U.S. patent application number 11/601607 was filed with the patent office on 2008-05-29 for antibodies to novel lipoxygenase proteins.
This patent application is currently assigned to Lexicon Genetics Incorporated. Invention is credited to Glenn Friedrich, Michael Nehls, Arthur T. Sands, C. Alexander Turner, Brian Zambrowicz.
Application Number | 20080125578 11/601607 |
Document ID | / |
Family ID | 26826972 |
Filed Date | 2008-05-29 |
United States Patent
Application |
20080125578 |
Kind Code |
A1 |
Turner; C. Alexander ; et
al. |
May 29, 2008 |
Antibodies to novel lipoxygenase proteins
Abstract
The nucleotide and corresponding amino acid sequences are
reported for a novel class of mammalian lipoxygenase proteins. The
novel lipoxygenase encoding polynucleotides were obtained from
human gene trap clones and human cDNA libraries.
Inventors: |
Turner; C. Alexander; (The
Woodlands, TX) ; Zambrowicz; Brian; (The Woodlands,
TX) ; Nehls; Michael; (Stockdorf, DE) ;
Friedrich; Glenn; (Houston, TX) ; Sands; Arthur
T.; (The Woodlands, TX) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Lexicon Genetics
Incorporated
|
Family ID: |
26826972 |
Appl. No.: |
11/601607 |
Filed: |
November 16, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10422264 |
Apr 23, 2003 |
7144730 |
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11601607 |
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09547435 |
Apr 12, 2000 |
6582957 |
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10422264 |
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60128817 |
Apr 12, 1999 |
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60150454 |
Aug 24, 1999 |
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Current U.S.
Class: |
530/387.3 ;
530/387.9; 530/388.1 |
Current CPC
Class: |
A01K 2217/05 20130101;
C12N 9/0069 20130101 |
Class at
Publication: |
530/387.3 ;
530/387.9; 530/388.1 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1-6. (canceled)
7. An isolated antibody or epitope-binding fragment thereof that
specifically binds a polypeptide encoded by a first polynucleotide
capable of hybridizing to a second polynucleotide in 0.5 M
NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
65.degree. C. with washing in 0.1.times.SSC/0.1% SDS at 68.degree.
C., wherein the second polynucleotide is complementary to the
entire nucleic acid sequence of SEQ ID NO: 1, and wherein the
polypeptide has lipoxygenase activity.
8. The isolated antibody or epitope-binding fragment thereof of
claim 7, wherein the antibody or epitope-binding fragment thereof
specifically binds a polypeptide having the amino acid sequence of
SEQ ID NO: 2.
9. The isolated antibody of claim 7, wherein the antibody or
epitope-binding fragment thereof is a monoclonal antibody or
epitope-binding fragment thereof.
10. The isolated antibody or epitope-binding fragment thereof of
claim 7, wherein the antibody or epitope-binding fragment thereof
is a humanized antibody or epitope-binding fragment thereof.
11. The isolated antibody or epitope-binding fragment thereof of
claim 7, wherein the antibody or epitope-binding fragment thereof
is a chimeric antibody or epitope-binding fragment thereof.
12. The isolated antibody or epitope-binding fragment thereof of
claim 7, wherein the antibody or epitope-binding fragment thereof
is an antibody fragment selected from a Fab fragment and a
F(ab').sub.2 fragment.
13. The isolated antibody or epitope-binding fragment thereof of
claim 7, wherein the antibody or epitope-binding fragment thereof
is a single-chain antibody.
14. The isolated antibody or epitope-binding fragment thereof of
claim 7, wherein the antibody or epitope-binding fragment thereof
further comprises a detectable label.
Description
[0001] The present application claims priority to U.S. Provisional
Application Ser. Nos. 60/128,817 and 60/150,454, all of which are
incorporated herein by reference in their entirety for any
purpose.
1. INTRODUCTION
[0002] The present invention relates to the discovery,
identification, and characterization of novel human polynucleotides
that encode proteins that share sequence similarity with
lipoxygenases. The invention encompasses the described
polynucleotides, host cell expression systems, the encoded
proteins, fusion proteins, polypeptides and peptides, antibodies to
the encoded proteins and peptides, and genetically engineered
animals that lack the disclosed genes or over-express the disclosed
genes, or antagonists and agonists of the proteins, and other
compounds that modulate the expression or activity of the proteins
encoded by the disclosed polynucleotides that can be used for
diagnosis, drug screening, clinical trial monitoring, or the
treatment of physiological or behavioral disorders.
2. BACKGROUND OF THE INVENTION
[0003] Lipoxygenases are enzymes that mediate the oxidation of
lipid substrates. As such, lipoxygenases are involved in the
synthesis of leukotrienes. Leukotrienes influence a variety of
biological processes, and can serve as, inter alia, potent
chemotactic agents, and mediators of inflammation, smooth muscle
contraction, etc. Accordingly, lipoxygenases represent a key target
for the regulation of a variety of biological pathways and
conditions.
3. SUMMARY OF THE INVENTION
[0004] The present invention relates to the discovery,
identification, and characterization of nucleotides that encode
novel human lipoxygenase proteins, and the corresponding amino acid
sequences of these proteins. The novel human proteins (NHPs)
described for the first time herein share structural similarity
with animal and plant lipoxygenase proteins. As such, the novel
genes represent a new class of lipoxygenase proteins with a range
of homologues and orthologs that transcend a broad range of phyla
and species.
[0005] The invention comprises (a) polypeptides with SEQ ID NOS:2,
4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28; (b) homologues
and allelic variants of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, and 28; (c) fragments of SEQ ID NOS:2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, and 28 of any size, for example,
from 4 amino acids to less than the full-length of a polypeptide of
SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28
and any number between; (d) fragments of SEQ ID NOS:2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, and 28 that correspond to a
functional domain (for example, a catalytic domain, a signal
sequence, a ligand binding domain, a regulatory domain, etc.); (e)
fusion proteins comprising a polypeptide sequence of any one of (a)
through (d); (f) mutant polypeptides (including engineered and
naturally occurring mutants) comprising a polypeptide sequence of
any one of (a) through (d), including, but not limited to, deletion
mutants, insertion mutants, substitution mutants, and mutant
polypeptides in which all or a part of at least one of the domains
is deleted or altered (e.g., a mutant of the active site with
altered substrate specificity).
[0006] The invention further comprises (g) polynucleotides with SEQ
ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29;
(h) polynucleotides encoding any one of the polypeptides of the
invention including, but not limited to, polypeptides specifically
described in (a) through (f) above; (i) polynucleotides capable of
hybridizing to a second polynucleotide that is complementary to a
polynucleotide described in (g) and/or (h) above under conditions
of low, medium, or high stringency; (j) oligonucleotides
corresponding to a segment of a polynucleotide described in (g)
through (i) above and such oligonucleotides having any size from 2
nucleotides through less than the full-length polynucleotide and
any length inbetween.
[0007] In certain embodiments, the novel human nucleic acid
sequences described herein, encode proteins/open reading frames
(ORFs) of 711, 489, 556, 334, 615, 460, 291, 69, 139, 195, 110,
867, 645, and 771 amino acids in length (see SEQ ID NOS:2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 respectively).
[0008] The invention further comprises antibodies to any one of the
polypeptides or polynucleotides of the invention. The invention
also comprises host cells that are engineered to contain and/or
express any one of the polynucleotides and/or polypeptides of the
invention.
[0009] The invention also comprises agonists and antagonists of the
described NHPs, including small molecules, large molecules, mutant
NHPs, or portions thereof that compete with native NHP, and
antibodies. The invention further comprises nucleotide sequences
that can be used to inhibit the expression of the described NHPs
(e.g., antisense and ribozyme oligonucleotides and/or
polynucleotides, and gene or regulatory sequence replacement
constructs) or to enhance the expression of the described NHP genes
(e.g., expression constructs that place the described gene under
the control of a strong promoter system), and transgenic animals
that express a NHP transgene, or "knock-outs" (which can be
conditional) that do not express functional NHP.
[0010] Further, the present invention also relates to methods for
identifying compounds that modulate, i.e., act as agonists or
antagonists of, NHP expression and/or NHP product activity that
utilize purified preparations of the described NHPs and/or NHP
product, or cells expressing the same. Such compounds can be used
as therapeutic agents for the treatment of any of a wide variety of
symptoms associated with biological disorders or imbalances.
4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES
[0011] The Sequence Listing provides the sequences of 14
lipoxygenase-like ORFs that are encoded by the described NHP
polynucleotides (SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 25, 27, and 29) and the amino acid sequences (SEQ ID NOS:2, 4,
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28) encoded
thereby.
5. DETAILED DESCRIPTION OF THE INVENTION
[0012] Lipoxygenases oxidize, or oxygenate, lipids to produce
leukotrienes. Depending on the leukotriene synthesized, a wide
variety of biological functions can be affected. Typically,
leukotrienes will bind cognate receptors an trigger a biological
effect (such as, for example, signal transduction). Interfering
with lipoxygenase activity ultimately effects leukotriene
production and downstream leukotriene-mediated processes.
Alternatively, enhancing lipoxygenase activity in vivo, can boost
the effects/activity levels the corresponding biological processes.
Various lipoxygenase activities can be found in a variety of cells
and tissues in both animals and plants. Three predominant types of
lipoxygenases include the 5-, 12-, and 15-lipoxygenases, and each
type of lipoxygenase can have additional forms depending upon the
tissues or cells in which they are expressed.
[0013] The 5-, 12-, and 15-lipoxygenases, and the leukotrienes they
produce, have been implicated with a variety of diseases and
disorders. Given that leukotrienes can modulate inflammatory
reactions, they have been associated with a spectrum of mammalian
diseases including, but not limited to, asthma, eye diseases,
anaphylaxis, lung disease, hematological disorders, infectious
diseases, granulomatosis, abscess, pancreatitis, prostatitis,
hepatitis, atherosclerosis, heart disease, graft rejection,
thrombosis, restenosis, ulcers, kidney disease, hypertension,
dermatoses, cramping, autoimmune disorders (lupus, scleroderma,
Crohn's disease, rheumatoid arthritis, etc.), granulomatosis,
hyperproliferative diseases, cancer, nausea, headache, metastases,
inflammatory bowel disorder, allergy, cancer, arthritis, eczema,
melanoma, erythema, acne, psoriasis, shingles, infectious disease,
and diabetes. Accordingly, one embodiment of the present invention
are processes for identifying compounds useful for the treatment of
one or more of the above diseases and disorders that include the
use of one or more of the described lipoxygenase-like genes,
proteins, or a novel portion thereof.
[0014] Given the biological importance of lipoxygenases, the genes
encoding such proteins (and the proteins encoded thereby as well as
inhibitors thereof) have been subjected to intense
scientific/commercial scrutiny (see, for example, U.S. Pat. Nos.
5,036,105, 5,162,365, 5,504,097, 5,066,679, 5,830,453, 4,761,403,
5,589,506, 5,026,729, and 5,861,268) (all of which are herein
incorporated by reference in their entirety).
[0015] The presently described NHPs share significant similarity
with previously described human lipoxygenases. Expression studies
using RT-PCR detect NHP transcripts in, inter alia, neural tissue
(i.e., brain, spinal cord, etc.), skin, testis, prostate, adrenal
gland, cervix, salivary gland, pancreas, heart, lymphoid cells
(lymph node, spleen, thymus), and mammary glands. Northern analysis
showed a predominant signal in testis, with less predominant, but
longer, transcripts detectable in testis, lymph node, and spinal
cord. A full length cDNA of a NHP coding region (with 5' and 3'
extensions) was isolated from a human brain cDNA library (Edge
BioSystems, Gaithersburg, Md.) and sequenced (SEQ ID NO: 29). A
possible murine ortholog of the described NHPs is predominantly
expressed in skin (Kinzig et al., 1999, Genomics 58:158-164).
[0016] The invention encompasses the use of the described NHP
nucleotides, NHPs and peptides, as well as antibodies, preferably
monoclonal antibodies, or binding fragments, domains, or fusion
proteins thereof, or anti-idiotypic variants derived therefrom,
that bind NHPs, other antagonists that inhibit binding activity or
expression, or agonists that activate NHP activity or increase NHP
expression, in the diagnosis and/or treatment of disease.
[0017] In particular, the invention described in the subsections
below encompasses NHP polypeptides or peptides corresponding to
functional domains of NHPs, mutated, truncated or deleted NHPs
(e.g., NHPs missing one or more functional domains or portions
thereof), NHP fusion proteins (e.g., a NHP or a functional domain
of a NHP fused to an unrelated protein or peptide such as an
immunoglobulin constant region, i.e., IgFc), nucleotide sequences
encoding such products, and host cell expression systems that can
produce such NHP products.
[0018] The invention also encompasses antibodies and anti-idiotypic
antibodies (including Fab fragments), antagonists and agonists of
the NHP, as well as compounds or nucleotide constructs that inhibit
expression of a NHP gene (transcription factor inhibitors,
antisense and ribozyme molecules, or gene or regulatory sequence
replacement constructs), or promote expression of NHP (e.g.,
expression constructs in which NHP coding sequences are operatively
associated with expression control elements such as promoters,
promoter/enhancers, etc.). The invention also relates to host cells
and animals genetically engineered to express the NHPs (or mutant
variants thereof) or to inhibit or "knock-out" expression of an
animal homolog of an endogenous NHP gene.
[0019] The NHPs or peptides, NHP fusion proteins, NHP nucleotide
sequences, antibodies, antagonists and agonists can be useful for
the detection of mutant NHPs or inappropriately expressed NHPs for
the diagnosis of disease. The NHP proteins or peptides, NHP fusion
proteins, NHP nucleotide sequences, host cell expression systems,
antibodies, antagonists, agonists and genetically engineered cells
and animals can be used for screening for drugs (or high throughput
screening of combinatorial libraries) effective in the treatment of
the symptomatic or phenotypic manifestations of perturbing the
normal function of NHP in the body.
[0020] The use of engineered host cells and/or animals offers an
advantage in that such systems allow for both the identification of
compounds that interact with an NHP, and also provide information
regarding the biological significance of the NHP.
[0021] Finally, NHP products (especially soluble derivatives such
as peptides corresponding to the NHP), and NHP fusion protein
products (such as NHP-Ig fusion proteins, i.e., fusions of a NHP,
or a domain of a NHP, to an IgFc), antibodies and anti-idiotypic
antibodies (including Fab fragments), antagonists or agonists
(including compounds that modulate signal transduction which may
act on downstream targets in a NHP-associated leukotriene pathway)
can be used to directly treat diseases or disorders.
[0022] Nucleotide constructs encoding such NHP products can be
delivered to host cells that subsequently express the products in
vivo; these genetically engineered cells function as "bioreactors"
in the body delivering a continuous supply of a NHP, a NHP peptide,
or a NHP fusion protein to the body. Nucleotide constructs encoding
functional NHPs, mutant NHPs, as well as antisense and ribozyme
molecules can also be used in "gene therapy" approaches for the
modulation of NHP expression. Thus, the invention also encompasses
pharmaceutical formulations and methods for treating biological
disorders.
[0023] Various aspects of the invention are described in greater
detail in the subsections below.
[0024] 5.1. NHP Polynucleotides
[0025] The cDNA sequences (SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, and 29) and deduced amino acid sequences
(SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and
28) corresponding to the described. NHPs are presented in the
Sequence Listing. The NHP ORFs were obtained from human testis and
brain cDNA libraries using probes and/or primers generated from
human gene trapped sequence tags.
[0026] The NHP sequences of the present invention include: (a) the
human DNA sequences presented in the Sequence Listing and
additionally contemplate any nucleotide sequence encoding a
contiguous and functional NHP that hybridizes to a complement of
the DNA sequences presented in the Sequence Listing under highly
stringent conditions, e.g., hybridization to filter-bound DNA in
0.5 M NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
65.degree. C., and washing in 0.1.times.SSC/0.1% SDS at 68.degree.
C. (Ausubel F. M. et al., eds., 1989, Current Protocols in
Molecular Biology, Vol. I, Green Publishing Associates, Inc., and
John Wiley & sons, Inc., New York, at p. 2.10.3) and encodes a
functionally equivalent gene product. Additionally contemplated are
any nucleotide sequences that hybridize to the complement of the
DNA sequences that encode and express an amino acid sequence
presented in the Sequence Listing under moderately stringent
conditions, e.g., washing in 0.2.times.SSC/0.1% SDS at 42.degree.
C. (Ausubel et al., 1989, supra), yet which still encode a
functionally equivalent NHP product. Functional equivalents of NHP
include naturally occurring NHPs present in other species, and
mutant NHPs whether naturally occurring or engineered. The
invention also includes degenerate variants of the disclosed
sequences.
[0027] The invention also includes nucleic acid molecules,
preferably DNA molecules, that hybridize to, and are therefore the
complements of, the described NHP nucleotide sequences. Such
hybridization conditions may be highly stringent or less highly
stringent, as described above. In instances wherein the nucleic
acid molecules are deoxyoligonucleotides ("DNA oligos"), such
molecules (and particularly about 16 to about 100 base long, about
20 to about 80, or about 34 to about 45 base long, or any variation
or combination of sizes represented therein that incorporate a
contiguous region of sequence first disclosed in the present
Sequence Listing, can be used in conjunction with the polymerase
chain reaction (PCR) to screen libraries, isolate clones, and
prepare cloning and sequencing templates, etc.
[0028] Alternatively, the oligonucleotides can be used singly or in
chip format as hybridization probes. For example, a series of the
described NHP oligonucleotide sequences, or the complements
thereof, can be used to represent all or a portion of the described
NHP sequences. The oligonucleotides, typically between about 16 to
about 40 (or any whole number within the stated range) nucleotides
in length, may partially overlap each other and/or the NHP sequence
may be represented using oligonucleotides that do not overlap.
Accordingly, the described NHP polynucleotide sequences shall
typically comprise at least about two or three distinct
oligonucleotide sequences of at least about 18, and preferably
about 25, nucleotides in length that are first disclosed in the
described Sequence Listing. Such oligonucleotide sequences may
begin at any nucleotide present within a sequence in the Sequence
Listing and proceed in either a sense (5'-to-3') orientation
vis-a-vis the described sequence or in an antisense orientation.
For oligonucleotide probes, highly stringent conditions may refer,
e.g., to washing in 6.times.SSC/0.05% sodium pyrophosphate at
37.degree. C. (for 14-base oligos), 48.degree. C. (for 17-base
oligos), 55.degree. C. (for 20-base oligos), and 60.degree. C. (for
23-base oligos). These nucleic acid molecules may encode or act as
NHP gene antisense molecules, useful, for example, in NHP gene
regulation (for and/or as antisense primers in amplification
reactions of NHP gene nucleic acid sequences). With respect to NHP
gene regulation, such techniques can be used to regulate biological
functions. For example, it has been reported that lipoxygenase mRNA
can be translationally "silenced" by a differentiation control
element in the 3' untranslated region (UTR) of the transcript in
erythroid cells (Ostareck et al., 1997, Cell, 89:597-606). Further,
such sequences may be used as part of ribozyme and/or triple helix
sequences, also useful for NHP gene regulation.
[0029] Lipoxygenase antisense oligonucleotides 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-(carboxyhydrbxylmethyl)uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0030] 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.
[0031] In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group consisting of a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
[0032] In yet another embodiment, the antisense oligonucleotide is
an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier et al., 1987, Nucl.
Acids Res. 15:6625-6641). The oligonucleotide is a
2'-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.
15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987,
FEBS Lett. 215:327-330).
[0033] Oligonucleotides of the present 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.
(1988, Nucl. Acids Res. 16:3209), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:7448-7451), etc.
[0034] Low stringency conditions are well known to those of skill
in the art, and will vary predictably depending on the specific
organisms from which the library and the labeled sequences are
derived. For guidance regarding such conditions see, for example,
Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual (and
periodic updates thereof), Cold Springs Harbor Press, N.Y.; and
Ausubel et al., 1989, Current Protocols in Molecular Biology, Green
Publishing Associates and Wiley Interscience, N.Y.
[0035] Alternatively, suitably labeled NHP nucleotide probes may be
used to screen a human genomic library using appropriately
stringent conditions, or by PCR. The identification and
characterization of human genomic clones is helpful for identifying
polymorphisms, determining the genomic structure of a given
locus/allele, and designing diagnostic tests. For example,
sequences derived from regions adjacent to the intron/exon
boundaries of the human gene can be used to design primers for use
in amplification assays to detect mutations within the exons,
introns, splice sites (e.g., splice acceptor and/or donor sites),
etc., that can be used in diagnostics and pharmacogenomics.
[0036] Further, a NHP gene homolog may be isolated from nucleic
acid of the organism of interest by performing PCR using two
degenerate oligonucleotide primer pools designed on the basis of
amino acid sequences within the NHP product disclosed herein. The
template for the reaction may be total RNA, mRNA, and/or cDNA
obtained by reverse transcription of mRNA prepared from, for
example, human or non-human cell lines or tissue, such as choroid
plexus, known or suspected to express a NHP gene allele.
[0037] The PCR product may be subcloned and sequenced to ensure
that the amplified sequences represent the sequence of the desired
NHP gene. The PCR fragment may then be used to isolate a full
length cDNA clone by a variety of methods. For example, the
amplified fragment may be labeled and used to screen a cDNA
library, such as a bacteriophage cDNA library. Alternatively, the
labeled fragment may be used to isolate genomic clones via the
screening of a genomic library.
[0038] PCR technology may also be utilized to isolate full length
cDNA sequences. For example, RNA may be isolated, following
standard procedures, from an appropriate cellular or tissue source
(i.e., one known, or suspected, to express a NHP gene, such as, for
example, skin, testis, or brain tissue). A reverse transcription
(RT) reaction may be performed on the RNA using an oligonucleotide
primer specific for the most 5' end of the amplified fragment for
the priming of first strand synthesis. The resulting RNA/DNA hybrid
may then be "tailed" using a standard terminal transferase
reaction, the hybrid may be digested with RNase H, and second
strand synthesis may then be primed with a complementary primer.
Thus, cDNA sequences upstream of the amplified fragment may easily
be isolated. For a review of cloning strategies which may be used,
see e.g., Sambrook et al., 1989, supra.
