U.S. patent application number 10/632983 was filed with the patent office on 2004-04-08 for 36 human secreted proteins.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Brewer, Laurie A., Duan, Roxanne, Ebner, Reinhard, Florence, Kimberly, Olsen, Henrik S., Rosen, Craig A., Ruben, Steven M..
Application Number | 20040067518 10/632983 |
Document ID | / |
Family ID | 27536070 |
Filed Date | 2004-04-08 |
United States Patent
Application |
20040067518 |
Kind Code |
A1 |
Olsen, Henrik S. ; et
al. |
April 8, 2004 |
36 human secreted proteins
Abstract
The present invention relates to 36 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 disorders
related to these novel human secreted proteins.
Inventors: |
Olsen, Henrik S.;
(Gaithersburg, MD) ; Ruben, Steven M.;
(Brookeville, MD) ; Rosen, Craig A.;
(Laytonsville, MD) ; Brewer, Laurie A.; (St. Paul,
MN) ; Ebner, Reinhard; (Gaithersburg, MD) ;
Duan, Roxanne; (Bethesda, MD) ; Florence,
Kimberly; (Rockville, MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
27536070 |
Appl. No.: |
10/632983 |
Filed: |
August 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10632983 |
Aug 4, 2003 |
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09716129 |
Nov 17, 2000 |
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6632920 |
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09716129 |
Nov 17, 2000 |
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09382572 |
Aug 25, 1999 |
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09382572 |
Aug 25, 1999 |
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PCT/US99/03939 |
Feb 24, 1999 |
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60076053 |
Feb 26, 1998 |
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60076051 |
Feb 26, 1998 |
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60076054 |
Feb 26, 1998 |
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60076052 |
Feb 26, 1998 |
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60076057 |
Feb 26, 1998 |
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Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
A01K 2217/075 20130101;
A61P 11/06 20180101; A61K 38/00 20130101; A61P 25/00 20180101; A61P
15/00 20180101; C07K 14/47 20130101; A61P 37/06 20180101; A61K
48/00 20130101; A01K 2217/05 20130101; A61P 29/00 20180101; A61P
19/02 20180101; A61P 7/00 20180101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/47 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence at least 95% identical to a sequence
selected from the group consisting of: (a) a polynucleotide
fragment of SEQ ID NO:X or a polynucleotide fragment of the cDNA
sequence included in ATCC Deposit No:Z, which is hybridizable to
SEQ ID NO:X; (b) a polynucleotide encoding a polypeptide fragment
of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA
sequence included in ATCC Deposit No:Z, which is hybridizable to
SEQ ID NO:X; (c) a polynucleotide encoding a polypeptide domain of
SEQ ID NO:Y or a polypeptide domain encoded by the cDNA sequence
included in ATCC Deposit No:Z, which is hybridizable to SEQ ID
NO:X; (d) a polynucleotide encoding a polypeptide epitope of SEQ ID
NO:Y or a polypeptide epitope encoded by the cDNA sequence included
in ATCC Deposit No:Z, which is hybridizable to SEQ ID NO:X; (e) a
polynucleotide encoding a polypeptide of SEQ ID NO:Y or the cDNA
sequence included in ATCC Deposit No:Z, which is hybridizable to
SEQ ID NO:X, having biological activity; (f) a polynucleotide which
is a variant of SEQ ID NO:X; (g) a polynucleotide which is an
allelic variant of SEQ ID NO:X; (b) a polynucleotide which encodes
a species homologue of the SEQ ID NO:Y; (i) a polynucleotide
capable of hybridizing under stringent conditions to any one of the
polynucleotides specified in (a)-(h), wherein said polynucleotide
does not hybridize under stringent conditions to a nucleic acid
molecule having a nucleotide sequence of only A residues or of only
T residues.
2. The isolated nucleic acid molecule of claim 1, wherein the
polynucleotide fragment comprises a nucleotide sequence encoding a
secreted protein.
3. The isolated nucleic acid molecule of claim 1, wherein the
polynucleotide fragment comprises a nucleotide sequence encoding
the sequence identified as SEQ ID NO:Y or the polypeptide encoded
by the cDNA sequence included in ATCC Deposit No:Z, which is
hybridizable to SEQ ID NO:X.
4. The isolated nucleic acid molecule of claim 1, wherein the
polynucleotide fragment comprises the entire nucleotide sequence of
SEQ ID NO:X or the cDNA sequence included in ATCC Deposit No:Z,
which is hybridizable to SEQ ID NO:X.
5. The isolated nucleic acid molecule of claim 2, wherein the
nucleotide sequence comprises sequential nucleotide deletions from
either the C-terminus or the N-terminus.
6. The isolated nucleic acid molecule of claim 3, wherein the
nucleotide sequence comprises sequential nucleotide deletions from
either the C-terminus or the N-terminus.
7. A recombinant vector comprising the isolated nucleic acid
molecule of claim 1.
8. A method of making a recombinant host cell comprising the
isolated nucleic acid molecule of claim 1.
9. A recombinant host cell produced by the method of claim 8.
10. The recombinant host cell of claim 9 comprising vector
sequences.
11. An isolated polypeptide comprising an amino acid sequence at
least 95% identical to a sequence selected from the group
consisting of: (a) a polypeptide fragment of SEQ ID NO:Y or the
encoded sequence included in ATCC Deposit No:Z; (b) a polypeptide
fragment of SEQ ID NO:Y or the encoded sequence included in ATCC
Deposit No:Z, having biological activity; (c) a polypeptide domain
of SEQ ID NO:Y or the encoded sequence included in ATCC Deposit
No:Z; (d) a polypeptide epitope of SEQ ID NO:Y or the encoded
sequence included in ATCC Deposit No:Z; (e) a secreted form of SEQ
ID NO:Y or the encoded sequence included in ATCC Deposit No:Z; (f)
a full length protein of SEQ ID NO:Y or the encoded sequence
included in ATCC Deposit No:Z; (g) a variant of SEQ ID NO:Y; (h) an
allelic variant of SEQ ID NO:Y; or (i) a species homologue of the
SEQ ID NO:Y.
12. The isolated polypeptide of claim 11, wherein the secreted form
or the full length protein comprises sequential amino acid
deletions from either the C-terminus or the N-terminus.
13. An isolated antibody that binds specifically to the isolated
polypeptide of claim 11.
14. A recombinant host cell that expresses the isolated polypeptide
of claim 11.
15. A method of making an isolated polypeptide comprising: (a)
culturing the recombinant host cell of claim 14 under conditions
such that said polypeptide is expressed; and (b) recovering said
polypeptide.
16. The polypeptide produced by claim 15.
17. A method for preventing, treating, or ameliorating a medical
condition, comprising administering to a mammalian subject a
therapeutically effective amount of the polypeptide of claim
11.
18. A method of diagnosing a pathological condition or a
susceptibility to a pathological condition in a subject comprising:
(a) determining the presence or absence of a mutation in the
polynucleotide of claim 1; and (b) diagnosing a pathological
condition or a susceptibility to a pathological condition based on
the presence or absence of said mutation.
19. A method of diagnosing a pathological condition or a
susceptibility to a pathological condition in a subject comprising:
(a) determining the presence or amount of expression of the
polypeptide of claim 11 in a biological sample; and (b) diagnosing
a pathological condition or a susceptibility to a pathological
condition based on the presence or amount of expression of the
polypeptide.
20. A method for identifying a binding partner to the polypeptide
of claim 11 comprising: (a) contacting the polypeptide of claim 11
with a binding partner; and (b) determining whether the binding
partner effects an activity of the polypeptide.
21. The gene corresponding to the cDNA sequence of SEQ ID NO:Y.
22. A method of identifying an activity in a biological assay,
wherein the method comprises: (a) expressing SEQ ID NO:X in a cell;
(b) isolating the supernatant; (c) detecting an activity in a
biological assay; and (d) identifying the protein in the
supernatant having the activity.
23. The product produced by the method of claim 20.
24. A method for preventing, treating, or ameliorating a medical
condition, comprising administering to a mammalian subject a
therapeutically effective amount of the polynucleotide of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/716,129, filed Nov. 17, 2000, which is a continuation of
U.S. application Ser. No. 09/382,572, filed Aug. 25, 1999 (now
abandoned), which is a continuation-in-part of International
Application No. PCT/US99/03939, filed Feb. 24, 1999, which claims
benefit under 35 U.S.C. .sctn. 119(e) of U.S. Provisional
Application Nos. 60/076,053, 60/076,051, 60/076,054, 60/076,052,
and 60/076,057, all filed Feb. 26, 1998. International Application
No. PCT/US99/03939 and U.S. Provisional Application Nos.
60/076,053, 60/076,051, 60/076,054, 60/076,052, and 60/076,057 are
all hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to newly identified polynucleotides
and the polypeptides encoded by these polynucleotides, uses of such
polynucleotides and polypeptides, 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 disorders 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 disorders and
conditions related to the polypeptides and polynucleotides, and
therapeutic methods for treating such disorders and conditions. The
invention further relates to screening methods for identifying
binding partners of the polypeptides.
DETAILED DESCRIPTION
[0008] Definitions
[0009] The following definitions are provided to facilitate
understanding of certain terms used throughout this
specification.
[0010] 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.
[0011] 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.
[0012] 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).
[0013] 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.
[0014] 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.
[0015] 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 sodium 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.
[0016] 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 .mu.g/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).
[0017] 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.
[0018] 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 digo dT as a primer).
[0019] 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.
[0020] 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).)
[0021] "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.
[0022] "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.)
[0023] Polynucleotides and Polypeptides of the Invention
[0024] Features of Protein Encoded by Gene No: 1
[0025] Preferred polypeptides of the invention comprise the
following amino acid sequence: HSSLPHFSSRI (SEQ ID NO: 85).
Polynucleotides encoding these polypeptides are also provided.
[0026] Contact of cells with supernatant expressing the product of
this gene induces the expression of osteocalcin in human SOAS-2
osteoblastic cells. Osteocalcin is a protein attributed to
influencing the bone mineralization of skeletal tissue, and is also
thought to be useful in inhibiting osteoblast function. Thus,
polynucleotides and polypeptides have uses which include, but are
not limited to, inducing the expression of osteocalcin.
[0027] The polypeptide of this gene has been determined to have a
transmembrane domain at about amino acid position 31-48 of the
amino acid sequence referenced in Table 1 for this gene. Moreover,
a cytoplasmic tail encompassing amino acids 49-55 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.
[0028] This gene is expressed primarily in in B-cell lymphoma, and
to a lesser extent in brain tissue.
[0029] Therefore, polynucleotides and polypeptides of the invention
are 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 haemopoietic system and brain,
including cancer. Similarly, polypeptides and antibodies directed
to these polypeptides are 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 central nervous system, expression
of this gene at significantly higher or lower levels is routinely
detected in certain tissues or cell types (e.g., immune, central
nervous system, 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.
[0030] The tissue distribution in B-cell lymphoma and brain tissue
indicates that polynucleotides and polypeptides corresponding to
this gene are useful for the diagnosis, treatment, and/or
prevention of disorders of the haemopoietic system and the central
nervous system, as well as cancers thereof. The expression of this
gene in B-cell lymphoma indicates that this gene product is
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).
[0031] 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 is 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. Alternatively, the tissue distribution in brain
tissue indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection/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. 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.
[0032] 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 is 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 1427 of SEQ ID NO:11, b is an integer
of 15 to 1441, 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.
[0033] Features of Protein Encoded by Gene No: 2
[0034] Preferred polypeptides of the invention comprise the
following amino acid sequence: RDSNGRGDSSLLKFVCPVPLKK (SEQ ID NO:
86). Polynucleotides encoding these polypeptides are also
provided.
[0035] The gene encoding the disclosed cDNA is thought to reside on
chromosome 2.
[0036] Accordingly, polynucleotides related to this invention are
useful as a marker in linkage analysis for chromosome 2.
[0037] This gene is expressed primarily in brain, fetal tissue,
ovarian cancer, colon and hepatocellular tumor and to a lesser
extent in several other tissues and organs.
[0038] Therefore, polynucleotides and polypeptides of the invention
are 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 developmental diseases and cancers.
Similarly, polypeptides and antibodies directed to these
polypeptides are 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 developing systems, expression of this gene at
significantly higher or lower levels is routinely detected in
certain tissues or cell types (e.g., neurological, developing,
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.
[0039] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 49 as residues: Ile-35 to
Lys-40. Polynucleotides encoding said polypeptides are also
provided.
[0040] The tissue distribution in brain and embryonic tissues
indicates that polynucleotides and polypeptides corresponding to
this gene are useful for the diagnosis, treatment, and/or
prevention of disorders of the neural system, as well as cancer.
The tissue distribution in brain tissue indicates that
polynucleotides and polypeptides corresponding to this gene are
useful for the detection/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, 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 diagnosis and
treatment 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. 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.
[0041] 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 is 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 2146 of SEQ ID NO:12, b is an integer
of 15 to 2160, 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.
[0042] Features of Protein Encoded by Gene No: 3
[0043] Preferred polypeptides of the invention comprise the
following amino acid sequence: IPEYTFRRRWFH (SEQ ID NO: 87).
Polynucleotides encoding these polypeptides are also provided.
[0044] This gene is expressed primarily in breast lymph nodes,
T-cells, bone marrow, brain tissue, haemopoietic cells and
cancerous tissues.
[0045] Therefore, polynucleotides and polypeptides of the invention
are 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 and diseases of the haemopoietic and central
nervous systems. Similarly, polypeptides and antibodies directed to
these polypeptides are 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 and central nervous systems, expression of this
gene at significantly higher or lower levels is routinely detected
in certain tissues or cell types (e.g., immune, neurological,
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.
[0046] The tissue distribution in haemopoietic and neural tissues
indicates that polynucleotides and polypeptides corresponding to
this gene are useful for the diagnosis, treatment, and/or
prevention of disorders of the haemopoietic system, the central
nervous system, and cancers thereof, particularly leukemias.
Expression of this gene product in normal and cancerous lymph nodes
indicates a role in the regulation of the proliferation; survival;
differentiation; and/or activation of potentially all hematopoietic
cell lineages, including blood stem cells. This gene product is
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).
[0047] 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 is 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. Alternatively, the tissue distribution in brain
tissue indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection/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. 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.
[0048] 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 is 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 1188 of SEQ ID NO:13, b is an integer
of 15 to 1202, 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.
[0049] Features of Protein Encoded by Gene No: 4
[0050] Preferred polypeptides of the invention comprise the
following amino acid sequence: LCVSMKIEWGRESCEKK (SEQ ID NO: 88).
Polynucleotides encoding these polypeptides are also provided.
[0051] This gene is expressed primarily in leukemia cells and fetal
tissue.
[0052] Therefore, polynucleotides and polypeptides of the invention
are 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 haemopoietic system and cancer.
Similarly, polypeptides and antibodies directed to these
polypeptides are 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, expression of this gene at significantly
higher or lower levels is 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.
[0053] The tissue distribution in leukemia cells indicates that
polynucleotides and polypeptides corresponding to this gene are
useful for the diagnosis, treatment, and/or prevention of disorders
of the haemopoietic system and cancers thereof, particularly
leukemia, as well as cancers of other tissues where expression has
been observed. Furthermore, this gene product is 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).
[0054] 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 is 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. 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.
[0055] 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 are
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 is useful in the detection, treatment, and/or prevention of
degenerative or proliferative conditions and diseases. The protein
is 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.
[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: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 is 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 1540 of SEQ ID NO:14, b is an integer
of 15 to 1554, 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.
[0057] Features of Protein Encoded by Gene No: 5
[0058] Preferred polypeptides of the invention comprise the
following amino acid sequence: RLKTTRAYSSQFWRPEVQNQGVRKV (SEQ ID
NO: 89). Polynucleotides encoding these polypeptides are also
provided.
[0059] This gene is expressed primarily in CD-34 Positive cord
blood cells.
[0060] Therefore, polynucleotides and polypeptides of the invention
are 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 disorders of the immune and haemopoietic
system, in addition to developing cells and tissues. Similarly,
polypeptides and antibodies directed to these polypeptides are
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 haemopoietic system, expression of this gene at significantly
higher or lower levels is routinely detected in certain tissues or
cell types (e.g., immune, 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.
[0061] The tissue distribution in CD34 (+) cells indicates that
polynucleotides and polypeptides corresponding to this gene are
useful for the diagnosis, treatment, and/or prevention of disorders
of the haemopoietic and immune systems, including cancers thereof.
Furthermore, expression of this gene product in CD34 (+) cells
indicates a role in the regulation of the proliferation; survival;
differentiation; and/or activation of potentially all hematopoietic
cell lineages, including blood stem cells. This gene product is
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).
[0062] 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 is 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.
[0063] 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 is 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:15, 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:15, and where b is greater
than or equal to a+14.
[0064] Features of Protein Encoded by Gene No: 6
[0065] Preferred polypeptides of the invention comprise the
following amino acid sequence:
LTLCLPRSLYALPQCPGPHVHPCPALLWDRAGLPLPLPGCIHGRSQVPWHEL- HSPA
AFNQGMMGMCTYPTPPLGRVMLRCGFLTVPRLSQEAWVWVPTVGAGVISYLRR
PPFLPVLCAPTPTLELPRFSVFVKELTLCCLPLSQCPCHSCEPAAGEVGADLCVAG (SEQ ID
NO: 90), LTLCLPRSLYALPQCPGPHVHPCPALLWDRAGLPLPLPGCI (SEQ ID NO: 91),
HGRSQVPWHELHSPAAFNQGMMGMCTYPTPPLGRVMLR (SEQ ID NO: 92),
CGFLTVPRLSQEAWVWVPTVGAGVISYLRRPPFLPVLCAPT (SEQ ID NO: 93), and/or
PTLELPRFSVFVKELTLCCLPLSQCPCHSCEPAAGEVGADLCVAG (SEQ ID NO: 94).
Polynucleotides encoding these polypeptides are also provided.
[0066] This gene is expressed primarily in lymph nodes, thymus,
chronic synovitis tissues, immune cells (e.g., T-cells), ovarian
tumor and to a lesser extent in several other tissues and
organs.
[0067] Therefore, polynucleotides and polypeptides of the invention
are 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 are
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 haemopoietic system, expression of this gene at significantly
higher or lower levels is 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.
[0068] The tissue distribution in immune tissues indicates that
polynucleotides and polypeptides corresponding to this gene are
useful for the diagnosis, treatment, and/or prevention of
inflammatory and haemopoietic disorders. Furthermore, expression of
this gene product in thymus and lymph nodes indicates a role in the
regulation of the proliferation; survival; differentiation; and/or
activation of potentially all hematopoietic cell lineages,
including blood stem cells. This gene product is 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).
[0069] 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 is 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.
[0070] 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 is 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 1043 of SEQ ID NO:16, b is an integer
of 15 to 1057, 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.
[0071] Features of Protein Encoded by Gene No: 7
[0072] Preferred polypeptides of the invention comprise the
following amino acid sequence:
IRHETFRVRGCSISRALSPFPLPFPHPGRSGWSGPEAK (SEQ ID NO: 95).
Polynucleotides encoding these polypeptides are also provided.
[0073] The gene encoding the disclosed cDNA is thought to reside on
chromosome 6. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
6.
[0074] The polypeptide of this gene has been determined to have
transmembrane domains at about amino acid positions 298-332,
139-167, 405-432, 42-74, 361-386, 100-124, 495-517, 189-211 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.
[0075] This gene is expressed primarily in embryonic, placental and
umbilical vein tissues, osteoblasts, T-cell lymphoma, colon, brain,
osteoclastoma, hepatocellular tumor and to a lesser extent in
several other tissues and organs, including cancerous tissues.
[0076] Therefore, polynucleotides and polypeptides of the invention
are 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 developing organs, growth disorders, and
cancer(s). Similarly, polypeptides and antibodies directed to these
polypeptides are 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 and fetal systems, highly vascularized tissues and
cancer, expression of this gene at significantly higher or lower
levels is routinely detected in certain tissues or cell types
(e.g., embryonic, placental, developing, 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.
[0077] The tissue distribution in placental and embryonic tissues
indicates that polynucleotides and polypeptides corresponding to
this gene are useful for the diagnosis, treatment, and/or
prevention of diseases of developing, embryonic, and/or fetal
systems, as well as growth disorders and cancer(s). Furthermore,
the tissue distribution in placental tissue indicates that
polynucleotides and polypeptides corresponding to this gene are
useful for the diagnosis and/or treatment of disorders of the
placenta. Specific expression within the placenta indicates that
this gene product may play a role in the proper establishment and
maintenance of placental function. Alternately, this gene product
is 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.
[0078] Expression of this gene product in a vascular-rich tissue
such as the placenta also indicates that this gene product is
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. It 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. Likewise, 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 diagnosis and treatment 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.
[0079] The tissue distribution in brain indicates polynucleotides
and polypeptides corresponding to this gene are 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.
[0080] Potentially, this gene product is 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.
[0081] 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 is 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 2066 of SEQ ID NO:17, b is an integer
of 15 to 2080, 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.
[0082] Features of Protein Encoded by Gene No: 8
[0083] The translation product of this gene shares some sequence
homology to beta-transducin (see, e.g., Genbank accession number
AAB52945 (AF000265.1); all references available through this
accession are hereby incorporated by reference herein.).
[0084] This gene is expressed primarily in the haemopoietic system,
the central nervous system, and fetal tissue, as well as in
cancer(s), and to a lesser extent in several other tissues and
organs.
[0085] Therefore, polynucleotides and polypeptides of the invention
are 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, haemopoietic diseases, developmental disorders, central
nervous system, and cancer. Similarly, polypeptides and antibodies
directed to these polypeptides are 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,
haemopoietic system, and cancer, expression of this gene at
significantly higher or lower levels is routinely detected in
certain tissues or cell types (e.g., immune, neurological,
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.
[0086] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 55 as residues: Lys-37 to
Ile-45. Polynucleotides encoding said polypeptides are also
provided.
[0087] The tissue distribution in immune and neurological tissues
indicates that polynucleotides and polypeptides corresponding to
this gene are useful for the diagnosis, treatment, and/or
prevention of disorders of the haemopoietic system, the central
nervous system, and cancer(s). This gene product is 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).
[0088] 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 is 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. Alternatively, the tissue distribution
indicates that polynucleotides and polypeptides corresponding to
this gene are useful for the detection/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. 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.
[0089] 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 is 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 588 of SEQ ID NO:18, b is an integer
of 15 to 602, 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.
[0090] Features of Protein Encoded by Gene No: 9
[0091] The polypeptide of this gene has been determined to have a
transmembrane domain at about amino acid position 7-36 of the amino
acid sequence referenced in Table 1 for this gene. Moreover, a
cytoplasmic tail encompassing amino acids 26-83 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.
[0092] This gene is expressed primarily in fetal skin.
[0093] Therefore, polynucleotides and polypeptides of the invention
are 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 and skin abnormalities. Similarly, polypeptides
and antibodies directed to these polypeptides are 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 epithelial structures,
expression of this gene at significantly higher or lower levels is
routinely detected in certain tissues or cell types (e.g.,
epithelial, and 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.
[0094] The tissue distribution in fetal epithelium indicates that
polynucleotides and polypeptides corresponding to this gene are
useful for the study and/or treatment of growth and skin disorders.
Furthermore, the tissue distribution indicates that polynucleotides
and polypeptides corresponding to this gene are useful for the
treatment, diagnosis, and/or prevention of various skin disorders
including 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 fungoides, 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.
Moreover, 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, althletes foot, and
ringworm). 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.
[0095] 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 is 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 615 of SEQ ID NO:19, b is an integer
of 15 to 629, 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.
[0096] Features of Protein Encoded by Gene No: 10
[0097] When tested against sensory neuronal cell lines,
supernatants removed from cells containing this gene activated the
EGR1 assay. Thus, it is likely that this gene activates sensory
neuron cells, and to a lesser extent other neuronal 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.
[0098] The polypeptide of this gene has been determined to have a
transmembrane domain at about amino acid position 101-117 of the
amino acid sequence referenced in Table 1 for this gene. Moreover,
a cytoplasmic tail encompassing amino acids 1-100 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.
[0099] This gene is expressed primarily in epileptic frontal cortex
tissue of the brain, and to a lesser extent in fetal heart
tissue.
[0100] Therefore, polynucleotides and polypeptides of the invention
are 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 cardiovascular abnormalities.
Similarly, polypeptides and antibodies directed to these
polypeptides are 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 vascular systems, expression of this gene
at significantly higher or lower levels is routinely detected in
certain tissues or cell types (e.g., neurological, vascular,
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.
[0101] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 57 as residues: Pro-43 to
Pro-50, Asn-65 to Gly-70. Polynucleotides encoding said
polypeptides are also provided.
[0102] The tissue distribution in frontal cortex tissue of the
brain and fetal heart tissue indicates that polynucleotides and
polypeptides corresponding to this gene are useful for the study
and/or treatment of nervous system and cardiovascular disorders.
The tissue distribution in frontal cortex tissue, in conjunction
with the observed biological activity data, indicates that
polynucleotides and polypeptides corresponding to this gene are
useful for the diagnosis and/or treatment of disorders of the brain
and nervous system. Elevated expression of this gene product within
the frontal cortex of the brain indicates that it is 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. Alternatively, the tissue distribution in
fetal heart tissue indicates that the protein product of this gene
is useful for the diagnosis and treatment of conditions and
pathologies of the cardiovascular system, such as heart disease,
restenosis, atherosclerosis, stoke, angina, thrombosis, and wound
healing. 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: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 is 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 2053 of SEQ ID NO:20, b is an integer
of 15 to 2067, 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.
[0104] Features of Protein Encoded by Gene No: 11
[0105] Preferred polypeptides of the invention comprise the
following amino acid sequence:
1 (SEQ ID NO: 96) PDSRPEARGDHVVRPSRGLRVTGATRSIMGPWGEPELLVWR-
PEAVASEPP VPVGLEVKLGALVLLLVLTLLCSLVPICVLRRPGANHEGSASRQKAL- SLV
SCFAGGVFLATCLLDLLPDYLAAIDEALAALHVTLQFPLQEFILA, (SEQ ID NO: 97)
PDSRPEARGDHVVRPSRGLRVTGATRSIMGPWGEP, (SEQ ID NO: 98)
ELLVWRPEAVASEPPVPVGLEVKLGALVLLLVLTLLC- , (SEQ ID NO: 99)
SLVPICVLRRPGANHEGSASRQKALSLVSCF- AGGVF, and/or (SEQ ID NO: 100)
LATCLLDLLPDYLAAIDEALAALHVTLQFPLQEFILA.
[0106] Polynucleotides encoding these polypeptides are also
provided.
[0107] The gene encoding the disclosed cDNA is thought to reside on
chromosome 1. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
1.
[0108] The translation product of this gene shares sequence
homology with the polypeptide sequence of a novel protein ETI-1,
which has cytostatic activity. ETI-1 is thought to be useful as an
antitumour agent. Based on the sequence similarity, the translation
product of this gene is expected to share biological activities
with cytostatic proteins. Such activities are known in the art and
described elsewhere herein.
[0109] 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 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.
[0110] The polypeptide of this gene has been determined to have a
transmembrane domains at about amino acid positions 63-83, 90-110,
121-139, 161-177 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 IIa membrane proteins.
[0111] The translation product of this gene shares some homology to
CGI-08 protein (see, e.g., Genbank accession number AAD277717
(AF132942.1); all references available through this accession are
hereby incorporated by reference herein.).
[0112] This gene is expressed primarily in fetal tissue, endocrine
organs and cancerous tissues, such as pancreas, lung, endometrial
tumors, as well as neuroblastomas, and to a lesser extent in
various normal and transformed cell types.
[0113] Therefore, polynucleotides and polypeptides of the invention
are 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, hormonal abnormalities and tumors. Similarly,
polypeptides and antibodies directed to these polypeptides are
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 and immune systems, expression of this gene at
significantly higher or lower levels is routinely detected in
certain tissues or cell types (e.g., endocrine, 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.
[0114] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 58 as residues: Lys-17 to
Glu-27, Gln-40 to Gly-47. Polynucleotides encoding said
polypeptides are also provided.
[0115] The tissue distribution in cancerous tissues such as
pancreatic, lung and endometrial tumors, in conjunction with the
observed biological activity data and the homology to a protein
known to have cytostatic activity, indicates that polynucleotides
and polypeptides corresponding to this gene are useful for the
study and/or treatment of tumors of various tissue types, such as
pancreas and endometrium, as well as cancers of other tissues where
expression has been observed. 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.
[0116] 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 are
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 is useful in the detection, treatment, and/or prevention of
degenerative or proliferative conditions and diseases. The protein
is 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. 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.
[0117] 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 is 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 983 of SEQ ID NO:21, b is an integer
of 15 to 997, 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.
[0118] Features of Protein Encoded by Gene No: 12
[0119] Preferred polypeptides of the invention comprise the
following amino acid sequence: KYILSSPLLDSLAEHKNLVWKSFLPRNF (SEQ ID
NO:101). Polynucleotides encoding these polypeptides are also
provided.
[0120] This gene is expressed primarily in leukemic spleen
tissue.
[0121] Therefore, polynucleotides and polypeptides of the invention
are 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, leukemia and other cancers. Similarly, polypeptides and
antibodies directed to these polypeptides are 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 is 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.
[0122] The tissue distribution in leukemic spleen tissue indicates
that polynucleotides and polypeptides corresponding to this gene
are useful for study and treatment of immune disorders and
neoplasias, as well as cancers of other tissues where expression
has been observed. 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.
[0123] 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 is 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 1369 of SEQ ID NO:22, b is an integer
of 15 to 1383, 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.
[0124] Features of Protein Encoded by Gene No: 13
[0125] Preferred polypeptides of the invention comprise the
following amino acid sequence:
YGKVVDLAPLHLDARISLSTLQQQLGQPEKALEALEPMYDPDTLAQDANAAQ- XE
LKLLLHRSTLLFSQGK (SEQ ID NO:102). Polynucleotides encoding these
polypeptides are also provided.
[0126] The gene encoding the disclosed cDNA is thought to reside on
chromosome 2. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
2.
[0127] The translation product of this gene shares sequence
homology to a transcription factor IIIC102 (see, e.g, Genbank
accesssion number AAD41479 (AF133123) and Mol. Cell. Biol.
19:4944-4952, 1999; all references and information available
through this accession and reference are hereby incorporated by
reference herein.) which is thought to functionally interact with
TFIIIB and RNA Polymerase III.
[0128] This gene is expressed primarily in haemopoietic tissues
such as fetal liver/spleen, osteoblasts, tongue, testes, tonsils,
T-cell lymphoma and to a lesser extent in several other normal and
transformed cell types.
[0129] Therefore, polynucleotides and polypeptides of the invention
are 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 growth abnormalities. Similarly,
polypeptides and antibodies directed to these polypeptides are
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 is routinely detected in certain tissues or cell types
(e.g., immune, 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.
[0130] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 60 as residues: Ser-31 to
Arg-36, Ser-44 to Glu-55, Asp-112 to Glu-119, Lys-132 to Asn-139,
Asn-148 to Leu-154, Thr-214 to Leu-220, Gly-260 to Ser-265.
Polynucleotides encoding said polypeptides are also provided.
[0131] The tissue distribution in immune tissues such as fetal
liver/spleen indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the study and/or
treatment of immune disorders. 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. This gene product is 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).
[0132] 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 is 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.
[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 is 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 1499 of SEQ ID NO:23, b is an integer
of 15 to 1513, 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] Preferred polypeptides of the invention comprise the
following amino acid sequence:
DFMETFPDFCLPLAPHYLGKAALWAMCPGRAWAGCGPVLRTSHLGPHSALPS- W
CNICXQAIVGAGRQRGLSEDPTCASHWDTKTGLVPSCGAGKGI (SEQ ID NO:103),
DFMETFPDFCLPLAPHYLGKAALWAMCPGRAWAGCGPVLRTSHL (SEQ ID NO: 104),
and/or GPHSALPSWCNICXQAIVGAGRQRGLSEDPTCASHWDTKTGLVPSCGAGKGI (SEQ ID
NO:105). Polynucleotides encoding these polypeptides are also
provided.
[0136] The gene encoding the disclosed cDNA is thought to reside on
chromosome 7. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
7.
[0137] This gene is expressed primarily in cerebellum tissue, and
to a lesser extent in fetal liver, synovial sarcoma, osteoclastoma,
and glioblastoma.
[0138] Therefore, polynucleotides and polypeptides of the invention
are 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 brain tumors. Similarly,
polypeptides and antibodies directed to these polypeptides are
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 and immune systems, expression of this gene at
significantly higher or lower levels is routinely detected in
certain tissues or cell types (e.g., nervous, 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.
[0139] The tissue distribution and in cerebellum tissue and fetal
liver tissue indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the study and/or
treatment of immune, neurodegenerative and cognitive disorders and
neoplasias. The tissue distribution in cerebellum tissue indicates
that polynucleotides and polypeptides corresponding to this gene
are useful for the detection/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. Alternatively, expression of this
gene product in fetal liver indicates a role in the regulation of
the proliferation; survival; differentiation; and/or activation of
potentially all hematopoietic cell lineages, including blood stem
cells. This gene product is 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).
[0140] 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 is 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.
[0141] 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 is 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 1030 of SEQ ID NO:24, b is an integer
of 15 to 1044, 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.
[0142] Features of Protein Encoded by Gene No: 15
[0143] Preferred polypeptides of the invention comprise the
following amino acid sequence:
RLPQRGQWAWVLQDALGIAFCLYMLKTIRLPTFKACTLLLLVLFLYDIFFVF- ITPF
LTKSGSSIMVEVATGPSDSATREKLPMVLKVPRLNSSPLALCDRPFSLLGFGDILVP
GLLVAYCHRFDIQVQSSRVYFVACTIAYGVGLLVTFVALALMQRGQPALLYLVP
CTLVTSCAVALWRRELGVFWTGSGFAKVLPPSPWAPAPADGPQPPKDSATPLSPQ
PPSEEPATSPWPAEQSPKSRTSEEMGAGAPMREPGSPAESEGRDQAQPSPVTQPGA SA (SEQ ID
NO:106), RLPQRGQWAWVLQDALGIAFCLYMLKTIRLPTFKACTLLLLVL (SEQ ID NO:
107), FLYDIFFVFITPFLTKSGSSIMVEVATGPSDSATREKLPMVLKV (SEQ ID NO:
108), PRLNSSPLALCDRPFSLLGFGDILVPGLLVAYCHRFDIQVQSSR (SEQ ID NO:
109), VYFVACTIAYGVGLLVTFVALALMQRGQPALLYLVPCTLVTSC (SEQ ID NO:110),
AVALWRRELGVFWTGSGFAKVLPPSPWAPAPADGPQPPKD (SEQ ID NO:111),
SATPLSPQPPSEEPATSPWPAEQSPKSRTSEEMGAGAPMRE (SEQ ID NO: 112), and/or
PGSPAESEGRDQAQPSPVTQPGASA (SEQ ID NO:113). Polynucleotides encoding
these polypeptides are also provided.
[0144] The gene encoding the disclosed cDNA is thought to reside on
chromosome 19. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
19.
[0145] The polypeptide of this gene has been determined to have
transmembrane domains at about amino acid positions 217-268,
293-316, 241-268, 169-194 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.
[0146] This gene is expressed primarily in eosinophils, brain,
placental tissue, bone marrow, tumors of the pancreas and testes
and to a lesser extent in various other normal and transformed
tissues.
[0147] Therefore, polynucleotides and polypeptides of the invention
are 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, placental, neurological and cancerous abnormalities.
Similarly, polypeptides and antibodies directed to these
polypeptides are 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 placenta and central nervous system, expression of this gene at
significantly higher or lower levels is routinely detected in
certain tissues or cell types (e.g., neurological, placental, lung,
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.
[0148] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 62 as residues: Gly-127 to
Asp-134, Gly-194 to Arg-201, His-205 to Glu-217, Pro-275 to
Arg-280, Pro-287 to Gln-294, Arg-315 to Arg-325. Polynucleotides
encoding said polypeptides are also provided.
[0149] The tissue distribution in brain tissue indicates that
polynucleotides and polypeptides corresponding to this gene are
useful for the study and/or treatment of central nervous system
disorders and neoplasias. Furthermore, the tissue distribution in
brain tissue indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection/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.
[0150] Alternatively, the tissue distribution in placental tissue
indicates that polynucleotides and polypeptides corresponding to
this gene are useful for the diagnosis and/or treatment of
disorders of the placenta. Specific expression within the placenta
indicates that this gene product may play a role in the proper
establishment and maintenance of placental function. Alternately,
this gene product is 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 this gene product is 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. It 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. The tissue
distribution in immune tissue (bone marrow and eosinophils)
indicates polynucleotides and polypeptides corresponding to this
gene are 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 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 is 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).
[0151] Since the gene is expressed in cells of lymphoid origin, the
natural gene product is involved in immune functions. Therefore it
is also 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.
[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: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 is 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 2561 of SEQ ID NO:25, b is an integer
of 15 to 2575, 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.
[0153] Features of Protein Encoded by Gene No: 16
[0154] Preferred polypeptides of the invention comprise the
following amino acid sequence: ESSGLPALGPRRRPWEQRWSDPITLK (SEQ ID
NO:114), and/or
LTLALDEIRLLKKDLGLIEMKKTDSEKRFGSVSFGRSCRLIPHALASWLQTLILCFC CRIC (SEQ
ID NO:115). Polynucleotides encoding these polypeptides are also
provided.
[0155] The gene encoding the disclosed cDNA is thought to reside on
chromosome 12. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
12.
[0156] The translation product of this gene shares sequence
homology to a type II membrane protein (see, e.g., Genbank
accession number BAA76498 (AB015631.1); all references available
through this accession are hereby incorporated by reference
herein.).
[0157] This gene is expressed primarily in placental and embryonic
tissues, PHA activated T-cells, uterus, osteoarthritis, lung,
adipose tissue and to a lesser extent in many other tissues.
[0158] Therefore, polynucleotides and polypeptides of the invention
are 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 and immune disorders, osteoarthritis,
respiratory and endocrine disorders. Similarly, polypeptides and
antibodies directed to these polypeptides are 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 and immune
systems, expression of this gene at significantly higher or lower
levels is routinely detected in certain tissues or cell types
(e.g., developing, 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.
[0159] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 63 as residues: Arg-21 to
Leu-26. Polynucleotides encoding said polypeptides are also
provided.
[0160] The tissue distribution in placental and embryonic tissues,
as well as in T-cells, indicates that polynucleotides and
polypeptides corresponding to this gene are useful for the study
and/or diagnosis of growth and immune disorders. Furthermore, the
tissue distribution in placental and embryonic tissues indicates
that polynucleotides and polypeptides corresponding to this gene
are useful for the diagnosis and/or treatment of disorders of the
placenta. Specific expression within the placenta indicates that
this gene product may play a role in the proper establishment and
maintenance of placental function. Alternately, this gene product
is 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.
[0161] Expression of this gene product in a vascular-rich tissue
such as the placenta also indicates that this gene product is
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. It 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. 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 diagnosis and treatment 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 this gene product in
T-cells indicates a role in the regulation of the proliferation;
survival; differentiation; and/or activation of potentially all
hematopoietic cell lineages, including blood stem cells. This gene
product is 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).
[0162] 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 is 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. The tissue distribution in adipose tissue
indicates that polynucleotides and polypeptides corresponding to
this gene are useful for the treatment of obesity and other
metabolic and endocrine conditions or disorders. Furthermore, the
protein product of 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. 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.
[0163] 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 is 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 704 of SEQ ID NO:26, b is an integer
of 15 to 718, 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.
[0164] Features of Protein Encoded by Gene No: 17
[0165] Preferred polypeptides of the invention comprise the
following amino acid sequence: GRPTRPVMAIQSLHPCPSELCCRACVXFYHWA
(SEQ ID NO:116). Polynucleotides encoding these polypeptides are
also provided.
[0166] The polypeptide of this gene has been determined to have a
transmembrane domain at about amino acid position 1-24 of the amino
acid sequence referenced in Table 1 for this gene. Moreover, a
cytoplasmic tail encompassing amino acids 21-62 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
[0167] This gene is expressed primarily in immune cells (e.g.,
activated monocytes, primary dendritic cells, and GM-CSF stimulated
macrophages), testes, ovary tumor and to a lesser extent in
activated T-cells and adult brain.
[0168] Therefore, polynucleotides and polypeptides of the invention
are 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 regulation and immune disorders, neural and
endocrine disorders, cancer. Similarly, polypeptides and antibodies
directed to these polypeptides are 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 is 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.
[0169] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 64 as residues: Thr-24 to
Gly-42, Glu-53 to Gly-58. Polynucleotides encoding said
polypeptides are also provided.
[0170] The tissue distribution in immune cells and leukocytes such
as monocytes, macrophage, primary dendritic cells, and T-cells,
indicates that polynucleotides and polypeptides corresponding to
this gene are useful for the study, diagnosis and/or treatment of
immune dysfuntion and other immune disorders. This gene product is
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).
[0171] 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 is 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. Expression of this gene product in T cells and
primary dendritic cells also strongly indicates a role for this
protein in immune function and immune surveillance.
[0172] The tissue distribution in brain indicates polynucleotides
and polypeptides corresponding to this gene are 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.
