lys His Tyr Ser His Cys
Phe Glu Ser 35 40 45 194 66 PRT Homo sapiens 194 Met Ser Leu Ile
Leu Leu Gly Ser Pro Ile Ile Pro Leu Trp Ser Tyr 1 5 10 15 Thr Ser
Ala Thr Gln Ala Ala Ala Leu Val Thr Ser His Val Trp Lys 20 25 30
Pro Ser Leu Glu Ala His Gln Ile Asn Ile Ser Pro Glu Pro Ser Ile 35
40 45 His Tyr Asp Arg Trp His Thr Gln Ser Asn Cys Ser Leu Ile Asn
Ser 50 55 60 Leu Gln 65 195 57 PRT Homo sapiens 195 Met Lys Gln Thr
Tyr Trp Gln Thr His Ile Leu Leu Val Leu Thr Leu 1 5 10 15 Tyr Phe
Ile Val Leu Ala Tyr Ser Pro Phe Leu Arg Phe Leu Leu Arg 20 25 30
Asn Ile Gly Thr His Pro Leu Leu Cys Ala Glu Gly Ile Thr Ser Phe 35
40 45 Phe Leu Ser Tyr Lys Asn Met Leu Tyr 50 55 196 52 PRT Homo
sapiens 196 Met Gly Pro Asn Phe Val Val Leu Cys Leu Asn Leu Leu Gln
Asp Thr 1 5 10 15 Leu Ala Tyr Ala Thr Ala Leu Leu Asn Glu Lys Glu
Gln Ser Gly Ser 20 25 30 Ser Asn Gly Ser Glu Ser Ser Pro Ala Asn
Glu Asn Gly Asp Arg His 35 40 45 Leu Gln Gln Val 50 197 43 PRT Homo
sapiens 197 Met Ile Val Ile Ala Val Ser Leu Ser Leu Phe Cys Asp Val
Val Ser 1 5 10 15 Ser Glu Cys Met Ser Cys Phe Thr Pro Lys Phe Ala
Asp Ile Val Ala 20 25 30 Asn Ala Tyr Gln Asn Glu Ser Tyr Ile Phe
Ile 35 40 198 52 PRT Homo sapiens 198 Met Leu Leu Pro Val Asn Thr
Leu Leu Tyr Ile Leu Leu Thr Pro Leu 1 5 10 15 Cys Phe Phe Tyr Gly
Thr Ser Arg Pro Pro Tyr Leu Glu Leu Val Thr 20 25 30 Leu Leu Lys
Lys Lys Lys Gln Ser Val Gly Phe Ser Val Cys Ile Leu 35 40 45 Glu
Ala Gly Arg 50 199 40 PRT Homo sapiens 199 Met Ile Ile Val Leu Phe
Ser Leu Ser Phe Leu Pro Leu Leu Pro Ser 1 5 10 15 Leu Leu Leu Ser
Ser Tyr Leu Cys Leu Phe Phe Phe Pro Ser Gln Ser 20 25 30 Pro Ser
Ser Phe Phe Phe His Leu 35 40 200 71 PRT Homo sapiens SITE (25) Xaa
equals any of the naturally occurring L-amino acids 200 Met Thr Glu
Gly His Val Phe Cys Phe Ala Leu Cys Cys Val Leu Val 1 5 10 15 Phe
Leu Ser Met Thr Leu Leu Val Xaa Ser Leu Glu Lys Thr Asn Ala 20 25
30 Gly Gly Val Ile Ala Trp Gly Cys Ile Ser Val Ser Val Gln Thr Gln
35 40 45 Thr Phe Ser Ser Pro Thr Ser Tyr Gln Thr Leu Phe Ile Ala
Cys Lys 50 55 60 Leu Trp Asn Pro Arg Lys Leu 65 70 201 59 PRT Homo
sapiens SITE (37) Xaa equals any of the naturally occurring L-amino
acids 201 Met Ile Gly Leu Thr Ile Ile Ala Cys Phe Ala Val Ile Val
Ser Ala 1 5 10 15 Lys Arg Ala Val Glu Arg His Glu Ser Leu Thr Ser
Trp Asn Leu Ala 20 25 30 Lys Lys Ala Lys Xaa Arg Glu Glu Ala Ala
Leu Ala Ala Gln Ala Lys 35 40 45 Ala Asn Asp Ile Leu Ser Asp Lys
Val Phe Thr 50 55 202 80 PRT Homo sapiens 202 Met Leu Thr Gly Ser
His Pro Gln Thr His Thr Cys Trp Leu Gly Thr 1 5 10 15 Arg Leu Trp
Val Val Leu Ser Cys Leu Ala Ser Leu Thr Val Ser Asp 20 25 30 Cys
Pro Glu His Gln Val Ser Ser Cys Ile Ser Ser Trp Pro Gly Glu 35 40
45 His Ser Val Ser Phe Gln Pro Phe Pro Pro Phe Pro His Ser Leu Gly
50 55 60 Gly Thr Glu Val Gly Val Glu Glu Ser Gln Met Ala Gly Val
Gly Ile 65 70 75 80 203 70 PRT Homo sapiens 203 Met Ile Ser Gly Val
Leu Ile Phe Asn Leu Ile Ala Ser Ser Trp Val 1 5 10 15 Leu Cys Phe
Pro Leu Cys Asp Leu Ser Cys Gln Lys Thr Leu Arg Ile 20 25 30 Phe
Phe Ala Ser Phe Phe His Ala Val Cys Val His Val Ser Cys Thr 35 40
45 Ser Trp Gln Pro Leu Val Leu Phe Ile Lys Trp Trp Val Val Gly Cys
50 55 60 Ser Pro Ala Val Ser Leu 65 70 204 78 PRT Homo sapiens 204
Met Leu His Met Phe Leu Leu Leu Leu Tyr Phe Phe Lys Asn Ser Lys 1 5
10 15 Ser Leu Phe Met Cys His Trp Ile Asn Leu Ser Asp Asn Val Ser
His 20 25 30 Lys Asn Leu Leu Asp Arg Leu Phe Phe Ser Cys Thr Leu
Asn Gly Gly 35 40 45 Val Glu Val Ser Gly Glu Gln Trp Ile Thr Lys
Ser Lys Leu Trp Lys 50 55 60 Ile Val Lys Arg Met Glu Lys Leu Asn
Thr Arg Tyr Gln Lys 65 70 75 205 115 PRT Homo sapiens 205 Met Cys
Met Ser Val Gly Ala His Ile Cys Val Cys Val Cys Met Cys 1 5 10 15
Val Leu His Val Cys Gly Glu Val Ser Ser Val Arg Ala Cys Asp Ser 20
25 30 Trp Asp Leu His Ser Cys Val Leu Pro Gln Arg Pro Gln Pro Gly
Gln 35 40 45 Ala Leu Thr Phe Cys Ala Pro Cys Ile Glu Pro Val Cys
Cys Gly Cys 50 55 60 Leu Trp Pro Pro Met Gly Asn Ser Gly Glu Leu
Ala Gly Gly Cys Ala 65 70 75 80 Gln Ser Pro Gly Cys Cys Tyr Cys His
Ser Ala Gln Leu Gly Gln Ala 85 90 95 Val Ala Pro Glu Gly Val Arg
Arg Glu Leu Trp Glu His Leu Tyr Ser 100 105 110 Val Leu Lys 115 206
50 PRT Homo sapiens 206 Met Pro Gly Cys Trp Val Leu Glu Leu Val Asp
His Trp Leu Ala Ser 1 5 10 15 Leu Trp Leu Val Val Ala Val Thr Glu
Cys Ala Ala Arg Pro Glu Trp 20 25 30 Leu Phe Trp Leu Cys Pro Pro
Ser Cys Ser Met Pro Gly Gly Gly Gly 35 40 45 Asp Thr 50 207 57 PRT
Homo sapiens 207 Met Lys Phe Tyr Ala Val Leu Leu Ser Ile Cys Leu
Leu Leu Ser Cys 1 5 10 15 Trp Cys Ala Cys His Val Arg Asp Cys Asn
Leu Ile Cys Leu Phe Ser 20 25 30 Thr Val Lys Ala Ile Thr Arg Glu
Leu Leu Gln Leu Pro Ser Tyr Val 35 40 45 Lys Arg Phe Phe Phe Asn
Ser Leu Arg 50 55 208 56 PRT Homo sapiens 208 Met Leu Val Ala Pro
Phe Asn Leu Leu Phe Glu Met Ala Pro Phe Asn 1 5 10 15 Ile Phe Leu
Phe Pro Gln Trp Gly Leu Leu Trp Leu Met Leu Tyr Leu 20 25 30 Leu
Tyr Val Phe Gln Ala Ser Leu Arg Thr Pro Glu Leu Thr Trp Glu 35 40
45 Arg Val Arg Ser Gln Val Asp Gln 50 55 209 49 PRT Homo sapiens
209 Met Leu Leu Thr Cys Ile Leu Leu His Leu Trp Ile Val Val Asp Ser
1 5 10 15 Val Ile Tyr Met Lys Pro Thr Ser Arg Asp Gly Cys Leu Leu
Ser Ala 20 25 30 Leu Gln Met Ala Arg Ser Leu Ile Ile Gln Leu Asn
His Ser Ser Ser 35 40 45 Asn 210 44 PRT Homo sapiens 210 Met Pro
Leu Cys Gly Leu Tyr Cys Leu Arg Ile Leu Met Phe Pro Leu 1 5 10 15
Arg Ser Ala Asn Ser Val Pro Leu Gln Cys Leu Pro Pro Ser Ser Leu 20
25 30 Ala Asn Lys Asp Ser His Phe Arg Ala Pro Arg Lys 35 40 211 44
PRT Homo sapiens SITE (18) Xaa equals any of the naturally
occurring L-amino acids SITE (25) Xaa equals any of the naturally
occurring L-amino acids 211 Met Ser Pro Ser Pro Arg Trp Gly Phe Leu
Cys Val Leu Phe Thr Ala 1 5 10 15 Val Xaa Pro Ala Pro Ser Thr Ala
Xaa Val Gln Asp Lys Cys Pro Val 20 25 30 Asn Thr Trp Glu Ala Met
Gln Ala Cys Val His Gly 35 40 212 160 PRT Homo sapiens SITE (136)
Xaa equals any of the naturally occurring L-amino acids 212 Met Ala
Phe Thr Phe Ala Ala Phe Cys Tyr Met Leu Ser Leu Val Leu 1 5 10 15
Cys Ala Ala Leu Ile Phe Phe Ala Ile Trp His Ile Ile Ala Phe Asp 20
25 30 Glu Leu Arg Thr Asp Phe Lys Ser Pro Ile Asp Gln Cys Asn Pro
Val 35 40 45 His Ala Arg Glu Arg Leu Arg Asn Ile Glu Arg Ile Cys
Phe Leu Leu 50 55 60 Arg Lys Leu Val Leu Pro Glu Tyr Ser Ile His
Ser Leu Phe Cys Ile 65 70 75 80 Met Phe Leu Cys Ala Gln Glu Trp Leu
Thr Leu Gly Leu Asn Val Pro 85 90 95 Leu Leu Phe Tyr His Phe Trp
Arg Tyr Phe His Cys Pro Ala Asp Ser 100 105 110 Ser Glu Leu Ala Tyr
Asp Pro Pro Val Val Met Asn Ala Asp Thr Leu 115 120 125 Ser Tyr Cys
Gln Lys Glu Ala Xaa Cys Lys Leu Ala Phe Tyr Leu Leu 130 135 140 Ser
Phe Phe Tyr Tyr Leu Tyr Cys Met Ile Tyr Thr Leu Val Ser Ser 145 150
155 160 213 198 PRT Homo sapiens 213 Met Tyr Arg Glu Arg Leu Arg
Thr Leu Leu Val Ile Ala Val Val Met 1 5 10 15 Ser Leu Leu Asn Ala
Leu Ser Thr Ser Gly Gly Ser Ile Ser Trp Asn 20 25 30 Asp Phe Val
His Glu Met Leu Ala Lys Gly Glu Val Gln Arg Val Gln 35 40 45 Val
Val Pro Glu Ser Asp Val Val Glu Val Tyr Leu His Pro Gly Ala 50 55
60 Val Val Phe Gly Arg Pro Arg Leu Ala Leu Met Tyr Arg Met Gln Val
65 70 75 80 Ala Asn Ile Asp Lys Phe Glu Glu Lys Leu Arg Ala Ala Glu
Asp Glu 85 90 95 Leu Asn Ile Glu Ala Lys Asp Arg Ile Pro Val Ser
Tyr Lys Arg Thr 100 105 110 Gly Phe Phe Gly Lys Cys Pro Val Leu Cys
Gly Asp Asp Gly Ser Gly 115 120 125 Pro Gly His Pro Val Val Cys Phe
Pro Ser Gly Arg Asp Asp Trp Arg 130 135 140 His Arg Arg Arg Trp Thr
Ser Arg Ser Arg Leu Leu Cys Trp Lys Ala 145 150 155 160 Leu Met Gly
Ser Val Gly Ala Asp His Thr Arg Glu Leu Arg Lys Pro 165 170 175 Ser
Gly Ser His Arg Pro Pro Phe Asn Val Val Ile Pro Trp Trp Trp 180 185
190 Lys Gln Asp Asp Gly Pro 195 214 59 PRT Homo sapiens 214 Met Asn
Ser Thr Leu Cys Val Val Leu Ser Leu Met Cys Met Asn Ser 1 5 10 15
Thr Leu Cys Val Val Leu Ser Leu Thr His Ser Cys Pro Ser Pro Gln 20
25 30 Val Pro Lys Val His Tyr Met Ile Phe Met Pro Leu His Leu His
Ser 35 40 45 Leu Ala Leu Thr Gln Leu Ile Ile Ile Tyr Lys 50 55 215
84 PRT Homo sapiens SITE (71) Xaa equals any of the naturally
occurring L-amino acids 215 Met Gly Cys Ile Pro Leu Ile Lys Ser Ile
Ser Asp Trp Arg Val Ile 1 5 10 15 Ala Leu Ala Ala Leu Trp Phe Cys
Leu Ile Gly Leu Ile Cys Gln Ala 20 25 30 Leu Cys Ser Glu Asp Gly
His Lys Arg Arg Ile Leu Thr Leu Gly Leu 35 40 45 Gly Phe Leu Val
Ile Pro Phe Leu Pro Ala Ser Asn Leu Phe Phe Arg 50 55 60 Val Gly
Phe Val Val Ala Xaa Cys Ser Ser Thr Ser Pro Ala Leu Gly 65 70 75 80
Thr Val Cys Cys 216 81 PRT Homo sapiens 216 Met Val Val Ala Gly Val
Val Val Leu Ile Leu Ala Leu Val Leu Ala 1 5 10 15 Trp Leu Ser Thr
Tyr Val Ala Asp Ser Gly Ser Asn Gln Leu Leu Gly 20 25 30 Ala Ile
Val Ser Ala Gly Asp Thr Ser Val Leu His Leu Gly His Val 35 40 45
Asp His Leu Val Ala Gly Gln Gly Asn Pro Glu Pro Thr Glu Leu Pro 50
55 60 His Pro Ser Glu Asp Lys Gln Val Gln Ala Ala Ala Val Gln Arg
Pro 65 70 75 80 Pro 217 90 PRT Homo sapiens 217 Met Met Val Trp Asn
Leu Phe Pro Cys Phe Pro Pro Leu Leu Leu Leu 1 5 10 15 Gln Phe Ile
Asp Cys Gln Gln Ser Ser Glu Ile Glu Gln Gly Phe Thr 20
25 30 Arg Ser Leu Leu Gly His Pro Ile Phe Phe Cys Pro Asp Pro Cys
Trp 35 40 45 Gln Ser Cys Met Asn Cys Val Ile Leu Leu Ser Ala Phe
Phe Phe Leu 50 55 60 Phe Asp Lys Met Asp Ile Lys Asn Ser Cys Cys
Ala Lys Val Ser Ser 65 70 75 80 Leu Leu Gln Glu Glu Asn Gln Phe Phe
Phe 85 90 218 335 PRT Homo sapiens 218 Met Lys Lys Glu Leu Pro Val
Asp Ser Cys Leu Pro Arg Ser Leu Glu 1 5 10 15 Leu His Pro Gln Lys
Met Asp Pro Lys Arg Gln His Ile Gln Leu Leu 20 25 30 Ser Ser Leu
Thr Glu Cys Leu Thr Val Asp Pro Leu Ser Ala Ser Val 35 40 45 Trp
Arg Gln Leu Tyr Pro Lys His Leu Ser Gln Ser Ser Leu Leu Leu 50 55
60 Glu His Leu Leu Ser Ser Trp Glu Gln Ile Pro Lys Lys Val Gln Lys
65 70 75 80 Ser Leu Gln Glu Thr Ile Gln Ser Leu Lys Leu Thr Asn Gln
Glu Leu 85 90 95 Leu Arg Lys Gly Ser Ser Asn Asn Gln Asp Val Val
Thr Cys Asp Met 100 105 110 Ala Cys Lys Gly Leu Leu Gln Gln Val Gln
Gly Pro Arg Leu Pro Trp 115 120 125 Thr Arg Leu Leu Leu Leu Leu Leu
Val Phe Ala Val Gly Phe Leu Cys 130 135 140 His Asp Leu Arg Ser His
Ser Ser Phe Gln Ala Ser Leu Thr Gly Arg 145 150 155 160 Leu Leu Arg
Ser Ser Gly Phe Leu Pro Ala Ser Gln Gln Ala Cys Ala 165 170 175 Lys
Leu Tyr Ser Tyr Ser Leu Gln Gly Tyr Ser Trp Leu Gly Glu Thr 180 185
190 Leu Pro Leu Trp Gly Ser His Leu Leu Thr Val Val Arg Pro Ser Leu
195 200 205 Gln Leu Ala Trp Ala His Thr Asn Ala Thr Val Ser Phe Leu
Ser Ala 210 215 220 His Cys Ala Ser His Leu Ala Trp Phe Gly Asp Ser
Leu Thr Ser Leu 225 230 235 240 Ser Gln Arg Leu Gln Ile Gln Leu Pro
Asp Ser Val Asn Gln Leu Leu 245 250 255 Arg Tyr Leu Arg Glu Leu Pro
Leu Leu Phe His Gln Asn Val Leu Leu 260 265 270 Pro Leu Trp His Leu
Leu Leu Glu Ala Leu Ala Trp Ala Gln Glu His 275 280 285 Cys His Glu
Ala Cys Arg Gly Glu Val Thr Trp Asp Cys Met Lys Thr 290 295 300 Gln
Leu Ser Glu Ala Val His Trp Thr Trp Leu Cys Leu Gln Asp Ile 305 310
315 320 Thr Val Ala Phe Leu Asp Trp Ala Leu Ala Leu Ile Ser Gln Gln
325 330 335 219 229 PRT Homo sapiens 219 Met Asp Pro Asp Arg Ala
Phe Ile Cys Gly Glu Ser Arg Gln Phe Ala 1 5 10 15 Gln Cys Leu Ile
Phe Gly Phe Leu Phe Leu Thr Ser Gly Met Leu Ile 20 25 30 Ser Val
Leu Gly Ile Trp Val Pro Gly Cys Gly Ser Asn Trp Ala Gln 35 40 45
Glu Pro Leu Asn Glu Thr Asp Thr Gly Asp Ser Glu Pro Arg Met Cys 50
55 60 Gly Phe Leu Ser Leu Gln Ile Met Gly Pro Leu Ile Val Leu Val
Gly 65 70 75 80 Leu Cys Phe Phe Val Val Ala His Val Lys Lys Arg Asn
Thr Leu Asn 85 90 95 Ala Gly Gln Asp Ala Ser Glu Arg Glu Glu Gly
Gln Ile Gln Ile Met 100 105 110 Glu Pro Val Gln Val Thr Val Gly Asp
Ser Val Ile Ile Phe Pro Pro 115 120 125 Pro Pro Pro Pro Tyr Phe Pro
