U.S. patent application number 10/813588 was filed with the patent office on 2005-03-10 for novel human proteins and polynucleotides encoding the same.
Invention is credited to Donoho, Gregory, Friedrich, Glenn, Nehls, Michael C., Sands, Arthur T., Turner, C. Alexander JR., Zambrowicz, Brian.
Application Number | 20050053969 10/813588 |
Document ID | / |
Family ID | 32329762 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050053969 |
Kind Code |
A1 |
Donoho, Gregory ; et
al. |
March 10, 2005 |
Novel human proteins 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) ; 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: |
32329762 |
Appl. No.: |
10/813588 |
Filed: |
March 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10813588 |
Mar 30, 2004 |
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09691344 |
Oct 18, 2000 |
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6743907 |
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60160285 |
Oct 19, 1999 |
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60183583 |
Feb 18, 2000 |
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Current U.S.
Class: |
435/6.16 ;
536/23.5 |
Current CPC
Class: |
C07K 14/47 20130101 |
Class at
Publication: |
435/006 ;
536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
1-5. (cancelled)
6. An isolated nucleic acid molecule comprising a nucleotide
sequence encoding an amino acid sequence drawn from the group
consisting of SEQ ID NOS: 2, 4, and 6.
7. The isolated nucleic acid molecule of claim 6, wherein said
nucleotide sequence is that of SEQ ID NO: 1.
8. The isolated nucleic acid molecule of claim 6, wherein said
nucleotide sequence is that of SEQ ID NO: 3.
9. The isolated nucleic acid molecule of claim 6, wherein said
nucleotide sequence is that of SEQ ID NO: 5.
10. An expression vector comprising a nucleotide sequence encoding
an amino acid sequence drawn from the group consisting of SEQ ID
NOS: 2, 4, and 6.
11. The expression vector of claim 10, wherein said nucleotide
sequence is that of SEQ ID NO:1.
12. The expression vector of claim 10, wherein said nucleotide
sequence is that of SEQ IID NO:3.
13. The expression vector of claim 10, wherein said nucleotide
sequence is that of SEQ ID NO:5.
14. A host cell comprising the expression vector of claim 10.
15. The host cell of claim 14 wherein the expression vector
comprises said nucleotide sequence of SEQ ID NO: 1.
16. The host cell of claim 14 wherein the expression vector
comprises said nucleotide sequence of SEQ ID NO:3.
17. The host cell of claim 14 wherein the expression vector
comprises said nucleotide sequence of SEQ ID NO:5.
18. An isolated polypeptide comprising an amino acid sequence drawn
from the group consisting of SEQ ID NOS: 2, 4, and 6.
19. The polypeptide of claim 18 wherein said amino acid sequence is
that of SEQ ID NO:2.
20. The polypeptide of claim 18 wherein said amino acid sequence is
that of SEQ ID NO:4.
21. The polypeptide of claim 18 wherein said amino acid sequence is
that of SEQ ID NO:6.
Description
[0001] The present application is a continuation of co-pending U.S.
application Ser. No. 09/691,344 filed on October 18, 2000 that
invention claims the benefit of U.S. Provisional Application Nos.
60/160,285 and 60/183,583 which were filed Oct. 19, 1999 and Feb.
18, 2000 respectively and are herein incorporated in their
entirety.
1. INTRODUCTION
[0002] The present invention relates to the discovery,
identification, and characterization of novel human polynucleotides
encoding proteins sharing sequence similarity with mammalian
proteins having CUB domains. 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
sequences, 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 disorders, or diseases.
2. BACKGROUND OF THE INVENTION
[0003] The CUB domain is an extracellular domain (ECD) present in
variety of diverse proteins such as bone morphogenetic protein 1,
proteinases, spermadhesins, complement subcomponents, and neuronal
recognition molecules. Given the importance of these functions, CUB
proteins have been associated with, inter alia, regulating
development, modulating cellular processes, and preventing
infectious disease.
3. 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 CUB
domain proteins, coagulation factors V and XIII, milk fat
globule-EGF factor 8, transcriptional repressor AE-binding
protein-1, and neuropilins 1 and 2 (which, like the presently
described protein, contain both CUB and discoidin domains).
[0005] The novel human nucleic acid (cDNA) sequences described
herein, encode proteins/open reading frames (ORFs) of 487, 586, and
539 amino acids in length (see SEQ ID NOS: 2, 4, and 6
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 NHPs, 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 NHPs (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.
Several knockout ES cell lines have been produced that contain a
gene trap mutation in a murine ortholog/homolog of the disclosed
NHPs.
