U.S. patent application number 09/728711 was filed with the patent office on 2002-05-23 for novel nucleic acids and polypeptides.
Invention is credited to Asundi, Vinod, Drmanac, Radoje T., Goodrich, Ryle, Liu, Chenghua, Ren, Feiyan, Tang, Y. Tom, Wehrman, Tom, Xue, Aidong J., Zhang, Jie, Zhao, Qing A., Zhou, Ping.
Application Number | 20020061567 09/728711 |
Document ID | / |
Family ID | 27066367 |
Filed Date | 2002-05-23 |
United States Patent
Application |
20020061567 |
Kind Code |
A1 |
Tang, Y. Tom ; et
al. |
May 23, 2002 |
Novel nucleic acids and polypeptides
Abstract
The present invention provides novel nucleic acids, novel
polypeptide sequences encoded by these nucleic acids and uses
thereof.
Inventors: |
Tang, Y. Tom; (San Jose,
CA) ; Liu, Chenghua; (San Jose, CA) ; Zhou,
Ping; (Cupertino, CA) ; Asundi, Vinod; (Foster
City, CA) ; Ren, Feiyan; (Cupertino, CA) ;
Zhang, Jie; (Campbell, CA) ; Zhao, Qing A.;
(San Jose, CA) ; Xue, Aidong J.; (Sunnyvale,
CA) ; Goodrich, Ryle; (San Jose, CA) ;
Wehrman, Tom; (Stanford, CA) ; Drmanac, Radoje
T.; (Palo Alto, CA) |
Correspondence
Address: |
Ivor R. Elrifi
Mintz, Levin, Cohn, Ferris, Glovsky and Popeo, P.C
One Financial Center
Boston
MA
02111
US
|
Family ID: |
27066367 |
Appl. No.: |
09/728711 |
Filed: |
November 30, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09728711 |
Nov 30, 2000 |
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09649167 |
Aug 23, 2000 |
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09649167 |
Aug 23, 2000 |
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09540217 |
Mar 31, 2000 |
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Current U.S.
Class: |
435/183 ;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/8107 20130101; C07K 14/47 20130101 |
Class at
Publication: |
435/183 ;
435/69.1; 435/325; 435/320.1; 536/23.2 |
International
Class: |
C12N 009/00; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising a nucleotide sequence
selected from the group consisting of SEQ ID NO: 1-10, a mature
protein coding portion of SEQ ID NO: 1-10, an active domain of SEQ
ID NO: 1-10, and complementary sequences thereof.
2. An isolated polynucleotide encoding a polypeptide with
biological activity, wherein said polynucleotide hybridizes to the
polynucleotide of claim 1 under stringent hybridization
conditions.
3. An isolated polynucleotide encoding a polypeptide with
biological activity, wherein said polynucleotide has greater than
about 90% sequence identity with the polynucleotide of claim 1.
4. The polynucleotide of claim 1 wherein said polynucleotide is
DNA.
5. An isolated polynucleotide of claim 1 wherein said
polynucleotide comprises the complementary sequences.
6. A vector comprising the polynucleotide of claim 1.
7. An expression vector comprising the polynucleotide of claim
1.
8. A host cell genetically engineered to comprise the
polynucleotide of claim 1.
9. A host cell genetically engineered to comprise the
polynucleotide of claim 1 operatively associated with a regulatory
sequence that modulates expression of the polynucleotide in the
host cell.
10. An isolated polypeptide, wherein the polypeptide is selected
from the group consisting of: (a) a polypeptide encoded by any one
of the polynucleotides of claim 1; and (b) a polypeptide encoded by
a polynucleotide hybridizing under stringent conditions with any
one of SEQ ID NO: 1-10.
11. A composition comprising the polypeptide of claim 10 and a
carrier.
12. An antibody directed against the polypeptide of claim 10.
13. A method for detecting the polynucleotide of claim 1 in a
sample, comprising: a) contacting the sample with a compound that
binds to and forms a complex with the polynucleotide of claim 1 for
a period sufficient to form the complex; and b) detecting the
complex, so that if a complex is detected, the polynucleotide of
claim 1 is detected.
14. A method for detecting the polynucleotide of claim 1 in a
sample, comprising: a) contacting the sample under stringent
hybridization conditions with nucleic acid primers that anneal to
the polynucleotide of claim 1 under such conditions; b) amplifying
a product comprising at least a portion of the polynucleotide of
claim 1; and c) detecting said product and thereby the
polynucleotide of claim 1 in the sample.
15. The method of claim 14, wherein the polynucleotide is an RNA
molecule and the method further comprises reverse transcribing an
annealed RNA molecule into a cDNA polynucleotide.
16. A method for detecting the polypeptide of claim 10 in a sample,
comprising: a) contacting the sample with a compound that binds to
and forms a complex with the polypeptide under conditions and for a
period sufficient to form the complex; and b) detecting formation
of the complex, so that if a complex formation is detected, the
polypeptide of claim 10 is detected.
17. A method for identifying a compound that binds to the
polypeptide of claim 10, comprising: a) contacting the compound
with the polypeptide of claim 10 under conditions sufficient to
form a polypeptide/compound complex; and b) detecting the complex,
so that if the polypeptide/compound complex is detected, a compound
that binds to the polypeptide of claim 10 is identified.
18. A method for identifying a compound that binds to the
polypeptide of claim 10, comprising: a) contacting the compound
with the polypeptide of claim 10, in a cell, under conditions
sufficient to form a polypeptide/compound complex, wherein the
complex drives expression of a reporter gene sequence in the cell;
and b) detecting the complex by detecting reporter gene sequence
expression, so that if the polypeptide/compound complex is
detected, a compound that binds to the polypeptide of claim 10 is
identified.
19. A method of producing the polypeptide of claim 10, comprising,
a) culturing a host cell comprising a polynucleotide sequence
selected from the group consisting of a polynucleotide sequence of
SEQ ID NO: 1-10, a mature protein coding portion of SEQ ID NO:
1-10, an active domain of SEQ ID NO: 1-10, complementary sequences
thereof and a polynucleotide sequence hybridizing under stringent
conditions to SEQ ID NO: 1-10, under conditions sufficient to
express the polypeptide in said cell; and b) isolating the
polypeptide from the cell culture or cells of step (a).
20. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of any one of the polypeptides
from the Sequence Listing, the mature protein portion thereof, or
the active domain thereof.
21. The polypeptide of claim 20 wherein the polypeptide is provided
on a polypeptide array.
22. A collection of polynucleotides, wherein the collection
comprising the sequence information of at least one of SEQ ID NO:
1-10.
23. The collection of claim 22, wherein the collection is provided
on a nucleic acid array.
24. The collection of claim 23, wherein the array detects
full-matches to any one of the polynucleotides in the
collection.
25. The collection of claim 23, wherein the array detects
mismatches to any one of the polynucleotides in the collection.
26. The collection of claim 22, wherein the collection is provided
in a computer-readable format.
27. A method of treatment comprising administering to a mammalian
subject in need thereof a therapeutic amount of a composition
comprising a polypeptide of claim 10 or 20 and a pharmaceutically
acceptable carrier.
28. A method of treatment comprising administering to a mammalian
subject in need thereof a therapeutic amount of a composition
comprising an antibody that specifically binds to a polypeptide of
claim 10 or 20 and a pharmaceutically acceptable carrier.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
U.S. application Ser. No. 09/649,167, filed Aug. 23, 2000, Attorney
Docket No. 790CIP, which in turn is a continuation-in-part
application of U.S. application Ser. No. 09/540,217, filed Mar. 31,
2000, Attorney Docket No. 790, both which are incorporated herein
by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field
[0003] The present invention provides novel polynucleotides and
proteins encoded by such polynucleotides, along with uses for these
polynucleotides and proteins, for example in therapeutic,
diagnostic and research methods.
[0004] 2. Background
[0005] Technology aimed at the discovery of protein factors
(including e.g., cytokines, such as lymphokines, interferons, CSFs,
chemokines, and interleukins) has matured rapidly over the past
decade. The now routine hybridization cloning and expression
cloning techniques clone novel polynucleotides "directly" in the
sense that they rely on information directly related to the
discovered protein (i.e., partial DNA/amino acid sequence of the
protein in the case of hybridization cloning; activity of the
protein in the case of expression cloning). More recent "indirect"
cloning techniques such as signal sequence cloning, which isolates
DNA sequences based on the presence of a now well-recognized
secretory leader sequence motif, as well as various PCR-based or
low stringency hybridization-based cloning techniques, have
advanced the state of the art by making available large numbers of
DNA/amino acid sequences for proteins that are known to have
biological activity, for example, by virtue of their secreted
nature in the case of leader sequence cloning, by virtue of their
cell or tissue source in the case of PCR-based techniques, or by
virtue of structural similarity to other genes of known biological
activity.
[0006] Identified polynucleotide and polypeptide sequences have
numerous applications in, for example, diagnostics, forensics, gene
mapping; identification of mutations responsible for genetic
disorders or other traits, to assess biodiversity, and to produce
many other types of data and products dependent on DNA and amino
acid sequences.
SUMMARY OF THE INVENTION
[0007] The compositions of the present invention include novel
isolated polypeptides, novel isolated polynucleotides encoding such
polypeptides, including recombinant DNA molecules, cloned genes or
degenerate variants thereof, especially naturally occurring
variants such as allelic variants, antisense polynucleotide
molecules, and antibodies that specifically recognize one or more
epitopes present on such polypeptides, as well as hybridomas
producing such antibodies.
[0008] The compositions of the present invention additionally
include vectors, including expression vectors, containing the
polynucleotides of the invention, cells genetically engineered to
contain such polynucleotides and cells genetically engineered to
express such polynucleotides.
[0009] The present invention relates to a collection or library of
at least one novel nucleic acid sequence assembled from expressed
sequence tags (ESTs) isolated mainly by sequencing by hybridization
(SBH), and in some cases, sequences obtained from one or more
public databases. The invention relates also to the proteins
encoded by such polynucleotides, along with therapeutic, diagnostic
and research utilities for these polynucleotides and proteins.
These nucleic acid sequences are designated as SEQ ID NO: 1-10 and
are provided in the Sequence Listing. In the nucleic acids provided
in the Sequence Listing, A is adenine; C is cytosine; G is guanine;
T is thymine; and N is any of the four bases. In the amino acids
provided in the Sequence Listing, * corresponds to the stop
codon.
[0010] The nucleic acid sequences of the present invention also
include, nucleic acid sequences that hybridize to the complement of
SEQ ID NO: 1-10 under stringent hybridization conditions; nucleic
acid sequences which are allelic variants or species homologues of
any of the nucleic acid sequences recited above, or nucleic acid
sequences that encode a peptide comprising a specific domain or
truncation of the peptides encoded by SEQ ID NO: 1-10. A
polynucleotide comprising a nucleotide sequence having at least 90%
identity to an identifying sequence of SEQ ID NO: 1-10 or a
degenerate variant or fragment thereof. The identifying sequence
can be 100 base pairs in length.
[0011] The nucleic acid sequences of the present invention also
include the sequence information from the nucleic acid sequences of
SEQ ID NO: 1-10. The sequence information can be a segment of any
one of SEQ ID NO: 1-10 that uniquely identifies or represents the
sequence information of SEQ ID NO: 1-10.
[0012] A collection as used in this application can be a collection
of only one polynucleotide. The collection of sequence information
or identifying information of each sequence can be provided on a
nucleic acid array. In one embodiment, segments of sequence
information is provided on a nucleic acid array to detect the
polynucleotide that contains the segment. The array can be designed
to detect full-match or mismatch to the polynucleotide that
contains the segment. The collection can also be provided in a
computer-readable format.
[0013] This invention also includes the reverse or direct
complement of any of the nucleic acid sequences recited above;
cloning or expression vectors containing the nucleic acid
sequences; and host cells or organisms transformed with these
expression vectors. Nucleic acid sequences (or their reverse or
direct complements) according to the invention have numerous
applications in a variety of techniques known to those skilled in
the art of molecular biology, such as use as hybridization probes,
use as primers for PCR, use in an array, use in computer-readable
media, use in sequencing full-length genes, use for chromosome and
gene mapping, use in the recombinant production of protein, and use
in the generation of anti-sense DNA or RNA, their chemical analogs
and the like.
[0014] In a preferred embodiment, the nucleic acid sequences of SEQ
ID NO: 1-10 or novel segments or parts of the nucleic acids of the
invention are used as primers in expression assays that are well
known in the art. In a particularly preferred embodiment, the
nucleic acid sequences of SEQ ID NO: 1-10 or novel segments or
parts of the nucleic acids provided herein are used in diagnostics
for identifying expressed genes or, as well known in the art and
exemplified by Vollrath et al., Science 258:52-59 (1992), as
expressed sequence tags for physical mapping of the human
genome.
[0015] The isolated polynucleotides of the invention include, but
are not limited to, a polynucleotide comprising any one of the
nucleotide sequences set forth in SEQ ID NO: 1-10; a polynucleotide
comprising any of the full length protein coding sequences of SEQ
ID NO: 1-10; and a polynucleotide comprising any of the nucleotide
sequences of the mature protein coding sequences of SEQ ID NO:
1-10. The polynucleotides of the present invention also include,
but are not limited to, a polynucleotide that hybridizes under
stringent hybridization conditions to (a) the complement of any one
of the nucleotide sequences set forth in SEQ ID NO: 1-10; (b) a
nucleotide sequence encoding any one of the amino acid sequences
set forth in the Sequence Listing; (c) a polynucleotide which is an
allelic variant of any polynucleotides recited above; (d) a
polynucleotide which encodes a species homolog (e.g. orthologs) of
any of the proteins recited above; or (e) a polynucleotide that
encodes a polypeptide comprising a specific domain or truncation of
any of the polypeptides comprising an amino acid sequence set forth
in the Sequence Listing.
[0016] The isolated polypeptides of the invention include, but are
not limited to, a polypeptide comprising any of the amino acid
sequences set forth in the Sequence Listing; or the corresponding
full length or mature protein. Polypeptides of the invention also
include polypeptides with biological activity that are encoded by
(a) any of the polynucleotides having a nucleotide sequence set
forth in SEQ ID NO: 1-10; or (b) polynucleotides that hybridize to
the complement of the polynucleotides of (a) under stringent
hybridization conditions. Biologically or immunologically active
variants of any of the polypeptide sequences in the Sequence
Listing, and "substantial equivalents" thereof (e.g., with at least
about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid
sequence identity) that preferably retain biological activity are
also contemplated. The polypeptides of the invention may be wholly
or partially chemically synthesized but are preferably produced by
recombinant means using the genetically engineered cells (e.g. host
cells) of the invention.
[0017] The invention also provides compositions comprising a
polypeptide of the invention. Polypeptide compositions of the
invention may further comprise an acceptable carrier, such as a
hydrophilic, e.g., pharmaceutically acceptable, carrier.
[0018] The invention also provides host cells transformed or
transfected with a polynucleotide of the invention.
[0019] The invention also relates to methods for producing a
polypeptide of the invention comprising growing a culture of the
host cells of the invention in a suitable culture medium under
conditions permitting expression of the desired polypeptide, and
purifying the polypeptide from the culture or from the host cells.
Preferred embodiments include those in which the protein produced
by such process is a mature form of the protein.
[0020] Polynucleotides according to the invention have numerous
applications in a variety of techniques known to those skilled in
the art of molecular biology. These techniques include use as
hybridization probes, use as oligomers, or primers, for PCR, use
for chromosome and gene mapping, use in the recombinant production
of protein, and use in generation of anti-sense DNA or RNA, their
chemical analogs and the like. For example, when the expression of
an mRNA is largely restricted to a particular cell or tissue type,
polynucleotides of the invention can be used as hybridization
probes to detect the presence of the particular cell or tissue mRNA
in a sample using, e.g., in situ hybridization.
[0021] In other exemplary embodiments, the polynucleotides are used
in diagnostics as expressed sequence tags for identifying expressed
genes or, as well known in the art and exemplified by Vollrath et
al., Science 258:52-59 (1992), as expressed sequence tags for
physical mapping of the human genome.
[0022] The polypeptides according to the invention can be used in a
variety of conventional procedures and methods that are currently
applied to other proteins. For example, a polypeptide of the
invention can be used to generate an antibody that specifically
binds the polypeptide. Such antibodies, particularly monoclonal
antibodies, are useful for detecting or quantitating the
polypeptide in tissue. The polypeptides of the invention can also
be used as molecular weight markers, and as a food supplement.
[0023] Methods are also provided for preventing, treating, or
ameliorating a medical condition which comprises the step of
administering to a mammalian subject a therapeutically effective
amount of a composition comprising a polypeptide of the present
invention and a pharmaceutically acceptable carrier.
[0024] In particular, the polypeptides and polynucleotides of the
invention can be utilized, for example, in methods for the
prevention and/or treatment of disorders involving aberrant protein
expression or biological activity.
[0025] The present invention further relates to methods for
detecting the presence of the polynucleotides or polypeptides of
the invention in a sample. Such methods can, for example, be
utilized as part of prognostic and diagnostic evaluation of
disorders as recited herein and for the identification of subjects
exhibiting a predisposition to such conditions. The invention
provides a method for detecting the polynucleotides of the
invention in a sample, comprising contacting the sample with a
compound that binds to and forms a complex with the polynucleotide
of interest for a period sufficient to form the complex and under
conditions sufficient to form a complex and detecting the complex
such that if a complex is detected, the polynucleotide of interest
is detected. The invention also provides a method for detecting the
polypeptides of the invention in a sample comprising contacting the
sample with a compound that binds to and forms a complex with the
polypeptide under conditions and for a period sufficient to form
the complex and detecting the formation of the complex such that if
a complex is formed, the polypeptide is detected.
[0026] The invention also provides kits comprising polynucleotide
probes and/or monoclonal antibodies, and optionally quantitative
standards, for carrying out methods of the invention. Furthermore,
the invention provides methods for evaluating the efficacy of
drugs, and monitoring the progress of patients, involved in
clinical trials for the treatment of disorders as recited
above.
[0027] The invention also provides methods for the identification
of compounds that modulate (i.e., increase or decrease) the
expression or activity of the polynucleotides and/or polypeptides
of the invention. Such methods can be utilized, for example, for
the identification of compounds that can ameliorate symptoms of
disorders as recited herein. Such methods can include, but are not
limited to, assays for identifying compounds and other substances
that interact with (e.g., bind to) the polypeptides of the
invention. The invention provides a method for identifying a
compound that binds to the polypeptides of the invention comprising
contacting the compound with a polypeptide of the invention in a
cell for a time sufficient to form a polypeptide/compound complex,
wherein the complex drives expression of a reporter gene sequence
in the cell; and detecting the complex by detecting the reporter
gene sequence expression such that if expression of the reporter
gene is detected the compound the binds to a polypeptide of the
invention is identified.
[0028] The methods of the invention also provides methods for
treatment which involve the administration of the polynucleotides
or polypeptides of the invention to individuals exhibiting symptoms
or tendencies. In addition, the invention encompasses methods for
treating diseases or disorders as recited herein comprising
administering compounds and other substances that modulate the
overall activity of the target gene products. Compounds and other
substances can effect such modulation either on the level of target
gene/protein expression or target protein activity.
[0029] The polypeptides of the present invention and the
polynucleotides encoding them are also useful for the same
functions known to one of skill in the art as the polypeptides and
polynucleotides to which they have homology (set forth in Table 2);
for which they have a signature region (as set forth in Table 3);
or for which they have homology to a gene family (as set forth in
Table 4). If no homology is set forth for a sequence, then the
polypeptides and polynucleotides of the present invention are
useful for a variety of applications, as described herein,
including use in arrays for detection.
DETAILED DESCRIPTION OF THE INVENTION
[0030] DEFINITIONS
[0031] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an" and "the" include plural
references unless the context clearly dictates otherwise.
[0032] The term "active" refers to those forms of the polypeptide
which retain the biologic and/or immunologic activities of any
naturally occurring polypeptide. According to the invention, the
terms "biologically active" or "biological activity" refer to a
protein or peptide having structural, regulatory or biochemical
functions of a naturally occurring molecule. Likewise
"immunologically active" or "immunological activity" refers to the
capability of the natural, recombinant or synthetic polypeptide to
induce a specific immune response in appropriate animals or cells
and to bind with specific antibodies.
[0033] The term "activated cells" as used in this application are
those cells which are engaged in extracellular or intracellular
membrane trafficking, including the export of secretory or
enzymatic molecules as part of a normal or disease process.
[0034] The terms "complementary" or "complementarity" refer to the
natural binding of polynucleotides by base pairing. For example,
the sequence 5'-AGT-3' binds to the complementary sequence
3'-TCA-5'. Complementarity between two single-stranded molecules
may be "partial" such that only some of the nucleic acids bind or
it may be "complete" such that total complementarity exists between
the single stranded molecules. The degree of complementarity
between the nucleic acid strands has significant effects on the
efficiency and strength of the hybridization between the nucleic
acid strands.
[0035] The term "embryonic stem cells (ES)" refers to a cell that
can give rise to many differentiated cell types in an embryo or an
adult, including the germ cells. The term "germ line stem cells
(GSCs)" refers to stem cells derived from primordial stem cells
that provide a steady and continuous source of germ cells for the
production of gametes. The term "primordial germ cells (PGCs)"
refers to a small population of cells set aside from other cell
lineages particularly from the yolk sac, mesenteries, or gonadal
ridges during embryogenesis that have the potential to
differentiate into germ cells and other cells. PGCs are the source
from which GSCs and ES cells are derived The PGCs, the GSCs and the
ES cells are capable of self-renewal. Thus these cells not only
populate the germ line and give rise to a plurality of terminally
differentiated cells that comprise the adult specialized organs,
but are able to regenerate themselves.
[0036] The term "expression modulating fragment," EMF, means a
series of nucleotides which modulates the expression of an operably
linked ORF or another EMF.
[0037] As used herein, a sequence is said to "modulate the
expression of an operably linked sequence" when the expression of
the sequence is altered by the presence of the EMF. EMFs include,
but are not limited to, promoters, and promoter modulating
sequences (inducible elements). One class of EMFs are nucleic acid
fragments which induce the expression of an operably linked ORF in
response to a specific regulatory factor or physiological
event.
[0038] The terms "nucleotide sequence" or "nucleic acid" or
"polynucleotide" or "oligonculeotide" are used interchangeably and
refer to a heteropolymer of nucleotides or the sequence of these
nucleotides. These phrases also refer to DNA or RNA of genomic or
synthetic origin which may be single-stranded or double-stranded
and may represent the sense or the antisense strand, to peptide
nucleic acid (PNA) or to any DNA-like or RNA-like material. In the
sequences herein A is adenine, C is cytosine, T is thymine, G is
guanine and N is A, C, G or T (U). It is contemplated that where
the polynucleotide is RNA, the T (thymine) in the sequences
provided herein is substituted with U (uracil). Generally, nucleic
acid segments provided by this invention may be assembled from
fragments of the genome and short oligonucleotide linkers, or from
a series of oligonucleotides, or from individual nucleotides, to
provide a synthetic nucleic acid which is capable of being
expressed in a recombinant transcriptional unit comprising
regulatory elements derived from a microbial or viral operon, or a
eukaryotic gene.
[0039] The terms "oligonucleotide fragment" or a "polynucleotide
fragment", "portion," or "segment" or "probe" or "primer" are used
interchangeably and refer to a sequence of nucleotide residues
which are at least about 5 nucleotides, more preferably at least
about 7 nucleotides, more preferably at least about 9 nucleotides,
more preferably at least about 11 nucleotides and most preferably
at least about 17 nucleotides. The fragment is preferably less than
about 500 nucleotides, preferably less than about 200 nucleotides,
more preferably less than about 100 nucleotides, more preferably
less than about 50 nucleotides and most preferably less than 30
nucleotides. Preferably the probe is from about 6 nucleotides to
about 200 nucleotides, preferably from about 15 to about 50
nucleotides, more preferably from about 17 to 30 nucleotides and
most preferably from about 20 to 25 nucleotides. Preferably the
fragments can be used in polymerase chain reaction (PCR), various
hybridization procedures or microarray procedures to identify or
amplify identical or related parts of mRNA or DNA molecules. A
fragment or segment may uniquely identify each polynucleotide
sequence of the present invention. Preferably the fragment
comprises a sequence substantially similar to any one of SEQ ID
NOs:1-10.
[0040] Probes may, for example, be used to determine whether
specific mRNA molecules are present in a cell or tissue or to
isolate similar nucleic acid sequences from chromosomal DNA as
described by Walsh et al. (Walsh, P. S. et al., 1992, PCR Methods
Appl 1:241-250). They may be labeled by nick translation, Klenow
fill-in reaction, PCR, or other methods well known in the art.
Probes of the present invention, their preparation and/or labeling
are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel,
F. M. et al., 1989, Current Protocols in Molecular Biology, John
Wiley & Sons, New York N.Y., both of which are incorporated
herein by reference in their entirety.
[0041] The nucleic acid sequences of the present invention also
include the sequence information from the nucleic acid sequences of
SEQ ID NOs: 1-10. The sequence information can be a segment of any
one of SEQ ID NOs: 1-10 that uniquely identifies or represents the
sequence information of that sequence of SEQ ID NO: 1-10. One such
segment can be a twenty-mer nucleic acid sequence because the
probability that a twenty-mer is fully matched in the human genome
is 1 in 300. In the human genome, there are three billion base
pairs in one set of chromosomes. Because 4.sup.20 possible
twenty-mers exist, there are 300 times more twenty-mers than there
are base pairs in a set of human chromosomes. Using the same
analysis, the probability for a seventeen-mer to be fully matched
in the human genome is approximately 1 in 5. When these segments
are used in arrays for expression studies, fifteen-mer segments can
be used. The probability that the fifteen-mer is fully matched in
the expressed sequences is also approximately one in five because
expressed sequences comprise less than approximately 5% of the
entire genome sequence.
[0042] Similarly, when using sequence information for detecting a
single mismatch, a segment can be a twenty-five mer. The
probability that the twenty-five mer would appear in a human genome
with a single mismatch is calculated by multiplying the probability
for a full match (1.div.4.sup.25) times the increased probability
for mismatch at each nucleotide position (3.times.25). The
probability that an eighteen mer with a single mismatch can be
detected in an array for expression studies is approximately one in
five. The probability that a twenty-mer with a single mismatch can
be detected in a human genome is approximately one in five.
[0043] The term "open reading frame," ORF, means a series of
nucleotide triplets coding for amino acids without any termination
codons and is a sequence translatable into protein.
[0044] The terms "operably linked" or "operably associated" refer
to functionally related nucleic acid sequences. For example, a
promoter is operably associated or operably linked with a coding
sequence if the promoter controls the transcription of the coding
sequence. While operably linked nucleic acid sequences can be
contiguous and in the same reading frame, certain genetic elements
e.g. repressor genes are not contiguously linked to the coding
sequence but still control transcription/translation of the coding
sequence.
[0045] The term "pluripotent" refers to the capability of a cell to
differentiate into a number of differentiated cell types that are
present in an adult organism. A pluripotent cell is restricted in
its differentiation capability in comparison to a totipotent
cell.
[0046] The terms "polypeptide" or "peptide" or "amino acid
sequence" refer to an oligopeptide, peptide, polypeptide or protein
sequence or fragment thereof and to naturally occurring or
synthetic molecules. A polypeptide "fragment," "portion," or
"segment" is a stretch of amino acid residues of at least about 5
amino acids, preferably at least about 7 amino acids, more
preferably at least about 9 amino acids and most preferably at
least about 17 or more amino acids. The peptide preferably is not
greater than about 200 amino acids, more preferably less than 150
amino acids and most preferably less than 100 amino acids.
Preferably the peptide is from about 5 to about 200 amino acids. To
be active, any polypeptide must have sufficient length to display
biological and/or immunological activity.
[0047] The term "naturally occurring polypeptide" refers to
polypeptides produced by cells that have not been genetically
engineered and specifically contemplates various polypeptides
arising from post-translational modifications of the polypeptide
including, but not limited to, acetylation, carboxylation,
glycosylation, phosphorylation, lipidation and acylation.
[0048] The term "translated protein coding portion" means a
sequence which encodes for the full length protein which may
include any leader sequence or any processing sequence.
[0049] The term "mature protein coding sequence" means a sequence
which encodes a peptide or protein without a signal or leader
sequence. The "mature protein portion" means that portion of the
protein which does not include a signal or leader sequence. The
peptide may have been produced by processing in the cell which
removes any leader/signal sequence. The mature protein portion may
or may not include the initial methionine residue. The methionine
residue may be removed from the protein during processing in the
cell. The peptide may be produced synthetically or the protein may
have been produced using a polynucleotide only encoding for the
mature protein coding sequence.
[0050] The term "derivative" refers to polypeptides chemically
modified by such techniques as ubiquitination, labeling (e.g., with
radionuclides or various enzymes), covalent polymer attachment such
as pegylation (derivatization with polyethylene glycol) and
insertion or substitution by chemical synthesis of amino acids such
as ornithine, which do not normally occur in human proteins.
[0051] The term "variant" (or "analog") refers to any polypeptide
differing from naturally occurring polypeptides by amino acid
insertions, deletions, and substitutions, created using, e g.,
recombinant DNA techniques. Guidance in determining which amino
acid residues may be replaced, added or deleted without abolishing
activities of interest, may be found by comparing the sequence of
the particular polypeptide with that of homologous peptides and
minimizing the number of amino acid sequence changes made in
regions of high homology (conserved regions) or by replacing amino
acids with consensus sequence.
[0052] Alternatively, recombinant variants encoding these same or
similar polypeptides may be synthesized or selected by making use
of the "redundancy" in the genetic code. Various codon
substitutions, such as the silent changes which produce various
restriction sites, may be introduced to optimize cloning into a
plasmid or viral vector or expression in a particular prokaryotic
or eukaryotic system. Mutations in the polynucleotide sequence may
be reflected in the polypeptide or domains of other peptides added
to the polypeptide to modify the properties of any part of the
polypeptide, to change characteristics such as ligand-binding
affinities, interchain affinities, or degradation/turnover
rate.
[0053] Preferably, amino acid "substitutions" are the result of
replacing one amino acid with another amino acid having similar
structural and/or chemical properties, i.e., conservative amino
acid replacements. "Conservative" 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.
"Insertions" or "deletions" are preferably in the range of about 1
to 20 amino acids, more preferably 1 to 10 amino acids. The
variation allowed may be experimentally determined by
systematically making insertions, deletions, or substitutions of
amino acids in a polypeptide molecule using recombinant DNA
techniques and assaying the resulting recombinant variants for
activity.
[0054] Alternatively, where alteration of function is desired,
insertions, deletions or non-conservative alterations can be
engineered to produce altered polypeptides. Such alterations can,
for example, alter one or more of the biological functions or
biochemical characteristics of the polypeptides of the invention.
For example, such alterations may change polypeptide
characteristics such as ligand-binding affinities, interchain
affinities, or degradation/turnover rate. Further, such alterations
can be selected so as to generate polypeptides that are better
suited for expression, scale up and the like in the host cells
chosen for expression. For example, cysteine residues can be
deleted or substituted with another amino acid residue in order to
eliminate disulfide bridges.
[0055] The terms "purified" or "substantially purified" as used
herein denotes that the indicated nucleic acid or polypeptide is
present in the substantial absence of other biological
macromolecules, e.g., polynucleotides, proteins, and the like. In
one embodiment, the polynucleotide or polypeptide is purified such
that it constitutes at least 95% by weight, more preferably at
least 99% by weight, of the indicated biological macromolecules
present (but water, buffers, and other small molecules, especially
molecules having a molecular weight of less than 1000 daltons, can
be present).
[0056] The term "isolated" as used herein refers to a nucleic acid
or polypeptide separated from at least one other component (e.g.,
nucleic acid or polypeptide) present with the nucleic acid or
polypeptide in its natural source. In one embodiment, the nucleic
acid or polypeptide is found in the presence of (if anything) only
a solvent, buffer, ion, or other component normally present in a
solution of the same. The terms "isolated" and "purified" do not
encompass nucleic acids or polypeptides present in their natural
source.
[0057] The term "recombinant," when used herein to refer to a
polypeptide or protein, means that a polypeptide or protein is
derived from recombinant (e.g., microbial, insect, or mammalian)
expression systems. "Microbial" refers to recombinant polypeptides
or proteins made in bacterial or fungal (e.g., yeast) expression
systems. As a product, "recombinant microbial" defines a
polypeptide or protein essentially free of native endogenous
substances and unaccompanied by associated native glycosylation.
Polypeptides or proteins expressed in most bacterial cultures,
e.g., E. coli, will be free of glycosylation modifications;
polypeptides or proteins expressed in yeast will have a
glycosylation pattern in general different from those expressed in
mammalian cells.
[0058] The term "recombinant expression vehicle or vector" refers
to a plasmid or phage or virus or vector, for expressing a
polypeptide from a DNA (RNA) sequence. An expression vehicle can
comprise a transcriptional unit comprising an assembly of (1) a
genetic element or elements having a regulatory role in gene
expression, for example, promoters or enhancers, (2) a structural
or coding sequence which is transcribed into mRNA and translated
into protein, and (3) appropriate transcription initiation and
termination sequences. Structural units intended for use in yeast
or eukaryotic expression systems preferably include a leader
sequence enabling extracellular secretion of translated protein by
a host cell. Alternatively, where recombinant protein is expressed
without a leader or transport sequence, it may include an amino
terminal methionine residue. This residue may or may not be
subsequently cleaved from the expressed recombinant protein to
provide a final product.
[0059] The term "recombinant expression system" means host cells
which have stably integrated a recombinant transcriptional unit
into chromosomal DNA or carry the recombinant transcriptional unit
extrachromosomally. Recombinant expression systems as defined
herein will express heterologous polypeptides or proteins upon
induction of the regulatory elements linked to the DNA segment or
synthetic gene to be expressed. This term also means host cells
which have stably integrated a recombinant genetic element or
elements having a regulatory role in gene expression, for example,
promoters or enhancers. Recombinant expression systems as defined
herein will express polypeptides or proteins endogenous to the cell
upon induction of the regulatory elements linked to the endogenous
DNA segment or gene to be expressed. The cells can be prokaryotic
or eukaryotic.
[0060] The term "secreted" includes a protein that is transported
across or through a membrane, including transport as a result of
signal sequences in its amino acid sequence when it is expressed in
a suitable host cell. "Secreted" proteins include without
limitation proteins secreted wholly (e.g., soluble proteins) or
partially (e.g., receptors) from the cell in which they are
expressed. "Secreted" proteins also include without limitation
proteins that are transported across the membrane of the
endoplasmic reticulum. "Secreted" proteins are also intended to
include proteins containing non-typical signal sequences (e.g.
Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992)
Cytokine 4(2):134 -143) and factors released from damaged cells
(e.g. Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al.
(1998) Annu. Rev. Immunol. 16:27-55)
[0061] Where desired, an expression vector may be designed to
contain a "signal or leader sequence" which will direct the
polypeptide through the membrane of a cell. Such a sequence may be
naturally present on the polypeptides of the present invention or
provided from heterologous protein sources by recombinant DNA
techniques.
[0062] The term "stringent" is used to refer to conditions that are
commonly understood in the art as stringent. Stringent conditions
can include highly stringent conditions (i.e., 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.), and moderately stringent
conditions (i.e., washing in 0.2.times.SSC/0.1% SDS at 42.degree.
C.). Other exemplary hybridization conditions are described herein
in the examples.
[0063] In instances of hybridization of deoxyoligonucleotides,
additional exemplary stringent hybridization conditions include
washing in 6.times.SSC/0.05% sodium pyrophosphate at 37.degree. C.
(for 14-base oligonucleotides), 48.degree. C. (for 17-base oligos),
55.degree. C. (for 20-base oligonucleotides), and 60.degree. C.
(for 23-base oligonucleotides).
[0064] As used herein, "substantially equivalent" can refer both to
nucleotide and amino acid sequences, for example a mutant sequence,
that varies from a reference sequence by one or more substitutions,
deletions, or additions, the net effect of which does not result in
an adverse functional dissimilarity between the reference and
subject sequences. Typically, such a substantially equivalent
sequence varies from one of those listed herein by no more than
about 35% (i.e., the number of individual residue substitutions,
additions, and/or deletions in a substantially equivalent sequence,
as compared to the corresponding reference sequence, divided by the
total number of residues in the substantially equivalent sequence
is about 0.35 or less). Such a sequence is said to have 65%
sequence identity to the listed sequence. In one embodiment, a
substantially equivalent, e.g., mutant, sequence of the invention
varies from a listed sequence by no more than 30% (70% sequence
identity); in a variation of this embodiment, by no more than 25%
(75% sequence identity); and in a further variation of this
embodiment, by no more than 20% (80% sequence identity) and in a
further variation of this embodiment, by no more than 10% (90%
sequence identity) and in a further variation of this embodiment,
by no more that 5% (95% sequence identity). Substantially
equivalent, e.g., mutant, amino acid sequences according to the
invention preferably have at least 80% sequence identity with a
listed amino acid sequence, more preferably at least 90% sequence
identity. Substantially equivalent nucleotide sequences of the
invention can have lower percent sequence identities, taking into
account, for example, the redundancy or degeneracy of the genetic
code. Preferably, nucleotide sequence has at least about 65%
identity, more preferably at least about 75% identity, and most
preferably at least about 95% identity. For the purposes of the
present invention, sequences having substantially equivalent
biological activity and substantially equivalent expression
characteristics are considered substantially equivalent. For the
purposes of determining equivalence, truncation of the mature
sequence (e.g., via a mutation which creates a spurious stop codon)
should be disregarded. Sequence identity may be determined, e.g.,
using the Jotun Hein method (Hein, J. (1990) Methods Enzymol.
183:626-645). Identity between sequences can also be determined by
other methods known in the art, e.g. by varying hybridization
conditions.
[0065] The term "totipotent" refers to the capability of a cell to
differentiate into all of the cell types of an adult organism.
[0066] The term "transformation" means introducing DNA into a
suitable host cell so that the DNA is replicable, either as an
extrachromosomal element, or by chromosomal integration. The term
"transfection" refers to the taking up of an expression vector by a
suitable host cell, whether or not any coding sequences are in fact
expressed. The term "infection" refers to the introduction of
nucleic acids into a suitable host cell by use of a virus or viral
vector.
[0067] As used herein, an "uptake modulating fragment," UMF, means
a series of nucleotides which mediate the uptake of a linked DNA
fragment into a cell. UMFs can be readily identified using known
UMFs as a target sequence or target motif with the computer-based
systems described below. The presence and activity of a UMF can be
confirmed by attaching the suspected UME to a marker sequence. The
resulting nucleic acid molecule is then incubated with an
appropriate host under appropriate conditions and the uptake of the
marker sequence is determined. As described above, a UMF will
increase the frequency of uptake of a linked marker sequence.
[0068] Each of the above terms is meant to encompass all that is
described for each, unless the context dictates otherwise.
[0069] NUCLEIC ACIDS OF THE INVENTION
[0070] Nucleotide sequences of the invention are set forth in the
Sequence Listing.
[0071] The isolated polynucleotides of the invention include a
polynucleotide comprising the nucleotide sequences of SEQ ID NO:
1-10; a polynucleotide encoding any one of the peptide sequences of
SEQ ID NO: 1-10; and a polynucleotide comprising the nucleotide
sequence encoding the mature protein coding sequence of the
polynucleotides of any one of SEQ ID NO: 1-10. The polynucleotides
of the present invention also include, but are not limited to, a
polynucleotide that hybridizes under stringent conditions to (a)
the complement of any of the nucleotides sequences of SEQ ID NO:
1-10; (b) nucleotide sequences encoding any one of the amino acid
sequences set forth in the Sequence Listing; (c) a polynucleotide
which is an allelic variant of any polynucleotide recited above;
(d) a polynucleotide which encodes a species homolog of any of the
proteins recited above; or (e) a polynucleotide that encodes a
polypeptide comprising a specific domain or truncation of the
polypeptides of SEQ ID NO: 1-10. Domains of interest may depend on
the nature of the encoded polypeptide; e.g., domains in
receptor-like polypeptides include ligand-binding, extracellular,
transmembrane, or cytoplasmic domains, or combinations thereof;
domains in immunoglobulin-like proteins include the variable
immunoglobulin-like domains; domains in enzyme-like polypeptides
include catalytic and substrate binding domains; and domains in
ligand polypeptides include receptor-binding domains.
[0072] The polynucleotides of the invention include naturally
occurring or wholly or partially synthetic DNA, e.g., cDNA and
genomic DNA, and RNA, e.g., mRNA. The polynucleotides may include
all of the coding region of the cDNA or may represent a portion of
the coding region of the cDNA.
