U.S. patent application number 11/158218 was filed with the patent office on 2005-10-20 for 13245, a novel human myotonic dystrophy type protein kinase and uses therefor.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Kapeller-Libermann, Rosana.
Application Number | 20050233375 11/158218 |
Document ID | / |
Family ID | 22914754 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050233375 |
Kind Code |
A1 |
Kapeller-Libermann, Rosana |
October 20, 2005 |
13245, a novel human myotonic dystrophy type protein kinase and
uses therefor
Abstract
The invention provides isolated nucleic acids molecules,
designated 13245 nucleic acid molecules, which encode a novel
myotonic dystrophy type protein kinase. The invention also provides
antisense nucleic acid molecules, recombinant expression vectors
containing 13245 nucleic acid molecules, host cells into which the
expression vectors have been introduced, and non-human transgenic
animals in which a 13245 gene has been introduced or disrupted. The
invention still further provides isolated 13245 proteins, fusion
proteins, antigenic peptides and anti-13245 antibodies. Diagnostic
methods utilizing compositions of the invention are also
provided.
Inventors: |
Kapeller-Libermann, Rosana;
(Chestnut Hill, MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
40 Landsdowne Street
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
22914754 |
Appl. No.: |
11/158218 |
Filed: |
June 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11158218 |
Jun 21, 2005 |
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10017216 |
Oct 23, 2001 |
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60242429 |
Oct 23, 2000 |
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Current U.S.
Class: |
435/6.14 ;
435/183; 435/320.1; 435/325; 435/69.1; 435/7.1; 530/388.26;
536/23.2; 800/8 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 9/12 20130101; C12N 9/1205 20130101; A01K 2217/05
20130101 |
Class at
Publication: |
435/006 ;
435/007.1; 435/069.1; 435/320.1; 435/325; 435/183; 530/388.26;
536/023.2; 800/008 |
International
Class: |
C12Q 001/68; G01N
033/53; A01K 067/00; C07H 021/04; C12N 009/00; C12N 015/09 |
Claims
1-7. (canceled)
8. An isolated polypeptide selected from the group consisting of:
a) a polypeptide which is encoded by a nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to
the nucleotide sequence of either of SEQ ID NOs: 1 and 3, wherein
the polypeptide has kinase activity; b) a polypeptide which is
encoded by a nucleic acid molecule comprising a fragment of at
least 1400 nucleotides of the nucleotide sequence of either of SEQ
ID NO: 1 or SEQ ID NO:3, wherein the fragment has kinase activity;
c) a polypeptide comprising the amino acid sequence of SEQ ID NO:
2; d) a polypeptide comprising an amino acid sequence which is at
least 95% identical to the amino acid sequence of SEQ ID NO:2,
wherein the polypeptide has kinase activity; and e) a fragment of a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
wherein the fragment comprises at least 1000 contiguous amino acids
of SEQ ID NO: 2; wherein the fragment has kinase activity.
9. The isolated polypeptide of claim 8 comprising the amino acid
sequence of SEQ ID NO: 2.
10. The polypeptide of claim 8, further comprising a heterologous
amino acid sequence.
11-49. (canceled)
50. An isolated polypeptide selected from the group consisting of:
a) a polypeptide which is encoded by a nucleic acid molecule
consisting of a nucleotide sequence which is at least 95% identical
to the nucleotide sequence of either of SEQ ID NOs: 1 and 3,
wherein the polypeptide has kinase activity; b) a polypeptide
fragment which is encoded by a nucleic acid molecule consisting of
a fragment of at least 1400 nucleotides of the nucleotide sequence
of either of SEQ ID NO: 1 or SEQ ID NO:3, wherein the polypeptide
fragment has kinase activity; c) a polypeptide consisting of the
amino acid sequence of SEQ ID NO: 2; d) a polypeptide consisting of
an amino acid sequence which is at least 95% identical to the amino
acid sequence of SEQ ID NO:2, wherein the polypeptide has kinase
activity. and e) a fragment of a polypeptide comprising the amino
acid sequence of SEQ ID NO: 2, wherein the fragment consists of at
least 1000 contiguous amino acids of SEQ ID NO: 2; wherein the
fragment has kinase activity.
51. The isolated polypeptide of claim 50 consisting of the amino
acid sequence of SEQ ID NO: 2.
52. The isolated polypeptide of claim 8, wherein the polypeptide is
encoded by the nucleotide sequence comprising SEQ ID NO: 3.
53. The isolated polypeptide of claim 50, wherein the polypeptide
is encoded by the nucleotide sequence consisting of SEQ ID NO: 3.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/017,216, filed Oct. 23, 2001, which claims the benefit
of U.S. Provisional Application No. 60/242,429, filed Oct. 23,
2000, now abandoned. Each of these applications is hereby
incorporated in its entirety by reference herein.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable
REFERENCE TO MICROFICHE APPENDIX
[0003] Not Applicable
BACKGROUND OF THE INVENTION
[0004] Protein phosphorylation, for example at serine, threonine,
and tyrosine residues, is a key regulatory mechanism for a variety
of cellular processes. Protein phosphorylation is influenced
primarily by enzymes of two types, namely protein kinases (PKs) and
protein phosphatases (PPs). PKs catalyze addition of a phosphate
moiety to a protein amino acid residue (generally a serine,
threonine, or tyrosine residue), and PPs catalyze removal of such
moieties. The catalytic activities of PKs and PPs are, in turn,
influenced by the state of the cell and the environment in which it
finds itself.
[0005] Myotonic dystrophy type PKs (MDPKs) are associated with
modulation of cell morphology, shape, and contractility. MDPKs are
also known to modulate the activity of skeletal muscle
voltage-gated sodium channels, but not cardiac muscle voltage-gated
sodium channels. MDPKs thus have a role in a variety of
musculodegenerative and other musculoskeletal disorders including,
for example, muscular dystrophy (MD) of various types (e.g.,
Duchenne's MD, limb-girdle MD, Becker MD, facioscapulohumerol MD,
mitochondrial myopathy, and congenital myopathy) and myotonic
dystrophies (e.g., Steinert's disease and Thomsen's disease).
[0006] Numerous MDPKs have been described, and many more are
believed to exist. In view of the widespread and critical nature of
MDPK activities in normal and pathological physiological processes,
a need exists for identification of further members of this protein
family. The present invention satisfies this need by providing a
novel human MDPK.
SUMMARY OF THE INVENTION
[0007] The present invention is based, in part, on the discovery of
a novel gene encoding a MDPK, the gene being referred to herein as
"13245". The nucleotide sequence of a cDNA encoding 13245 is shown
in SEQ ID NO: 1, and the amino acid sequence of a 13245 polypeptide
is shown in SEQ ID NO: 2. In addition, the nucleotide sequence of
the coding region is depicted in SEQ ID NO: 3.
[0008] Accordingly, in one aspect, the invention features a nucleic
acid molecule that encodes a 13245 protein or polypeptide, e.g., a
biologically active portion of the 13245 protein. In a preferred
embodiment the isolated nucleic acid molecule encodes a polypeptide
having the amino acid sequence SEQ ID NO: 2. In other embodiments,
the invention provides isolated 13245 nucleic acid molecules having
the nucleotide sequence of either of SEQ ID NOs: 1 and 3.
[0009] In still other embodiments, the invention provides nucleic
acid molecules that have sequences that are substantially identical
(e.g., naturally occurring allelic variants) to the nucleotide
sequence of either of SEQ ID NOs: 1 and 3. In other embodiments,
the invention provides a nucleic acid molecule which hybridizes
under stringent hybridization conditions with a nucleic acid
molecule having a sequence comprising the nucleotide sequence of
either of SEQ ID NOs: 1 and 3, wherein the nucleic acid encodes a
full length 13245 protein or an active fragment thereof.
[0010] In a related aspect, the invention further provides nucleic
acid constructs that include a 13245 nucleic acid molecule
described herein. In certain embodiments, the nucleic acid
molecules of the invention are operatively linked to native or
heterologous regulatory sequences. Also included are vectors and
host cells containing the 13245 nucleic acid molecules of the
invention, e.g., vectors and host cells suitable for producing
13245 nucleic acid molecules and polypeptides.
[0011] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for
detection of 13245-encoding nucleic acids.
[0012] In still another related aspect, isolated nucleic acid
molecules that are antisense to a 13245-encoding nucleic acid
molecule are provided.
[0013] In another aspect, the invention features 13245
polypeptides, and biologically active or antigenic fragments
thereof that are useful, e.g., as reagents or targets in assays
applicable to treatment and diagnosis of 13245-mediated or related
disorders (e.g., MDPK-mediated disorders such as those described
herein). In another embodiment, the invention provides 13245
polypeptides having protein kinase activity. Preferred polypeptides
are 13245 proteins including at least one pkinase domain, and
preferably having a 13245 activity, e.g., a 13245 activity as
described herein. Preferred polypeptides are 13245 proteins
including at least one CNH domain and at least one pkinase domain.
Other preferred polypeptides are 13245 proteins including at least
one CNH domain, at least one pkinase domain, at least one phorbol
ester/diacylglycerol binding domain, at least one PH domain, and at
least one leucine zipper domain.
[0014] In other embodiments, the invention provides 13245
polypeptides, e.g., a 13245 polypeptide having the amino acid
sequence shown in SEQ ID NO: 2, an amino acid sequence that is
substantially identical to the amino acid sequence shown in SEQ ID
NO: 2, or an amino acid sequence encoded by a nucleic acid molecule
having a nucleotide sequence which hybridizes under stringent
hybridization conditions to a nucleic acid molecule comprising the
nucleotide sequence of either of SEQ ID NOs: 1 and 3, wherein the
nucleic acid encodes a full length 13245 protein or an active
fragment thereof.
[0015] In a related aspect, the invention further provides nucleic
acid constructs that include a 13245 nucleic acid molecule
described herein.
[0016] In a related aspect, the invention provides 13245
polypeptides or fragments operatively linked to non-13245
polypeptides to form fusion proteins.
[0017] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably, specifically bind, 13245 polypeptides.
[0018] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the 13245 polypeptides or nucleic acids.
[0019] In still another aspect, the invention provides a process
for modulating 13245 polypeptide or nucleic acid expression or
activity, e.g., using the screened compounds. In certain
embodiments, the methods involve treatment of conditions related to
aberrant activity or expression of the 13245 polypeptides or
nucleic acids, such as conditions involving aberrant or deficient
protein phosphorylation or aberrant or deficient cell process
regulation (e.g., aberrant or deficient cell signaling or aberrant
or deficient muscular function).
[0020] The invention also provides assays for determining the
activity of or the presence or absence of 13245 polypeptides or
nucleic acid molecules in a biological sample, including for
disease diagnosis.
[0021] In further aspect the invention provides assays for
determining the presence or absence of a genetic alteration in a
13245 polypeptide or nucleic acid molecule, including for disease
diagnosis.
[0022] The invention also includes a method of modulating the
ability of a cell to catalyze interconversion of the phosphorylated
and de-phosphorylated forms a GTPase protein. The method comprises
modulating 13245 protein activity in the cell. The activity of
13245 protein can be modulated by inhibiting expression of the
13245 gene in the cell, for example by administering to the cell an
antisense oligonucleotide which hybridizes under stringent
conditions with a transcript (e.g., an mRNA) of the 13245 gene, an
antisense oligonucleotide which hybridizes under stringent
conditions with a polynucleotide having the nucleotide sequence SEQ
ID NO: 1, or an antisense oligonucleotide which hybridizes under
stringent conditions with a polynucleotide having the nucleotide
sequence SEQ ID NO: 3. Alternatively the activity of 13245 protein
can be inhibited without significantly affecting 13245 gene
expression in the cell. For example, the activity of 13245 protein
can be inhibited by administering to the cell an agent which
inhibits an activity of 13245 protein, such as an antibody which
specifically binds with 13245 protein. In a related aspect, the
activity of 13245 can be modulated by enhancing expression of 13245
in the cell. For example, expression of 13245 in a cell can be
enhanced by administering to the cell an agent that enhances
expression of 13245, such as an expression vector encoding 13245
protein.
[0023] The invention further includes a method for assessing
whether a test compound is useful for modulating at least one
phenomenon selected from the group consisting of (1)
interconversion of the phosphorylated and de-phosphorylated forms
of a serine, threonine, or tyrosine residue of a GTPase protein;
(2) cell contractility; (3) cell growth; (4) cell conductivity; (5)
entry of a cell into the cell cycle; (6) progression of a cell
through the cell cycle; (7) mitogenesis; (8) cell metabolism; (9)
gene transcription; (11) cytokinesis; (12) cell shape; (13) cell
movement; (14) integration of a viral genome into a host cell
genome; (15) maintenance of a viral genome within a host cell
genome; (16) a cytological change in a virus-infected host cell;
(17) virus production in a virus-infected host cell; (18)
interaction of a virion with a membrane of a virus-infected host
cell; and (19) encapsulation of a virion within a portion of a
membrane of a virus-infected host cell. The method comprises:
[0024] a) adding the test compound to a first composition
comprising a polypeptide that has an amino acid sequence at least
90% identical to SEQ ID NO: 2 and that exhibits a 13245 activity;
and
[0025] b) comparing the 13245 activity in the first composition and
in a second composition that is substantially identical to the
first composition except that it does not comprise the test
compound. A difference in the 13245 activity in the first and
second compositions is an indication that the test compound is
useful for modulating the phenomenon.
[0026] The invention also includes another method for assessing
whether a test compound is useful for modulating at least one
phenomenon selected from the group consisting of (1)
interconversion of the phosphorylated and de-phosphorylated forms
of a serine, threonine, or tyrosine residue of a GTPase protein;
(2) cell contractility; (3) cell growth; (4) cell conductivity; (5)
entry of a cell into the cell cycle; (6) progression of a cell
through the cell cycle; (7) mitogenesis; (8) cell metabolism; (9)
gene transcription; (11) cytokinesis; (12) cell shape; (13) cell
movement; (14) integration of a viral genome into a host cell
genome; (15) maintenance of a viral genome within a host cell
genome; (16) a cytological change in a virus-infected host cell;
(17) virus production in a virus-infected host cell; (18)
interaction of a virion with a membrane of a virus-infected host
cell; and (19) encapsulation of a virion within a portion of a
membrane of a virus-infected host cell. This method comprises:
[0027] a) adding the test compound to a first composition
comprising a cell which comprises a nucleic acid that encodes a
polypeptide that has an amino acid sequence at least 90% identical
to SEQ ID NO: 2 and that exhibits a 13245 activity; and
[0028] b) comparing 13245 activity in the first composition and in
a second composition that is substantially identical to the first
composition except that it does not comprise the test compound.
[0029] A difference in the 13245 activity in the first and second
compositions is an indication that the test compound is useful for
modulating the phenomenon.
[0030] Compounds identified using these methods can be used to make
a pharmaceutical composition for modulating the phenomenon, for
example by combining it with a pharmaceutically acceptable carrier.
Such compositions can be used to modulate the phenomenon in a
human.
[0031] The invention includes another method for identifying a
compound useful for modulating at least one phenomenon selected
from the group consisting of (1) interconversion of the
phosphorylated and de-phosphorylated forms of a serine, threonine,
or tyrosine residue of a GTPase protein; (2) cell contractility;
(3) cell growth; (4) cell conductivity; (5) entry of a cell into
the cell cycle; (6) progression of a cell through the cell cycle;
(7) mitogenesis; (8) cell metabolism; (9) gene transcription; (11)
cytokinesis; (12) cell shape; (13) cell movement; (14) integration
of a viral genome into a host cell genome; (15) maintenance of a
viral genome within a host cell genome; (16) a cytological change
in a virus-infected host cell; (17) virus production in a
virus-infected host cell; (18) interaction of a virion with a
membrane of a virus-infected host cell; and (19) encapsulation of a
virion within a portion of a membrane of a virus-infected host
cell. This method comprises:
[0032] a) contacting the test compound and a polypeptide selected
from the group consisting of
[0033] i) a polypeptide which is encoded by a nucleic acid molecule
comprising a portion having a nucleotide sequence which is at least
90% identical to one of SEQ ID NOs: 1 and 3; and
[0034] ii) a fragment of a polypeptide having either an amino acid
sequence comprising SEQ ID NO: 2, wherein the fragment comprises at
least 25 contiguous amino acid residues of SEQ ID NO: 2
[0035] or a cell that expresses the polypeptide; and
[0036] b) determining whether the polypeptide binds with the test
compound. Binding of the polypeptide and the test compound is an
indication that the test compound is useful for modulating the
phenomenon.
[0037] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 depicts a cDNA sequence (SEQ ID NO: 1) and predicted
amino acid sequence (SEQ ID NO: 2) of human 13245. The
methionine-initiated open reading frame of human 13245 (without the
5'- and 3'-non-translated regions) starts at nucleotide 19 of SEQ
ID NO: 1, and the coding region (not including the terminator
codon; shown in SEQ ID NO: 3) extends through nucleotide 6178 of
SEQ ID NO: 1.
[0039] FIG. 2 depicts a hydropathy plot of human 13245. Relatively
hydrophobic residues are shown above the dashed horizontal line,
and relative hydrophilic residues are below the dashed horizontal
line. The cysteine residues (cys) are indicated by short vertical
lines below the hydropathy trace. The numbers corresponding to the
amino acid sequence of human 13245 are indicated. Polypeptides of
the invention include fragments which include: all or part of a
hydrophobic sequence, i.e., a sequence above the dashed line, e.g.,
the sequence of about residues 195-210 of SEQ ID NO: 2; all or part
of a hydrophilic sequence, i.e., a sequence below the dashed line,
e.g., the sequence of residues 455-475 of SEQ ID NO: 2; a sequence
which includes a cysteine residue; or a glycosylation site.
[0040] FIG. 3 is an alignment of the amino acid sequence (SEQ ID
NO: 2) of 13245, murine rho/rac-interacting citron kinase
("AAC72823"; GENBANK.RTM. accession no. AAC72823; SEQ ID NO: 4),
murine citron-K kinase ("AAC27933"; GENBANK.RTM. accession no.
AAC27933; SEQ ID NO: 5), murine citron protein ("P49025";
GENBANK.RTM. accession no. P49025; SEQ ID NO: 6), and human citron
protein ("014578"; GENBANK.RTM. accession no. 014578; SEQ ID NO:
7). The alignment was made using the Multalin v4ersion 5.4.1
software using the blosum62 symbol comparison table, a gap weight
of 12, and a gap length weight of 2.
DETAILED DESCRIPTION
[0041] The human 13245 cDNA sequence (FIG. 1; SEQ ID NO: 1), which
is approximately 6575 nucleotide residues long including
non-translated regions, contains a predicted methionine-initiated
coding sequence of about 6160 nucleotide residues, excluding
termination codon (i.e., nucleotide residues 19-6178 of SEQ ID NO:
1; also shown in SEQ ID NO: 3). The coding sequence encodes a 2053
amino acid protein having the amino acid sequence SEQ ID NO: 2.
[0042] Human 13245 contains the following regions or other
structural features: a predicted pkinase domain (PF00069) at about
amino acid residues 97-360 of SEQ ID NO: 2, a predicted protein
kinase C terminal domain at residues 361-390 of SEQ ID NO: 2, a
serine/threonine protein kinase active site signature sequence at
residues 217-229 of SEQ ID NO: 2, predicted leucine zipper domains
at residues 838-859, 975-996, 1041-1062, and 1143-1164 of SEQ ID
NO: 2, a predicted carbamoyl-phosphate synthase sub-domain
signature sequence at residues 1156-1163 of SEQ ID NO: 2, a
predicted phorbol ester/diacylglycerol binding domain at residues
1389-1437 of SEQ IUD NO: 2, a predicted pleckstrin homology (PH)
domain at residues 1470-1525 of SEQ ID NO: 2, and a predicted CNH
domain at residues 1568-1865 of SEQ ID NO: 2.
[0043] The human 13245 protein has predicted N-glycosylation sites
(Pfam accession number PS00001) at about amino acid residues
819-822, 1571-1574, 1694-1697, 1717-1720, and 2026-2029 of SEQ ID
NO: 2; predicted cAMP-/cGMP-dependent protein kinase
phosphorylation sites (Pfam accession number PS00004) at about
amino acid residues 78-81, 477-480, 579-582, 601-604, 680-683,
1322-1325, and 1366-1369 of SEQ ID NO: 2; predicted protein kinase
C phosphorylation sites (Pfam accession number PS00005) at about
amino acid residues 93-95, 248-250, 308-310, 378-380, 487-489,
498-500, 516-518, 546-548, 577-579, 824-826, 872-874, 1025-1027,
1033-1035, 1096-1098, 1144-1146, 1170-1172, 1215-1217, 1268-1270,
1314-1316, 1335-1337, 1363-1365, 1376-1378, 1542-1544, 1724-1726,
1892-1894, 1910-1912, 1963-1965, and 1977-1979 of SEQ ID NO: 2;
predicted casein kinase II phosphorylation sites (Pfam accession
number PS00006) located at about amino acid residues 83-86, 93-96,
140-143, 361-364, 381-384, 386-389, 410-413, 436-439, 445-448,
480-483, 487-490, 501-504, 529-532, 867-870, 908-911, 935-938,
940-943, 973-976, 1015-1018, 1025-1028, 1046-4049, 1081-1084,
1142-1145, 1170-1173, 1218-1221, 1308-1311, 1314-1317, 1370-1373,
1736-1739, 1794-1797, 1864-1867, 1882-1885, 1904-1907, 1964-1967,
and 2012-2015 of SEQ ID NO: 2; a predicted tyrosine kinase
phosphorylation site at residues 741-747 of SEQ ID NO: 2; predicted
N-myristoylation sites (Pfam accession number PS00008) at about
amino acid residues 50-5, 1202-1207, 1532-1537, 1584-1589,
1675-1680, and 1999-2004 of SEQ ID NO: 2; a predicted amidation
site (Pfam accession number PS00009) at about amino acid residues
134-136 of SEQ ID NO: 2.