Glu Ser Ser Ala Ser Ala Val Ala Glu 130 135 140 Ser Pro Gly Thr Asn
Ser Leu Leu Pro Asn Glu Asn Pro Pro Ser Tyr 145 150 155 160 Tyr Ser
Ile Phe Asn Tyr Gly Thr Pro Thr Ser Glu Gly Ala Ala Ser 165 170 175
Glu Arg Asp Cys Glu Ser Ile Tyr Thr Ile Ser Gly Thr Asn Ser Ser 180
185 190 Ser Glu Ala Ser His Thr Pro His Leu Pro Ser Glu Leu Pro Pro
Arg 195 200 205 Tyr Glu Glu Lys Glu Asn Ala Ala Ala Thr Phe Leu Pro
Leu Ser Ser 210 215 220 Glu Pro Ser Pro Pro 225 220 62 PRT Homo
sapiens 220 Met Ser Ile Ser Leu Ser Ser Leu Ile Leu Leu Pro Ile Trp
Ile Asn 1 5 10 15 Met Ala Gln Ile Gln Gln Gly Gly Pro Asp Glu Lys
Glu Lys Thr Thr 20 25 30 Ala Leu Lys Asp Leu Leu Ser Arg Ile Asp
Leu Asp Glu Leu Met Lys 35 40 45 Lys Asp Glu Pro Pro Leu Asp Phe
Leu Ile Pro Trp Lys Val 50 55 60 221 170 PRT Homo sapiens SITE
(163) Xaa equals any of the naturally occurring L-amino acids 221
Met Ala Ala Gly Pro Gly Thr His Leu Ser Leu Phe Trp Ala Arg Ile 1 5
10 15 Ala Thr Leu Ala Val Trp Ala Ala Ala Ala Leu Val Thr Val Pro
Thr 20 25 30 Ala Val Phe Gly Val Glu Gly Glu Val Cys Gly Val Arg
Leu Cys Leu 35 40 45 Leu Arg Phe Pro Ser Arg Tyr Trp Leu Gly Ala
Tyr Gln Leu Gln Arg 50 55 60 Val Val Leu Ala Phe Met Val Pro Leu
Gly Val Ile Thr Thr Ser Tyr 65 70 75 80 Leu Leu Leu Leu Ala Phe Leu
Gln Arg Arg Gln Arg Arg Arg Gln Asp 85 90 95 Ser Arg Val Val Ala
Arg Ser Val Arg Ile Leu Val Ala Ser Phe Phe 100 105 110 Leu Cys Trp
Phe Pro Asn His Val Val Thr Leu Trp Gly Val Leu Val 115 120 125 Lys
Phe Asp Leu Val Pro Trp Asn Ser Thr Phe Tyr Thr Ile Gln Thr 130 135
140 Tyr Val Phe Pro Val Thr Thr Cys Leu Ala His Ser Asn Ser Cys Leu
145 150 155 160 Asn Pro Xaa Ala Tyr Val Leu Ser Arg Ile 165 170 222
42 PRT Homo sapiens SITE (18) Xaa equals any of the naturally
occurring L-amino acids SITE (37) Xaa equals any of the naturally
occurring L-amino acids 222 Met Ala Gly Cys Leu Gly Ser Tyr Leu Leu
Val Met Ile Leu Ile Leu 1 5 10 15 Cys Xaa Ala His Phe Phe Ile Cys
Gly Asn Glu Asp Asn Arg Val Leu 20 25 30 Arg Tyr Asn Leu Xaa Thr
Met Ser Val Thr 35 40 223 56 PRT Homo sapiens 223 Met Cys Ile Ser
Gly Cys Leu Phe His Cys Ser Ile Cys Leu Phe Phe 1 5 10 15 Met Leu
Val Pro Tyr Cys Phe Asp Tyr Cys Leu Val Met Tyr Phe Glu 20 25 30
Ile Lys Thr Cys Gly Tyr Leu Leu Leu Cys Ser Pro Cys Gln Asp Tyr 35
40 45 Ser Arg Ser Phe Val Ala Ser Ser 50 55 224 96 PRT Homo sapiens
224 Met Tyr Arg Glu Arg Leu Arg Thr Leu Leu Val Ile Ala Val Val Met
1 5 10 15 Ser Leu Leu Asn Ala Leu Ser Thr Ser Gly Gly Ser Ile Ser
Trp Asn 20 25 30 Asp Phe Val His Glu Met Leu Ala Lys Gly Glu Val
Gln Arg Val Gln 35 40 45 Val Val Pro Glu Ser Asp Val Val Glu Val
Tyr Leu His Pro Gly Ala 50 55 60 Val Val Phe Gly Arg Pro Arg Leu
Ala Leu Met Tyr Arg Met Gln Leu 65 70 75 80 Gln Ile Leu Thr Ser Leu
Lys Arg Ser Phe Glu Gln Leu Lys Met Ser 85 90 95 225 22 PRT Homo
sapiens 225 Trp Ala Gly Thr Gln Glu Pro Thr Gly Leu Pro Ser Thr Leu
Ser Arg 1 5 10 15 Ser Glu Ser Trp Asp His 20 226 171 PRT Homo
sapiens 226 Glu Ile Ile His Asn Leu Pro Thr Ser Arg Met Ala Ala Arg
Thr Lys 1 5 10 15 Lys Lys Asn Asp Ile Ile Asn Ile Lys Val Pro Ala
Asp Cys Asn Thr 20 25 30 Arg Met Ser Tyr Tyr Tyr Lys Gly Ser Gly
Lys Arg Gly Glu Met Glu 35 40 45 Ser Trp Leu Val Met Ser Ser Trp
Ser Ile Leu Asp Phe Glu Phe Leu 50 55 60 Glu Ala Arg Pro Gln Leu
Phe Asn Leu Val Tyr Thr Glu His Ser Thr 65 70 75 80 Tyr Ser Gly Arg
His Tyr Thr Arg Glu Arg Gly Gly Phe Met Val Phe 85 90 95 Lys Asn
Ser Tyr Ser Gln Leu Leu Leu Lys Arg Lys Asp Ser Leu Cys 100 105 110
Ala Phe Ile Gln Pro Met Ala Leu Asn Ile Ile His Val Pro Met Ser 115
120 125 Ser Lys Cys Ile Phe Pro Ala Gln Ser Gly Pro Ser Thr Phe Arg
Ser 130 135 140 Leu Trp Trp Cys Pro His Pro Ile Ser Lys Cys Gln Leu
Gly Leu Tyr 145 150 155 160 Ser Ser Gln Ile Arg Asp Ile Pro Tyr Leu
Ala 165 170 227 35 PRT Homo sapiens 227 Glu Ile Ile His Asn Leu Pro
Thr Ser Arg Met Ala Ala Arg Thr Lys 1 5 10 15 Lys Lys Asn Asp Ile
Ile Asn Ile Lys Val Pro Ala Asp Cys Asn Thr 20 25 30 Arg Met Ser 35
228 36 PRT Homo sapiens 228 Tyr Tyr Tyr Lys Gly Ser Gly Lys Arg Gly
Glu Met Glu Ser Trp Leu 1 5 10 15 Val Met Ser Ser Trp Ser Ile Leu
Asp Phe Glu Phe Leu Glu Ala Arg 20 25 30 Pro Gln Leu Phe 35 229 36
PRT Homo sapiens 229 Asn Leu Val Tyr Thr Glu His Ser Thr Tyr Ser
Gly Arg His Tyr Thr 1 5 10 15 Arg Glu Arg Gly Gly Phe Met Val Phe
Lys Asn Ser Tyr Ser Gln Leu 20 25 30 Leu Leu Lys Arg 35 230 35 PRT
Homo sapiens 230 Lys Asp Ser Leu Cys Ala Phe Ile Gln Pro Met Ala
Leu Asn Ile Ile 1 5 10 15 His Val Pro Met Ser Ser Lys Cys Ile Phe
Pro Ala Gln Ser Gly Pro 20 25 30 Ser Thr Phe 35 231 29 PRT Homo
sapiens 231 Arg Ser Leu Trp Trp Cys Pro His Pro Ile Ser Lys Cys Gln
Leu Gly 1 5 10 15 Leu Tyr Ser Ser Gln Ile Arg Asp Ile Pro Tyr Leu
Ala 20 25 232 533 PRT Homo sapiens SITE (473) Xaa equals any of the
naturally occurring L-amino acids 232 Glu Ala Cys Gly Ala Ala Ala
Met Ala Ala Leu Thr Ile Ala Thr Gly 1 5 10 15 Thr Gly Asn Trp Phe
Ser Ala Leu Ala Leu Gly Val Thr Leu Leu Lys 20 25 30 Cys Leu Leu
Ile Pro Thr Tyr His Ser Thr Asp Phe Glu Val His Arg 35 40 45 Asn
Trp Leu Ala Ile Thr His Ser Leu Pro Ile Ser Gln Trp Tyr Tyr 50 55
60 Glu Ala Thr Ser Glu Trp Thr Leu Asp Tyr Pro Pro Phe Phe Ala Trp
65 70 75 80 Phe Glu Tyr Ile Leu Ser His Val Ala Lys Tyr Phe Asp Gln
Glu Met 85 90 95 Leu Asn Val His Asn Leu Asn Tyr Ser Ser Ser Arg
Thr Leu Leu Phe 100 105 110 Gln Arg Phe Ser Val Ile Phe Met Asp Val
Leu Phe Val Tyr Ala Val 115 120 125 Arg Glu Cys Cys Lys Cys Ile Asp
Gly Lys Lys Val Gly Lys Glu Leu 130 135 140 Thr Glu Lys Pro Lys Phe
Ile Leu Ser Val Leu Leu Leu Trp Asn Phe 145 150 155 160 Gly Leu Leu
Ile Val Asp His Ile His Phe Gln Tyr Asn Gly Phe Leu 165 170 175 Phe
Gly Leu Met Leu Leu Ser Ile Ala Arg Leu Phe Gln Lys Arg His 180 185
190 Met Glu Gly Ala Phe Leu Phe Ala Val Leu Leu His Phe Lys His Ile
195 200 205 Tyr Leu Tyr Val Ala Pro Ala Tyr Gly Val Tyr Leu Leu Arg
Ser Tyr 210 215 220 Cys Phe Thr Ala Asn Lys Pro Asp Gly Ser Ile Arg
Trp Lys Ser Phe 225 230 235 240 Ser Phe Val Arg Val Ile Ser Leu Gly
Leu Val Val Phe Leu Val Ser 245 250 255 Ala Leu Ser Leu Gly Pro Phe
Leu Ala Leu Asn Gln Leu Pro Gln Val 260 265 270 Phe Ser Arg Leu Phe
Pro Phe Lys Arg Gly Leu Cys His Ala Tyr Trp 275 280 285 Ala Pro Asn
Phe Trp Ala Leu Tyr Asn Ala Leu Asp Lys Val Leu Ser 290 295 300 Val
Ile Gly Leu Lys Leu Lys Phe Leu Asp Pro Asn Asn Ile Pro Lys 305 310
315 320 Ala Ser Met Thr Ser Gly Leu Val Gln Gln Phe Gln His Thr Val
Leu 325 330 335 Pro Ser Val Thr Pro Leu Ala Thr Leu Ile Cys Thr Leu
Ile Ala Ile 340 345 350 Leu Pro Ser Ile Phe Cys Leu Trp Phe Lys Pro
Gln Gly Pro Arg Gly 355 360 365 Phe Leu Arg Cys Leu Thr Leu Cys Ala
Leu Ser Ser Phe Met Phe Gly 370 375 380 Trp His Val His Glu Lys Ala
Ile Leu Leu Ala Ile Leu Pro Met Ser 385 390 395 400 Leu Leu Ser Val
Gly Lys Ala Gly Asp Ala Ser Ile Phe Leu Ile Leu 405 410 415 Thr Thr
Thr Gly His Tyr Ser Leu Phe Pro Leu Leu Phe Thr Ala Pro 420 425 430
Glu Leu Pro Ile Lys Ile Leu Leu Met Leu Leu Phe Thr Ile Tyr Ser 435
440 445 Ile Ser Ser Leu Lys Thr Leu Phe Arg Lys Glu Lys Pro Leu Phe
Asn 450 455 460 Trp Met Glu Thr Phe Tyr Leu Leu Xaa Leu Gly Pro Leu
Glu Val Cys 465 470 475 480 Cys Glu Phe Val Phe Pro Phe Thr Ser Trp
Lys Val Lys Tyr Pro Phe 485 490 495 Ile Pro Leu Leu Leu Thr Ser Val
Tyr Cys Ala Val Gly Ile Thr Tyr 500 505 510 Ala Trp Phe Lys Leu Tyr
Val Ser Val Leu Ile Asp Ser Ala Ile Gly 515 520 525 Lys Thr Lys Lys
Gln 530 233 460 PRT Homo sapiens 233 Met Phe Thr Ile Lys Leu Leu
Leu Phe Ile Val Pro Leu Val Ile Ser 1 5 10 15 Ser Arg Ile Asp Gln
Asp Asn Ser Ser Phe Asp Ser Leu Ser Pro Glu 20 25 30 Pro Lys Ser
Arg Phe Ala Met Leu Asp Asp Val Lys Ile Leu Ala Asn 35 40 45 Gly
Leu Leu Gln Leu Gly His Gly Leu Lys Asp Phe Val His Lys Thr 50 55
60 Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln
65 70 75 80 Ser Phe Tyr Asp Leu Ser Leu Gln Thr Ser Glu Ile Lys Glu
Glu Glu 85 90 95 Lys Glu Leu Arg Arg Thr Thr Tyr Lys Leu Gln Val
Lys Asn Glu Glu 100 105 110 Val Lys Asn Met Ser Leu Glu Leu Asn Ser
Lys Leu Glu Ser Leu Leu 115 120 125 Glu Glu Lys Ile Leu Leu Gln Gln
Lys Val Lys Tyr Leu Glu Glu Gln 130 135 140 Leu Thr Asn Leu Ile Gln
Asn Gln Pro Glu Thr Pro Glu His Pro Glu 145 150 155 160 Val Thr Ser
Leu Lys Thr Phe Val Glu Lys Gln Asp Asn Ser Ile Lys 165 170 175 Asp
Leu Leu Gln Thr Val Glu Asp Gln Tyr Lys Gln Leu Asn Gln Gln 180 185
190 His Ser Gln Ile Lys Glu Ile Glu Asn Gln Leu Arg Arg Thr Ser Ile
195 200 205 Gln Glu Pro Thr Glu Ile Ser Leu Ser Ser Lys Pro Arg Ala
Pro Arg 210 215 220 Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn
Val Lys His Asp 225 230 235 240 Gly Ile Pro Ala Glu Cys Thr Thr Ile
Tyr Asn Arg Gly Glu His Thr 245 250 255 Ser Gly Met Tyr Ala Ile Arg
Pro Ser Asn Ser Gln Val Phe His Val 260 265 270 Tyr Cys Asp Val Ile
Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg 275 280 285 Ile Asp Gly
Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr 290 295 300 Gly
Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile 305 310
315 320 Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu
Glu 325 330 335 Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe
Tyr Leu Gly 340 345 350 Asn His Glu Thr Asn Tyr Thr Leu His Leu Val
Ala Ile Thr Gly Asn 355 360 365 Val Pro Asn Ala Ile Pro Glu Asn Lys
Asp Leu Val Phe Ser Thr Trp 370 375 380 Asp His Lys Ala Lys Gly His
Phe Asn Cys Pro Glu Gly Tyr Ser Gly 385 390 395
400 Gly Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys
405 410 415 Tyr Asn Lys Pro Arg Ala Lys Ser Lys Pro Glu Arg Arg Arg
Gly Leu 420 425 430 Ser Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile
Lys Ser Thr Lys 435 440 445 Met Leu Ile His Pro Thr Asp Ser Glu Ser
Phe Glu 450 455 460 234 37 PRT Homo sapiens 234 Met Phe Thr Ile Lys
Leu Leu Leu Phe Ile Val Pro Leu Val Ile Ser 1 5 10 15 Ser Arg Ile
Asp Gln Asp Asn Ser Ser Phe Asp Ser Leu Ser Pro Glu 20 25 30 Pro
Lys Ser Arg Phe 35 235 34 PRT Homo sapiens 235 Ala Met Leu Asp Asp
Val Lys Ile Leu Ala Asn Gly Leu Leu Gln Leu 1 5 10 15 Gly His Gly
Leu Lys Asp Phe Val His Lys Thr Lys Gly Gln Ile Asn 20 25 30 Asp
Ile 236 35 PRT Homo sapiens 236 Phe Gln Lys Leu Asn Ile Phe Asp Gln
Ser Phe Tyr Asp Leu Ser Leu 1 5 10 15 Gln Thr Ser Glu Ile Lys Glu
Glu Glu Lys Glu Leu Arg Arg Thr Thr 20 25 30 Tyr Lys Leu 35 237 36
PRT Homo sapiens 237 Gln Val Lys Asn Glu Glu Val Lys Asn Met Ser
Leu Glu Leu Asn Ser 1 5 10 15 Lys Leu Glu Ser Leu Leu Glu Glu Lys
Ile Leu Leu Gln Gln Lys Val 20 25 30 Lys Tyr Leu Glu 35 238 36 PRT
Homo sapiens 238 Glu Gln Leu Thr Asn Leu Ile Gln Asn Gln Pro Glu
Thr Pro Glu His 1 5 10 15 Pro Glu Val Thr Ser Leu Lys Thr Phe Val
Glu Lys Gln Asp Asn Ser 20 25 30 Ile Lys Asp Leu 35 239 35 PRT Homo
sapiens 239 Leu Gln Thr Val Glu Asp Gln Tyr Lys Gln Leu Asn Gln Gln
His Ser 1 5 10 15 Gln Ile Lys Glu Ile Glu Asn Gln Leu Arg Arg Thr
Ser Ile Gln Glu 20 25 30 Pro Thr Glu 35 240 35 PRT Homo sapiens 240
Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro Arg Thr Thr Pro Phe Leu 1 5
10 15 Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp Gly Ile Pro Ala
Glu 20 25 30 Cys Thr Thr 35 241 36 PRT Homo sapiens 241 Ile Tyr Asn
Arg Gly Glu His Thr Ser Gly Met Tyr Ala Ile Arg Pro 1 5 10 15 Ser
Asn Ser Gln Val Phe His Val Tyr Cys Asp Val Ile Ser Gly Ser 20 25
30 Pro Trp Thr Leu 35 242 36 PRT Homo sapiens 242 Ile Gln His Arg
Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu 1 5 10 15 Asn Tyr
Lys Tyr Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly 20 25 30
Leu Glu Lys Ile 35 243 35 PRT Homo sapiens 243 Tyr Ser Ile Val Lys
Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu 1 5 10 15 Asp Trp Lys
Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly 20 25 30 Asn
His Glu 35 244 35 PRT Homo sapiens 244 Thr Asn Tyr Thr Leu His Leu
Val Ala Ile Thr Gly Asn Val Pro Asn 1 5 10 15 Ala Ile Pro Glu Asn
Lys Asp Leu Val Phe Ser Thr Trp Asp His Lys 20 25 30 Ala Lys Gly 35
245 36 PRT Homo sapiens 245 His Phe Asn Cys Pro Glu Gly Tyr Ser Gly
Gly Trp Trp Trp His Asp 1 5 10 15 Glu Cys Gly Glu Asn Asn Leu Asn
Gly Lys Tyr Asn Lys Pro Arg Ala 20 25 30 Lys Ser Lys Pro 35 246 34
PRT Homo sapiens 246 Glu Arg Arg Arg Gly Leu Ser Trp Lys Ser Gln
Asn Gly Arg Leu Tyr 1 5 10 15 Ser Ile Lys Ser Thr Lys Met Leu Ile
His Pro Thr Asp Ser Glu Ser 20 25 30 Phe Glu 247 36 PRT Homo
sapiens 247 Leu Pro Pro Arg Gly Pro Ala Thr Phe Gly Ser Pro Gly Cys
Pro Pro 1 5 10 15 Ala Asn Ser Pro Pro Ser Ala Pro Ala Thr Pro Glu
Pro Ala Arg Ala 20 25 30 Pro Glu Arg Val 35 248 44 PRT Homo sapiens
248 Gly Thr Arg Ala Gly Val Ser Lys Tyr Thr Gly Gly Arg Gly Val Thr
1 5 10 15 Trp Ala Pro Ser Ser Ala Ala Val Pro Arg Ile Ser Ser Ala
Thr Met 20 25 30 Arg Met Gly Leu Thr Ser Phe Ser Thr Thr Gly Ala 35
40 249 306 PRT Homo sapiens SITE (293) Xaa equals any of the
naturally occurring L-amino acids 249 Trp Gln Ser Gly His Arg Leu
Trp Gln Leu Glu Trp Pro Pro Pro Pro 1 5 10 15 Leu Ser Ala Asp Glu
His Pro Trp Glu Gly Pro Leu Pro Gly Thr Ser 20 25 30 Pro Ser Pro
Lys Phe Ser Met Pro Ser Pro Val Pro His Gly His His 35 40 45 Arg
Pro Thr Leu Thr Met Thr Arg Ser Trp Arg Ile Phe Phe Asn Asn 50 55
60 Ile Ala Tyr Arg Ser Ser Ser Ala Asn Arg Leu Phe Arg Val Ile Arg
65 70 75 80 Arg Glu His Gly Asp Pro Leu Ile Glu Glu Leu Asn Pro Gly
Asp Ala 85 90 95 Leu Glu Pro Glu Gly Arg Gly Thr Gly Gly Val Val
Thr Asp Phe Asp 100 105 110 Gly Asp Gly Met Leu Asp Leu Ile Leu Ser
His Gly Glu Ser Met Ala 115 120 125 Gln Pro Leu Ser Val Phe Arg Gly
Asn Gln Gly Phe Asn Asn Asn Trp 130 135 140 Leu Arg Val Val Pro Arg
Thr Arg Phe Gly Ala Phe Ala Arg Gly Ala 145 150 155 160 Lys Val Val
Leu Tyr Thr Lys Lys Ser Gly Ala His Leu Arg Ile Ile 165 170 175 Asp
Gly Gly Ser Gly Tyr Leu Cys Glu Met Glu Pro Val Ala His Phe 180 185
190 Gly Leu Gly Lys Asp Glu Ala Ser Ser Val Glu Val Thr Trp Pro Asp
195 200 205 Gly Lys Met Val Ser Arg Asn Val Ala Ser Gly Glu Met Asn
Ser Val 210 215 220 Leu Glu Ile Leu Tyr Pro Arg Asp Glu Asp Thr Leu
Gln Asp Pro Ala 225 230 235 240 Pro Leu Glu Cys Gly Gln Gly Phe Ser
Gln Gln Glu Asn Gly His Cys 245 250 255 Met Asp Thr Asn Glu Cys Ile
Gln Phe Pro Phe Val Cys Pro Arg Asp 260 265 270 Lys Pro Val Cys Val
Asn Thr Tyr Gly Ser Tyr Arg Cys Arg Thr Asn 275 280 285 Lys Lys Cys
Ser Xaa Gly Leu Arg Val Pro Thr Arg Met Ala His Thr 290 295 300 Gly
Leu 305 250 36 PRT Homo sapiens 250 Trp Gln Ser Gly His Arg Leu Trp
Gln Leu Glu Trp Pro Pro Pro Pro 1 5 10 15 Leu Ser Ala Asp Glu His
Pro Trp Glu Gly Pro Leu Pro Gly Thr Ser 20 25 30 Pro Ser Pro Lys 35
251 35 PRT Homo sapiens 251 Phe Ser Met Pro Ser Pro Val Pro His Gly
His His Arg Pro Thr Leu 1 5 10 15 Thr Met Thr Arg Ser Trp Arg Ile
Phe Phe Asn Asn Ile Ala Tyr Arg 20 25 30 Ser Ser Ser 35 252 37 PRT
Homo sapiens 252 Ala Asn Arg Leu Phe Arg Val Ile Arg Arg Glu His
Gly Asp Pro Leu 1 5 10 15 Ile Glu Glu Leu Asn Pro Gly Asp Ala Leu
Glu Pro Glu Gly Arg Gly 20 25 30 Thr Gly Gly Val Val 35 253 34 PRT
Homo sapiens 253 Thr Asp Phe Asp Gly Asp Gly Met Leu Asp Leu Ile
Leu Ser His Gly 1 5 10 15 Glu Ser Met Ala Gln Pro Leu Ser Val Phe
Arg Gly Asn Gln Gly Phe 20 25 30 Asn Asn 254 35 PRT Homo sapiens
254 Asn Trp Leu Arg Val Val Pro Arg Thr Arg Phe Gly Ala Phe Ala Arg
1 5 10 15 Gly Ala Lys Val Val Leu Tyr Thr Lys Lys Ser Gly Ala His
Leu Arg 20 25 30 Ile Ile Asp 35 255 36 PRT Homo sapiens 255 Gly Gly
Ser Gly Tyr Leu Cys Glu Met Glu Pro Val Ala His Phe Gly 1 5 10 15
Leu Gly Lys Asp Glu Ala Ser Ser Val Glu Val Thr Trp Pro Asp Gly 20
25 30 Lys Met Val Ser 35 256 35 PRT Homo sapiens 256 Arg Asn Val
Ala Ser Gly Glu Met Asn Ser Val Leu Glu Ile Leu Tyr 1 5 10 15 Pro
Arg Asp Glu Asp Thr Leu Gln Asp Pro Ala Pro Leu Glu Cys Gly 20 25
30 Gln Gly Phe 35 257 36 PRT Homo sapiens 257 Ser Gln Gln Glu Asn
Gly His Cys Met Asp Thr Asn Glu Cys Ile Gln 1 5 10 15 Phe Pro Phe
Val Cys Pro Arg Asp Lys Pro Val Cys Val Asn Thr Tyr 20 25 30 Gly
Ser Tyr Arg 35 258 22 PRT Homo sapiens SITE (9) Xaa equals any of
the naturally occurring L-amino acids 258 Cys Arg Thr Asn Lys Lys
Cys Ser Xaa Gly Leu Arg Val Pro Thr Arg 1 5 10 15 Met Ala His Thr
Gly Leu 20 259 9 PRT Homo sapiens 259 Gln Ser Pro Ile Asp Ile Gln
Thr Asp 1 5 260 18 PRT Homo sapiens 260 Leu His Asn Asn Gly His Thr
Val Gln Leu Ser Leu Pro Ser Thr Leu 1 5 10 15 Tyr Leu 261 11 PRT
Homo sapiens 261 Tyr Val Ala Ala Gln Leu His Leu His Trp Gly 1 5 10
262 11 PRT Homo sapiens 262 Ala Glu Leu His Ile Val His Tyr Asp Ser
Asp 1 5 10 263 16 PRT Homo sapiens 263 Gly Gln His Trp Thr Tyr Glu
Gly Pro His Gly Gln Asp His Trp Pro 1 5 10 15 264 14 PRT Homo
sapiens 264 Gln Ser Pro Ile Asp Ile Gln Thr Asp Ser Val Thr Phe Asp
1 5 10 265 15 PRT Homo sapiens 265 Leu His Asn Asn Gly His Thr Val
Gln Leu Ser Leu Pro Ser Thr 1 5 10 15 266 12 PRT Homo sapiens 266
Lys Tyr Val Ala Ala Gln Leu His Leu His Trp Gly 1 5 10 267 13 PRT
Homo sapiens 267 Ala Glu Leu His Ile Val His Tyr Asp Ser Asp Ser
Tyr 1 5 10 268 1667 DNA Homo sapiens 268 ggccgcgccg ccgctgccgc
cgccgcgcgc gattctgctt ctcagaagat gcactattat 60 agatactcta
acgccaaggt cagctgctgg tacaagtacc tccttttcag ctacaacatc 120
atcttctgat tggctggagt tgtcttcctt ggagtcgggc tgtgggcatg gagcgaaaag
180 ggtgtgctgt ccgacctcac caaagtgacc cggatgcatg gaatcgaccc
tgtggtgctg 240 gtcctgatgg tgggcgtggt gatgttcacc ctggggttcg
ccggctgcgt gggggctctg 300 cgggagaata tctgcttgct caactttttc
tgtggcacca tcgtgctcat cttcttcctg 360 gagctggctg tggccgtgct
ggccttcctg ttccaggact gggtgaggga ccggttccgg 420 gagttcttcg
agagcaacat caagtcctac cgggacgata tcgatctgca aaacctcatc 480
gactcccttc agaaagctaa ccagtgctgt ggcgcatatg gccctgaaag actgggacct
540 cagacgtcta cttcaattgc agcggtgcca gctacagccg agagaatgcg
gggtcccctt 600 ctcctgctgc gtgccagatc ctgcgcaaaa agttgtgaac
acacagtgtg gatatgatgt 660 caggattcag ctgaagagca agtgggatga
gtccatcttc acgaaaggct gcatccaggc 720 gctggaaagc tggctcccgc
ggaacattta cattgtggct ggcgtcttca tcgccatctc 780 gctgttgcag
atatttggca tcttcctggc aaggacgctg atctcagaca tcgaggcagt 840
gaaggccggc catcacttct gaggagcaga gttgagggag ccgagctgag ccacgctggg
900 aggccagagc ctttctctgc catcagccct acgtccagag ggagaggagc
cgacaccccc 960 agagccagtg ccccatctta agcatcagcg tgacgtgacc
tctctgtttc tgcttgctgg 1020 tgctgaagac caagggtccc ccttgttacc
tgcccaaact tgtgactgca tccctctgga 1080 gtctacccag agacagagaa
tgtgtcttta tgtgggagtg gtgactctga aagacagaga 1140 gggctcctgt
ggctgccagg agggcttgac tcagaccccc tgcagctcaa gcatgtctgc 1200
aggacacctg gtccccctct cccagtggca tcccaaacat ctgctttggg tccatcccac
1260 atctgtgggt gggcccgtgg gtaagaaggg aaccccacag gcgtggaaca
gggcatcctc 1320 tctcccatcc aagcaaagcc agcatggggg cctgcccgta
acgggaggcg gacgtggccc 1380 cgctgggcct ctgagtgcca gcgcagtctg
ctgggacatg cacatatcag gggttgtttg 1440 caggatcctc agccatgttc
aagtgaagta agcctgagcc agtgcgtgga ctggtgccac 1500 gggagtgcct
tgtccactgt ccccctgtgt ccaccagcta ttctcctggc gccggaactg 1560
cctctggtct tgatagcatt aagccctgat tggccggtgg cgcggtgggc atggttcttc
1620 actgagagcc ggctctcctt ttcttaaagt gtgtaaatag tttattt 1667 269
270 PRT Homo sapiens 269 Met His Tyr Tyr Arg Tyr Ser Asn Ala Lys
Val Ser Cys Trp Tyr Lys 1 5 10 15 Tyr Leu Leu Phe Ser Tyr Asn Ile
Ile Phe Trp Leu Ala Gly Val Val 20 25 30 Phe Leu Gly Val Gly Leu
Trp Ala Trp Ser Glu Lys Gly Val Leu Ser 35 40 45 Asp Leu Thr Lys
Val Thr Arg Met His Gly Ile Asp Pro Val Val Leu 50 55 60 Val Leu
Met Val Gly Val Val Met Phe Thr Leu Gly Phe Ala Gly Cys 65 70 75 80
Val Gly Ala Leu Arg Glu Asn Ile Cys Leu Leu Asn Phe Phe Cys Gly 85
90 95 Thr Ile Val Leu Ile Phe Phe Leu Glu Leu Ala Val Ala Val Leu
Ala 100 105 110 Phe Leu Phe Gln Asp Trp Val Arg Asp Arg Phe Arg Glu
Phe Phe Glu 115 120 125 Ser Asn Ile Lys Ser Tyr Arg Asp Asp Ile Asp
Leu Gln Asn Leu Ile 130 135 140 Asp Ser Leu Gln Lys Ala Asn Gln Cys
Cys Gly Ala Tyr Gly Pro Glu 145 150 155 160 Asp Trp Asp Leu Asn Val
Tyr Phe Asn Cys Ser Gly Ala Ser Tyr Ser 165 170 175 Arg Glu Lys Cys
Gly Val Pro Phe Ser Cys Cys Val Pro Asp Pro Ala 180 185 190 Gln Lys
Val Val Asn Thr Gln Cys Gly Tyr Asp Val Arg Ile Gln Leu 195 200 205
Lys Ser Lys Trp Asp Glu Ser Ile Phe Thr Lys Gly Cys Ile Gln Ala 210
215 220 Leu Glu Ser Trp Leu Pro Arg Asn Ile Tyr Ile Val Ala Gly Val
Phe 225 230 235 240 Ile Ala Ile Ser Leu Leu Gln Ile Phe Gly Ile Phe
Leu Ala Arg Thr 245 250 255 Leu Ile Ser Asp Ile Glu Ala Val Lys Ala
Gly His His Phe 260 265 270 270 277 PRT Homo sapiens 270 Ser Gly
Asn Leu Gly Ser Ala Asp Gly Trp Ala Tyr Ile Asp Val Glu 1 5 10 15
Val Arg Arg Pro Trp Ala Phe Val Gly Pro Gly Cys Ser Arg Ser Ser 20
25 30 Gly Asn Gly Ser Thr Ala Tyr Gly Leu Val Gly Ser Pro Arg Trp
Leu 35 40 45 Ser Pro Phe His Thr Gly Gly Ala Val Ser Leu Pro Arg
Arg Pro Arg 50 55 60 Gly Pro Gly Pro Val Leu Gly Val Ala Arg Pro
Cys Leu Arg Cys Val 65 70 75 80 Leu Arg Pro Glu His Tyr Glu Pro Gly
Ser His Tyr Ser Gly Phe Ala 85 90 95 Gly Arg Asp Ala Ser Arg Ala
Phe Val Thr Gly Asp Cys Ser Glu Ala 100 105 110 Gly Leu Val Asp Asp
Val Ser Asp Leu Ser Ala Ala Glu Met Leu Thr 115 120 125 Leu His Asn
Trp Leu Ser Phe Tyr Glu Lys Asn Tyr Val Cys Val Gly 130 135 140 Arg
Val Thr Gly Arg Phe Tyr Gly Glu Asp Gly Leu Pro Thr Pro Ala 145 150
155 160 Leu Thr Gln Val Glu Ala Ala Ile Thr Arg Gly Leu Glu Ala Asn
Lys 165 170 175 Leu Gln Leu Gln Glu Lys Gln Thr Phe Pro Pro Cys Asn
Ala Glu Trp 180 185 190 Ser Ser Ala Arg Gly Ser Arg Leu Trp Cys Ser
Gln Lys Ser Gly Gly 195 200 205 Val Ser Arg Asp Trp Ile Gly Val Pro
Arg Lys Leu Tyr Lys Pro Gly 210 215 220 Ala Lys Glu Pro Arg Cys Val
Cys Val Arg Thr Thr Gly Pro Pro Ser 225 230 235 240 Gly Gln Met Pro
Asp Asn Pro Pro His Arg Asn Arg Gly Asp Leu Asp 245 250 255 His Pro
Asn Leu Ala Glu Tyr Thr Gly Cys Pro Pro Leu Ala Ile Thr 260 265 270
Cys Ser Phe Pro Leu 275 271 36 PRT Homo sapiens 271 Ser Gly Asn Leu
Gly Ser Ala Asp Gly Trp Ala Tyr Ile Asp Val Glu 1 5 10 15 Val Arg
Arg Pro Trp Ala Phe Val Gly Pro Gly Cys Ser Arg Ser Ser 20 25 30
Gly Asn Gly Ser 35 272 36 PRT Homo sapiens 272 Thr Ala Tyr Gly Leu
Val Gly Ser Pro Arg Trp Leu Ser Pro Phe His 1 5 10 15 Thr Gly Gly
Ala Val Ser Leu Pro Arg Arg Pro Arg Gly Pro Gly Pro 20 25 30 Val
Leu Gly Val 35 273 36 PRT Homo sapiens 273 Ala Arg Pro Cys Leu Arg
Cys Val Leu Arg Pro Glu His Tyr Glu Pro 1 5 10 15 Gly Ser His Tyr
Ser Gly Phe Ala Gly Arg Asp Ala Ser Arg Ala Phe 20 25 30 Val Thr
Gly Asp 35 274 36 PRT Homo sapiens 274 Cys Ser Glu Ala
Gly Leu Val Asp Asp Val Ser Asp Leu Ser Ala Ala 1 5 10 15 Glu Met
Leu Thr Leu His Asn Trp Leu Ser Phe Tyr Glu Lys Asn Tyr 20 25 30
Val Cys Val Gly 35 275 36 PRT Homo sapiens 275 Arg Val Thr Gly Arg
Phe Tyr Gly Glu Asp Gly Leu Pro Thr Pro Ala 1 5 10 15 Leu Thr Gln
Val Glu Ala Ala Ile Thr Arg Gly Leu Glu Ala Asn Lys 20 25 30 Leu
Gln Leu Gln 35 276 36 PRT Homo sapiens 276 Glu Lys Gln Thr Phe Pro
Pro Cys Asn Ala Glu Trp Ser Ser Ala Arg 1 5 10 15 Gly Ser Arg Leu
Trp Cys Ser Gln Lys Ser Gly Gly Val Ser Arg Asp 20 25 30 Trp Ile
Gly Val 35 277 29 PRT Homo sapiens 277 Pro Arg Lys Leu Tyr Lys Pro
Gly Ala Lys Glu Pro Arg Cys Val Cys 1 5 10 15 Val Arg Thr Thr Gly
Pro Pro Ser Gly Gln Met Pro Asp 20 25 278 32 PRT Homo sapiens 278
Asn Pro Pro His Arg Asn Arg Gly Asp Leu Asp His Pro Asn Leu Ala 1 5
10 15 Glu Tyr Thr Gly Cys Pro Pro Leu Ala Ile Thr Cys Ser Phe Pro
Leu 20 25 30 279 171 PRT Homo sapiens 279 Ser Gln Leu Leu Pro Gly
Ser Val Pro Gly Trp Ala Ala His Pro Leu 1 5 10 15 Arg Arg Thr Val
Leu Ser Pro Ser Gln His Thr His Asn Ser Ser His 20 25 30 Arg Met
Lys Ala Asn Cys Glu Val Ser Ala Ser Gln Arg Leu Thr Gly 35 40 45