[0039] A cDNA of a mutant NHP gene may be isolated, for example, by
using PCR. In this case, the first cDNA strand may be synthesized
by hybridizing an oligo-dT oligonucleotide to mRNA isolated from
tissue known or suspected to be expressed in an individual
putatively carrying a mutant NHP allele, and by extending the new
strand with reverse transcriptase. The second strand of the cDNA is
then synthesized using an oligonucleotide that hybridizes
specifically to the 5' end of the normal gene. Using these two
primers, the product is then amplified via PCR, optionally cloned
into a suitable vector, and subjected to DNA sequence analysis
through methods well known to those of skill in the art. By
comparing the DNA sequence of the mutant NHP allele to that of the
normal NHP allele, the mutation(s) responsible for the loss or
alteration of function of the mutant NHP gene product can be
ascertained.
[0040] Alternatively, a genomic library can be constructed using
DNA obtained from an individual suspected of or known to carry the
mutant NHP allele, or a cDNA library can be constructed using RNA
from a tissue known, or suspected, to express the mutant NHP
allele. A normal NHP gene, or any suitable fragment thereof, can
then be labeled and used as a probe to identify the corresponding
mutant NHP allele in such libraries. Clones containing the mutant
NHP gene sequences may then be purified and subjected to sequence
analysis according to methods well known to those of skill in the
art.
[0041] Additionally, an expression library can be constructed
utilizing cDNA synthesized from, for example, RNA isolated from a
tissue known, or suspected, to express a mutant NHP allele in an
individual suspected of or known to carry such a mutant allele. In
this manner, gene products made by the putatively mutant tissue may
be expressed and screened using standard antibody screening
techniques in conjunction with antibodies raised against the normal
NHP product, as described, below, in Section 5.3. (For screening
techniques, see, for example, Harlow, E. and Lane, eds., 1988,
"Antibodies: A Laboratory Manual", Cold Spring Harbor Press, Cold
Spring Harbor.)
[0042] Additionally, screening can be accomplished by screening
with labeled NHP fusion proteins, such as, for example, AP-NHP or
NHP-AP fusion proteins. In cases where a NHP mutation results in an
expressed gene product with altered function (e.g., as a result of
a missense or a frameshift mutation), a polyclonal set of
antibodies to NHP are likely to cross-react with the mutant NHP
gene product. Library clones detected via their reaction with such
labeled antibodies can be purified and subjected to sequence
analysis according to methods well known to those of skill in the
art.
[0043] An additional method of "screening" for NHP-related
sequences (both nucleotide an amino acid) involves electronic
methods of storing, retrieving, and analyzing the described
sequences and derivatives thereof. Accordingly, an additional
embodiment of the present invention includes computer readable
electronic data storage medium, or any system incorporating the
same, that comprises a representation of any contiguous stretch of
sequence first disclosed in the Sequence Listing.
[0044] The invention also encompasses nucleotide sequences that
encode mutant NHPs, peptide fragments of the NHPs, truncated NHPs,
and NHP fusion proteins. These include, but are not limited to
nucleotide sequences encoding mutant NHPs described in section 5.2
infra; polypeptides or peptides corresponding to one or more
domains of the NHP or portions of these domains; truncated NHPs in
which one or more of the domains is deleted, or a truncated
nonfunctional NHP. Nucleotides encoding fusion proteins may
include, but are not limited to, full length NHP sequences,
truncated NHPs, or nucleotides encoding peptide fragments of a NHP
fused to an unrelated protein or peptide, such as for example, a
NHP domain fused to an Ig Fc domain which increases the stability
and half life of the resulting fusion protein (e.g., NHP-Ig) in the
bloodstream; or an enzyme such as a fluorescent protein or a
luminescent protein which can be used as a marker.
[0045] The invention also encompasses: (a) DNA vectors that contain
any of the foregoing NHP coding sequences and/or their complements
(i.e., antisense); (b) DNA expression vectors that contain any of
the foregoing NHP coding sequences operatively associated with a
regulatory element that directs the expression of the coding
sequences; (c) genetically engineered host cells that contain any
of the foregoing NHP coding sequences operatively associated with a
regulatory element that directs the expression of the coding
sequences in the host cell; and (d) genetically engineered host
cells that express an endogenous NHP gene under the control of an
exogenously introduced regulatory element (i.e., gene activation).
As used herein, regulatory elements include but are not limited to
inducible and non-inducible promoters, enhancers, operators and
other elements known to those skilled in the art that drive and
regulate expression. Such regulatory elements include but are not
limited to the cytomegalovirus hCMV immediate early gene,
regulatable, viral (particularly retroviral LTR promoters) the
early or late promoters of SV40 adenovirus, the lac system, the trp
system, the TAC system, the TRC system, the major operator and
promoter regions of phage lambda, the control regions of fd coat
protein, the promoter for 3-phosphoglycerate kinase (PGK), the
promoters of acid phosphatase, and the promoters of the yeast
.alpha.-mating factors.
[0046] 5.2. NHP Polypeptides
[0047] NHPs, polypeptides, peptide fragments, mutated, truncated,
or deleted forms of the NHPs, and/or NHP fusion proteins can be
prepared for a variety of uses, including but not limited to the
generation of antibodies, as reagents in diagnostic assays, the
identification of other cellular gene products related to a NHP, as
reagents in assays for screening for compounds that can be as
pharmaceutical reagents useful in the therapeutic treatment of
mental, biological, or medical disorders and disease.
[0048] The Sequence Listing discloses the amino acid sequences
encoded by the described NHP genes. The NHPs have initiator
methionines in DNA sequence contexts consistent with translation
initiation sites. The sequence data presented herein indicate that
alternative forms (e.g., variants arising from alternative
splicing, promoters, etc.) of the NHPs may exist (which may or may
not be tissue specific).
[0049] The NHP amino acid sequences of the invention include the
amino acid sequences presented in the Sequence Listing as well as
analogues and derivatives thereof. Further, corresponding NHP
homologues from other species are encompassed by the invention. In
fact, any NHP protein encoded by the NHP nucleotide sequences
described in Section 5.1, above, are within the scope of the
invention, as are any novel polynucleotide sequences encoding all
or any novel portion of an amino acid sequence presented in the
Sequence Listing. The degenerate nature of the genetic code is well
known, and, accordingly, each amino acid presented in the Sequence
Listing, is generically representative of the well known nucleic
acid "triplet" codon, or in many cases codons, that can encode the
amino acid. As such, as contemplated herein, the amino acid
sequences presented in the Sequence Listing, when taken together
with the genetic code (see, for example, Table 4-1 at page 109 of
"Molecular Cell Biology", 1986, J. Darnell et al. eds., Scientific
American Books, New York, N.Y., herein incorporated by reference)
are generically representative of all the various permutations and
combinations of nucleic acid sequences that can encode such amino
acid sequences.
[0050] The invention also encompasses proteins that are
functionally equivalent to the NHPs encoded by the nucleotide
sequences described in Section 5.1, as judged by any of a number of
criteria, including, but not limited to, the ability to mediate
lipoxygenase activity, the ability to effect an identical or
complementary leukotriene pathway, a change in cellular metabolism
(e.g., ion flux, tyrosine phosphorylation, etc.), or change in
phenotype when the NHP equivalent is similarly expressed or mutated
in an appropriate cell type (such as the amelioration, prevention
or delay of a biochemical, biophysical, or overt phenotype. Such
functionally equivalent NHP proteins include, but are not limited
to, additions or substitutions of amino acid residues within the
amino acid sequence encoded by the NHP nucleotide sequences
described above, in Section 5.1, but which result in a silent
change, thus producing a functionally equivalent gene product.
Amino acid substitutions can be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues involved. For
example, nonpolar (hydrophobic) amino acids include alanine,
leucine, isoleucine, valine, proline, phenylalanine, tryptophan,
and methionine; polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine;
positively charged (basic) amino acids include arginine, lysine,
and histidine; and negatively charged (acidic) amino acids include
aspartic acid and glutamic acid.
[0051] While random mutations can be made to NHP DNA (using random
mutagenesis techniques well known to those skilled in the art) and
the resulting mutant NHPs tested for activity, site-directed
mutations of the NHP coding sequence can be engineered (using
site-directed mutagenesis techniques well known to those skilled in
the art) to generate mutant NHPs with increased function, e.g.,
higher lipoxygenase activity, decreased function, and/or increased
physiological half-life. One starting point for such analysis is to
align the disclosed human sequences with corresponding gene/protein
sequences from, for example, other mammals in order to identify
amino acid sequence motifs that are conserved between different
species. Non-conservative changes can be engineered at variable
positions to alter function, signal transduction capability, or
both. Alternatively, where alteration of function is desired,
deletion or non-conservative alterations of the conserved regions
(i.e., identical amino acids) can be engineered.
[0052] Other mutations to the NHP coding sequence can be made to
generate NHPs that are better suited for expression, scale up, etc.
in the host cells chosen. For example, cysteine residues can be
deleted or substituted with another amino acid in order to
eliminate disulfide bridges; N-linked glycosylation sites can be
altered or eliminated to achieve, for example, expression of a
homogeneous product that is more easily recovered and purified from
yeast hosts which are known to hyperglycosylate-linked sites. To
this end, a variety of amino acid substitutions at one or both of
the first or third amino acid positions of any one or more of the
glycosylation recognition sequences (N-X-S or N-X-T), and/or an
amino acid deletion at the second position of any one or more such
recognition sequences will prevent glycosylation of the NHP at the
modified tripeptide sequence. (See, e.g., Miyajima et al., 1986,
EMBO J. 5(6):1193-1197).
[0053] Peptides corresponding to one or more domains of a NHP,
truncated or deleted NHPs, as well as fusion proteins in which a
full length NHP, a NHP peptide, or truncated NHP is fused to an
unrelated protein, are also within the scope of the invention and
can be designed on the basis of the presently disclosed NHP
nucleotide and NHP amino acid sequences. Such fusion proteins
include, but are not limited to, IgFc fusions which stabilize the
NHP protein or peptide and prolong half-life in vivo; or fusions to
any amino acid sequence that allows the fusion protein to be
anchored to the cell membrane; or fusions to an enzyme, fluorescent
protein, or luminescent protein which provide a marker
function.
[0054] While the NHPs and peptides can be chemically synthesized
(e.g., see Creighton, 1983, Proteins: Structures and Molecular
Principles, W.H. Freeman & Co., N.Y.), large polypeptides
derived from a NHP and full length NHPs can be advantageously
produced by recombinant DNA technology using techniques well known
in the art for expressing nucleic acid containing NHP gene
sequences and/or coding sequences. Such methods can be used to
construct expression vectors containing a NHP nucleotide sequences
described in Section 5.1 and appropriate transcriptional and
translational control signals. These methods include, for example,
in vitro recombinant DNA techniques, synthetic techniques, and in
vivo genetic recombination. See, for example, the techniques
described in Sambrook et al, 1989, supra, and Ausubel et al., 1989,
supra. For example, recombinant lipoxygenase has been successfully
produced in insect cells (using baculo virus) and purified using
nickel affinity chromatography. Alternatively, RNA corresponding to
all or a portion of a transcript encoded by a NHP nucleotide
sequence may be chemically synthesized using, for example,
synthesizers. See, for example, the techniques described in
"Oligonucleotide Synthesis", 1984, Gait, M. J. ed., IRL Press,
Oxford, which is incorporated by reference herein in its
entirety.
[0055] A variety of host-expression vector systems may be utilized
to express the NHP nucleotide sequences of the invention. Where the
NHP peptide or polypeptide is a soluble derivative, the peptide or
polypeptide can be recovered from the culture, or from the host
cell in cases where the NHP peptide or polypeptide is not secreted,
and from the culture media in cases where the NHP peptide or
polypeptide has been engineered to be secreted by the cells.
However, such expression systems also encompass engineered host
cells that express a NHP, or functional equivalent, in situ, i.e.,
anchored in the cell membrane. One study has indicated that the
majority of one type of 15S-lipoxygenase protein can typically be
found in soluble cytoplasmic cell fractions, but the majority of
lipoxygenase activity can be found in the membrane fraction.
[0056] Purification or enrichment of NHP from such expression
systems can be accomplished using appropriate detergents and lipid
micelles and methods well known to those skilled in the art.
However, such engineered host cells themselves may be used in
situations where it is important to not only to retain the
structural and functional characteristics of the NHP, but to assess
biological activity, e.g., in drug screening assays.
[0057] The expression systems that may be used for purposes of the
invention include but are not limited to microorganisms such as
bacteria (e.g., E. coli, B. subtilis) transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing NHP nucleotide sequences; yeast (e.g., Saccharomyces,
Pichia) transformed with recombinant yeast expression vectors
containing NHP nucleotide sequences; insect cell systems infected
with recombinant virus expression vectors (e.g., baculo virus)
containing NHP sequences; plant cell systems infected with
recombinant virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid)
containing NHP nucleotide sequences; or mammalian cell systems
(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression
constructs containing promoters derived from the genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian
viruses (e.g., the adenovirus late promoter; the vaccinia virus
7.5K promoter).
[0058] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the NHP
product being expressed. For example, when a large quantity of such
a protein is to be produced for the identification of molecules
that inhibit or enhance NHP activity, for the generation of
pharmaceutical compositions comprising NHP, or for raising
antibodies to a NHP, vectors that direct the expression of high
levels of fusion protein products that are readily purified may be
desirable. Such vectors include, but are not limited, to the E.
coli expression vector pUR278 (Ruther et al., 1983, EMBO J.
2:1791), in which a NHP coding sequence may be ligated individually
into the vector in-frame with the lacZ coding region so that a
fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,
Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J.
Biol. Chem. 264:5503-5509); and the like. pGEX vectors may also be
used to express foreign polypeptides as fusion proteins with
glutathione S-transferase (GST). In general, such fusion proteins
are soluble and can easily be purified from lysed cells by
adsorption to glutathione-agarose beads followed by elution in the
presence of free glutathione. The PGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the
cloned target gene product can be released from the GST moiety.
[0059] In an insect system, Autographa californica nuclear
polyhidrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. A NHP gene
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter). Successful insertion of NHP gene coding sequence will
result in inactivation of the polyhedrin gene and production of
non-occluded recombinant virus (i.e., virus lacking the
proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted gene is expressed (e.g., see Smith et
al., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).
[0060] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the NHP nucleotide sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing a NHP
product in infected hosts (e.g., See Logan & Shenk, 1984, Proc.
Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may
also be required for efficient translation of inserted NHP
nucleotide sequences. These signals include the ATG initiation
codon and adjacent sequences. In cases where an entire NHP gene or
cDNA, including its own initiation codon and adjacent sequences, is
inserted into the appropriate expression vector, no additional
translational control signals may be needed. However, in cases
where only a portion of a NHP coding sequence is inserted,
exogenous translational control signals, including, perhaps, the
ATG initiation codon, must be provided. Furthermore, the initiation
codon must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert.
[0061] These exogenous translational control signals and initiation
codons can be of a variety of origins, both natural and synthetic.
The efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (See Bittner et al., 1987, Methods in Enzymol.
153:516-544).
[0062] In addition, a host cell strain may be chosen that modulates
the expression of the inserted sequences, or modifies and processes
the gene product in the specific fashion desired. Such
modifications. (e.g., glycosylation) and processing (e.g.,
cleavage) of protein products may be important for the function of
the protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include, but are not limited to, CHO, VERO, BHK, HeLa,
COS, MDCK, 293, 3T3, WI38, and in particular, human cell lines.
[0063] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the NHP sequences described above may be
engineered. Rather than using expression vectors that contain viral
origins of replication, host cells can be transformed with DNA
controlled by appropriate expression control elements (e.g.,
promoter, enhancer sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines that express a NHP product. Such engineered cell lines may be
particularly useful in screening and evaluation of compounds that
affect the endogenous activity of the NHP product.
[0064] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler, et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes
can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for the following genes: dhfr, which confers
resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci.
USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA
78:1527); gpt, which confers resistance to mycophenolic acid
(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072);
neo, which confers resistance to the aminoglycoside G-418
(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro,
which confers resistance to hygromycin (Santerre, et al., 1984,
Gene 30:147).
[0065] Alternatively, any fusion protein may be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. For example, a system described by Janknecht et al.
allows for the ready purification of non-denatured fusion proteins
expressed in human cell lines (Janknecht, et al., 1991, Proc. Natl.
Acad. Sci. USA 88: 8972-8976). In this system, the gene of interest
is subcloned into a vaccinia recombination plasmid such that the
gene's open reading frame is translationally fused to an
amino-terminal tag consisting of six histidine residues. Extracts
from cells infected with recombinant vaccinia virus are loaded onto
Ni.sup.2+ nitriloacetic acid-agarose columns and histidine-tagged
proteins are selectively eluted with imidazole-containing
buffers.
[0066] NHP products can also be expressed in transgenic animals.
Animals of any species, including, but not limited to, worms, mice,
rats, rabbits, guinea pigs, pigs, micro-pigs, birds, goats, and
non-human primates, e.g., baboons, monkeys, and chimpanzees may be
used to generate NHP transgenic animals.
[0067] Any technique known in the art may be used to introduce a
NHP transgene into animals to produce the founder lines of
transgenic animals. Such techniques include, but are not limited to
pronuclear microinjection (Hoppe, P. C. and Wagner, T. E., 1989,
U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into
germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci.,
USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson
et al., 1989, Cell 56:313-321); electroporation of embryos (Lo,
1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated gene
transfer (Lavitrano et al., 1989, Cell 57:717-723); etc. For a
review of such techniques, see Gordon, 1989, Transgenic Animals,
Intl. Rev. Cytol. 115:171-229, which is incorporated by reference
herein in its entirety.
[0068] The present invention provides for transgenic animals that
carry the NHP 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 somatic cell transgenic animals. The transgene
may be integrated as a single transgene or 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.,
1992, Proc. Natl. Acad. Sci. USA 89:6232-6236. 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.
[0069] When it is desired that the NHP gene transgene be integrated
into the chromosomal site of the endogenous NHP gene, gene
targeting is preferred. Briefly, when such a technique is to be
utilized, vectors containing some nucleotide sequences homologous
to the endogenous NHP 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 NHP gene (i.e., "knockout" animals).
[0070] The transgene may also be selectively introduced into a
particular cell type, thus inactivating the endogenous NHP gene in
only that cell type, by following, for example, the teaching of Gu
et al., 1994, Science, 265:103-106. 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.
[0071] Once transgenic animals have been generated, the expression
of the recombinant NHP gene may be assayed utilizing standard
techniques. Initial screening may be accomplished by Southern blot
analysis or PCR techniques to analyze animal tissues to assay
whether 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 RT-PCR.
Samples of NHP gene-expressing tissue, may also be evaluated
immunocytochemically using antibodies specific for the NHP
transgene product.
[0072] 5.3. Antibodies to NHPs
[0073] Antibodies that specifically recognize one or more epitopes
of a NHP, or epitopes of conserved variants of a NHP, or peptide
fragments of a NHP are also encompassed by the invention. Such
antibodies include but are not limited to polyclonal antibodies,
monoclonal antibodies (mAbs), humanized or chimeric antibodies,
single chain antibodies, Fab fragments, F(ab').sub.2 fragments,
fragments produced by a Fab expression library, anti-idiotypic
(anti-Id) antibodies, and epitope-binding fragments of any of the
above.
[0074] The antibodies of the invention may be used, for example, in
the detection of NHP in a biological sample and may, therefore, be
utilized as part of a diagnostic or prognostic technique whereby
patients may be tested for abnormal amounts of NHP. Such antibodies
may also be utilized in conjunction with, for example, compound
screening schemes, as described, below, in Section 5.5, for the
evaluation of the effect of test compounds on expression and/or
activity of a NHP gene product. Additionally, such antibodies can
be used in conjunction gene therapy to, for example, evaluate the
normal and/or engineered NHP-expressing cells prior to their
introduction into the patient. Such antibodies may additionally be
used as a method for the inhibition of abnormal NHP activity. Thus,
such antibodies, domains thereof, or peptides therefrom, may be
utilized as part of treatment methods.
[0075] For the production of antibodies, various host animals may
be immunized by injection with the NHP, an NHP peptide (e.g., one
corresponding to a functional domain of an NHP), truncated NHP
polypeptides (NHP in which one or more domains have been deleted),
functional equivalents of the NHP or mutants of the NHP. Such host
animals may include but are not limited to rabbits, mice, goats,
and rats, to name but a few. Various adjuvants may be used to
increase the immunological response, depending on the host species,
including but not limited to Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies
are heterogeneous populations of antibody molecules derived from
the sera of the immunized animals.
[0076] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, may be obtained by any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique of Kohler and Milstein, (1975,
Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell
hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72;
Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and
the EBV-hybridoma technique (Cole et al., 1985, Monoclonal
Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such
antibodies may be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. The hybridoma producing the
mAb of this invention may be cultivated in vitro or in vivo.
Production of high titers of mAbs in vivo makes this the presently
preferred method of production.
[0077] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608;
Takeda et al., 1985, Nature, 314:452-454) by splicing the genes
from a mouse antibody molecule of appropriate antigen specificity
together with genes from a human antibody molecule of appropriate
biological activity can be used. A chimeric antibody is a molecule
in which different portions are derived from different animal
species, such as those having a variable region derived from a
murine mAb and a human immunoglobulin constant region.
[0078] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988,
Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci.
USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be
adapted to produce single chain antibodies against NHP gene
products. Single chain antibodies are formed by linking the heavy
and light chain fragments of the Fv region via an amino acid
bridge, resulting in a single chain polypeptide.
[0079] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, such fragments include,
but are not limited to: the F(ab').sub.2 fragments which can be
produced by pepsin digestion of the antibody molecule and the Fab
fragments which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments. Alternatively, Fab expression
libraries may be constructed (Huse et al., 1989, Science,
246:1275-1281) to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity.
[0080] Antibodies to a NHP can, in turn, be utilized to generate
anti-idiotype antibodies that "mimic" a given NHP, using techniques
well known to those skilled in the art. (See, e.g., Greenspan &
Bona, 1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.
147(8):2429-2438). For example antibodies which bind to a NHP
domain and competitively inhibit the binding of NHP to its cognate
receptor can be used to generate anti-idiotypes that "mimic" the
NHP and, therefore, bind and activate or neutralize a receptor.
Such anti-idiotypic antibodies or Fab fragments of such
anti-idiotypes can be used in therapeutic regimens involving the
NHP signaling pathway.
[0081] 5.4. Diagnosis of Abnormalities Related to a NHP
[0082] A variety of methods can be employed for the diagnostic and
prognostic evaluation of disorders related to NHP function, and for
the identification of subjects having a predisposition to such
disorders.