[0073] The present invention also provides genes corresponding to
the cDNA sequences disclosed herein. The corresponding genes can be
isolated in accordance with known methods using the sequence
information disclosed herein. Such methods include the preparation
of probes or primers from the disclosed sequence information for
identification and/or amplification of genes in appropriate genomic
libraries or other sources of genomic materials. Further 5' and 3'
sequence can be obtained using methods known in the art. For
example, full length cDNA or genomic DNA that corresponds to any of
the polynucleotides of SEQ ID NO: 1-10 can be obtained by screening
appropriate cDNA or genomic DNA libraries under suitable
hybridization conditions using any of the polynucleotides of SEQ ID
NO: 1-10 or a portion thereof as a probe. Alternatively, the
polynucleotides of SEQ ID NO: 1-10 may be used as the basis for
suitable primer(s) that allow identification and/or amplification
of genes in appropriate genomic DNA or cDNA libraries.
[0074] The nucleic acid sequences of the invention can be assembled
from ESTs and sequences (including cDNA and genomic sequences)
obtained from one or more public databases, such as dbEST, gbpri,
and UniGene. The EST sequences can provide identifying sequence
information, representative fragment or segment information, or
novel segment information for the full-length gene.
[0075] The polynucleotides of the invention also provide
polynucleotides including nucleotide sequences that are
substantially equivalent to the polynucleotides recited above.
Polynucleotides according to the invention can have, e.g., at least
about 65%, at least about 70%, at least about 75%, at least about
80%, more typically at least about 90%, and even more typically at
least about 95%, sequence identity to a polynucleotide recited
above.
[0076] Included within the scope of the nucleic acid sequences of
the invention are nucleic acid sequence fragments that hybridize
under stringent conditions to any of the nucleotide sequences of
SEQ ID NO: 1-10, or complements thereof, which fragment is greater
than about 5 nucleotides, preferably 7 nucleotides, more preferably
greater than 9 nucleotides and most preferably greater than 17
nucleotides. Fragments of, e.g. 15, 17, or 20 nucleotides or more
that are selective for (i.e. specifically hybridize to any one of
the polynucleotides of the invention) are contemplated. Probes
capable of specifically hybridizing to a polynucleotide can
differentiate polynucleotide sequences of the invention from other
polynucleotide sequences in the same family of genes or can
differentiate human genes from genes of other species, and are
preferably based on unique nucleotide sequences.
[0077] The sequences falling within the scope of the present
invention are not limited to these specific sequences, but also
include allelic and species variations thereof. Allelic and species
variations can be routinely determined by comparing the sequence
provided in SEQ ID NO: 1-10, a representative fragment thereof, or
a nucleotide sequence at least 90% identical, preferably 95%
identical, to SEQ ID NOs: 1-10 with a sequence from another isolate
of the same species. Furthermore, to accommodate codon variability,
the invention includes nucleic acid molecules coding for the same
amino acid sequences as do the specific ORFs disclosed herein. In
other words, in the coding region of an ORF, substitution of one
codon for another codon that encodes the same amino acid is
expressly contemplated.
[0078] The nearest neighbor or homology result for the nucleic
acids of the present invention, including SEQ ID NOs: 1-10, can be
obtained by searching a database using an algorithm or a program.
Preferably, a BLAST which stands for Basic Local Alignment Search
Tool is used to search for local sequence alignments (Altshul, S.
F. J Mol. Evol. 36 290-300 (1993) and Altschul S. F. et al. J. Mol.
Biol. 21:403-410 (1990)). Alternatively a FASTA version 3 search
against Genpept, using Fastxy algorithm.
[0079] Species homologs (or orthologs) of the disclosed
polynucleotides and proteins are also provided by the present
invention. Species homologs may be isolated and identified by
making suitable probes or primers from the sequences provided
herein and screening a suitable nucleic acid source from the
desired species.
[0080] The invention also encompasses allelic variants of the
disclosed polynucleotides or proteins; that is, naturally-occurring
alternative forms of the isolated polynucleotide which also encode
proteins which are identical, homologous or related to that encoded
by the polynucleotides.
[0081] The nucleic acid sequences of the invention are further
directed to sequences which encode variants of the described
nucleic acids. These amino acid sequence variants may be prepared
by methods known in the art by introducing appropriate nucleotide
changes into a native or variant polynucleotide. There are two
variables in the construction of amino acid sequence variants: the
location of the mutation and the nature of the mutation. Nucleic
acids encoding the amino acid sequence variants are preferably
constructed by mutating the polynucleotide to encode an amino acid
sequence that does not occur in nature. These nucleic acid
alterations can be made at sites that differ in the nucleic acids
from different species (variable positions) or in highly conserved
regions (constant regions). Sites at such locations will typically
be modified in series, e.g., by substituting first with
conservative choices (e.g., hydrophobic amino acid to a different
hydrophobic amino acid) and then with more distant choices (e.g.,
hydrophobic amino acid to a charged amino acid), and then deletions
or insertions may be made at the target site. Amino acid sequence
deletions generally range from about 1 to 30 residues, preferably
about 1 to 10 residues, and are typically contiguous. Amino acid
insertions include amino- and/or carboxyl-terminal fusions ranging
in length from one to one hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Intrasequence insertions may range generally from about 1 to 10
amino residues, preferably from 1 to 5 residues. Examples of
terminal insertions include the heterologous signal sequences
necessary for secretion or for intracellular targeting in different
host cells and sequences such as FLAG or poly-histidine sequences
useful for purifying the expressed protein.
[0082] In a preferred method, polynucleotides encoding the novel
amino acid sequences are changed via site-directed mutagenesis.
This method uses oligonucleotide sequences to alter a
polynucleotide to encode the desired amino acid variant, as well as
sufficient adjacent nucleotides on both sides of the changed amino
acid to form a stable duplex on either side of the site of being
changed. In general, the techniques of site-directed mutagenesis
are well known to those of skill in the art and this technique is
exemplified by publications such as, Edelman et al., DNA 2:183
(1983). A versatile and efficient method for producing
site-specific changes in a polynucleotide sequence was published by
Zoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may
also be used to create amino acid sequence variants of the novel
nucleic acids. When small amounts of template DNA are used as
starting material, primer(s) that differs slightly in sequence from
the corresponding region in the template DNA can generate the
desired amino acid variant. PCR amplification results in a
population of product DNA fragments that differ from the
polynucleotide template encoding the polypeptide at the position
specified by the primer. The product DNA fragments replace the
corresponding region in the plasmid and this gives a polynucleotide
encoding the desired amino acid variant.
[0083] A further technique for generating amino acid variants is
the cassette mutagenesis technique described in Wells et al., Gene
34:315 (1985); and other mutagenesis techniques well known in the
art, such as, for example, the techniques in Sambrook et al.,
supra, and Current Protocols in Molecular Biology, Ausubel et al.
Due to the inherent degeneracy of the genetic code, other DNA
sequences which encode substantially the same or a functionally
equivalent amino acid sequence may be used in the practice of the
invention for the cloning and expression of these novel nucleic
acids. Such DNA sequences include those which are capable of
hybridizing to the appropriate novel nucleic acid sequence under
stringent conditions.
[0084] Polynucleotides encoding preferred polypeptide truncations
of the invention can be used to generate polynucleotides encoding
chimeric or fusion proteins comprising one or more domains of the
invention and heterologous protein sequences.
[0085] The polynucleotides of the invention additionally include
the complement of any of the polynucleotides recited above. The
polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic)
or RNA. Methods and algorithms for obtaining such polynucleotides
are well known to those of skill in the art and can include, for
example, methods for determining hybridization conditions that can
routinely isolate polynucleotides of the desired sequence
identities.
[0086] In accordance with the invention, polynucleotide sequences
comprising the mature protein coding sequences corresponding to any
one of SEQ ID NO: 1-10, or functional equivalents thereof, may be
used to generate recombinant DNA molecules that direct the
expression of that nucleic acid, or a functional equivalent
thereof, in appropriate host cells. Also included are the cDNA
inserts of any of the clones identified herein.
[0087] A polynucleotide according to the invention can be joined to
any of a variety of other nucleotide sequences by well-established
recombinant DNA techniques (see Sambrook J et al. (1989) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).
Useful nucleotide sequences for joining to polynucleotides include
an assortment of vectors, e.g., plasmids, cosmids, lambda phage
derivatives, phagemids, and the like, that are well known in the
art. Accordingly, the invention also provides a vector including a
polynucleotide of the invention and a host cell containing the
polynucleotide. In general, the vector contains an origin of
replication functional in at least one organism, convenient
restriction endonuclease sites, and a selectable marker for the
host cell. Vectors according to the invention include expression
vectors, replication vectors, probe generation vectors, and
sequencing vectors. A host cell according to the invention can be a
prokaryotic or eukaryotic cell and can be a unicellular organism or
part of a multicellular organism.
[0088] The present invention further provides recombinant
constructs comprising a nucleic acid having any of the nucleotide
sequences of SEQ ID NOs: 1-10 or a fragment thereof or any other
polynucleotides of the invention. In one embodiment, the
recombinant constructs of the present invention comprise a vector,
such as a plasmid or viral vector, into which a nucleic acid having
any of the nucleotide sequences of SEQ ID NOs: 1-10 or a fragment
thereof is inserted, in a forward or reverse orientation. In the
case of a vector comprising one of the ORFs of the present
invention, the vector may further comprise regulatory sequences,
including for example, a promoter, operably linked to the ORF.
Large numbers of suitable vectors and promoters are known to those
of skill in the art and are commercially available for generating
the recombinant constructs of the present invention. The following
vectors are provided by way of example. Bacterial: pBs,
phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a,
pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540,
pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
[0089] The isolated polynucleotide of the invention may be operably
linked to an expression control sequence such as the pMT2 or pED
expression vectors disclosed in Kaufman et al., Nucleic Acids Res.
19, 4485-4490 (1991), in order to produce the protein
recombinantly. Many suitable expression control sequences are known
in the art. General methods of expressing recombinant proteins are
also known and are exemplified in R. Kaufman, Methods in Enzymology
185, 537-566 (1990). As defined herein "operably linked" means that
the isolated polynucleotide of the invention and an expression
control sequence are situated within a vector or cell in such a way
that the protein is expressed by a host cell which has been
transformed (transfected) with the ligated
polynucleotide/expression control sequence.
[0090] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lac, lacZ, T3, T7,
gpt, lambda PR, and trc. Eukaryotic promoters include CMV immediate
early, HSV thymidine kinase, early and late SV40, LTRs from
retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art. Generally, recombinant expression
vectors will include origins of replication and selectable markers
permitting transformation of the host cell, e.g., the ampicillin
resistance gene of E. coli and S. cerevisiae TRP1 gene, and a
promoter derived from a highly-expressed gene to direct
transcription of a downstream structural sequence. Such promoters
can be derived from operons encoding glycolytic enzymes such as
3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or
heat shock proteins, among others. The heterologous structural
sequence is assembled in appropriate phase with translation
initiation and termination sequences, and preferably, a leader
sequence capable of directing secretion of translated protein into
the periplasmic space or extracellular medium. Optionally, the
heterologous sequence can encode a fusion protein including an
amino terminal identification peptide imparting desired
characteristics, e.g., stabilization or simplified purification of
expressed recombinant product. Useful expression vectors for
bacterial use are constructed by inserting a structural DNA
sequence encoding a desired protein together with suitable
translation initiation and termination signals in operable reading
phase with a functional promoter. The vector will comprise one or
more phenotypic selectable markers and an origin of replication to
ensure maintenance of the vector and to, if desirable, provide
amplification within the host. Suitable prokaryotic hosts for
transformation include E. coli, Bacillus subtilis, Salmonella
typhimurium and various species within the genera Pseudomonas,
Streptomyces, and Staphylococcus, although others may also be
employed as a matter of choice.
[0091] As a representative but non-limiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed. Following
transformation of a suitable host strain and growth of the host
strain to an appropriate cell density, the selected promoter is
induced or derepressed by appropriate means (e.g., temperature
shift or chemical induction) and cells are cultured for an
additional period. Cells are typically harvested by centrifugation,
disrupted by physical or chemical means, and the resulting crude
extract retained for further purification.
[0092] Polynucleotides of the invention can also be used to induce
immune responses. For example, as described in Fan et al., Nat.
Biotech. 17:870-872 (1999), incorporated herein by reference,
nucleic acid sequences encoding a polypeptide may be used to
generate antibodies against the encoded polypeptide following
topical administration of naked plasmid DNA or following injection,
and preferably intramuscular injection of the DNA. The nucleic acid
sequences are preferably inserted in a recombinant expression
vector and may be in the form of naked DNA.
[0093] HOSTS
[0094] The present invention further provides host cells
genetically engineered to contain the polynucleotides of the
invention. For example, such host cells may contain nucleic acids
of the invention introduced into the host cell using known
transformation, transfection or infection methods. The present
invention still further provides host cells genetically engineered
to express the polynucleotides of the invention, wherein such
polynucleotides are in operative association with a regulatory
sequence heterologous to the host cell which drives expression of
the polynucleotides in the cell.
[0095] Knowledge of nucleic acid sequences allows for modification
of cells to permit, or increase, expression of endogenous
polypeptide. Cells can be modified (e.g., by homologous
recombination) to provide increased polypeptide expression by
replacing, in whole or in part, the naturally occurring promoter
with all or part of a heterologous promoter so that the cells
express the polypeptide at higher levels. The heterologous promoter
is inserted in such a manner that it is operatively linked to the
encoding sequences. See, for example, PCT International Publication
No. WO94/12650, PCT International Publication No. WO92/20808, and
PCT International Publication No. WO91/09955. It is also
contemplated that, in addition to heterologous promoter DNA,
amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional
CAD gene which encodes carbamyl phosphate synthase, aspartate
transcarbamylase, and dihydroorotase) and/or intron DNA may be
inserted along with the heterologous promoter DNA. If linked to the
coding sequence, amplification of the marker DNA by standard
selection methods results in co-amplification of the desired
protein coding sequences in the cells.
[0096] The host cell can be a higher eukaryotic host cell, such as
a mammalian cell, a lower eukaryotic host cell, such as a yeast
cell, or the host cell can be a prokaryotic cell, such as a
bacterial cell. Introduction of the recombinant construct into the
host cell can be effected by calcium phosphate transfection, DEAE,
dextran mediated transfection, or electroporation (Davis, L. et
al., Basic Methods in Molecular Biology (1986)). The host cells
containing one of the polynucleotides of the invention, can be used
in conventional manners to produce the gene product encoded by the
isolated fragment (in the case of an ORF) or can be used to produce
a heterologous protein under the control of the EMF.
[0097] Any host/vector system can be used to express one or more of
the ORFs of the present invention. These include, but are not
limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS
cells, 293 cells, and Sf9 cells, as well as prokaryotic host such
as E. coli and B. subtilis. The most preferred cells are those
which do not normally express the particular polypeptide or protein
or which expresses the polypeptide or protein at low natural level.
Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which
is hereby incorporated by reference.
[0098] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell 23:175 (1981). Other cell lines capable
of expressing a compatible vector are, for example, the C127,
monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney
293 cells, human epidermal A431 cells, human Colo205 cells, 3T3
cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell strains derived from in vitro culture of
primary tissue, primary explants, HeLa cells, mouse L cells, BHK,
HL-60, U937, HaK or Jurkat cells. Mammalian expression vectors will
comprise an origin of replication, a suitable promoter and also any
necessary ribosome binding sites, polyadenylation site, splice
donor and acceptor sites, transcriptional termination sequences,
and 5' flanking nontranscribed sequences. DNA sequences derived
from the SV40 viral genome, for example, SV40 origin, early
promoter, enhancer, splice, and polyadenylation sites may be used
to provide the required nontranscribed genetic elements.
Recombinant polypeptides and proteins produced in bacterial culture
are usually isolated by initial extraction from cell pellets,
followed by one or more salting-out, aqueous ion exchange or size
exclusion chromatography steps. Protein refolding steps can be
used, as necessary, in completing configuration of the mature
protein. Finally, high performance liquid chromatography (HPLC) can
be employed for final purification steps. Microbial cells employed
in expression of proteins can be disrupted by any convenient
method, including freeze-thaw cycling, sonication, mechanical
disruption, or use of cell lysing agents.
[0099] Alternatively, it may be possible to produce the protein in
lower eukaryotes such as yeast or insects or in prokaryotes such as
bacteria. Potentially suitable yeast strains include Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any yeast strain capable of expressing heterologous
proteins. Potentially suitable bacterial strains include
Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any
bacterial strain capable of expressing heterologous proteins. If
the protein is made in yeast or bacteria, it may be necessary to
modify the protein produced therein, for example by phosphorylation
or glycosylation of the appropriate sites, in order to obtain the
functional protein. Such covalent attachments may be accomplished
using known chemical or enzymatic methods.
[0100] In another embodiment of the present invention, cells and
tissues may be engineered to express an endogenous gene comprising
the polynucleotides of the invention under the control of inducible
regulatory elements, in which case the regulatory sequences of the
endogenous gene may be replaced by homologous recombination. As
described herein, gene targeting can be used to replace a gene's
existing regulatory region with a regulatory sequence isolated from
a different gene or a novel regulatory sequence synthesized by
genetic engineering methods. Such regulatory sequences may be
comprised of promoters, enhancers, scaffold-attachment regions,
negative regulatory elements, transcriptional initiation sites,
regulatory protein binding sites or combinations of said sequences.
Alternatively, sequences which affect the structure or stability of
the RNA or protein produced may be replaced, removed, added, or
otherwise modified by targeting. These sequence include
polyadenylation signals, mRNA stability elements, splice sites,
leader sequences for enhancing or modifying transport or secretion
properties of the protein, or other sequences which alter or
improve the function or stability of protein or RNA molecules.
[0101] The targeting event may be a simple insertion of the
regulatory sequence, placing the gene under the control of the new
regulatory sequence, e.g., inserting a new promoter or enhancer or
both upstream of a gene. Alternatively, the targeting event may be
a simple deletion of a regulatory element, such as the deletion of
a tissue-specific negative regulatory element. Alternatively, the
targeting event may replace an existing element; for example, a
tissue-specific enhancer can be replaced by an enhancer that has
broader or different cell-type specificity than the naturally
occurring elements. Here, the naturally occurring sequences are
deleted and new sequences are added. In all cases, the
identification of the targeting event may be facilitated by the use
of one or more selectable marker genes that are contiguous with the
targeting DNA, allowing for the selection of cells in which the
exogenous DNA has integrated into the host cell genome. The
identification of the targeting event may also be facilitated by
the use of one or more marker genes exhibiting the property of
negative selection, such that the negatively selectable marker is
linked to the exogenous DNA, but configured such that the
negatively selectable marker flanks the targeting sequence, and
such that a correct homologous recombination event with sequences
in the host cell genome does not result in the stable integration
of the negatively selectable marker. Markers useful for this
purpose include the Herpes Simplex Virus thymidine kinase (TK) gene
or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt)
gene.
[0102] The gene targeting or gene activation techniques which can
be used in accordance with this aspect of the invention are more
particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S.
Pat. No. 5,578,461 to Sherwin et al.; International Application No.
PCT/US92/09627 (WO93/09222) by Selden et al.; and International
Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al.,
each of which is incorporated by reference herein in its
entirety.
[0103] POLYPEPTIDES OF THE INVENTION
[0104] The isolated polypeptides of the invention include, but are
not limited to, a polypeptide comprising: the amino acid sequences
set forth as any one of SEQ ID NO: 1-10 or an amino acid sequence
encoded by any one of the nucleotide sequences SEQ ID NOs: 1-10 or
the corresponding full length or mature protein. Polypeptides of
the invention also include polypeptides preferably with biological
or immunological activity that are encoded by: (a) a polynucleotide
having any one of the nucleotide sequences set forth in SEQ ID NOs:
1-10 or (b) polynucleotides encoding any one of the amino acid
sequences set forth as SEQ ID NO: 1-10 or (c) polynucleotides that
hybridize to the complement of the polynucleotides of either (a) or
(b) under stringent hybridization conditions. The invention also
provides biologically active or immunologically active variants of
any of the amino acid sequences set forth as SEQ ID NO: 1-10 or the
corresponding full length or mature protein; and "substantial
equivalents" thereof (e.g., with at least about 65%, at least about
70%, at least about 75%, at least about 80%, at least about 85%, at
least about 90%, typically at least about 95%, more typically at
least about 98%, or most typically at least about 99% amino acid
identity) that retain biological activity. Polypeptides encoded by
allelic variants may have a similar, increased, or decreased
activity compared to polypeptides comprising SEQ ID NO: 1-10.
[0105] Fragments of the proteins of the present invention which are
capable of exhibiting biological activity are also encompassed by
the present invention. Fragments of the protein may be in linear
form or they may be cyclized using known methods, for example, as
described in H. U. Saragovi, et al., Bio/Technology 10, 773-778
(1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114,
9245-9253 (1992), both of which are incorporated herein by
reference. Such fragments may be fused to carrier molecules such as
immunoglobulins for many purposes, including increasing the valency
of protein binding sites.
[0106] The present invention also provides both full-length and
mature forms (for example, without a signal sequence or precursor
sequence) of the disclosed proteins. The protein coding sequence is
identified in the sequence listing by translation of the disclosed
nucleotide sequences. The mature form of such protein may be
obtained by expression of a full-length polynucleotide in a
suitable mammalian cell or other host cell. The sequence of the
mature form of the protein is also determinable from the amino acid
sequence of the full-length form. Where proteins of the present
invention are membrane bound, soluble forms of the proteins are
also provided. In such forms, part or all of the regions causing
the proteins to be membrane bound are deleted so that the proteins
are fully secreted from the cell in which they are expressed.
[0107] Protein compositions of the present invention may further
comprise an acceptable carrier, such as a hydrophilic, e.g.,
pharmaceutically acceptable, carrier.
[0108] The present invention further provides isolated polypeptides
encoded by the nucleic acid fragments of the present invention or
by degenerate variants of the nucleic acid fragments of the present
invention. By "degenerate variant" is intended nucleotide fragments
which differ from a nucleic acid fragment of the present invention
(e.g., an ORF) by nucleotide sequence but, due to the degeneracy of
the genetic code, encode an identical polypeptide sequence.
Preferred nucleic acid fragments of the present invention are the
ORFs that encode proteins.
[0109] A variety of methodologies known in the art can be utilized
to obtain any one of the isolated polypeptides or proteins of the
present invention. At the simplest level, the amino acid sequence
can be synthesized using commercially available peptide
synthesizers. The synthetically-constructed protein sequences, by
virtue of sharing primary, secondary or tertiary structural and/or
conformational characteristics with proteins may possess biological
properties in common therewith, including protein activity. This
technique is particularly useful in producing small peptides and
fragments of larger polypeptides. Fragments are useful, for
example, in generating antibodies against the native polypeptide.
Thus, they may be employed as biologically active or immunological
substitutes for natural, purified proteins in screening of
therapeutic compounds and in immunological processes for the
development of antibodies.
[0110] The polypeptides and proteins of the present invention can
alternatively be purified from cells which have been altered to
express the desired polypeptide or protein. As used herein, a cell
is said to be altered to express a desired polypeptide or protein
when the cell, through genetic manipulation, is made to produce a
polypeptide or protein which it normally does not produce or which
the cell normally produces at a lower level. One skilled in the art
can readily adapt procedures for introducing and expressing either
recombinant or synthetic sequences into eukaryotic or prokaryotic
cells in order to generate a cell which produces one of the
polypeptides or proteins of the present invention.
[0111] The invention also relates to methods for producing a
polypeptide comprising growing a culture of host cells of the
invention in a suitable culture medium, and purifying the protein
from the cells or the culture in which the cells are grown. For
example, the methods of the invention include a process for
producing a polypeptide in which a host cell containing a suitable
expression vector that includes a polynucleotide of the invention
is cultured under conditions that allow expression of the encoded
polypeptide. The polypeptide can be recovered from the culture,
conveniently from the culture medium, or from a lysate prepared
from the host cells and further purified. Preferred embodiments
include those in which the protein produced by such process is a
full length or mature form of the protein.
[0112] In an alternative method, the polypeptide or protein is
purified from bacterial cells which naturally produce the
polypeptide or protein. One skilled in the art can readily follow
known methods for isolating polypeptides and proteins in order to
obtain one of the isolated polypeptides or proteins of the present
invention. These include, but are not limited to,
immunochromatography, HPLC, size-exclusion chromatography,
ion-exchange chromatography, and immuno-affinity chromatography.
See, e.g., Scopes, Protein Purification: Principles and Practice,
Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A
Laboratory Manual; Ausubel et al., Current Protocols in Molecular
Biology. Polypeptide fragments that retain biological/immunological
activity include fragments comprising greater than about 100 amino
acids, or greater than about 200 amino acids, and fragments that
encode specific protein domains.
[0113] The purified polypeptides can be used in in vitro binding
assays which are well known in the art to identify molecules which
bind to the polypeptides. These molecules include but are not
limited to, for e.g., small molecules, molecules from combinatorial
libraries, antibodies or other proteins. The molecules identified
in the binding assay are then tested for antagonist or agonist
activity in in vivo tissue culture or animal models that are well
known in the art. In brief, the molecules are titrated into a
plurality of cell cultures or animals and then tested for either
cell/animal death or prolonged survival of the animal/cells.
[0114] In addition, the peptides of the invention or molecules
capable of binding to the peptides may be complexed with toxins,
e.g., ricin or cholera, or with other compounds that are toxic to
cells. The toxin-binding molecule complex is then targeted to a
tumor or other cell by the specificity of the binding molecule for
SEQ ID NO: 1-10.
[0115] The protein of the invention may also be expressed as a
product of transgenic animals, e.g., as a component of the milk of
transgenic cows, goats, pigs, or sheep which are characterized by
somatic or germ cells containing a nucleotide sequence encoding the
protein.
[0116] The proteins provided herein also include proteins
characterized by amino acid sequences similar to those of purified
proteins but into which modification are naturally provided or
deliberately engineered. For example, modifications, in the peptide
or DNA sequence, can be made by those skilled in the art using
known techniques. Modifications of interest in the protein
sequences may include the alteration, substitution, replacement,
insertion or deletion of a selected amino acid residue in the
coding sequence. For example, one or more of the cysteine residues
may be deleted or replaced with another amino acid to alter the
conformation of the molecule. Techniques for such alteration,
substitution, replacement, insertion or deletion are well known to
those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584).
Preferably, such alteration, substitution, replacement, insertion
or deletion retains the desired activity of the protein. Regions of
the protein that are important for the protein function can be
determined by various methods known in the art including the
alanine-scanning method which involved systematic substitution of
single or strings of amino acids with alanine, followed by testing
the resulting alanine-containing variant for biological activity.
This type of analysis determines the importance of the substituted
amino acid(s) in biological activity. Regions of the protein that
are important for protein function may be determined by the eMATRIX
program.
[0117] Other fragments and derivatives of the sequences of proteins
which would be expected to retain protein activity in whole or in
part and are useful for screening or other immunological
methodologies may also be easily made by those skilled in the art
given the disclosures herein. Such modifications are encompassed by
the present invention.
[0118] The protein may also be produced by operably linking the
isolated polynucleotide of the invention to suitable control
sequences in one or more insect expression vectors, and employing
an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially
available in kit form from, e.g., Invitrogen, San Diego, Calif.,
U.S.A. (the MaxBat.TM. kit), and such methods are well known in the
art, as described in Summers and Smith, Texas Agricultural
Experiment Station Bulletin No. 1555 (1987), incorporated herein by
reference. As used herein, an insect cell capable of expressing a
polynucleotide of the present invention is "transformed."
[0119] The protein of the invention may be prepared by culturing
transformed host cells under culture conditions suitable to express
the recombinant protein. The resulting expressed protein may then
be purified from such culture (i.e., from culture medium or cell
extracts) using known purification processes, such as gel
filtration and ion exchange chromatography. The purification of the
protein may also include an affinity column containing agents which
will bind to the protein; one or more column steps over such
affinity resins as concanavalin A-agarose, heparin-toyopearl.TM. or
Cibacrom blue 3GA Sepharose.TM.; one or more steps involving
hydrophobic interaction chromatography using such resins as phenyl
ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
[0120] Alternatively, the protein of the invention may also be
expressed in a form which will facilitate purification. For
example, it may be expressed as a fusion protein, such as those of
maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin (TRX), or as a His tag. Kits for expression and
purification of such fusion proteins are commercially available
from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway,
N.J.) and Invitrogen, respectively. The protein can also be tagged
with an epitope and subsequently purified by using a specific
antibody directed to such epitope. One such epitope ("FLAG.RTM.")
is commercially available from Kodak (New Haven, Conn.).
[0121] Finally, one or more reverse-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify the protein. Some or all of the
foregoing purification steps, in various combinations, can also be
employed to provide a substantially homogeneous isolated
recombinant protein. The protein thus purified is substantially
free of other mammalian proteins and is defined in accordance with
the present invention as an "isolated protein."
[0122] The polypeptides of the invention include analogs
(variants). This embraces fragments, as well as peptides in which
one or more amino acids has been deleted, inserted, or substituted.
Also, analogs of the polypeptides of the invention embrace fusions
of the polypeptides or modifications of the polypeptides of the
invention, wherein the polypeptide or analog is fused to another
moiety or moieties, e.g., targeting moiety or another therapeutic
agent. Such analogs may exhibit improved properties such as
activity and/or stability. Examples of moieties which may be fused
to the polypeptide or an analog include, for example, targeting
moieties which provide for the delivery of polypeptide to
pancreatic cells, e.g., antibodies to pancreatic cells, antibodies
to immune cells such as T-cells, monocytes, dendritic cells,
granulocytes, etc., as well as receptor and ligands expressed on
pancreatic or immune cells. Other moieties which may be fused to
the polypeptide include therapeutic agents which are used for
treatment, for example, immunosuppressive drugs such as
cyclosporin, SK506, azathioprine, CD3 antibodies and steroids.
Also, polypeptides may be fused to immune modulators, and other
cytokines such as alpha or beta interferon.
[0123] DETERMINING POLYPEPTIDE AND POLYNUCLEOTIDE IDENTITY AND
SIMILARITY
[0124] Preferred identity and/or similarity are designed to give
the largest match between the sequences tested. Methods to
determine identity and similarity are codified in computer programs
including, but are not limited to, the GCG program package,
including GAP (Devereux, J., et al., Nucleic Acids Research
12(1):387 (1984); Genetics Computer Group, University of Wisconsin,
Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et
al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S. F.
et al., Nucleic Acids Res. vol. 25, pp. 3389-3402, herein
incorporated by reference), eMatrix software (Wu et al., J. Comp.
Biol., Vol. 6, pp. 219-235 (1999), herein incorporated by
reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol. 4,
pp. 202-209, herein incorporated by reference), pFam software
(Sonnhammer et al., Nucleic Acids Res., Vol. 26(1), pp. 320-322
(1998), herein incorporated by reference) and the Kyte-Doolittle
hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31
(982), incorporated herein by reference). The BLAST programs are
publicly available from the National Center for Biotechnology
Information (NCBI) and other sources (BLAST Manual, Altschul, S.,
et al. NCB NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J.
Mol. Biol. 215:403-410 (1990).
[0125] GENE THERAPY
[0126] Mutations in the polynucleotides of the invention gene may
result in loss of normal function of the encoded protein. The
invention thus provides gene therapy to restore normal activity of
the polypeptides of the invention; or to treat disease states
involving polypeptides of the invention. Delivery of a functional
gene encoding polypeptides of the invention to appropriate cells is
effected ex vivo, in situ, or in vivo by use of vectors, and more
particularly viral vectors (e.g., adenovirus, adeno-associated
virus, or a retrovirus), or ex vivo by use of physical DNA transfer
methods (e.g., liposomes or chemical treatments). See, for example,
Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20
(1998). For additional reviews of gene therapy technology see
Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific
American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).
Introduction of any one of the nucleotides of the present invention
or a gene encoding the polypeptides of the present invention can
also be accomplished with extrachromosomal substrates (transient
expression) or artificial chromosomes (stable expression). Cells
may also be cultured ex vivo in the presence of proteins of the
present invention in order to proliferate or to produce a desired
effect on or activity in such cells. Treated cells can then be
introduced in vivo for therapeutic purposes. Alternatively, it is
contemplated that in other human disease states, preventing the
expression of or inhibiting the activity of polypeptides of the
invention will be useful in treating the disease states. It is
contemplated that antisense therapy or gene therapy could be
applied to negatively regulate the expression of polypeptides of
the invention.
[0127] Other methods inhibiting expression of a protein include the
introduction of antisense molecules to the nucleic acids of the
present invention, their complements, or their translated RNA
sequences, by methods known in the art. Further, the polypeptides
of the present invention can be inhibited by using targeted
deletion methods, or the insertion of a negative regulatory element
such as a silencer, which is tissue specific.
[0128] The present invention still further provides cells
genetically engineered in vivo to express the polynucleotides of
the invention, wherein such polynucleotides are in operative
association with a regulatory sequence heterologous to the host
cell which drives expression of the polynucleotides in the cell.
These methods can be used to increase or decrease the expression of
the polynucleotides of the present invention.
[0129] Knowledge of DNA sequences provided by the invention allows
for modification of cells to permit, increase, or decrease,
expression of endogenous polypeptide. Cells can be modified (e.g.,
by homologous recombination) to provide increased polypeptide
expression by replacing, in whole or in part, the naturally
occurring promoter with all or part of a heterologous promoter so
that the cells express the protein at higher levels. The
heterologous promoter is inserted in such a manner that it is
operatively linked to the desired protein encoding sequences. See,
for example, PCT International Publication No. WO 94/12650, PCT
International Publication No. WO 92/20808, and PCT International
Publication No. WO 91/09955. It is also contemplated that, in
addition to heterologous promoter DNA, amplifiable marker DNA
(e.g., ada, dhfr, and the multifunctional CAD gene which encodes
carbamyl phosphate synthase, aspartate transcarbamylase, and
dihydroorotase) and/or intron DNA may be inserted along with the
heterologous promoter DNA. If linked to the desired protein coding
sequence, amplification of the marker DNA by standard selection
methods results in co-amplification of the desired protein coding
sequences in the cells.
[0130] In another embodiment of the present invention, cells and
tissues may be engineered to express an endogenous gene comprising
the polynucleotides of the invention under the control of inducible
regulatory elements, in which case the regulatory sequences of the
endogenous gene may be replaced by homologous recombination. As
described herein, gene targeting can be used to replace a gene's
existing regulatory region with a regulatory sequence isolated from
a different gene or a novel regulatory sequence synthesized by
genetic engineering methods. Such regulatory sequences may be
comprised of promoters, enhancers, scaffold-attachment regions,
negative regulatory elements, transcriptional initiation sites,
regulatory protein binding sites or combinations of said sequences.
Alternatively, sequences which affect the structure or stability of
the RNA or protein produced may be replaced, removed, added, or
otherwise modified by targeting. These sequences include
polyadenylation signals, mRNA stability elements, splice sites,
leader sequences for enhancing or modifying transport or secretion
properties of the protein, or other sequences which alter or
improve the function or stability of protein or RNA molecules.
[0131] The targeting event may be a simple insertion of the
regulatory sequence, placing the gene under the control of the new
regulatory sequence, e.g., inserting a new promoter or enhancer or
both upstream of a gene. Alternatively, the targeting event may be
a simple deletion of a regulatory element, such as the deletion of
a tissue-specific negative regulatory element. Alternatively, the
targeting event may replace an existing element; for example, a
tissue-specific enhancer can be replaced by an enhancer that has
broader or different cell-type specificity than the naturally
occurring elements. Here, the naturally occurring sequences are
deleted and new sequences are added. In all cases, the
identification of the targeting event may be facilitated by the use
of one or more selectable marker genes that are contiguous with the
targeting DNA, allowing for the selection of cells in which the
exogenous DNA has integrated into the cell genome. The
identification of the targeting event may also be facilitated by
the use of one or more marker genes exhibiting the property of
negative selection, such that the negatively selectable marker is
linked to the exogenous DNA, but configured such that the
negatively selectable marker flanks the targeting sequence, and
such that a correct homologous recombination event with sequences
in the host cell genome does not result in the stable integration
of the negatively selectable marker. Markers useful for this
purpose include the Herpes Simplex Virus thymidine kinase (TK) gene
or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt)
gene.
[0132] The gene targeting or gene activation techniques which can
be used in accordance with this aspect of the invention are more
particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S.
Pat. No.5,578,461 to Sherwin et al.; International Application No.
PCT/US92/09627 (WO93/09222) by Selden et al.; and International
Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al.,
each of which is incorporated by reference herein in its
entirety.
[0133] TRANSGENIC ANIMALS
[0134] In preferred methods to determine biological functions of
the polypeptides of the invention in vivo, one or more genes
provided by the invention are either over expressed or inactivated
in the germ line of animals using homologous recombination
[Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene
is over expressed, under the regulatory control of exogenous or
endogenous promoter elements, are known as transgenic animals.
Animals in which an endogenous gene has been inactivated by
homologous recombination are referred to as "knockout" animals.
Knockout animals, preferably non-human mammals, can be prepared as
described in U.S. Pat. No. 5,557,032, incorporated herein by
reference. Transgenic animals are useful to determine the roles
polypeptides of the invention play in biological processes, and
preferably in disease states. Transgenic animals are useful as
model systems to identify compounds that modulate lipid metabolism.
Transgenic animals, preferably non-human mammals, are produced
using methods as described in U.S. Pat. No 5,489,743 and PCT
Publication No. WO94/28122, incorporated herein by reference.
[0135] Transgenic animals can be prepared wherein all or part of a
promoter of the polynucleotides of the invention is either
activated or inactivated to alter the level of expression of the
polypeptides of the invention. Inactivation can be carried out
using homologous recombination methods described above. Activation
can be achieved by supplementing or even replacing the homologous
promoter to provide for increased protein expression. The
homologous promoter can be supplemented by insertion of one or more
heterologous enhancer elements known to confer promoter activation
in a particular tissue.
[0136] The polynucleotides of the present invention also make
possible the development, through, e.g., homologous recombination
or knock out strategies, of animals that fail to express
polypeptides of the invention or that express a variant
polypeptide. Such animals are useful as models for studying the in
vivo activities of polypeptide as well as for studying modulators
of the polypeptides of the invention.
[0137] In preferred methods to determine biological functions of
the polypeptides of the invention in vivo, one or more genes
provided by the invention are either over expressed or inactivated
in the germ line of animals using homologous recombination
[Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene
is over expressed, under the regulatory control of exogenous or
endogenous promoter elements, are known as transgenic animals.
Animals in which an endogenous gene has been inactivated by
homologous recombination are referred to as "knockout" animals.
Knockout animals, preferably non-human mammals, can be prepared as
described in U.S. Pat. No. 5,557,032, incorporated herein by
reference. Transgenic animals are useful to determine the roles
polypeptides of the invention play in biological processes, and
preferably in disease states. Transgenic animals are useful as
model systems to identify compounds that modulate lipid metabolism.
Transgenic animals, preferably non-human mammals, are produced
using methods as described in U.S. Pat. No 5,489,743 and PCT
Publication No. WO94/28122, incorporated herein by reference.
[0138] Transgenic animals can be prepared wherein all or part of
the polynucleotides of the invention promoter is either activated
or inactivated to alter the level of expression of the polypeptides
of the invention. Inactivation can be carried out using homologous
recombination methods described above. Activation can be achieved
by supplementing or even replacing the homologous promoter to
provide for increased protein expression. The homologous promoter
can be supplemented by insertion of one or more heterologous
enhancer elements known to confer promoter activation in a
particular tissue.
[0139] USES AND BIOLOGICAL ACTIVITY
[0140] The polynucleotides and proteins of the present invention
are expected to exhibit one or more of the uses or biological
activities (including those associated with assays cited herein)
identified herein. Uses or activities described for proteins of the
present invention may be provided by administration or use of such
proteins or of polynucleotides encoding such proteins (such as, for
example, in gene therapies or vectors suitable for introduction of
DNA). The mechanism underlying the particular condition or
pathology will dictate whether the polypeptides of the invention,
the polynucleotides of the invention or modulators (activators or
inhibitors) thereof would be beneficial to the subject in need of
treatment. Thus, "therapeutic compositions of the invention"
include compositions comprising isolated polynucleotides (including
recombinant DNA molecules, cloned genes and degenerate variants
thereof) or polypeptides of the invention (including full length
protein, mature protein and truncations or domains thereof), or
compounds and other substances that modulate the overall activity
of the target gene products, either at the level of target
gene/protein expression or target protein activity. Such modulators
include polypeptides, analogs, (variants), including fragments and
fusion proteins, antibodies and other binding proteins; chemical
compounds that directly or indirectly activate or inhibit the
polypeptides of the invention (identified, e.g., via drug screening
assays as described herein); antisense polynucleotides and
polynucleotides suitable for triple helix formation; and in
particular antibodies or other binding partners that specifically
recognize one or more epitopes of the polypeptides of the
invention.
[0141] The polypeptides of the present invention may likewise be
involved in cellular activation or in one of the other
physiological pathways described herein.