Arg Ile Arg His Pro Arg Gly Leu Leu Gln Asn Ser Pro Arg Ser Arg 50
55 60 Lys Leu Trp Met Arg Leu Gly Leu Arg Ser Arg Tyr Ser Gly Thr
Gln 65 70 75 80 Ala Arg Ser Ala Pro Ala Gly Gly His Ile Val Asp Thr
Ala Glu Gln 85 90 95 Arg Gln Val Gln Ala Arg Val Pro Trp Ala Ala
Ala Val Ala Arg Gln 100 105 110 Leu Leu Arg Tyr Glu Lys Ala Lys Ala
Ser Ala Gly Thr Pro Pro Ala 115 120 125 His Lys Pro Cys Cys His Tyr
Arg Cys Cys Gly Tyr Ser Gln Ala Gln 130 135 140 Gln Lys Pro Thr Ala
Ser Ala Pro Gln His Leu Tyr Arg Pro Thr Arg 145 150 155 160 Pro His
Phe Arg Gly Cys Arg Ser Ile Ser Val 165 170 280 13 PRT Homo sapiens
280 Leu Leu Leu Cys Pro Trp Trp Leu Cys Phe Asp Trp Ser 1 5 10 281
270 PRT Homo sapiens 281 Met Gly Cys Ile Pro Leu Ile Lys Ser Ile
Ser Asp Trp Arg Val Ile 1 5 10 15 Ala Leu Ala Ala Leu Trp Phe Cys
Leu Ile Gly Leu Ile Cys Gln Ala 20 25 30 Leu Cys Ser Glu Asp Gly
His Lys Arg Arg Ile Leu Thr Leu Gly Leu 35 40 45 Gly Phe Leu Val
Ile Pro Phe Leu Pro Ala Ser Asn Leu Phe Phe Arg 50 55 60 Val Gly
Phe Val Val Ala Glu Cys Val Leu Tyr Leu Pro Ser Ile Gly 65 70 75 80
Tyr Cys Val Leu Leu Thr Phe Gly Phe Gly Ala Leu Ser Lys His Thr 85
90 95 Lys Lys Lys Lys Leu Ile Ala Ala Val Val Leu Gly Ile Leu Phe
Ile 100 105 110 Asn Thr Leu Arg Cys Val Leu Arg Thr Ala Lys Trp Arg
Ser Glu Glu 115 120 125 Gln Leu Phe Arg Ser Ala Leu Ser Val Cys Pro
Leu Asn Ala Lys Val 130 135 140 His Tyr Asn Ile Gly Lys Asn Leu Ala
Asp Lys Gly Asn Gln Thr Ala 145 150 155 160 Ala Ile Arg Tyr Tyr Arg
Glu Ala Val Arg Leu Asn Pro Lys Tyr Val 165 170 175 His Ala Met Asn
Asn Leu Gly Asn Ile Leu Lys Glu Arg Asn Glu Leu 180 185 190 Gln Glu
Ala Glu Glu Leu Leu Ser Leu Ala Val Gln Ile Gln Pro Asp 195 200 205
Phe Ala Ala Ala Trp Met Asn Leu Gly Ile Val Gln Asn Ser Leu Lys 210
215 220 Arg Phe Glu Thr Ala Glu Gln Asn Tyr Arg Thr Ala Ile Lys His
Arg 225 230 235 240 Arg Lys Tyr Pro Asp Cys Tyr Tyr Asn Leu Gly Arg
Leu Val Arg Thr 245 250 255 Gly Cys Pro Val Pro Val Glu Gly Lys Met
Gly Tyr Phe Ser 260 265 270 282 38 PRT Homo sapiens 282 Met Gly Cys
Ile Pro Leu Ile Lys Ser Ile Ser Asp Trp Arg Val Ile 1 5 10 15 Ala
Leu Ala Ala Leu Trp Phe Cys Leu Ile Gly Leu Ile Cys Gln Ala 20 25
30 Leu Cys Ser Glu Asp Gly 35 283 38 PRT Homo sapiens 283 His Lys
Arg Arg Ile Leu Thr Leu Gly Leu Gly Phe Leu Val Ile Pro 1 5 10 15
Phe Leu Pro Ala Ser Asn Leu Phe Phe Arg Val Gly Phe Val Val Ala 20
25 30 Glu Cys Val Leu Tyr Leu 35 284 38 PRT Homo sapiens 284 Pro
Ser Ile Gly Tyr Cys Val Leu Leu Thr Phe Gly Phe Gly Ala Leu 1 5 10
15 Ser Lys His Thr Lys Lys Lys Lys Leu Ile Ala Ala Val Val Leu Gly
20 25 30 Ile Leu Phe Ile Asn Thr 35 285 38 PRT Homo sapiens 285 Leu
Arg Cys Val Leu Arg Thr Ala Lys Trp Arg Ser Glu Glu Gln Leu 1 5 10
15 Phe Arg Ser Ala Leu Ser Val Cys Pro Leu Asn Ala Lys Val His Tyr
20 25 30 Asn Ile Gly Lys Asn Leu 35 286 38 PRT Homo sapiens 286 Ala
Asp Lys Gly Asn Gln Thr Ala Ala Ile Arg Tyr Tyr Arg Glu Ala 1 5 10
15 Val Arg Leu Asn Pro Lys Tyr Val His Ala Met Asn Asn Leu Gly Asn
20 25 30 Ile Leu Lys Glu Arg Asn 35 287 38 PRT Homo sapiens 287 Glu
Leu Gln Glu Ala Glu Glu Leu Leu Ser Leu Ala Val Gln Ile Gln 1 5 10
15 Pro Asp Phe Ala Ala Ala Trp Met Asn Leu Gly Ile Val Gln Asn Ser
20 25 30 Leu Lys Arg Phe Glu Thr 35 288 42 PRT Homo sapiens 288 Ala
Glu Gln Asn Tyr Arg Thr Ala Ile Lys His Arg Arg Lys Tyr Pro 1 5 10
15 Asp Cys Tyr Tyr Asn Leu Gly Arg Leu Val Arg Thr Gly Cys Pro Val
20 25 30 Pro Val Glu Gly Lys Met Gly Tyr Phe Ser 35 40 289 16 PRT
Homo sapiens 289 Leu Ile Lys Ser Ile Ser Asp Trp Arg Val Ile Ala
Leu Ala Ala Leu 1 5 10 15 290 15 PRT Homo sapiens 290 Arg Asp Asn
Asp Tyr Leu Leu His Gly His Arg Pro Pro Met Phe 1 5 10 15 291 24
PRT Homo sapiens 291 Ser Phe Arg Ala Cys Phe Lys Ser Ile Phe Arg
Ile His Thr Glu Thr 1 5 10 15 Gly Asn Ile Trp Thr His Leu Leu 20
292 29 PRT Homo sapiens 292 Gly Phe Val Leu Phe Leu Phe Leu Gly Ile
Leu Thr Met Leu Arg Pro 1 5 10 15 Asn Met Tyr Phe Met Ala Pro Leu
Gln Glu Lys Val Val 20 25 293 457 PRT Homo sapiens 293 Thr Gly Pro
Glu Phe Pro Gly Ser Asn Ser Thr Val Ala Arg Arg Ile 1 5 10 15 Lys
Asp Leu Ala Ala Asp Ile Glu Glu Glu Leu Val Cys Arg Leu Lys 20 25
30 Ile Cys Asp Gly Phe Ser Leu Gln Leu Asp Glu Ser Ala Asp Val Ser
35 40 45 Gly Leu Ala Val Leu Leu Val Phe Val Arg Tyr Arg Phe Asn
Lys Ser 50 55 60 Ile Glu Glu Asp Leu Leu Leu Cys Glu Ser Leu Gln
Ser Asn Ala Thr 65 70 75 80 Gly Glu Glu Ile Phe Asn Cys Ile Asn Ser
Phe Met Gln Lys His Glu 85 90 95 Ile Glu Trp Glu Lys Cys Val Asp
Val Cys Ser Asp Ala Ser Arg Ala 100 105 110 Val Asp Gly Lys Ile Ala
Glu Ala Val Thr Leu Ile Lys Tyr Val Ala 115 120 125 Pro Glu Ser Thr
Ser Ser His Cys Leu Leu Tyr Arg His Ala Leu Ala 130 135 140 Val Lys
Ile Met Pro Thr Ser Leu Lys Asn Val Leu Asp Gln Ala Val 145 150 155
160 Gln Ile Ile Asn Tyr Ile Lys Ala Arg Pro His Gln Ser Arg Leu Leu
165 170 175 Lys Ile Leu Cys Glu Glu Met Gly Ala Gln His Thr Ala Leu
Leu Leu 180 185 190 Asn Thr Glu Val Arg Trp Leu Ser Arg Gly Lys Val
Leu Val Arg Leu 195 200 205 Phe Glu Leu Arg Arg Glu Leu Leu Val Phe
Met Asp Ser Ala Phe Arg 210 215 220 Leu Ser Asp Cys Leu Thr Asn Ser
Ser Trp Leu Leu Arg Leu Ala Tyr 225 230 235 240 Leu Ala Asp Ile Phe
Thr Lys Leu Asn Glu Val Asn Leu Ser Met Gln 245 250 255 Gly Lys Asn
Val Thr Val Phe Thr Val Phe Asp Lys Met Ser Ser Leu 260 265 270 Leu
Arg Lys Leu Glu Phe Trp Ala Ser Ser Val Glu Glu Glu Asn Phe 275 280
285 Asp Cys Phe Pro Thr Leu Ser Asp Phe Leu Thr Glu Ile Asn Ser Thr
290 295 300 Val Asp Lys Asp Ile Cys Ser Ala Ile Val Gln His Leu Arg
Gly Leu 305 310 315 320 Arg Ala Thr Leu Leu Lys Tyr Phe Pro Val Thr
Asn Asp Asn Asn Ala 325 330 335 Trp Val Arg Asn Pro Phe Thr Val Thr
Val Lys Pro Ala Ser Leu Val 340 345 350 Ala Arg Asp Tyr Glu Ser Leu
Ile Asp Leu Thr Ser Asp Ser Gln Val 355 360 365 Lys Gln Asn Phe Ser
Glu Leu Ser Leu Asn Asp Phe Trp Ser Ser Leu 370 375 380 Ile Gln Glu
Tyr Pro Ser Ile Ala Arg Arg Ala Val Arg Val Leu Leu 385 390 395 400
Pro Phe Ala Thr Met His Leu Cys Glu Thr Gly Phe Ser Tyr Tyr Ala 405
410 415 Ala Thr Lys Thr Lys Tyr Arg Lys Arg Leu Asp Ala Ala Pro His
Met 420 425 430 Arg Ile Arg Leu Ser Asn Ile Thr Pro Asn Ile Lys Arg
Ile Cys Asp 435 440 445 Lys Lys Thr Gln Lys His Cys Ser His 450 455
294 31 PRT Homo sapiens 294 Asp Ile Glu Glu Glu Leu Val Cys Arg Leu
Lys Ile Cys Asp Gly Phe 1 5 10 15 Ser Leu Gln Leu Asp Glu Ser Ala
Asp Val Ser Gly Leu Ala Val 20 25 30 295 36 PRT Homo sapiens 295
Asn Ser Phe Met Gln Lys His Glu Ile Glu Trp Glu Lys Cys Val Asp 1 5
10 15 Val Cys Ser Asp Ala Ser Arg Ala Val Asp Gly Lys Ile Ala Glu
Ala 20 25 30 Val Thr Leu Ile 35 296 36 PRT Homo sapiens 296 Leu Asp
Gln Ala Val Gln Ile Ile Asn Tyr Ile Lys Ala Arg Pro His 1 5 10 15
Gln Ser Arg Leu Leu Lys Ile Leu Cys Glu Glu Met Gly Ala Gln His 20
25 30 Thr Ala Leu Leu 35 297 49 PRT Homo sapiens 297 Ser Ala Phe
Arg Leu Ser Asp Cys Leu Thr Asn Ser Ser Trp Leu Leu 1 5 10 15 Arg
Leu Ala Tyr Leu Ala Asp Ile Phe Thr Lys Leu Asn Glu Val Asn 20 25
30 Leu Ser Met Gln Gly Lys Asn Val Thr Val Phe Thr Val Phe Asp Lys
35 40 45 Met 298 32 PRT Homo sapiens 298 Ser Asp Phe Leu Thr Glu
Ile Asn Ser Thr Val Asp Lys Asp Ile Cys 1 5 10 15 Ser Ala Ile Val
Gln His Leu Arg Gly Leu Arg Ala Thr Leu Leu Lys 20 25 30 299 38 PRT
Homo sapiens 299 Ser Asp Ser Gln Val Lys Gln Asn Phe Ser Glu Leu
Ser Leu Asn Asp 1 5 10 15 Phe Trp Ser Ser Leu Ile Gln Glu Tyr Pro
Ser Ile Ala Arg Arg Ala 20 25 30 Val Arg Val Leu Leu Pro 35 300 325
PRT Homo sapiens SITE (171) Xaa equals any of the naturally
occurring L-amino acids SITE (222) Xaa equals any of the naturally
occurring L-amino acids 300 Asp Pro Arg Val Arg Glu Cys Leu Gln Asp
Trp Ala Ser Phe Leu Arg 1 5 10 15 Leu Ala Ile Pro Ser Met Leu Met
Leu Cys Met Glu Trp Trp Ala Tyr 20 25 30 Glu Val Gly Ser Phe Leu
Ser Gly Ile Leu Gly Met Val Glu Leu Gly 35 40 45 Ala Gln Ser Ile
Val Tyr Glu Leu Ala Ile Ile Val Tyr Met Val Pro 50 55 60 Ala Gly
Phe Ser Val Ala Ala Ser Val Arg Val Gly Asn Ala Leu Gly 65 70 75 80
Ala Gly Asp Met Glu Gln Ala Arg Lys Ser Ser Thr Val Ser Leu Leu 85
90 95 Ile Thr Val Leu Phe Ala Val Ala Phe Ser Val Leu Leu Leu Ser
Cys 100 105 110 Lys Asp His Val Gly Tyr Ile Phe Thr Thr Asp Arg Asp
Ile Ile Asn 115 120 125 Leu Val Ala Gln Val Val Pro Ile Tyr Ala Val
Ser His Leu Phe Glu 130 135 140 Ala Leu Ala Cys Thr Ser Gly Gly Val
Leu Arg Gly Ser Gly Asn Gln 145 150 155 160 Lys Val Gly Ala Ile Val
Asn Thr Ile Gly Xaa Tyr Val Val Gly Leu 165 170 175 Pro Ile Gly Ile
Ala Leu Met Phe Ala Thr Thr Leu Gly Val Met Gly 180 185 190 Leu Trp
Ser Gly Ile Ile Ile Cys Thr Val Phe Gln Ala Val Cys Phe 195 200 205
Leu Gly Phe Ile Ile Gln Leu Asn Trp Lys Lys Ala Cys Xaa Gln Ala 210
215 220 Gln Val His Ala Asn Leu Lys Val Asn Asn Val Pro Arg Ser Gly
Asn 225 230 235 240 Ser Ala Leu Pro Gln Asp Pro Leu His Pro Gly Cys
Pro Glu Asn Leu 245 250 255 Glu Gly Ile Leu Thr Asn Asp Val Gly Lys
Thr Gly Glu Pro Gln Ser 260 265 270 Asp Gln Gln Met Arg Gln Glu Glu
Pro Leu Pro Glu His Pro Gln Asp 275 280 285 Gly Ala Lys Leu Ser Arg
Lys Gln Leu Val Leu Arg Arg Gly Leu Leu 290 295 300 Leu Leu Gly Val
Phe Leu Ile Leu Leu Val Gly Ile Leu Val Arg Phe 305 310 315 320 Tyr
Val Arg Ile Gln 325 301 328 PRT Homo sapiens 301 Gly Thr Arg Ile
His Thr Ile Leu Val Tyr Gln Glu Ser Asn Arg Lys 1 5 10 15 Met Asp
Ser Val Asp Pro Ala Ser Ser Gln Ala Met Glu Leu Ser Asp 20 25 30
Val Thr Leu Ile Glu Gly Val Gly Asn Glu Val Met Val Val Ala Gly 35
40 45 Val Val Val Leu Ile Leu Ala Leu Val Leu Ala Trp Leu Ser Thr
Tyr 50 55 60 Val Ala Asp Ser Gly Ser Asn Gln Leu Leu Gly Ala Ile
Val Ser Ala 65 70 75 80 Gly Asp Thr Ser Val Leu His Leu Gly His Val
Asp His Leu Val Ala 85 90 95 Gly Gln Gly Asn Pro Glu Pro Thr Glu
Leu Pro His Pro Ser Glu Gly 100 105 110 Asn Asp Glu Lys Ala Glu Glu
Ala Gly Glu Gly Arg Gly Asp Ser Thr 115 120 125 Gly Glu Ala Gly Ala
Gly Gly Gly Val Glu Pro Ser Leu Glu His Leu 130 135 140 Leu Asp Ile
Gln Gly Leu Pro Lys Arg Gln Ala Gly Ala Gly Ser Ser 145 150 155 160
Ser Pro Glu Ala Pro Leu Arg Ser Glu Asp Ser Thr Cys Leu Pro Pro 165
170 175 Ser Pro Gly Leu Ile Thr Val Arg Leu Lys Phe Leu Asn Asp Thr
Glu 180 185 190 Glu Leu Ala Val Ala Arg Pro Glu Asp Thr Val Gly Ala
Leu Lys Ser 195 200 205 Lys Tyr Phe Pro Gly Gln Glu Ser Gln Met Lys
Leu Ile Tyr Gln Gly 210 215 220 Arg Leu Leu Gln Asp Pro Ala Arg Thr
Leu Arg Ser Leu Asn Ile Thr 225 230 235 240 Asp Asn Cys Val Ile His
Cys His Arg Ser Pro Pro Gly Ser Ala Val 245 250 255 Pro Gly Pro Ser
Ala Ser Leu Ala Pro Ser Ala Thr Glu Pro Pro Ser 260 265 270 Leu Gly
Val Asn Val Gly Ser Leu Met Val Pro Val Phe Val Val Leu 275 280 285
Leu Gly Val Val Trp Tyr Phe Arg Ile Asn Tyr Arg Gln Phe Phe Thr 290
295 300 Ala Pro Ala Thr Val Ser Leu Val Gly Val Thr Val Phe Phe Ser
Phe 305 310 315 320 Leu Val Phe Gly Met Tyr Gly Arg 325 302 26 PRT
Homo sapiens 302 Asp Ser Arg Ile Ser Leu Leu Val Asn Asn Ala Gly
Val Gly Ala Thr 1 5 10
15 Ala Ser Leu Leu Glu Ser Asp Ala Asp Lys 20 25 303 159 PRT Homo
sapiens SITE (110) Xaa equals any of the naturally occurring
L-amino acids 303 Met Asp Ala Met Ile Leu Leu Asn Val Leu Ala Leu
Thr Arg Leu Ala 1 5 10 15 Lys Ala Ala Ala Thr Asn Phe Val Ala Gln
Gly Arg Gly Thr Ile Ile 20 25 30 Asn Ile Gly Ser Ile Val Ala Leu
Ala Pro Lys Val Leu Asn Gly Val 35 40 45 Tyr Gly Gly Thr Lys Ala
Phe Val Gln Ala Phe Ser Glu Ser Leu Gln 50 55 60 His Glu Leu Ser
Asp Lys Gly Val Val Val Gln Val Val Leu Pro Gly 65 70 75 80 Ala Thr
Ala Thr Glu Phe Trp Asp Ile Ala Gly Leu Pro Val Asn Asn 85 90 95
Leu Pro Glu Ala Met Val Met Thr Thr Glu Asn Leu Val Xaa Ala Ala 100
105 110 Leu Ala Gly Leu Ala Gln Gly Glu Ala Val Thr Ile Pro Ser Leu
Pro 115 120 125 Asp Ser Ala Asp Trp Asp Thr Tyr Glu Arg Ala Arg Leu