[0173] Potentially, this gene product is involved in synapse
formation, neurotransmission, learning, cognition, homeostasis, or
neuronal differentiation or survival. The tissue distribution in
testes tissue indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis and/or
treatment of male reproductive and endocrine disorders. It may also
prove to be valuable in the diagnosis and treatment of testicular
cancer, as well as cancers of other tissues where expression 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.
[0174] 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 is 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 640 of SEQ ID NO:27, b is an integer
of 15 to 654, 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.
[0175] Features of Protein Encoded by Gene No: 18
[0176] The translation product of this gene shares sequence
homology with NADH ubiquinone oxidoreductase B115 complex (see,
e.g., Genbank accession number AAD05421.1 (AF044957); all
references available through this accession are hereby incorporated
by reference herein.) which is thought to be important in cellular
respiration and metabolism.
[0177] Preferred polypeptides of the invention comprise the
following amino acid sequence: NSKNTRNERSFLKLFRNIHDIPLTVLENK (SEQ
ID NO:117). Polynucleotides encoding these polypeptides are also
provided.
[0178] This gene is expressed primarily in fetal liver/spleen,
infant brain tissues, spinal cord, prostate, multiple sclerosis and
to a lesser extent in fetal kidney and adipose tissues.
[0179] Therefore, polynucleotides and polypeptides of the invention
are 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 and developmental disorders, and adult metabolic
disorders. Similarly, polypeptides and antibodies directed to these
polypeptides are 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 developmental and metabolic systems, expression of this gene at
significantly higher or lower levels is routinely detected in
certain tissues or cell types (e.g., immune, renal, neurological,
metabolic, 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.
[0180] The tissue distribution in a number of fetal and infant
tissues, as well as the homology to NADH ubiquinone oxireductase
B15 complex, indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the study, diagnosis
and/or treatment of various developmental and growth disorders, and
adult metabolic disorders, such as Tay-Sach's Disease,
phenylkenonuria, galactosemia, hyperlipidemias, porphyrias, and
Hurler's syndrome. Furthermore, the tissue distribution in adipose
tissue indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the treatment of obesity
and other metabolic and endocrine conditions or disorders.
[0181] The tissue distribution in brain indicates polynucleotides
and polypeptides corresponding to this gene are 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.
[0182] Potentially, this gene product is involved in synapse
formation, neurotransmission, learning, cognition, homeostasis, or
neuronal differentiation or survival. Furthermore, the protein
product of 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.
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: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 is 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:28, 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:28, and where b is greater
than or equal to a+14.
[0184] Features of Protein Encoded by Gene No: 19
[0185] The translation product of this gene shares sequence
similarity with neuronal olfactomedin-related ER localized protein
isolated from the rat (Genbank accession no.
gi.vertline.442370).
[0186] Preferred polypeptides of the invention comprise the
following amino acid sequence: PRVRGEGNRCWTQGALCHRM (SEQ ID
NO:118). Polynucleotides encoding these polypeptides are also
provided.
[0187] In another embodiment, polypeptides comprising the amino
acid sequence of the open reading frame upstream of the predicted
signal peptide are contemplated by the present invention.
Specifically, polypeptides of the invention comprise the following
amino acid sequence:
PRVRGEGNRCWTQGALCHRMMVALRGASALLVLFLAAFLPPPQCTQDPAMVHY
IYQRFRVLEQGLEKCTQ
ATRAYIQEFQEFSKNISVMLGRCQTYTSEYKSAVGNLALRVERAQREIDYIQYLRE
ADECIESEDKTLAE MLLQEAEEEKKIRTLLNASCDNMLMGIKSLK KKMMDTHGSWMKDAVYNSPK
VYLLIGSRNNTVWEFAN
IRAFMEDNTKPAPRKQILTLSWQGTGQVIYKGFLFFHNQATSNEIIKYNLQKRTVE
DRMLLPGGVGRALV
YQHSPSTYIDLAVDEHGLWAIHSGPGTHSHLVLTKIEPGTLGVEHSWDTPCRSQD
AEASFLLCGVLYVVY STGGQGPHRITCIYDPLGTISEEDLPNLFFPKRPRSHSM
NPRDKQLYAWNEGN QIIYKLQTKRKLTL K (SEQ ID NO:119). Polynucleotides
encoding these polypeptides are also provided.
[0188] The gene encoding the disclosed cDNA is thought to reside on
chromosome 11. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
11.
[0189] This gene is expressed primarily in synovial sarcoma tissue,
and to a lesser extent in fetal dura mater, adipose, and hogkins'
lymphoma tissues.
[0190] Therefore, polynucleotides and polypeptides of the invention
are 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, cancers and other metabolic disorders. Similarly,
polypeptides and antibodies directed to these polypeptides are
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
metabolic system, expression of this gene at significantly higher
or lower levels is routinely detected in certain tissues or cell
types (e.g., musculo-skeletal, metabolic, 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.
[0191] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 66 as residues: Tyr-76 to
Lys-81, Glu-92 to Asp-99, Glu-125 to Ile-132, Asp-197 to Arg-204,
Gln-241 to Glu-246, Pro-310 to Ala-316, Thr-332 to His-338, Pro-361
to Ser-366, Leu-392 to Thr-399. Polynucleotides encoding said
polypeptides are also provided.
[0192] The tissue distribution in musculo-skeletal and metabolic
tissues, and the sequence similarity to neuronal
olfactomedin-related ER localized protein, indicates that
polynucleotides and polypeptides corresponding to this gene are
useful for the study, diagnosis and/or treatment of cancers and
other metabolic disorders. Representative uses are described here
and elsewhere herein. Furthermore, the tissue distribution
indicates that polynucleotides and polypeptides corresponding to
this gene are 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 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. 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 diagnosis and
treatment 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. 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.
[0193] 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 is 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 2006 of SEQ ID NO:29, b is an integer
of 15 to 2020, 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.
[0194] Features of Protein Encoded by Gene No: 20
[0195] The translation product of this gene shares sequence
homology with tissue plasminogen activator gene enhancer element
which is thought to play a role in blood clotting.
[0196] Preferred polypeptides of the invention comprise the
following amino acid sequence: FPCICLSGLLDLLIWRPFSEELTKTFG (SEQ ID
NO:120). Polynucleotides encoding these polypeptides are also
provided.
[0197] In another embodiment, polypeptides comprising the amino
acid sequence of the open reading frame upstream of the predicted
signal peptide are contemplated by the present invention.
Specifically, polypeptides of the invention comprise the following
amino acid sequence: FPCICLSGLLDLLIWRPFSEELTKTFGMVSLLSS
YLLLLELLSKRSLFLQWYLFFGLQCCSSFLCRKNESQC- FTRLKERSAGSV (SEQ ID
NO:121). Polynucleotides encoding these polypeptides are also
provided.
[0198] This gene is expressed primarily in rhabdomyosarcoma and to
a lesser extent in lymphocytic leukemia.
[0199] Therefore, polynucleotides and polypeptides of the invention
are 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, cancers and clotting diseases and/or disorders, such as
hemophelia. Similarly, polypeptides and antibodies directed to
these polypeptides are 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 metabolic and blood clotting systems, expression of this gene
at significantly higher or lower levels is routinely detected in
certain tissues or cell types (e.g., hematopoietic, developmental,
metabolic disorders, and cancerous and wounded tissues) or bodily
fluids (e.g., 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.
[0200] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 67 as residues: Cys-38 to
Cys-45, Leu-49 to Ala-54. Polynucleotides encoding said
polypeptides are also provided.
[0201] The homology to the conserved tissue plasminogen activator
gene enhancer element indicates that the protein product of this
gene is useful for the study, treatment and diagnosis of cancers
and blood clotting disorders. Representative uses are described
here and elsewhere herein. The protein is useful for the treatment,
detection, and/or prevention of metabolic and developmental
disorders which include, but are not limited to diabetes, or
placental aberrations. The protein is also useful for the treating
and ameliorating cardiovascular conditions such as coronary artery
disease, atherosclerosis, or arteriosclerosis. Moreover, the
protein is useful in the detection, treatment, and/or prevention of
vascular conditions, which include, but are not limited to,
microvascular disease, vascular leak syndrome, aneurysm, stroke,
atherosclerosis, arteriosclerosis, or embolism. Alternatively, the
expression within lymphocytic leukemia cells, combined with its
homology to tissue plasminogen activator indicates this gene
product is 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).
[0202] Since the gene is expressed in cells of lymphoid origin, the
natural gene product is involved in immune functions. Therefore it
is 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 (i.e. through
modulating integrin function). 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.
[0203] 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 is 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 1069 of SEQ ID NO:30, b is an integer
of 15 to 1083, 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.
[0204] Features of Protein Encoded by Gene No: 21
[0205] The gene encoding the disclosed cDNA is believed to reside
on chromosome 12. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
12.
[0206] This gene is expressed primarily in fetal liver, fetal
spleen and infant brain and to a lesser extent in human epididymus
tissue.
[0207] Therefore, polynucleotides and polypeptides of the invention
are 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, hematopoietic, immune, CNS, and/or
reproductive diseases and/or disorders. Similarly, polypeptides and
antibodies directed to these polypeptides are 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, developmental,
and central nervous systems, expression of this gene at
significantly higher or lower levels is routinely detected in
certain tissues or cell types (e.g., developmental, hematopoietic,
immune, CNS, reproductive, and cancerous and wounded tissues) or
bodily fluids (e.g., lymph, serum, plasma, amniotic fluid, urine,
bile, 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.
[0208] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 68 as residues: Pro-29 to
Pro-35. Polynucleotides encoding said polypeptides are also
provided.
[0209] The tissue distribution in fetal liver, fetal spleen and
infant brain indicates that the protein product of this gene is
useful for the study, diagnosis and treatment of reproductive,
developmental, and CNS disorders. Moreover, the 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.
[0210] 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 are
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 is useful in the detection, treatment, and/or prevention of
degenerative or proliferative conditions and diseases. The protein
is 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.
[0211] 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 is 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 1566 of SEQ ID NO:31, b is an integer
of 15 to 1580, 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.
[0212] Features of Protein Encoded by Gene No: 22
[0213] The translation product of this gene shares sequence
homology with human beta-casein which is thought to be important as
a milk component. This sequence encodes a homolog of the human milk
protein, beta-casein. This sequence can be used in the production
of recombinant human beta-casein for use as a constituent of infant
formulae. Beta-casein is a phosphorylated protein which is present
in milk of several species including humans in which it is the
major casein subunit. This protein is believed to enhance calcium
adsorbtion by chelating calcium to its phosphorylated residues and
thereby keeping it in an adsorbable form. Human beta-casein is
easily digestible by newborn infants and the digestive products
have been found to play an important part in calcium uptake, and
thus in the mineralisation of the skeleton. A digestion product of
human beta-casein has been found to have opiod activity and is
involved in the sleeping patterns of breast-fed infants.
[0214] Preferred polypeptides of the invention comprise the
following amino acid sequence: KDTCTRMXIAALFTIAKIWNQPKX (SEQ ID
NO:122), RHMHTYVYCGTIHNSKDLEPTQMXDXIKKMWHLYTTKYYAAIKKD (SEQ ID
NO:123), RKCGTYIPRNTMQP (SEQ ID NO:124), and/or
KRTEFMSFXGTWMKLEAIILSKLTQEEKTKHLMF- SLISGS (SEQ ID NO:125).
Polynucleotides encoding these polypeptides are also provided.
[0215] This gene is expressed primarily in adult heart.
[0216] Therefore, polynucleotides and polypeptides of the invention
are 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 and/or vascular disorders and diseases,
particularly deficiency in milk production, atherosclerosis, or
coronary artery disease. Similarly, polypeptides and antibodies
directed to these polypeptides are 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 and
mammary glands, expression of this gene at significantly higher or
lower levels is routinely detected in certain tissues or cell types
(e.g., reproductive, vascular, and cancerous and wounded tissues)
or bodily fluids (e.g., 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.
[0217] The homology to human beta-casein indicates that the protein
product of this gene is useful as a constituent of infant formulae.
Representative uses are described here and elsewhere herein.
Moreover, the protein is useful for the detection, treatment,
and/or prevention of aberrant mammary gland function and diseases,
in addition to a possible use in the developmental of novel protein
expression for the isolation of heterologous proteins using the
beta-casein enhancer, promoter, and encoding nucleotide sequences.
Alternatively, the expression in adult heart indicates the protein
is useful in the detection, treatment, and/or prevention of a
variety of vascular disorders and condtions, which include, but are
not limited to miscrovascular disease, vascular leak syndrome,
aneurysm, stroke, embolism, thrombosis, 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.
[0218] 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 is 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 782 of SEQ ID NO:32, b is an integer
of 15 to 796, 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.
[0219] Features of Protein Encoded by Gene No: 23
[0220] The translation product of this gene shares sequence
homology with a rat protein, dendrin, which is distributed in the
dendrites of neurons of the forebrain.
[0221] Preferred polypeptides of the invention comprise the
following amino acid sequence: PKSDTSPASSR (SEQ ID NO:126),
PKSDTSPASSRLCWD (SEQ ID NO: 127),
YVPSFLPKATGSIPSRKGGVGSEKPEVPLQTYKEIVHCCEEQVLTLATEQTYAVEG
ETPINRLSLLLSGR
VRVSQDGQFLHYIFPYQFMDSPEWESLQPSEEGVFQVTLTAETSCSYISWPRKSLH
LLLTKERYISCLFS
ALLGYDISEKLYTLNDKLFAKFGLRFDIRLPSLYHVLGPTAADAGPESEKGDEEVC
EPAVSPPQATPTSL
QQTPPCSTPPATTNFPAPPTRARLSRPDSGILASRIPLQSYSQVISRGQAPLAPTHTP EL (SEQ
ID NO:128), ATGSIPSRKGGVGSEKPEVPL (SEQ ID NO:129),
IVHCCEEQVLTLATEQTYAVEGETP (SEQ ID NO:130), QDGQFLHYIFPYQFMDSPEWESL
(SEQ ID NO:131), TLTAETSCSYISWPRKSLHLLLT (SEQ ID NO:132),
DISEKLYTLNDKLFAKFGLRFDIRL (SEQ ID NO:133),
SLYHVLGPTAADAGPESEKGDEEVCE (SEQ ID NO:134), and/or
TTNFPAPPTRARLSRPDSGILASRIPLQ (SEQ ID NO:135). Polynucleotides
encoding these polypeptides are also provided.
[0222] In another embodiment, polypeptides comprising the amino
acid sequence of the open reading frame upstream of the predicted
signal peptide are contemplated by the present invention.
Specifically, polypeptides of the invention comprise the following
amino acid sequence: PKSDTSPASSR
LCWDMTSRRSSTLSMTSSLLSLGCALTSAFPASTMSWVPLLQMLDQSPRRVMRK
SVSQLCPLLRPHPPLS
SKHPLVLPLQLPPTFLHLLPGPGCPGQTVAYWLLEFLSRATLKLYPGDRPLWLQPT
RLNFKDHWTIFSVA SAALFCVHRMATDRHASFPTHWKAHRQGERGHRRCQHCRYSKDLK (SEQ
ID NO:136). Polynucleotides encoding these polypeptides are also
provided.
[0223] The gene encoding the disclosed cDNA is believed to reside
on chromosome 3. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
3.
[0224] This gene is expressed primarily in the heart and and to a
lesser extent, in tonsil.
[0225] Therefore, polynucleotides and polypeptides of the invention
are 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 diseases and/or disorders and
conditions, particularly congestive heart failure. Similarly,
polypeptides and antibodies directed to these polypeptides are
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 and neural sysytems, expression of this gene at
significantly higher or lower levels is routinely detected in
certain tissues or cell types (e.g., cardiovascular, vascular,
immune, hematopoletic, 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.
[0226] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 70 as residues: Met-1 to
Ser-7, Asp-41 to Met-48, Pro-61 to Ser-67, Pro-121 to Trp-130,
His-161 to Lys-181. Polynucleotides encoding said polypeptides are
also provided.
[0227] The homology to dendrin indicates that the protein product
of this gene is useful for the detection, treament, and/or
prevention of neuronal diseases such as memory loss. Representative
uses are described in the "Regeneration" and "Hyperproliferative
Disorders" sections below, in Example 11, 15, and 18, and elsewhere
herein. Moreover, polynucleotides and polypeptides corresponding to
this gene are useful for the detection, treatment, and/or
prevention of neurodegenerative disease states, behavioral
disorders, or inflammatory conditions which include, but are not
limited to 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 gerie product
in regions of the brain indicates it plays a role in normal neural
function.
[0228] Potentially, this gene product is involved in synapse
formation, neurotransmission, learning, cognition, homeostasis, or
neuronal differentiation or survival. Alternatively, the expression
in heart tissue indicates the protein is useful in the detection,
treatment, and/or prevention of a variety of vascular disorders and
condtions, 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.
[0229] 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 is 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:33, 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:33, and where b is greater
than or equal to a+14.
[0230] Features of Protein Encoded by Gene No: 24
[0231] Preferred polypeptides of the invention comprise the
following amino acid sequence: YFSHGICSHA (SEQ ID NO:137).
Polynucleotides encoding these polypeptides are also provided.
[0232] This gene is expressed primarily in peripheral mononuclear
cells, and to a lesser extent in other white blood cells such as
neutrophils and lymphocytes from lymphomas.
[0233] Therefore, polynucleotides and polypeptides of the invention
are 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 or immune disorders and conditions,
particularly leukemia and lymphomas. Similarly, polypeptides and
antibodies directed to these polypeptides are 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 is routinely
detected in certain tissues or cell types (e.g., hematopoietic,
immune, 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.
[0234] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 71 as residues: Leu-41 to
Pro-48. Polynucleotides encoding said polypeptides are also
provided.
[0235] The tissue distribution in blood cells indicates that the
protein product of this gene is useful for diagnosis and treatment
of blood diseases such as leukemia and 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. Moreover, the tissue distribution
indicates polynucleotides and polypeptides corresponding to this
gene are 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 hematopoletic lineages. The uses include
bone marrow cell ex-vivo culture, bone marrow transplantation, bone
marrow reconstitution, radiotherapy or chemotherapy of
neoplasia.
[0236] 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.
[0237] 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 is 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 1050 of SEQ ID NO:34, b is an integer
of 15 to 1064, 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.
[0238] Features of Protein Encoded by Gene No: 25
[0239] The translation product of this gene was shown to have
homology to a conserved ubiquitin thiolesterase (deubiquitinating
enzyme) (See Genbank Accession No.
sp.vertline.Q09738.vertline.UBPA_SCHPO) which is thought to be
important in protein metabolism, processing, and/or regulation.
[0240] Preferred polypeptides of the invention comprise the
following amino acid sequence:
NSEDISQTRQELGLCISQRCLSDRKKSRRSGVWVRACT MQFMKHVFPRLISPRRP (SEQ ID
NO:138), PTRHFCGTSSCLTGTAVRCRAPAP VWSVRCPHCFRSSDAWVDPGIPDRYLQAYLL
(SEQ ID NO:139), GEAMDAEXAVAPPGCSHLGSFKVD-
NWKQNLRAIYQCFVWSGTAEARKRKAKSC ICHVCGVHLNRLHSCLY
CVFFGCFTKKHIHEHAKAKRHNLAID- LMYGGIYCFLCQDYIYDKDMEIIAKEEQR
KAWKMQGVGEKFSTW EPTKRELELLKHNPKRRKITSNCTIGLR-
GLINLGNTCFMNCIVQALTHTPLLRDFFL SDRHRCEMQSPSS
CLVCEMSSLFQEFGRVGRPGNSGPVPAGVP- SIVSPE (SEQ ID NO:140),
VAPPGCSHLGSFKVDNWKQNLRAI (SEQ ID NO:141), TAEARKRKAKSCICHVCGVHLNR
(SEQ ID NO:142), FTKKHIHEHAKAKRHNLAIDLMY (SEQ ID NO:143),
YDKDMEIIAKEEQRKAWKMQG (SEQ ID NO:144), ELLKHNPKRRKITSNCTIGLRGLIN-
LGN (SEQ ID NO:145), GNTCFMNCIVQALTHTPLLRDFFLSD (SEQ ID NO:146),
and/or EFGRVGRPGNSGPVPAGVPS (SEQ ID NO:147). Polynucleotides
encoding these polypeptides are also provided.
[0241] This gene is expressed primarily in L428 cells, and to a
lesser extent, in osteoblasts.
[0242] Therefore, polynucleotides and polypeptides of the invention
are 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 diseases and/or disorders, particularly
osteosarcoma or other bone diseases. Similarly, polypeptides and
antibodies directed to these polypeptides are 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 system, expression
of this gene at significantly higher or lower levels is routinely
detected in certain tissues or cell types (e.g., skeletal,
metabolic, developmental, and cancerous and wounded tissues) or
bodily fluids (e.g., 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.
[0243] The tissue distribution in osteoblasts indicates that the
protein product of this gene is useful for diagnosis and treatment
of bone disorders such as osteoporosis. Representative uses are
described in the "Regeneration" and "Hyperproliferative Disorders"
sections below, in Example 11, 15, and 18, and elsewhere herein.
Moreover, the homology to the ubiquitin thiolesterase indicates the
protein can be used to arrest proliferation of haematopoietic cells
for treating or preventing e.g. cancer especially leukaemias or
lymphomas (in addition to other proliferative conditions in other
cells or cell types). The protein can also be used to stimulate
haematopoietic cell proliferation e.g. to produce blood cells for
replacing blood cell depletion due to disease or condition e.g.
immune suppression from AIDS or therapy such as chemotherapy or
dialysis. The protein may also be used to suppress the immune
system e.g. during organ or cell transplantation. The
polynucleotides of the present invention can be used to transform
cells for screening agents which inhibit DUB enzyme activity.
Furthermore, the protein is useful for the detection and treatment
of disorders and conditions affecting the skeletal system, in
particular bone cancer, 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 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.
[0244] 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 is 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 741 of SEQ ID NO:35, b is an integer
of 15 to 755, 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.
[0245] Features of Protein Encoded by Gene No: 26
[0246] The polypeptide of this gene has been determined to have a
transmembrane domain at about amino acid position 27-43 of the
amino acid sequence referenced in Table 1 for this gene. Moreover,
a cytoplasmic tail encompassing amino acids 44 to 74 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.
[0247] In another embodiment, polypeptides comprising the amino
acid sequence of the open reading frame upstream of the predicted
signal peptide are contemplated by the present invention.
Specifically, polypeptides of the invention comprise the following
amino acid sequence: NSEDISQTRQELGLCISQRCLSDRKKSRRSGVWVRACT
MQFMKHVFPRLISPRRPMVQFEVIFLLFGLCFSS- SSSRLVGSQVENFSPTPCIFQAFR
CSSLAHSMSLS (SEQ ID NO:148). Polynucleotides encoding these
polypeptides are also provided.
[0248] This gene is expressed primarily in cells from Hodgkin's
lymphoma.
[0249] Therefore, polynucleotides and polypeptides of the invention
are 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 or immune diseases and condtions,
particularly Hodgkin's lymphoma. Similarly, polypeptides and
antibodies directed to these polypeptides are 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 is routinely
detected in certain tissues or cell types (e.g., hematopoietic,
immune, and cancerous and wounded tissues) or bodily fluids (e.g.,
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.
[0250] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 73 as residues: Arg-47 to
His-52, Gly-64 to Leu-71. Polynucleotides encoding said
polypeptides are also provided.
[0251] The tissue distribution in Hodgkin's lymphoma indicates that
the protein product of this gene is useful for diagnosis and
treatment of hematopoietic and immune disorders and conditions.
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.
[0252] 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.
[0253] 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 is 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 590 of SEQ ID NO:36, b is an integer
of 15 to 604, 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.
[0254] Features of Protein Encoded by Gene No: 27
[0255] The translation product of this gene shares sequence
homology with `AP2` tumor-specific DNA, which is thought to be
important in detecting insertions or deletions in DNA sequences in
tumor cells. Such mutations are markers of cancer and can be used
in the diagnosis of cancer, esp. colorectal, stomach or pancreatic
tumours.
[0256] Preferred polypeptides of the invention comprise the
following amino acid sequence: AFPWPTS (SEQ ID NO:149).
Polynucleotides encoding these polypeptides are also provided.
[0257] This gene is expressed primarily in breast lymph nodes from
a breast cancer patient, and to a lesser extent in adrenal gland
and tonsils.
[0258] Therefore, polynucleotides and polypeptides of the invention
are 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 and/or immune diseases and disorders,
particularly cancers, such as breast cancer, colorectal cancer, and
pancreatic cancer. Similarly, polypeptides and antibodies directed
to these polypeptides are 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 and digestive system, expression of
this gene at significantly higher or lower levels is routinely
detected in certain tissues or cell types (e.g., reproductive,
immune, and cancerous and wounded tissues) or bodily fluids (e.g.,
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] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 74 as residues: Ile-25 to
Trp-30. Polynucleotides encoding said polypeptides are also
provided.
[0260] The tissue distribution breast lymph nodes, combined with
the homology to `AP2 tumor-specific DNA sequence` indicates that
the protein product or the DNA sequnce of this gene is useful for
detecting insertions or deletions in DNA sequences in tumor cells.
Such mutations are markers of cancer and can be used in the
diagnosis of cancer, esp. colorectal, stomach and pancreatic
tumours. Representative uses are described in the
"Hyperproliferative Disorders" and "Regeneration" sections below
and elsewhere herein. Expression within 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 and treatment of cancer and other proliferative
disorders. Similarly, developmental tissues rely on decisions
involving cell differentiation and/or apoptosis in pattern
formation.
[0261] 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).
Therefore, the polynucleotides and polypeptides of the present
invention are 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 is useful in the detection,
treatment, and/or prevention of degenerative or proliferative
conditions and diseases, particularly those of the immune and/or
hemaopoietic systems. 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.
[0262] 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 is 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 798 of SEQ ID NO:37, b is an integer
of 15 to 812, 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.
[0263] Features of Protein Encoded by Gene No: 28
[0264] The translation product of this gene was shown to have
homology to a conserved reverse transcriptase homolog which may
implicate this protein as playing a role in various DNA processing
and modulatory activities (See Genbank Accession
No.bbs.vertline.80120).
[0265] Preferred polypeptides of the invention comprise the
following amino acid sequence: ESNFFYPYDSQLALLSSVTCSAS (SEQ ID
NO:150), KLKMFAFYVQVLNQSKSIFVYSRNLIFFIHMIVSWPSFLQLPAVHQCHQSSVHICG
VSGLFPSSNYQCL SLCQNHTVLIITTL (SEQ ID NO:151),
SILNVIPNLSKQSFEEFDRLILKYMQKSKSKRIA KILLSNKKTCPTKY (SEQ ID NO: 152),
LPQILRWLKYHQSVWGKQTPVTLHYLTLDLIQEFTP (SEQ ID NO:153), and/or
IFVYSRNLIFFIHMIVSWPSFLQLPAVHQCHQS (SEQ ID NO:154). Polynucleotides
encoding these polypeptides are also provided.
[0266] This gene is expressed primarily in B-cell lymphoma.
[0267] Therefore, polynucleotides and polypeptides of the invention
are 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 or hematopoietic disorders and condtions,
particularly B-cell lymphoma. Similarly, polypeptides and
antibodies directed to these polypeptides are 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 is routinely
detected in certain tissues or cell types (e.g., immune,
hematopoietic, developmental, 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.
[0268] The tissue distribution in B-cell lymphoma tissue indicates
that the protein product of this gene is useful for diagnosis and
treatment of immune or hematopoietic 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. 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 is 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).
[0269] Since the gene is expressed in cells of lymphoid origin, the
natural gene product is involved in immune functions. Therefore it
is 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,
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.
In addition, the homology to a reverse transcriptase protein
indicates the protein may play a vital role in DNA metabolism,
processing, and/or regulatory roles which would have utility in
treating or detecting developmental and proliferative disorders and
conditions. The protein is useful in, but not limited to, the
inhibition or enhancement of apoptosis, transcription, translation,
trafficing, and other cellular functions. 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.
[0270] 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 is 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 1135 of SEQ ID NO:38, b is an integer
of 15 to 1149, 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.
[0271] Features of Protein Encoded by Gene No: 29
[0272] Preferred polypeptides of the invention comprise the
following amino acid sequence:
PTGNDLVYVFPCLLSVFSRMEEPSVFCLFFPLSILISSASRTFPGTQQVFSI- VHGVTD
VSAKKVQSQGRM
TSTGLDFNLLPAWFPSPTSLQPTEDLFQTGSLSRSFFCSKAFSSSPLSPGGSPN- ALTS
VKEHLVSPAFLA SHSCTAESFPRVDVIHAVPIAWIPAPLHPIQLINSWFFFFFFFF (SEQ ID
NO:155), DLVYVFPCLLSVFSRMEEPSVFCL (SEQ ID NO:156),
ISSASRTFPGTQQVFSIVHGVTDV (SEQ ID NO:157), FNLLPAWFPSPTSLQPTEDL (SEQ
ID NO:158), FCSKAFSSSPLSPGGSPNALTSVKE (SEQ ID NO:159), and/or
TAESFPRVDVIHAVPIAWIPAPL (SEQ ID NO:160). Polynucleotides encoding
these polypeptides are also provided.
[0273] This gene is expressed primarily in B-cell lymphoma.
[0274] Therefore, polynucleotides and polypeptides of the invention
are 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 or hematopoietic disorders, particularly B-cell
lymphoma. Similarly, polypeptides and antibodies directed to these
polypeptides are 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 is 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.
[0275] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 76 as residues: Glu-16 to
Arg-21. Polynucleotides encoding said polypeptides are also
provided.
[0276] The tissue distribution in B-cell lymphoma tissue indicates
that the protein product of this gene is useful for diagnosis and
treatment of immune or hematopoietic disorders and conditions.
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. This gene product is 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).
[0277] Since the gene is expressed in cells of lymphoid origin, the
natural gene product is involved in immune functions. Therefore it
is 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,
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.
[0278] 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 is 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 1073 of SEQ ID NO:39, b is an integer
of 15 to 1087, 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.
[0279] Features of Protein Encoded by Gene No: 30
[0280] The translation product of this gene was shown to have
homology to histone family of proteins. Based on the sequence
similarity, the translation product of this gene is expected to
share biological activities with histone proteins, in addition to
other proteins having DNA binding activity. Such activities are
known in the art and described elsewhere herein.
[0281] Preferred polypeptides of the invention comprise the
following amino acid sequence: FSFLKPLCAPRAPWLWLPPSSKSRVHVGPGDFRS
(SEQ ID NO:161),
VCGTGGLEPNLAWVRVDNGSFPSSSPSVPLEHPGCGCLLHPRAESMLGQETSDPC
PGAASGFVFPQWAG
LGLLVHLYPSLSYAALACCVSGLYSLPFTQALGNQPSFXQERQRRSMPLLWAS (SEQ ID
NO:162), HAGRKTVK (SEQ ID NO:163),
SFYAKMPMERKALEMVEKCLDKYFQHLCDDLEVFAAHAGRKTVKPED- LELLMR RQGLVTDQ
(SEQ ID NO:164), PMERKALEMVEKCLDKYFQ (SEQ ID NO:165),
EVFAAHAGRKTVKPEDLELLMR (SEQ ID NO:166),
SFPSSSPSVPLEHPGCGCLLHPRAESMLGQE (SEQ ID NO:167), and/or
YPSLSYAALACCVSGLYSLPFTQALGN (SEQ ID NO:168). Polynucleotides
encoding these polypeptides are also provided.
[0282] In another embodiment, polypeptides comprising the amino
acid sequence of the open reading frame upstream of the predicted
signal peptide are contemplated by the present invention.
Specifically, polypeptides of the invention comprise the following
amino acid sequence:
2 (SEQ ID NO: 169) FSFLKPLCAPRAPWLWLPPSSKSRVHVGPGDFRSMSWCCL-
WLCLSSVGR TGSAGPSLPFSELCSLGLLRLRPVFSPLHSGPGKPAQFLAGEAEEVN- AFA
LGFLSTSSGVSGEDEVEPLH
DGVEEAEKKMEEEGVSVSEMEATGAQGPSRVEEAEGHTEVTEAEGSQGTA
EADGPGASSGDEDASGRAAS PESASSTPESLQARRHHQFLEPAPAPGAAVLSSEPA-
EPLLVRHPPRPRTT GPRPRQDPHKAGLSHYVKLF
SFYAKMPMERKALEMVEKCLDKYFQHLCDDLEVFAAHAGRKTVKPEDLEL
LMRRQGLVTDQVSLHVLVERHLPLEYRQLLIPCAYSGNSVFPAQ.
[0283] Polynucleotides encoding these polypeptides are also
provided.
[0284] The gene encoding the disclosed cDNA is believed to reside
on chromosome 16. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
16.
[0285] This gene is expressed primarily in epididymus and, to a
lesser extent, in placenta and fetal liver/spleen.
[0286] Therefore, polynucleotides and polypeptides of the invention
are 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, hematopoietic, and/or immune disorders
and conditions, particularly male infertility. Similarly,
polypeptides and antibodies directed to these polypeptides are
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 is routinely detected in certain tissues or
cell types (e.g., reproductive, hematopoietic, immune, and
cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum, plasma, urine, seminal fluid, 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.
[0287] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 77 as residues: His-44 to
Pro-50, Glu-90 to Glu-96, Gln-111 to Glu-117, Ser-143 to Gly-151,
Ala-154 to Leu-166, Pro-199 to Ala-216, Gly-264 to Asp-272.
Polynucleotides encoding said polypeptides are also provided.
[0288] The tissue distribution in epididimus and placental tissue
indicates that the protein product of this gene is useful for
diagnosis and treatment of various reproductive disorders and
conditions which include, but are not limited to infertility.
Representative uses are described in the "Hyperproliferative
Disorders" and "Regeneration" sections below and elsewhere herein.
Moreover, the protein is useful for the treatment and diagnosis of
conditions concerning proper testicular function (e.g. endocrine
function, sperm maturation), as well as cancer. Therefore, this
gene product is useful in the treatment of impotence, and could be
useful as a contraceptive, either directly or indirectly.
Considering the homology to histone proteins, this gene product is
also useful in assays designed to identify binding agents, as such
agents (antagonists) are 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 is expressed,
particularly at low levels, in other tissues of the body.
Therefore, this gene product is 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.
[0289] 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 is 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 1262 of SEQ ID NO:40, b is an integer
of 15 to 1276, 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.
[0290] Features of Protein Encoded by Gene No: 31
[0291] The translation product of this gene was shown to have
homology to serine/threonine phosphatase proteins. Based on the
sequence similarity, the translation product of this gene is
expected to share biological activities with proteins involved in
signal transduction and/or the cell cycle. Such activities are
known in the art and described elsewhere herein.
[0292] Preferred polypeptides of the invention comprise the
following amino acid sequence: APGGVNSEGRGQHLPPPXL AVCLKLHL (SEQ ID
NO:170). Polynucleotides encoding these polypeptides are also
provided.
[0293] In another embodiment, polypeptides comprising the amino
acid sequence of the open reading frame upstream of the predicted
signal peptide are contemplated by the present invention.
Specifically, polypeptides of the invention comprise the following
amino acid sequence: APGGVNSEGRGQHLPPPXL
AVCLKLHLMSLPIPWLSLPPCPILGQPAGLLLWLFRPFSQCCQCPWEGRASLR- HPN
GPSGCREAEAWPQR
SLLRQQLQQAHPLPTLPTPERLPEQMLFPSSSSKPFSLLSLTIWARLVGRLTNRI- CPV
PPGSVASSMSLQ AGRCGNPVVLPQPMPPGLLCMNECSLVPGLGRGQVNSRV (SEQ ID
NO:171). Polynucleotides encoding these polypeptides are also
provided.
[0294] The gene encoding the disclosed cDNA is believed to reside
on chromosome 9. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
9.
[0295] This gene is expressed primarily in breast and infant brain
and, to a lesser extent, in neutrophils, fetal spleen, and
activated monocytes.
[0296] Therefore, polynucleotides and polypeptides of the invention
are 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, developmental, immune, and/or
hematopoletic disorders, particularly breast cancer. Similarly,
polypeptides and antibodies directed to these polypeptides are
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 metabolic systems, expression of this gene at significantly
higher or lower levels is routinely detected in certain tissues or
cell types (e.g., reproductive, developmental, immune,
hematopoietic, and cancerous and wounded tissues) or bodily fluids
(e.g., lymph, serum, plasma, urine, amniotic fluid, breast milk,
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.
[0297] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 78 as residues: Pro-37 to
Ala-42, Leu-44 to Cys-53, Glu-57 to Leu-65, Pro-79 to Pro-85.
Polynucleotides encoding said polypeptides are also provided.
[0298] The tissue distribution in breast tissue and neutrophils
indicates that the protein product of this gene is useful for
diagnosis and treatment of certain cancers including those of
reproductive and immune cell origin. 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. Similarly, polynucleotides and polypeptides
corresponding to this gene are 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. The uses include bone marrow cell ex-vivo
culture, bone marrow transplantation, bone marrow reconstitution,
radiotherapy or chemotherapy of neoplasia.
[0299] 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.
Moreover, the expression within various fetal and infant tissues
indicates this protein may play a role in the regulation of
cellular division, and may show utility in the diagnosis and
treatment of cancer and other proliferative disorders. Similarly,
developmental tissues rely on decisions involving cell
differentiation and/or apoptosis in pattern formation.
[0300] 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).
Therefore, the polynucleotides and polypeptides of the present
invention are 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 is useful in the detection,
treatment, and/or prevention of degenerative or proliferative
conditions and 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.
[0301] 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 is 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 2069 of SEQ ID NO:41, b is an integer
of 15 to 2083, 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.
[0302] Features of Protein Encoded by Gene No: 32
[0303] Preferred polypeptides of the invention comprise the
following amino acid sequence:
NSAEPAWVPVCARGGGAGCGRRRGRRFCAAGAVPAAERGGENGS (SEQ ID NO:172),
SLVPALK EVVVLWRRQMVLYLVWAFIPESWLNSLGLTYWPQKYWAVALPVYLLIAIVIGYV
LLFGINMMSTSPLDSI HTITDNYAKNQQQKKYQEEAIPALRDISISEVNQMFFLAAKELYTKN
(SEQ ID NO: 173), MVLYLVWAFIPESWLNSLGLTYWPQKYW (SEQ ID NO:174),
YWAVALPVYLLIAIVIGYVLLFGIN (SEQ ID NO:175), and/or
QQQKKYQEEAIPALRDISISEV (SEQ ID NO:176). Polynucleotides encoding
these polypeptides are also provided.
[0304] In another embodiment, polypeptides comprising the amino
acid sequence of the open reading frame upstream of the predicted
signal peptide are contemplated by the present invention.
Specifically, polypeptides of the invention comprise the following
amino acid sequence:
NSAEPAWVPVCARGGGAGCGRRRGRRFCAAGAVPAAERGGENGSMVSRSTSLT
LIVFLFHRLSKAPGKMV ENSPSPLPERAIYGFVLFLSSQFGFKNLKGSRVC (SEQ ID
NO:177). Polynucleotides encoding these polypeptides are also
provided.
[0305] This gene is expressed primarily in fetal liver/spleen.
[0306] Therefore, polynucleotides and polypeptides of the invention
are 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, hematopoietic, and/or developmental disorders
and conditions, particularly haemopoiesis, leukemias, and
lymphomas. Similarly, polypeptides and antibodies directed to these
polypeptides are 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, expression of this gene at significantly
higher or lower levels is routinely detected in certain tissues or
cell types (e.g., immune, hematopoietic, developmental, and
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.
[0307] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 79 as residues: Asn-28 to
Pro-34. Polynucleotides encoding said polypeptides are also
provided.
[0308] The tissue distribution in fetal liver/spleen indicates that
the protein product of this gene is useful for diagnosis and
treatment of disorders involving haemopoiesis. 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, polynucleotides and polypeptides
corresponding to this gene are 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. The uses include bone marrow cell ex-vivo
culture, bone marrow transplantation, bone marrow reconstitution,
radiotherapy or chemotherapy of neoplasia.
[0309] 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.
Expression within fetal tissue indicates this protein may play a
role in the regulation of cellular division, and may show utility
in the diagnosis and treatment of cancer and other proliferative
disorders. Similarly, developmental tissues rely on decisions
involving cell differentiation and/or apoptosis in pattern
formation.
[0310] 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).
Therefore, the polynucleotides and polypeptides of the present
invention are 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 is useful in the detection,
treatment, and/or prevention of degenerative or proliferative
conditions and 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.
[0311] 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 is 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 1002 of SEQ ID NO:42, b is an integer
of 15 to 1016, 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.
[0312] Features of Protein Encoded by Gene No: 33
[0313] Preferred polypeptides of the invention comprise the
following amino acid sequence: LSPRLFDAGILLWGASVNVTJWEVRXAQSSAS
(SEQ ID NO:178). Polynucleotides encoding these polypeptides are
also provided. n another embodiment, polypeptides comprising the
amino acid sequence of the open reading frame upstream of the
predicted signal peptide are contemplated by the present invention.
Specifically, polypeptides of the invention comprise the following
amino acid sequence: LSPRLFDAGILLWGASVNVTIWEVRXAQS-
SASMLPSAWGPLQVASFFLLSFXFCFL
SSSPHLGRQETHXVVLEDDEGAPCPAEDELALQDNGFLSKNEVLRT- RCLGSRSGS
ASATPPTTSGTARAARPPSQC (SEQ ID NO:179). Polynucleotides encoding
these polypeptides are also provided.
