U.S. patent application number 10/843130 was filed with the patent office on 2004-10-14 for novel human proteases and polynucleotides encoding the same.
Invention is credited to Donoho, Gregory, Friedrich, Glenn, Hilbun, Erin, Nehls, Michael C., Sands, Arthur T., Turner, C. Alexander JR., Zambrowicz, Brian.
Application Number | 20040203059 10/843130 |
Document ID | / |
Family ID | 22598145 |
Filed Date | 2004-10-14 |
United States Patent
Application |
20040203059 |
Kind Code |
A1 |
Donoho, Gregory ; et
al. |
October 14, 2004 |
Novel human proteases and polynucleotides encoding the same
Abstract
Novel human polynucleotide and polypeptide sequences are
disclosed that can be used in therapeutic, diagnostic, and
pharmacogenomic applications.
Inventors: |
Donoho, Gregory; (The
Woodlands, TX) ; Hilbun, Erin; (Spring, TX) ;
Turner, C. Alexander JR.; (The Woodlands, TX) ;
Nehls, Michael C.; (Stockdorf, DE) ; Friedrich,
Glenn; (Houston, TX) ; Zambrowicz, Brian; (The
Woodlands, TX) ; Sands, Arthur T.; (The Woodlands,
TX) |
Correspondence
Address: |
Lance K. Ishimoto
LEXICON GENETICS INCORPORATED
8800 Technology Forest Place
The Woodlands
TX
77381
US
|
Family ID: |
22598145 |
Appl. No.: |
10/843130 |
Filed: |
May 11, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10843130 |
May 11, 2004 |
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10200910 |
Jul 22, 2002 |
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6777221 |
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10200910 |
Jul 22, 2002 |
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09710099 |
Nov 10, 2000 |
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6441154 |
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60165260 |
Nov 12, 1999 |
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Current U.S.
Class: |
435/6.16 ;
536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 9/48 20130101 |
Class at
Publication: |
435/006 ;
536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
1. (Cancelled)
2. (Cancelled)
3. An isolated nucleic acid molecule comprising a nucleotide
sequence encoding the amino acid sequence disclosed in SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.
4. (Cancelled)
5. An isolated polypeptide comprising the amino acid sequence of
SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID
NO:14.
6. An antibody that specifically recognizes a polypeptide
comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.
Description
[0001] The present application claims the benefit of U.S.
Provisional Application No. 60/165,260 which was filed on Nov. 12,
1999 and is herein incorporated by reference in its entirety.
INTRODUCTION
[0002] The present invention relates to the discovery,
identification, and characterization of novel human polynucleotides
encoding proteins that share sequence similarity with animal
proteases. 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
either lack or over express the disclosed genes, antagonists and
agonists of the proteins, and other compounds that modulate the
expression or activity of the proteins encoded by the disclosed
genes that can be used for diagnosis, drug screening, clinical
trial monitoring and the treatment of physiological disorders.
BACKGROUND OF THE INVENTION
[0003] Proteases are enzymes that cleave polypeptide sequences. In
particular, carboxypeptidases hydrolyze the peptide bonds at the
carboxy-terminal end of an amino acid chain, and have been
identified in a wide variety of cells and animals. Peptidases have
been implicated in a wide variety of cellular functions including,
but not limited to, digestion, coagulation, diabetes, prostate
cancer, gynecological disorders, neurological disorders, and
obesity. Accordingly, peptidases represent key targets/players for
the regulation of a variety of physiological processes and
pathways.
SUMMARY OF THE INVENTION
[0004] The present invention relates to the discovery,
identification, and characterization of nucleotides that encode
novel human 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
proteases, and especially carboxypeptidases. As such, the described
NHPs represent a new family of protease-related proteins with a
range of homologues and orthologs that transcend phyla and a broad
range of species.
[0005] The novel human nucleic acid sequences described herein,
encode proteins/open reading frames (ORFs) of 351, 314, 436, 399,
351, 314, and 69 amino acids in length (see SEQ ID NOS: 2, 4, 6, 8,
10, 12 and 14 respectively).
[0006] The invention also encompasses agonists and antagonists of
the described NHPs, including small molecules, large molecules,
mutant NHPs, or portions thereof, that compete with native NHP,
peptides, and antibodies, as well as nucleotide sequences that can
be used to inhibit the expression of the described NHPs (e.g.,
antisense and ribozyme molecules, 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 a functional
NHP.
[0007] Further, the present invention also relates to processes of
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.
DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES
[0008] The Sequence Listing provides the sequences of several
protease ORFs that encode the described NHP amino acid
sequences.
DETAILED DESCRIPTION OF THE INVENTION
[0009] The NHPs, described for the first time herein, are novel
proteins that are expressed in, inter alia, human cell lines, and
human prostate, testis, and placenta cells. The described sequences
were compiled from gene trapped cDNAs and clones isolated from a
human test is cDNA library. The present invention encompasses the
nucleotides presented in the Sequence Listing, host cells
expressing such nucleotides, the expression products of such
nucleotides, and: (a) nucleotides that encode mammalian homologs of
the described genes, including the specifically described NHPs, and
the NHP products; (b) nucleotides that encode one or more portions
of the NHPs that correspond to functional domains, and the
polypeptide products specified by such nucleotide sequences,
including but not limited to the novel regions of any active
domain(s); (c) isolated nucleotides that encode mutant versions,
engineered or naturally occurring, of the described NHPs in which
all or a part of at least one domain is deleted or altered, and the
polypeptide products specified by such nucleotide sequences,
including but not limited to soluble proteins and peptides in which
all or a portion of the signal sequence in deleted; (d) nucleotides
that encode chimeric fusion proteins containing all or a portion of
a coding region of an NHP, or one of its domains (e.g., a
receptor/ligand binding domain, accessory protein/self-association
domain, etc.) fused to another peptide or polypeptide; or (e)
therapeutic or diagnostic derivatives of the described
polynucleotides such as oligonucleotides, antisense
polynucleotides, ribozymes, dsRNA, or gene therapy constructs
comprising a sequence first disclosed in the Sequence Listing.
[0010] As discussed above, the present invention includes: (a) the
human DNA sequences presented in the Sequence Listing (and vectors
comprising the same) and additionally contemplates any nucleotide
sequence encoding a contiguous NHP open reading frame (ORF) that
hybridizes to a complement of a DNA sequence 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 sequence 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 still encode a functionally equivalent NHP product.
Functional equivalents of a NHP include naturally occurring NHPs
present in other species and mutant NHPs whether naturally
occurring or engineered (by site directed mutagenesis, gene
shuffling, directed evolution as described in, for example, U.S.
Pat. No. 5,837,458). The invention also includes degenerate nucleic
acid variants of the disclosed NHP polynucleotide sequences.