Ala Leu Gly 130 135 140 Pro Asn Leu Ser His Arg Glu Pro Ala Ala Arg
Tyr Gly Leu Lys 145 150 155 304 146 PRT Homo sapiens 304 Gly Thr
Pro Ala Gly Thr Gly Pro Glu Phe Pro Gly Arg Pro Thr Arg 1 5 10 15
Pro Ser Arg Thr Glu Ser Ala Gln Thr Thr Gln His Ser Pro Leu Arg 20
25 30 Pro Leu Trp Arg Leu Lys Arg Asp Ser Ser Pro Cys His Pro Gln
Thr 35 40 45 Arg Ala Asp Trp Gly Val Cys Pro Pro Trp Gly Gly Ala
Ala Gln Gly 50 55 60 Leu Arg Pro Gly Cys His Leu Ala Pro Arg Arg
Cys Leu Cys Pro Gly 65 70 75 80 Ser Cys Cys Pro Trp His Trp Ala Glu
Ala Gln Trp Ser Phe Leu Trp 85 90 95 Arg Gly Leu Trp Gly Leu Arg
Thr Leu Pro Thr Ala Leu Arg Ala Ser 100 105 110 Pro Ala Ala Ser Gly
Thr Val Thr Tyr Ser Ala Cys Leu Gly Thr Ser 115 120 125 Cys Leu Leu
Arg Ala Pro Cys Trp Arg Leu Arg Thr Cys Arg Gln Ser 130 135 140 Trp
Cys 145 305 28 PRT Homo sapiens 305 Gly Thr Pro Ala Gly Thr Gly Pro
Glu Phe Pro Gly Arg Pro Thr Arg 1 5 10 15 Pro Ser Arg Thr Glu Ser
Ala Gln Thr Thr Gln His 20 25 306 30 PRT Homo sapiens 306 Ser Pro
Leu Arg Pro Leu Trp Arg Leu Lys Arg Asp Ser Ser Pro Cys 1 5 10 15
His Pro Gln Thr Arg Ala Asp Trp Gly Val Cys Pro Pro Trp 20 25 30
307 30 PRT Homo sapiens 307 Gly Gly Ala Ala Gln Gly Leu Arg Pro Gly
Cys His Leu Ala Pro Arg 1 5 10 15 Arg Cys Leu Cys Pro Gly Ser Cys
Cys Pro Trp His Trp Ala 20 25 30 308 30 PRT Homo sapiens 308 Glu
Ala Gln Trp Ser Phe Leu Trp Arg Gly Leu Trp Gly Leu Arg Thr 1 5 10
15 Leu Pro Thr Ala Leu Arg Ala Ser Pro Ala Ala Ser Gly Thr 20 25 30
309 28 PRT Homo sapiens 309 Val Thr Tyr Ser Ala Cys Leu Gly Thr Ser
Cys Leu Leu Arg Ala Pro 1 5 10 15 Cys Trp Arg Leu Arg Thr Cys Arg
Gln Ser Trp Cys 20 25 310 507 PRT Homo sapiens 310 Met Pro Val Pro
Trp Phe Leu Leu Ser Leu Ala Leu Gly Arg Ser Pro 1 5 10 15 Val Val
Leu Ser Leu Glu Arg Leu Val Gly Pro Gln Asp Ala Thr His 20 25 30
Cys Ser Pro Gly Leu Ser Cys Arg Leu Trp Asp Ser Asp Ile Leu Cys 35
40 45 Leu Pro Gly Asp Ile Val Pro Ala Pro Gly Pro Val Leu Ala Pro
Thr 50 55 60 His Leu Gln Thr Glu Leu Val Leu Arg Cys Gln Lys Glu
Thr Asp Cys 65 70 75 80 Asp Leu Cys Leu Arg Val Ala Val His Leu Ala
Val His Gly His Trp 85 90 95 Glu Glu Pro Glu Asp Glu Glu Lys Phe
Gly Gly Ala Ala Asp Leu Gly 100 105 110 Val Glu Glu Pro Arg Asn Ala
Ser Leu Gln Ala Gln Val Val Leu Ser 115 120 125 Phe Gln Ala Tyr Pro
Thr Ala Arg Cys Val Leu Leu Glu Val Gln Val 130 135 140 Pro Ala Ala
Leu Val Gln Phe Gly Gln Ser Val Gly Ser Val Val Tyr 145 150 155 160
Asp Cys Phe Glu Ala Ala Leu Gly Ser Glu Val Arg Ile Trp Ser Tyr 165
170 175 Thr Gln Pro Arg Tyr Glu Lys Glu Leu Asn His Thr Gln Gln Leu
Pro 180 185 190 Asp Cys Arg Gly Leu Glu Val Trp Asn Ser Ile Pro Ser
Cys Trp Ala 195 200 205 Leu Pro Trp Leu Asn Val Ser Ala Asp Gly Asp
Asn Val His Leu Val 210 215 220 Leu Asn Val Ser Glu Glu Gln His Phe
Gly Leu Ser Leu Tyr Trp Asn 225 230 235 240 Gln Val Gln Gly Pro Pro
Lys Pro Arg Trp His Lys Asn Leu Thr Gly 245 250 255 Pro Gln Ile Ile
Thr Leu Asn His Thr Asp Leu Val Pro Cys Leu Cys 260 265 270 Ile Gln
Val Trp Pro Leu Glu Pro Asp Ser Val Arg Thr Asn Ile Cys 275 280 285
Pro Phe Arg Glu Asp Pro Arg Ala His Gln Asn Leu Trp Gln Ala Ala 290
295 300 Arg Leu Arg Leu Leu Thr Leu Gln Ser Trp Leu Leu Asp Ala Pro
Cys 305 310 315 320 Ser Leu Pro Ala Glu Ala Ala Leu Cys Trp Arg Ala
Pro Gly Gly Asp 325 330 335 Pro Cys Gln Pro Leu Val Pro Pro Leu Ser
Trp Glu Asn Val Thr Val 340 345 350 Asp Lys Val Leu Glu Phe Pro Leu
Leu Lys Gly His Pro Asn Leu Cys 355 360 365 Val Gln Val Asn Ser Ser
Glu Lys Leu Gln Leu Gln Glu Cys Leu Trp 370 375 380 Ala Asp Ser Leu
Gly Pro Leu Lys Asp Asp Val Leu Leu Leu Glu Thr 385 390 395 400 Arg
Gly Pro Gln Asp Asn Arg Ser Leu Cys Ala Leu Glu Pro Ser Gly 405 410
415 Cys Thr Ser Leu Pro Ser Lys Ala Ser Thr Arg Ala Ala Arg Leu Gly
420 425 430 Glu Tyr Leu Leu Gln Asp Leu Gln Ser Gly Gln Cys Leu Gln
Leu Trp 435 440 445 Asp Asp Asp Leu Gly Ala Leu Trp Ala Cys Pro Met
Asp Lys Tyr Ile 450 455 460 His Lys Arg Trp Ala Leu Val Trp Leu Ala
Cys Leu Leu Phe Arg Arg 465 470 475 480 Ala Leu Ser Leu Ile Leu Leu
Leu Lys Lys Asp His Ala Lys Gly Trp 485 490 495 Leu Arg Leu Leu Lys
Gln Asp Val Arg Ser Gly 500 505 311 11 PRT Homo sapiens 311 Pro Pro
Arg Pro Ser Thr Ser Gly Gln Trp Gly 1 5 10 312 11 PRT Homo sapiens
312 Arg Arg Ser Pro Phe Thr Ser Ala Gln Thr Gly 1 5 10 313 23 PRT
Homo sapiens 313 Gly Thr Gly Trp Asp Phe Gly Leu Ala Ala Val Cys
Leu Arg Ala Ala 1 5 10 15 Glu Val Ala Gly Ser Phe Lys 20 314 146
PRT Homo sapiens 314 Gly Tyr Arg Arg Val Phe Glu Glu Tyr Met Arg
Val Ile Ser Gln Arg 1 5 10 15 Tyr Pro Asp Ile Arg Ile Glu Gly Glu
Asn Tyr Leu Pro Gln Pro Ile 20 25 30 Tyr Arg His Ile Ala Ser Phe
Leu Ser Val Phe Lys Leu Val Leu Ile 35 40 45 Gly Leu Ile Ile Val
Gly Lys Asp Pro Phe Ala Phe Phe Gly Met Gln 50 55 60 Ala Pro Ser
Ile Trp Gln Trp Gly Gln Glu Asn Lys Val Tyr Ala Cys 65 70 75 80 Met
Met Val Phe Phe Leu Ser Asn Met Ile Glu Asn Gln Cys Met Ser 85 90
95 Thr Gly Ala Phe Glu Ile Thr Leu Asn Asp Val Pro Val Trp Ser Lys
100 105 110 Leu Glu Ser Gly His Leu Pro Ser Met Gln Gln Leu Val Gln
Ile Leu 115 120 125 Asp Asn Glu Met Lys Leu Asn Val His Met Asp Ser
Ile Pro His His 130 135 140 Arg Ser 145 315 34 PRT Homo sapiens 315
Gly Tyr Arg Arg Val Phe Glu Glu Tyr Met Arg Val Ile Ser Gln Arg 1 5
10 15 Tyr Pro Asp Ile Arg Ile Glu Gly Glu Asn Tyr Leu Pro Gln Pro
Ile 20 25 30 Tyr Arg 316 34 PRT Homo sapiens 316 His Ile Ala Ser
Phe Leu Ser Val Phe Lys Leu Val Leu Ile Gly Leu 1 5 10 15 Ile Ile
Val Gly Lys Asp Pro Phe Ala Phe Phe Gly Met Gln Ala Pro 20 25 30
Ser Ile 317 34 PRT Homo sapiens 317 Trp Gln Trp Gly Gln Glu Asn Lys
Val Tyr Ala Cys Met Met Val Phe 1 5 10 15 Phe Leu Ser Asn Met Ile
Glu Asn Gln Cys Met Ser Thr Gly Ala Phe 20 25 30 Glu Ile 318 36 PRT
Homo sapiens 318 Thr Leu Asn Asp Val Pro Val Trp Ser Lys Leu Glu
Ser Gly His Leu 1 5 10 15 Pro Ser Met Gln Gln Leu Val Gln Ile Leu
Asp Asn Glu Met Lys Leu 20 25 30 Asn Val His Met 35 319 8 PRT Homo
sapiens 319 Asp Ser Ile Pro His His Arg Ser 1 5 320 30 PRT Homo
sapiens 320 Gly Arg Ala Arg Gly Arg Pro Pro Gly Pro Glu Ala Ala Pro
Ala Ser 1 5 10 15 Leu Ser Val Ser Leu Arg Arg Glu Val His Ser Arg
Gly Glu 20 25 30 321 333 PRT Homo sapiens SITE (15) Xaa equals any
of the naturally occurring L-amino acids SITE (16) Xaa equals any
of the naturally occurring L-amino acids SITE (20) Xaa equals any
of the naturally occurring L-amino acids 321 Gln Thr Pro Phe Thr
Cys Thr Leu Ile His Arg His Ala Cys Xaa Xaa 1 5 10 15 Pro Val Arg
Xaa Ser Arg Val Asp Pro Arg Val Arg Gly Lys Gln Ala 20 25 30 Leu
Ile Trp Leu Leu Gly Val His Gly Glu Arg Ile Pro Asn Ala Pro 35 40
45 Tyr Val Leu Glu Asp Phe Val Glu Asn Val Lys Ser Glu Thr Phe Pro
50 55 60 Ala Val Lys Met Glu Leu Leu Thr Ala Leu Leu Arg Leu Phe
Leu Ser 65 70 75 80 Arg Pro Ala Glu Cys Gln Asp Met Leu Gly Arg Leu
Leu Tyr Tyr Cys 85 90 95 Ile Glu Glu Glu Lys Asp Met Ala Val Arg
Asp Arg Gly Leu Phe Tyr 100 105 110 Tyr Arg Leu Leu Leu Val Gly Ile
Asp Glu Val Lys Arg Ile Leu Cys 115 120 125 Ser Pro Lys Ser Asp Pro
Thr Leu Gly Leu Leu Glu Asp Pro Ala Glu 130 135 140 Arg Pro Val Asn
Ser Trp Ala Ser Asp Phe Asn Thr Leu Val Pro Val 145 150 155 160 Tyr
Gly Lys Ala His Trp Ala Thr Ile Ser Lys Cys Gln Gly Ala Glu 165 170
175 Arg Cys Asp Pro Glu Leu Pro Lys Thr Ser Ser Phe Ala Ala Ser Gly
180 185 190 Pro Leu Ile Pro Glu Glu Asn Lys Glu Arg Val Gln Glu Leu
Pro Asp 195 200 205 Ser Gly Ala Leu Met Leu Val Pro Asn Arg Gln Leu
Thr Ala Asp Tyr 210 215 220 Phe Glu Lys Thr Trp Leu Ser Leu Lys Val
Ala His Gln Gln Val Leu 225 230 235 240 Pro Trp Arg Gly Glu Phe His
Pro Asp Thr Leu Gln Met Ala Leu Gln 245 250 255 Val Val Asn Ile Gln
Thr Ile Ala Met Ser Arg Ala Gly Ser Arg Pro 260 265 270 Trp Lys Ala
Tyr Leu Ser Ala Gln Asp Asp Thr Gly Cys Leu Phe Leu 275 280 285 Thr
Glu Leu Leu Leu Glu Pro Gly Asn Ser Glu Met Gln Ile Ser Val 290 295
300 Lys Gln Asn Glu Ala Arg Thr Glu Thr Leu Asn Ser Phe Ile Ser Val
305 310 315 320 Leu Glu Thr Val Ile Gly Thr Ile Glu Glu Ile Lys Ser
325 330 322 12 PRT Homo sapiens 322 Cys Glu Asn Thr Glu Gly Gly Tyr
Arg Cys Ile Cys 1 5 10 323 12 PRT Homo sapiens 323 Cys Asp Cys Gln
Ala Gly Tyr Gly Gly Glu Ala Cys 1 5 10 324 14 PRT Homo sapiens 324
Cys Ile Cys Ala Glu Gly Tyr Lys Gln Met Glu Gly Ile Cys 1 5 10 325
27 PRT Homo sapiens 325 Asp Ile Asp Glu Cys Gly Thr Glu Gly Ala Asn
Cys Gly Ala Asp Gln 1 5 10 15 Phe Cys Val Asn Thr Glu Gly Ser Tyr
Glu Cys 20 25 326 26 PRT Homo sapiens 326 Asp Val Asp Glu Cys Glu
Thr Glu Val Cys Pro Gly Glu Asn Lys Gln 1 5 10 15 Cys Glu Asn Thr
Glu Gly Gly Tyr Arg Cys 20 25 327 34 PRT Homo sapiens 327 Cys Asp
Cys Gln Ala Gly Tyr Gly Gly Glu Ala Cys Gly Gln Cys Gly 1 5 10 15
Leu Gly Tyr Phe Glu Ala Glu Arg Asn Ala Ser His Leu Val Cys Ser 20
25 30 Ala Cys 328 389 PRT Homo sapiens 328 Met Ile Ser Leu Pro Gly
Pro Leu Val Thr Asn Leu Leu Arg Phe Leu 1 5 10 15 Phe Leu Gly Leu
Ser Ala Leu Ala Pro Pro Ser Arg Ala Gln Leu Gln 20 25 30 Leu His
Leu Pro Ala Asn Arg Leu Gln Ala Val Glu Gly Gly Glu Val 35 40 45
Val Leu Pro Ala Trp Tyr Thr Leu His Gly Glu Val Ser Ser Ser Gln 50
55 60 Pro Trp Glu Val Pro Phe Val Met Trp Phe Phe Lys Gln Lys Glu
Lys 65 70 75 80 Glu Asp Gln Val Leu Ser Tyr Ile Asn Gly Val Thr Thr
Ser Lys Pro 85 90 95 Gly Val Ser Leu Val Tyr Ser Met Pro Ser Arg
Asn Leu Ser Leu Arg 100 105 110 Leu Glu Gly Leu Gln Glu Lys Asp Ser
Gly Pro Tyr Ser Cys Ser Val 115 120 125 Asn Val Gln Asn Lys Gln Gly
Lys Ser Arg Gly His Ser Ile Lys Thr 130 135 140 Leu Glu Leu Asn Val
Leu Val Pro Pro Ala Pro Pro Ser Cys Arg Leu 145 150 155 160 Gln Gly
Val Pro His Val Gly Ala Asn Val Thr Leu Ser Cys Gln Ser 165 170 175
Pro Arg Ser Lys Pro Ala Val Gln Tyr Gln Trp Asp Arg Gln Leu Pro 180
185 190 Ser Phe Gln Thr Phe Phe Ala Pro Ala Leu Asp Val Ile Arg Gly
Ser 195 200 205 Leu Ser Leu Thr Asn Leu Ser Ser Ser Met Ala Gly Val
Tyr Val Cys 210 215 220 Lys Ala His Asn Glu Val Gly Thr Ala Gln Cys
Asn Val Thr Leu Glu 225 230 235 240 Val Ser Thr Gly Pro Gly Ala Ala
Val Val Ala Gly Ala Val Val Gly 245 250 255 Thr Leu Val Gly Leu Gly
Leu Leu Ala Gly Leu Val Leu Leu Tyr His 260 265 270 Arg Arg Gly Lys
Ala Leu Glu Glu Pro Ala Asn Asp Ile Lys Glu Asp 275 280 285 Ala Ile
Ala Pro Arg Thr Leu Pro Trp Pro Lys Ser Ser Asp Thr Ile 290 295 300
Ser Lys Asn Gly Thr Leu Ser Ser Val Thr Ser Ala Arg Ala Leu Arg 305
310 315 320 Pro Pro His Gly Pro Pro Arg Pro Gly Ala Leu Thr Pro Thr
Pro Ser 325 330 335 Leu Ser Ser Gln Ala Leu Pro Ser Pro Arg Leu Pro
Thr Thr Asp Gly 340 345 350 Ala His Pro Gln Pro Ile Ser Pro Ile Pro
Gly Gly Val Ser Ser Ser 355 360 365 Gly Leu Ser Arg Met Gly Ala Val
Pro Val Met Val Pro Ala Gln Ser 370 375 380 Gln Ala Gly Ser Leu 385
329 35 PRT Homo sapiens 329 Met Ile Ser Leu Pro Gly Pro Leu Val Thr
Asn Leu Leu Arg Phe Leu 1 5 10 15 Phe Leu Gly Leu Ser Ala Leu Ala
Pro Pro Ser Arg Ala Gln Leu Gln 20 25 30 Leu His Leu 35 330 35 PRT
Homo sapiens 330 Pro Ala Asn Arg Leu Gln Ala Val Glu Gly Gly Glu
Val Val Leu Pro 1 5 10 15 Ala Trp Tyr Thr Leu His Gly Glu Val Ser
Ser Ser Gln Pro Trp Glu 20 25 30 Val Pro Phe 35 331 35 PRT Homo
sapiens 331 Val Met Trp Phe Phe Lys Gln Lys Glu Lys Glu Asp Gln Val
Leu Ser 1 5 10 15 Tyr Ile Asn Gly Val Thr Thr Ser Lys Pro Gly Val
Ser Leu Val Tyr 20 25 30 Ser Met Pro 35 332 35 PRT Homo sapiens 332
Ser Arg Asn Leu Ser Leu Arg Leu Glu Gly Leu Gln Glu Lys Asp Ser 1
5
10 15 Gly Pro Tyr Ser Cys Ser Val Asn Val Gln Asn Lys Gln Gly Lys
Ser 20 25 30 Arg Gly His 35 333 35 PRT Homo sapiens 333 Ser Ile Lys
Thr Leu Glu Leu Asn Val Leu Val Pro Pro Ala Pro Pro 1 5 10 15 Ser
Cys Arg Leu Gln Gly Val Pro His Val Gly Ala Asn Val Thr Leu 20 25
30 Ser Cys Gln 35 334 35 PRT Homo sapiens 334 Ser Pro Arg Ser Lys