[0007] Further, the present invention also relates to processes for
identifying compounds that modulate, i.e., act as agonists or
antagonists, of NHP expression and/or NHP activity that utilize
purified preparations of the described NHPs and/or NHP products, 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
[0008] The Sequence Listing provides the sequences of several NHP
ORFs encoding the described NHP amino acid sequences. SEQ ID NO:7
describes a NHP ORF and flanking sequences.
5. 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, pituitary, fetal brain, brain, thymus, spleen,
lymph node, trachea, kidney, fetal liver, thyroid, adrenal gland,
salivary gland, stomach, small intestine, colon, muscle, heart,
mammary gland, adipose, skin, esophagus, bladder, cervix, rectum,
and testis cells.
[0010] The described sequences were compiled from gene trapped
cDNAs, genomic sequence, and clones isolated from human brain,
adipose, testis, and placenta cDNA libraries (Edge Biosystems,
Gaithersburg, Md., and Clontech, Palo Alto, Calif.). 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 NHP products; (b) nucleotides that
encode one or more portions of a NHP 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 is
deleted; (d) nucleotides that encode chimeric fusion proteins
containing all or a portion of a coding region of a NHP, or one of
its domains (e.g., a receptor or 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.
[0011] 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), or a
contiguous exon splice junction first described in the Sequence
Listing, 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.
[0012] Additionally contemplated are polynucleotides encoding a NHP
ORF, or its functional equivalent, encoded by a polynucleotide
sequence that is about 99, 95, 90, or about 85 percent similar or
identical to corresponding regions of the nucleotide sequences of
the Sequence Listing (as measured by BLAST sequence comparison
analysis using, for example, the GCG sequence analysis package
using standard default settings).
[0013] 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.
[0014] 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 a 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.
[0015] 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.
[0016] 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-N-6-isopente- nyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0017] 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.
[0018] 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.
[0019] 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'-O-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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] Further, a NHP 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 human or
non-human cell lines or tissue known or suspected to express an
allele of a NHP gene. 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 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 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,
connective tissue disorders, infertility, 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 can
be expressed and screened using standard antibody screening
techniques in conjunction with antibodies raised against 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 by screening
with 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 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 a 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 NHPs or NHP
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 the endogenous receptor
for a NHP, but can also identify compounds that trigger
NHP-mediated activities or pathways.
[0032] Finally, the NHP products can be used as therapeutics. For
example, soluble derivatives such as NHP peptides/domains
corresponding to 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 a NHP could activate or
effectively antagonize the endogenous NHP receptor. 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 NHP, 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.
5.1 The NHP Sequences
[0034] The cDNA sequences and corresponding deduced amino acid
sequences of the described NHPs are presented in the Sequence
Listing. SEQ ID NO:7 describes a NHP ORF as well as flanking
regions. The NHP nucleotides were obtained from human cDNA
libraries using probes and/or primers generated from human gene
trapped sequence tags, and genomic sequence. Expression analysis
has provided evidence that the described NHP can be expressed a
variety of human cells as well as gene trapped human cells.
5.2 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.
[0036] The Sequence Listing discloses the amino acid sequences
encoded by the described NHP polynucleotides. The NHPs display an
initiator methionines in DNA sequence contexts consistent with a
translation initiation site, and several of the ORFs display a
consensus signal sequence which can indicate that the described NHP
ORFs are secreted proteins, or can be membrane associated.
[0037] 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 NHPs encoded by a NHP nucleotide sequence 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 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.
[0039] A variety of host-expression vector systems can be used to
express the NHP nucleotide sequences of the invention. Where, as in
the present instance, a NHP peptide or NHP polypeptide is thought
to be a soluble or secreted molecule, the peptide or polypeptide
can be recovered from the culture media. Such expression systems
also encompass engineered host cells that express NHP, or
functional equivalent, in situ. 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 a NHP, but to
assess biological activity, e.g., in drug screening assays.
[0040] 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 encoding 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).
[0041] 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 generation of pharmaceutical
compositions of and/or containing a 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 (Pharmacia
or American Type Culture Collection) can 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.
[0042] 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 coding
sequence can 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 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
sequence is expressed (e.g., see Smith et al., 1983, J. Virol. 46:
584; Smith, U.S. Pat. No. 4,215,051).
[0043] 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 sequence
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).
[0044] 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.
[0045] 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 can 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 a NHP product. Such engineered cell lines may
be particularly useful in screening and evaluation of compounds
that affect the endogenous activity of a NHP product.
[0046] 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).
[0047] Alternatively, any fusion protein can 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.