[0011] Additionally contemplated are polynucleotides encoding NHP
ORFs, or their functional equivalents, encoded by polynucleotide
sequences that are about 99, 95, 90, or about 85 percent identical
or similar to corresponding regions of SEQ ID NO:1 (as measured by
BLAST sequence comparison analysis using, for example, the GCG
sequence analysis package using default parameters).
[0012] The invention also includes nucleic acid molecules,
preferably DNA molecules, that hybridize to, and are therefore the
complements of, the described NHP gene nucleotide sequences. Such
hybridization conditions may be highly stringent or less highly
stringent, as described above. In instances where the nucleic acid
molecules are deoxyoligonucleotides ("DNA oligos"), such molecules
are generally about 16 to about 100 bases long, or about 20 to
about 80, or about 34 to about 45 bases long, or any variation or
combination of sizes represented therein that incorporate a
contiguous region of sequence first disclosed in the Sequence
Listing. Such oligonucleotides can be used in conjunction with the
polymerase chain reaction (PCR) to screen libraries, isolate
clones, and prepare cloning and sequencing templates, etc.
[0013] Alternatively, such NHP oligonucleotides can be used as
hybridization probes for screening libraries, and assessing gene
expression patterns (particularly using a micro array or
high-throughput "chip" format). Additionally, 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 each 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.
[0014] 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. Further, such sequences may be used as part of ribozyme
and/or triple helix sequences that are also useful for NHP gene
regulation.
[0015] Inhibitory antisense or double stranded oligonucleotides can
additionally comprise at least one modified base moiety which is
selected from the group including but not limited to
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluraci- l, 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-isopenten- yladenine,
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.
[0016] The antisense oligonucleotide can also comprise at least one
modified sugar moiety selected from the group including but not
limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0017] In yet another embodiment, the antisense oligonucleotide
will comprise 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.
[0018] 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). Alternatively, double stranded RNA can be
used to disrupt the expression and function of a targeted NHP.
[0019] Oligonucleotides of the invention can 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 can be synthesized by the method of Stein et al.
(1988, Nucl. Acids Res. 16:3209), and 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.
[0020] 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.
[0021] Alternatively, suitably labeled NHP nucleotide probes can 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 (including, but not limited to, nucleotide repeats,
microsatellite alleles, single nucleotide polymorphisms, or coding
single nucleotide 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.
[0022] Further, a NHP gene homolog can be isolated from nucleic
acid from an organism of interest by performing PCR using two
degenerate or "wobble" oligonucleotide primer pools designed on the
basis of amino acid sequences within the NHP products 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 known or
suspected to express an allele of a NHP gene.
[0023] The PCR product can be subcloned and sequenced to ensure
that the amplified sequences represent the sequence of the desired
NHP gene. The PCR fragment can then be used to isolate a full
length cDNA clone by a variety of methods. For example, the
amplified fragment can be labeled and used to screen a cDNA
library, such as a bacteriophage cDNA library. Alternatively, the
labeled fragment can be used to isolate genomic clones via the
screening of a genomic library.
[0024] PCR technology can also be used to isolate full length cDNA
sequences. For example, RNA can 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, testis tissue). A reverse transcription (RT) reaction can
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 can be isolated. For a review of
cloning strategies that can be used, see e.g., Sambrook et al.,
1989, supra.
[0025] A cDNA encoding a mutant NHP gene can be isolated, for
example, by using PCR. In this case, the first cDNA strand can 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 a corresponding normal NHP allele, the mutation(s)
responsible for the loss or alteration of function of the mutant
NHP gene product can be ascertained.
[0026] Alternatively, a genomic library can be constructed using
DNA obtained from an individual suspected of or known to carry a
mutant NHP allele (e.g., a person manifesting a NHP-associated
phenotype such as, for example, obesity, high blood pressure,
etc.), or a cDNA library can be constructed using RNA from a tissue
known, or suspected, to express a 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 mutant NHP gene sequences can
then be purified and subjected to sequence analysis according to
methods well known to those skilled in the art.
[0027] 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 a normal
NHP product, as described below. (For screening techniques, see,
for example, Harlow, E. and Lane, eds., 1988, "Antibodies: A
Laboratory Manual", Cold Spring Harbor Press, Cold Spring
Harbor.)
[0028] Additionally, screening can be accomplished using labeled
NHP fusion proteins, such as, for example, alkaline phosphatase-NHP
or NHP-alkaline phosphatase 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),
polyclonal antibodies to a NHP are likely to cross-react with a
corresponding 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 in
the art.
[0029] 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 (for example, baculo virus as described in U.S. Pat. No.
5,869,336 herein incorporated by reference); (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 human cytomegalovirus (hCMV) immediate early gene, regulatable,
viral elements (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.
[0030] The present 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 the
expression of a NHP (e.g., expression constructs in which NHP
coding sequences are operatively associated with expression control
elements such as promoters, promoter/enhancers, etc.).
[0031] The NHPs or NHP 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. The use of engineered host cells and/or animals may offer an
advantage in that such systems allow not only for the
identification of compounds that bind to an endogenous
receptor/ligand of a NHP, but can also identify compounds that
trigger NHP-mediated activity.
[0032] Finally, the NHP products can be used as therapeutics. For
example, soluble versions or derivatives of a NHP, or
peptides/domains corresponding a NHP, NHP fusion protein products
(especially NHP-Ig fusion proteins, i.e., fusions of a NHP, or a
domain of a NHP, to an IgFc), NHP antibodies and anti-idiotypic
antibodies (including Fab fragments), antagonists or agonists
(including compounds that modulate or act on downstream targets in
a NHP-mediated pathway) can be used to directly treat diseases or
disorders. For instance, the administration of an effective amount
of soluble NHP, or a NHP-IgFc fusion protein or an anti-idiotypic
antibody (or its Fab) that mimics the NHP could activate or
effectively antagonize NHP function. Nucleotide constructs encoding
such NHP products can be used to genetically engineer host cells to
express such 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.
[0033] Various aspects of the invention are described in greater
detail in the subsections below.
THE NHP SEQUENCES
[0034] The cDNA sequences and the corresponding deduced amino acid
sequences of the described NHPs are presented in the Sequence
Listing. The NHP genes were obtained from a human testis cDNA
library using probes and/or primers generated from human gene
trapped sequence tags. Expression analysis has provided evidence
that the described NHPs can be expressed, for example, in a variety
of human cell types and that the described NHPs share significant
similarity to a variety of proteases, and especially
carboxypeptidase A, and particularly A1 or A2, from, inter alia,
humans, mice, and rats. Several polymorphisms were identified
during this project including a T-to-C transition at, for example,
base number 1007 of SEQ ID NO:5 (changing a L to a S), a G-to-T
transversion at position 1014 of SEQ ID NO:5 (changing a E to a D),
and a translationally silent T-to-C transition at position 1,158 of
SEQ ID NO:5. SEQ ID NO: 15 describes a full length NHP ORF with
flanking 5' and 3' sequences.
