U.S. patent application number 10/960789 was filed with the patent office on 2005-06-30 for novel human transporter proteins and polynucleotides encoding the same.
Invention is credited to Hu, Yi, Kieke, James Alvin, Revelli, Jean-Pierre, Turner, C. Alexander JR., Walke, D. Wade, Wilganowski, Nathaniel L., Zambrowicz, Brian.
Application Number | 20050142588 10/960789 |
Document ID | / |
Family ID | 22683078 |
Filed Date | 2005-06-30 |
United States Patent
Application |
20050142588 |
Kind Code |
A1 |
Walke, D. Wade ; et
al. |
June 30, 2005 |
Novel human transporter 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: |
Walke, D. Wade; (Spring,
TX) ; Wilganowski, Nathaniel L.; (Houston, TX)
; Hu, Yi; (The Woodlands, TX) ; Kieke, James
Alvin; (Houston, TX) ; Zambrowicz, Brian; (The
Woodlands, TX) ; Revelli, Jean-Pierre; (Spring,
TX) ; Turner, C. Alexander JR.; (The Woodlands,
TX) |
Correspondence
Address: |
Lance K. Ishimoto
Lexicon Genetics Incorporated
8800 Technology Forest Place
The Woodlands
TX
77381
US
|
Family ID: |
22683078 |
Appl. No.: |
10/960789 |
Filed: |
October 7, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10960789 |
Oct 7, 2004 |
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10345884 |
Jan 15, 2003 |
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10345884 |
Jan 15, 2003 |
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09795927 |
Feb 28, 2001 |
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6531309 |
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60185956 |
Feb 29, 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
What is claimed is:
1. An isolated nucleic acid molecule comprising at least 24
contiguous bases of nucleotide sequence first disclosed in SEQ ID
NO: 1.
2. An isolated nucleic acid molecule comprising a nucleotide
sequence that,: (a) encodes the amino acid sequence shown in SEQ ID
NO: 2; and (b) hybridizes under stringent conditions to the
nucleotide sequence of SEQ ID NO:1 or the complement thereof.
3. An isolated nucleic acid molecule comprising a nucleotide
sequence that encodes the amino acid sequence shown in SEQ ID
NO:2.
4. An isolated nucleic acid molecule comprising a nucleotide
sequence that: (a) encodes the amino acid sequence shown in SEQ ID
NO:7; and (b) hybridizes under stringent conditions to the
nucleotide sequence of SEQ ID NO:6 or the complement thereof.
5. An isolated nucleic acid molecule comprising a nucleotide
sequence that encodes the amino acid sequence shown in SEQ ID
NO:7.
6. An isolated nucleic acid molecule comprising a nucleotide
sequence that: (a) encodes the amino acid sequence shown in SEQ ID
NO:10; and (b) hybridizes under stringent conditions to the
nucleotide sequence of SEQ ID NO:9 or the complement thereof.
7. An isolated nucleic acid molecule comprising a nucleotide
sequence that encodes the amino acid sequence shown in SEQ ID
NO:10.
Description
[0001] The present application claims the benefit of U.S.
Provisional Application No. 60/185,956 which was filed on Feb. 29,
2000 and is herein incorporated by reference in its entirety.
1. INTRODUCTION
[0002] The present invention relates to the discovery,
identification, and characterization of novel human polynucleotides
encoding proteins that share sequence similarity with mammalian
transporter proteins. 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
polynucleotides, 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, and
treatment of diseases and disorders.
2. BACKGROUND OF THE INVENTION
[0003] Transporter proteins are integral membrane proteins that
mediate or facilitate the passage of materials across the lipid
bilayer. Given that the transport of materials across the membrane
can play an important physiological role, transporter proteins are
good drug targets. Additionally, one of the mechanisms of drug
resistance involves diseased cells using cellular transporter
systems to export chemotherapeutic agents from the cell. Such
mechanisms are particularly relevant to cells manifesting
resistance to a multiplicity of drugs.
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 mammalian ion
transporters, calcium transporters (particularly calcium
transporting ATPases), sulfate transporters, and zinc
transporters.
[0005] The novel human nucleic acid sequences described herein,
encode alternative proteins/open reading frames (ORFs) of 1,177 and
374 amino acids in length (calcium-transporting ATPase, SEQ ID NOS:
2 and 4), 970 (sulfate transporter, SEQ ID NO:7), and 507 (zinc
transporter, SEQ ID NO:10) amino acids in length.
[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 polynucleotides (e.g., expression constructs that
place the described polynucleotide 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. Knock-out mice can be produced in several
ways, one of which involves the use of mouse embryonic stem cells
("ES cells") lines that contain gene trap mutations in a murine
homolog of at least one of the described NHPs. When the unique NHP
sequences described in SEQ ID NOS:1-11 are "knocked-out" they
provide a method of identifying phenotypic expression of the
particular gene as well as a method of assigning function to
previously unknown genes. Additionally, the unique NHP sequences
described in SEQ ID NOS:1-11 are useful for the identification of
coding sequence and the mapping a unique gene to a particular
chromosome.
[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 product, or
cells expressing the same. Such compounds can be used as
therapeutic agents for the treatment of any of a wide variety of
symptoms associated with biological disorders or imbalances.
4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES
[0008] The Sequence Listing provides the sequences of the described
NHP ORFs that encode the described NHP amino acid sequences. SEQ ID
NOS 5, 8, and 11 describe nucleotides encoding NHP ORFs along with
regions of flanking sequence.
5. DETAILED DESCRIPTION OF THE INVENTION
[0009] The NHPs described for the first time herein are novel
proteins that may be expressed in, inter alia, human cell lines,
fetal brain, pituitary, cerebellum, thymus, spleen, lymph node,
bone marrow, trachea, kidney, fetal liver, liver, prostate, testis,
thyroid, adrenal gland, salivary gland, stomach, small intestine,
colon, adipose, rectum, pericardium, bone marrow, placenta, and
gene trapped human cells. More particularly, the NHP that is
similar to sulfate transporters (and the down-regulated in adenoma,
or DRA, gene) is predominantly found in bone marrow and testis, and
the zinc transporter-like NHP can be found expressed in the
placenta.
[0010] 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 polynucleotides,
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 (or hydrophobic transmembrane) sequence is 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 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) 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 a DNA sequence that encodes and
expresses 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 encodes 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 NHP
ORFs, or their functional equivalents, encoded by polynucleotide
sequences that are 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 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. An oligonucleotide or polynucleotide sequence first
disclosed in at least a portion of one or more of the sequences of
SEQ ID NOS: 1-11 can be used as a hybridization probe in
conjunction with a solid support matrix/substrate (resins, beads,
membranes, plastics, polymers, metal or metallized substrates,
crystalline or polycrystalline substrates, etc.). Of particular
note are spatially addressable arrays (i.e., gene chips, microtiter
plates, etc.) of oligonucleotides and polynucleotides, or
corresponding oligopeptides and polypeptides, wherein at least one
of the biopolymers present on the spatially addressable array
comprises an oligonucleotide or polynucleotide sequence first
disclosed in at least one of the sequences of SEQ ID NOS: 1-11, or
an amino acid sequence encoded thereby. Methods for attaching
biopolymers to, or synthesizing biopolymers on, solid support
matrices, and conducting binding studies thereon are disclosed in,
inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752, 5,744,305,
4,631,211, 5,445,934, 5,252,743, 4,713,326, 5,424,186, and
4,689,405 the disclosures of which are herein incorporated by
reference in their entirety.
[0015] Addressable arrays comprising sequences first disclosed in
SEQ ID NOS:1-11 can be used to identify and characterize the
temporal and tissue specific expression of a gene. These
addressable arrays incorporate oligonucleotide sequences of
sufficient length to confer the required specificity, yet be within
the limitations of the production technology. The length of these
probes is within a range of between about 8 to about 2000
nucleotides. Preferably the probes consist of 60 nucleotides and
more preferably 25 nucleotides from the sequences first disclosed
in SEQ ID NOS:1-11.
[0016] For example, a series of the described oligonucleotide
sequences, or-the-complements thereof, can be used in chip format
to represent all or a portion of the described sequences. The
oligonucleotides, typically between about 16 to about 40 (or any
whole number within the stated range) nucleotides in length can
partially overlap each other and/or the sequence may be represented
using oligonucleotides that do not overlap. Accordingly, the
described polynucleotide sequences shall typically comprise at
least about two or three distinct oligonucleotide sequences of at
least about 8 nucleotides in length that are each first disclosed
in the described Sequence Listing. Such oligonucleotide sequences
can 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.
[0017] Microarray-based analysis allows the discovery of broad
patterns of genetic activity, providing new understanding of gene
functions and generating novel and unexpected insight into
transcriptional processes and biological mechanisms. The use of
addressable arrays comprising sequences first disclosed in SEQ ID
NOS:1-11 provides detailed information about transcriptional
changes involved in a specific pathway, potentially leading to the
identification of novel components or gene functions that manifest
themselves as novel phenotypes.
[0018] Probes consisting of sequences first disclosed in SEQ ID
NOS:1-11 can also be used in the identification, selection and
validation of novel molecular targets for drug discovery. The use
of these unique sequences permits the direct confirmation of drug
targets and recognition of drug dependent changes in gene
expression that are modulated through pathways distinct from the
drugs intended target. These unique sequences therefore also have
utility in defining and monitoring both drug action and
toxicity.
[0019] As an example of utility, the sequences first disclosed in
SEQ ID NOS:1-11 can be utilized in microarrays or other assay
formats, to screen collections of genetic material from patients
who have a particular medical condition. These investigations can
also be carried out using the sequences first disclosed in SEQ ID
NOS:1-11 in silico and by comparing previously collected genetic
databases and the disclosed sequences using computer software known
to those in the art.
[0020] Thus the sequences first disclosed in SEQ ID NOS:1-11 can be
used to identify mutations associated with a particular disease and
also as a diagnostic or prognostic assay.
[0021] Although the presently described sequences have been
specifically described using nucleotide sequence, it should be
appreciated that each of the sequences can uniquely be described
using any of a wide variety of additional structural attributes, or
combinations thereof. For example, a given sequence can be
described by the net composition of the nucleotides present within
a given region of the sequence in conjunction with the presence of
one or more specific oligonucleotide sequence(s) first disclosed in
the SEQ ID NOS: 1-11. Alternatively, a restriction map specifying
the relative positions of restriction endonuclease digestion sites,
or various palindromic or other specific oligonucleotide sequences
can be used to structurally describe a given sequence.