5.3 Antibodies to NHP Products
[0048] 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.
[0049] The antibodies of the invention may be used, for example, in
the detection of a 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 for the evaluation of the effect of test
compounds on expression and/or activity of a NHP sequence product.
Additionally, such antibodies can be used in conjunction gene
therapy to, for example, evaluate the normal and/or engineered
NHP-expres sing 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.
[0050] For the production of antibodies, various host animals may
be immunized by injection with a NHP, an NHP peptide (e.g., one
corresponding to a functional domain of a NHP), truncated NHP
polypeptides (NHP in which one or more domains have been deleted),
functional equivalents of a NHP or mutated variants of a 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.
[0051] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, can 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.
[0052] 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.
[0053] 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 sequence
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.
[0054] 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.
[0055] 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 a 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 a NHP signaling
pathway.
[0056] 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. 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
7 1 1464 DNA homo sapiens 1 atgacatcta agaattatcc cgggacctac
cccaatcaca ctgtttgcga aaagacaatt 60 acagtaccaa aggggaaaag
actgattctg aggttgggag atttggatat cgaatcccag 120 acctgtgctt
ctgactatct tctcttcacc agctcttcag atcaatatgg tccatactgt 180
ggaagtatga ctgttcccaa agaactcttg ttgaacacaa gtgaagtaac cgtccgcttt
240 gagagtggat cccacatttc tggccggggt tttttgctga cctatgcgag
cagcgaccat 300 ccagatttaa taacatgttt ggaacgagct agccattatt
tgaagacaga atacagcaaa 360 ttctgcccag ctggttgtag agacgtagca
ggagacattt ctgggaatat ggtagatgga 420 tatagagata cctctttatt
gtgcaaagct gccatccatg caggaataat tgctgatgaa 480 ctaggtggcc
agatcagtgt gcttcagcgc aaagggatca gtcgatatga agggattctg 540
gccaatggtg ttctttcgag ggatggttcc ctgtcagaca agcgatttct gtttacctcc
600 aatggttgca gcagatcctt gagttttgaa cctgacgggc aaatcagagc
ttcttcctca 660 tggcagtcgg tcaatgagag tggagaccaa gttcactggt
ctcctggcca agcccgactt 720 caggaccaag gcccatcatg ggcttcgggc
gacagtagca acaaccacaa accacgagag 780 tggctggaga tcgatttggg
ggagaaaaag aaaataacag gaattaggac cacaggatct 840 acacagtcga
acttcaactt ttatgttaag agttttgtga tgaacttcaa aaacaataat 900
tctaagtgga agacctataa aggaattgtg aataatgaag aaaaggtgtt tcagggtaac
960 tctaactttc gggacccagt gcaaaacaat ttcatccctc ccatcgtggc
cagatatgtg 1020 cgggttgtcc cccagacatg gcaccagagg atagccttga
aggtggagct cattggttgc 1080 cagattacac aaggtaatga ttcattggtg