[0044] For general information regarding PFAM identifiers, PS
prefix and PF prefix domain identification numbers, refer to
Sonnhammer et al. (1997, Protein 28:405-420) and
http://www.psc.edu/general/software/packag- es/pfam/pfam.html.
[0045] The 13245 protein contains a significant number of
structural characteristics in common with members of the MDPK
family. The term "family" when referring to the protein and nucleic
acid molecules of the invention means two or more proteins or
nucleic acid molecules having a common structural domain or motif
and having sufficient amino acid or nucleotide sequence homology as
defined herein. Such family members can be naturally or
non-naturally occurring and can be from either the same or
different species. For example, a family can contain a first
protein of human origin as well as other distinct proteins of human
origin, or alternatively, can contain homologues of non-human
origin, e.g., MDPK proteins for any species described in the art.
Members of a family can also have common functional
characteristics.
[0046] A 13245 polypeptide can include a pkinase domain. As used
herein, the term "pkinase domain" refers to a protein domain having
an amino acid sequence of about 200-300 amino acid residues in
length, preferably, at least about 225-300 amino acids, more
preferably about 264 amino acid residues and has a bit score for
the alignment of the sequence to the pkinase domain (HMM) of at
least 100 or greater, preferably 150 or greater, and more
preferably 200 or greater. The pkinase domain has been assigned the
PFAM accession PF00069 (http://genome.wustl.edu/Pfam/html).
[0047] A 13245 polypeptide can include a pkinase domain. As used
herein, the term "CNH domain" refers to a protein domain having an
amino acid sequence of about 250-350 amino acid residues in length,
preferably, at least about 275-325 amino acids, more preferably
about 298 amino acid residues and has a bit score for the alignment
of the sequence to the pkinase domain (HMM) of at least 100 or
greater, preferably 200 or greater, and more preferably 300 or
greater. The pkinase domain has been assigned the PFAM accession
PF00780 (http://genome.wustl.edu/Pfamhtml).
[0048] In a preferred embodiment, 13245 polypeptide or protein has
a pkinase domain or a region which includes at least about 200-300,
more preferably about 225-300, or 264 amino acid residues and has
at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with
a pkinase domain, e.g., the pkinase domain of human 13245 (e.g.,
residues 97-360 of SEQ ID NO: 2).
[0049] In another preferred embodiment, 13245 polypeptide or
protein has a CNH domain or a region which includes at least about
250-350, more preferably about 275-325, or 298 amino acid residues
and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100%
homology with a CNH domain, e.g., the CNH domain of human 13245
(e.g., residues 1568-1865 of SEQ ID NO: 2).
[0050] To identify the presence of a pkinase or CNH domain profile
in a 13245 receptor, the amino acid sequence of the protein is
searched against a database of HMMs (e.g., the Pfam database,
release 2.1) using the default parameters
(http://www.sanger.ac.uk/Software/Pfa/H_search). For example, the
hmmsf program, which is available as part of the HMMER package of
search programs, is a family specific default program for PF00069
or PF00780 and score of 100 is the default threshold score for
determining a hit. For example, using ORFAnalyzer software, a
pkinase domain profile was identified in the amino acid sequence of
SEQ ID NO: 2 (e.g., amino acids 53-303 of SEQ ID NO: 2).
Accordingly, a 13245 protein having at least about 60-70%, more
preferably about 70-80%, or about 80-90% homology with the pkinase
domain profile or the CNH domain profile of human 13245 is within
the scope of the invention.
[0051] While not being bound by any particular theory of operation,
13245 protein is believed to be, in at least one embodiment, a
nuclear membrane protein having its carboxyl-terminal domain
oriented within the nuclear envelope. In this embodiment, 13245
protein is capable of transmitting signaling information from the
cytoplasm to the nucleus, whereby, for example, gene transcription
can be regulated.
[0052] In one embodiment of the invention, a 13245 polypeptide
includes at least one pkinase domain. In another embodiment, the
13245 polypeptide includes at least one pkinase domain and at least
one CNH domain. The 13245 molecules of the present invention can
further include one or more of the N-glycosylation,
cAMP-/cGMP-dependent protein kinase phosphorylation, protein kinase
C phosphorylation, casein kinase II phosphorylation, tyrosine
kinase phosphorylation, N-myristoylation, amidation, leucine
zipper, serine/threonine protein kinase active site,
carbamoyl-phosphate synthase sub-domain signature, protein kinase C
terminal domain phorbol ester/diacylglycerol binding, and
pleckstrin homology domains and sites described herein, and
preferably comprises most or all of them.
[0053] As shown in FIG. 3, 13245 protein exhibits significant
sequence homology with several proteins related to citron protein,
which is known to interact with GTPase enzymes of the Rho family.
These GTPases include, for example, Rho-A, -B, and -C, Rac-1 and
-2, and CDC42. These GTPases regulate cell structure, including
cell shape, cell contraction, cell movement, distribution of
structural proteins (e.g., actin) within the cell, formation of
focal adhesions, cytokinesis, and cell division. These GTPases are
also known to modulate gene expression in a
phosphorylation-dependent manner. Proteins that are able to
interact with, catalyze interconversion of phosphorylated and
non-phosphorylated Rho GTPase isoforms, or both, are able to
modulate these cellular processes.
[0054] Occurrence of pkinase, pkinase_C, a protein kinase
ATP-binding region signature, a serine/threonine protein kinase
active site signature, a tyrosine kinase phosphorylation site,
multiple potential phosphorylation sites, and similarity with
citron proteins indicates that 13245 protein is able to interact
with Rho GTPases and catalyze interconversion of their
phosphorylated and non-phosphorylated forms. Ability of 13245 to
modulate the phosphorylation state of Rho GTPases indicates that
13245 is able to modulate one or more of cell shape, cell
contraction, cell movement, distribution of structural proteins
(e.g., actin) within the cell, formation of focal adhesions,
cytokinesis, and cell division in cells in which it is expressed.
Furthermore, these characteristics indicate that 13245 protein is
involved in disorders in which one or more of these processes are
aberrant. 13245 molecules described herein can therefore be used to
predict, diagnose, inhibit, prevent, alleviate, or cure these
disorders. Examples of disorders in which one or more of these
processes are aberrant include tumorigenesis, tumor growth, tumor
metastasis, and viral infection of a cell.
[0055] Expression of 13245 is greater in peripheral blood cells
than in many other cell types, and enhanced expression of 13245 is
observed in HIV-1-infected cells, including cells of the CCRF cell
line that have been infected with this virus. HIV-1-infected cells
undergo various morphological changes, and the pattern of gene
expression in HIV-1-infected cells differs from the pattern
observed in non-infected cells of the same type. These observations
indicate that 13245 has a role in the effects of HIV-1 infection on
host cells (e.g., peripheral blood mononuclear cells). Modulating
13245 expression, activity, or both in HIV-1-infected cells can
modulate the processes listed above, thereby alleviating or
reversing the effects of HIV-1 infection. By way of example 13245
molecules described herein can be used to inhibit insertion of the
HIV-1 genome into the host cell genome, inhibit or reverse
maintenance of the HIV-1 genome within the host cell genome,
inhibit or reverse cytological changes induced by HIV-1 infection,
inhibit HIV-1 virus production in infected cells, inhibit
interaction of HIV-1 virions with the host cell cytoplasmic
membrane, inhibit encapsulation of HIV-1 virus particles in
portions of the host cell membrane, or inhibit HIV-1 virus release
from infected cells. 13245 molecules can therefore be used to treat
individuals who are infected with HIV-1 (e.g., individuals
afflicted with acquired immune deficiency syndrome) or with other
pathogenic viruses or to inhibit transmission of the virus from one
individual to another.
[0056] Because the 13245 polypeptides of the invention can modulate
13245-mediated activities, they can be used to develop novel
diagnostic and therapeutic agents for 13245-mediated or related
disorders, as described below.
[0057] As used herein, a "13245 activity," "biological activity of
13245," or "functional activity of 13245," refers to an activity
exerted by a 13245 protein, polypeptide or nucleic acid molecule
on, for example, a 13245-responsive cell or on a 13245 substrate
(e.g., a protein substrate such as a skeletal muscle voltage-gated
sodium channel protein) as determined in vivo or in vitro. In one
embodiment, a 13245 activity is a direct activity, such as
association with a 13245 target molecule. A "target molecule" or
"binding partner" of a 13245 protein is a molecule (e.g., a protein
or nucleic acid) with which the 13245 protein binds or interacts in
nature. In an exemplary embodiment, such a target molecule is a
13245 receptor. A 13245 activity can also be an indirect activity,
such as a cellular signaling activity mediated by interaction of
the 13245 protein with a 13245 receptor.
[0058] The 13245 molecules of the present invention are predicted
to have similar biological activities as MDPK family members. For
example, the 13245 proteins of the present invention can have one
or more of the following activities:
[0059] (1) catalyzing formation of a covalent bond within or
between an amino acid residue and a phosphate moiety;
[0060] (2) modulating cell contractility;
[0061] (3) modulating cell growth;
[0062] (4) modulating cell conductivity;
[0063] (5) modulating entry of a cell into the cell cycle;
[0064] (6) modulating progression of a cell through the cell
cycle;
[0065] (7) modulating mitogenesis;
[0066] (8) modulating cell metabolism;
[0067] (9) modulating gene transcription;
[0068] (10) catalyzing interconversion of phosphorylated and
non-phosphorylated forms of a GTPase, such as a Rho GTPase;
[0069] (11) modulating cytokinesis;
[0070] (12) modulating cell shape;
[0071] (13) modulating cell movement (e.g., tumor metastasis);
[0072] (14) modulating integration of a viral genome into a host
cell genome;
[0073] (15) modulating maintenance of a viral genome within a host
cell genome;
[0074] (16) modulating cytological changes in a virus-infected host
cell;
[0075] (17) modulating virus production in a virus-infected host
cell;
[0076] (18) modulating interaction of a virion with a membrane of a
virus-infected host cell; and
[0077] (19) modulating encapsulation of a virion within a portion
of a membrane of a virus-infected host cell.
[0078] Thus, 13245 molecules described herein can act as novel
diagnostic targets and therapeutic agents for prognosticating,
diagnosing, preventing, inhibiting, alleviating, or curing
MDPK-related disorders.
[0079] Other activities, as described below, include the ability to
modulate function, survival, morphology, proliferation and/or
differentiation of cells of tissues in which 13245 molecules are
expressed. Thus, the 13245 molecules can act as novel diagnostic
targets and therapeutic agents for controlling disorders involving
aberrant activities of these cells.
[0080] The 13245 molecules can also act as novel diagnostic targets
and therapeutic agents for controlling various disorders, including
skeletal muscle disorders (e.g., muscular and myotonic dystrophies
as described herein and in the art).
[0081] The 13245 protein, fragments thereof, and derivatives and
other variants of the sequence in SEQ ID NO: 2 thereof are
collectively referred to as "polypeptides or proteins of the
invention" or "13245 polypeptides or proteins". Nucleic acid
molecules encoding such polypeptides or proteins are collectively
referred to as "nucleic acids of the invention" or "13245 nucleic
acids." 13245 molecules refer to 13245 nucleic acids, polypeptides,
and antibodies.
[0082] As used herein, the term "nucleic acid molecule" includes
DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules
(e.g., an mRNA) and analogs of the DNA or RNA generated, e.g., by
the use of nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0083] The term "isolated or purified nucleic acid molecule"
includes nucleic acid molecules that are separated from other
nucleic acid molecules that are present in the natural source of
the nucleic acid. For example, with regards to genomic DNA, the
term "isolated" includes nucleic acid molecules that are separated
from the chromosome with which the genomic DNA is naturally
associated. Preferably, an "isolated" nucleic acid is free of
sequences that naturally flank the nucleic acid (i.e., sequences
located at the 5'- and/or 3'-ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
For example, in various embodiments, the isolated nucleic acid
molecule can contain less than about 5 kilobases, 4 kilobases, 3
kilobases, 2 kilobases, 1 kilobase, 0.5 kilobase or 0.1 kilobase of
5'- and/or 3'-nucleotide sequences which naturally flank the
nucleic acid molecule in genomic DNA of the cell from which the
nucleic acid is derived. Moreover, an "isolated" nucleic acid
molecule, such as a cDNA molecule, can be substantially free of
other cellular material, or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized.
[0084] As used herein, the term "hybridizes under stringent
conditions" describes conditions for hybridization and washing.
Stringent conditions are known to those skilled in the art and can
be found in available references (e.g., Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1-6.3.6).
Aqueous and non-aqueous methods are described in that reference and
either can be used. A preferred example of stringent hybridization
conditions are hybridization in 6.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by one or more
washes in 0.2.times.SSC, 0.1% (w/v) SDS at 50.degree. C. Another
example of stringent hybridization conditions are hybridization in
6.times.SSC at about 45.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.1% (w/v) SDS at 55.degree. C. A further example
of stringent hybridization conditions are hybridization in
6.times.SSC at about 45.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.1% (w/v) SDS at 60.degree. C. Preferably,
stringent hybridization conditions are hybridization in 6.times.SSC
at about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% (w/v) SDS at 65.degree. C. Particularly
preferred stringency conditions (and the conditions that should be
used if the practitioner is uncertain about what conditions should
be applied to determine if a molecule is within a hybridization
limitation of the invention) are 0.5 molar sodium phosphate, 7%
(w/v) SDS at 65.degree. C., followed by one or more washes at
0.2.times.SSC, 1% (w/v) SDS at 65.degree. C. Preferably, an
isolated nucleic acid molecule of the invention that hybridizes
under stringent conditions to the sequence of SEQ ID NO: 1 or SEQ
ID NO: 3, corresponds to a naturally-occurring nucleic acid
molecule.
[0085] As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural
protein).
[0086] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules which include an open reading frame
encoding a 13245 protein, preferably a mammalian 13245 protein, and
can further include non-coding regulatory sequences and
introns.
[0087] An "isolated" or "purified" polypeptide or protein is
substantially free of cellular material or other contaminating
proteins from the cell or tissue source from which the protein is
derived, or substantially free from chemical precursors or other
chemicals when chemically synthesized. In one embodiment, the
language "substantially free" means preparation of 13245 protein
having less than about 30%, 20%, 10% and more preferably 5% (by dry
weight), of non-13245 protein (also referred to herein as a
"contaminating protein"), or of chemical precursors or non-13245
chemicals. When the 13245 protein or biologically active portion
thereof is recombinantly produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, more preferably less than about
10%, and most preferably less than about 5% of the volume of the
protein preparation. The invention includes isolated or purified
preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry
weight.
[0088] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of 13245 (e.g., the sequence
of either of SEQ ID NOs: 1 and 3) without abolishing or, more
preferably, without substantially altering a biological activity,
whereas an "essential" amino acid residue results in such a change.
For example, amino acid residues that are conserved among the
polypeptides of the present invention, e.g., those present in the
pkinase domain are predicted to be particularly non-amenable to
alteration.
[0089] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), non-polar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a 13245 protein is
preferably replaced with another amino acid residue from the same
side chain family. Alternatively, in another embodiment, mutations
can be introduced randomly along all or part of a 13245 coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for 13245 biological activity to identify
mutants that retain activity. Following mutagenesis of SEQ ID NO: 1
or 3, the encoded protein can be expressed recombinantly and the
activity of the protein can be determined.
[0090] As used herein, a "biologically active portion" of a 13245
protein includes a fragment of a 13245 protein that participates in
an interaction between a 13245 molecule and a non-13245 molecule.
Biologically active portions of a 13245 protein include peptides
comprising amino acid sequences sufficiently homologous to or
derived from the amino acid sequence of the 13245 protein, e.g.,
the amino acid sequence shown in SEQ ID NO: 2, which include less
amino acids than the full length 13245 proteins, and exhibit at
least one activity of a 13245 protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the 13245 protein, e.g., a domain or motif capable of
catalyzing an activity described herein, such as covalent addition
of a phosphate moiety to a protein amino acid residue (e.g., to a
serine or threonine hydroxyl group).
[0091] A biologically active portion of a 13245 protein can be a
polypeptide that for example, 10, 25, 50, 100, 200, 300, or 400,
500, 1000, 1500, or 2000 or more amino acids in length.
Biologically active portions of a 13245 protein can be used as
targets for developing agents that modulate a 13245-mediated
activity, e.g., a biological activity described herein.
[0092] Calculations of homology or sequence identity between
sequences (the terms are used interchangeably herein) are performed
as follows.
[0093] To determine the percent identity of two amino acid
sequences, or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, 90%, 100% of the length
of the reference sequence (e.g., when aligning a second sequence to
the 13245 amino acid sequence of SEQ ID NO: 2, 100 amino acid
residues, preferably at least 200, 300, 400, 500, 1000, 1500, or
2000 or more amino acid residues are aligned). The amino acid
residues or nucleotides at corresponding amino acid positions or
nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0094] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman et al. (1970, J. Mol. Biol. 48:444-453) algorithm which
has been incorporated into the GAP program in the GCG software
package (available at http://www.gcg.com), using either a BLOSUM 62
matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8,
6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another
preferred embodiment, the percent identity between two nucleotide
sequences is determined using the GAP program in the GCG software
package (available at http://www.gcg.com), using a NWSgapdna.CMP
matrix and a gap weight of 40, 50, 60, 70, or 80 and a length
weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of
parameters (and the one that should be used if the practitioner is
uncertain about what parameters should be applied to determine if a
molecule is within a sequence identity or homology limitation of
the invention) are a BLOSUM 62 scoring matrix with a gap penalty of
12, a gap extend penalty of 4, and a frameshift gap penalty of
5.
[0095] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of Meyers et al.
(1989, CABIOS, 4:11-17) which has been incorporated into the ALIGN
program (version 2.0), using a PAM120 weight residue table, a gap
length penalty of 12 and a gap penalty of 4.
[0096] The nucleic acid and protein sequences described herein can
be used as a "query sequence" to perform a search against public
databases to, for example, identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al. (1990, J. Mol.
Biol. 215:403-410). BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to 13245 nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to 13245 protein molecules of the invention. To obtain
gapped alignments for comparison purposes, gapped BLAST can be
utilized as described in Altschul et al. (1997, Nucl. Acids Res.
25:3389-3402). When using BLAST and gapped BLAST programs, the
default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See <http://www.ncbi.nlm.nih.gov>.
[0097] "Malexpression or aberrant expression," as used herein,
refers to a non-wild-type pattern of gene expression, at the RNA or
protein level. It includes: expression at non-wild-type levels,
i.e., over- or under-expression; a pattern of expression that
differs from wild-type in terms of the time or stage at which the
gene is expressed, e.g., increased or decreased expression (as
compared with wild-type) at a predetermined developmental period or
stage; a pattern of expression that differs from wild-type in terms
of decreased expression (as compared with wild-type) in a
predetermined cell type or tissue type; a pattern of expression
that differs from wild-type in terms of the splicing size, amino
acid sequence, post-transitional modification, or biological
activity of the expressed polypeptide; a pattern of expression that
differs from wild-type in terms of the effect of an environmental
stimulus or extracellular stimulus on expression of the gene, e.g.,
a pattern of increased or decreased expression (as compared with
wild-type) in the presence of an increase or decrease in the
strength of the stimulus.
[0098] "Subject," as used herein, can refer to a mammal, e.g., a
human, or to an experimental or animal or disease model. The
subject can also be a non-human animal, e.g., a horse, cow, goat,
or other domestic animal.
[0099] A "purified preparation of cells," as used herein, refers
to, in the case of plant or animal cells, an in vitro preparation
of cells and not an entire intact plant or animal. In the case of
cultured cells or microbial cells, it consists of a preparation of
at least 10%, and more preferably, 50% of the subject cells.
[0100] Various aspects of the invention are described in further
detail below.
[0101] Isolated Nucleic Acid Molecules
[0102] In one aspect, the invention provides, an isolated or
purified, nucleic acid molecule that encodes a 13245 polypeptide
described herein, e.g., a full-length 13245 protein or a fragment
thereof, e.g., a biologically active portion of 13245 protein. Also
included is a nucleic acid fragment suitable for use as a
hybridization probe, which can be used, e.g., to a identify nucleic
acid molecule encoding a polypeptide of the invention, 13245 mRNA,
and fragments suitable for use as primers, e.g., PCR primers for
the amplification or mutation of nucleic acid molecules.
[0103] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO: 1,
or a portion thereof. In one embodiment, the nucleic acid molecule
includes sequences encoding the human 13245 protein (i.e., "the
coding region," from nucleotides 19-6178 of SEQ ID NO: 1), as well
as 5'-non-translated sequences (nucleotides 1-18 of SEQ ID NO: 1)
or 3'-non-translated sequences (nucleotides 6179-6575 of SEQ ID NO:
1). Alternatively, the nucleic acid molecule can include only the
coding region of SEQ ID NO: 1 (e.g., nucleotides 19-6178,
corresponding to SEQ ID NO: 3) and, e.g., no flanking sequences
which normally accompany the subject sequence. In another
embodiment, the nucleic acid molecule encodes a sequence
corresponding to the 2053 amino acid residue protein of SEQ ID NO:
2.