Such-restriction maps, which are typically generated by widely
available computer programs (e.g., the University of Wisconsin GCG
sequence analysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann
Arbor, Mich., etc.), can optionally be used in conjunction with one
or more discrete nucleotide sequence(s) present in the sequence
that can be described by the relative position of the sequence
relatve to one or more additional sequence(s) or one or more
restriction sites present in the disclosed sequence.
[0022] 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.
[0023] Inhibitory antisense or double stranded oligdnucleotides 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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
human or non-human cell lines or tissue known or suspected to
express an allele of a NHP gene.
[0031] 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.
[0032] 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). 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.
[0033] 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.
[0034] 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.
[0035] 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 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,
N.Y.).
[0036] 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 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.
[0037] 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 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.
[0038] 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.).
[0039] 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 the endogenous receptor
for an NHP, but can also identify compounds that trigger
NHP-mediated activities or pathways.
[0040] Finally, the NHP products can be used as therapeutics. For
example, soluble derivatives such as NHP peptides/domains
corresponding to NHPs, 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 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 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.
[0041] Various aspects of the invention are described in greater
detail in the subsections below.
5.1 THE NHP SEQUENCES
[0042] The cDNA sequences and the corresponding deduced amino acid
sequences of the described NHPs are presented in the Sequence
Listing. The NHP nucleotides were obtained from clustered human
gene trapped sequences, testis and mammary transcript RACE
products, ESTs, and human brain, testis, trachea, pituitary,
thymus, and mammary gland cDNA libraries (Edge Biosystems,
Gaithersburg, Md.).
[0043] SEQ ID NOS:1-5 describe sequences that are similar to
eucaryotic ATP-driven ion pumps such as calcium transporting
ATPases, and which can be found expressed in a variety of human
cells and tissues. The described sequences were assembled using
gene trapped sequences and clones isolated from human kidney, lymph
node, and thymus cDNA libraries (Edge Biosystems, Gaithersburg,
Md.).
[0044] SEQ ID NOS:6-8 describe sequences that are similar to, inter
alia, sulfate transporter and cotransporter proteins, and can be
found expressed in human bone marrow and testis. Several
polymorphisms were found in this NHP including, but not limited to,
possible A-to-G transitions at nucleotide positions corresponding
to nucleotides 589, 692, 917, 1,164, and 2,390 of, for example SEQ
ID NO:8 which be silent or can result in the met corresponding to
amino acid position 73 of SEQ ID NO:7 converting to a val (e.g.,
met 73 converting to val 73), val 148 converting to ile, asn 230
converting to lys, ile 562 converting to val. An additional C-to-T
transition was identified that converts ala 777 to val. SEQ ID
NOS:6-8 can be expressed in bone marrow and predominantly in testis
cells. These NHPs were assembled from gene trapped sequences and
clones from a human testis cDNA library (Edge Biosystems,
Gaithersburg, Md.).
[0045] SEQ ID NOS:9-11 describe sequences that are similar to zinc
transporters and vesicular transporters, can be found expressed in,
inter alia, placenta and adrenal gland, and these NHP sequences
were assembled using gene trapped sequences and clones from human
adrenal and placenta cDNA libraries (Edge Biosystems, Gaithersburg,
Md.).
[0046] Transporters and transporter related multidrug resistance
(MDR) sequences, as well as uses and applications that are germane
to the described NHPs, are described in U.S. Pat. Nos. 5,198,344
and 5,866,699 which are herein incorporated by reference in their
entirety.
5.2 NHPS AND NHP POLYPEPTIDES
[0047] NHPs, polypeptides, peptide fragments, mutated, truncated,
or deleted forms of the NHPS, and/or NHP fusion proteins can be
prepared for a variety of uses. These uses include but are not
limited to the generation of antibodies, as reagents in diagnostic
assays, the identification of other cellular gene products related
to a NHP, as reagents in assays for screening for compounds that
can be as pharmaceutical reagents useful in the therapeutic
treatment of mental, biological, or medical disorders and diseases.
Given the similarity information and expression data, the described
NHPs can be targeted (by drugs, oligos, antibodies, etc,) in order
to treat disease, or to therapeutically augment the efficacy of,
for example, chemotherapeutic agents used in, the treatment of
breast or prostate cancer.
[0048] The Sequence Listing discloses the amino acid sequences
encoded by the described NHP polynucleotides. The NHPs typically
display have initiator methionines in DNA sequence contexts
consistent with a translation initiation site.
[0049] The NHP amino acid sequences of the invention include the
amino acid sequence 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.
[0050] 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.
[0051] 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, the NHP peptide or polypeptide is thought to
be membrane protein, the hydrophobic regions of the protein can be
excised and the resulting soluble peptide or polypeptide can be
recovered from the culture media. Such expression systems also
encompass engineered host cells that express a 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 the NHP, but to assess biological
activity, e.g., in drug screening assays.
[0052] The expression systems that may be used for purposes of the
invention include but are not limited to microorganisms such as
bacteria (e.g., E. coli, B. subtilis) transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing NHP nucleotide sequences; yeast (e.g., Saccharomyces,
Pichia) transformed with recombinant yeast expression vectors
containing NHP nucleotide sequences; insect cell systems infected
with recombinant virus expression vectors (e.g., 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).
[0053] 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 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 (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.
[0054] 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 may be cloned individually into non-essential regions (for
example the pdlyhedrin 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).
[0055] 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).
[0056] 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.
[0057] 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 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.