[0314] The gene encoding the disclosed cDNA is believed to reside
on chromosome 10. Accordingly, polynucleotides related to this
invention are useful as a marker in linkage analysis for chromosome
10.
[0315] This gene is expressed primarily in Jurkat T-cell G1 phase
and to a lesser extent in 12 Week Old Early Stage Human.
[0316] Therefore, polynucleotides and polypeptides of the invention
are 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, hematopoietic, and/or developmental diseases
and conditions. Similarly, polypeptides and antibodies directed to
these polypeptides are 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 is routinely detected in certain tissues or cell
types (e.g., immune, hematopoietic, developmental, and 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.
[0317] The tissue distribution in Jurkat cells indicates that the
protein product of this gene is useful for diagnosis and treatment
of certain immune disorders, especially involving Jurkat 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. Moreover, this gene product
is 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).
[0318] Since the gene is expressed in cells of lymphoid origin, the
natural gene product is involved in immune functions. Therefore it
is 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,
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. Expression within embryonic 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 and treatment of cancer and other
proliferative disorders. Similarly, developmental tissues rely on
decisions involving cell differentiation and/or apoptosis in
pattern formation.
[0319] 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).
Therefore, the polynucleotides and polypeptides of the present
invention are 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 is useful in the detection,
treatment, and/or prevention of degenerative or proliferative
conditions and 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.
[0320] 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 is 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 2183 of SEQ ID NO:43, b is an integer
of 15 to 2197, 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.
[0321] Features of Protein Encoded by Gene No: 34
[0322] Preferred polypeptides of the invention comprise the
following amino acid sequence: NLTSDPRPLALPPPCGDFIKVTSFSPGLETHT
(SEQ ID NO:180),
EQQRLRDRETQTGXDSRAKTQRGEDGESERGRWRLREGEDGDSEREEDGDSER
WRLRSMESQRGEDGHSGGWRVRRMETHRKGRMESQERLETGEGIETQ
RGEDGDSEGGRWRLKEDGNPGERRT- EMRQRLGEAG (SEQ ID NO:181),
GHGVAGXCLPQPLLPPSPPDYDERSHLHDTFTQMTHALQELAAAQG- SFEVAFPDA
AEKMKKVFTQLKEAQACIPPCEGLQEFARRFLCSGCYSRVCDLPLDCPVQDVTVT
RGDQAMFSCIVNFQLPKEEITYSWKFAGGGLRTQDLSYFRDMPRAEGYLARIRPA
QLTHRGTFSCVIKQDQRPLARLYFFLNVTGRPRGRRQSCRPRSGKCCAGRRGMPS (SEQ ID
NO:182), and/or GDHPHFISVLGKVQREGRRGPEGQAEGQTERNSQRRKAQRP (SEQ ID
NO:183). Polynucleotides encoding these polypeptides are also
provided.
[0323] In another embodiment, polypeptides comprising the amino
acid sequence of the open reading frame upstream of the predicted
signal peptide are contemplated by the present invention.
Specifically, polypeptides of the invention comprise the following
amino acid sequence: NLTSDPRPLALPPPCGDFIKVTSFSPGLETHTMALLA
LASAVPSALLALAVFRVPAWACLLCFTTYSERLRI- CQMFVGMRSPSLKSVRRPSRP
PSRASLTPKSVRRP STLHQCPGEGAEGGQERPRGSG (SEQ ID NO:184).
Polynucleotides encoding these polypeptides are also provided.
[0324] This gene is expressed primarily in uterine cancer and, to a
lesser extent, in macrophage and adult testis.
[0325] Therefore, polynucleotides and polypeptides of the invention
are 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 conditions, particularly
uterine and testicular cancer. Similarly, polypeptides and
antibodies directed to these polypeptides are 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 and immune
systems, expression of this gene at significantly higher or lower
levels is 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, seminal 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.
[0326] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 81 as residues: Ser-54 to
Arg-64, Lys-70 to Thr-77, Gly-88 to Ser-96. Polynucleotides
encoding said polypeptides are also provided.
[0327] The tissue distribution in uterine and testis tissue
indicates that the protein product of this gene is useful for
diagnosis and treatment of certain cancers, including uterine
cancer. Representative uses are described in the
"Hyperproliferative Disorders" and "Regeneration" sections below
and elsewhere herein. Moreover, the tissue distribution in testis
tissue indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the treatment and
diagnosis of conditions concerning proper testicular function (e.g.
endocrine function, sperm maturation), as well as cancer.
Therefore, this gene product is 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) are 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 is expressed,
particularly at low levels, in other tissues of the body.
Therefore, this gene product is 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.
[0328] 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 is 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 1985 of SEQ ID NO:44, b is an integer
of 15 to 1999, 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.
[0329] Features of Protein Encoded by Gene No: 35
[0330] Preferred polypeptides of the invention comprise the
following amino acid sequence: MLVYQNQAQFSSN (SEQ ID NO:185).
Polynucleotides encoding these polypeptides are also provided.
[0331] In another embodiment, polypeptides comprising the amino
acid sequence of the open reading frame upstream of the predicted
signal peptide are contemplated by the present invention.
Specifically, polypeptides of the invention comprise the following
amino acid sequence: MLVYQNQAQFSSNMWLNFSDVHTYLSSIALLC
FCLSGVLCCICNNSVFHIQQYILIIITFPLVVI (SEQ ID NO:186). Polynucleotides
encoding these polypeptides are also provided.
[0332] This gene is expressed primarily in the cerebellum.
[0333] Therefore, polynucleotides and polypeptides of the invention
are 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. Similarly, polypeptides
and antibodies directed to these polypeptides are 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 is
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.
[0334] The tissue distribution in cerebellum indicates that the
protein product of this gene is useful for the diagnosis and
treatment of neurodegenerative disorders. Moreover, polynucleotides
and polypeptides corresponding to this gene are 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.
[0335] Potentially, this gene product is 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.
[0336] 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 is 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 1505 of SEQ ID NO:45, b is an integer
of 15 to 1519, 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.
[0337] Features of Protein Encoded by Gene No: 36
[0338] This gene is expressed primarily in cerebellum and whole
brain and to a lesser extent in pineal gland, fetal liver/spleen,
and ovary.
[0339] Therefore, polynucleotides and polypeptides of the invention
are 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 are
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 is routinely detected in certain tissues or
cell types (e.g., neural, endocrine, 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.
[0340] Preferred polypeptides of the present invention comprise
immunogenic epitopes shown in SEQ ID NO: 83 as residues: His-3 to
Phe-11, Pro-35 to Arg-40. Polynucleotides encoding said
polypeptides are also provided.
[0341] The tissue distribution in cerebellum and whole brain
indicates that the protein product of this gene is useful for the
diagnosis and treatment of neurodegenerative disorders involving
the cerebellum or other brain regions. Moreover, polynucleotides
and polypeptides corresponding to this gene are 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.
[0342] Potentially, this gene product is 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.
[0343] 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 is 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 1175 of SEQ ID NO:46, b is an integer
of 15 to 1189, 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.
3 5' NT NT of AA First Last 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 HCE1Q30 209626 Uni-ZAP XR 11 1441 1 1441 137
137 48 1 30 31 55 Feb. 12, 1998 2 HAGBP70 209626 Uni-ZAP XR 12 2160
163 2142 360 360 49 1 34 35 40 Feb. 12, 1998 3 HBCAY27 209626
Uni-ZAP XR 13 1202 447 1202 580 580 50 1 18 19 93 Feb. 12, 1998 4
HCACU58 209626 Uni-ZAP XR 14 1554 1 1554 137 137 51 1 30 31 83 Feb.
12, 1998 5 HCWLD74 209626 ZAP Express 15 1540 1 1540 138 138 52 1
21 22 65 Feb. 12, 1998 6 HDPFP29 209626 pCMVSport 16 1057 1 1057
293 293 53 1 30 31 52 Feb. 12, 1998 3.0 7 HDPPH47 209626 pCMVSport
17 2080 105 2080 116 116 54 1 35 36 540 Feb. 12, 1998 3.0 8 HFEAN33
209626 Uni-ZAP XR 18 602 1 602 25 25 55 1 26 27 177 Feb. 12, 1998 9
HFEAT91 209626 Uni-ZAP XR 19 629 1 629 21 21 56 1 32 33 83 Feb. 12,
1998 10 HFPAO71 209626 Uni-ZAP XR 20 2067 364 2067 414 414 57 1 33
34 131 Feb. 12, 1998 11 HLWAA17 209626 pCMVSport 21 997 246 997 436
436 58 1 15 16 187 Feb. 12, 1998 3.0 12 HLYCQ18 209626 pSport1 22
1383 1 1383 126 126 59 1 34 35 40 Feb. 12, 1998 13 HOSFG70 209626
Uni-ZAP XR 23 1513 203 1513 257 257 60 1 23 24 338 Feb. 12, 1998 14
HSSAJ29 209626 Uni-ZAP XR 24 1044 1 1044 103 103 61 1 25 26 47 Feb.
12, 1998 15 HUSIF44 209626 pSport1 25 2575 1 2575 27 27 62 1 25 26
336 Feb. 12, 1998 15 HUSIF44 209626 pSport1 47 2584 1 2584 29 29 84
1 28 29 132 Feb. 12, 1998 16 H6EDX46 209626 Uni-ZAP XR 26 718 1 718
128 128 63 1 20 21 84 Feb. 12, 1998 17 HABAG37 209626 pSport1 27
654 1 639 97 97 64 1 31 32 62 Feb. 12, 1998 18 HACBD91 209626
Uni-ZAP XR 28 1445 1 1445 117 117 65 1 42 43 49 Feb. 12, 1998 19
HADEH21 209626 pSport1 29 2020 1 2020 61 61 66 1 25 26 401 Feb. 12,
1998 20 HAGHD57 209626 Uni-ZAP XR 30 1083 97 1083 402 402 67 1 24
25 57 Feb. 12, 1998 21 HAGHR69 209626 Uni-ZAP XR 31 1580 1 1450 11
11 68 1 21 22 72 Feb. 12, 1998 22 HAHDB16 209626 Uni-ZAP XR 32 796
1 796 93 93 69 1 20 21 50 Feb. 12, 1998 23 HAHDR32 209626 Uni-ZAP
XR 33 1256 365 1256 435 435 70 1 25 26 181 Feb. 12, 1998 24 HAJAW93
209626 pCMVSport 34 1064 45 1064 218 218 71 1 30 31 48 Feb. 12,
1998 3.0 25 HAJBR69 209626 pCMVSport 35 755 1 755 262 262 72 1 19
20 53 Feb. 12, 1998 3.0 26 HAMGO32 209626 pCMVSport 36 604 1 604
119 119 73 1 22 23 74 Feb. 12, 1998 3.0 27 HATBR65 209626 Uni-ZAP
XR 37 812 1 812 252 252 74 1 16 17 64 Feb. 12, 1998 28 HBJLD29
209626 Uni-ZAP XR 38 1149 1 1149 142 142 75 1 29 30 43 Feb. 12,
1998 29 HBJNB13 209626 Uni-ZAP XR 39 1087 1 1087 12 12 76 1 17 18
52 Feb. 12, 1998 30 HCE2F54 209626 Uni-ZAP XR 40 1276 19 1256 166
166 77 1 19 20 319 Feb. 12, 1998 31 HCE3C52 209626 Uni-ZAP XR 41
2083 119 2074 236 236 78 1 33 34 171 Feb. 12, 1998 32 HCEEA88
209626 Uni-ZAP XR 42 1016 1 1016 134 134 79 1 23 24 60 Feb. 12,
1998 33 HCEFE96 209626 Uni-ZAP XR 43 2197 1 2197 121 121 80 1 26 27
100 Feb. 12, 1998 34 HCEIF12 209626 Uni-ZAP XR 44 1999 1 1999 198
198 81 1 15 16 97 Feb. 12, 1998 35 HCEOR67 209626 Uni-ZAP XR 45
1519 1 1519 155 155 82 1 27 28 52 Feb. 12, 1998 36 HCEVB76 209626
Uni-ZAP XR 46 1189 1 1189 105 105 83 1 37 38 40 Feb. 12, 1998
[0344] 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.
[0345] 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.
[0346] "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."
[0347] 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.
[0348] 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."
[0349] 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.
[0350] 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).
[0351] 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.
[0352] 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.
[0353] 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.
[0354] 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.
[0355] 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.
[0356] 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.
[0357] 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.
[0358] Signal Sequences
[0359] 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 th4e 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.
[0360] 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.
[0361] 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.
[0362] 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.
[0363] 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.
[0364] Polynucleotide and Polypeptide Variants
[0365] 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 the deposited
clone.
[0366] The present invention also encompasses variants of the
polypeptide sequence disclosed in SEQ ID NO:Y and/or encoded by the
deposited clone.
[0367] "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.
[0368] 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 nuclotide 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.
[0369] 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).
[0370] 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 fragement specified as described
herein.
[0371] 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 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 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 identity
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.
[0372] 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 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.
[0373] 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/alignement 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
sequnce are manually corrected for. No other manual corrections are
to made for the purposes of the present invention.
[0374] 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.
[0375] 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.
[0376] 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.
[0377] 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.
[0378] 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 m-RNA
to those preferred by a bacterial host such as E. coli).
[0379] 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.
[0380] 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).)
[0381] 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.
[0382] 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.
[0383] 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.
[0384] 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.
[0385] 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.
[0386] 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.
[0387] 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.
[0388] 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).)
[0389] 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 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.
[0390] Polynucleotide and Polypeptide Fragments
[0391] The present invention is also directed to polynucleotide
fragments of the polynucleotides of the invention.
[0392] 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 portioon
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.
[0393] 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.
[0394] 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.
[0395] 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.
[0396] 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.
[0397] 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.
[0398] Epitopes & Antibodies
[0399] The present invention is also directed to polypeptide
fragments comprising, or alternatively consisting of, an epitope of
the polypeptide sequence shown in SEQ ID NO:Y, or the polypeptide
sequence encoded by the cDNA contained in a deposited clone.
Polynucleotides encoding these epitopes (such as, for example, the
sequence disclosed in SEQ ID NO:X) are also encompassed by the
invention, is the nucleotide sequences of the complementary strand
of the polynucleotides encoding these epitopes. And polynucleotides
which hybridize to the complementary strand under stringent
hybridization conditions or lower stringency conditions.
[0400] In the present invention, "epitopes" refer to polypeptide
fragments having antigenic or immunogenic activity in an animal,
especially in a human. A preferred embodiment of the present
invention relates to a polypeptide fragment comprising an epitope,
as well as the polynucleotide encoding this fragment. A region of a
protein molecule to which an antibody can bind is defined as an
"antigenic epitope." In contrast, an "immunogenic epitope" is
defined as a part of a protein that elicits an antibody response.
(See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA
81:3998-4002 (1983).)
[0401] Fragments which function as epitopes may be produced by any
conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad.
Sci. USA 82:5131-5135 (1985) further described in U.S. Pat. No.
4,631,211.)
[0402] 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
15, at least 20, at least 25, 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. Antigenic epitopes are useful, for
example, to raise antibodies, including monoclonal antibodies, that
specifically bind the epitope. (See, for instance, Wilson et al.,
Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666
(1983).)
[0403] 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).) A preferred immunogenic
epitope includes the secreted protein. The immunogenic epitopes may
be presented together with a carrier protein, such as an albumin,
to an animal system (such as rabbit or mouse) or, if it is long
enough (at least about 25 amino acids), 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.)
[0404] 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 of 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.gs of peptide or carrier protein
and Freund's adjuvant. 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.
[0405] As one of skill in the art will appreciate, and discussed
above, the polypeptides of the present invention comprising an
immunogenic or antigenic epitope can be fused to heterologous
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, any
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.
This has been shown, e.g., 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., EPA 0,394,827; Traunecker et al.,
Nature, 331:84-86 (1988). Fusion proteins that have a
disulfide-linked dimeric structure due to the IgG portion can also
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 to aid in detection and
purification of the expressed polypeptide.
[0406] 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 corresponding to SEQ ID NO:Y thereby
effectively generating 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, 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 corresponding to SEQ ID
NO:X and corresponding polypeptides may be achieved by DNA
shuffling. DNA shuffling involves the assembly of two or more DNA
segments into a desired molecule corresponding to SEQ ID NO:X
polynucleotides of the invention by homologous, or site-specific,
recombination. In another embodiment, polynucleotides corresponding
to SEQ ID NO:X and corresponding 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 coding polynucleotide
corresponding to SEQ ID NO:X, or the polypeptide encoded thereby
may be recombined with one or more components, motifs, sections,
parts, domains, fragments, etc. of one or more heterologous
molecules.
[0407] Antibodies
[0408] The present invention further relates to antibodies and
T-cell antigen receptors (TCR) which specifically bind the
polypeptides of the present invention. The antibodies of the
present invention include IgG (including IgG1, IgG2, IgG3, and
IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY. As
used herein, the term "antibody" (Ab) is meant to include whole
antibodies, including single-chain whole antibodies, and
antigen-binding fragments thereof. 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
V.sub.L or V.sub.H domain. The antibodies may be from any animal
origin including birds and mammals. Preferably, the antibodies are
human, murine, rabbit, goat, guinea pig, camel, horse, or
chicken.
[0409] Antigen-binding antibody fragments, including single-chain
antibodies, may comprise the variable region(s) alone or in
combination with the entire or partial of the following: hinge
region, CH1, CH2, and CH3 domains. Also included in the invention
are any combinations of variable region(s) and hinge region, CH1,
CH2, and CH3 domains. The present invention further includes
monoclonal, polyclonal, chimeric, humanized, and human monoclonal
and human polyclonal antibodies which specifically bind the
polypeptides of the present invention. The present invention
further includes antibodies which are anti-idiotypic to the
antibodies of the present invention.
[0410] 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 heterologous
compositions, such as a heterologous polypeptide or solid support
material. See, e.g., 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. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648;
Kostelny et al., J. Immunol. 148:1547-1553 (1992).
[0411] 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 are recognized or specifically bound
by the antibody. 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.
[0412] 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 the polypeptides
of the present invention are included. 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.
Further included in the present invention are antibodies which only
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. Preferred binding affinities include those
with a dissociation constant or Kd less than 5.times.10.sup.-6M,
10.sup.-6M, 5.times.10.sup.-7M, 10.sup.-7M, 5.times.10.sup.-8M,
10.sup.-8M, 5.times.10.sup.-9M, 10.sup.-9M, 5.times.10.sup.-10M,
10.sup.-10M, 5.times.10.sup.-11M, 10.sup.-11M, 5.times.10.sup.-12M,
10.sup.-12M, 5.times.10.sup.-13M, 10.sup.-13 M,
5.times.10.sup.-14M, 10.sup.-14M, 5.times.10.sup.-15M, and
10.sup.-15M.
[0413] Antibodies of the present invention have uses that include,
but are not limited to, methods known in the art 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 in the
entirety).
[0414] 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, or toxins. See, e.g., WO 92/08495; WO
91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 0 396
387.
[0415] The antibodies of the present invention may be prepared by
any suitable method known in the art. For example, a polypeptide of
the present invention or an antigenic fragment thereof can be
administered to an animal in order to induce the production of sera
containing polyclonal antibodies. The term "monoclonal antibody" is
not a 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.
Monoclonal antibodies can be prepared using a wide variety of
techniques known in the art including the use of hybridoma,
recombinant, and phage display technology.
[0416] Hybridoma techniques include those known in the art and
taught 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). Fab and F(ab')2 fragments may be produced by
proteolytic cleavage, using enzymes such as papain (to produce Fab
fragments) or pepsin (to produce F(ab')2 fragments).
[0417] Alternatively, antibodies of the present invention can be
produced through the application of recombinant DNA and phage
display technology or through synthetic chemistry using methods
known in the art. For example, the antibodies of the present
invention can be prepared using various phage display methods known
in the art. In phage display methods, functional antibody domains
are displayed on the surface of a phage particle which carries
polynucleotide sequences encoding them. Phage with a desired
binding property are selected from a repertoire or combinatorial
antibody library (e.g. human or murine) by selecting directly with
antigen, typically antigen bound or captured to a solid surface or
bead. Phage used in these methods are typically filamentous phage
including fd and M13 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
1879-18 (1997); Burton et al., Advances in Immunology 57:191-280
(1994); PCT/GB91/01134; 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
and 5,733,743 (said references incorporated by reference in their
entireties).
[0418] 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. 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 WO 92/22324;
Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et
al., AJRI34:26-34 (1995); and Better et al., Science 240:1041-1043
(1988) (said references incorporated by reference in their
entireties).
[0419] 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, L. 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. 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; and U.S. Pat. No. 5,807,715.
Antibodies can be humanized using a variety of techniques including
CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101;
and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519
596; Padlan E. A., Molecular Immunology 28(4/5):489-498 (1991);
Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska.
et al., PNAS 91:969-9.73 (1994)), and chain shuffling (U.S. Pat.
No. 5,565,332). Human antibodies can be made by a variety of
methods known in the art including phage display methods described
above. See also, U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806,
and 5,814,318; and WO 98/46645, WO 98/50433, WO 98/24893, WO
98/16654, WO 96/34096, WO 96/33735, and WO 91/10741 (said
references incorporated by reference in their entireties).
[0420] Further included in the present invention are antibodies
recombinantly fused or chemically conjugated (including both
covalently and non-covalently conjugations) to a polypeptide of the
present invention. The antibodies may be specific for antigens
other than polypeptides 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 WO 93/21232; EP 0 439 095; 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) (said references incorporated by reference in
their entireties).
[0421] 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 hinge region, CH1 domain, CH2 domain, and CH3
domain or any combination of whole domains or portions thereof. The
polypeptides of the present invention 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. 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 0 307 434, EP 0 367 166; WO
96/04388, WO 91/06570; Ashkenazi et al., PNAS 88:10535-10539
(1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et
al., PNAS 89:11337-11341 (1992) (said references incorporated by
reference in their entireties).
[0422] The invention further relates to antibodies which 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. Included
are both receptor-specific antibodies and ligand-specific
antibodies. Included are 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. Also include are
receptor-specific antibodies which both prevent ligand binding and
receptor activation. Likewise, included 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 are antibodies which
activate the receptor. These antibodies may act as agonists for
either all or less than all of the biological activities affected
by ligand-mediated receptor activation. The antibodies may be
specified as agonists or antagonists for biological activities
comprising specific activities disclosed herein. The above antibody
agonists can be made using methods known in the art. See e.g., 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., Cytokinde 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) (said references incorporated by reference in
their entireties).
[0423] As discussed above, 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 ligand can be
used to generate anti-idiotypes that "mimic" the polypeptide
mutimerization 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.
[0424] Fusion Proteins
[0425] 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.
[0426] 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.
[0427] 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.
[0428] Moreover, polypeptides of the present invention, including
fragments, and specifically epitopes, can be combined with parts of
the constant domain of immunoglobulins (IgG), 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).)
[0429] 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).)
[0430] 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).)
[0431] Thus, any of these above fusions can be engineered using the
polynucleotides or the polypeptides of the present invention.
[0432] Vectors, Host Cells, and Protein Production
[0433] 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.
[0434] 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.
[0435] 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.
[0436] 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; 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.
[0437] 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. Other suitable vectors will be readily
apparent to the skilled artisan.
[0438] 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.
[0439] 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.
[0440] 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.
[0441] 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
(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), the disclosures of each
of which are incorporated by reference in their entireties).
[0442] 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).
[0443] 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.
[0444] 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.
[0445] 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.
[0446] 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).
[0447] 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.
[0448] 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.
[0449] 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, pharmaceutical compositions)
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.
[0450] 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.
[0451] 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.
[0452] 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.
[0453] 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.
[0454] 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.
[0455] 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.
[0456] 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.
[0457] 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).
[0458] 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).
[0459] Uses of the Polynucleotides
[0460] 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.
[0461] 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.
[0462] 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.
[0463] 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.
[0464] 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).
[0465] 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.
[0466] 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.
[0467] 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.
[0468] 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.
[0469] In addition to the foregoing, a polynucleotide can be used
to control gene expression through triple helix formation or
antisense DNA or RNA. Both methods rely on binding of the
polynucleotide to DNA or RNA. For these techniques, preferred
polynucleotides are usually 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
disease.
[0470] 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.
[0471] 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.
[0472] The polynucleotides of the present invention can also be
used as an alternative to R-FLP, 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.
[0473] 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, 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.
[0474] 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.
[0475] 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.
[0476] Uses of the Polypeptides
[0477] Each of the polypeptides identified herein can be used in
numerous ways. The following description should be considered
exemplary and utilizes known techniques.
[0478] 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.
[0479] 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.
[0480] 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 99 mTc. 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).)
[0481] 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.
[0482] Moreover, polypeptides of the present invention can be used
to treat 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), to inhibit the activity of a
polypeptide (e.g., an oncogene), 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).
[0483] Similarly, antibodies directed to a polypeptide of the
present invention can also be used to treat 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).
[0484] 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.
[0485] Gene Therapy Methods
[0486] Another aspect of the present invention is to gene therapy
methods for treating 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.
[0487] 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.
[0488] 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.
[0489] 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.
[0490] 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.
[0491] 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.
[0492] 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.
[0493] 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.
[0494] For the naked 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.
[0495] 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.
[0496] 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.
[0497] 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.
[0498] 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.
[0499] 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, Feigner
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).
[0500] 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-(trimet- hylammonio)propane) liposomes.
Preparation of DOTMA liposomes is explained in the literature, see,
e.g., Feigner 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.
[0501] 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.
[0502] 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 15 EC. 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.
[0503] 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 Ca.sup.2+-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.
[0504] 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.
[0505] 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.
[0506] In certain embodiments, cells are be 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.
[0507] 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, T19-14.times.,
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.
[0508] 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.
[0509] In certain other embodiments, cells are engineered, er 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)).
[0510] 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.
[0511] 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, for example, the HARP promoter of
the present invention, 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.
[0512] 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.
[0513] 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.
[0514] 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.
[0515] 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.
[0516] 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.
[0517] 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.
[0518] 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.
[0519] 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,
VEGP-1, 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.
[0520] 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.
[0521] 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)).
[0522] 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.
[0523] 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.
[0524] 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.
[0525] 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.
[0526] 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
[0527] Biological Activities
[0528] 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.
[0529] Immune Activity
[0530] The polynucleotides or polypeptides, or agonists or
antagonists of the present invention may be useful in treating
deficiencies or disorders of the immune system, by 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 deficiencies or disorders may be genetic, somatic,
such as cancer or some autoimmune disorders, acquired (e.g., by
chemotherapy or toxins), or infectious. Moreover, a polynucleotides
or polypeptides, or agonists or antagonists of the present
invention can be used as a marker or detector of a particular
immune system disease or disorder.
[0531] A polynucleotides or polypeptides, or agonists or
antagonists of the present invention may be useful in treating or
detecting deficiencies or disorders of hematopoietic cells. A
polynucleotides or polypeptides, 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 those disorders associated with a
decrease in certain (or many) types hematopoietic cells. Examples
of immunologic deficiency syndromes include, but are not limited
to: blood protein disorders (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.
[0532] Moreover, a polynucleotides or polypeptides, 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, a polynucleotides or polypeptides, or
agonists or antagonists of the present invention could be used to
treat blood coagulation disorders (e.g., afibrinogenemia, factor
deficiencies), blood platelet disorders (e.g. thrombocytopenia), or
wounds resulting from trauma, surgery, or other causes.
Alternatively, a polynucleotides or polypeptides, 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 of
heart attacks (infarction), strokes, or scarring.
[0533] A polynucleotides or polypeptides, or agonists or
antagonists of the present invention may also be useful in treating
or detecting 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 a polynucleotides or polypeptides, or
agonists or antagonists of the present invention that inhibits an
immune response, particularly the proliferation, differentiation,
or chemotaxis of T-cells, may be an effective therapy in preventing
autoimmune disorders.
[0534] Examples of autoimmune disorders that can be treated or
detected by the present invention include, but are not limited to:
Addison's Disease, hemolytic anemia, antiphospholipid syndrome,
rheumatoid arthritis, dermatitis, allergic encephalomyelitis,
glomerulonephritis, Goodpasture's Syndrome, Graves' 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.
[0535] Similarly, allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated by a polynucleotides or polypeptides, or agonists
or antagonists of the present invention. Moreover, these molecules
can be used to treat anaphylaxis, hypersensitivity to an antigenic
molecule, or blood group incompatibility.
[0536] A polynucleotides or polypeptides, or agonists or
antagonists of the present invention may also be used to treat
and/or prevent organ rejection or graft-versus-host disease (GVHD).
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. The
administration of a polynucleotides or polypeptides, or agonists or
antagonists of the present invention that inhibits an immune
response, particularly the proliferation, differentiation, or
chemotaxis of T-cells, may be an effective therapy in preventing
organ rejection or GVHD.
[0537] Similarly, a polynucleotides or polypeptides, or agonists or
antagonists of the present invention may also be used to modulate
inflammation. For example, the polypeptide or polynucleotide may
inhibit the proliferation and differentiation of cells involved in
an inflammatory response. These molecules can be used to treat
inflammatory conditions, both chronic and acute conditions,
including 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, or resulting from over production of cytokines (e.g., TNF
or IL-1.)
[0538] Hyperproliferative Disorders
[0539] A polynucleotides or polypeptides, or agonists or
antagonists of the invention can be used to treat or detect
hyperproliferative disorders, 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.
[0540] For example, by increasing an immune response, particularly
increasing antigenic qualities of the hyperproliferative disorder
or by proliferating, differentiating, or mobilizing T-cells,
hyperproliferative disorders can be treated. 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
hyperproliferative disorders, such as a chemotherapeutic agent.
[0541] Examples of hyperproliferative disorders that can be treated
or detected by a polynucleotides or polypeptides, or agonists or
antagonists of the present invention include, but are not limited
to neoplasms located in the: 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.
[0542] Similarly, other hyperproliferative disorders can also be
treated or detected by a polynucleotides or polypeptides, or
agonists or antagonists of the present 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.
[0543] Cardiovascular Disorders
[0544] Polynucleotides or polypeptides, or agonists or antagonists
of the invention may be used to treat cardiovascular disorders,
including peripheral artery disease, such as limb ischemia.
[0545] Cardiovascular disorders 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, cortriatriatum, 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.
[0546] Cardiovascular disorders 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.
[0547] 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.
[0548] 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.
[0549] 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.
[0550] Myocardial ischemias include coronary disease, such as
angina pectoris, coronary aneurysm, coronary arteriosclerosis,
coronary thrombosis, coronary vasospasm, myocardial infarction and
myocardial stunning.
[0551] 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 disorders, diabetic angiopathies, diabetic
retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids,
hepatic veno-occlusive disease, hypertension, hypotension,
ischemia, peripheral vascular diseases, phlebitis, pulmonary
veno-occlusive 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.
[0552] Aneurysms include dissecting aneurysms, false aneurysms,
infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral
aneurysms, coronary aneurysms, heart aneurysms, and iliac
aneurysms.
[0553] 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.
[0554] Cerebrovascular disorders 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.
[0555] 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.
[0556] 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.
[0557] Polynucleotides or polypeptides, or agonists or antagonists
of the invention, are especially effective for the treatment of
critical limb ischemia and coronary disease. As shown in the
Examples, administration of polynucleotides and polypeptides of the
invention to an experimentally induced ischemia rabbit hindlimb may
restore blood pressure ratio, blood flow, angiographic score, and
capillary density.
[0558] 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
pharmaceutical composition, described in more detail below. Methods
of delivering polynucleotides of the invention are described in
more detail herein.
[0559] Anti-Angiogenesis Activity
[0560] 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
disorders, 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).
[0561] The present invention provides for treatment of diseases or
disorders associated with neovascularization by administration of
the polynucleotides or polypeptides, or agonists or antagonists of
the 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)).
[0562] Ocular disorders associated with neovascularization which
can be treated with the polynucleotides or polypeptides or agonists
or antagonists of the invention 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).
[0563] Additionally, disorders which can be treated with the
polynucleotides and polypeptides of the present invention
(including agonist and/or antagonists) 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.
[0564] Moreover, disorders and/or states, which can be treated with
be treated with polynucleotides or polypeptides or agonists or
antagonists of the present invention, 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.
[0565] Diseases at the Cellular Level
[0566] Diseases associated with increased cell survival or the
inhibition of apoptosis that could be treated or detected 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 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, 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.
[0567] Additional diseases or conditions associated with increased
cell survival that could be treated or detected 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.
[0568] Diseases associated with increased apoptosis that could be
treated or detected by the polynucleotides or polypeptides, and/or
agonists 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.
[0569] Wound Healing and Epithelial Cell Proliferation
[0570] 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, burns 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
[0571] 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.
[0572] 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.
[0573] 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.
[0574] 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 bums, (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.
[0575] 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 bums, that cause necrosis of the bronchiolar
epithelium and alveoli could be effectively treated 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.
[0576] 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).
[0577] 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.
[0578] Infectious Disease
[0579] A polypeptide or polynucleotide and/or agonist or antagonist
of the present invention can be used to treat or detect 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. 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.
[0580] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated or detected by a
polynucleotide or polypeptide and/or agonist or antagonist of the
present invention. Examples of viruses, include, but are not
limited to the following DNA and RNA viral families: Arbovirus,
Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae,
Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae,
Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as,
Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus
(e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae),
Orthomyxoviridae (e.g., Influenza), Papovaviridae, Parvovnidae,
Picomaviridae, Poxyiridae (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,
encephalitis, eye infections (e.g., conjunctivitis, keratitis),
chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active,
Delta), 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. A polypeptide or
polynucleotide, and/or agonist or antagonist of the present
invention can be used to treat or detect any of these symptoms or
diseases.
[0581] Similarly, bacterial or fungal agents that can cause disease
or symptoms and that can be treated or detected by a polynucleotide
or polypeptide and/or agonist or antagonist of the present
invention include, but not limited to, the following Gram-Negative
and Gram-positive bacterial families and fungi: Actinomycetales
(e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis,
Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae,
Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis,
Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses,
Enterobacteriaceae (Kiebsiella, Salmonella, Serratia, Yersinia),
Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis,
Listeria, Mycoplasmatales, Neisseriaceae (e.g., Acinetobacter,
Gonorrhea, Menigococcal), Pasteurellacea Infections (e.g.,
Actinobacillus, Heamophilus, Pasteurella), Pseudomonas,
Rickettsiaceae, Chlamydiaceae, Syphilis, and Staphylococcal. 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,
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. A polypeptide or polynucleotide
and/or agonist or antagonist of the present invention can be used
to treat or detect any of these symptoms or diseases.
[0582] Moreover, parasitic agents causing disease or symptoms that
can be treated or detected by a polynucleotide or polypeptide
and/or agonist or antagonist of the present invention include, but
not limited to, the following families: Amebiasis, Babesiosis,
Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine,
Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis,
Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas.
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. A
polypeptide or polynucleotide and/or agonist or antagonist of the
present invention can be used to treat or detect any of these
symptoms or diseases.
[0583] Preferably, treatment 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.
[0584] Regeneration
[0585] 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.
[0586] 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.
[0587] 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 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.
[0588] 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 using
this method include central and peripheral nervous system diseases,
neuropathies, or mechanical and traumatic disorders (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 using the polynucleotide or
polypeptide and/or agonist or antagonist of the present
invention.
[0589] Chemotaxis
[0590] 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.
[0591] 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 inflammation, infection, hyperproliferative disorders, 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 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 wounds.
[0592] 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 to treat
disorders. Thus, a polynucleotide or polypeptide and/or agonist or
antagonist of the present invention could be used as an inhibitor
of chemotaxis.
[0593] Binding Activity
[0594] 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.
[0595] 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.
[0596] 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.
[0597] 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.
[0598] 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.
[0599] 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.
[0600] 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.
[0601] 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.
[0602] 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.
[0603] 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-I), 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).
[0604] 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.
[0605] 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.
[0606] 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.
[0607] All of these above assays can be used as diagnostic or
prognostic markers. The molecules discovered using these assays can
be used to treat 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.
[0608] 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.
[0609] Drug Screening
[0610] 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.
[0611] 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.
[0612] 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.
[0613] 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.
[0614] 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.
[0615] Antisense And Ribozyme (Antagonists)
[0616] 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.
[0617] 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.
[0618] 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 know 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 (Bernoist 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.
[0619] 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.
[0620] 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.
[0621] 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 Apr. 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.
[0622] 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-carboxymethylaminomet-
hyluracil, 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-N-6-isopente- nyladenine,
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.
[0623] 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.
[0624] 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.
[0625] 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-O-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)).
[0626] 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.
[0627] 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.
[0628] 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 fully 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.
[0629] 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.
[0630] 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.
[0631] 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.
[0632] The antagonist/agonist may also be employed to prevent the
growth of scar tissue during wound healing.
[0633] The antagonist/agonist may also be employed to treat the
diseases described herein.
[0634] Other Activities
[0635] 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.
[0636] The polypeptide may also be employed for treating wounds due
to injuries, burns, 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.
[0637] The polypeptide of the present invention may also be
employed stimulate neuronal growth and to treat and prevent
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.
[0638] The polypeptide of the present invention may be also be
employed to prevent skin aging due to sunburn by stimulating
keratinocyte growth.
[0639] 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.
[0640] The polypeptide of the invention may also be employed to
maintain organs before transplantation or for supporting cell
culture of primary tissues.
[0641] The polypeptide of the present invention may also be
employed for inducing tissue of mesodermal origin to differentiate
in early embryos.
[0642] 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.
[0643] 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.
[0644] 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 disorders), 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.
[0645] 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.
[0646] Other Preferred Embodiments
[0647] 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.
[0648] 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.
[0649] 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.
[0650] 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.
[0651] 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.
[0652] 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.
[0653] 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.
[0654] 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.
[0655] 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.
[0656] 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.
[0657] 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.
[0658] 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.
[0659] 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.
[0660] 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.
[0661] 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.
[0662] 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.
[0663] 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.
[0664] 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.
[0665] 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.
[0666] 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.
[0667] 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.
[0668] 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.
[0669] 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.
[0670] 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.
[0671] 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.
[0672] 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.
[0673] 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.
[0674] 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.
[0675] 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.
[0676] 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.
[0677] 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.
[0678] 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.
[0679] 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.
[0680] 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.
[0681] 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.
[0682] 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.
[0683] 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.
[0684] 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.
[0685] 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.
[0686] In any of these methods, the step of detecting said
polypeptide molecules includes using an antibody.
[0687] 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.
[0688] 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.
[0689] 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.
[0690] 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.
[0691] 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.
[0692] 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.
[0693] 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
[0694] 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."
4 Vector Used to Construct Library Corresponding Deposited 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
[0695] 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.
[0696] 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 DH10B, 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.
[0697] 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.
[0698] 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.
[0699] 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 .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, N.Y. (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.
[0700] 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 .mu.l of reaction mixture with 0.5 .mu.g of the above cDNA
template. A convenient reaction mixture is 1.5-5 mM MgCl.sub.2,
0.01% (w/v) gelatin, 20 .mu.M each of dATP, dCTP, dGTP, dTTP, 25
.mu.mol 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.
[0701] 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).)
[0702] 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.
[0703] 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.
[0704] 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
[0705] 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
[0706] 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.
[0707] Multiple Tissue Northern (MTN) blots containing various
human tissues (H) or human immune system tissues (IM) (Clontech)
are examined with the labeled probe using ExpressHyb.TM.
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
[0708] 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
[0709] 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.
[0710] 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.
[0711] Clones containing the desired constructs are grown overnight
(O/N) in liquid culture in LB media supplemented with both Amp (100
.mu.g/ml) and Kan (25 .mu.g/ml). The O/N culture is used to
inoculate a large culture at a ratio of 1:100 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.
[0712] 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.times.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).
[0713] 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.
[0714] 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
6M-1M 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.
[0715] 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 (lacIq). The
origin of replication (oriC) is derived from pUC19 (LTI,
Gaithersburg, Md.). The promoter sequence and operator sequences
are made synthetically.
[0716] 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.
[0717] 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
[0718] 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.
[0719] Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10C 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.
[0720] The cells are then lysed by passing the solution through a
microfluidizer (Microfluidics, 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.
[0721] 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.
[0722] 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.
[0723] To clarify the refolded polypeptide solution, a previously
prepared tangential filtration unit equipped with 0.16 .mu.m
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.
[0724] 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.
[0725] 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 .mu.g 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
[0726] 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,
Xba I 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.
[0727] Many other baculovirus vectors can be used in place of the
vector above, such as pAc373, pVL941, and pAcMI1, 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).
[0728] 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).
[0729] 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.
[0730] 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.).
[0731] The fragment and the dephosphorylated plasmid are ligated
together with T4 DNA ligase. E. coli AB101 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.
[0732] Five .mu.g of a plasmid containing the polynucleotide is
co-transfected with 1.0 .mu.g 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 .mu.g of BaculoGold.TM. virus DNA and 5
.mu.g of the plasmid are mixed in a sterile well of a microtiter
plate containing 50 .mu.l of serum-free Grace's medium (Life
Technologies Inc., Gaithersburg, Md.). Afterwards, 10 .mu.l
Lipofectin plus 90 .mu.l 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.degree. 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.degree. C.
for four days.
[0733] 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 .mu.l 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.
[0734] 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 .mu.Ci of .sup.35S-methionine and 5 .mu.Ci
.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).