[0083] Such methods may, for example, utilize reagents such as the
NHP nucleotide sequences described in Section 5.1, and NHP
antibodies, as described, in Section 5.3. Specifically, such
reagents may be used, for example, for: (1) the detection of the
presence of NHP gene mutations, or the detection of either over- or
under-expression of NHP mRNA relative to a given phenotype; (2) the
detection of either an over- or an under-abundance of NHP gene
product relative to a given phenotype; and (3) the detection of
perturbations or abnormalities in any potential signal
transduction, metabolic, or catabolic: pathway mediated by a
NHP.
[0084] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
specific NHP nucleotide sequence or NHP antibody reagent described
herein, which may be conveniently used, e.g., in clinical settings,
to diagnose patients exhibiting inflammatory disorders.
[0085] For the detection of NHP mutations, any nucleated cell can
be used as a starting source for genomic nucleic acid. For the
detection of NHP gene expression or NHP gene products, any cell
type or tissue in which the NHP gene is expressed, such as, for
example, skin, testis, or brain cells, may be utilized.
[0086] Nucleic acid-based detection techniques are described,
below, in Section 5.4.1. Peptide detection techniques are
described, below, in Section 5.4.2.
[0087] 5.4.1. Detection of NHP Polynucleotides
[0088] Mutations within a NHP gene can be detected by utilizing a
number of techniques. Nucleic acid from any nucleated cell can be
used as the starting point for such assay techniques, and may be
isolated according to standard nucleic acid preparation procedures
which are well known to those of skill in the art.
[0089] DNA may be used in hybridization or amplification assays of
biological samples to detect abnormalities involving NHP gene
structure, including point mutations, insertions, deletions and
chromosomal rearrangements. Such assays may include, but are not
limited to, Southern analyses, single stranded conformational
polymorphism analyses (SSCP), and PCR analyses.
[0090] Such diagnostic methods for the detection of NHP
gene-specific mutations can involve for example, contacting and
incubating nucleic acids including recombinant DNA molecules,
cloned genes or degenerate variants thereof, obtained from a
sample, e.g., derived from a patient sample or other appropriate
cellular source, with one or more labeled nucleic acid reagents
including recombinant DNA molecules, cloned genes or degenerate
variants thereof, as described in Section 5.1, under conditions
favorable for the specific annealing of these reagents to their
complementary sequences within a given NHP gene. Preferably, the
lengths of these nucleic acid reagents are at least 15 to 30
nucleotides. After incubation, all non-annealed nucleic acids are
removed from the nucleic acid:NHP molecule hybrid. The presence of
nucleic acids which have hybridized, if any such molecules exist,
is then detected. Using such a detection scheme, the nucleic acid
from the cell type or tissue of interest can be immobilized, for
example, to a solid support such as a membrane, or a plastic
surface such as that on a microtiter plate or polystyrene beads. In
this case, after incubation, non-annealed, labeled nucleic acid
reagents of the type described in Section 5.1 are easily removed.
Detection of the remaining, annealed, labeled NHP nucleic acid
reagents is accomplished using standard techniques well-known to
those in the art. The NHP gene sequences to which the nucleic acid
reagents have annealed can be compared to the annealing pattern
expected from a normal NHP gene sequence in order to determine
whether a NHP gene mutation is present.
[0091] Alternative diagnostic methods for the detection of NHP gene
specific nucleic acid molecules, in patient samples or other
appropriate cell sources, may involve their amplification, e.g., by
PCR (the experimental embodiment set forth in Mullis, K. B., 1987,
U.S. Pat. No. 4,683,202), followed by the detection of the
amplified molecules using techniques well known to those of skill
in the art. The resulting amplified sequences can be compared to
those which would be expected if the nucleic acid being amplified
contained only normal copies of a NHP gene in order to determine
whether a NHP gene mutation exists.
[0092] Additionally, well-known genotyping techniques can be
performed to identify individuals carrying NHP gene mutations. Such
techniques include, for example, the use of restriction fragment
length polymorphisms (RFLPs), which involve sequence variations in
one of the recognition sites for the specific restriction enzyme
used.
[0093] Additionally, improved methods for analyzing DNA
polymorphisms which can be utilized for the identification of NHP
gene mutations have been described which capitalize on the presence
of variable numbers of short, tandemly repeated DNA sequences
between the restriction enzyme sites. For example, Weber (U.S. Pat.
No. 5,075,217, which is incorporated herein by reference in its
entirety) describes a DNA marker based on length polymorphisms in
blocks of (dC-dA)n-(dG-dT)n short tandem repeats. The average
separation of (dC-dA)n-(dG-dT)n blocks is estimated to be
30,000-60,000 bp. Markers which are so closely spaced exhibit a
high frequency co-inheritance, and are extremely useful in the
identification of genetic mutations, such as, for example,
mutations within a given NHP gene, and the diagnosis of diseases
and disorders related to NHP mutations.
[0094] Also, Caskey et al. (U.S. Pat. No. 5,364,759, which is
incorporated herein by reference in its entirety) describe a DNA
profiling assay for detecting short tri and tetra nucleotide repeat
sequences. The process includes extracting the DNA of interest,
such as the NHP gene, amplifying the extracted DNA, and labeling
the repeat sequences to form a genotypic map of the individual's
DNA.
[0095] An additional embodiment of the present invention involves
identifying the association between NHPs, or NHP variants, and
disease. Using such associations, individuals can be identified
that harbor NHP variants or display NHP expression profiles that
correlate with a given disease (e.g., dermatoses, asthma, IBD,
etc.). Once such a genetic diagnosis has been established using
single nucleotide polymorphisms (SNPs), coding SNPs (cSNPs), etc.,
an appropriate treatment regimen can be tailored to the
patient.
[0096] The level of NHP gene expression can also be assayed by
detecting and measuring NHP transcription. For example, RNA from a
cell type or tissue known, or suspected to express the NHP gene,
such as skin, testis, or brain, may be isolated and tested
utilizing hybridization or PCR techniques such as are described,
above. The isolated cells can be derived from cell culture or from
a patient. The analysis of cells taken from culture may be a
necessary step in the assessment of cells to be used as part of a
cell-based gene therapy technique or, alternatively, to test the
effect of compounds on the expression of the NHP gene. Such
analyses may reveal both quantitative and qualitative aspects of
the expression pattern of the NHP gene, including activation or
inactivation of NHP gene expression. A preferred method of
conducting such screening assays uses the described sequences as
part of a larger array of sequences (i.e., microchip arrays,
etc.).
[0097] In one embodiment of such a detection scheme, cDNAs are
synthesized from the RNAs of interest (e.g., by reverse
transcription of the RNA molecule into cDNA). A sequence within the
cDNA is then used as the template for a nucleic acid amplification
reaction, such as a PCR amplification reaction, or the like. The
nucleic acid reagents used as synthesis initiation reagents (e.g.,
primers) in the reverse transcription and nucleic acid
amplification steps of this method are chosen from among the NHP
nucleic acid reagents described in Section 5.1. The preferred
lengths of such nucleic acid reagents are at least 9-30
nucleotides. For detection of the amplified product, the nucleic
acid amplification may be performed using radioactively or
non-radioactively labeled nucleotides. Alternatively, enough
amplified product may be made such that the product may be
visualized by standard ethidium bromide staining, by utilizing any
other suitable nucleic acid staining method, or by sequencing.
[0098] Additionally, it is possible to perform such NHP gene
expression assays "in situ", i.e., directly upon tissue sections.
(fixed and/or frozen) of patient tissue obtained from biopsies or
resections, such that no nucleic acid purification is necessary.
Nucleic acid reagents such as those described in Section 5.1 may be
used as probes and/or primers for such in situ procedures (See, for
example, Nuovo, G. J., 1992, "PCR In Situ Hybridization: Protocols
And Applications", Raven Press, NY).
[0099] Alternatively, if a sufficient quantity of the appropriate
cells can be obtained, standard Northern analysis can be performed
to determine the level of mRNA expression of the NHP gene.
[0100] 5.4.2. Detection of NHP Polypeptides
[0101] Antibodies directed against wild type or mutant NHP products
or conserved variants or peptide fragments thereof, which are
discussed, above, in Section 5.3, may also be used as diagnostics
and prognostics, as described herein. Such diagnostic methods, may
be used to detect abnormalities in the level of NHP gene
expression, or abnormalities in the structure and/or temporal,
tissue, cellular, or subcellular location of the NHP, and may be
performed in vivo or in vitro, such as, for example, on biopsy
tissue.
[0102] For example, antibodies directed to epitopes of an NHP can
be used in vivo to detect the pattern and level of expression of
the NHP in the body. Such antibodies can be labeled, e.g., with a
radio-opaque or other appropriate compound and injected into a
subject in order to visualize binding to the NHP expressed in the
body using methods such as X-rays, CAT-scans, or MRI. Labeled
antibody fragments, e.g., the Fab or single chain antibody
comprising the smallest portion of the antigen binding region, are
preferred for this purpose to promote crossing the blood-brain
barrier and permit labeling of NHPs expressed in the brain.
[0103] Additionally, any NHP fusion protein or NHP conjugated
protein whose presence can be detected, can be administered. For
example, NHP fusion or conjugated proteins labeled with a
radio-opaque or other appropriate compound can be administered and
visualized in vivo, as discussed, above for labeled antibodies.
Further such NHP fusion proteins (such as AP-NHP or NHP-AP) can be
utilized for in vitro diagnostic procedures.
[0104] Alternatively, immunoassays or fusion protein detection
assays, as described above, can be utilized on biopsy and autopsy
samples in vitro to permit assessment of the expression pattern of
the NHP. Such assays are not confined to the use of antibodies that
define a NHP domain, but can include the use of antibodies directed
to epitopes of any domain of a NHP. The use of each or all of these
labeled antibodies will yield useful information regarding
translation and intracellular transport of the NHP to the cell
surface and can identify defects in processing.
[0105] The tissue or cell type to be analyzed will generally
include those which are known, or suspected, to express the NHP
gene, such as, for example, epithelial cells, brain cells, etc. The
protein isolation methods employed herein may, for example, be such
as those described in Harlow and Lane (Harlow, E. and Lane, D.,
1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated
herein by reference in its entirety. The isolated cells can be
derived from cell culture or from a patient. The analysis of cells
taken from culture may be a necessary step in the assessment of
cells that could be used as part of a cell-based gene therapy
technique or, alternatively, to test the effect of compounds on the
expression of a NHP gene.
[0106] For example, antibodies, or fragments of antibodies, such as
those described, above, in Section 5.3, useful in the present
invention may be used to quantitatively or qualitatively detect the
presence of NHP products or conserved variants or peptide fragments
thereof. This can be accomplished, for example, by
immunofluorescence techniques employing a fluorescently labeled
antibody (see below, this Section) coupled with light microscopic,
flow cytometric, or fluorimetric detection. Such techniques are
especially preferred if such NHP products are at least transiently
present on the cell surface.
[0107] The antibodies (or fragments thereof) or NHP fusion or
conjugated proteins useful in the present invention may,
additionally, be employed histologically, as in immunofluorescence,
immunoelectron microscopy or non-immuno assays, for in situ
detection of NHP gene products or conserved variants or peptide
fragments thereof, or to assay NHP binding (in the case of labeled
NHP-fusion protein).
[0108] In situ detection may be accomplished by removing a
histological specimen from a patient, and applying thereto a
labeled antibody or fusion protein of the present invention. The
antibody (or fragment) or fusion protein is preferably applied by
overlaying the labeled antibody (or fragment) onto a biological
sample. Through the use of such a procedure, it is possible to
determine not only the presence of the NHP product, or conserved
variants or peptide fragments, or NHP binding, but also its
distribution in the examined tissue. Using the present invention,
those of ordinary skill will readily perceive that any of a wide
variety of histological methods (such as staining procedures) can
be modified in order to achieve such in situ detection.
[0109] Immunoassays and non-immunoassays for NHP products, or
conserved variants or peptide fragments thereof, will typically
comprise incubating a sample, such as a biological fluid, a tissue
extract, freshly harvested cells, or lysates of cells which have
been incubated in cell culture, in the presence of a detectably
labeled antibody capable of identifying NHP products or conserved
variants or peptide fragments thereof, and detecting the bound
antibody by any of a number of techniques well-known in the
art.
[0110] Alternatively, the labeled antibody can be directed against
an antigenic tag that has been directly or indirectly attached to a
NHP.
[0111] The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support which is capable of
immobilizing cells, cell particles or soluble proteins. The support
may then be washed with suitable buffers followed by treatment with
the detectably labeled NHP antibody or NHP receptor fusion protein.
The solid phase support may then be washed with the buffer a second
time to remove unbound antibody or fusion protein. The amount of
bound label on solid support may then be detected by conventional
means.
[0112] By "solid phase support or carrier" is intended any support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
[0113] The binding activity of a given lot of NHP antibody or NHP
ligand fusion protein may be determined according to well known
methods. Those skilled in the art will be able to determine
operative and optimal assay conditions for each determination by
employing routine experimentation.
[0114] With respect to antibodies, one of the ways in which the NHP
antibody can be detectably labeled is by linking the same to an
enzyme and use in an enzyme immunoassay (EIA) (Voller, A., "The
Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic
Horizons 2:1-7, Microbiological Associates Quarterly Publication,
Walkersville, Md.); Voller, A. et al., 1978, J. Clin. Pathol.
31:507-520; Butler, J. E., 1981, Meth. Enzymol. 73:482-523; Maggio,
E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.;
Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin,
Tokyo). The enzyme that is bound to the antibody will react with an
appropriate substrate, preferably a chromogenic substrate, in such
a manner as to produce a chemical moiety which can be detected, for
example, by spectrophotometric, fluorimetric or by visual means.
Enzymes which can be used to detectably label the antibody include,
but are not limited to, malate dehydrogenase, staphylococcal
nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase,
alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. The detection can be accomplished by
colorimetric methods which employ a chromogenic substrate for the
enzyme. Detection may also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0115] Detection may also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect NHP
through the use of a radioimmunoassay (RIA) (see, for example,
Weintraub, B., Principles of Radioimmunoassays, Seventh Training
Course on Radioligand AssayTechniques, The Endocrine Society,
March, 1986, which is incorporated by reference herein). The
radioactive isotope can be detected by such means as the use of a
gamma counter or a scintillation counter or by autoradiography.
[0116] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wave length, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
[0117] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0118] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0119] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in, which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0120] 5.5. Screening Assays for Compounds that Modulate NHP
Expression or Activity
[0121] The following assays are designed to identify compounds that
interact with (e.g., bind to) NHPs, compounds that interfere NHP
activity, compounds that modulate the activity of NHP gene
expression (i.e., modulate the level of NHP gene expression) or
compounds that modulate the levels of NHP in the body. Assays may
additionally be utilized that identify compounds that bind to NHP
gene regulatory sequences (e.g., promoter sequences) and,
consequently, can modulate NHP gene expression. See e.g., Platt, K.
A., 1994, J. Biol. Chem. 269:28558-28562, which is incorporated
herein by reference in its entirety.
[0122] The compounds that can be screened in accordance with the
invention include but are not limited to peptides, antibodies and
fragments thereof, and other organic compounds (e.g.,
peptidomimetics) that bind to a NHP and inhibit, hinder, or enhance
lipoxygenase activity.
[0123] Such compounds may include, but are not limited to, peptides
such as, for example, soluble peptides, including but not limited
to members of random peptide libraries; (see, e.g., Lam, K. S. et
al., 1991, Nature 354:82-84; Houghten, R. et al., 1991, Nature
354:84-86), and combinatorial chemistry-derived molecular libraries
made of D- and/or L-configuration amino acids, phosphopeptides
(including, but not limited to members of random or partially
degenerate, directed phosphopeptide libraries; see, e.g., Songyang,
Z. et al., 1993, Cell 72:767-778), antibodies (including, but not
limited to, polyclonal, monoclonal, humanized, anti-idiotypic,
chimeric or single chain antibodies, and FAb, F(ab').sub.2 and FAb
expression library fragments, and epitope-binding fragments
thereof), and small organic or inorganic molecules.
[0124] Other compounds which can be screened in accordance with the
invention include but are not limited to small organic molecules
that are able to cross the blood-brain barrier, gain entry into an
appropriate cell (e.g., in the choroid plexus, pituitary, the
hypothalamus, etc.) and affect the expression of a NHP gene or some
other gene involved in a NHP mediated pathway (e.g., by interacting
with the regulatory region or transcription factors involved in
gene expression or translation); or such compounds that affect
NHP-mediated leukotriene synthesis, or catabolic, inflammatory, or
metabolic pathways.
[0125] Computer modeling and searching technologies permit
identification of compounds, or the improvement of already
identified compounds, that can modulate NHP expression or activity.
Having identified such a compound or composition, the active sites
or regions are identified. Such active sites might typically be
ligand or substrate binding sites. The active site can be
identified using methods known in the art including, for example,
from the amino acid sequences of peptides, from the nucleotide
sequences of nucleic acids, or from study of complexes of the
relevant compound or composition with its natural ligand. In the
latter case, chemical or X-ray crystallographic methods can be used
to find the active site by finding where on the factor the
complexed ligand is found.
[0126] Next, the three dimensional geometric structure of the
active site is determined. This can be done by known methods,
including X-ray crystallography, which can determine a complete
molecular structure. On the other hand, solid or liquid phase NMR
can be used to determine certain intra-molecular distances. Any
other experimental method of structure determination can be used to
obtain partial or complete geometric structures. The geometric
structures may be measured with a complexed ligand, natural or
artificial, which may increase the accuracy of the active site
structure determined.
[0127] If an incomplete or insufficiently accurate structure is
determined, the methods of computer based numerical modeling can be
used to complete the structure or improve its accuracy. Any
recognized modeling method may be used, including parameterized
models specific to particular biopolymers such as proteins or
nucleic acids, molecular dynamics models based on computing
molecular motions, statistical mechanics models based on thermal
ensembles, or combined models. For most types of models, standard
molecular force fields, representing the forces between constituent
atoms and groups, are necessary, and can be selected from force
fields known in physical chemistry. The incomplete or less accurate
experimental structures can serve as constraints on the complete
and more accurate structures computed by these modeling
methods.
[0128] Finally, having determined the structure of the active site
(or binding site), either experimentally, by modeling, or by a
combination, candidate modulating compounds can be identified by
searching databases containing compounds along with information on
their molecular structure. Such a search seeks compounds having
structures that match the determined active site structure and that
interact with the groups defining the active site. Such a search
can be manual, but is preferably computer assisted. These compounds
found from this search are potential NHP modulating compounds.
[0129] Alternatively, these methods can be used to identify
improved modulating compounds from an already known modulating
compound or ligand. The composition of the known compound can be
modified and the structural effects of modification can be
determined using the experimental and computer modeling methods
described above applied to the new composition. The altered
structure is then compared to the active site structure of the
compound to determine if an improved fit or interaction results. In
this manner systematic variations in composition, such as by
varying side groups, can be quickly evaluated to obtain modified
modulating compounds or ligands of improved specificity or
activity.
[0130] Further experimental and computer modeling methods useful to
identify modulating compounds based upon identification of the
active sites (or binding sites) of a NHP, and related transduction
and transcription factors will be apparent to those of skill in the
art.
[0131] Examples of molecular modeling systems are the CHARMm and
QUANTA programs (Polygen Corporation, Waltham, Mass.). CHARMm
performs the energy minimization and molecular dynamics functions.
QUANTA performs the construction, graphic modeling and analysis of
molecular structure. QUANTA allows interactive construction,
modification, visualization, and analysis of the behavior of
molecules with each other.
[0132] A number of articles review computer modeling of drugs
interactive with specific proteins, such as Rotivinen, et al.,
1988, Acta Pharmaceutical Fennica 97:159-166; Ripka, New Scientist
54-57 (Jun. 16, 1988); McKinaly and Rossmann, 1989, Annu. Rev.
Pharmacol. Toxiciol. 29:111-122; Perry and Davies, OSAR:
Quantitative Structure-Activity Relationships in Drug Design pp.
189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R.
Soc. Lond. 236:125-140 and 141-162; and, with respect to a model
receptor for nucleic acid components, Askew, et al., 1989, J. Am.
Chem. Soc. 111:1082-1090. Other computer programs that screen and
graphically depict chemicals are available from companies such as
BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga,
Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario).
Although these are primarily designed for application to drugs
specific to particular proteins, they can be adapted to design of
drugs specific to regions of DNA or RNA, once that region is
identified.
[0133] Although described above with reference to design and
generation of compounds which could alter binding, one could also
screen libraries of known compounds, including natural products or
synthetic chemicals, and biologically active materials, including
proteins, for compounds which are inhibitors or activators.
[0134] Cell-based systems can also be used to identify compounds
that bind (or mimic) NHPs as well as assess the altered activity
associated with such binding in living cells. One tool of
particular interest for such assays is green fluorescent protein
which is described, inter alia, in U.S. Pat. No. 5,625,048, herein
incorporated by reference. Cells that may be used in such cellular
assays include, but are not limited to, leukocytes, or cell lines
derived from leukocytes, lymphocytes, stem cells, including
embryonic stem cells, and the like. In addition, expression host
cells (e.g., B95 cells, COS cells, CHO cells, OMK cells,
fibroblasts, Sf9 cells) genetically engineered to express a
functional NHP of interest and to respond to activation by the
test, or natural, ligand, as measured by a chemical or phenotypic
change, or induction of another host cell gene, can be used as an
end point in the assay.
[0135] Compounds identified via assays such as those described
herein may be useful, for example, in elucidating the biological
function of a NHP product. Such compounds can be administered to a
patient at therapeutically effective doses to treat any of a
variety of physiological or mental disorders. A therapeutically
effective dose refers to that amount of the compound sufficient to
result in any amelioration, impediment, prevention, or alteration
of any biological symptom.
[0136] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0137] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0138] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
Thus, the compounds and their physiologically acceptable salts and
solvates may be formulated for administration by inhalation or
insufflation (either through the mouth or the nose) or oral,
buccal, parenteral, intracranial, intrathecal, topical (skin
creams, ointments, etc.), or rectal administration.
[0139] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0140] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound.
[0141] For buccal administration the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0142] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0143] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0144] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0145] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0146] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0147] 5.5.1. In Vitro Screening Assays for Compounds that Bind to
NHPs
[0148] In vitro systems may be designed to identify compounds
capable of interacting with (e.g., binding to) NHPs. The compounds
identified can be useful, for example, in modulating the activity
of wild type and/or mutant NHP products; can be useful in
elaborating the biological function of the NHP; can be utilized in
screens for identifying compounds that disrupt normal NHP
interactions; or may themselves disrupt or activate such
interactions.