[0104] In another embodiment, an isolated nucleic acid molecule of
the invention includes a nucleic acid molecule which is a
complement of the nucleotide sequence shown in either of SEQ ID
NOs: 1 and 3, and a portion of either of these sequences. In other
embodiments, the nucleic acid molecule of the invention is
sufficiently complementary to the nucleotide sequence shown in
either of SEQ ID NOs: 1 and 3 that it can hybridize with a nucleic
acid having that sequence, thereby forming a stable duplex.
[0105] In one embodiment, an isolated nucleic acid molecule of the
invention includes a nucleotide sequence which is at least about
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% or more homologous to the entire length of the
nucleotide sequence shown in either of SEQ ID NOs: 1 and 3, or a
portion, preferably of the same length, of either of these
nucleotide sequences.
[0106] 13245 Nucleic Acid Fragments
[0107] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of one of SEQ ID NOs: 1 and 3.
For example, such a nucleic acid molecule can include a fragment
that can be used as a probe or primer or a fragment encoding a
portion of a 13245 protein, e.g., an immunogenic or biologically
active portion of a 13245 protein. A fragment can comprise
nucleotides corresponding to residues 97-360 of SEQ ID NO: 2, which
encodes a pkinase domain of human 13245, or to residues 1568-1865
of SEQ ID NO: 2, which encodes a CNH domain of human 13245. The
nucleotide sequence determined from the cloning of the 13245 gene
facilitates generation of probes and primers for use in identifying
and/or cloning other 13245 family members, or fragments thereof, as
well as 13245 homologues, or fragments thereof, from other
species.
[0108] In another embodiment, a nucleic acid includes a nucleotide
sequence that includes part, or all, of the coding region and
extends into either (or both) the 5'- or 3'-non-coding region.
Other embodiments include a fragment that includes a nucleotide
sequence encoding an amino acid fragment described herein. Nucleic
acid fragments can encode a specific domain or site described
herein or fragments thereof, particularly fragments thereof that
are at least about 250 amino acids in length. Fragments also
include nucleic acid sequences corresponding to specific amino acid
sequences described above or fragments thereof. Nucleic acid
fragments should not to be construed as encompassing those
fragments that may have been disclosed prior to the invention.
[0109] A nucleic acid fragment can include a sequence corresponding
to a domain, region, or functional site described herein. A nucleic
acid fragment can also include one or more domain, region, or
functional site described herein.
[0110] 13245 probes and primers are provided. Typically a
probe/primer is an isolated or purified oligonucleotide. The
oligonucleotide typically includes a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 7, 12
or 15, preferably about 20 or 25, more preferably about 30, 35, 40,
45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or
antisense sequence of either of SEQ ID NOs: 1 and 3, or a naturally
occurring allelic variant or mutant of either of SEQ ID NOs: 1 and
3.
[0111] In a preferred embodiment the nucleic acid is a probe which
is at least 5 or 10, and less than 200, more preferably less than
100, or less than 50, base pairs in length. It should be identical,
or differ by 1, or fewer than 5 or 10 bases, from a sequence
disclosed herein. If alignment is needed for this comparison the
sequences should be aligned for maximum homology. "Looped" out
sequences from deletions or insertions, or mismatches, are
considered differences.
[0112] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid that encodes a pkinase domain
at about amino acid residues 97 to 360 of SEQ ID NO: 2 or the
predicted CNH domain at about amino acid residues 1568 to 1865 of
SEQ ID NO: 2.
[0113] In another embodiment a set of primers is provided, e.g.,
primers suitable for use in a PCR, which can be used to amplify a
selected region of a 13245 sequence. The primers should be at least
5, 10, or 50 base pairs in length and less than 100, or less than
200, base pairs in length. The primers should be identical, or
differs by one base from a sequence disclosed herein or from a
naturally occurring variant. Primers suitable for amplifying all or
a portion of any of the following regions are provided: e.g., one
or more a pkinase domain and the predicted CNH domain, as defined
above relative to SEQ ID NO: 2.
[0114] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0115] A nucleic acid fragment encoding a "biologically active
portion of a 13245 polypeptide" can be prepared by isolating a
portion of the nucleotide sequence of either of SEQ ID NOs: 1 and
3, which encodes a polypeptide having a 13245 biological activity
(e.g., the biological activities of the 13245 proteins are
described herein), expressing the encoded portion of the 13245
protein (e.g., by recombinant expression in vitro) and assessing
the activity of the encoded portion of the 13245 protein. For
example, a nucleic acid fragment encoding a biologically active
portion of 13245 includes a pkinase domain, e.g., amino acid
residues 97 to 360 of SEQ ID NO: 2, or a CNH domain, e.g., amino
acid residues 1568 to 1865 of SEQ ID NO: 2. A nucleic acid fragment
encoding a biologically active portion of a 13245 polypeptide can
comprise a nucleotide sequence that is greater than 25 or more
nucleotides in length.
[0116] In one embodiment, a nucleic acid includes one that has a
nucleotide sequence which is greater than 260, 300, 400, 500, 600,
700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500,
3000, 4000, 5000, or 6000 or more nucleotides in length and that
hybridizes under stringent hybridization conditions with a nucleic
acid molecule having the sequence of either of SEQ ID NOs: 1 and
3.
[0117] 13245 Nucleic Acid Variants
[0118] The invention further encompasses nucleic acid molecules
having a sequence that differs from the nucleotide sequence shown
in either of SEQ ID NOs: 1 and 3. Such differences can be
attributable to degeneracy of the genetic code (i.e., differences
which result in a nucleic acid that encodes the same 13245 proteins
as those encoded by the nucleotide sequence disclosed herein). In
another embodiment, an isolated nucleic acid molecule of the
invention encodes a protein having an amino acid sequence which
differs by at least 1, but by fewer than 5, 10, 20, 50, or 100,
amino acid residues from SEQ ID NO: 2. If alignment is needed for
this comparison the sequences should be aligned for maximum
homology. "Looped" out sequences from deletions or insertions, or
mismatches, are considered differences.
[0119] Nucleic acids of the inventor can be chosen for having
codons, which are preferred, or non-preferred, for a particular
expression system. For example, the nucleic acid can be one in
which at least one codon, at preferably at least 10%, or 20% of the
codons has been altered such that the sequence is optimized for
expression in E. coli, yeast, human, insect, or CHO cells.
[0120] Nucleic acid variants can be naturally occurring, such as
allelic variants (same locus), homologs (different locus), and
orthologs (different organism) or can be non-naturally occurring.
Non-naturally occurring variants can be made by mutagenesis
techniques, including those applied to polynucleotides, cells, or
organisms. The variants can contain nucleotide substitutions,
deletions, inversions and insertions. Variation can occur in either
or both the coding and non-coding regions. The variations can
produce both conservative and non-conservative amino acid
substitutions (as compared in the encoded product).
[0121] In a preferred embodiment, the nucleic acid has a sequence
that differs from that of either of SEQ ID NOs: 1 and 3, e.g., as
follows: by at least one, but by fewer than 10, 20, 30, or 40,
nucleotide residues; or by at least one but by fewer than 1%, 5%,
10% or 20% of the nucleotide residues in the subject nucleic acid.
If necessary for this analysis the sequences should be aligned for
maximum homology. "Looped" out sequences from deletions or
insertions, or mismatches, are considered differences.
[0122] Orthologs, homologs, and allelic variants can be identified
using methods known in the art. These variants comprise a
nucleotide sequence encoding a polypeptide that is 50%, at least
about 55%, typically at least about 70-75%, more typically at least
about 80-85%, and most typically at least about 90-95% or more
identical to the nucleotide sequence shown in either of SEQ ID NOs:
1 and 3, or a fragment of either of these sequences. Such nucleic
acid molecules can readily be identified as being able to hybridize
under stringent conditions, to the nucleotide sequence shown in
either of SEQ ID NOs: 1 and 3, or a fragment of either of these
sequences. Nucleic acid molecules corresponding to orthologs,
homologs, and allelic variants of the 13245 cDNAs of the invention
can further be isolated by mapping to the same chromosome or locus
as the 13245 gene.
[0123] Preferred variants include those that are correlated with
any of the 13245 biological activities described herein, e.g.,
catalyzing formation of a covalent bond between an amino acid
residue of a protein (e.g., a serine or threonine residue) and a
phosphate moiety.
[0124] Allelic variants of 13245 (e.g., human 13245) include both
functional and non-functional proteins. Functional allelic variants
are naturally occurring amino acid sequence variants of the 13245
protein within a population that maintain the ability to mediate
any of the 13245 biological activities described herein.
[0125] Functional allelic variants will typically contain only
conservative substitution of one or more amino acids of SEQ ID NO:
2, or substitution, deletion or insertion of non-critical residues
in non-critical regions of the protein. Non-functional allelic
variants are naturally-occurring amino acid sequence variants of
the 13245 (e.g., human 13245) protein within a population that do
not have the ability to mediate any of the 13245 biological
activities described herein. Non-functional allelic variants will
typically contain a non-conservative substitution, a deletion, or
insertion, or premature truncation of the amino acid sequence of
SEQ ID NO: 2, or a substitution, insertion, or deletion in critical
residues or critical regions of the protein.
[0126] Moreover, nucleic acid molecules encoding other 13245 family
members and, thus, which have a nucleotide sequence which differs
from the 13245 sequences of either of SEQ ID NOs: 1 and 3 are
within the scope of the invention.
[0127] Antisense Nucleic Acid Molecules, Ribozymes and Modified
13245 Nucleic Acid Molecules
[0128] In another aspect, the invention features, an isolated
nucleic acid molecule that is antisense to 13245. An "antisense"
nucleic acid can include a nucleotide sequence that is
complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. The antisense
nucleic acid can be complementary to an entire 13245 coding strand,
or to only a portion thereof (e.g., the coding region of human
13245 corresponding to SEQ ID NO: 3). In another embodiment, the
antisense nucleic acid molecule is antisense to a "non-coding
region" of the coding strand of a nucleotide sequence encoding
13245 (e.g., the 5'- and 3'-non-translated regions).
[0129] An antisense nucleic acid can be designed such that it is
complementary to the entire coding region of 13245 mRNA, but more
preferably is an oligonucleotide that is antisense to only a
portion of the coding or non-coding region of 13245 mRNA. For
example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of 13245 mRNA, e.g.,
between the -10 and +10 regions of the target gene nucleotide
sequence of interest. An antisense oligonucleotide can be, for
example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, or 80 or more nucleotide residues in length.
[0130] An antisense nucleic acid of the invention can be
constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used. The antisense nucleic acid also can be
produced biologically using an expression vector into which a
nucleic acid has been sub-cloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0131] The antisense nucleic acid molecules of the invention are
typically administered to a subject (e.g., by direct injection at a
tissue site), or generated in situ such that they hybridize with or
bind to cellular mRNA and/or genomic DNA encoding a 13245 protein
to thereby inhibit expression of the protein, e.g., by inhibiting
transcription and/or translation. Alternatively, antisense nucleic
acid molecules can be modified to target selected cells and then
administered systemically. For systemic administration, antisense
molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell surface, e.g.,
by linking the antisense nucleic acid molecules to peptides or
antibodies that bind to cell surface receptors or antigens. The
antisense nucleic acid molecules can also be delivered to cells
using the vectors described herein. To achieve sufficient
intracellular concentrations of the antisense molecules, vector
constructs in which the antisense nucleic acid molecule is placed
under the control of a strong pol 11 or pol III promoter are
preferred.
[0132] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an alpha-anomeric nucleic acid
molecule. An alpha-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual beta-units, the strands run parallel to each other
(Gaultier et al., 1987, Nucl. Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987,
FEBS Lett. 215:327-330).
[0133] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. A ribozyme having specificity for a
13245-encoding nucleic acid can include one or more sequences
complementary to the nucleotide sequence of a 13245 cDNA disclosed
herein (i.e., SEQ ID NO: 1 or SEQ ID NO: 3), and a sequence having
known catalytic sequence responsible for mRNA cleavage (see, for
example, U.S. Pat. No. 5,093,246 or Haselhoff et al. (1988, Nature
334:585-591). For example, a derivative of a Tetrahymena L-19 IVS
RNA can be constructed in which the nucleotide sequence of the
active site is complementary to the nucleotide sequence to be
cleaved in a 13245-encoding mRNA (e.g., U.S. Pat. No. 4,987,071;
and U.S. Pat. No. 5,116,742). Alternatively, 13245 mRNA can be used
to select a catalytic RNA having a specific ribonuclease activity
from a pool of RNA molecules (e.g., Bartel et al., 1993, Science
261:1411-1418).
[0134] 13245 gene expression can be inhibited by targeting
nucleotide sequences complementary to the regulatory region of the
13245 (e.g., the 13245 promoter and/or enhancers) to form triple
helical structures that prevent transcription of the 13245 gene in
target cells (Helene, 1991, Anticancer Drug Des. 6:569-584; Helene,
et al., 1992, Ann. N.Y. Acad. Sci. 660:27-36; Maher, 1992,
Bioassays 14:807-815). The potential sequences that can be targeted
for triple helix formation can be increased by creating a so-called
"switchback" nucleic acid molecule. Switchback molecules are
synthesized in an alternating 5' to 3', 3' to 5' manner, such that
they hybridize with first one strand of a duplex and then the
other, eliminating the necessity for a sizeable stretch of either
purines or pyrimidines to be present on one strand of a duplex.
[0135] The invention also provides detectably labeled
oligonucleotide primer and probe molecules. Typically, such labels
are chemiluminescent, fluorescent, radioactive, or
calorimetric.
[0136] A 13245 nucleic acid molecule can be modified at the base
moiety, sugar moiety or phosphate backbone to improve, e.g., the
stability, hybridization, or solubility of the molecule. For
example, the deoxyribose phosphate backbone of the nucleic acid
molecules can be modified to generate peptide nucleic acids (Hyrup
et al., 1996, Bioorg. Med. Chem. 4:5-23). As used herein, the terms
"peptide nucleic acid" (PNA) refers to a nucleic acid mimic, e.g.,
a DNA mimic, in which the deoxyribose phosphate backbone is
replaced by a pseudopeptide backbone and only the four natural
nucleobases are retained. The neutral backbone of a PNA can allow
for specific hybridization to DNA and RNA under conditions of low
ionic strength. The synthesis of PNA oligomers can be performed
using standard solid phase peptide synthesis protocols as described
in Hyrup et al. (1996, supra; Perry-OKeefe et al., Proc. Natl.
Acad. Sci. USA 93:14670-14675).
[0137] PNAs of 13245 nucleic acid molecules can be used in
therapeutic and diagnostic applications. For example, PNAs can be
used as antisense or anti-gene agents for sequence-specific
modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs
of 13245 nucleic acid molecules can also be used in the analysis of
single base pair mutations in a gene, (e.g., by PNA-directed PCR
clamping); as `artificial restriction enzymes` when used in
combination with other enzymes, (e.g., S1 nucleases, as described
in Hyrup et al., 1996, supra); or as probes or primers for DNA
sequencing or hybridization (Hyrup et al., 1996, supra;
Perry-OKeefe, supra).
[0138] In other embodiments, the oligonucleotide can include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.
USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA
84:648-652; PCT publication number WO 88/09810) or the blood-brain
barrier (see, e.g., PCT publication number WO 89/10134). In
addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (e.g., Krol et al., 1988,
Bio-Techniques 6:958-976) or intercalating agents (e.g., Zon, 1988,
Pharm. Res. 5:539-549). To this end, the oligonucleotide can be
conjugated to another molecule, (e.g., a peptide, hybridization
triggered cross-linking agent, transport agent, or
hybridization-triggered cleavage agent).
[0139] The invention also includes molecular beacon oligonucleotide
primer and probe molecules having at least one region which is
complementary to a 13245 nucleic acid of the invention, two
complementary regions, one having a fluorophore and the other
having a quencher, such that the molecular beacon is useful for
quantitating the presence of the 13245 nucleic acid of the
invention in a sample. Molecular beacon nucleic acids are
described, for example, in U.S. Pat. No. 5,854,033, U.S. Pat. No.
5,866,336, and U.S. Pat. No. 5,876,930.
[0140] Isolated 13245 Polypeptides
[0141] In another aspect, the invention features, an isolated 13245
protein, or fragment, e.g., a biologically active portion, for use
as immunogens or antigens to raise or test (or more generally to
bind) anti-13245 antibodies. 13245 protein can be isolated from
cells or tissue sources using standard protein purification
techniques. 13245 protein or fragments thereof can be produced by
recombinant DNA techniques or synthesized chemically.
[0142] Polypeptides of the invention include those that arise as a
result of the existence of multiple genes, alternative
transcription events, alternative RNA splicing events, and
alternative translational and post-translational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same post-translational
modifications present when the polypeptide is expressed in a native
cell, or in systems which result in the alteration or omission of
post-translational modifications, e.g., glycosylation or cleavage,
present when expressed in a native cell.
[0143] In a preferred embodiment, a 13245 polypeptide has one or
more of the following characteristics:
[0144] (1) it catalyzes formation of a covalent bond within or
between an amino acid residue and a phosphate moiety;
[0145] (2) it modulates cell contractility;
[0146] (3) it modulates cell growth;
[0147] (4) it modulates cell conductivity;
[0148] (5) it modulates entry of a cell into the cell cycle;
[0149] (6) it modulates progression of a cell through the cell
cycle;
[0150] (7) it modulates mitogenesis;
[0151] (8) it modulates cell metabolism; and
[0152] (9) it modulates gene transcription;
[0153] (10) it catalyzes interconversion of phosphorylated and
non-phosphorylated forms of a GTPase, such as a Rho GTPase;
[0154] (11) it modulates cytokinesis;
[0155] (12) it modulates cell shape;
[0156] (13) it modulates cell movement (e.g., tumor
metastasis);
[0157] (14) it modulates integration of a viral genome into a host
cell genome;
[0158] (15) it modulates maintenance of a viral genome within a
host cell genome;
[0159] (16) it modulates cytological changes in a virus-infected
host cell;
[0160] (17) it modulates virus production in a virus-infected host
cell;
[0161] (18) it modulates interaction of a virion with a membrane of
a virus-infected host cell;
[0162] (19) it modulates encapsulation of a virion within a portion
of a membrane of a virus-infected host cell
[0163] (20) it has a molecular weight, amino acid composition or
other physical characteristic of a 13245 protein of SEQ ID NO:
2;
[0164] (21) it has an overall sequence similarity (identity) of at
least 60-65%, preferably at least 70%, more preferably at least 75,
80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%
or more, with a portion of SEQ ID NO: 2;
[0165] (22) it has a CNH domain which is preferably about 70%, 80%,
90%, 95%, 96%, 97%, 98%, or 99% or more, identical with amino acid
residues 1568-1865 of SEQ ID NO: 2; or
[0166] (23) it has at least one pkinase domain which is preferably
about 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more, identical
with amino acid residues 97-360 of SEQ ID NO: 2.
[0167] In a preferred embodiment, the 13245 protein or fragment
thereof differs only insubstantially, if at all, from the
corresponding sequence in SEQ ID NO: 2. In one embodiment, it
differs by at least one, but by fewer than 15, 10 or 5 amino acid
residues. In another, it differs from the corresponding sequence in
SEQ ID NO: 2 by at least one residue but fewer than 20%, 15%, 10%
or 5% of the residues differ from the corresponding sequence in SEQ
ID NO: 2 (if this comparison requires alignment the sequences
should be aligned for maximum homology. "Looped" out sequences from
deletions or insertions, or mismatches, are considered
differences). The differences are, preferably, differences or
changes at a non-essential amino acid residues or involve a
conservative substitution of one residue for another. In a
preferred embodiment the differences are not in residues 97-360 or
1568-1865 of SEQ ID NO: 2.
[0168] Other embodiments include a protein that has one or more
changes in amino acid sequence, relative to SEQ ID NO: 2 (e.g., a
change in an amino acid residue which is not essential for
activity). Such 13245 proteins differ in amino acid sequence from
SEQ ID NO: 2, yet retain biological activity.
[0169] In one embodiment, the protein includes an amino acid
sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%
or more homologous to SEQ ID NO: 2.
[0170] A 13245 protein or fragment is provided which has an amino
acid sequence which varies from SEQ ID NO: 2 in one or both of the
regions corresponding to residues 1-96, 361-1567, and 1866-2053 of
SEQ ID NO: 2 by at least one, but by fewer than 15, 10 or 5 amino
acid residues, but which does not differ from SEQ ID NO: 2 in the
region corresponding to residues 97-360 and 1568-1865 of SEQ ID NO:
2 (if this comparison requires alignment the sequences should be
aligned for maximum homology. "Looped" out sequences from deletions
or insertions, or mismatches, are considered differences). In some
embodiments the difference is at a non-essential residue or is a
conservative substitution, while in others the difference is at an
essential residue or is a non-conservative substitution.
[0171] A biologically active portion of a 13245 protein should
include the 13245 pkinase domain, the 13245 CNH domain, or both.
Moreover, other biologically active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of a native 13245 protein.
[0172] In a preferred embodiment, the 13245 protein has the amino
acid sequence SEQ ID NO: 2. In other embodiments, the 13245 protein
is substantially identical to SEQ ID NO: 2. In yet another
embodiment, the 13245 protein is substantially identical to SEQ ID
NO: 2 and retains the functional activity of the protein of SEQ ID
NO: 2.