[0142] RESEARCH USES AND UTILITIES
[0143] The polynucleotides provided by the present invention can be
used by the research community for various purposes. The
polynucleotides can be used to express recombinant protein for
analysis, characterization or therapeutic use; as markers for
tissues in which the corresponding protein is preferentially
expressed (either constitutively or at a particular stage of tissue
differentiation or development or in disease states); as molecular
weight markers on gels; as chromosome markers or tags (when
labeled) to identify chromosomes or to map related gene positions;
to compare with endogenous DNA sequences in patients to identify
potential genetic disorders; as probes to hybridize and thus
discover novel, related DNA sequences; as a source of information
to derive PCR primers for genetic fingerprinting; as a probe to
"subtract-out" known sequences in the process of discovering other
novel polynucleotides; for selecting and making oligomers for
attachment to a "gene chip" or other support, including for
examination of expression patterns; to raise anti-protein
antibodies using DNA immunization techniques; and as an antigen to
raise anti-DNA antibodies or elicit another immune response. Where
the polynucleotide encodes a protein which binds or potentially
binds to another protein (such as, for example, in a
receptor-ligand interaction), the polynucleotide can also be used
in interaction trap assays (such as, for example, that described in
Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides
encoding the other protein with which binding occurs or to identify
inhibitors of the binding interaction.
[0144] The polypeptides provided by the present invention can
similarly be used in assays to determine biological activity,
including in a panel of multiple proteins for high-throughput
screening; to raise antibodies or to elicit another immune
response; as a reagent (including the labeled reagent) in assays
designed to quantitatively determine levels of the protein (or its
receptor) in biological fluids; as markers for tissues in which the
corresponding polypeptide is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation
or development or in a disease state); and, of course, to isolate
correlative receptors or ligands. Proteins involved in these
binding interactions can also be used to screen for peptide or
small molecule inhibitors or agonists of the binding
interaction.
[0145] Any or all of these research utilities are capable of being
developed into reagent grade or kit format for commercialization as
research products.
[0146] Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include without limitation "Molecular Cloning: A Laboratory
Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J.,
E. F. Fritsch and T. Maniatis eds., 1989, and "Methods in
Enzymology: Guide to Molecular Cloning Techniques", Academic Press,
Berger, S. L. and A. R. Kimmel eds., 1987.
[0147] NUTRITIONAL USES
[0148] Polynucleotides and polypeptides of the present invention
can also be used as nutritional sources or supplements. Such uses
include without limitation use as a protein or amino acid
supplement, use as a carbon source, use as a nitrogen source and
use as a source of carbohydrate. In such cases the polypeptide or
polynucleotide of the invention can be added to the feed of a
particular organism or can be administered as a separate solid or
liquid preparation, such as in the form of powder, pills,
solutions, suspensions or capsules. In the case of microorganisms,
the polypeptide or polynucleotide of the invention can be added to
the medium in or on which the microorganism is cultured.
[0149] CYTOKINE AND CELL PROLIFERATION/DIFFERENTIATION ACTIVITY
[0150] A polypeptide of the present invention may exhibit activity
relating to cytokine, cell proliferation (either inducing or
inhibiting) or cell differentiation (either inducing or inhibiting)
activity or may induce production of other cytokines in certain
cell populations. A polynucleotide of the invention can encode a
polypeptide exhibiting such attributes. Many protein factors
discovered to date, including all known cytokines, have exhibited
activity in one or more factor-dependent cell proliferation assays,
and hence the assays serve as a convenient confirmation of cytokine
activity. The activity of therapeutic compositions of the present
invention is evidenced by any one of a number of routine factor
dependent cell proliferation assays for cell lines including,
without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G,
M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e,
CMK, HUVEC, and Caco. Therapeutic compositions of the invention can
be used in the following:
[0151] Assays for T-cell or thymocyte proliferation include without
limitation those described in: Current Protocols in Immunology, Ed
by J. E. Coligan, A. M. Kruisbeek, D. B. Margulies, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai
et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J.
Inmmunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular
Immunology 133:327-341, 1991; Bertagnolli, et al., I. Immunol.
149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761,
1994.
[0152] Assays for cytokine production and/or proliferation of
spleen cells, lymph node cells or thymocytes include, without
limitation, those described in: Polyclonal T cell stimulation,
Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in
Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John
Wiley and Sons, Toronto. 1994; and Measurement of mouse and human
interleukin-.gamma., Schreiber, R. D. In Current Protocols in
Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John
Wiley and Sons, Toronto. 1994.
[0153] Assays for proliferation and differentiation of
hematopoietic and lymphopoietic cells include, without limitation,
those described in: Measurement of Human and Murine Interleukin 2
and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In
Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.
6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al.,
J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature
336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci.
U.S.A. 80:2931-2938, 1983; Measurement of mouse and human
interleukin 6--Nordan, R. In Current Protocols in Immunology. J. E.
Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto.
1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861,
1986; Measurement of human Interleukin 11--Bennett, F., Giannotti,
J., Clark, S. C. and Turner, K. J. In Current Protocols in
Immunology. J. E. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and
Sons, Toronto. 1991; Measurement of mouse and human Interleukin
9--Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J. In
Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp.
6.13.1, John Wiley and Sons, Toronto. 1991.
[0154] Assays for T-cell clone responses to antigens (which will
identify, among others, proteins that affect APC-T cell
interactions as well as direct T-cell effects by measuring
proliferation and cytokine production) include, without limitation,
those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W
Strober, Pub. Greene Publishing Associates and Wiley-Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter
6, Cytokines and their cellular receptors; Chapter 7, Immunologic
studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA
77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411,
1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol. 140:508-512, 1988.
[0155] STEM CELL GROWTH FACTOR ACTIVITY
[0156] A polypeptide of the present invention may exhibit stem cell
growth factor activity and be involved in the proliferation,
differentiation and survival of pluripotent and totipotent stem
cells including primordial germ cells, embryonic stem cells,
hematopoietic stem cells and/or germ line stem cells.
Administration of the polypeptide of the invention to stem cells in
vivo or ex vivo is expected to maintain and expand cell populations
in a totipotential or pluripotential state which would be useful
for re-engineering damaged or diseased tissues, transplantation,
manufacture of bio-pharmaceuticals and the development of
bio-sensors. The ability to produce large quantities of human cells
has important working applications for the production of human
proteins which currently must be obtained from non-human sources or
donors, implantation of cells to treat diseases such as
Parkinson's, Alzheimer's and other neurodegenerative diseases;
tissues for grafting such as bone marrow, skin, cartilage, tendons,
bone, muscle (including cardiac muscle), blood vessels, cornea,
neural cells, gastrointestinal cells and others; and organs for
transplantation such as kidney, liver, pancreas (including islet
cells), heart and lung.
[0157] It is contemplated that multiple different exogenous growth
factors and/or cytokines may be administered in combination with
the polypeptide of the invention to achieve the desired effect,
including any of the growth factors listed herein, other stem cell
maintenance factors, and specifically including stem cell factor
(SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any
of the interleukins, recombinant soluble IL-6 receptor fused to
IL-6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CFS,
GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF-4),
platelet-derived growth factor (PDGF), neural growth factors and
basic fibroblast growth factor (bFGF).
[0158] Since totipotent stem cells can give rise to virtually any
mature cell type, expansion of these cells in culture will
facilitate the production of large quantities of mature cells.
Techniques for culturing stem cells are known in the art and
administration of polypeptides of the invention, optionally with
other growth factors and/or cytokines, is expected to enhance the
survival and proliferation of the stem cell populations. This can
be accomplished by direct administration of the polypeptide of the
invention to the culture medium. Alternatively, stroma cells
transfected with a polynucleotide that encodes for the polypeptide
of the invention can be used as a feeder layer for the stem cell
populations in culture or in vivo. Stromal support cells for feeder
layers may include embryonic bone marrow fibroblasts, bone marrow
stromal cells, fetal liver cells, or cultured embryonic fibroblasts
(see U.S. Pat. No. 5,690,926).
[0159] Stem cells themselves can be transfected with a
polynucleotide of the invention to induce autocrine expression of
the polypeptide of the invention. This will allow for generation of
undifferentiated totipotential/pluripotential stem cell lines that
are useful as is or that can then be differentiated into the
desired mature cell types. These stable cell lines can also serve
as a source of undifferentiated totipotential/pluripotential mRNA
to create cDNA libraries and templates for polymerase chain
reaction experiments. These studies would allow for the isolation
and identification of differentially expressed genes in stem cell
populations that regulate stem cell proliferation and/or
maintenance.
[0160] Expansion and maintenance of totipotent stem cell
populations will be useful in the treatment of many pathological
conditions. For example, polypeptides of the present invention may
be used to manipulate stem cells in culture to give rise to
neuroepithelial cells that can be used to augment or replace cells
damaged by illness, autoimmune disease, accidental damage or
genetic disorders. The polypeptide of the invention may be useful
for inducing the proliferation of neural cells and for the
regeneration of nerve and brain tissue, i.e. for the treatment of
central and peripheral nervous system diseases and neuropathies, as
well as mechanical and traumatic disorders which involve
degeneration, death or trauma to neural cells or nerve tissue. In
addition, the expanded stem cell populations can also be
genetically altered for gene therapy purposes and to decrease host
rejection of replacement tissues after grafting or
implantation.
[0161] Expression of the polypeptide of the invention and its
effect on stem cells can also be manipulated to achieve controlled
differentiation of the stem cells into more differentiated cell
types. A broadly applicable method of obtaining pure populations of
a specific differentiated cell type from undifferentiated stem cell
populations involves the use of a cell-type specific promoter
driving a selectable marker. The selectable marker allows only
cells of the desired type to survive. For example, stem cells can
be induced to differentiate into cardiomyocytes (Wobus et al.,
Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.
Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder,
L. W. In: Principles of Tissue Engineering eds. Lanza et al.,
Academic Press (1997)). Alternatively, directed differentiation of
stem cells can be accomplished by culturing the stem cells in the
presence of a differentiation factor such as retinoic acid and an
antagonist of the polypeptide of the invention which would inhibit
the effects of endogenous stem cell factor activity and allow
differentiation to proceed.
[0162] In vitro cultures of stem cells can be used to determine if
the polypeptide of the invention exhibits stem cell growth factor
activity. Stem cells are isolated from any one of various cell
sources (including hematopoietic stem cells and embryonic stem
cells) and cultured on a feeder layer, as described by Thompson et
al. Proc. Natl. Acad. Sci, U.S.A., 92: 7844-7848 (1995), in the
presence of the polypeptide of the invention alone or in
combination with other growth factors or cytokines. The ability of
the polypeptide of the invention to induce stem cells proliferation
is determined by colony formation on semi-solid support e.g. as
described by Bernstein et al., Blood, 77: 2316-2321 (1991).
[0163] HEMATOPOIESIS REGULATING ACTIVITY
[0164] A polypeptide of the present invention may be involved in
regulation of hematopoiesis and, consequently, in the treatment of
myeloid or lymphoid cell disorders. Even marginal biological
activity in support of colony forming cells or of factor-dependent
cell lines indicates involvement in regulating hematopoiesis, e.g.
in supporting the growth and proliferation of erythroid progenitor
cells alone or in combination with other cytokines, thereby
indicating utility, for example, in treating various anemias or for
use in conjunction with irradiation/chemotherapy to stimulate the
production of erythroid precursors and/or erythroid cells; in
supporting the growth and proliferation of myeloid cells such as
granulocytes and monocytes/macrophages (i.e., traditional CSF
activity) useful, for example, in conjunction with chemotherapy to
prevent or treat consequent myelo-suppression; in supporting the
growth and proliferation of megakaryocytes and consequently of
platelets thereby allowing prevention or treatment of various
platelet disorders such as thrombocytopenia, and generally for use
in place of or complimentary to platelet transfusions; and/or in
supporting the growth and proliferation of hematopoietic stem cells
which are capable of maturing to any and all of the above-mentioned
hematopoietic cells and therefore find therapeutic utility in
various stem cell disorders (such as those usually treated with
transplantation, including, without limitation, aplastic anemia and
paroxysmal nocturnal hemoglobinuria), as well as in repopulating
the stem cell compartment post irradiation/chemotherapy, either
in-vivo or ex-vivo (i.e., in conjunction with bone marrow
transplantation or with peripheral progenitor cell transplantation
(homologous or heterologous)) as normal cells or genetically
manipulated for gene therapy.
[0165] Therapeutic compositions of the invention can be used in the
following:
[0166] Suitable assays for proliferation and differentiation of
various hematopoietic lines are cited above.
[0167] Assays for embryonic stem cell differentiation (which will
identify, among others, proteins that influence embryonic
differentiation hematopoiesis) include, without limitation, those
described in: Johansson et al. Cellular Biology 15:141-151, 1995;
Keller et al., Molecular and Cellular Biology 13:473-486, 1993;
McClanahan et al., Blood 81:2903-2915, 1993.
[0168] Assays for stem cell survival and differentiation (which
will identify, among others, proteins that regulate
lympho-hematopoiesis) include, without limitation, those described
in: Methylcellulose colony forming assays, Freshney, M. G. In
Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.
265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al.,
Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive
hematopoietic colony forming cells with high proliferative
potential, McNiece, I. K. and Briddell, R. A. In Culture of
Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,
Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental
Hematology 22:353-359, 1994; Cobblestone area forming cell assay,
Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I.
Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York,
N.Y. 1994; Long term bone marrow cultures in the presence of
stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of
Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-179,
Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating
cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R.
I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New
York, N.Y. 1994.
[0169] TISSUE GROWTH ACTIVITY
[0170] A polypeptide of the present invention also may be involved
in bone, cartilage, tendon, ligament and/or nerve tissue growth or
regeneration, as well as in wound healing and tissue repair and
replacement, and in healing of burns, incisions and ulcers.
[0171] A polypeptide of the present invention which induces
cartilage and/or bone growth in circumstances where bone is not
normally formed, has application in the healing of bone fractures
and cartilage damage or defects in humans and other animals.
Compositions of a polypeptide, antibody, binding partner, or other
modulator of the invention may have prophylactic use in closed as
well as open fracture reduction and also in the improved fixation
of artificial joints. De novo bone formation induced by an
osteogenic agent contributes to the repair of congenital, trauma
induced, or oncologic resection induced craniofacial defects, and
also is useful in cosmetic plastic surgery.
[0172] A polypeptide of this invention may also be involved in
attracting bone-forming cells, stimulating growth of bone-forming
cells, or inducing differentiation of progenitors of bone-forming
cells. Treatment of osteoporosis, osteoarthritis, bone degenerative
disorders, or periodontal disease, such as through stimulation of
bone and/or cartilage repair or by blocking inflammation or
processes of tissue destruction (collagenase activity, osteoclast
activity, etc.) mediated by inflammatory processes may also be
possible using the composition of the invention.
[0173] Another category of tissue regeneration activity that may
involve the polypeptide of the present invention is tendon/ligament
formation. Induction of tendon/ligament-like tissue or other tissue
formation in circumstances where such tissue is not normally
formed, has application in the healing of tendon or ligament tears,
deformities and other tendon or ligament defects in humans and
other animals. Such a preparation employing a tendon/ligament-like
tissue inducing protein may have prophylactic use in preventing
damage to tendon or ligament tissue, as well as use in the improved
fixation of tendon or ligament to bone or other tissues, and in
repairing defects to tendon or ligament tissue. De novo
tendon/ligament-like tissue formation induced by a composition of
the present invention contributes to the repair of congenital,
trauma induced, or other tendon or ligament defects of other
origin, and is also useful in cosmetic plastic surgery for
attachment or repair of tendons or ligaments. The compositions of
the present invention may provide environment to attract tendon- or
ligament-forming cells, stimulate growth of tendon- or
ligament-forming cells, induce differentiation of progenitors of
tendon- or ligament-forming cells, or induce growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to
effect tissue repair. The compositions of the invention may also be
useful in the treatment of tendinitis, carpal tunnel syndrome and
other tendon or ligament defects. The compositions may also include
an appropriate matrix and/or sequestering agent as a carrier as is
well known in the art.
[0174] The compositions of the present invention may also be useful
for proliferation of neural cells and for regeneration of nerve and
brain tissue, i.e. for the treatment of central and peripheral
nervous system diseases and neuropathies, as well as mechanical and
traumatic disorders, which involve degeneration, death or trauma to
neural cells or nerve tissue. More specifically, a composition may
be used in the treatment of diseases of the peripheral nervous
system, such as peripheral nerve injuries, peripheral neuropathy
and localized neuropathies, and central nervous system diseases,
such as Alzheimer's, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further
conditions which may be treated in accordance with the present
invention include mechanical and traumatic disorders, such as
spinal cord disorders, head trauma and cerebrovascular diseases
such as stroke. Peripheral neuropathies resulting from chemotherapy
or other medical therapies may also be treatable using a
composition of the invention.
[0175] Compositions of the invention may also be useful to promote
better or faster closure of non-healing wounds, including without
limitation pressure ulcers, ulcers associated with vascular
insufficiency, surgical and traumatic wounds, and the like.
[0176] Compositions of the present invention may also be involved
in the generation or regeneration of other tissues, such as organs
(including, for example, pancreas, liver, intestine, kidney, skin,
endothelium), muscle (smooth, skeletal or cardiac) and vascular
(including vascular endothelium) tissue, or for promoting the
growth of cells comprising such tissues. Part of the desired
effects may be by inhibition or modulation of fibrotic scarring may
allow normal tissue to regenerate. A polypeptide of the present
invention may also exhibit angiogenic activity.
[0177] A composition of the present invention may also be useful
for gut protection or regeneration and treatment of lung or liver
fibrosis, reperfusion injury in various tissues, and conditions
resulting from systemic cytokine damage.
[0178] A composition of the present invention may also be useful
for promoting or inhibiting differentiation of tissues described
above from precursor tissues or cells; or for inhibiting the growth
of tissues described above.
[0179] Therapeutic compositions of the invention can be used in the
following:
[0180] Assays for tissue generation activity include, without
limitation, those described in: International Patent Publication
No. WO95/16035 (bone, cartilage, tendon); International Patent
Publication No. WO95/05846 (nerve, neuronal); International Patent
Publication No. WO91/07491 (skin, endothelium).
[0181] Assays for wound healing activity include, without
limitation, those described in: Winter, Epidermal Wound Healing,
pps. 71-112 (Maibach, H. I. and Rovee, D. T., eds.), Year Book
Medical Publishers, Inc., Chicago, as modified by Eaglstein and
Mertz, J. Invest. Dennatol 71:382-84 (1978).
[0182] IMMUNE STIMULATING OR SUPPRESSING ACTIVITY
[0183] A polypeptide of the present invention may also exhibit
immune stimulating or immune suppressing activity, including
without limitation the activities for which assays are described
herein. A polynucleotide of the invention can encode a polypeptide
exhibiting such activities. A protein may be useful in the
treatment of various immune deficiencies and disorders (including
severe combined immunodeficiency (SCID)), e.g., in regulating (up
or down) growth and proliferation of T and/or B lymphocytes, as
well as effecting the cytolytic activity of NK cells and other cell
populations. These immune deficiencies may be genetic or be caused
by viral (e.g., HIV) as well as bacterial or fungal infections, or
may result from autoimmune disorders. More specifically, infectious
diseases causes by viral, bacterial, fungal or other infection may
be treatable using a protein of the present invention, including
infections by HIV, hepatitis viruses, herpes viruses, mycobacteria,
Leishmania spp., malaria spp. and various fungal infections such as
candidiasis. Of course, in this regard, proteins of the present
invention may also be useful where a boost to the immune system
generally may be desirable, i.e., in the treatment of cancer.
[0184] Autoimmune disorders which may be treated using a protein of
the present invention include, for example, connective tissue
disease, multiple sclerosis, systemic lupus erythematosus,
rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent
diabetes mellitis, myasthenia gravis, graft-versus-host disease and
autoimmune inflammatory eye disease. Such a protein (or antagonists
thereof, including antibodies) of the present invention may also to
be useful in the treatment of allergic reactions and conditions
(e.g., anaphylaxis, serum sickness, drug reactions, food allergies,
insect venom allergies, mastocytosis, allergic rhinitis,
hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic
dermatitis, allergic contact dermatitis, erythema multiforme,
Stevens-Johnson syndrome, allergic conjunctivitis, atopic
keratoconjunctivitis, venereal keratoconjunctivitis, giant
papillary conjunctivitis and contact allergies), such as asthma
(particularly allergic asthma) or other respiratory problems. Other
conditions, in which immune suppression is desired (including, for
example, organ transplantation), may also be treatable using a
protein (or antagonists thereof) of the present invention. The
therapeutic effects of the polypeptides or antagonists thereof on
allergic reactions can be evaluated by in vivo animals models such
as the cumulative contact enhancement test (Lastbom et al.,
Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al.,
Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr
et al., Arch. Toxocol. 73: 501-9), and murine local lymph node
assay (Kimber et al., J. Toxicol. Environ. Health 53: 563-79).
[0185] Using the proteins of the invention it may also be possible
to modulate immune responses, in a number of ways. Down regulation
may be in the form of inhibiting or blocking an immune response
already in progress or may involve preventing the induction of an
immune response. The functions of activated T cells may be
inhibited by suppressing T cell responses or by inducing specific
tolerance in T cells, or both. Immunosuppression of T cell
responses is generally an active, non-antigen-specific, process
which requires continuous exposure of the T cells to the
suppressive agent. Tolerance, which involves inducing
non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and
persists after exposure to the tolerizing agent has ceased.
Operationally, tolerance can be demonstrated by the lack of a T
cell response upon reexposure to specific antigen in the absence of
the tolerizing agent.
[0186] Down regulating or preventing one or more antigen functions
(including without limitation B lymphocyte antigen functions (such
as, for example, B7)), e.g., preventing high level lymphokine
synthesis by activated T cells, will be useful in situations of
tissue, skin and organ transplantation and in graft-versus-host
disease (GVHD). For example, blockage of T cell function should
result in reduced tissue destruction in tissue transplantation.
Typically, in tissue transplants, rejection of the transplant is
initiated through its recognition as foreign by T cells, followed
by an immune reaction that destroys the transplant. The
administration of a therapeutic composition of the invention may
prevent cytokine synthesis by immune cells, such as T cells, and
thus acts as an immunosuppressant. Moreover, a lack of
costimulation may also be sufficient to anergize the T cells,
thereby inducing tolerance in a subject. Induction of long-term
tolerance by B lymphocyte antigen-blocking reagents may avoid the
necessity of repeated administration of these blocking reagents. To
achieve sufficient immunosuppression or tolerance in a subject, it
may also be necessary to block the function of a combination of B
lymphocyte antigens.
[0187] The efficacy of particular therapeutic compositions in
preventing organ transplant rejection or GVHD can be assessed using
animal models that are predictive of efficacy in humans. Examples
of appropriate systems which can be used include allogeneic cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice,
both of which have been used to examine the immunosuppressive
effects of CTLA4Ig fusion proteins in vivo as described in Lenschow
et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl.
Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of
GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York,
1989, pp. 846-847) can be used to determine the effect of
therapeutic compositions of the invention on the development of
that disease.
[0188] Blocking antigen function may also be therapeutically useful
for treating autoimmune diseases. Many autoimmune disorders are the
result of inappropriate activation of T cells that are reactive
against self tissue and which promote the production of cytokines
and autoantibodies involved in the pathology of the diseases.
Preventing the activation of autoreactive T cells may reduce or
eliminate disease symptoms. Administration of reagents which block
stimulation of T cells can be used to inhibit T cell activation and
prevent production of autoantibodies or T cell-derived cytokines
which may be involved in the disease process. Additionally,
blocking reagents may induce antigen-specific tolerance of
autoreactive T cells which could lead to long-term relief from the
disease. The efficacy of blocking reagents in preventing or
alleviating autoimmune disorders can be determined using a number
of well-characterized animal models of human autoimmune diseases.
Examples include murine experimental autoimmune encephalitis,
systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice,
murine autoimmune collagen arthritis, diabetes mellitus in NOD mice
and BB rats, and murine experimental myasthenia gravis (see Paul
ed., Fundamental Immunology, Raven Press, New York, 1989, pp.
840-856).
[0189] Upregulation of an antigen function (e.g., a B lymphocyte
antigen function), as a means of up regulating immune responses,
may also be useful in therapy. Upregulation of immune responses may
be in the form of enhancing an existing immune response or
eliciting an initial immune response. For example, enhancing an
immune response may be useful in cases of viral infection,
including systemic viral diseases such as influenza, the common
cold, and encephalitis.
[0190] Alternatively, anti-viral immune responses may be enhanced
in an infected patient by removing T cells from the patient,
costimulating the T cells in vitro with viral antigen-pulsed APCs
either expressing a peptide of the present invention or together
with a stimulatory form of a soluble peptide of the present
invention and reintroducing the in vitro activated T cells into the
patient. Another method of enhancing anti-viral immune responses
would be to isolate infected cells from a patient, transfect them
with a nucleic acid encoding a protein of the present invention as
described herein such that the cells express all or a portion of
the protein on their surface, and reintroduce the transfected cells
into the patient. The infected cells would now be capable of
delivering a costimulatory signal to, and thereby activate, T cells
in vivo.
[0191] A polypeptide of the present invention may provide the
necessary stimulation signal to T cells to induce a T cell mediated
immune response against the transfected tumor cells. In addition,
tumor cells which lack MHC class I or MHC class II molecules, or
which fail to reexpress sufficient mounts of MHC class I or MHC
class II molecules, can be transfected with nucleic acid encoding
all or a portion of (e.g., a cytoplasmic-domain truncated portion)
of an MHC class I alpha chain protein and .beta..sub.2
microglobulin protein or an MHC class II alpha chain protein and an
MHC class II beta chain protein to thereby express MEC class I or
MHC class II proteins on the cell surface. Expression of the
appropriate class I or class II MHC in conjunction with a peptide
having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2,
B7-3) induces a T cell mediated immune response against the
transfected tumor cell. Optionally, a gene encoding an antisense
construct which blocks expression of an MHC class II associated
protein, such as the invariant chain, can also be cotransfected
with a DNA encoding a peptide having the activity of a B lymphocyte
antigen to promote presentation of tumor associated antigens and
induce tumor specific immunity. Thus, the induction of a T cell
mediated immune response in a human subject may be sufficient to
overcome tumor-specific tolerance in the subject.
[0192] The activity of a protein of the invention may, among other
means, be measured by the following methods:
[0193] Suitable assays for thymocyte or splenocyte cytotoxicity
include, without limitation, those described in: Current Protocols
in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.
Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 3, In Vitro assays for
Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies
in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974,
1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al.,
I. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.
140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998;
Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et
al., J. Immunol. 153:3079-3092, 1994.
[0194] Assays for T-cell-dependent immunoglobulin responses and
isotype switching (which will identify, among others, proteins that
modulate T-cell dependent antibody responses and that affect
Th1/Th2 profiles) include, without limitation, those described in:
Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell
function: In vitro antibody production, Mond, J. J. and Brunswick,
M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol
1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
[0195] Mixed lymphocyte reaction (MLR) assays (which will identify,
among others, proteins that generate predominantly Th1 and CTL
responses) include, without limitation, those described in: Current
Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D.
H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 3, In Vitro assays for
Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies
in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et
al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783, 1992.
[0196] Dendritic cell-dependent assays (which will identify, among
others, proteins expressed by dendritic cells that activate naive
T-cells) include, without limitation, those described in: Guery et
al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal
of Immunology 154:5071-5079, 1995; Porgador et al., Journal of
Experimental Medicine 182:255-260, 1995; Nair et al., Journal of
Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine
169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical
Investigation 94:797-807, 1994; and Inaba et al., Journal of
Experimental Medicine 172:631-640, 1990.
[0197] Assays for lymphocyte survival/apoptosis (which will
identify, among others, proteins that prevent apoptosis after
superantigen induction and proteins that regulate lymphocyte
homeostasis) include, without limitation, those described in:
Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al.,
Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research
53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry
14:891-897, 1993; Gorczyca et al., International Journal of
Oncology 1:639-648, 1992.
[0198] Assays for proteins that influence early steps of T-cell
commitment and development include, without limitation, those
described in: Antica et al., Blood 84:111-117, 1994; Fine et al.,
Cellular Immunology 155:111-122, 1994; Galy et al., Blood
85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA
88:7548-7551, 1991.
[0199] ACTIVIN/INHIBIN ACTIVITY
[0200] A polypeptide of the present invention may also exhibit
activin- or inhibin-related activities. A polynucleotide of the
invention may encode a polypeptide exhibiting such characteristics.
Inhibins are characterized by their ability to inhibit the release
of follicle stimulating hormone (FSH), while activins and are
characterized by their ability to stimulate the release of follicle
stimulating hormone (FSH). Thus, a polypeptide of the present
invention, alone or in heterodimers with a member of the inhibin
family, may be useful as a contraceptive based on the ability of
inhibins to decrease fertility in female mammals and decrease
spermatogenesis in male mammals. Administration of sufficient
amounts of other inhibins can induce infertility in these mammals.
Alternatively, the polypeptide of the invention, as a homodimer or
as a heterodimer with other protein subunits of the inhibin group,
may be useful as a fertility inducing therapeutic, based upon the
ability of activin molecules in stimulating FSH release from cells
of the anterior pituitary. See, for example, U.S. Pat. No.
4,798,885. A polypeptide of the invention may also be useful for
advancement of the onset of fertility in sexually immature mammals,
so as to increase the lifetime reproductive performance of domestic
animals such as, but not limited to, cows, sheep and pigs.
[0201] The activity of a polypeptide of the invention may, among
other means, be measured by the following methods.
[0202] Assays for activin/inhibin activity include, without
limitation, those described in: Vale et al., Endocrinology
91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et
al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663,
1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095,
1986.
[0203] CHEMOTACTIC/CHEMOKINETIC ACTIVITY
[0204] A polypeptide of the present invention may be involved in
chemotactic or chemokinetic activity for mammalian cells,
including, for example, monocytes, fibroblasts, neutrophils,
T-cells, mast cells, eosinophils, epithelial and/or endothelial
cells. A polynucleotide of the invention can encode a polypeptide
exhibiting such attributes. Chemotactic and chemokinetic receptor
activation can be used to mobilize or attract a desired cell
population to a desired site of action. Chemotactic or chemokinetic
compositions (e.g. proteins, antibodies, binding partners, or
modulators of the invention) provide particular advantages in
treatment of wounds and other trauma to tissues, as well as in
treatment of localized infections. For example, attraction of
lymphocytes, monocytes or neutrophils to tumors or sites of
infection may result in improved immune responses against the tumor
or infecting agent.
[0205] A protein or peptide has chemotactic activity for a
particular cell population if it can stimulate, directly or
indirectly, the directed orientation or movement of such cell
population. Preferably, the protein or peptide has the ability to
directly stimulate directed movement of cells. Whether a particular
protein has chemotactic activity for a population of cells can be
readily determined by employing such protein or peptide in any
known assay for cell chemotaxis.
[0206] Therapeutic compositions of the invention can be used in the
following:
[0207] Assays for chemotactic activity (which will identify
proteins that induce or prevent chemotaxis) consist of assays that
measure the ability of a protein to induce the migration of cells
across a membrane as well as the ability of a protein to induce the
adhesion of one cell population to another cell population.
Suitable assays for movement and adhesion include, without
limitation, those described in: Current Protocols in Immunology, Ed
by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta
Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest.
95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et
al Eur. J. Immunol. 25:1744-1748; Gruber et al. J. of Immunol.
152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768,
1994.
[0208] HEMOSTATIC AND THROMBOLYTIC ACTIVITY
[0209] A polypeptide of the invention may also be involved in
hemostatis or thrombolysis or thrombosis. A polynucleotide of the
invention can encode a polypeptide exhibiting such attributes.
Compositions may be useful in treatment of various coagulation
disorders (including hereditary disorders, such as hemophilias) or
to enhance coagulation and other hemostatic events in treating
wounds resulting from trauma, surgery or other causes. A
composition of the invention may also be useful for dissolving or
inhibiting formation of thromboses and for treatment and prevention
of conditions resulting therefrom (such as, for example, infarction
of cardiac and central nervous system vessels (e.g., stroke).
[0210] Therapeutic compositions of the invention can be used in the
following:
[0211] Assay for hemostatic and thrombolytic activity include,
without limitation, those described in: Linet et al., J. Clin.
Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res.
45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991);
Schaub, Prostaglandins 35:467-474, 1988.
[0212] CANCER DIAGNOSIS AND THERAPY
[0213] Polypeptides of the invention may be involved in cancer cell
generation, proliferation or metastasis. Detection of the presence
or amount of polynucleotides or polypeptides of the invention may
be useful for the diagnosis and/or prognosis of one or more types
of cancer. For example, the presence or increased expression of a
polynucleotide/polypeptide of the invention may indicate a
hereditary risk of cancer, a precancerous condition, or an ongoing
malignancy. Conversely, a defect in the gene or absence of the
polypeptide may be associated with a cancer condition.
Identification of single nucleotide polymorphisms associated with
cancer or a predisposition to cancer may also be useful for
diagnosis or prognosis.
[0214] Cancer treatments promote tumor regression by inhibiting
tumor cell proliferation, inhibiting angiogenesis (growth of new
blood vessels that is necessary to support tumor growth) and/or
prohibiting metastasis by reducing tumor cell motility or
invasiveness. Therapeutic compositions of the invention may be
effective in adult and pediatric oncology including in solid phase
tumors/malignancies, locally advanced tumors, human soft tissue
sarcomas, metastatic cancer, including lymphatic metastases, blood
cell malignancies including multiple myeloma, acute and chronic
leukemias, and lymphomas, head and neck cancers including mouth
cancer, larynx cancer and thyroid cancer, lung cancers including
small cell carcinoma and non-small cell cancers, breast cancers
including small cell carcinoma and ductal carcinoma,
gastrointestinal cancers including esophageal cancer, stomach
cancer, colon cancer, colorectal cancer and polyps associated with
colorectal neoplasia, pancreatic cancers, liver cancer, urologic
cancers including bladder cancer and prostate cancer, malignancies
of the female genital tract including ovarian carcinoma, uterine
(including endometrial) cancers, and solid tumor in the ovarian
follicle, kidney cancers including renal cell carcinoma, brain
cancers including intrinsic brain tumors, neuroblastoma, astrocytic
brain tumors, gliomas, metastatic tumor cell invasion in the
central nervous system, bone cancers including osteomas, skin
cancers including malignant melanoma, tumor progression of human
skin keratinocytes, squamous cell carcinoma, basal cell carcinoma,
hemangiopericytoma and Karposi's sarcoma.
[0215] Polypeptides, polynucleotides, or modulators of polypeptides
of the invention (including inhibitors and stimulators of the
biological activity of the polypeptide of the invention) may be
administered to treat cancer. Therapeutic compositions can be
administered in therapeutically effective dosages alone or in
combination with adjuvant cancer therapy such as surgery,
chemotherapy, radiotherapy, thermotherapy, and laser therapy, and
may provide a beneficial effect, e.g. reducing tumor size, slowing
rate of tumor growth, inhibiting metastasis, or otherwise improving
overall clinical condition, without necessarily eradicating the
cancer.
[0216] The composition can also be administered in therapeutically
effective amounts as a portion of an anti-cancer cocktail. An
anti-cancer cocktail is a mixture of the polypeptide or modulator
of the invention with one or more anti-cancer drugs in addition to
a pharmaceutically acceptable carrier for delivery. The use of
anti-cancer cocktails as a cancer treatment is routine. Anti-cancer
drugs that are well known in the art and can be used as a treatment
in combination with the polypeptide or modulator of the invention
include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin,
Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin
(cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside),
Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl,
Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine,
5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide),
Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide
acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine
HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna,
Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide,
Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate,
Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate,
Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2,
Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine
sulfate.
[0217] In addition, therapeutic compositions of the invention may
be used for prophylactic treatment of cancer. There are hereditary
conditions and/or environmental situations (e.g. exposure to
carcinogens) known in the art that predispose an individual to
developing cancers. Under these circumstances, it may be beneficial
to treat these individuals with therapeutically effective doses of
the polypeptide of the invention to reduce the risk of developing
cancers.
[0218] In vitro models can be used to determine the effective doses
of the polypeptide of the invention as a potential cancer
treatment. These in vitro models include proliferation assays of
cultured tumor cells, growth of cultured tumor cells in soft agar
(see Freshney, (1987) Culture of Animal Cells: A Manual of Basic
Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor
systems in nude mice as described in Giovanella et al., J. Natl.
Can. Inst., 52: 921-30 (1974), mobility and invasive potential of
tumor cells in Boyden Chamber assays as described in Pilkington et
al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays
such as induction of vascularization of the chick chorioallantoic
membrane or induction of vascular endothelial cell migration as
described in Ribatta et al., Intl. J. Dev. Biol., 40: 1189-97
(1999) and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999),
respectively. Suitable tumor cells lines are available, e.g. from
American Type Tissue Culture Collection catalogs.
[0219] RECEPTOR/LIGAND ACTIVITY
[0220] A polypeptide of the present invention may also demonstrate
activity as receptor, receptor ligand or inhibitor or agonist of
receptor/ligand interactions. A polynucleotide of the invention can
encode a polypeptide exhibiting such characteristics. Examples of
such receptors and ligands include, without limitation, cytokine
receptors and their ligands, receptor kinases and their ligands,
receptor phosphatases and their ligands, receptors involved in
cell-cell interactions and their ligands (including without
limitation, cellular adhesion molecules (such as selectins,
integrins and their ligands) and receptor/ligand pairs involved in
antigen presentation, antigen recognition and development of
cellular and humoral immune responses. Receptors and ligands are
also useful for screening of potential peptide or small molecule
inhibitors of the relevant receptor/ligand interaction. A protein
of the present invention (including, without limitation, fragments
of receptors and ligands) may themselves be useful as inhibitors of
receptor/ligand interactions.
[0221] The activity of a polypeptide of the invention may, among
other means, be measured by the following methods:
[0222] Suitable assays for receptor-ligand activity include without
limitation those described in: Current Protocols in Immunology, Ed
by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion
under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl.
Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med.
168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160
1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994;
Stitt et al., Cell 80:661-670, 1995.
[0223] By way of example, the polypeptides of the invention may be
used as a receptor for a ligand(s) thereby transmitting the
biological activity of that ligand(s). Ligands may be identified
through binding assays, affinity chromatography, dihybrid screening
assays, BIAcore assays, gel overlay assays, or other methods known
in the art.
[0224] Studies characterizing drugs or proteins as agonist or
antagonist or partial agonists or a partial antagonist require the
use of other proteins as competing ligands. The polypeptides of the
present invention or ligand(s) thereof may be labeled by being
coupled to radioisotopes, calorimetric molecules or a toxin
molecules by conventional methods. ("Guide to Protein Purification"
Murray P. Deutscher (ed) Methods in Enzymology Vol. 182 (1990)
Academic Press, Inc. San Diego). Examples of radioisotopes include,
but are not limited to, tritium and carbon-14 . Examples of
calorimetric molecules include, but are not limited to, fluorescent
molecules such as fluorescamine, or rhodamine or other colorimetric
molecules. Examples of toxins include, but are not limited, to
ricin.
[0225] DRUG SCREENING
[0226] This invention is particularly useful for screening chemical
compounds by using the novel polypeptides or binding fragments
thereof in any of a variety of drug screening techniques. The
polypeptides or fragments employed in such a test may either be
free in solution, affixed to a solid support, borne on a cell
surface or located intracellularly. One method of drug screening
utilizes eukaryotic or prokaryotic host cells which are stably
transformed with recombinant nucleic acids expressing the
polypeptide or a fragment thereof. Drugs are screened against such
transformed cells in competitive binding assays. Such cells, either
in viable or fixed form, can be used for standard binding assays.
One may measure, for example, the formation of complexes between
polypeptides of the invention or fragments and the agent being
tested or examine the diminution in complex formation between the
novel polypeptides and an appropriate cell line, which are well
known in the art.
[0227] Sources for test compounds that may be screened for ability
to bind to or modulate (i.e., increase or decrease) the activity of
polypeptides of the invention include (1) inorganic and organic
chemical libraries, (2) natural product libraries, and (3)
combinatorial libraries comprised of either random or mimetic
peptides, oligonucleotides or organic molecules.
[0228] Chemical libraries may be readily synthesized or purchased
from a number of commercial sources, and may include structural
analogs of known compounds or compounds that are identified as
"hits" or "leads" via natural product screening.
[0229] The sources of natural product libraries are microorganisms
(including bacteria and fungi), animals, plants or other
vegetation, or marine organisms, and libraries of mixtures for
screening may be created by: (1) fermentation and extraction of
broths from soil, plant or marine microorganisms or (2) extraction
of the organisms themselves. Natural product libraries include
polyketides, non-ribosomal peptides, and (non-naturally occurring)
variants thereof. For a review, see Science 282:63-68 (1998).