[0735] 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
[0736] 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).
[0737] 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), pBC12MI (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.
[0738] 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.
[0739] 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.
[0740] 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.
[0741] 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.
[0742] 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.)
[0743] 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.
[0744] 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.
[0745] 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 .mu.g 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 nM, 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 .mu.M, 2 .mu.M, 5 .mu.M, 10 mM,
20 mM). The same procedure is repeated until clones are obtained
which grow at a concentration of 100-200 .mu.M. 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
[0746] 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.
[0747] 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:
[0748] 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.
[0749] 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.)
[0750] Human IgG Fc region:
5 (SEQ ID NO: 1) GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCC-
CACCGTGC CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCA- AA
ACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGG
TGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG
TAAATGAGTGCGACGGCCGCGACTCTAGAGGAT
Example 10
Production of an Antibody from a Polypeptide
[0751] 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.
[0752] 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 .mu.g/ml of
streptomycin.
[0753] 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 (SP2O), 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.
[0754] 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.
[0755] 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.
[0756] 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
[0757] The following protocol produces a supernatant containing a
polypeptide to be tested. This supernatant can then be used in the
Screening Assays described in Examples 13-20.
[0758] 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 1 ml 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.
[0759] 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.
[0760] 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 R.sub.T 15-45 minutes. After about 20
minutes, use a multi-channel pipetter to add 1.50 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.
[0761] 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.
[0762] 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 KCl; 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 NaE.sub.2PO.sub.4--H.sub.2O; 71.02 mg/L
of Na.sub.2HPO4; 0.4320 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-Phenylalamine; 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 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.
[0763] 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.
[0764] 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.
[0765] 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
[0766] 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.
[0767] 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.
[0768] 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.
[0769] 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)).
[0770] 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.
[0771] 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.
6 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) I1-10 + ? ? - 1, 3 gp130 family IL-6 (Pleiotrophic)
+ + + ? 1, 3 GAS (IRF1 > Lys6 > IFP) I1-11 (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)
[0772] 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:
7 (SEQ ID NO: 3) 5':GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAA-
TGATTTCC CCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3'
[0773] The downstream primer is complementary to the SV40 promoter
and is flanked with a Hind III site:
5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NO:4)
[0774] 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/Hind III
and subcloned into BLSK2-. (Stratagene.) Sequencing with forward
and reverse primers confirms that the insert contains the following
sequence:
8 (SEQ ID NO: 5) 5':CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGAT-
TTCCCCGA AATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAG- TC
CCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCA
TTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGG
CCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGA
GGCCTAGGCTTTTGCAAAAAGCTT:3'
[0775] 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.
[0776] 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.
[0777] 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.
[0778] 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-B/EGR, GAS/NF-KB, 11-2/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
[0779] The following protocol is used to assess T-cell activity by
identifying factors, determining whether supernate 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.
[0780] 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.
[0781] 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.
[0782] 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 1 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.
[0783] 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.
[0784] 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.
[0785] 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).
[0786] 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.
[0787] 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.
[0788] 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.
[0789] 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
[0790] 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.
[0791] 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.
[0792] 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 mm.
[0793] 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.
[0794] 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.
[0795] 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).
[0796] 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
[0797] 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.
[0798] Particularly, the following protocol is used to assess
neuronal activity in PC12 cell lines. PC12 cells (rat
phenochrombcytoma 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.
[0799] 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:
9 (SEQ ID NO: 6) 5' GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3' (SEQ ID NO:
7) 5' GCGAAGCTTCGCGACTCCCCGGATCCGC- CTC-3'
[0800] 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 EGRL
promoter.
[0801] 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.
[0802] 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.
[0803] 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.
[0804] 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.
[0805] 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.
[0806] 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
[0807] NF-.kappa.B (Nuclear Factor .kappa.B) 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-.kappa.B regulates the expression of genes
involved in immune cell activation, control of apoptosis
(NF-.kappa.B appears to shield cells from apoptosis), B and T-cell
development, anti-viral and antimicrobial responses, and multiple
stress responses.
[0808] In non-stimulated conditions, NF-.kappa.B is retained in the
cytoplasm with I-.kappa.B (Inhibitor .kappa.B). However, upon
stimulation, I-.kappa.B is phosphorylated and degraded, causing
NF-.kappa.B to shuttle to the nucleus, thereby activating
transcription of target genes. Target genes activated by
NF-.kappa.B include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.
[0809] Due to its central role and ability to respond to a range of
stimuli, reporter constructs utilizing the NF-.kappa.B promoter
element are used to screen the supernatants produced in Example 11.
Activators or inhibitors of NF-.kappa.B would be useful in treating
diseases. For example, inhibitors of NF-.kappa.B could be used to
treat those diseases related to the acute or chronic activation of
NF-.kappa.B, such as rheumatoid arthritis.
[0810] To construct a vector containing the NF-.kappa.B promoter
element, a PCR based strategy is employed. The upstream primer
contains four tandem copies of the NF-.kappa.B 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:
10 (SEQ ID NO: 9) 5':GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGA-
CTTTCCGGG ACTTTCCATCCTGCCATCTCAATTAG:3'
[0811] The downstream primer is complementary to the 3' end of the
SV40 promoter and is flanked with a Hind III site: 5':
GCGGCAAGCTTTTTGCAAAGCCT- AGGC:3'(SEQ ID NO:4)
[0812] 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 Hind
III and subcloned into BLSK2-. (Stratagene) Sequencing with the T7
and T3 primers confirms the insert contains the following
sequence:
11 (SEQ ID NO: 10) 5':CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTT-
CCGGGACTTT CCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAAC- TCCG
CCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGG
CTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTG
AGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGC AAAAAGCTT:3'
[0813] Next, replace the SV40 minimal promoter element present in
the pSEAP2-promoter plasmid (Clontech) with this NF-.kappa.B/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.
[0814] In order to generate stable mammalian cell lines, the
NF-.kappa.B/SV40/SEAP cassette is removed from the above
NF-.kappa.B/SEAP vector using restriction enzymes SalI and NotI,
and inserted into a vector containing neomycin resistance.
Particularly, the NF-.kappa.B/SV40/SEAP cassette was inserted into
pGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1
with SalI and NotI.
[0815] Once NF-.kappa.B/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
[0816] 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.
[0817] Prime a dispenser with the 2.5.times. Dilution Buffer and
dispense 15 .mu.l of 2.5.times. dilution buffer into Optiplates
containing 35 .mu.l of a supernatant. Seal the plates with a
plastic sealer and incubate at 65 degrees C. for 30 min. Separate
the Optiplates to avoid uneven heating.
[0818] Cool the samples to room temperature for 15 minutes. Empty
the dispenser and prime with the Assay Buffer. Add 50 .mu.l Assay
Buffer and incubate at room temperature 5 min. Empty the dispenser
and prime with the Reaction Buffer (see the table below). Add 50
.mu.l 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.
[0819] Read the relative light unit in the luminometer. Set H12 as
blank, and print the results. An increase in chemiluminescence
indicates reporter activity.
12 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
[0820] 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.
[0821] 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.
[0822] 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.
[0823] 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.
[0824] 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.
[0825] 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.
[0826] 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++ concentration.
Example 19
High-Throughput Screening Assay Identifying Tyrosine Kinase
Activity
[0827] 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.
[0828] 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, lck,
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).
[0829] 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.
[0830] 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.
[0831] 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 Na4P207 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.
[0832] 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.
[0833] 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.
[0834] 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.
[0835] The tyrosine kinase assay reaction is then terminated by
adding 10 ul of 120 mm EDTA and place the reactions on ice.
[0836] 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.
[0837] 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
[0838] 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.
[0839] 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.
[0840] 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.
[0841] 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
[0842] 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).
[0843] 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.
[0844] 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.
[0845] 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.
[0846] 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
[0847] 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.
[0848] 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.
[0849] 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.
[0850] 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.
[0851] 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
Formulating a Polypeptide
[0852] The secreted polypeptide composition 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 secreted
polypeptide 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.
[0853] As a general proposition, the total pharmaceutically
effective amount of secreted polypeptide 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
secreted polypeptide 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.
[0854] Pharmaceutical compositions containing the secreted protein
of the invention 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,
intrasternal, subcutaneous and intraarticular injection and
infusion.
[0855] Compositions of the invention are also suitably administered
by sustained-release systems. Suitable examples of
sustained-release compositions 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).
[0856] 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).
[0857] Sustained-release compositions also include liposomally
entrapped compositions 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 XXX polypeptide my be 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
XXX polypeptide therapy.
[0858] In yet an additional embodiment, the compositions 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)).
[0859] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0860] For parenteral administration, in one embodiment, the
secreted polypeptide 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 polypeptides.
[0861] Generally, the formulations are prepared by contacting the
polypeptide 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.
[0862] 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.
[0863] The secreted polypeptide 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.
[0864] Any polypeptide to be used for therapeutic administration
can be sterile. Sterility is readily accomplished by filtration
through sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutic polypeptide compositions 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.
[0865] Polypeptides 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 polypeptide solution, and
the resulting mixture is lyophilized. The infusion solution is
prepared by reconstituting the lyophilized polypeptide using
bacteriostatic Water-for-Injection.
[0866] 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.
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 polypeptides of the present
invention may be employed in conjunction with other therapeutic
compounds.
[0867] The compositions of the invention may be administered alone
or in combination with other therapeutic agents. Therapeutic agents
that may be administered in combination with the compositions 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.
[0868] In one embodiment, the compositions 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
compositions 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-IBBL, 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.
[0869] Conventional nonspecific immunosuppressive agents, that may
be administered in combination with the compositions 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.
[0870] In a further embodiment, the compositions of the invention
are administered in combination with an antibiotic agent.
Antibiotic agents that may be administered with the compositions of
the invention include, but are not limited to, tetracycline,
metronidazole, amoxicillin, beta-lactamases, aminoglycosides,
macrolides, quinolones, fluoroquinolones, cephalosporins,
erythromycin, ciprofloxacin, and streptomycin.
[0871] In an additional embodiment, the compositions of the
invention are administered alone or in combination with an
anti-inflammatory agent. Anti-inflammatory agents that may be
administered with the compositions 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.
[0872] In another embodiment, compostions of the invention are
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with the
compositions 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).
[0873] In an additional embodiment, the compositions of the
invention are administered in combination with cytokines. Cytokines
that may be administered with the compositions of the invention
include, but are not limited to, IL2, U-3, IL4, IL5, IL6, IL7,
IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and
TNF-alpha.
[0874] In an additional embodiment, the compositions of the
invention are administered in combination with angiogenic proteins.
Angiogenic proteins that may be administered with the compositions
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-682110; Platelet Derived Growth
Factor-B (PDGF-B), as disclosed in European Patent Number
EP-282317; Placental Growth Factor (PDGF-A), as disclosed in
International Publication Number WO 92/06194; Placental Growth
Factor-2 (P1GF-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-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.
[0875] In an additional embodiment, the compositions of the
invention are administered in combination with Fibroblast Growth
Factors. Fibroblast Growth Factors that may be administered with
the compositions 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.
[0876] In additional embodiments, the compositions 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
[0877] 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
pharmaceutical composition comprising an amount of the polypeptide
to increase the activity level of the polypeptide in such an
individual.
[0878] 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
[0879] 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).
[0880] 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
[0881] 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.
[0882] 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.
[0883] 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.
[0884] 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 HB 101, which are then plated
onto agar containing kanamycin for the purpose of confirming that
the vector has the gene of interest properly inserted.
[0885] 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).
[0886] 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.
[0887] 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
[0888] 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.
[0889] 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.
[0890] 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.
[0891] 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.
[0892] 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.
[0893] 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.
[0894] 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 pUC18
(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.
[0895] 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.degree. 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.
[0896] 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.
[0897] 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 Therapy--In Vivo
[0898] 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).
[0899] 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.
[0900] 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.
[0901] 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.
[0902] 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.
[0903] 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.
[0904] 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.
[0905] 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.
[0906] 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
[0907] 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.
[0908] 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.
[0909] 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)).
[0910] 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.
[0911] 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.
[0912] 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.
[0913] 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 conditions and/or disorders associated with aberrant
expression, and in screening for compounds effective in
ameliorating such conditions and/or disorders.
Example 30
Knock-Out Animals
[0914] 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.
[0915] 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.
[0916] Alternatively, the cells can be incorporated into a matrix
and implanted in the body, e.g., 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).
[0917] 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.
[0918] 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 conditions and/or disorders
associated with aberrant expression, and in screening for compounds
effective in ameliorating such conditions and/or disorders.
Example 31
Isolation of Antibody Fragments Directed Against Polypeptides of
the Invention from a Library of scFvs
[0919] Naturally occurring V-genes isolated from human PBLs are
constructed into a large library of antibody fragments which
contain reactivities against a polypeptide having the amino acid
sequence of SEQ ID NO:Y to which the donor may or may not have been
exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein in
its entirety by reference).
[0920] Rescue of the library.
[0921] A library of scFvs is constructed from the RNA of human PBLs
as described in WO92/01047. To rescue phage displaying antibody
fragments, approximately 10.sup.9 E. coli harboring the phagemid
are used to inoculate 50 ml of 2.times.TY containing 1% glucose and
100 micrograms/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
inoculate 50 ml of 2.times.TY-AMP-GLU, 2.times.108 TU of delta gene
3 helper (M13 delta gene III, see WO92/01047) 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 of 2.times.TY containing 100
micrograms/ml ampicillin and 50 micrograms/ml kanamycin and grown
overnight. Phage are prepared as described in WO92/01047.
[0922] 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 M13 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 were spun down (EEC-Centra 8, 4000 revs/min for 10
min), resuspended in 300 ml 2.times.TY broth containing 100
micrograms ampicillin/ml and 25 micrograms 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 micrometer filter
(Minisart NML; Sartorius) to give a final concentration of
approximately 10.sup.13 transducing units/ml (ampicillin-resistant
clones).
[0923] Panning the Library.
[0924] Immunotubes (Nunc) are coated overnight in PBS with 4 ml of
either 100 micrograms/ml or 10 micrograms/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 10.sup.13 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 1.0M 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 micrograms/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.
[0925] Characterization of Binders.
[0926] Eluted phage from the third and fourth 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 microtiter plates coated with either 10 picograms/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., WO92/01047) and then by sequencing.
[0927] 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.
[0928] 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
186 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
MISC_FEATURE (3) Xaa equals any of the twenty naturally occurring
L-amino acids 2 Trp Ser Xaa Trp Ser 1 5 3 86 DNA Artificial
sequence Primer 3 gcgcctcgag atttccccga aatctagatt tccccgaaat
gatttccccg aaatgatttc 60 cccgaaatat ctgccatctc aattag 86 4 27 DNA
Artificial sequence Primer 4 gcggcaagct ttttgcaaag cctaggc 27 5 271
DNA Artificial sequence synthetic GAS containing promoter element 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 Artificial sequence Primer 6 gcgctcgagg
gatgacagcg atagaacccc gg 32 7 31 DNA Artificial sequence Primer 7
gcgaagcttc gcgactcccc ggatccgcct c 31 8 12 DNA Artificial sequence
Primer 8 ggggactttc cc 12 9 73 DNA Artificial sequence Primer 9
gcggcctcga ggggactttc ccggggactt tccggggact ttccgggact ttccatcctg
60 ccatctcaat tag 73 10 256 DNA Artificial sequence vector
containing the NF-KB promoter element 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 1441 DNA Homo sapiens 11
ggcacgagtt ttatttatct tgcctagggt gtgttgggct tcataaatct gtggattggt
60 atctttcgtc agttctgtaa aagtctcagg tactctttgt taacattcgt
ctctgcccca 120 tttttcttct agaattatga tcaaacatgc actaattaga
ccttttattg tattctcttt 180 gcttcttagg ctatgctctg aaaatttatt
ttgtcctaat actcagttta tagttctgtc 240 ttgtttccaa tctgttgtta
aatccctcct ttcaatttta aatctcagtt actgtatttt 300 ttaattctag
aaggtcgttt ggttctttca aatccactag atcaagtcac tctctgtgac 360
ttcctacgac ctatattcta gcctttgcct tttattattt tttaacattg taagcaaagt
420 tgttttatag ttgataattc ttgtatctgt ggtctttgtc tcttcatact
agctgttatt 480 tcttaacagt tttccctgat cccaccttac ttcctcatag
atttgattat ctttgattat 540 gtgctgctta ttttttttaa agataattat
ctgtattaat atgagaccta gattttaaga 600 tacttcccct caggattgtg
ctgtgtgcct gggcacattg caagtccaaa ttactttaaa 660 gtatcctaga
acaccagagg tgaaaattca tgccattctc gtgcatcttt accttatggg 720
tatagcccat tgggaatcct tagtgtcggg agggagttct attacacctt ccacttcggg
780 cagatcctgg gctttgacat tcgtcctctt catcataacc aaatatcaga
cttgcctaat 840 gcgtaaatgc ccttggggta aagcatctgt gttctgctta
cctctcaggg ttcctgcttt 900 tccctttagc tttggttcca tcacttgtgt
gttcaaccag tatgggaaac tgcctctaga 960 agcagtgttt taaaatttta
gtctaaccat aaaaatagag aatgcttacc ctctggatat 1020 aaatgccata
gcgataactt tgaactccta cattacatgc cttttatgta aggcaaacct 1080
cagtacacat tgagagacag tgtcatatac ttattaagat cacaggctct aaaatcagac
1140 tgccttgttt aaatcctggc cctgccatgt agtagtaacc tgtgttaatt
tatgcaagat 1200 acgtaatctc tctgtgcctt ggtttcttcg tttaaaaagc
agaataatag cttgcctcat 1260 gtggtcatta tgaaaaacaa acaaggccgg
gcaccgtggc ttacgcctgt agtctcagca 1320 ctttgggagg ctgaggtggg
cagatgacct gaggtcggga gtttgagacc agcctgccca 1380 acatggagga
accccgtctc tactgaaaaa aaaaaaagag agagagagag agagaactag 1440 t 1441
12 2160 DNA Homo sapiens 12 ttttgaattc tatagattgt cttggaagga
tactgtgtga tgggtcaggc acacagtaat 60 tggagacttt taatgtatgt
aatatttcat agattgcatg ctattaatca tctgtgaggg 120 tagtattttt
tgttttattg taagtttccc tcttttttta taaattaaaa gatggttggt 180
attaggaatt tcaaatgaat gcagaaaatc ttacatgctg tgtactatta atattataac
240 agacgatcca agtccaaaat ctgaccaata aagcaaccat tttatcaaga
tagagggatt 300 ctaatgggag aggggattct tccctcctga agtttgtgtg
tccagtcccc ttaaaaaaaa 360 tgaatagttg tcttttcttg tgcatattaa
tactcgaaag tgccatggtg gtattaatga 420 aagtacactt tattgttgcc
tttgaactta cggccaaggc aataaatcag aaacaaaaat 480 agtgccaatg
tgtcaaaatc gacatctgag agattcagcc tcccatttgg aataaatatg 540
aatcttctaa gctatcttgt ttaatatttt ccatcattta gctacttcct atctccctca
600 gaggcgcctg ctgttcccat tttagagttg acagtggcct gctaattttg
ctatgttcct 660 aaaagttact gggtgtgaga cattttcatc ccctcctttt
tcctactgct ggtgtttatt 720 atccagctag acaatatttt atgcatattt
accgtgatgt ctggaccgta cctgtgctcc 780 ttggcagttt atgttgaaga
taactaaaga tttttctctt tgggaggcat caaaatgatg 840 gtagtttgct
tttatctttt tatgttcatt ttcttttagt aggtgacctt tctgcattaa 900
gaactgtttt tatcttttac taccttttct tttctccttt gtggagacag catgacatgt
960 cctgaaggtc acctttgcct ttgaaaaagg tttgatggag gaattcacag
gtgactgaca 1020 agtctttgaa aagaatggga tctgctcact tctggtcttt
ttggccggga actcctgatt 1080 ggtgttaagg tggtaatttc ccccatataa