[0173] 13245 Chimeric or Fusion Proteins
[0174] In another aspect, the invention provides 13245 chimeric or
fusion proteins. As used herein, a 13245 "chimeric protein" or
"fusion protein" includes a 13245 polypeptide linked to a non-13245
polypeptide. A "non-13245 polypeptide" refers to a polypeptide
having an amino acid sequence corresponding to a protein which is
not substantially homologous to the 13245 protein, e.g., a protein
which is different from the 13245 protein and which is derived from
the same or a different organism. The 13245 polypeptide of the
fusion protein can correspond to all or a portion e.g., a fragment
described herein of a 13245 amino acid sequence. In a preferred
embodiment, a 13245 fusion protein includes at least one or more
biologically active portions of a 13245 protein. The non-13245
polypeptide can be fused to the amino or carboxyl terminus of the
13245 polypeptide.
[0175] The fusion protein can include a moiety that has a high
affinity for a ligand. For example, the fusion protein can be a
GST-13245 fusion protein in which the 13245 sequences are fused to
the carboxyl terminus of the GST sequences. Such fusion proteins
can facilitate the purification of recombinant 13245.
Alternatively, the fusion protein can be a 13245 protein containing
a heterologous signal sequence at its amino terminus. In certain
host cells (e.g., mammalian host cells), expression and/or
secretion of 13245 can be increased through use of a heterologous
signal sequence.
[0176] Fusion proteins can include all or a part of a serum
protein, e.g., an IgG constant region, or human serum albumin.
[0177] The 13245 fusion proteins of the invention can be
incorporated into pharmaceutical compositions and administered to a
subject in vivo. The 13245 fusion proteins can be used to affect
the bioavailability of a 13245 substrate. 13245 fusion proteins can
be useful therapeutically for the treatment of disorders caused by,
for example, (i) aberrant modification or mutation of a gene
encoding a 13245 protein; (ii) mis-regulation of the 13245 gene;
and (iii) aberrant post-translational modification of a 13245
protein.
[0178] Moreover, the 13245-fusion proteins of the invention can be
used as immunogens to produce anti-13245 antibodies in a subject,
to purify 13245 ligands and in screening assays to identify
molecules that inhibit the interaction of 13245 with a 13245
substrate.
[0179] Expression vectors are commercially available that already
encode a fusion moiety (e.g., a GST polypeptide). A 13245-encoding
nucleic acid can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the 13245 protein.
[0180] Variants of 13245 Proteins
[0181] In another aspect, the invention also features a variant of
a 13245 polypeptide, e.g., which functions as an agonist (mimetics)
or as an antagonist. Variants of the 13245 proteins can be
generated by mutagenesis, e.g., discrete point mutation, the
insertion or deletion of sequences or the truncation of a 13245
protein. An agonist of the 13245 proteins can retain substantially
the same, or a subset, of the biological activities of the
naturally occurring form of a 13245 protein. An antagonist of a
13245 protein can inhibit one or more of the activities of the
naturally occurring form of the 13245 protein by, for example,
competitively modulating a 13245-mediated activity of a 13245
protein. Thus, specific biological effects can be elicited by
treatment with a variant of limited function. Preferably, treatment
of a subject with a variant having a subset of the biological
activities of the naturally occurring form of the protein has fewer
side effects in a subject relative to treatment with the naturally
occurring form of the 13245 protein.
[0182] Variants of a 13245 protein can be identified by screening
combinatorial libraries of mutants, e.g., truncation mutants, of a
13245 protein for agonist or antagonist activity.
[0183] Libraries of fragments e.g., amino-terminal,
carboxyl-terminal, or internal fragments, of a 13245 protein coding
sequence can be used to generate a variegated population of
fragments for screening and subsequent selection of variants of a
13245 protein.
[0184] Variants in which a cysteine residue is added or deleted or
in which a residue that is glycosylated is added or deleted are
particularly preferred.
[0185] Methods for screening gene products of combinatorial
libraries made by point mutations or truncation, and for screening
cDNA libraries for gene products having a selected property.
Recursive ensemble mutagenesis (REM), a technique which enhances
the frequency of functional mutants in the libraries, can be used
in combination with the screening assays to identify 13245 variants
(Arkin et al., 1992, Proc. Natl. Acad. Sci. USA 89:7811-7815;
Delgrave et al., 1993, Protein Engr. 6:327-331).
[0186] Cell based assays can be exploited to analyze a variegated
13245 library. For example, a library of expression vectors can be
transfected into a cell line, e.g., a cell line, which ordinarily
responds to 13245 in a substrate-dependent manner. The transfected
cells are then contacted with 13245 and the effect of the
expression of the mutant on signaling by the 13245 substrate can be
detected, e.g., by measuring changes in cell growth and/or
enzymatic activity. Plasmid DNA can then be recovered from the
cells that score for inhibition, or alternatively, potentiation of
signaling by the 13245 substrate, and the individual clones further
characterized.
[0187] In another aspect, the invention features a method of making
a 13245 polypeptide, e.g., a peptide having a non-wild-type
activity, e.g., an antagonist, agonist, or super agonist of a
naturally-occurring 13245 polypeptide, e.g., a naturally-occurring
13245 polypeptide. The method includes: altering the sequence of a
13245 polypeptide, e.g., altering the sequence, e.g., by
substitution or deletion of one or more residues of a non-conserved
region, a domain or residue disclosed herein, and testing the
altered polypeptide for the desired activity.
[0188] In another aspect, the invention features a method of making
a fragment or analog of a 13245 polypeptide a biological activity
of a naturally occurring 13245 polypeptide. The method includes:
altering the sequence, e.g., by substitution or deletion of one or
more residues, of a 13245 polypeptide, e.g., altering the sequence
of a non-conserved region, or a domain or residue described herein,
and testing the altered polypeptide for the desired activity.
[0189] Anti-13245 Antibodies
[0190] In another aspect, the invention provides an anti-13245
antibody. The term "antibody" as used herein refers to an
immunoglobulin molecule or immunologically active portion thereof,
i.e., an antigen-binding portion. Examples of immunologically
active portions of immunoglobulin molecules include F(ab) and
F(ab).sub.2 fragments which can be generated by treating the
antibody with an enzyme such as pepsin.
[0191] The antibody can be a polyclonal, monoclonal, recombinant,
e.g., a chimeric or humanized, fully-human, non-human, e.g.,
murine, or single chain antibody. In a preferred embodiment, it has
effector function and can fix complement. The antibody can be
coupled to a toxin or imaging agent.
[0192] A full-length 13245 protein or, antigenic peptide fragment
of 13245 can be used as an immunogen or can be used to identify
anti-13245 antibodies made with other immunogens, e.g., cells,
membrane preparations, and the like. The antigenic peptide of 13245
should include at least 8 amino acid residues of the amino acid
sequence shown in SEQ ID NO: 2 and encompasses an epitope of 13245.
Preferably, the antigenic peptide includes at least 10 amino acid
residues, more preferably at least 15 amino acid residues, even
more preferably at least 20 amino acid residues, and most
preferably at least 30 amino acid residues.
[0193] Fragments of 13245 which include about residues 195-210 of
SEQ ID NO: 2 can be used to make antibodies, e.g., for use as
immunogens or to characterize the specificity of an antibody,
against hydrophobic regions of the 13245 protein. Similarly, a
fragment of 13245 which include about residues 455-475 of SEQ ID
NO: 2 can be used to make an antibody against a hydrophilic region
of the 13245 protein.
[0194] Antibodies reactive with, or specific for, any of these
regions, or other regions or domains described herein are
provided.
[0195] Preferred epitopes encompassed by the antigenic peptide are
regions of 13245 are located on the surface of the protein, e.g.,
hydrophilic regions, as well as regions with high antigenicity. For
example, an Emini surface probability analysis of the human 13245
protein sequence can be used to indicate the regions that have a
particularly high probability of being localized to the surface of
the 13245 protein and are thus likely to constitute surface
residues useful for targeting antibody production.
[0196] In a preferred embodiment the antibody binds an epitope on
any domain or region on 13245 proteins described herein.
[0197] Chimeric, humanized, but most preferably, completely human
antibodies are desirable for applications which include repeated
administration, e.g., therapeutic treatment (and some diagnostic
applications) of human patients.
[0198] The anti-13245 antibody can be a single chain antibody. A
single-chain antibody (scFV) can be engineered (e.g., Colcher et
al., 1999, Ann. N.Y. Acad. Sci. 880:263-280; Reiter, 1996, Clin.
Cancer Res. 2:245-252). The single chain antibody can be dimerized
or multimerized to generate multivalent antibodies having
specificities for different epitopes of the same target 13245
protein.
[0199] In a preferred embodiment, the antibody has reduced or no
ability to bind an Fc receptor. For example, it can be an isotype,
subtype, fragment or other mutant, which does not support binding
to an Fc receptor, e.g., it can have a mutated or deleted Fc
receptor binding region.
[0200] An anti-13245 antibody (e.g., monoclonal antibody) can be
used to isolate 13245 by standard techniques, such as affinity
chromatography or immunoprecipitation. Moreover, an anti-13245
antibody can be used to detect 13245 protein (e.g., in a cellular
lysate or cell supernatant) in order to evaluate the abundance and
pattern of expression of the protein. Anti-13245 antibodies can be
used diagnostically to monitor protein levels in tissue as part of
a clinical testing procedure, e.g., to, for example, determine the
efficacy of a given treatment regimen. Detection can be facilitated
by coupling (i.e., physically linking) the antibody to a detectable
substance (i.e., antibody labeling). Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, beta-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0201] Recombinant Expression Vectors, Host Cells and Genetically
Engineered Cells
[0202] In another aspect, the invention includes, vectors,
preferably expression vectors, containing a nucleic acid encoding a
polypeptide described herein. As used herein, the term "vector"
refers to a nucleic acid molecule capable of transporting another
nucleic acid to which it has been linked and can include a plasmid,
cosmid or viral vector. The vector can be capable of autonomous
replication or it can integrate into a host DNA. Viral vectors
include, e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses.
[0203] A vector can include a 13245 nucleic acid in a form suitable
for expression of the nucleic acid in a host cell. Preferably the
recombinant expression vector includes one or more regulatory
sequences operatively linked to the nucleic acid sequence to be
expressed. The term "regulatory sequence" includes promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals). Regulatory sequences include those that
direct constitutive expression of a nucleotide sequence, as well as
tissue-specific regulatory and/or inducible sequences. The design
of the expression vector can depend on such factors as the choice
of the host cell to be transformed, the level of expression of
protein desired, and the like. The expression vectors of the
invention can be introduced into host cells to thereby produce
proteins or polypeptides, including fusion proteins or
polypeptides, encoded by nucleic acids as described herein (e.g.,
13245 proteins, mutant forms of 13245 proteins, fusion proteins,
and the like).
[0204] The recombinant expression vectors of the invention can be
designed for expression of 13245 proteins in prokaryotic or
eukaryotic cells. For example, polypeptides of the invention can be
expressed in E. coli, insect cells (e.g., using baculovirus
expression vectors), yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel (1990, Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego).
Alternatively, the recombinant expression vector can be transcribed
and translated in vitro, for example using T7 promoter regulatory
sequences and T7 polymerase.
[0205] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
protein to enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include
Factor Xa, thrombin and enterokinase. Typical fusion expression
vectors include pGEX (Pharmacia Biotech Inc; Smith et al., 1988,
Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and
pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione
S-transferase (GST), maltose E binding protein, or protein A,
respectively, to the target recombinant protein.
[0206] Purified fusion proteins can be used in 13245 activity
assays, (e.g., direct assays or competitive assays described in
detail below), or to generate antibodies specific for 13245
proteins. In a preferred embodiment, a fusion protein expressed in
a retroviral expression vector of the present invention can be used
to infect bone marrow cells that are subsequently transplanted into
irradiated recipients. The pathology of the subject recipient is
then examined after sufficient time has passed (e.g., six
weeks).
[0207] To maximize recombinant protein expression in E. coli, the
protein is expressed in a host bacterial strain with an impaired
capacity to proteolytically cleave the recombinant protein
(Gottesman, 1990, Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, 119-128). Another strategy is to
alter the nucleic acid sequence of the nucleic acid to be inserted
into an expression vector so that the individual codons for each
amino acid are those preferentially utilized in E. coli (Wada et
al., 1992, Nucl. Acids Res. 20:2111-2118). Such alteration of
nucleic acid sequences of the invention can be carried out by
standard DNA synthesis techniques.
[0208] The 13245 expression vector can be a yeast expression
vector, a vector for expression in insect cells, e.g., a
baculovirus expression vector, or a vector suitable for expression
in mammalian cells.
[0209] When used in mammalian cells, the expression vector's
control functions are often provided by viral regulatory elements.
For example, commonly used viral promoters are derived from
polyoma, adenovirus 2, cytomegalovirus and simian virus 40
(SV40).
[0210] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al., 1987, Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame et al., 1988,
Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto et al., 1989, EMBO J. 8:729-733) and
immunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen et
al., 1983, Cell 33:741-748), neuron-specific promoters (e.g., the
neurofilament promoter; Byrne et al., 1989, Proc. Natl. Acad. Sci.
USA 86:5473-5477), pancreas-specific promoters (Edlund et al.,
1985, Science 230:912-916), and mammary gland-specific promoters
(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European
Patent Application publication number 264,166).
Developmentally-regulated promoters are also encompassed, for
example, the murine hox promoters (Kessel et al., 1990, Science
249:374-379) and the alpha-fetoprotein promoter (Campes et al.,
1989, Genes Dev. 3:537-546).
[0211] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. Regulatory sequences
(e.g., viral promoters and/or enhancers) operatively linked to a
nucleic acid cloned in the antisense orientation can be chosen
which direct the constitutive, tissue specific or cell type
specific expression of antisense RNA in a variety of cell types.
The antisense expression vector can be in the form of a recombinant
plasmid, phagemid or attenuated virus. For a discussion of the
regulation of gene expression using antisense genes, see Weintraub,
H. et al. (1986, Trends Genet. 1:Review).
[0212] Another aspect the invention provides a host cell which
includes a nucleic acid molecule described herein, e.g., a 13245
nucleic acid molecule within a recombinant expression vector or a
13245 nucleic acid molecule containing sequences which allow it to
homologously recombine into a specific site of the host cell's
genome. The terms "host cell" and "recombinant host cell" are used
interchangeably herein. Such terms refer not only to the particular
subject cell, but also to the progeny or potential progeny of such
a cell. Because certain modifications can occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are included within the scope of the term as used herein.
[0213] A host cell can be any prokaryotic or eukaryotic cell. For
example, a 13245 protein can be expressed in bacterial cells such
as E. coli, insect cells, yeast or mammalian cells (such as Chinese
hamster ovary (CHO) cells) or COS cells. Other suitable host cells
are known to those skilled in the art.
[0214] Vector DNA can be introduced into host cells via
conventional transformation or transfection techniques. As used
herein, the terms "transformation" and "transfection" are intended
to refer to a variety of art-recognized techniques for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation.
[0215] A host cell of the invention can be used to produce (i.e.,
express) a 13245 protein. Accordingly, the invention further
provides methods for producing a 13245 protein using the host cells
of the invention. In one embodiment, the method includes culturing
the host cell of the invention (into which a recombinant expression
vector encoding a 13245 protein has been introduced) in a suitable
medium such that a 13245 protein is produced. In another
embodiment, the method further includes isolating a 13245 protein
from the medium or the host cell.
[0216] In another aspect, the invention features, a cell or
purified preparation of cells which include a 13245 transgene, or
which otherwise mal-express 13245. The cell preparation can consist
of human or non-human cells, e.g., rodent cells, e.g., mouse or rat
cells, rabbit cells, or pig cells. In preferred embodiments, the
cell or cells include a 13245 transgene, e.g., a heterologous form
of a 13245, e.g., a gene derived from humans (in the case of a
non-human cell). The 13245 transgene can be mal-expressed, e.g.,
over-expressed or under-expressed. In other preferred embodiments,
the cell or cells include a gene that mal-expresses an endogenous
13245, e.g., a gene the expression of which is disrupted, e.g., a
knockout. Such cells can serve as a model for studying disorders
that are related to mutated or mal-expressed 13245 alleles or for
use in drug screening.
[0217] In another aspect, the invention includes, a human cell,
e.g., a hematopoietic stem cell, transformed with nucleic acid that
encodes a subject 13245 polypeptide.
[0218] Also provided are cells, preferably human cells, e.g., human
hematopoietic or fibroblast cells, in which an endogenous 13245 is
under the control of a regulatory sequence that does not normally
control expression of the endogenous 13245 gene. The expression
characteristics of an endogenous gene within a cell, e.g., a cell
line or microorganism, can be modified by inserting a heterologous
DNA regulatory element into the genome of the cell such that the
inserted regulatory element is operably linked to the endogenous
13245 gene. For example, an endogenous 13245 gene that is
"transcriptionally silent," e.g., not normally expressed, or
expressed only at very low levels, can be activated by inserting a
regulatory element that is capable of promoting the expression of a
normally expressed gene product in that cell. Techniques such as
targeted homologous recombination, can be used to insert the
heterologous DNA as described (e.g., U.S. Pat. No. 5,272,071; PCT
publication number WO 91/06667).
[0219] Transgenic Animals
[0220] The invention provides non-human transgenic animals. Such
animals are useful for studying the function and/or activity of a
13245 protein and for identifying and/or evaluating modulators of
13245 activity. As used herein, a "transgenic animal" is a
non-human animal, preferably a mammal, more preferably a rodent
such as a rat or mouse, in which one or more of the cells of the
animal includes a transgene. Other examples of transgenic animals
include non-human primates, sheep, dogs, cows, goats, chickens,
amphibians, and the like. A transgene is exogenous DNA or a
rearrangement, e.g., a deletion of endogenous chromosomal DNA,
which preferably is integrated into or occurs in the genome of the
cells of a transgenic animal. A transgene can direct the expression
of an encoded gene product in one or more cell types or tissues of
the transgenic animal, other transgenes, e.g., a knockout, reduce
expression. Thus, a transgenic animal can be one in which an
endogenous 13245 gene has been altered, e.g., by homologous
recombination between the endogenous gene and an exogenous DNA
molecule introduced into a cell of the animal (e.g., an embryonic
cell of the animal, prior to development of the animal).
[0221] Intronic sequences and polyadenylation signals can also be
included in the transgene to increase the efficiency of expression
of the transgene. A tissue-specific regulatory sequence(s) can be
operably linked to a transgene of the invention to direct
expression of a 13245 protein to particular cells. A transgenic
founder animal can be identified based upon the presence of a 13245
transgene in its genome and/or expression of 13245 mRNA in tissues
or cells of the animals. A transgenic founder animal can then be
used to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene encoding a 13245 protein
can further be bred to other transgenic animals carrying other
transgenes.
[0222] 13245 proteins or polypeptides can be expressed in
transgenic animals or plants, e.g., a nucleic acid encoding the
protein or polypeptide can be introduced into the genome of an
animal. In preferred embodiments the nucleic acid is placed under
the control of a tissue specific promoter, e.g., a milk- or
egg-specific promoter, and recovered from the milk or eggs produced
by the animal. Suitable animals are mice, pigs, cows, goats, and
sheep.
[0223] The invention also includes a population of cells from a
transgenic animal, as discussed, e.g., below.
[0224] Uses
[0225] The nucleic acid molecules, proteins, protein homologues,
and antibodies described herein can be used in one or more of the
following methods: a) screening assays; b) predictive medicine
(e.g., diagnostic assays, prognostic assays, monitoring clinical
trials, and pharmacogenetics); and c) methods of treatment (e.g.,
therapeutic and prophylactic). The isolated nucleic acid molecules
of the invention can be used, for example, to express a 13245
protein (e.g., via a recombinant expression vector in a host cell
in gene therapy applications), to detect a 13245 mRNA (e.g., in a
biological sample), to detect a genetic alteration in a 13245 gene
and to modulate 13245 activity, as described further below. The
13245 proteins can be used to treat disorders characterized by
insufficient or excessive production of a 13245 substrate or
production of 13245 inhibitors. In addition, the 13245 proteins can
be used to screen for naturally occurring 13245 substrates, to
screen for drugs or compounds which modulate 13245 activity, as
well as to treat disorders characterized by insufficient or
excessive production of 13245 protein or production of 13245
protein forms which have decreased, aberrant or unwanted activity
compared to 13245 wild-type protein. Exemplary disorders include
those in which protein phosphorylation is aberrant (e.g., muscular
and myotonic dystrophies). Moreover, the anti-13245 antibodies of
the invention can be used to detect and isolate 13245 proteins,
regulate the bioavailability of 13245 proteins, and modulate 13245
activity.
[0226] A method of evaluating a compound for the ability to
interact with, e.g., bind to, a subject 13245 polypeptide is
provided. The method includes: contacting the compound with the
subject 13245 polypeptide; and evaluating the ability of the
compound to interact with, e.g., to bind or form a complex with,
the subject 13245 polypeptide. This method can be performed in
vitro, e.g., in a cell free system, or in vivo, e.g., in a
two-hybrid interaction trap assay. This method can be used to
identify naturally-occurring molecules that interact with a subject
13245 polypeptide. It can also be used to find natural or synthetic
inhibitors of a subject 13245 polypeptide. Screening methods are
discussed in more detail below.
[0227] Screening Assays
[0228] The invention provides screening methods (also referred to
herein as "assays") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., proteins, peptides,
peptidomimetics, peptoids, small molecules or other drugs) which
bind with 13245 proteins, have a stimulatory or inhibitory effect
on, for example, 13245 expression or 13245 activity, or have a
stimulatory or inhibitory effect on, for example, the expression or
activity of a 13245 substrate. Compounds thus identified can be
used to modulate the activity of target gene products (e.g., 13245
genes) in a therapeutic protocol, to elaborate the biological
function of the target gene product, or to identify compounds that
disrupt normal target gene interactions.