[0230] Combinatorial libraries are composed of large numbers of
peptides, oligonucleotides or organic compounds and can be readily
prepared by traditional automated synthesis methods, PCR, cloning
or proprietary synthetic methods. Of particular interest are
peptide and oligonucleotide combinatorial libraries. Still other
libraries of interest include peptide, protein, peptidomimetic,
multiparallel synthetic collection, recombinatorial, and
polypeptide libraries. For a review of combinatorial chemistry and
libraries created therefrom, see Myers, Curr. Opin. Biotechnol.
8:701-707 (1997). For reviews and examples of peptidomimetic
libraries, see Al-Obeidi et al., Mol. Biotechnol, 9(3):205-23
(1998); Hruby et al., Curr Opin Chem Biol, 1(1):114-19 (1997Dorner
et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated
dipeptides).
[0231] Identification of modulators through use of the various
libraries described herein permits modification of the candidate
"hit" (or "lead") to optimize the capacity of the "hit" to bind a
polypeptide of the invention. The molecules identified in the
binding assay are then tested for antagonist or agonist activity in
in vivo tissue culture or animal models that are well known in the
art. In brief, the molecules are titrated into a plurality of cell
cultures or animals and then tested for either cell/animal death or
prolonged survival of the animal/cells.
[0232] The binding molecules thus identified may be complexed with
toxins, e.g., ricin or cholera, or with other compounds that are
toxic to cells such as radioisotopes. The toxin-binding molecule
complex is then targeted to a tumor or other cell by the
specificity of the binding molecule for a polypeptide of the
invention. Alternatively, the binding molecules may be complexed
with imaging agents for targeting and imaging purposes.
[0233] ASSAY FOR RECEPTOR ACTIVITY
[0234] The invention also provides methods to detect specific
binding of a polypeptide e.g. a ligand or a receptor. The art
provides numerous assays particularly useful for identifying
previously unknown binding partners for receptor polypeptides of
the invention. For example, expression cloning using mammalian or
bacterial cells, or dihybrid screening assays can be used to
identify polynucleotides encoding binding partners. As another
example, affinity chromatography with the appropriate immobilized
polypeptide of the invention can be used to isolate polypeptides
that recognize and bind polypeptides of the invention. There are a
number of different libraries used for the identification of
compounds, and in particular small molecules, that modulate (i.e.,
increase or decrease) biological activity of a polypeptide of the
invention. Ligands for receptor polypeptides of the invention can
also be identified by adding exogenous ligands, or cocktails of
ligands to two cells populations that are genetically identical
except for the expression of the receptor of the invention: one
cell population expresses the receptor of the invention whereas the
other does not. The response of the two cell populations to the
addition of ligands(s) are then compared. Alternatively, an
expression library can be co-expressed with the polypeptide of the
invention in cells and assayed for an autocrine response to
identify potential ligand(s). As still another example, BIAcore
assays, gel overlay assays, or other methods known in the art can
be used to identify binding partner polypeptides, including, (1)
organic and inorganic chemical libraries, (2) natural product
libraries, and (3) combinatorial libraries comprised of random
peptides, oligonucleotides or organic molecules.
[0235] The role of downstream intracellular signaling molecules in
the signaling cascade of the polypeptide of the invention can be
determined. For example, a chimeric protein in which the
cytoplasmic domain of the polypeptide of the invention is fused to
the extracellular portion of a protein, whose ligand has been
identified, is produced in a host cell. The cell is then incubated
with the ligand specific for the extracellular portion of the
chimeric protein, thereby activating the chimeric receptor. Known
downstream proteins involved in intracellular signaling can then be
assayed for expected modifications i.e. phosphorylation. Other
methods known to those in the art can also be used to identify
signaling molecules involved in receptor activity.
[0236] ANTI-INFLAMMATORY ACTIVITY
[0237] Compositions of the present invention may also exhibit
anti-inflammatory activity. The anti-inflammatory activity may be
achieved by providing a stimulus to cells involved in the
inflammatory response, by inhibiting or promoting cell-cell
interactions (such as, for example, cell adhesion), by inhibiting
or promoting chemotaxis of cells involved in the inflammatory
process, inhibiting or promoting cell extravasation, or by
stimulating or suppressing production of other factors which more
directly inhibit or promote an inflammatory response. Compositions
with such activities can be used to treat inflammatory conditions
including chronic or acute conditions), including without
limitation intimation associated with infection (such as septic
shock, sepsis or systemic inflammatory response syndrome (SIRS)),
ischemia-reperfusion injury, endotoxin lethality, arthritis,
complement-mediated hyperacute rejection, nephritis, cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's
disease or resulting from over production of cytokines such as TNF
or IL-1. Compositions of the invention may also be useful to treat
anaphylaxis and hypersensitivity to an antigenic substance or
material. Compositions of this invention may be utilized to prevent
or treat conditions such as, but not limited to, sepsis, acute
pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid
arthritis, chronic inflammatory arthritis, pancreatic cell damage
from diabetes mellitus type 1, graft versus host disease,
inflammatory bowel disease, inflamation associated with pulmonary
disease, other autoimmune disease or inflammatory disease, an
antiproliferative agent such as for acute or chronic mylegenous
leukemia or in the prevention of premature labor secondary to
intrauterine infections.
[0238] LEUKEMIAS
[0239] Leukemias and related disorders may be treated or prevented
by administration of a therapeutic that promotes or inhibits
function of the polynucleotides and/or polypeptides of the
invention. Such leukemias and related disorders include but are not
limited to acute leukemia, acute lymphocytic leukemia, acute
myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic,
monocytic, erythroleukemia, chronic leukemia, chronic myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia (for a
review of such disorders, see Fishman et al., 1985, Medicine, 2d
Ed., J. B. Lippincott Co., Pa.).
[0240] NERVOUS SYSTEM DISORDERS
[0241] Nervous system disorders, involving cell types which can be
tested for efficacy of intervention with compounds that modulate
the activity of the polynucleotides and/or polypeptides of the
invention, and which can be treated upon thus observing an
indication of therapeutic utility, include but are not limited to
nervous system injuries, and diseases or disorders which result in
either a disconnection of axons, a diminution or degeneration of
neurons, or demyelination. Nervous system lesions which may be
treated in a patient (including human and non-human mammalian
patients) according to the invention include but are not limited to
the following lesions of either the central (including spinal cord,
brain) or peripheral nervous systems:
[0242] (i) traumatic lesions, including lesions caused by physical
injury or associated with surgery, for example, lesions which sever
a portion of the nervous system, or compression injuries;
[0243] (ii) ischemic lesions, in which a lack of oxygen in a
portion of the nervous system results in neuronal injury or death,
including cerebral infarction or ischemia, or spinal cord
infarction or ischemia;
[0244] (iii) infectious lesions, in which a portion of the nervous
system is destroyed or injured as a result of infection, for
example, by an abscess or associated with infection by human
immunodeficiency virus, herpes zoster, or herpes simplex virus or
with Lyme disease, tuberculosis, syphilis;
[0245] (iv) degenerative lesions, in which a portion of the nervous
system is destroyed or injured as a result of a degenerative
process including but not limited to degeneration associated with
Parkinson's disease, Alzheimer's disease, Huntington's chorea, or
amyotrophic lateral sclerosis;
[0246] (v) lesions associated with nutritional diseases or
disorders, in which a portion of the nervous system is destroyed or
injured by a nutritional disorder or disorder of metabolism
including but not limited to, vitamin B12 deficiency, folic acid
deficiency, Wernicke disease, tobacco-alcohol amblyopia,
Marchiafava-Bignami disease (primary degeneration of the corpus
callosum), and alcoholic cerebellar degeneration;
[0247] (vi) neurological lesions associated with systemic diseases
including but not limited to diabetes (diabetic neuropathy, Bell's
palsy), systemic lupus erythematosus, carcinoma, or
sarcoidosis;
[0248] (vii) lesions caused by toxic substances including alcohol,
lead, or particular neurotoxins; and
[0249] (viii) demyelinated lesions in which a portion of the
nervous system is destroyed or injured by a demyelinating disease
including but not limited to multiple sclerosis, human
immunodeficiency virus-associated myelopathy, transverse myelopathy
or various etiologies, progressive multifocal leukoencephalopathy,
and central pontine myelinolysis.
[0250] Therapeutics which are useful according to the invention for
treatment of a nervous system disorder may be selected by testing
for biological activity in promoting the survival or
differentiation of neurons. For example, and not by way of
limitation, therapeutics which elicit any of the following effects
may be useful according to the invention:
[0251] (i) increased survival time of neurons in culture;
[0252] (ii) increased sprouting of neurons in culture or in
vivo;
[0253] (iii) increased production of a neuron-associated molecule
in culture or in vivo, e.g., choline acetyltransferase or
acetylcholinesterase with respect to motor neurons; or
[0254] (iv) decreased symptoms of neuron dysfunction in vivo.
[0255] Such effects may be measured by any method known in the art.
In preferred, non-limiting embodiments, increased survival of
neurons may be measured by the method set forth in Arakawa et al.
(1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons
may be detected by methods set forth in Pestronk et al. (1980, Exp.
Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci.
4:17-42); increased production of neuron-associated molecules may
be measured by bioassay, enzymatic assay, antibody binding,
Northern blot assay, etc., depending on the molecule to be
measured; and motor neuron dysfunction may be measured by assessing
the physical manifestation of motor neuron disorder, e.g.,
weakness, motor neuron conduction velocity, or functional
disability.
[0256] In specific embodiments, motor neuron disorders that may be
treated according to the invention include but are not limited to
disorders such as infarction, infection, exposure to toxin, trauma,
surgical damage, degenerative disease or malignancy that may affect
motor neurons as well as other components of the nervous system, as
well as disorders that selectively affect neurons such as
amyotrophic lateral sclerosis, and including but not limited to
progressive spinal muscular atrophy, progressive bulbar palsy,
primary lateral sclerosis, infantile and juvenile muscular atrophy,
progressive bulbar paralysis of childhood (Fazio-Londe syndrome),
poliomyelitis and the post polio syndrome, and Hereditary
Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
[0257] OTHER ACTIVITIES
[0258] A polypeptide of the invention may also exhibit one or more
of the following additional activities or effects: inhibiting the
growth, infection or function of, or killing, infectious agents,
including, without limitation, bacteria, viruses, fungi and other
parasites; effecting (suppressing or enhancing) bodily
characteristics, including, without limitation, height, weight,
hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ or body part size or shape (such as, for
example, breast augmentation or diminution, change in bone form or
shape); effecting biorhythms or circadian cycles or rhythms;
effecting the fertility of male or female subjects; effecting the
metabolism, catabolism, anabolism, processing, utilization, storage
or elimination of dietary fat, lipid, protein, carbohydrate,
vitamins, minerals, co-factors or other nutritional factors or
component(s); effecting behavioral characteristics, including,
without limitation, appetite, libido, stress, cognition (including
cognitive disorders), depression (including depressive disorders)
and violent behaviors; providing analgesic effects or other pain
reducing effects; promoting differentiation and growth of embryonic
stem cells in lineages other than hematopoietic lineages; hormonal
or endocrine activity; in the case of enzymes, correcting
deficiencies of the enzyme and treating deficiency-related
diseases; treatment of hyperproliferative disorders (such as, for
example, psoriasis); immunoglobulin-like activity (such as, for
example, the ability to bind antigens or complement); and the
ability to act as an antigen in a vaccine composition to raise an
immune response against such protein or another material or entity
which is cross-reactive with such protein.
[0259] IDENTIFICATION OF POLYMORPHISMS
[0260] The demonstration of polymorphisms makes possible the
identification of such polymorphisms in human subjects and the
pharmacogenetic use of this information for diagnosis and
treatment. Such polymorphisms may be associated with, e.g.,
differential predisposition or susceptibility to various disease
states (such as disorders involving inflammation or immune
response) or a differential response to drug administration, and
this genetic information can be used to tailor preventive or
therapeutic treatment appropriately. For example, the existence of
a polymorphism associated with a predisposition to inflammation or
autoimmune disease makes possible the diagnosis of this condition
in humans by identifying the presence of the polymorphism.
[0261] Polymorphisms can be identified in a variety of ways known
in the art which all generally involve obtaining a sample from a
patient, analyzing DNA from the sample, optionally involving
isolation or amplification of the DNA, and identifying the presence
of the polymorphism in the DNA. For example, PCR may be used to
amplify an appropriate fragment of genomic DNA which may then be
sequenced. Alternatively, the DNA may be subjected to
allele-specific oligonucleotide hybridization (in which appropriate
oligonucleotides are hybridized to the DNA under conditions
permitting detection of a single base mismatch) or to a single
nucleotide extension assay (in which an oligonucleotide that
hybridizes immediately adjacent to the position of the polymorphism
is extended with one or more labeled nucleotides). In addition,
traditional restriction fragment length polymorphism analysis
(using restriction enzymes that provide differential digestion of
the genomic DNA depending on the presence or absence of the
polymorphism) may be performed. Arrays with nucleotide sequences of
the present invention can be used to detect polymorphisms. The
array can comprise modified nucleotide sequences of the present
invention in order to detect the nucleotide sequences of the
present invention. In the alternative, any one of the nucleotide
sequences of the present invention can be placed on the array to
detect changes from those sequences.
[0262] Alternatively a polymorphism resulting in a change in the
amino acid sequence could also be detected by detecting a
corresponding change in amino acid sequence of the protein, e.g.,
by an antibody specific to the variant sequence.
[0263] ARTHRITIS AND INFLAMMATION
[0264] The immunosuppressive effects of the compositions of the
invention against rheumatoid arthritis is determined in an
experimental animal model system. The experimental model system is
adjuvant induced arthritis in rats, and the protocol is described
by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et
al., 1963, Int. Arch. Allergy Appl. Immunol., 23:129. Induction of
the disease can be caused by a single injection, generally
intradermally, of a suspension of killed Mycobacterium tuberculosis
in complete Freund's adjuvant (CFA). The route of injection can
vary, but rats may be injected at the base of the tail with an
adjuvant mixture. The polypeptide is administered in phosphate
buffered solution (PBS) at a dose of about 1-5 mg/kg. The control
consists of administering PBS only.
[0265] The procedure for testing the effects of the test compound
would consist of intradermally injecting killed Mycobacterium
tuberculosis in CFA followed by immediately administering the test
compound and subsequent treatment every other day until day 24. At
14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium
CFA, an overall arthritis score may be obtained as described by J.
Holoskitz above. An analysis of the data would reveal that the test
compound would have a dramatic affect on the swelling of the joints
as measured by a decrease of the arthritis score.
[0266] THERAPEUTIC METHODS
[0267] The compositions (including polypeptide fragments, analogs,
variants and antibodies or other binding partners or modulators
including antisense polynucleotides) of the invention have numerous
applications in a variety of therapeutic methods. Examples of
therapeutic applications include, but are not limited to, those
exemplified herein.
EXAMPLE
[0268] One embodiment of the invention is the administration of an
effective amount of the polypeptides or other composition of the
invention to individuals affected by a disease or disorder that can
be modulated by regulating the peptides of the invention. While the
mode of administration is not particularly important, parenteral
administration is preferred. An exemplary mode of administration is
to deliver an intravenous bolus. The dosage of the polypeptides or
other composition of the invention will normally be determined by
the prescribing physician. It is to be expected that the dosage
will vary according to the age, weight, condition and response of
the individual patient. Typically, the amount of polypeptide
administered per dose will be in the range of about 0.01.mu.g/kg to
100 mg/kg of body weight, with the preferred dose being about
0.1.mu.g/kg to 10 .mu.g/kg of patient body weight. For parenteral
administration, polypeptides of the invention will be formulated in
an injectable form combined with a pharmaceutically acceptable
parenteral vehicle. Such vehicles are well known in the art and
examples include water, saline, Ringer's solution, dextrose
solution, and solutions consisting of small amounts of the human
serum albumin. The vehicle may contain minor amounts of additives
that maintain the isotonicity and stability of the polypeptide or
other active ingredient. The preparation of such solutions is
within the skill of the art.
[0269] PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION
[0270] A protein or other composition of the present invention
(from whatever source derived, including without limitation from
recombinant and non-recombinant sources and including antibodies
and other binding partners of the polypeptides of the invention)
may be administered to a patient in need, by itself, or in
pharmaceutical compositions where it is mixed with suitable
carriers or excipient(s) at doses to treat or ameliorate a variety
of disorders. Such a composition may optionally contain (in
addition to protein or other active ingredient and a carrier)
diluents, fillers, salts, buffers, stabilizers, solubilizers, and
other materials well known in the art. The term "pharmaceutically
acceptable" means a non-toxic material that does not interfere with
the effectiveness of the biological activity of the active
ingredient(s). The characteristics of the carrier will depend on
the route of administration. The pharmaceutical composition of the
invention may also contain cytokines, lymphokines, or other
hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, EL-10, IL-11, IL-12, IL-13,
IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem cell factor, and erythropoietin. In further
compositions, proteins of the invention may be combined with other
agents beneficial to the treatment of the disease or disorder in
question. These agents include various growth factors such as
epidermal growth factor (EGF), platelet-derived growth factor
(PDGF), transforming growth factors (TGF-.alpha. and TGF-.beta.),
insulin-like growth factor (IGF), as well as cytokines described
herein.
[0271] The pharmaceutical composition may further contain other
agents which either enhance the activity of the protein or other
active ingredient or complement its activity or use in treatment.
Such additional factors and/or agents may be included in the
pharmaceutical composition to produce a synergistic effect with
protein or other active ingredient of the invention, or to minimize
side effects. Conversely, protein or other active ingredient of the
present invention may be included in formulations of the particular
clotting factor, cytokine, lymphokine, other hematopoietic factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent
to minimize side effects of the clotting factor, cytokine,
lymphokine, other hematopoietic factor, thrombolytic or
anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra,
IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive
agents). A protein of the present invention may be active in
multimers (e.g., heterodimers or homodimers) or complexes with
itself or other proteins. As a result, pharmaceutical compositions
of the invention may comprise a protein of the invention in such
multimeric or complexed form.
[0272] As an alternative to being included in a pharmaceutical
composition of the invention including a first protein, a second
protein or a therapeutic agent may be concurrently administered
with the first protein (e.g., at the same time, or at differing
times provided that therapeutic concentrations of the combination
of agents is achieved at the treatment site). Techniques for
formulation and administration of the compounds of the instant
application may be found in "Remington's Pharmaceutical Sciences,"
Mack Publishing Co., Easton, Pa., latest edition. A therapeutically
effective dose further refers to that amount of the compound
sufficient to result in amelioration of symptoms, e.g., treatment,
healing, prevention or amelioration of the relevant medical
condition, or an increase in rate of treatment, healing, prevention
or amelioration of such conditions. When applied to an individual
active ingredient, administered alone, a therapeutically effective
dose refers to that ingredient alone. When applied to a
combination, a therapeutically effective dose refers to combined
amounts of the active ingredients that result in the therapeutic
effect, whether administered in combination, serially or
simultaneously.
[0273] In practicing the method of treatment or use of the present
invention, a therapeutically effective amount of protein or other
active ingredient of the present invention is administered to a
mammal having a condition to be treated. Protein or other active
ingredient of the present invention may be administered in
accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing
cytokines, lymphokines or other hematopoietic factors. When
co-administered with one or more cytokines, lymphokines or other
hematopoietic factors, protein or other active ingredient of the
present invention may be administered either simultaneously with
the cytokine(s), lymphokine(s), other hematopoietic factor(s),
thrombolytic or anti-thrombotic factors, or sequentially. If
administered sequentially, the attending physician will decide on
the appropriate sequence of administering protein or other active
ingredient of the present invention in combination with
cytokine(s), lymphokine(s), other hematopoietic factor(s),
thrombolytic or anti-thrombotic factors.
[0274] ROUTES OF ADMINISTRATION
[0275] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections. Administration of protein or other active
ingredient of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can
be carried out in a variety of conventional ways, such as oral
ingestion, inhalation, topical application or cutaneous,
subcutaneous, intraperitoneal, parenteral or intravenous injection.
Intravenous administration to the patient is preferred.
[0276] Alternately, one may administer the compound in a local
rather than systemic manner, for example, via injection of the
compound directly into a arthritic joints or in fibrotic tissue,
often in a depot or sustained release formulation. In order to
prevent the scarring process frequently occurring as complication
of glaucoma surgery, the compounds may be administered topically,
for example, as eye drops. Furthermore, one may administer the drug
in a targeted drug delivery system, for example, in a liposome
coated with a specific antibody, targeting, for example, arthritic
or fibrotic tissue. The liposomes will be targeted to and taken up
selectively by the afflicted tissue.
[0277] The polypeptides of the invention are administered by any
route that delivers an effective dosage to the desired site of
action. The determination of a suitable route of administration and
an effective dosage for a particular indication is within the level
of skill in the art. Preferably for wound treatment, one
administers the therapeutic compound directly to the site. Suitable
dosage ranges for the polypeptides of the invention can be
extrapolated from these dosages or from similar studies in
appropriate animal models. Dosages can then be adjusted as
necessary by the clinician to provide maximal therapeutic
benefit.
[0278] COMPOSITIONS/FORMULATIONS
[0279] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in a conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the
active compounds into preparations which can be used
pharmaceutically. These pharmaceutical compositions may be
manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes. Proper formulation is dependent upon the route of
administration chosen. When a therapeutically effective amount of
protein or other active ingredient of the present invention is
administered orally, protein or other active ingredient of the
present invention will be in the form of a tablet, capsule, powder,
solution or elixir. When administered in tablet form, the
pharmaceutical composition of the invention may additionally
contain a solid carrier such as a gelatin or an adjuvant. The
tablet, capsule, and powder contain from about 5 to 95% protein or
other active ingredient of the present invention, and preferably
from about 25 to 90% protein or other active ingredient of the
present invention. When administered in liquid form, a liquid
carrier such as water, petroleum, oils of animal or plant origin
such as peanut oil, mineral oil, soybean oil, or sesame oil, or
synthetic oils may be added. The liquid form of the pharmaceutical
composition may further contain physiological saline solution,
dextrose or other saccharide solution, or glycols such as ethylene
glycol, propylene glycol or polyethylene glycol. When administered
in liquid form, the pharmaceutical composition contains from about
0.5 to 90% by weight of protein or other active ingredient of the
present invention, and preferably from about 1 to 50% protein or
other active ingredient of the present invention.
[0280] When a therapeutically effective amount of protein or other
active ingredient of the present invention is administered by
intravenous, cutaneous or subcutaneous injection, protein or other
active ingredient of the present invention will be in the form of a
pyrogen-free, parenterally acceptable aqueous solution. The
preparation of such parenterally acceptable protein or other active
ingredient solutions, having due regard to pH, isotonicity,
stability, and the like, is within the skill in the art. A
preferred pharmaceutical composition for intravenous, cutaneous, or
subcutaneous injection should contain, in addition to protein or
other active ingredient of the present invention, an isotonic
vehicle such as Sodium Chloride Injection, Ringer's Injection,
Dextrose Injection, Dextrose and Sodium Chloride Injection,
Lactated Ringer's Injection, or other vehicle as known in the art.
The pharmaceutical composition of the present invention may also
contain stabilizers, preservatives, buffers, antioxidants, or other
additives known to those of skill in the art. For injection, the
agents of the invention may be formulated in aqueous solutions,
preferably in physiologically compatible buffers such as Hanks's
solution, Ringer's solution, or physiological saline buffer. For
transmucosal administration, penetrants appropriate to the barrier
to be permeated are used in the formulation. Such penetrants are
generally known in the art.
[0281] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a patient to be treated.
Pharmaceutical preparations for oral use can be obtained from a
solid excipient, optionally grinding a resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sugars,
including lactose, sucrose, mannitol, or sorbitol; cellulose
preparations such as, for example, maize starch, wheat starch, rice
starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate. Dragee cores are provided with suitable coatings. For
this purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0282] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration. For buccal
administration, the compositions may take the form of tablets or
lozenges formulated in conventional manner.
[0283] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch. The compounds may
be formulated for parenteral administration by injection, e.g., by
bolus injection or continuous infusion. Formulations for injection
may be presented in unit dosage form, e.g., in ampules or in
multi-dose containers, with an added preservative. The compositions
may take such forms as suspensions, solutions or emulsions in oily
or aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents.
[0284] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions. Alternatively,
the active ingredient may be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0285] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides. In addition to the formulations described previously,
the compounds may also be formulated as a depot preparation. Such
long acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0286] A pharmaceutical carrier for the hydrophobic compounds of
the invention is a co-solvent system comprising benzyl alcohol, a
nonpolar surfactant, a water-miscible organic polymer, and an
aqueous phase. The co-solvent system may be the VPD co-solvent
system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the
nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol
300, made up to volume in absolute ethanol. The VPD co-solvent
system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in
water solution. This co-solvent system dissolves hydrophobic
compounds well, and itself produces low toxicity upon systemic
administration. Naturally, the proportions of a co-solvent system
may be varied considerably without destroying its solubility and
toxicity characteristics. Furthermore, the identity of the
co-solvent components may be varied: for example, other
low-toxicity nonpolar surfactants may be used instead of
polysorbate 80; the fraction size of polyethylene glycol may be
varied; other biocompatible polymers may replace polyethylene
glycol, e.g. polyvinyl pyrrolidone; and other sugars or
polysaccharides may substitute for dextrose. Alternatively, other
delivery systems for hydrophobic pharmaceutical compounds may be
employed. Liposomes and emulsions are well known examples of
delivery vehicles or carriers for hydrophobic drugs. Certain
organic solvents such as dimethylsulfoxide also may be employed,
although usually at the cost of greater toxicity. Additionally, the
compounds may be delivered using a sustained-release system, such
as semipermeable matrices of solid hydrophobic polymers containing
the therapeutic agent. Various types of sustained-release materials
have been established and are well known by those skilled in the
art. Sustained-release capsules may, depending on their chemical
nature, release the compounds for a few weeks up to over 100 days.
Depending on the chemical nature and the biological stability of
the therapeutic reagent, additional strategies for protein or other
active ingredient stabilization may be employed.
[0287] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene glycols.
Many of the active ingredients of the invention may be provided as
salts with pharmaceutically compatible counter ions. Such
pharmaceutically acceptable base addition salts are those salts
which retain the biological effectiveness and properties of the
free acids and which are obtained by reaction with inorganic or
organic bases such as sodium hydroxide, magnesium hydroxide,
ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino
acids, sodium acetate, potassium benzoate, triethanol amine and the
like.
[0288] The pharmaceutical composition of the invention may be in
the form of a complex of the protein(s) or other active
ingredient(s) of present invention along with protein or peptide
antigens. The protein and/or peptide antigen will deliver a
stimulatory signal to both B and T lymphocytes. B lymphocytes will
respond to antigen through their surface immunoglobulin receptor. T
lymphocytes will respond to antigen through the T cell receptor
(TCR) following presentation of the antigen by MHC proteins. MHC
and structurally related proteins including those encoded by class
I and class II MHC genes on host cells will serve to present the
peptide antigen(s) to T lymphocytes. The antigen components could
also be supplied as purified MHC-peptide complexes alone or with
co-stimulatory molecules that can directly signal T cells.
Alternatively antibodies able to bind surface immunoglobulin and
other molecules on B cells as well as antibodies able to bind the
TCR and other molecules on T cells can be combined with the
pharmaceutical composition of the invention.
[0289] The pharmaceutical composition of the invention may be in
the form of a liposome in which protein of the present invention is
combined, in addition to other pharmaceutically acceptable
carriers, with amphipathic agents such as lipids which exist in
aggregated form as micelles, insoluble monolayers, liquid crystals,
or lamellar layers in aqueous solution. Suitable lipids for
liposomal formulation include, without limitation, monoglycerides,
diglycerides, sulfatides, lysolecithins, phospholipids, saponin,
bile acids, and the like. Preparation of such liposomal
formulations is within the level of skill in the art, as disclosed,
for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and
4,737,323, all of which are incorporated herein by reference.
[0290] The amount of protein or other active ingredient of the
present invention in the pharmaceutical composition of the present
invention will depend upon the nature and severity of the condition
being treated, and on the nature of prior treatments which the
patient has undergone. Ultimately, the attending physician will
decide the amount of protein or other active ingredient of the
present invention with which to treat each individual patient.
Initially, the attending physician will administer low doses of
protein or other active ingredient of the present invention and
observe the patient's response. Larger doses of protein or other
active ingredient of the present invention may be administered
until the optimal therapeutic effect is obtained for the patient,
and at that point the dosage is not increased further. It is
contemplated that the various pharmaceutical compositions used to
practice the method of the present invention should contain about
0.01 .mu.g to about 100 mg (preferably about 0.1 .mu.g to about 10
mg, more preferably about 0.1 .mu.g to about 1 mg) of protein or
other active ingredient of the present invention per kg body
weight. For compositions of the present invention which are useful
for bone, cartilage, tendon or ligament regeneration, the
therapeutic method includes administering the composition
topically, systematically, or locally as an implant or device. When
administered, the therapeutic composition for use in this invention
is, of course, in a pyrogen-free, physiologically acceptable form.
Further, the composition may desirably be encapsulated or injected
in a viscous form for delivery to the site of bone, cartilage or
tissue damage. Topical administration may be suitable for wound
healing and tissue repair. Therapeutically useful agents other than
a protein or other active ingredient of the invention which may
also optionally be included in the composition as described above,
may alternatively or additionally, be administered simultaneously
or sequentially with the composition in the methods of the
invention. Preferably for bone and/or cartilage formation, the
composition would include a matrix capable of delivering the
protein-containing or other active ingredient-containing
composition to the site of bone and/or cartilage damage, providing
a structure for the developing bone and cartilage and optimally
capable of being resorbed into the body. Such matrices may be
formed of materials presently in use for other implanted medical
applications.
[0291] The choice of matrix material is based on biocompatibility,
biodegradability, mechanical properties, cosmetic appearance and
interface properties. The particular application of the
compositions will define the appropriate formulation. Potential
matrices for the compositions may be biodegradable and chemically
defined calcium sulfate, tricalcium phosphate, hydroxyapatite,
polylactic acid, polyglycolic acid and polyanhydrides. Other
potential materials are biodegradable and biologically
well-defined, such as bone or dermal collagen. Further matrices are
comprised of pure proteins or extracellular matrix components.
Other potential matrices are nonbiodegradable and chemically
defined, such as sintered hydroxyapatite, bioglass, aluminates, or
other ceramics. Matrices may be comprised of combinations of any of
the above mentioned types of material, such as polylactic acid and
hydroxyapatite or collagen and tricalcium phosphate. The
bioceramics may be altered in composition, such as in
calcium-aluminate-phosphate and processing to alter pore size,
particle size, particle shape, and biodegradability. Presently
preferred is a 50:50 (mole weight) copolymer of lactic acid and
glycolic acid in the form of porous particles having diameters
ranging from 150 to 800 microns. In some applications, it will be
useful to utilize a sequestering agent, such as carboxymethyl
cellulose or autologous blood clot, to prevent the protein
compositions from disassociating from the matrix.
[0292] A preferred family of sequestering agents is cellulosic
materials such as alkylcelluloses (including
hydroxyalkylcelluloses), including methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most
preferred being cationic salts of carboxymethylcellulose (CMC).
Other preferred sequestering agents include hyaluronic acid, sodium
alginate, poly(ethylene glycol), polyoxyethylene oxide,
carboxyvinyl polymer and poly(vinyl alcohol). The amount of
sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt
% based on total formulation weight, which represents the amount
necessary to prevent desorption of the protein from the polymer
matrix and to provide appropriate handling of the composition, yet
not so much that the progenitor cells are prevented from
infiltrating the matrix, thereby providing the protein the
opportunity to assist the osteogenic activity of the progenitor
cells. In further compositions, proteins or other active
ingredients of the invention may be combined with other agents
beneficial to the treatment of the bone and/or cartilage defect,
wound, or tissue in question. These agents include various growth
factors such as epidermal growth factor (EGF), platelet derived
growth factor (PDGF), transforming growth factors (TGF-.alpha. and
TGF-.beta.), and insulin-like growth factor (IGF).
[0293] The therapeutic compositions are also presently valuable for
veterinary applications. Particularly domestic animals and
thoroughbred horses, in addition to humans, are desired patients
for such treatment with proteins or other active ingredients of the
present invention. The dosage regimen of a protein-containing
pharmaceutical composition to be used in tissue regeneration will
be determined by the attending physician considering various
factors which modify the action of the proteins, e.g., amount of
tissue weight desired to be formed, the site of damage, the
condition of the damaged tissue, the size of a wound, type of
damaged tissue (e.g., bone), the patient's age, sex, and diet, the
severity of any infection, time of administration and other
clinical factors. The dosage may vary with the type of matrix used
in the reconstitution and with inclusion of other proteins in the
pharmaceutical composition. For example, the addition of other
known growth factors, such as IGF I (insulin like growth factor I),
to the final composition, may also effect the dosage. Progress can
be monitored by periodic assessment of tissue/bone growth and/or
repair, for example, X-rays, histomorphometric determinations and
tetracycline labeling.
[0294] Polynucleotides of the present invention can also be used
for gene therapy. Such polynucleotides can be introduced either in
vivo or ex vivo into cells for expression in a mammalian subject.
Polynucleotides of the invention may also be administered by other
known methods for introduction of nucleic acid into a cell or
organism (including, without limitation, in the form of viral
vectors or naked DNA). Cells may also be cultured ex vivo in the
presence of proteins of the present invention in order to
proliferate or to produce a desired effect on or activity in such
cells. Treated cells can then be introduced in vivo for therapeutic
purposes.
[0295] EFFECTIVE DOSAGE
[0296] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
More specifically, a therapeutically effective amount means an
amount effective to prevent development of or to alleviate the
existing symptoms of the subject being treated. Determination of
the effective amount is well within the capability of those skilled
in the art, especially in light of the detailed disclosure provided
herein. For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from
appropriate in vitro assays. For example, a dose can be formulated
in animal models to achieve a circulating concentration range that
can be used to more accurately determine useful doses in humans.
For example, a dose can be formulated in animal models to achieve a
circulating concentration range that includes the IC.sub.50 as
determined in cell culture (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of the protein's
biological activity). Such information can be used to more
accurately determine useful doses in humans.
[0297] A therapeutically effective dose refers to that amount of
the compound that results in amelioration of symptoms or a
prolongation of survival in a patient. Toxicity and therapeutic
efficacy of such compounds can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio between LD.sub.50 and ED.sub.50. Compounds
which exhibit high therapeutic indices are preferred. The data
obtained from these cell culture assays and animal studies can be
used in formulating a range of dosage for use in human. The dosage
of such compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. The
exact formulation, route of administration and dosage can be chosen
by the individual physician in view of the patient's condition.
See, e.g., Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p.1. Dosage amount and interval may be
adjusted individually to provide plasma levels of the active moiety
which are sufficient to maintain the desired effects, or minimal
effective concentration (MEC). The MEC will vary for each compound
but can be estimated from in vitro data. Dosages necessary to
achieve the MEC will depend on individual characteristics and route
of administration. However, HPLC assays or bioassays can be used to
determine plasma concentrations.
[0298] Dosage intervals can also be determined using MEC value.
Compounds should be administered using a regimen which maintains
plasma levels above the MEC for 10-90% of the time, preferably
between 30-90% and most preferably between 50-90%. In cases of
local administration or selective uptake, the effective local
concentration of the drug may not be related to plasma
concentration.
[0299] An exemplary dosage regimen for polypeptides or other
compositions of the invention will be in the range of about 0.01
.mu.g/kg to 100 mg/kg of body weight daily, with the preferred dose
being about 0.1 .mu.g/kg to 25 mg/kg of patient body weight daily,
varying in adults and children. Dosing may be once daily, or
equivalent doses may be delivered at longer or shorter
intervals.
[0300] The amount of composition administered will, of course, be
dependent on the subject being treated, on the subject's age and
weight, the severity of the affliction, the manner of
administration and the judgment of the prescribing physician.
[0301] PACKAGING
[0302] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may, for example,
comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. Compositions comprising a compound of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition.
[0303] ANTIBODIES
[0304] Another aspect of the invention is an antibody that
specifically binds the polypeptide of the invention. Such
antibodies include monoclonal and polyclonal antibodies, single
chain antibodies, chimeric antibodies, bifunctional/bispecific
antibodies, humanized antibodies, human antibodies, and
complementary determining region (CDR)-grafted antibodies,
including compounds which include CDR and/or antigen-binding
sequences, which specifically recognize a polypeptide of the
invention. Preferred antibodies of the invention are human
antibodies which are produced and identified according to methods
described in WO93/11236, published Jun. 20, 1993, which is
incorporated herein by reference in its entirety. Antibody
fragments, including Fab, Fab', F(ab').sub.2, and F.sub.v, are also
provided by the invention. The term "specific for" indicates that
the variable regions of the antibodies of the invention recognize
and bind polypeptides of the invention exclusively (i.e., able to
distinguish the polypeptide of the invention from other similar
polypeptides despite sequence identity, homology, or similarity
found in the family of polypeptides), but may also interact with
other proteins (for example, S. aureus protein A or other
antibodies in ELISA techniques) through interactions with sequences
outside the variable region of the antibodies, and in particular,
in the constant region of the molecule. Screening assays to
determine binding specificity of an antibody of the invention are
well known and routinely practiced in the art. For a comprehensive
discussion of such assays, see Harlow et al. (Eds), Antibodies A
Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring
Harbor, N.Y. (1988), Chapter 6. Antibodies that recognize and bind
fragments of the polypeptides of the invention are also
contemplated, provided that the antibodies are first and foremost
specific for, as defined above, full length polypeptides of the
invention. As with antibodies that are specific for full length
polypeptides of the invention, antibodies of the invention that
recognize fragments are those which can distinguish polypeptides
from the same family of polypeptides despite inherent sequence
identity, homology, or similarity found in the family of proteins.
Antibodies of the invention can be produced using any method well
known and routinely practiced in the art.
[0305] Non-human antibodies may be humanized by any methods known
in the art. In one method, the non-human CDRs are inserted into a
human antibody or consensus antibody framework sequence. Further
changes can then be introduced into the antibody framework to
modulate affinity or immunogenicity.
[0306] Antibodies of the invention are useful for, for example,
therapeutic purposes (by modulating activity of a polypeptide of
the invention), diagnostic purposes to detect or quantitate a
polypeptide of the invention, as well as purification of a
polypeptide of the invention. Kits comprising an antibody of the
invention for any of the purposes described herein are also
comprehended. In general, a kit of the invention also includes a
control antigen for which the antibody is immunospecific. The
invention further provides a hybridoma that produces an antibody
according to the invention. Antibodies of the invention are useful
for detection and/or purification of the polypeptides of the
invention.
[0307] Polypeptides of the invention may also be used to immunize
animals to obtain polyclonal and monoclonal antibodies which
specifically react with the protein. Such antibodies may be
obtained using either the entire protein or fragments thereof as an
immunogen. The peptide immunogens additionally may contain a
cysteine residue at the carboxyl terminus, and are conjugated to a
hapten such as keyhole limpet hemocyanin (KLH). Methods for
synthesizing such peptides are known in the art, for example, as in
R. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); J. L.
Krstenansky, et al., FEBS Lett. 211, 10 (1987).
[0308] Monoclonal antibodies binding to the protein of the
invention may be useful diagnostic agents for the immunodetection
of the protein. Neutralizing monoclonal antibodies binding to the
protein may also be useful therapeutics for both conditions
associated with the protein and also in the treatment of some forms
of cancer where abnormal expression of the protein is involved. In
the case of cancerous cells or leukemic cells, neutralizing
monoclonal antibodies against the protein may be useful in
detecting and preventing the metastatic spread of the cancerous
cells, which may be mediated by the protein. In general, techniques
for preparing polyclonal and monoclonal antibodies as well as
hybridomas capable of producing the desired antibody are well known
in the art (Campbell, A. M., Monoclonal Antibodies Technology:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St.
Groth et al., J. Inmunol. 35:1-21 (1990); Kohler and Milstein,
Nature 256:495-497 (1975)), the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., Immunology Today 4:72 (1983);
Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc. (1985), pp. 77-96).
[0309] Any animal (mouse, rabbit, etc.) which is known to produce
antibodies can be immunized with a peptide or polypeptide of the
invention. Methods for immunization are well known in the art. Such
methods include subcutaneous or intraperitoneal injection of the
polypeptide. One skilled in the art will recognize that the amount
of the protein encoded by the ORF of the present invention used for
immunization will vary based on the animal which is immunized, the
antigenicity of the peptide and the site of injection. The protein
that is used as an immunogen may be modified or administered in an
adjuvant in order to increase the protein's antigenicity. Methods
of increasing the antigenicity of a protein are well known in the
art and include, but are not limited to, coupling the antigen with
a heterologous protein (such as globulin or .beta.-galactosidase)
or through the inclusion of an adjuvant during immunization.