[0149] The principle of the assays used to identify compounds that
bind to NHPs, involves preparing a reaction mixture of an NHP and
the test compound under conditions and for a time sufficient to
allow the two components to interact and bind, thus forming a
complex which can be removed and/or detected in the reaction
mixture.
[0150] The NHP species used can vary depending upon the goal of the
screening assay. For example, where compounds that directly
interact with the NHP are sought, full-length NHP, peptides
corresponding to the NHP, or fusion proteins containing NHPs can be
used.
[0151] The screening assays can be conducted in a variety of ways.
For example, one method to conduct such an assay would involve
anchoring the NHP, polypeptide, peptide, or fusion protein
therefrom, or the test substance onto a solid phase and detecting
NHP/test compound complexes anchored on the solid phase at the end
of the reaction. In one embodiment of such a method, the NHP
reactant may be anchored onto a solid surface, and the test
compound, which is not anchored, may be labeled, either directly or
indirectly.
[0152] In practice, microtiter plates may conveniently be utilized
as the solid phase. The anchored component may be immobilized by
non-covalent or covalent attachments. Non-covalent attachment may
be accomplished by simply coating the solid surface with a solution
of the protein and drying. Alternatively, an immobilized antibody,
preferably a monoclonal antibody, specific for the protein to be
immobilized may be used to anchor the protein to the solid surface.
The surfaces may be prepared in advance and stored.
[0153] In order to conduct the assay, the nonimmobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously nonimmobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
nonimmobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the previously nonimmobilized
component (the antibody, in turn, may be directly labeled or
indirectly labeled with a labeled anti-Ig antibody).
[0154] Alternatively, a reaction can be conducted in a liquid
phase, the reaction products separated from unreacted components,
and complexes detected; e.g., using an immobilized antibody
specific for a NHP protein, polypeptide, peptide or fusion protein
or the test compound to anchor any complexes formed in solution,
and a labeled antibody specific for the other component of the
possible complex to detect anchored complexes.
[0155] Alternatively, cell-based assays can be used to identify
compounds that interact with NHP. To this end, cell lines that
express a NHP or cell lines (e.g., COS cells, CHO cells,
fibroblasts, etc.) that have been genetically engineered to express
NHP (e.g., by transfection or transduction of suitably engineered
NHP DNA) can be used. Interaction of the test compound with, for
example, a NHP expressed by the host cell can be determined by
comparison to cells that do not express the NHP.
[0156] 5.5.2. Assays for Intracellular Proteins that are Associated
with NHP
[0157] Any method suitable for detecting protein-protein
interactions may be employed for identifying membrane proteins or
intracellular proteins that directly or indirectly interact with a
NHP. For direct interactions, the traditional methods that can be
employed include, but are not limited to, co-immunoprecipitation,
crosslinking and co-purification through gradients or
chromatographic columns of cell lysates or proteins obtained from
cell lysates and a NHP to identify proteins in the lysate that
interact with the NHP. For these assays, the NHP component can be a
full length NHP, a soluble derivative of a NHP, a NHP peptide, or a
NHP fusion protein. Once isolated, such an intracellular protein
can be identified and can, in turn, be used, in conjunction with
standard techniques, to identify proteins with which it interacts.
For example, at least a portion of the amino acid sequence of an
intracellular protein which interacts with a NHP can be ascertained
using techniques known in the art, such as Edman degradation. (See,
e.g., Creighton, 1983, "Proteins: Structures and Molecular
Principles", W.H. Freeman & Co., N.Y., pp. 3449). The amino
acid sequence obtained may be used as a guide for the generation of
oligonucleotide mixtures that can be used to screen for gene
sequences encoding such intracellular proteins. Screening may be
accomplished, for example, by standard hybridization or PCR
techniques. Techniques for the generation of oligonucleotide
mixtures and the screening are well-known. (See, e.g., Ausubel,
supra, and PCR Protocols: A Guide to Methods and Applications,
1990, Innis, M. et al., eds. Academic Press, Inc., New York).
[0158] Additionally, methods can be employed that result in the
simultaneous identification of genes that encode transmembrane or
intracellular proteins that interact with the NHP. These methods
include, for example, probing expression libraries, in a manner
similar to the well known technique of antibody probing of
.lamda.gt11 libraries, using labeled NHP protein, or an NHP
polypeptide, peptide or fusion protein, e.g., an NHP polypeptide or
NHP domain fused to a marker (e.g., an enzyme, flour, luminescent
protein, or dye), or an Ig-Fc domain.
[0159] One method that detects protein interactions in vivo, the
two-hybrid system, is described in detail for illustration only and
not by way of limitation. One version of this system has been
described (Chien et al., 1991, Proc. Natl. Acad. Sci. USA,
88:9578-9582) and is commercially available from Clontech (Palo
Alto, Calif.).
[0160] Briefly, utilizing such a system, plasmids are constructed
that encode two hybrid proteins: one plasmid consists of
nucleotides encoding the DNA-binding domain of a transcription
activator protein fused to a nucleotide sequence encoding a NHP, or
NHP polypeptide, peptide, or fusion protein therefrom, and the
other plasmid consists of nucleotides encoding the transcription
activator protein's activation domain fused to a cDNA encoding an
unknown protein which has been recombined into this plasmid as part
of a cDNA library. The DNA-binding domain fusion plasmid and the
cDNA library are transformed into a strain of the yeast
Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS
or lacZ) whose regulatory region contains the transcription
activator's binding site. Either hybrid protein alone cannot
activate transcription of the reporter gene: the DNA-binding domain
hybrid cannot because it does not provide activation function and
the activation domain hybrid cannot because it cannot localize to
the activator's binding sites. Interaction of the two hybrid
proteins reconstitutes the functional activator protein and results
in expression of the reporter gene, which is detected by an assay
for the reporter gene product.
[0161] The two-hybrid system or related methodology may be used to
screen activation domain libraries for proteins that interact with
the "bait" gene product. By way of example, and not by way of
limitation, a NHP can be used as the bait product. Total genomic or
cDNA sequences are fused to the DNA encoding an activation domain.
This library and a plasmid encoding a hybrid of a bait NHP gene
product fused to the DNA-binding domain are cotransformed into a
yeast reporter strain, and the resulting transformants are screened
for those that express the reporter gene. For example, and not by
way of limitation, a bait NHP gene sequence, such as the open
reading frame of a NHP (or a domain of a NHP) can be cloned into a
vector such that it is translationally fused to the DNA encoding
the DNA-binding domain of the GAL4 protein. These colonies are
purified and the library plasmids responsible for reporter gene
expression are isolated. DNA sequencing is then used to identify
the proteins encoded by the library plasmids.
[0162] A cDNA library of the cell line from which proteins that
interact with bait NHP gene product are to be detected can be made
using methods routinely practiced in the art. According to the
particular system described herein, for example, the cDNA fragments
can be inserted into a vector such that they are translationally
fused to the transcriptional activation domain of GAL4. This
library can be co-transformed along with the bait NHP gene-GAL4
fusion plasmid into a yeast strain which contains a lacZ gene
driven by a promoter which contains GAL4 activation sequence. A
cDNA encoded protein, fused to GAL4 transcriptional activation
domain, that interacts with bait NHP gene product will reconstitute
an active GAL4 protein and thereby drive expression of the HIS3
gene. Colonies which express HIS3 can be detected by their growth
on petri dishes containing semi-solid agar based media lacking
histidine. The cDNA can then be purified from these strains, and
used to produce and isolate the bait NHP gene-interacting protein
using techniques routinely practiced in the art.
[0163] 5.5.3. Assays for Compounds that Interfere with
NHP/Intracellular Macromolecule or NHP/Transmembrane Macromolecule
Interaction
[0164] Macromolecules that interact with NHPs are referred to, for
purposes of this discussion, as "binding partners". These binding
partners are likely to be involved in NHP mediated biological
pathways. Therefore, it is desirable to identify compounds that
interfere with or disrupt the interaction of such binding partners
which may be useful in regulating or augmenting NHP activity in the
body and/or controlling disorders associated with NHP activity (or
a deficiency thereof).
[0165] The basic principle of the assay systems used to identify
compounds that interfere with the interaction between a NHP or NHP
receptor (collectively, the NHP moiety), and its binding partner or
partners involves preparing a reaction mixture containing NHP, or
NHP polypeptides, peptides or fusion proteins as described in
Sections 5.5.1 and 5.5.2 above, and the binding partner under
conditions and for a time sufficient to allow the two to interact
and bind, thus forming a complex. In order to test a compound for
inhibitory activity, the reaction mixture is prepared in the
presence and absence of the test compound. The test compound may be
initially included in the reaction mixture, or may be added at a
time subsequent to the addition of the NHP moiety and its binding
partner. Control reaction mixtures are incubated without the test
compound or with a placebo. The formation of any complexes between
the NHP moiety and the binding partner is then detected. The
formation of a complex in the control reaction, but not in the
reaction mixture containing the test compound, indicates that the
compound interferes with the interaction of the NHP moiety and the
interactive binding partner. Additionally, complex formation within
reaction mixtures containing the test compound and normal NHP
protein may also be compared to complex formation within reaction
mixtures containing the test compound and a mutant NHP. This
comparison may be important in those cases wherein it is desirable
to identify compounds that specifically disrupt interactions of
mutant, or mutated, NHPs but not normal NHPs, or other
lipoxygenases.
[0166] The assay for compounds that interfere with the interaction
of the NHP and binding partners can be conducted in a heterogeneous
or homogeneous format. Heterogeneous assays involve anchoring
either the NHP moiety or the binding partner onto a solid phase and
detecting complexes anchored on the solid phase at the end of the
reaction. In homogeneous assays, the entire reaction is carried out
in a liquid phase. In either approach, the order of addition of
reactants can be varied to obtain different information about the
compounds being tested. For example, test compounds that interfere
with the interaction by competition can be identified by conducting
the reaction in the presence of the test substance; i.e., by adding
the test substance to the reaction mixture prior to, or
simultaneously with, a NHP moiety and interactive binding partner.
Alternatively, test compounds that disrupt preformed complexes,
e.g. compounds with higher binding constants that displace one of
the components from the complex, can be tested by adding the test
compound to the reaction mixture after complexes have been formed.
The various formats are described briefly below.
[0167] In a heterogeneous assay system, either a NHP moiety or an
interactive binding partner, is anchored onto a solid surface,
while the non-anchored species is labeled, either directly or
indirectly. In practice, microtiter plates are conveniently
utilized. The anchored species may be immobilized by non-covalent
or covalent attachments. Non-covalent attachment may be
accomplished simply by coating the solid surface with a solution of
the NHP moiety or binding partner and drying. Alternatively, an
immobilized antibody specific for the species to be anchored may be
used to anchor the species to the solid surface. The surfaces may
be prepared in advance and stored.
[0168] In order to conduct the assay, the partner of the
immobilized species is exposed to the coated surface with or
without the test compound. After the reaction is complete,
unreacted components are removed (e.g., by washing) and any
complexes formed will remain immobilized on the solid surface. The
detection of complexes anchored on the solid surface can be
accomplished in a number of ways. Where the non-immobilized species
is pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the non-immobilized
species is not pre-labeled, an indirect label can be used to detect
complexes anchored on the surface; e.g., using a labeled antibody
specific for the initially non-immobilized species (the antibody,
in turn, may be directly labeled or indirectly labeled with a
labeled anti-Ig antibody). Depending upon the order of addition of
reaction components, test compounds which inhibit complex formation
or which disrupt preformed complexes can be detected.
[0169] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected; e.g., using an immobilized antibody specific for one of
the binding components to anchor any complexes formed in solution,
and a labeled antibody specific for the other partner to detect
anchored complexes. Again, depending upon the order of addition of
reactants to the liquid phase, test compounds which inhibit complex
or which disrupt preformed complexes can be identified.
[0170] In an alternate embodiment of the invention, a homogeneous
assay can be used. In this approach, a preformed complex of a NHP
moiety and an interactive binding partner is prepared in which
either the NHP moiety or its binding partners is labeled, but the
signal generated by the label is quenched due to formation of the
complex (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein which
utilizes this approach for immunoassays). The addition of a test
substance that competes with and displaces one of the species from
the preformed complex will result in the generation of a signal
above background. In this way, test substances which disrupt
NHP/intracellular binding partner interaction can be
identified.
[0171] In a particular embodiment, a NHP fusion can be prepared for
immobilization. For example, a NHP or a peptide fragment can be
fused to a glutathione-S-transferase (GST) gene using a fusion
vector, such as pGEX-5X-1, in such a manner that its binding
activity is maintained in the resulting fusion protein. The
interactive binding partner can be purified and used to raise a
monoclonal antibody, using methods routinely practiced in the art
and described above, in Section 5.3. This antibody can be labeled
with the radioactive isotope .sup.125I, for example, by methods
routinely practiced in the art. In a heterogeneous assay, e.g., the
GST-NHP fusion protein can be anchored to glutathione-agarose
beads. The interactive binding partner can then be added in the
presence or absence of the test compound in a manner that allows
interaction and binding to occur. At the end of the reaction
period, unbound material can be washed away, and the labeled
monoclonal antibody can be added to the system and allowed to bind
to the complexed components. The interaction between a NHP moiety
and the interactive binding partner can be detected by measuring
the amount of radioactivity that remains associated with the
glutathione-agarose beads. A successful inhibition of the
interaction by the test compound will result in a decrease in
measured radioactivity.
[0172] Alternatively, the GST-NHP moiety fusion protein and the
interactive binding partner can be mixed together in liquid in the
absence of the solid glutathione-agarose beads. The test compound
can be added either during or after the species are allowed to
interact. This mixture can then be added to the glutathione-agarose
beads and unbound material is washed away. Again the extent of
inhibition of the NHP moiety/binding partner interaction can be
detected by adding the labeled antibody and measuring the
radioactivity associated with the beads.
[0173] In another embodiment of the invention, these same
techniques can be employed using peptide fragments that correspond
to the binding domains of a NHP moiety and/or the interactive or
binding partner (in cases where the binding partner is a protein),
in place of one or both of the full length proteins. Any number of
methods routinely practiced in the art can be used to identify and
isolate the binding sites. These methods include, but are not
limited to, mutagenesis of the gene encoding one of the proteins
and screening for disruption of binding in a co-immunoprecipitation
assay. Compensatory mutations in the gene encoding the second
species in the complex can then be selected. Sequence analysis of
the genes encoding the respective proteins will reveal the
mutations that correspond to the region of the protein involved in
interactive binding. Alternatively, one protein can be anchored to
a solid surface using methods described above, and allowed to
interact with and bind to its labeled binding partner, which has
been treated with a proteolytic enzyme, such as trypsin. After
washing, a relatively short, labeled peptide comprising the binding
domain may remain associated with the solid material, which can be
isolated and identified by amino acid sequencing. Also, once the
gene coding for the intracellular binding partner is obtained,
short gene segments can be engineered to express peptide fragments
of the protein, which can then be tested for binding activity and
purified or synthesized.
[0174] For example, and not by way of limitation, a NHP moiety can
be anchored to a solid material as described, above, by making a
GST-NHP moiety fusion protein and allowing it to bind to
glutathione agarose beads. The interactive binding partner can be
labeled with a radioactive isotope, such as .sup.35S, and cleaved
with a proteolytic enzyme such as trypsin. Cleavage products can
then be added to the anchored GST-NHP moiety fusion protein and
allowed to bind. After washing away unbound peptides, labeled bound
material, representing the intracellular binding partner binding
domain, can be eluted, purified, and analyzed for amino acid
sequence by well-known methods. Peptides so identified can be
produced synthetically or fused to appropriate facilitative
proteins using recombinant DNA technology.
[0175] 6.0. Reference to Microorganism Deposits
[0176] The following plasmid has been deposited at the American
Type Culture Collection (ATCC), Manassas, Va., USA, under the terms
of the Budapest Treaty on the International Recognition of the
Deposit of Microorganisms for the Purposes of Patent Procedure and
Regulations thereunder (Budapest Treaty) and is thus maintained and
made available according to the terms of the Budapest Treaty.
Availability of such plasmid is not to be construed as a license to
practice the invention in contravention of the rights granted under
the authority of any government in accordance with its patent
laws.
[0177] The deposited plasmid has been assigned the indicated ATCC
deposit number
TABLE-US-00001 Plasmid ATCC No. LEXENZ17D PTA-503
[0178] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims. All publications, patents,
and patent applications referenced herein are incorporated by
reference in their entirety.