[0229] In one embodiment, the invention provides assays for
screening candidate or test compounds that are substrates of a
13245 protein or polypeptide or a biologically active portion
thereof. In another embodiment, the invention provides assays for
screening candidate or test compounds that bind to or modulate the
activity of a 13245 protein or polypeptide or a biologically active
portion thereof.
[0230] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries; peptoid
libraries (libraries of molecules having the functionalities of
peptides, but with a novel, non-peptide backbone which are
resistant to enzymatic degradation but which nevertheless remain
bioactive; e.g., Zuckermann et al., 1994, J. Med. Chem.
37:2678-2685); spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution; the `one-bead one-compound` library method; and
synthetic library methods using affinity chromatography selection.
The biological library and peptoid library approaches are limited
to peptide libraries, while the other four approaches are
applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds (Lam, 1997, Anticancer Drug Des.
12:145).
[0231] Examples of methods for the synthesis of molecular libraries
have been described (e.g., DeWitt et al., 1993, Proc. Natl. Acad.
Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA
91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et
al., 1993, Science 261:1303; Carrell et al., 1994, Angew. Chem.
Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed.
Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem. 37:1233).
[0232] Libraries of compounds can be presented in solution (e.g.,
Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991,
Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),
bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. No.
5,223,409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA
89:1865-1869), or on phage (Scott et al., 1990, Science
249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al.,
1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991, J.
Mol. Biol. 222:301-310; U.S. Pat. No. 5,223,409).
[0233] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a 13245 protein or biologically active portion
thereof is contacted with a test compound, and the ability of the
test compound to modulate 13245 activity is determined. Determining
the ability of the test compound to modulate 13245 activity can be
accomplished by monitoring, for example, changes in enzymatic
activity. The cell, for example, can be of mammalian origin.
[0234] The ability of the test compound to modulate 13245 binding
to a compound, e.g., a 13245 substrate, or to bind to 13245 can
also be evaluated. This can be accomplished, for example, by
coupling the compound, e.g., the substrate, with a radioisotope or
enzymatic label such that binding of the compound, e.g., the
substrate, to 13245 can be determined by detecting the labeled
compound, e.g., substrate, in a complex. Alternatively, 13245 could
be coupled with a radioisotope or enzymatic label to monitor the
ability of a test compound to modulate 13245 binding to a 13245
substrate in a complex. For example, compounds (e.g., 13245
substrates) can be labeled with .sup.125I, .sup.35S, .sup.14C, or
.sup.3H, either directly or indirectly, and the radioisotope
detected by direct counting of radio-emission or by scintillation
counting. Alternatively, compounds can be enzymatically labeled
with, for example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
[0235] The ability of a compound (e.g., a 13245 substrate) to
interact with 13245 with or without the labeling of any of the
interactants can be evaluated. For example, a microphysiometer can
be used to detect the interaction of a compound with 13245 without
the labeling of either the compound or the 13245 (McConnell et al.,
1992, Science 257:1906-1912). As used herein, a "microphysiometer"
(e.g., Cytosensor) is an analytical instrument that measures the
rate at which a cell acidifies its environment using a
light-addressable potentiometric sensor (LAPS). Changes in this
acidification rate can be used as an indicator of the interaction
between a compound and 13245.
[0236] In yet another embodiment, a cell-free assay is provided in
which a 13245 protein or biologically active portion thereof is
contacted with a test compound and the ability of the test compound
to bind to the 13245 protein or biologically active portion thereof
is evaluated. Preferred biologically active portions of the 13245
proteins to be used in assays of the present invention include
fragments that participate in interactions with non-13245
molecules, e.g., fragments with high surface probability
scores.
[0237] Soluble and/or membrane-bound forms of isolated proteins
(e.g., 13245 proteins or biologically active portions thereof) can
be used in the cell-free assays of the invention. When
membrane-bound forms of the protein are used, it can be desirable
to utilize a solubilizing agent. Examples of such solubilizing
agents include non-ionic detergents such as n-octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton.RTM. X-100, Triton.RTM. X-114,
Thesit.RTM., Isotridecypoly(ethylene glycol ether)n,
3-{(3-cholamidopropyl)dimethylamminio}-propane sulfonate (CHAPS),
3-{(3-cholamidopropyl)dimethylamminio}-2-hydroxy-1-propane
sulfonate (CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane
sulfonate.
[0238] Cell-free assays involve preparing a reaction mixture of the
target gene protein and the test compound under conditions and for
a time sufficient to allow the two components to interact and bind,
thus forming a complex that can be removed and/or detected.
[0239] The interaction between two molecules can also be detected,
e.g., using fluorescence energy transfer (FET; e.g., U.S. Pat. No.
5,631,169; U.S. Pat. No. 4,868,103). A fluorophore label is
selected such that a first donor molecule's emitted fluorescent
energy will be absorbed by a fluorescent label on a second,
`acceptor` molecule, which in turn is able to fluoresce due to the
absorbed energy. Alternately, the `donor` protein molecule can
simply utilize the natural fluorescent energy of tryptophan
residues. Labels are chosen that emit different wavelengths of
light, such that the `acceptor` molecule label can be
differentiated from that of the `donor`. Since the efficiency of
energy transfer between the labels is related to the distance
separating the molecules, the spatial relationship between the
molecules can be assessed. In a situation in which binding occurs
between the molecules, the fluorescent emission of the `acceptor`
molecule label in the assay should be maximal. An FET binding event
can be conveniently measured through standard fluorometric
detection means well known in the art (e.g., using a
fluorimeter).
[0240] In another embodiment, determining the ability of the 13245
protein to bind to a target molecule can be accomplished using
real-time biomolecular interaction analysis (BIA; e.g., Sjolander
et al., 1991, Anal. Chem. 63:2338-2345; Szabo et al., 1995, Curr.
Opin. Struct. Biol. 5:699-705). "Surface plasmon resonance" (SPR)
or "BIA" detects biospecific interactions in real time, without
labeling any of the interactants (e.g., BIAcore). Changes in the
mass at the binding surface (indicative of a binding event) result
in alterations of the refractive index of light near the surface
(the optical phenomenon of SPR), resulting in a detectable signal
that can be used as an indication of real-time reactions between
biological molecules.
[0241] In one embodiment, the target gene product or the test
substance is anchored onto a solid phase. The target gene
product/test compound complexes anchored on the solid phase can be
detected at the end of the reaction. Preferably, the target gene
product can be anchored onto a solid surface, and the test
compound, (which is not anchored), can be labeled, either directly
or indirectly, with detectable labels discussed herein.
[0242] It can be desirable to immobilize either 13245, an
anti-13245 antibody or its target molecule to facilitate separation
of complexed from non-complexed forms of one or both of the
proteins, as well as to accommodate automation of the assay.
Binding of a test compound to a 13245 protein, or interaction of a
13245 protein with a target molecule in the presence and absence of
a candidate compound, can be accomplished in any vessel suitable
for containing the reactants. Examples of such vessels include
microtiter plates, test tubes, and micro-centrifuge tubes. In one
embodiment, a fusion protein can be provided which adds a domain
that allows one or both of the proteins to be bound to a matrix.
For example, glutathione-S-transferase/13245 fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione Sepharose.TM. beads (Sigma Chemical, St. Louis,
Mo.) or glutathione-derivatized microtiter plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or 13245 protein, and the mixture
incubated under conditions conducive for complex formation (e.g.,
at physiological conditions for salt and pH). Following incubation,
the beads or microtiter plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described above. Alternatively, the complexes can be dissociated
from the matrix, and the level of 13245 binding or activity
determined using standard techniques.
[0243] Other techniques for immobilizing either a 13245 protein or
a target molecule on matrices include using conjugation of biotin
and streptavidin. Biotinylated 13245 protein or target molecules
can be prepared from biotin-N-hydroxy-succinimide using techniques
known in the art (e.g., biotinylation kit, Pierce Chemicals,
Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
[0244] In order to conduct the assay, the non-immobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, non-reacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously non-immobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
non-immobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the immobilized component (the
antibody, in turn, can be directly labeled or indirectly labeled
with, e.g., a labeled anti-Ig antibody).
[0245] In one embodiment, this assay is performed utilizing
antibodies reactive with 13245 protein or target molecules but
which do not interfere with binding of the 13245 protein to its
target molecule. Such antibodies can be derivatized to the wells of
the plate, and unbound target or 13245 protein trapped in the wells
by antibody conjugation. Methods for detecting such complexes, in
addition to those described above for the GST-immobilized
complexes, include immunodetection of complexes using antibodies
reactive with the 13245 protein or target molecule, as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the 13245 protein or target molecule.
[0246] Alternatively, cell free assays can be conducted in a liquid
phase. In such an assay, the reaction products are separated from
non-reacted components, by any of a number of standard techniques,
including, but not limited to: differential centrifugation (e.g.,
Rivas et al., 1993, Trends Biochem. Sci. 18:284-287);
chromatography (e.g., gel filtration chromatography or ion-exchange
chromatography); electrophoresis (e.g., Ausubel et al., eds., 1999,
Current Protocols in Molecular Biology, J. Wiley, New York); and
immunoprecipitation (e.g., Ausubel, supra). Such resins and
chromatographic techniques are known to one skilled in the art
(e.g., Heegaard, 1998, J. Mol. Recognit. 11:141-148; Hage et al.,
1997, J. Chromatogr. B Biomed. Sci. Appl. 699:499-525). Further,
fluorescence energy transfer can also be conveniently utilized, as
described herein, to detect binding without further purification of
the complex from solution.
[0247] In a preferred embodiment, the assay includes contacting the
13245 protein or biologically active portion thereof with a known
compound which binds 13245 to form an assay mixture, contacting the
assay mixture with a test compound, and determining the ability of
the test compound to interact with a 13245 protein, wherein
determining the ability of the test compound to interact with a
13245 protein includes determining the ability of the test compound
to preferentially bind to 13245 or biologically active portion
thereof, or to modulate the activity of a target molecule, as
compared to the known compound.
[0248] The target gene products of the invention can, in vivo,
interact with one or more cellular or extracellular macromolecules,
such as proteins. For the purposes of this discussion, such
cellular and extracellular macromolecules are referred to herein as
"binding partners." Compounds that disrupt such interactions can be
useful in regulating the activity of the target gene product. Such
compounds can include, but are not limited to molecules such as
antibodies, peptides, and small molecules. The preferred target
genes/products for use in this embodiment are the 13245 genes
herein identified. In an alternative embodiment, the invention
provides methods for determining the ability of the test compound
to modulate the activity of a 13245 protein through modulation of
the activity of a downstream effector of a 13245 target molecule.
For example, the activity of the effector molecule on an
appropriate target can be determined, or the binding of the
effector to an appropriate target can be determined, as previously
described.
[0249] To identify compounds that interfere with the interaction
between the target gene product and its cellular or extracellular
binding partner(s), a reaction mixture containing the target gene
product and the binding partner is prepared, under conditions and
for a time sufficient, to allow the two products to form complex.
In order to test an inhibitory agent, the reaction mixture is
provided in the presence and absence of the test compound. The test
compound can be initially included in the reaction mixture, or can
be added at a time subsequent to the addition of the target gene
and its cellular or extracellular binding partner. Control reaction
mixtures are incubated without the test compound or with a placebo.
The formation of any complexes between the target gene product and
the cellular or extracellular binding partner is then detected. The
formation of a complex in the control reaction, but not in the
reaction mixture containing the test compound, indicates that the
compound interferes with the interaction of the target gene product
and the interactive binding partner. Additionally, complex
formation within reaction mixtures containing the test compound and
normal target gene product can also be compared to complex
formation within reaction mixtures containing the test compound and
mutant target gene product. This comparison can be important in
those cases wherein it is desirable to identify compounds that
disrupt interactions of mutant but not normal target gene
products.
[0250] These assays can be conducted in a heterogeneous or
homogeneous format. Heterogeneous assays involve anchoring either
the target gene product or the binding partner onto a solid phase,
and detecting complexes anchored on the solid phase at the end of
the reaction. In homogeneous assays, the entire reaction is carried
out in a liquid phase. In either approach, the order of addition of
reactants can be varied to obtain different information about the
compounds being tested. For example, test compounds that interfere
with the interaction between the target gene products and the
binding partners, e.g., by competition, can be identified by
conducting the reaction in the presence of the test substance.
Alternatively, test compounds that disrupt preformed complexes,
e.g., compounds with higher binding constants that displace one of
the components from the complex, can be tested by adding the test
compound to the reaction mixture after complexes have been formed.
The various formats are briefly described below.
[0251] In a heterogeneous assay system, either the target gene
product or the interactive cellular or extracellular binding
partner, is anchored onto a solid surface (e.g., a microtiter
plate), while the non-anchored species is labeled, either directly
or indirectly. The anchored species can be immobilized by
non-covalent or covalent attachments. Alternatively, an immobilized
antibody specific for the species to be anchored can be used to
anchor the species to the solid surface.
[0252] In order to conduct the assay, the partner of the
immobilized species is exposed to the coated surface with or
without the test compound. After the reaction is complete,
non-reacted components are removed (e.g., by washing) and any
complexes formed will remain immobilized on the solid surface.
Where the non-immobilized species is pre-labeled, the detection of
label immobilized on the surface indicates that complexes were
formed. Where the non-immobilized species is not pre-labeled, an
indirect label can be used to detect complexes anchored on the
surface; e.g., using a labeled antibody specific for the initially
non-immobilized species (the antibody, in turn, can be directly
labeled or indirectly labeled with, e.g., a labeled anti-Ig
antibody). Depending upon the order of addition of reaction
components, test compounds that inhibit complex formation or that
disrupt preformed complexes can be detected.
[0253] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from non-reacted components, and complexes
detected; e.g., using an immobilized antibody specific for one of
the binding components to anchor any complexes formed in solution,
and a labeled antibody specific for the other partner to detect
anchored complexes. Again, depending upon the order of addition of
reactants to the liquid phase, test compounds that inhibit complex
or that disrupt preformed complexes can be identified.
[0254] In an alternate embodiment of the invention, a homogeneous
assay can be used. For example, a preformed complex of the target
gene product and the interactive cellular or extracellular binding
partner product is prepared in that either the target gene products
or their binding partners are labeled, but the signal generated by
the label is quenched due to complex formation (e.g., U.S. Pat. No.
4,109,496 that utilizes this approach for immunoassays). The
addition of a test substance that competes with and displaces one
of the species from the preformed complex will result in the
generation of a signal above background. In this way, test
substances that disrupt target gene product-binding partner
interaction can be identified.
[0255] In yet another aspect, the 13245 proteins can be used as
"bait proteins" in a two-hybrid assay or three-hybrid assay (e.g.,
U.S. Pat. No. 5,283,317; Zervos et al., 1993, Cell 72:223-232;
Madura et al., 1993, J. Biol. Chem. 268:12046-12054; Bartel et al.,
1993, Biotechniques 14:920-924; Iwabuchi et al., 1993, Oncogene
8:1693-1696; PCT publication number WO 94/10300), to identify other
proteins, which bind to or interact with 13245 ("13245-binding
proteins" or "13245-bp") and are involved in 13245 activity. Such
13245-bps can be activators or inhibitors of signals by the 13245
proteins or 13245 targets as, for example, downstream elements of a
13245-mediated signaling pathway.
[0256] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a 13245
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
(Alternatively, the 13245 protein can be fused to the activator
domain). If the "bait" and the "prey" proteins are able to interact
in vivo forming a 13245-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) that is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene that encodes the protein that interacts with
the 13245 protein.
[0257] In another embodiment, modulators of 13245 expression are
identified. For example, a cell or cell free mixture is contacted
with a candidate compound and the expression of 13245 mRNA or
protein evaluated relative to the level of expression of 13245 mRNA
or protein in the absence of the candidate compound. When
expression of 13245 mRNA or protein is greater in the presence of
the candidate compound than in its absence, the candidate compound
is identified as a stimulator of 13245 mRNA or protein expression.
Alternatively, when expression of 13245 mRNA or protein is less
(i.e., statistically significantly less) in the presence of the
candidate compound than in its absence, the candidate compound is
identified as an inhibitor of 13245 mRNA or protein expression. The
level of 13245 mRNA or protein expression can be determined by
methods described herein for detecting 13245 mRNA or protein.
[0258] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a cell
free assay, and the ability of the agent to modulate the activity
of a 13245 protein can be confirmed in vivo, e.g., in an animal
such as an animal model for a disease.
[0259] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein (e.g., a 13245 modulating agent, an antisense
13245 nucleic acid molecule, a 13245-specific antibody, or a
13245-binding partner) in an appropriate animal model to determine
the efficacy, toxicity, side effects, or mechanism of action, of
treatment with such an agent. Furthermore, novel agents identified
by the above-described screening assays can be used for treatments
as described herein.
[0260] Detection Assays
[0261] Portions or fragments of the nucleic acid sequences
identified herein can be used as polynucleotide reagents. For
example, these sequences can be used to: (i) map their respective
genes on a chromosome, e.g., to locate gene regions associated with
genetic disease or to associate 13245 with a disease; (ii) identify
an individual from a minute biological sample (tissue typing); and
(iii) aid in forensic identification of a biological sample. These
applications are described in the subsections below.
[0262] Chromosome Mapping
[0263] The 13245 nucleotide sequences or portions thereof can be
used to map the location of the 13245 genes on a chromosome. This
process is called chromosome mapping. Chromosome mapping is useful
in correlating the 13245 sequences with genes associated with
disease.
[0264] Briefly, 13245 genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 base pairs in length) from
the 13245 nucleotide sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 3).
These primers can then be used for PCR screening of somatic cell
hybrids containing individual human chromosomes. Only those hybrids
containing the human gene corresponding to the 13245 sequences will
yield an amplified fragment.
[0265] A panel of somatic cell hybrids in which each cell line
contains either a single human chromosome or a small number of
human chromosomes, and a full set of mouse chromosomes, can allow
easy mapping of individual genes to specific human chromosomes
(DEustachio et al., 1983, Science 220:919-924).
[0266] Other mapping strategies e.g., in situ hybridization as
described (Fan et al., 1990, Proc. Natl. Acad. Sci. USA
87:6223-6227), pre-screening with labeled flow-sorted chromosomes,
and pre-selection by hybridization to chromosome specific cDNA
libraries can be used to map 13245 to a chromosomal location.
[0267] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. The FISH technique can be
used with a DNA sequence as short as 500 or 600 bases. However,
clones larger than 1,000 bases have a higher likelihood of binding
to a unique chromosomal location with sufficient signal intensity
for simple detection. Preferably 1,000 bases, and more preferably
2,000 bases will suffice to get good results at a reasonable amount
of time. For a review of FISH, see Verma et al. (1988, Human
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New
York).
[0268] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to non-coding regions
of the genes are typically preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0269] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data (such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), as described (e.g.,
Egeland et al., 1987, Nature, 325:783-787).
[0270] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the 13245 gene, can be determined. If a mutation is observed in
some or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
[0271] Tissue Typing
[0272] 13245 sequences can be used to identify individuals from
biological samples using, e.g., restriction fragment length
polymorphism (RFLP). In this technique, an individual's genomic DNA
is digested with one or more restriction enzymes, the fragments
separated, e.g., in a Southern blot, and probed to yield bands for
identification. The sequences of the present invention are useful
as additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[0273] Furthermore, the sequences of the present invention can also
be used to determine the actual base-by-base DNA sequence of
selected portions of an individual's genome. Thus, the 13245
nucleotide sequence described herein can be used to prepare PCR
primers homologous to the 5'- and 3'-ends of the sequence. These
primers can then be used to amplify an individual's DNA and
subsequently sequence it. Panels of corresponding DNA sequences
from individuals, prepared in this manner, can provide unique
individual identifications, as each individual will have a unique
set of such DNA sequences due to allelic differences.
[0274] Allelic variation occurs to some degree in the coding
regions of these sequences, and to a greater degree in the
non-coding regions. Each of the sequences described herein can, to
some degree, be used as a standard against which DNA from an
individual can be compared for identification purposes. Because
greater numbers of polymorphisms occur in the non-coding regions,
fewer sequences are necessary to differentiate individuals. The
non-coding sequences of SEQ ID NO: 1 can provide positive
individual identification with a panel of perhaps 10 to 1,000
primers which each yield a non-coding amplified sequence of 100
bases. If predicted coding sequences are used, such as those in SEQ
ID NO: 3, a more appropriate number of primers for positive
individual identification would be 500-2,000.
[0275] If a panel of reagents from 13245 nucleotide sequences
described herein is used to generate a unique identification
database for an individual, those same reagents can later be used
to identify tissue from that individual. Using the unique
identification database, positive identification of the individual,
living or dead, can be made from extremely small tissue
samples.
[0276] Use of Partial 13245 Sequences in Forensic Biology
[0277] DNA-based identification techniques can also be used in
forensic biology. To make such an identification, PCR technology
can be used to amplify DNA sequences taken from very small
biological samples such as tissues, e.g., hair or skin, or body
fluids, e.g., blood, saliva, or semen found at a crime scene. The
amplified sequence can then be compared to a standard, thereby
allowing identification of the origin of the biological sample.
[0278] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e., another DNA
sequence that is unique to a particular individual). As mentioned
above, actual nucleotide sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to
non-coding regions of SEQ ID NO: 1 (e.g., fragments having a length
of at least 20 nucleotide residues, preferably at least 30
nucleotide residues) are particularly appropriate for this use.