[0058] 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).
[0059] 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.
[0060] Also encompassed by the present invention are fusion
proteins that direct the NHP to a target organ and/or facilitate
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
or lipid complex based delivery systems. Such technologies are
described in Liposomes:A Practical Approach, New,RRC ed., Oxford
University Press, New York and 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.
Additionally embodied are novel protein constructs engineered in
such a way that they facilitate transport of the NHP to the target
site or desired organ. This goal may be achieved by coupling of the
NHP to a cytokine or other ligand that provides targeting
specificity, and/or to a protein transducing domain (see generally
U.S. applications Ser. No. 60/111,701 and 60/056,713, both of which
are herein incorporated by reference, for examples of such
transducing sequences) to facilitate passage across cellular
membranes if needed and can optionally be engineered to include
nuclear localization sequences when desired.
5.3 ANTIBODIES TO NHP PRODUCTS
[0061] 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.
[0062] The antibodies of the invention may be used, for example, in
the detection of NHP in a biological sample and may, therefore, be
utilized as part of a diagnostic or prognostic technique whereby
patients may be tested for abnormal amounts of NHP. Such antibodies
may also be utilized in conjunction with, for example, compound
screening schemes 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.
[0063] 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 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 liimpet 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.
[0064] 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.
[0065] 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. Also encompassed by
the present invention is the use of fully humanized monoclonal
antibodies as described in U.S. Pat. No. 6,150,584 and respective
disclosures which are herein incorporated by reference in their
entirety.
[0066] 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.
[0067] 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.
[0068] Antibodies to a NHP can, in turn, be utilized to generate
anti-idiotype antibodies that "mimic" a given NHP, using techniques
well known to those skilled in the art. (See, e.g., Greenspan &
Bona, 1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.
147(8):2429-2438). For example antibodies which bind to a NHP
domain and competitively inhibit the binding of NHP to its cognate
receptor can be used to generate anti-idiotypes that "mimic" the
NHP and, therefore, bind and activate or neutralize a receptor.
Such anti-idiotypic antibodies or Fab fragments of such
anti-idiotypes can be used in therapeutic regimens involving a NHP
mediated pathway.
[0069] 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
11 1 3534 DNA homo sapiens 1 atgtggcgct ggatccggca gcagctgggt
tttgacccac cacatcagag tgacacaaga 60 accatctacg tagccaacag
gtttcctcag aatggccttt acacacctca gaaatttata 120 gataacagga
tcatttcatc taagtacact gtgtggaatt ttgttccaaa aaatttattt 180
gaacagttca gaagagtggc aaacttttat tttcttatta tatttttggt tcagcttatg
240 attgatacac ctaccagtcc agttaccagt ggacttccat tattctttgt
gataacagta 300 actgccataa agcagggata tgaagattgg ttacggcata
actcagataa tgaagtaaat 360 ggagctcctg tttatgttgt tcgaagtggt
ggccttgtaa aaactagatc aaaaaacatt 420 cgggtgggtg atattgttcg
aatagccaaa gatgaaattt ttcctgcaga cttggtgctt 480 ctgtcctcag
atcgactgga tggttcctgt cacgttacaa ctgctagttt ggacggagaa 540
actaacctga agacacatgt ggcagttcca gaaacagcat tattacaaac agttgccaat
600 ttggacactc tagtagctgt aatagaatgc cagcaaccag aagcagactt
atacagattc 660 atgggacgaa tgatcataac ccaacaaatg gaagaaattg
taagacctct ggggccggag 720 agtctcctgc ttcgtggagc cagattaaaa
aacacaaaag aaatttttgg tgttgcggta 780 tacactggaa tggaaactaa
gatggcatta aattacaaga gcaaatcaca gaaacgatct 840 gcagtagaaa
agtcaatgaa tacatttttg ataatttatc tagtaattct tatatctgaa 900
gctgtcatca gcactatctt gaagtataca tggcaagctg aagaaaaatg ggatgaacct
960 tggtataacc aaaaaacaga acatcaaaga aatagcagta agattctgag
atttatttca 1020 gacttccttg cttttttggt tctctacaat ttcatcattc
caatttcatt atatgtgaca 1080 gtcgaaatgc agaaatttct tggatcattt
tttattggct gggatcttga tctgtatcat 1140 gaagaatcag atcagaaagc
tcaagtcaat acttccgatc tgaatgaaga gcttggacag 1200 gtagagtacg
tgtttacaga taaaactggt acactgacag aaaatgagat gcagtttcgg 1260
gaatgttcaa ttaatggcat gaaataccaa gaaattaatg gtagacttgt acccgaagga
1320 ccaacaccag actcttcaga aggaaactta tcttatctta gtagtttatc
ccatcttaac 1380 aacttatccc atcttacaac cagttcctct ttcagaacca
gtcctgaaaa tgaaactgaa 1440 ctaattaaag aacatgatct cttctttaaa
gcagtcagtc tctgtcacac tgtacagatt 1500 agcaatgttc aaactgactg
cactggtgat ggtccctggc aatccaacct ggcaccatcg 1560 cagttggagt
actatgcatc ttcaccagat gaaaaggctc tagtagaagc tgctgcaagg 1620
attggtattg tgtttattgg caattctgaa gaaactatgg aggttaaaac tcttggaaaa
1680 ctggaacggt acaaactgct tcatattctg gaatttgatt cagatcgtag
gagaatgagt 1740 gtaattgttc aggcaccttc aggtgagaag ttattatttg
ctaaaggagc tgagtcatca 1800 attctcccta aatgtatagg tggagaaata
gaaaaaacca gaattcatgt agatgaattt 1860 gctttgaaag ggctaagaac
tctgtgtata gcatatagaa aatttacatc aaaagagtat 1920 gaggaaatag
ataaacgcat atttgaagcc aggactgcct tgcagcagcg ggaagagaaa 1980
ttggcagctg ttttccagtt catagagaaa gacctgatat tacttggagc cacagcagta
2040 gaagacagac tacaagataa agttcgagaa actattgaag cattgagaat
ggctggtatc 2100 aaagtatggg tacttactgg ggataaacat gaaacagctg
ttagtgtgag tttatcatgt 2160 ggccattttc atagaaccat gaacatcctt
gaacttataa accagaaatc agacagcgag 2220 tgtgctgaac aattgaggca
gcttgccaga agaattacag aggatcatgt gattcagcat 2280 gggctggtag
tggatgggac cagcctatct cttgcactca gggagcatga aaaactattt 2340
atggaagttt gcagaaattg ttcagctgta ttatgctgtc gtatggctcc actgcagaaa
2400 gcaaaagtaa taagactaat aaaaatatca cctgagaaac ctataacatt
ggctgttggt 2460 gatggtgcta atgacgtaag catgatacaa gaagcccatg
ttggcatagg aatcatgggt 2520 aaagaaggaa gacaggctgc aagaaacagt
gactatgcaa tagccagatt taagttcctc 2580 tccaaattgc tttttgttca
tggtcatttt tattatatta gaatagctac ccttgtacag 2640 tatttttttt
ataagaatgt gtgctttatc acaccccagt ttttatatca gttctactgt 2700
ttgttttctc agcaaacatt gtatgacagc gtgtacctga ctttatacaa tatttgtttt
2760 acttccctac ctattctgat atatagtctt ttggaacagc atgtagaccc
tcatgtgtta 2820 caaaataagc ccacccttta tcgagacatt agtaaaaacc
gcctcttaag tattaaaaca 2880 tttctttatt ggaccatcct gggcttcagt
catgccttta ttttcttttt tggatcctat 2940 ttactaatag ggaaagatac
atctctgctt ggaaatggcc agatgttygg aaactggaca 3000 tttggcactt
tggtcttcac agtcatggtt attacagtca cagtaaagat ggctctggaa 3060
actcattttt ggacttggat caaccatctc gttacctggg gatctattat attttatttt
3120 gtattttcct tgttttatgg agggattctc tggccatttt tgggctccca
gaatatgtat 3180 tttgtgttta ttcagctcct gtcaagtggt tctgcttggt
ttgccataat cctcatggtt 3240 gttacatgtc tatttcttga tatcataaag
aaggtctttg accgacacct ccaccctaca 3300 agtactgaaa aggcacagct
tactgaaaca aatgcaggta tcaagtgctt ggactccatg 3360 tgctgtttcc
cggaaggaga agcagcgtgt gcatctgttg gaagaatgct ggaacgagtt 3420
ataggaagat gtagtccaac ccacatcagc agatcatgga gtgcatcgga tcctttctat
3480 accaacgaca ggagcatctt gactctctcc acaatggact catctacttg ttaa
3534 2 1177 PRT homo sapiens 2 Met Trp Arg Trp Ile Arg Gln Gln Leu
Gly Phe Asp Pro Pro His Gln 1 5 10 15 Ser Asp Thr Arg Thr Ile Tyr
Val Ala Asn Arg Phe Pro Gln Asn Gly 20 25 30 Leu Tyr Thr Pro Gln