[0310] For monoclonal antibodies, spleen cells from the immunized
animals are removed, fused with myeloma cells, such as SP2/0-Ag14
myeloma cells, and allowed to become monoclonal antibody producing
hybridoma cells. Any one of a number of methods well known in the
art can be used to identify the hybridoma cell which produces an
antibody with the desired characteristics. These include screening
the hybridomas with an ELISA assay, Western blot analysis, or
radioimmunoassay (Lutz et al., Exp. Cell Research. 175:109-124
(1988)). Hybridomas secreting the desired antibodies are cloned and
the class and subclass is determined using procedures known in the
art (Campbell, A. M., Monoclonal Antibody Technology: Laboratory
Techniques in Biochemistry and Molecular Biology, Elsevier Science
Publishers, Amsterdam, The Netherlands (1984)). Techniques
described for the production of single chain antibodies (U.S. Pat.
No. 4,946,778) can be adapted to produce single chain antibodies to
proteins of the present invention.
[0311] For polyclonal antibodies, antibody-containing antiserum is
isolated from the immunized animal and is screened for the presence
of antibodies with the desired specificity using one of the
above-described procedures. The present invention further provides
the above-described antibodies in delectably labeled form.
Antibodies can be delectably labeled through the use of
radioisotopes, affinity labels (such as biotin, avidin, etc.),
enzymatic labels (such as horseradish peroxidase, alkaline
phosphatase, etc.) fluorescent labels (such as FITC or rhodamine,
etc.), paramagnetic atoms, etc. Procedures for accomplishing such
labeling are well-known in the art, for example, see (Sternberger,
L. A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A.
et al., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol.
109:129 (1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)).
[0312] The labeled antibodies of the present invention can be used
for in vitro, in vivo, and in situ assays to identify cells or
tissues in which a fragment of the polypeptide of interest is
expressed. The antibodies may also be used directly in therapies or
other diagnostics. The present invention further provides the
above-described antibodies immobilized on a solid support. Examples
of such solid supports include plastics such as polycarbonate,
complex carbohydrates such as agarose and Sepharose.RTM., acrylic
resins and such as polyacrylamide and latex beads. Techniques for
coupling antibodies to such solid supports are well known in the
art (Weir, D. M. et al., "Handbook of Experimental Immunology" 4th
Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10
(1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N.Y.
(1974)). The immobilized antibodies of the present invention can be
used for in vitro, in vivo, and in situ assays as well as for
immuno-affinity purification of the proteins of the present
invention.
[0313] COMPUTER READABLE SEQUENCES
[0314] In one application of this embodiment, a nucleotide sequence
of the present invention can be recorded on computer readable
media. As used herein, "computer readable media" refers to any
medium which can be read and accessed directly by a computer. Such
media include, but are not limited to: magnetic storage media, such
as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. A skilled artisan can readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide sequence of the present
invention. As used herein, "recorded" refers to a process for
storing information on computer readable medium. A skilled artisan
can readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide sequence information of the present
invention.
[0315] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide sequence of the present invention.
The choice of the data storage structure will generally be based on
the means chosen to access the stored information. In addition, a
variety of data processor programs and formats can be used to store
the nucleotide sequence information of the present invention on
computer readable medium. The sequence information can be
represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. A
skilled artisan can readily adapt any number of data processor
structuring formats (e.g. text file or database) in order to obtain
computer readable medium having recorded thereon the nucleotide
sequence information of the present invention.
[0316] By providing any of the nucleotide sequences SEQ ID NOs:
1-10 or a representative fragment thereof; or a nucleotide sequence
at least 95% identical to any of the nucleotide sequences of SEQ ID
NOs: 1-10 in computer readable form, a skilled artisan can
routinely access the sequence information for a variety of
purposes. Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium. The examples which follow demonstrate how
software which implements the BLAST (Altschul et al., J. Mol. Biol.
215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem.
17:203-207 (1993)) search algorithms on a Sybase system is used to
identify open reading frames (ORFs) within a nucleic acid sequence.
Such ORFs may be protein encoding fragments and may be useful in
producing commercially important proteins such as enzymes used in
fermentation reactions and in the production of commercially useful
metabolites.
[0317] As used herein, "a computer-based system" refers to the
hardware means, software means, and data storage means used to
analyze the nucleotide sequence information of the present
invention. The minimum hardware means of the computer-based systems
of the present invention comprises a central processing unit (CPU),
input means, output means, and data storage means. A skilled
artisan can readily appreciate that any one of the currently
available computer-based systems are suitable for use in the
present invention. As stated above, the computer-based systems of
the present invention comprise a data storage means having stored
therein a nucleotide sequence of the present invention and the
necessary hardware means and software means for supporting and
implementing a search means. As used herein, "data storage means"
refers to memory which can store nucleotide sequence information of
the present invention, or a memory access means which can access
manufactures having recorded thereon the nucleotide sequence
information of the present invention.
[0318] As used herein, "search means" refers to one or more
programs which are implemented on the computer-based system to
compare a target sequence or target structural motif with the
sequence information stored within the data storage means. Search
means are used to identify fragments or regions of a known sequence
which match a particular target sequence or target motif. A variety
of known algorithms are disclosed publicly and a variety of
commercially available software for conducting search means are and
can be used in the computer-based systems of the present invention.
Examples of such software includes, but is not limited to,
Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA
(NPOLYPEPTIDEIA). A skilled artisan can readily recognize that any
one of the available algorithms or implementing software packages
for conducting homology searches can be adapted for use in the
present computer-based systems. As used herein, a "target sequence"
can be any nucleic acid or amino acid sequence of six or more
nucleotides or two or more amino acids. A skilled artisan can
readily recognize that the longer a target sequence is, the less
likely a target sequence will be present as a random occurrence in
the database. The most preferred sequence length of a target
sequence is from about 10 to 300 amino acids, more preferably from
about 30 to 100 nucleotide residues. However, it is well recognized
that searches for commercially important fragments, such as
sequence fragments involved in gene expression and protein
processing, may be of shorter length.
[0319] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[0320] TRIPLE HELIX FORMATION
[0321] In addition, the fragments of the present invention, as
broadly described, can be used to control gene expression through
triple helix formation or antisense DNA or RNA, both of which
methods are based on the binding of a polynucleotide sequence to
DNA or RNA. Polynucleotides suitable for use in these methods are
preferably 20 to 40 bases in length and are designed to be
complementary to a region of the gene involved in transcription
(triple helix--see Lee et al., Nucl. Acids Res. 6:3073 (1979);
Cooney et al., Science 15241:456 (1988); and Dervan et al., Science
251:1360 (1991)) or to the mRNA itself (antisense--Olmno, J.
Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).
Triple helix-formation optimally results in a shut-off of RNA
transcription from DNA, while antisense RNA hybridization blocks
translation of an mRNA molecule into polypeptide. Both techniques
have been demonstrated to be effective in model systems.
Information contained in the sequences of the present invention is
necessary for the design of an antisense or triple helix
oligonucleotide.
[0322] DIAGNOSTIC ASSAYS AND KITS
[0323] The present invention further provides methods to identify
the presence or expression of one of the ORFs of the present
invention, or homolog thereof, in a test sample, using a nucleic
acid probe or antibodies of the present invention, optionally
conjugated or otherwise associated with a suitable label.
[0324] In general, methods for detecting a polynucleotide of the
invention can comprise contacting a sample with a compound that
binds to and forms a complex with the polynucleotide for a period
sufficient to form the complex, and detecting the complex, so that
if a complex is detected, a polynucleotide of the invention is
detected in the sample. Such methods can also comprise contacting a
sample under stringent hybridization conditions with nucleic acid
primers that anneal to a polynucleotide of the invention under such
conditions, and amplifying annealed polynucleotides, so that if a
polynucleotide is amplified, a polynucleotide of the invention is
detected in the sample.
[0325] In general, methods for detecting a polypeptide of the
invention can comprise contacting a sample with a compound that
binds to and forms a complex with the polypeptide for a period
sufficient to form the complex, and detecting the complex, so that
if a complex is detected, a polypeptide of the invention is
detected in the sample.
[0326] In detail, such methods comprise incubating a test sample
with one or more of the antibodies or one or more of the nucleic
acid probes of the present invention and assaying for binding of
the nucleic acid probes or antibodies to components within the test
sample.
[0327] Conditions for incubating a nucleic acid probe or antibody
with a test sample vary. Incubation conditions depend on the format
employed in the assay, the detection methods employed, and the type
and nature of the nucleic acid probe or antibody used in the assay.
One skilled in the art will recognize that any one of the commonly
available hybridization, amplification or immunological assay
formats can readily be adapted to employ the nucleic acid probes or
antibodies of the present invention. Examples of such assays can be
found in Chard, T., An Introduction to Radioimmunoassay and Related
Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands
(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,
Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3
(1985); Tijssen, P., Practice and Theory of immunoassays:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The
test samples of the present invention include cells, protein or
membrane extracts of cells, or biological fluids such as sputum,
blood, serum, plasma, or urine. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts used as
the sample to be assayed. Methods for preparing protein extracts or
membrane extracts of cells are well known in the art and can be
readily be adapted in order to obtain a sample which is compatible
with the system utilized.
[0328] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention. Specifically, the invention
provides a compartment kit to receive, in close confinement, one or
more containers which comprises: (a) a first container comprising
one of the probes or antibodies of the present invention; and (b)
one or more other containers comprising one or more of the
following: wash reagents, reagents capable of detecting presence of
a bound probe or antibody.
[0329] In detail, a compartment kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers or strips of
plastic or paper. Such containers allows one to efficiently
transfer reagents from one compartment to another compartment such
that the samples and reagents are not cross-contaminated, and the
agents or solutions of each container can be added in a
quantitative fashion from one compartment to another. Such
containers will include a container which will accept the test
sample, a container which contains the antibodies used in the
assay, containers which contain wash reagents (such as phosphate
buffered saline, Tris-buffers, etc.), and containers which contain
the reagents used to detect the bound antibody or probe. Types of
detection reagents include labeled nucleic acid probes, labeled
secondary antibodies, or in the alternative, if the primary
antibody is labeled, the enzymatic, or antibody binding reagents
which are capable of reacting with the labeled antibody. One
skilled in the art will readily recognize that the disclosed probes
and antibodies of the present invention can be readily incorporated
into one of the established kit formats which are well known in the
art.
[0330] MEDICAL IMAGING
[0331] The novel polypeptides and binding partners of the invention
are useful in medical imaging of sites expressing the molecules of
the invention (e.g., where the polypeptide of the invention is
involved in the immune response, for imaging sites of inflammation
or infection). See, e.g., Kunkel et al., U.S. Pat. No. 5,413,778.
Such methods involve chemical attachment of a labeling or imaging
agent, administration of the labeled polypeptide to a subject in a
pharmaceutically acceptable carrier, and imaging the labeled
polypeptide in vivo at the target site.
[0332] SCREENING ASSAYS
[0333] Using the isolated proteins and polynucleotides of the
invention, the present invention further provides methods of
obtaining and identifying agents which bind to a polypeptide
encoded by an ORF corresponding to any of the nucleotide sequences
set forth in SEQ ID NOs: 1-10, or bind to a specific domain of the
polypeptide encoded by the nucleic acid. In detail, said method
comprises the steps of:
[0334] (a) contacting an agent with an isolated protein encoded by
an ORF of the present invention, or nucleic acid of the invention;
and
[0335] (b) determining whether the agent binds to said protein or
said nucleic acid.
[0336] In general, therefore, such methods for identifying
compounds that bind to a polynucleotide of the invention can
comprise contacting a compound with a polynucleotide of the
invention for a time sufficient to form a polynucleotide/compound
complex, and detecting the complex, so that if a
polynucleotide/compound complex is detected, a compound that binds
to a polynucleotide of the invention is identified.
[0337] Likewise, in general, therefore, such methods for
identifying compounds that bind to a polypeptide of the invention
can comprise contacting a compound with a polypeptide of the
invention for a time sufficient to form a polypeptide/compound
complex, and detecting the complex, so that if a
polypeptide/compound complex is detected, a compound that binds to
a polynucleotide of the invention is identified.
[0338] Methods for identifying compounds that bind to a polypeptide
of the invention can also comprise contacting a compound with a
polypeptide of the invention in a cell for a time sufficient to
form a polypeptide/compound complex, wherein the complex drives
expression of a receptor gene sequence in the cell, and detecting
the complex by detecting reporter gene sequence expression, so that
if a polypeptide/compound complex is detected, a compound that
binds a polypeptide of the invention is identified.
[0339] Compounds identified via such methods can include compounds
which modulate the activity of a polypeptide of the invention (that
is, increase or decrease its activity, relative to activity
observed in the absence of the compound). Alternatively, compounds
identified via such methods can include compounds which modulate
the expression of a polynucleotide of the invention (that is,
increase or decrease expression relative to expression levels
observed in the absence of the compound). Compounds, such as
compounds identified via the methods of the invention, can be
tested using standard assays well known to those of skill in the
art for their ability to modulate activity/expression.
[0340] The agents screened in the above assay can be, but are not
limited to, peptides, carbohydrates, vitamin derivatives, or other
pharmaceutical agents. The agents can be selected and screened at
random or rationally selected or designed using protein modeling
techniques.
[0341] For random screening, agents such as peptides,
carbohydrates, pharmaceutical agents and the like are selected at
random and are assayed for their ability to bind to the protein
encoded by the ORF of the present invention. Alternatively, agents
may be rationally selected or designed. As used herein, an agent is
said to be "rationally selected or designed" when the agent is
chosen based on the configuration of the particular protein. For
example, one skilled in the art can readily adapt currently
available procedures to generate peptides, pharmaceutical agents
and the like, capable of binding to a specific peptide sequence, in
order to generate rationally designed antipeptide peptides, for
example see Hurby et al., Application of Synthetic Peptides:
Antisense Peptides," In Synthetic Peptides, A User's Guide, W. H.
Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry
28:9230-8 (1989), or pharmaceutical agents, or the like.
[0342] In addition to the foregoing, one class of agents of the
present invention, as broadly described, can be used to control
gene expression through binding to one of the ORFs or EMFs of the
present invention. As described above, such agents can be randomly
screened or rationally designed/selected. Targeting the ORF or EMF
allows a skilled artisan to design sequence specific or element
specific agents, modulating the expression of either a single ORF
or multiple ORFs which rely on the same EMF for expression control.
One class of DNA binding agents are agents which contain base
residues which hybridize or form a triple helix formation by
binding to DNA or RNA. Such agents can be based on the classic
phosphodiester, ribonucleic acid backbone, or can be a variety of
sulfhydryl or polymeric derivatives which have base attachment
capacity.
[0343] Agents suitable for use in these methods preferably contain
20 to 40 bases and are designed to be complementary to a region of
the gene involved in transcription (triple helix--see Lee et al.,
Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456
(1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA
itself (antisense--Okano, J. Neurochem. 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation
optimally results in a shut-off of RNA transcription from DNA,
while antisense RNA hybridization blocks translation of an mRNA
molecule into polypeptide. Both techniques have been demonstrated
to be effective in model systems. Information contained in the
sequences of the present invention is necessary for the design of
an antisense or triple helix oligonucleotide and other DNA binding
agents.
[0344] Agents which bind to a protein encoded by one of the ORFs of
the present invention can be used as a diagnostic agent. Agents
which bind to a protein encoded by one of the ORFs of the present
invention can be formulated using known techniques to generate a
pharmaceutical composition.
[0345] USE OF NUCLEIC ACIDS AS PROBES
[0346] Another aspect of the subject invention is to provide for
polypeptide-specific nucleic acid hybridization probes capable of
hybridizing with naturally occurring nucleotide sequences. The
hybridization probes of the subject invention may be derived from
any of the nucleotide sequences SEQ ID NOs: 1-10. Because the
corresponding gene is only expressed in a limited number of
tissues, a hybridization probe derived from of any of the
nucleotide sequences SEQ ID NOs: 1-10 can be used as an indicator
of the presence of RNA of cell type of such a tissue in a
sample.
[0347] Any suitable hybridization technique can be employed, such
as, for example, in situ hybridization. PCR as described in U.S.
Pat. Nos. 4,683,195 and 4,965,188 provides additional uses for
oligonucleotides based upon the nucleotide sequences. Such probes
used in PCR may be of recombinant origin, may be chemically
synthesized, or a mixture of both. The probe will comprise a
discrete nucleotide sequence for the detection of identical
sequences or a degenerate pool of possible sequences for
identification of closely related genomic sequences.
[0348] Other means for producing specific hybridization probes for
nucleic acids include the cloning of nucleic acid sequences into
vectors for the production of mRNA probes. Such vectors are known
in the art and are commercially available and may be used to
synthesize RNA probes in vitro by means of the addition of the
appropriate RNA polymerase as T7 or SP6 RNA polymerase and the
appropriate radioactively labeled nucleotides. The nucleotide
sequences may be used to construct hybridization probes for mapping
their respective genomic sequences. The nucleotide sequence
provided herein may be mapped to a chromosome or specific regions
of a chromosome using well known genetic and/or chromosomal mapping
techniques. These techniques include in situ hybridization, linkage
analysis against known chromosomal markers, hybridization screening
with libraries or flow-sorted chromosomal preparations specific to
known chromosomes, and the like. The technique of fluorescent in
situ hybridization of chromosome spreads has been described, among
other places, in Verma et al (1988) Human Chromosomes: A Manual of
Basic Techniques, Pergamon Press, New York N.Y.
[0349] Fluorescent in situ hybridization of chromosomal
preparations and other physical chromosome mapping techniques may
be correlated with additional genetic map data. Examples of genetic
map data can be found in the 1994 Genome Issue of Science (265:
1981f). Correlation between the location of a nucleic acid on a
physical chromosomal map and a specific disease (or predisposition
to a specific disease) may help delimit the region of DNA
associated with that genetic disease. The nucleotide sequences of
the subject invention may be used to detect differences in gene
sequences between normal, carrier or affected individuals.
[0350] PREPARATION OF SUPPORT BOUND OLIGONUCLEOTIDES
[0351] Oligonucleotides, i.e., small nucleic acid segments, may be
readily prepared by, for example, directly synthesizing the
oligonucleotide by chemical means, as is commonly practiced using
an automated oligonucleotide synthesizer.
[0352] Support bound oligonucleotides may be prepared by any of the
methods known to those of skill in the art using any suitable
support such as glass, polystyrene or Teflon. One strategy is to
precisely spot oligonucleotides synthesized by standard
synthesizers. Immobilization can be achieved using passive
adsorption (Inouye & Hondo, (1990) J. Clin. Microbiol. 28(6)
1469-72); using UV light (Nagata et al., 1985; Dahlen et al., 1987;
Morrissey & Collins, (1989) Mol. Cell Probes 3(2) 189-207) or
by covalent binding of base modified DNA (Keller et al., 1988;
1989); all references being specifically incorporated herein.
[0353] Another strategy that may be employed is the use of the
strong biotin-streptavidin interaction as a linker. For example,
Broude et al. (1994) Proc. Natl. Acad. Sci. USA 91(8) 3072-6,
describe the use of biotinylated probes, although these are duplex
probes, that are immobilized on streptavidin-coated magnetic beads.
Streptavidin-coated beads may be purchased from Dynal, Oslo. Of
course, this same linking chemistry is applicable to coating any
surface with streptavidin. Biotinylated probes may be purchased
from various sources, such as, e.g., Operon Technologies (Alameda,
Calif.).
[0354] Nunc Laboratories (Naperville, Ill.) is also selling
suitable material that could be used. Nunc Laboratories have
developed a method by which DNA can be covalently bound to the
microwell surface termed Covalink NH. CovaLink NH is a polystyrene
surface grafted with secondary amino groups (>NH) that serve as
bridge-heads for further covalent coupling. CovaLink Modules may be
purchased from Nunc Laboratories. DNA molecules may be bound to
CovaLink exclusively at the 5'-end by a phosphoramidate bond,
allowing immobilization of more than 1 pmol of DNA (Rasmussen et
al., (1991) Anal. Biochem. 198(1) 138-42).
[0355] The use of CovaLink NH strips for covalent binding of DNA
molecules at the 5'-end has been described (Rasmussen et al.,
(1991). in this technology, a phosphoramidate bond is employed (Chu
et al., (1983) Nucleic Acids Res. 11(8) 6513-29). This is
beneficial as immobilization using only a single covalent bond is
preferred. The phosphoramidate bond joins the DNA to the CovaLink
NH secondary amino groups that are positioned at the end of spacer
arms covalently grafted onto the polystyrene surface through a 2 nm
long spacer arm. To link an oligonucleotide to CovaLink NH via an
phosphoramidate bond, the oligonucleotide terminus must have a
5'-end phosphate group. It is, perhaps, even possible for biotin to
be covalently bound to CovaLink and then streptavidin used to bind
the probes.
[0356] More specifically, the linkage method includes dissolving
DNA in water (7.5 ng/ul) and denaturing for 10 min. at 95.degree.
C. and cooling on ice for 10 min. Ice-cold 0.1 M 1-methylimidazole,
pH 7.0 (1-MeIm.sub.7), is then added to a final concentration of 10
mM 1-MeIm.sub.7. A ss DNA solution is then dispensed into CovaLink
NH strips (75 ul/well) standing on ice.
[0357] Carbodiimide 0.2 M
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in
10 mM 1-MeIm.sub.7, is made fresh and 25 ul added per well. The
strips are incubated for 5 hours at 50.degree. C. After incubation
the strips are washed using, e.g., Nunc-Immuno Wash; first the
wells are washed 3 times, then they are soaked with washing
solution for 5 min., and finally they are washed 3 times (where in
the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50.degree.
C.).
[0358] It is contemplated that a further suitable method for use
with the present invention is that described in PCT Patent
Application WO 90/03382 (Southern & Maskos), incorporated
herein by reference. This method of preparing an oligonucleotide
bound to a support involves attaching a nucleoside 3'-reagent
through the phosphate group by a covalent phosphodiester link to
aliphatic hydroxyl groups carried by the support. The
oligonucleotide is then synthesized on the supported nucleoside and
protecting groups removed from the synthetic oligonucleotide chain
under standard conditions that do not cleave the oligonucleotide
from the support. Suitable reagents include nucleoside
phosphoramidite and nucleoside hydrogen phosphorate.
[0359] An on-chip strategy for the preparation of DNA probe for the
preparation of DNA probe arrays may be employed. For example,
addressable laser-activated photodeprotection may be employed in
the chemical synthesis of oligonucleotides directly on a glass
surface, as described by Fodor et al. (1991) Science 251(4995)
767-73, incorporated herein by reference. Probes may also be
immobilized on nylon supports as described by Van Ness et al.
(1991) Nucleic Acids Res. 19(12) 3345-50; or linked to Teflon using
the method of Duncan & Cavalier (1988) Anal. Biochem. 169(1)
104-8; all references being specifically incorporated herein.
[0360] To link an oligonucleotide to a nylon support, as described
by Van Ness et al. (1991), requires activation of the nylon surface
via alkylation and selective activation of the 5'-amine of
oligonucleotides with cyanuric chloride.
[0361] One particular way to prepare support bound oligonucleotides
is to utilize the light-generated synthesis described by Pease et
al., (1994) PNAS USA 91(11) 5022-6, incorporated herein by
reference). These authors used current photolithographic techniques
to generate arrays of immobilized oligonucleotide probes (DNA
chips). These methods, in which light is used to direct the
synthesis of oligonucleotide probes in high-density, miniaturized
arrays, utilize photolabile 5'-protected N-acyl-deoxynucleoside
phosphoramidites, surface linker chemistry and versatile
combinatorial synthesis strategies. A matrix of 256 spatially
defined oligonucleotide probes may be generated in this manner.
[0362] PREPARATION OF NUCLEIC ACID FRAGMENTS
[0363] The nucleic acids may be obtained from any appropriate
source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected
chromosome bands, cosmid or YAC inserts, and RNA, including mRNA
without any amplification steps. For example, Sambrook et al.
(1989) describes three protocols for the isolation of high
molecular weight DNA from mammalian cells (p. 9.14-9.23).
[0364] DNA fragments may be prepared as clones in M13, plasmid or
lambda vectors and/or prepared directly from genomic DNA or cDNA by
PCR or other amplification methods. Samples may be prepared or
dispensed in multiwell plates. About 100-1000 ng of DNA samples may
be prepared in 2-500 ml of final volume.
[0365] The nucleic acids would then be fragmented by any of the
methods known to those of skill in the art including, for example,
using restriction enzymes as described at 9.24-9.28 of Sambrook et
al. (1989), shearing by ultrasound and NaOH treatment.
[0366] Low pressure shearing is also appropriate, as described by
Schriefer et al. (1990) Nucleic Acids Res. 18(24) 7455-6,
incorporated herein by reference). In this method, DNA samples are
passed through a small French pressure cell at a variety of low to
intermediate pressures. A lever device allows controlled
application of low to intermediate pressures to the cell. The
results of these studies indicate that low-pressure shearing is a
useful alternative to sonic and enzymatic DNA fragmentation
methods.
[0367] One particularly suitable way for fragmenting DNA is
contemplated to be that using the two base recognition
endonuclease, CviJI, described by Fitzgerald et al. (1992) Nucleic
Acids Res. 20(14) 3753-62. These authors described an approach for
the rapid fragmentation and fractionation of DNA into particular
sizes that they contemplated to be suitable for shotgun cloning and
sequencing.
[0368] The restriction endonuclease CviJI normally cleaves the
recognition sequence PuGCPy between the G and C to leave blunt
ends. Atypical reaction conditions, which alter the specificity of
this enzyme (CviJI**), yield a quasi-random distribution of DNA
fragments form the small molecule pUC19 (2688 base pairs).
Fitzgerald et al. (1992) quantitatively evaluated the randomness of
this fragmentation strategy, using a CviJI** digest of pUC19 that
was size fractionated by a rapid gel filtration method and directly
ligated, without end repair, to a lac Z minus M13 cloning vector.
Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy
and PuGCPu, in addition to PuGCPy sites, and that new sequence data
is accumulated at a rate consistent with random fragmentation.
[0369] As reported in the literature, advantages of this approach
compared to sonication and agarose gel fractionation include:
smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug);
and fewer steps are involved (no preligation, end repair, chemical
extraction, or agarose gel electrophoresis and elution are
needed
[0370] Irrespective of the manner in which the nucleic acid
fragments are obtained or prepared, it is important to denature the
DNA to give single stranded pieces available for hybridization.
This is achieved by incubating the DNA solution for 2-5 minutes at
80-90.degree. C. The solution is then cooled quickly to 2.degree.
C. to prevent renaturation of the DNA fragments before they are
contacted with the chip. Phosphate groups must also be removed from
genomic DNA by methods known in the art.
[0371] PREPARATION OF DNA ARRAYS
[0372] Arrays may be prepared by spotting DNA samples on a support
such as a nylon membrane. Spotting may be performed by using arrays
of metal pins (the positions of which correspond to an array of
wells in a microtiter plate) to repeated by transfer of about 20 nl
of a DNA solution to a nylon membrane. By offset printing, a
density of dots higher than the density of the wells is achieved.
One to 25 dots may be accommodated in 1mm.sup.2, depending on the
type of label used. By avoiding spotting in some preselected number
of rows and columns, separate subsets (subarrays) may be formed.
Samples in one subarray may be the same genomic segment of DNA (or
the same gene) from different individuals, or may be different,
overlapped genomic clones. Each of the subarrays may represent
replica spotting of the same samples. In one example, a selected
gene segment may be amplified from 64 patients. For each patient,
the amplified gene segment may be in one 96-well plate (all 96
wells containing the same sample). A plate for each of the 64
patients is prepared. By using a 96-pin device, all samples may be
spotted on one 8.times.12 cm membrane. Subarrays may contain 64
samples, one from each patient. Where the 96 subarrays are
identical, the dot span may be 1 mm.sup.2 and there may be a 1 mm
space between subarrays.
[0373] Another approach is to use membranes or plates (available
from NUNC, Naperville, Ill.) which may be partitioned by physical
spacers e.g. a plastic grid molded over the membrane, the grid
being similar to the sort of membrane applied to the bottom of
multiwell plates, or hydrophobic strips. A fixed physical spacer is
not preferred for imaging by exposure to flat phosphor-storage
screens or x-ray films.
[0374] The present invention is illustrated in the following
examples. Upon consideration of the present disclosure, one of
skill in the art will appreciate that many other embodiments and
variations may be made in the scope of the present invention.
Accordingly, it is intended that the broader aspects of the present
invention not be limited to the disclosure of the following
examples. The present invention is not to be limited in scope by
the exemplified embodiments which are intended as illustrations of
single aspects of the invention, and compositions and methods which
are functionally equivalent are within the scope of the invention.
Indeed, numerous modifications and variations in the practice of
the invention are expected to occur to those skilled in the art
upon consideration of the present preferred embodiments.
Consequently, the only limitations which should be placed upon the
scope of the invention are those which appear in the appended
claims.
[0375] All references cited within the body of the instant
specification are hereby incorporated by reference in their
entirety.
1 5.0 EXAMPLES 5.1 EXAMPLE 1
[0376] Novel Nucleic Acid Sequences Obtained From Various
Libraries
[0377] A plurality of novel nucleic acids were obtained from cDNA
libraries prepared from various human tissues and in some cases
isolated from a genomic library derived from human chromosome using
standard PCR, SBH sequence signature analysis and Sanger sequencing
techniques. The inserts of the library were amplified with PCR
using primers specific for the vector sequences which flank the
inserts. Clones from cDNA libraries were spotted on nylon membrane
filters and screened with oligonucleotide probes (e.g., 7 mers) to
obtain signature sequences. The clones were clustered into groups
of similar or identical sequences. Representative clones were
selected for sequencing.
[0378] In some cases, the 5' sequence of the amplified inserts was
then deduced using a typical Sanger sequencing protocol. PCR
products were purified and subjected to fluorescent dye terminator
cycle sequencing. Single pass gel sequencing was done using a 377
Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid
sequences. In some cases RACE (Random Amplification of cDNA Ends)
was performed to further extend the sequence in the 5'
direction.
Example 2
[0379] Novel Nucleic Acids
[0380] The novel nucleic acids of the present invention of the
invention were assembled from sequences that were obtained from a
cDNA library by methods described in Example 1 above, and in some
cases sequences obtained from one or more public databases. The
nucleic acids were assembled using an EST sequence as a seed. Then
a recursive algorithm was used to extend the seed EST into an
extended assemblage, by pulling additional sequences from different
databases (i.e., Hyseq's database containing EST sequences, dbEST
version 114, gb pri 114, and UniGene version 101) that belong to
this assemblage. The algorithm terminated when there was no
additional sequences from the above databases that would extend the
assemblage. Inclusion of component sequences into the assemblage
was based on a BLASTN hit to the extending assemblage with BLAST
score greater than 300 and percent identity greater than 95%.
[0381] Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full
length gene cDNA sequence and its corresponding protein sequence
were generated from the assemblage. Any frame shifts and incorrect
stop codons were corrected by hand editing. During editing, the
sequence was checked using FASTY and/or BLAST against Genbank
(i.e., dbEST version 120, gb pri 120, UniGene version 120, Genpept
release 120). Other computer programs which may have been used in
the editing process were phredPhrap and Consed (University of
Washington) and ed-ready, ed-ext and cg-zip-2 (Hyseq, Inc.). The
full-length nucleotide and amino acid sequences, including splice
variants resulting from these procedures are shown in the Sequence
Listing as SEQ ID NOS: 1-10.
[0382] Table 1 shows the various tissue sources of SEQ ID NO:
1-10.
[0383] The homology for SEQ ID NO: 1-10 were obtained by a BLASTP
version 2.0al 19MP-WashU search against Genpept release 120 and the
amino acid version of Geneseq released on Oct. 26, 2000, using
BLAST algorithm. The results showed homologues for SEQ ID NO: 1-10
from Genpept. The homologues with identifiable functions for SEQ ID
NO: 1-10 are shown in Table 2 below.
[0384] Using eMatrix software package (Stanford University,
Stanford, Calif.) (Wu et al., J. Comp. Biol., Vol. 6 pp. 219-235
(1999) herein incorporated by reference), all the sequences were
examined to determine whether they had identifiable signature
regions. Table 3 shows the signature region found in the indicated
polypeptide sequences, the description of the signature, the
eMatrix p-value(s) and the position(s) of the signature within the
polypeptide sequence.
[0385] Using the pFam software program (Sonnhammer et al., Nucleic
Acids Res., Vol. 26(1) pp. 320-322 (1998) herein incorporated by
reference) all the polypeptide sequences were examined for domains
with homology to certain peptide domains. Table 4 shows the name of
the domain found, the description, the p-value and the pFam score
for the identified domain within the sequence.
[0386] The nucleotide sequence within the sequences that codes for
signal peptide sequences and their cleavage sites can be determine
from using Neural Network SignalP V1.1 program (from Center for
Biological Sequence Analysis, The Technical University of Denmark).
The process for identifying prokaryotic and eukaryotic signal
peptides and their cleavage sites are also disclosed by Henrik
Nielson, Jacob Engelbrecht, Soren Brunak, and Gunnar von Heijne in
the publication "Identification of prokaryotic and eukaryotic
signal peptides and prediction of their cleavage sites" Protein
Engineering, Vol. 10, no. 1, pp. 1-6 (1997), incorporated herein by
reference. A maximum S score and a mean S score, as described in
the Nielson et as reference, was obtained for the polypeptide
sequences. Table 5 shows the position of the signal peptide in each
of the polypeptides and the maximum score and mean score associated
with that signal peptide.
2TABLE 1 LIBRARY/ HYSEQ LIBRARY SEQ ID TISSUE ORIGIN RNA SOURCE
NAME NOS: adult brain GIBCO ABD003 5-6 adult brain Clontech ABR008
5-6 cultured Strategene ADP001 5-6 preadipocytes adult kidney GIBCO
AKD001 2 adult liver Invitrogen ALV002 5-6 adult ovary Invitrogen
AOV001 5-6 10 adult spleen GIBCO ASP001 5-6 bone marrow Clontech
BMD002 2 5-6 adult cervix BioChain CVX001 8 fetal brain Clontech
FBR006 2-3 fetal brain Invitrogen FBT002 3 5-7 fetal kidney
Clontech FKD002 5-6 fetal liver- Columbia University FLS001 5-6
spleen fetal liver- Columbia University FLS002 2 5-6 spleen fetal
liver- Columbia University FLS003 5-6 spleen fetal muscle
Invitrogen FMS002 5-6 fetal skin Invitrogen FSK001 10 umbilical
cord BioChain FUC001 3 infant brain Columbia University IB2002 8
infant brain Columbia University IB2003 4 infant brain Columbia
University IBM002 2 lung tumor Invitrogen LGT002 5-6 8 lymphocytes
ATCC LPC001 5-6 mammary gland Invitrogen MMG001 1 4-6 induced
neuron Strategene NTD001 9 cells rectum Invitrogen REC001 8
salivary gland Clontech SAL001 10 skeletal Clontech SKM001 1 muscle
thyroid gland Clontech THR001 2 trachea Clontech TRC001 2
[0387]
3TABLE 2 CORRESPONDING SEQ ID NO. IN SMITH- SEQ ID U.S.S.N
ACCESSION WATERMAN % NO: 09/649,167 NUMBER DESCRIPTION SCORE
IDENTITY 1 6138 AL136365 Homo sapiens 1715 100 bA143M15.2 (novel DM
DNA binding domain containing protein) 2 6221 AE003742 Drosophila
189 28 melanogaster CG13827 gene product 3 8371 4 11110 AF231024
Homo sapiens 15953 100 11111 protocadherin 11112 Flamingo 2 5 15219
AC016795 Arabidopsis 449 37 15220 thaliana putative 15221 ubiquitin
carboxyl- terminal hydrolase 6 15219 AC016795 Arabidopsis 449 37
15220 thaliana putative 15221 ubiquitin carboxyl- terminal
hydrolase 7 16198 D29822 Callithrix jacchus 267 70 16199 Cytochrome
P-450 16200 16201 8 16629 Z29332_aa1 Human receptor 2189 99 16630
tyrosine kinase coding region. 9 21383 Y36307 Human secreted 1053
100 protein encoded by gene 84 10 23036 AF242769 Homo sapiens 1521
100 mesenchymal stem cell protein DSC54
[0388]
4TABLE 3 ACCES- SEQ ID SION NO: NO. DESCRIPTION RESULTS* 1 DM01803
1 HERPESVIRUS DM01803I 15.63 GLYCOPROTEIN H. 8.226e-07 210-246 2
PR00519 5-HYDROXYTRYPTAMINE PR00519C 9.73 5B RECEPTOR 7.202e-06
130-145 SIGNATURE 3 DM00522 499 kw TRYPSIN DM00522B 9.43 KINASE
KUNITZ 7.923e-06 50-64 PANCREATIC. 4 PR00517 5-HYDROXYTRYPTAMINE
PR00517E 10.98 2C RECEPTOR 9.270e-08 88-105 SIGNATURE 5 PF00602
Influenza RNA- PF00602F 11.46 dependant RNA 8.275e-07 polymerase
subunit 1118-1173 PB1. 6 PF00602 Influenza RNA- PF00602F 11.46
dependant RNA 8.275e-07 polymerase subunit 1140-1195 PB1. 7 BL01095
Chitinases family 18 BL01095C 10.76 proteins. 1.000e-05 82-94 9
PR00669 INHIBIN ALPHA CHAIN PR00669B 8.27 SIGNATURE 9.910e-06
164-181 10 PR00513 5-HYDROXYTRYPTAMINE PR00513D 11.06 1B RECEPTOR
2.991e-06 54-72 SIGNATURE *Results include in order: accession
number subtype; raw score; p-value; position of signature in amino
acid sequence.