[0279] The 13245 nucleotide sequences described herein can further
be used to provide polynucleotide reagents, e.g., labeled or
label-able probes which can be used in, for example, an in situ
hybridization technique, to identify a specific tissue, e.g., a
tissue containing hematopoietic cells. This can be very useful in
cases where a forensic pathologist is presented with a tissue of
unknown origin. Panels of such 13245 probes can be used to identify
tissue by species and/or by organ type.
[0280] In a similar fashion, these reagents, e.g., 13245 primers or
probes can be used to screen tissue culture for contamination
(i.e., to screen for the presence of a mixture of different types
of cells in a culture).
[0281] Predictive Medicine
[0282] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual.
[0283] Generally, the invention provides a method of determining if
a subject is at risk for a disorder related to a lesion in, or the
malexpression of, a gene that encodes a 13245 polypeptide.
[0284] Such disorders include, e.g., a disorder associated with the
malexpression of a 13245 polypeptide, e.g., an immune disorder or a
neoplastic disorder.
[0285] The method includes one or more of the following:
[0286] (i) detecting, in a tissue of the subject, the presence or
absence of a mutation which affects the expression of the 13245
gene, or detecting the presence or absence of a mutation in a
region which controls the expression of the gene, e.g., a mutation
in the 5'-control region;
[0287] (ii) detecting, in a tissue of the subject, the presence or
absence of a mutation which alters the structure of the 13245
gene;
[0288] (iii) detecting, in a tissue of the subject, the
malexpression of the 13245 gene at the mRNA level, e.g., detecting
a non-wild-type level of a mRNA; and
[0289] (iv) detecting, in a tissue of the subject, the
malexpression of the gene at the protein level, e.g., detecting a
non-wild-type level of a 13245 polypeptide.
[0290] In preferred embodiments the method includes: ascertaining
the existence of at least one of: a deletion of one or more
nucleotides from the 13245 gene; an insertion of one or more
nucleotides into the gene, a point mutation, e.g., a substitution
of one or more nucleotides of the gene, a gross chromosomal
rearrangement of the gene, e.g., a translocation, inversion, or
deletion.
[0291] For example, detecting the genetic lesion can include: (i)
providing a probe/primer including an oligonucleotide containing a
region of nucleotide sequence which hybridizes to a sense or
antisense sequence from SEQ ID NO: 1, or naturally occurring
mutants thereof, or 5'- or 3'-flanking sequences naturally
associated with the 13245 gene; (ii) exposing the probe/primer to
nucleic acid of the tissue; and detecting the presence or absence
of the genetic lesion by hybridization of the probe/primer to the
nucleic acid, e.g., by in situ hybridization.
[0292] In preferred embodiments, detecting the malexpression
includes ascertaining the existence of at least one of: an
alteration in the level of a messenger RNA transcript of the 13245
gene; the presence of a non-wild-type splicing pattern of a
messenger RNA transcript of the gene; or a non-wild-type level of
13245 RNA or protein.
[0293] Methods of the invention can be used for prenatal screening
or to determine if a subject's offspring will be at risk for a
disorder.
[0294] In preferred embodiments the method includes determining the
structure of a 13245 gene, an abnormal structure being indicative
of risk for the disorder.
[0295] In preferred embodiments the method includes contacting a
sample form the subject with an antibody to the 13245 protein or a
nucleic acid, which hybridizes specifically with the gene. These
and other embodiments are discussed below.
[0296] Diagnostic and Prognostic Assays
[0297] The presence, level, or absence of 13245 protein or nucleic
acid in a biological sample can be evaluated by obtaining a
biological sample from a test subject and contacting the biological
sample with a compound or an agent capable of detecting 13245
protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes
13245 protein such that the presence of 13245 protein or nucleic
acid is detected in the biological sample. The term "biological
sample" includes tissues, cells and biological fluids isolated from
a subject, as well as tissues, cells and fluids present within a
subject. A preferred biological sample is serum. The level of
expression of the 13245 gene can be measured in a number of ways,
including, but not limited to: measuring the mRNA encoded by the
13245 genes; measuring the amount of protein encoded by the 13245
genes; or measuring the activity of the protein encoded by the
13245 genes.
[0298] The level of mRNA corresponding to the 13245 gene in a cell
can be determined both by in situ and by in vitro formats.
[0299] The isolated mRNA can be used in hybridization or
amplification assays that include, but are not limited to, Southern
or Northern analyses, polymerase chain reaction analyses and probe
arrays. One preferred diagnostic method for the detection of mRNA
levels involves contacting the isolated mRNA with a nucleic acid
molecule (probe) that can hybridize to the mRNA encoded by the gene
being detected. The nucleic acid probe can be, for example, a
full-length 13245 nucleic acid, such as the nucleic acid of SEQ ID
NO: 1, or a portion thereof, such as an oligonucleotide of at least
7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient
to specifically hybridize under stringent conditions to 13245 mRNA
or genomic DNA. Other suitable probes for use in the diagnostic
assays are described herein.
[0300] In one format, mRNA (or cDNA) is immobilized on a surface
and contacted with the probes, for example by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probes are immobilized on a surface and the mRNA (or cDNA) is
contacted with the probes, for example, in a two-dimensional gene
chip array. A skilled artisan can adapt known mRNA detection
methods for use in detecting the level of mRNA encoded by the 13245
genes.
[0301] The level of mRNA in a sample that is encoded by 13245 can
be evaluated with nucleic acid amplification, e.g., by RT-PCR (U.S.
Pat. No. 4,683,202), ligase chain reaction (Barany, 1991, Proc.
Natl. Acad. Sci. USA 88:189-193), self-sustained sequence
replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA
87:1874-1878), transcriptional amplification system (Kwoh et al.,
1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase
(Lizardi et al., 1988, Bio/Technology 6:1197), rolling circle
replication (U.S. Pat. No. 5,854,033) or any other nucleic acid
amplification method, followed by the detection of the amplified
molecules using techniques known in the art. As used herein,
amplification primers are defined as being a pair of nucleic acid
molecules that can anneal to 5'- or 3'-regions of a 13245 gene
(plus and minus strands, respectively, or vice-versa) and contain a
short region in between. In general, amplification primers are from
about 10 to 30 nucleotides in length and flank a region from about
50 to 200 nucleotides in length. Under appropriate conditions and
with appropriate reagents, such primers permit the amplification of
a nucleic acid molecule comprising the nucleotide sequence between
the primers.
[0302] For in situ methods, a cell or tissue sample can be
prepared/processed and immobilized on a support, typically a glass
slide, and then contacted with a probe that can hybridize to mRNA
that encodes the 13245 gene being analyzed.
[0303] In another embodiment, the methods include further
contacting a control sample with a compound or agent capable of
detecting 13245 mRNA, or genomic DNA, and comparing the presence of
13245 mRNA or genomic DNA in the control sample with the presence
of 13245 mRNA or genomic DNA in the test sample.
[0304] A variety of methods can be used to determine the level of
protein encoded by 13245. In general, these methods include
contacting an agent that selectively binds to the protein, such as
an antibody with a sample, to evaluate the level of protein in the
sample. In a preferred embodiment, the antibody bears a detectable
label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab').sub.2) can be used. The term "labeled," with regard to the
probe or antibody, is intended to encompass direct labeling of the
probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with a detectable
substance. Examples of detectable substances are provided
herein.
[0305] The detection methods can be used to detect 13245 protein in
a biological sample in vitro as well as in vivo. In vitro
techniques for detection of 13245 protein include enzyme linked
immunosorbent assays (ELISAs), immunoprecipitations,
immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay
(RIA), and Western blot analysis. In vivo techniques for detection
of 13245 protein include introducing into a subject a labeled
anti-13245 antibody. For example, the antibody can be labeled with
a radioactive marker whose presence and location in a subject can
be detected by standard imaging techniques.
[0306] In another embodiment, the methods further include
contacting the control sample with a compound or agent capable of
detecting 13245 protein, and comparing the presence of 13245
protein in the control sample with the presence of 13245 protein in
the test sample.
[0307] The invention also includes kits for detecting the presence
of 13245 in a biological sample. For example, the kit can include a
compound or agent capable of detecting 13245 protein or mRNA in a
biological sample, and a standard. The compound or agent can be
packaged in a suitable container. The kit can further comprise
instructions for using the kit to detect 13245 protein or nucleic
acid.
[0308] For antibody-based kits, the kit can include: (1) a first
antibody (e.g., attached to a solid support) which binds to a
polypeptide corresponding to a marker of the invention; and,
optionally, (2) a second, different antibody which binds to either
the polypeptide or the first antibody and is conjugated to a
detectable agent.
[0309] For oligonucleotide-based kits, the kit can include: (1) an
oligonucleotide, e.g., a detectably-labeled oligonucleotide, which
hybridizes to a nucleic acid sequence encoding a polypeptide
corresponding to a marker of the invention or (2) a pair of primers
useful for amplifying a nucleic acid molecule corresponding to a
marker of the invention. The kit can also includes a buffering
agent, a preservative, or a protein-stabilizing agent. The kit can
also includes components necessary for detecting the detectable
agent (e.g., an enzyme or a substrate). The kit can also contain a
control sample or a series of control samples that can be assayed
and compared to the test sample contained. Each component of the
kit can be enclosed within an individual container and all of the
various containers can be within a single package, along with
instructions for interpreting the results of the assays performed
using the kit.
[0310] The diagnostic methods described herein can identify
subjects having, or at risk of developing, a disease or disorder
associated with malexpressed, aberrant or unwanted 13245 expression
or activity. As used herein, the term "unwanted" includes an
unwanted phenomenon involved in a biological response such as
induction of an inappropriate immune response or deregulated cell
proliferation.
[0311] In one embodiment, a disease or disorder associated with
aberrant or unwanted 13245 expression or activity is identified. A
test sample is obtained from a subject and 13245 protein or nucleic
acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level,
e.g., the presence or absence, of 13245 protein or nucleic acid is
diagnostic for a subject having or at risk of developing a disease
or disorder associated with aberrant or unwanted 13245 expression
or activity. As used herein, a "test sample" refers to a biological
sample obtained from a subject of interest, including a biological
fluid (e.g., serum), cell sample, or tissue.
[0312] The prognostic assays described herein can be used to
determine whether a subject can be administered an agent (e.g., an
agonist, antagonist, peptidomimetic, protein, peptide, nucleic
acid, small molecule, or other drug candidate) to treat a disease
or disorder associated with aberrant or unwanted 13245 expression
or activity. For example, such methods can be used to determine
whether a subject can be effectively treated with an agent that
modulates 13245 expression or activity.
[0313] The methods of the invention can also be used to detect
genetic alterations in a 13245 gene, thereby determining if a
subject with the altered gene is at risk for a disorder
characterized by misregulation in 13245 protein activity or nucleic
acid expression, such as a disorder associated with tumorigenesis
or induction of an inappropriate immune response. In preferred
embodiments, the methods include detecting, in a sample from the
subject, the presence or absence of a genetic alteration
characterized by at least one of an alteration affecting the
integrity of a gene encoding a 13245 protein, or the malexpression
of the 13245 gene. For example, such genetic alterations can be
detected by ascertaining the existence of at least one of 1) a
deletion of one or more nucleotides from a 13245 gene; 2) an
addition of one or more nucleotides to a 13245 gene; 3) a
substitution of one or more nucleotides of a 13245 gene, 4) a
chromosomal rearrangement of a 13245 gene; 5) an alteration in the
level of a messenger RNA transcript of a 13245 gene, 6) aberrant
modification of a 13245 gene, such as of the methylation pattern of
the genomic DNA, 7) the presence of a non-wild-type splicing
pattern of a messenger RNA transcript of a 13245 gene, 8) a
non-wild-type level of a 13245 protein, 9) allelic loss of a 13245
gene, and 10) inappropriate post-translational modification of a
13245 protein.
[0314] An alteration can be detected without a probe/primer in a
polymerase chain reaction, such as anchor PCR or RACE-PCR, or,
alternatively, in a ligation chain reaction (LCR), the latter of
which can be particularly useful for detecting point mutations in
the 13245 gene. This method can include the steps of collecting a
sample of cells from a subject, isolating nucleic acid (e.g.,
genomic, mRNA or both) from the sample, contacting the nucleic acid
sample with one or more primers which specifically hybridize to a
13245 gene under conditions such that hybridization and
amplification of the 13245 gene occurs (if present), and detecting
the presence or absence of an amplification product, or detecting
the size of the amplification product and comparing the length to a
control sample. It is anticipated that PCR and/or LCR can be
desirable to use as a preliminary amplification step in conjunction
with any of the techniques used for detecting mutations described
herein.
[0315] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh et
al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), or other
nucleic acid amplification methods, followed by the detection of
the amplified molecules using techniques known to those of skill in
the art.
[0316] In another embodiment, mutations in a 13245 gene from a
sample cell can be identified by detecting alterations in
restriction enzyme cleavage patterns. For example, sample and
control DNA is isolated, amplified (optionally), digested with one
or more restriction endonucleases, and fragment length sizes are
determined, e.g., by gel electrophoresis, and compared. Differences
in fragment length sizes between sample and control DNA indicates
mutations in the sample DNA. Moreover, the use of sequence specific
ribozymes (e.g., U.S. Pat. No. 5,498,531) can be used to score for
the presence of specific mutations by development or loss of a
ribozyme cleavage site.
[0317] In other embodiments, genetic mutations in 13245 can be
identified by hybridizing a sample to control nucleic acids, e.g.,
DNA or RNA, by, e.g., two-dimensional arrays, or, e.g., chip based
arrays. Such arrays include a plurality of addresses, each of which
is positionally distinguishable from the other. A different probe
is located at each address of the plurality. The arrays can have a
high density of addresses, e.g., can contain hundreds or thousands
of oligonucleotides probes (Cronin et al., 1996, Hum. Mutat.
7:244-255; Kozal et al., 1996, Nature Med. 2:753-759). For example,
genetic mutations in 13245 can be identified in two-dimensional
arrays containing light-generated DNA probes as described (Cronin
et al., supra). Briefly, a first hybridization array of probes can
be used to scan through long stretches of DNA in a sample and
control to identify base changes between the sequences by making
linear arrays of sequential overlapping probes. This step allows
the identification of point mutations. This step is followed by a
second hybridization array that allows the characterization of
specific mutations by using smaller, specialized probe arrays
complementary to all variants or mutations detected. Each mutation
array is composed of parallel probe sets, one complementary to the
wild-type gene and the other complementary to the mutant gene.
[0318] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
13245 gene and detect mutations by comparing the sequence of the
sample 13245 with the corresponding wild-type (control) sequence.
Automated sequencing procedures can be utilized when performing the
diagnostic assays (1995, Biotechniques 19:448), including
sequencing by mass spectrometry.
[0319] Other methods for detecting mutations in the 13245 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al., 1985, Science 230:1242; Cotton et al., 1988, Proc. Natl.
Acad. Sci. USA 85:4397; Saleeba et al., 1992, Meth. Enzymol.
217:286-295).
[0320] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in 13245
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et
al., 1994, Carcinogenesis 15:1657-1662; U.S. Pat. No.
5,459,039).
[0321] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in 13245 genes. For
example, single strand conformation polymorphism (SSCP) can be used
to detect differences in electrophoretic mobility between mutant
and wild-type nucleic acids (Orita et al., 1989, Proc. Natl. Acad.
Sci. USA 86:2766; Cotton, 1993, Mutat. Res. 285:125-144; Hayashi,
1992, Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA
fragments of sample and control 13245 nucleic acids will be
denatured and allowed to re-nature. The secondary structure of
single-stranded nucleic acids varies according to sequence, the
resulting alteration in electrophoretic mobility enables the
detection of even a single base change. The DNA fragments can be
labeled or detected with labeled probes. The sensitivity of the
assay can be enhanced by using RNA (rather than DNA), in which the
secondary structure is more sensitive to a change in sequence. In a
preferred embodiment, the subject method utilizes heteroduplex
analysis to separate double stranded heteroduplex molecules on the
basis of changes in electrophoretic mobility (Keen et al., 1991,
Trends Genet 7:5).
[0322] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al., 1985, Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 base pairs of high-melting GC-rich DNA by PCR. In
a further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem
265:12753).
[0323] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension (Saiki et al., 1986, Nature 324:163; Saiki et al., 1989,
Proc. Natl. Acad. Sci. USA 86:6230).
[0324] Alternatively, allele specific amplification technology that
depends on selective PCR amplification can be used in conjunction
with the instant invention. Oligonucleotides used as primers for
specific amplification can carry the mutation of interest in the
center of the molecule (so that amplification depends on
differential hybridization; Gibbs et al., 1989, Nucl. Acids Res.
17:2437-2448) or at the extreme 3'-end of one primer where, under
appropriate conditions, mismatch can prevent, or reduce polymerase
extension (Prossner, 1993, Tibtech 11:238). In addition, it can be
desirable to introduce a novel restriction site in the region of
the mutation to create cleavage-based detection (Gasparini et al.,
1992, Mol. Cell Probes 6:1). It is anticipated that in certain
embodiments, amplification can also be performed using Taq ligase
for amplification (Barany, 1991, Proc. Natl. Acad. Sci. USA
88:189). In such cases, ligation will occur only if there is a
perfect match at the 3'-end of the 5'-sequence making it possible
to detect the presence of a known mutation at a specific site by
looking for the presence or absence of amplification.
[0325] The methods described herein can be performed, for example,
using pre-packaged diagnostic kits comprising at least one probe
nucleic acid or antibody reagent described herein, which can be
conveniently used, e.g., in clinical settings to diagnose patients
exhibiting symptoms or family history of a disease or illness
involving a 13245 gene.
[0326] Use of 13245 Molecules as Surrogate Markers
[0327] The 13245 molecules of the invention are also useful as
markers of disorders or disease states, as markers for precursors
of disease states, as markers for predisposition of disease states,
as markers of drug activity, or as markers of the pharmacogenomic
profile of a subject. Using the methods described herein, the
presence, absence and/or quantity of the 13245 molecules of the
invention can be detected, and can be correlated with one or more
biological states in vivo. For example, the 13245 molecules of the
invention can serve as surrogate markers for one or more disorders
or disease states or for conditions leading up to disease states.
As used herein, a "surrogate marker" is an objective biochemical
marker which correlates with the absence or presence of a disease
or disorder, or with the progression of a disease or disorder
(e.g., with the presence or absence of a tumor). The presence or
quantity of such markers is independent of the disease. Therefore,
these markers can serve to indicate whether a particular course of
treatment is effective in lessening a disease state or disorder.
Surrogate markers are of particular use when the presence or extent
of a disease state or disorder is difficult to assess through
standard methodologies (e.g., early stage tumors), or when an
assessment of disease progression is desired before a potentially
dangerous clinical endpoint is reached (e.g., an assessment of
cardiovascular disease can be made using cholesterol levels as a
surrogate marker, and an analysis of HIV infection can be made
using HIV RNA levels as a surrogate marker, well in advance of the
undesirable clinical outcomes of myocardial infarction or
fully-developed AIDS). Examples of the use of surrogate markers
have been described (e.g., Koomen et al., 2000, J. Mass. Spectrom.
35:258-264; James, 1994, AIDS Treat. News Arch. 209).
[0328] The 13245 molecules of the invention are also useful as
pharmacodynamic markers. As used herein, a "pharmacodynamic marker"
is an objective biochemical marker which correlates specifically
with drug effects. The presence or quantity of a pharmacodynamic
marker is not related to the disease state or disorder for which
the drug is being administered; therefore, the presence or quantity
of the marker is indicative of the presence or activity of the drug
in a subject. For example, a pharmacodynamic marker can be
indicative of the concentration of the drug in a biological tissue,
in that the marker is either expressed or transcribed or not
expressed or transcribed in that tissue in relationship to the
level of the drug. In this fashion, the distribution or uptake of
the drug can be monitored by the pharmacodynamic marker. Similarly,
the presence or quantity of the pharmacodynamic marker can be
related to the presence or quantity of the metabolic product of a
drug, such that the presence or quantity of the marker is
indicative of the relative breakdown rate of the drug in vivo.
Pharmacodynamic markers are of particular use in increasing the
sensitivity of detection of drug effects, particularly when the
drug is administered in low doses. Since even a small amount of a
drug can be sufficient to activate multiple rounds of marker (e.g.,
a 13245 marker) transcription or expression, the amplified marker
can be in a quantity which is more readily detectable than the drug
itself. Also, the marker can be more easily detected due to the
nature of the marker itself; for example, using the methods
described herein, anti-13245 antibodies can be employed in an
immune-based detection system for a 13245 protein marker, or
13245-specific radiolabeled probes can be used to detect a 13245
mRNA marker. Furthermore, the use of a pharmacodynamic marker can
offer mechanism-based prediction of risk due to drug treatment
beyond the range of possible direct observations. Examples of the
use of pharmacodynamic markers have been described (e.g., U.S. Pat.
No. 6,033,862; Hattis et al., 1991, Env. Health Perspect. 90:
229-238; Schentag, 1999, Am. J. Health-Syst. Pharm. 56 Suppl. 3:
S21-S24; Nicolau, 1999, Am, J. Health-Syst. Pharm. 56 Suppl. 3:
S16-S20).