NHPS AND NHP POLYPEPTIDES
[0035] 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. These uses include, but are not
limited to, the generation of antibodies, as reagents in diagnostic
assays, for 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.
Several uses and applications for plasma carboxypeptidases similar
to those described herein are described in U.S. Pat. No. 5,593,674,
the disclosure of which is herein incorporated by reference in its
entirety.
[0036] 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 a translation
initiation site and a hydrophobic signal-like sequence is present
near the N-terminal region of the protein. The sequence data
presented herein indicate that alternatively spliced forms of the
NHPs exist (which may or may not be tissue specific).
[0037] The NHP amino acid sequences of the invention include the
nucleotide and 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 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.
[0038] The invention also encompasses proteins that are
functionally equivalent to the NHPs encoded by the presently
described nucleotide sequences as judged by any of a number of
criteria, including, but not limited to, the ability to bind and
cleave a substrate of a NHP, or the ability to effect an identical
or complementary downstream pathway, or a change in cellular
metabolism (e.g., proteolytic activity, ion flux, tyrosine
phosphorylation, etc.). 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, but which result in a
silent change, thus producing a functionally equivalent gene
product. Amino acid substitutions may 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.
[0039] Also encompassed by the present invention are novel protein
constructs engineered in such a way that they facilitate transport
of the NHP to the target site, to the desired organ, across the
cell membrane and/or to the nucleus where the NHP can exert its
function activity. This goal may be achieved by coupling of the NHP
to a cytokine or other ligand that would direct the NHP to the
target organ and facilitate receptor mediated transport across the
membrane into the cytosol. Conjugation of NHPs to antibody
molecules or their Fab fragments could be used to target cells
bearing a particular epitope. Attaching the appropriate signal
sequence to the NHP would also transport the NHP to the desired
location within the cell. Alternatively targeting of NHP or its
nucleic acid sequence might be achieved using liposome based
delivery systems. Such technologies are described in U.S. Pat. Nos.
4,594,595, 5,459,127, 5,948,767 and 6,110,490 and their respective
disclosures which are herein incorporated by reference in their
entirety.
[0040] A variety of host-expression vector systems can be used to
express the NHP nucleotide sequences of the invention. The
presently described NHPs are similar to plasma carboxypeptidases
and are likely soluble proteins. Where the NHP peptide or
polypeptide to be expressed is a soluble NHP protein, or a NHP
peptide derived from a substantially nonhydrophobic domain of a
NHP, or a truncated or deleted NHP the peptide or polypeptide can
be recovered from the culture, i.e., from the host cell in cases
where the NHP peptide or polypeptide is not secreted, or from the
culture media in cases where the NHP peptide or polypeptide is
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.
Purification or enrichment of a 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 not only to retain the structural and functional
characteristics of the NHP, but to assess biological activity,
e.g., in drug screening assays.
[0041] The expression systems that can 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., Saccharomiyces,
Pichia) transformed with recombinant yeast expression vectors
containing NHP nucleotide sequences; insect cell systems infected
with recombinant virus expression vectors (e.g., baculovirus)
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).
[0042] In bacterial systems, a number of expression vectors can 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 generation of pharmaceutical
compositions of or containing 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.
[0043] 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).
[0044] 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. 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).
[0045] 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.
[0046] 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 which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines which express the 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.
[0047] 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).
[0048] 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.
ANTIBODIES TO NHP PRODUCTS
[0049] 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. Antibodies, and applications are uses thereof, similar to
those contemplated herein are described in U.S. Pat. No. 5,474,901
the disclosure of which is herein incorporated by reference in its
entirety.
[0050] The antibodies of the invention can 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, 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 may, therefore, be utilized as part of treatment
methods.
[0051] For the production of antibodies, various host animals may
be immunized by injection with the NHP, an NHP peptide (e.g., one
corresponding the 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 mutated variant of the NHP.
Such host animals may include but are not limited to pigs, 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 adjuvant (complete
and incomplete), mineral salts such as aluminum hydroxide or
aluminum phosphate, surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and Corynebacterium parvum. Alternatively, the
immune response could be enhanced by combination and or coupling
with molecules such as keyhole limpet hemocyanin, tetanus toxoid,
diptheria toxoid, ovalbumin, cholera toxin or fragments thereof.
Polyclonal antibodies are heterogeneous populations of antibody
molecules derived from the sera of the immunized animals.
[0052] 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.
[0053] 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. Such
technologies are described in U.S. Pat. Nos. 6,075,181 and
5,877,397 and their respective disclosures which are herein
incorporated by reference in their entirety.
[0054] 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.
[0055] 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.
[0056] 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/ligand can be used to generate anti-idiotypes that "mimic"
the NHP and, therefore, bind and activate or neutralize a receptor,
cofactor, ligand, or binding partner. Such anti-idiotypic
antibodies or Fab fragments of such anti-idiotypes can be used in
therapeutic regimens involving a NHP mediated pathway.
[0057] 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 are from the foregoing
description. Such modifications are intended to fall within the
scope of the appended claims. All cited publications, patents, and
patent applications are herein incorporated by reference in their
entirety.