Lys Phe Ile Asp Asn Arg Ile Ile Ser Ser Lys 35 40 45 Tyr Thr Val
Trp Asn Phe Val Pro Lys Asn Leu Phe Glu Gln Phe Arg 50 55 60 Arg
Val Ala Asn Phe Tyr Phe Leu Ile Ile Phe Leu Val Gln Leu Met 65 70
75 80 Ile Asp Thr Pro Thr Ser Pro Val Thr Ser Gly Leu Pro Leu Phe
Phe 85 90 95 Val Ile Thr Val Thr Ala Ile Lys Gln Gly Tyr Glu Asp
Trp Leu Arg 100 105 110 His Asn Ser Asp Asn Glu Val Asn Gly Ala Pro
Val Tyr Val Val Arg 115 120 125 Ser Gly Gly Leu Val Lys Thr Arg Ser
Lys Asn Ile Arg Val Gly Asp 130 135 140 Ile Val Arg Ile Ala Lys Asp
Glu Ile Phe Pro Ala Asp Leu Val Leu 145 150 155 160 Leu Ser Ser Asp
Arg Leu Asp Gly Ser Cys His Val Thr Thr Ala Ser 165 170 175 Leu Asp
Gly Glu Thr Asn Leu Lys Thr His Val Ala Val Pro Glu Thr 180 185 190
Ala Leu Leu Gln Thr Val Ala Asn Leu Asp Thr Leu Val Ala Val Ile 195
200 205 Glu Cys Gln Gln Pro Glu Ala Asp Leu Tyr Arg Phe Met Gly Arg
Met 210 215 220 Ile Ile Thr Gln Gln Met Glu Glu Ile Val Arg Pro Leu
Gly Pro Glu 225 230 235 240 Ser Leu Leu Leu Arg Gly Ala Arg Leu Lys
Asn Thr Lys Glu Ile Phe 245 250 255 Gly Val Ala Val Tyr Thr Gly Met
Glu Thr Lys Met Ala Leu Asn Tyr 260 265 270 Lys Ser Lys Ser Gln Lys
Arg Ser Ala Val Glu Lys Ser Met Asn Thr 275 280 285 Phe Leu Ile Ile
Tyr Leu Val Ile Leu Ile Ser Glu Ala Val Ile Ser 290 295 300 Thr Ile
Leu Lys Tyr Thr Trp Gln Ala Glu Glu Lys Trp Asp Glu Pro 305 310 315
320 Trp Tyr Asn Gln Lys Thr Glu His Gln Arg Asn Ser Ser Lys Ile Leu
325 330 335 Arg Phe Ile Ser Asp Phe Leu Ala Phe Leu Val Leu Tyr Asn
Phe Ile 340 345 350 Ile Pro Ile Ser Leu Tyr Val Thr Val Glu Met Gln
Lys Phe Leu Gly 355 360 365 Ser Phe Phe Ile Gly Trp Asp Leu Asp Leu
Tyr His Glu Glu Ser Asp 370 375 380 Gln Lys Ala Gln Val Asn Thr Ser
Asp Leu Asn Glu Glu Leu Gly Gln 385 390 395 400 Val Glu Tyr Val Phe
Thr Asp Lys Thr Gly Thr Leu Thr Glu Asn Glu 405 410 415 Met Gln Phe
Arg Glu Cys Ser Ile Asn Gly Met Lys Tyr Gln Glu Ile 420 425 430 Asn
Gly Arg Leu Val Pro Glu Gly Pro Thr Pro Asp Ser Ser Glu Gly 435 440
445 Asn Leu Ser Tyr Leu Ser Ser Leu Ser His Leu Asn Asn Leu Ser His
450 455 460 Leu Thr Thr Ser Ser Ser Phe Arg Thr Ser Pro Glu Asn Glu
Thr Glu 465 470 475 480 Leu Ile Lys Glu His Asp Leu Phe Phe Lys Ala
Val Ser Leu Cys His 485 490 495 Thr Val Gln Ile Ser Asn Val Gln Thr
Asp Cys Thr Gly Asp Gly Pro 500 505 510 Trp Gln Ser Asn Leu Ala Pro
Ser Gln Leu Glu Tyr Tyr Ala Ser Ser 515 520 525 Pro Asp Glu Lys Ala
Leu Val Glu Ala Ala Ala Arg Ile Gly Ile Val 530 535 540 Phe Ile Gly
Asn Ser Glu Glu Thr Met Glu Val Lys Thr Leu Gly Lys 545 550 555 560
Leu Glu Arg Tyr Lys Leu Leu His Ile Leu Glu Phe Asp Ser Asp Arg 565
570 575 Arg Arg Met Ser Val Ile Val Gln Ala Pro Ser Gly Glu Lys Leu
Leu 580 585 590 Phe Ala Lys Gly Ala Glu Ser Ser Ile Leu Pro Lys Cys
Ile Gly Gly 595 600 605 Glu Ile Glu Lys Thr Arg Ile His Val Asp Glu
Phe Ala Leu Lys Gly 610 615 620 Leu Arg Thr Leu Cys Ile Ala Tyr Arg
Lys Phe Thr Ser Lys Glu Tyr 625 630 635 640 Glu Glu Ile Asp Lys Arg
Ile Phe Glu Ala Arg Thr Ala Leu Gln Gln 645 650 655 Arg Glu Glu Lys
Leu Ala Ala Val Phe Gln Phe Ile Glu Lys Asp Leu 660 665 670 Ile Leu
Leu Gly Ala Thr Ala Val Glu Asp Arg Leu Gln Asp Lys Val 675 680 685
Arg Glu Thr Ile Glu Ala Leu Arg Met Ala Gly Ile Lys Val Trp Val 690
695 700 Leu Thr Gly Asp Lys His Glu Thr Ala Val Ser Val Ser Leu Ser
Cys 705 710 715 720 Gly His Phe His Arg Thr Met Asn Ile Leu Glu Leu
Ile Asn Gln Lys 725 730 735 Ser Asp Ser Glu Cys Ala Glu Gln Leu Arg
Gln Leu Ala Arg Arg Ile 740 745 750 Thr Glu Asp His Val Ile Gln His
Gly Leu Val Val Asp Gly Thr Ser 755 760 765 Leu Ser Leu Ala Leu Arg
Glu His Glu Lys Leu Phe Met Glu Val Cys 770 775 780 Arg Asn Cys Ser
Ala Val Leu Cys Cys Arg Met Ala Pro Leu Gln Lys 785 790 795 800 Ala
Lys Val Ile Arg Leu Ile Lys Ile Ser Pro Glu Lys Pro Ile Thr 805 810
815 Leu Ala Val Gly Asp Gly Ala Asn Asp Val Ser Met Ile Gln Glu Ala
820 825 830 His Val Gly Ile Gly Ile Met Gly Lys Glu Gly Arg Gln Ala
Ala Arg 835 840 845 Asn Ser Asp Tyr Ala Ile Ala Arg Phe Lys Phe Leu
Ser Lys Leu Leu 850 855 860 Phe Val His Gly His Phe Tyr Tyr Ile Arg
Ile Ala Thr Leu Val Gln 865 870 875 880 Tyr Phe Phe Tyr Lys Asn Val
Cys Phe Ile Thr Pro Gln Phe Leu Tyr 885 890 895 Gln Phe Tyr Cys Leu
Phe Ser Gln Gln Thr Leu Tyr Asp Ser Val Tyr 900 905 910 Leu Thr Leu
Tyr Asn Ile Cys Phe Thr Ser Leu Pro Ile Leu Ile Tyr 915 920 925 Ser
Leu Leu Glu Gln His Val Asp Pro His Val Leu Gln Asn Lys Pro 930 935
940 Thr Leu Tyr Arg Asp Ile Ser Lys Asn Arg Leu Leu Ser Ile Lys Thr
945 950 955 960 Phe Leu Tyr Trp Thr Ile Leu Gly Phe Ser His Ala Phe
Ile Phe Phe 965 970 975 Phe Gly Ser Tyr Leu Leu Ile Gly Lys Asp Thr
Ser Leu Leu Gly Asn 980 985 990 Gly Gln Met Phe Gly Asn Trp Thr Phe
Gly Thr Leu Val Phe Thr Val 995 1000 1005 Met Val Ile Thr Val Thr
Val Lys Met Ala Leu Glu Thr His Phe Trp 1010 1015 1020 Thr Trp Ile
Asn His Leu Val Thr Trp Gly Ser Ile Ile Phe Tyr Phe 1025 1030 1035
1040 Val Phe Ser Leu Phe Tyr Gly Gly Ile Leu Trp Pro Phe Leu Gly
Ser 1045 1050 1055 Gln Asn Met Tyr Phe Val Phe Ile Gln Leu Leu Ser
Ser Gly Ser Ala 1060 1065 1070 Trp Phe Ala Ile Ile Leu Met Val Val
Thr Cys Leu Phe Leu Asp Ile 1075 1080 1085 Ile Lys Lys Val Phe Asp
Arg His Leu His Pro Thr Ser Thr Glu Lys 1090 1095 1100 Ala Gln Leu
Thr Glu Thr Asn Ala Gly Ile Lys Cys Leu Asp Ser Met 1105 1110 1115
1120 Cys Cys Phe Pro Glu Gly Glu Ala Ala Cys Ala Ser Val Gly Arg
Met 1125 1130 1135 Leu Glu Arg Val Ile Gly Arg Cys Ser Pro Thr His
Ile Ser Arg Ser 1140 1145 1150 Trp Ser Ala Ser Asp Pro Phe Tyr Thr
Asn Asp Arg Ser Ile Leu Thr 1155 1160 1165 Leu Ser Thr Met Asp Ser
Ser Thr Cys 1170 1175 3 1125 DNA homo sapiens 3 atgtggcgct
ggatccggca gcagctgggt tttgacccac cacatcagag tgacacaaga 60
accatctacg tagccaacag gtttcctcag aatggccttt acacacctca gaaatttata
120 gataacagga tcatttcatc taagtacact gtgtggaatt ttgttccaaa
aaatttattt 180 gaacagttca gaagagtggc aaacttttat tttcttatta
tatttttggt tcagcttatg 240 attgatacac ctaccagtcc agttaccagt
ggacttccat tattctttgt gataacagta 300 actgccataa agcagggata
tgaagattgg ttacggcata actcagataa tgaagtaaat 360 ggagctcctg
tttatgttgt tcgaagtggt ggccttgtaa aaactagatc aaaaaacatt 420
cgggtgggtg atattgttcg aatagccaaa gatgaaattt ttcctgcaga cttggtgctt
480 ctgtcctcag atcgactgga tggttcctgt cacgttacaa ctgctagttt
ggacggagaa 540 actaacctga agacacatgt ggcagttcca gaaacagcat
tattacaaac agttgccaat 600 ttggacactc tagtagctgt aatagaatgc
cagcaaccag aagcagactt atacagattc 660 atgggacgaa tgatcataac
ccaacaaatg gaagaaattg taagacctct ggggccggag 720 agtctcctgc
ttcgtggagc cagattaaaa aacacaaaag aaatttttgg tgttgcggta 780
tacactggaa tggaaactaa gatggcatta aattacaaga gcaaatcaca gaaacgatct
840 gcagtagaaa agtcaatgaa tacatttttg ataatttatc tagtaattct
tatatctgaa 900 gctgtcatca gcactatctt gaagtataca tggcaagctg
aagaaaaatg ggatgaacct 960 tggtataacc aaaaaacaga acatcaaaga
aatagcaatt ctgagattta tttcagactt 1020 ccttgctttt ttggttctct
acaatttcat cattccaatt tcattatatg tgacagtcga 1080 atgcagaaa
tttcttggat cattttttat tggctgggat cttga 1125 4 374 PRT homo sapiens
4 Met Trp Arg Trp Ile Arg Gln Gln Leu Gly Phe Asp Pro Pro His Gln 1
5 10 15 Ser Asp Thr Arg Thr Ile Tyr Val Ala Asn Arg Phe Pro Gln Asn
Gly 20 25 30 Leu Tyr Thr Pro Gln Lys Phe Ile Asp Asn Arg Ile Ile
Ser Ser Lys 35 40 45 Tyr Thr Val Trp Asn Phe Val Pro Lys Asn Leu
Phe Glu Gln Phe Arg 50 55 60 Arg Val Ala Asn Phe Tyr Phe Leu Ile
Ile Phe Leu Val Gln Leu Met 65 70 75 80 Ile Asp Thr Pro Thr Ser Pro
Val Thr Ser Gly Leu Pro Leu Phe Phe 85 90 95 Val Ile Thr Val Thr