gatttagaat cactgagttt gagctagatg 1140 aaatttttaa aatttctggt
tgtctcatta gactgatgag gtgagttttc ttcttcatat 1200 gaacagctag
ttaataacag cagagttctc actcagtgct cagtacttaa ttttccactg 1260
caccacaact gtcttaacta aatgtgctgt atttttcttt aaaagttaag agttctattt
1320 ggtgttttca ggaatatacg tgaaaagaca tgccatgttt tggtaaatac
catcagagtt 1380 gtgtaaaggc gtgtactaag tgcaatctta atttgtggaa
ataatcttca tttacccctc 1440 ctaaaactac actcagtata aacactttcc
cataaggtgt gtgcagtaaa aatgttatat 1500 tactccaaca ctggcaggag
cacagcacag cagccttatt ggagagagcc ttataaaagt 1560 gattaaatgg
aggcattgag ctcattacct ttaagtttac tttgtgctga cctttgttcc 1620
tgttttgaga atctcatata attattaaaa aaaaaaaaca attaaaacga aacggcgggg
1680 cctagctgtg tataaatgat ccttgctgaa tatcttaagg ttttttgtaa
gaaaaaagaa 1740 aaaccaacaa aaaaagctta ttttcacatt aaaatgaaac
ctcttttgca acttaagaat 1800 tctatggaaa agcagttttt atcatatttt
gtgtccatgc accatttttc ttaaaatggc 1860 ttacaaaaaa gaatgtaaac
aatttgtgat ctggccagtt gtacttttag ctcccagagg 1920 gagagttggt
ggtattatga gttgagtaaa aaccatccag gggaacttga gggagcagtc 1980
tgttgccagt aatgttcctt gtgtgccatt aaaccacctc cagatgagtg gaggaacatc
2040 actttttaat tttttaattg tatttggaat tgttgccgtg tactaagaac
ttgacctaaa 2100 taaaatccca caaagtataa aaaaaaaaaa aaaaaaaaaa
aagggcggcc gctcgcgatc 2160 13 1202 DNA Homo sapiens 13 gggtcgaccc
acgcgtccgg tttttttaga aatagaagtg cttacagatt tgtttgagca 60
gaatagatac ccatgttcag agagagagag agagagccat tgagaggcas agagtgccga
120 atgtgaattc tgtgtaaatt gaaaagttac ggtccccgtc cataaggaga
tggctctggt 180 tgtcttgaat ttagacagct ttccagagga ggaggttctc
tcctgcagca gtgtgggaaa 240 aaaatctacc agatttggac cagaatgact
gcaatttagg tcagaacatg atacttagag 300 ggaaaaaagt aatcatggct
tgaatagctg ccctaaggcc agctacaggg ctgcagggtc 360 tgtggacctt
tcttgtcaga actcaggatc cttaggagtc cccacaaggg catggaggct 420
gtggccatca tgagtggaag aaccagcttc caaaggaacc taggattgtt ctggccctgg
480 ctccaggtgc atatctctgc tttccgtgag cattgaatgg gaaatcacgt
gttagcagaa 540 taaatcccag aatacacatt caggaggaga tggttccaca
tggcacgtaa gtcctttgcc 600 ttattgatgt ttgtctggca gatgtcctta
agccttccca tcaagggctt tattctaagg 660 gtggctaact ggctatttaa
acctcatctc aatagtgtct gtctaggatg gcaaaatcac 720 accaggttct
gctgggcaaa tctcccaggt ggcgtattgc tagaggagag cgccactgca 780
gaggacacac tgtcttggcc actggctttg caaaccatag tggaggaggg agtctggggt
840 caccaaccac ttcctggctg actgcctgag ccacattata caagactgtt
tgttgactta 900 gtaatactga agccagagct caccaagatt ctgacatcaa
gcatgaaagt gactaattct 960 ttcawttccc agcactttga caaaggggac
tctcttaaaa ctcctcatcg gacgttagtg 1020 gaagtcgact catgtatcta
aacatgtagc cagcgtaagt tcttggtcag attgatctaa 1080 tgactttttt
agcgtgtgtg tgtgtgtgtg tgtgtgtgtt taatcttttt aagtttgggt 1140
gaagtttgac cttctagaat tcctgtttat tcctgagcaa aaaaaaaaaa aaaaaactcg
1200 ag 1202 14 1554 DNA Homo sapiens misc_feature (695) n equals
a,t,g, or c 14 gctttaatag tgtacactta cacatctgga aggaagagag
ttccatatgg cagggatgat 60 tgggacagga gggatctttt gataactttg
tgtgagcatg aaaatcgaat ggggaaggga 120 gagctgtgaa aaaaaaatgt
tatctctttt tttttgcttc tggaaaccca gctttttggt 180 cagccgtctt
gtgatttggc tgggcctggt ttgtgggggt cgctctctga gttgggtagc 240
tcttggagaa gattatctgg gaactcccat ccttatccca aacatacacc aaacctgccc
300 ccatccacca ttatgggaat tagtaccaga gcatccttgc agattagttc
tcattttctc 360 tctttgtgag cacacacaca tcaggtagag ttccagaaac
ccagctttag gacactgttc 420 acatatcaca ggaggagcaa ggacatgaat
acaagagagc tctttcctga ccagcagtgg 480 gargtggttg tactatctat
ttawttgttt attwatttat ttattttttg agatggartc 540 tccttctgtc
acccaggctg gagtgcagtg gcatgatctc ggctcactgc aatctctgcc 600
tcctgggttc aagcagtcct cctgcctcag ccccccaagt agctgsgatt acaggctgca
660 ccaccatgcc ccgctaattt ttgtattttt agtanagatg gggtttcacc
atgttggcca 720 ggctggtctg taactcctga mctcaggtga tccacctgcc
ttagcctccc aaggtgctgg 780 gattacaggt gtgagccacc gtgcccggsc
tggttccact atttattaaa atgtatatat 840 gtgttttyca cttttttggt
aggcatttta ttgntaataa tttggaaatt aaaaaaattt 900 ctccacaagc
ttattttttg tggagacaag gtctccctgt gttgcctagg ctggtcttga 960
attcctgggc taagtgattg gtctgccttg gcctctcaaa gtgctgggga ttacaggcat
1020 aagtcaccat gccctgtttg scagcaagkt ttawackgct ctttttggta
gggawwtkct 1080 maggtwcagt gatagagaac atgkagttgt ggtgggawac
agtggctyat gactgtatcc 1140 gcactttggg aggctgaggc aggaggattg
cttgaggctg agagttgagn acaggcctgg 1200 gcaacatagc aagacacctt
ctctaaaatg aaaaaaatta gctggatgtg gtgtcatgta 1260 cctgtagtcc
cagttgcttg ggaggctgag gcaggaggat cacttgagcc tgggtgttca 1320
agataggcct ggtcaacaca gcaagacccc ttctctaaaa atgaaaataa aaaaattagc
1380 tggttgtggt ggcatgtacc tgtagtccca gttacttggg aggctgagac
aggaggattg 1440 cttgagccag gggtttgagg ctgcagtgag ctatgactgc
tcccctgcac cccaggctgg 1500 gtgacagagt gagacccagt ctctaaaata
aaaaaaaaaa aaaaaaaact cgta 1554 15 1540 DNA Homo sapiens
misc_feature (651) n equals a,t,g, or c 15 agaattcggc acgagggcat
attactttcc taggactgcc acaacaaact attaccaact 60 agcggcttaa
aacaacaaga gcttattcct cacagttctg gaggccagaa gtccaaaacc 120
aaggtgtcag gaaggtcatg ctctctccaa agtctccaag gatgctcctt ccttgcctcc
180 tccagcctct ggtcgtggcc aacatcccga gggttccttg gcttgcagat
gaatcactta 240 atcccacccc catcatcaca tggcagtccc cctgtgtagc
tcagctctgt ccaaatttcc 300 cctttcctac aaggacatta gtcactggat
tatgacacag ctcatcttaa ctggattata 360 tctgcaaaga ccctgttata
tctgcaaaga cgagttaaca ttcacatgtt ccaggggaga 420 tatgaatttt
aaggggacag tattggaccc agtataggag ggcaggcagc agcgagggag 480
ccagggaggg ctggcctgac ttgagcctgt ttgaaaagca tcatcctcct accaagactg
540 ggggctgctg gttctgacaa ggtttgcagg atcagctggg atgatgggtt
scamccaytc 600 cttcgagyta cgttggaccc ctgggcccac ttacagcaag
gagcttgccc ntycgtgtag 660 ctctycgtca gtgtgggaaa atctgartga
gccagagaag ggtgagattc cccctgcaga 720 gcaggcagta ctgagcaaat
ccaggatcca gaactccagt tctaatcctg gctcttgcct 780 gctttcctgt
gtgaccctgg ggaagtggtt ttccctctct gagactctcc ttccccatgt 840
gagtcacaag ggctgggcct agctgacccc caaggccctt acatgagtgg atagttgcat
900 tttaaacctg gtgctcccca ggataaggga gtcaacccca aggagactgg
ggtttctcct 960 gagcctggcc cctggggatg agcactcact gtggaaaaag
ctggccactt cttagccctt 1020 gtcatgggca gaaaacatgc ccctccagcc
ccaccagcac caacacacag ccaagctcac 1080 tgtttcattt ttagagagaa
atcagggctt tcggtgcagc tgantgacac agacaagggg 1140 cggggggaca
tgaaagggag cgggcaagga cggaaattac acttctccta gcaacctggt 1200
tctgcagctc ctaggcctgg ggccgcgtga tacatgccat tcccaattaa cgggatgtta
1260 aatatacccc ggctcagcct gccccatgct gagccccgcc tggggcagtg
cagggagcca 1320 tgtgatggtg tagagcactc tgcaacaccc catattcatg
ttcccactcc tagggccccg 1380 ctcggtcccc aggaggccag agcggtcctg
ccctctgcct gagcatggct cagctccagc 1440 ctccacttgc cctcccctat
gctggccagc tcgggggtct gcaggcagcc tgtggggcag 1500 ggccagttgg
ccaaactctc caagccagaa gcccctcgag 1540 16 1057 DNA Homo sapiens 16
tcgacccacg cgtccgctga gattacaggt gtgagccacc aggctcagcc ccctaagatt
60 tgaaacactt taaatggccc atggtagggt tcctgctagg ataaaacatt
aagcggctgt 120 taaaagaaat aaaaggagga cacgtctctg tgcactggtg
tggacaaatc tccaagtcac 180 tgcaaaatgg aaaaagtata agatgctctt
tccctgaacc tcaagggtcc cgcccctctc 240 actttcaggt ctctggacct
ctgactgaca ctgtgcctgc ccaggtccct gtatgcactg 300 ccacagtgcc
ctgggcccca tgtccacccc tgtcctgccc ttctctggga tagggctggc 360
cttcctctgc ctctgcctgg ctgcatccat ggtcgatctc aagtgccttg gcatgaactc
420 cactctcctg cagccttcaa tcaaggaatg atggggatgt gtacataccc
caccccaccc 480 cttggcaggg tgatgctgag gtgtggattt ttaacagttc
ccagactttc ccaggaggct 540 tgggtttggg tgcccacagt gggagctggt
gtgatatcat accttcgccg gccgcctttc 600 cttcctgttc tctgtgcccc
tactcccact ctagagctgc cccgtttctc tgttttcgtg 660 aaagagctga
ccctgtgctg cctcccactc tcccaatgcc cctgccactc ctgtgagcct 720
gctgctggtg aggtcggtgc tgacctctgt gttgctggat aatgagtcat ctatctctgg
780 aggagaagaa aggcaggtcc tccacagccc tgataaaatc tccaagtctc
ccagtttcgg 840 gtccctctcc tgggatgcag acccactgcc tgcccagctg
gtacgatcca catgccctct 900 tcttgggaat aggggcatgg gaaagtgact
aaagatactg ttctggctgc tgtgttcact 960 gtgagtaata aactgtccat
ttctccgaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020 aaaaaaaaaa
aaaaaaaaaa aaaaaaaggg cggccgc 1057 17 2080 DNA Homo sapiens
misc_feature (145) n equals a,t,g, or c 17 aattcggcac gagaccttta
gggtgcgcgg gtgcagtata tctcgcgctc tctccccttt 60 ccccctcccc
tttccccacc ccgggcgctc aggttggtct ggaccggaag cgaagatggc 120
gacttctggc gcggcctcgg cgganctggt gatcggctgg tgcatattcg gcctcttact
180 actggctatt ttggcattct gctggatata tgttcgtaaa taccaaagtc
ggcgggaaag 240 tgaagttgtc tccaccataa cagcaatttt ttctctagca
attgcactta tcacatcagc 300 acttctacca gtggatatat ttttggtttc
ttacatgaaa aatcaaaatg gtacatttaa 360 ggactgggct aatgctaatg
tcagcagaca gattgaggac actgtattat acggttacta 420 tactttatat
tctgttatat tgttctgtgt gttcttctgg atcccttttg tctacttcta 480
ttatgaagaa aaggatgatg atgatactag taaatgtact caaattaaaa cggcactcaa
540 gtatactttg ggatttgttg tgatttgtgc actgcttctt ttagttggtg
cctttgttcc 600 attgaatgtt cccaataaca aaaattctac agagtgggaa
aaagtgaagt ccctatttga 660 agaacttgga agtagtcatg gtttagctgc
attgtcattt tctatcagtt ctctgacctt 720 gattggaatg ttggcagcta
taacttacac agcctatggc atgtctgcgt tacctttaaa 780 tctgataaaa
ggcactagaa gcgctgctta tgaacgtttg gaaaacactg aagacattga 840
agaagtagaa caacacattc aaacgattaa atcaaaaagc aaagatggtc gacctttgcc
900 agcaagggat aaacgcgcct taaaacaatt tgaagaaagg ttacgaacac
ttaagaagag 960 agagaggcat ttagaattca ttgaaaacag ctggtggaca
aaattttgtg gcgctctgcg 1020 tcccctgaag atcgtctggg gaatattttt
catcttagtt gcattgctgt ttgtaatttc 1080 tctcttcttg tcaaatttag
ataaagctct tcattcagct ggaatagatt ctggtttcat 1140 aatttttgga
gctaacctga gtaatccact gaatatgctt ttgcctttac tacaaacagt 1200
tttccctctt gattatattc ttataacaat tattattatg tactttattt ttacttcaat
1260 ggcaggaatt cgaaatattg gcatatggtt cttttggatt agattatata
aaatcagaag 1320 aggtagaacc aggccccaag cactcctttt tctctgcatg
atacttctgc ttattgtcct 1380 tcacactagc tacatgattt atagtcttgc
tccccaatat gttatgtatg gaagccaaaa 1440 ttacttaata gagactaata
taacttctga taatcataaa ggcaattcaa ccctttctgt 1500 gccaaagaga
tgtgatgcag awgctcctga agatcagtgt actgttaccc ggacatacct 1560
attccttcac aagttctggt tcttcagtgc tgcttactat tttggtaact gggcctttct
1620 tggggtattt ttgattggat taattgtatc ctgttgtaaa gggaagaaat
cggttattga 1680 aggagtagat gaagattcag acataagtga tgatgagccc
tctgtctatt ctgcttgaca 1740 gccttctgtc ttaaaggttt tataatgctg
actgaatatc tgttatgcat ttttaaagta 1800 ttaaactaac attaggattt
gctaactagc tttcatcaaa aatgggagca tggctataag 1860 acaactatat
tttattatat gttttctgaa gtaacattgt atcatagatt aacattttaa 1920
attaccataa tcatgctatg taaatataag actactggct ttgtgaggga atgtttgtgc
1980 aaaatttttt cctctaatgt ataatagtgt taaattgatt aaaaatcttc
cagaattaaa 2040 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gggcggccgc 2080 18
602 DNA Homo sapiens 18 aattcggcac agkttgtgtt tctmatgttc caggtccggc
caggctggca gctcctgctg 60 gtcatgtttt cctcatgtgc tgtttccaac
cagctcttgg tctggtaccc agcaactgcc 120 ttagcagaca acaaacctgt
agcacctgac cgacgaatca gtgggcatgt gggcatcatc 180 ttcagcatgt
catacctgga aagcaaggga ttgctggcta cagyttcaga agaccgaagc 240
gttcgtatct ggaaggtggg cgacctgcga gtgcctgggg gtcgggtgca gaatattggg
300 cactgctttg ggcacagcgc ccgtgtgtgg caggtcaagc ttctagagaa
ttaccttatc 360 agtgcaggag aggattgtgt ctgcttggtg tggagccatg
aaggtgagat cctccaggcc 420 tttcggggac accaggatgt gtacccggtt
gtagtaggag ctgaaatcca tgctgagctg 480 taccaggaac ttgcatatct
agagacagag actgagtcac tggcccatct ctttgctctt 540 gtccccaggc
cagaataaag aatagagtgt aaaaaaaaaa aaaaaaaaaa aaaaaactcg 600 ag 602
19 629 DNA Homo sapiens misc_feature (533) n equals a,t,g, or c 19
gcacactttg accacttgtg atgagcctta tttgggagca aggtcttcaa ctttgtggtt
60 tttgtttgtt ttatttggtt ttctgcttct gcattagttc acttagggtg
atggctttca 120 gctgctycca tgttgcttgc tgcaaaggat atgattttgt
ycttttttat ggctgtgtag 180 tattccatgg tgtatatgga ccacattttc
tttatccaat ccaccatata tgggcaccta 240 ggttgattcc atgtctttgc
tattgtgaat agcactgtga tgaacataga agtggattaa 300 atttcttttt
cttgacagtc tcctaattta tgcttgtaca tatatttttc tctcatgcct 360
tgaggttttt aaaagtcctc tcctctttct catggcaata cttttactaa agtacatttc
420 ctgggaatcc ttagggttcc
ccttattttg aataggctga atattttcat atgtttggtg 480 atttttatct
tttaatcctt taataggttt gaaagtctct cttgatatgg gtngctcaga 540
taggctccat cgtagagtct agaaatcatc ctatgatttt tttttgccca ttcctaggtt
600 aaaaaaaaaa aaaaaaaaaa aaactcgag 629 20 2067 DNA Homo sapiens 20
aattcggcac gaggaaaaac aaaagttttt taaaacaata aaagttaaca gtcaataatg
60 tgtttgtcgg caagaagccc tctgttaata atggtctaaa caaataagac
attgtttttc 120 tccaataaag aaatccagag gcaggcagta gctggctttg
attcagcctc tgactgtcac 180 tgtcagggcc ccaggcccca tgagcctttc
gtctttcctg catgttggct tatcttctca 240 tgcttgtgac ttcctggttg
caacacggct gctgcaacac cagacatctt gcctgtcttc 300 aaggcaggaa
ggagggggaa actatcgcct accagctatt tttcttacct tagctcctcc 360
atgtcttgga tcaaaagcat ctctttgaac ctctccctca ggcataccct gaaatgctgt
420 ggactttaac cttttttctg ttgcaaaggt cgctcacatc tccctggttg
tttggtcttc 480 tcttccttgg ctctagtaac acagcagtct gttgcttcct
aggacaactt ataatgggac 540 ccaaagggga aagaggattt cccgggcctc
caggaagatg tctttgtgga cccactatga 600 atgtgaataa cccttcctac
ggggaatctg tgtatgggcc cagttccccg cgagttcctg 660 tggtaaggct
ttctgggaga agtctggggt ggttatccgt gaggacctct cacctgatcc 720
ttatggggct ttgtaaaatc ctttcagtaa aactaacttt ttttcacgac tctgagtaca
780 ccctcattat aggaaattgg aaaatatgag aaaatcaaga ggaaaaccaa
attgtccatt 840 tgattgtgag tccattttgg ggtattttct ttgtcttatt
aaaatctaac ttttatatgg 900 ttgagattat attgtataaa aatgtacttt
tggccgggca tggtggctta tgcctgtaat 960 cccagcactt tgggaggcca
aggtgggtgg attataaggt caggagttcg agatcagcct 1020 ggccgataca
gtgaaacccc atctctacta aaaaatatat ttaaaaaatt agccgggcgc 1080
ggtggtgcac gcctgttgtc tcagctactt gggaggctga ggtgggagaa tcgcttgaac
1140 ccaggaggcg gagattgcag tgagctgaga tagcaccact gcactccagc
ctgggcaaca 1200 gagcgagact ccgtctcaaa aaaagttata ctttgktatc
ttagttgaaa tcctgccatg 1260 tttccacact ctataaataa cattttaaac
tttttattag ggaaaatttc aaatacatat 1320 aaaagcagaa caaatagtgt
aatgaacccc tgtgtaccct tcacccaact ttaataatga 1380 tcaactcatg
gcgagcctgt gtccttgttt tctctttatg cctactcact cctgcccatt 1440
ctctgttgta ttattttgaa gtaaaccttg gacatctgtt catcataatc atccatctag
1500 tgtggctgtg ctacaattta cttaaccagt gttggtgttt aaccaaccta
ttgcttattg 1560 gccaccccca agctttttac taatgtaaat aatgctgtaa
agaatatctt tgagtaggat 1620 aattttaaga atcacttcca gatgtcaaat
tacttgacta tatgacattg ccttttaact 1680 taagtcttgg gaacgtttta
aatatttaaa aatgttaaat ccgaggccgg gcgcggtggc 1740 tcatgcctgt
aatcccagaa ctttgggagg ccgaggtggg tggatcacct tgaggtcagg 1800
agctcgcaac cagcctggcc aacatggcga aaccctatct ctactaaaaa tacaaaagtt
1860 agccaggcat tgtggtgcac acctgtaatc ccacctactc gagaggctga
ggcaggagaa 1920 ttgcttgaac ccgggaggca gaggttgcaa tgagccgaga
tcacgctact tcactccagc 1980 ctgggcaacc gcgtgagact ccatctcaaa
aacaaaagaa aaaaaaaaaw aaaaaaaccg 2040 gcacgagggg gggcccgtac ccaatcg
2067 21 997 DNA Homo sapiens misc_feature (963) n equals a,t,g, or
c 21 cccgactcta ggccggaagc gcgcggagac catgtagtga gaccctcgcg
aggtctgaga 60 gtcactggag ctaccagaag catcatgggg ccctggggag
agccagagct cctggtgtgg 120 cgccccgagg cggtagcttc agagcctcca
gtgcctgtgg ggctggaggt gaagttgggg 180 gccctggtgc tgctgctggt
gctcaccctc ctctgcagcc tggtgcccat ctgtgtgctg 240 cgccggccag
gagctaacca tgaaggctca gcttcccgcc agaaagccct gagcctagta 300
agctgtttcg cggggggcgt ctttttggcc acttgtctcc tggacctgct gcctgactac
360 ctggctgcca tagatgaggc cctggcagcc ttgcacgtga cgctccagtt
cccactgcaa 420 gagttcatcc tggccatggg cttcttcctg gtcctggtga
tggagcagat cacactggct 480 tacaaggagc agtcagggcc gtcacctctg
gaggaaacaa gggctctgct gggaacagtg 540 aatggtgggc cgcagcattg
gcatgatggg ccaggggtcc cacaggcgag tggagcccca 600 gcaaccccct
cagccttgcg tgcctgtgta ctggtgttct ccctggccct ccactccgtg 660
ttcgaggggc tggcggtagg gctgcagcga gaccgggctc gggccatgga gctgtgcctg
720 gctttgctgc tccacaaggg catcctggct gtcagcctgt ccctgcggct
gttgcagagc 780 caccttaggg cacaggtggt ggctggctgt gggatcctct
tctcatgcat gacacctcta 840 ggcatcgggc tgggtgcagc tctggcagag
tcggcaggac ctctgcacca gctggcccag 900 tctgtgctag agggcatggc
agctggcacc tttytytata tcacctttyt ggaaatcctg 960 ctntttcatc
ccaaatttaa gggggtttca agaagaa 997 22 1383 DNA Homo sapiens
misc_feature (556) n equals a,t,g, or c 22 ggtgcaaaga acatagaata
ttttgaaaaa cataagactg aaaatacatt ctgagctcac 60 ctttgcttga
tagtttggct gaacataaaa atctagtttg gaaatctttt ttgcctagaa 120
attttatgac attttcccca ttgtcttcta ccttctggtg gtcttccaga tttcactgtg
180 aaatgctgtg gtttgtatct ttacttgtca cttttactgc acactcagtt
gaatactctc 240 aatattaaag ctcatgccct ccagtttggg catattttga
tgaatatttt gtgaaaattc 300 cttgcctttt ccaacttcta gaagctgcct
ctacactttg attctttggg ctctttcttt 360 ttttctccac cttcaaagcc
agcagcatag cacttccaaa tttctctctg cttctgccct 420 agtactaata
ttaagtgagg tctccttgtt tcaaagaaaa tggatgtcaa taaagcactg 480
atgcatcagc aaatagtttt aaactccctg gakgwatatc tagtcttcca gaatacctct
540 cttctctact agagtntaga tntattcatt tactcatcay tcmatcattt
aamaaacatt 600 ttctaagaaa ctgctttgcc tttgggactg ccctaggywc
tggaatataa tagtgagcat 660 gacattgttt gaacttttaa agcagcttac
agttaaatag gtgaaacaaa ccaatataca 720 aggacttgcc atatataaca
aatacttttg tagagctaag tatagaatgt aaaagaaagg 780 aaatagctca
gtcttggagg gggaaaggag atttctcagt gacctgggac acttgaagaa 840
taagtaggag tcatccaaac aaagaacagg caaagctatt gatatagcat gtacaaagac
900 ccagaagtga aagaaggtat agtattttca gagaaattac tcatgtaatc
tgagacttag 960 acaagtagag acagagatga atctggagag gcaaagtaat
gaaggacctt atgaattggg 1020 tgaccataga atatactatc caaacctata
cacttttgaa aatgaaagag atacccgggt 1080 gcagtggctc atgcctgtaa
tcccagcact ttgggaggcc aagcaagaca ggccgatcat 1140 ctgaggtcag
gagtttgaga ccagcctgac caacatggtg aaaccccatc actactaaca 1200
ttacaaaatt agccaggcat ggtagctcac acctgtattc ccagctactc aggaggctga
1260 ggtagtagaa tcgcttgaac ccgggaagtg gaagttgtca gtgagccaag
attgtgccat 1320 tgcactctag cctgggtggc agagcgaaac tctgtctcaa
aaaaaaaaaa aaaaagggcg 1380 gcc 1383 23 1513 DNA Homo sapiens
misc_feature (1502) n equals a,t,g, or c 23 gcagaatgtt taaaggcctt
aggtatatgg agcgagctgc tgaaastatg gcaaggtggt 60 tgatctggcc
ccactccatt tggatgcaag gatttcactt tctacccttc agcagcagct 120
gggccagcct gagaaagctc tggaagctct ggaaccaatg tatgatccag atactttagc
180 acaggatgca aatgctgcac agcrggaact gaagttattg cttcatcgtt
ctactctgtt 240 gttttcacaa ggcaaaatgt atggttatgt ggatacctta
cttactatgt tagccatgct 300 tttaaaggta gcaatgaatc gagcccaagt
ttgtttgata tccagttcca agtctggaga 360 gaggcatctt tatcttatta
aagtatcgag agacaaaata tcagacagca atgaccaaga 420 gtcagcaaat
tgtgatgcaa aagcaatatt tgctgtgctc acaagcgtct tgacaaagga 480
tgactggtgg aatcttctgt tgaaggccat atactcctta tgtgacctat cccgatttca
540 agaggctgag ttgcttgtag attcctcatt ggaatattac tcattttatg
atgacaggca 600 aaaacgcaaa gaactagaat actttggtct gtctgctgca
attctggaca aaaatttcag 660 aaaggcatac aactatatca ggataatggt
aatggaaaat gtcaataaac cccagctctg 720 gaacattttc aatcaagtta
ccatgcactc ccaagatgta cgacatcatc gcttctgtct 780 ccgtttgatg
ctgaaaaacc cagaaaatca tgccctatgt gtcttaaatg gacacaatgc 840
atttgtatct ggtagtttta agcatgcgct tggacagtat gtgcaagcct ttcgcactca
900 ccctgacgaa cctctctata gcttctgtat aggcctaacc tttattcata
tggcatctca 960 gaagtatgtg ttacggagac atgctcttat tgtacagggc
ttttcctttc ttaatcgata 1020 cctcagttta cgtgggccct gccaggaatc
attctacaat ttgggccgtg gccttcatca 1080 gttggggctg attcatcttg
caatccacta ttatcagaag gccctggagc tccctccact 1140 tgtggtagag
ggtatagaac ttgaccagtt agacttacga agagatattg cctacaactt 1200
gtctctcatc tatcagagca gtgggaatac cggaatggct caaacgcttt tgtataccta
1260 ttgttctata taaagcaccg caactgagaa cagagcaatg gcagctgctg
tgtgaggacc 1320 agtgtcttct gtctcagggc ttattatttg taactccaaa
atagaaatga caatttcaga 1380 attacctaac aaacagtgta tttattttta
atatgtgata atgatcttgt ggtatatatg 1440 caaaattatt cctacaaaaa
aaaaaaaaaa aaactcgtag ggggggcccg gtacccaatc 1500 cnaatttttc cnc
1513 24 1044 DNA Homo sapiens 24 ggattttcag agacaaaggt ccaagttagg
agacgtaatt actcagtgct ttgaagggac 60 atccaaggtg ctcactctta
gccatagccg ttggtttcct ggatgctgac tgtgaagatt 120 ctaaagtgct
tcctagggtg ggcggtggtg gcaggaggcc ttggacggag tcaggccaga 180
cccagcctcc tgtttaatag gctgagccca agcgtccctc agatgcgaat ccaacagcct
240 tggtgagttg taagatttca tggaaacttt ccctgacttc tgtctccccc
ttgctcccca 300 ttacctggga aaggcagctt tgtgggccat gtgtcccgga
agggcctggg ctggctgtgg 360 cccagtgctc aggaccagcc atcttggccc
tcacagcgcc ctgcccagtt ggtgtaatat 420 ttgtyttcaa gccattgttg
gagcaggcag gcaaaggggg ctttctgagg atccaacgtg 480 tgccagccac
tgggatacaa agacaggcct ggttcctagc tgtggggctg ggaagggtat 540
ctgacatcaa tggtggcacc tggcagagga cacacagaca acagcaggca gcatggactt
600 ttatgtttgt agcttgagct ggttttaatt ggaagctctg tgatttacat
aatcacttac 660 aatctctgta aataaggaac tatttatgag gaattgtaaa
tttcctctct cccccttctt 720 accctgtctg tgatcttgtc tgtgatgcag
taatgatatt ccactctagg ttcccatgat 780 cagtggtgaa atatagtgat
tttcacctgt gcttccattc tgaagttctg gaaagaagta 840 ctggatggac
tgaagtccag gacaacgtyc caaagaaagg cagagtccag gtaggcttgg 900
aggaccaagc cctggatgag cactggaggg cagaggcctc agtgtccagc actgtgccct
960 gcacatggaa agcccctacg tttgtggaat gaatgaataa taaaaatgtt
ttcataagtg 1020 aaaaaaaaaa aaaaaaaact cgag 1044 25 2575 DNA Homo
sapiens 25 ccacgcgtcc gcgggcaccg gccgacatgg cggcagcggt ggcggctgcg
ctggcgcggc 60 ttttggcggc ctttctgctc ctcgcggccc aggtggcctg
tgagtacggc atggtgcacg 120 tggtctccca ggccgggggc cccgaaggca
aagactactg catcctctac aacccgcagt 180 gggcccatct tccgcacgac
ctcagcaagg catctttcct gcagctgcgc aactggacgg 240 cctccctgct
ctgctccgca gccgacctcc ccgcccgtgg cttcagcaac cagatcccgc 300
tggtggcgcg ggggaactgc accttctatg agaaagtgag gctggcccag ggcagcggag
360 cacgcgggct gctcatcgtc agcagggaga ggctggtccc cccggggggt
aataagacgc 420 agtatgatga gattggcatt cccgtggccc tgctcagcta
caaagacatg ctggacatct 480 tcacgcgttt cggccgcacg gtgagggcgg
cgctgtatgc gcctaaggag ccggtgctgg 540 actacaacat ggtcatcatc
ttcatcatgg ctgtgggcac cgtcgccatc ggcggctact 600 gggccgggag
tcgggacgtg aagaaaaggt acatgaagca caagcgcgac gatgggcccg 660
agaagcagga ggacgaggcg gtggacgtga cgccggtgat gacctgcgtg tttgtggtga
720 tgtgctgctc catgctggtg ctgctctact acttctacga cctcctcgtg
tgcgtggtca 780 tcgggatctt ctgcctggcc tccgccaccg gcctctacag
ctgcctggcg ccctgtgtgc 840 ggcggctgcc cttcggcaag tgcaggatcc
ccaacaacag cctgccctac ttccacaagc 900 gcccgcaggc ccgtatgctg
ctcctggcgc tcttctgcgt ggccgtcagc gtggtgtggg 960 gcgtcttccg
caacgaggac agtgggcctg ggtcctccag gatgccctgg gcatcgcctt 1020
ctgcctctac atgctgaaga ccatccgtct gcccaccttc aaggcctgca cgctgctgct
1080 gctggtgctg ttcctctacg acatcttctt cgtgttcatc acgcccttcc
tgaccaagag 1140 tgggagcagc atcatggtgg aggtggccac tgggccctcg
gactcagcca cccgtgagaa 1200 gctgcccatg gtcctgaagg tgcccaggct
gaactcctca cctctggccc tgtgtgaccg 1260 gcccttctcc ctcctgggtt
tcggagacat tttggtgcca gggctgctgg tggcctactg 1320 ccacaggttt
gacatccagg tacagtcctc cagggtatac ttcgtggcct gcaccatcgc 1380
ctatggcgtt ggcctccttg tgacattcgt ggcactggcc ctgatgcagc gtggccagcc
1440 cgctctcctc tacctggtgc cctgcacgct ggtgacgagc tgcgctgtgg
cgctctggcg 1500 ccgggagctg ggcgtgttct ggacgggcag cggctttgcg
aaagtcctac ctccatctcc 1560 gtgggcccca gcaccagccg acggcccgca
gcctcccaaa gactctgcca cgccactctc 1620 cccgcagccg cccagcgaag
aaccagccac atccccctgg cctgctgagc agtccccaaa 1680 atcacgcacg
tccgaggaga tgggggctgg agcccccatg cgggagcctg ggagcccagc 1740
tgaatccgag ggccgggacc aggcccagcc gtccccggta acccagcctg gcgcctcggc
1800 ctaggggagg ggtgagacgc tcgctgccgt gcccgccaca ccaagatgtt
ggggctgcct 1860 ggcgcccatg gagacagaca gacagacgct tgtcccccgg
gaccgaggcc tgtgccgtcc 1920 ccacccgccc caacatggtg cttatccttg
ccgagacccc tgcagtcgtg cccgcgccca 1980 gcccagctgc cccggctgca
cgcctgctgc tcccagctcg cccggctgcc acaagctttc 2040 tgcgggtcca
tcctccccgc aggaggaggg gtccgtcctt cgcaggcctt gcccggcctc 2100
tctgcagacc ctcaagcgtc gtctgcatga gtgagcaggc gtgggtggac tttggccgcg
2160 gccacacttg gtgctcacca gctgcttcgg ccttcaggtg acctccctcc
ccacggcatc 2220 ctgctctccg ggtggaagag cagctttttg tctcccagaa
ggcatcgctt ttccctcttg 2280 agcagatcgg agcccctggg aggtttggaa
gctgcctcca agcctaggac acggaccggt 2340 ggccggggcg gcctctggcc
cctgacgctg gctgagacag gcccgtgggg cggggttttg 2400 gggcgtgaac
aaggctggca gtaagtggac aagctgctcc cctggctaag gccctgccct 2460
gccctcagcc agaggtgcct ggccatgcct gcacactcct ccccatttta ataaatggtc
2520 gcaacttcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa
2575 26 718 DNA Homo sapiens misc_feature (21) n equals a,t,g, or c
26 aactagggat cccccgggct ncaggaattc gccactggrg ccaaagtgag
agtccagcgg 60 tcttccagcg cttgggccac ggcggcggcc ctgggagcag
aggtggagcg accccattac 120 gctaaagatg aaaggctggg gttggctggc
cctgcttctg ggggccctgc tgggaaccgc 180 ctgggctcgg aggagccagg
atctccactg tggagcatgc agggctctgg tggatgaact 240 agaatgggaa
attgcccagg tggaccccaa gaagaccatt cagatgggat ctttccggat 300
caatccagat ggcagccagt cagtggtgga ggtaactgtt actgttcccc caaacaaagt
360 agctcactct ggctttggat gaaattcgac tgcttaaaaa ggaccttggt
ttaatagaaa 420 tgaagaaaac agactcagaa aaaagatttg gctctgtctc
atttggaaga agctgcaggc 480 ttattcccca tgcacttgct tcctggctgc
aaaccttaat actttgtttc tgctgtagaa 540 tttgttagca aacagggagt
cctgatcagc acccttctcc acatccacat gactggtttt 600 taatgtagca
ctgtggtata catgcaaaca tccgttcaaa atctgagtcg gagctaaaaa 660
aaaaaaaaaa aaaactcrag ggggggcccg agtacccaat tsgccctaga agaggcga 718
27 654 DNA Homo sapiens misc_feature (613) n equals a,t,g, or c 27
ggtcgaccca cgcgtccggt catggccatc cagagcctgc acccttgccc ctcagagctc
60 tgctgcaggg cctgcgtgas yttttaccac tgggcgatgg tggctgtgac
gggcggcgtg 120 ggcgtggccg ctgccctgtg tctctgtagc ctcctgctgt
ggccgacccg cctgcgacgc 180 tcccgaggcg gagaacaccg aacacccagt
gaaggtgagg ggatcagcac ggcgccgcca 240 ccgtgctgga acgagactca
gccacaagga ggtgcgaagc tctgacccag gccacagtgc 300 ggatgcacct
tgaggatgtc acgctcagtg agagacacca gacacagaag ggtacgctgt 360
gatcccactt ctatgaaatg tccaggacag accaatccac agaatcaggg agaggattcg
420 tgggtgccgg gactggggag ggggacctgg gggtgactag gtgacataat
ggggacaggg 480 ctgccttctg ggtgatgaga atgttctgga atcagatggg
atggctgcac ggcgtggtga 540 aggtactgaa cgccacctca ctgtaagacg
gtagattttg tattttacca caataaacaa 600 aacaaaacaa aanmaaaaaa
aanaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 654 28 1445 DNA Homo sapiens
28 ggcacgaggg atttgaacaa gatcattaga attcaaaaaa caccagaaat
gaaagatctt 60 tcctgaagct gtttaggaat attcatgata tacccttaac
tgttctagag aacaaaatgc 120 gtctgtgctc cttcacaaaa gtccctatga
atttgtttct caatgtgatc cttcttaagt 180 tctataactt tttgttttca
ttaattttag gaaaatcctg ccttgcttcg ttgggcctat 240 gcaagaacaa
taaatgtcta tcctaatttc agacccactc ctaaaaactc actcatggga 300
gctctgtgtg gatttgggcc cctcatcttc atttattata ttatcaaaac tgagagggta
360 agtattcaga ccagatgttt agtatttgag tgataggttc actttctagg
gaccagctgc 420 agctccttct cttgaagatt gccaccagtg cccctcccac
cttggggctg tcctctgcct 480 tcccttcctc tcttctttta tctttattcc
tttccagcag gagttaaaac agaaagtttt 540 cagtcacctt tgtctatttt
tgttagttca tttgtttttt aaaaagatga tgtttattgg 600 gttaagtatt
agcagaatac ataaatcatt tagtacgttt cctgtttgcg tgaattctat 660
ttatgttggt cacattttgc aaattaatgt taaaacctat taatactcta cgggacagag
720 aagcacaagc tgcctgtgtg gggaatagct gccgtcagca gcctgggtat
atgattggag 780 agaaagtcaa gctgatcttt ggcaccaaac cattccacat
ctggtactaa accctgagct 840 gcagccccca ggcttgtgtt gccactggag
cccactcgtc tagctttgtc tttaactggc 900 ccatctgcat tcccattaga
gttcgtgtat tttgattatc tggtgaatga tctacttaac 960 agaaaggtag
tccacatttt cccagaaagt gtttgcattt tgctttcaat atatggtttt 1020
atgggataat atatttctaa tgactaaaat gtgagtaaga tgtttttgaa taggagcatt
1080 ttcttactgt gtctttagtt cctcggatta ctgtttcttc gcacactccc
tgggctttag 1140 acagtgggat tgcaattagg tttggagtgt ttcattctgt
ttgtcagttg tacggtgggt 1200 tgtgccaaaa tgcagttttt cttacctttt
ttatttattt atttttatct aatatagcca 1260 actggcagaa tatattgtct
ttaatgtact ttttttctgt ctttacagga taggaaagaa 1320 aaacttatcc
aggaaggaaa attggatcga acatttcacc tctcatatta agtctggcaa 1380
tgatgactat atgtattcct gcctaaataa atcatctatt aatcattaaa aaaaaaaaaa
1440 aaaaa 1445 29 2020 DNA Homo sapiens 29 ccacgcgtcc ggggtgaggg
caacagatgc tggacccagg gagctctctg ccacaggatg 60 atggtggccc
ttcgaggagc ttctgcattg ctggttctgt tccttgcagc ttttctgccc 120
ccgccgcagt gtacccagga cccagccatg gtgcattaca tctaccagcg ctttcgagtc
180 ttggagcaag ggctggaaaa atgtacccaa gcaacgaggg catacattca
agaattccaa 240 gagttctcaa aaaatatatc tgtcatgctg ggaagatgtc
agacctacac aagtgagtac 300 aagagtgcag tgggtaactt ggcactgaga
gttgaacgtg cccaacggga gattgactac 360 atacaatacc ttcgagaggc
tgacgagtgc atcgaatcag aggacaagac actggcagaa 420 atgttgctcc
aagaagctga agaagagaaa aagatccgga ctctgctgaa tgcaagctgt 480
gacaacatgc tgatgggcat aaagtctttg aaaatagtga agaagatgat ggacacacat
540 ggctcttgga tgaaagatgc tgtctataac tctccaaagg tgtacttatt
aattggatcc 600 agaaacaaca ctgtttggga atttgcaaac atacgggcat
tcatggagga taacaccaag 660 ccagctcccc ggaagcaaat cctaacactt
tcctggcagg gaacaggcca agtgatctac 720 aaaggttttc tattttttca
taaccaagca acttctaatg agataatcaa atataacctg 780 cagaagagga
ctgtggaaga tcgaatgctg ctcccaggag gggtaggccg agcattggtt 840
taccagcact ccccctcaac ttacattgac ctggctgtgg atgagcatgg gctctgggcc
900 atccactctg ggccaggcac ccatagccat ttggttctca caaagattga
gccgggcaca 960 ctgggagtgg agcattcatg ggatacccca tgcagaagcc
aggatgctga agcctcattc 1020 ctcttgtgtg gggttctcta tgtggtctac
agtactgggg gccagggccc tcatcgcatc 1080 acctgcatct atgatccact
gggcactatc agtgaggagg acttgcccaa cttgttcttc 1140 cccaagagac
caagaagtca ctccatgatc cattacaacc ccagagataa gcagctctat 1200
gcctggaatg aaggaaacca gatcatttac aaactacaga caaagagaaa gctgactctg
1260 aagtaatgca ttacagctgt gagaaagagc actgtggctt tggcagctgt
tctacaggac 1320 agtgaggcta tagccccttc acaatatagt atccctctaa
tcacacacag gaagagtgtg 1380 tagaagtgga aatacgtatg cctcctttcc
caaatgtcac tgccttaggt atcttccaag 1440 agcttagatg agagcatatc