Sequence CWU 1
1
15 1 1056 DNA homo sapiens 1 atgcagggca cccctggagg cgggacgcgc
cctgggccat cccccgtgga caggcggaca 60 ctcctggtct tcagctttat
cctggcagca gctttgggcc aaatgaattt cacaggggac 120 caggttcttc
gagtcctggc caaagatgag aagcagcttt cacttctcgg ggatctggag 180
ggcctgaaac cccagaaggt ggacttctgg cgtggcccag ccaggcccag cctccctgtg
240 gatatgagag ttcctttctc tgaactgaaa gacatcaaag cttatctgga
gtctcatgga 300 cttgcttaca gcatcatgat aaaggacatc caggtgctgc
tggatgagga aagacaggcc 360 atggcgaaat cccgccggct ggagcgcagc
accaacagct tcagttactc atcataccac 420 accctggagg agatatatag
ctggattgac aactttgtaa tggagcattc cgatattgtc 480 tcaaaaattc
agattggcaa cagctttgaa aaccagtcca ttcttgtcct gaagttcagc 540
actggaggtt ctcggcaccc agccatctgg atcgacactg gaattcactc ccgggagtgg
600 atcacccatg ccaccggcat ctggactgcc aataagattg tcagtgatta
tggcaaagac 660 cgtgtcctga cagacatact gaatgccatg gacatcttca
tagagctcgt cacaaaccct 720 gatgggtttg cttttaccca cagcatgaac
cgcttatggc ggaagaacaa gtccatcaga 780 cctggaatct tctgcatcgg
cgtggatctc aacaggaact ggaagtcggg ttttggagga 840 aatggttcta
acagcaaccc ctgctcagaa acttatcacg ggccctcccc tcagtcggag 900
ccggaggtgg ctgccatagt gaacttcatc acagcccatg gcaacttcaa ggctctgatc
960 tccatccaca gctactctca gatgcttatg tacccttacg gccgattgct
ggagcccgtt 1020 tcaaatcaga gggagttggt gagactggct gcttag 1056 2 351
PRT homo sapiens 2 Met Gln Gly Thr Pro Gly Gly Gly Thr Arg Pro Gly
Pro Ser Pro Val 1 5 10 15 Asp Arg Arg Thr Leu Leu Val Phe Ser Phe
Ile Leu Ala Ala Ala Leu 20 25 30 Gly Gln Met Asn Phe Thr Gly Asp
Gln Val Leu Arg Val Leu Ala Lys 35 40 45 Asp Glu Lys Gln Leu Ser
Leu Leu Gly Asp Leu Glu Gly Leu Lys Pro 50 55 60 Gln Lys Val Asp
Phe Trp Arg Gly Pro Ala Arg Pro Ser Leu Pro Val 65 70 75 80 Asp Met
Arg Val Pro Phe Ser Glu Leu Lys Asp Ile Lys Ala Tyr Leu 85 90 95
Glu Ser His Gly Leu Ala Tyr Ser Ile Met Ile Lys Asp Ile Gln Val 100
105 110 Leu Leu Asp Glu Glu Arg Gln Ala Met Ala Lys Ser Arg Arg Leu
Glu 115 120 125 Arg Ser Thr Asn Ser Phe Ser Tyr Ser Ser Tyr His Thr
Leu Glu Glu 130 135 140 Ile Tyr Ser Trp Ile Asp Asn Phe Val Met Glu
His Ser Asp Ile Val 145 150 155 160 Ser Lys Ile Gln Ile Gly Asn Ser
Phe Glu Asn Gln Ser Ile Leu Val 165 170 175 Leu Lys Phe Ser Thr Gly
Gly Ser Arg His Pro Ala Ile Trp Ile Asp 180 185 190 Thr Gly Ile His
Ser Arg Glu Trp Ile Thr His Ala Thr Gly Ile Trp 195 200 205 Thr Ala
Asn Lys Ile Val Ser Asp Tyr Gly Lys Asp Arg Val Leu Thr 210 215 220
Asp Ile Leu Asn Ala Met Asp Ile Phe Ile Glu Leu Val Thr Asn Pro 225
230 235 240 Asp Gly Phe Ala Phe Thr His Ser Met Asn Arg Leu Trp Arg
Lys Asn 245 250 255 Lys Ser Ile Arg Pro Gly Ile Phe Cys Ile Gly Val
Asp Leu Asn Arg 260 265 270 Asn Trp Lys Ser Gly Phe Gly Gly Asn Gly
Ser Asn Ser Asn Pro Cys 275 280 285 Ser Glu Thr Tyr His Gly Pro Ser
Pro Gln Ser Glu Pro Glu Val Ala 290 295 300 Ala Ile Val Asn Phe Ile
Thr Ala His Gly Asn Phe Lys Ala Leu Ile 305 310 315 320 Ser Ile His
Ser Tyr Ser Gln Met Leu Met Tyr Pro Tyr Gly Arg Leu 325 330 335 Leu
Glu Pro Val Ser Asn Gln Arg Glu Leu Val Arg Leu Ala Ala 340 345 350
3 945 DNA homo sapiens 3 atgcagggca cccctggagg cgggacgcgc
cctgggccat cccccgtgga caggcggaca 60 ctcctggtct tcagctttat
cctggcagca gctttgggcc aaatgaattt cacaggggac 120 caggttcttc
gagtcctggc caaagatgag aagcagcttt cacttctcgg ggatctggag 180
ggcctgaaac cccagaaggt ggacttctgg cgtggcccag ccaggcccag cctccctgtg
240 gatatgagag ttcctttctc tgaactgaaa gacatcaaag cttatctgga
gtctcatgga 300 cttgcttaca gcatcatgat aaaggacatc caggtgctgc
tggatgagga aagacaggcc 360 atggcgaaat cccgccggct ggagcgcagc
accaacagct tcagttactc atcataccac 420 accctggagg agatatatag
ctggattgac aactttgtaa tggagcattc cgatattgtc 480 tcaaaaattc
agattggcaa cagctttgaa aaccagtcca ttcttgtcct gaagttcagc 540
actggaggtt ctcggcaccc agccatctgg atcgacactg gaattcactc ccgggagtgg
600 atcacccatg ccaccggcat ctggactgcc aataagaacc gcttatggcg
gaagaacaag 660 tccatcagac ctggaatctt ctgcatcggc gtggatctca
acaggaactg gaagtcgggt 720 tttggaggaa atggttctaa cagcaacccc
tgctcagaaa cttatcacgg gccctcccct 780 cagtcggagc cggaggtggc
tgccatagtg aacttcatca cagcccatgg caacttcaag 840 gctctgatct
ccatccacag ctactctcag atgcttatgt acccttacgg ccgattgctg 900
gagcccgttt caaatcagag ggagttggtg agactggctg cttag 945 4 314 PRT
homo sapiens 4 Met Gln Gly Thr Pro Gly Gly Gly Thr Arg Pro Gly Pro
Ser Pro Val 1 5 10 15 Asp Arg Arg Thr Leu Leu Val Phe Ser Phe Ile
Leu Ala Ala Ala Leu 20 25 30 Gly Gln Met Asn Phe Thr Gly Asp Gln