Sequence CWU 1
1
2912701DNAHomo sapiens 1atggcagtgt accgcctgtg tgtgaccact ggtccctacc
tgagggccgg cacactggac 60aacatctctg tcacactggt gggcacgtgt ggtgaaagcc
ccaagcagcg gctagatcga 120atgggcaggg acttcgcccc tggatcggta
cagaagtaca aggtgcgttg cacagcggag 180ctgggtgagc tcttgctgct
gcgtgtacac aaggagcgct acgctttctt ccgcaaggac 240tcttggtact
gtagccgcat ctgtgtcacc gaaccggatg gtagtgtatc ccacttcccc
300tgctatcagt ggattgaagg ctactgcacc gtggagctga ggccaggaac
agcaagaact 360atttgtcagg actctcttcc cctcctcctg gatcacagga
cacgggagct ccgggcccga 420caagaatgct accgctggaa gatctatgcc
cctggcttcc cctgcatggt agacgtcaac 480agctttcagg agatggagtc
agacaagaaa tttgccttga caaagacgac aacttgtgta 540gaccagggtg
acagcagtgg gaatcggtac ctgcccggct tccccatgaa aattgacatc
600ccatccctga tgtacatgga gcccaatgtt cgatactcag ccaccaagac
gatctcgctg 660ctcttcaatg ccatccctgc gtccttggga atgaagcttc
gagggctgtt ggatcgcaag 720ggctcctgga agaagctgga tgacatgcag
aacatcttct ggtgccataa gaccttcacg 780acaaagtatg tcacagagca
ctggtgtgaa gatcacttct ttgggtacca gtacctgaat 840ggtgtcaatc
ccgtcatgct ccactgcatc tctagcttgc ccagcaagct gcctgtcacc
900aatgacatgg tggccccctt gctgggacag gacacatgcc tgcagacaga
gctagagagg 960gggaacatct tcctagcgga ctactggatc ctggcggagg
cccccaccca ctgcctaaac 1020ggccgccagc agtacgtggc cgccccactg
tgcctgctgt ggctcagccc ccagggggcg 1080ctggtgccct tggccatcca
gctcagccag acccccgggc ctgacagccc catcttcctg 1140cccactgact
ccgaatggga ctggctgctg gccaagacgt gggtgcgcaa ctctgagttc
1200ctggtgcacg aaaacaacac gcactttctg tgcacgcatt tgctgtgcga
ggccttcgcc 1260atggccacgc tgcgccagct gccgctctgc caccccatct
acaagctcct actcccccac 1320actcgataca cgctgcaggt gaacaccatc
gcgagggcca cgctgctcaa ccccgagggc 1380ctcgtggacc aggtcacgtc
catcgggagg caaggcctca tctacctcat gagcacgggc 1440ctggcccact
tcacctacac caatttctgc cttccggaca gcctgcgggc ccgcggcgtc
1500ctggctatcc ccaactacca ctaccgagac gacggcctga agatctgggc
ggccattgag 1560agctttgtct cagaaatcgt gggctactat tatcccagtg
acgcatctgt gcagcaggat 1620tcggagctgc aggcctggac tggcgagatt
tttgctcagg cgttcctggg ccgggaaagc 1680tcaggtttcc caagccggct
gtgcacccca ggagagatgg tgaagttcct cactgcaatc 1740atcttcaatt
gctctgccca gcacgctgct gtcaacagtg ggcagcatga ctttggggcc
1800tggatgccca atgctccatc atccatgagg cagcccccac cccagaccaa
ggggaccacc 1860accctgaaga cttacctaga caccctccct gaagtgaaca
tcagctgtaa caacctcctc 1920ctcttctggt tggttagcca agaacccaag
gaccagaggc ccctgggcac ctacccagat 1980gagcacttca cagaggaggc
cccgaggcgg agcatcgccg ccttccagag ccgcctggcc 2040cagatctcaa
gggacatcca ggagcggaac cagggtctgg cactgcccta cacctacctg
2100gaccctcccc tcattgagaa cagcgtctcc atctaaccac ccccaaatac
cacccaagaa 2160gaaagaaagg tccaagcatg aggaggacca gttcctcagg
tcctccagac ccttccatcc 2220tccctgttct cagttcacct gaaccttctc
ttctgcacat ggagactttt gcagccaaga 2280tggctctgac atcatacaaa
ctgggccctg agctgtgaga gaccagcaca gcagcgtcca 2340ggttaaaagc
cgctgaccaa agtccaatgc acaatagccc ctccgaaagg aaggaaccgc
2400ttcacttctt gccccacttg gggcagcctc ttgttccagc ctcttggaat
gcccagcttg 2460ggtttctgag cttttctccc tcatcctccc caatccccaa
actccttctc ctaccatgcc 2520tttctacgtt ctctttcttc caagcctaga
gccaccagcc cagcttcctt ctctggaaaa 2580gcctggaaac tgggcacaga
aggactgtgt gcctggctaa catgtggtcc cctttgtccc 2640tagcaccttt
aaggggaggg gaagaattgg agggcagctt gcctggaccc ctaacggctg 2700t
27012711PRTHomo sapiens 2Met Ala Val Tyr Arg Leu Cys Val Thr Thr
Gly Pro Tyr Leu Arg Ala 1 5 10 15Gly Thr Leu Asp Asn Ile Ser Val
Thr Leu Val Gly Thr Cys Gly Glu 20 25 30Ser Pro Lys Gln Arg Leu Asp
Arg Met Gly Arg Asp Phe Ala Pro Gly 35 40 45Ser Val Gln Lys Tyr Lys
Val Arg Cys Thr Ala Glu Leu Gly Glu Leu 50 55 60Leu Leu Leu Arg Val
His Lys Glu Arg Tyr Ala Phe Phe Arg Lys Asp65 70 75 80Ser Trp Tyr
Cys Ser Arg Ile Cys Val Thr Glu Pro Asp Gly Ser Val 85 90 95Ser His
Phe Pro Cys Tyr Gln Trp Ile Glu Gly Tyr Cys Thr Val Glu 100 105
110Leu Arg Pro Gly Thr Ala Arg Thr Ile Cys Gln Asp Ser Leu Pro Leu
115 120 125Leu Leu Asp His Arg Thr Arg Glu Leu Arg Ala Arg Gln Glu
Cys Tyr 130 135 140Arg Trp Lys Ile Tyr Ala Pro Gly Phe Pro Cys Met
Val Asp Val Asn145 150 155 160Ser Phe Gln Glu Met Glu Ser Asp Lys
Lys Phe Ala Leu Thr Lys Thr 165 170 175Thr Thr Cys Val Asp Gln Gly
Asp Ser Ser Gly Asn Arg Tyr Leu Pro 180 185 190Gly Phe Pro Met Lys
Ile Asp Ile Pro Ser Leu Met Tyr Met Glu Pro 195 200 205Asn Val Arg
Tyr Ser Ala Thr Lys Thr Ile Ser Leu Leu Phe Asn Ala 210 215 220Ile
Pro Ala Ser Leu Gly Met Lys Leu Arg Gly Leu Leu Asp Arg Lys225 230
235 240Gly Ser Trp Lys Lys Leu Asp Asp Met Gln Asn Ile Phe Trp Cys
His 245 250 255Lys Thr Phe Thr Thr Lys Tyr Val Thr Glu His Trp Cys
Glu Asp His 260 265 270Phe Phe Gly Tyr Gln Tyr Leu Asn Gly Val Asn
Pro Val Met Leu His 275 280 285Cys Ile Ser Ser Leu Pro Ser Lys Leu
Pro Val Thr Asn Asp Met Val 290 295 300Ala Pro Leu Leu Gly Gln Asp
Thr Cys Leu Gln Thr Glu Leu Glu Arg305 310 315 320Gly Asn Ile Phe
Leu Ala Asp Tyr Trp Ile Leu Ala Glu Ala Pro Thr 325 330 335His Cys
Leu Asn Gly Arg Gln Gln Tyr Val Ala Ala Pro Leu Cys Leu 340 345
350Leu Trp Leu Ser Pro Gln Gly Ala Leu Val Pro Leu Ala Ile Gln Leu
355 360 365Ser Gln Thr Pro Gly Pro Asp Ser Pro Ile Phe Leu Pro Thr
Asp Ser 370 375 380Glu Trp Asp Trp Leu Leu Ala Lys Thr Trp Val Arg
Asn Ser Glu Phe385 390 395 400Leu Val His Glu Asn Asn Thr His Phe
Leu Cys Thr His Leu Leu Cys 405 410 415Glu Ala Phe Ala Met Ala Thr
Leu Arg Gln Leu Pro Leu Cys His Pro 420 425 430Ile Tyr Lys Leu Leu
Leu Pro His Thr Arg Tyr Thr Leu Gln Val Asn 435 440 445Thr Ile Ala
Arg Ala Thr Leu Leu Asn Pro Glu Gly Leu Val Asp Gln 450 455 460Val
Thr Ser Ile Gly Arg Gln Gly Leu Ile Tyr Leu Met Ser Thr Gly465 470
475 480Leu Ala His Phe Thr Tyr Thr Asn Phe Cys Leu Pro Asp Ser Leu
Arg 485 490 495Ala Arg Gly Val Leu Ala Ile Pro Asn Tyr His Tyr Arg
Asp Asp Gly 500 505 510Leu Lys Ile Trp Ala Ala Ile Glu Ser Phe Val
Ser Glu Ile Val Gly 515 520 525Tyr Tyr Tyr Pro Ser Asp Ala Ser Val
Gln Gln Asp Ser Glu Leu Gln 530 535 540Ala Trp Thr Gly Glu Ile Phe
Ala Gln Ala Phe Leu Gly Arg Glu Ser545 550 555 560Ser Gly Phe Pro
Ser Arg Leu Cys Thr Pro Gly Glu Met Val Lys Phe 565 570 575Leu Thr
Ala Ile Ile Phe Asn Cys Ser Ala Gln His Ala Ala Val Asn 580 585
590Ser Gly Gln His Asp Phe Gly Ala Trp Met Pro Asn Ala Pro Ser Ser
595 600 605Met Arg Gln Pro Pro Pro Gln Thr Lys Gly Thr Thr Thr Leu
Lys Thr 610 615 620Tyr Leu Asp Thr Leu Pro Glu Val Asn Ile Ser Cys
Asn Asn Leu Leu625 630 635 640Leu Phe Trp Leu Val Ser Gln Glu Pro
Lys Asp Gln Arg Pro Leu Gly 645 650 655Thr Tyr Pro Asp Glu His Phe
Thr Glu Glu Ala Pro Arg Arg Ser Ile 660 665 670Ala Ala Phe Gln Ser
Arg Leu Ala Gln Ile Ser Arg Asp Ile Gln Glu 675 680 685Arg Asn Gln
Gly Leu Ala Leu Pro Tyr Thr Tyr Leu Asp Pro Pro Leu 690 695 700Ile
Glu Asn Ser Val Ser Ile705 71031470DNAHomo sapiens 3atggcagtgt
accgcctgtg tgtgaccact ggtccctacc tgagggccgg cacactggac 60aacatctctg
tcacactggt gggcacgtgt ggtgaaagcc ccaagcagcg gctagatcga
120atgggcaggg acttcgcccc tggatcggta cagaagtaca aggtgcgttg
cacagcggag 180ctgggtgagc tcttgctgct gcgtgtacac aaggagcgct
acgctttctt ccgcaaggac 240tcttggtact gtagccgcat ctgtgtcacc
gaaccggatg gtagtgtatc ccacttcccc 300tgctatcagt ggattgaagg
ctactgcacc gtggagctga ggccaggaac agcaagaact 360atttgtcagg
actctcttcc cctcctcctg gatcacagga cacgggagct ccgggcccga
420caagaatgct accgctggaa gatctatgcc cctggcttcc cctgcatggt
agacgtcaac 480agctttcagg agatggagtc agacaagaaa tttgccttga
caaagacgac aacttgtgta 540gaccagggtg acagcagtgg gaatcggtac
ctgcccggct tccccatgaa aattgacatc 600ccatccctga tgtacatgga
gcccaatgtt cgatactcag ccaccaagac gatctcgctg 660ctcttcaatg
ccatccctgc gtccttggga atgaagcttc gagggctgtt ggatcgcaag
720ggctcctgga agaagctgga tgacatgcag aacatcttct ggtgccataa
gaccttcacg 780acaaagtatg tcacagagca ctggtgtgaa gatcacttct
ttgggtacca gtacctgaat 840ggtgtcaatc ccgtcatgct ccactgcatc
tctagcttgc ccagcaagct gcctgtcacc 900aatgacatgg tggccccctt
gctgggacag gacacatgcc tgcagacaga gctagagagg 960gggaacatct
tcctagcgga ctactggatc ctggcggagg cccccaccca ctgcctaaac
1020ggccgccagc agtacgtggc cgccccactg tgcctgctgt ggctcagccc
ccagggggcg 1080ctggtgccct tggccatcca gctcagccag acccccgggc
ctgacagccc catcttcctg 1140cccactgact ccgaatggga ctggctgctg
gccaagacgt gggtgcgcaa ctctgagttc 1200ctggtgcacg aaaacaacac
gcactttctg tgcacgcatt tgctgtgcga ggccttcgcc 1260atggccacgc
tgcgccagct gccgctctgc caccccatct acaagctcct actcccccac
1320actcgataca cgctgcaggt gaacaccatc gcgagggcca cgctgctcaa
ccccgagggc 1380ctcgtggacc agcctgcggg cccgcggcgt cctggctatc
cccaactacc actaccgaga 1440cgacggcctg aagatctggg cggccattga
14704489PRTHomo sapiens 4Met Ala Val Tyr Arg Leu Cys Val Thr Thr
Gly Pro Tyr Leu Arg Ala 1 5 10 15Gly Thr Leu Asp Asn Ile Ser Val
Thr Leu Val Gly Thr Cys Gly Glu 20 25 30Ser Pro Lys Gln Arg Leu Asp
Arg Met Gly Arg Asp Phe Ala Pro Gly 35 40 45Ser Val Gln Lys Tyr Lys
Val Arg Cys Thr Ala Glu Leu Gly Glu Leu 50 55 60Leu Leu Leu Arg Val
His Lys Glu Arg Tyr Ala Phe Phe Arg Lys Asp65 70 75 80Ser Trp Tyr
Cys Ser Arg Ile Cys Val Thr Glu Pro Asp Gly Ser Val 85 90 95Ser His
Phe Pro Cys Tyr Gln Trp Ile Glu Gly Tyr Cys Thr Val Glu 100 105
110Leu Arg Pro Gly Thr Ala Arg Thr Ile Cys Gln Asp Ser Leu Pro Leu
115 120 125Leu Leu Asp His Arg Thr Arg Glu Leu Arg Ala Arg Gln Glu
Cys Tyr 130 135 140Arg Trp Lys Ile Tyr Ala Pro Gly Phe Pro Cys Met
Val Asp Val Asn145 150 155 160Ser Phe Gln Glu Met Glu Ser Asp Lys
Lys Phe Ala Leu Thr Lys Thr 165 170 175Thr Thr Cys Val Asp Gln Gly
Asp Ser Ser Gly Asn Arg Tyr Leu Pro 180 185 190Gly Phe Pro Met Lys
Ile Asp Ile Pro Ser Leu Met Tyr Met Glu Pro 195 200 205Asn Val Arg
Tyr Ser Ala Thr Lys Thr Ile Ser Leu Leu Phe Asn Ala 210 215 220Ile
Pro Ala Ser Leu Gly Met Lys Leu Arg Gly Leu Leu Asp Arg Lys225 230
235 240Gly Ser Trp Lys Lys Leu Asp Asp Met Gln Asn Ile Phe Trp Cys
His 245 250 255Lys Thr Phe Thr Thr Lys Tyr Val Thr Glu His Trp Cys
Glu Asp His 260 265 270Phe Phe Gly Tyr Gln Tyr Leu Asn Gly Val Asn
Pro Val Met Leu His 275 280 285Cys Ile Ser Ser Leu Pro Ser Lys Leu
Pro Val Thr Asn Asp Met Val 290 295 300Ala Pro Leu Leu Gly Gln Asp
Thr Cys Leu Gln Thr Glu Leu Glu Arg305 310 315 320Gly Asn Ile Phe
Leu Ala Asp Tyr Trp Ile Leu Ala Glu Ala Pro Thr 325 330 335His Cys
Leu Asn Gly Arg Gln Gln Tyr Val Ala Ala Pro Leu Cys Leu 340 345
350Leu Trp Leu Ser Pro Gln Gly Ala Leu Val Pro Leu Ala Ile Gln Leu
355 360 365Ser Gln Thr Pro Gly Pro Asp Ser Pro Ile Phe Leu Pro Thr
Asp Ser 370 375 380Glu Trp Asp Trp Leu Leu Ala Lys Thr Trp Val Arg
Asn Ser Glu Phe385 390 395 400Leu Val His Glu Asn Asn Thr His Phe
Leu Cys Thr His Leu Leu Cys 405 410 415Glu Ala Phe Ala Met Ala Thr
Leu Arg Gln Leu Pro Leu Cys His Pro 420 425 430Ile Tyr Lys Leu Leu
Leu Pro His Thr Arg Tyr Thr Leu Gln Val Asn 435 440 445Thr Ile Ala
Arg Ala Thr Leu Leu Asn Pro Glu Gly Leu Val Asp Gln 450 455 460Pro
Ala Gly Pro Arg Arg Pro Gly Tyr Pro Gln Leu Pro Leu Pro Arg465 470
475 480Arg Arg Pro Glu Asp Leu Gly Gly His 48552236DNAHomo sapiens
5atggtagacg tcaacagctt tcaggagatg gagtcagaca agaaatttgc cttgacaaag
60acgacaactt gtgtagacca gggtgacagc agtgggaatc ggtacctgcc cggcttcccc
120atgaaaattg acatcccatc cctgatgtac atggagccca atgttcgata
ctcagccacc 180aagacgatct cgctgctctt caatgccatc cctgcgtcct
tgggaatgaa gcttcgaggg 240ctgttggatc gcaagggctc ctggaagaag
ctggatgaca tgcagaacat cttctggtgc 300cataagacct tcacgacaaa
gtatgtcaca gagcactggt gtgaagatca cttctttggg 360taccagtacc
tgaatggtgt caatcccgtc atgctccact gcatctctag cttgcccagc
420aagctgcctg tcaccaatga catggtggcc cccttgctgg gacaggacac
atgcctgcag 480acagagctag agagggggaa catcttccta gcggactact
ggatcctggc ggaggccccc 540acccactgcc taaacggccg ccagcagtac
gtggccgccc cactgtgcct gctgtggctc 600agcccccagg gggcgctggt
gcccttggcc atccagctca gccagacccc cgggcctgac 660agccccatct
tcctgcccac tgactccgaa tgggactggc tgctggccaa gacgtgggtg
720cgcaactctg agttcctggt gcacgaaaac aacacgcact ttctgtgcac
gcatttgctg 780tgcgaggcct tcgccatggc cacgctgcgc cagctgccgc
tctgccaccc catctacaag 840ctcctactcc cccacactcg atacacgctg
caggtgaaca ccatcgcgag ggccacgctg 900ctcaaccccg agggcctcgt
ggaccaggtc acgtccatcg ggaggcaagg cctcatctac 960ctcatgagca
cgggcctggc ccacttcacc tacaccaatt tctgccttcc ggacagcctg
1020cgggcccgcg gcgtcctggc tatccccaac taccactacc gagacgacgg
cctgaagatc 1080tgggcggcca ttgagagctt tgtctcagaa atcgtgggct
actattatcc cagtgacgca 1140tctgtgcagc aggattcgga gctgcaggcc
tggactggcg agatttttgc tcaggcgttc 1200ctgggccggg aaagctcagg
tttcccaagc cggctgtgca ccccaggaga gatggtgaag 1260ttcctcactg
caatcatctt caattgctct gcccagcacg ctgctgtcaa cagtgggcag
1320catgactttg gggcctggat gcccaatgct ccatcatcca tgaggcagcc
cccaccccag 1380accaagggga ccaccaccct gaagacttac ctagacaccc
tccctgaagt gaacatcagc 1440tgtaacaacc tcctcctctt ctggttggtt
agccaagaac ccaaggacca gaggcccctg 1500ggcacctacc cagatgagca
cttcacagag gaggccccga ggcggagcat cgccgccttc 1560cagagccgcc
tggcccagat ctcaagggac atccaggagc ggaaccaggg tctggcactg
1620ccctacacct acctggaccc tcccctcatt gagaacagcg tctccatcta
accaccccca 1680aataccaccc aagaagaaag aaaggtccaa gcatgaggag
gaccagttcc tcaggtcctc 1740cagacccttc catcctccct gttctcagtt
cacctgaacc ttctcttctg cacatggaga 1800cttttgcagc caagatggct
ctgacatcat acaaactggg ccctgagctg tgagagacca 1860gcacagcagc
gtccaggtta aaagccgctg accaaagtcc aatgcacaat agcccctccg
1920aaaggaagga accgcttcac ttcttgcccc acttggggca gcctcttgtt
ccagcctctt 1980ggaatgccca gcttgggttt ctgagctttt ctccctcatc
ctccccaatc cccaaactcc 2040ttctcctacc atgcctttct acgttctctt
tcttccaagc ctagagccac cagcccagct 2100tccttctctg gaaaagcctg
gaaactgggc acagaaggac tgtgtgcctg gctaacatgt 2160ggtccccttt
gtccctagca cctttaaggg gaggggaaga attggagggc agcttgcctg
2220gacccctaac ggctgt 22366556PRTHomo sapiens 6Met Val Asp Val Asn
Ser Phe Gln Glu Met Glu Ser Asp Lys Lys Phe 1 5 10 15Ala Leu Thr
Lys Thr Thr Thr Cys Val Asp Gln Gly Asp Ser Ser Gly 20 25 30Asn Arg
Tyr Leu Pro Gly Phe Pro Met Lys Ile Asp Ile Pro Ser Leu 35 40 45Met
Tyr Met Glu Pro Asn Val Arg Tyr Ser Ala Thr Lys Thr Ile Ser 50 55
60Leu Leu Phe Asn Ala Ile Pro Ala Ser Leu Gly Met Lys Leu Arg Gly65
70 75 80Leu Leu Asp Arg Lys Gly Ser Trp Lys Lys Leu Asp Asp Met Gln
Asn 85 90 95Ile Phe Trp Cys His Lys Thr Phe Thr Thr Lys Tyr Val Thr
Glu His 100 105 110Trp Cys Glu Asp His Phe Phe Gly Tyr Gln Tyr Leu
Asn Gly Val Asn 115 120 125Pro Val Met Leu His Cys Ile Ser Ser Leu
Pro Ser Lys Leu Pro Val 130 135 140Thr Asn Asp Met Val Ala Pro Leu
Leu Gly Gln Asp Thr Cys Leu Gln145 150 155 160Thr Glu Leu Glu Arg
Gly Asn Ile Phe Leu Ala Asp Tyr Trp Ile Leu 165 170 175Ala Glu Ala
Pro Thr His Cys Leu Asn Gly Arg Gln
Gln Tyr Val Ala 180 185 190Ala Pro Leu Cys Leu Leu Trp Leu Ser Pro
Gln Gly Ala Leu Val Pro 195 200 205Leu Ala Ile Gln Leu Ser Gln Thr
Pro Gly Pro Asp Ser Pro Ile Phe 210 215 220Leu Pro Thr Asp Ser Glu
Trp Asp Trp Leu Leu Ala Lys Thr Trp Val225 230 235 240Arg Asn Ser
Glu Phe Leu Val His Glu Asn Asn Thr His Phe Leu Cys 245 250 255Thr
His Leu Leu Cys Glu Ala Phe Ala Met Ala Thr Leu Arg Gln Leu 260 265
270Pro Leu Cys His Pro Ile Tyr Lys Leu Leu Leu Pro His Thr Arg Tyr