Pro Ala Val Gln Tyr Gln Trp Asp Arg Gln Leu 1 5 10 15 Pro Ser Phe
Gln Thr Phe Phe Ala Pro Ala Leu Asp Val Ile Arg Gly 20 25 30 Ser
Leu Ser 35 335 35 PRT Homo sapiens 335 Leu Thr Asn Leu Ser Ser Ser
Met Ala Gly Val Tyr Val Cys Lys Ala 1 5 10 15 His Asn Glu Val Gly
Thr Ala Gln Cys Asn Val Thr Leu Glu Val Ser 20 25 30 Thr Gly Pro 35
336 35 PRT Homo sapiens 336 Gly Ala Ala Val Val Ala Gly Ala Val Val
Gly Thr Leu Val Gly Leu 1 5 10 15 Gly Leu Leu Ala Gly Leu Val Leu
Leu Tyr His Arg Arg Gly Lys Ala 20 25 30 Leu Glu Glu 35 337 35 PRT
Homo sapiens 337 Pro Ala Asn Asp Ile Lys Glu Asp Ala Ile Ala Pro
Arg Thr Leu Pro 1 5 10 15 Trp Pro Lys Ser Ser Asp Thr Ile Ser Lys
Asn Gly Thr Leu Ser Ser 20 25 30 Val Thr Ser 35 338 35 PRT Homo
sapiens 338 Ala Arg Ala Leu Arg Pro Pro His Gly Pro Pro Arg Pro Gly
Ala Leu 1 5 10 15 Thr Pro Thr Pro Ser Leu Ser Ser Gln Ala Leu Pro
Ser Pro Arg Leu 20 25 30 Pro Thr Thr 35 339 39 PRT Homo sapiens 339
Asp Gly Ala His Pro Gln Pro Ile Ser Pro Ile Pro Gly Gly Val Ser 1 5
10 15 Ser Ser Gly Leu Ser Arg Met Gly Ala Val Pro Val Met Val Pro
Ala 20 25 30 Gln Ser Gln Ala Gly Ser Leu 35 340 36 PRT Homo sapiens
340 341 27 PRT Homo sapiens 341 342 12 PRT Homo sapiens 342 343 11
PRT Homo sapiens 343 344 13 PRT Homo sapiens 344 345 21 PRT Homo
sapiens 345 346 23 PRT Homo sapiens 346 Asn Ser Ala Arg Val Glu Phe
Phe Ile Pro Pro Leu Arg Ile Thr Gln 1 5 10 15 Lys Val Arg Ser Thr
Lys Ser 20 347 123 PRT Homo sapiens 347 Met Met Val Trp Asn Leu Phe
Pro Cys Phe Pro Pro Leu Leu Leu Leu 1 5 10 15 Gln Phe Ile Asp Cys
Gln Gln Ser Ser Glu Ile Glu Gln Gly Phe Thr 20 25 30 Arg Ser Leu
Leu Gly His Pro Ile Phe Phe Cys Pro Asp Pro Cys Trp 35 40 45 Gln
Ser Cys Met Asn Cys Val Ile Leu Ser Val Leu Ser Phe Phe Phe 50 55
60 Leu Ile Arg Trp Ile Ser Lys Ile Val Ala Val Gln Lys Leu Glu Ser
65 70 75 80 Ser Ser Arg Arg Lys Pro Ile Leu Phe Leu Ile Ile Ser Cys
Glu Ile 85 90 95 Ala Ser Phe Ile His Leu Phe Leu Ser Gln Met Ser
Ala Glu Cys Cys 100 105 110 Cys Phe Tyr Leu Val Ile Leu Ile Cys Lys
Tyr 115 120 348 28 PRT Homo sapiens 348 Met Met Val Trp Asn Leu Phe
Pro Cys Phe Pro Pro Leu Leu Leu Leu 1 5 10 15 Gln Phe Ile Asp Cys
Gln Gln Ser Ser Glu Ile Glu 20 25 349 28 PRT Homo sapiens 349 Gln
Gly Phe Thr Arg Ser Leu Leu Gly His Pro Ile Phe Phe Cys Pro 1 5 10
15 Asp Pro Cys Trp Gln Ser Cys Met Asn Cys Val Ile 20 25 350 35 PRT
Homo sapiens 350 Leu Ser Val Leu Ser Phe Phe Phe Leu Ile Arg Trp
Ile Ser Lys Ile 1 5 10 15 Val Ala Val Gln Lys Leu Glu Ser Ser Ser
Arg Arg Lys Pro Ile Leu 20 25 30 Phe Leu Ile 35 351 32 PRT Homo
sapiens 351 Ile Ser Cys Glu Ile Ala Ser Phe Ile His Leu Phe Leu Ser
Gln Met 1 5 10 15 Ser Ala Glu Cys Cys Cys Phe Tyr Leu Val Ile Leu
Ile Cys Lys Tyr 20 25 30 352 59 PRT Homo sapiens 352 Lys Val Asp
Thr Pro Arg Arg His Phe Cys Pro Glu Ile Ser Phe Phe 1 5 10 15 Leu
Thr Pro Leu Pro Gln Ser Ala Arg Asn Ser Thr Val Arg Asn Ala 20 25
30 Leu Ser Gly Leu Lys Asn Leu Thr Pro Ala Met Ile Ser Thr Val Ser
35 40 45 Lys Gln Asp Thr Ser Lys Leu Gly Glu Glu Glu 50 55 353 26
PRT Homo sapiens 353 Pro Thr Arg Pro Pro Thr Arg Pro Leu Ser Phe
Thr Phe Thr Lys Gln 1 5 10 15 Thr Ser Ser Thr Cys Leu Ser Leu His
Phe 20 25 354 50 PRT Homo sapiens 354 Leu Glu Cys Val Leu Leu Ile
Cys Phe Arg Ala Met Ser Ala Ile Tyr 1 5 10 15 Thr His Thr Ser Ile
Gly Asn Ala Gln Lys Leu Phe Thr Asp Gly Ser 20 25 30 Ala Phe Arg
Arg Val Arg Glu Pro Leu Pro Lys Glu Gly Lys Ser Trp 35 40 45 Pro
Gln 50 355 22 PRT Homo sapiens 355 Lys Gln Asn Leu Thr Asn Leu Asp
Val Pro Val Gln Tyr His Val Ala 1 5 10 15 Leu Ser Asp Lys Val Lys
20 356 117 PRT Homo sapiens SITE (71) Xaa equals any of the
naturally occurring L-amino acids 356 Pro Ser Cys Pro Pro Glu Met
Lys Lys Glu Leu Pro Val Asp Ser Cys 1 5 10 15 Leu Pro Arg Ser Leu
Glu Leu His Pro Gln Lys Met Asp Pro Lys Arg 20 25 30 Gln His Ile
Gln Leu Leu Ser Ser Leu Thr Glu Cys Leu Thr Val Asp 35 40 45 Pro
Leu Ser Ala Ser Val Trp Arg Gln Leu Tyr Pro Lys His Leu Ser 50 55
60 Gln Ser Ser Leu Leu Leu Xaa His Leu Leu Ser Ser Trp Glu Gln Ile
65 70 75 80 Pro Lys Lys Val Gln Lys Ser Leu Gln Glu Thr Ile Gln Ser
Leu Lys 85 90 95 Leu Thr Asn Gln Glu Leu Leu Arg Lys Gly Ser Ser
Asn Asn Gln Asp 100 105 110 Val Val Thr Cys Asp 115 357 103 PRT
Homo sapiens 357 Lys Ala Pro Tyr Ser Trp Leu Ala Asp Ser Trp Pro
His Pro Ser Arg 1 5 10 15 Ser Pro Ser Ala Gln Glu Pro Arg Gly Ser
Cys Cys Pro Ser Asn Pro 20 25 30 Asp Pro Asp Asp Arg Tyr Tyr Asn
Glu Ala Gly Ile Ser Leu Tyr Leu 35 40 45 Ala Gln Thr Ala Arg Gly
Thr Ala Ala Pro Gly Glu Gly Pro Val Tyr 50 55 60 Ser Thr Ile Asp
Pro Ala Gly Glu Glu Leu Gln Thr Phe His Gly Gly 65 70 75 80 Phe Pro
Gln His Pro Ser Gly Asp Leu Gly Pro Trp Ser Gln Tyr Ala 85 90 95
Pro Pro Glu Trp Ser Gln Gly 100 358 43 PRT Homo sapiens 358 Leu Gln
Gln Thr Met Gln Ala Met Leu His Phe Gly Gly Arg Leu Ala 1 5 10 15
Gln Ser Leu Arg Gly Thr Ser Lys Glu Ala Ala Ser Asp Pro Ser Asp 20
25 30 Ser Pro Asn Leu Pro Thr Pro Gly Ser Trp Trp 35 40 359 45 PRT
Homo sapiens 359 Glu Gln Leu Thr Gln Ala Ser Arg Val Tyr Ala Ser
Gly Gly Thr Glu 1 5 10 15 Gly Phe Pro Leu Ser Arg Trp Ala Pro Gly
Arg His Gly Thr Ala Ala 20 25 30 Glu Glu Gly Ala Gln Glu Arg Pro
Leu Pro Thr Asp Glu 35 40 45 360 45 PRT Homo sapiens 360 Met Ala
Pro Gly Arg Gly Leu Trp Leu Gly Arg Leu Phe Gly Val Pro 1 5 10 15
Gly Gly Pro Ala Glu Asn Glu Asn Gly Ala Leu Lys Ser Arg Arg Pro 20
25 30 Ser Ser Trp Leu Pro Pro Thr Val Ser Val Leu Ala Leu 35 40 45
361 44 PRT Homo sapiens 361 Val Lys Arg Gly Ala Pro Pro Glu Met Pro
Ser Pro Gln Glu Leu Glu 1 5 10 15 Ala Ser Ala Pro Arg Met Val Gln
Thr His Arg Ala Val Arg Ala Leu 20 25 30 Cys Asp His Thr Ala Ala
Arg Pro Asp Gln Leu Ser 35 40 362 38 PRT Homo sapiens 362 Phe Arg
Arg Gly Glu Val Leu Arg Val Ile Thr Thr Val Asp Glu Asp 1 5 10 15
Trp Leu Arg Cys Gly Arg Asp Gly Met Glu Gly Leu Val Pro Val Gly 20
25 30 Tyr Thr Ser Leu Val Leu 35 363 215 PRT Homo sapiens 363 Leu
Gln Gln Thr Met Gln Ala Met Leu His Phe Gly Gly Arg Leu Ala 1 5 10
15 Gln Ser Leu Arg Gly Thr Ser Lys Glu Ala Ala Ser Asp Pro Ser Asp
20 25 30 Ser Pro Asn Leu Pro Thr Pro Gly Ser Trp Trp Glu Gln Leu
Thr Gln 35 40 45 Ala Ser Arg Val Tyr Ala Ser Gly Gly Thr Glu Gly
Phe Pro Leu Ser 50 55 60 Arg Trp Ala Pro Gly Arg His Gly Thr Ala
Ala Glu Glu Gly Ala Gln 65 70 75 80 Glu Arg Pro Leu Pro Thr Asp Glu
Met Ala Pro Gly Arg Gly Leu Trp 85 90 95 Leu Gly Arg Leu Phe Gly
Val Pro Gly Gly Pro Ala Glu Asn Glu Asn 100 105 110 Gly Ala Leu Lys
Ser Arg Arg Pro Ser Ser Trp Leu Pro Pro Thr Val 115 120 125 Ser Val
Leu Ala Leu Val Lys Arg Gly Ala Pro Pro Glu Met Pro Ser 130 135 140
Pro Gln Glu Leu Glu Ala Ser Ala Pro Arg Met Val Gln Thr His Arg 145
150 155 160 Ala Val Arg Ala Leu Cys Asp His Thr Ala Ala Arg Pro Asp
Gln Leu 165 170 175 Ser Phe Arg Arg Gly Glu Val Leu Arg Val Ile Thr
Thr Val Asp Glu 180 185 190 Asp Trp Leu Arg Cys Gly Arg Asp Gly Met
Glu Gly Leu Val Pro Val 195 200 205 Gly Tyr Thr Ser Leu Val Leu 210
215 364 72 PRT Homo sapiens SITE (7) Xaa equals any of the
naturally occurring L-amino acids 364 Ala Arg Ala Cys Pro Arg Xaa
Gly Ala Ala Val Glu Lys Leu Gly Gly 1 5 10 15 Lys Pro Val Gln Pro
Asp Ser Lys Pro Thr Cys Cys Ser Gln Val Lys 20 25 30 Ala Glu Gly
Leu Ile Phe Ala Gly Leu Thr Gly Leu Lys Leu Leu Pro 35 40 45 Ser
Ser Leu Gln Arg Ala Val Phe Val Arg Gln Cys Leu Gly Phe Trp 50 55
60 Asn Asp Gly Ser Arg Ala Leu Gln 65 70 365 136 PRT Homo sapiens
SITE (130) Xaa equals any of the naturally occurring L-amino acids
365 Met Ser Pro Asn Leu Asn Ala Thr His Thr Ser Ala Gln Thr Pro Gly
1 5 10 15 Phe Met Glu Arg Lys Thr Thr His Thr Val Ala Gln Ala Leu
Ser His 20 25 30 Ala Val Arg Thr Ile Arg Gly Ala Arg Ser Pro Leu
Arg Pro Asp Ala 35 40 45 Ser Arg Thr Pro Thr Ser Cys Gln Met Ser
Thr Gln Ser Leu Leu Ile 50 55 60 Cys Lys Ala Arg Leu Pro Ser Phe
Gln Asn Pro Arg His Cys Leu Thr 65 70 75 80 Lys Thr Ala Leu Cys Lys
Glu Leu Gly Ser Asn Leu Ser Pro Val Arg 85 90 95 Pro Ala Lys Ile
Ser Pro Ser Ala Leu Thr Cys Glu Gln His Val Gly 100 105 110 Leu Glu
Ser Gly Trp Thr Gly Phe Pro Pro Ser Phe Ser Thr Ala Ala 115 120 125
Pro Xaa Leu Gly Gln Ala Arg Ala 130 135 366 31 PRT Homo sapiens 366
Phe Gln Ser Val Tyr His Met Lys Leu Gln Ser Ser Asn Leu Pro Ala 1 5
10 15 Ser Val Tyr Gly Asn Asn Leu Asn Cys Ile Asn Ser Ser Ser Ser
20 25 30 367 241 PRT Homo sapiens 367 Gly Leu Ser Ile His Asp Gly
Thr Trp Lys Ser Ala Ile Tyr Gly Phe 1 5 10 15 Gly Asp Gln Ser Asn
Leu Arg Lys Leu Arg Asn Val Ser Asn Leu Lys 20 25 30 Pro Val Pro
Leu Ile Gly Pro Lys Leu Lys Arg Arg Trp Pro Ile Ser 35 40 45 Tyr
Cys Arg Glu Leu Lys Gly Tyr Ser Ile Pro Phe Met Gly Ser Asp 50 55
60 Val Ser Val Val Arg Arg Thr Gln Arg Tyr Leu Tyr Glu Asn Leu Glu
65 70 75 80 Glu Ser Pro Val Gln Tyr Ala Ala Tyr Val Thr Val Gly Gly
Ile Thr 85 90 95 Ser Val Ile Lys Leu Met Phe Ala Gly Leu Phe Phe
Leu Phe Phe Val 100 105 110 Arg Phe Gly Ile Gly Arg Gln Leu Leu Ile
Lys Phe Pro Trp Phe Phe 115 120 125 Ser Phe Gly Tyr Phe Ser Lys Gln
Gly Pro Thr Gln Lys Gln Ile Asp 130 135 140 Ala Ala Ser Phe Thr Leu
Thr Phe Phe Gly Gln Gly Tyr Ser Gln Gly 145 150 155 160 Thr Gly Thr
Asp Lys Asn Lys Pro Asn Ile Lys Ile Cys Thr Gln Val 165 170 175 Lys
Gly Pro Glu Ala Gly Tyr Val Ala Thr Pro Ile Ala Met Val Gln 180 185
190 Ala Ala Met Thr Leu Leu Ser Asp Ala Ser His Leu Pro Lys Ala Gly
195 200 205 Gly Val Phe Thr Pro Gly Ala Ala Phe Ser Lys Thr Lys Leu
Ile Asp 210 215 220 Arg Leu Asn Lys His Gly Ile Glu Phe Ser Val Ile
Ser Ser Ser Glu 225 230 235 240 Val 368 62 PRT Homo sapiens 368 Met
Asp Pro Asp Arg Ala Phe Ile Cys Gly Glu Ser Arg Gln Phe Ala 1 5 10
15 Gln Cys Leu Ile Phe Gly Phe Leu Phe Leu Thr Ser Gly Met Leu Ile
20 25 30 Ser Val Leu Gly Ile Trp Val Pro Gly Cys Gly Ser Asn Trp
Ala Gln 35 40 45 Glu Pro Leu Asn Glu Thr Asp Thr Gly Asp Ser Glu
Pro Arg 50 55 60 369 229 PRT Homo sapiens 369 Met Asp Pro Asp Arg
Ala Phe Ile Cys Gly Glu Ser Arg Gln Phe Ala 1 5 10 15 Gln Cys Leu
Ile Phe Gly Phe Leu Phe Leu Thr Ser Gly Met Leu Ile 20 25 30 Ser
Val Leu Gly Ile Trp Val Pro Gly Cys Gly Ser Asn Trp Ala Gln 35 40
45 Glu Pro Leu Asn Glu Thr Asp Thr Gly Asp Ser Glu Pro Arg Met Cys
50 55 60 Gly Phe Leu Ser Leu Gln Ile Met Gly Pro Leu Ile Val Leu
Val Gly 65 70 75 80 Leu Cys Phe Phe Val Val Ala His Val Lys Lys Arg
Asn Thr Leu Asn 85 90 95 Ala Gly Gln Asp Ala Ser Glu Arg Glu Glu
Gly Gln Ile Gln Ile Met 100 105 110 Glu Pro Val Gln Val Thr Val Gly
Asp Ser Val Ile Ile Phe Pro Pro 115 120 125 Pro Pro Pro Pro Tyr Phe
Pro Glu Ser Ser Ala Ser Ala Val Ala Glu 130 135 140 Ser Pro Gly Thr
Asn Ser Leu Leu Pro Asn Glu Asn Pro Pro Ser Tyr 145 150 155 160 Tyr
Ser Ile Phe Asn Tyr Gly Thr Pro Thr Ser Glu Gly Ala Ala Ser 165 170
175 Glu Arg Asp Cys Glu Ser Ile Tyr Thr Ile Ser Gly Thr Asn Ser Ser
180 185 190 Ser Glu Ala Ser His Thr Pro His Leu Pro Ser Glu Leu Pro
Pro Arg 195 200 205 Tyr Glu Glu Lys Glu Asn Ala Ala Ala Thr Phe Leu
Pro Leu Ser Ser 210 215 220 Glu Pro Ser Pro Pro 225 370 37 PRT Homo
sapiens 370 Phe Asp Phe Ile Ala Ser Leu Leu Lys Ala Asn Arg Leu Ser
Leu Gln 1 5 10 15 Thr Cys Glu Leu Leu Leu Ala Ala Ala Leu Leu Pro
Ser Glu Arg Tyr 20 25 30 Lys Ala Ile Ser Ile 35 371 63 PRT Homo
sapiens 371 Met Asn Lys Lys Ala Glu Leu Lys Pro Ser Ala Leu Pro Gly
Trp Ala 1 5 10 15 Asn Val Trp Lys Leu Met Cys Leu Val Thr Val Cys
Ala Ser Leu Ile 20 25 30 Ile Thr Ser Asp Ser Val Val Ser Thr Val
Arg Leu Lys Gly Ser Cys 35 40 45 Glu Asp Tyr Leu Gly Leu Ser Cys
Gly Asn Thr Ser His Ala Tyr 50 55 60 372 434 PRT Homo sapiens 372
Met Ser Ala Asp Gly Ala Glu Ala Asp Gly Ser Thr Gln Val Thr Val 1 5
10 15 Glu Glu Pro Val Gln Gln Pro Ser Val Val Asp Arg Val Ala Ser
Met 20 25 30 Pro Leu Ile Ser Ser Thr Cys Asp Met Val Ser Ala Ala