Val Leu Arg Val Leu Ala Lys 35 40 45 Asp Glu Lys Gln Leu Ser Leu
Leu Gly Asp Leu Glu Gly Leu Lys Pro 50 55 60 Gln Lys Val Asp Phe
Trp Arg Gly Pro Ala Arg Pro Ser Leu Pro Val 65 70 75 80 Asp Met Arg
Val Pro Phe Ser Glu Leu Lys Asp Ile Lys Ala Tyr Leu 85 90 95 Glu
Ser His Gly Leu Ala Tyr Ser Ile Met Ile Lys Asp Ile Gln Val 100 105
110 Leu Leu Asp Glu Glu Arg Gln Ala Met Ala Lys Ser Arg Arg Leu Glu
115 120 125 Arg Ser Thr Asn Ser Phe Ser Tyr Ser Ser Tyr His Thr Leu
Glu Glu 130 135 140 Ile Tyr Ser Trp Ile Asp Asn Phe Val Met Glu His
Ser Asp Ile Val 145 150 155 160 Ser Lys Ile Gln Ile Gly Asn Ser Phe
Glu Asn Gln Ser Ile Leu Val 165 170 175 Leu Lys Phe Ser Thr Gly Gly
Ser Arg His Pro Ala Ile Trp Ile Asp 180 185 190 Thr Gly Ile His Ser
Arg Glu Trp Ile Thr His Ala Thr Gly Ile Trp 195 200 205 Thr Ala Asn
Lys Asn Arg Leu Trp Arg Lys Asn Lys Ser Ile Arg Pro 210 215 220 Gly
Ile Phe Cys Ile Gly Val Asp Leu Asn Arg Asn Trp Lys Ser Gly 225 230
235 240 Phe Gly Gly Asn Gly Ser Asn Ser Asn Pro Cys Ser Glu Thr Tyr
His 245 250 255 Gly Pro Ser Pro Gln Ser Glu Pro Glu Val Ala Ala Ile
Val Asn Phe 260 265 270 Ile Thr Ala His Gly Asn Phe Lys Ala Leu Ile
Ser Ile His Ser Tyr 275 280 285 Ser Gln Met Leu Met Tyr Pro Tyr Gly
Arg Leu Leu Glu Pro Val Ser 290 295 300 Asn Gln Arg Glu Leu Val Arg
Leu Ala Ala 305 310 5 1311 DNA homo sapiens 5 atgcagggca cccctggagg
cgggacgcgc cctgggccat cccccgtgga caggcggaca 60 ctcctggtct
tcagctttat cctggcagca gctttgggcc aaatgaattt cacaggggac 120
caggttcttc gagtcctggc caaagatgag aagcagcttt cacttctcgg ggatctggag
180 ggcctgaaac cccagaaggt ggacttctgg cgtggcccag ccaggcccag
cctccctgtg 240 gatatgagag ttcctttctc tgaactgaaa gacatcaaag
cttatctgga gtctcatgga 300 cttgcttaca gcatcatgat aaaggacatc
caggtgctgc tggatgagga aagacaggcc 360 atggcgaaat cccgccggct
ggagcgcagc accaacagct tcagttactc atcataccac 420 accctggagg
agatatatag ctggattgac aactttgtaa tggagcattc cgatattgtc 480
tcaaaaattc agattggcaa cagctttgaa aaccagtcca ttcttgtcct gaagttcagc
540 actggaggtt ctcggcaccc agccatctgg atcgacactg gaattcactc
ccgggagtgg 600 atcacccatg ccaccggcat ctggactgcc aataagattg
tcagtgatta tggcaaagac 660 cgtgtcctga cagacatact gaatgccatg
gacatcttca tagagctcgt cacaaaccct 720 gatgggtttg cttttaccca
cagcatgaac cgcttatggc ggaagaacaa gtccatcaga 780 cctggaatct
tctgcatcgg cgtggatctc aacaggaact ggaagtcggg ttttggagga 840
aatggttcta acagcaaccc ctgctcagaa acttatcacg ggccctcccc tcagtcggag
900 ccggaggtgg ctgccatagt gaacttcatc acagcccatg gcaacttcaa
ggctctgatc 960 tccatccaca gctactctca gatgcttatg tacccttacg
gccgattgct ggagcccgtt 1020 tcaaatcaga gggagttgta cgatcttgcc
aaggatgcgg tggaggcctt gtataaggtc 1080 catgggatcg agtacatttt
tggcagcatc agcaccaccc tctatgtggc cagtgggatc 1140 accgtcgact
gggcctatga cagtggcatc aagtacgcct tcagctttga gctccgggac 1200
actgggcagt atggcttcct gctgccggcc acacagatca tccccacggc ccaggagacg
1260 tggatggcgc ttcggaccat catggagcac accctgaatc acccctacta g 1311
6 436 PRT homo sapiens 6 Met Gln Gly Thr Pro Gly Gly Gly Thr Arg
Pro Gly Pro Ser Pro Val 1 5 10 15 Asp Arg Arg Thr Leu Leu Val Phe
Ser Phe Ile Leu Ala Ala Ala Leu 20 25 30 Gly Gln Met Asn Phe Thr
Gly Asp Gln Val Leu Arg Val Leu Ala Lys 35 40 45 Asp Glu Lys Gln
Leu Ser Leu Leu Gly Asp Leu Glu Gly Leu Lys Pro 50 55 60 Gln Lys
Val Asp Phe Trp Arg Gly Pro Ala Arg Pro Ser Leu Pro Val 65 70 75 80
Asp Met Arg Val Pro Phe Ser Glu Leu Lys Asp Ile Lys Ala Tyr Leu 85
90 95 Glu Ser His Gly Leu Ala Tyr Ser Ile Met Ile Lys Asp Ile Gln
Val 100 105 110 Leu Leu Asp Glu Glu Arg Gln Ala Met Ala Lys Ser Arg
Arg Leu Glu 115 120 125 Arg Ser Thr Asn Ser Phe Ser Tyr Ser Ser Tyr
His Thr Leu Glu Glu 130 135 140 Ile Tyr Ser Trp Ile Asp Asn Phe Val
Met Glu His Ser Asp Ile Val 145 150 155 160 Ser Lys Ile Gln Ile Gly
Asn Ser Phe Glu Asn Gln Ser Ile Leu Val 165 170 175 Leu Lys Phe Ser
Thr Gly Gly Ser Arg His Pro Ala Ile Trp Ile Asp 180 185 190 Thr Gly
Ile His Ser Arg Glu Trp Ile Thr His Ala Thr Gly Ile Trp 195 200 205
Thr Ala Asn Lys Ile Val Ser Asp Tyr Gly Lys Asp Arg Val Leu Thr 210
215 220 Asp Ile Leu Asn Ala Met Asp Ile Phe Ile Glu Leu Val Thr Asn
Pro 225 230 235 240 Asp Gly Phe Ala Phe Thr His Ser Met Asn Arg Leu
Trp Arg Lys Asn 245 250 255 Lys Ser Ile Arg Pro Gly Ile Phe Cys Ile
Gly Val Asp Leu Asn Arg 260 265 270 Asn Trp Lys Ser Gly Phe Gly Gly
Asn Gly Ser Asn Ser Asn Pro Cys 275 280 285 Ser Glu Thr Tyr His Gly