tggcgcaaga caagtcaaag caccagtgtt 1140 tcaactaaga aagaagatga
gacaatcaca aggcccatcc cctcggaaga aacatccaca 1200 ggaataaaca
ttacaacggt ggctattcca ttggtgctcc ttgttgtcct ggtgtttgct 1260
ggaatgggga tctttgcagc ctttagaaag aagaagaaga aaggaagtcc gtatggatca
1320 gcggaggctc agaaaacaga ctgttggaag cagattaaat atccctttgc
cagacatcag 1380 tcagctgagt ttaccatcag ctatgataat gagaaggaga
tgacacaaaa gttagatctc 1440 atcacaagtg atatggcagg ttaa 1464 2 487
PRT homo sapiens 2 Met Thr Ser Lys Asn Tyr Pro Gly Thr Tyr Pro Asn
His Thr Val Cys 1 5 10 15 Glu Lys Thr Ile Thr Val Pro Lys Gly Lys
Arg Leu Ile Leu Arg Leu 20 25 30 Gly Asp Leu Asp Ile Glu Ser Gln
Thr Cys Ala Ser Asp Tyr Leu Leu 35 40 45 Phe Thr Ser Ser Ser Asp
Gln Tyr Gly Pro Tyr Cys Gly Ser Met Thr 50 55 60 Val Pro Lys Glu
Leu Leu Leu Asn Thr Ser Glu Val Thr Val Arg Phe 65 70 75 80 Glu Ser
Gly Ser His Ile Ser Gly Arg Gly Phe Leu Leu Thr Tyr Ala 85 90 95
Ser Ser Asp His Pro Asp Leu Ile Thr Cys Leu Glu Arg Ala Ser His 100
105 110 Tyr Leu Lys Thr Glu Tyr Ser Lys Phe Cys Pro Ala Gly Cys Arg
Asp 115 120 125 Val Ala Gly Asp Ile Ser Gly Asn Met Val Asp Gly Tyr
Arg Asp Thr 130 135 140 Ser Leu Leu Cys Lys Ala Ala Ile His Ala Gly
Ile Ile Ala Asp Glu 145 150 155 160 Leu Gly Gly Gln Ile Ser Val Leu
Gln Arg Lys Gly Ile Ser Arg Tyr 165 170 175 Glu Gly Ile Leu Ala Asn
Gly Val Leu Ser Arg Asp Gly Ser Leu Ser 180 185 190 Asp Lys Arg Phe
Leu Phe Thr Ser Asn Gly Cys Ser Arg Ser Leu Ser 195 200 205 Phe Glu
Pro Asp Gly Gln Ile Arg Ala Ser Ser Ser Trp Gln Ser Val 210 215 220
Asn Glu Ser Gly Asp Gln Val His Trp Ser Pro Gly Gln Ala Arg Leu 225
230 235 240 Gln Asp Gln Gly Pro Ser Trp Ala Ser Gly Asp Ser Ser Asn
Asn His 245 250 255 Lys Pro Arg Glu Trp Leu Glu Ile Asp Leu Gly Glu
Lys Lys Lys Ile 260 265 270 Thr Gly Ile Arg Thr Thr Gly Ser Thr Gln
Ser Asn Phe Asn Phe Tyr 275 280 285 Val Lys Ser Phe Val Met Asn Phe
Lys Asn Asn Asn Ser Lys Trp Lys 290 295 300 Thr Tyr Lys Gly Ile Val
Asn Asn Glu Glu Lys Val Phe Gln Gly Asn 305 310 315 320 Ser Asn Phe
Arg Asp Pro Val Gln Asn Asn Phe Ile Pro Pro Ile Val 325 330 335 Ala
Arg Tyr Val Arg Val Val Pro Gln Thr Trp His Gln Arg Ile Ala 340 345
350 Leu Lys Val Glu Leu Ile Gly Cys Gln Ile Thr Gln Gly Asn Asp Ser
355 360 365 Leu Val Trp Arg Lys Thr Ser Gln Ser Thr Ser Val Ser Thr
Lys Lys 370 375 380 Glu Asp Glu Thr Ile Thr Arg Pro Ile Pro Ser Glu
Glu Thr Ser Thr 385 390 395 400 Gly Ile Asn Ile Thr Thr Val Ala Ile
Pro Leu Val Leu Leu Val Val 405 410 415 Leu Val Phe Ala Gly Met Gly
Ile Phe Ala Ala Phe Arg Lys Lys Lys 420 425 430 Lys Lys Gly Ser Pro
Tyr Gly Ser Ala Glu Ala Gln Lys Thr Asp Cys 435 440 445 Trp Lys Gln
Ile Lys Tyr Pro Phe Ala Arg His Gln Ser Ala Glu Phe 450 455 460 Thr
Ile Ser Tyr Asp Asn Glu Lys Glu Met Thr Gln Lys Leu Asp Leu 465 470
475 480 Ile Thr Ser Asp Met Ala Gly 485 3 1761 DNA homo sapiens 3
atgggattcg gtgcggggca gcgactgcgc cccgtcccgg cgccgcgctc gtccgcagag