Tyr Ala Ser 35
40 45 Thr Lys Glu Ser Tyr Pro His Val Lys Thr Val Cys Asp Ala Ala
Glu 50 55 60 Lys Gly Val Arg Thr Leu Thr Ala Ala Ala Val Ser Gly
Ala Gln Pro 65 70 75 80 Ile Leu Ser Lys Leu Glu Pro Gln Ile Ala Ser
Ala Ser Glu Tyr Ala 85 90 95 His Arg Gly Leu Asp Lys Leu Glu Glu
Asn Leu Pro Ile Leu Gln Gln 100 105 110 Pro Thr Glu Lys Val Leu Ala
Asp Thr Lys Glu Leu Val Ser Ser Lys 115 120 125 Val Ser Gly Ala Gln
Glu Met Val Ser Ser Ala Lys Asp Thr Val Ala 130 135 140 Thr Gln Leu
Ser Glu Ala Val Asp Ala Thr Arg Gly Ala Val Gln Ser 145 150 155 160
Gly Val Asp Lys Thr Lys Ser Val Val Thr Gly Gly Val Gln Ser Val 165
170 175 Met Gly Ser Arg Leu Gly Gln Met Val Leu Ser Gly Val Asp Thr
Val 180 185 190 Leu Gly Lys Ser Glu Glu Trp Ala Asp Asn His Leu Pro
Leu Thr Asp 195 200 205 Ala Glu Leu Ala Arg Ile Ala Thr Ser Leu Asp
Gly Phe Asp Val Ala 210 215 220 Ser Val Gln Gln Gln Arg Gln Glu Gln
Ser Tyr Phe Val Arg Leu Gly 225 230 235 240 Ser Leu Ser Glu Arg Leu
Arg Gln His Ala Tyr Glu His Ser Leu Gly 245 250 255 Lys Leu Arg Ala
Thr Lys Gln Arg Ala Gln Glu Ala Leu Leu Gln Leu 260 265 270 Ser Gln
Ala Leu Ser Leu Met Glu Thr Val Lys Gln Gly Val Asp Gln 275 280 285
Lys Leu Val Glu Gly Gln Glu Lys Leu His Gln Met Trp Leu Ser Trp 290
295 300 Asn Gln Lys Gln Leu Gln Gly Pro Glu Lys Glu Pro Pro Lys Pro
Glu 305 310 315 320 Gln Val Glu Ser Arg Ala Leu Thr Met Phe Arg Asp
Ile Ala Gln Gln 325 330 335 Leu Gln Ala Thr Cys Thr Ser Leu Gly Ser
Ser Ile Gln Gly Leu Pro 340 345 350 Thr Asn Val Lys Asp Gln Val Gln
Gln Ala Arg Arg Gln Val Glu Asp 355 360 365 Leu Gln Ala Thr Phe Ser
Ser Ile His Ser Phe Gln Asp Leu Ser Ser 370 375 380 Ser Ile Leu Ala
Gln Ser Arg Glu Arg Val Ala Ser Ala Arg Glu Ala 385 390 395 400 Leu
Asp His Met Val Glu Tyr Val Ala Gln Asn Thr Pro Val Thr Trp 405 410
415 Leu Val Gly Pro Phe Ala Pro Gly Ile Thr Glu Lys Ala Pro Glu Glu
420 425 430 Lys Lys 373 66 PRT Homo sapiens 373 Met Leu Cys Lys Ser
Leu Leu Tyr Cys Val Val Ser Tyr Leu Tyr Tyr 1 5 10 15 Phe Val Phe
Ile Tyr Phe Phe Pro Val Phe Leu Ile Cys Ser Trp Leu 20 25 30 Glu
Leu Gln Met Trp Asn Leu Gln Ile Gly Arg Ala Asp Cys Phe Gln 35 40
45 Asn Thr Leu Val Tyr Val Leu Ser Leu Cys Leu Gln Tyr Lys Asn His
50 55 60 Pro Ala 65 374 25 PRT Homo sapiens 374 Ile Asp Leu Ser Phe
Pro Ser Thr Asn Val Ser Leu Glu Asp Arg Asn 1 5 10 15 Thr Thr Lys
Pro Ser Val Asn Val Gly 20 25 375 12 PRT Homo sapiens 375 Val Ala
His Ala Cys Asn Pro Ser Thr Leu Gly Gly 1 5 10 376 17 PRT Homo
sapiens 376 Gly Gly Gln Ile Thr Arg Ser Gly Asp Gln Asp Gln Pro Asp
Gln His 1 5 10 15 Gly 377 12 PRT Homo sapiens 377 Gly Phe Thr Met
Leu Val Arg Leu Val Leu Ile Ser 1 5 10 378 28 PRT Homo sapiens 378
Pro Arg Asp Leu Pro Thr Ser Ala Ser Gln Ser Ala Gly Ile Thr Gly 1 5
10 15 Met Ser His Pro Ala Arg Pro Lys Leu Leu Phe Asn 20 25 379 46
PRT Homo sapiens 379 Pro Phe Trp Ala Ala Glu Ser Ala Leu Asp Phe
His Trp Pro Phe Gly 1 5 10 15 Gly Ala Leu Cys Lys Met Val Leu Thr
Ala Thr Val Leu Asn Val Tyr 20 25 30 Ala Ser Ile Phe Leu Ile Thr
Ala Leu Ser Val Ala Arg Tyr 35 40 45 380 12 PRT Homo sapiens 380
Thr His Ala Asp Lys Asn Gln Val Arg Asn Ser Asn 1 5 10 381 15 PRT
Homo sapiens 381 Gln Phe Leu Ser Trp Glu Gln Cys Thr Gly Asn Thr
Glu Ser Gln 1 5 10 15 382 13 PRT Homo sapiens SITE (9) Xaa equals
any of the naturally occurring L-amino acids 382 Val Arg Arg Pro
Lys Ala Lys Gly Xaa Gln Thr Ser Asn 1 5 10 383 19 PRT Homo sapiens
383 Pro Thr Gln Leu Asn Lys His Lys Pro Thr Thr Lys Glu Arg Arg Arg
1 5 10 15 Lys Gly Leu 384 9 PRT Homo sapiens 384 Leu Ile Ser Lys
His Glu Asn Ile Tyr 1 5 385 27 PRT Homo sapiens SITE (5) Xaa equals
any of the naturally occurring L-amino acids SITE (6) Xaa equals
any of the naturally occurring L-amino acids SITE (8) Xaa equals
any of the naturally occurring L-amino acids SITE (22) Xaa equals
any of the naturally occurring L-amino acids 385 Thr Leu Tyr Ile
Xaa Xaa Met Xaa Thr Gln Thr Trp Arg Asp Gln Gly 1 5 10 15 Arg Cys
Gly Arg Asp Xaa Ile Asn Cys Ile Val 20 25 386 33 PRT Homo sapiens
386 Ser Leu Cys Thr Pro Gly Arg Gly Trp Glu Glu Ser Trp Gly Ser Ser
1 5 10 15 Leu Pro Asn Leu Thr Gly Trp Ser Val Ser Ser Leu Asp Asn
Asn Asp 20 25 30 Val 387 204 PRT Homo sapiens SITE (107) Xaa equals
any of the naturally occurring L-amino acids 387 Met Gln Val Ala
Leu Lys Glu Asp Leu Asp Ala Leu Lys Glu Lys Phe 1 5 10 15 Arg Thr
Met Glu Ser Asn Gln Lys Ser Ser Phe Gln Glu Ile Pro Lys 20 25 30
Leu Asn Glu Glu Leu Leu Ser Lys Gln Lys Gln Leu Glu Lys Ile Glu 35
40 45 Ser Gly Glu Met Gly Leu Asn Lys Val Trp Ile Asn Ile Thr Glu
Met 50 55 60 Asn Lys Gln Ile Ser Leu Leu Thr Ser Ala Val Asn His
Leu Lys Ala 65 70 75 80 Asn Val Lys Ser Ala Ala Asp Leu Ile Ser Leu
Pro Thr Thr Val Glu 85 90 95 Gly Leu Gln Lys Ser Val Ala Ser Ile
Gly Xaa Thr Leu Asn Ser Val 100 105 110 His Leu Ala Val Glu Ala Leu
Gln Lys Thr Val Asp Glu His Lys Lys 115 120 125 Thr Met Glu Leu Leu
Gln Ser Asp Met Asn Gln His Phe Leu Lys Glu 130 135 140 Thr Pro Gly
Ser Asn Gln Ile Ile Pro Ser Pro Ser Ala Thr Ser Glu 145 150 155 160
Leu Asp Asn Lys Thr His Ser Glu Asn Leu Lys Gln Met Gly Asp Arg 165
170 175 Ser Ala Thr Leu Lys Arg Gln Ser Leu Asp Gln Val Thr Asn Arg
Thr 180 185 190 Asp Thr Val Lys Ile Gln Ser Ile Lys Lys Glu Gly 195
200 388 43 PRT Homo sapiens 388 Met Gln Val Ala Leu Lys Glu Asp Leu
Asp Ala Leu Lys Glu Lys Phe 1 5 10 15 Arg Thr Met Glu Ser Asn Gln
Lys Ser Ser Phe Gln Glu Ile Pro Lys 20 25 30 Leu Asn Glu Glu Leu
Leu Ser Lys Gln Lys Gln 35 40 389 43 PRT Homo sapiens 389 Leu Glu
Lys Ile Glu Ser Gly Glu Met Gly Leu Asn Lys Val Trp Ile 1 5 10 15
Asn Ile Thr Glu Met Asn Lys Gln Ile Ser Leu Leu Thr Ser Ala Val 20
25 30 Asn His Leu Lys Ala Asn Val Lys Ser Ala Ala 35 40 390 43 PRT
Homo sapiens SITE (21) Xaa equals any of the naturally occurring
L-amino acids 390 Asp Leu Ile Ser Leu Pro Thr Thr Val Glu Gly Leu
Gln Lys Ser Val 1 5 10 15 Ala Ser Ile Gly Xaa Thr Leu Asn Ser Val
His Leu Ala Val Glu Ala 20 25 30 Leu Gln Lys Thr Val Asp Glu His
Lys Lys Thr 35 40 391 43 PRT Homo sapiens 391 Met Glu Leu Leu Gln
Ser Asp Met Asn Gln His Phe Leu Lys Glu Thr 1 5 10 15 Pro Gly Ser
Asn Gln Ile Ile Pro Ser Pro Ser Ala Thr Ser Glu Leu 20 25 30 Asp
Asn Lys Thr His Ser Glu Asn Leu Lys Gln 35 40 392 32 PRT Homo
sapiens 392 Met Gly Asp Arg Ser Ala Thr Leu Lys Arg Gln Ser Leu Asp
Gln Val 1 5 10 15 Thr Asn Arg Thr Asp Thr Val Lys Ile Gln Ser Ile
Lys Lys Glu Gly 20 25 30 393 258 PRT Homo sapiens SITE (161) Xaa
equals any of the naturally occurring L-amino acids 393 Asp Ser Glu
Ser Ser Ser Glu Glu Glu Glu Glu Phe Gly Val Val Gly 1 5 10 15 Asn
Arg Ser Arg Phe Ala Lys Gly Asp Tyr Leu Arg Cys Cys Lys Ile 20 25
30 Cys Tyr Pro Leu Cys Gly Phe Val Ile Leu Ala Ala Cys Val Val Ala
35 40 45 Cys Val Gly Leu Val Trp Met Gln Val Ala Leu Lys Glu Asp
Leu Asp 50 55 60 Ala Leu Lys Glu Lys Phe Arg Thr Met Glu Ser Asn
Gln Lys Ser Ser 65 70 75 80 Phe Gln Glu Ile Pro Lys Leu Asn Glu Glu
Leu Leu Ser Lys Gln Lys 85 90 95 Gln Leu Glu Lys Ile Glu Ser Gly
Glu Met Gly Leu Asn Lys Val Trp 100 105 110 Ile Asn Ile Thr Glu Met
Asn Lys Gln Ile Ser Leu Leu Thr Ser Ala 115 120 125 Val Asn His Leu
Lys Ala Asn Val Lys Ser Ala Ala Asp Leu Ile Ser 130 135 140 Leu Pro
Thr Thr Val Glu Gly Leu Gln Lys Ser Val Ala Ser Ile Gly 145 150 155
160 Xaa Thr Leu Asn Ser Val His Leu Ala Val Glu Ala Leu Gln Lys Thr
165 170 175 Val Asp Glu His Lys Lys Thr Met Glu Leu Leu Gln Ser Asp
Met Asn 180 185 190 Gln His Phe Leu Lys Glu Thr Pro Gly Ser Asn Gln
Ile Ile Pro Ser 195 200 205 Pro Ser Ala Thr Ser Glu Leu Asp Asn Lys
Thr His Ser Glu Asn Leu 210 215 220 Lys Gln Met Gly Asp Arg Ser Ala
Thr Leu Lys Arg Gln Ser Leu Asp 225 230 235 240 Gln Val Thr Asn Arg
Thr Asp Thr Val Lys Ile Gln Ser Ile Lys Lys 245 250 255 Glu Gly 394
12 PRT Homo sapiens 394 Ser Pro Gln Phe Leu Ser Ser Lys Ser Leu Pro
Thr 1 5 10 395 107 PRT Homo sapiens 395 Gly Pro Pro Ser Pro Arg Gly
Leu Pro Ser Leu Pro Leu His Leu Pro 1 5 10 15 Ala Pro Arg Arg Tyr
Leu Gln Ser Arg Tyr Ala Cys Ser Gln Ser Ser 20 25 30 Val Ser Ala
Ala Ala Arg Arg Trp Gly Ser Gly Trp Met Ala Trp Asp 35 40 45 Pro
Trp Asn Gln Ala Ser Gly Arg Tyr Ala Arg Ile Thr Leu Leu Ser 50 55
60 Val Gln Ala Cys His Gln Pro Thr Val Trp Pro Arg Ala Gly His Ser
65 70 75 80 Leu Pro Glu Arg Tyr Ser Leu His Pro His Asn Gly Asp Ser
Thr His 85 90 95 Leu Ser Gly Leu Leu Thr Val Lys Cys Gly Ala 100
105 396 37 PRT Homo sapiens 396 Gly Pro Pro Ser Pro Arg Gly Leu Pro
Ser Leu Pro Leu His Leu Pro 1 5 10 15 Ala Pro Arg Arg Tyr Leu Gln
Ser Arg Tyr Ala Cys Ser Gln Ser Ser 20 25 30 Val Ser Ala Ala Ala 35
397 33 PRT Homo sapiens 397 Arg Arg Trp Gly Ser Gly Trp Met Ala Trp
Asp Pro Trp Asn Gln Ala 1 5 10 15 Ser Gly Arg Tyr Ala Arg Ile Thr
Leu Leu Ser Val Gln Ala Cys His 20 25 30 Gln 398 37 PRT Homo
sapiens 398 Pro Thr Val Trp Pro Arg Ala Gly His Ser Leu Pro Glu Arg
Tyr Ser 1 5 10 15 Leu His Pro His Asn Gly Asp Ser Thr His Leu Ser
Gly Leu Leu Thr 20 25 30 Val Lys Cys Gly Ala 35 399 173 PRT Homo
sapiens SITE (130) Xaa equals any of the naturally occurring
L-amino acids SITE (152) Xaa equals any of the naturally occurring
L-amino acids 399 Gly Pro Pro Ser Pro Arg Gly Leu Pro Ser Leu Pro
Leu His Leu Pro 1 5 10 15 Ala Pro Arg Arg Tyr Leu Gln Ser Arg Tyr
Ala Cys Ser Gln Ser Ser 20 25 30 Val Ser Ala Ala Ala Arg Arg Trp
Gly Ser Gly Trp Met Ala Trp Asp 35 40 45 Pro Trp Asn Gln Ala Ser
Gly Arg Tyr Ala Arg Ile Thr Leu Leu Ser 50 55 60 Val Gln Ala Cys
His Gln Pro Thr Val Trp Pro Arg Ala Gly His Ser 65 70 75 80 Leu Pro
Glu Arg Tyr Ser Leu His Pro His Asn Gly Asp Ser Thr His 85 90 95
Leu Ser Gly Leu Leu Thr Val Lys Cys Gly Ala Met Ala Gly Phe Ala 100
105 110 Ser Tyr Pro Trp Ser Asp Phe Pro Trp Cys Trp Val Val Cys Phe
Ser 115 120 125 Phe Xaa Phe Phe Phe Leu Arg Gln Ser Glu Ser Leu Ser
Gln Lys Lys 130 135 140 Arg Gln Val Ala Asp Glu Leu Xaa Phe Gly Gln
Ser Lys Arg Asp Ser 145 150 155 160 Asp Gly Gly Trp Met Leu Arg Ser
Ser Ala Gly Asn Ser 165 170 400 119 PRT Homo sapiens SITE (46) Xaa
equals any of the naturally occurring L-amino acids SITE (52) Xaa
equals any of the naturally occurring L-amino acids SITE (110) Xaa
equals any of the naturally occurring L-amino acids 400 Met Glu Ser
Cys Ser Val Val Gln Ala Gly Val Lys Trp Cys Asp Leu 1 5 10 15 Gly
Ser Leu Gln Pro Pro Pro Arg Phe Lys Gln Phe Ser Trp Glu Val 20 25
30 Glu Val Ala Val Ser Arg Asp His Thr Ile Ala Leu Gln Xaa Gly Gly
35 40 45 Gln Ser Lys Xaa Leu Ser Gln Lys Lys Glu Lys Lys Tyr Val
Leu Asn 50 55 60 Ala Thr Phe Leu Asn Phe Tyr Phe Cys Arg Asp Lys
Val Leu Leu Cys 65 70 75 80 Cys Pro Gly Trp Ser His Ile Val Gly Leu
Lys Gln Ser Ser His Leu 85 90 95 Gly Leu Arg Lys Cys Trp Asp Tyr
Arg His Gly Pro Leu Xaa Leu Ala 100 105 110 Leu Cys His Phe Val Cys
Lys 115 401 18 PRT Homo sapiens 401 Asn Gln Glu Asn Ser Leu Gln Thr
Asn Ser Tyr Leu Asp Ser Thr Glu 1 5 10 15 Ser Lys 402 31 PRT Homo
sapiens SITE (17) Xaa equals any of the naturally occurring L-amino
acids SITE (19) Xaa equals any of the naturally occurring L-amino
acids SITE (30) Xaa equals any of the naturally occurring L-amino
acids 402 Gln Lys Arg Ala Cys Phe Pro Phe Ala Phe Cys Arg Asp Cys
Gln Phe 1 5 10 15 Xaa Glu Xaa Ser Pro Ala Met Leu Pro Val Gln Pro
Ala Xaa Leu 20 25 30 403 11 PRT Homo sapiens 403 Val Ser Ala His
Gly Ile Trp Leu Phe Arg Ser 1 5 10 404 49 PRT Homo sapiens SITE
(35) Xaa equals any of the naturally occurring L-amino acids SITE
(37) Xaa equals any of the naturally occurring L-amino acids SITE
(48) Xaa equals any of the naturally occurring L-amino acids 404
Lys His Ala Ala Pro Pro Ala Ser Leu Ser Leu Ser Leu Leu Leu His 1 5
10 15 His Gly Gln Lys Arg Ala Cys Phe Pro Phe Ala Phe Cys Arg Asp