Ala Ile Lys Gln Gly Tyr Glu Asp Trp Leu Arg 100 105 110 His Asn Ser
Asp Asn Glu Val Asn Gly Ala Pro Val Tyr Val Val Arg 115 120 125 Ser
Gly Gly Leu Val Lys Thr Arg Ser Lys Asn Ile Arg Val Gly Asp 130 135
140 Ile Val Arg Ile Ala Lys Asp Glu Ile Phe Pro Ala Asp Leu Val Leu
145 150 155 160 Leu Ser Ser Asp Arg Leu Asp Gly Ser Cys His Val Thr
Thr Ala Ser 165 170 175 Leu Asp Gly Glu Thr Asn Leu Lys Thr His Val
Ala Val Pro Glu Thr 180 185 190 Ala Leu Leu Gln Thr Val Ala Asn Leu
Asp Thr Leu Val Ala Val Ile 195 200 205 Glu Cys Gln Gln Pro Glu Ala
Asp Leu Tyr Arg Phe Met Gly Arg Met 210 215 220 Ile Ile Thr Gln Gln
Met Glu Glu Ile Val Arg Pro Leu Gly Pro Glu 225 230 235 240 Ser Leu
Leu Leu Arg Gly Ala Arg Leu Lys Asn Thr Lys Glu Ile Phe 245 250 255
Gly Val Ala Val Tyr Thr Gly Met Glu Thr Lys Met Ala Leu Asn Tyr 260
265 270 Lys Ser Lys Ser Gln Lys Arg Ser Ala Val Glu Lys Ser Met Asn
Thr 275 280 285 Phe Leu Ile Ile Tyr Leu Val Ile Leu Ile Ser Glu Ala
Val Ile Ser 290 295 300 Thr Ile Leu Lys Tyr Thr Trp Gln Ala Glu Glu
Lys Trp Asp Glu Pro 305 310 315 320 Trp Tyr Asn Gln Lys Thr Glu His
Gln Arg Asn Ser Asn Ser Glu Ile 325 330 335 Tyr Phe Arg Leu Pro Cys
Phe Phe Gly Ser Leu Gln Phe His His Ser 340 345 350 Asn Phe Ile Ile
Cys Asp Ser Arg Asn Ala Glu Ile Ser Trp Ile Ile 355 360 365 Phe Tyr
Trp Leu Gly Ser 370 5 7277 DNA homo sapiens 5 gccgcgggat gggaacgcgg
cgcggggagt gaggcagtgg cggcggcggc ggtaagcgga 60 acttcggccc
gaggggctcg cccgctcccg cctctgtctt gtcggcctcc acctgcagcc 120
ccgcggcccc cgcgccccgc gggacccgga cggcgacgac gggggaatgt ggcgctggat
180 ccggcagcag ctgggttttg acccaccaca tcagagtgac acaagaacca
tctacgtagc 240 caacaggttt cctcagaatg gcctttacac acctcagaaa
tttatagata acaggatcat 300 ttcatctaag tacactgtgt ggaattttgt
tccaaaaaat ttatttgaac agttcagaag 360 agtggcaaac ttttattttc
ttattatatt tttggttcag cttatgattg atacacctac 420 cagtccagtt
accagtggac ttccattatt ctttgtgata acagtaactg ccataaagca 480
gggatatgaa gattggttac ggcataactc agataatgaa gtaaatggag ctcctgttta
540 tgttgttcga agtggtggcc ttgtaaaaac tagatcaaaa aacattcggg
tgggtgatat 600 tgttcgaata gccaaagatg aaatttttcc tgcagacttg
gtgcttctgt cctcagatcg 660 actggatggt tcctgtcacg ttacaactgc
tagtttggac ggagaaacta acctgaagac 720 acatgtggca
gttccagaaa cagcattatt acaaacagtt gccaatttgg acactctagt 780
agctgtaata gaatgccagc aaccagaagc agacttatac agattcatgg gacgaatgat
840 cataacccaa caaatggaag aaattgtaag acctctgggg ccggagagtc
tcctgcttcg 900 tggagccaga ttaaaaaaca caaaagaaat ttttggtgtt
gcggtataca ctggaatgga 960 aactaagatg gcattaaatt acaagagcaa
atcacagaaa cgatctgcag tagaaaagtc 1020 aatgaataca tttttgataa
tttatctagt aattcttata tctgaagctg tcatcagcac 1080 tatcttgaag
tatacatggc aagctgaaga aaaatgggat gaaccttggt ataaccaaaa 1140
aacagaacat caaagaaata gcagtaagat tctgagattt atttcagact tccttgcttt
1200 tttggttctc tacaatttca tcattccaat ttcattatat gtgacagtcg
aaatgcagaa 1260 atttcttgga tcatttttta ttggctggga tcttgatctg
tatcatgaag aatcagatca 1320 gaaagctcaa gtcaatactt ccgatctgaa
tgaagagctt ggacaggtag agtacgtgtt 1380 tacagataaa actggtacac
tgacagaaaa tgagatgcag tttcgggaat gttcaattaa 1440 tggcatgaaa
taccaagaaa ttaatggtag acttgtaccc gaaggaccaa caccagactc 1500
ttcagaagga aacttatctt atcttagtag tttatcccat cttaacaact tatcccatct
1560 tacaaccagt tcctctttca gaaccagtcc tgaaaatgaa actgaactaa
ttaaagaaca 1620 tgatctcttc tttaaagcag tcagtctctg tcacactgta
cagattagca atgttcaaac 1680 tgactgcact ggtgatggtc cctggcaatc
caacctggca ccatcgcagt tggagtacta 1740 tgcatcttca ccagatgaaa
aggctctagt agaagctgct gcaaggattg gtattgtgtt 1800 tattggcaat
tctgaagaaa ctatggaggt taaaactctt ggaaaactgg aacggtacaa 1860
actgcttcat attctggaat ttgattcaga tcgtaggaga atgagtgtaa ttgttcaggc
1920 accttcaggt gagaagttat tatttgctaa aggagctgag tcatcaattc
tccctaaatg 1980 tataggtgga gaaatagaaa aaaccagaat tcatgtagat
gaatttgctt tgaaagggct 2040 aagaactctg tgtatagcat atagaaaatt
tacatcaaaa gagtatgagg aaatagataa 2100 acgcatattt gaagccagga
ctgccttgca gcagcgggaa gagaaattgg cagctgtttt 2160 ccagttcata
gagaaagacc tgatattact tggagccaca gcagtagaag acagactaca 2220
agataaagtt cgagaaacta ttgaagcatt gagaatggct ggtatcaaag tatgggtact
2280 tactggggat aaacatgaaa cagctgttag tgtgagttta tcatgtggcc
attttcatag 2340 aaccatgaac atccttgaac ttataaacca gaaatcagac
agcgagtgtg ctgaacaatt 2400 gaggcagctt gccagaagaa ttacagagga
tcatgtgatt cagcatgggc tggtagtgga 2460 tgggaccagc ctatctcttg
cactcaggga gcatgaaaaa ctatttatgg aagtttgcag 2520 aaattgttca
gctgtattat gctgtcgtat ggctccactg cagaaagcaa aagtaataag 2580
actaataaaa atatcacctg agaaacctat aacattggct gttggtgatg gtgctaatga
2640 cgtaagcatg atacaagaag cccatgttgg cataggaatc atgggtaaag
aaggaagaca 2700 ggctgcaaga aacagtgact atgcaatagc cagatttaag
ttcctctcca aattgctttt 2760 tgttcatggt catttttatt atattagaat
agctaccctt gtacagtatt ttttttataa 2820 gaatgtgtgc tttatcacac
cccagttttt atatcagttc tactgtttgt tttctcagca 2880 aacattgtat
gacagcgtgt acctgacttt atacaatatt tgttttactt ccctacctat 2940
tctgatatat agtcttttgg aacagcatgt agaccctcat gtgttacaaa ataagcccac
3000 cctttatcga gacattagta aaaaccgcct cttaagtatt aaaacatttc
tttattggac 3060 catcctgggc ttcagtcatg cctttatttt cttttttgga
tcctatttac taatagggaa 3120 agatacatct ctgcttggaa atggccagat
gttyggaaac tggacatttg gcactttggt 3180 cttcacagtc atggttatta
cagtcacagt aaagatggct ctggaaactc atttttggac 3240 ttggatcaac
catctcgtta cctggggatc tattatattt tattttgtat tttccttgtt 3300
ttatggaggg attctctggc catttttggg ctcccagaat atgtattttg tgtttattca
3360 gctcctgtca agtggttctg cttggtttgc cataatcctc atggttgtta
catgtctatt 3420 tcttgatatc ataaagaagg tctttgaccg acacctccac
cctacaagta ctgaaaaggc 3480 acagcttact gaaacaaatg caggtatcaa
gtgcttggac tccatgtgct gtttcccgga 3540 aggagaagca gcgtgtgcat
ctgttggaag aatgctggaa cgagttatag gaagatgtag 3600 tccaacccac
atcagcagat catggagtgc atcggatcct ttctatacca acgacaggag 3660
catcttgact ctctccacaa tggactcatc tacttgttaa aggggcagta gtactttgtg
3720 ggagccagtt cacctccttt cctaaaattc agtgtgatca ccctgttaat
ggccacacta 3780 gctctgaaat taatttccaa aatctttgta gtagttcata
cccactcaga gttataatgg 3840 caaacaaaca gaaagcatta gtacaagccc
ctcccaacac ccttaatttg aatctgaaca 3900 tgttaaaatt tgagaataaa
gagacatttt tcatctcttt gtctggtttg tcccttgtgc 3960 ttatgggact
cctaatggca tttcagtctg ttgctgaggc cattatattt taatataaat 4020
gtagaaaaaa gagagaaatc ttagtaaaga gtatttttta gtattagctt gattattgac
4080 tcttctattt aaatctgctt ctgtaaatta tgctgaaagt ttgccttgag
aactctattt 4140 ttttattaga gttatattta aagcttttca tgggaaaagt
taatgtgaat actgaggaat 4200 tttggtccct cagtgacctg tgttgttaat
tcattaatgc attctgagtt cacagagcaa 4260 attaggagaa tcatttccaa
ccattattta ctgcagtatg gggagtaaat ttataccaat 4320 tcctctaact
gtactgtaac acagcctgta aagttagcca tataaatgca agggtatatc 4380
atatatacaa atcaggaatc aggtccgttc accgaacttc aaattgatgt ttactaatat
4440 ttttgtgaca gagtataaag accctatagt gggtaaatta gatactatta
gcatattatt 4500 aatttaatgt ctttatcatt ggatcttttg catgctttaa
tctggttaac atatttaaat 4560 ttgctttttt tctctttacc tgaaggctct
gtgtatagta tttcatgaca tcgttgtaca 4620 gtttaactat atcaataaaa
agtttggaca gtatttaaat attgcaaata tgtttaatta 4680 tacaaatcag
aatagtatgg gtaattaaat gaatacaaaa agaagagcct ctttctgcag 4740
ccgacttaga catgctcttc cctttctata agctagattt tagaataaag ggtttcagtt
4800 aataatctta ttttcaggtt atgtcatcta acttatagca aactaccaca
atacagtgag 4860 ttctgccagt gtcccagtac aaggcatatt tcaggtgtgg
ctgtggaatg taaaaatgct 4920 caacttgtat caggtaatgt tagcaataaa
ttaaatgcta agaatgatta atcgggtaca 4980 tgttactgta attaactcat
tgcacttcaa aacctaactt ccatcctgaa tttatcaagt 5040 agttcagtat
tgtcatttgt ttttgtttta ttgaaaagta atgttgtctt aagatttaga 5100