60 gaggcggccc ggcccgggca gctgcggctc gggatccgtc gaggggaggc
cgagcttgcc 120 aagctggcgc ccagcggggt catggtgccc ggcgcccgcg
gcggcggcgc actggcgcgg 180 gctgccgggc ggggcctcct ggctttgctg
ctcgcggtct ccgccccgct ccggctgcag 240 gcggaggagc tgggtgatgg
ctgtggacac ctagtgactt atcaggatag tggcacaatg 300 acatctaaga
attatcccgg gacctacccc aatcacactg tttgcgaaaa gacaattaca 360
gtaccaaagg ggaaaagact gattctgagg ttgggagatt tggatatcga atcccagacc
420 tgtgcttctg actatcttct cttcaccagc tcttcagatc aatatggtcc
atactgtgga 480 agtatgactg ttcccaaaga actcttgttg aacacaagtg
aagtaaccgt ccgctttgag 540 agtggatccc acatttctgg ccggggtttt
ttgctgacct atgcgagcag cgaccatcca 600 gatttaataa catgtttgga
acgagctagc cattatttga agacagaata cagcaaattc 660 tgcccagctg
gttgtagaga cgtagcagga gacatttctg ggaatatggt agatggatat 720
agagatacct ctttattgtg caaagctgcc atccatgcag gaataattgc tgatgaacta
780 ggtggccaga tcagtgtgct tcagcgcaaa gggatcagtc gatatgaagg
gattctggcc 840 aatggtgttc tttcgaggga tggttccctg tcagacaagc
gatttctgtt tacctccaat 900 ggttgcagca gatccttgag ttttgaacct
gacgggcaaa tcagagcttc ttcctcatgg 960 cagtcggtca atgagagtgg
agaccaagtt cactggtctc ctggccaagc ccgacttcag 1020 gaccaaggcc
catcatgggc ttcgggcgac agtagcaaca accacaaacc acgagagtgg 1080
ctggagatcg atttggggga gaaaaagaaa ataacaggaa ttaggaccac aggatctaca
1140 cagtcgaact tcaactttta tgttaagagt tttgtgatga acttcaaaaa
caataattct 1200 aagtggaaga cctataaagg aattgtgaat aatgaagaaa
aggtgtttca gggtaactct 1260 aactttcggg acccagtgca aaacaatttc
atccctccca tcgtggccag atatgtgcgg 1320 gttgtccccc agacatggca
ccagaggata gccttgaagg tggagctcat tggttgccag 1380 attacacaag
gtaatgattc attggtgtgg cgcaagacaa gtcaaagcac cagtgtttca 1440
actaagaaag aagatgagac aatcacaagg cccatcccct cggaagaaac atccacagga
1500 ataaacatta caacggtggc tattccattg gtgctccttg ttgtcctggt
gtttgctgga 1560 atggggatct ttgcagcctt tagaaagaag aagaagaaag
gaagtccgta tggatcagcg 1620 gaggctcaga aaacagactg ttggaagcag
attaaatatc cctttgccag acatcagtca 1680 gctgagttta ccatcagcta
tgataatgag aaggagatga cacaaaagtt agatctcatc 1740 acaagtgata
tggcaggtta a 1761 4 586 PRT homo sapiens 4 Met Gly Phe Gly Ala Gly
Gln Arg Leu Arg Pro Val Pro Ala Pro Arg 1 5 10 15 Ser Ser Ala Glu
Glu Ala Ala Arg Pro Gly Gln Leu Arg Leu Gly Ile 20 25 30 Arg Arg
Gly Glu Ala Glu Leu Ala Lys Leu Ala Pro Ser Gly Val Met 35 40 45
Val Pro Gly Ala Arg Gly Gly Gly Ala Leu Ala Arg Ala Ala Gly Arg 50
55 60 Gly Leu Leu Ala Leu Leu Leu Ala Val Ser Ala Pro Leu Arg Leu
Gln 65 70 75 80 Ala Glu Glu Leu Gly Asp Gly Cys Gly His Leu Val Thr
Tyr Gln Asp 85 90 95 Ser Gly Thr Met Thr Ser Lys Asn Tyr Pro Gly
Thr Tyr Pro Asn His 100 105 110 Thr Val Cys Glu Lys Thr Ile Thr Val
Pro Lys Gly Lys Arg Leu Ile 115 120 125 Leu Arg Leu Gly Asp Leu Asp
Ile Glu Ser Gln Thr Cys Ala Ser Asp 130 135 140 Tyr Leu Leu Phe Thr
Ser Ser Ser Asp Gln Tyr Gly Pro Tyr Cys Gly 145 150 155 160 Ser Met
Thr Val Pro Lys Glu Leu Leu Leu Asn Thr Ser Glu Val Thr 165 170 175