275 280 285Thr Leu Gln Val Asn Thr Ile Ala Arg Ala Thr Leu Leu Asn
Pro Glu 290 295 300Gly Leu Val Asp Gln Val Thr Ser Ile Gly Arg Gln
Gly Leu Ile Tyr305 310 315 320Leu Met Ser Thr Gly Leu Ala His Phe
Thr Tyr Thr Asn Phe Cys Leu 325 330 335Pro Asp Ser Leu Arg Ala Arg
Gly Val Leu Ala Ile Pro Asn Tyr His 340 345 350Tyr Arg Asp Asp Gly
Leu Lys Ile Trp Ala Ala Ile Glu Ser Phe Val 355 360 365Ser Glu Ile
Val Gly Tyr Tyr Tyr Pro Ser Asp Ala Ser Val Gln Gln 370 375 380Asp
Ser Glu Leu Gln Ala Trp Thr Gly Glu Ile Phe Ala Gln Ala Phe385 390
395 400Leu Gly Arg Glu Ser Ser Gly Phe Pro Ser Arg Leu Cys Thr Pro
Gly 405 410 415Glu Met Val Lys Phe Leu Thr Ala Ile Ile Phe Asn Cys
Ser Ala Gln 420 425 430His Ala Ala Val Asn Ser Gly Gln His Asp Phe
Gly Ala Trp Met Pro 435 440 445Asn Ala Pro Ser Ser Met Arg Gln Pro
Pro Pro Gln Thr Lys Gly Thr 450 455 460Thr Thr Leu Lys Thr Tyr Leu
Asp Thr Leu Pro Glu Val Asn Ile Ser465 470 475 480Cys Asn Asn Leu
Leu Leu Phe Trp Leu Val Ser Gln Glu Pro Lys Asp 485 490 495Gln Arg
Pro Leu Gly Thr Tyr Pro Asp Glu His Phe Thr Glu Glu Ala 500 505
510Pro Arg Arg Ser Ile Ala Ala Phe Gln Ser Arg Leu Ala Gln Ile Ser
515 520 525Arg Asp Ile Gln Glu Arg Asn Gln Gly Leu Ala Leu Pro Tyr
Thr Tyr 530 535 540Leu Asp Pro Pro Leu Ile Glu Asn Ser Val Ser
Ile545 550 55571005DNAHomo sapiens 7atggtagacg tcaacagctt
tcaggagatg gagtcagaca agaaatttgc cttgacaaag 60acgacaactt gtgtagacca
gggtgacagc agtgggaatc ggtacctgcc cggcttcccc 120atgaaaattg
acatcccatc cctgatgtac atggagccca atgttcgata ctcagccacc
180aagacgatct cgctgctctt caatgccatc cctgcgtcct tgggaatgaa
gcttcgaggg 240ctgttggatc gcaagggctc ctggaagaag ctggatgaca
tgcagaacat cttctggtgc 300cataagacct tcacgacaaa gtatgtcaca
gagcactggt gtgaagatca cttctttggg 360taccagtacc tgaatggtgt
caatcccgtc atgctccact gcatctctag cttgcccagc 420aagctgcctg
tcaccaatga catggtggcc cccttgctgg gacaggacac atgcctgcag
480acagagctag agagggggaa catcttccta gcggactact ggatcctggc
ggaggccccc 540acccactgcc taaacggccg ccagcagtac gtggccgccc
cactgtgcct gctgtggctc 600agcccccagg gggcgctggt gcccttggcc
atccagctca gccagacccc cgggcctgac 660agccccatct tcctgcccac
tgactccgaa tgggactggc tgctggccaa gacgtgggtg 720cgcaactctg
agttcctggt gcacgaaaac aacacgcact ttctgtgcac gcatttgctg
780tgcgaggcct tcgccatggc cacgctgcgc cagctgccgc tctgccaccc
catctacaag 840ctcctactcc cccacactcg atacacgctg caggtgaaca
ccatcgcgag ggccacgctg 900ctcaaccccg agggcctcgt ggaccagcct
gcgggcccgc ggcgtcctgg ctatccccaa 960ctaccactac cgagacgacg
gcctgaagat ctgggcggcc attga 10058334PRTHomo sapiens 8Met Val Asp
Val Asn Ser Phe Gln Glu Met Glu Ser Asp Lys Lys Phe 1 5 10 15Ala
Leu Thr Lys Thr Thr Thr Cys Val Asp Gln Gly Asp Ser Ser Gly 20 25
30Asn Arg Tyr Leu Pro Gly Phe Pro Met Lys Ile Asp Ile Pro Ser Leu
35 40 45Met Tyr Met Glu Pro Asn Val Arg Tyr Ser Ala Thr Lys Thr Ile
Ser 50 55 60Leu Leu Phe Asn Ala Ile Pro Ala Ser Leu Gly Met Lys Leu
Arg Gly65 70 75 80Leu Leu Asp Arg Lys Gly Ser Trp Lys Lys Leu Asp
Asp Met Gln Asn 85 90 95Ile Phe Trp Cys His Lys Thr Phe Thr Thr Lys
Tyr Val Thr Glu His 100 105 110Trp Cys Glu Asp His Phe Phe Gly Tyr
Gln Tyr Leu Asn Gly Val Asn 115 120 125Pro Val Met Leu His Cys Ile
Ser Ser Leu Pro Ser Lys Leu Pro Val 130 135 140Thr Asn Asp Met Val
Ala Pro Leu Leu Gly Gln Asp Thr Cys Leu Gln145 150 155 160Thr Glu
Leu Glu Arg Gly Asn Ile Phe Leu Ala Asp Tyr Trp Ile Leu 165 170
175Ala Glu Ala Pro Thr His Cys Leu Asn Gly Arg Gln Gln Tyr Val Ala
180 185 190Ala Pro Leu Cys Leu Leu Trp Leu Ser Pro Gln Gly Ala Leu
Val Pro 195 200 205Leu Ala Ile Gln Leu Ser Gln Thr Pro Gly Pro Asp
Ser Pro Ile Phe 210 215 220Leu Pro Thr Asp Ser Glu Trp Asp Trp Leu
Leu Ala Lys Thr Trp Val225 230 235 240Arg Asn Ser Glu Phe Leu Val
His Glu Asn Asn Thr His Phe Leu Cys 245 250 255Thr His Leu Leu Cys
Glu Ala Phe Ala Met Ala Thr Leu Arg Gln Leu 260 265 270Pro Leu Cys
His Pro Ile Tyr Lys Leu Leu Leu Pro His Thr Arg Tyr 275 280 285Thr
Leu Gln Val Asn Thr Ile Ala Arg Ala Thr Leu Leu Asn Pro Glu 290 295
300Gly Leu Val Asp Gln Pro Ala Gly Pro Arg Arg Pro Gly Tyr Pro
Gln305 310 315 320Leu Pro Leu Pro Arg Arg Arg Pro Glu Asp Leu Gly
Gly His 325 33091848DNAHomo sapiens 9atggcagtgt accgcctgtg
tgtgaccact ggtccctacc tgagggccgg cacactggac 60aacatctctg tcacactggt
gggcacgtgt ggtgaaagcc ccaagcagcg gctagatcga 120atgggcaggg
acttcgcccc tggatcggta cagaagtaca aggtgcgttg cacagcggag
180ctgggtgagc tcttgctgct gcgtgtacac aaggagcgct acgctttctt
ccgcaaggac 240tcttggtact gtagccgcat ctgtgtcacc gaaccggatg
gtagtgtatc ccacttcccc 300tgctatcagt ggattgaagg ctactgcacc
gtggagctga ggccaggaac agcaagaact 360atttgtcagg actctcttcc
cctcctcctg gatcacagga cacgggagct ccgggcccga 420caagaatgct
accgctggaa gatctatgcc cctggcttcc cctgcatggt agacgtcaac
480agctttcagg agatggagtc agacaagaaa tttgccttga caaagacgac
aacttgtgta 540gaccagggtg acagcagtgg gaatcggtac ctgcccggct
tccccatgaa aattgacatc 600ccatccctga tgtacatgga gcccaatgtt
cgatactcag ccaccaagac gatctcgctg 660ctcttcaatg ccatccctgc
gtccttggga atgaagcttc gagggctgtt ggatcgcaag 720ggctcctgga
agaagctgga tgacatgcag aacatcttct ggtgccataa gaccttcacg
780acaaagtatg tcacagagca ctggtgtgaa gatcacttct ttgggtacca
gtacctgaat 840ggtgtcaatc ccgtcatgct ccactgcatc tctagcttgc
ccagcaagct gcctgtcacc 900aatgacatgg tggccccctt gctgggacag
gacacatgcc tgcagacaga gctagagagg 960gggaacatct tcctagcgga
ctactggatc ctggcggagg cccccaccca ctgcctaaac 1020ggccgccagc
agtacgtggc cgccccactg tgcctgctgt ggctcagccc ccagggggcg
1080ctggtgccct tggccatcca gctcagccag acccccgggc ctgacagccc
catcttcctg 1140cccactgact ccgaatggga ctggctgctg gccaagacgt
gggtgcgcaa ctctgagttc 1200ctggtgcacg aaaacaacac gcactttctg
tgcacgcatt tgctgtgcga ggccttcgcc 1260atggccacgc tgcgccagct
gccgctctgc caccccatct acaagctcct actcccccac 1320actcgataca
cgctgcaggt gaacaccatc gcgagggcca cgctgctcaa ccccgagggc
1380ctcgtggacc aggtcacgtc catcgggagg caaggcctca tctacctcat
gagcacgggc 1440ctggcccact tcacctacac caatttctgc cttccggaca
gcctgcgggc ccgcggcgtc 1500ctggctatcc ccaactacca ctaccgagac
gacggcctga agatctgggc ggccattgag 1560agctttgtct cagaaatcgt
gggctactat tatcccagtg acgcatctgt gcagcaggat 1620tcggagctgc
aggcctggac tggcgagatt tttgctcagg cgttcctggg ccgggaaagc
1680tcaggtttcc caagccggct gtgcacccca ggagagatgg tgaagttcct
cactgcaatc 1740atcttcaatt gctctgccca gcacgctgct gtcaacagtg
ggcaggacgg cagaggtgga 1800atcagggatg gtgaagaggg aggtgatact
ccccttctgg ccaactga 184810615PRTHomo sapiens 10Met Ala Val Tyr Arg
Leu Cys Val Thr Thr Gly Pro Tyr Leu Arg Ala 1 5 10 15Gly Thr Leu
Asp Asn Ile Ser Val Thr Leu Val Gly Thr Cys Gly Glu 20 25 30Ser Pro
Lys Gln Arg Leu Asp Arg Met Gly Arg Asp Phe Ala Pro Gly 35 40 45Ser
Val Gln Lys Tyr Lys Val Arg Cys Thr Ala Glu Leu Gly Glu Leu 50 55
60Leu Leu Leu Arg Val His Lys Glu Arg Tyr Ala Phe Phe Arg Lys Asp65
70 75 80Ser Trp Tyr Cys Ser Arg Ile Cys Val Thr Glu Pro Asp Gly Ser
Val 85 90 95Ser His Phe Pro Cys Tyr Gln Trp Ile Glu Gly Tyr Cys Thr
Val Glu 100 105 110Leu Arg Pro Gly Thr Ala Arg Thr Ile Cys Gln Asp
Ser Leu Pro Leu 115 120 125Leu Leu Asp His Arg Thr Arg Glu Leu Arg
Ala Arg Gln Glu Cys Tyr 130 135 140Arg Trp Lys Ile Tyr Ala Pro Gly
Phe Pro Cys Met Val Asp Val Asn145 150 155 160Ser Phe Gln Glu Met
Glu Ser Asp Lys Lys Phe Ala Leu Thr Lys Thr 165 170 175Thr Thr Cys
Val Asp Gln Gly Asp Ser Ser Gly Asn Arg Tyr Leu Pro 180 185 190Gly
Phe Pro Met Lys Ile Asp Ile Pro Ser Leu Met Tyr Met Glu Pro 195 200
205Asn Val Arg Tyr Ser Ala Thr Lys Thr Ile Ser Leu Leu Phe Asn Ala
210 215 220Ile Pro Ala Ser Leu Gly Met Lys Leu Arg Gly Leu Leu Asp
Arg Lys225 230 235 240Gly Ser Trp Lys Lys Leu Asp Asp Met Gln Asn
Ile Phe Trp Cys His 245 250 255Lys Thr Phe Thr Thr Lys Tyr Val Thr
Glu His Trp Cys Glu Asp His 260 265 270Phe Phe Gly Tyr Gln Tyr Leu
Asn Gly Val Asn Pro Val Met Leu His 275 280 285Cys Ile Ser Ser Leu
Pro Ser Lys Leu Pro Val Thr Asn Asp Met Val 290 295 300Ala Pro Leu
Leu Gly Gln Asp Thr Cys Leu Gln Thr Glu Leu Glu Arg305 310 315
320Gly Asn Ile Phe Leu Ala Asp Tyr Trp Ile Leu Ala Glu Ala Pro Thr
325 330 335His Cys Leu Asn Gly Arg Gln Gln Tyr Val Ala Ala Pro Leu
Cys Leu 340 345 350Leu Trp Leu Ser Pro Gln Gly Ala Leu Val Pro Leu
Ala Ile Gln Leu 355 360 365Ser Gln Thr Pro Gly Pro Asp Ser Pro Ile
Phe Leu Pro Thr Asp Ser 370 375 380Glu Trp Asp Trp Leu Leu Ala Lys
Thr Trp Val Arg Asn Ser Glu Phe385 390 395 400Leu Val His Glu Asn
Asn Thr His Phe Leu Cys Thr His Leu Leu Cys 405 410 415Glu Ala Phe
Ala Met Ala Thr Leu Arg Gln Leu Pro Leu Cys His Pro 420 425 430Ile
Tyr Lys Leu Leu Leu Pro His Thr Arg Tyr Thr Leu Gln Val Asn 435 440
445Thr Ile Ala Arg Ala Thr Leu Leu Asn Pro Glu Gly Leu Val Asp Gln
450 455 460Val Thr Ser Ile Gly Arg Gln Gly Leu Ile Tyr Leu Met Ser
Thr Gly465 470 475 480Leu Ala His Phe Thr Tyr Thr Asn Phe Cys Leu
Pro Asp Ser Leu Arg 485 490 495Ala Arg Gly Val Leu Ala Ile Pro Asn
Tyr His Tyr Arg Asp Asp Gly 500 505 510Leu Lys Ile Trp Ala Ala Ile
Glu Ser Phe Val Ser Glu Ile Val Gly 515 520 525Tyr Tyr Tyr Pro Ser
Asp Ala Ser Val Gln Gln Asp Ser Glu Leu Gln 530 535 540Ala Trp Thr
Gly Glu Ile Phe Ala Gln Ala Phe Leu Gly Arg Glu Ser545 550 555
560Ser Gly Phe Pro Ser Arg Leu Cys Thr Pro Gly Glu Met Val Lys Phe
565 570 575Leu Thr Ala Ile Ile Phe Asn Cys Ser Ala Gln His Ala Ala
Val Asn 580 585 590Ser Gly Gln Asp Gly Arg Gly Gly Ile Arg Asp Gly
Glu Glu Gly Gly 595 600 605Asp Thr Pro Leu Leu Ala Asn 610
615111383DNAHomo sapiens 11atggtagacg tcaacagctt tcaggagatg
gagtcagaca agaaatttgc cttgacaaag 60acgacaactt gtgtagacca gggtgacagc
agtgggaatc ggtacctgcc cggcttcccc 120atgaaaattg acatcccatc
cctgatgtac atggagccca atgttcgata ctcagccacc 180aagacgatct
cgctgctctt caatgccatc cctgcgtcct tgggaatgaa gcttcgaggg
240ctgttggatc gcaagggctc ctggaagaag ctggatgaca tgcagaacat
cttctggtgc 300cataagacct tcacgacaaa gtatgtcaca gagcactggt
gtgaagatca cttctttggg 360taccagtacc tgaatggtgt caatcccgtc
atgctccact gcatctctag cttgcccagc 420aagctgcctg tcaccaatga
catggtggcc cccttgctgg gacaggacac atgcctgcag 480acagagctag
agagggggaa catcttccta gcggactact ggatcctggc ggaggccccc
540acccactgcc taaacggccg ccagcagtac gtggccgccc cactgtgcct
gctgtggctc 600agcccccagg gggcgctggt gcccttggcc atccagctca
gccagacccc cgggcctgac 660agccccatct tcctgcccac tgactccgaa
tgggactggc tgctggccaa gacgtgggtg 720cgcaactctg agttcctggt
gcacgaaaac aacacgcact ttctgtgcac gcatttgctg 780tgcgaggcct
tcgccatggc cacgctgcgc cagctgccgc tctgccaccc catctacaag
840ctcctactcc cccacactcg atacacgctg caggtgaaca ccatcgcgag
ggccacgctg 900ctcaaccccg agggcctcgt ggaccaggtc acgtccatcg
ggaggcaagg cctcatctac 960ctcatgagca cgggcctggc ccacttcacc
tacaccaatt tctgccttcc ggacagcctg 1020cgggcccgcg gcgtcctggc
tatccccaac taccactacc gagacgacgg cctgaagatc 1080tgggcggcca
ttgagagctt tgtctcagaa atcgtgggct actattatcc cagtgacgca
1140tctgtgcagc aggattcgga gctgcaggcc tggactggcg agatttttgc
tcaggcgttc 1200ctgggccggg aaagctcagg tttcccaagc cggctgtgca
ccccaggaga gatggtgaag 1260ttcctcactg caatcatctt caattgctct
gcccagcacg ctgctgtcaa cagtgggcag 1320gacggcagag gtggaatcag
ggatggtgaa gagggaggtg atactcccct tctggccaac 1380tga
138312460PRTHomo sapiens 12Met Val Asp Val Asn Ser Phe Gln Glu Met
Glu Ser Asp Lys Lys Phe 1 5 10 15Ala Leu Thr Lys Thr Thr Thr Cys
Val Asp Gln Gly Asp Ser Ser Gly 20 25 30Asn Arg Tyr Leu Pro Gly Phe
Pro Met Lys Ile Asp Ile Pro Ser Leu 35 40 45Met Tyr Met Glu Pro Asn
Val Arg Tyr Ser Ala Thr Lys Thr Ile Ser 50 55 60Leu Leu Phe Asn Ala
Ile Pro Ala Ser Leu Gly Met Lys Leu Arg Gly65 70 75 80Leu Leu Asp
Arg Lys Gly Ser Trp Lys Lys Leu Asp Asp Met Gln Asn 85 90 95Ile Phe
Trp Cys His Lys Thr Phe Thr Thr Lys Tyr Val Thr Glu His 100 105
110Trp Cys Glu Asp His Phe Phe Gly Tyr Gln Tyr Leu Asn Gly Val Asn
115 120 125Pro Val Met Leu His Cys Ile Ser Ser Leu Pro Ser Lys Leu
Pro Val 130 135 140Thr Asn Asp Met Val Ala Pro Leu Leu Gly Gln Asp
Thr Cys Leu Gln145 150 155 160Thr Glu Leu Glu Arg Gly Asn Ile Phe
Leu Ala Asp Tyr Trp Ile Leu 165 170 175Ala Glu Ala Pro Thr His Cys
Leu Asn Gly Arg Gln Gln Tyr Val Ala 180 185 190Ala Pro Leu Cys Leu
Leu Trp Leu Ser Pro Gln Gly Ala Leu Val Pro 195 200 205Leu Ala Ile
Gln Leu Ser Gln Thr Pro Gly Pro Asp Ser Pro Ile Phe 210 215 220Leu
Pro Thr Asp Ser Glu Trp Asp Trp Leu Leu Ala Lys Thr Trp Val225 230
235 240Arg Asn Ser Glu Phe Leu Val His Glu Asn Asn Thr His Phe Leu
Cys 245 250 255Thr His Leu Leu Cys Glu Ala Phe Ala Met Ala Thr Leu
Arg Gln Leu 260 265 270Pro Leu Cys His Pro Ile Tyr Lys Leu Leu Leu
Pro His Thr Arg Tyr 275 280 285Thr Leu Gln Val Asn Thr Ile Ala Arg
Ala Thr Leu Leu Asn Pro Glu 290 295 300Gly Leu Val Asp Gln Val Thr
Ser Ile Gly Arg Gln Gly Leu Ile Tyr305 310 315 320Leu Met Ser Thr
Gly Leu Ala His Phe Thr Tyr Thr Asn Phe Cys Leu 325 330 335Pro Asp
Ser Leu Arg Ala Arg Gly Val Leu Ala Ile Pro Asn Tyr His 340 345
350Tyr Arg Asp Asp Gly Leu Lys Ile Trp Ala Ala Ile Glu Ser Phe Val
355 360 365Ser Glu Ile Val Gly Tyr Tyr Tyr Pro Ser Asp Ala Ser Val
Gln Gln 370 375 380Asp Ser Glu Leu Gln Ala Trp Thr Gly Glu Ile Phe
Ala Gln Ala Phe385 390 395 400Leu Gly Arg Glu Ser Ser Gly Phe Pro
Ser Arg Leu Cys Thr Pro Gly 405 410 415Glu Met Val Lys Phe Leu Thr
Ala Ile Ile Phe Asn Cys Ser Ala Gln 420
425 430His Ala Ala Val Asn Ser Gly Gln Asp Gly Arg Gly Gly Ile Arg
Asp 435 440 445Gly Glu Glu Gly Gly Asp Thr Pro Leu Leu Ala Asn 450
455 460131441DNAHomo sapiens 13atggccacgc tgcgccagct gccgctctgc
caccccatct acaagctcct actcccccac 60actcgataca cgctgcaggt gaacaccatc
gcgagggcca cgctgctcaa ccccgagggc 120ctcgtggacc aggtcacgtc
catcgggagg caaggcctca tctacctcat gagcacgggc 180ctggcccact
tcacctacac caatttctgc cttccggaca gcctgcgggc ccgcggcgtc
240ctggctatcc ccaactacca ctaccgagac gacggcctga agatctgggc
ggccattgag 300agctttgtct cagaaatcgt gggctactat tatcccagtg
acgcatctgt gcagcaggat 360tcggagctgc aggcctggac tggcgagatt
tttgctcagg cgttcctggg ccgggaaagc 420tcaggtttcc caagccggct
gtgcacccca ggagagatgg tgaagttcct cactgcaatc 480atcttcaatt
gctctgccca gcacgctgct gtcaacagtg ggcagcatga ctttggggcc
540tggatgccca atgctccatc atccatgagg cagcccccac cccagaccaa
ggggaccacc 600accctgaaga cttacctaga caccctccct gaagtgaaca
tcagctgtaa caacctcctc 660ctcttctggt tggttagcca agaacccaag
gaccagaggc ccctgggcac ctacccagat 720gagcacttca cagaggaggc
cccgaggcgg agcatcgccg ccttccagag ccgcctggcc 780cagatctcaa
gggacatcca ggagcggaac cagggtctgg cactgcccta cacctacctg
840gaccctcccc tcattgagaa cagcgtctcc atctaaccac ccccaaatac
cacccaagaa 900gaaagaaagg tccaagcatg aggaggacca gttcctcagg
tcctccagac ccttccatcc 960tccctgttct cagttcacct gaaccttctc
ttctgcacat ggagactttt gcagccaaga 1020tggctctgac atcatacaaa
ctgggccctg agctgtgaga gaccagcaca gcagcgtcca 1080ggttaaaagc
cgctgaccaa agtccaatgc acaatagccc ctccgaaagg aaggaaccgc
1140ttcacttctt gccccacttg gggcagcctc ttgttccagc ctcttggaat
gcccagcttg 1200ggtttctgag cttttctccc tcatcctccc caatccccaa
actccttctc ctaccatgcc 1260tttctacgtt ctctttcttc caagcctaga
gccaccagcc cagcttcctt ctctggaaaa 1320gcctggaaac tgggcacaga
aggactgtgt gcctggctaa catgtggtcc cctttgtccc 1380tagcaccttt
aaggggaggg gaagaattgg agggcagctt gcctggaccc ctaacggctg 1440t