atcaggaaag tttcaacaat gtccattact cccccaaacc 1500 tcctggctct
caaggatgac cacattctga tacagcctac ttcaagcctt ttgttttact 1560
gctccccagc atttactgta actctgccat cttccctccc acaattagag ttgtatgcca
1620 gcccctaata ttcaccactg gcttttctct cccctggcct ttgctgaagc
tcttccctct 1680 ttttcaaatg tctattgata ttctcccatt ttcactgccc
aactaaaata
ctattaatat 1740 ttctttcttt tctttccttt tttttgagac aaggtctcac
tatgttgccc aggctggtct 1800 caaactccag agctcaagag atcctcctgc
ctcagcctcc taagtacctg ggattacagg 1860 catgtgccac cacacctggc
ttaaaatact atttcttatt gaggtttaac ctctatttcc 1920 cctagccctg
tccttccact aagcttggta gatgtaataa taaagtgaaa atattaacat 1980
ttgaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2020 30 1083 DNA Homo
sapiens 30 aattcggcag agccctgaac cccgcacccc accctcgagg ccagaaatcg
gttgcctctg 60 gggacctgag aagcgagacc actcggcgcc ctgacttgca
aagttggggt ctttattggc 120 ctccgggatt ctgctcctgg cggtttctcc
aggctggtga tgggcaagcc gggtgtacca 180 agtccaggat gcacatgagg
agcgtttgta gcagtcactg aatcacctca tgactagcgg 240 ggcaggcctc
taattcaccg caggatttcc ggtaggttgg attgtggggt tggtgtttgc 300
actccaaaga gktgctgtga tttccctgta tctgtctttc tggcttgtta gatcttctca
360 tttggcgtcc tttctccgaa gagttaacca agacgtttgg catggtttcc
ttgctttcct 420 cctatctttt gctgctagag ctgctttcga aaagaagtct
tttcttgcag tggtatcttt 480 tctttgggtt acagtgttgt tcatcctttc
tttgccgaaa gaatgaatcc cagtgcttca 540 caaggttaaa ggaaagatct
gctggtagtg tttagtcttt gttctgagct gatatgtgtt 600 agtagctttt
tgtttttaaa ttttattagt aaaatttcac cagtgaacca gaagctcttt 660
ttttctgttg tgaaatgcta gctttaagat ttctgagaac tttgtgtcaa agaaatcttt
720 gaaaagttac tgaagtatac agagaggttc acaattttaa atgtgcaggt
ggtccgggcg 780 cggtagatca cacctgtaat cccagcactt tgggacgcca
aggtgggcgg atcacttgag 840 cccaggattt ccagaccagc ctgggcaacg
tgccaaaacc ctatctctac taaaattaca 900 aaagttagct gtgtgtggtg
gtgtgtgcct gtagtcccag ctacctggta ggctgaggtg 960 ggaggatcac
cagagcccag gaggttgaga ttgcagtgag ccgtgatcat ggcagtgcac 1020
tcccgcctgg gtggcagagt gagaccctgt ctccaaaaaa aaaaaaaaaa aaaaaaactc
1080 gag 1083 31 1580 DNA Homo sapiens misc_feature (1513) n equals
a,t,g, or c 31 gctggggaag atgctgcgtc cagcgttacc gtggctgtac
cttggcctct gcagcctcct 60 ggtgggggag gcagaggccc cgagccccgt
ggatccgctg gagcggagcc ggccgtacgc 120 ggtgctgcga gggcagaacc
tggtgttgat gggaaccatt ttcagcatcc tgctggtgac 180 tgtcatcctt
atggcatttt gtgtctacaa gcccattcgg cgtcggtgac agccagacaa 240
gttcttcaat gagtatttgg gaataggata agttgtgttg cacacaggcc agtggagaag
300 ttggaaccaa aactttccta cttggaaatg acctttggtc tggacagttg
gtaaatgcta 360 aatgaattag aagaaaacat gtactagaca ttattttttc
ctaacactgt agcgcaaata 420 attggcccct gagtccgctt ctcagtgttt
ctgactgtac ttgttaaaag taagacctga 480 aagctccaaa ggtcagtgta
aagatggagt gttcatgaga aagaaaacat ggtaaccttg 540 tgagtgcctg
taagaaccac actgtaaaga actcatcatt aatgcttgaa aatgttatta 600
agaaggagac ttaccatgca gacattccct atttaagaac catttggtta cagtgggtta
660 agaatcacag attttttttt ttaatctcac ctgagttagc ctagaatgcg
ctggttgcaa 720 agtggtgtca gctgtgggga tcttgggccc tcgttcctca
cctgcatcct gccctgcact 780 caggtgctcc ccctgaagtc agggtcacat
caggtagacc tgttactata tgcacctttg 840 gcctggaatg ctctgaagtt
ggactggaaa tgttactagg ttggcctgtt acaaaaagga 900 ccccatcctg
cttaaacaca ttgatctccc ttgccctgca tttgagtctt tctagcccac 960
ggtctgaaac ttgaggcagc tttccagatt tggaatgtaa aaggctcagt gggcactctg
1020 ttcatccctg ggtggggagg gcccagccaa cagaagtgca tgtccactgt
gcgggccagt 1080 gtgtgtttac acaaatttca tctcagcttt gaaaatgctg
ctattagttt ccactgttgg 1140 tgaactggat tttttcctcc tattgaaatg
atactttcat acttataaag ctgtcgtcaa 1200 tatttatttc aaggtgctag
atttaatttt gttattaaat tgaaatgctt atcttgtgtt 1260 caagcacagc
actgatttta acaacctgca tttaatgtga agtaaccgaa gtaggatact 1320
gtaactgtgt aaggattttg tttgtaatct tgtaacattg aaccattgaa atgttcagtt
1380 ctttgctttt gagcaaaacg tcaattaaaa ctaaagtaaa atcctatata
ttgttttact 1440 ccaccagtta tttcccaagt gtttgaaatg caggtgtgtg
tctgaatttg gatctaatcc 1500 acttaaagga ggnctgtgga ggggaaattc
cttttttgag gncgggtttt gggtcccctt 1560 gcccggggaa agggttcccg 1580 32
796 DNA Homo sapiens misc_feature (748) n equals a,t,g, or c 32
tcggcccgag aagaaatgtg acgcactctc accaagatgc tgaagctgac attcatcaat
60 aagcagctgt gcatccacta ggcatttggt aaatgttaac ttatctaccg
aggtggtgtt 120 ttcttagcct cccacctcct tgctgtggag cagcttcatg
taccatgatg catattcaga 180 tcattcttaa tactcatatt ttgatagaga
ggtttttagg ttttctttta aaccaagttt 240 attgagataa actactttgg
taggatatgg aacttaggaa taatggtatg aaactagaca 300 gctttttttt
ttttattaca ctttaagttc tgggatatgt gttcagaaca tgcaggtttg 360
ttacataggt atacacgtgc catggtggtt tgctgcaccc atcaacctgt catctgtatt
420 cggtgtttct cctaattcta tcccwcccct acccccctgc ccccaaaaag
gccccagtgt 480 gtgatggtcc cctccctgtg tccatgtgtt ctcattgttc
aactcccact tatgagtgag 540 aacatgaggt gtttggtttt ttcttcctgt
gttagtttgc tgagaatgat ggcttccagc 600 ttcatccatg tccctkcaaa
ggacatgaac tcagtccttt tttatggctg catagtattt 660 cgtggtatat
aagtgccaca ttttctttat ycagtctayc atttgggttg gttccaaatc 720
tttgctattg tgaatagtgc cgcaatanac atacgtgtgc atgtgtcttt aaaaaaaaaa
780 aaaaaaaaaa ctcgag 796 33 1256 DNA Homo sapiens 33 ctatgttcca
tcattccttc ccaaagccac cggaagcatt ccttctagga aaggtggagt 60
cggtagtgag aagccggagg tgcccctaca gacatacaag gagattgttc actgctgyga
120 ggagcaggtc ttaactctgg ccactgaaca gacctatgct gtggagggtg
agacacccat 180 caaccgcctg tccctgctgc tctctggccg ggttcgtgtg
agccaggatg ggcagtttct 240 gcactacatc tttccatacc agttcatgga
ctctcctgag tgggaatcac tacagccttc 300 tgaggagggg gtgttccagg
tcactctgac tgctgagacc tcatgtagct acatttcctg 360 gccccggaaa
agtctccatc ttcttctgac caaagagcga tacatctcct gcctcttctc 420
ggctctgctg ggatatgaca tctcggagaa gctctacact ctcaatgaca agctctttgc
480 taagtttggg ctgcgctttg acatccgcct tcccagcctc taccatgtcc
tgggtcccac 540 tgctgcagat gctggaccag agtccgagaa gggtgatgag
gaagtctgtg agccagctgt 600 gtcccctcct caggccacac ccacctctct
ccagcaaaca cccccttgtt ctacccctcc 660 agctaccacc aactttcctg
cacctcctac ccgggccagg ttgtccaggc cagacagtgg 720 catactggct
tctagaattc ctctccagag ctactctcaa gttatatcca ggggacaggc 780
ccctttggct ccaacccaca cgcctgaact ttaaggatca ttggactatc ttctctgtgg
840 ccagcgcagc tctcttctgt gttcacagaa tggccactga taggcaygcc
tcttttccca 900 cccactggaa ggctcacagg caaggtgaga gaggacacag
aaggtgccaa cactgtcgct 960 acagtaagga cctgaagtga ctttgagaaa
ttcaccctca caaaccttcc ttcaggagca 1020 ggcattggta gtgcagaggc
acagattccg tcctttacca gctgcagaat cttgggcaag 1080 ttacatagcc
tctgtgagcc tcatcggtaa acagtggggg ttatgaaacc cacctcacag 1140
ggttgttgtg aggatccaat gagttgattt aggtaagcac ctagcacatg ccgtggcacc
1200 aagtaagcac tcaataaatc actcaactcc ttaaaaaaaa aaaaaaaaaa ctcgag
1256 34 1064 DNA Homo sapiens misc_feature (462) n equals a,t,g, or
c 34 cttcagcctg ggcaacagag caagaacctg tctcagtcaa tcaataaatg
tatgtatata 60 tatatatgta tatatgtcag accaccgtct gaaattgctg
ttcatgattg gaaatcgaac 120 tggaaacccg aaggcaggag atgtgtgctc
ccttgggatg tatggggaaa tcacacagag 180 ctgttagtac ttcagtcatg
ggatttgctc tcatgctatg catatgggcc tcacaacttg 240 taaatgccac
tggaagatgg cttatctaag gttccttatt ttgtggtctt tcccccttag 300
ttctgcagtg agtggggcaa agcgtgtcac tgaccttttg aatggaaaac actggaagcc
360 ttagcgttct taattcctga aatgttcatt tttwcttcta agcaactggg
cttcasagga 420 gattagggca ggcaataaca gtgttgacac cagggcaact
gntttcycct gttatgggat 480 tatwcaacat ctgctttctg ctaagctcca
tggaaggcac agaggaaaca cagcagagtc 540 catgccttag agactttgta
cctgatgaat tgagtggtat caggacaatg ctatttaatg 600 tttgatccat
cccttctcta agcacatctc agatttctgt gctacctgat ttaacccttt 660
cagttcatag aacccagaag gataaggtga aaagatagac cgggaaaagt aatgcaagtg
720 gccaagagta gcttccactt caaagttcct catgtgtgtg tgctaacatt
gtgacttctg 780 ttcagtcatt gtcagtataa actgtacatt ggaatcattt
gtagcttttt aaaaaatgcc 840 tatgcctcac cctagaccta ccacatcaaa
atctcaggat agagtctcaa gctaaaaagc 900 ctctatttga gccaggctta
ttggcacctg cctgtagtcc cgtactcaga aggctgaagt 960 gagaggatcg
cttgaactca ggagtttaac gccagcagag gcaatagggc aaaatagcga 1020
gatctcatct ctttaaaaaa aaaaaanntn aaaagggcgg ccgc 1064 35 755 DNA
Homo sapiens misc_feature (1) n equals a,t,g, or c 35 natttcccgt
tcagttattc cggtgacact atagaaggta cgcctgcagg taccggtccg 60
gaattcccgg gtcgacccac gcgtccgaac tcctgaaaca gtgaggacat ctcacagacc
120 agacaggagc tggggctctg catctcacag cggtgcctgt cagacaggaa
gaagtcccgc 180 agaagtggcg tgtgggtcag ggcctgcacg atgcagttca
tgaagcatgt gttcccaagg 240 ttgatcagcc cacgcagacc tatggtgcag
ttcgaggtga tctttctcct tttcgggttg 300 tgcttcagca gttcaagctc
ccgtttggtt ggttcccaag ttgaaaactt ctctccaacg 360 ccttgcattt
tccaagcttt tcgctgctcc tccttggcga ttatttccat gtctttgtca 420
tagatgtagt cctggcacag aaaacagtag atgcctccgt acatcagatc aatggccagg
480 ttgtgccgct tcgccttcgc atgctcgtga atatgcttct ttgtgaaaca
gccgaagaag 540 acacagtaga ggcaggaatg cagcctgttg aggtggacgc
cacagacatg gcagatacag 600 gacttggcct tgcgcttgcg ggcctcagcc
gtgccgctcc acacgaagca ctggtagatg 660 gcccgcaggt tctgcttcca
gttgtccacc ttgaagctgc ccaggtgcga gcagcccggc 720 ggcgctaccg
ccnnctcggc gtccatggcc tcgcc 755 36 604 DNA Homo sapiens 36
ggcctcccaa agtgctggga ttacaggcgt gagcaagatt atatttcttt aataaagaat
60 acgtgacttt tatttaagtt gtcagattta ttggaaatgt tgttcgtaat
atcttttgat 120 gtctgtggta cctgtaatga ttcctttttt actactactg
ttttttttct ctctctcttc 180 cacccaccat ccccatttac tttatttttc
catctttata ttttcaggct ctttattggt 240 cagaattctt agttgtagaa
aagagagttc acaccaggta ctcttaagca gaaaatgttt 300 tattaagggg
cacagacagc acagacagct tacaaaagtg taggggaccc aaacagaaac 360
cctttaattt tactgattag aatggggagc tcaacagagg caactattaa aatgtgcaga
420 aggtattcag aagattgtga gcaggcatat atgagagatt tcattacaag
ctctctacag 480 taaccaacca taatgcagaa tagaaattca ttattttgga
cttttgctac ctgtcaattt 540 aactgttacg tttttacgga cttgttaaaa
aagtgattag atagactgtt ttagattttc 600 ctta 604 37 812 DNA Homo
sapiens misc_feature (17) n equals a,t,g, or c 37 gaccattttt
agccaanctt ggaattaacc ctcacttaag ggaacaaaag ctggagcttc 60
caccgcgttg gcggccgctc tagaactagt ggatcccccg ggctgcanga attcggccac
120 gagaggactt ccccacctca tgcagctatt tgggccgtgg cgtctgaaat
ttattatttc 180 agagtcaccc ctttratgac cttggcagtg ractgcagtc
atctgtttag gcctttccat 240 ggcccacgtc aatgccgtta tttctgtttg
ttgcacattt gatttccttg ttgttggcat 300 ttagaaggcc ccctgcttcc
cagatcacac cacgggcatg gaccacagag attgcatctt 360 gtgagtctgt
agaaatggtc aaggccttgt cctctcttag gtccagagct caggtgaatg 420
cagattttcc cggccatctg tgctgaagtc cctgtgggga ggctcctggc tggtttcctg
480 taggtagaca gctacacgtc ctgcccttca ttggcttctt ttcatgaagc
tcctgccatc 540 tacaaaacat gtctcccttc ttgaatcaca tctctgttat
tgaagctctg gaagtcaacc 600 gggcgtggtg gctatgccta taatcccagc
attttgggat gccggggcgg gtggatcacc 660 tgaggtcagg agttcgggac
cagcctggcc aacatggcga aaccccgtct ctaatacaag 720 tgcaaaaatt
ggccaggcgt ggtggtcact gtgctccagc ctgggtgaca gagcgagctc 780
cgtctcaaaa aaaaaaaaaa aaaaaactcg ag 812 38 1149 DNA Homo sapiens 38
gtaaaagtta aaaatgtttg ccttttatgt tcaggtttta aatcaatcta aaagtatttt
60 tgtatacagt aggaatctaa ttttttttat ccatatgata gtcagttggc
cctcctttct 120 tcagttacct gcagtgcatc aatgtcatca gtcaagtgtc
catatatgtg gtgtttctgg 180 gcttttcccc tcttccaatt atcagtgttt
atccctgtgt caaaatcaca cagtattaat 240 tattacaact ttatagtaag
tcttaatatt tagtagggca agtcttttta tttgataaga 300 gtatctttgc
tattctttta cttttactct tctgcatata ttttagaatc agcctgtcaa 360
attcctcaaa agactgtttg gataaatttg gaattacatt caatatagat caatttggga
420 ataaccaaga gttttgggat attgagtctt cattcattaa catagctttt
ctcttcattt 480 atttaggtct tattttttca tagagcttta aaaggtttac
tataaaatct ttaaggggta 540 aactcttgaa taaggtccag agttaaatag
tggagagtta caggtgtctg tttgccccaa 600 acagactgat ggtatttcag
ccatctcagg atttggggaa gtcagcacaa ggatcaaaag 660 actgagaaag
tttaacttct cctctaggat gtttttagta cacacattat atttataagg 720
aaacatattg taaataatag agtgataatg gccacttacc tgagccttat tataaacaga
780 aacaaatatc caaccaatgc catagagcag aagtacagcc tgaaaccaag
aagaaaaaga 840 gcaattaaat caggatctca tctaggccaa gcttgaaaag
gaagcttatt tcctatttgt 900 cttctgccta atgaatgtgt cattactggg
gaccttacca ataaaggtgc ttggatacat 960 tttccagcac agaaacttaa
tttgcaggaa catgatcttt acttgtaaaa ggatacattc 1020 ttaattcggg
atggttattt gagctgattt tcaaggcatt tatttttaat ttatttgtca 1080
gatgggaata aaggtggttt ataattaaat ttaaaagata gtttaaaaaa aaaaaaaaaa
1140 aaactcgag 1149 39 1087 DNA Homo sapiens 39 gcccactgga
aatgatcttg tttatgtgtt tccttgttta ctgtctgtct tcagtagaat 60
ggaagagcca tcggtatttt gtcttttttt ccccttgtcc attcttatat cctcagcttc
120 tagaacattc cctggaacac agcaagtgtt cagtattgtt catggagtga
cagatgtctc 180 agccaagaag gtacaatcac agggaagaat gacttcaact
ggtcttgact tcaacctgct 240 tccagcctgg ttcccttctc ccacctccct
acagcccaca gaagatcttt tccaaactgg 300 aagtctgtcc aggtcattct
tctgctctaa ggctttcagt agctccccct tatccccagg 360 aggaagtcca
aatgccttaa caagtgtcaa ggaacacttg gtgagccctg ctttccttgc 420
tagtcactcg tgcacagctg agtctttccc cagagtggat gtcattcatg ctgttcccat
480 tgcctggatt cctgctcctc tccaccccat tcaactgata aactcgtggt
tttttttttt 540 tttttttttt ttttgagack gagtctcgat ctatcaccca
cgctggagyg cagaggttgc 600 tgtgagctga ggtggtgcta gtgccctcca
gcctggatga cagagtgaga ctcggtctca 660 ataaataaat aaataataaa
gaagaaacca acccttttga ccccggggtc tcagactttt 720 agccaccaca
tcagcgagtt atggtgtttg ttatagcagc tctgggaaac taatgcatgt 780
tttcagtgac attttagccc cttctgaatg gtctggatgg ctggttgaca gctaagttca
840 gacccgatgc aaaagcgcag tctatgtagg aatgtcccct gtggacagct
gctgtgtagc 900 caaggtgggt tacatactag gaaagggccc tgggggcccc
cacagggagc taactctatt 960 gacgggggac ccaggtaaca gatgcaggca
tttgctgtga gtcacaagac actgatgtgt 1020 gtttgcttgc ctgggcaaca
tagtgagacc ccgtctctat taaaaaaaaa aaaaaaaaaa 1080 actcgag 1087 40
1276 DNA Homo sapiens 40 gtgagtgtgt ggcactggtg gcctggagcc
aaatttagct tgggtgagag ttgacaatgg 60 tagttttcct tcctcaagcc
cctctgtgcc cctagagcac cctggctgtg gctgcctcct 120 tcatccaaga
gcagagtcca tgttgggcca ggagacttca gatccatgtc ctggtgctgc 180
ctctggcttt gtctttcctc agtgggcagg actgggtctg ctggtccatc tttacccttc
240 tctgagctat gcagccttgg cctgctgcgt ctccggcctg tattctctcc
ccttcactca 300 ggccctggga aaccagccca gtttctkgca ggagaggcag
aggaggtcaa tgcctttgct 360 ctgggcttcc tgagcaccag cagtggtgtc
tctggagaag atgaagtaga gcccttacac 420 gatggagttg aagaggcaga
gaaaaagatg gaagaagaag gtgtgagtgt gagtgaaatg 480 gaggcaacag
gagcacaagg acccagcagg gtagaagagg ctgagggaca cacagaggtg 540
acagaagcag agggatccca ggggactgct gaggctgacg ggccaggagc atcttcaggg
600 gatgaggatg cctctggcag ggcagcaagt ccagagtcgg cctccagcac
ccctgagtct 660 ctccaggcca ggcgacatca tcagtttctt gagccagccc
cagcgcctgg tgctgcagtc 720 ttatcttcag agcctgcaga gcctctgttg
gtcaggcatc cccctaggcc ccggaccacc 780 ggccccaggc cccggcaaga
tccccacaag gctggactga gccactatgt gaaactcttt 840 agcttctatg
ccaagatgcc catggagagg aaggctcttg agatggtgga gaagtgccta 900
gataaatatt tccagcatct ttgtgatgat ctggaggtat ttgctgctca tgctggccgc
960 aagactgtga agccagagga cctggagctg ctgatgcggc ggcagggcct
ggtcactgac 1020 caagtctcac tgcacgtgct agtggagcgg cacctgcccc
tggagtaccg gcagctgctc 1080 atcccctgtg catacagtgg caactctgtc
ttccctgccc agtagtggcc aggcttcaac 1140 actttccctg tcccacctgg
ggactcttgc ccccacatat ttctccaggt ctcctcccca 1200 cccccccagc
atcaataaag tgtcataaac agaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260
attggggggg ggcccc 1276 41 2083 DNA Homo sapiens 41 gccctttacc
cccaacccca ggccactggg cccttcccac accacttggg gagctgagaa 60
gaggaggctg gagtaaggga ggacttgatc atccaagaaa tactttttat tgctgggagt
120 cttctgaacc tcaccaaact gaggccagag ctgagctcct gggggagtta
attcagaggg 180 gagaggccag cacctccctc ctccatygct cgctgtgtgc
cttaaactcc atctcatgtc 240 cctccccatc ccctggcttt ccctccctcc
ttgccccatc ctgggccagc cagcagggct 300 cctcctctgg ctcttcagac
ctttcagcca gtgctgtcag tgcccctggg agggaagggc 360 atccctgagg
cacccgaatg gtccctcagg gtgcagggag gcagaagcct ggccacagag 420
gagcctccta aggcagcagc tgcagcaagc gcaccctctc cccactctcc ccacgccaga
480 gcggcttcca gagcagatgc tgtttccatc ctcctcgtca aaaccattct
cgctgctgag 540 cttgacaatc tgggcaaggc ttgtggggcg cttgacaaac
agaatctgcc ctgtgccgcc 600 tggttccgtg gcctccagca tgagcctgca
ggcagggcgc tgcgggaacc cagttgtgct 660 gccccagccc atgcctccgg
gtctgctgtg catgaatgag tgctcacttg tcccgggttt 720 aggacgtggt
caagtgaaca gcagggtcta actgtgctta cttagcccag ttcaaacaga 780
acaaaggaaa aatatagaaa gcaacatctg ttgatcattt aggttttttt ttaaaccacc
840 atgtcacttt gagtccttca tgggtttttg aacagcattt atcaagaaga
aaatgtgggc 900 tttttcccct ctcccgtgtt ttgtttgtcc tgtagataga
gggaggaaag ccgtgcagtg 960 gcaggcggga ccccctctgg tggcgggacc
ccctcttgcg gtggtcttgc ggggccagcc 1020 gggacctgtc actttattat
ttaaggagtg tgtgtgtaga gtcgctggct tattaacagt 1080 attgtgtgtg
ggttgggttt ttagtttgtt ccttcttttt gaagtccctt catttcaatc 1140
cttgactctc tctccccttc ccttgcccag ctctgttgaa tgctgctgtg cgcgtgtgag
1200 ggccgctctg cacacagggc ccttgggttg tgtgaactga aattctccct
gtatttgtga 1260 gactcgcagg agtccccatc tgtagcacag gcaatgccag
tgccatgctg cagcctcaga 1320 aaccaggcct ctcactccag cagcaggcag
aaccgtgtct gtggtcgggt gctgtccaca 1380 gctctgtctg ccttgttctt
gggcttgagc tggatagagg tggggtctct tcaccttccc 1440 tgaattcaga
acagaccctg tgcctggccc cagtgtgccc aggcaattcc ccaggccctc 1500
attgggagcc cttggtgttc tgagcagcag ggcccaggca gcacatgagc agtgcccagg
1560 ggctccctgc gtgaggacgg caaggtgcga tgtatgtcta acttattgat
ggcaggcagc 1620 cccctgtgcc ccctaagcct ggccctggtt attgctgagc
tctgtgctca gtgctgcggc 1680 ctggccgtgg ctcgtctgtt cctttggggg
gcccgggcgg gttgtgggaa tcagtcttca 1740 cagacagacg tgagccaggc
ggaggactcg ttccttgcag aggtcagtcc tcacctgcag 1800 gtgtcggggt
ggrggggggc aaggaggggc aggcacacac catgtctgac ctgaacccga 1860
ttctggggag catcttcccg ctccggcccc acgacctcca cagggttaca ttgtaatata
1920 tatgccccag ctaacctgtc tgatggtggc atcttcctgc agacatttca
aacatgtaac 1980 ttttatatga aaaaaaataa acacagatga aagctgccca
aaaaaaaaaa aaaaaaaaaa 2040 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa agg 2083 42 1016 DNA Homo sapiens 42 taattcggca
gagccagcct gggtgcctgt atgcgctcga ggcgggggcg cgggttgcgg 60
acggaggcgc gggaggcggt tctgcgcggc gggggccgta cccgcggcgg agcgaggagg
120 cgagaatgga tcaatggtgt cacggagcac atcgctgacg ctgattgtgt
tccttttcca 180 cagattgtct aaagccccag gaaaaatggt ggaaaattca
ccgtcgccat
tgccagaaag 240 agcgatttat ggctttgttc ttttcttaag ctcccaattt
ggcttcaaaa atctgaaggg 300 atctcgtgtt tgctgagtgt tccgtgtgcc
agcactagcc taggaggttt tagtctctac 360 aaatatttat acagcaccag
acgcgatggc aaagtggata aagcctggtc cctgccctta 420 aggaggtcgt
ggtcttgtgg aggagacaga tggtacttta cctcgtgtgg gcctttattc 480
ctgaatcttg gctaaactct ttaggtttaa cctattggcc tcaaaaatat tgggcagttg
540 cattacctgt ctacctcctt attgctatag taattggcta cgtgctcttg
tttgggatta 600 acatgatgag tacctctcca ctcgactcca tccatacaat
cacagataac tatgcaaaaa 660 atcaacagca gaagaaatac caagaggagg
ccattccagc cttaagagat atttctatta 720 gtgaagtaaa ccaaatgttc
tttcttgcag ccaaagaact ttacaccaaa aactgaactg 780 tgtgtaacca
tagtaacacc aagcacgtat ttatttataa gtttttgcca ttataatttt 840
gaccataaat taatttgacc atctctctta ttaatagaga agtaaaaaat gtaagttgac
900 cttctcttag attatgttca atgaatattg taaatgttca agtattgtta
atgaatagaa 960 taaatacaat attgcattcc cataaaaaaa aaaaaaaaaa
aaaaaaaaac tcgtag 1016 43 2197 DNA Homo sapiens 43 ggtttgagaa
actgtagtac ctagctttct ccaaggctgt ttgatgctgg gatcctcttg 60
tggggggcgt ctgtcaacgt caccatttgg gaagtgcgca ragctcarag cagcgcttcc
120 atgcttcctt ctgcttgggg gcccctccag gtagcttctt tcttcttgct
atcttttyct 180 ttctgttttc tctcctcttc cccgcacctt gggaggcagg
agacccactt kgtkgtgctk 240 gargatgatg agggcgcccc gtgcccagca
gaggatgagc tggccctgca ggacaacggg 300 ttcctgagca agaatgaggt
gctgcgcaca aggtgtctcg gctcacggag cggctccgca 360 agcgctaccc
caccaacaac ttcgggaact gcacgggctg ctcggccacc ttctcagtgc 420
tgaagaagag gcggagctgc agtaattgtg gaaacagctt ctgctctcga tgctgctcct
480 tcaaggtgcc caagtcctcc atgggggcca cagccctgaa gcccagaggg
agactgtgtt 540 tgtgtgtgcc tcgtgtaacc agaccttgag caagtgagaa
gagaggccag ggtccaacca 600 ggcacccgtc cttggggcca gcagtagacc
ccccactctc cccacccctg gcccactgtg 660 gtgtgtgctg ggcaaatgtg
gcctgaatgc taggtaggct tccccttcct tcctcactct 720 ctccagctgg
attctggagc tgttctccat ccatgagagt ggctggcaat ggctgctctc 780
aatcccttga gggagaagag cccctggagg gcctggcatg tttgccctgc tctgcctggg
840 actgagcgag tggacttagg gctgggcagg cagtagccac cagagggcag
cagcgaacta 900 ggccaggcct gactggggtc tgaagatcag ggtcagtgtg
gctatgcctg ggaattccag 960 acctgaggtt gggaaaagag gtttttctcc
tgcagggtac tgggccaggc cctcagcctc 1020 agagagcctg cagaagggct
tgggagtgcc acaccccatc tctgctgatt gaatgtccct 1080 ccaggcacca
ggatctcatc atttccccat cagagggtgt ggccaggcct aacaagacca 1140
tgggtgcttc tagaaacagg gttgaagttc ccagattccc tgagaggaga atgtgtatag
1200 gagggtttgg ctgagtcctt cagcgttaag tggaggaaag cttggggaag
ccccaatagc 1260 tggacagacc tcagcctccc ctcgaagaca cctcaattca
cagactctca gcccacacaa 1320 tgccccagtg tccccagctc cgctggagca
gctgcagggc acttggatca caacttctgc 1380 accctctgtc cagagtctag
ggcagtcctc cactggccca gcactccagt ttcctttccc 1440 tgcctcttgt
ccaatggagt gggaggccag gtgagtggag cagaggtcct gaagcccttg 1500
acccctgggg gcctgggtag tgtaggatct cgctgggctg ggtcctggat tccagggcta
1560 ttccctggag gacagtctca gttatgggat aaggccccct gggggtctcc
atttctttcc 1620 aacagtttca tgttcactac tggactctta cgggctcagt
atctctccct tagccatgag 1680 ctggctcagg catcccttcc cttccctgga
gctgccctgc ctttctcaag tatttattta 1740 tttattgcat ggttcctggg
aacatgtggc acaagtaatg ggatgaggag gaattggggg 1800 tgggggtctt
ctacctagga ctcttccctg gagtcatggg ctgcctggga cccaggaccc 1860
atgagggggc tgagaggttt ctacactcga ggagcagggg tccagagagg caggctgggg
1920 aggcaaggga cccatcctag gcccgctttc ttgccgagcc aagcagctta
gctggggctg 1980 tgcagccagg ggcttaccca ggccagtgga ggtgccacag
ccctggggag ccagacaggc 2040 tttggtatcg tatcgcctct gtgtcctttt
aagagaggag agttcagtac cccgtgcttt 2100 ctttacactg gagaggaact
aaaaggatct ctgtgtctat ggagaattgt caataaaaag 2160 gcctcaagct
tmaaaaaaaa aaaaaaaaaa ctcgtag 2197 44 1999 DNA Homo sapiens
misc_feature (965) n equals a,t,g, or c 44 ggcacgagcc caaccacaca
cctggggaat tgctggcctg acttctgacc cctgactcct 60 catacccttc
ctccagagca tgacatttga ccaccaactg aaacctgacc tctgacccca 120
gaccactggc ccttcccccg ccctgtggtg acttcataaa ggttactagc ttctcccctg
180 gccttgagac ccacacgatg gccctgctgg ctctggccag tgccgtcccg
tctgccctgc 240 tggccctggc tgtcttcagg gtgcccgcct gggcctgtct
cctctgcttc acaacctact 300 ctgagcgcct ccgcatctgc cagatgtttg
ttgggatgcg gagcccaagc ttgaagagtg 360 tgaggaggcc ttcacggccg
ccttccaggg cctctctgac accgaaatca gtgaggagac 420 catccacact
tcatcagtgt cctggggaag gtgcagaggg agggcaggag aggcccagag 480
ggtcaggctg agggacagac agagagaaac agtcagagga gaaaggctca aagaccatga
540 gaacaacaga gacttaggga cagagagaca cagacagggg asgacagcag
ggcaaagact 600 cagagagggg aggatggaga gtcagagagg ggaagatgga
gactcagaga gggggaggat 660 ggagactcag agagagagga agatggagac
tcggaaagat ggagactcag gagtatggag 720 agtcagagag gggaggatgg
acactcggga ggatggagag tcaggaggat ggagactcat 780 agaaagggga
ggatggagag tcaggagagg ttggagactg gagagggaat agagacccag 840
agaggggagg atggagactc agagggtgga agatggagac tcaaagagga tggaaaccca
900 ggagagagga ggacagagat gaggcagaga ctaggggaag caggatagcg
actggtcggg 960 ggcanagact canggaggat agagacttgg gagggactca
ggaagcatag cgactgtggg 1020 gcaaagagtc agagagggga ggatacagac
ttgggagggc agagactcag aaacagaatg 1080 ttcgcattag ggacatggtg
ttgcggggan ctgcctcccc cagcccctgc tccctccctc 1140 accgccagac
tatgatgaga gaagccacct gcatgacacc ttcacccaga tgacccatgc 1200
cctgcaggag ctggctgctg cccagggatc ctttgaggtt gccttccctg atgctgcgga
1260 gaaaatgaag aaggtcttta cacagcttaa agaagcccag gcttgcatcc
ctccctgcga 1320 aggtctccag gagttcgccc ggcgtttcct ctgcagcggg
tgctactcta gggtctgcga 1380 cctcccgctg gactgcccag ttcaggatgt
gacagtgact cggggcgacc aggctatgtt 1440 ttcttgcatc gtaaacttcc
agctgccaaa ggaggagatc acctattcct ggaagttcgc 1500 aggaggaggt
ctccggactc aggacttgtc ctatttccga gatatgccgc gggccgaagg 1560
atacctggcg cggatccggc cggctcagct cacgcaccgc gggacgttct cctgcgtgat
1620 caagcaagac cagcgccccc tggcccggct ctacttcttt cttaacgtga
cgggccgccc 1680 ccgcgggcgg agacagagtt gcaggcctcg ttccgggaag
tgctgcgctg ggcgccgcgg 1740 gatgccgagc tgatcgagcc ctggaggccc
agcctgggcg agctgctggc caggcccgag 1800 gctctgacgc ccagcaatct
gttcctgctt gcagtcctcg gggccctcgc atcagcgagt 1860 gcgacagtgt
tggcgtggat gttctttcga tggtactgca gtggcaacta acaaaggtat 1920
ctttcctcct tccctatcct atttccatcc tgaaaataaa gaatatattt caactctaaa
1980 aaaaaaaaaa aaaaaaaaa 1999 45 1519 DNA Homo sapiens 45
cggcacgagg taaattctgc cttcacccag tatatctttc caatgtagga tgatttattt
60 ttcattacat ttgttctaag attttgtagt gattactcta gtgtttgtca
tgtaaatgtt 120 agtttatcaa aatcaggctc agttctccag taatatgtgg
ttaaatttca gtgatgtaca 180 cacatatttg tcctctatag ctctattatg
tttttgcctt tctggggtac tttgttgcat 240 atgtaacaac tcagtgttcc
acattcaaca atatattctt ataattatta cttttccact 300 ggtagtcatt
tagttcagtt tttatggtgt tatttataaa tataatacat gtatgttaaa 360
tttttctatg ttgtaggcct aacaatccat tactacttat tactttacac aattgacttg
420 taaatccata acaaggagaa aggtgaagaa acacatattc agtttgtctt
ttccacttat 480 atagtcttgc atggtgctct ctgctttttc ctgtggattt
tgacagactc aataattttt 540 tttttttttg agatggagtc ttcctctgtt
gtcccaggct ggagtgcggt ggctctatct 600 tggccactgc agcctccacc
tgccgggttc aaatgattgt cctgcctcag ccccccaagt 660 agctgggact
acaggcgtgt gccaccacac ccggctaatt tttgtatttt tttttttttt 720
tgtagagaca gggttttgcc atgtttgcca gggtgctctc aaactcctga cctctagtga
780 tccaccagcc tcggcctccc aaagtgctga gattacagac gcgagccacc
gcacctggca 840 ctttggtctt gtagggctgg tgtgccagga aaaattctct
caatgttcat atttttaagc 900 ctaggaaaat atttgttttg tctttcattt
tgaacatgaa cattgttgga ttctggttaa 960 ggtgtgtttt ggagcagagc
atgatgaggg actgggtgag gggcttccca gtgaaaggag 1020 gaacagaacc
tagcaaaagc aaatatgtct gtttcactcc aatcacttat tacaataacc 1080
tctattactt aatatcaaaa aaaaagcaat gatcaatatg ttgaaaggga tggaagaagc
1140 aagtatgatg tttgaccata atagcattca ttgcttggga aactaaagac
aaaacaactg 1200 gaaaactatt aacactgttg gccaggtgtg gtggctcatg
cctgtcatcc cagcactttg 1260 ggaggctggg gtggatggat cgcttgagcc
caggagttcg agaccagcct gggcaacatg 1320 gcgaaacctc atctctacaa
aaaatacaaa aaattacccg ggtctggtgg tgtgttcctg 1380 tggtcccagc
tactcgggag gctgagatgg aaggattgct tgagcctggg aagtcgatgc 1440
tgcagtgagc caggattgca ccactgcact ctagcctgag tgacagagtg agaccctgtc
1500 taaaaaaaaa aaaaaaaaa 1519 46 1189 DNA Homo sapiens 46
ggcacgagat ttaatacaaa gtttgctttg agacttttca gcatatgatc ttttttccat
60 aaacttgtac agtgcaaaag acattttgaa taccatgatc gatgatgtcc
catgcttcga 120 ggaaaaccaa acactttccg cctctcttgc aaaatccatt
cctcatgctg accctcctca 180 cgatggctgt gtcagcccag ccccttccct
tctccaggcc cagataactc ttccacaaac 240 aagatgagag ccactcggga
aaagagccat agtcaactgg gagggcctac atctggatgg 300 cggtggaaaa
acttgagggt ttggggttca aagtcagccc atcccacctg gcaaaatcct 360
cctggaagga ggaccttcaa gagcgcatca cctgaatgtc atgaagaagt atctctgaat
420 gtatccagga gaggaactgc ataaccaaag gggtgaccag ccctcagatg
tgcttattgg 480 attccagtac aaacgccacc aaagccagcc cactgctctc
ctacaaggaa ggaaagatct 540 gcacgtgtaa aacatggggc agccttggaa
catggtgttt tttggagttt cctttctcac 600 agttttccat ctccccactt
ctttgatcag tcatgtgtcc gtgacctcgt tccatgacat 660 caggatagct
gtgtttgcac accatgctgc atgttcattt ggagccagga ggggttctca 720
gtggagcctg gcttagggaa cagggagcga tggaagaatg ccaacattag cgttggtctt
780 ctcttgtcag gaatgaagga tgcttgcaca catgcacccc ctcactctca
cacttgcaca 840 catacacaca cacacacacg aaatggttgg tttgtcaaaa
ctcactgtag tacataaagc 900 ttgcactctg cgtcctatat ctagcagcat
ggggtacgtt tggcagttca ctccattagg 960 gggtaaataa tttatgacca
ttcatctgtt tttatgaatt tttttatcta gacaataatt 1020 gtaaataaag
aactcaccat ctctgttcat ttaatactat gcaatggtta tgctttcaat 1080
cgctggctct tctgactcgt gcagtgtggt tctgaaatgt ttgtggttta aaaaaaaaag
1140 caaaaaacac tcaacagaac atagtaaata taaaaaaaaa aaaaaaaaa 1189 47
2584 DNA Homo sapiens misc_feature (1389) n equals a,t,g, or c 47
acccacgcgt ccgcgggcac cggccgacat ggcggcagcg gtggcggctg cgctggcgcg
60 gcttttggcg gcctttctgc tcctcgcggc ccaggtggcc tgtgagtacg
gcatggtgca 120 cgtggtctcc caggccgggg gccccgaagg caaagactac
tgcatcctct acaacccgca 180 gtgggcccat cttccgcacg acctcagcaa
ggcatctttc ctgcagctgc gcaactggac 240 ggcctccctg ctctgctccg
cagccgacct ccccgcccgt ggcttcagca accagatccc 300 gctggtggcg
cgggggaact gcaccttcta tgagaaagtg aggctggccc agggcagcgg 360
acacgcgggc tgctcatcgt cagcagggag argctggtcc ccccgggggg taataagacg
420 catatgatga gattggcatt cccgtggccc tgctcagcta caaagacatg
ctggacatct 480 tcacgcgttt cggccgcacg gtgagggcgg cgctgtatgc
gcctaaggag ccggtgctgg 540 actacaacat ggtcatcatc ttcatcatgg
ctgtgggsac cgtcgccatc ggcggctact 600 gggccgggak tcgggacgtg
aagaaaaggt acatgaagca caagcgcgac gatgggcccg 660 agaagcagga
ggacgaggcg gtggacgtga cgccggtgat gacctgcgtg tttgtggtga 720
tgtgctgctc catgctggtg ctgctctact ayttctacga cctcctcgtg trcgtggtca
780 tcgggatctt ctgcctggcc tccgccaccg gcctctacag ctgcctggcg
ccctgtgtgc 840 ggcggctgcc cttcggcaag tgcaggatcc ccaacaacag
cctgccctac ttccacaagc 900 gcccgcaggc ccgtatgctg ctcctggcgc
tcttctgcgt ggscgtcagc gtggtgtggg 960 gcgtcttccg caacgargac
cagtgggcct gggtcctcca ggatgccctg ggcatcgcct 1020 tctgcctcta
catgctgaag accatccgtc tgcccacctt caaggcctgc acgctgctgc 1080
tgctggtgct gttcctctac gacatcttct tcgtgttcat cacgcccttc ctgaccaaga
1140 gtgggagcag catcatggtg gaggtggcca ctgggccctc ggactcagcc
acccgtgaga 1200 agctgcccat ggtcctgaag gtgcccaggc tgaactcctc
acctctggcc ctgtgtgacc 1260 ggcccttctc cctcctgggt ttcggagaca
ttttggtgcc agggctgctg gtggcctact 1320 gccacaggtt tgacatccag
gtacagtcct ccagggtata cttcgtggcc tgcaccatcg 1380 cctatggynt
tggcctcctt gtgacattcg tggcactggc cctgatgcag cgtggccagc 1440
ccgctctcct ctacctggtg ccctgcacgc tggtgacgag ctgcgctgtg gcgctctggc
1500 gcygkgagct gggcgtgttc tggacgggca gcggctttgc gaaagtccta
cctccatctc 1560 cgtgggcccc agcaccagcc gacggcccgc agcctcccaa