Val Arg Phe Glu Ser Gly Ser His Ile Ser Gly Arg Gly Phe Leu Leu 180
185 190 Thr Tyr Ala Ser Ser Asp His Pro Asp Leu Ile Thr Cys Leu Glu
Arg 195 200 205 Ala Ser His Tyr Leu Lys Thr Glu Tyr Ser Lys Phe Cys
Pro Ala Gly 210 215 220 Cys Arg Asp Val Ala Gly Asp Ile Ser Gly Asn
Met Val Asp Gly Tyr 225 230 235 240 Arg Asp Thr Ser Leu Leu Cys Lys
Ala Ala Ile His Ala Gly Ile Ile 245 250 255 Ala Asp Glu Leu Gly Gly
Gln Ile Ser Val Leu Gln Arg Lys Gly Ile 260 265 270 Ser Arg Tyr Glu
Gly Ile Leu Ala Asn Gly Val Leu Ser Arg Asp Gly 275 280 285 Ser Leu
Ser Asp Lys Arg Phe Leu Phe Thr Ser Asn Gly Cys Ser Arg 290 295 300
Ser Leu Ser Phe Glu Pro Asp Gly Gln Ile Arg Ala Ser Ser Ser Trp 305
310 315 320 Gln Ser Val Asn Glu Ser Gly Asp Gln Val His Trp Ser Pro
Gly Gln 325 330 335 Ala Arg Leu Gln Asp Gln Gly Pro Ser Trp Ala Ser
Gly Asp Ser Ser 340 345 350 Asn Asn His Lys Pro Arg Glu Trp Leu Glu
Ile Asp Leu Gly Glu Lys 355 360 365 Lys Lys Ile Thr Gly Ile Arg Thr
Thr Gly Ser Thr Gln Ser Asn Phe 370 375 380 Asn Phe Tyr Val Lys Ser
Phe Val Met Asn Phe Lys Asn Asn Asn Ser 385 390 395 400 Lys Trp Lys
Thr Tyr Lys Gly Ile Val Asn Asn Glu Glu Lys Val Phe 405 410 415 Gln
Gly Asn Ser Asn Phe Arg Asp Pro Val Gln Asn Asn Phe Ile Pro 420 425
430 Pro Ile Val Ala Arg Tyr Val Arg Val Val Pro Gln Thr Trp His Gln
435 440 445 Arg Ile Ala Leu Lys Val Glu Leu Ile Gly Cys Gln Ile Thr
Gln Gly 450 455 460 Asn Asp Ser Leu Val Trp Arg Lys Thr Ser Gln Ser
Thr Ser Val Ser 465 470 475 480 Thr Lys Lys Glu Asp Glu Thr Ile Thr
Arg Pro Ile Pro Ser Glu Glu 485 490 495 Thr Ser Thr Gly Ile Asn Ile
Thr Thr Val Ala Ile Pro Leu Val Leu 500 505 510 Leu Val Val Leu Val
Phe Ala Gly Met Gly Ile Phe Ala Ala Phe Arg 515 520 525 Lys Lys Lys
Lys Lys Gly Ser Pro Tyr Gly Ser Ala Glu Ala Gln Lys 530 535 540 Thr
Asp Cys Trp Lys Gln Ile Lys Tyr Pro Phe Ala Arg His Gln Ser 545 550
555 560 Ala Glu Phe Thr Ile Ser Tyr Asp Asn Glu Lys Glu Met Thr Gln
Lys 565 570 575 Leu Asp Leu Ile Thr Ser Asp Met Ala Gly 580 585 5
1620 DNA homo sapiens 5 atggtgcccg gcgcccgcgg cggcggcgca ctggcgcggg
ctgccgggcg gggcctcctg 60 gctttgctgc tcgcggtctc cgccccgctc
cggctgcagg cggaggagct gggtgatggc 120 tgtggacacc tagtgactta
tcaggatagt ggcacaatga catctaagaa ttatcccggg 180 acctacccca
atcacactgt ttgcgaaaag acaattacag taccaaaggg gaaaagactg 240
attctgaggt tgggagattt ggatatcgaa tcccagacct gtgcttctga ctatcttctc
300 ttcaccagct cttcagatca atatggtcca tactgtggaa gtatgactgt
tcccaaagaa 360 ctcttgttga acacaagtga agtaaccgtc cgctttgaga
gtggatccca catttctggc 420 cggggttttt tgctgaccta tgcgagcagc
gaccatccag atttaataac atgtttggaa 480 cgagctagcc attatttgaa
gacagaatac agcaaattct gcccagctgg ttgtagagac 540 gtagcaggag
acatttctgg gaatatggta gatggatata gagatacctc tttattgtgc 600
aaagctgcca tccatgcagg aataattgct gatgaactag gtggccagat cagtgtgctt
660 cagcgcaaag ggatcagtcg atatgaaggg attctggcca atggtgttct
ttcgagggat 720 ggttccctgt cagacaagcg atttctgttt acctccaatg