[0329] The 13245 molecules of the invention are also useful as
pharmacogenomic markers. As used herein, a "pharmacogenomic marker"
is an objective biochemical marker which correlates with a specific
clinical drug response or susceptibility in a subject (e.g., McLeod
et al., 1999, Eur. J. Cancer 35:1650-1652). The presence or
quantity of the pharmacogenomic marker is related to the predicted
response of the subject to a specific drug or class of drugs prior
to administration of the drug. By assessing the presence or
quantity of one or more pharmacogenomic markers in a subject, a
drug therapy which is most appropriate for the subject, or which is
predicted to have a greater degree of success, can be selected. For
example, based on the presence or quantity of RNA, or protein
(e.g., 13245 protein or RNA) for specific tumor markers in a
subject, a drug or course of treatment can be selected that is
optimized for the treatment of the specific tumor likely to be
present in the subject. Similarly, the presence or absence of a
specific sequence mutation in 13245 DNA can correlate 13245 drug
response. The use of pharmacogenomic markers therefore permits the
application of the most appropriate treatment for each subject
without having to administer the therapy.
[0330] Pharmaceutical Compositions
[0331] The nucleic acid and polypeptides, fragments thereof, as
well as anti-13245 antibodies (also referred to herein as "active
compounds") of the invention can be incorporated into
pharmaceutical compositions. Such compositions typically include
the nucleic acid molecule, protein, or antibody and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" includes solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. Supplementary active compounds can
also be incorporated into the compositions.
[0332] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e.g., inhalation), transdermal (topical),
transmucosal, and rectal administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0333] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It should be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including an agent in the composition that
delays absorption, for example, aluminum monostearate and
gelatin.
[0334] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying, which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0335] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash. Pharmaceutically compatible binding agents
and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder, such as microcrystalline cellulose, gum
tragacanth or gelatin; an excipient, such as starch or lactose; a
disintegrating agent, such as alginic acid, Primogel.TM., or corn
starch; a lubricant, such as magnesium stearate or Sterotes.TM.; a
glidant, such as colloidal silicon dioxide; a sweetening agent,
such as sucrose or saccharin; or a flavoring agent, such as
peppermint, methyl salicylate, or orange flavoring.
[0336] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser that contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0337] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0338] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0339] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells using monoclonal antibodies directed
towards viral antigens) can also be used as pharmaceutically
acceptable carriers. These can be prepared according to described
methods (e.g., U.S. Pat. No. 4,522,811).
[0340] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
[0341] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.5O/ED.sub.50. Compounds
that exhibit high therapeutic indices are preferred. While
compounds that exhibit toxic side effects can be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0342] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma can
be measured, for example, by high performance liquid
chromatography.
[0343] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 milligrams per kilogram body weight, preferably
about 0.01 to 25 milligrams per kilogram body weight, more
preferably about 0.1 to 20 milligrams per kilogram body weight, and
even more preferably about 1 to 10 milligrams per kilogram, 2 to 9
milligrams per kilogram, 3 to 8 milligrams per kilogram, 4 to 7
milligrams per kilogram, or 5 to 6 milligrams per kilogram body
weight. The protein or polypeptide can be administered one time per
week for between about 1 to 10 weeks, preferably between 2 to 8
weeks, more preferably between about 3 to 7 weeks, and even more
preferably for about 4, 5, or 6 weeks. The skilled artisan will
appreciate that certain factors can influence the dosage and timing
required to effectively treat a subject, including but not limited
to the severity of the disease or disorder, previous treatments,
the general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of a protein, polypeptide, or antibody can include
a single treatment or, preferably, can include a series of
treatments.
[0344] For antibodies, the preferred dosage is 0.1 milligrams per
kilogram of body weight (generally 10 to 20 milligrams per
kilogram). If the antibody is to act in the brain, a dosage of 50
to 100 milligrams per kilogram is usually appropriate. Generally,
partially human antibodies and fully human antibodies have a longer
half-life within the human body than other antibodies. Accordingly,
lower dosages and less frequent administration is often possible.
Modifications such as lipidation can be used to stabilize
antibodies and to enhance uptake and tissue penetration (e.g., into
the brain). A method for the lipidation of antibodies is described
by Cruikshank et al. (1997, J. AIDS Hum. Retrovir. 14:193).
[0345] The present invention encompasses agents that modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics (e.g., peptoids), amino
acids, amino acid analogs, polynucleotides, polynucleotide analogs,
nucleotides, nucleotide analogs, organic or inorganic compounds
(i.e., including hetero-organic and organo-metallic compounds)
having a molecular weight less than about 10,000 grams per mole,
organic or inorganic compounds having a molecular weight less than
about 5,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 1,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 500
grams per mole, and salts, esters, and other pharmaceutically
acceptable forms of such compounds.
[0346] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram. It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. When one or more of these small molecules is to be
administered to an animal (e.g., a human) in order to modulate
expression or activity of a polypeptide or nucleic acid of the
invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0347] An antibody (or fragment thereof) can be conjugated to a
therapeutic moiety such as a cytotoxin, a therapeutic agent or a
radioactive metal ion. A cytotoxin or cytotoxic agent includes any
agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0348] The conjugates of the invention can be used for modifying a
given biological response, and the drug moiety is not to be
construed as limited to classical chemical therapeutic agents. For
example, the drug moiety can be a protein or polypeptide possessing
a desired biological activity. Such proteins can include, for
example, a toxin such as abrin, ricin A, gelonin, pseudomonas
exotoxin, or diphtheria toxin; a protein such as tumor necrosis
factor, alpha-interferon, beta-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator; or,
biological response modifiers such as, for example, lymphokines,
interleukins-1, -2, and -6, granulocyte macrophage colony
stimulating factor, granulocyte colony stimulating factor, or other
growth factors.
[0349] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980.
[0350] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (e.g., Chen et al., 1994, Proc. Natl. Acad.
Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene
therapy vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded. Alternatively, where the complete
gene delivery vector can be produced intact from recombinant cells,
e.g., retroviral vectors, the pharmaceutical preparation can
include one or more cells which produce the gene delivery
system.
[0351] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0352] Methods of Treatment
[0353] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant or unwanted 13245 expression or activity. With regards to
both prophylactic and therapeutic methods of treatment, such
treatments can be specifically tailored or modified, based on
knowledge obtained from the field of pharmacogenomics.
"Pharmacogenomics," as used herein, refers to the application of
genomics technologies such as gene sequencing, statistical
genetics, and gene expression analysis to drugs in clinical
development and on the market. More specifically, the term refers
the study of how a patient's genes determine his or her response to
a drug (e.g., a patient's "drug response phenotype," or "drug
response genotype".) Thus, another aspect of the invention provides
methods for tailoring an individual's prophylactic or therapeutic
treatment with either the 13245 molecules of the present invention
or 13245 modulators according to that individual's drug response
genotype. Pharmacogenomics allows a clinician or physician to
target prophylactic or therapeutic treatments to patients who will
most benefit from the treatment and to avoid treatment of patients
who will experience toxic drug-related side effects.
[0354] Treatment is defined as the application or administration of
a therapeutic agent to a patient, or application or administration
of a therapeutic agent to an isolated tissue or cell line from a
patient, who has a disease, a symptom of disease or a
predisposition toward a disease, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve or affect
the disease, the symptoms of disease or the predisposition toward
disease.
[0355] A therapeutic agent includes, but is not limited to, small
molecules, peptides, antibodies, ribozymes and antisense
oligonucleotides.
[0356] In one aspect, the invention provides a method for
preventing a disease or condition in a subject associated with an
aberrant or unwanted 13245 expression or activity, by administering
to the subject a 13245 or an agent which modulates 13245
expression, or at least one 13245 activity. Subjects at risk for a
disease which is caused or contributed to by aberrant or unwanted
13245 expression or activity can be identified by, for example, any
or a combination of diagnostic or prognostic assays as described
herein. Administration of a prophylactic agent can occur prior to
the manifestation of symptoms characteristic of the 13245
aberrance, such that a disease or disorder is prevented or,
alternatively, delayed in its progression. Depending on the type of
13245 aberrance, for example, a 13245 protein, 13245 agonist or
13245 antagonist agent can be used for treating the subject. The
appropriate agent can be determined based on screening assays
described herein.
[0357] It is possible that some 13245 disorders can be caused, at
least in part, by an abnormal level of gene product, or by the
presence of a gene product exhibiting abnormal activity. As such,
the reduction in the level and/or activity of such gene products
would bring about the amelioration of disorder symptoms.
[0358] As discussed, successful treatment of 13245 disorders can be
brought about by techniques that serve to inhibit the expression or
activity of target gene products. For example, compounds, e.g., an
agent identified using an assays described above, that proves to
exhibit negative modulatory activity, can be used in accordance
with the invention to prevent and/or ameliorate symptoms of 13245
disorders. Such molecules can include, but are not limited to
peptides, phosphopeptides, small organic or inorganic molecules, or
antibodies (including, for example, polyclonal, monoclonal,
humanized, anti-idiotypic, chimeric or single chain antibodies, and
Fab, F(ab).sub.2 and Fab expression library fragments, scFV
molecules, and epitope-binding fragments thereof).
[0359] Further, antisense and ribozyme molecules that inhibit
expression of the target gene can also be used in accordance with
the invention to reduce the level of target gene expression, thus
effectively reducing the level of target gene activity. Still
further, triple helix molecules can be utilized in reducing the
level of target gene activity. Antisense, ribozyme and triple helix
molecules are discussed above.
[0360] It is possible that the use of antisense, ribozyme, and/or
triple helix molecules to reduce or inhibit mutant gene expression
can also reduce or inhibit the transcription (triple helix) and/or
translation (antisense, ribozyme) of mRNA produced by normal target
gene alleles, such that the concentration of normal target gene
product present can be lower than is necessary for a normal
phenotype. In such cases, nucleic acid molecules that encode and
express target gene polypeptides exhibiting normal target gene
activity can be introduced into cells via gene therapy method.
Alternatively, in instances in that the target gene encodes an
extracellular protein, it can be preferable to co-administer normal
target gene protein into the cell or tissue in order to maintain
the requisite level of cellular or tissue target gene activity.
[0361] Another method by which nucleic acid molecules can be
utilized in treating or preventing a disease characterized by 13245
expression is through the use of aptamer molecules specific for
13245 protein. Aptamers are nucleic acid molecules having a
tertiary structure that permits them to specifically bind to
protein ligands (e.g., Osborne et al., 1997, Curr. Opin. Chem.
Biol. 1:5-9; Patel, 1997, Curr. Opin. Chem. Biol. 1:32-46). Since
nucleic acid molecules can in many cases be more conveniently
introduced into target cells than therapeutic protein molecules can
be, aptamers offer a method by which 13245 protein activity can be
specifically decreased without the introduction of drugs or other
molecules which can have pluripotent effects.
[0362] Antibodies can be generated that are both specific for
target gene product and that reduce target gene product activity.
Such antibodies may, therefore, by administered in instances
whereby negative modulatory techniques are appropriate for the
treatment of 13245 disorders.
[0363] In circumstances wherein injection of an animal or a human
subject with a 13245 protein or epitope for stimulating antibody
production is harmful to the subject, it is possible to generate an
immune response against 13245 through the use of anti-idiotypic
antibodies (e.g., Herlyn, 1999, Ann. Med. 31:66-78;
Bhattacharya-Chatterjee et al., 1998, Cancer Treat. Res. 94:51-68).
If an anti-idiotypic antibody is introduced into a mammal or human
subject, it should stimulate the production of anti-anti-idiotypic
antibodies, which should be specific to the 13245 protein. Vaccines
directed to a disease characterized by 13245 expression can also be
generated in this fashion.
[0364] In instances where the target antigen is intracellular and
whole antibodies are used, internalizing antibodies can be
preferred. Lipofectin or liposomes can be used to deliver the
antibody or a fragment of the Fab region that binds to the target
antigen into cells. Where fragments of the antibody are used, the
smallest inhibitory fragment that binds to the target antigen is
preferred. For example, peptides having an amino acid sequence
corresponding to the Fv region of the antibody can be used.
Alternatively, single chain neutralizing antibodies that bind to
intracellular target antigens can also be administered. Such single
chain antibodies can be administered, for example, by expressing
nucleotide sequences encoding single-chain antibodies within the
target cell population (e.g., Marasco et al., 1993, Proc. Natl.
Acad. Sci. USA 90:7889-7893).
[0365] The identified compounds that inhibit target gene
expression, synthesis and/or activity can be administered to a
patient at therapeutically effective doses to prevent, treat or
ameliorate 13245 disorders. A therapeutically effective dose refers
to that amount of the compound sufficient to result in amelioration
of symptoms of the disorders.
[0366] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
that exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects can be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0367] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound that achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma can
be measured, for example, by high performance liquid
chromatography.
[0368] Another example of determination of effective dose for an
individual is the ability to directly assay levels of "free" and
"bound" compound in the serum of the test subject. Such assays can
utilize antibody mimics and/or "biosensors" that have been created
through molecular imprinting techniques. The compound which is able
to modulate 13245 activity is used as a template, or "imprinting
molecule," to spatially organize polymerizable monomers prior to
their polymerization with catalytic reagents. The subsequent
removal of the imprinted molecule leaves a polymer matrix that
contains a repeated "negative image" of the compound and is able to
selectively rebind the molecule under biological assay conditions.
Detailed reviews of this technique appear in the art (Ansell et
al., 1996, Curr. Opin. Biotechnol. 7:89-94; Shea, 1994, Trends
Polymer Sci. 2:166-173). Such "imprinted" affinity matrixes are
amenable to ligand-binding assays, whereby the immobilized
monoclonal antibody component is replaced by an appropriately
imprinted matrix (e.g., a matrix described in Vlatakis et al.,
1993, Nature 361:645-647. Through the use of isotope-labeling, the
"free" concentration of compound which modulates the expression or
activity of 13245 can be readily monitored and used in calculations
of IC.sub.50.
[0369] Such "imprinted" affinity matrixes can also be designed to
include fluorescent groups whose photon-emitting properties
measurably change upon local and selective binding of target
compound. These changes can be readily assayed in real time using
appropriate fiber optic devices, in turn allowing the dose in a
test subject to be quickly optimized based on its individual
IC.sub.50. A rudimentary example of such a "biosensor" is discussed
in Kriz et al. (1995, Anal. Chem. 67:2142-2144).
[0370] Another aspect of the invention pertains to methods of
modulating 13245 expression or activity for therapeutic purposes.
Accordingly, in an exemplary embodiment, the modulatory method of
the invention involves contacting a cell with a 13245 or agent that
modulates one or more of the activities of 13245 protein activity
associated with the cell. An agent that modulates 13245 protein
activity can be an agent as described herein, such as a nucleic
acid or a protein, a naturally-occurring target molecule of a 13245
protein (e.g., a 13245 substrate or receptor), a 13245 antibody, a
13245 agonist or antagonist, a peptidomimetic of a 13245 agonist or
antagonist, or other small molecule.
[0371] In one embodiment, the agent stimulates one or 13245
activities. Examples of such stimulatory agents include active
13245 protein and a nucleic acid molecule encoding 13245. In
another embodiment, the agent inhibits one or more 13245
activities. Examples of such inhibitory agents include antisense
13245 nucleic acid molecules, anti-13245 antibodies, and 13245
inhibitors. These modulatory methods can be performed in vitro
(e.g., by culturing the cell with the agent) or, alternatively, in
vivo (e.g., by administering the agent to a subject). As such, the
present invention provides methods of treating an individual
afflicted with a disease or disorder characterized by aberrant or
unwanted expression or activity of a 13245 protein or nucleic acid
molecule. In one embodiment, the method involves administering an
agent (e.g., an agent identified by a screening assay described
herein), or combination of agents that modulates (e.g.,
up-regulates or down-regulates) 13245 expression or activity. In
another embodiment, the method involves administering a 13245
protein or nucleic acid molecule as therapy to compensate for
reduced, aberrant, or unwanted 13245 expression or activity.
[0372] Stimulation of 13245 activity is desirable in situations in
which 13245 is abnormally down-regulated and/or in which increased
13245 activity is likely to have a beneficial effect. For example,
stimulation of 13245 activity is desirable in situations in which a
13245 is down-regulated and/or in which increased 13245 activity is
likely to have a beneficial effect. Likewise, inhibition of 13245
activity is desirable in situations in which 13245 is abnormally
up-regulated and/or in which decreased 13245 activity is likely to
have a beneficial effect.
[0373] Pharmacogenomics
[0374] The 13245 molecules of the present invention, as well as
agents, or modulators which have a stimulatory or inhibitory effect
on 13245 activity (e.g., 13245 gene expression) as identified by a
screening assay described herein can be administered to individuals
to treat (prophylactically or therapeutically) 13245-associated
disorders associated with aberrant or unwanted 13245 activity
(e.g., disorders associated with tumorigenesis or induction of an
inappropriate immune response). In conjunction with such treatment,
pharmacogenomics (i.e., the study of the relationship between an
individual's genotype and that individual's response to a foreign
compound or drug) can be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by
altering the relation between dose and blood concentration of the
pharmacologically active drug. Thus, a physician or clinician can
consider applying knowledge obtained in relevant pharmacogenomics
studies in determining whether to administer a 13245 molecule or
13245 modulator as well as tailoring the dosage and/or therapeutic
regimen of treatment with a 13245 molecule or 13245 modulator.
[0375] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons (e.g.,
Eichelbaum et al., 1996, Clin. Exp. Pharmacol. Physiol. 23:983-985;
Linder et al., 1997, Clin. Chem. 43:254-266). In general, two types
of pharmacogenetic conditions can be differentiated. Genetic
conditions transmitted as a single factor altering the way drugs
act on the body (altered drug action) or genetic conditions
transmitted as single factors altering the way the body acts on
drugs (altered drug metabolism). These pharmacogenetic conditions
can occur either as rare genetic defects or as naturally-occurring
polymorphisms. For example, glucose-6-phosphate dehydrogenase
deficiency (G6PD) is a common inherited enzymopathy in which the
main clinical complication is hemolysis after ingestion of oxidant
drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and
consumption of fava beans.
[0376] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association," relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants). Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high-resolution map can be generated from a
combination of some ten million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP can be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that can be common among
such genetically similar individuals.
[0377] Alternatively, a method termed the "candidate gene approach"
can be utilized to identify genes that predict drug response.
According to this method, if a gene that encodes a drug's target is
known (e.g., a 13245 protein of the present invention), all common
variants of that gene can be fairly easily identified in the
population and it can be determined if having one version of the
gene versus another is associated with a particular drug
response.
[0378] Alternatively, a method termed "gene expression profiling,"
can be utilized to identify genes that predict drug response. For
example, the gene expression of an animal dosed with a drug (e.g.,
a 13245 molecule or 13245 modulator of the present invention) can
give an indication whether gene pathways related to toxicity have
been turned on.
[0379] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment of an individual. This knowledge, when applied to dosing
or drug selection, can avoid adverse reactions or therapeutic
failure and thus enhance therapeutic or prophylactic efficiency
when treating a subject with a 13245 molecule or 13245 modulator,
such as a modulator identified by one of the exemplary screening
assays described herein.
[0380] The present invention further provides methods for
identifying new agents, or combinations, that are based on
identifying agents that modulate the activity of one or more of the
gene products encoded by one or more of the 13245 genes of the
present invention, wherein these products can be associated with
resistance of the cells to a therapeutic agent. Specifically, the
activity of the proteins encoded by the 13245 genes of the present
invention can be used as a basis for identifying agents for
overcoming agent resistance. By blocking the activity of one or
more of the resistance proteins, target cells, e.g., cells of the
immune system, will become sensitive to treatment with an agent
that the unmodified target cells were resistant to.
[0381] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a 13245 protein can be applied in
clinical trials. For example, the effectiveness of an agent
determined by a screening assay as described herein to increase
13245 gene expression, protein levels, or up-regulate 13245
activity, can be monitored in clinical trials of subjects
exhibiting decreased 13245 gene expression, protein levels, or
down-regulated 13245 activity. Alternatively, the effectiveness of
an agent determined by a screening assay to decrease 13245 gene
expression, protein levels, or down-regulate 13245 activity, can be
monitored in clinical trials of subjects exhibiting increased 13245
gene expression, protein levels, or up-regulated 13245 activity. In
such clinical trials, the expression or activity of a 13245 gene,
and preferably, other genes that have been implicated in, for
example, a 13245-associated disorder can be used as a "read out" or
markers of the phenotype of a particular cell.
Other Embodiments
[0382] In another aspect, the invention features, a method of
analyzing a plurality of capture probes. The method can be used,
e.g., to analyze gene expression. The method includes: providing a
two-dimensional array having a plurality of addresses, each address
of the plurality being positionally distinguishable from each other
address of the plurality, and each address of the plurality having
a unique capture probe, e.g., a nucleic acid or peptide sequence;
contacting the array with a 13245, preferably purified, nucleic
acid, preferably purified, polypeptide, preferably purified, or
antibody, and thereby evaluating the plurality of capture probes.
Binding, e.g., in the case of a nucleic acid, hybridization with a
capture probe at an address of the plurality, is detected, e.g., by
signal generated from a label attached to the 13245 nucleic acid,
polypeptide, or antibody.
[0383] The capture probes can be a set of nucleic acids from a
selected sample, e.g., a sample of nucleic acids derived from a
control or non-stimulated tissue or cell.
[0384] The method can include contacting the 13245 nucleic acid,
polypeptide, or antibody with a first array having a plurality of
capture probes and a second array having a different plurality of
capture probes. The results of hybridization can be compared, e.g.,
to analyze differences in expression between a first and second
sample. The first plurality of capture probes can be from a control
sample, e.g., a wild-type, normal, or non-diseased, non-stimulated,
sample, e.g., a biological fluid, tissue, or cell sample. The
second plurality of capture probes can be from an experimental
sample, e.g., a mutant type, at risk, disease-state or
disorder-state, or stimulated, sample, e.g., a biological fluid,
tissue, or cell sample.