agactctgcc acgccactct 1620 ccccgcagcc gcccagcgaa gaaccagcca
catccccctg gcctgctgag cagtccccaa 1680 aatcacgcac gtccgaggag
atgggggctg gagccatgct gggascctgg gagcccagct 1740 gaatccgagg
gccgggacca ggccagccgt ccccggtaac ccagcctggc gcctcggcct 1800
aggggagggg tgagacgctc gctgccgtgc ccgccacacc aagatgttgg ggctgcctgg
1860 cgcccactgg agacagacag acagacgcyt gtcccccggg accgaggcct
gtgccgtccc 1920 cacccgcccc aacatggtgc ttatccttgc cgagacccct
gcagtccgtg cccgcgccca 1980 gcccagctgc cccggctgca cgcctgctgc
tcccagctcg cccggctgcc acaagctctc 2040 tgcgggtcca tcctccccgc
aggaggaggg gtccgtcctc gcaggccytg cccggcctct 2100 ctgcagaccc
tcaagcgtcg tctgcatgag tgagcaggcg tgggtggact ctggccgcgg 2160
ccacacttgg tgctcaccag ctgcttcggc cttcaggtga cctccctccc cacggcatcc
2220 tgctctccgg gtggaagagc agctttctgt ctcccagaag gcatcgcttt
tccctcttga 2280 gcagatcgga gcccctggga ggtttggaag ctgcctccaa
gcctaggaca cggaccggtg 2340 gccggggcgg cctctggccc ctgacgctgg
ctgagacagg cccgtggggc ggggttttgg 2400 ggcgtgaaca aggctggcag
taagtggaca agctgctccc ctggctaagg ccctgccctg 2460 ccctcagcca
gaggtgcctg gccatgcctg cacactcctc cccattttaa taaatggtcg 2520
caacttcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagggcgg
2580 ccgc 2584 48 55 PRT Homo sapiens 48 Met Ile Lys His Ala Leu
Ile Arg Pro Phe Ile Val Phe Ser Leu Leu 1 5 10 15 Leu Arg Leu Cys
Ser Glu Asn Leu Phe Cys Pro Asn Thr Gln Phe Ile 20 25 30 Val Leu
Ser Cys Phe Gln Ser Val Val Lys Ser Leu Leu Ser Ile Leu 35 40 45
Asn Leu Ser Tyr Cys Ile Phe 50 55 49 40 PRT Homo sapiens 49 Met Asn
Ser Cys Leu Phe Leu Cys Ile Leu Ile Leu Glu Ser Ala Met 1 5 10 15
Val Val Leu Met Lys Val His Phe Ile Val Ala Phe Glu Leu Thr Ala 20
25 30 Lys Ala Ile Asn Gln Lys Gln Lys 35 40 50 93 PRT Homo sapiens
50 Met Ala Arg Lys Ser Phe Ala Leu Leu Met Phe Val Trp Gln Met Ser
1 5 10 15 Leu Ser Leu Pro Ile Lys Gly Phe Ile Leu Arg Val Ala Asn
Trp Leu 20 25 30 Phe Lys Pro His Leu Asn Ser Val Cys Leu Gly Trp
Gln Asn His Thr 35 40 45 Arg Phe Cys Trp Ala Asn Leu Pro Gly Gly
Val Leu Leu Glu Glu Ser 50 55 60 Ala Thr Ala Glu Asp Thr Leu Ser
Trp Pro Leu Ala Leu Gln Thr Ile 65 70 75 80 Val Glu Glu Gly Val Trp
Gly His Gln Pro Leu Pro Gly 85 90 51 83 PRT Homo sapiens 51 Met Leu
Ser Leu Phe Phe Cys Phe Trp Lys Pro Ser Phe Leu Val Ser 1 5 10 15
Arg Leu Val Ile Trp Leu Gly Leu Val Cys Gly Gly Arg Ser Leu Ser 20
25 30 Trp Val Ala Leu Gly Glu Asp Tyr Leu Gly Thr Pro Ile Leu Ile
Pro 35 40 45 Asn Ile His Gln Thr Cys Pro His Pro Pro Leu Trp Glu
Leu Val Pro 50 55 60 Glu His Pro Cys Arg Leu Val Leu Ile Phe Ser
Leu Cys Glu His Thr 65 70 75 80 His Ile Arg 52 65 PRT Homo sapiens
52 Met Leu Ser Pro Lys Ser Pro Arg Met Leu Leu Pro Cys Leu Leu Gln
1 5 10 15 Pro Leu Val Val Ala Asn Ile Pro Arg Val Pro Trp Leu Ala
Asp Glu 20 25 30 Ser Leu Asn Pro Thr Pro Ile Ile Thr Trp Gln Ser
Pro Cys Val Ala 35 40 45 Gln Leu Cys Pro Asn Phe Pro Phe Pro Thr
Arg Thr Leu Val Thr Gly 50 55 60 Leu 65 53 52 PRT Homo sapiens 53
Met His Cys His Ser Ala Leu Gly Pro Met Ser Thr Pro Val Leu Pro 1 5
10 15 Phe Ser Gly Ile Gly Leu Ala Phe Leu Cys Leu Cys Leu Ala Ala
Ser 20 25 30 Met Val Asp Leu Lys Cys Leu Gly Met Asn Ser Thr Leu
Leu Gln Pro 35 40 45 Ser Ile Lys Glu 50 54 540 PRT Homo sapiens
MISC_FEATURE (10) Xaa equals any of the naturally occurring L-amino
acids 54 Met Ala Thr Ser Gly Ala Ala Ser Ala Xaa Leu Val Ile Gly
Trp Cys 1 5 10 15 Ile Phe Gly Leu Leu Leu Leu Ala Ile Leu Ala Phe
Cys Trp Ile Tyr 20 25 30 Val Arg Lys Tyr Gln Ser Arg Arg Glu Ser
Glu Val Val Ser Thr Ile 35 40 45 Thr Ala Ile Phe Ser Leu Ala Ile
Ala Leu Ile Thr Ser Ala Leu Leu 50 55 60 Pro Val Asp Ile Phe Leu
Val Ser Tyr Met Lys Asn Gln Asn Gly Thr 65 70 75 80 Phe Lys Asp Trp
Ala Asn Ala Asn Val Ser Arg Gln Ile Glu Asp Thr 85 90 95 Val Leu
Tyr Gly Tyr Tyr Thr Leu Tyr Ser Val Ile Leu Phe Cys Val 100 105 110
Phe Phe Trp Ile Pro Phe Val Tyr Phe Tyr Tyr Glu Glu Lys Asp Asp 115
120 125 Asp Asp Thr Ser Lys Cys Thr Gln Ile Lys Thr Ala Leu Lys Tyr
Thr 130 135 140 Leu Gly Phe Val Val Ile Cys Ala Leu Leu Leu Leu Val
Gly Ala Phe 145 150 155 160 Val Pro Leu Asn Val Pro Asn Asn Lys Asn
Ser Thr Glu Trp Glu Lys 165 170 175 Val Lys Ser Leu Phe Glu Glu Leu
Gly Ser Ser His Gly Leu Ala Ala 180 185 190 Leu Ser Phe Ser Ile Ser
Ser Leu Thr Leu Ile Gly Met Leu Ala Ala 195 200 205 Ile Thr Tyr Thr
Ala Tyr Gly Met Ser Ala Leu Pro Leu Asn Leu Ile 210 215 220 Lys Gly
Thr Arg Ser Ala Ala Tyr Glu Arg Leu Glu Asn Thr Glu Asp 225 230 235
240 Ile Glu Glu Val Glu Gln His Ile Gln Thr Ile Lys Ser Lys Ser Lys
245 250 255 Asp Gly Arg Pro Leu Pro Ala Arg Asp Lys Arg Ala Leu Lys
Gln Phe 260 265 270 Glu Glu Arg Leu Arg Thr Leu Lys Lys Arg Glu Arg
His Leu Glu Phe 275 280 285 Ile Glu Asn Ser Trp Trp Thr Lys Phe Cys
Gly Ala Leu Arg Pro
Leu 290 295 300 Lys Ile Val Trp Gly Ile Phe Phe Ile Leu Val Ala Leu
Leu Phe Val 305 310 315 320 Ile Ser Leu Phe Leu Ser Asn Leu Asp Lys
Ala Leu His Ser Ala Gly 325 330 335 Ile Asp Ser Gly Phe Ile Ile Phe
Gly Ala Asn Leu Ser Asn Pro Leu 340 345 350 Asn Met Leu Leu Pro Leu
Leu Gln Thr Val Phe Pro Leu Asp Tyr Ile 355 360 365 Leu Ile Thr Ile
Ile Ile Met Tyr Phe Ile Phe Thr Ser Met Ala Gly 370 375 380 Ile Arg
Asn Ile Gly Ile Trp Phe Phe Trp Ile Arg Leu Tyr Lys Ile 385 390 395
400 Arg Arg Gly Arg Thr Arg Pro Gln Ala Leu Leu Phe Leu Cys Met Ile
405 410 415 Leu Leu Leu Ile Val Leu His Thr Ser Tyr Met Ile Tyr Ser
Leu Ala 420 425 430 Pro Gln Tyr Val Met Tyr Gly Ser Gln Asn Tyr Leu
Ile Glu Thr Asn 435 440 445 Ile Thr Ser Asp Asn His Lys Gly Asn Ser
Thr Leu Ser Val Pro Lys 450 455 460 Arg Cys Asp Ala Xaa Ala Pro Glu
Asp Gln Cys Thr Val Thr Arg Thr 465 470 475 480 Tyr Leu Phe Leu His
Lys Phe Trp Phe Phe Ser Ala Ala Tyr Tyr Phe 485 490 495 Gly Asn Trp
Ala Phe Leu Gly Val Phe Leu Ile Gly Leu Ile Val Ser 500 505 510 Cys
Cys Lys Gly Lys Lys Ser Val Ile Glu Gly Val Asp Glu Asp Ser 515 520
525 Asp Ile Ser Asp Asp Glu Pro Ser Val Tyr Ser Ala 530 535 540 55
177 PRT Homo sapiens MISC_FEATURE (67) Xaa equals any of the
naturally occurring L-amino acids 55 Met Phe Gln Val Arg Pro Gly
Trp Gln Leu Leu Leu Val Met Phe Ser 1 5 10 15 Ser Cys Ala Val Ser
Asn Gln Leu Leu Val Trp Tyr Pro Ala Thr Ala 20 25 30 Leu Ala Asp
Asn Lys Pro Val Ala Pro Asp Arg Arg Ile Ser Gly His 35 40 45 Val
Gly Ile Ile Phe Ser Met Ser Tyr Leu Glu Ser Lys Gly Leu Leu 50 55
60 Ala Thr Xaa Ser Glu Asp Arg Ser Val Arg Ile Trp Lys Val Gly Asp
65 70 75 80 Leu Arg Val Pro Gly Gly Arg Val Gln Asn Ile Gly His Cys
Phe Gly 85 90 95 His Ser Ala Arg Val Trp Gln Val Lys Leu Leu Glu
Asn Tyr Leu Ile 100 105 110 Ser Ala Gly Glu Asp Cys Val Cys Leu Val
Trp Ser His Glu Gly Glu 115 120 125 Ile Leu Gln Ala Phe Arg Gly His
Gln Asp Val Tyr Pro Val Val Val 130 135 140 Gly Ala Glu Ile His Ala
Glu Leu Tyr Gln Glu Leu Ala Tyr Leu Glu 145 150 155 160 Thr Glu Thr
Glu Ser Leu Ala His Leu Phe Ala Leu Val Pro Arg Pro 165 170 175 Glu
56 83 PRT Homo sapiens MISC_FEATURE (36) Xaa equals any of the
naturally occurring L-amino acids 56 Met Ser Leu Ile Trp Glu Gln
Gly Leu Gln Leu Cys Gly Phe Cys Leu 1 5 10 15 Phe Tyr Leu Val Phe
Cys Phe Cys Ile Ser Ser Leu Arg Val Met Ala 20 25 30 Phe Ser Cys
Xaa His Val Ala Cys Cys Lys Gly Tyr Asp Phe Val Leu 35 40 45 Phe
Tyr Gly Cys Val Val Phe His Gly Val Tyr Gly Pro His Phe Leu 50 55
60 Tyr Pro Ile His His Ile Trp Ala Pro Arg Leu Ile Pro Cys Leu Cys
65 70 75 80 Tyr Cys Glu 57 131 PRT Homo sapiens 57 Met Leu Trp Thr
Leu Thr Phe Phe Leu Leu Gln Arg Ser Leu Thr Ser 1 5 10 15 Pro Trp
Leu Phe Gly Leu Leu Phe Leu Gly Ser Ser Asn Thr Ala Val 20 25 30
Cys Cys Phe Leu Gly Gln Leu Ile Met Gly Pro Lys Gly Glu Arg Gly 35
40 45 Phe Pro Gly Pro Pro Gly Arg Cys Leu Cys Gly Pro Thr Met Asn
Val 50 55 60 Asn Asn Pro Ser Tyr Gly Glu Ser Val Tyr Gly Pro Ser
Ser Pro Arg 65 70 75 80 Val Pro Val Val Arg Leu Ser Gly Arg Ser Leu
Gly Trp Leu Ser Val 85 90 95 Arg Thr Ser His Leu Ile Leu Met Gly
Leu Cys Lys Ile Leu Ser Val 100 105 110 Lys Leu Thr Phe Phe His Asp
Ser Glu Tyr Thr Leu Ile Ile Gly Asn 115 120 125 Trp Lys Ile 130 58
187 PRT Homo sapiens MISC_FEATURE (167) Xaa equals any of the
naturally occurring L-amino acids 58 Met Gly Phe Phe Leu Val Leu
Val Met Glu Gln Ile Thr Leu Ala Tyr 1 5 10 15 Lys Glu Gln Ser Gly
Pro Ser Pro Leu Glu Glu Thr Arg Ala Leu Leu 20 25 30 Gly Thr Val
Asn Gly Gly Pro Gln His Trp His Asp Gly Pro Gly Val 35 40 45 Pro
Gln Ala Ser Gly Ala Pro Ala Thr Pro Ser Ala Leu Arg Ala Cys 50 55
60 Val Leu Val Phe Ser Leu Ala Leu His Ser Val Phe Glu Gly Leu Ala
65 70 75 80 Val Gly Leu Gln Arg Asp Arg Ala Arg Ala Met Glu Leu Cys
Leu Ala 85 90 95 Leu Leu Leu His Lys Gly Ile Leu Ala Val Ser Leu
Ser Leu Arg Leu 100 105 110 Leu Gln Ser His Leu Arg Ala Gln Val Val
Ala Gly Cys Gly Ile Leu 115 120 125 Phe Ser Cys Met Thr Pro Leu Gly
Ile Gly Leu Gly Ala Ala Leu Ala 130 135 140 Glu Ser Ala Gly Pro Leu
His Gln Leu Ala Gln Ser Val Leu Glu Gly 145 150 155 160 Met Ala Ala
Gly Thr Phe Xaa Tyr Ile Thr Phe Leu Glu Ile Leu Leu 165 170 175 Phe
His Pro Lys Phe Lys Gly Val Ser Arg Arg 180 185 59 40 PRT Homo
sapiens 59 Met Thr Phe Ser Pro Leu Ser Ser Thr Phe Trp Trp Ser Ser
Arg Phe 1 5 10 15 His Cys Glu Met Leu Trp Phe Val Ser Leu Leu Val
Thr Phe Thr Ala 20 25 30 His Ser Val Glu Tyr Ser Gln Tyr 35 40 60
338 PRT Homo sapiens 60 Met Tyr Gly Tyr Val Asp Thr Leu Leu Thr Met
Leu Ala Met Leu Leu 1 5 10 15 Lys Val Ala Met Asn Arg Ala Gln Val
Cys Leu Ile Ser Ser Ser Lys 20 25 30 Ser Gly Glu Arg His Leu Tyr
Leu Ile Lys Val Ser Arg Asp Lys Ile 35 40 45 Ser Asp Ser Asn Asp
Gln Glu Ser Ala Asn Cys Asp Ala Lys Ala Ile 50 55 60 Phe Ala Val
Leu Thr Ser Val Leu Thr Lys Asp Asp Trp Trp Asn Leu 65 70 75 80 Leu
Leu Lys Ala Ile Tyr Ser Leu Cys Asp Leu Ser Arg Phe Gln Glu 85 90
95 Ala Glu Leu Leu Val Asp Ser Ser Leu Glu Tyr Tyr Ser Phe Tyr Asp
100 105 110 Asp Arg Gln Lys Arg Lys Glu Leu Glu Tyr Phe Gly Leu Ser
Ala Ala 115 120 125 Ile Leu Asp Lys Asn Phe Arg Lys Ala Tyr Asn Tyr
Ile Arg Ile Met 130 135 140 Val Met Glu Asn Val Asn Lys Pro Gln Leu
Trp Asn Ile Phe Asn Gln 145 150 155 160 Val Thr Met His Ser Gln Asp
Val Arg His His Arg Phe Cys Leu Arg 165 170 175 Leu Met Leu Lys Asn
Pro Glu Asn His Ala Leu Cys Val Leu Asn Gly 180 185 190 His Asn Ala
Phe Val Ser Gly Ser Phe Lys His Ala Leu Gly Gln Tyr 195 200 205 Val
Gln Ala Phe Arg Thr His Pro Asp Glu Pro Leu Tyr Ser Phe Cys 210 215
220 Ile Gly Leu Thr Phe Ile His Met Ala Ser Gln Lys Tyr Val Leu Arg
225 230 235 240 Arg His Ala Leu Ile Val Gln Gly Phe Ser Phe Leu Asn
Arg Tyr Leu 245 250 255 Ser Leu Arg Gly Pro Cys Gln Glu Ser Phe Tyr
Asn Leu Gly Arg Gly 260 265 270 Leu His Gln Leu Gly Leu Ile His Leu
Ala Ile His Tyr Tyr Gln Lys 275 280 285 Ala Leu Glu Leu Pro Pro Leu
Val Val Glu Gly Ile Glu Leu Asp Gln 290 295 300 Leu Asp Leu Arg Arg
Asp Ile Ala Tyr Asn Leu Ser Leu Ile Tyr Gln 305 310 315 320 Ser Ser
Gly Asn Thr Gly Met Ala Gln Thr Leu Leu Tyr Thr Tyr Cys 325 330 335
Ser Ile 61 47 PRT Homo sapiens 61 Met Leu Thr Val Lys Ile Leu Lys
Cys Phe Leu Gly Trp Ala Val Val 1 5 10 15 Ala Gly Gly Leu Gly Arg
Ser Gln Ala Arg Pro Ser Leu Leu Phe Asn 20 25 30 Arg Leu Ser Pro
Ser Val Pro Gln Met Arg Ile Gln Gln Pro Trp 35 40 45 62 336 PRT
Homo sapiens 62 Met Ala Ala Ala Val Ala Ala Ala Leu Ala Arg Leu Leu
Ala Ala Phe 1 5 10 15 Leu Leu Leu Ala Ala Gln Val Ala Cys Glu Tyr
Gly Met Val His Val 20 25 30 Val Ser Gln Ala Gly Gly Pro Glu Gly
Lys Asp Tyr Cys Ile Leu Tyr 35 40 45 Asn Pro Gln Trp Ala His Leu
Pro His Asp Leu Ser Lys Ala Ser Phe 50 55 60 Leu Gln Leu Arg Asn
Trp Thr Ala Ser Leu Leu Cys Ser Ala Ala Asp 65 70 75 80 Leu Pro Ala
Arg Gly Phe Ser Asn Gln Ile Pro Leu Val Ala Arg Gly 85 90 95 Asn
Cys Thr Phe Tyr Glu Lys Val Arg Leu Ala Gln Gly Ser Gly Ala 100 105
110 Arg Gly Leu Leu Ile Val Ser Arg Glu Arg Leu Val Pro Pro Gly Gly
115 120 125 Asn Lys Thr Gln Tyr Asp Glu Ile Gly Ile Pro Val Ala Leu
Leu Ser 130 135 140 Tyr Lys Asp Met Leu Asp Ile Phe Thr Arg Phe Gly
Arg Thr Val Arg 145 150 155 160 Ala Ala Leu Tyr Ala Pro Lys Glu Pro
Val Leu Asp Tyr Asn Met Val 165 170 175 Ile Ile Phe Ile Met Ala Val
Gly Thr Val Ala Ile Gly Gly Tyr Trp 180 185 190 Ala Gly Ser Arg Asp
Val Lys Lys Arg Tyr Met Lys His Lys Arg Asp 195 200 205 Asp Gly Pro
Glu Lys Gln Glu Asp Glu Ala Val Asp Val Thr Pro Val 210 215 220 Met
Thr Cys Val Phe Val Val Met Cys Cys Ser Met Leu Val Leu Leu 225 230
235 240 Tyr Tyr Phe Tyr Asp Leu Leu Val Cys Val Val Ile Gly Ile Phe
Cys 245 250 255 Leu Ala Ser Ala Thr Gly Leu Tyr Ser Cys Leu Ala Pro
Cys Val Arg 260 265 270 Arg Leu Pro Phe Gly Lys Cys Arg Ile Pro Asn
Asn Ser Leu Pro Tyr 275 280 285 Phe His Lys Arg Pro Gln Ala Arg Met
Leu Leu Leu Ala Leu Phe Cys 290 295 300 Val Ala Val Ser Val Val Trp
Gly Val Phe Arg Asn Glu Asp Ser Gly 305 310 315 320 Pro Gly Ser Ser
Arg Met Pro Trp Ala Ser Pro Ser Ala Ser Thr Cys 325 330 335 63 84
PRT Homo sapiens 63 Met Lys Gly Trp Gly Trp Leu Ala Leu Leu Leu Gly
Ala Leu Leu Gly 1 5 10 15 Thr Ala Trp Ala Arg Arg Ser Gln Asp Leu
His Cys Gly Ala Cys Arg 20 25 30 Ala Leu Val Asp Glu Leu Glu Trp
Glu Ile Ala Gln Val Asp Pro Lys 35 40 45 Lys Thr Ile Gln Met Gly
Ser Phe Arg Ile Asn Pro Asp Gly Ser Gln 50 55 60 Ser Val Val Glu
Val Thr Val Thr Val Pro Pro Asn Lys Val Ala His 65 70 75 80 Ser Gly
Phe Gly 84 64 62 PRT Homo sapiens 64 Met Val Ala Val Thr Gly Gly
Val Gly Val Ala Ala Ala Leu Cys Leu 1 5 10 15 Cys Ser Leu Leu Leu
Trp Pro Thr Arg Leu Arg Arg Ser Arg Gly Gly 20 25 30 Glu His Arg
Thr Pro Ser Glu Gly Glu Gly Ile Ser Thr Ala Pro Pro 35 40 45 Pro
Cys Trp Asn Glu Thr Gln Pro Gln Gly Gly Ala Lys Leu 50 55 60 65 49
PRT Homo sapiens 65 Met Arg Leu Cys Ser Phe Thr Lys Val Pro Met Asn
Leu Phe Leu Asn 1 5 10 15 Val Ile Leu Leu Lys Phe Tyr Asn Phe Leu
Phe Ser Leu Ile Leu Gly 20 25 30 Lys Ser Cys Leu Ala Ser Leu Gly
Leu Cys Lys Asn Asn Lys Cys Leu 35 40 45 Ser 49 66 401 PRT Homo
sapiens 66 Met Val Ala Leu Arg Gly Ala Ser Ala Leu Leu Val Leu Phe
Leu Ala 1 5 10 15 Ala Phe Leu Pro Pro Pro Gln Cys Thr Gln Asp Pro
Ala Met Val His 20 25 30 Tyr Ile Tyr Gln Arg Phe Arg Val Leu Glu
Gln Gly Leu Glu Lys Cys 35 40 45 Thr Gln Ala Thr Arg Ala Tyr Ile
Gln Glu Phe Gln Glu Phe Ser Lys 50 55 60 Asn Ile Ser Val Met Leu
Gly Arg Cys Gln Thr Tyr Thr Ser Glu Tyr 65 70 75 80 Lys Ser Ala Val
Gly Asn Leu Ala Leu Arg Val Glu Arg Ala Gln Arg 85 90 95 Glu Ile
Asp Tyr Ile Gln Tyr Leu Arg Glu Ala Asp Glu Cys Ile Glu 100 105 110
Ser Glu Asp Lys Thr Leu Ala Glu Met Leu Leu Gln Glu Ala Glu Glu 115
120 125 Glu Lys Lys Ile Arg Thr Leu Leu Asn Ala Ser Cys Asp Asn Met
Leu 130 135 140 Met Gly Ile Lys Ser Leu Lys Ile Val Lys Lys Met Met
Asp Thr His 145 150 155 160 Gly Ser Trp Met Lys Asp Ala Val Tyr Asn
Ser Pro Lys Val Tyr Leu 165 170 175 Leu Ile Gly Ser Arg Asn Asn Thr
Val Trp Glu Phe Ala Asn Ile Arg 180 185 190 Ala Phe Met Glu Asp Asn
Thr Lys Pro Ala Pro Arg Lys Gln Ile Leu 195 200 205 Thr Leu Ser Trp
Gln Gly Thr Gly Gln Val Ile Tyr Lys Gly Phe Leu 210 215 220 Phe Phe
His Asn Gln Ala Thr Ser Asn Glu Ile Ile Lys Tyr Asn Leu 225 230 235
240 Gln Lys Arg Thr Val Glu Asp Arg Met Leu Leu Pro Gly Gly Val Gly
245 250 255 Arg Ala Leu Val Tyr Gln His Ser Pro Ser Thr Tyr Ile Asp
Leu Ala 260 265 270 Val Asp Glu His Gly Leu Trp Ala Ile His Ser Gly
Pro Gly Thr His 275 280 285 Ser His Leu Val Leu Thr Lys Ile Glu Pro
Gly Thr Leu Gly Val Glu 290 295 300 His Ser Trp Asp Thr Pro Cys Arg
Ser Gln Asp Ala Glu Ala Ser Phe 305 310 315 320 Leu Leu Cys Gly Val
Leu Tyr Val Val Tyr Ser Thr Gly Gly Gln Gly 325 330 335 Pro His Arg
Ile Thr Cys Ile Tyr Asp Pro Leu Gly Thr Ile Ser Glu 340 345 350 Glu
Asp Leu Pro Asn Leu Phe Phe Pro Lys Arg Pro Arg Ser His Ser 355 360
365 Met Ile His Tyr Asn Pro Arg Asp Lys Gln Leu Tyr Ala Trp Asn Glu
370 375 380 Gly Asn Gln Ile Ile Tyr Lys Leu Gln Thr Lys Arg Lys Leu
Thr Leu 385 390 395 400 Lys 67 57 PRT Homo sapiens 67 Met Val Ser
Leu Leu Ser Ser Tyr Leu Leu Leu Leu Glu Leu Leu Ser 1 5 10 15 Lys
Arg Ser Leu Phe Leu Gln Trp Tyr Leu Phe Phe Gly Leu Gln Cys 20 25
30 Cys Ser Ser Phe Leu Cys Arg Lys Asn Glu Ser Gln Cys Phe Thr Arg
35 40 45 Leu Lys Glu Arg Ser Ala Gly Ser Val 50 55 68 72 PRT Homo
sapiens 68 Met Leu Arg Pro Ala Leu Pro Trp Leu Tyr Leu Gly Leu Cys
Ser Leu 1 5 10 15 Leu Val Gly Glu Ala Glu Ala Pro Ser Pro Val Asp
Pro Leu Glu Arg 20 25 30 Ser Arg Pro Tyr Ala Val Leu Arg Gly Gln
Asn Leu Val Leu Met Gly 35 40 45 Thr Ile Phe Ser Ile Leu Leu Val
Thr Val Ile Leu Met Ala Phe Cys 50 55 60 Val Tyr Lys Pro Ile Arg
Arg Arg 65 70 69 50 PRT Homo sapiens 69 Met Leu Thr Tyr Leu Pro Arg
Trp Cys Phe Leu Ser Leu Pro Pro Pro 1 5 10 15 Cys Cys Gly Ala Ala
Ser Cys Thr Met Met His Ile Gln Ile Ile Leu 20 25 30 Asn Thr His
Ile Leu Ile Glu Arg Phe Leu Gly Phe Leu Leu Asn Gln 35 40 45 Val
Tyr 50 70 181 PRT Homo sapiens 70 Met Thr Ser Arg Arg Ser Ser Thr
Leu Ser Met Thr Ser Ser Leu Leu 1
5 10 15 Ser Leu Gly Cys Ala Leu Thr Ser Ala Phe Pro Ala Ser Thr Met
Ser 20 25 30 Trp Val Pro Leu Leu Gln Met Leu Asp Gln Ser Pro Arg
Arg Val Met 35 40 45 Arg Lys Ser Val Ser Gln Leu Cys Pro Leu Leu
Arg Pro His Pro Pro 50 55 60 Leu Ser Ser Lys His Pro Leu Val Leu
Pro Leu Gln Leu Pro Pro Thr 65 70 75 80 Phe Leu His Leu Leu Pro Gly
Pro Gly Cys Pro Gly Gln Thr Val Ala 85 90 95 Tyr Trp Leu Leu Glu
Phe Leu Ser Arg Ala Thr Leu Lys Leu Tyr Pro 100 105 110 Gly Asp Arg
Pro Leu Trp Leu Gln Pro Thr Arg Leu Asn Phe Lys Asp 115 120 125 His
Trp Thr Ile Phe Ser Val Ala Ser Ala Ala Leu Phe Cys Val His 130 135
140 Arg Met Ala Thr Asp Arg His Ala Ser Phe Pro Thr His Trp Lys Ala
145 150 155 160 His Arg Gln Gly Glu Arg Gly His Arg Arg Cys Gln His
Cys Arg Tyr 165 170 175 Ser Lys Asp Leu Lys 180 71 48 PRT Homo
sapiens 71 Met His Met Gly Leu Thr Thr Cys Lys Cys His Trp Lys Met
Ala Tyr 1 5 10 15 Leu Arg Phe Leu Ile Leu Trp Ser Phe Pro Leu Ser
Ser Ala Val Ser 20 25 30 Gly Ala Lys Arg Val Thr Asp Leu Leu Asn
Gly Lys His Trp Lys Pro 35 40 45 72 53 PRT Homo sapiens 72 Met Val
Gln Phe Glu Val Ile Phe Leu Leu Phe Gly Leu Cys Phe Ser 1 5 10 15
Ser Ser Ser Ser Arg Leu Val Gly Ser Gln Val Glu Asn Phe Ser Pro 20
25 30 Thr Pro Cys Ile Phe Gln Ala Phe Arg Cys Ser Ser Leu Ala Ile
Ile 35 40 45 Ser Met Ser Leu Ser 50 73 74 PRT Homo sapiens 73 Met
Ser Val Val Pro Val Met Ile Pro Phe Leu Leu Leu Leu Phe Phe 1 5 10
15 Phe Ser Leu Ser Ser Thr His His Pro His Leu Leu Tyr Phe Ser Ile
20 25 30 Phe Ile Phe Ser Gly Ser Leu Leu Val Arg Ile Leu Ser Cys
Arg Lys 35 40 45 Glu Ser Ser His Gln Val Leu Leu Ser Arg Lys Cys
Phe Ile Lys Gly 50 55 60 His Arg Gln His Arg Gln Leu Thr Lys Val 65
70 74 74 64 PRT Homo sapiens 74 Met Pro Leu Phe Leu Phe Val Ala His
Leu Ile Ser Leu Leu Leu Ala 1 5 10 15 Phe Arg Arg Pro Pro Ala Ser
Gln Ile Thr Pro Arg Ala Trp Thr Thr 20 25 30 Glu Ile Ala Ser Cys
Glu Ser Val Glu Met Val Lys Ala Leu Ser Ser 35 40 45 Leu Arg Ser
Arg Ala Gln Val Asn Ala Asp Phe Pro Gly His Leu Cys 50 55 60 75 43
PRT Homo sapiens 75 Met Ser Ser Val Lys Cys Pro Tyr Met Trp Cys Phe
Trp Ala Phe Pro 1 5 10 15 Leu Phe Gln Leu Ser Val Phe Ile Pro Val
Ser Lys Ser His Ser Ile 20 25 30 Asn Tyr Tyr Asn Phe Ile Val Ser
Leu Asn Ile 35 40 76 52 PRT Homo sapiens 76 Met Ile Leu Phe Met Cys
Phe Leu Val Tyr Cys Leu Ser Ser Val Glu 1 5 10 15 Trp Lys Ser His
Arg Tyr Phe Val Phe Phe Ser Pro Cys Pro Phe Leu 20 25 30 Tyr Pro
Gln Leu Leu Glu His Ser Leu Glu His Ser Lys Cys Ser Val 35 40 45
Leu Phe Met Glu 50 77 319 PRT Homo sapiens 77 Met Ser Trp Cys Cys
Leu Trp Leu Cys Leu Ser Ser Val Gly Arg Thr 1 5 10 15 Gly Ser Ala
Gly Pro Ser Leu Pro Phe Ser Glu Leu Cys Ser Leu Gly 20 25 30 Leu
Leu Arg Leu Arg Pro Val Phe Ser Pro Leu His Ser Gly Pro Gly 35 40
45 Lys Pro Ala Gln Phe Leu Ala Gly Glu Ala Glu Glu Val Asn Ala Phe
50 55 60 Ala Leu Gly Phe Leu Ser Thr Ser Ser Gly Val Ser Gly Glu
Asp Glu 65 70 75 80 Val Glu Pro Leu His Asp Gly Val Glu Glu Ala Glu
Lys Lys Met Glu 85 90 95 Glu Glu Gly Val Ser Val Ser Glu Met Glu
Ala Thr Gly Ala Gln Gly 100 105 110 Pro Ser Arg Val Glu Glu Ala Glu
Gly His Thr Glu Val Thr Glu Ala 115 120 125 Glu Gly Ser Gln Gly Thr
Ala Glu Ala Asp Gly Pro Gly Ala Ser Ser 130 135 140 Gly Asp Glu Asp
Ala Ser Gly Arg Ala Ala Ser Pro Glu Ser Ala Ser 145 150 155 160 Ser
Thr Pro Glu Ser Leu Gln Ala Arg Arg His His Gln Phe Leu Glu 165 170
175 Pro Ala Pro Ala Pro Gly Ala Ala Val Leu Ser Ser Glu Pro Ala Glu
180 185 190 Pro Leu Leu Val Arg His Pro Pro Arg Pro Arg Thr Thr Gly
Pro Arg 195 200 205 Pro Arg Gln Asp Pro His Lys Ala Gly Leu Ser His
Tyr Val Lys Leu 210 215 220 Phe Ser Phe Tyr Ala Lys Met Pro Met Glu
Arg Lys Ala Leu Glu Met 225 230 235 240 Val Glu Lys Cys Leu Asp Lys
Tyr Phe Gln His Leu Cys Asp Asp Leu 245 250 255 Glu Val Phe Ala Ala
His Ala Gly Arg Lys Thr Val Lys Pro Glu Asp 260 265 270 Leu Glu Leu
Leu Met Arg Arg Gln Gly Leu Val Thr Asp Gln Val Ser 275 280 285 Leu
His Val Leu Val Glu Arg His Leu Pro Leu Glu Tyr Arg Gln Leu 290 295
300 Leu Ile Pro Cys Ala Tyr Ser Gly Asn Ser Val Phe Pro Ala Gln 305
310 315 319 78 171 PRT Homo sapiens 78 Met Ser Leu Pro Ile Pro Trp
Leu Ser Leu Pro Pro Cys Pro Ile Leu 1 5 10 15 Gly Gln Pro Ala Gly
Leu Leu Leu Trp Leu Phe Arg Pro Phe Ser Gln 20 25 30 Cys Cys Gln
Cys Pro Trp Glu Gly Arg Ala Ser Leu Arg His Pro Asn 35 40 45 Gly
Pro Ser Gly Cys Arg Glu Ala Glu Ala Trp Pro Gln Arg Ser Leu 50 55
60 Leu Arg Gln Gln Leu Gln Gln Ala His Pro Leu Pro Thr Leu Pro Thr
65 70 75 80 Pro Glu Arg Leu Pro Glu Gln Met Leu Phe Pro Ser Ser Ser
Ser Lys 85 90 95 Pro Phe Ser Leu Leu Ser Leu Thr Ile Trp Ala Arg
Leu Val Gly Arg 100 105 110 Leu Thr Asn Arg Ile Cys Pro Val Pro Pro
Gly Ser Val Ala Ser Ser 115 120 125 Met Ser Leu Gln Ala Gly Arg Cys
Gly Asn Pro Val Val Leu Pro Gln 130 135 140 Pro Met Pro Pro Gly Leu
Leu Cys Met Asn Glu Cys Ser Leu Val Pro 145 150 155 160 Gly Leu Gly
Arg Gly Gln Val Asn Ser Arg Val 165 170 79 60 PRT Homo sapiens 79
Met Val Ser Arg Ser Thr Ser Leu Thr Leu Ile Val Phe Leu Phe His 1 5
10 15 Arg Leu Ser Lys Ala Pro Gly Lys Met Val Glu Asn Ser Pro Ser
Pro 20 25 30 Leu Pro Glu Arg Ala Ile Tyr Gly Phe Val Leu Phe Leu
Ser Ser Gln 35 40 45 Phe Gly Phe Lys Asn Leu Lys Gly Ser Arg Val
Cys 50 55 60 80 100 PRT Homo sapiens MISC_FEATURE (20) Xaa equals
any of the naturally occurring L-amino acids 80 Met Leu Pro Ser Ala
Trp Gly Pro Leu Gln Val Ala Ser Phe Phe Leu 1 5 10 15 Leu Ser Phe
Xaa Phe Cys Phe Leu Ser Ser Ser Pro His Leu Gly Arg 20 25 30 Gln
Glu Thr His Xaa Val Val Leu Glu Asp Asp Glu Gly Ala Pro Cys 35 40
45 Pro Ala Glu Asp Glu Leu Ala Leu Gln Asp Asn Gly Phe Leu Ser Lys
50 55 60 Asn Glu Val Leu Arg Thr Arg Cys Leu Gly Ser Arg Ser Gly
Ser Ala 65 70 75 80 Ser Ala Thr Pro Pro Thr Thr Ser Gly Thr Ala Arg
Ala Ala Arg Pro 85 90 95 Pro Ser Gln Cys 100 81 97 PRT Homo sapiens
81 Met Ala Leu Leu Ala Leu Ala Ser Ala Val Pro Ser Ala Leu Leu Ala
1 5 10 15 Leu Ala Val Phe Arg Val Pro Ala Trp Ala Cys Leu Leu Cys
Phe Thr 20 25 30 Thr Tyr Ser Glu Arg Leu Arg Ile Cys Gln Met Phe
Val Gly Met Arg 35 40 45 Ser Pro Ser Leu Lys Ser Val Arg Arg Pro
Ser Arg Pro Pro Ser Arg 50 55 60 Ala Ser Leu Thr Pro Lys Ser Val
Arg Arg Pro Ser Thr Leu His Gln 65 70 75 80 Cys Pro Gly Glu Gly Ala
Glu Gly Gly Gln Glu Arg Pro Arg Gly Ser 85 90 95 Gly 82 52 PRT Homo
sapiens 82 Met Trp Leu Asn Phe Ser Asp Val His Thr Tyr Leu Ser Ser
Ile Ala 1 5 10 15 Leu Leu Cys Phe Cys Leu Ser Gly Val Leu Cys Cys
Ile Cys Asn Asn 20 25 30 Ser Val Phe His Ile Gln Gln Tyr Ile Leu
Ile Ile Ile Thr Phe Pro 35 40 45 Leu Val Val Ile 50 83 40 PRT Homo
sapiens 83 Met Ser His Ala Ser Arg Lys Thr Lys His Phe Pro Pro Leu
Leu Gln 1 5 10 15 Asn Pro Phe Leu Met Leu Thr Leu Leu Thr Met Ala
Val Ser Ala Gln 20 25 30 Pro Leu Pro Phe Ser Arg Pro Arg 35 40 84
132 PRT Homo sapiens MISC_FEATURE (122) Xaa equals any of the
naturally occurring L-amino acids 84 Met Ala Ala Ala Val Ala Ala
Ala Leu Ala Arg Leu Leu Ala Ala Phe 1 5 10 15 Leu Leu Leu Ala Ala
Gln Val Ala Cys Glu Tyr Gly Met Val His Val 20 25 30 Val Ser Gln
Ala Gly Gly Pro Glu Gly Lys Asp Tyr Cys Ile Leu Tyr 35 40 45 Asn
Pro Gln Trp Ala His Leu Pro His Asp Leu Ser Lys Ala Ser Phe 50 55
60 Leu Gln Leu Arg Asn Trp Thr Ala Ser Leu Leu Cys Ser Ala Ala Asp
65 70 75 80 Leu Pro Ala Arg Gly Phe Ser Asn Gln Ile Pro Leu Val Ala
Arg Gly 85 90 95 Asn Cys Thr Phe Tyr Glu Lys Val Arg Leu Ala Gln
Gly Ser Gly His 100 105 110 Ala Gly Cys Ser Ser Ser Ala Gly Arg Xaa
Trp Ser Pro Arg Gly Val 115 120 125 Ile Arg Arg Ile 130 85 11 PRT
Homo sapiens 85 His Ser Ser Leu Pro His Phe Ser Ser Arg Ile 1 5 10
86 22 PRT Homo sapiens 86 Arg Asp Ser Asn Gly Arg Gly Asp Ser Ser
Leu Leu Lys Phe Val Cys 1 5 10 15 Pro Val Pro Leu Lys Lys 20 87 12
PRT Homo sapiens 87 Ile Pro Glu Tyr Thr Phe Arg Arg Arg Trp Phe His
1 5 10 88 17 PRT Homo sapiens 88 Leu Cys Val Ser Met Lys Ile Glu
Trp Gly Arg Glu Ser Cys Glu Lys 1 5 10 15 Lys 89 25 PRT Homo
sapiens 89 Arg Leu Lys Thr Thr Arg Ala Tyr Ser Ser Gln Phe Trp Arg
Pro Glu 1 5 10 15 Val Gln Asn Gln Gly Val Arg Lys Val 20 25 90 165
PRT Homo sapiens 90 Leu Thr Leu Cys Leu Pro Arg Ser Leu Tyr Ala Leu
Pro Gln Cys Pro 1 5 10 15 Gly Pro His Val His Pro Cys Pro Ala Leu
Leu Trp Asp Arg Ala Gly 20 25 30 Leu Pro Leu Pro Leu Pro Gly Cys
Ile His Gly Arg Ser Gln Val Pro 35 40 45 Trp His Glu Leu His Ser
Pro Ala Ala Phe Asn Gln Gly Met Met Gly 50 55 60 Met Cys Thr Tyr
Pro Thr Pro Pro Leu Gly Arg Val Met Leu Arg Cys 65 70 75 80 Gly Phe
Leu Thr Val Pro Arg Leu Ser Gln Glu Ala Trp Val Trp Val 85 90 95
Pro Thr Val Gly Ala Gly Val Ile Ser Tyr Leu Arg Arg Pro Pro Phe 100
105 110 Leu Pro Val Leu Cys Ala Pro Thr Pro Thr Leu Glu Leu Pro Arg
Phe 115 120 125 Ser Val Phe Val Lys Glu Leu Thr Leu Cys Cys Leu Pro
Leu Ser Gln 130 135 140 Cys Pro Cys His Ser Cys Glu Pro Ala Ala Gly
Glu Val Gly Ala Asp 145 150 155 160 Leu Cys Val Ala Gly 165 91 41
PRT Homo sapiens 91 Leu Thr Leu Cys Leu Pro Arg Ser Leu Tyr Ala Leu
Pro Gln Cys Pro 1 5 10 15 Gly Pro His Val His Pro Cys Pro Ala Leu
Leu Trp Asp Arg Ala Gly 20 25 30 Leu Pro Leu Pro Leu Pro Gly Cys
Ile 35 40 92 38 PRT Homo sapiens 92 His Gly Arg Ser Gln Val Pro Trp
His Glu Leu His Ser Pro Ala Ala 1 5 10 15 Phe Asn Gln Gly Met Met
Gly Met Cys Thr Tyr Pro Thr Pro Pro Leu 20 25 30 Gly Arg Val Met
Leu Arg 35 93 41 PRT Homo sapiens 93 Cys Gly Phe Leu Thr Val Pro
Arg Leu Ser Gln Glu Ala Trp Val Trp 1 5 10 15 Val Pro Thr Val Gly
Ala Gly Val Ile Ser Tyr Leu Arg Arg Pro Pro 20 25 30 Phe Leu Pro
Val Leu Cys Ala Pro Thr 35 40 94 45 PRT Homo sapiens 94 Pro Thr Leu
Glu Leu Pro Arg Phe Ser Val Phe Val Lys Glu Leu Thr 1 5 10 15 Leu
Cys Cys Leu Pro Leu Ser Gln Cys Pro Cys His Ser Cys Glu Pro 20 25
30 Ala Ala Gly Glu Val Gly Ala Asp Leu Cys Val Ala Gly 35 40 45 95
38 PRT Homo sapiens 95 Ile Arg His Glu Thr Phe Arg Val Arg Gly Cys
Ser Ile Ser Arg Ala 1 5 10 15 Leu Ser Pro Phe Pro Leu Pro Phe Pro
His Pro Gly Arg Ser Gly Trp 20 25 30 Ser Gly Pro Glu Ala Lys 35 96
145 PRT Homo sapiens 96 Pro Asp Ser Arg Pro Glu Ala Arg Gly Asp His
Val Val Arg Pro Ser 1 5 10 15 Arg Gly Leu Arg Val Thr Gly Ala Thr
Arg Ser Ile Met Gly Pro Trp 20 25 30 Gly Glu Pro Glu Leu Leu Val
Trp Arg Pro Glu Ala Val Ala Ser Glu 35 40 45 Pro Pro Val Pro Val
Gly Leu Glu Val Lys Leu Gly Ala Leu Val Leu 50 55 60 Leu Leu Val
Leu Thr Leu Leu Cys Ser Leu Val Pro Ile Cys Val Leu 65 70 75 80 Arg
Arg Pro Gly Ala Asn His Glu Gly Ser Ala Ser Arg Gln Lys Ala 85 90
95 Leu Ser Leu Val Ser Cys Phe Ala Gly Gly Val Phe Leu Ala Thr Cys
100 105 110 Leu Leu Asp Leu Leu Pro Asp Tyr Leu Ala Ala Ile Asp Glu
Ala Leu 115 120 125 Ala Ala Leu His Val Thr Leu Gln Phe Pro Leu Gln
Glu Phe Ile Leu 130 135 140 Ala 145 97 35 PRT Homo sapiens 97 Pro
Asp Ser Arg Pro Glu Ala Arg Gly Asp His Val Val Arg Pro Ser 1 5 10
15 Arg Gly Leu Arg Val Thr Gly Ala Thr Arg Ser Ile Met Gly Pro Trp
20 25 30 Gly Glu Pro 35 98 37 PRT Homo sapiens 98 Glu Leu Leu Val
Trp Arg Pro Glu Ala Val Ala Ser Glu Pro Pro Val 1 5 10 15 Pro Val
Gly Leu Glu Val Lys Leu Gly Ala Leu Val Leu Leu Leu Val 20 25 30
Leu Thr Leu Leu Cys 35 99 36 PRT Homo sapiens 99 Ser Leu Val Pro
Ile Cys Val Leu Arg Arg Pro Gly Ala Asn His Glu 1 5 10 15 Gly Ser
Ala Ser Arg Gln Lys Ala Leu Ser Leu Val Ser Cys Phe Ala 20 25 30
Gly Gly Val Phe 35 100 37 PRT Homo sapiens 100 Leu Ala Thr Cys Leu
Leu Asp Leu Leu Pro Asp Tyr Leu Ala Ala Ile 1 5 10 15 Asp Glu Ala
Leu Ala Ala Leu His Val Thr Leu Gln Phe Pro Leu Gln 20 25 30 Glu
Phe Ile Leu Ala 35 101 28 PRT Homo sapiens 101 Lys Tyr Ile Leu Ser
Ser Pro Leu Leu Asp Ser Leu Ala Glu His Lys 1 5 10 15 Asn Leu Val
Trp Lys Ser Phe Leu Pro Arg Asn Phe 20 25 102 70 PRT Homo sapiens
MISC_FEATURE (53) Xaa equals any of the naturally occurring L-amino
acids 102 Tyr Gly Lys Val Val Asp Leu Ala Pro Leu His Leu Asp Ala
Arg Ile 1 5 10 15 Ser Leu Ser Thr Leu Gln Gln Gln Leu Gly Gln Pro
Glu Lys Ala Leu 20 25 30 Glu Ala Leu Glu Pro Met Tyr Asp Pro Asp
Thr Leu Ala Gln Asp Ala 35 40 45 Asn Ala Ala Gln Xaa Glu Leu Lys
Leu Leu Leu His Arg Ser Thr Leu 50
55 60 Leu Phe Ser Gln Gly Lys 65 70 103 96 PRT Homo sapiens
MISC_FEATURE (58) Xaa equals any of the naturally occurring L-amino
acids 103 Asp Phe Met Glu Thr Phe Pro Asp Phe Cys Leu Pro Leu Ala
Pro His 1 5 10 15 Tyr Leu Gly Lys Ala Ala Leu Trp Ala Met Cys Pro
Gly Arg Ala Trp 20 25 30 Ala Gly Cys Gly Pro Val Leu Arg Thr Ser