[0389]
5TABLE 4 SEQ ID pFAM NO: pFAM NAME DESCRIPTION p-value SCORE 1
DM-domain DM DNA binding domain 4.2e-25 96.8 4 7tm_2 7
transmembrane receptor 6.4e-90 312.2 (Secretin family) 5 UCH-2
Ubiquitin carboxyl-terminal 0.0003 17.5 hydrolase family 2 6 UCH-2
Ubiquitin carboxyl-terminal 0.0003 17.5 hydrolase family 2 7 p450
Cytochrome P450 0.0014 15.4 8 pkinase Eukaryotic protein kinase
8.6e-39 142.3 domain
[0390]
6TABLE 5 POSITION OF SIGNAL maxS SEQ ID IN AMINO ACID (MAXIMUM
meanS NO: SEQUENCE SCORE) (MEAN SCORE) 4 1-20 0.971 0.941 7 1-31
0.936 0.729 9 1-30 0.970 0.574
[0391]
Sequence CWU 1
1
10 1 2157 DNA Homo sapiens CDS (1)..(1020) 1 atg aac ggc tac ggc
tcc ccc tac ctg tac atg ggc ggc ccg gtg tcg 48 Met Asn Gly Tyr Gly
Ser Pro Tyr Leu Tyr Met Gly Gly Pro Val Ser 1 5 10 15 cag ccg cca
cgg gcg ccc ctg cag cgc acg ccc aag tgc gcg cgc tgc 96 Gln Pro Pro
Arg Ala Pro Leu Gln Arg Thr Pro Lys Cys Ala Arg Cys 20 25 30 cgc
aac cat ggc gtc ctg tcc tgg ctc aag ggc cac aag cgt tac tgc 144 Arg
Asn His Gly Val Leu Ser Trp Leu Lys Gly His Lys Arg Tyr Cys 35 40
45 cgc ttc aag gac tgc acc tgc gag aag tgc atc ctc atc atc gag cgg
192 Arg Phe Lys Asp Cys Thr Cys Glu Lys Cys Ile Leu Ile Ile Glu Arg
50 55 60 cag cgg gtc atg gct gcg cag gtg gcg ctg cgc cgg cag cag
gcc aac 240 Gln Arg Val Met Ala Ala Gln Val Ala Leu Arg Arg Gln Gln
Ala Asn 65 70 75 80 gag agc ttg gag agc ctc atc ccc gac tcg ctg cgc
gct ctg cca ggg 288 Glu Ser Leu Glu Ser Leu Ile Pro Asp Ser Leu Arg
Ala Leu Pro Gly 85 90 95 ccc ccg ccg ccg ggg gac gcc gtc gcc gcc
ccg cag ccg ccg cca gcc 336 Pro Pro Pro Pro Gly Asp Ala Val Ala Ala
Pro Gln Pro Pro Pro Ala 100 105 110 tct cag ccg tcg cag ccg cag ccg
ccg cgc cct gct gcc gag ttg gcc 384 Ser Gln Pro Ser Gln Pro Gln Pro
Pro Arg Pro Ala Ala Glu Leu Ala 115 120 125 gcg gcc gcc gcg ctg cgt
tgg act gcc gag ccg cag ccc ggg gct ctg 432 Ala Ala Ala Ala Leu Arg
Trp Thr Ala Glu Pro Gln Pro Gly Ala Leu 130 135 140 cag gcg cag ctc
gcc aag cca gat ttg act gaa gaa cga ctt gga gac 480 Gln Ala Gln Leu
Ala Lys Pro Asp Leu Thr Glu Glu Arg Leu Gly Asp 145 150 155 160 ggc
aag tcg gca gac aat aca gag gtc ttc agt gac aaa gac act gac 528 Gly
Lys Ser Ala Asp Asn Thr Glu Val Phe Ser Asp Lys Asp Thr Asp 165 170
175 cag agg agt tcc cca gat gtg gca aag agt aag ggc tgc ttc acc cct
576 Gln Arg Ser Ser Pro Asp Val Ala Lys Ser Lys Gly Cys Phe Thr Pro
180 185 190 gag agc cct gag ata gtg tcc gtg gag gaa ggg gga tac gct
gtc cag 624 Glu Ser Pro Glu Ile Val Ser Val Glu Glu Gly Gly Tyr Ala
Val Gln 195 200 205 aaa aac gga ggc aac ccc gag agc cgc cct gac agc
ccc aag tgt cac 672 Lys Asn Gly Gly Asn Pro Glu Ser Arg Pro Asp Ser
Pro Lys Cys His 210 215 220 gcg gag cag aat cac ctc ctg att gag ggc
ccc tcg ggg act gtt tct 720 Ala Glu Gln Asn His Leu Leu Ile Glu Gly
Pro Ser Gly Thr Val Ser 225 230 235 240 ctg ccc ttc agc ttg aaa gcc
aac aga ccg ccg ctt gaa gtg tta aaa 768 Leu Pro Phe Ser Leu Lys Ala
Asn Arg Pro Pro Leu Glu Val Leu Lys 245 250 255 aag ata ttc ccc aac
cag aag cca acg gtg ctt gag ctc atc ctc aag 816 Lys Ile Phe Pro Asn
Gln Lys Pro Thr Val Leu Glu Leu Ile Leu Lys 260 265 270 ggc tgt ggc
ggg gac ctg gtg agc gcc gtg gaa gtc ctt ctg tcc agc 864 Gly Cys Gly
Gly Asp Leu Val Ser Ala Val Glu Val Leu Leu Ser Ser 275 280 285 cga
tcc tca gtc acg gga gca gag cga act tcc gca gaa cct gag agt 912 Arg
Ser Ser Val Thr Gly Ala Glu Arg Thr Ser Ala Glu Pro Glu Ser 290 295
300 cta gcg ttg ccc tcc aat ggg cac atc ttt gaa cac acc ttg agc tcc
960 Leu Ala Leu Pro Ser Asn Gly His Ile Phe Glu His Thr Leu Ser Ser
305 310 315 320 tac ccc atc tcg tct tcc aat ggt ctg tgg gat cag cct
ttc gag tcc 1008 Tyr Pro Ile Ser Ser Ser Asn Gly Leu Trp Asp Gln
Pro Phe Glu Ser 325 330 335 cag aca cgt tga ggt tttctgccga
ctctagcaac gttgtcccca gtcccttggc 1063 Gln Thr Arg * 340 tgggcctctg
cagccccctt tcccccagcc accccggtac ccgctgatgc tgaggaatac 1123
tttggcgaga agccagtcga gccccttttt gcccaatgat gtcaccctgt ggaacaccat
1183 gacgctgcag cagcagtatc agctgaggtc ccagtatgtc agtcctttcc
ccagtaactc 1243 taccagcgtc ttcagaagct cgcccgtcct tcctgcccgc
gccacggaag accctcggat 1303 ttccatccct gatgatgggt gtccatttgt
gtcaaagcag tccatttaca ccgaggacga 1363 ctatgacgag aggtctgact
cctcagactc tagaacactc aacacatcat cttaaagtgg 1423 tgctggatgg
gtggtggcca ggtgacattt tctgtgcgtt ttgaccctga ggcatctgag 1483
gagaggccac atcttgtgta tgccctttcc ttctgtttga caaggtgact gtgcttgatt
1543 ctatacatta gcaataaaaa cataacttat ttaacttctt gcacttcact
ggaaaatgcc 1603 aaatagctct gttctgtggc tttagtgctg aatgtttatt
gtaaaagaga gtctaatgtt 1663 aagaatagtc ttgggaaggc tgggtccgtg
gaagatttat ttggggatgt aaagctgaag 1723 gtcagccttg cacctaaacc
caacctggaa tgttaaatga aataatatac ttgaatgcaa 1783 ttttgtaaaa
gtggattcct caggatatgt gaaacctaaa ggaagtggtt ctgttgcaaa 1843
tggactataa acagggacat tatattctta cactaaaaat ccttgcattt taaagagaga
1903 tgcacttaag aatagagtga actgctcata tgcttattta agcttggaca
gttttcagag 1963 acaaacccca ttaagaatta ctcttttcac atggctgaat
cgaaacatgt gtaatgtcaa 2023 tgtaaaacca atcacagctg tgaactgcat
gaaatgtatt gtgaaacgaa cacaagatta 2083 agctttgtca ggttaatgta
gcatgttaag gactctagaa aaaaataaac taaggagacg 2143 agaaaaaaaa aaaa
2157 2 1330 DNA Homo sapiens CDS (478)..(1101) 2 tggttcttcc
tttattctta attcttaact gtgatggatc cttgcaatga aagaattaaa 60
aagggcgggt gcggtggggc acgcctgtaa ttccagcact ttggaaggtc gacgtgggac
120 gattgcttaa gcccagggct caaggccagc ctgggtaaca tgaacaattc
ttaacgtcca 180 aagataacgt ctttccttaa cgtcgaaaga tgccttgttg
tgatattaca gcagtcattc 240 ctgctcacta cgggcaggca gacagtgcag
aaaaggtgaa gcacaaaaac taaaaaggaa 300 aatctctcct tgaaccactg
cgaacagatc cgagtgctgg ggtactgctg ccagctggtt 360 ggtggagttc
tggttgaaca gtgtcccgcc aggtccgaag tggggacacg tctgttggtg 420
gtgtccaccc aactcagcca ctgcaggacc atcttgcgac tctttgatga cctggcc 477
atg ttt gtc tac act aag caa tat ggc ctg ggg gca cag gag gag gac 525
Met Phe Val Tyr Thr Lys Gln Tyr Gly Leu Gly Ala Gln Glu Glu Asp 1 5
10 15 gcc ttt gtc cgc tgt gtc tcc gtc cta ggg aac ctg gct gac cag
ctc 573 Ala Phe Val Arg Cys Val Ser Val Leu Gly Asn Leu Ala Asp Gln
Leu 20 25 30 tac tac ccc tgt gag cac gtg gcc tgg gcg gct gat gcc
cgg gtc ctc 621 Tyr Tyr Pro Cys Glu His Val Ala Trp Ala Ala Asp Ala
Arg Val Leu 35 40 45 cac gtg gac tct tct cgg tgg tgg acg ctg agt
aca acc ctg tgg gcc 669 His Val Asp Ser Ser Arg Trp Trp Thr Leu Ser
Thr Thr Leu Trp Ala 50 55 60 ctc tct ctg ctc ctg ggg gtt gcc agg
tcc ctg tgg atg ctg ctg aaa 717 Leu Ser Leu Leu Leu Gly Val Ala Arg
Ser Leu Trp Met Leu Leu Lys 65 70 75 80 ctg aga cag agg ctg cgg agc
ccc acg gcg ccc ttc acc agc ccg ctg 765 Leu Arg Gln Arg Leu Arg Ser
Pro Thr Ala Pro Phe Thr Ser Pro Leu 85 90 95 ccc cgg ggc aag cgg
agg gcc atg gag gcg cag atg cag tcg gag gcg 813 Pro Arg Gly Lys Arg
Arg Ala Met Glu Ala Gln Met Gln Ser Glu Ala 100 105 110 ctg tca ctt
ctc agc aac ctg gcc gac ctg gcc aac gcc gtg cac tgg 861 Leu Ser Leu
Leu Ser Asn Leu Ala Asp Leu Ala Asn Ala Val His Trp 115 120 125 ctg
ccc cgg ggc gtg ctg tgg gcc ggc cgc ttc ccg ccg tgg cta gtg 909 Leu
Pro Arg Gly Val Leu Trp Ala Gly Arg Phe Pro Pro Trp Leu Val 130 135
140 ggc ctc atg ggc acc acc tcc tgc ctg cca gca tgt acc agg cgg ccc
957 Gly Leu Met Gly Thr Thr Ser Cys Leu Pro Ala Cys Thr Arg Arg Pro
145 150 155 160 ggg ccg gcg gcc agg ccg agg cca cta ccc cct gac act
gcc gga aga 1005 Gly Pro Ala Ala Arg Pro Arg Pro Leu Pro Pro Asp
Thr Ala Gly Arg 165 170 175 gca cag gga cac agc cag agc cca cgg agg
gcc ctt ccc gca aag cag 1053 Ala Gln Gly His Ser Gln Ser Pro Arg
Arg Ala Leu Pro Ala Lys Gln 180 185 190 aag ccg cca ggg cag ggg ccg
ggg ctc tca tgg agc tgc ctg tgg tga 1101 Lys Pro Pro Gly Gln Gly
Pro Gly Leu Ser Trp Ser Cys Leu Trp * 195 200 205 aacccatggg
cagggtgggt ggagggactg atgcgaggtc ccccaggcct cagcctggag 1161
ctcctgagcc gtgacagttt tcaggggccg tgcagggctt cagcagcacc tcctgcctgc
1221 cagctggcgt cttgtatcca aatcaagatg aggaagaggg ggcctgtcgt
gccttgagaa 1281 agctggaacg ggaatcaatt aaacattgtg gtgctggaaa
aaaaaaaaa 1330 3 992 DNA Homo sapiens CDS (314)..(691) 3 tggtctctgc
ccctcctcag atggcctgtt aggtaactca cctgtgtgtg tgtgcatggt 60
ccttcttgag taatggctgc acaggaagga aattacctag tggccactgg aacaggagcc
120 tccggcagcc atctttatgt ccttgggctg agactccagg ctgcccagca
gaggataagg 180 tggcctctga gaagcctctt ttttgtcctc tgaccttggg
actccacact cctgcttcca 240 tggatttgaa ggttccaaca tcccccttct
ccacggtctc cagcaggcac agcccaggtc 300 tgacggggag gtg atg ccc acg ctg
gac atg gcc ttg ttc gac tgg acc 349 Met Pro Thr Leu Asp Met Ala Leu
Phe Asp Trp Thr 1 5 10 gat tat gaa gac tta aaa cct gat ggt tgg ccc
tct gca aag aag aaa 397 Asp Tyr Glu Asp Leu Lys Pro Asp Gly Trp Pro
Ser Ala Lys Lys Lys 15 20 25 gag aaa cac cgc ggt aaa ctc tcc agt
gat ggt aac gaa aca tca cca 445 Glu Lys His Arg Gly Lys Leu Ser Ser
Asp Gly Asn Glu Thr Ser Pro 30 35 40 gcc gaa ggg gaa cca tgc gac
cat cac caa tac tgc ctg aca ggg act 493 Ala Glu Gly Glu Pro Cys Asp
His His Gln Tyr Cys Leu Thr Gly Thr 45 50 55 60 tgc tgc gac ctg cgg
gag cat ttc tgc aca ccc cac aac cga ggc ctg 541 Cys Cys Asp Leu Arg
Glu His Phe Cys Thr Pro His Asn Arg Gly Leu 65 70 75 aac aac aaa
tgc ttc gat gac tgc atg tgt gtg gaa ggg ctg cgc tgc 589 Asn Asn Lys
Cys Phe Asp Asp Cys Met Cys Val Glu Gly Leu Arg Cys 80 85 90 tat
gcc aaa ttc cac cgg aac cgc agg gtt aca cgg agg aaa ggg cgc 637 Tyr
Ala Lys Phe His Arg Asn Arg Arg Val Thr Arg Arg Lys Gly Arg 95 100
105 tgt gtg gag ccc gag acg gcc aac ggc gac cag gga tcc ttc atc aac
685 Cys Val Glu Pro Glu Thr Ala Asn Gly Asp Gln Gly Ser Phe Ile Asn
110 115 120 gtc tag cggccccgcg ggactgggga ctgagcccag gaggtttgca
caagccgggc 741 Val * 125 gatttgtttg taactagcag tgggagatca
agttggggaa cagatggctg aggctgcaga 801 ctcaggccca ggacactcaa
ccccaggagg ggagccgctc ggcgaatgag ctgggtgggt 861 gcccaggagc
cggcccgcag cacctgcaca cacgaagtcc ggacccacgc agcctccatc 921
ccgcgtgtct tgctctccgc gatggcaatg ccgagagtgc cctctactgt ccgactccag
981 cactgcaaca g 992 4 11389 DNA Homo sapiens CDS (1)..(9045) 4 atg
gcg ccg ccg ccg ccg ccc gtg ctg ccc gtg ctg ctg ctc ctg gcc 48 Met
Ala Pro Pro Pro Pro Pro Val Leu Pro Val Leu Leu Leu Leu Ala 1 5 10
15 gcc gcc gcc gcc ctg ccg gcg atg ggg ctg cga gcg gcc gcc tgg gag
96 Ala Ala Ala Ala Leu Pro Ala Met Gly Leu Arg Ala Ala Ala Trp Glu
20 25 30 ccg cgc gta ccc ggc ggg acc cgc gcc ttc gcc ctc cgg ccc
ggc tgt 144 Pro Arg Val Pro Gly Gly Thr Arg Ala Phe Ala Leu Arg Pro
Gly Cys 35 40 45 acc tac gcg gtg ggc gcc gct tgc acg ccc cgg gcg
ccg cgg gag ctg 192 Thr Tyr Ala Val Gly Ala Ala Cys Thr Pro Arg Ala
Pro Arg Glu Leu 50 55 60 ctg gac gtg ggc cgc gat ggg cgg ctg gca
gga cgt cgg cgc gtc tcg 240 Leu Asp Val Gly Arg Asp Gly Arg Leu Ala
Gly Arg Arg Arg Val Ser 65 70 75 80 ggc gcg ggg cgc ccg ctg ccg ctg
caa gtc cgc ttg gtg gcc cgc agt 288 Gly Ala Gly Arg Pro Leu Pro Leu
Gln Val Arg Leu Val Ala Arg Ser 85 90 95 gcc ccg acg gcg ctg agc
cgc cgc ctg cgg gcg cgc acg cac ctt ccc 336 Ala Pro Thr Ala Leu Ser
Arg Arg Leu Arg Ala Arg Thr His Leu Pro 100 105 110 ggc tgc gga gcc
cgt gcc cgg ctc tgc gga acc ggt gcc cgg ctc tgc 384 Gly Cys Gly Ala
Arg Ala Arg Leu Cys Gly Thr Gly Ala Arg Leu Cys 115 120 125 ggg gcg
ctc tgc ttc ccc gtc ccc ggc ggc tgc gcg gcc gcg cag cat 432 Gly Ala
Leu Cys Phe Pro Val Pro Gly Gly Cys Ala Ala Ala Gln His 130 135 140
tcg gcg ctc gca gct ccg acc acc tta ccc gcc tgc cgc tgc ccg ccg 480
Ser Ala Leu Ala Ala Pro Thr Thr Leu Pro Ala Cys Arg Cys Pro Pro 145
150 155 160 cgc ccc agg ccc cgc tgt ccc ggc cgt ccc atc tgc ctg ccg
ccg ggc 528 Arg Pro Arg Pro Arg Cys Pro Gly Arg Pro Ile Cys Leu Pro
Pro Gly 165 170 175 ggc tcg gtc cgc ctg cgt ctg ctg tgc gcc ctg cgg
cgc gcg gct ggc 576 Gly Ser Val Arg Leu Arg Leu Leu Cys Ala Leu Arg
Arg Ala Ala Gly 180 185 190 gcc gtc cgg gtg gga ctg gcg ctg gag gcc
gcc acc gcg ggg acg ccc 624 Ala Val Arg Val Gly Leu Ala Leu Glu Ala
Ala Thr Ala Gly Thr Pro 195 200 205 tcc gcg tcg cca tcc cca tcg ccg
ccc ctg ccg ccg aac ttg ccc gaa 672 Ser Ala Ser Pro Ser Pro Ser Pro
Pro Leu Pro Pro Asn Leu Pro Glu 210 215 220 gcc cgg gcg ggg ccg gcg
cga cgg gcc cgg cgg ggc acg agc ggc aga 720 Ala Arg Ala Gly Pro Ala
Arg Arg Ala Arg Arg Gly Thr Ser Gly Arg 225 230 235 240 ggg agc ctg
aag ttt ccg atg ccc aac tac cag gtg gcg ttg ttt gag 768 Gly Ser Leu
Lys Phe Pro Met Pro Asn Tyr Gln Val Ala Leu Phe Glu 245 250 255 aac
gaa ccg gcg ggc acc ctc atc ctc cag ctg cac gcg cac tac acc 816 Asn
Glu Pro Ala Gly Thr Leu Ile Leu Gln Leu His Ala His Tyr Thr 260 265
270 atc gag ggc gag gag gag cgc gtg agc tat tac atg gag ggg ctg ttc
864 Ile Glu Gly Glu Glu Glu Arg Val Ser Tyr Tyr Met Glu Gly Leu Phe
275 280 285 gac gag cgc tcc cgg ggc tac ttc cga atc gac tct gcc acg
ggc gcc 912 Asp Glu Arg Ser Arg Gly Tyr Phe Arg Ile Asp Ser Ala Thr
Gly Ala 290 295 300 gtg agc acg gac agc gta ctg gac cgc gag acc aag
gag acg cac gtc 960 Val Ser Thr Asp Ser Val Leu Asp Arg Glu Thr Lys
Glu Thr His Val 305 310 315 320 ctc agg gtg aaa gcc gtg gac tac agt
acg ccg ccg cgc tcg gcc acc 1008 Leu Arg Val Lys Ala Val Asp Tyr
Ser Thr Pro Pro Arg Ser Ala Thr 325 330 335 acc tac atc act gtc ttg
gtc aaa gac acc aac gac cac agc ccg gtc 1056 Thr Tyr Ile Thr Val
Leu Val Lys Asp Thr Asn Asp His Ser Pro Val 340 345 350 ttc gag cag
tcg gag tac cgc gag cgc gtg cgg gag aac ctg gag gtg 1104 Phe Glu
Gln Ser Glu Tyr Arg Glu Arg Val Arg Glu Asn Leu Glu Val 355 360 365
ggc tac gag gtg ctg acc atc cgc gcc agc gac cgc gac tcg ccc atc
1152 Gly Tyr Glu Val Leu Thr Ile Arg Ala Ser Asp Arg Asp Ser Pro
Ile 370 375 380 aac gcc aac ttg cgt tac cgc gtg ttg ggg ggc gcg tgg
gac gtc ttc 1200 Asn Ala Asn Leu Arg Tyr Arg Val Leu Gly Gly Ala
Trp Asp Val Phe 385 390 395 400 cag ctc aac gag agc tct ggc gtg gtg
agc aca cgg gcg gtg ctg gac 1248 Gln Leu Asn Glu Ser Ser Gly Val
Val Ser Thr Arg Ala Val Leu Asp 405 410 415 cgg gag gag gcg gcc gag
tac cag ctc ctg gtg gag gcc aac gac cag 1296 Arg Glu Glu Ala Ala
Glu Tyr Gln Leu Leu Val Glu Ala Asn Asp Gln 420 425 430 ggg cgc aat
ccg ggc ccg ctc agt gcc acg gcc acc gtg tac atc gag 1344 Gly Arg
Asn Pro Gly Pro Leu Ser Ala Thr Ala Thr Val Tyr Ile Glu 435 440 445
gtg gag gac gag aac gac aac tac ccc cag ttc agc gag cag aac tac
1392 Val Glu Asp Glu Asn Asp Asn Tyr Pro Gln Phe Ser Glu Gln Asn
Tyr 450 455 460 gtg gtc cag gtg ccc gag gac gtg ggg ctc aac acg gct
gtg ctg cga 1440 Val Val Gln Val Pro Glu Asp Val Gly Leu Asn Thr
Ala Val Leu Arg 465 470 475 480 gtg cag gcc acg gac cgg gac cag ggc
cag aac gcg gcc att cac tac 1488 Val Gln Ala Thr Asp Arg Asp Gln
Gly Gln Asn Ala Ala Ile His Tyr 485 490 495 agc atc ctc agc ggg aac
gtg gcc ggc cag ttc tac ctg cac tcg ctg 1536 Ser Ile Leu Ser Gly
Asn Val Ala Gly Gln Phe Tyr Leu His Ser Leu 500 505 510 agc ggg atc
ctg gat gtg atc aac ccc ttg gat ttc gag gat gtc cag 1584 Ser Gly
Ile Leu Asp Val Ile Asn Pro Leu Asp Phe Glu Asp Val Gln 515 520 525
aaa tac tcg ctg agc att aag gcc cag gat ggg ggc cgg ccc ccg ctc
1632 Lys Tyr Ser Leu Ser Ile Lys Ala Gln Asp Gly Gly Arg Pro Pro
Leu 530 535 540 atc aat tct tca ggg gtg gtg tct gtg cag gtg ctg gat
gtc aac gac 1680 Ile Asn Ser Ser Gly Val Val Ser Val Gln Val Leu
Asp Val Asn Asp 545 550 555 560 aac gag cct atc ttt gtg agc agc ccc
ttc cag gcc acg gtg ctg gag 1728 Asn Glu
Pro Ile Phe Val Ser Ser Pro Phe Gln Ala Thr Val Leu Glu 565 570 575
aat gtg ccc ctg ggc tac ccc gtg gtg cac att cag gcg gtg gac gcg
1776 Asn Val Pro Leu Gly Tyr Pro Val Val His Ile Gln Ala Val Asp
Ala 580 585 590 gac tct gga gag aac gcc cgg ctg cac tat cgc ctg gtg
gac acg gcc 1824 Asp Ser Gly Glu Asn Ala Arg Leu His Tyr Arg Leu
Val Asp Thr Ala 595 600 605 tcc acc ttt ctg ggg ggc ggc agc gct ggg
cct aag aat cct gcc ccc 1872 Ser Thr Phe Leu Gly Gly Gly Ser Ala
Gly Pro Lys Asn Pro Ala Pro 610 615 620 acc cct gac ttc ccc ttc cag
atc cac aac agc tcc ggt tgg atc aca 1920 Thr Pro Asp Phe Pro Phe
Gln Ile His Asn Ser Ser Gly Trp Ile Thr 625 630 635 640 gtg tgt gcc
gag ctg gac cgc gag gag gtg gag cac tac agc ttc ggg 1968 Val Cys
Ala Glu Leu Asp Arg Glu Glu Val Glu His Tyr Ser Phe Gly 645 650 655
gtg gag gcg gtg gac cac ggc tcg ccc ccc atg agc tcc tcc acc agc
2016 Val Glu Ala Val Asp His Gly Ser Pro Pro Met Ser Ser Ser Thr
Ser 660 665 670 gtg tcc atc acg gtg ctg gac gtg aat gac aac gac ccg
gtg ttc acg 2064 Val Ser Ile Thr Val Leu Asp Val Asn Asp Asn Asp
Pro Val Phe Thr 675 680 685 cag ccc acc tac gag ctt cgt ctg aat gag
gat gcg gcc gtg ggg agc 2112 Gln Pro Thr Tyr Glu Leu Arg Leu Asn
Glu Asp Ala Ala Val Gly Ser 690 695 700 agc gtg ctg acc ctg cag gcc
cgc gac cgt gac gcc aac agt gtg att 2160 Ser Val Leu Thr Leu Gln
Ala Arg Asp Arg Asp Ala Asn Ser Val Ile 705 710 715 720 acc tac cag
ctc aca ggc ggc aac acc cgg aac cgc ttt gca ctc agc 2208 Thr Tyr
Gln Leu Thr Gly Gly Asn Thr Arg Asn Arg Phe Ala Leu Ser 725 730 735
agc cag aga ggg ggc ggc ctc atc acc ctg gcg cta cct ctg gac tac
2256 Ser Gln Arg Gly Gly Gly Leu Ile Thr Leu Ala Leu Pro Leu Asp
Tyr 740 745 750 aag cag gag cag cag tac gtg ctg gcg gtg aca gca tcc
gac ggc aca 2304 Lys Gln Glu Gln Gln Tyr Val Leu Ala Val Thr Ala
Ser Asp Gly Thr 755 760 765 cgg tcg cac act gcg cat gtc cta atc aac
gtc act gat gcc aac acc 2352 Arg Ser His Thr Ala His Val Leu Ile
Asn Val Thr Asp Ala Asn Thr 770 775 780 cac agg cct gtc ttt cag agc
tcc cat tac aca gtg agt gtc agt gag 2400 His Arg Pro Val Phe Gln
Ser Ser His Tyr Thr Val Ser Val Ser Glu 785 790 795 800 gac agg cct
gtg ggc acc tcc att gct acc ctc agt gcc aac gat gag 2448 Asp Arg
Pro Val Gly Thr Ser Ile Ala Thr Leu Ser Ala Asn Asp Glu 805 810 815
gac aca gga gag aat gcc cgc atc acc tac gtg att cag gac ccc gtg
2496 Asp Thr Gly Glu Asn Ala Arg Ile Thr Tyr Val Ile Gln Asp Pro
Val 820 825 830 ccg cag ttc cgc att gac ccc gac agt ggc acc atg tac
acc atg atg 2544 Pro Gln Phe Arg Ile Asp Pro Asp Ser Gly Thr Met
Tyr Thr Met Met 835 840 845 gag ctg gac tat gag aac cag gtc gcc tac
acg ctg acc atc atg gcc 2592 Glu Leu Asp Tyr Glu Asn Gln Val Ala
Tyr Thr Leu Thr Ile Met Ala 850 855 860 cag gac aac ggc atc ccg cag
aaa tca gac acc acc acc cta gag atc 2640 Gln Asp Asn Gly Ile Pro
Gln Lys Ser Asp Thr Thr Thr Leu Glu Ile 865 870 875 880 ctc atc ctc
gat gcc aat gac aat gca ccc cag ttc ctg tgg gat ttc 2688 Leu Ile
Leu Asp Ala Asn Asp Asn Ala Pro Gln Phe Leu Trp Asp Phe 885 890 895
tac cag ggt tcc atc ttt gag gat gct cca ccc tcg acc agc atc ctc
2736 Tyr Gln Gly Ser Ile Phe Glu Asp Ala Pro Pro Ser Thr Ser Ile
Leu 900 905 910 cag gtc tct gcc acg gac cgg gac tca ggt ccc aat ggg
cgt ctg ctg 2784 Gln Val Ser Ala Thr Asp Arg Asp Ser Gly Pro Asn
Gly Arg Leu Leu 915 920 925 tac acc ttc cag ggt ggg gac gac ggc gat
ggg gac ttc tac atc gag 2832 Tyr Thr Phe Gln Gly Gly Asp Asp Gly
Asp Gly Asp Phe Tyr Ile Glu 930 935 940 ccc acg tcc ggt gtg att cgc
acc cag cgc cgg ctg gac cgg gag aat 2880 Pro Thr Ser Gly Val Ile
Arg Thr Gln Arg Arg Leu Asp Arg Glu Asn 945 950 955 960 gtg gcc gtg
tac aac ctt tgg gct ctg gct gtg gat cgg ggc agt ccc 2928 Val Ala
Val Tyr Asn Leu Trp Ala Leu Ala Val Asp Arg Gly Ser Pro 965 970 975
act ccc ctt agc gcc tcg gta gaa atc cag gtg acc atc ttg gac att
2976 Thr Pro Leu Ser Ala Ser Val Glu Ile Gln Val Thr Ile Leu Asp
Ile 980 985 990 aat gac aat gcc ccc atg ttt gag aag gac gaa ctg gag
ctg ttt gtt 3024 Asn Asp Asn Ala Pro Met Phe Glu Lys Asp Glu Leu
Glu Leu Phe Val 995 1000 1005 gag gag aac aac cca gtg ggg tcg gtg
gtg gca aag att cgt gct aac 3072 Glu Glu Asn Asn Pro Val Gly Ser
Val Val Ala Lys Ile Arg Ala Asn 1010 1015 1020 gac cct gat gaa ggc
cct aat gcc cag atc atg tat cag att gtg gaa 3120 Asp Pro Asp Glu
Gly Pro Asn Ala Gln Ile Met Tyr Gln Ile Val Glu 1025 1030 1035 1040
ggg gac atg cgg cat ttc ttc cag ctg gac ctg ctc aac ggg gac ctg
3168 Gly Asp Met Arg His Phe Phe Gln Leu Asp Leu Leu Asn Gly Asp
Leu 1045 1050 1055 cgt gcc atg gtg gag ctg gac ttt gag gtc cgg cgg
gag tat gtg ctg 3216 Arg Ala Met Val Glu Leu Asp Phe Glu Val Arg
Arg Glu Tyr Val Leu 1060 1065 1070 gtg gtg cag gcc acg tcg gct ccg
ctg gtg agc cga gcc acg gtg cac 3264 Val Val Gln Ala Thr Ser Ala
Pro Leu Val Ser Arg Ala Thr Val His 1075 1080 1085 atc ctt ctc gtg
gac cag aat gac aac ccg cct gtg ctg ccc gac ttc 3312 Ile Leu Leu
Val Asp Gln Asn Asp Asn Pro Pro Val Leu Pro Asp Phe 1090 1095 1100
cag atc ctc ttc aac aac tat gtc acc aac aag tcc aac agt ttc ccc
3360 Gln Ile Leu Phe Asn Asn Tyr Val Thr Asn Lys Ser Asn Ser Phe
Pro 1105 1110 1115 1120 acc ggc gtg atc ggc tgc atc ccg gcc cat gac
ccc gac gtg tca gac 3408 Thr Gly Val Ile Gly Cys Ile Pro Ala His
Asp Pro Asp Val Ser Asp 1125 1130 1135 agc ctc aac tac acc ttc gtg
cag ggc aac gag ctg cgc ctg ttg ctg 3456 Ser Leu Asn Tyr Thr Phe
Val Gln Gly Asn Glu Leu Arg Leu Leu Leu 1140 1145 1150 ctg gac ccc
gcc acg ggc gaa ctg cag ctc agc cgc gac ctg gac aac 3504 Leu Asp
Pro Ala Thr Gly Glu Leu Gln Leu Ser Arg Asp Leu Asp Asn 1155 1160
1165 aac cgg ccg ctg gag gcg ctc atg gag gtg tct gtg tct gat ggc
atc 3552 Asn Arg Pro Leu Glu Ala Leu Met Glu Val Ser Val Ser Asp
Gly Ile 1170 1175 1180 cac agc gtc acg gcc ttc tgc acc ctg cgt gtc
acc atc atc acg gac 3600 His Ser Val Thr Ala Phe Cys Thr Leu Arg
Val Thr Ile Ile Thr Asp 1185 1190 1195 1200 gac atg ctg acc aac agc
atc act gtc cgc ctg gag aac atg tcc cag 3648 Asp Met Leu Thr Asn
Ser Ile Thr Val Arg Leu Glu Asn Met Ser Gln 1205 1210 1215 gag aag
ttc ctg tcc ccg ctg ctg gcc ctc ttc gtg gag ggg gtg gcc 3696 Glu
Lys Phe Leu Ser Pro Leu Leu Ala Leu Phe Val Glu Gly Val Ala 1220
1225 1230 gcc gtg ctg tcc acc acc aag gac gac gtc ttc gtc ttc aac
gtc cag 3744 Ala Val Leu Ser Thr Thr Lys Asp Asp Val Phe Val Phe
Asn Val Gln 1235 1240 1245 aac gac acc gac gtc agc tcc aac atc ctg
aac gtg acc ttc tcg gcg 3792 Asn Asp Thr Asp Val Ser Ser Asn Ile
Leu Asn Val Thr Phe Ser Ala 1250 1255 1260 ctg ctg cct ggc ggc gtc
cgc ggc cag ttc ttc ccg tcg gag gac ctg 3840 Leu Leu Pro Gly Gly
Val Arg Gly Gln Phe Phe Pro Ser Glu Asp Leu 1265 1270 1275 1280 cag
gag cag atc tac ctg aat cgg acg ctg ctg acc acc atc tcc acg 3888
Gln Glu Gln Ile Tyr Leu Asn Arg Thr Leu Leu Thr Thr Ile Ser Thr
1285 1290 1295 cag cgc gtg ctg ccc ttc gac gac aac atc tgc ctg cgc
gag ccc tgc 3936 Gln Arg Val Leu Pro Phe Asp Asp Asn Ile Cys Leu
Arg Glu Pro Cys 1300 1305 1310 gag aac tac atg aag tgc gtg tcc gtt
ctg cga ttc gac agc tcc gcg 3984 Glu Asn Tyr Met Lys Cys Val Ser
Val Leu Arg Phe Asp Ser Ser Ala 1315 1320 1325 ccc ttc ctc agc tcc
acc acc gtg ctc ttc cgg ccc atc cac ccc atc 4032 Pro Phe Leu Ser
Ser Thr Thr Val Leu Phe Arg Pro Ile His Pro Ile 1330 1335 1340 aac
ggc ctg cgc tgc cgc tgc ccg ccc ggc ttc acc ggc gac tac tgc 4080
Asn Gly Leu Arg Cys Arg Cys Pro Pro Gly Phe Thr Gly Asp Tyr Cys
1345 1350 1355 1360 gag acg gag atc gac ctc tgc tac tcc gac ccg tgc
ggc gcc aac ggc 4128 Glu Thr Glu Ile Asp Leu Cys Tyr Ser Asp Pro
Cys Gly Ala Asn Gly 1365 1370 1375 cgc tgc cgc agc cgc gag ggc ggc
tac acc tgc gag tgc ttc gag gac 4176 Arg Cys Arg Ser Arg Glu Gly
Gly Tyr Thr Cys Glu Cys Phe Glu Asp 1380 1385 1390 ttc act gga gag
cac tgt gag gtg gat gcc cgc tca ggc cgc tgt gcc 4224 Phe Thr Gly
Glu His Cys Glu Val Asp Ala Arg Ser Gly Arg Cys Ala 1395 1400 1405
aac ggg gtg tgc aag aac ggg ggc acc tgc gtg aac ctg ctc atc ggc
4272 Asn Gly Val Cys Lys Asn Gly Gly Thr Cys Val Asn Leu Leu Ile
Gly 1410 1415 1420 ggc ttc cac tgc gtg tgt cct cct ggc gag tat gag
agg ccc tac tgt 4320 Gly Phe His Cys Val Cys Pro Pro Gly Glu Tyr
Glu Arg Pro Tyr Cys 1425 1430 1435 1440 gag gtg acc acc agg agc ttc
ccg ccc cag tcc ttc gtc acc ttc cgg 4368 Glu Val Thr Thr Arg Ser
Phe Pro Pro Gln Ser Phe Val Thr Phe Arg 1445 1450 1455 ggc ctg aga
cag cgc ttc cac ttc acc atc tcc ctc acg ttt gcc act 4416 Gly Leu
Arg Gln Arg Phe His Phe Thr Ile Ser Leu Thr Phe Ala Thr 1460 1465
1470 cag gaa agg aac ggc ttg ctt ctc tac aac ggc cgc ttc aat gag
aag 4464 Gln Glu Arg Asn Gly Leu Leu Leu Tyr Asn Gly Arg Phe Asn
Glu Lys 1475 1480 1485 cac gac ttc atc gcc ctg gag atc gtg gac gag
cag gtg cag ctc acc 4512 His Asp Phe Ile Ala Leu Glu Ile Val Asp
Glu Gln Val Gln Leu Thr 1490 1495 1500 ttc tct gca ggc gag aca aca
acg acc gtg gca ccg aag gtt ccc agt 4560 Phe Ser Ala Gly Glu Thr
Thr Thr Thr Val Ala Pro Lys Val Pro Ser 1505 1510 1515 1520 ggt gtg
agt gac ggg cgg tgg cac tct gtg cag gtg cag tac tac aac 4608 Gly
Val Ser Asp Gly Arg Trp His Ser Val Gln Val Gln Tyr Tyr Asn 1525
1530 1535 aag ccc aat att ggc cac ctg ggc ctg ccc cat ggg ccg tcc
ggg gaa 4656 Lys Pro Asn Ile Gly His Leu Gly Leu Pro His Gly Pro
Ser Gly Glu 1540 1545 1550 aag atg gcc gtg gtg aca gtg gat gat tgt
gac aca acc atg gct gtg 4704 Lys Met Ala Val Val Thr Val Asp Asp
Cys Asp Thr Thr Met Ala Val 1555 1560 1565 cgc ttt gga aag gac atc
ggg aac tac agc tgc gct gcc cag ggc act 4752 Arg Phe Gly Lys Asp
Ile Gly Asn Tyr Ser Cys Ala Ala Gln Gly Thr 1570 1575 1580 cag acc
ggc tcc aag aag tcc ctg gat ctg acc ggc cct cta ctc ctg 4800 Gln
Thr Gly Ser Lys Lys Ser Leu Asp Leu Thr Gly Pro Leu Leu Leu 1585
1590 1595 1600 ggg ggt gtc ccc aac ctg cca gaa gac ttc cca gtg cac
aac cgg cag 4848 Gly Gly Val Pro Asn Leu Pro Glu Asp Phe Pro Val
His Asn Arg Gln 1605 1610 1615 ttc gtg ggc tgc atg cgg aac ctg tca
gtc gac ggc aaa aat gtg gac 4896 Phe Val Gly Cys Met Arg Asn Leu
Ser Val Asp Gly Lys Asn Val Asp 1620 1625 1630 atg gcc gga ttc atc
gcc aac aat ggc acc cgg gaa ggc tgc gct gct 4944 Met Ala Gly Phe
Ile Ala Asn Asn Gly Thr Arg Glu Gly Cys Ala Ala 1635 1640 1645 cgg
agg aac ttc tgc gat ggg agg cgg tgt cag aat gga ggc acc tgt 4992
Arg Arg Asn Phe Cys Asp Gly Arg Arg Cys Gln Asn Gly Gly Thr Cys
1650 1655 1660 gtc aac agg tgg aat atg tat ctg tgt gag tgt cca ctc
cga ttc ggc 5040 Val Asn Arg Trp Asn Met Tyr Leu Cys Glu Cys Pro
Leu Arg Phe Gly 1665 1670 1675 1680 ggg aag aac tgt gag caa gcc atg
cct cac ccc cag ctc ttc agc ggt 5088 Gly Lys Asn Cys Glu Gln Ala
Met Pro His Pro Gln Leu Phe Ser Gly 1685 1690 1695 gag agc gtc gtg
tcc tgg agt gac ctg aac atc atc atc tct gtg ccc 5136 Glu Ser Val
Val Ser Trp Ser Asp Leu Asn Ile Ile Ile Ser Val Pro 1700 1705 1710
tgg tac ctg ggg ctc atg ttc cgg acc cgg aag gag gac agc gtt ctg
5184 Trp Tyr Leu Gly Leu Met Phe Arg Thr Arg Lys Glu Asp Ser Val
Leu 1715 1720 1725 atg gag gcc acc agt ggt ggg ccc acc agc ttt cgc
ctc cag atc ctg 5232 Met Glu Ala Thr Ser Gly Gly Pro Thr Ser Phe