agtgattatt agcttgagaa ctattaccca gctctaagca aataatgatt gtatacatat
5160 taagataatg gttaaatgcg gttttaccaa gttttccctt gaaaatgtaa
ttcctttatg 5220 gagatttatt gtgcagccct aagcttcctt cccatttcat
gaatataagg cttctagaat 5280 tggactggca ggggaaagaa tggtagagac
agaaattaag actttatcct tgtttgcttg 5340 taaactatta ttttcttgct
aatgtaacat ttgtctgttc cagtgatgta aggatattaa 5400 gttattaagc
taaatattaa ttttcaaaaa tagtccttct ttaacttaga tatttcatag 5460
ctggatttag gaagatctgt tattctggaa gtactaaaaa gaataataca acgtacaatg
5520 tctgcattca ctaattcatg ttccagaaga ggaaataatg aagatatact
cagtagagta 5580 ctaggtggga ggatatggaa atttgctcat aaaatctctt
ataaaacgtg catataacaa 5640 aatgacaccc agtaggcctg cattacattt
acatgaccgt gtttatttgc catcaaataa 5700 actgagtact gacaccagac
aaagactcca aagtcataaa atagcctatg accaactgca 5760 gcaagacagg
aggtcagctc gcctataatg gtgcttaaag tgtgattgat gtaattttct 5820
gtactcacca tttgaagtta gttaaggaga actttatttt tttaaaaaaa gtaaatggca
5880 accactagtg tgctcatcct gaactgttac tccaaatcca ctccgttttt
aaagcaaaat 5940 tatcttgtga ttttaagaaa agagttttct atttatttaa
gaaagtaaca atgcagtctg 6000 caagctttca gtagttttct agtgctatat
tcatcctgta aaactcttac tacgtaacca 6060 gtaatcacaa ggaaagtgtc
ccctttgcat atttctttaa aattctttct ttggaaagta 6120 tgatgttgat
aattaactta cccttatctg ccaaaaccag agcaaaatgc taaatacgtt 6180
attgctaatc agtggtctca aatcgatttg cctccctttg cctcgtctga gggctgtaag
6240 cctgaagata gtggcaagca ccaagtcagt ttccaaaatt gcccctcagc
tgctttaagt 6300 gactcagcac cctgcctcag cttcagcagg cstaggctca
ccctgggcgg agcaaagtat 6360 gggccaggga gaactacagc tacgaagacc
tgctgtcgag ttgagaaaag gggagaattt 6420 atggtctgaa ttttctaact
gtcctctttc ttgggtctaa agctcataat acacaaaggc 6480 ttccagacct
gagccacacc caggccctat cctgaacagg agactaaaca gaggcaaatc 6540
aaccctagga aatacttgca ttctgcccta cggttagtac caggactgag gtcatttcta
6600 ctggaaaaga ttgtgagatt gaacttatct gatcgcttga gactcctaat
aggcaggagt 6660 caaggccact agaaaattga cagttaagag ccaaaagttt
ttaaaatatg ctactctgaa 6720 aaatctcgtg aaggctgtag gaaaagggag
aatcttccat gttggtgttt ttcctgtaaa 6780 gatcagtttg gggtatgata
taagcaggta ttaataaaaa taacacacca aagagttacg 6840 taaaacatgt
tttattaatt ttggtcccca cgtacagaca ttttatttct attttgaaat 6900
gagttatcta ttttcataaa agtaaaacac tattaaagtg ctgttttatg tgaaataact
6960 tgaatgttgt tcctataaaa aatagatcat aactcatgat atgtttgtaa
tcatggtaat 7020 ttagattttt atgaggaatg agtatctgga aatattgtag
caatacttgg tttaaaattt 7080 tggacctgag acactgtggc tgtctaatgt
aatcctttaa aaattctctg cattgtcagt 7140 aaatgtagta tattattgta
cagctactca taatttttta aagtttatga agttatattt 7200 tcaaataaa
aactttccta tataattaaa aaaaaaaaaa aaaaaaaaaa aaaaaacaaa 7260
aaaaaaaaa aaaaaaa 7277 6 2913 DNA homo sapiens 6 atggcacaac
tagagaggag cgccatctct ggcttcagct ctaagtccag gcgaaactca 60
ttcgcatatg atgttaagcg tgaagtatac aatgaggaga cctttcaaca ggaacacaaa
120 aggaaggcct cctcttctgg gaacatgaac atcaacatca ccaccttcag
acaccacgtc 180 cagtgccgct gctcatggca caggttccta cgatgcrtgc
ttacaatctt tcccttccta 240 gaatggatgt gtatgtatcg attaaaggat
tggcttctgg gagacttact tgctggtata 300 agtgttggcc ttgtgcaagt
tccccaaggc ctgacactta gtttgctggc aaggcaactg 360 attcctcctc
tcaacatcgc ttatgcagct ttctgttctt cggtaatcta tgtaattttt 420
ggatcgtgtc atcaaatgtc cgttggttcc ttcttcctgg tgagtgctct gctgatcaac
480 gttctgaaag tgagcccatt caacaacggt caactggtca tgggatcttt
cgtcaagaat 540 gagttttcgg ccccctccta ccttatgggc tataataaat
ccttgagtgt ggtggcaacc 600 acaacttttc tgactgggat tattcagcta
ataatgggcg tattgggttt gggcttcatt 660 gccacttacc ttccggagtc
tgcaatgaat gcttacctgg ctgctgtggc acttcatatc 720 atgctgtccc
agctgacttt catctttggg attatgatta gtttccatgc cggtcccatc 780
tccttcttct atgacataat taattactgt gtagctctcc caaaagcgaa ttccaccagc
840 attctagtat ttctaactgt tgttgttgct ctgcgaatca acaaatgtat
cagaatttct 900 ttcaatcagt atcccattga gtttcccatg gaattatttc
tgattattgg cttcactgtg 960 attgcaaaca agataagcat ggccacagaa
accagccaga cgcttattga catgattcct 1020 tatagctttc tgcttcctgt
aacaccagat ttcagccttc ttcccaagat aattttacaa 1080 gccttctcct
tatctttggt gagctccttt ctgctcatat ttctgggcaa gaagattgcc 1140
agtcttcaca attacagtgt caattccaac caggatttaa tagccatcgg cctttgcaat
1200 gtcgtcagtt catttttcag atcttgtgtg tttactggtg ctattgctag
gactattatc 1260 caggataaat ctggaggaag acaacagttt gcatctctgg
taggcgcagg tgtgatgctg 1320 ctcctgatgg tgaagatggg acactttttc
tacacactgc caaatgctgt gctggctggt 1380 attattctga gcaacgtcat
tccctacctt gaaaccattt ctaacctacc cagcctgtgg 1440 aggcaggacc
aatatgactg tgctctttgg atgatgacat tctcatcttc aattttcctg 1500
ggactggaca ttggactaat tatctcagta gtttctgctt tcttcatcac cactgttcgt
1560 tcacacagag ctaagattct tctcctgggt caaatcccta acaccaacat
ttatagaagc 1620 atcaatgatt atcgggagat catcaccatt cctggggtga
aaatcttcca gtgctgcagc 1680 tcaattacat ttgtaaatgt ttactaccta
aagcataagc tgttaaaaga ggttgatatg 1740 gtaaaggtgc ctcttaaaga
agaagaaatt ttcagcttgt ttaattcaag tgacaccaat 1800 ctacaaggag
gaaagatttg caggtgtttc tgcaactgtg atgatctgga gccgctgccc 1860
aggattcttt acacagagcg atttgaaaat aaactggatc ccgaagcatc ctccattaac
1920 ctgattcact gctcacattt tgagagcatg aacacaagcc aaactgcatc
cgaagaccaa 1980 gtgccataca cagtatcgtc cgtgtctcag aaaaatcaag
ggcaacagta tgaggaggtg 2040 gaggaagttt ggcttcctaa taactcatca
agaaacagct caccaggact gcctgatgtg 2100 gcggaaagcc aggggaggag
atcactcatc ccttactcag atgcgtctct actgcccagt 2160 gtccacacca
tcatcctgga tttctccatg gtacactacg tggattcacg ggggttagtc 2220
gtattaagac agatatgcaa tgcctttcaa aacgccaaca ttttgatact cattgcaggg
2280 tgtcactctt ccatagtcag ggcatttgag aggaatgatt tctttgacgc
tggcatcacc 2340 aagacccagc tgttcctcag cgttcacgac gccgtgctgt
ttgccttgtc aaggaaggtc 2400 ataggctcct ctgagttaag catcgatgaa
tccgagacag tgatacggga aacctactca 2460 gaaacagaca agaatgacaa
ttcaagatat aaaatgagca gcagttttct aggaagccaa 2520 aaaaatgtaa
gtccaggctt catcaagatc caacagcctg tagaagagga gtcggagttg 2580
gatttggagc tggaatcaga acaagaggct gggctgggtc tggacctaga cctggatcgg
2640 gagctggagc ctgaaatgga gcccaaggct gagaccgaga ccaagaccca
gaccgagatg 2700 gagccccagc ctgagactga gcctgagatg gagcccaacc
ccaaatctag gccaagagct 2760 cacacttttc ctcagcagcg ttactggcct
atgtatcatc cgtctatggc ttccacccag 2820 tctcagactc agactcggac
atggtcagtg gagaggagac gccatcctat ggattcatac 2880 caccagagg
gcaacagcaa tgaagatgtc tag 2913 7 970 PRT homo sapiens VARIANT
(1)...(970) Xaa = Any Amino Acid 7 Met Ala Gln Leu Glu Arg Ser Ala
Ile Ser Gly Phe Ser Ser Lys Ser 1 5 10 15 Arg Arg Asn Ser Phe Ala
Tyr Asp Val Lys Arg Glu Val Tyr Asn Glu 20 25 30 Glu Thr Phe Gln
Gln Glu His Lys Arg Lys Ala Ser Ser Ser Gly Asn 35 40 45 Met Asn
Ile Asn Ile Thr Thr Phe Arg His His Val Gln Cys Arg Cys 50 55 60
Ser Trp His Arg Phe Leu Arg Cys Met Leu Thr Ile Phe Pro Phe Leu 65
70 75 80 Glu Trp Met Cys Met Tyr Arg Leu Lys Asp Trp Leu Leu Gly
Asp Leu 85 90 95 Leu Ala Gly Ile Ser Val Gly Leu Val Gln Val Pro
Gln Gly Leu Thr 100 105 110 Leu Ser Leu Leu Ala Arg Gln Leu Ile Pro
Pro Leu Asn Ile Ala Tyr 115 120 125 Ala Ala Phe Cys Ser Ser Val Ile
Tyr Val Ile Phe Gly Ser Cys His 130 135 140 Gln Met Ser Val Gly Ser
Phe Phe Leu Val Ser Ala Leu Leu Ile Asn 145 150 155 160 Val Leu Lys
Val Ser Pro Phe Asn Asn Gly Gln Leu Val Met Gly Ser 165 170 175 Phe
Val Lys Asn Glu Phe Ser Ala Pro Ser Tyr Leu Met Gly Tyr Asn 180 185
190 Lys Ser Leu Ser Val Val Ala Thr Thr Thr Phe Leu Thr Gly Ile Ile
195 200 205 Gln Leu Ile Met Gly Val Leu Gly Leu Gly Phe Ile Ala Thr
Tyr Leu 210 215 220 Pro Glu Ser Ala Met Asn Ala Tyr Leu Ala Ala Val
Ala Leu His Ile 225 230 235 240 Met Leu Ser Gln Leu Thr Phe Ile Phe
Gly Ile Met Ile Ser Phe His 245 250 255 Ala Gly Pro Ile Ser Phe Phe
Tyr Asp Ile Ile Asn Tyr Cys Val Ala 260 265 270 Leu Pro Lys Ala Asn