144114291PRTHomo sapiens 14Met Ala Thr Leu Arg Gln Leu Pro Leu Cys
His Pro Ile Tyr Lys Leu 1 5 10 15Leu Leu Pro His Thr Arg Tyr Thr
Leu Gln Val Asn Thr Ile Ala Arg 20 25 30Ala Thr Leu Leu Asn Pro Glu
Gly Leu Val Asp Gln Val Thr Ser Ile 35 40 45Gly Arg Gln Gly Leu Ile
Tyr Leu Met Ser Thr Gly Leu Ala His Phe 50 55 60Thr Tyr Thr Asn Phe
Cys Leu Pro Asp Ser Leu Arg Ala Arg Gly Val65 70 75 80Leu Ala Ile
Pro Asn Tyr His Tyr Arg Asp Asp Gly Leu Lys Ile Trp 85 90 95Ala Ala
Ile Glu Ser Phe Val Ser Glu Ile Val Gly Tyr Tyr Tyr Pro 100 105
110Ser Asp Ala Ser Val Gln Gln Asp Ser Glu Leu Gln Ala Trp Thr Gly
115 120 125Glu Ile Phe Ala Gln Ala Phe Leu Gly Arg Glu Ser Ser Gly
Phe Pro 130 135 140Ser Arg Leu Cys Thr Pro Gly Glu Met Val Lys Phe
Leu Thr Ala Ile145 150 155 160Ile Phe Asn Cys Ser Ala Gln His Ala
Ala Val Asn Ser Gly Gln His 165 170 175Asp Phe Gly Ala Trp Met Pro
Asn Ala Pro Ser Ser Met Arg Gln Pro 180 185 190Pro Pro Gln Thr Lys
Gly Thr Thr Thr Leu Lys Thr Tyr Leu Asp Thr 195 200 205Leu Pro Glu
Val Asn Ile Ser Cys Asn Asn Leu Leu Leu Phe Trp Leu 210 215 220Val
Ser Gln Glu Pro Lys Asp Gln Arg Pro Leu Gly Thr Tyr Pro Asp225 230
235 240Glu His Phe Thr Glu Glu Ala Pro Arg Arg Ser Ile Ala Ala Phe
Gln 245 250 255Ser Arg Leu Ala Gln Ile Ser Arg Asp Ile Gln Glu Arg
Asn Gln Gly 260 265 270Leu Ala Leu Pro Tyr Thr Tyr Leu Asp Pro Pro
Leu Ile Glu Asn Ser 275 280 285Val Ser Ile 29015210DNAHomo sapiens
15atggccacgc tgcgccagct gccgctctgc caccccatct acaagctcct actcccccac
60actcgataca cgctgcaggt gaacaccatc gcgagggcca cgctgctcaa ccccgagggc
120ctcgtggacc agcctgcggg cccgcggcgt cctggctatc cccaactacc
actaccgaga 180cgacggcctg aagatctggg cggccattga 2101669PRTHomo
sapiens 16Met Ala Thr Leu Arg Gln Leu Pro Leu Cys His Pro Ile Tyr
Lys Leu 1 5 10 15Leu Leu Pro His Thr Arg Tyr Thr Leu Gln Val Asn
Thr Ile Ala Arg 20 25 30Ala Thr Leu Leu Asn Pro Glu Gly Leu Val Asp
Gln Pro Ala Gly Pro 35 40 45Arg Arg Pro Gly Tyr Pro Gln Leu Pro Leu
Pro Arg Arg Arg Pro Glu 50 55 60Asp Leu Gly Gly His6517420DNAHomo
sapiens 17atggtgaagt tcctcactgc aatcatcttc aattgctctg cccagcacgc
tgctgtcaac 60agtgggcagc atgactttgg ggcctggatg cccaatgctc catcatccat
gaggcagccc 120ccaccccaga ccaaggggac caccaccctg aagacttacc
tagacaccct ccctgaagtg 180aacatcagct gtaacaacct cctcctcttc
tggttggtta gccaagaacc caaggaccag 240aggcccctgg gcacctaccc
agatgagcac ttcacagagg aggccccgag gcggagcatc 300gccgccttcc
agagccgcct ggcccagatc tcaagggaca tccaggagcg gaaccagggt
360ctggcactgc cctacaccta cctggaccct cccctcattg agaacagcgt
ctccatctaa 42018139PRTHomo sapiens 18Met Val Lys Phe Leu Thr Ala
Ile Ile Phe Asn Cys Ser Ala Gln His 1 5 10 15Ala Ala Val Asn Ser
Gly Gln His Asp Phe Gly Ala Trp Met Pro Asn 20 25 30Ala Pro Ser Ser
Met Arg Gln Pro Pro Pro Gln Thr Lys Gly Thr Thr 35 40 45Thr Leu Lys
Thr Tyr Leu Asp Thr Leu Pro Glu Val Asn Ile Ser Cys 50 55 60Asn Asn
Leu Leu Leu Phe Trp Leu Val Ser Gln Glu Pro Lys Asp Gln65 70 75
80Arg Pro Leu Gly Thr Tyr Pro Asp Glu His Phe Thr Glu Glu Ala Pro
85 90 95Arg Arg Ser Ile Ala Ala Phe Gln Ser Arg Leu Ala Gln Ile Ser
Arg 100 105 110Asp Ile Gln Glu Arg Asn Gln Gly Leu Ala Leu Pro Tyr
Thr Tyr Leu 115 120 125Asp Pro Pro Leu Ile Glu Asn Ser Val Ser Ile
130 13519588DNAHomo sapiens 19atggccacgc tgcgccagct gccgctctgc
caccccatct acaagctcct actcccccac 60actcgataca cgctgcaggt gaacaccatc
gcgagggcca cgctgctcaa ccccgagggc 120ctcgtggacc aggtcacgtc
catcgggagg caaggcctca tctacctcat gagcacgggc 180ctggcccact
tcacctacac caatttctgc cttccggaca gcctgcgggc ccgcggcgtc
240ctggctatcc ccaactacca ctaccgagac gacggcctga agatctgggc
ggccattgag 300agctttgtct cagaaatcgt gggctactat tatcccagtg
acgcatctgt gcagcaggat 360tcggagctgc aggcctggac tggcgagatt
tttgctcagg cgttcctggg ccgggaaagc 420tcaggtttcc caagccggct
gtgcacccca ggagagatgg tgaagttcct cactgcaatc 480atcttcaatt
gctctgccca gcacgctgct gtcaacagtg ggcaggacgg cagaggtgga
540atcagggatg gtgaagaggg aggtgatact ccccttctgg ccaactga
58820195PRTHomo sapiens 20Met Ala Thr Leu Arg Gln Leu Pro Leu Cys
His Pro Ile Tyr Lys Leu 1 5 10 15Leu Leu Pro His Thr Arg Tyr Thr
Leu Gln Val Asn Thr Ile Ala Arg 20 25 30Ala Thr Leu Leu Asn Pro Glu
Gly Leu Val Asp Gln Val Thr Ser Ile 35 40 45Gly Arg Gln Gly Leu Ile
Tyr Leu Met Ser Thr Gly Leu Ala His Phe 50 55 60Thr Tyr Thr Asn Phe
Cys Leu Pro Asp Ser Leu Arg Ala Arg Gly Val65 70 75 80Leu Ala Ile
Pro Asn Tyr His Tyr Arg Asp Asp Gly Leu Lys Ile Trp 85 90 95Ala Ala
Ile Glu Ser Phe Val Ser Glu Ile Val Gly Tyr Tyr Tyr Pro 100 105
110Ser Asp Ala Ser Val Gln Gln Asp Ser Glu Leu Gln Ala Trp Thr Gly
115 120 125Glu Ile Phe Ala Gln Ala Phe Leu Gly Arg Glu Ser Ser Gly
Phe Pro 130 135 140Ser Arg Leu Cys Thr Pro Gly Glu Met Val Lys Phe
Leu Thr Ala Ile145 150 155 160Ile Phe Asn Cys Ser Ala Gln His Ala
Ala Val Asn Ser Gly Gln Asp 165 170 175Gly Arg Gly Gly Ile Arg Asp
Gly Glu Glu Gly Gly Asp Thr Pro Leu 180 185 190Leu Ala Asn
19521898DNAHomo sapiens 21atgcccaatg ctccatcatc catgaggcag
cccccacccc agaccaaggg gaccaccacc 60ctgaagactt acctagacac cctccctgaa
gtgaacatca gctgtaacaa cctcctcctc 120ttctggttgg ttagccaaga
acccaaggac cagaggcccc tgggcaccta cccagatgag 180cacttcacag
aggaggcccc gaggcggagc atcgccgcct tccagagccg cctggcccag
240atctcaaggg acatccagga gcggaaccag ggtctggcac tgccctacac
ctacctggac 300cctcccctca ttgagaacag cgtctccatc taaccacccc
caaataccac ccaagaagaa 360agaaaggtcc aagcatgagg aggaccagtt
cctcaggtcc tccagaccct tccatcctcc 420ctgttctcag ttcacctgaa
ccttctcttc tgcacatgga gacttttgca gccaagatgg 480ctctgacatc
atacaaactg ggccctgagc tgtgagagac cagcacagca gcgtccaggt
540taaaagccgc tgaccaaagt ccaatgcaca atagcccctc cgaaaggaag
gaaccgcttc 600acttcttgcc ccacttgggg cagcctcttg ttccagcctc
ttggaatgcc cagcttgggt 660ttctgagctt ttctccctca tcctccccaa
tccccaaact ccttctccta ccatgccttt 720ctacgttctc tttcttccaa
gcctagagcc accagcccag cttccttctc tggaaaagcc 780tggaaactgg
gcacagaagg actgtgtgcc tggctaacat gtggtcccct ttgtccctag
840cacctttaag gggaggggaa gaattggagg gcagcttgcc tggaccccta acggctgt
89822110PRTHomo sapiens 22Met Pro Asn Ala Pro Ser Ser Met Arg Gln
Pro Pro Pro Gln Thr Lys 1 5 10 15Gly Thr Thr Thr Leu Lys Thr Tyr
Leu Asp Thr Leu Pro Glu Val Asn 20 25 30Ile Ser Cys Asn Asn Leu Leu
Leu Phe Trp Leu Val Ser Gln Glu Pro 35 40 45Lys Asp Gln Arg Pro Leu
Gly Thr Tyr Pro Asp Glu His Phe Thr Glu 50 55 60Glu Ala Pro Arg Arg
Ser Ile Ala Ala Phe Gln Ser Arg Leu Ala Gln65 70 75 80Ile Ser Arg
Asp Ile Gln Glu Arg Asn Gln Gly Leu Ala Leu Pro Tyr 85 90 95Thr Tyr
Leu Asp Pro Pro Leu Ile Glu Asn Ser Val Ser Ile 100 105
110232604DNAHomo sapiens 23atggggagga acagatcttg ggggacattg
gggagtgggc ggacaagcac tccagggcat 60cagtccgggc cgctgaccag gcggagggca
gtgtcccaat tatacaggcg ttacctcctt 120ttctccatct cagcatctga
tccctccctc cgcagtggaa cccaggctcc tgatatccat 180ctgggtgagc
cagccagagg gaccggctgt gtcagaggca agcaaacaag tattagagtg
240caagactgtg ggcggagaga ggaagcccga gccgccagca gggagcttcg
gagagagaaa 300gcccaggaac atcccagaga gagctgggcc catcctcagc
cctacccagc cccgcagccc 360ctagccctcc gcccagaaac ccagccctgt
ccggcgtgcc gctcttctcc tccaggccgg 420ctgctgctgc ggccagcgtt
gccggggcat cccttcctcc ttcccatcat ggcagtgtac 480cgcctgtgtg
tgaccactgg tccctacctg agggccggca cactggacaa catctctgtc
540acactggtgg gcacgtgtgg tgaaagcccc aagcagcggc tagatcgaat
gggcagggac 600ttcgcccctg gatcggtaca gaagtacaag gtgcgttgca
cagcggagct gggtgagctc 660ttgctgctgc gtgtacacaa ggagcgctac
gctttcttcc gcaaggactc ttggtactgt 720agccgcatct gtgtcaccga
accggatggt agtgtatccc acttcccctg ctatcagtgg 780attgaaggct
actgcaccgt ggagctgagg ccaggaacag caagaactat ttgtcaggac
840tctcttcccc tcctcctgga tcacaggaca cgggagctcc gggcccgaca
agaatgctac 900cgctggaaga tctatgcccc tggcttcccc tgcatggtag
acgtcaacag ctttcaggag 960atggagtcag acaagaaatt tgccttgaca
aagacgacaa cttgtgtaga ccagggtgac 1020agcagtggga atcggtacct
gcccggcttc cccatgaaaa ttgacatccc atccctgatg 1080tacatggagc
ccaatgttcg atactcagcc accaagacga tctcgctgct cttcaatgcc
1140atccctgcgt ccttgggaat gaagcttcga gggctgttgg atcgcaaggg
ctcctggaag 1200aagctggatg acatgcagaa catcttctgg tgccataaga
ccttcacgac aaagtatgtc 1260acagagcact ggtgtgaaga tcacttcttt
gggtaccagt acctgaatgg tgtcaatccc 1320gtcatgctcc actgcatctc
tagcttgccc agcaagctgc ctgtcaccaa tgacatggtg 1380gcccccttgc
tgggacagga cacatgcctg cagacagagc tagagagggg gaacatcttc
1440ctagcggact actggatcct ggcggaggcc cccacccact gcctaaacgg
ccgccagcag 1500tacgtggccg ccccactgtg cctgctgtgg ctcagccccc
agggggcgct ggtgcccttg 1560gccatccagc tcagccagac ccccgggcct
gacagcccca tcttcctgcc cactgactcc 1620gaatgggact ggctgctggc
caagacgtgg gtgcgcaact ctgagttcct ggtgcacgaa 1680aacaacacgc
actttctgtg cacgcatttg ctgtgcgagg ccttcgccat ggccacgctg
1740cgccagctgc cgctctgcca ccccatctac aagctcctac tcccccacac
tcgatacacg 1800ctgcaggtga acaccatcgc gagggccacg ctgctcaacc
ccgagggcct cgtggaccag 1860gtcacgtcca tcgggaggca aggcctcatc
tacctcatga gcacgggcct ggcccacttc 1920acctacacca atttctgcct
tccggacagc ctgcgggccc gcggcgtcct ggctatcccc 1980aactaccact
accgagacga cggcctgaag atctgggcgg ccattgagag ctttgtctca
2040gaaatcgtgg gctactatta tcccagtgac gcatctgtgc agcaggattc
ggagctgcag 2100gcctggactg gcgagatttt tgctcaggcg ttcctgggcc
gggaaagctc aggtttccca 2160agccggctgt gcaccccagg agagatggtg
aagttcctca ctgcaatcat cttcaattgc 2220tctgcccagc acgctgctgt
caacagtggg cagcatgact ttggggcctg gatgcccaat 2280gctccatcat
ccatgaggca gcccccaccc cagaccaagg ggaccaccac cctgaagact
2340tacctagaca ccctccctga agtgaacatc agctgtaaca acctcctcct
cttctggttg 2400gttagccaag aacccaagga ccagaggccc ctgggcacct
acccagatga gcacttcaca 2460gaggaggccc cgaggcggag catcgccgcc
ttccagagcc gcctggccca gatctcaagg 2520gacatccagg agcggaacca
gggtctggca ctgccctaca cctacctgga ccctcccctc 2580attgagaaca
gcgtctccat ctaa 260424867PRTHomo sapiens 24Met Gly Arg Asn Arg Ser
Trp Gly Thr Leu Gly Ser Gly Arg Thr Ser 1 5 10 15Thr Pro Gly His
Gln Ser Gly Pro Leu Thr Arg Arg Arg Ala Val Ser 20 25 30Gln Leu Tyr
Arg Arg Tyr Leu Leu Phe Ser Ile Ser Ala Ser Asp Pro 35 40 45Ser Leu
Arg Ser Gly Thr Gln Ala Pro Asp Ile His Leu Gly Glu Pro 50 55 60Ala
Arg Gly Thr Gly Cys Val Arg Gly Lys Gln Thr Ser Ile Arg Val65 70 75
80Gln Asp Cys Gly Arg Arg Glu Glu Ala Arg Ala Ala Ser Arg Glu Leu
85 90 95Arg Arg Glu Lys Ala Gln Glu His Pro Arg Glu Ser Trp Ala His
Pro 100 105 110Gln Pro Tyr Pro Ala Pro Gln Pro Leu Ala Leu Arg Pro
Glu Thr Gln 115 120 125Pro Cys Pro Ala Cys Arg Ser Ser Pro Pro Gly
Arg Leu Leu Leu Arg 130 135 140Pro Ala Leu Pro Gly His Pro Phe Leu
Leu Pro Ile Met Ala Val Tyr145 150 155 160Arg Leu Cys Val Thr Thr
Gly Pro Tyr Leu Arg Ala Gly Thr Leu Asp 165 170 175Asn Ile Ser Val
Thr Leu Val Gly Thr Cys Gly Glu Ser Pro Lys Gln 180 185 190Arg Leu
Asp Arg Met Gly Arg Asp Phe Ala Pro Gly Ser Val Gln Lys 195 200
205Tyr Lys Val Arg Cys Thr Ala Glu Leu Gly Glu Leu Leu Leu Leu Arg
210 215 220Val His Lys Glu Arg Tyr Ala Phe Phe Arg Lys Asp Ser Trp
Tyr Cys225 230 235 240Ser Arg Ile Cys Val Thr Glu Pro Asp Gly Ser
Val Ser His Phe Pro 245 250 255Cys Tyr Gln Trp Ile Glu Gly Tyr Cys
Thr Val Glu Leu Arg Pro Gly 260 265 270Thr Ala Arg Thr Ile Cys Gln
Asp Ser Leu Pro Leu Leu Leu Asp His 275 280 285Arg Thr Arg Glu Leu
Arg Ala Arg Gln Glu Cys Tyr Arg Trp Lys Ile 290 295 300Tyr Ala Pro
Gly Phe Pro Cys Met Val Asp Val Asn Ser Phe Gln Glu305 310 315
320Met Glu Ser Asp Lys Lys Phe Ala Leu Thr Lys Thr Thr Thr Cys Val
325 330 335Asp Gln Gly Asp Ser Ser Gly Asn Arg Tyr Leu Pro Gly Phe
Pro Met 340 345 350Lys Ile Asp Ile Pro Ser Leu Met Tyr Met Glu Pro
Asn Val Arg Tyr 355 360 365Ser Ala Thr Lys Thr Ile Ser Leu Leu Phe
Asn Ala Ile Pro Ala Ser 370 375 380Leu Gly Met Lys Leu Arg Gly Leu
Leu Asp Arg Lys Gly Ser Trp Lys385 390 395 400Lys Leu Asp Asp Met
Gln Asn Ile Phe Trp Cys His Lys Thr Phe Thr 405 410 415Thr Lys Tyr
Val Thr Glu His Trp Cys Glu Asp His Phe Phe Gly Tyr 420 425 430Gln
Tyr Leu Asn Gly Val Asn Pro Val Met Leu His Cys Ile Ser Ser 435 440
445Leu Pro Ser Lys Leu Pro Val Thr Asn Asp Met Val Ala Pro Leu Leu
450 455 460Gly Gln Asp Thr Cys Leu Gln Thr Glu Leu Glu Arg Gly Asn
Ile Phe465 470 475 480Leu Ala Asp Tyr Trp Ile Leu Ala Glu Ala Pro
Thr His Cys Leu Asn 485 490 495Gly Arg Gln Gln Tyr Val Ala Ala Pro
Leu Cys Leu Leu Trp Leu Ser 500 505 510Pro Gln Gly Ala Leu Val Pro
Leu Ala Ile Gln Leu Ser Gln Thr Pro 515 520 525Gly Pro Asp Ser Pro
Ile Phe Leu Pro Thr Asp Ser Glu Trp Asp Trp 530 535 540Leu Leu Ala
Lys Thr Trp Val Arg Asn Ser Glu Phe Leu Val His Glu545 550 555
560Asn Asn Thr His Phe Leu Cys Thr His Leu Leu Cys Glu Ala Phe Ala
565
570 575Met Ala Thr Leu Arg Gln Leu Pro Leu Cys His Pro Ile Tyr Lys
Leu 580 585 590Leu Leu Pro His Thr Arg Tyr Thr Leu Gln Val Asn Thr
Ile Ala Arg 595 600 605Ala Thr Leu Leu Asn Pro Glu Gly Leu Val Asp
Gln Val Thr Ser Ile 610 615 620Gly Arg Gln Gly Leu Ile Tyr Leu Met
Ser Thr Gly Leu Ala His Phe625 630 635 640Thr Tyr Thr Asn Phe Cys
Leu Pro Asp Ser Leu Arg Ala Arg Gly Val 645 650 655Leu Ala Ile Pro
Asn Tyr His Tyr Arg Asp Asp Gly Leu Lys Ile Trp 660 665 670Ala Ala
Ile Glu Ser Phe Val Ser Glu Ile Val Gly Tyr Tyr Tyr Pro 675 680
685Ser Asp Ala Ser Val Gln Gln Asp Ser Glu Leu Gln Ala Trp Thr Gly
690 695 700Glu Ile Phe Ala Gln Ala Phe Leu Gly Arg Glu Ser Ser Gly
Phe Pro705 710 715 720Ser Arg Leu Cys Thr Pro Gly Glu Met Val Lys
Phe Leu Thr Ala Ile 725 730 735Ile Phe Asn Cys Ser Ala Gln His Ala
Ala Val Asn Ser Gly Gln His 740 745 750Asp Phe Gly Ala Trp Met Pro
Asn Ala Pro Ser Ser Met Arg Gln Pro 755 760 765Pro Pro Gln Thr Lys
Gly Thr Thr Thr Leu Lys Thr Tyr Leu Asp Thr 770 775 780Leu Pro Glu
Val Asn Ile Ser Cys Asn Asn Leu Leu Leu Phe Trp Leu785 790 795
800Val Ser Gln Glu Pro Lys Asp Gln Arg Pro Leu Gly Thr Tyr Pro Asp
805 810 815Glu His Phe Thr Glu Glu Ala Pro Arg Arg Ser Ile Ala Ala
Phe Gln 820 825 830Ser Arg Leu Ala Gln Ile Ser Arg Asp Ile Gln Glu
Arg Asn Gln Gly 835 840 845Leu Ala Leu Pro Tyr Thr Tyr Leu Asp Pro
Pro Leu Ile Glu Asn Ser 850 855 860Val Ser Ile865251938DNAHomo
sapiens 25atggggagga acagatcttg ggggacattg gggagtgggc ggacaagcac
tccagggcat 60cagtccgggc cgctgaccag gcggagggca gtgtcccaat tatacaggcg
ttacctcctt 120ttctccatct cagcatctga tccctccctc cgcagtggaa
cccaggctcc tgatatccat 180ctgggtgagc cagccagagg gaccggctgt
gtcagaggca agcaaacaag tattagagtg 240caagactgtg ggcggagaga
ggaagcccga gccgccagca gggagcttcg gagagagaaa 300gcccaggaac
atcccagaga gagctgggcc catcctcagc cctacccagc cccgcagccc
360ctagccctcc gcccagaaac ccagccctgt ccggcgtgcc gctcttctcc
tccaggccgg 420ctgctgctgc ggccagcgtt gccggggcat cccttcctcc
ttcccatcat ggcagtgtac 480cgcctgtgtg tgaccactgg tccctacctg
agggccggca cactggacaa catctctgtc 540acactggtgg gcacgtgtgg
tgaaagcccc aagcagcggc tagatcgaat gggcagggac 600ttcgcccctg
gatcggtaca gaagtacaag gtgcgttgca cagcggagct gggtgagctc
660ttgctgctgc gtgtacacaa ggagcgctac gctttcttcc gcaaggactc
ttggtactgt 720agccgcatct gtgtcaccga accggatggt agtgtatccc
acttcccctg ctatcagtgg 780attgaaggct actgcaccgt ggagctgagg
ccaggaacag caagaactat ttgtcaggac 840tctcttcccc tcctcctgga
tcacaggaca cgggagctcc gggcccgaca agaatgctac 900cgctggaaga