Pro Ser Pro Gln Ser Glu Pro Glu Val Ala 290 295 300 Ala Ile Val Asn
Phe Ile Thr Ala His Gly Asn Phe Lys Ala Leu Ile 305 310 315 320 Ser
Ile His Ser Tyr Ser Gln Met Leu Met Tyr Pro Tyr Gly Arg Leu 325 330
335 Leu Glu Pro Val Ser Asn Gln Arg Glu Leu Tyr Asp Leu Ala Lys Asp
340 345 350 Ala Val Glu Ala Leu Tyr Lys Val His Gly Ile Glu Tyr Ile
Phe Gly 355 360 365 Ser Ile Ser Thr Thr Leu Tyr Val Ala Ser Gly Ile
Thr Val Asp Trp 370 375 380 Ala Tyr Asp Ser Gly Ile Lys Tyr Ala Phe
Ser Phe Glu Leu Arg Asp 385 390 395 400 Thr Gly Gln Tyr Gly Phe Leu
Leu Pro Ala Thr Gln Ile Ile Pro Thr 405 410 415 Ala Gln Glu Thr Trp
Met Ala Leu Arg Thr Ile Met Glu His Thr Leu 420 425 430 Asn His Pro
Tyr 435 7 1200 DNA homo sapiens 7 atgcagggca cccctggagg cgggacgcgc
cctgggccat cccccgtgga caggcggaca 60 ctcctggtct tcagctttat
cctggcagca gctttgggcc aaatgaattt cacaggggac 120 caggttcttc
gagtcctggc caaagatgag aagcagcttt cacttctcgg ggatctggag 180
ggcctgaaac cccagaaggt ggacttctgg cgtggcccag ccaggcccag cctccctgtg
240 gatatgagag ttcctttctc tgaactgaaa gacatcaaag cttatctgga
gtctcatgga 300 cttgcttaca gcatcatgat aaaggacatc caggtgctgc
tggatgagga aagacaggcc 360 atggcgaaat cccgccggct ggagcgcagc
accaacagct tcagttactc atcataccac 420 accctggagg agatatatag
ctggattgac aactttgtaa tggagcattc cgatattgtc 480 tcaaaaattc
agattggcaa cagctttgaa aaccagtcca ttcttgtcct gaagttcagc 540
actggaggtt ctcggcaccc agccatctgg atcgacactg gaattcactc ccgggagtgg
600 atcacccatg ccaccggcat ctggactgcc aataagaacc gcttatggcg
gaagaacaag 660 tccatcagac ctggaatctt ctgcatcggc gtggatctca
acaggaactg gaagtcgggt 720 tttggaggaa atggttctaa cagcaacccc
tgctcagaaa cttatcacgg gccctcccct 780 cagtcggagc cggaggtggc
tgccatagtg aacttcatca cagcccatgg caacttcaag 840 gctctgatct
ccatccacag ctactctcag atgcttatgt acccttacgg ccgattgctg 900
gagcccgttt caaatcagag ggagttgtac gatcttgcca aggatgcggt ggaggccttg
960 tataaggtcc atgggatcga gtacattttt ggcagcatca gcaccaccct
ctatgtggcc 1020 agtgggatca ccgtcgactg ggcctatgac agtggcatca
agtacgcctt cagctttgag 1080 ctccgggaca ctgggcagta tggcttcctg
ctgccggcca cacagatcat ccccacggcc 1140 caggagacgt ggatggcgct
tcggaccatc atggagcaca ccctgaatca cccctactag 1200 8 399 PRT homo
sapiens 8 Met Gln Gly Thr Pro Gly Gly Gly Thr Arg Pro Gly Pro Ser
Pro Val 1 5 10 15 Asp Arg Arg Thr Leu Leu Val Phe Ser Phe Ile Leu
Ala Ala Ala Leu 20 25 30 Gly Gln Met Asn Phe Thr Gly Asp Gln Val
Leu Arg Val Leu Ala Lys 35 40 45 Asp Glu Lys Gln Leu Ser Leu Leu
Gly Asp Leu Glu Gly Leu Lys Pro 50 55 60 Gln Lys Val Asp Phe Trp
Arg Gly Pro Ala Arg Pro Ser Leu Pro Val 65 70 75 80 Asp Met Arg Val
Pro Phe Ser Glu Leu Lys Asp Ile Lys Ala Tyr Leu 85 90 95 Glu Ser
His Gly Leu Ala Tyr Ser Ile Met Ile Lys Asp Ile Gln Val 100 105 110
Leu Leu Asp Glu Glu Arg Gln Ala Met Ala Lys Ser Arg Arg Leu Glu 115
120 125 Arg Ser Thr Asn Ser Phe Ser Tyr Ser Ser Tyr His Thr Leu Glu
Glu 130 135 140 Ile Tyr Ser Trp Ile Asp Asn Phe Val Met Glu His Ser
Asp Ile Val 145 150 155 160 Ser Lys Ile Gln Ile Gly Asn Ser Phe Glu
Asn Gln Ser Ile Leu Val 165 170 175 Leu Lys Phe Ser Thr Gly Gly Ser
Arg His Pro Ala Ile Trp Ile Asp 180 185 190 Thr Gly Ile His Ser Arg
Glu Trp Ile Thr His Ala Thr Gly Ile Trp 195 200 205 Thr Ala Asn Lys
Asn Arg Leu Trp Arg Lys Asn Lys Ser Ile Arg Pro 210 215 220 Gly Ile
Phe Cys Ile Gly Val Asp Leu Asn Arg Asn Trp Lys Ser Gly 225 230 235
240 Phe Gly Gly Asn Gly Ser Asn Ser Asn Pro Cys Ser Glu Thr Tyr His
245 250 255 Gly Pro Ser Pro Gln Ser Glu Pro Glu Val Ala Ala Ile Val
Asn Phe 260 265 270 Ile Thr Ala His Gly Asn Phe Lys Ala Leu Ile Ser
Ile His Ser Tyr 275 280 285 Ser Gln Met Leu Met Tyr Pro Tyr Gly Arg
Leu Leu Glu Pro Val Ser 290 295 300 Asn Gln Arg Glu Leu Tyr Asp Leu
Ala Lys Asp Ala Val Glu Ala Leu 305 310 315 320 Tyr Lys Val His Gly
Ile Glu Tyr Ile Phe Gly Ser Ile Ser Thr Thr 325 330 335 Leu Tyr Val
Ala Ser Gly Ile Thr Val Asp Trp Ala Tyr Asp Ser Gly 340 345 350 Ile
Lys Tyr Ala Phe Ser Phe Glu Leu Arg Asp Thr Gly Gln Tyr Gly 355 360
365 Phe Leu Leu Pro Ala Thr Gln Ile Ile Pro Thr Ala Gln Glu Thr Trp
370 375 380 Met Ala Leu Arg Thr Ile Met Glu His Thr Leu Asn His Pro
Tyr 385 390 395 9 1056 DNA homo sapiens 9 atgcagggca cccctggagg
cgggacgcgc cctgggccat cccccgtgga caggcggaca 60 ctcctggtct
tcagctttat cctggcagca gctttgggcc aaatgaattt cacaggggac 120
caggttcttc gagtcctggc caaagatgag aagcagcttt cacttctcgg ggatctggag
180 ggcctgaaac cccagaaggt ggacttctgg cgtggcccag ccaggcccag