Cys 20 25 30 Gln Phe Xaa Glu Xaa Ser Pro Ala Met Leu Pro Val Gln
Pro Ala Xaa 35 40 45 Leu 405 101 PRT Homo sapiens 405 Met Cys Asp
Asn Leu Ile Met Leu Arg Thr Leu Met Arg Tyr Ile Val 1 5 10 15 Phe
Leu Ser Leu Gln Cys Leu Trp Gly Gln Gly Thr His Ser Ser Cys 20 25
30 Tyr Pro Pro Ser Pro Leu Arg Leu Pro Leu Phe Phe Phe Leu Asp Ile
35 40 45 Lys Leu Gly Ile Ser Asn Trp Pro Val Val Met Gln Ser Cys
Phe Ala 50 55 60 Leu Tyr Leu Ala Gly Leu Ile Cys Leu Thr Arg Ser
His Glu Ala Ile 65 70 75 80 Gly Arg Ser Ser Leu Ser Pro Ser Ser Ser
Ala Pro Lys Val Val Ala 85 90 95 Arg Gly Val Pro Ser 100 406 138
PRT Homo sapiens 406 Met Leu Val Leu Met Thr Leu Phe Leu Leu Leu
Tyr Tyr Arg Tyr Val 1 5 10 15 Tyr Gly Phe Gly Val
Cys Val Tyr Val His Ile Tyr Ala His Ile Tyr 20 25 30 Thr His Thr
His Ile Tyr Asn Gln Leu Ser Ile Ala Tyr Ser Ser Leu 35 40 45 Ile
Ile Tyr Ile Leu Tyr Ser Asn Phe Ser Asn Thr Pro Thr Lys Ser 50 55
60 Phe Ser Pro Pro Tyr Gln Tyr Tyr Asn Val Pro Asp Asn Asn Ile Thr
65 70 75 80 Asn Pro Ala Leu Thr Pro Thr Asp Phe Phe Glu Asn Lys Gln
Leu Leu 85 90 95 His Ala Ile Ser Phe Leu Tyr Ser Pro Thr Gly Phe
Leu Gln Pro Pro 100 105 110 Ala His Pro Val Gln Leu Arg Thr Ser Thr
Thr Leu Tyr Gly Asn His 115 120 125 Arg Gly Gln Thr Gly Cys Ser Gln
Leu Asp 130 135 407 67 PRT Homo sapiens 407 Ser Asn Thr Pro Thr Lys
Ser Phe Ser Pro Pro Tyr Gln Tyr Tyr Asn 1 5 10 15 Val Pro Asp Asn
Asn Ile Thr Asn Pro Ala Leu Thr Pro Thr Asp Phe 20 25 30 Phe Glu
Asn Lys Gln Leu Leu His Ala Ile Ser Phe Leu Tyr Ser Pro 35 40 45
Thr Gly Phe Leu Gln Pro Pro Ala His Pro Val Gln Leu Arg Thr Ser 50
55 60 Thr Thr Leu 65 408 12 PRT Homo sapiens 408 Met Glu Met Asn
Tyr Cys Gly Ser Arg Val Leu Tyr 1 5 10 409 61 PRT Homo sapiens 409
Leu Gly Ser Pro Ile Ile Pro Leu Trp Ser Tyr Thr Ser Ala Thr Gln 1 5
10 15 Ala Ala Ala Leu Val Thr Ser His Val Trp Lys Pro Ser Leu Glu
Ala 20 25 30 His Gln Ile Asn Ile Ser Pro Glu Pro Ser Ile His Tyr
Asp Arg Trp 35 40 45 His Thr Gln Ser Asn Cys Ser Leu Ile Asn Ser
Leu Gln 50 55 60 410 12 PRT Homo sapiens 410 Ile Pro Glu Glu Ala
Ser Cys Phe Pro Ser Ala Val 1 5 10 411 17 PRT Homo sapiens 411 Glu
Ile Leu Phe Gly Lys Leu Lys Ser Lys Ala Ala Leu Cys Thr Gln 1 5 10
15 Gly 412 19 PRT Homo sapiens 412 His Ala Asp Arg Tyr Thr Cys Cys
Arg Cys Leu Ser Pro Phe Ser Leu 1 5 10 15 Ala Gly Leu 413 15 PRT
Homo sapiens 413 Leu Ser Asp Pro Leu Leu Leu Pro Asp Cys Ser Phe
Ser Phe Asn 1 5 10 15 414 25 PRT Homo sapiens 414 Lys Ala Val Ala
Tyr Ala Asn Val Ser Cys Arg Arg Phe Lys His Lys 1 5 10 15 Thr Thr
Lys Leu Gly Pro Ile Gln Trp 20 25 415 26 PRT Homo sapiens 415 Pro
Ser Ser Gln Ser Pro Glu Pro Pro Gln Pro Leu Ser Leu Phe Val 1 5 10
15 Thr Arg Leu Pro Asn Leu Tyr Asp Phe Pro 20 25 416 19 PRT Homo
sapiens 416 Ser Arg Gln Ile Ile Cys Thr Asn Leu Cys Lys Cys Thr Pro
Ile Cys 1 5 10 15 Phe Leu Phe 417 15 PRT Homo sapiens 417 Lys Gly
Ser Leu Pro Trp Arg Leu Leu Leu Pro Leu Asn Gly Pro 1 5 10 15 418
19 PRT Homo sapiens 418 Leu Cys Arg Leu Val Phe Glu Ser Ser Ala Gly
His Val Ser Val Cys 1 5 10 15 His Ser Phe 419 11 PRT Homo sapiens
419 Met Leu Leu Pro Val Asn Thr Leu Leu Tyr Ile 1 5 10 420 14 PRT
Homo sapiens 420 Leu Leu Thr Pro Leu Cys Phe Phe Tyr Gly Thr Ser
Arg Pro 1 5 10 421 7 PRT Homo sapiens 421 Pro Tyr Leu Glu Leu Val
Thr 1 5 422 13 PRT Homo sapiens 422 Leu Leu Lys Lys Lys Lys Gln Ser
Val Gly Phe Ser Val 1 5 10 423 7 PRT Homo sapiens 423 Cys Ile Leu
Glu Ala Gly Arg 1 5 424 11 PRT Homo sapiens 424 Met Gly Phe Ser Ala
Pro Thr Pro Gly Pro Leu 1 5 10 425 11 PRT Homo sapiens 425 Phe Asp
Leu Arg Arg Leu Ile Leu Ser Ile Val 1 5 10 426 17 PRT Homo sapiens
426 Ala Phe Cys Pro His Val Thr Pro Cys Lys Tyr Ala Val Ile His Thr
1 5 10 15 Val 427 11 PRT Homo sapiens 427 Asn Thr Pro Leu Leu Phe
Leu Trp Asp Leu Gln 1 5 10 428 17 PRT Homo sapiens 428 Ala Thr Ile
Phe Arg Thr Ser Tyr Leu Ile Lys Lys Glu Lys Thr Val 1 5 10 15 Cys
429 17 PRT Homo sapiens 429 Trp Leu Leu Ser Leu His Leu Gly Gly Arg
Glu Val Arg Ala Gly Ala 1 5 10 15 Pro 430 11 PRT Homo sapiens 430
Gln Thr Leu Gln Glu Gly Ser Leu His Ser Ile 1 5 10 431 95 PRT Homo
sapiens 431 Met Gly Phe Ser Ala Pro Thr Pro Gly Pro Leu Phe Asp Leu
Arg Arg 1 5 10 15 Leu Ile Leu Ser Ile Val Ala Phe Cys Pro His Val
Thr Pro Cys Lys 20 25 30 Tyr Ala Val Ile His Thr Val Asn Thr Pro
Leu Leu Phe Leu Trp Asp 35 40 45 Leu Gln Ala Thr Ile Phe Arg Thr
Ser Tyr Leu Ile Lys Lys Glu Lys 50 55 60 Thr Val Cys Trp Leu Leu
Ser Leu His Leu Gly Gly Arg Glu Val Arg 65 70 75 80 Ala Gly Ala Pro
Gln Thr Leu Gln Glu Gly Ser Leu His Ser Ile 85 90 95 432 33 PRT
Homo sapiens 432 Tyr Trp Val Ser Ile Ser Gln Arg Ser Val Cys Gln
Gln Ala Arg Thr 1 5 10 15 Ser Ile Phe Phe Lys Asp Gly Leu Ser Arg
Glu Lys Tyr Ser Asn Asn 20 25 30 Gly 433 160 PRT Homo sapiens 433
Leu Ser Val Arg Ala Pro Gly Val Pro Ala Ala Arg Pro Arg Leu Ser 1 5
10 15 Ser Ala Arg Gln Ala Gly Ala Gly Arg Gly Glu Leu Arg Gly Gln
Arg 20 25 30 Leu Trp Leu Gly Pro Glu Cys Gly Cys Gly Ala Gly Gln
Ala Gly Ser 35 40 45 Met Leu Arg Ala Val Gly Ser Leu Leu Arg Leu
Gly Arg Gly Leu Thr 50 55 60 Val Arg Cys Gly Pro Gly Ala Pro Leu
Glu Ala Thr Arg Arg Pro Ala 65 70 75 80 Pro Ala Leu Pro Pro Arg Gly
Leu Pro Cys Tyr Ser Ser Gly Gly Ala 85 90 95 Pro Ser Asn Ser Gly
Pro Gln Gly His Gly Glu Ile His Arg Val Pro 100 105 110 Thr Gln Arg
Arg Pro Ser Gln Phe Asp Lys Lys Ile Leu Leu Trp Thr 115 120 125 Gly
Arg Phe Lys Ser Met Glu Glu Ile Pro Pro Arg Ile Pro Pro Glu 130 135
140 Met Ile Asp Thr Ala Arg Asn Lys Ala Arg Val Lys Ala Cys Tyr Ile
145 150 155 160 434 36 PRT Homo sapiens 434 Leu Ser Val Arg Ala Pro
Gly Val Pro Ala Ala Arg Pro Arg Leu Ser 1 5 10 15 Ser Ala Arg Gln
Ala Gly Ala Gly Arg Gly Glu Leu Arg Gly Gln Arg 20 25 30 Leu Trp
Leu Gly 35 435 34 PRT Homo sapiens 435 Pro Glu Cys Gly Cys Gly Ala
Gly Gln Ala Gly Ser Met Leu Arg Ala 1 5 10 15 Val Gly Ser Leu Leu
Arg Leu Gly Arg Gly Leu Thr Val Arg Cys Gly 20 25 30 Pro Gly 436 34
PRT Homo sapiens 436 Ala Pro Leu Glu Ala Thr Arg Arg Pro Ala Pro
Ala Leu Pro Pro Arg 1 5 10 15 Gly Leu Pro Cys Tyr Ser Ser Gly Gly
Ala Pro Ser Asn Ser Gly Pro 20 25 30 Gln Gly 437 27 PRT Homo
sapiens 437 His Gly Glu Ile His Arg Val Pro Thr Gln Arg Arg Pro Ser
Gln Phe 1 5 10 15 Asp Lys Lys Ile Leu Leu Trp Thr Gly Arg Phe 20 25
438 29 PRT Homo sapiens 438 Lys Ser Met Glu Glu Ile Pro Pro Arg Ile
Pro Pro Glu Met Ile Asp 1 5 10 15 Thr Ala Arg Asn Lys Ala Arg Val
Lys Ala Cys Tyr Ile 20 25 439 57 PRT Homo sapiens 439 Cys Ser Pro
Gly Gln Asp Glu Met Gln Asp Glu Thr Trp Cys Ser Gly 1 5 10 15 Gln
Ser Glu Thr Val Asn Glu Ala Lys Gln Leu Arg Thr Thr His Ser 20 25
30 Arg Val Pro Asn Gln Gln Val Cys Val Cys Gly Trp Leu Pro Val Asn
35 40 45 Ile Ser Pro His Ser Pro Leu Lys Lys 50 55 440 147 PRT Homo
sapiens 440 Met Ser Gly Asp Val Cys Val Phe Gly Tyr Ala His Leu His
Ser Gln 1 5 10 15 Thr Lys His Ser Gly Ser Gln Gly Trp Val Leu Ile
Tyr Leu Phe Ala 20 25 30 Met Gln Lys Ile Ser Cys Thr Lys Leu Pro
Leu Leu Arg Asn Leu Lys 35 40 45 Leu Asn Leu Val Trp Leu Ser Gln
Gly Trp Val Phe Phe Lys Gly Leu 50 55 60 Trp Gly Glu Met Leu Thr
Gly Ser His Pro Gln Thr His Thr Cys Trp 65 70 75 80 Leu Gly Thr Arg
Leu Trp Val Val Leu Ser Cys Leu Ala Ser Leu Thr 85 90 95 Val Ser
Asp Cys Pro Glu His Gln Val Ser Ser Cys Ile Ser Ser Trp 100 105 110
Pro Gly Glu His Ser Val Ser Phe Gln Pro Phe Pro Pro Phe Pro His 115
120 125 Ser Leu Gly Gly Thr Glu Val Gly Val Glu Glu Ser Gln Met Ala
Gly 130 135 140 Val Gly Ile 145 441 15 PRT Homo sapiens 441 Leu Asn
Ile Leu Ile Ser Leu Thr Val Ser Ser His Cys Lys Leu 1 5 10 15 442
13 PRT Homo sapiens 442 Ile Asn Tyr His Ser Gly Phe Ile His Gln Phe
Leu Ala 1 5 10 443 11 PRT Homo sapiens 443 Met Ala Asn Asn Ser Leu
Ser Ser Gln Phe Ile 1 5 10 444 65 PRT Homo sapiens 444 Ile Ser Gly
Val Leu Ile Phe Asn Leu Ile Ala Ser Ser Trp Val Leu 1 5 10 15 Cys
Phe Pro Leu Cys Asp Leu Ser Cys Gln Lys Thr Leu Arg Ile Phe 20 25
30 Phe Ala Ser Phe Phe His Ala Val Cys Val His Val Ser Cys Thr Ser
35 40 45 Trp Gln Pro Leu Val Leu Phe Ile Lys Trp Trp Val Val Gly
Cys Ser 50 55 60 Pro 65 445 23 PRT Homo sapiens 445 Cys Asp Leu Ser
Cys Gln Lys Thr Leu Arg Ile Phe Phe Ala Ser Phe 1 5 10 15 Phe His
Ala Val Cys Val His 20 446 9 PRT Homo sapiens 446 Glu Leu Ala Ile
Gly Glu Ser Cys Ser 1 5 447 17 PRT Homo sapiens 447 Pro Val Ile Trp
Pro Asp Gly Lys Arg Ile Val Leu Leu Ala Glu Val 1 5 10 15 Ser 448
27 PRT Homo sapiens 448 Phe Tyr Tyr Phe Trp Arg Gln Gly Gly Ser Cys
Phe Val Gln Thr Gly 1 5 10 15 Val Gln Trp Cys Asp His Gly Ser Leu
Gln Leu 20 25 449 10 PRT Homo sapiens 449 Thr Pro Gly Arg Gln Ser
Lys Thr Pro Ser 1 5 10 450 34 PRT Homo sapiens 450 Tyr Phe Ile Ile
Phe Gly Asp Arg Glu Gly Leu Ala Leu Phe Arg Leu 1 5 10 15 Glu Cys
Ser Gly Val Ile Met Ala His Cys Asn Phe Glu Leu Leu Gly 20 25 30
Asp Arg 451 10 PRT Homo sapiens 451 Cys Phe Leu Ser Val Ser Phe Gln
Trp Asn 1 5 10 452 17 PRT Homo sapiens 452 Val Thr Ile Ala Gln Val
Gly Ile Phe Val Cys Phe Val His Cys Cys 1 5 10 15 Thr 453 17 PRT
Homo sapiens 453 Pro Gly Gln Val Pro Ser Lys His Leu Gly Ser Asn
Ala Ser Val Arg 1 5 10 15 Ala 454 22 PRT Homo sapiens 454 Asp Glu
Gly Ala Lys Val Gln Arg Arg Pro Trp Gly Ser Gln Thr His 1 5 10 15
Ser Pro Val Leu Phe Leu 20 455 18 PRT Homo sapiens 455 Leu Thr Arg
Pro Gly Leu Trp Gly Ser Leu Leu Pro Val Gln Gln Gln 1 5 10 15 Arg
Gly 456 15 PRT Homo sapiens 456 Cys Ala Ser Leu Gly Val Leu Arg Ala
Asn Arg Ser Pro Cys Val 1 5 10 15 457 18 PRT Homo sapiens 457 Ser
Trp Leu Glu Val Thr Thr Leu Ser Ala Pro Gly Pro Val Ile Thr 1 5 10
15 Thr Tyr 458 18 PRT Homo sapiens SITE (9) Xaa equals any of the
naturally occurring L-amino acids 458 Pro Gly Gln Trp Val Arg Glu
Ile Xaa Leu Val Gly Arg Ala Val Ala 1 5 10 15 Arg Val 459 16 PRT
Homo sapiens SITE (6) Xaa equals any of the naturally occurring
L-amino acids 459 Leu Thr Trp Pro Pro Xaa Gly Pro Met Gly Thr Val
Trp Pro Gly Phe 1 5 10 15 460 17 PRT Homo sapiens 460 Met Ala Asp
Ile Pro Gly Thr Phe Leu Ala Leu Gly Cys His Gly Gln 1 5 10 15 Arg
461 15 PRT Homo sapiens 461 Val Gly Arg Gly Ser Trp Ala Ser Gly Trp
Thr Asn Gln Ser Ala 1 5 10 15 462 16 PRT Homo sapiens 462 Pro Asp
His Pro Leu Pro Val Gly Leu Leu Glu Ala Trp Arg Val Glu 1 5 10 15
463 142 PRT Homo sapiens SITE (72) Xaa equals any of the naturally
occurring L-amino acids SITE (87) Xaa equals any of the naturally
occurring L-amino acids 463 Trp Gly Ser Gln Thr His Ser Pro Val Leu
Phe Leu Leu Thr Arg Pro 1 5 10 15 Gly Leu Trp Gly Ser Leu Leu Pro
Val Gln Gln Gln Arg Gly Cys Ala 20 25 30 Ser Leu Gly Val Leu Arg
Ala Asn Arg Ser Pro Cys Val Ser Trp Leu 35 40 45 Glu Val Thr Thr
Leu Ser Ala Pro Gly Pro Val Ile Thr Thr Tyr Pro 50 55 60 Gly Gln
Trp Val Arg Glu Ile Xaa Leu Val Gly Arg Ala Val Ala Arg 65 70 75 80
Val Leu Thr Trp Pro Pro Xaa Gly Pro Met Gly Thr Val Trp Pro Gly 85
90 95 Phe Met Ala Asp Ile Pro Gly Thr Phe Leu Ala Leu Gly Cys His
Gly 100 105 110 Gln Arg Val Gly Arg Gly Ser Trp Ala Ser Gly Trp Thr
Asn Gln Xaa 115 120 125 Ser Ala Phe Pro Ala Gly Pro Pro Asp His Pro
Leu Pro Val 130 135 140 464 94 PRT Homo sapiens SITE (84) Xaa
equals any of the naturally occurring L-amino acids 464 Leu Ala Arg
Ala Asp Pro Pro Gly Cys Arg Arg Arg Gly Trp Arg Pro 1 5 10 15 Ser
Ser Ala Glu Leu Gln Leu Arg Leu Leu Thr Pro Thr Phe Glu Gly 20 25
30 Ile Asn Gly Leu Leu Leu Lys Gln His Leu Val Gln Asn Pro Val Arg
35 40 45 Leu Trp Gln Leu Leu Gly Gly Thr Phe Tyr Phe Asn Thr Ser
Arg Leu 50 55 60 Lys Gln Lys Asn Lys Glu Lys Asp Lys Ser Lys Gly
Lys Ala Pro Glu 65 70 75 80 Glu Asp Glu Xaa Glu Arg Arg Arg Arg Glu
Arg Asp Asp Gln 85 90 465 12 PRT Homo sapiens 465 Phe Leu Arg Phe
Trp Cys Thr Cys His Val Ser Ser 1 5 10
* * * * *
References