Ser Thr Ser Ile Leu Val Phe Leu Thr Val Val 275 280 285 Val Ala Leu
Arg Ile Asn Lys Cys Ile Arg Ile Ser Phe Asn Gln Tyr 290 295 300 Pro
Ile Glu Phe Pro Met Glu Leu Phe Leu Ile Ile Gly Phe Thr Val 305 310
315 320 Ile Ala Asn Lys Ile Ser Met Ala Thr Glu Thr Ser Gln Thr Leu
Ile 325 330 335 Asp Met Ile Pro Tyr Ser Phe Leu Leu Pro Val Thr Pro
Asp Phe Ser 340 345 350 Leu Leu Pro Lys Ile Ile Leu Gln Ala Phe Ser
Leu Ser Leu Val Ser 355 360 365 Ser Phe Leu Leu Ile Phe Leu Gly Lys
Lys Ile Ala Ser Leu His Asn 370 375 380 Tyr Ser Val Asn Ser Asn Gln
Asp Leu Ile Ala Ile Gly Leu Cys Asn 385 390 395 400 Val Val Ser Ser
Phe Phe Arg Ser Cys Val Phe Thr Gly Ala Ile Ala 405 410 415 Arg Thr
Ile Ile Gln Asp Lys Ser Gly Gly Arg Gln Gln Phe Ala Ser 420 425 430
Leu Val Gly Ala Gly Val Met Leu Leu Leu Met Val Lys Met Gly His 435
440 445 Phe Phe Tyr Thr Leu Pro Asn Ala Val Leu Ala Gly Ile Ile Leu
Ser 450 455 460 Asn Val Ile Pro Tyr Leu Glu Thr Ile Ser Asn Leu Pro
Ser Leu Trp 465 470 475 480 Arg Gln Asp Gln Tyr Asp Cys Ala Leu Trp
Met Met Thr Phe Ser Ser 485 490 495 Ser Ile Phe Leu Gly Leu Asp Ile
Gly Leu Ile Ile Ser Val Val Ser 500 505 510 Ala Phe Phe Ile Thr Thr
Val Arg Ser His Arg Ala Lys Ile Leu Leu 515 520 525 Leu Gly Gln Ile
Pro Asn Thr Asn Ile Tyr Arg Ser Ile Asn Asp Tyr 530 535 540 Arg Glu
Ile Ile Thr Ile Pro Gly Val Lys Ile Phe Gln Cys Cys Ser 545 550 555
560 Ser Ile Thr Phe Val Asn Val Tyr Tyr Leu Lys His Lys Leu Leu Lys
565 570 575 Glu Val Asp Met Val Lys Val Pro Leu Lys Glu Glu Glu Ile
Phe Ser 580 585 590 Leu Phe Asn Ser Ser Asp Thr Asn Leu Gln Gly Gly
Lys Ile Cys Arg 595 600 605 Cys Phe Cys Asn Cys Asp Asp Leu Glu Pro
Leu Pro Arg Ile Leu Tyr 610 615 620 Thr Glu Arg Phe Glu Asn Lys Leu
Asp Pro Glu Ala Ser Ser Ile Asn 625 630 635 640 Leu Ile His Cys Ser
His Phe Glu Ser Met Asn Thr Ser Gln Thr Ala 645 650 655 Ser Glu Asp
Gln Val Pro Tyr Thr Val Ser Ser Val Ser Gln Lys Asn 660 665 670 Gln
Gly Gln Gln Tyr Glu Glu Val Glu Glu Val Trp Leu Pro Asn Asn 675 680
685 Ser Ser Arg Asn Ser Ser Pro Gly Leu Pro Asp Val Ala Glu Ser Gln
690 695 700 Gly Arg Arg Ser Leu Ile Pro Tyr Ser Asp Ala Ser Leu Leu
Pro Ser 705 710 715 720 Val His Thr Ile Ile Leu Asp Phe Ser Met Val
His Tyr Val Asp Ser 725 730 735 Arg Gly Leu Val Val Leu Arg Gln Ile
Cys Asn Ala Phe Gln Asn Ala 740 745 750 Asn Ile Leu Ile Leu Ile Ala
Gly Cys His Ser Ser Ile Val Arg Ala 755 760 765 Phe Glu Arg Asn Asp
Phe Phe Asp Ala Gly Ile Thr Lys Thr Gln Leu 770 775 780 Phe Leu Ser
Val His Asp Ala Val Leu Phe Ala Leu Ser Arg Lys Val 785 790 795 800
Ile Gly Ser Ser Glu Leu Ser Ile Asp Glu Ser Glu Thr Val Ile Arg 805
810 815 Glu Thr Tyr Ser Glu Thr Asp Lys Asn Asp Asn Ser Arg Tyr Lys
Met 820 825 830 Ser Ser Ser Phe Leu Gly Ser Gln Lys Asn Val Ser Pro
Gly Phe Ile 835 840 845 Lys Ile Gln Gln Pro Val Glu Glu Glu Ser Glu
Leu Asp Leu Glu Leu 850 855 860 Glu Ser Glu Gln Glu Ala Gly Leu Gly
Leu Asp Leu Asp Leu Asp Arg 865 870 875 880 Glu Leu Glu Pro Glu Met
Glu Pro Lys Ala Glu Thr Glu Thr
Lys Thr 885 890 895 Gln Thr Glu Met Glu Pro Gln Pro Glu Thr Glu Pro
Glu Met Glu Pro 900 905 910 Asn Pro Lys Ser Arg Pro Arg Ala His Thr
Phe Pro Gln Gln Arg Tyr 915 920 925 Trp Pro Met Tyr His Pro Ser Met
Ala Ser Thr Gln Ser Gln Thr Gln 930 935 940 Thr Arg Thr Trp Ser Val
Glu Arg Arg Arg His Pro Met Asp Ser Tyr 945 950 955 960 Ser Pro Glu
Gly Asn Ser Asn Glu Asp Val 965 970 8 3749 DNA homo sapiens 8
ttttccaact ccccatctcc tccctcctca gattaaaaga agttatatgg actttgtgat
60 gttttctgcc gctttgtgaa gtaggcctta tttctcttgt cctttcgtac
agggaggaat 120 ttgaagtaga tagaaaccga cctggattac tccggtctga
actcagatca cgtaggactt 180 taatcgttga acaaacgaac ctttaatagc
ggctgcacca tcgggatgtc ctgatccaac 240 atcgaggtcg taaaccctat
tgttgatatg gactctagaa taggattgcg ctgttatccc 300 tagggtaact
tgttccgttg gtcaagttat tggatcaatt gagtatagta gttcgctttg 360
actggtgaag tcttggcatg tactgctcgg aggttgggtt ctgctccgag gtcgccccaa
420 ccgaaatttt taatgcagga gcgcccgcac tcccgccccc gccaaggagc
caggaatggc 480 acaactagag aggagcgcca tctctggctt cagctctaag
tccaggcgaa actcattcgc 540 atatgatgtt aagcgtgaag tatacaatga
ggagaccttt caacaggaac acaaaaggaa 600 ggcctcctct tctgggaaca
tgaacatcaa catcaccacc ttcagacacc acgtccagtg 660 ccgctgctca
tggcacaggt tcctacgatg crtgcttaca atctttccct tcctagaatg 720
gatgtgtatg tatcgattaa aggattggct tctgggagac ttacttgctg gtataagtgt
780 tggccttgtg caagttcccc aaggcctgac acttagtttg ctggcaaggc
aactgattcc 840 tcctctcaac atcgcttatg cagctttctg ttcttcggta
atctatgtaa tttttggatc 900 gtgtcatcaa atgtccgttg gttccttctt
cctggtgagt gctctgctga tcaacgttct 960 gaaagtgagc ccattcaaca
acggtcaact ggtcatggga tctttcgtca agaatgagtt 1020 ttcggccccc
tcctacctta tgggctataa taaatccttg agtgtggtgg caaccacaac 1080
ttttctgact gggattattc agctaataat gggcgtattg ggtttgggct tcattgccac
1140 ttaccttccg gagtctgcaa tgaatgctta cctggctgct gtggcacttc
atatcatgct 1200 gtcccagctg actttcatct ttgggattat gattagtttc
catgccggtc ccatctcctt 1260 cttctatgac ataattaatt actgtgtagc
tctcccaaaa gcgaattcca ccagcattct 1320 agtatttcta actgttgttg
ttgctctgcg aatcaacaaa tgtatcagaa tttctttcaa 1380 tcagtatccc
attgagtttc ccatggaatt atttctgatt attggcttca ctgtgattgc 1440
aaacaagata agcatggcca cagaaaccag ccagacgctt attgacatga ttccttatag
1500 ctttctgctt cctgtaacac cagatttcag ccttcttccc aagataattt
tacaagcctt 1560 ctccttatct ttggtgagct cctttctgct catatttctg
ggcaagaaga ttgccagtct 1620 tcacaattac agtgtcaatt ccaaccagga
tttaatagcc atcggccttt gcaatgtcgt 1680 cagttcattt ttcagatctt
gtgtgtttac tggtgctatt gctaggacta ttatccagga 1740 taaatctgga
ggaagacaac agtttgcatc tctggtaggc gcaggtgtga tgctgctcct 1800
gatggtgaag atgggacact ttttctacac actgccaaat gctgtgctgg ctggtattat
1860 tctgagcaac gtcattccct accttgaaac catttctaac ctacccagcc
tgtggaggca 1920 ggaccaatat gactgtgctc tttggatgat gacattctca
tcttcaattt tcctgggact 1980 ggacattgga ctaattatct cagtagtttc
tgctttcttc atcaccactg ttcgttcaca 2040 cagagctaag attcttctcc
tgggtcaaat ccctaacacc aacatttata gaagcatcaa 2100 tgattatcgg
gagatcatca ccattcctgg ggtgaaaatc ttccagtgct gcagctcaat 2160
tacatttgta aatgtttact acctaaagca taagctgtta aaagaggttg atatggtaaa
2220 ggtgcctctt aaagaagaag aaattttcag cttgtttaat tcaagtgaca
ccaatctaca 2280 aggaggaaag atttgcaggt gtttctgcaa ctgtgatgat
ctggagccgc tgcccaggat 2340 tctttacaca gagcgatttg aaaataaact
ggatcccgaa gcatcctcca ttaacctgat 2400 tcactgctca cattttgaga
gcatgaacac aagccaaact gcatccgaag accaagtgcc 2460 atacacagta
tcgtccgtgt ctcagaaaaa tcaagggcaa cagtatgagg aggtggagga 2520
agtttggctt cctaataact catcaagaaa cagctcacca ggactgcctg atgtggcgga
2580 aagccagggg aggagatcac tcatccctta ctcagatgcg tctctactgc
ccagtgtcca 2640 caccatcatc ctggatttct ccatggtaca ctacgtggat
tcacgggggt tagtcgtatt 2700 aagacagata tgcaatgcct ttcaaaacgc
caacattttg atactcattg cagggtgtca 2760 ctcttccata gtcagggcat
ttgagaggaa tgatttcttt gacgctggca tcaccaagac 2820 ccagctgttc
ctcagcgttc acgacgccgt gctgtttgcc ttgtcaagga aggtcatagg 2880
ctcctctgag ttaagcatcg atgaatccga gacagtgata cgggaaacct actcagaaac
2940 agacaagaat gacaattcaa gatataaaat gagcagcagt tttctaggaa
gccaaaaaaa 3000 tgtaagtcca ggcttcatca agatccaaca gcctgtagaa
gaggagtcgg agttggattt 3060 ggagctggaa tcagaacaag aggctgggct
gggtctggac ctagacctgg atcgggagct 3120 ggagcctgaa atggagccca
aggctgagac cgagaccaag acccagaccg agatggagcc 3180 ccagcctgag
actgagcctg agatggagcc caaccccaaa tctaggccaa gagctcacac 3240
ttttcctcag cagcgttact ggcctatgta tcatccgtct atggcttcca cccagtctca
3300 gactcagact cggacatggt cagtggagag gagacgccat cctatggatt
catactcacc 3360 agagggcaac agcaatgaag atgtctagga gatgaactag