Arg Leu Gln Ile Leu 1730 1735 1740 aac aac tac ctc cag ttt gag gtg
tcc cac ggc ccc tcc gat gtg gag 5280 Asn Asn Tyr Leu Gln Phe Glu
Val Ser His Gly Pro Ser Asp Val Glu 1745 1750 1755 1760 tcc gtg atg
ctg tcc ggg ttg cgg gtg acc gac ggg gag tgg cac cac 5328 Ser Val
Met Leu Ser Gly Leu Arg Val Thr Asp Gly Glu Trp His His 1765 1770
1775 ctg ctg atc gag ctg aag aat gtt aag gag gac agt gag atg aag
cac 5376 Leu Leu Ile Glu Leu Lys Asn Val Lys Glu Asp Ser Glu Met
Lys His 1780 1785 1790 ctg gtc acc atg acc ttg gac tat ggg atg gac
cag aac aag gca gat 5424 Leu Val Thr Met Thr Leu Asp Tyr Gly Met
Asp Gln Asn Lys Ala Asp 1795 1800 1805 atc ggg ggc atg ctt ccc ggg
ctg acg gta agg agc gtg gtg gtc gga 5472 Ile Gly Gly Met Leu Pro
Gly Leu Thr Val Arg Ser Val Val Val Gly 1810 1815 1820 ggc gcc tct
gaa gac aag gtc tcc gtg cgc cgt gga ttc cga ggc tgc 5520 Gly Ala
Ser Glu Asp Lys Val Ser Val Arg Arg Gly Phe Arg Gly Cys 1825 1830
1835 1840 atg cag gga gtg agg atg ggg ggg acg ccc acc aac gtc gcc
acc ctg 5568 Met Gln Gly Val Arg Met Gly Gly Thr Pro Thr Asn Val
Ala Thr Leu 1845 1850 1855 aac atg aac aac gca ctc aag gtc agg gtg
aag gac ggc tgt gat gtg 5616 Asn Met Asn Asn Ala Leu Lys Val Arg
Val Lys Asp Gly Cys Asp Val 1860 1865 1870 gac gac ccc tgt acc tcg
agc ccc tgt ccc ccc aat agc cgc tgc cac 5664 Asp Asp Pro Cys Thr
Ser Ser Pro Cys Pro Pro Asn Ser Arg Cys His 1875 1880 1885 gac gcc
tgg gag gac tac agc tgc gtc tgt gac aaa ggg tac ctt gga 5712 Asp
Ala Trp Glu Asp Tyr Ser Cys Val Cys Asp Lys Gly Tyr Leu Gly 1890
1895 1900 ata aac tgt gtg gat gcc tgt cac ctg aac ccc tgc gag aac
atg ggg 5760 Ile Asn Cys Val Asp Ala Cys His Leu Asn Pro Cys Glu
Asn Met Gly 1905 1910 1915 1920 gcc tgc gtg cgc tcc ccc ggc tcc ccg
cag ggc tac gtg tgc gag tgt 5808 Ala Cys Val Arg Ser Pro Gly Ser
Pro Gln Gly Tyr Val Cys Glu Cys 1925 1930 1935 ggg ccc agt cac tac
ggg ccg tac tgt gag aac aaa ctc gac ctt ccg 5856 Gly Pro Ser His
Tyr Gly Pro Tyr Cys Glu Asn Lys Leu Asp Leu Pro 1940 1945 1950 tgc
ccc aga ggc tgg tgg ggg aac ccc gtc tgt gga ccc tgc cac tgt 5904
Cys Pro Arg Gly Trp Trp Gly Asn Pro Val Cys Gly Pro Cys His Cys
1955 1960 1965 gcc gtc agc aaa ggc ttt gat ccc gac tgt aat aag acc
aac ggc cag 5952 Ala Val Ser Lys Gly Phe Asp Pro Asp Cys Asn Lys
Thr Asn Gly Gln 1970 1975 1980 tgc caa tgc aag gag aat tac tac aag
ctc cta gcc cag gac acc tgt 6000 Cys Gln Cys Lys Glu Asn Tyr Tyr
Lys Leu Leu Ala Gln Asp Thr Cys 1985 1990 1995 2000 ctg ccc tgc gac
tgc ttc ccc cat ggc tcc cac agc cgc act tgc gac 6048 Leu Pro Cys
Asp Cys Phe Pro His Gly Ser His Ser Arg Thr Cys Asp 2005 2010 2015
atg gcc acc ggg cag tgt gcc tgc aag ccc ggc gtc atc ggc cgc cag
6096 Met Ala Thr Gly Gln Cys Ala Cys Lys Pro Gly Val Ile Gly Arg
Gln 2020 2025 2030 tgc aac cgc tgc gac aac ccg ttt gcc gag gtc acc
acg ctc ggc tgt 6144 Cys Asn Arg Cys Asp Asn Pro Phe Ala Glu Val
Thr Thr Leu Gly Cys 2035 2040 2045 gaa gtg atc tac aat ggc tgt ccc
aaa gca ttt gag gcc ggc atc tgg 6192 Glu Val Ile Tyr Asn Gly Cys
Pro Lys Ala Phe Glu Ala Gly Ile Trp 2050 2055 2060 tgg cca cag acc
aag ttc ggg cag ccg gct gcg gtg cca tgc cct aag 6240 Trp Pro Gln
Thr Lys Phe Gly Gln Pro Ala Ala Val Pro Cys Pro Lys 2065 2070 2075
2080 gga tcc gtt gga aat gcg gtc cga cac tgc agc
ggg gag aag ggc tgg 6288 Gly Ser Val Gly Asn Ala Val Arg His Cys
Ser Gly Glu Lys Gly Trp 2085 2090 2095 ctg ccc cca gag ctc ttt aac
tgt acc acc atc tcc ttc gtg gac ctc 6336 Leu Pro Pro Glu Leu Phe
Asn Cys Thr Thr Ile Ser Phe Val Asp Leu 2100 2105 2110 agg gcc atg
aat gag aag ctg agc cgc aat gag acg cag gtg gac ggc 6384 Arg Ala
Met Asn Glu Lys Leu Ser Arg Asn Glu Thr Gln Val Asp Gly 2115 2120
2125 gcc agg gcc ctg cag ctg gtg agg gcg ctg cgc agt gct aca cag
cac 6432 Ala Arg Ala Leu Gln Leu Val Arg Ala Leu Arg Ser Ala Thr
Gln His 2130 2135 2140 acg ggc acg ctc ttt ggc aat gac gtg cgc acg
gcc tac cag ctg ctg 6480 Thr Gly Thr Leu Phe Gly Asn Asp Val Arg
Thr Ala Tyr Gln Leu Leu 2145 2150 2155 2160 ggc cac gtc ctt cag cac
gag agc tgg cag cag ggc ttc gac ctg gca 6528 Gly His Val Leu Gln
His Glu Ser Trp Gln Gln Gly Phe Asp Leu Ala 2165 2170 2175 gcc acg
cag gac gcc gac ttt cac gag gac gtc atc cac tcg ggc agc 6576 Ala
Thr Gln Asp Ala Asp Phe His Glu Asp Val Ile His Ser Gly Ser 2180
2185 2190 gcc ctc ctg gcc cca gcc acc agg gcg gcg tgg gag cag atc
cag cgg 6624 Ala Leu Leu Ala Pro Ala Thr Arg Ala Ala Trp Glu Gln
Ile Gln Arg 2195 2200 2205 agc gag ggc ggc acg gca cag ctg ctc cgg
cgc ctc gag ggc tac ttc 6672 Ser Glu Gly Gly Thr Ala Gln Leu Leu
Arg Arg Leu Glu Gly Tyr Phe 2210 2215 2220 agc aac gtg gca cgc aac
gtg cgg cgg acg tac ctg cgg ccc ttc gtc 6720 Ser Asn Val Ala Arg
Asn Val Arg Arg Thr Tyr Leu Arg Pro Phe Val 2225 2230 2235 2240 atc
gtc acc gcc aac atg att ctt gct gtc gac atc ttt gac aag ttc 6768
Ile Val Thr Ala Asn Met Ile Leu Ala Val Asp Ile Phe Asp Lys Phe
2245 2250 2255 aac ttt acg gga gcc agg gtc ccg cga ttc gac acc atc
cat gaa gag 6816 Asn Phe Thr Gly Ala Arg Val Pro Arg Phe Asp Thr
Ile His Glu Glu 2260 2265 2270 ttc ccc agg gag ctg gag tcc tcc gtc
tcc ttc cca gcc gac ttc ttc 6864 Phe Pro Arg Glu Leu Glu Ser Ser
Val Ser Phe Pro Ala Asp Phe Phe 2275 2280 2285 aga cca cct gaa gaa
aaa gaa ggc ccc ctg ctg agg ccg gct ggc cgg 6912 Arg Pro Pro Glu
Glu Lys Glu Gly Pro Leu Leu Arg Pro Ala Gly Arg 2290 2295 2300 agg
acc acc ccg cag acc acg cgc ccg ggg cct ggc acc gag agg gag 6960
Arg Thr Thr Pro Gln Thr Thr Arg Pro Gly Pro Gly Thr Glu Arg Glu
2305 2310 2315 2320 gcc ccg atc agc agg cgg agg cga cac cct gat gac
gct ggc cag ttc 7008 Ala Pro Ile Ser Arg Arg Arg Arg His Pro Asp
Asp Ala Gly Gln Phe 2325 2330 2335 gcc gtc gct ctg gtc atc att tac
cgc acc ctg ggg cag ctc ctg ccc 7056 Ala Val Ala Leu Val Ile Ile
Tyr Arg Thr Leu Gly Gln Leu Leu Pro 2340 2345 2350 gag cgc tac gac
ccc gac cgt cgc agc ctc cgg ttg cct cac cgg ccc 7104 Glu Arg Tyr
Asp Pro Asp Arg Arg Ser Leu Arg Leu Pro His Arg Pro 2355 2360 2365
atc att aat acc ccg atg gtg agc acg ctg gtg tac agc gag ggg gct
7152 Ile Ile Asn Thr Pro Met Val Ser Thr Leu Val Tyr Ser Glu Gly
Ala 2370 2375 2380 ccg ctc ccg aga ccc ctg gag agg ccc gtc ctg gtg
gag ttc gcc ctg 7200 Pro Leu Pro Arg Pro Leu Glu Arg Pro Val Leu
Val Glu Phe Ala Leu 2385 2390 2395 2400 ctg gag gtg gag gag cga acc
aag cct gtc tgc gtg ttc tgg aac cac 7248 Leu Glu Val Glu Glu Arg
Thr Lys Pro Val Cys Val Phe Trp Asn His 2405 2410 2415 tcc ctg gcc
gtt ggt ggg acg gga ggg tgg tct gcc cgg ggc tgc gag 7296 Ser Leu
Ala Val Gly Gly Thr Gly Gly Trp Ser Ala Arg Gly Cys Glu 2420 2425
2430 ctc ctg tcc agg aac cgg aca cat gtc gcc tgc cag tgc agc cac
aca 7344 Leu Leu Ser Arg Asn Arg Thr His Val Ala Cys Gln Cys Ser
His Thr 2435 2440 2445 gcc agc ttt gcg gtg ctc atg gat atc tcc agg
cgt gag aac ggg gag 7392 Ala Ser Phe Ala Val Leu Met Asp Ile Ser
Arg Arg Glu Asn Gly Glu 2450 2455 2460 gtc ctg cct ctg aag att gtc
acc tat gcc gct gtg tcc ttg tca ctg 7440 Val Leu Pro Leu Lys Ile
Val Thr Tyr Ala Ala Val Ser Leu Ser Leu 2465 2470 2475 2480 gca gcc
ctg ctg gtg gcc ttc gtc ctc ctg agc ctg gtc cgc atg ctg 7488 Ala
Ala Leu Leu Val Ala Phe Val Leu Leu Ser Leu Val Arg Met Leu 2485
2490 2495 cgc tcc aac ctg cac agc att cac aag cac ctc gcc gtg gcg
ctc ttc 7536 Arg Ser Asn Leu His Ser Ile His Lys His Leu Ala Val
Ala Leu Phe 2500 2505 2510 ctc tct cag ctg gtg ttc gtg att ggg atc
aac cag acg gaa aac ccg 7584 Leu Ser Gln Leu Val Phe Val Ile Gly
Ile Asn Gln Thr Glu Asn Pro 2515 2520 2525 ttt ctg tgc aca gtg gtt
gcc atc ctc ctc cac tac atc tac atg agc 7632 Phe Leu Cys Thr Val
Val Ala Ile Leu Leu His Tyr Ile Tyr Met Ser 2530 2535 2540 acc ttt
gcc tgg acc ctc gtg gag agc ctg cat gtc tac cgc atg ctg 7680 Thr
Phe Ala Trp Thr Leu Val Glu Ser Leu His Val Tyr Arg Met Leu 2545
2550 2555 2560 acc gag gtg cgc aac atc gac acg ggg ccc atg cgg ttc
tac tac gtc 7728 Thr Glu Val Arg Asn Ile Asp Thr Gly Pro Met Arg
Phe Tyr Tyr Val 2565 2570 2575 gtg ggc tgg ggc atc ccg gcc att gtc
aca gga ctg gcg gtc ggc ctg 7776 Val Gly Trp Gly Ile Pro Ala Ile
Val Thr Gly Leu Ala Val Gly Leu 2580 2585 2590 gac ccc cag ggc tac
ggg aac ccc gac ttc tgc tgg ctg tcg ctt caa 7824 Asp Pro Gln Gly
Tyr Gly Asn Pro Asp Phe Cys Trp Leu Ser Leu Gln 2595 2600 2605 gac
acc ctg att tgg agc ttt gcg ggg ccc atc gga gct gtt ata atc 7872
Asp Thr Leu Ile Trp Ser Phe Ala Gly Pro Ile Gly Ala Val Ile Ile
2610 2615 2620 atc aac aca gtc act tct gtc cta tct gca aag gtt tcc
tgc caa aga 7920 Ile Asn Thr Val Thr Ser Val Leu Ser Ala Lys Val
Ser Cys Gln Arg 2625 2630 2635 2640 aag cac cat tat tat ggg aaa aaa
ggg atc gtc tcc ctg ctg agg acc 7968 Lys His His Tyr Tyr Gly Lys
Lys Gly Ile Val Ser Leu Leu Arg Thr 2645 2650 2655 gca ttc ctc ctg
ctg ctg ctc atc agc gcc acc tgg ctg ctg ggg ctg 8016 Ala Phe Leu
Leu Leu Leu Leu Ile Ser Ala Thr Trp Leu Leu Gly Leu 2660 2665 2670
ctg gct gtg aac cgc gat gca ctg agc ttt cac tac ctc ttc gcc atc
8064 Leu Ala Val Asn Arg Asp Ala Leu Ser Phe His Tyr Leu Phe Ala
Ile 2675 2680 2685 ttc agc ggc tta cag ggc ccc ttc gtc ctc ctt ttc
cac tgc gtg ctc 8112 Phe Ser Gly Leu Gln Gly Pro Phe Val Leu Leu
Phe His Cys Val Leu 2690 2695 2700 aac cag gag gtc cgg aag cac ctg
aag ggc gtg ctc ggc ggg agg aag 8160 Asn Gln Glu Val Arg Lys His
Leu Lys Gly Val Leu Gly Gly Arg Lys 2705 2710 2715 2720 ctg cac ctg
gag gac tcc gcc acc acc agg gcc acc ctg ctg acg cgc 8208 Leu His
Leu Glu Asp Ser Ala Thr Thr Arg Ala Thr Leu Leu Thr Arg 2725 2730
2735 tcc ctc aac tgc aac acc acc ttc ggt gac ggg cct gac atg ctg
cgc 8256 Ser Leu Asn Cys Asn Thr Thr Phe Gly Asp Gly Pro Asp Met
Leu Arg 2740 2745 2750 aca gac ttg ggc gag tcc acc gcc tcg ctg gac
agc atc gtc agg gat 8304 Thr Asp Leu Gly Glu Ser Thr Ala Ser Leu
Asp Ser Ile Val Arg Asp 2755 2760 2765 gaa ggg atc cag aag ctc ggc
gtg tcc tct ggg ctg gtg agg ggc agc 8352 Glu Gly Ile Gln Lys Leu
Gly Val Ser Ser Gly Leu Val Arg Gly Ser 2770 2775 2780 cac gga gag
cca gac gcg tcc ctc atg ccc agg agc tgc aag gat ccc 8400 His Gly
Glu Pro Asp Ala Ser Leu Met Pro Arg Ser Cys Lys Asp Pro 2785 2790
2795 2800 cct ggc cac gat tcc gac tca gat agc gag ctg tcc ctg gat
gag cag 8448 Pro Gly His Asp Ser Asp Ser Asp Ser Glu Leu Ser Leu
Asp Glu Gln 2805 2810 2815 agc agc tct tac gcc tcc tca cac tcg tca
gac agc gag gac gat ggg 8496 Ser Ser Ser Tyr Ala Ser Ser His Ser
Ser Asp Ser Glu Asp Asp Gly 2820 2825 2830 gtg gga gct gag gaa aaa
tgg gac ccg gcc agg ggc gcc gtc cac agc 8544 Val Gly Ala Glu Glu
Lys Trp Asp Pro Ala Arg Gly Ala Val His Ser 2835 2840 2845 acc ccc
aaa ggg gac gct gtg gcc aac cac gtt ccg gcc ggc tgg ccc 8592 Thr
Pro Lys Gly Asp Ala Val Ala Asn His Val Pro Ala Gly Trp Pro 2850
2855 2860 gac cag agc ctg gct gag agt gac agt gag gac ccc agc ggc
aag ccc 8640 Asp Gln Ser Leu Ala Glu Ser Asp Ser Glu Asp Pro Ser
Gly Lys Pro 2865 2870 2875 2880 cgc ctg aag gtg gag acc aag gtc agc
gtg gag ctg cac cgc gag gag 8688 Arg Leu Lys Val Glu Thr Lys Val
Ser Val Glu Leu His Arg Glu Glu 2885 2890 2895 cag ggc agt cac cgt
gga gag tac ccc ccg gac cag gag agc ggg ggc 8736 Gln Gly Ser His
Arg Gly Glu Tyr Pro Pro Asp Gln Glu Ser Gly Gly 2900 2905 2910 gca
gcc agg ctt gct agc agc cag ccc cca gag cag agg aaa ggc atc 8784
Ala Ala Arg Leu Ala Ser Ser Gln Pro Pro Glu Gln Arg Lys Gly Ile
2915 2920 2925 ttg aaa aat aaa gtc acc tac ccg ccg ccg ctg acg ctg
acg gag cag 8832 Leu Lys Asn Lys Val Thr Tyr Pro Pro Pro Leu Thr
Leu Thr Glu Gln 2930 2935 2940 acg ctg aag ggc cgg ctc cgg gag aag
ctg gcc gac tgt gag cag agc 8880 Thr Leu Lys Gly Arg Leu Arg Glu
Lys Leu Ala Asp Cys Glu Gln Ser 2945 2950 2955 2960 ccc aca tcc tcg
cgc acg tct tcc ctg ggc tct ggc ggc ccc gac tgc 8928 Pro Thr Ser
Ser Arg Thr Ser Ser Leu Gly Ser Gly Gly Pro Asp Cys 2965 2970 2975
gcc atc aca gtc aag agc cct ggg agg gag ccg ggg cgt gac cac ctc
8976 Ala Ile Thr Val Lys Ser Pro Gly Arg Glu Pro Gly Arg Asp His
Leu 2980 2985 2990 aac ggg gtg gcc atg aat gtg cgc act ggg agc gcc
cag gcc gat ggc 9024 Asn Gly Val Ala Met Asn Val Arg Thr Gly Ser
Ala Gln Ala Asp Gly 2995 3000 3005 tcc gac tct gag aaa ccg tga gg
caagcccgtc accccacaca ggctgcggca 9077 Ser Asp Ser Glu Lys Pro *
3010 3015 tcaccctcag accttggagc ccaaggggcc actgcccttg aagtggagtg
ggcccagagt 9137 gtggcggtcc ccatggtggc agccccccga ctgatcatcc
agacacaaag gtcttggttc 9197 tcccaggagc tcagggcctg tcagacctgg
tgacaagtgc caaaggccac aggcatgagg 9257 gaggcgtgga ccactgggcc
agcaccgctg agtcctaaga ctgcagtcaa agccagaact 9317 gagaggggac
cccagactgg gcccagaggc tggccagagt tcaggaacgc cgggcacaga 9377
ccaaagaccg cggtccagcc ccgcccaggc gggcatctca tggcagtgcg gacccgtggc
9437 tggcagcccg ggcagtcctt tgcaaaggca ccccttgtct taaaatcact
tcgctatgtg 9497 ggaaaggtgg agatactttt atatatttgt atgggactct
gaggaggtgc aacctgtata 9557 tatattgcat tcgtgctgac tttgttatcc
cgagagatcc atgcaatgat ctcttgctgt 9617 cttctctgtc aagattgcac
agttgtactt gaatctggca tgtgttgacg aaactggtgc 9677 cccagcagat
caaaggtggg aaatacgtca gcagtggggc taaaaccaag cggctagaag 9737
ccctacagct gccttcggcc aggaagtgag gatggtgtgg gccctccccg ccggccccct
9797 gggtccccag tgttcgctgt gtgtgcgttt gtcctctgct gccatctgcc
ccggctgtgt 9857 gaattcaaga cagggcagtg cagcactagg caggtgtgag
gagccctgct gaggtcactg 9917 tggggcacgg ttgccacacg gctgtcattt
ttcacctggt cattctgtga ccaccacccc 9977 ctcccctcac cgcctcccag
gtggcccggg agctgcaggt ggggatggct ttgtcctttg 10037 ctcctgctcc
ccgtgggacc tgggacctta aagcgttgca ggttcctgat ttggacagag 10097
gtgtggggcc ttccaggccg ttacatacct cctgccaatt ctctaactct ctgagactgc
10157 gaggatctcc aggcagggtt ctcccctctg gagtctgacc aattacttca
ttttgcttca 10217 aatggccaat tgtgcagagg gacaaagcca cagccacact
cttcaacggt taccaaactg 10277 tttttggaaa ttcacaccaa ggtcgggccc
actgcaggca gctggcacag cgtggcccga 10337 ggggctgtgg aacgggtccc
ggaactgtca gacatgtttg attttagcgt ttcctttgtt 10397 cttcaaatca
ggtgcccaaa taagtgatca gcacagctgc ttccaaatag gagaaaccat 10457
aaaataggat gaaaatcaag taaaatgcaa agatgtccac actgttttaa acttgaccct
10517 gatgaaaatg tgagcactgt tagcagatgc ctatgggaga ggaaaagcgt
atctgaaaat 10577 ggtccaggac aggaggatga aatgagatcc cagagtcctc
acacctgaat gaattataca 10637 tgtgccttac caggtgagtg gtctttcgaa
gataaaaaac tctagtccct ttaaacgttt 10697 gcccctggcg tttcctaagt
acgaaaaggt ttttaagtct tcgaacagtc tcctttcatg 10757 actttaacag
gattctgccc cctgaggtgt aatttttttg ttctattttt ttccacgtac 10817
tccacagcca acatcacgag gtgtaatttt taatttgatc agaactgtta ccaaaaaaca
10877 actgtcagtt ttattgagat gggaaaaatg taaacctatt tttattactt
aagactttat 10937 gggagagatt agacactgga ggtttttaac agaacgtgta
tttattaatg ttcaaaacac 10997 tggaattaca aatgagaaga gtctacaata
aattaagatt tttgaatttg tacttctgcg 11057 gtgctggttt ttctccacaa
acacccccgc ccctccccat gcccagggtg gccgtggaag 11117 ggacggttta
cggacgtgca gctgagctgt ccgtgtccca tgctccctca gccagtggaa 11177
cgtgccggaa ctttttgtcc attccctagt aggcctgcca cagcctagat gggcagtttt
11237 tgtctttcac caaatttgag gacttttttt ttttgccatt atttcttcag
ttttcttttc 11297 ttgcactgat ctttctcctc tccttctgtg actccagtga
ctcagacgtt agacctcttg 11357 atgttttccc actggtccct gaggctctgt tc
11389 5 4087 DNA Homo sapiens CDS (113)..(4087) 5 aattcccggg
tcgacgattt cgtggcggcg cccagttggg gcgggttcgt tcgcttcgcg 60
ttttggccag ggcgggggtc tgggctttag gcaggtagta tttagtttca ca atg 115
Met 1 ttt ggg gac ctg ttt gaa gag gag tat tcc act gtg tct aat aat
cag 163 Phe Gly Asp Leu Phe Glu Glu Glu Tyr Ser Thr Val Ser Asn Asn
Gln 5 10 15 tat gga aaa ggg aag aaa tta aag act aaa gct ttg gag cca
cct gct 211 Tyr Gly Lys Gly Lys Lys Leu Lys Thr Lys Ala Leu Glu Pro
Pro Ala 20 25 30 cct aga gaa ttc acc aat tta agc gga atc aga aat
cag ggt gga acc 259 Pro Arg Glu Phe Thr Asn Leu Ser Gly Ile Arg Asn
Gln Gly Gly Thr 35 40 45 tgt tac ctc aat tcc ctt ctt cag act ctt
cat ttc aca cct gaa ttc 307 Cys Tyr Leu Asn Ser Leu Leu Gln Thr Leu
His Phe Thr Pro Glu Phe 50 55 60 65 aga gaa gct cta ttt tct ctt ggc
cca gaa gag ctt ggt ttg ttt gaa 355 Arg Glu Ala Leu Phe Ser Leu Gly
Pro Glu Glu Leu Gly Leu Phe Glu 70 75 80 gat aag gat aaa ccc gat
gca aag gtt cga atc atc cct tta cag tta 403 Asp Lys Asp Lys Pro Asp
Ala Lys Val Arg Ile Ile Pro Leu Gln Leu 85 90 95 cag cgc ttg ttt
gct cag ctt ctg ctc tta gac cag gaa gct gca tcc 451 Gln Arg Leu Phe
Ala Gln Leu Leu Leu Leu Asp Gln Glu Ala Ala Ser 100 105 110 aca gca
gac ctc act gac agc ttt ggg tgg acc agt aat gag gaa atg 499 Thr Ala
Asp Leu Thr Asp Ser Phe Gly Trp Thr Ser Asn Glu Glu Met 115 120 125
agg caa cat gat gtg cag gaa ctg aat cga atc ctc ttc agc gct ttg 547
Arg Gln His Asp Val Gln Glu Leu Asn Arg Ile Leu Phe Ser Ala Leu 130
135 140 145 gaa act tct tta gtt ggg acc tcc ggt cat gac ctc atc tat
cgt ctg 595 Glu Thr Ser Leu Val Gly Thr Ser Gly His Asp Leu Ile Tyr
Arg Leu 150 155 160 tac cat gga acc att gtt aac cag att gtt tgt aaa
gaa tgt aag aac 643 Tyr His Gly Thr Ile Val Asn Gln Ile Val Cys Lys
Glu Cys Lys Asn 165 170 175 gtt agc gag agg cag gaa gac ttc tta gat
cta aca gta gca gtc aaa 691 Val Ser Glu Arg Gln Glu Asp Phe Leu Asp
Leu Thr Val Ala Val Lys 180 185 190 aat gta tcc ggt ttg gaa gat gct
ctc tgg aac atg tat gta gaa gag 739 Asn Val Ser Gly Leu Glu Asp Ala
Leu Trp Asn Met Tyr Val Glu Glu 195 200 205 gaa gtt ttt gat tgt gac
aac ttg tac cac tgt gga act tgt gac agg 787 Glu Val Phe Asp Cys Asp
Asn Leu Tyr His Cys Gly Thr Cys Asp Arg 210 215 220 225 ctg gtt aaa
gca gca aag tcg gcc aaa tta cgt aag ctg cct cct ttt 835 Leu Val Lys
Ala Ala Lys Ser Ala Lys Leu Arg Lys Leu Pro Pro Phe 230 235 240 ctt
act gtt tca tta cta aga ttt aat ttt gat ttt gtg aaa tgc gaa 883 Leu
Thr Val Ser Leu Leu Arg Phe Asn Phe Asp Phe Val Lys Cys Glu 245 250
255 cgc tac aag gaa act agc tgt tat aca ttc cct ctc cgg att aat ctc
931 Arg Tyr Lys Glu Thr Ser Cys Tyr Thr Phe Pro Leu Arg Ile Asn Leu
260 265 270 aag ccc ttt tgt gaa cag agt gaa ttg gat gac tta gaa tat
ata tat 979 Lys Pro Phe Cys Glu Gln Ser Glu Leu Asp Asp Leu Glu Tyr
Ile Tyr 275 280 285 gac ctc ttc tca gtt att ata cac aaa ggt ggc tgc
tac gga ggc cat 1027 Asp Leu Phe Ser Val Ile Ile His Lys Gly Gly
Cys Tyr Gly Gly His 290 295 300 305 tac cat gta tat att aaa gat gtt
gat cat ttg gga aac tgg cag ttt 1075 Tyr His Val Tyr Ile Lys Asp
Val Asp His Leu Gly Asn Trp Gln Phe 310 315 320 caa
gag gaa aaa agt aaa cca gat gtg aat ctg aaa gat ctc cag agt 1123
Gln Glu Glu Lys Ser Lys Pro Asp Val Asn Leu Lys Asp Leu Gln Ser 325
330 335 gaa gaa gag att gat cat cca ctg atg att cta aaa gca atc tta
tta 1171 Glu Glu Glu Ile Asp His Pro Leu Met Ile Leu Lys Ala Ile
Leu Leu 340 345 350 gag gag aat aat cta att cct gtt gat cag ctg ggc
cag aaa ctt ttg 1219 Glu Glu Asn Asn Leu Ile Pro Val Asp Gln Leu
Gly Gln Lys Leu Leu 355 360 365 aaa aag ata gga ata tct tgg aac aag
aag tac aga aaa cag cat gga 1267 Lys Lys Ile Gly Ile Ser Trp Asn
Lys Lys Tyr Arg Lys Gln His Gly 370 375 380 385 cca ttg cgg aag ttc
tta cag ctc cat tct cag ata ttt cta ctc agt 1315 Pro Leu Arg Lys
Phe Leu Gln Leu His Ser Gln Ile Phe Leu Leu Ser 390 395 400 tca gat
gaa agt aca gtt cgt ctc ttg aag aat agt tct ctc cag gct 1363 Ser
Asp Glu Ser Thr Val Arg Leu Leu Lys Asn Ser Ser Leu Gln Ala 405 410
415 gag tct gat ttc caa agg aat gac cag caa att ttc aag atg ctt cct
1411 Glu Ser Asp Phe Gln Arg Asn Asp Gln Gln Ile Phe Lys Met Leu
Pro 420 425 430 cca gaa tcc cca ggt tta aac aat agc atc tcc tgt ccc
cac tgg ttt 1459 Pro Glu Ser Pro Gly Leu Asn Asn Ser Ile Ser Cys
Pro His Trp Phe 435 440 445 gat ata aat gat tct aaa gtc cag cca atc
agg gaa aag gat att gaa 1507 Asp Ile Asn Asp Ser Lys Val Gln Pro
Ile Arg Glu Lys Asp Ile Glu 450 455 460 465 cag caa ttt cag ggt aaa
gaa agt gcc tac atg ttg ttt tat cgg aaa 1555 Gln Gln Phe Gln Gly
Lys Glu Ser Ala Tyr Met Leu Phe Tyr Arg Lys 470 475 480 tcc cag ttg
cag aga ccc cct gaa gct cga gct aat cca aga tat ggg 1603 Ser Gln
Leu Gln Arg Pro Pro Glu Ala Arg Ala Asn Pro Arg Tyr Gly 485 490 495
gtt cca tgt cat tta ctg aat gaa atg gat gca gct aac att gaa ctg
1651 Val Pro Cys His Leu Leu Asn Glu Met Asp Ala Ala Asn Ile Glu
Leu 500 505 510 caa acc aaa agg gca gaa tgt gat tct gca aac aat act
ttt gaa ttg 1699 Gln Thr Lys Arg Ala Glu Cys Asp Ser Ala Asn Asn
Thr Phe Glu Leu 515 520 525 cat ctt cac ctg ggc cct cag tat cat ttc
ttc aat ggg gct ctg cac 1747 His Leu His Leu Gly Pro Gln Tyr His
Phe Phe Asn Gly Ala Leu His 530 535 540 545 cca gta gtc tct caa aca
gaa agc gtg tgg gat ttg acc ttt gat aaa 1795 Pro Val Val Ser Gln
Thr Glu Ser Val Trp Asp Leu Thr Phe Asp Lys 550 555 560 aga aaa act
tta gga gat ctc cgg cag tca ata ttt cag ctg tta gaa 1843 Arg Lys
Thr Leu Gly Asp Leu Arg Gln Ser Ile Phe Gln Leu Leu Glu 565 570 575
ttt tgg gaa gga gac atg gtt ctt agt gtt gca aag ctt gta cca gca
1891 Phe Trp Glu Gly Asp Met Val Leu Ser Val Ala Lys Leu Val Pro
Ala 580 585 590 gga ctt cac att tac cag tca ctt ggc ggg gat gaa ctg
aca ctg tgt 1939 Gly Leu His Ile Tyr Gln Ser Leu Gly Gly Asp Glu
Leu Thr Leu Cys 595 600 605 gaa act gaa att gct gat ggg gaa gac atc
ttt gtg tgg aat ggg gtg 1987 Glu Thr Glu Ile Ala Asp Gly Glu Asp
Ile Phe Val Trp Asn Gly Val 610 615 620 625 gag gtt ggt gga gtc cac
att caa act ggt att gac tgc gaa cct cta 2035 Glu Val Gly Gly Val
His Ile Gln Thr Gly Ile Asp Cys Glu Pro Leu 630 635 640 ctt tta aat
gtt ctt cat cta gac aca agc agt gat gga gaa aag tgt 2083 Leu Leu
Asn Val Leu His Leu Asp Thr Ser Ser Asp Gly Glu Lys Cys 645 650 655
tgt cag gtg ata gaa tct cca cat gtc ttt cca gct aat gca gaa gtg
2131 Cys Gln Val Ile Glu Ser Pro His Val Phe Pro Ala Asn Ala Glu
Val 660 665 670 ggc act gtc ctc aca gcc tta gca atc cca gca ggt gtc
atc ttc atc 2179 Gly Thr Val Leu Thr Ala Leu Ala Ile Pro Ala Gly
Val Ile Phe Ile 675 680 685 aac agt gct gga tgt cca ggt ggg gag ggt
tgg acg gcc atc ccc aag 2227 Asn Ser Ala Gly Cys Pro Gly Gly Glu
Gly Trp Thr Ala Ile Pro Lys 690 695 700 705 gaa gac atg agg aag acg
ttc agg gag caa ggg ctc aga aat gga agc 2275 Glu Asp Met Arg Lys
Thr Phe Arg Glu Gln Gly Leu Arg Asn Gly Ser 710 715 720 tca att tta
att cag gat tct cat gat gat aac agc ttg ttg acc aag 2323 Ser Ile
Leu Ile Gln Asp Ser His Asp Asp Asn Ser Leu Leu Thr Lys 725 730 735
gaa gag aaa tgg gtc act agt atg aat gag att gac tgg ctc cac gtt
2371 Glu Glu Lys Trp Val Thr Ser Met Asn Glu Ile Asp Trp Leu His
Val 740 745 750 aaa aat tta tgc cag tta gaa tct gaa gag aag caa gtt
aaa ata tca 2419 Lys Asn Leu Cys Gln Leu Glu Ser Glu Glu Lys Gln
Val Lys Ile Ser 755 760 765 gca act gtt aac aca atg gtg ttt gat att
cga att aaa gcc ata aag 2467 Ala Thr Val Asn Thr Met Val Phe Asp
Ile Arg Ile Lys Ala Ile Lys 770 775 780 785 gaa tta aaa tta atg aag
gaa cta gct gac aac agc tgt ttg aga cct 2515 Glu Leu Lys Leu Met
Lys Glu Leu Ala Asp Asn Ser Cys Leu Arg Pro 790 795 800 att gat aga
aat ggg aag ctt ctt tgt cca gtg ccg gac agc tat act 2563 Ile Asp
Arg Asn Gly Lys Leu Leu Cys Pro Val Pro Asp Ser Tyr Thr 805 810 815
ttg aag gaa gca gaa ttg aag atg gga agt tca ttg gga ctg tgt ctt
2611 Leu Lys Glu Ala Glu Leu Lys Met Gly Ser Ser Leu Gly Leu Cys
Leu 820 825 830 gga aaa gca cca agt tcg tct cag ttg ttc ctg ttt ttt
gca atg ggg 2659 Gly Lys Ala Pro Ser Ser Ser Gln Leu Phe Leu Phe
Phe Ala Met Gly 835 840 845 agt gac gtt caa cct ggg aca gaa atg gaa
atc gta gta gaa gaa aca 2707 Ser Asp Val Gln Pro Gly Thr Glu Met
Glu Ile Val Val Glu Glu Thr 850 855 860 865 ata tct gtg aga gat tgt
tta aag tta atg ctg aag aaa tct ggc cta 2755 Ile Ser Val Arg Asp
Cys Leu Lys Leu Met Leu Lys Lys Ser Gly Leu 870 875 880 caa gga gat
gcc tgg cat tta cga aaa atg gat tgg tgc tat gaa gct 2803 Gln Gly
Asp Ala Trp His Leu Arg Lys Met Asp Trp Cys Tyr Glu Ala 885 890 895
gga gag cct tta tgt gaa gaa gat gca aca ctg aaa gaa ctt ctg ata
2851 Gly Glu Pro Leu Cys Glu Glu Asp Ala Thr Leu Lys Glu Leu Leu
Ile 900 905 910 tgt tct gga gat act ttg ctt tta att gaa gga caa ctt
cct cct ctg 2899 Cys Ser Gly Asp Thr Leu Leu Leu Ile Glu Gly Gln
Leu Pro Pro Leu 915 920 925 ggt ttc ctg aag gtg ccc atc tgg tgg tac
cag ctt cag ggt ccc tca 2947 Gly Phe Leu Lys Val Pro Ile Trp Trp
Tyr Gln Leu Gln Gly Pro Ser 930 935 940 945 gga cac tgg gag agt cat
cag gac cag acc aac tgt act tcg tct tgg 2995 Gly His Trp Glu Ser
His Gln Asp Gln Thr Asn Cys Thr Ser Ser Trp 950 955 960 ggc aga gtt
tgg aga gcc act tcc agc caa ggt gct tct ggg aac gag 3043 Gly Arg
Val Trp Arg Ala Thr Ser Ser Gln Gly Ala Ser Gly Asn Glu 965 970 975
cct gcg caa gtt tct ctc ctc tac ttg gga gac ata gag atc tca gaa
3091 Pro Ala Gln Val Ser Leu Leu Tyr Leu Gly Asp Ile Glu Ile Ser
Glu 980 985 990 gat gcc acg ctg gcg gag ctg aag tct cag gcc atg acc
ttg cct cct 3139 Asp Ala Thr Leu Ala Glu Leu Lys Ser Gln Ala Met
Thr Leu Pro Pro 995 1000 1005 ttc ctg gag ttc ggt gtc ccg tcc cca
gcc cac ctc aga gcc tgg acg 3187 Phe Leu Glu Phe Gly Val Pro Ser
Pro Ala His Leu Arg Ala Trp Thr 1010 1015 1020 1025 gtg gag agg aag
cgc cca ggc agg ctt tta cga act gac cgg cag cca 3235 Val Glu Arg
Lys Arg Pro Gly Arg Leu Leu Arg Thr Asp Arg Gln Pro 1030 1035 1040
ctc agc ccc cag gac gtg ctg ctg agg aca cag gtg cgc atc cct ggt
3283 Leu Ser Pro Gln Asp Val Leu Leu Arg Thr Gln Val Arg Ile Pro
Gly 1045 1050 1055 gag agg acc tat gcc cct gcc ctg gac ctg gtg tgg
aac gcg gcc cag 3331 Glu Arg Thr Tyr Ala Pro Ala Leu Asp Leu Val
Trp Asn Ala Ala Gln 1060 1065 1070 ggt ggg act gcc ggc tcc ctg agg
cag aga gtt gcc gat ttc tat cgt 3379 Gly Gly Thr Ala Gly Ser Leu
Arg Gln Arg Val Ala Asp Phe Tyr Arg 1075 1080 1085 ctt ccc gtg gag
aag att gaa att gcc aaa tac ttt ccc gaa aag ttc 3427 Leu Pro Val
Glu Lys Ile Glu Ile Ala Lys Tyr Phe Pro Glu Lys Phe 1090 1095 1100
1105 gag tgg ctt ccg ata tct agc tgg aac caa caa ata acc aag agg
aaa 3475 Glu Trp Leu Pro Ile Ser Ser Trp Asn Gln Gln Ile Thr Lys
Arg Lys 1110 1115 1120 aag aaa aaa aaa caa gat tat ttg caa ggg gca
ccg tat tac ttg aaa 3523 Lys Lys Lys Lys Gln Asp Tyr Leu Gln Gly
Ala Pro Tyr Tyr Leu Lys 1125 1130 1135 gac gga gat act att ggt gtt
aag ccc ttt aaa gtc ccc ctg ttt ggc 3571 Asp Gly Asp Thr Ile Gly
Val Lys Pro Phe Lys Val Pro Leu Phe Gly 1140 1145 1150 cac tcg cag
tgg aag ctt ctg gag ctg aag gga aag aat ctc ctg att 3619 His Ser
Gln Trp Lys Leu Leu Glu Leu Lys Gly Lys Asn Leu Leu Ile 1155 1160
1165 gac gac gat gat gat ttc agt aca atc aga gat gac act gga aaa
gaa 3667 Asp Asp Asp Asp Asp Phe Ser Thr Ile Arg Asp Asp Thr Gly