cctccctgtg 240 gatatgagag ttcctttctc tgaactgaaa gacatcaaag
cttatctgga gtctcatgga 300 cttgcttaca gcatcatgat aaaggacatc
caggtgctgc tggatgagga aagacaggcc 360 atggcgaaat cccgccggct
ggagcgcagc accaacagct tcagttactc atcataccac 420 accctggagg
agatatatag ctggattgac aactttgtaa tggagcattc cgatattgtc 480
tcaaaaattc agattggcaa cagctttgaa aaccagtcca ttcttgtcct gaagttcagc
540 actggaggtt ctcggcaccc agccatctgg atcgacactg gaattcactc
ccgggagtgg 600 atcacccatg ccaccggcat ctggactgcc aataagattg
tcagtgatta tggcaaagac 660 cgtgtcctga cagacatact gaatgccatg
gacatcttca tagagctcgt cacaaaccct 720 gatgggtttg cttttaccca
cagcatgaac cgcttatggc ggaagaacaa gtccatcaga 780 cctggaatct
tctgcatcgg cgtggatctc aacaggaact ggaagtcggg ttttggagga 840
aatggttcta acagcaaccc ctgctcagaa acttatcacg ggccctcccc tcagtcggag
900 ccggaggtgg ctgccatagt gaacttcatc acagcccatg gcaacttcaa
ggctctgatc 960 tccatccaca gctactctca gatgcttatg tacccttacg
gccgattgct ggagcccgtt 1020 tcaaatcaga gggagttggt gagactggct gcttag
1056 10 351 PRT homo
sapiens 10 Met Gln Gly Thr Pro Gly Gly Gly Thr Arg Pro Gly Pro Ser
Pro Val 1 5 10 15 Asp Arg Arg Thr Leu Leu Val Phe Ser Phe Ile Leu
Ala Ala Ala Leu 20 25 30 Gly Gln Met Asn Phe Thr Gly Asp Gln Val
Leu Arg Val Leu Ala Lys 35 40 45 Asp Glu Lys Gln Leu Ser Leu Leu
Gly Asp Leu Glu Gly Leu Lys Pro 50 55 60 Gln Lys Val Asp Phe Trp
Arg Gly Pro Ala Arg Pro Ser Leu Pro Val 65 70 75 80 Asp Met Arg Val
Pro Phe Ser Glu Leu Lys Asp Ile Lys Ala Tyr Leu 85 90 95 Glu Ser
His Gly Leu Ala Tyr Ser Ile Met Ile Lys Asp Ile Gln Val 100 105 110
Leu Leu Asp Glu Glu Arg Gln Ala Met Ala Lys Ser Arg Arg Leu Glu 115
120 125 Arg Ser Thr Asn Ser Phe Ser Tyr Ser Ser Tyr His Thr Leu Glu
Glu 130 135 140 Ile Tyr Ser Trp Ile Asp Asn Phe Val Met Glu His Ser
Asp Ile Val 145 150 155 160 Ser Lys Ile Gln Ile Gly Asn Ser Phe Glu
Asn Gln Ser Ile Leu Val 165 170 175 Leu Lys Phe Ser Thr Gly Gly Ser
Arg His Pro Ala Ile Trp Ile Asp 180 185 190 Thr Gly Ile His Ser Arg
Glu Trp Ile Thr His Ala Thr Gly Ile Trp 195 200 205 Thr Ala Asn Lys
Ile Val Ser Asp Tyr Gly Lys Asp Arg Val Leu Thr 210 215 220 Asp Ile
Leu Asn Ala Met Asp Ile Phe Ile Glu Leu Val Thr Asn Pro 225 230 235
240 Asp Gly Phe Ala Phe Thr His Ser Met Asn Arg Leu Trp Arg Lys Asn
245 250 255 Lys Ser Ile Arg Pro Gly Ile Phe Cys Ile Gly Val Asp Leu
Asn Arg 260 265 270 Asn Trp Lys Ser Gly Phe Gly Gly Asn Gly Ser Asn
Ser Asn Pro Cys 275 280 285 Ser Glu Thr Tyr His Gly Pro Ser Pro Gln
Ser Glu Pro Glu Val Ala 290 295 300 Ala Ile Val Asn Phe Ile Thr Ala
His Gly Asn Phe Lys Ala Leu Ile 305 310 315 320 Ser Ile His Ser Tyr
Ser Gln Met Leu Met Tyr Pro Tyr Gly Arg Leu 325 330 335 Leu Glu Pro
Val Ser Asn Gln Arg Glu Leu Val Arg Leu Ala Ala 340 345 350 11 945
DNA homo sapiens 11 atgcagggca cccctggagg cgggacgcgc cctgggccat
cccccgtgga caggcggaca 60 ctcctggtct tcagctttat cctggcagca
gctttgggcc aaatgaattt cacaggggac 120 caggttcttc gagtcctggc
caaagatgag aagcagcttt cacttctcgg ggatctggag 180 ggcctgaaac
cccagaaggt ggacttctgg cgtggcccag ccaggcccag cctccctgtg 240
gatatgagag ttcctttctc tgaactgaaa gacatcaaag cttatctgga gtctcatgga
300 cttgcttaca gcatcatgat aaaggacatc caggtgctgc tggatgagga
aagacaggcc 360 atggcgaaat cccgccggct ggagcgcagc accaacagct
tcagttactc atcataccac 420 accctggagg agatatatag ctggattgac
aactttgtaa tggagcattc cgatattgtc 480 tcaaaaattc agattggcaa
cagctttgaa aaccagtcca ttcttgtcct gaagttcagc 540 actggaggtt
ctcggcaccc agccatctgg atcgacactg gaattcactc ccgggagtgg 600
atcacccatg ccaccggcat ctggactgcc aataagaacc gcttatggcg gaagaacaag
660 tccatcagac ctggaatctt ctgcatcggc gtggatctca acaggaactg
gaagtcgggt 720 tttggaggaa atggttctaa cagcaacccc tgctcagaaa
cttatcacgg gccctcccct 780 cagtcggagc cggaggtggc tgccatagtg
aacttcatca cagcccatgg caacttcaag 840 gctctgatct ccatccacag
ctactctcag atgcttatgt acccttacgg ccgattgctg 900 gagcccgttt
caaatcagag ggagttggtg agactggctg cttag 945 12 314 PRT homo sapiens
12 Met Gln Gly Thr Pro Gly Gly Gly Thr Arg Pro Gly Pro Ser Pro Val
1 5 10 15 Asp Arg Arg Thr Leu Leu Val Phe Ser Phe Ile Leu Ala Ala
Ala Leu 20 25 30 Gly Gln Met Asn Phe Thr Gly Asp Gln Val Leu Arg
Val Leu Ala Lys 35 40 45 Asp Glu Lys Gln Leu Ser Leu Leu Gly Asp
Leu Glu Gly Leu Lys Pro 50 55 60 Gln Lys Val Asp Phe Trp Arg Gly
Pro Ala Arg Pro Ser Leu Pro Val 65 70 75 80 Asp Met Arg Val Pro Phe
Ser Glu Leu Lys Asp Ile Lys Ala Tyr Leu 85 90 95 Glu Ser His Gly