His Leu Gly Pro His Ser 35 40 45 Ala Leu Pro Ser Trp Cys Asn Ile
Cys Xaa Gln Ala Ile Val Gly Ala 50 55 60 Gly Arg Gln Arg Gly Leu
Ser Glu Asp Pro Thr Cys Ala Ser His Trp 65 70 75 80 Asp Thr Lys Thr
Gly Leu Val Pro Ser Cys Gly Ala Gly Lys Gly Ile 85 90 95 104 44 PRT
Homo sapiens 104 Asp Phe Met Glu Thr Phe Pro Asp Phe Cys Leu Pro
Leu Ala Pro His 1 5 10 15 Tyr Leu Gly Lys Ala Ala Leu Trp Ala Met
Cys Pro Gly Arg Ala Trp 20 25 30 Ala Gly Cys Gly Pro Val Leu Arg
Thr Ser His Leu 35 40 105 52 PRT Homo sapiens MISC_FEATURE (14) Xaa
equals any of the naturally occurring L-amino acids 105 Gly Pro His
Ser Ala Leu Pro Ser Trp Cys Asn Ile Cys Xaa Gln Ala 1 5 10 15 Ile
Val Gly Ala Gly Arg Gln Arg Gly Leu Ser Glu Asp Pro Thr Cys 20 25
30 Ala Ser His Trp Asp Thr Lys Thr Gly Leu Val Pro Ser Cys Gly Ala
35 40 45 Gly Lys Gly Ile 50 106 280 PRT Homo sapiens 106 Arg Leu
Pro Gln Arg Gly Gln Trp Ala Trp Val Leu Gln Asp Ala Leu 1 5 10 15
Gly Ile Ala Phe Cys Leu Tyr Met Leu Lys Thr Ile Arg Leu Pro Thr 20
25 30 Phe Lys Ala Cys Thr Leu Leu Leu Leu Val Leu Phe Leu Tyr Asp
Ile 35 40 45 Phe Phe Val Phe Ile Thr Pro Phe Leu Thr Lys Ser Gly
Ser Ser Ile 50 55 60 Met Val Glu Val Ala Thr Gly Pro Ser Asp Ser
Ala Thr Arg Glu Lys 65 70 75 80 Leu Pro Met Val Leu Lys Val Pro Arg
Leu Asn Ser Ser Pro Leu Ala 85 90 95 Leu Cys Asp Arg Pro Phe Ser
Leu Leu Gly Phe Gly Asp Ile Leu Val 100 105 110 Pro Gly Leu Leu Val
Ala Tyr Cys His Arg Phe Asp Ile Gln Val Gln 115 120 125 Ser Ser Arg
Val Tyr Phe Val Ala Cys Thr Ile Ala Tyr Gly Val Gly 130 135 140 Leu
Leu Val Thr Phe Val Ala Leu Ala Leu Met Gln Arg Gly Gln Pro 145 150
155 160 Ala Leu Leu Tyr Leu Val Pro Cys Thr Leu Val Thr Ser Cys Ala
Val 165 170 175 Ala Leu Trp Arg Arg Glu Leu Gly Val Phe Trp Thr Gly
Ser Gly Phe 180 185 190 Ala Lys Val Leu Pro Pro Ser Pro Trp Ala Pro
Ala Pro Ala Asp Gly 195 200 205 Pro Gln Pro Pro Lys Asp Ser Ala Thr
Pro Leu Ser Pro Gln Pro Pro 210 215 220 Ser Glu Glu Pro Ala Thr Ser
Pro Trp Pro Ala Glu Gln Ser Pro Lys 225 230 235 240 Ser Arg Thr Ser
Glu Glu Met Gly Ala Gly Ala Pro Met Arg Glu Pro 245 250 255 Gly Ser
Pro Ala Glu Ser Glu Gly Arg Asp Gln Ala Gln Pro Ser Pro 260 265 270
Val Thr Gln Pro Gly Ala Ser Ala 275 280 107 43 PRT Homo sapiens 107
Arg Leu Pro Gln Arg Gly Gln Trp Ala Trp Val Leu Gln Asp Ala Leu 1 5
10 15 Gly Ile Ala Phe Cys Leu Tyr Met Leu Lys Thr Ile Arg Leu Pro
Thr 20 25 30 Phe Lys Ala Cys Thr Leu Leu Leu Leu Val Leu 35 40 108
44 PRT Homo sapiens 108 Phe Leu Tyr Asp Ile Phe Phe Val Phe Ile Thr
Pro Phe Leu Thr Lys 1 5 10 15 Ser Gly Ser Ser Ile Met Val Glu Val
Ala Thr Gly Pro Ser Asp Ser 20 25 30 Ala Thr Arg Glu Lys Leu Pro
Met Val Leu Lys Val 35 40 109 44 PRT Homo sapiens 109 Pro Arg Leu
Asn Ser Ser Pro Leu Ala Leu Cys Asp Arg Pro Phe Ser 1 5 10 15 Leu
Leu Gly Phe Gly Asp Ile Leu Val Pro Gly Leu Leu Val Ala Tyr 20 25
30 Cys His Arg Phe Asp Ile Gln Val Gln Ser Ser Arg 35 40 110 43 PRT
Homo sapiens 110 Val Tyr Phe Val Ala Cys Thr Ile Ala Tyr Gly Val
Gly Leu Leu Val 1 5 10 15 Thr Phe Val Ala Leu Ala Leu Met Gln Arg
Gly Gln Pro Ala Leu Leu 20 25 30 Tyr Leu Val Pro Cys Thr Leu Val
Thr Ser Cys 35 40 111 40 PRT Homo sapiens 111 Ala Val Ala Leu Trp
Arg Arg Glu Leu Gly Val Phe Trp Thr Gly Ser 1 5 10 15 Gly Phe Ala
Lys Val Leu Pro Pro Ser Pro Trp Ala Pro Ala Pro Ala 20 25 30 Asp
Gly Pro Gln Pro Pro Lys Asp 35 40 112 41 PRT Homo sapiens 112 Ser
Ala Thr Pro Leu Ser Pro Gln Pro Pro Ser Glu Glu Pro Ala Thr 1 5 10
15 Ser Pro Trp Pro Ala Glu Gln Ser Pro Lys Ser Arg Thr Ser Glu Glu
20 25 30 Met Gly Ala Gly Ala Pro Met Arg Glu 35 40 113 25 PRT Homo
sapiens 113 Pro Gly Ser Pro Ala Glu Ser Glu Gly Arg Asp Gln Ala Gln
Pro Ser 1 5 10 15 Pro Val Thr Gln Pro Gly Ala Ser Ala 20 25 114 26
PRT Homo sapiens 114 Glu Ser Ser Gly Leu Pro Ala Leu Gly Pro Arg
Arg Arg Pro Trp Glu 1 5 10 15 Gln Arg Trp Ser Asp Pro Ile Thr Leu
Lys 20 25 115 61 PRT Homo sapiens 115 Leu Thr Leu Ala Leu Asp Glu
Ile Arg Leu Leu Lys Lys Asp Leu Gly 1 5 10 15 Leu Ile Glu Met Lys
Lys Thr Asp Ser Glu Lys Arg Phe Gly Ser Val 20 25 30 Ser Phe Gly
Arg Ser Cys Arg Leu Ile Pro His Ala Leu Ala Ser Trp 35 40 45 Leu
Gln Thr Leu Ile Leu Cys Phe Cys Cys Arg Ile Cys 50 55 60 116 32 PRT
Homo sapiens MISC_FEATURE (27) Xaa equals any of the naturally
occurring L-amino acids 116 Gly Arg Pro Thr Arg Pro Val Met Ala Ile
Gln Ser Leu His Pro Cys 1 5 10 15 Pro Ser Glu Leu Cys Cys Arg Ala
Cys Val Xaa Phe Tyr His Trp Ala 20 25 30 117 29 PRT Homo sapiens
117 Asn Ser Lys Asn Thr Arg Asn Glu Arg Ser Phe Leu Lys Leu Phe Arg
1 5 10 15 Asn Ile His Asp Ile Pro Leu Thr Val Leu Glu Asn Lys 20 25
118 20 PRT Homo sapiens 118 Pro Arg Val Arg Gly Glu Gly Asn Arg Cys
Trp Thr Gln Gly Ala Leu 1 5 10 15 Cys His Arg Met 20 119 421 PRT
Homo sapiens 119 Pro Arg Val Arg Gly Glu Gly Asn Arg Cys Trp Thr
Gln Gly Ala Leu 1 5 10 15 Cys His Arg Met Met Val Ala Leu Arg Gly
Ala Ser Ala Leu Leu Val 20 25 30 Leu Phe Leu Ala Ala Phe Leu Pro
Pro Pro Gln Cys Thr Gln Asp Pro 35 40 45 Ala Met Val His Tyr Ile
Tyr Gln Arg Phe Arg Val Leu Glu Gln Gly 50 55 60 Leu Glu Lys Cys
Thr Gln Ala Thr Arg Ala Tyr Ile Gln Glu Phe Gln 65 70 75 80 Glu Phe
Ser Lys Asn Ile Ser Val Met Leu Gly Arg Cys Gln Thr Tyr 85 90 95
Thr Ser Glu Tyr Lys Ser Ala Val Gly Asn Leu Ala Leu Arg Val Glu 100
105 110 Arg Ala Gln Arg Glu Ile Asp Tyr Ile Gln Tyr Leu Arg Glu Ala
Asp 115 120 125 Glu Cys Ile Glu Ser Glu Asp Lys Thr Leu Ala Glu Met
Leu Leu Gln 130 135 140 Glu Ala Glu Glu Glu Lys Lys Ile Arg Thr Leu
Leu Asn Ala Ser Cys 145 150 155 160 Asp Asn Met Leu Met Gly Ile Lys
Ser Leu Lys Ile Val Lys Lys Met 165 170 175 Met Asp Thr His Gly Ser
Trp Met Lys Asp Ala Val Tyr Asn Ser Pro 180 185 190 Lys Val Tyr Leu
Leu Ile Gly Ser Arg Asn Asn Thr Val Trp Glu Phe 195 200 205 Ala Asn
Ile Arg Ala Phe Met Glu Asp Asn Thr Lys Pro Ala Pro Arg 210 215 220
Lys Gln Ile Leu Thr Leu Ser Trp Gln Gly Thr Gly Gln Val Ile Tyr 225
230 235 240 Lys Gly Phe Leu Phe Phe His Asn Gln Ala Thr Ser Asn Glu
Ile Ile 245 250 255 Lys Tyr Asn Leu Gln Lys Arg Thr Val Glu Asp Arg
Met Leu Leu Pro 260 265 270 Gly Gly Val Gly Arg Ala Leu Val Tyr Gln
His Ser Pro Ser Thr Tyr 275 280 285 Ile Asp Leu Ala Val Asp Glu His
Gly Leu Trp Ala Ile His Ser Gly 290 295 300 Pro Gly Thr His Ser His
Leu Val Leu Thr Lys Ile Glu Pro Gly Thr 305 310 315 320 Leu Gly Val
Glu His Ser Trp Asp Thr Pro Cys Arg Ser Gln Asp Ala 325 330 335 Glu
Ala Ser Phe Leu Leu Cys Gly Val Leu Tyr Val Val Tyr Ser Thr 340 345
350 Gly Gly Gln Gly Pro His Arg Ile Thr Cys Ile Tyr Asp Pro Leu Gly
355 360 365 Thr Ile Ser Glu Glu Asp Leu Pro Asn Leu Phe Phe Pro Lys
Arg Pro 370 375 380 Arg Ser His Ser Met Ile His Tyr Asn Pro Arg Asp
Lys Gln Leu Tyr 385 390 395 400 Ala Trp Asn Glu Gly Asn Gln Ile Ile
Tyr Lys Leu Gln Thr Lys Arg 405 410 415 Lys Leu Thr Leu Lys 420 120
27 PRT Homo sapiens 120 Phe Pro Cys Ile Cys Leu Ser Gly Leu Leu Asp
Leu Leu Ile Trp Arg 1 5 10 15 Pro Phe Ser Glu Glu Leu Thr Lys Thr
Phe Gly 20 25 121 84 PRT Homo sapiens 121 Phe Pro Cys Ile Cys Leu
Ser Gly Leu Leu Asp Leu Leu Ile Trp Arg 1 5 10 15 Pro Phe Ser Glu
Glu Leu Thr Lys Thr Phe Gly Met Val Ser Leu Leu 20 25 30 Ser Ser
Tyr Leu Leu Leu Leu Glu Leu Leu Ser Lys Arg Ser Leu Phe 35 40 45
Leu Gln Trp Tyr Leu Phe Phe Gly Leu Gln Cys Cys Ser Ser Phe Leu 50
55 60 Cys Arg Lys Asn Glu Ser Gln Cys Phe Thr Arg Leu Lys Glu Arg
Ser 65 70 75 80 Ala Gly Ser Val 122 24 PRT Homo sapiens
MISC_FEATURE (8) Xaa equals any of the naturally occurring L-amino
acids 122 Lys Asp Thr Cys Thr Arg Met Xaa Ile Ala Ala Leu Phe Thr
Ile Ala 1 5 10 15 Lys Ile Trp Asn Gln Pro Lys Xaa 20 123 45 PRT
Homo sapiens MISC_FEATURE (24) Xaa equals any of the naturally
occurring L-amino acids 123 Arg His Met His Thr Tyr Val Tyr Cys Gly
Thr Ile His Asn Ser Lys 1 5 10 15 Asp Leu Glu Pro Thr Gln Met Xaa
Asp Xaa Ile Lys Lys Met Trp His 20 25 30 Leu Tyr Thr Thr Lys Tyr
Tyr Ala Ala Ile Lys Lys Asp 35 40 45 124 14 PRT Homo sapiens 124
Arg Lys Cys Gly Thr Tyr Ile Pro Arg Asn Thr Met Gln Pro 1 5 10 125
40 PRT Homo sapiens MISC_FEATURE (9) Xaa equals any of the
naturally occurring L-amino acids 125 Lys Arg Thr Glu Phe Met Ser
Phe Xaa Gly Thr Trp Met Lys Leu Glu 1 5 10 15 Ala Ile Ile Leu Ser
Lys Leu Thr Gln Glu Glu Lys Thr Lys His Leu 20 25 30 Met Phe Ser
Leu Ile Ser Gly Ser 35 40 126 11 PRT Homo sapiens 126 Pro Lys Ser
Asp Thr Ser Pro Ala Ser Ser Arg 1 5 10 127 15 PRT Homo sapiens 127
Pro Lys Ser Asp Thr Ser Pro Ala Ser Ser Arg Leu Cys Trp Asp 1 5 10
15 128 270 PRT Homo sapiens 128 Tyr Val Pro Ser Phe Leu Pro Lys Ala
Thr Gly Ser Ile Pro Ser Arg 1 5 10 15 Lys Gly Gly Val Gly Ser Glu
Lys Pro Glu Val Pro Leu Gln Thr Tyr 20 25 30 Lys Glu Ile Val His
Cys Cys Glu Glu Gln Val Leu Thr Leu Ala Thr 35 40 45 Glu Gln Thr
Tyr Ala Val Glu Gly Glu Thr Pro Ile Asn Arg Leu Ser 50 55 60 Leu
Leu Leu Ser Gly Arg Val Arg Val Ser Gln Asp Gly Gln Phe Leu 65 70
75 80 His Tyr Ile Phe Pro Tyr Gln Phe Met Asp Ser Pro Glu Trp Glu
Ser 85 90 95 Leu Gln Pro Ser Glu Glu Gly Val Phe Gln Val Thr Leu
Thr Ala Glu 100 105 110 Thr Ser Cys Ser Tyr Ile Ser Trp Pro Arg Lys
Ser Leu His Leu Leu 115 120 125 Leu Thr Lys Glu Arg Tyr Ile Ser Cys
Leu Phe Ser Ala Leu Leu Gly 130 135 140 Tyr Asp Ile Ser Glu Lys Leu
Tyr Thr Leu Asn Asp Lys Leu Phe Ala 145 150 155 160 Lys Phe Gly Leu
Arg Phe Asp Ile Arg Leu Pro Ser Leu Tyr His Val 165 170 175 Leu Gly
Pro Thr Ala Ala Asp Ala Gly Pro Glu Ser Glu Lys Gly Asp 180 185 190
Glu Glu Val Cys Glu Pro Ala Val Ser Pro Pro Gln Ala Thr Pro Thr 195
200 205 Ser Leu Gln Gln Thr Pro Pro Cys Ser Thr Pro Pro Ala Thr Thr
Asn 210 215 220 Phe Pro Ala Pro Pro Thr Arg Ala Arg Leu Ser Arg Pro
Asp Ser Gly 225 230 235 240 Ile Leu Ala Ser Arg Ile Pro Leu Gln Ser
Tyr Ser Gln Val Ile Ser 245 250 255 Arg Gly Gln Ala Pro Leu Ala Pro
Thr His Thr Pro Glu Leu 260 265 270 129 21 PRT Homo sapiens 129 Ala
Thr Gly Ser Ile Pro Ser Arg Lys Gly Gly Val Gly Ser Glu Lys 1 5 10
15 Pro Glu Val Pro Leu 20 130 25 PRT Homo sapiens 130 Ile Val His
Cys Cys Glu Glu Gln Val Leu Thr Leu Ala Thr Glu Gln 1 5 10 15 Thr
Tyr Ala Val Glu Gly Glu Thr Pro 20 25 131 23 PRT Homo sapiens 131
Gln Asp Gly Gln Phe Leu His Tyr Ile Phe Pro Tyr Gln Phe Met Asp 1 5
10 15 Ser Pro Glu Trp Glu Ser Leu 20 132 23 PRT Homo sapiens 132
Thr Leu Thr Ala Glu Thr Ser Cys Ser Tyr Ile Ser Trp Pro Arg Lys 1 5
10 15 Ser Leu His Leu Leu Leu Thr 20 133 25 PRT Homo sapiens 133
Asp Ile Ser Glu Lys Leu Tyr Thr Leu Asn Asp Lys Leu Phe Ala Lys 1 5
10 15 Phe Gly Leu Arg Phe Asp Ile Arg Leu 20 25 134 26 PRT Homo
sapiens 134 Ser Leu Tyr His Val Leu Gly Pro Thr Ala Ala Asp Ala Gly
Pro Glu 1 5 10 15 Ser Glu Lys Gly Asp Glu Glu Val Cys Glu 20 25 135
28 PRT Homo sapiens 135 Thr Thr Asn Phe Pro Ala Pro Pro Thr Arg Ala
Arg Leu Ser Arg Pro 1 5 10 15 Asp Ser Gly Ile Leu Ala Ser Arg Ile
Pro Leu Gln 20 25 136 196 PRT Homo sapiens 136 Pro Lys Ser Asp Thr
Ser Pro Ala Ser Ser Arg Leu Cys Trp Asp Met 1 5 10 15 Thr Ser Arg
Arg Ser Ser Thr Leu Ser Met Thr Ser Ser Leu Leu Ser 20 25 30 Leu
Gly Cys Ala Leu Thr Ser Ala Phe Pro Ala Ser Thr Met Ser Trp 35 40
45 Val Pro Leu Leu Gln Met Leu Asp Gln Ser Pro Arg Arg Val Met Arg
50 55 60 Lys Ser Val Ser Gln Leu Cys Pro Leu Leu Arg Pro His Pro
Pro Leu 65 70 75 80 Ser Ser Lys His Pro Leu Val Leu Pro Leu Gln Leu
Pro Pro Thr Phe 85 90 95 Leu His Leu Leu Pro Gly Pro Gly Cys Pro
Gly Gln Thr Val Ala Tyr 100 105 110 Trp Leu Leu Glu Phe Leu Ser Arg
Ala Thr Leu Lys Leu Tyr Pro Gly 115 120 125 Asp Arg Pro Leu Trp Leu
Gln Pro Thr Arg Leu Asn Phe Lys Asp His 130 135 140 Trp Thr Ile Phe
Ser Val Ala Ser Ala Ala Leu Phe Cys Val His Arg 145 150 155 160 Met
Ala Thr Asp Arg His Ala Ser Phe Pro Thr His Trp Lys Ala His 165 170
175 Arg Gln Gly Glu Arg Gly His Arg Arg Cys Gln His Cys Arg Tyr Ser
180 185 190 Lys Asp Leu Lys
195 137 10 PRT Homo sapiens 137 Tyr Phe Ser His Gly Ile Cys Ser His
Ala 1 5 10 138 55 PRT Homo sapiens 138 Asn Ser Glu Asp Ile Ser Gln
Thr Arg Gln Glu Leu Gly Leu Cys Ile 1 5 10 15 Ser Gln Arg Cys Leu
Ser Asp Arg Lys Lys Ser Arg Arg Ser Gly Val 20 25 30 Trp Val Arg
Ala Cys Thr Met Gln Phe Met Lys His Val Phe Pro Arg 35 40 45 Leu
Ile Ser Pro Arg Arg Pro 50 55 139 55 PRT Homo sapiens 139 Pro Thr
Arg His Phe Cys Gly Thr Ser Ser Cys Leu Thr Gly Thr Ala 1 5 10 15
Val Arg Cys Arg Ala Pro Ala Pro Val Trp Ser Val Arg Cys Pro His 20
25 30 Cys Phe Arg Ser Ser Asp Ala Trp Val Asp Pro Gly Ile Pro Asp
Arg 35 40 45 Tyr Leu Gln Ala Tyr Leu Leu 50 55 140 246 PRT Homo
sapiens MISC_FEATURE (8) Xaa equals any of the naturally occurring
L-amino acids 140 Gly Glu Ala Met Asp Ala Glu Xaa Ala Val Ala Pro
Pro Gly Cys Ser 1 5 10 15 His Leu Gly Ser Phe Lys Val Asp Asn Trp
Lys Gln Asn Leu Arg Ala 20 25 30 Ile Tyr Gln Cys Phe Val Trp Ser
Gly Thr Ala Glu Ala Arg Lys Arg 35 40 45 Lys Ala Lys Ser Cys Ile
Cys His Val Cys Gly Val His Leu Asn Arg 50 55 60 Leu His Ser Cys
Leu Tyr Cys Val Phe Phe Gly Cys Phe Thr Lys Lys 65 70 75 80 His Ile
His Glu His Ala Lys Ala Lys Arg His Asn Leu Ala Ile Asp 85 90 95
Leu Met Tyr Gly Gly Ile Tyr Cys Phe Leu Cys Gln Asp Tyr Ile Tyr 100
105 110 Asp Lys Asp Met Glu Ile Ile Ala Lys Glu Glu Gln Arg Lys Ala
Trp 115 120 125 Lys Met Gln Gly Val Gly Glu Lys Phe Ser Thr Trp Glu
Pro Thr Lys 130 135 140 Arg Glu Leu Glu Leu Leu Lys His Asn Pro Lys
Arg Arg Lys Ile Thr 145 150 155 160 Ser Asn Cys Thr Ile Gly Leu Arg
Gly Leu Ile Asn Leu Gly Asn Thr 165 170 175 Cys Phe Met Asn Cys Ile
Val Gln Ala Leu Thr His Thr Pro Leu Leu 180 185 190 Arg Asp Phe Phe
Leu Ser Asp Arg His Arg Cys Glu Met Gln Ser Pro 195 200 205 Ser Ser
Cys Leu Val Cys Glu Met Ser Ser Leu Phe Gln Glu Phe Gly 210 215 220
Arg Val Gly Arg Pro Gly Asn Ser Gly Pro Val Pro Ala Gly Val Pro 225
230 235 240 Ser Ile Val Ser Pro Glu 245 141 24 PRT Homo sapiens 141
Val Ala Pro Pro Gly Cys Ser His Leu Gly Ser Phe Lys Val Asp Asn 1 5
10 15 Trp Lys Gln Asn Leu Arg Ala Ile 20 142 23 PRT Homo sapiens
142 Thr Ala Glu Ala Arg Lys Arg Lys Ala Lys Ser Cys Ile Cys His Val
1 5 10 15 Cys Gly Val His Leu Asn Arg 20 143 23 PRT Homo sapiens
143 Phe Thr Lys Lys His Ile His Glu His Ala Lys Ala Lys Arg His Asn
1 5 10 15 Leu Ala Ile Asp Leu Met Tyr 20 144 21 PRT Homo sapiens
144 Tyr Asp Lys Asp Met Glu Ile Ile Ala Lys Glu Glu Gln Arg Lys Ala
1 5 10 15 Trp Lys Met Gln Gly 20 145 28 PRT Homo sapiens 145 Glu
Leu Leu Lys His Asn Pro Lys Arg Arg Lys Ile Thr Ser Asn Cys 1 5 10
15 Thr Ile Gly Leu Arg Gly Leu Ile Asn Leu Gly Asn 20 25 146 26 PRT
Homo sapiens 146 Gly Asn Thr Cys Phe Met Asn Cys Ile Val Gln Ala
Leu Thr His Thr 1 5 10 15 Pro Leu Leu Arg Asp Phe Phe Leu Ser Asp
20 25 147 20 PRT Homo sapiens 147 Glu Phe Gly Arg Val Gly Arg Pro
Gly Asn Ser Gly Pro Val Pro Ala 1 5 10 15 Gly Val Pro Ser 20 148
108 PRT Homo sapiens 148 Asn Ser Glu Asp Ile Ser Gln Thr Arg Gln
Glu Leu Gly Leu Cys Ile 1 5 10 15 Ser Gln Arg Cys Leu Ser Asp Arg
Lys Lys Ser Arg Arg Ser Gly Val 20 25 30 Trp Val Arg Ala Cys Thr
Met Gln Phe Met Lys His Val Phe Pro Arg 35 40 45 Leu Ile Ser Pro
Arg Arg Pro Met Val Gln Phe Glu Val Ile Phe Leu 50 55 60 Leu Phe
Gly Leu Cys Phe Ser Ser Ser Ser Ser Arg Leu Val Gly Ser 65 70 75 80
Gln Val Glu Asn Phe Ser Pro Thr Pro Cys Ile Phe Gln Ala Phe Arg 85
90 95 Cys Ser Ser Leu Ala Ile Ile Ser Met Ser Leu Ser 100 105 149 7
PRT Homo sapiens 149 Ala Phe Pro Trp Pro Thr Ser 1 5 150 23 PRT
Homo sapiens 150 Glu Ser Asn Phe Phe Tyr Pro Tyr Asp Ser Gln Leu
Ala Leu Leu Ser 1 5 10 15 Ser Val Thr Cys Ser Ala Ser 20 151 83 PRT
Homo sapiens 151 Lys Leu Lys Met Phe Ala Phe Tyr Val Gln Val Leu
Asn Gln Ser Lys 1 5 10 15 Ser Ile Phe Val Tyr Ser Arg Asn Leu Ile
Phe Phe Ile His Met Ile 20 25 30 Val Ser Trp Pro Ser Phe Leu Gln
Leu Pro Ala Val His Gln Cys His 35 40 45 Gln Ser Ser Val His Ile
Cys Gly Val Ser Gly Leu Phe Pro Ser Ser 50 55 60 Asn Tyr Gln Cys
Leu Ser Leu Cys Gln Asn His Thr Val Leu Ile Ile 65 70 75 80 Thr Thr
Leu 152 48 PRT Homo sapiens 152 Ser Ile Leu Asn Val Ile Pro Asn Leu
Ser Lys Gln Ser Phe Glu Glu 1 5 10 15 Phe Asp Arg Leu Ile Leu Lys
Tyr Met Gln Lys Ser Lys Ser Lys Arg 20 25 30 Ile Ala Lys Ile Leu
Leu Ser Asn Lys Lys Thr Cys Pro Thr Lys Tyr 35 40 45 153 36 PRT
Homo sapiens 153 Leu Pro Gln Ile Leu Arg Trp Leu Lys Tyr His Gln
Ser Val Trp Gly 1 5 10 15 Lys Gln Thr Pro Val Thr Leu His Tyr Leu
Thr Leu Asp Leu Ile Gln 20 25 30 Glu Phe Thr Pro 35 154 33 PRT Homo
sapiens 154 Ile Phe Val Tyr Ser Arg Asn Leu Ile Phe Phe Ile His Met
Ile Val 1 5 10 15 Ser Trp Pro Ser Phe Leu Gln Leu Pro Ala Val His
Gln Cys His Gln 20 25 30 Ser 155 184 PRT Homo sapiens 155 Pro Thr
Gly Asn Asp Leu Val Tyr Val Phe Pro Cys Leu Leu Ser Val 1 5 10 15
Phe Ser Arg Met Glu Glu Pro Ser Val Phe Cys Leu Phe Phe Pro Leu 20
25 30 Ser Ile Leu Ile Ser Ser Ala Ser Arg Thr Phe Pro Gly Thr Gln
Gln 35 40 45 Val Phe Ser Ile Val His Gly Val Thr Asp Val Ser Ala
Lys Lys Val 50 55 60 Gln Ser Gln Gly Arg Met Thr Ser Thr Gly Leu
Asp Phe Asn Leu Leu 65 70 75 80 Pro Ala Trp Phe Pro Ser Pro Thr Ser
Leu Gln Pro Thr Glu Asp Leu 85 90 95 Phe Gln Thr Gly Ser Leu Ser
Arg Ser Phe Phe Cys Ser Lys Ala Phe 100 105 110 Ser Ser Ser Pro Leu
Ser Pro Gly Gly Ser Pro Asn Ala Leu Thr Ser 115 120 125 Val Lys Glu
His Leu Val Ser Pro Ala Phe Leu Ala Ser His Ser Cys 130 135 140 Thr
Ala Glu Ser Phe Pro Arg Val Asp Val Ile His Ala Val Pro Ile 145 150
155 160 Ala Trp Ile Pro Ala Pro Leu His Pro Ile Gln Leu Ile Asn Ser
Trp 165 170 175 Phe Phe Phe Phe Phe Phe Phe Phe 180 156 24 PRT Homo
sapiens 156 Asp Leu Val Tyr Val Phe Pro Cys Leu Leu Ser Val Phe Ser
Arg Met 1 5 10 15 Glu Glu Pro Ser Val Phe Cys Leu 20 157 24 PRT
Homo sapiens 157 Ile Ser Ser Ala Ser Arg Thr Phe Pro Gly Thr Gln
Gln Val Phe Ser 1 5 10 15 Ile Val His Gly Val Thr Asp Val 20 158 20
PRT Homo sapiens 158 Phe Asn Leu Leu Pro Ala Trp Phe Pro Ser Pro
Thr Ser Leu Gln Pro 1 5 10 15 Thr Glu Asp Leu 20 159 25 PRT Homo
sapiens 159 Phe Cys Ser Lys Ala Phe Ser Ser Ser Pro Leu Ser Pro Gly
Gly Ser 1 5 10 15 Pro Asn Ala Leu Thr Ser Val Lys Glu 20 25 160 23
PRT Homo sapiens 160 Thr Ala Glu Ser Phe Pro Arg Val Asp Val Ile
His Ala Val Pro Ile 1 5 10 15 Ala Trp Ile Pro Ala Pro Leu 20 161 34
PRT Homo sapiens 161 Phe Ser Phe Leu Lys Pro Leu Cys Ala Pro Arg
Ala Pro Trp Leu Trp 1 5 10 15 Leu Pro Pro Ser Ser Lys Ser Arg Val
His Val Gly Pro Gly Asp Phe 20 25 30 Arg Ser 162 122 PRT Homo
sapiens MISC_FEATURE (108) Xaa equals any of the naturally
occurring L-amino acids 162 Val Cys Gly Thr Gly Gly Leu Glu Pro Asn
Leu Ala Trp Val Arg Val 1 5 10 15 Asp Asn Gly Ser Phe Pro Ser Ser
Ser Pro Ser Val Pro Leu Glu His 20 25 30 Pro Gly Cys Gly Cys Leu
Leu His Pro Arg Ala Glu Ser Met Leu Gly 35 40 45 Gln Glu Thr Ser
Asp Pro Cys Pro Gly Ala Ala Ser Gly Phe Val Phe 50 55 60 Pro Gln
Trp Ala Gly Leu Gly Leu Leu Val His Leu Tyr Pro Ser Leu 65 70 75 80
Ser Tyr Ala Ala Leu Ala Cys Cys Val Ser Gly Leu Tyr Ser Leu Pro 85
90 95 Phe Thr Gln Ala Leu Gly Asn Gln Pro Ser Phe Xaa Gln Glu Arg
Gln 100 105 110 Arg Arg Ser Met Pro Leu Leu Trp Ala Ser 115 120 163
8 PRT Homo sapiens 163 His Ala Gly Arg Lys Thr Val Lys 1 5 164 61
PRT Homo sapiens 164 Ser Phe Tyr Ala Lys Met Pro Met Glu Arg Lys
Ala Leu Glu Met Val 1 5 10 15 Glu Lys Cys Leu Asp Lys Tyr Phe Gln
His Leu Cys Asp Asp Leu Glu 20 25 30 Val Phe Ala Ala His Ala Gly
Arg Lys Thr Val Lys Pro Glu Asp Leu 35 40 45 Glu Leu Leu Met Arg
Arg Gln Gly Leu Val Thr Asp Gln 50 55 60 165 19 PRT Homo sapiens
165 Pro Met Glu Arg Lys Ala Leu Glu Met Val Glu Lys Cys Leu Asp Lys
1 5 10 15 Tyr Phe Gln 166 22 PRT Homo sapiens 166 Glu Val Phe Ala
Ala His Ala Gly Arg Lys Thr Val Lys Pro Glu Asp 1 5 10 15 Leu Glu
Leu Leu Met Arg 20 167 31 PRT Homo sapiens 167 Ser Phe Pro Ser Ser
Ser Pro Ser Val Pro Leu Glu His Pro Gly Cys 1 5 10 15 Gly Cys Leu
Leu His Pro Arg Ala Glu Ser Met Leu Gly Gln Glu 20 25 30 168 27 PRT
Homo sapiens 168 Tyr Pro Ser Leu Ser Tyr Ala Ala Leu Ala Cys Cys
Val Ser Gly Leu 1 5 10 15 Tyr Ser Leu Pro Phe Thr Gln Ala Leu Gly
Asn 20 25 169 353 PRT Homo sapiens 169 Phe Ser Phe Leu Lys Pro Leu
Cys Ala Pro Arg Ala Pro Trp Leu Trp 1 5 10 15 Leu Pro Pro Ser Ser
Lys Ser Arg Val His Val Gly Pro Gly Asp Phe 20 25 30 Arg Ser Met
Ser Trp Cys Cys Leu Trp Leu Cys Leu Ser Ser Val Gly 35 40 45 Arg
Thr Gly Ser Ala Gly Pro Ser Leu Pro Phe Ser Glu Leu Cys Ser 50 55
60 Leu Gly Leu Leu Arg Leu Arg Pro Val Phe Ser Pro Leu His Ser Gly
65 70 75 80 Pro Gly Lys Pro Ala Gln Phe Leu Ala Gly Glu Ala Glu Glu
Val Asn 85 90 95 Ala Phe Ala Leu Gly Phe Leu Ser Thr Ser Ser Gly
Val Ser Gly Glu 100 105 110 Asp Glu Val Glu Pro Leu His Asp Gly Val
Glu Glu Ala Glu Lys Lys 115 120 125 Met Glu Glu Glu Gly Val Ser Val
Ser Glu Met Glu Ala Thr Gly Ala 130 135 140 Gln Gly Pro Ser Arg Val
Glu Glu Ala Glu Gly His Thr Glu Val Thr 145 150 155 160 Glu Ala Glu
Gly Ser Gln Gly Thr Ala Glu Ala Asp Gly Pro Gly Ala 165 170 175 Ser
Ser Gly Asp Glu Asp Ala Ser Gly Arg Ala Ala Ser Pro Glu Ser 180 185
190 Ala Ser Ser Thr Pro Glu Ser Leu Gln Ala Arg Arg His His Gln Phe
195 200 205 Leu Glu Pro Ala Pro Ala Pro Gly Ala Ala Val Leu Ser Ser
Glu Pro 210 215 220 Ala Glu Pro Leu Leu Val Arg His Pro Pro Arg Pro
Arg Thr Thr Gly 225 230 235 240 Pro Arg Pro Arg Gln Asp Pro His Lys
Ala Gly Leu Ser His Tyr Val 245 250 255 Lys Leu Phe Ser Phe Tyr Ala
Lys Met Pro Met Glu Arg Lys Ala Leu 260 265 270 Glu Met Val Glu Lys
Cys Leu Asp Lys Tyr Phe Gln His Leu Cys Asp 275 280 285 Asp Leu Glu
Val Phe Ala Ala His Ala Gly Arg Lys Thr Val Lys Pro 290 295 300 Glu
Asp Leu Glu Leu Leu Met Arg Arg Gln Gly Leu Val Thr Asp Gln 305 310
315 320 Val Ser Leu His Val Leu Val Glu Arg His Leu Pro Leu Glu Tyr
Arg 325 330 335 Gln Leu Leu Ile Pro Cys Ala Tyr Ser Gly Asn Ser Val
Phe Pro Ala 340 345 350 Gln 170 27 PRT Homo sapiens MISC_FEATURE
(18) Xaa equals any of the naturally occurring L-amino acids 170
Ala Pro Gly Gly Val Asn Ser Glu Gly Arg Gly Gln His Leu Pro Pro 1 5
10 15 Pro Xaa Leu Ala Val Cys Leu Lys Leu His Leu 20 25 171 198 PRT
Homo sapiens MISC_FEATURE (18) Xaa equals any of the naturally
occurring L-amino acids 171 Ala Pro Gly Gly Val Asn Ser Glu Gly Arg
Gly Gln His Leu Pro Pro 1 5 10 15 Pro Xaa Leu Ala Val Cys Leu Lys
Leu His Leu Met Ser Leu Pro Ile 20 25 30 Pro Trp Leu Ser Leu Pro
Pro Cys Pro Ile Leu Gly Gln Pro Ala Gly 35 40 45 Leu Leu Leu Trp
Leu Phe Arg Pro Phe Ser Gln Cys Cys Gln Cys Pro 50 55 60 Trp Glu
Gly Arg Ala Ser Leu Arg His Pro Asn Gly Pro Ser Gly Cys 65 70 75 80
Arg Glu Ala Glu Ala Trp Pro Gln Arg Ser Leu Leu Arg Gln Gln Leu 85
90 95 Gln Gln Ala His Pro Leu Pro Thr Leu Pro Thr Pro Glu Arg Leu
Pro 100 105 110 Glu Gln Met Leu Phe Pro Ser Ser Ser Ser Lys Pro Phe
Ser Leu Leu 115 120 125 Ser Leu Thr Ile Trp Ala Arg Leu Val Gly Arg
Leu Thr Asn Arg Ile 130 135 140 Cys Pro Val Pro Pro Gly Ser Val Ala
Ser Ser Met Ser Leu Gln Ala 145 150 155 160 Gly Arg Cys Gly Asn Pro
Val Val Leu Pro Gln Pro Met Pro Pro Gly 165 170 175 Leu Leu Cys Met
Asn Glu Cys Ser Leu Val Pro Gly Leu Gly Arg Gly 180 185 190 Gln Val
Asn Ser Arg Val 195 172 44 PRT Homo sapiens 172 Asn Ser Ala Glu Pro
Ala Trp Val Pro Val Cys Ala Arg Gly Gly Gly 1 5 10 15 Ala Gly Cys
Gly Arg Arg Arg Gly Arg Arg Phe Cys Ala Ala Gly Ala 20 25 30 Val
Pro Ala Ala Glu Arg Gly Gly Glu Asn Gly Ser 35 40 173 124 PRT Homo
sapiens 173 Ser Leu Val Pro Ala Leu Lys Glu Val Val Val Leu Trp Arg
Arg Gln 1 5 10 15 Met Val Leu Tyr Leu Val Trp Ala Phe Ile Pro Glu
Ser Trp Leu Asn 20 25 30 Ser Leu Gly Leu Thr Tyr Trp Pro Gln Lys
Tyr Trp Ala Val Ala Leu 35 40 45 Pro Val Tyr Leu Leu Ile Ala Ile
Val Ile Gly Tyr Val Leu Leu Phe 50 55 60 Gly Ile Asn Met Met Ser
Thr Ser Pro Leu Asp Ser Ile His Thr Ile 65 70 75 80 Thr Asp Asn Tyr
Ala Lys Asn Gln Gln Gln Lys Lys Tyr Gln Glu Glu 85 90 95 Ala Ile
Pro Ala Leu Arg Asp Ile Ser Ile Ser Glu Val Asn Gln Met 100 105 110
Phe Phe Leu Ala Ala Lys Glu Leu Tyr Thr Lys Asn 115 120 174 28 PRT
Homo sapiens 174 Met Val Leu Tyr Leu Val Trp Ala Phe Ile Pro Glu
Ser Trp Leu Asn 1 5 10 15 Ser Leu Gly Leu Thr Tyr Trp Pro Gln Lys
Tyr Trp 20
25 175 25 PRT Homo sapiens 175 Tyr Trp Ala Val Ala Leu Pro Val Tyr
Leu Leu Ile Ala Ile Val Ile 1 5 10 15 Gly Tyr Val Leu Leu Phe Gly
Ile Asn 20 25 176 22 PRT Homo sapiens 176 Gln Gln Gln Lys Lys Tyr
Gln Glu Glu Ala Ile Pro Ala Leu Arg Asp 1 5 10 15 Ile Ser Ile Ser
Glu Val 20 177 104 PRT Homo sapiens 177 Asn Ser Ala Glu Pro Ala Trp
Val Pro Val Cys Ala Arg Gly Gly Gly 1 5 10 15 Ala Gly Cys Gly Arg
Arg Arg Gly Arg Arg Phe Cys Ala Ala Gly Ala 20 25 30 Val Pro Ala
Ala Glu Arg Gly Gly Glu Asn Gly Ser Met Val Ser Arg 35 40 45 Ser
Thr Ser Leu Thr Leu Ile Val Phe Leu Phe His Arg Leu Ser Lys 50 55
60 Ala Pro Gly Lys Met Val Glu Asn Ser Pro Ser Pro Leu Pro Glu Arg
65 70 75 80 Ala Ile Tyr Gly Phe Val Leu Phe Leu Ser Ser Gln Phe Gly
Phe Lys 85 90 95 Asn Leu Lys Gly Ser Arg Val Cys 100 178 32 PRT
Homo sapiens MISC_FEATURE (26) Xaa equals any of the naturally
occurring L-amino acids 178 Leu Ser Pro Arg Leu Phe Asp Ala Gly Ile
Leu Leu Trp Gly Ala Ser 1 5 10 15 Val Asn Val Thr Ile Trp Glu Val
Arg Xaa Ala Gln Ser Ser Ala Ser 20 25 30 179 132 PRT Homo sapiens
MISC_FEATURE (26) Xaa equals any of the naturally occurring L-amino
acids 179 Leu Ser Pro Arg Leu Phe Asp Ala Gly Ile Leu Leu Trp Gly
Ala Ser 1 5 10 15 Val Asn Val Thr Ile Trp Glu Val Arg Xaa Ala Gln
Ser Ser Ala Ser 20 25 30 Met Leu Pro Ser Ala Trp Gly Pro Leu Gln
Val Ala Ser Phe Phe Leu 35 40 45 Leu Ser Phe Xaa Phe Cys Phe Leu
Ser Ser Ser Pro His Leu Gly Arg 50 55 60 Gln Glu Thr His Xaa Val
Val Leu Glu Asp Asp Glu Gly Ala Pro Cys 65 70 75 80 Pro Ala Glu Asp
Glu Leu Ala Leu Gln Asp Asn Gly Phe Leu Ser Lys 85 90 95 Asn Glu
Val Leu Arg Thr Arg Cys Leu Gly Ser Arg Ser Gly Ser Ala 100 105 110
Ser Ala Thr Pro Pro Thr Thr Ser Gly Thr Ala Arg Ala Ala Arg Pro 115
120 125 Pro Ser Gln Cys 130 180 32 PRT Homo sapiens 180 Asn Leu Thr
Ser Asp Pro Arg Pro Leu Ala Leu Pro Pro Pro Cys Gly 1 5 10 15 Asp
Phe Ile Lys Val Thr Ser Phe Ser Pro Gly Leu Glu Thr His Thr 20 25
30 181 135 PRT Homo sapiens MISC_FEATURE (14) Xaa equals any of the
naturally occurring L-amino acids 181 Glu Gln Gln Arg Leu Arg Asp
Arg Glu Thr Gln Thr Gly Xaa Asp Ser 1 5 10 15 Arg Ala Lys Thr Gln
Arg Gly Glu Asp Gly Glu Ser Glu Arg Gly Arg 20 25 30 Trp Arg Leu
Arg Glu Gly Glu Asp Gly Asp Ser Glu Arg Glu Glu Asp 35 40 45 Gly
Asp Ser Glu Arg Trp Arg Leu Arg Ser Met Glu Ser Gln Arg Gly 50 55
60 Glu Asp Gly His Ser Gly Gly Trp Arg Val Arg Arg Met Glu Thr His
65 70 75 80 Arg Lys Gly Arg Met Glu Ser Gln Glu Arg Leu Glu Thr Gly
Glu Gly 85 90 95 Ile Glu Thr Gln Arg Gly Glu Asp Gly Asp Ser Glu
Gly Gly Arg Trp 100 105 110 Arg Leu Lys Glu Asp Gly Asn Pro Gly Glu
Arg Arg Thr Glu Met Arg 115 120 125 Gln Arg Leu Gly Glu Ala Gly 130
135 182 220 PRT Homo sapiens MISC_FEATURE (7) Xaa equals any of the
naturally occurring L-amino acids 182 Gly His Gly Val Ala Gly Xaa
Cys Leu Pro Gln Pro Leu Leu Pro Pro 1 5 10 15 Ser Pro Pro Asp Tyr
Asp Glu Arg Ser His Leu His Asp Thr Phe Thr 20 25 30 Gln Met Thr
His Ala Leu Gln Glu Leu Ala Ala Ala Gln Gly Ser Phe 35 40 45 Glu
Val Ala Phe Pro Asp Ala Ala Glu Lys Met Lys Lys Val Phe Thr 50 55
60 Gln Leu Lys Glu Ala Gln Ala Cys Ile Pro Pro Cys Glu Gly Leu Gln
65 70 75 80 Glu Phe Ala Arg Arg Phe Leu Cys Ser Gly Cys Tyr Ser Arg
Val Cys 85 90 95 Asp Leu Pro Leu Asp Cys Pro Val Gln Asp Val Thr
Val Thr Arg Gly 100 105 110 Asp Gln Ala Met Phe Ser Cys Ile Val Asn
Phe Gln Leu Pro Lys Glu 115 120 125 Glu Ile Thr Tyr Ser Trp Lys Phe
Ala Gly Gly Gly Leu Arg Thr Gln 130 135 140 Asp Leu Ser Tyr Phe Arg
Asp Met Pro Arg Ala Glu Gly Tyr Leu Ala 145 150 155 160 Arg Ile Arg
Pro Ala Gln Leu Thr His Arg Gly Thr Phe Ser Cys Val 165 170 175 Ile
Lys Gln Asp Gln Arg Pro Leu Ala Arg Leu Tyr Phe Phe Leu Asn 180 185
190 Val Thr Gly Arg Pro Arg Gly Arg Arg Gln Ser Cys Arg Pro Arg Ser
195 200 205 Gly Lys Cys Cys Ala Gly Arg Arg Gly Met Pro Ser 210 215
220 183 41 PRT Homo sapiens 183 Gly Asp His Pro His Phe Ile Ser Val
Leu Gly Lys Val Gln Arg Glu 1 5 10 15 Gly Arg Arg Gly Pro Glu Gly
Gln Ala Glu Gly Gln Thr Glu Arg Asn 20 25 30 Ser Gln Arg Arg Lys
Ala Gln Arg Pro 35 40 184 129 PRT Homo sapiens 184 Asn Leu Thr Ser
Asp Pro Arg Pro Leu Ala Leu Pro Pro Pro Cys Gly 1 5 10 15 Asp Phe
Ile Lys Val Thr Ser Phe Ser Pro Gly Leu Glu Thr His Thr 20 25 30
Met Ala Leu Leu Ala Leu Ala Ser Ala Val Pro Ser Ala Leu Leu Ala 35
40 45 Leu Ala Val Phe Arg Val Pro Ala Trp Ala Cys Leu Leu Cys Phe
Thr 50 55 60 Thr Tyr Ser Glu Arg Leu Arg Ile Cys Gln Met Phe Val
Gly Met Arg 65 70 75 80 Ser Pro Ser Leu Lys Ser Val Arg Arg Pro Ser
Arg Pro Pro Ser Arg 85 90 95 Ala Ser Leu Thr Pro Lys Ser Val Arg
Arg Pro Ser Thr Leu His Gln 100 105 110 Cys Pro Gly Glu Gly Ala Glu
Gly Gly Gln Glu Arg Pro Arg Gly Ser 115 120 125 Gly 185 13 PRT Homo
sapiens 185 Met Leu Val Tyr Gln Asn Gln Ala Gln Phe Ser Ser Asn 1 5
10 186 65 PRT Homo sapiens 186 Met Leu Val Tyr Gln Asn Gln Ala Gln
Phe Ser Ser Asn Met Trp Leu 1 5 10 15 Asn Phe Ser Asp Val His Thr
Tyr Leu Ser Ser Ile Ala Leu Leu Cys 20 25 30 Phe Cys Leu Ser Gly
Val Leu Cys Cys Ile Cys Asn Asn Ser Val Phe 35 40 45 His Ile Gln
Gln Tyr Ile Leu Ile Ile Ile Thr Phe Pro Leu Val Val 50 55 60 Ile
65
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