gttgcagcag atccttgagt 780 tttgaacctg acgggcaaat cagagcttct
tcctcatggc agtcggtcaa tgagagtgga 840 gaccaagttc actggtctcc
tggccaagcc cgacttcagg accaaggccc atcatgggct 900 tcgggcgaca
gtagcaacaa ccacaaacca cgagagtggc tggagatcga tttgggggag 960
aaaaagaaaa taacaggaat taggaccaca ggatctacac agtcgaactt caacttttat
1020 gttaagagtt ttgtgatgaa cttcaaaaac aataattcta agtggaagac
ctataaagga 1080 attgtgaata atgaagaaaa ggtgtttcag ggtaactcta
actttcggga cccagtgcaa 1140 aacaatttca tccctcccat cgtggccaga
tatgtgcggg ttgtccccca gacatggcac 1200 cagaggatag ccttgaaggt
ggagctcatt ggttgccaga ttacacaagg taatgattca 1260 ttggtgtggc
gcaagacaag tcaaagcacc agtgtttcaa ctaagaaaga agatgagaca 1320
atcacaaggc ccatcccctc ggaagaaaca tccacaggaa taaacattac aacggtggct
1380 attccattgg tgctccttgt tgtcctggtg tttgctggaa tggggatctt
tgcagccttt 1440 agaaagaaga agaagaaagg aagtccgtat ggatcagcgg
aggctcagaa aacagactgt 1500 tggaagcaga ttaaatatcc ctttgccaga
catcagtcag ctgagtttac catcagctat 1560 gataatgaga aggagatgac
acaaaagtta gatctcatca caagtgatat ggcaggttaa 1620 6 539 PRT homo
sapiens 6 Met Val Pro Gly Ala Arg Gly Gly Gly Ala Leu Ala Arg Ala
Ala Gly 1 5 10 15 Arg Gly Leu Leu Ala Leu Leu Leu Ala Val Ser Ala
Pro Leu Arg Leu 20 25 30 Gln Ala Glu Glu Leu Gly Asp Gly Cys Gly
His Leu Val Thr Tyr Gln 35 40 45 Asp Ser Gly Thr Met Thr Ser Lys
Asn Tyr Pro Gly Thr Tyr Pro Asn 50 55 60 His Thr Val Cys Glu Lys
Thr Ile Thr Val Pro Lys Gly Lys Arg Leu 65 70 75 80 Ile Leu Arg Leu
Gly Asp Leu Asp Ile Glu Ser Gln Thr Cys Ala Ser 85 90 95 Asp Tyr
Leu Leu Phe Thr Ser Ser Ser Asp Gln Tyr Gly Pro Tyr Cys 100 105 110
Gly Ser Met Thr Val Pro Lys Glu Leu Leu Leu Asn Thr Ser Glu Val 115
120 125 Thr Val Arg Phe Glu Ser Gly Ser His Ile Ser Gly Arg Gly Phe
Leu 130 135 140 Leu Thr Tyr Ala Ser Ser Asp His Pro Asp Leu Ile Thr
Cys Leu Glu 145 150 155 160 Arg Ala Ser His Tyr Leu Lys Thr Glu Tyr
Ser Lys Phe Cys Pro Ala 165 170 175 Gly Cys Arg Asp Val Ala Gly Asp
Ile Ser Gly Asn Met Val Asp Gly 180 185 190 Tyr Arg Asp Thr Ser Leu
Leu Cys Lys Ala Ala Ile His Ala Gly Ile 195 200 205 Ile Ala Asp Glu
Leu Gly Gly Gln Ile Ser Val Leu Gln Arg Lys Gly 210 215 220 Ile Ser
Arg Tyr Glu Gly Ile Leu Ala Asn Gly Val Leu Ser Arg Asp 225 230 235
240 Gly Ser Leu Ser Asp Lys Arg Phe Leu Phe Thr Ser Asn Gly Cys Ser
245 250 255 Arg Ser Leu Ser Phe Glu Pro Asp Gly Gln Ile Arg Ala Ser
Ser Ser 260 265 270 Trp Gln Ser Val Asn Glu Ser Gly Asp Gln Val His
Trp Ser Pro Gly 275 280 285 Gln Ala Arg Leu Gln Asp Gln Gly Pro Ser
Trp Ala Ser Gly Asp Ser 290 295 300 Ser Asn Asn His Lys Pro Arg Glu
Trp Leu Glu Ile Asp Leu Gly Glu 305 310 315 320 Lys Lys Lys Ile Thr
Gly Ile Arg Thr Thr Gly Ser Thr Gln Ser Asn 325 330 335 Phe Asn Phe
Tyr Val Lys Ser Phe Val Met Asn Phe Lys Asn Asn Asn 340 345 350 Ser
Lys Trp Lys Thr Tyr Lys Gly Ile Val Asn Asn Glu Glu Lys Val 355 360
365 Phe Gln Gly Asn Ser Asn Phe Arg Asp Pro Val Gln Asn Asn Phe Ile
370 375 380 Pro Pro Ile Val Ala Arg Tyr Val Arg Val Val Pro Gln Thr