tctatgcccc tggcttcccc tgcatggtag acgtcaacag ctttcaggag
960atggagtcag acaagaaatt tgccttgaca aagacgacaa cttgtgtaga
ccagggtgac 1020agcagtggga atcggtacct gcccggcttc cccatgaaaa
ttgacatccc atccctgatg 1080tacatggagc ccaatgttcg atactcagcc
accaagacga tctcgctgct cttcaatgcc 1140atccctgcgt ccttgggaat
gaagcttcga gggctgttgg atcgcaaggg ctcctggaag 1200aagctggatg
acatgcagaa catcttctgg tgccataaga ccttcacgac aaagtatgtc
1260acagagcact ggtgtgaaga tcacttcttt gggtaccagt acctgaatgg
tgtcaatccc 1320gtcatgctcc actgcatctc tagcttgccc agcaagctgc
ctgtcaccaa tgacatggtg 1380gcccccttgc tgggacagga cacatgcctg
cagacagagc tagagagggg gaacatcttc 1440ctagcggact actggatcct
ggcggaggcc cccacccact gcctaaacgg ccgccagcag 1500tacgtggccg
ccccactgtg cctgctgtgg ctcagccccc agggggcgct ggtgcccttg
1560gccatccagc tcagccagac ccccgggcct gacagcccca tcttcctgcc
cactgactcc 1620gaatgggact ggctgctggc caagacgtgg gtgcgcaact
ctgagttcct ggtgcacgaa 1680aacaacacgc actttctgtg cacgcatttg
ctgtgcgagg ccttcgccat ggccacgctg 1740cgccagctgc cgctctgcca
ccccatctac aagctcctac tcccccacac tcgatacacg 1800ctgcaggtga
acaccatcgc gagggccacg ctgctcaacc ccgagggcct cgtggaccag
1860cctgcgggcc cgcggcgtcc tggctatccc caactaccac taccgagacg
acggcctgaa 1920gatctgggcg gccattga 193826645PRTHomo sapiens 26Met
Gly Arg Asn Arg Ser Trp Gly Thr Leu Gly Ser Gly Arg Thr Ser 1 5 10
15Thr Pro Gly His Gln Ser Gly Pro Leu Thr Arg Arg Arg Ala Val Ser
20 25 30Gln Leu Tyr Arg Arg Tyr Leu Leu Phe Ser Ile Ser Ala Ser Asp
Pro 35 40 45Ser Leu Arg Ser Gly Thr Gln Ala Pro Asp Ile His Leu Gly
Glu Pro 50 55 60Ala Arg Gly Thr Gly Cys Val Arg Gly Lys Gln Thr Ser
Ile Arg Val65 70 75 80Gln Asp Cys Gly Arg Arg Glu Glu Ala Arg Ala
Ala Ser Arg Glu Leu 85 90 95Arg Arg Glu Lys Ala Gln Glu His Pro Arg
Glu Ser Trp Ala His Pro 100 105 110Gln Pro Tyr Pro Ala Pro Gln Pro
Leu Ala Leu Arg Pro Glu Thr Gln 115 120 125Pro Cys Pro Ala Cys Arg
Ser Ser Pro Pro Gly Arg Leu Leu Leu Arg 130 135 140Pro Ala Leu Pro
Gly His Pro Phe Leu Leu Pro Ile Met Ala Val Tyr145 150 155 160Arg
Leu Cys Val Thr Thr Gly Pro Tyr Leu Arg Ala Gly Thr Leu Asp 165 170
175Asn Ile Ser Val Thr Leu Val Gly Thr Cys Gly Glu Ser Pro Lys Gln
180 185 190Arg Leu Asp Arg Met Gly Arg Asp Phe Ala Pro Gly Ser Val
Gln Lys 195 200 205Tyr Lys Val Arg Cys Thr Ala Glu Leu Gly Glu Leu
Leu Leu Leu Arg 210 215 220Val His Lys Glu Arg Tyr Ala Phe Phe Arg
Lys Asp Ser Trp Tyr Cys225 230 235 240Ser Arg Ile Cys Val Thr Glu
Pro Asp Gly Ser Val Ser His Phe Pro 245 250 255Cys Tyr Gln Trp Ile
Glu Gly Tyr Cys Thr Val Glu Leu Arg Pro Gly 260 265 270Thr Ala Arg
Thr Ile Cys Gln Asp Ser Leu Pro Leu Leu Leu Asp His 275 280 285Arg
Thr Arg Glu Leu Arg Ala Arg Gln Glu Cys Tyr Arg Trp Lys Ile 290 295
300Tyr Ala Pro Gly Phe Pro Cys Met Val Asp Val Asn Ser Phe Gln
Glu305 310 315 320Met Glu Ser Asp Lys Lys Phe Ala Leu Thr Lys Thr
Thr Thr Cys Val 325 330 335Asp Gln Gly Asp Ser Ser Gly Asn Arg Tyr
Leu Pro Gly Phe Pro Met 340 345 350Lys Ile Asp Ile Pro Ser Leu Met
Tyr Met Glu Pro Asn Val Arg Tyr 355 360 365Ser Ala Thr Lys Thr Ile
Ser Leu Leu Phe Asn Ala Ile Pro Ala Ser 370 375 380Leu Gly Met Lys
Leu Arg Gly Leu Leu Asp Arg Lys Gly Ser Trp Lys385 390 395 400Lys
Leu Asp Asp Met Gln Asn Ile Phe Trp Cys His Lys Thr Phe Thr 405 410
415Thr Lys Tyr Val Thr Glu His Trp Cys Glu Asp His Phe Phe Gly Tyr
420 425 430Gln Tyr Leu Asn Gly Val Asn Pro Val Met Leu His Cys Ile
Ser Ser 435 440 445Leu Pro Ser Lys Leu Pro Val Thr Asn Asp Met Val
Ala Pro Leu Leu 450 455 460Gly Gln Asp Thr Cys Leu Gln Thr Glu Leu
Glu Arg Gly Asn Ile Phe465 470 475 480Leu Ala Asp Tyr Trp Ile Leu
Ala Glu Ala Pro Thr His Cys Leu Asn 485 490 495Gly Arg Gln Gln Tyr
Val Ala Ala Pro Leu Cys Leu Leu Trp Leu Ser 500 505 510Pro Gln Gly
Ala Leu Val Pro Leu Ala Ile Gln Leu Ser Gln Thr Pro 515 520 525Gly
Pro Asp Ser Pro Ile Phe Leu Pro Thr Asp Ser Glu Trp Asp Trp 530 535
540Leu Leu Ala Lys Thr Trp Val Arg Asn Ser Glu Phe Leu Val His
Glu545 550 555 560Asn Asn Thr His Phe Leu Cys Thr His Leu Leu Cys
Glu Ala Phe Ala 565 570 575Met Ala Thr Leu Arg Gln Leu Pro Leu Cys
His Pro Ile Tyr Lys Leu 580 585 590Leu Leu Pro His Thr Arg Tyr Thr
Leu Gln Val Asn Thr Ile Ala Arg 595 600 605Ala Thr Leu Leu Asn Pro
Glu Gly Leu Val Asp Gln Pro Ala Gly Pro 610 615 620Arg Arg Pro Gly
Tyr Pro Gln Leu Pro Leu Pro Arg Arg Arg Pro Glu625 630 635 640Asp
Leu Gly Gly His 645272316DNAHomo sapiens 27atggggagga acagatcttg
ggggacattg gggagtgggc ggacaagcac tccagggcat 60cagtccgggc cgctgaccag
gcggagggca gtgtcccaat tatacaggcg ttacctcctt 120ttctccatct
cagcatctga tccctccctc cgcagtggaa cccaggctcc tgatatccat
180ctgggtgagc cagccagagg gaccggctgt gtcagaggca agcaaacaag
tattagagtg 240caagactgtg ggcggagaga ggaagcccga gccgccagca
gggagcttcg gagagagaaa 300gcccaggaac atcccagaga gagctgggcc
catcctcagc cctacccagc cccgcagccc 360ctagccctcc gcccagaaac
ccagccctgt ccggcgtgcc gctcttctcc tccaggccgg 420ctgctgctgc
ggccagcgtt gccggggcat cccttcctcc ttcccatcat ggcagtgtac
480cgcctgtgtg tgaccactgg tccctacctg agggccggca cactggacaa
catctctgtc 540acactggtgg gcacgtgtgg tgaaagcccc aagcagcggc
tagatcgaat gggcagggac 600ttcgcccctg gatcggtaca gaagtacaag
gtgcgttgca cagcggagct gggtgagctc 660ttgctgctgc gtgtacacaa
ggagcgctac gctttcttcc gcaaggactc ttggtactgt 720agccgcatct
gtgtcaccga accggatggt agtgtatccc acttcccctg ctatcagtgg
780attgaaggct actgcaccgt ggagctgagg ccaggaacag caagaactat
ttgtcaggac 840tctcttcccc tcctcctgga tcacaggaca cgggagctcc
gggcccgaca agaatgctac 900cgctggaaga tctatgcccc tggcttcccc
tgcatggtag acgtcaacag ctttcaggag 960atggagtcag acaagaaatt
tgccttgaca aagacgacaa cttgtgtaga ccagggtgac 1020agcagtggga
atcggtacct gcccggcttc cccatgaaaa ttgacatccc atccctgatg
1080tacatggagc ccaatgttcg atactcagcc accaagacga tctcgctgct
cttcaatgcc 1140atccctgcgt ccttgggaat gaagcttcga gggctgttgg
atcgcaaggg ctcctggaag 1200aagctggatg acatgcagaa catcttctgg
tgccataaga ccttcacgac aaagtatgtc 1260acagagcact ggtgtgaaga
tcacttcttt gggtaccagt acctgaatgg tgtcaatccc 1320gtcatgctcc
actgcatctc tagcttgccc agcaagctgc ctgtcaccaa tgacatggtg
1380gcccccttgc tgggacagga cacatgcctg cagacagagc tagagagggg
gaacatcttc 1440ctagcggact actggatcct ggcggaggcc cccacccact
gcctaaacgg ccgccagcag 1500tacgtggccg ccccactgtg cctgctgtgg
ctcagccccc agggggcgct ggtgcccttg 1560gccatccagc tcagccagac
ccccgggcct gacagcccca tcttcctgcc cactgactcc 1620gaatgggact
ggctgctggc caagacgtgg gtgcgcaact ctgagttcct ggtgcacgaa
1680aacaacacgc actttctgtg cacgcatttg ctgtgcgagg ccttcgccat
ggccacgctg 1740cgccagctgc cgctctgcca ccccatctac aagctcctac
tcccccacac tcgatacacg 1800ctgcaggtga acaccatcgc gagggccacg
ctgctcaacc ccgagggcct cgtggaccag 1860gtcacgtcca tcgggaggca
aggcctcatc tacctcatga gcacgggcct ggcccacttc 1920acctacacca
atttctgcct tccggacagc ctgcgggccc gcggcgtcct ggctatcccc
1980aactaccact accgagacga cggcctgaag atctgggcgg ccattgagag
ctttgtctca 2040gaaatcgtgg gctactatta tcccagtgac gcatctgtgc
agcaggattc ggagctgcag 2100gcctggactg gcgagatttt tgctcaggcg
ttcctgggcc gggaaagctc aggtttccca 2160agccggctgt gcaccccagg
agagatggtg aagttcctca ctgcaatcat cttcaattgc 2220tctgcccagc
acgctgctgt caacagtggg caggacggca gaggtggaat cagggatggt
2280gaagagggag gtgatactcc ccttctggcc aactga 231628771PRTHomo
sapiens 28Met Gly Arg Asn Arg Ser Trp Gly Thr Leu Gly Ser Gly Arg
Thr Ser 1 5 10 15Thr Pro Gly His Gln Ser Gly Pro Leu Thr Arg Arg
Arg Ala Val Ser 20 25 30Gln Leu Tyr Arg Arg Tyr Leu Leu Phe Ser Ile
Ser Ala Ser Asp Pro 35 40 45Ser Leu Arg Ser Gly Thr Gln Ala Pro Asp
Ile His Leu Gly Glu Pro 50 55 60Ala Arg Gly Thr Gly Cys Val Arg Gly
Lys Gln Thr Ser Ile Arg Val65 70 75 80Gln Asp Cys Gly Arg Arg Glu
Glu Ala Arg Ala Ala Ser Arg Glu Leu 85 90 95Arg Arg Glu Lys Ala Gln
Glu His Pro Arg Glu Ser Trp Ala His Pro 100 105 110Gln Pro Tyr Pro
Ala Pro Gln Pro Leu Ala Leu Arg Pro Glu Thr Gln 115 120 125Pro Cys
Pro Ala Cys Arg Ser Ser Pro Pro Gly Arg Leu Leu Leu Arg 130 135
140Pro Ala Leu Pro Gly His Pro Phe Leu Leu Pro Ile Met Ala Val
Tyr145 150 155 160Arg Leu Cys Val Thr Thr Gly Pro Tyr Leu Arg Ala
Gly Thr Leu Asp 165 170 175Asn Ile Ser Val Thr Leu Val Gly Thr Cys
Gly Glu Ser Pro Lys Gln 180 185 190Arg Leu Asp Arg Met Gly Arg Asp
Phe Ala Pro Gly Ser Val Gln Lys 195 200 205Tyr Lys Val Arg Cys Thr
Ala Glu Leu Gly Glu Leu Leu Leu Leu Arg 210 215 220Val His Lys Glu
Arg Tyr Ala Phe Phe Arg Lys Asp Ser Trp Tyr Cys225 230 235 240Ser
Arg Ile Cys Val Thr Glu Pro Asp Gly Ser Val Ser His Phe Pro 245 250
255Cys Tyr Gln Trp Ile Glu Gly Tyr Cys Thr Val Glu Leu Arg Pro Gly
260 265 270Thr Ala Arg Thr Ile Cys Gln Asp Ser Leu Pro Leu Leu Leu
Asp His 275 280 285Arg Thr Arg Glu Leu Arg Ala Arg Gln Glu Cys Tyr
Arg Trp Lys Ile 290 295 300Tyr Ala Pro Gly Phe Pro Cys Met Val Asp
Val Asn Ser Phe Gln Glu305 310 315 320Met Glu Ser Asp Lys Lys Phe
Ala Leu Thr Lys Thr Thr Thr Cys Val 325 330 335Asp Gln Gly Asp Ser
Ser Gly Asn Arg Tyr Leu Pro Gly Phe Pro Met 340 345 350Lys Ile Asp
Ile Pro Ser Leu Met Tyr Met Glu Pro Asn Val Arg Tyr 355 360 365Ser
Ala Thr Lys Thr Ile Ser Leu Leu Phe Asn Ala Ile Pro Ala Ser 370 375
380Leu Gly Met Lys Leu Arg Gly Leu Leu Asp Arg Lys Gly Ser Trp
Lys385 390 395 400Lys Leu Asp Asp Met Gln Asn Ile Phe Trp Cys His
Lys Thr Phe Thr 405 410 415Thr Lys Tyr Val Thr Glu His Trp Cys Glu
Asp His Phe Phe Gly Tyr 420 425 430Gln Tyr Leu Asn Gly Val Asn Pro
Val Met Leu His Cys Ile Ser Ser 435 440 445Leu Pro Ser Lys Leu Pro
Val Thr Asn Asp Met Val Ala Pro Leu Leu 450 455 460Gly Gln Asp Thr
Cys Leu Gln Thr Glu Leu Glu Arg Gly Asn Ile Phe465 470 475 480Leu
Ala Asp Tyr Trp Ile Leu Ala Glu Ala Pro Thr His Cys Leu Asn 485 490
495Gly Arg Gln Gln Tyr Val Ala Ala Pro Leu Cys Leu Leu Trp Leu Ser
500 505 510Pro Gln Gly Ala Leu Val Pro Leu Ala Ile Gln Leu Ser Gln
Thr Pro 515 520 525Gly Pro Asp Ser Pro Ile Phe Leu Pro Thr Asp Ser
Glu Trp Asp Trp 530 535 540Leu Leu Ala Lys Thr Trp Val Arg Asn Ser
Glu Phe Leu Val His Glu545 550 555 560Asn Asn Thr His Phe Leu Cys
Thr His Leu Leu Cys Glu Ala Phe Ala 565 570 575Met Ala Thr Leu Arg
Gln Leu Pro Leu Cys His Pro Ile Tyr Lys Leu 580 585 590Leu Leu Pro
His Thr Arg Tyr Thr Leu Gln Val Asn Thr Ile Ala Arg 595 600 605Ala
Thr Leu Leu Asn Pro Glu Gly Leu Val Asp Gln Val Thr Ser Ile 610 615
620Gly Arg Gln Gly Leu Ile Tyr Leu Met Ser Thr Gly Leu Ala His
Phe625 630 635 640Thr Tyr Thr Asn Phe Cys Leu Pro Asp Ser Leu Arg
Ala Arg Gly Val 645 650 655Leu Ala Ile Pro Asn Tyr His Tyr Arg Asp
Asp Gly Leu Lys Ile Trp 660 665 670Ala Ala Ile Glu Ser Phe Val Ser
Glu Ile Val Gly Tyr Tyr Tyr Pro 675 680 685Ser Asp Ala Ser Val Gln
Gln Asp Ser Glu Leu Gln Ala Trp Thr Gly 690 695 700Glu Ile Phe Ala
Gln Ala Phe Leu Gly Arg Glu Ser Ser Gly Phe Pro705 710 715 720Ser
Arg Leu Cys Thr Pro Gly Glu Met Val Lys Phe Leu Thr Ala Ile 725 730
735Ile Phe Asn Cys Ser Ala Gln His Ala Ala Val Asn Ser Gly Gln Asp
740 745 750Gly Arg Gly Gly Ile Arg Asp Gly Glu Glu Gly Gly Asp Thr
Pro Leu 755 760 765Leu Ala Asn 770293384DNAHomo sapiens
29caggtgccct gtgctcatct ctgcctggga atggggagga acagatcttg ggggacattg
60gggagtgggc ggacaagcac tccagggcat cagtccgggc cgctgaccag gcggagggca
120gtgtcccaat tatacaggcg ttacctcctt ttctccatct cagcatctga
tccctccctc 180cgcagtggaa cccaggctcc tgatatccat ctgggtgagc
cagccagagg gaccggctgt 240gtcagaggca
agcaaacaag tattagagtg caagactgtg ggcggagaga ggaagcccga
300gccgccagca gggagcttcg gagagagaaa gcccaggaac atcccagaga
gagctgggcc 360catcctcagc cctacccagc cccgcagccc ctagccctcc
gcccagaaac ccagccctgt 420ccggcgtgcc gctcttctcc tccaggccgg
ctgctgctgc ggccagcgtt gccggggcat 480cccttcctcc ttcccatcat
ggcagtgtac cgcctgtgtg tgaccactgg tccctacctg 540agggccggca
cactggacaa catctctgtc acactggtgg gcacgtgtgg tgaaagcccc
600aagcagcggc tagatcgaat gggcagggac ttcgcccctg gatcggtaca
gaagtacaag 660gtgcgttgca cagcggagct gggtgagctc ttgctgctgc
gtgtacacaa ggagcgctac 720gctttcttcc gcaaggactc ttggtactgt
agccgcatct gtgtcaccga accggatggt 780agtgtatccc acttcccctg
ctatcagtgg attgaaggct actgcaccgt ggagctgagg 840ccaggaacag
caagaactat ttgtcaggac tctcttcccc tcctcctgga tcacaggaca
900cgggagctcc gggcccgaca agaatgctac cgctggaaga tctatgcccc
tggcttcccc 960tgcatggtag acgtcaacag ctttcaggag atggagtcag
acaagaaatt tgccttgaca 1020aagacgacaa cttgtgtaga ccagggtgac
agcagtggga atcggtacct gcccggcttc 1080cccatgaaaa ttgacatccc
atccctgatg tacatggagc ccaatgttcg atactcagcc 1140accaagacga
tctcgctgct cttcaatgcc atccctgcgt ccttgggaat gaagcttcga
1200gggctgttgg atcgcaaggg ctcctggaag aagctggatg acatgcagaa
catcttctgg 1260tgccataaga ccttcacgac aaagtatgtc acagagcact
ggtgtgaaga tcacttcttt 1320gggtaccagt acctgaatgg tgtcaatccc
gtcatgctcc actgcatctc tagcttgccc 1380agcaagctgc ctgtcaccaa
tgacatggtg gcccccttgc tgggacagga cacatgcctg 1440cagacagagc
tagagagggg gaacatcttc ctagcggact actggatcct ggcggaggcc
1500cccacccact gcctaaacgg ccgccagcag tacgtggccg ccccactgtg
cctgctgtgg 1560ctcagccccc agggggcgct ggtgcccttg gccatccagc
tcagccagac ccccgggcct 1620gacagcccca tcttcctgcc cactgactcc
gaatgggact ggctgctggc caagacgtgg 1680gtgcgcaact ctgagttcct
ggtgcacgaa aacaacacgc actttctgtg cacgcatttg 1740ctgtgcgagg
ccttcgccat ggccacgctg cgccagctgc cgctctgcca ccccatctac
1800aagctcctac tcccccacac tcgatacacg ctgcaggtga acaccatcgc
gagggccacg 1860ctgctcaacc ccgagggcct cgtggaccag gtcacgtcca
tcgggaggca aggcctcatc 1920tacctcatga gcacgggcct ggcccacttc
acctacacca atttctgcct tccggacagc 1980ctgcgggccc gcggcgtcct
ggctatcccc aactaccact accgagacga cggcctgaag 2040atctgggcgg
ccattgagag ctttgtctca gaaatcgtgg gctactatta tcccagtgac
2100gcatctgtgc agcaggattc ggagctgcag gcctggactg gcgagatttt
tgctcaggcg 2160ttcctgggcc gggaaagctc aggtttccca agccggctgt
gcaccccagg agagatggtg 2220aagttcctca ctgcaatcat cttcaattgc
tctgcccagc acgctgctgt caacagtggg 2280cagcatgact ttggggcctg
gatgcccaat gctccatcat ccatgaggca gcccccaccc 2340cagaccaagg
ggaccaccac cctgaagact tacctagaca ccctccctga agtgaacatc
2400agctgtaaca acctcctcct cttctggttg gttagccaag aacccaagga
ccagaggccc 2460ctgggcacct acccagatga gcacttcaca gaggaggccc
cgaggcggag catcgccgcc 2520ttccagagcc gcctggccca gatctcaagg
gacatccagg agcggaacca gggtctggca 2580ctgccctaca cctacctgga
ccctcccctc attgagaaca gtgtctccat ctaaccaccc 2640ccaaatacca
cccaagaaga aagaaaggtc caagcatgag gaggaccagt tcctcaggtc
2700ctccagaccc ttccatcctc cctgttctca gttcacctga accttctctt
ctgcacatgg 2760agacttttgc agccaagatg gctctgacat catacaaact
gggccctgag ctgtgagaga 2820ccagcacagc agcgtccagg ttaaaagccg
ctgaccaaag tccaatgcac aatagcccct 2880ccgaaaggaa ggaaccgctt
cacttcttgc cccacttggg gcagcctctt gttccagcct 2940cttggaatgc
ccagcttggg tttctgagct tttctccctc atcctccccc atccccaaac
3000tccttctcct accatgcctt tctacgttct ctttcttcca agcctagagc
caccagccca 3060gcttccttct ctggaaaagc ctggaaactg ggcacagaag
gactgtgtgc ctgggtctaa 3120catgtggtcc cctttgtccc tagcaccttt
aaggggaggg gaagaattgg agggcagctt 3180gcctggaccc ctaacggctg
ttctcaggaa caggttccca ggcctggggt gtttgtggag 3240atctgtcttt
ctccaaagat ttcatccaac tcccctttca tcccactccc tttcatccca
3300tttttttctt tctgtccttg agcccagtga gttcaataaa aaccaaaata
tttggcaaaa 3360aaaaaaaaaa aaaaaaaaaa aaaa 3384
* * * * *