aaataagggg tcagataatg 3420 ctggcaaatc ctcctaccca aaaaggggtc
aattgtccag agacctagac tggatacgaa 3480 ctagcagtac ttccttcctg
actgtgactc ctactacctg ccagccttct tccttgctct 3540 gcgctgggat
catactccca aatcacatta ctaaatgcca acaattatct ctgaattccc 3600
tatccaggct cccctcattt caccttcagc atatattcta gtcatgaatt tccttcttca
3660 cacaccccac atctctgggc tttgtgccag accatctcta acttaatcct
ctcatccctg 3720 ttcccctttc tccaaagaga tgaagctca 3749 9 1524 DNA
homo sapiens 9 atggggtgtt ggggtcggaa ccggggccgg ctgctgtgca
tgctggcgct gaccttcatg 60 ttcatggtgc tggaggtggt ggtgagccgg
gtgacctcgt cgctggcgat gctctccgac 120 tccttccaca tgctgtcgga
cgtgctggcg ctggtggtgg cgctggtggc cgagcgcttc 180 gcccggcgga
cccacgccac ccagaagaac acgttcggct ggatccgagc cgaggtaatg 240
ggggctctgg tgaacgccat cttcctgact ggcctctgtt tcgccatcct gctggaggcc
300 atcgagcgct tcatcgagcc gcacgagatg cagcagccgc tggtggtcct
tggggtcggc 360 gtggccgggc tgctggtcaa cgtgctgggg ctctgcctct
tccaccatca cagcggcttc 420 agccaggact ccggccacgg ccactcgcac
gggggtcacg gccacggcca cggcctcccc 480 aaggggcctc gcgttaagag
cacccgcccc gggagcagcg acatcaacgt ggccccgggc 540 gagcagggtc
ccgaccagga ggagaccaac accctggtgg ccaataccag caactccaac 600
gggctgaaat tggaccccgc agacccagaa aaccccagaa gtggtgatac agtggaagta
660 caagtgaatg gaaatcttgt cagagaacct gaccatatgg aactggaaga
agatagggct 720 ggacaactta acatgcgtgg agtttttctg catgtccttg
gagatgcctt gggttcagtg 780 attgtagtag taaatgcctt agtcttttac
ttttcttgga aaggttgttc tgaaggggat 840 ttttgtgtga atccatgttt
ccctgacccc tgcaaagcat ttgtagaaat aattaatagt 900 actcatgcat
cactttatga ggctggtcct tgctgggtgc tatatttaga tccaactctt 960
tgtgttgtaa tggtttgtat acttctttac acaacctatc cattacttaa ggaatctgct
1020 cttattcttc tacaaactgt tcctaaacaa attgatatca gaaatttgat
aaaagaactt 1080 cgaaatgttg aaggagttga ggaagttcat gaattacatg
tttggcaact tgctggaagc 1140 agaatcattg ccactgctca cataaaatgt
gaagatccaa catcatacat ggaggtggct 1200 aaaaccatta aagacgtttt
tcataatcac ggaattcacg ctactaccat tcagcctgaa 1260 tttgctagtg
taggctctaa atcaagtgta gttccgtgtg aacttgcctg cagaacccag 1320
tgtgctttga agcaatgttg tgggacacta ccacaagccc cttatggaaa ggatgcagaa
1380 aagaccccag cagttagcat ttcttgttta gaacttagta acaatctaga
gaagaagccc 1440 aggaggacta aagctgaaaa catccctgct gttgtgatag
agattaaaaa catgccaaac 1500 aaacaacctg aatcatcttt gtga 1524 10 507
PRT homo sapiens 10 Met Gly Cys Trp Gly Arg Asn Arg Gly Arg Leu Leu
Cys Met Leu Ala 1 5 10 15 Leu Thr Phe Met Phe Met Val Leu Glu Val
Val Val Ser Arg Val Thr 20 25 30 Ser Ser Leu Ala Met Leu Ser Asp
Ser Phe His Met Leu Ser Asp Val 35 40 45 Leu Ala Leu Val Val Ala
Leu Val Ala Glu Arg Phe Ala Arg Arg Thr 50 55 60 His Ala Thr Gln
Lys Asn Thr Phe Gly Trp Ile Arg Ala Glu Val Met 65 70 75 80 Gly Ala
Leu Val Asn Ala Ile Phe Leu Thr Gly Leu Cys Phe Ala Ile 85 90 95
Leu Leu Glu Ala Ile Glu Arg Phe Ile Glu Pro His Glu Met Gln Gln 100
105 110 Pro Leu Val Val Leu Gly Val Gly Val Ala Gly Leu Leu Val Asn
Val 115 120 125 Leu Gly Leu Cys Leu Phe His His His Ser Gly Phe Ser
Gln Asp Ser 130 135 140 Gly His Gly His Ser His Gly Gly His Gly His
Gly His Gly Leu Pro 145 150 155 160 Lys Gly Pro Arg Val Lys Ser Thr
Arg Pro Gly Ser Ser Asp Ile Asn 165 170 175 Val Ala Pro Gly Glu Gln
Gly Pro Asp Gln Glu Glu Thr Asn Thr Leu 180 185 190 Val Ala Asn Thr
Ser Asn Ser Asn Gly Leu Lys Leu Asp Pro Ala Asp 195 200 205 Pro Glu
Asn Pro Arg Ser Gly Asp Thr Val Glu Val Gln Val Asn Gly 210 215 220
Asn Leu Val Arg Glu Pro Asp His Met Glu Leu Glu Glu Asp Arg Ala 225
230 235 240 Gly Gln Leu Asn Met Arg Gly Val Phe Leu His Val Leu Gly
Asp Ala 245 250 255 Leu Gly Ser Val Ile Val Val Val Asn Ala Leu Val
Phe Tyr Phe Ser 260 265 270 Trp Lys Gly Cys Ser Glu Gly Asp Phe Cys
Val Asn Pro Cys Phe Pro 275 280 285 Asp Pro Cys Lys Ala Phe Val Glu
Ile Ile Asn Ser Thr His Ala Ser 290 295 300 Leu Tyr Glu Ala Gly Pro
Cys Trp Val Leu Tyr Leu Asp Pro Thr Leu 305 310 315 320 Cys Val Val
Met Val Cys Ile Leu Leu Tyr Thr Thr Tyr Pro Leu Leu 325 330 335 Lys
Glu Ser Ala Leu Ile Leu Leu Gln Thr Val Pro Lys Gln Ile Asp 340 345
350 Ile Arg Asn Leu Ile Lys Glu Leu Arg Asn Val Glu Gly Val Glu Glu
355 360 365 Val His Glu Leu His Val Trp Gln Leu Ala Gly Ser Arg Ile
Ile Ala 370 375 380 Thr Ala His Ile Lys Cys Glu Asp Pro Thr Ser Tyr
Met Glu Val Ala 385 390 395 400 Lys Thr Ile Lys Asp Val Phe His Asn
His Gly Ile His Ala Thr Thr 405 410 415 Ile Gln Pro Glu Phe Ala Ser
Val Gly Ser Lys Ser Ser Val Val Pro 420 425 430 Cys Glu Leu Ala Cys
Arg Thr Gln Cys Ala Leu Lys Gln Cys Cys Gly 435 440 445 Thr Leu Pro
Gln Ala Pro Tyr Gly Lys Asp Ala Glu Lys Thr Pro Ala 450 455 460 Val
Ser Ile Ser Cys Leu Glu Leu Ser Asn Asn Leu Glu Lys Lys Pro 465 470
475 480 Arg Arg Thr Lys Ala Glu Asn Ile Pro Ala Val Val Ile Glu Ile
Lys 485 490 495 Asn Met Pro Asn Lys Gln Pro Glu Ser Ser Leu 500 505
11 2222 DNA homo sapiens 11 ctccggctgc ggctcttggt accccggctc
cgggagccca gctccccgcc accgccgccg 60 cctgggtgtg ggggctgctg
aggctgagcc gggcttcggc gccggctctg aggacggacg 120 cctgaggagc
tgcgcggcgc ggcgccgccg gctggcggag aacgcccaca ggcgcggggc 180
tcggcggctt gacccgggct tgtccccgtg cggccgcggg ggcccctcag cggtttcccg
240 aacggcccga ctcgggcgct cctccgtgtc gcggtcgccg accctccgcg
tcccgccaac 300 gccgccgctg caccagtctc cgggccgggc tcggcgggcc
ccgcagccgc agccatgggg 360 tgttggggtc ggaaccgggg ccggctgctg
tgcatgctgg cgctgacctt catgttcatg 420 gtgctggagg tggtggtgag
ccgggtgacc tcgtcgctgg cgatgctctc cgactccttc 480 cacatgctgt
cggacgtgct ggcgctggtg gtggcgctgg tggccgagcg cttcgcccgg 540
cggacccacg ccacccagaa gaacacgttc ggctggatcc gagccgaggt aatgggggct
600 ctggtgaacg ccatcttcct gactggcctc tgtttcgcca tcctgctgga
ggccatcgag 660 cgcttcatcg agccgcacga gatgcagcag ccgctggtgg
tccttggggt cggcgtggcc 720 gggctgctgg tcaacgtgct ggggctctgc
ctcttccacc atcacagcgg cttcagccag 780 gactccggcc acggccactc
gcacgggggt cacggccacg gccacggcct ccccaagggg 840 cctcgcgtta
agagcacccg ccccgggagc agcgacatca acgtggcccc gggcgagcag 900
ggtcccgacc aggaggagac caacaccctg gtggccaata ccagcaactc caacgggctg
960 aaattggacc ccgcagaccc agaaaacccc agaagtggtg atacagtgga
agtacaagtg 1020 aatggaaatc ttgtcagaga acctgaccat atggaactgg
aagaagatag ggctggacaa 1080 cttaacatgc gtggagtttt tctgcatgtc
cttggagatg ccttgggttc agtgattgta 1140 gtagtaaatg ccttagtctt
ttacttttct tggaaaggtt gttctgaagg ggatttttgt 1200 gtgaatccat
gtttccctga cccctgcaaa gcatttgtag aaataattaa tagtactcat 1260
gcatcacttt atgaggctgg tccttgctgg gtgctatatt tagatccaac tctttgtgtt
1320 gtaatggttt gtatacttct ttacacaacc tatccattac ttaaggaatc
tgctcttatt 1380 cttctacaaa ctgttcctaa acaaattgat atcagaaatt
tgataaaaga acttcgaaat 1440 gttgaaggag ttgaggaagt tcatgaatta
catgtttggc aacttgctgg aagcagaatc 1500 attgccactg ctcacataaa
atgtgaagat ccaacatcat acatggaggt ggctaaaacc 1560 attaaagacg
tttttcataa tcacggaatt cacgctacta ccattcagcc tgaatttgct 1620
agtgtaggct ctaaatcaag tgtagttccg tgtgaacttg cctgcagaac ccagtgtgct
1680 ttgaagcaat gttgtgggac actaccacaa gccccttatg gaaaggatgc
agaaaagacc 1740 ccagcagtta gcatttcttg tttagaactt agtaacaatc
tagagaagaa gcccaggagg 1800 actaaagctg aaaacatccc tgctgttgtg
atagagatta aaaacatgcc aaacaaacaa 1860 cctgaatcat ctttgtgagt
cttgaaaaag atgtgatatt tgacttttgc tttaaactgc 1920 aagaggaaaa
agactccact gaaattctaa gtttgccaag tagtgtaatt gaagtccttg 1980
tctggtcaca cagtttaatt ctatttttgt aagaacataa tgggactgca taacagagtt
2040 ctatattaca atttgtgatt attagtacag agtacagcta tgctgtgact
gttttggaaa 2100 gccagtttta acactatgtt acatttttgt ttaaagtaag
ttaaacctta tataacataa 2160 gacatttga tttctggatt tttcccatgg
ataaaaaatt aggggggata aaattaaaat 2220 g 2222
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