Lys Glu 1170 1175 1180 1185 aag cag aaa caa cgg gcc ctg ggg aga agg
aaa agt atc ttg gct gcc 3715 Lys Gln Lys Gln Arg Ala Leu Gly Arg
Arg Lys Ser Ile Leu Ala Ala 1190 1195 1200 cgt gtg cca ggg act gca
tgc cct ctg cca ctg ggg gcg tct ggg gaa 3763 Arg Val Pro Gly Thr
Ala Cys Pro Leu Pro Leu Gly Ala Ser Gly Glu 1205 1210 1215 gag agg
tcc cag gtg gag gtg gca ggg cag ctc tgc agc cct cag gtc 3811 Glu
Arg Ser Gln Val Glu Val Ala Gly Gln Leu Cys Ser Pro Gln Val 1220
1225 1230 ctg gtg ccc tca gag acc tgg gat atg gga gag ggg ctg cag
cgc tgg 3859 Leu Val Pro Ser Glu Thr Trp Asp Met Gly Glu Gly Leu
Gln Arg Trp 1235 1240 1245 ctg ctt cgg ggg ttt tcc tgt tta aag aaa
atg cac aac gtg tgc gtg 3907 Leu Leu Arg Gly Phe Ser Cys Leu Lys
Lys Met His Asn Val Cys Val 1250 1255 1260 1265 aac cgc agg tat gga
ggc agc ggc atg ccg ttg ctc cgc tgt ggg aga 3955 Asn Arg Arg Tyr
Gly Gly Ser Gly Met Pro Leu Leu Arg Cys Gly Arg 1270 1275 1280 cgg
ttg act ttc cca gac agc agc tta gcg gaa agc agc atc ccg aag 4003
Arg Leu Thr Phe Pro Asp Ser Ser Leu Ala Glu Ser Ser Ile Pro Lys
1285 1290 1295 acg gtg tcg cat gtc ctg agc ctg ctt gtc acc tgg gcc
tca cgg ttt 4051 Thr Val Ser His Val Leu Ser Leu Leu Val Thr Trp
Ala Ser Arg Phe 1300 1305 1310 ctc gtc agc ccc gtc act gat ggg aag
cag cac tga 4087 Leu Val Ser Pro Val Thr Asp Gly Lys Gln His * 1315
1320 1325 6 4153 DNA Homo sapiens CDS (113)..(4153) 6 aattcccggg
tcgacgattt cgtggcggcg cccagttggg gcgggttcgt tcgcttcgcg 60
ttttggccag ggcgggggtc tgggctttag gcaggtagta tttagtttca ca atg 115
Met 1 ttt ggg gac ctg ttt gaa gag gag tat tcc act gtg tct aat aat
cag 163 Phe Gly Asp Leu Phe Glu Glu Glu Tyr Ser Thr Val Ser Asn Asn
Gln 5 10 15 tat gga aaa ggg aag aaa tta aag act aaa gct ttg gag cca
cct gct 211 Tyr Gly Lys Gly Lys Lys Leu Lys Thr Lys Ala Leu Glu Pro
Pro Ala 20 25 30 cct aga gaa ttc acc aat tta agc gga atc aga aat
cag ggt gga acc 259 Pro Arg Glu Phe Thr Asn Leu Ser Gly Ile Arg Asn
Gln Gly Gly Thr 35 40 45 tgt tac ctc aat tcc ctt ctt cag act ctt
cat ttc aca cct gaa ttc 307 Cys Tyr Leu Asn Ser Leu Leu Gln Thr Leu
His Phe Thr Pro Glu Phe 50 55 60 65 aga gaa gct cta ttt tct ctt ggc
cca gaa gag ctt ggt ttg ttt gaa 355 Arg Glu Ala Leu Phe Ser Leu Gly
Pro Glu Glu Leu Gly Leu Phe Glu 70 75 80 gat aag gat aaa ccc gat
gca aag gtt cga atc atc cct tta cag tta 403 Asp Lys Asp Lys Pro Asp
Ala Lys Val Arg Ile Ile Pro Leu Gln Leu 85 90 95 cag cgc ttg ttt
gct cag ctt ctg ctc tta gac cag gaa gct gca tcc 451 Gln Arg Leu Phe
Ala Gln Leu Leu Leu Leu Asp Gln Glu Ala Ala Ser 100 105 110 aca gca
gac ctc act gac agc ttt ggg tgg acc agt aat gag gaa atg 499 Thr Ala
Asp Leu Thr Asp Ser Phe Gly Trp Thr Ser Asn Glu Glu Met 115 120 125
agg caa cat gat gtg cag gaa ctg aat cga atc ctc ttc agc gct ttg 547
Arg Gln His Asp Val Gln Glu Leu Asn Arg Ile Leu Phe Ser Ala Leu 130
135 140 145 gaa act tct tta gtt ggg acc tcc ggt cat gac ctc atc tat
cgt ctg 595 Glu Thr Ser Leu Val Gly Thr Ser Gly His Asp Leu Ile Tyr
Arg Leu 150 155 160 tac cat gga acc att gtt aac cag att gtt tgt aaa
gaa tgt aag aac 643 Tyr His Gly Thr Ile Val Asn Gln Ile Val Cys Lys
Glu Cys Lys Asn 165 170 175 gtt agc gag agg cag gaa gac ttc tta gat
cta aca gta gca gtc aaa 691 Val Ser Glu Arg Gln Glu Asp Phe Leu Asp
Leu Thr Val Ala Val Lys 180 185 190 aat gta tcc ggt ttg gaa gat gct
ctc tgg aac atg tat gta gaa gag 739 Asn Val Ser Gly Leu Glu Asp Ala
Leu Trp Asn Met Tyr Val Glu Glu 195 200 205 gaa gtt ttt gat tgt gac
aac ttg tac cac tgt gga act tgt gac agg 787 Glu Val Phe Asp Cys Asp
Asn Leu Tyr His Cys Gly Thr Cys Asp Arg 210 215 220 225 ctg gtt aaa
gca gca aag tcg gcc aaa tta cgt aag ctg cct cct ttt 835 Leu Val Lys
Ala Ala Lys Ser Ala Lys Leu Arg Lys Leu Pro Pro Phe 230 235 240 ctt
act gtt tca tta cta aga ttt aat ttt gat ttt gtg aaa tgc gaa 883 Leu
Thr Val Ser Leu Leu Arg Phe Asn Phe Asp Phe Val Lys Cys Glu 245 250
255 cgc tac aag gaa act agc tgt tat aca ttc cct ctc cgg att aat ctc
931 Arg Tyr Lys Glu Thr Ser Cys Tyr Thr Phe Pro Leu Arg Ile Asn Leu
260 265 270 aag ccc ttt tgt gaa cag agt gaa ttg gat gac tta gaa tat
ata tat 979 Lys Pro Phe Cys Glu Gln Ser Glu Leu Asp Asp Leu Glu Tyr
Ile Tyr 275 280 285 gac ctc ttc tca gtt att ata cac aaa ggt ggc tgc
tac gga ggc cat 1027 Asp Leu Phe Ser Val Ile Ile His Lys Gly Gly
Cys Tyr Gly Gly His 290 295 300 305 tac cat gta tat att aaa gat gtt
gat cat ttg gga aac tgg cag ttt 1075 Tyr His Val Tyr Ile Lys Asp
Val Asp His Leu Gly Asn Trp Gln Phe 310 315 320 caa gag gaa aaa agt
aaa cca gat gtg aat ctg aaa gat ctc cag agt 1123 Gln Glu Glu Lys
Ser Lys Pro Asp Val Asn Leu Lys Asp Leu Gln Ser 325 330 335 gaa gaa
gag att gat cat cca ctg atg att cta aaa gca atc tta tta 1171 Glu
Glu Glu Ile Asp His Pro Leu Met Ile Leu Lys Ala Ile Leu Leu 340 345
350 gag gag aat aat cta att cct gtt gat cag ctg ggc cag aaa ctt ttg
1219 Glu Glu Asn Asn Leu Ile Pro Val Asp Gln Leu Gly Gln Lys Leu
Leu 355 360 365 aaa aag ata gga ata tct tgg aac aag aag tac aga aaa
cag cat gga 1267 Lys Lys Ile Gly Ile Ser Trp Asn Lys Lys Tyr Arg
Lys Gln His Gly 370 375 380 385 cca ttg cgg aag ttc tta cag ctc cat
tct cag ata ttt cta ctc agt 1315 Pro Leu Arg Lys Phe Leu Gln Leu
His Ser Gln Ile Phe Leu Leu Ser 390 395 400 tca gat gaa agt aca gtt
cgt ctc ttg aag aat agt tct ctc cag gct 1363 Ser Asp Glu Ser Thr
Val Arg Leu Leu Lys Asn Ser Ser Leu Gln Ala 405 410 415 gag tct gat
ttc caa agg aat gac cag caa att ttc aag atg ctt cct 1411 Glu Ser
Asp Phe Gln Arg Asn Asp Gln Gln Ile Phe Lys Met Leu Pro 420 425 430
cca gaa tcc cca ggt tta aac aat agc atc tcc tgt ccc cac tgg ttt
1459 Pro Glu Ser Pro Gly Leu Asn Asn Ser Ile Ser Cys Pro His Trp
Phe 435 440 445 gat ata aat gat tct aaa gtc cag cca atc agg gaa aag
gat att gaa 1507 Asp Ile Asn Asp Ser Lys Val Gln Pro Ile Arg Glu
Lys Asp Ile Glu 450 455 460 465 cag caa ttt cag ggt aaa gaa agt gcc
tac atg ttg ttt tat cgg aaa 1555 Gln Gln Phe Gln Gly Lys Glu Ser
Ala Tyr Met Leu Phe Tyr Arg Lys 470 475 480 tcc cag ttg cag aga ccc
cct gaa gct cga gct aat cca aga tat ggg 1603 Ser Gln Leu Gln
Arg Pro Pro Glu Ala Arg Ala Asn Pro Arg Tyr Gly 485 490 495 gtt cca
tgt cat tta ctg aat gaa atg gat gca gct aac att gaa ctg 1651 Val
Pro Cys His Leu Leu Asn Glu Met Asp Ala Ala Asn Ile Glu Leu 500 505
510 caa acc aaa agg gca gaa tgt gat tct gca aac aat act ttt gaa ttg
1699 Gln Thr Lys Arg Ala Glu Cys Asp Ser Ala Asn Asn Thr Phe Glu
Leu 515 520 525 cat ctt cac ctg ggc cct cag tat cat ttc ttc aat ggg
gct ctg cac 1747 His Leu His Leu Gly Pro Gln Tyr His Phe Phe Asn
Gly Ala Leu His 530 535 540 545 cca gta gtc tct caa aca gaa agc gtg
tgg gat ttg acc ttt gat aaa 1795 Pro Val Val Ser Gln Thr Glu Ser
Val Trp Asp Leu Thr Phe Asp Lys 550 555 560 aga aaa act tta gga gat
ctc cgg cag tca ata ttt cag ctg tta gaa 1843 Arg Lys Thr Leu Gly
Asp Leu Arg Gln Ser Ile Phe Gln Leu Leu Glu 565 570 575 ttt tgg gaa
gga gac atg gtt ctt agt gtt gca aag ctt gta cca gca 1891 Phe Trp
Glu Gly Asp Met Val Leu Ser Val Ala Lys Leu Val Pro Ala 580 585 590
gga ctt cac att tac cag tca ctt ggc ggg gat gaa ctg aca ctg tgt
1939 Gly Leu His Ile Tyr Gln Ser Leu Gly Gly Asp Glu Leu Thr Leu
Cys 595 600 605 gaa act gaa att gct gat ggg gaa gac atc ttt gtg tgg
aat ggg gtg 1987 Glu Thr Glu Ile Ala Asp Gly Glu Asp Ile Phe Val
Trp Asn Gly Val 610 615 620 625 gag gtt ggt gga gtc cac att caa act
ggt att gac tgc gaa cct cta 2035 Glu Val Gly Gly Val His Ile Gln
Thr Gly Ile Asp Cys Glu Pro Leu 630 635 640 ctt tta aat gtt ctt cat
cta gac aca agc agt gat gga gaa aag tgt 2083 Leu Leu Asn Val Leu
His Leu Asp Thr Ser Ser Asp Gly Glu Lys Cys 645 650 655 tgt cag gtg
ata gaa tct cca cat gtc ttt cca gct aat gca gaa gtg 2131 Cys Gln
Val Ile Glu Ser Pro His Val Phe Pro Ala Asn Ala Glu Val 660 665 670
ggc act gtc ctc aca gcc tta gca atc cca gca ggt gtc atc ttc atc
2179 Gly Thr Val Leu Thr Ala Leu Ala Ile Pro Ala Gly Val Ile Phe
Ile 675 680 685 aac agt gct gga tgt cca ggt ggg gag ggt tgg acg gcc
atc ccc aag 2227 Asn Ser Ala Gly Cys Pro Gly Gly Glu Gly Trp Thr
Ala Ile Pro Lys 690 695 700 705 gaa gac atg agg aag acg ttc agg gag
caa ggg ctc aga aat gga agc 2275 Glu Asp Met Arg Lys Thr Phe Arg
Glu Gln Gly Leu Arg Asn Gly Ser 710 715 720 tca att tta att cag gat
tct cat gat gat aac agc ttg ttg acc aag 2323 Ser Ile Leu Ile Gln
Asp Ser His Asp Asp Asn Ser Leu Leu Thr Lys 725 730 735 gaa gag aaa
tgg gtc act agt atg aat gag att gac tgg ctc cac gtt 2371 Glu Glu
Lys Trp Val Thr Ser Met Asn Glu Ile Asp Trp Leu His Val 740 745 750
aaa aat tta tgc cag tta gaa tct gaa gag aag caa gtt aaa ata tca
2419 Lys Asn Leu Cys Gln Leu Glu Ser Glu Glu Lys Gln Val Lys Ile
Ser 755 760 765 gca act gtt aac aca atg gtg ttt gat att cga att aaa
gcc ata aag 2467 Ala Thr Val Asn Thr Met Val Phe Asp Ile Arg Ile
Lys Ala Ile Lys 770 775 780 785 gaa tta aaa tta atg aag gaa cta gct
gac aac agc tgt ttg aga cct 2515 Glu Leu Lys Leu Met Lys Glu Leu
Ala Asp Asn Ser Cys Leu Arg Pro 790 795 800 att gat aga aat ggg aag
ctt ctt tgt cca gtg ccg gac agc tat act 2563 Ile Asp Arg Asn Gly
Lys Leu Leu Cys Pro Val Pro Asp Ser Tyr Thr 805 810 815 ttg aag gaa
gca gaa ttg aag atg gga agt tca ttg gga ctg tgt ctt 2611 Leu Lys
Glu Ala Glu Leu Lys Met Gly Ser Ser Leu Gly Leu Cys Leu 820 825 830
gga aaa gca cca agt tcg tct cag ttg ttc ctg ttt ttt gca atg ggg
2659 Gly Lys Ala Pro Ser Ser Ser Gln Leu Phe Leu Phe Phe Ala Met
Gly 835 840 845 agt gac gtt caa cct ggg aca gaa atg gaa atc gta gta
gaa gaa aca 2707 Ser Asp Val Gln Pro Gly Thr Glu Met Glu Ile Val
Val Glu Glu Thr 850 855 860 865 ata tct gtg aga gat tgt tta aag tta
atg ctg aag aaa tct ggc cta 2755 Ile Ser Val Arg Asp Cys Leu Lys
Leu Met Leu Lys Lys Ser Gly Leu 870 875 880 caa gga gat gcc tgg cat
tta cga aaa atg gat tgg tgc tat gaa gct 2803 Gln Gly Asp Ala Trp
His Leu Arg Lys Met Asp Trp Cys Tyr Glu Ala 885 890 895 gga gag cct
tta tgt gaa gaa gat gca aca ctg aaa gaa ctt ctg ata 2851 Gly Glu
Pro Leu Cys Glu Glu Asp Ala Thr Leu Lys Glu Leu Leu Ile 900 905 910
tgt tct gga gat act ttg ctt tta att gaa gga caa ctt cct cct ctg
2899 Cys Ser Gly Asp Thr Leu Leu Leu Ile Glu Gly Gln Leu Pro Pro
Leu 915 920 925 ggt ttc ctg caa gtg ccc atc tgg tgg tac cag ctt cag
ggt ccc tca 2947 Gly Phe Leu Gln Val Pro Ile Trp Trp Tyr Gln Leu
Gln Gly Pro Ser 930 935 940 945 gga cac tgg gag agt cat cag gac cag
acc aac tgt act tcg tct tgg 2995 Gly His Trp Glu Ser His Gln Asp
Gln Thr Asn Cys Thr Ser Ser Trp 950 955 960 ggc aga gtt tgg aga gcc
act tcc agc caa ggt gct tct ggg aac gag 3043 Gly Arg Val Trp Arg
Ala Thr Ser Ser Gln Gly Ala Ser Gly Asn Glu 965 970 975 cct gcg caa
gtt tct ctc ctc tac ttg gga gac ata gag atc tca gaa 3091 Pro Ala
Gln Val Ser Leu Leu Tyr Leu Gly Asp Ile Glu Ile Ser Glu 980 985 990
gat gcc acg ctg gcg gag ctg aag tct cag gcc atg acc ttg cct cct
3139 Asp Ala Thr Leu Ala Glu Leu Lys Ser Gln Ala Met Thr Leu Pro
Pro 995 1000 1005 ttc ctg gag ttc ggt gtc ccg tcc cca gcc cac ctc
aga gcc tgg acg 3187 Phe Leu Glu Phe Gly Val Pro Ser Pro Ala His
Leu Arg Ala Trp Thr 1010 1015 1020 1025 gtg gag agg aag cgc cca ggc
agg ctt tta cga act gac cgg cag cca 3235 Val Glu Arg Lys Arg Pro
Gly Arg Leu Leu Arg Thr Asp Arg Gln Pro 1030 1035 1040 ctc agg gaa
tat aaa cta gga cgg aga att gag atc tgc tta gag ccc 3283 Leu Arg
Glu Tyr Lys Leu Gly Arg Arg Ile Glu Ile Cys Leu Glu Pro 1045 1050
1055 ctt cag aaa ggc gaa aac ttg ggc ccc cag gac gtg ctg ctg agg
aca 3331 Leu Gln Lys Gly Glu Asn Leu Gly Pro Gln Asp Val Leu Leu
Arg Thr 1060 1065 1070 cag gtg cgc atc cct ggt gag agg acc tat gcc
cct gcc ctg gac ctg 3379 Gln Val Arg Ile Pro Gly Glu Arg Thr Tyr
Ala Pro Ala Leu Asp Leu 1075 1080 1085 gtg tgg aac gcg gcc cag ggt
ggg act gcc ggc tcc ctg agg cag aga 3427 Val Trp Asn Ala Ala Gln
Gly Gly Thr Ala Gly Ser Leu Arg Gln Arg 1090 1095 1100 1105 gtt gcc
gat ttc tat cgt ctt ccc gtg gag aag att gaa att gcc aaa 3475 Val
Ala Asp Phe Tyr Arg Leu Pro Val Glu Lys Ile Glu Ile Ala Lys 1110
1115 1120 tac ttt ccc gaa aag ttc gag tgg ctt ccg ata tct agc tgg
aac caa 3523 Tyr Phe Pro Glu Lys Phe Glu Trp Leu Pro Ile Ser Ser
Trp Asn Gln 1125 1130 1135 caa ata acc aag agg aaa aag aaa aaa aaa
caa gat tat ttg caa ggg 3571 Gln Ile Thr Lys Arg Lys Lys Lys Lys
Lys Gln Asp Tyr Leu Gln Gly 1140 1145 1150 gca ccg tat tac ttg aaa
gac gga gat act att ggt gtt aag ccc ttt 3619 Ala Pro Tyr Tyr Leu
Lys Asp Gly Asp Thr Ile Gly Val Lys Pro Phe 1155 1160 1165 aaa gtc
ccc ctg ttt ggc cac tcg cag tgg aag ctt ctg gag ctg aag 3667 Lys
Val Pro Leu Phe Gly His Ser Gln Trp Lys Leu Leu Glu Leu Lys 1170
1175 1180 1185 gga aag aat ctc ctg att gac gac gat gat gat ttc agt
aca atc aga 3715 Gly Lys Asn Leu Leu Ile Asp Asp Asp Asp Asp Phe
Ser Thr Ile Arg 1190 1195 1200 gat gac act gga aaa gaa aag cag aaa
caa cgg gcc ctg ggg aga agg 3763 Asp Asp Thr Gly Lys Glu Lys Gln
Lys Gln Arg Ala Leu Gly Arg Arg 1205 1210 1215 aaa agt atc ttg gct
gcc cgt gtg cca ggg act gca tgc cct ctg cca 3811 Lys Ser Ile Leu
Ala Ala Arg Val Pro Gly Thr Ala Cys Pro Leu Pro 1220 1225 1230 ctg
ggg gcg tct ggg gaa gag agg tcc cag gtg gag gtg gca ggg cag 3859
Leu Gly Ala Ser Gly Glu Glu Arg Ser Gln Val Glu Val Ala Gly Gln
1235 1240 1245 ctc tgc agc cct cag gtc ctg gtg ccc tca gag acc tgg
gat atg gga 3907 Leu Cys Ser Pro Gln Val Leu Val Pro Ser Glu Thr
Trp Asp Met Gly 1250 1255 1260 1265 gag ggg ctg cag cgc tgg ctg ctt
cgg ggg ttt tcc tgt tta aag aaa 3955 Glu Gly Leu Gln Arg Trp Leu
Leu Arg Gly Phe Ser Cys Leu Lys Lys 1270 1275 1280 atg cac aac gtg
tgc gtg aac cgc agg tat gga ggc agc ggc atg ccg 4003 Met His Asn
Val Cys Val Asn Arg Arg Tyr Gly Gly Ser Gly Met Pro 1285 1290 1295
ttg ctc cgc tgt ggg aga cgg ttg act ttc cca gac agc agc tta gcg
4051 Leu Leu Arg Cys Gly Arg Arg Leu Thr Phe Pro Asp Ser Ser Leu
Ala 1300 1305 1310 gaa agc agc atc ccg aag acg gtg tcg cat gtc ctg
agc ctg ctt gtc 4099 Glu Ser Ser Ile Pro Lys Thr Val Ser His Val
Leu Ser Leu Leu Val 1315 1320 1325 acc tgg gcc tca cgg ttt ctc gtc
agc ccc gtc act gat ggg aag cag 4147 Thr Trp Ala Ser Arg Phe Leu
Val Ser Pro Val Thr Asp Gly Lys Gln 1330 1335 1340 1345 cac tga
4153 His * 7 776 DNA Homo sapiens CDS (147)..(446) 7 cagcaggttg
gctcacagca gaaggcaaag gccatcatca gctcccttta taagggaacg 60
gtcatgcact gggtgtgctg agagtgtcct gcctggtcct ctgtgcctgg tggggtggag
120 gtgccaggtg tgtccagagg agccca atg ggc agt gag gca gcc atg ggg
ctg 173 Met Gly Ser Glu Ala Ala Met Gly Leu 1 5 gat gca ctg gtg ccc
ctg gca gtg aca gtg gcc atc ttc ctg ctc ctg 221 Asp Ala Leu Val Pro
Leu Ala Val Thr Val Ala Ile Phe Leu Leu Leu 10 15 20 25 gtg gac ctg
atg cag cag cac caa cgc tgg act gca cgc tac ccg cca 269 Val Asp Leu
Met Gln Gln His Gln Arg Trp Thr Ala Arg Tyr Pro Pro 30 35 40 ggc
ccc ctg cca ctg ccc ggg ctg ggc aac ttg ctg cat gtg gac ttc 317 Gly
Pro Leu Pro Leu Pro Gly Leu Gly Asn Leu Leu His Val Asp Phe 45 50
55 cag aac ata tac acc ttc aac cag gtg agg gag gag gtc ctg agg atc
365 Gln Asn Ile Tyr Thr Phe Asn Gln Val Arg Glu Glu Val Leu Arg Ile
60 65 70 ccc cac cac cag caa aca tgg gtg gtg ggt gga gcc aca gtc
tgg aca 413 Pro His His Gln Gln Thr Trp Val Val Gly Gly Ala Thr Val
Trp Thr 75 80 85 aga agc cag gct gag aag ggg aag cag att tga
gggacttc ctgggggagg 464 Arg Ser Gln Ala Glu Lys Gly Lys Gln Ile *
90 95 100 gcatttatgc atggcatgaa agatggcatt ttccaaaggc caaggaagag
taaggcaagg 524 gcctggaggt ggaactgtac ttggcagtgg gcgtgcacgc
ccattggcca tcatatgtta 584 aggaccacaa ggtccctctt gtgacaccag
aatgaaaggg cctggcaaat ggaacaacca 644 gccaggggcc ctggttagcc
cttttatatc ccacaattat accctggggc tctcttgtcc 704 ctacaccccc
gtttccttct ctttgtttct ctacggaatc tcctgtcttg tcgctgtttt 764
cctttttttg gc 776 8 1621 DNA Homo sapiens CDS (38)..(1414) 8
gatatgtcca agaaaccacc ccctgataac gtctttg atg aca tgc caa gct tgg 55
Met Thr Cys Gln Ala Trp 1 5 cac gag gca gag gag gga cac gga att act
cac agc tgt gct gtg tgc 103 His Glu Ala Glu Glu Gly His Gly Ile Thr
His Ser Cys Ala Val Cys 10 15 20 att ctc tgt ggg cct agc agg gaa
ggg gac agc cct gtg gca atg ggc 151 Ile Leu Cys Gly Pro Ser Arg Glu
Gly Asp Ser Pro Val Ala Met Gly 25 30 35 atg aca cgg atg ctc ctg
gaa tgc agt ctc agt gac aag ttg tgt gtc 199 Met Thr Arg Met Leu Leu
Glu Cys Ser Leu Ser Asp Lys Leu Cys Val 40 45 50 atc cag gag aag
cag tat gaa gtg att atc gtc cca act ttg ttg gtt 247 Ile Gln Glu Lys
Gln Tyr Glu Val Ile Ile Val Pro Thr Leu Leu Val 55 60 65 70 act atc
ttc ctc atc ctt ctt ggg gtc atc ctg tgg ctt ttt atc aga 295 Thr Ile
Phe Leu Ile Leu Leu Gly Val Ile Leu Trp Leu Phe Ile Arg 75 80 85
gaa caa aga act caa cag cag cgt tct gga cct caa ggc att gcc cct 343
Glu Gln Arg Thr Gln Gln Gln Arg Ser Gly Pro Gln Gly Ile Ala Pro 90
95 100 gtt cct cca cct agg gac cta agc tgg gaa gca gga cat gga gga
aat 391 Val Pro Pro Pro Arg Asp Leu Ser Trp Glu Ala Gly His Gly Gly
Asn 105 110 115 gtg gct ttg cca ctt aag gag aca tcc gtg gaa aac ttt
ctg gga gct 439 Val Ala Leu Pro Leu Lys Glu Thr Ser Val Glu Asn Phe
Leu Gly Ala 120 125 130 acc aca cct gcc ctg gct aag ctg cag gtg ccg
cgg gag caa ctc tct 487 Thr Thr Pro Ala Leu Ala Lys Leu Gln Val Pro
Arg Glu Gln Leu Ser 135 140 145 150 gaa gtt ctg gag cag att tgc agt
ggt agc tgt ggg ccc atc ttt cga 535 Glu Val Leu Glu Gln Ile Cys Ser
Gly Ser Cys Gly Pro Ile Phe Arg 155 160 165 gcc aat atg aac act ggg
gac cct tct aag ccc aag agt gtt att ctc 583 Ala Asn Met Asn Thr Gly
Asp Pro Ser Lys Pro Lys Ser Val Ile Leu 170 175 180 aag gct tta aaa
gaa cca gct ggg ctc cat gag gta caa gat ttc tta 631 Lys Ala Leu Lys
Glu Pro Ala Gly Leu His Glu Val Gln Asp Phe Leu 185 190 195 ggg cga
atc aaa ttc cat caa tac ctg ggg aaa cac aaa aac ctg gtg 679 Gly Arg
Ile Lys Phe His Gln Tyr Leu Gly Lys His Lys Asn Leu Val 200 205 210
cag ctg gaa ggc tgc tgc act gaa aag ctg cca ctc tat atg gtg ttg 727
Gln Leu Glu Gly Cys Cys Thr Glu Lys Leu Pro Leu Tyr Met Val Leu 215
220 225 230 gag gat gtg gcc cag ggg gac ctg ctc ggc ttt ctc tgg acc
tgt cgg 775 Glu Asp Val Ala Gln Gly Asp Leu Leu Gly Phe Leu Trp Thr
Cys Arg 235 240 245 cgg gat gtg atg act atg gat ggt ctt ctc tat gat
ctc aca gaa aaa 823 Arg Asp Val Met Thr Met Asp Gly Leu Leu Tyr Asp
Leu Thr Glu Lys 250 255 260 caa gta tat cac atc gga aag cag gtc ctt
ttg gcg ctg gaa ttc ctg 871 Gln Val Tyr His Ile Gly Lys Gln Val Leu
Leu Ala Leu Glu Phe Leu 265 270 275 cag gag aag cat ttg ttc cat ggg
gat gtg gca gcc agg aat att ctg 919 Gln Glu Lys His Leu Phe His Gly
Asp Val Ala Ala Arg Asn Ile Leu 280 285 290 atg caa agt gat ctc act
gct aag ctc tgt gga tta ggc ctg gct tat 967 Met Gln Ser Asp Leu Thr
Ala Lys Leu Cys Gly Leu Gly Leu Ala Tyr 295 300 305 310 gaa gtt tac
acc cga ggg gcc atc tcc tct act caa acc ata cct ctc 1015 Glu Val
Tyr Thr Arg Gly Ala Ile Ser Ser Thr Gln Thr Ile Pro Leu 315 320 325
aag tgg ctt gcc cca gaa cgg ctt ctc ctg aga cct gct agc atc aga
1063 Lys Trp Leu Ala Pro Glu Arg Leu Leu Leu Arg Pro Ala Ser Ile
Arg 330 335 340 gca gat gtc tgg tct ttt ggg atc ctg ctc tat gag atg
gtg act cta 1111 Ala Asp Val Trp Ser Phe Gly Ile Leu Leu Tyr Glu
Met Val Thr Leu 345 350 355 gga gca cca ccg tat cct gaa gtc cct cct
acc agc atc cta gag cat 1159 Gly Ala Pro Pro Tyr Pro Glu Val Pro
Pro Thr Ser Ile Leu Glu His 360 365 370 ctc caa aga agg aaa atc atg
aag aga ccc agt agc tgc aca cat acc 1207 Leu Gln Arg Arg Lys Ile
Met Lys Arg Pro Ser Ser Cys Thr His Thr 375 380 385 390 atg tac agt
atc atg aag tcc tgc tgg cgc tgg cgt gag gct gac cgc 1255 Met Tyr
Ser Ile Met Lys Ser Cys Trp Arg Trp Arg Glu Ala Asp Arg 395 400 405
ccc tca cct aga gag ctg cgc ttg cgc cta gaa gct gcc att aaa act
1303 Pro Ser Pro Arg Glu Leu Arg Leu Arg Leu Glu Ala Ala Ile Lys
Thr 410 415 420 gca gat gac gag gct gtg tta caa gta cca gag ttg gtg
gta cct gaa 1351 Ala Asp Asp Glu Ala Val Leu Gln Val Pro Glu Leu
Val Val Pro Glu 425 430 435 ctg tat gca gct gtg gcc ggc atc aga gtg
gag agc ctc ttc tac aac 1399 Leu Tyr Ala Ala Val Ala Gly Ile Arg
Val Glu Ser Leu Phe Tyr Asn 440 445 450 tat agc atg ctt tga
agagtctcgg gcaagaaaca ttcatgcatg agtatatgtt 1454 Tyr Ser Met Leu *
455 cttggaatca attcctctaa gaacagagaa tggtctttcc cagggacaca
aagggagaaa 1514 tgggacatgg attcttgatc ttcctttaca catttctcgg
gaaatctgaa atgatgctgg 1574 atgggactct acacatcctg agctaagaca
tactgtcagt ctcactt 1621 9 829 DNA
Homo sapiens CDS (70)..(690) 9 gggctgggca gatgggtggg tgagttccct
ctccccagag ccatcggcca ggtaccaaag 60 ctcagctgt atg gat tcc caa cag
gag gac ctg cgc ttc cct ggg atg 108 Met Asp Ser Gln Gln Glu Asp Leu
Arg Phe Pro Gly Met 1 5 10 tgg gtc tca ttg tac ttt gga atc ctg ggg
ctg tgt tct gtg ata act 156 Trp Val Ser Leu Tyr Phe Gly Ile Leu Gly
Leu Cys Ser Val Ile Thr 15 20 25 gga ggg tgc att atc ttt ctg cac
tgg agg aag aac ttg agg cgg gaa 204 Gly Gly Cys Ile Ile Phe Leu His
Trp Arg Lys Asn Leu Arg Arg Glu 30 35 40 45 gag cat gcc cag cag tgg
gtg gag gtg atg aga gct gcc aca ttc acc 252 Glu His Ala Gln Gln Trp
Val Glu Val Met Arg Ala Ala Thr Phe Thr 50 55 60 tac agc cca ttg
ttg tac tgg att aac aag cga cgg cgc tac ggc atg 300 Tyr Ser Pro Leu
Leu Tyr Trp Ile Asn Lys Arg Arg Arg Tyr Gly Met 65 70 75 aat gca
gcc atc aac acg ggc cct gcc cct gct gtc acc aag act gag 348 Asn Ala
Ala Ile Asn Thr Gly Pro Ala Pro Ala Val Thr Lys Thr Glu 80 85 90
act gag gtc cag aat cca gat gtt ctg tgg gat ttg gac atc ccc gaa 396
Thr Glu Val Gln Asn Pro Asp Val Leu Trp Asp Leu Asp Ile Pro Glu 95
100 105 ggc agg agc cat gct gac caa gac agc aac ccc aag gcg gaa gcc
cct 444 Gly Arg Ser His Ala Asp Gln Asp Ser Asn Pro Lys Ala Glu Ala
Pro 110 115 120 125 gct ccc ctg caa cct gca ctg cag ctg gct cca cag
cag ccc cag gcc 492 Ala Pro Leu Gln Pro Ala Leu Gln Leu Ala Pro Gln
Gln Pro Gln Ala 130 135 140 aga tcc cca ttc cca ctt ccc atc ttt cag
gag gtg ccc ttt gcc cca 540 Arg Ser Pro Phe Pro Leu Pro Ile Phe Gln
Glu Val Pro Phe Ala Pro 145 150 155 ccc ttg tgc aac cta ccc ccc ctg
ctg aac cac tct gtc tcc tat cct 588 Pro Leu Cys Asn Leu Pro Pro Leu
Leu Asn His Ser Val Ser Tyr Pro 160 165 170 ttg gcc acc tgt cct gaa
agg aat gtt ctc ttc cat tcc ctc ctg aat 636 Leu Ala Thr Cys Pro Glu
Arg Asn Val Leu Phe His Ser Leu Leu Asn 175 180 185 ctg gcc cag gaa
gac cat agc ttc aat gcc aag cct ttt cct tca gaa 684 Leu Ala Gln Glu
Asp His Ser Phe Asn Ala Lys Pro Phe Pro Ser Glu 190 195 200 205 ctg
tag cctcctctca ctgaaggtgg gagctgcagg aatcaggtgc agagtaggaa 740 Leu
* atggaactaa cctcaggaag gtggtattga cagaggtcag gacccacctg gatgtcatgc
800 tatgaaacat taaaagaaaa aaaaaaaaa 829 10 1812 DNA Homo sapiens
CDS (358)..(1203) 10 gccgcccggc cgagcgcgga gcgcagccac tcgccgctgc
ccagggagcg cccaagatgt 60 ggggggaccg gggcggcagc ggccgtagca
gcgccaggga cgggggcacg cagcagcctc 120 cgctcgcccg cctgtcctga
cctgcctcgc ttgcccccaa agaatgtcag ccaagtccaa 180 ggggaacccc
tcctcgtcct gtccagccga gggaccgccg gcagcctcca aaaccaaggt 240
gaaggaacag atcaagatca tcgtggagga tttggaatta gtcctgggcg acctgaagga
300 cgtggccaag gaacttaagg agatgaagtc ccactctgtt gcccaggcta gagtgca
357 atg gca caa tct ggg ctc act gca acc tct gcc tcc cag gtt caa gct
405 Met Ala Gln Ser Gly Leu Thr Ala Thr Ser Ala Ser Gln Val Gln Ala
1 5 10 15 att ctc ctg cct cag cct gcc tca gtg cgc cac tac gcc tgg
gtg gtt 453 Ile Leu Leu Pro Gln Pro Ala Ser Val Arg His Tyr Ala Trp
Val Val 20 25 30 gac cag att gac acc ctg acc tct gac cta cag ctg
gag gat gag atg 501 Asp Gln Ile Asp Thr Leu Thr Ser Asp Leu Gln Leu
Glu Asp Glu Met 35 40 45 act gac agc tcc aaa acg gac acg ctg aat
agt agc tca agt ggc aca 549 Thr Asp Ser Ser Lys Thr Asp Thr Leu Asn
Ser Ser Ser Ser Gly Thr 50 55 60 aca gcc tcc agc cta gag aag atc
aaa gtg cag gct aat gca ccg ctt 597 Thr Ala Ser Ser Leu Glu Lys Ile
Lys Val Gln Ala Asn Ala Pro Leu 65 70 75 80 att aaa ccc cca gca cac
cca tct gct atc ctc acg gtc ctg aga aag 645 Ile Lys Pro Pro Ala His
Pro Ser Ala Ile Leu Thr Val Leu Arg Lys 85 90 95 cca aac cct cca
cca cct cct cca agg ttg aca cct gtg aag tgt gaa 693 Pro Asn Pro Pro
Pro Pro Pro Pro Arg Leu Thr Pro Val Lys Cys Glu 100 105 110 gac ccc
aaa agg gtg gtt cca act gcc aat cct gta aaa acc aat ggc 741 Asp Pro
Lys Arg Val Val Pro Thr Ala Asn Pro Val Lys Thr Asn Gly 115 120 125
acc ctt cta cga aat gga ggc tta cca ggt gga cct aac aaa att cca 789
Thr Leu Leu Arg Asn Gly Gly Leu Pro Gly Gly Pro Asn Lys Ile Pro 130
135 140 aat gga gat atc tgc tgc ata ccc aac agt aac ttg gac aag gct
cca 837 Asn Gly Asp Ile Cys Cys Ile Pro Asn Ser Asn Leu Asp Lys Ala
Pro 145 150 155 160 gtc cag ctt ctg atg cat aga cct gaa aaa gac aga
tgt ccc cag gca 885 Val Gln Leu Leu Met His Arg Pro Glu Lys Asp Arg
Cys Pro Gln Ala 165 170 175 ggg cct cga gaa cga gtt cgg ttt aat gaa
aaa gta cag tac cat ggc 933 Gly Pro Arg Glu Arg Val Arg Phe Asn Glu
Lys Val Gln Tyr His Gly 180 185 190 tat tgt cct gac tgt gat acc cgg
tat aac ata aaa aac agg gag gtc 981 Tyr Cys Pro Asp Cys Asp Thr Arg
Tyr Asn Ile Lys Asn Arg Glu Val 195 200 205 cac tta cac agt gaa cct
gtc cac cca ccg gga aag att cct cac caa 1029 His Leu His Ser Glu
Pro Val His Pro Pro Gly Lys Ile Pro His Gln 210 215 220 ggc cct ccc
ctc cct cct aca ccc cat ctc cct cct ttc cca cta gaa 1077 Gly Pro
Pro Leu Pro Pro Thr Pro His Leu Pro Pro Phe Pro Leu Glu 225 230 235
240 aat ggg gga atg gga ata agc cac agt aac agc ttc ccc cct atc aga
1125 Asn Gly Gly Met Gly Ile Ser His Ser Asn Ser Phe Pro Pro Ile
Arg 245 250 255 cct gca act gtg cct cct ccc act gca cca aaa cca cag
aag acg atc 1173 Pro Ala Thr Val Pro Pro Pro Thr Ala Pro Lys Pro
Gln Lys Thr Ile 260 265 270 ttg agg aag tca acc act aca acc gtg tga
t gtatgccatt aaaaaaattg 1224 Leu Arg Lys Ser Thr Thr Thr Thr Val *
275 280 tttttttaat tttctatatt ataaacataa aataagtaat gagcactttc
tactcaagca 1284 ataaaaagcc caaatatatt aatcctgcat tcagcaaagt
ggcataaaaa tcacctggta 1344 agtatgcagc acattgctta tatcctgggt
atgcattatt ttaaatgttg tatcattaaa 1404 aacctcagaa tgatgaaaaa
tatgaatgat gcattgtttt tgcaattgac ctatgacaaa 1464 ctgtgaacct
gcagatttca cctattttga tttactataa gagctgggat ttgattcatt 1524
ttatttatgc ctaagtcatc tatgcattaa catgtcatat tcttaacttt gatctaatgc
1584 tttttactag gaaattttaa tactgaagga ctattttatt atttttttct
aaagatgttt 1644 gtcactagtt tttcattatt aaatgctgag gccaatacca
agaagtttat tttctatatt 1704 atacaattat gaattacatg ctcagctata
tatgtaataa aatactttgg tctgtggaaa 1764 tattgttaaa tcaataaaca
atagtaaata atgacaaaaa aaaaaaaa 1812
* * * * *