Trp His 385 390 395 400 Gln Arg Ile Ala Leu Lys Val Glu Leu Ile Gly
Cys Gln Ile Thr Gln 405 410 415 Gly Asn Asp Ser Leu Val Trp Arg Lys
Thr Ser Gln Ser Thr Ser Val 420 425 430 Ser Thr Lys Lys Glu Asp Glu
Thr Ile Thr Arg Pro Ile Pro Ser Glu 435 440 445 Glu Thr Ser Thr Gly
Ile Asn Ile Thr Thr Val Ala Ile Pro Leu Val 450 455 460 Leu Leu Val
Val Leu Val Phe Ala Gly Met Gly Ile Phe Ala Ala Phe 465 470 475 480
Arg Lys Lys Lys Lys Lys Gly Ser Pro Tyr Gly Ser Ala Glu Ala Gln 485
490 495 Lys Thr Asp Cys Trp Lys Gln Ile Lys Tyr Pro Phe Ala Arg His
Gln 500 505 510 Ser Ala Glu Phe Thr Ile Ser Tyr Asp Asn Glu Lys Glu
Met Thr Gln 515 520 525 Lys Leu Asp Leu Ile Thr Ser Asp Met Ala Gly
530 535 7 1768 DNA homo sapiens 7 ggcggaggag ctgggtgatg gctgtggaca
cctagtgact tatcaggata gtggcacaat 60 gacatctaag aattatcccg
ggacctaccc caatcacact gtttgcgaaa agacaattac 120 agtaccaaag
gggaaaagac tgattctgag gttgggagat ttggatatcg aatcccagac 180
ctgtgcttct gactatcttc tcttcaccag ctcttcagat caatatggtc catactgtgg
240 aagtatgact gttcccaaag aactcttgtt gaacacaagt gaagtaaccg
tccgctttga 300 gagtggatcc cacatttctg gccggggttt tttgctgacc
tatgcgagca gcgaccatcc 360 agatttaata acatgtttgg aacgagctag
ccattatttg aagacagaat acagcaaatt 420 ctgcccagct ggttgtagag
acgtagcagg agacatttct gggaatatgg tagatggata 480 tagagatacc
tctttattgt gcaaagctgc catccatgca ggaataattg ctgatgaact 540
aggtggccag atcagtgtgc ttcagcgcaa agggatcagt cgatatgaag ggattctggc
600 caatggtgtt ctttcgaggg atggttccct gtcagacaag cgatttctgt
ttacctccaa 660 tggttgcagc agatccttga gttttgaacc tgacgggcaa
atcagagctt cttcctcatg 720 gcagtcggtc aatgagagtg gagaccaagt
tcactggtct cctggccaag cccgacttca 780 ggaccaaggc ccatcatggg
cttcgggcga cagtagcaac aaccacaaac cacgagagtg 840 gctggagatc
gatttggggg agaaaaagaa aataacagga attaggacca caggatctac 900
acagtcgaac ttcaactttt atgttaagag ttttgtgatg aacttcaaaa acaataattc
960 taagtggaag acctataaag gaattgtgaa taatgaagaa aaggtgtttc
agggtaactc 1020 taactttcgg gacccagtgc aaaacaattt catccctccc
atcgtggcca gatatgtgcg 1080 ggttgtcccc cagacatggc accagaggat
agccttgaag gtggagctca ttggttgcca 1140 gattacacaa ggtaatgatt
cattggtgtg gcgcaagaca agtcaaagca ccagtgtttc 1200 aactaagaaa
gaagatgaga caatcacaag gcccatcccc tcggaagaaa catccacagg 1260
aataaacatt acaacggtgg ctattccatt ggtgctcctt gttgtcctgg tgtttgctgg
1320 aatggggatc tttgcagcct ttagaaagaa gaagaagaaa ggaagtccgt
atggatcagc 1380 ggaggctcag aaaacagact gttggaagca gattaaatat
ccctttgcca gacatcagtc 1440 agctgagttt accatcagct atgataatga
gaaggagatg acacaaaagt tagatctcat 1500 cacaagtgat atggcaggtt
aactccgttg actgccaaaa tagcatcccc aacgtgcagc 1560 cctccgcatc
tatcagcagg ttgccccgga tggatctcag agatgaggat tggaacacca 1620
tgttctttcc caccctaaca acaacaaagg gcagtaaatt aaagtactct ttgtaaggta
1680 cagttaccga ttaatctaga gataaaatat tttcttaaaa atatatttca
ttaaacacct 1740 atgctgtctc tatgcaaaaa aaaaaaaa 1768
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