Leu Ala Tyr Ser Ile Met Ile Lys Asp Ile Gln Val 100 105 110 Leu Leu
Asp Glu Glu Arg Gln Ala Met Ala Lys Ser Arg Arg Leu Glu 115 120 125
Arg Ser Thr Asn Ser Phe Ser Tyr Ser Ser Tyr His Thr Leu Glu Glu 130
135 140 Ile Tyr Ser Trp Ile Asp Asn Phe Val Met Glu His Ser Asp Ile
Val 145 150 155 160 Ser Lys Ile Gln Ile Gly Asn Ser Phe Glu Asn Gln
Ser Ile Leu Val 165 170 175 Leu Lys Phe Ser Thr Gly Gly Ser Arg His
Pro Ala Ile Trp Ile Asp 180 185 190 Thr Gly Ile His Ser Arg Glu Trp
Ile Thr His Ala Thr Gly Ile Trp 195 200 205 Thr Ala Asn Lys Asn Arg
Leu Trp Arg Lys Asn Lys Ser Ile Arg Pro 210 215 220 Gly Ile Phe Cys
Ile Gly Val Asp Leu Asn Arg Asn Trp Lys Ser Gly 225 230 235 240 Phe
Gly Gly Asn Gly Ser Asn Ser Asn Pro Cys Ser Glu Thr Tyr His 245 250
255 Gly Pro Ser Pro Gln Ser Glu Pro Glu Val Ala Ala Ile Val Asn Phe
260 265 270 Ile Thr Ala His Gly Asn Phe Lys Ala Leu Ile Ser Ile His
Ser Tyr 275 280 285 Ser Gln Met Leu Met Tyr Pro Tyr Gly Arg Leu Leu
Glu Pro Val Ser 290 295 300 Asn Gln Arg Glu Leu Val Arg Leu Ala Ala
305 310 13 210 DNA homo sapiens 13 atgatgtttt tgaacaagaa gacrccccat
gggtgctgtg ctgtcctgag gcctgggcca 60 tggtgcccaa ggaaagcccc
tgaagctcac caggaggaag aagcatgcag ggcacccctg 120 gaggcgggac
gcgccctggg ccatcccccg tggacaggcg gacactcctg gtcttcagct 180
ttatcctggc agcagctttg ggccaaatga 210 14 69 PRT homo sapiens 14 Met
Met Phe Leu Asn Lys Lys Thr Pro His Gly Cys Cys Ala Val Leu 1 5 10
15 Arg Pro Gly Pro Trp Cys Pro Arg Lys Ala Pro Glu Ala His Gln Glu
20 25 30 Glu Glu Ala Cys Arg Ala Pro Leu Glu Ala Gly Arg Ala Leu
Gly His 35 40 45 Pro Pro Trp Thr Gly Gly His Ser Trp Ser Ser Ala
Leu Ser Trp Gln 50 55 60 Gln Leu Trp Ala Lys 65 15 2247 DNA homo
sapiens 15 ctctctctct cttttactct tactctttct ctctcactct ctctcttttc
ccacccttaa 60 gccaagtaca gggatagttg tctcatcatt ggtggcttaa
aatgatgttt ttgaacaaga 120 agacrcccca tgggtacttt tggtgactag
cactatctct gtktttttcc ttttaaattc 180 ctgagctatt gtttagcagt
acaccctttt atctccattg ctactgaagc tgaatgttac 240 ttgggtggaa
agcataactg ctttcttttc tatgtcctta aaccctttga taatgttact 300
gtttgagagt ccctgaagcc aggatactag aagagtctgg cttgtctgaa cagctgaact
360 acgaaataat ggagtagggc aggctttacc aagccaattc actcaagttg
tctcatctat 420 accccttcaa accctgtgag ctgtgactaa aagctgggct
ttccagcctc taggtgctgt 480 gctgtcctga ggcctgggcc atggtgccca
aggaaagccc ctgaagctca ccaggaggaa 540 gaagcatgca gggcacccct
ggaggcggga cgcgccctgg gccatccccc gtggacaggc 600 ggacactcct
ggtcttcagc tttatcctgg cagcagcttt gggccaaatg aatttcacag 660
gggaccaggt tcttcgagtc ctggccaaag atgagaagca gctttcactt ctcggggatc
720 tggagggcct gaaaccccag aaggtggact tctggcgtgg cccagccagg
cccagcctcc 780 ctgtggatat gagagttcct ttctctgaac tgaaagacat
caaagcttat ctggagtctc 840 atggacttgc ttacagcatc atgataaagg
acatccaggt gctgctggat gaggaaagac 900 aggccatggc gaaatcccgc
cggctggagc gcagcaccaa cagcttcagt tactcatcat 960 accacaccct
ggaggagata tatagctgga ttgacaactt tgtaatggag cattccgata 1020
ttgtctcaaa aattcagatt ggcaacagct ttgaaaacca gtccattctt gtcctgaagt
1080 tcagcactgg aggttctcgg cacccagcca tctggatcga cactggaatt
cactcccggg 1140 agtggatcac ccatgccacc ggcatctgga ctgccaataa
gattgtcagt gattatggca 1200 aagaccgtgt cctgacagac atactgaatg
ccatggacat cttcatagag ctcgtcacaa 1260 accctgatgg gtttgctttt
acccacagca tgaaccgctt atggcggaag aacaagtcca 1320 tcagacctgg
aatcttctgc atcggcgtgg atctcaacag gaactggaag tcgggttttg 1380
gaggaaatgg ttctaacagc aacccctgct cagaaactta tcacgggccc tcccctcagt
1440 cggagccgga ggtggctgcc atagtgaact tcatcacagc ccatggcaac
ttcaaggctc 1500 tgatctccat ccacagctac tctcagatgc ttatgtaccc
ttacggccga ttgctggagc 1560 ccgtttcaaa tcagagggag ttggtgagac
tggctgctta gggcctgggg agaagagacc 1620 gcttcacaga aaaatccata
tctgtcatac tcccagaggg ctcaggttgt tactctgaat 1680 gcaggggtct
gggctgattg accccatggt gcggggggtg gggtaggggg agcttgctgt 1740
tctcacgtgt gatcaagttc aaagctggaa atgctgtgct ccttctcaca agggccatct
1800 cacttcaact tcaggactgc taaatcatgc ttacgatctt gccaaggatg
cggtggaggc 1860 cttgtataag gtccatggga tcgagtacat ttttggcagc
atcagcacca ccctctatgt 1920 ggccagtggg atcaccgtcg actgggccta
tgacagtggc atcaagtacg ccttcagctt 1980 tgagctccgg gacactgggc
agtatggctt cctgctgccg gccacacaga tcatccccac 2040 ggcccaggag
acgtggatgg cgcttcggac catcatggag cacaccctga atcaccccta 2100
ctagcagcac gactgagggc aggaggctcc atccttctcc ccaaggtctg tggctcctcc
2160 cgaaacccaa gttatgcatc cccatcccca tgccctcatc ccgacctctt
agaaaataaa 2220 tacaagtttg aaaaaaaaaa aaaaaaa 2247
* * * * *