[0385] The plurality of capture probes can be a plurality of
nucleic acid probes each of which specifically hybridizes, with an
allele of 13245. Such methods can be used to diagnose a subject,
e.g., to evaluate risk for a disease or disorder, to evaluate
suitability of a selected treatment for a subject, to evaluate
whether a subject has a disease or disorder. 13245 is associated
with protein phosphorylation, thus it is useful for evaluating
disorders relating to aberrant protein phosphorylation, such as
tumorigenesis and inappropriate cell signaling.
[0386] The method can be used to detect SNPs, as described
above.
[0387] In another aspect, the invention features, a method of
analyzing a plurality of probes. The method is useful, e.g., for
analyzing gene expression. The method includes: providing a two
dimensional array having a plurality of addresses, each address of
the plurality being positionally distinguishable from each other
address of the plurality having a unique capture probe, e.g.,
wherein the capture probes are from a cell or subject which express
13245 or from a cell or subject in which a 13245 mediated response
has been elicited, e.g., by contact of the cell with 13245 nucleic
acid or protein, or administration to the cell or subject 13245
nucleic acid or protein; contacting the array with one or more
inquiry probe, wherein an inquiry probe can be a nucleic acid,
polypeptide, or antibody (which is preferably other than 13245
nucleic acid, polypeptide, or antibody); providing a
two-dimensional array having a plurality of addresses, each address
of the plurality being positionally distinguishable from each other
address of the plurality, and each address of the plurality having
a unique capture probe, e.g., wherein the capture probes are from a
cell or subject which does not express 13245 (or does not express
as highly as in the case of the 13245 positive plurality of capture
probes) or from a cell or subject which in which a 13245 mediated
response has not been elicited (or has been elicited to a lesser
extent than in the first sample); contacting the array with one or
more inquiry probes (which is preferably other than a 13245 nucleic
acid, polypeptide, or antibody), and thereby evaluating the
plurality of capture probes. Binding, e.g., in the case of a
nucleic acid, hybridization with a capture probe at an address of
the plurality, is detected, e.g., by signal generated from a label
attached to the nucleic acid, polypeptide, or antibody.
[0388] In another aspect, the invention features, a method of
analyzing a plurality of probes or a sample. The method is useful,
e.g., for analyzing gene expression. The method includes: providing
a two dimensional array having a plurality of addresses, each
address of the plurality being positionally distinguishable from
each other address of the plurality having a unique capture probe,
contacting the array with a first sample from a cell or subject
which express or malexpress 13245 or from a cell or subject in
which a 13245-mediated response has been elicited, e.g., by contact
of the cell with 13245 nucleic acid or protein, or administration
to the cell or subject 13245 nucleic acid or protein; providing a
two dimensional array having a plurality of addresses, each address
of the plurality being positionally distinguishable from each other
address of the plurality, and each address of the plurality having
a unique capture probe, and contacting the array with a second
sample from a cell or subject which does not express 13245 (or does
not express as highly as in the case of the 13245 positive
plurality of capture probes) or from a cell or subject which in
which a 13245 mediated response has not been elicited (or has been
elicited to a lesser extent than in the first sample); and
comparing the binding of the first sample with the binding of the
second sample. Binding, e.g., in the case of a nucleic acid,
hybridization with a capture probe at an address of the plurality,
is detected, e.g., by signal generated from a label attached to the
nucleic acid, polypeptide, or antibody. The same array can be used
for both samples or different arrays can be used. If different
arrays are used the plurality of addresses with capture probes
should be present on both arrays.
[0389] In another aspect, the invention features a method of
analyzing 13245, e.g., analyzing structure, function, or
relatedness to other nucleic acid or amino acid sequences. The
method includes: providing a 13245 nucleic acid or amino acid
sequence, e.g., nucleotide sequence from 13245 or a portion
thereof; comparing the 13245 sequence with one or more preferably a
plurality of sequences from a collection of sequences, e.g., a
nucleic acid or protein sequence database; to thereby analyze
13245.
[0390] The method can include evaluating the sequence identity
between a 13245 sequence and a database sequence. The method can be
performed by accessing the database at a second site, e.g., via the
internet.
[0391] In another aspect, the invention features, a set of
oligonucleotides, useful, e.g., for identifying SNPs, or
identifying specific alleles of 13245. The set includes a plurality
of oligonucleotides, each of which has a different nucleotide at an
interrogation position, e.g., an SNP or the site of a mutation. In
a preferred embodiment, the plurality of oligonucleotides are
identical in sequence with one another (except for differences in
length). The oligonucleotides can be provided with differential
labels, such that an oligonucleotide that hybridizes to one allele
provides a signal that is distinguishable from an oligonucleotide
that hybridizes to a second allele.
[0392] The sequence of a 13245 molecules is provided in a variety
of mediums to facilitate use thereof. A sequence can be provided as
a manufacture, other than an isolated nucleic acid or amino acid
molecule, which contains a 13245. Such a manufacture can provide a
nucleotide or amino acid sequence, e.g., an open reading frame, in
a form which allows examination of the manufacture using means not
directly applicable to examining the nucleotide or amino acid
sequences, or a subset thereof, as they exists in nature or in
purified form.
[0393] A 13245 nucleotide or amino acid sequence can be recorded on
computer readable media. As used herein, "computer readable media"
refers to any medium that 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.
[0394] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid 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.TM. and
Microsoft Word.TM., or represented in the form of an ASCII file,
stored in a database application, such as DB2, Sybase.TM.,
Oracle.TM., or the like. The 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.
[0395] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. A search is used to identify fragments or regions of the
sequences of the invention that match a particular target sequence
or target motif.
[0396] As used herein, a "target sequence" can be any DNA 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. Typical sequence
lengths of a target sequence are from about 10 to 100 amino acids
or from about 30 to 300 nucleotide residues. However, it is well
recognized that commercially important fragments, such as sequence
fragments involved in gene expression and protein processing, can
be of shorter length.
[0397] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. 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 include, but are not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
[0398] Thus, the invention features a method of making a computer
readable record of a sequence of a 13245 sequence that includes
recording the sequence on a computer readable matrix. In a
preferred embodiment, the record includes one or more of the
following: identification of an open reading frame; identification
of a domain, region, or site; identification of the start of
transcription; identification of the transcription terminator; the
full length amino acid sequence of the protein, or a mature form
thereof; the 5'-end of the translated region; or 5'and/or
3'-regulatory regions.
[0399] In another aspect, the invention features, a method of
analyzing a sequence. The method includes: providing a 13245
sequence or record, in computer readable form; comparing a second
sequence to the gene name sequence; thereby analyzing a sequence.
Comparison can include comparing to sequences for sequence identity
or determining if one sequence is included within the other, e.g.,
determining if the 13245 sequence includes a sequence being
compared. In a preferred embodiment, the 13245 or second sequence
is stored on a first computer, e.g., at a first site and the
comparison is performed, read, or recorded on a second computer,
e.g., at a second site. E.g., the 13245 or second sequence can be
stored in a public or proprietary database in one computer, and the
results of the comparison performed, read, or recorded on a second
computer. In a preferred embodiment the record includes one or more
of the following: identification of an ORF; identification of a
domain, region, or site; identification of the start of
transcription; identification of the transcription terminator; the
full length amino acid sequence of the protein, or a mature form
thereof; the 5'-end of the translated region; or 5'- and/or
3'-regulatory regions.
[0400] This invention is further illustrated by the following
examples that should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are incorporated herein by
reference.
EXAMPLES
Example 1
[0401] Identification and Characterization of Human 13245 cDNA
[0402] The human 13245 nucleotide sequence (FIG. 1; SEQ ID NO: 1),
which is approximately 6575 nucleotides in length including
non-translated regions, contains a predicted methionine-initiated
coding sequence at about nucleotide residues 19-6178. The coding
sequence encodes a 2053 amino acid protein (SEQ ID NO: 2).
Example 2
[0403] Tissue Distribution of 13245 mRNA
[0404] Northern blot hybridizations with various RNA samples can be
performed under standard conditions and washed under stringent
conditions, i.e., 0.2.times.SSC at 65.degree. C. A DNA probe
corresponding to all or a portion of the 13245 cDNA (SEQ ID NO: 1)
can be used. The DNA can, for example, be radioactively labeled
with .sup.32P-dCTP using the Prime-It.TM. Kit (Stratagene, La
Jolla, Calif.) according to the instructions of the supplier.
Filters containing mRNA from mouse hematopoietic and endocrine
tissues, and cancer cell lines (Clontech, Palo Alto, Calif.) can be
probed in ExpressHyb.TM. hybridization solution (Clontech) and
washed at high stringency according to manufacturer's
recommendations.
Example 3
[0405] Recombinant Expression of 13245 in Bacterial Cells
[0406] In this example, 13245 is expressed as a recombinant
glutathione-S-transferase (GST) fusion polypeptide in E. coli and
the fusion polypeptide is isolated and characterized. Specifically,
13245 nucleic acid sequences are fused to GST nucleic acid
sequences and this fusion construct is expressed in E. coli, e.g.,
strain PEB199. Expression of the GST-13245 fusion construct in
PEB199 is induced with IPTG. The recombinant fusion polypeptide is
purified from crude bacterial lysates of the induced PEB199 strain
by affinity chromatography on glutathione beads. Using
polyacrylamide gel electrophoretic analysis of the polypeptide
purified from the bacterial lysates, the molecular weight of the
resultant fusion polypeptide is determined.
Example 4
[0407] Expression of Recombinant 13245 Protein in COS Cells
[0408] To express the 13245 gene in COS cells, the pcDNA/Amp vector
by Invitrogen Corporation (San Diego, Calif.) is used. This vector
contains an SV40 origin of replication, an ampicillin resistance
gene, an E. coli replication origin, a CMV promoter followed by a
polylinker region, and an SV40 intron and polyadenylation site. A
DNA fragment encoding the entire 13245 protein and an HA tag
(Wilson et al., 1984, Cell 37:767) or a FLAG.RTM. tag fused
in-frame to its 3'-end of the fragment is cloned into the
polylinker region of the vector, thereby placing the expression of
the recombinant protein under the control of the CMV promoter.
[0409] To construct the plasmid, the 13245 DNA sequence is
amplified by PCR using two primers. The 5' primer contains the
restriction site of interest followed by approximately twenty
nucleotides of the 13245 coding sequence starting from the
initiation codon; the 3'-end sequence contains complementary
sequences to the other restriction site of interest, a translation
stop codon, the HA tag or FLAG.RTM. tag and the last 20 nucleotides
of the 13245 coding sequence. The PCR amplified fragment and the
pcDNA/Amp vector are digested with the appropriate restriction
enzymes and the vector is dephosphorylated using the CIAP enzyme
(New England Biolabs, Beverly, Mass.). Preferably the two
restriction sites chosen are different so that the 13245 gene is
inserted in the desired orientation. The ligation mixture is
transformed into E. coli cells (strains HB101, DHSalpha, SURE,
available from Stratagene Cloning Systems, La Jolla, Calif., can be
used), the transformed culture is plated on ampicillin media
plates, and resistant colonies are selected. Plasmid DNA is
isolated from transformants and examined by restriction analysis
for the presence of the correct fragment.
[0410] COS cells are subsequently transfected with the
13245-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium
chloride co-precipitation methods, DEAE-dextran-mediated
transfection, lipofection, or electroporation. Other suitable
methods for transfecting host cells can be found in Sambrook et
al., (1989, Molecular Cloning: A Laboratory Manual. 2nd ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The
expression of the 13245 polypeptide is detected by radiolabeling
(.sup.35S-methionine or .sup.35S-cysteine, available from NEN,
Boston, Mass., can be used) and immunoprecipitation (Harlow et al.,
1988, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.) using an HA-specific
monoclonal antibody. Briefly, the cells are labeled for 8 hours
with .sup.35S-methionine (or .sup.35S-cysteine). The culture media
are then collected and the cells are lysed using detergents (RIPA
buffer, 150 millimolar NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50
millimolar Tris, pH 7.5). Both the cell lysate and the culture
media are precipitated with an HA-specific monoclonal antibody.
Precipitated polypeptides are then analyzed by SDS-PAGE.
[0411] Alternatively, DNA containing the 13245 coding sequence is
cloned directly into the polylinker of the pcDNA/Amp vector using
the appropriate restriction sites. The resulting plasmid is
transfected into COS cells in the manner described above, and the
expression of the 13245 polypeptide is detected by radiolabeling
and immunoprecipitation using a 13245-specific monoclonal
antibody.
[0412] Equivalents
[0413] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
7 1 6574 DNA Homo sapiens 1 agagccgcca gtggggagat gttgaagttc
aaatatggag cgcggaatcc tttggatgct 60 ggtgctgctg aacccattgc
cagccgggcc tccaggctga atctgttctt ccaggggaaa 120 ccacccttta
tgactcaaca gcagatgtct cctctttccc gagaagggat attagatgcc 180
ctctttgttc tctttgaaga atgcagtcag cctgctctga tgaagattaa gcacgtgagc
240 aactttgtcc ggaagtattc cgacaccata gctgagttac aggagctcca
gccttcggca 300 aaggacttcg aagtcagaag tcttgtaggt tgtggtcact
ttgctgaagt gcaggtggta 360 agagagaaag caaccgggga catctatgct
atgaaagtga tgaagaagaa ggctttattg 420 gcccaggagc aggtttcatt
ttttgaggaa gagcggaaca tattatctcg aagcacaagc 480 ccgtggatcc
cccaattaca gtatgccttt caggacaaaa atcaccttta tctgatggag 540
gaatatcagc ctggagggga cttgctgtca cttttgaata gatatgagga ccagttagat
600 gaaaacctga tacagtttta cctagctgag ctgattttgg ctgttcacag
cgttcatctg 660 atgggatacg tgcatcgaga catcaagcct gagaacattc
tcgttgaccg cacaggacac 720 atcaagctgg tggattttgg atctgccgcg
aaaatgaatt caaacaagat ggtgaatgcc 780 aaactcccga ttgggacccc
agattacatg gctcctgaag tgctgactgt gatgaacggg 840 gatggaaaag
gcacctacgg cctggactgt gactggtggt cagtgggcgt gattgcctat 900
gagatgattt atgggagatc ccccttcgca gagggaacct ctgccagaac cttcaataac
960 attatgaatt tccagcggtt tttgaaattt ccagatgacc ccaaagtgag
cagtgacttt 1020 cttgatctga ttcaaagctt gttgtgcggc cagaaagaga
gactgaagtt tgaaggtctt 1080 tgctgccatc ctttcttctc taaaattgac
tggaacaaca ttcgtaactc tcctcccccc 1140 ttcgttccca ccctcaagtc
tgacgatgac acctccaatt ttgatgaacc agagaagaat 1200 tcgtgggttt
catcctctcc gtgccagctg agcccctcag gcttctcggg tgaagaactg 1260
ccgtttgtgg ggttttcgta cagcaaggca ctggggattc ttggtagatc tgagtctgtt
1320 gtgtcgggtc tggactcccc tgccaagact agctccatgg aaaagaaact
tctcatcaaa 1380 agcaaagagc tacaagactc tcaggacaag tgtcacaaga
tggagcagga aatgacccgg 1440 ttacatcgga gagtgtcaga ggtggaggct
gtgcttagtc agaaggaggt ggagctgaag 1500 gcctctgaga ctcagagatc
cctcctggag caggaccttg ctacctacat cacagaatgc 1560 agtagcttaa
agcgaagttt ggagcaagca cggatggagg tgtcccagga ggatgacaaa 1620
gcactgcagc ttctccatga tatcagagag cagagccgga agctccaaga aatcaaagag
1680 caggagtacc aggctcaagt ggaagaaatg aggttgatga tgaatcagtt
ggaagaggat 1740 cttgtctcag caagaagacg gagtgatctc tacgaatctg
agctgagaga gtctcggctt 1800 gctgctgaag aattcaagcg gaaagcgaca
gaatgtcagc ataaactgtt gaaggctaag 1860 gatcaaggga agcctgaagt
gggagaatat gcgaaactgg agaagatcaa tgctgagcag 1920 cagctcaaaa
ttcaggagct ccaagagaaa ctggagaagg ctgcaaagga gcgagccgag 1980
agggagctgg agaagctgca gaaccgagag gattcttctg aaggcatcag aaagaagctg
2040 gtggaagctg aggaacgccg ccattctctg gagaacaagg taaagagact
agagaccatg 2100 gagcgtagag aaaacagact gaaggatgac atccagacaa
aatcccaaca gatccagcag 2160 atggctgata aaattctgga gctcgaagag
aaacatcggg aggcccaagt ctcagcccag 2220 cacctagaag tgcacctgaa
acagaaagag cagcactatg aggaaaagat taaagtgttg 2280 gacaatcaga
taaagaaaga cctggctgac aaggagacac tggagaacat gatgcagaga 2340
cacgaggagg aggcccatga gaagggcaaa attctcagcg aacagaaggc gatgatcaat
2400 gctatggatt ccaagatcag atccctggaa cagaggattg tggaactgtc
tgaagccaat 2460 aaacttgcag caaatagcag tctttttacc caaaggaaca
tgaaggccca agaagagatg 2520 atttctgaac tcaggcaaca gaaattttac
ctggagacac aggctgggaa gttggaggcc 2580 cagaaccgaa aactggagga
gcagctggag aagatcagcc accaagacca cagtgacaag 2640 aatcggctgc
tggaactgga gacaagattg cgggaggtca gtctagagca cgaggagcag 2700
aaactggagc tcaagcgcca gctcacagag ctacagctct ccctgcagga gcgcgagtca
2760 cagttgacag ccctgcaggc tgcacgggcg gccctggaga gccagcttcg
ccaggcgaag 2820 acagagctgg aagagaccac agcagaagct gaagaggaga
tccaggcact cacggcacat 2880 agagatgaaa tccagcgcaa atttgatgct
cttcgtaaca gctgtactgt aatcacagac 2940 ctggaggagc agctaaacca
gctgaccgag gacaacgctg aactcaacaa ccaaaacttc 3000 tacttgtcca
aacaactcga tgaggcttct ggcgccaacg acgagattgt acaactgcga 3060
agtgaagtgg accatctccg ccgggagatc acggaacgag agatgcagct taccagccag
3120 aagcaaacga tggaggctct gaagaccacg tgcaccatgc tggaggaaca
ggtcatggat 3180 ttggaggccc taaacgatga gctgctagaa aaagagcggc
agtgggaggc ctggaggagc 3240 gtcctgggtg atgagaaatc ccagtttgag
tgtcgggttc gagagctgca gagaatgctg 3300 gacaccgaga aacagagcag
ggcgagagcc gatcagcgga tcaccgagtc tcgccaggtg 3360 gtggagctgg
cagtgaagga gcacaaggct gagattctcg ctctgcagca ggctctcaaa 3420
gagcagaagc tgaaggccga gagcctctct gacaagctca atgacctgga gaagaagcat
3480 gctatgcttg aaatgaatgc ccgaagctta cagcagaagc tggagactga
acgagagctc 3540 aaacagaggc ttctggaaga gcaagccaaa ttacagcagc
agatggacct gcagaaaaat 3600 cacattttcc gtctgactca aggactgcaa
gaagctctag atcgggctga tctactgaag 3660 acagaaagaa gtgacttgga
gtatcagctg gaaaacattc aggttctcta ttctcatgaa 3720 aaggtgaaaa
tggaaggcac tatttctcaa caaaccaaac tcattgattt tctgcaagcc 3780
aaaatggacc aacctgctaa aaagaaaaag ggtttattta gtcgacggaa agaggaccct
3840 gctttaccca cacaggttcc tctgcagtac aatgagctga agctggccct
ggagaaggag 3900 aaagctcgct gtgcagagct agaggaagcc cttcagaaga
cccgcatcga gctccggtcc 3960 gcccgggagg aagctgccca ccgcaaagca
acggaccacc cacacccatc cacgccagcc 4020 accgcgaggc agcagatcgc
catgtccgcc atcgtgcggt cgccagagca ccagcccagt 4080 gccatgagcc
tgctggcccc gccatccagc cgcagaaagg agtcttcaac tccagaggaa 4140
tttagtcggc gtcttaagga acgcatgcac cacaatattc ctcaccgatt caacgtagga
4200 ctgaacatgc gagccacaaa gtgtgctgtg tgtctggata ccgtgcactt
tggacgccag 4260 gcatccaaat gtctcgaatg tcaggtgatg tgtcacccca
agtgctccac gtgcttgcca 4320 gccacctgcg gcttgcctgc tgaatatgcc
acacacttca ccgaggcctt ctgccgtgac 4380 aaaatgaact ccccaggtct
ccagaccaag gagcccagca gcagcttgca cctggaaggg 4440 tggatgaagg
tgcccaggaa taacaaacga ggacagcaag gctgggacag gaagtacatt 4500
gtcctggagg gatcaaaagt cctcatttat gacaatgaag ccagagaagc tggacagagg
4560 ccggtggaag aatttgagct gtgccttccc gacggggatg tatctattca
tggtgccgtt 4620 ggtgcttccg aactcgcaaa tacagccaaa gcagaaaaag
cagaagctga tgctaaactg 4680 cttggaaact ccctgctgaa actggaaggt
gatgaccgtc tagacatgaa ctgcacgctg 4740 cccttcagtg accaggtggt
gttggtgggc accgaggaag ggctctacgc cctgaatgtc 4800 ttgaaaaact
ccctaaccca tgtcccagga attggagcag tcttccaaat ttatattatc 4860
aaggacctgg agaagctact catgatagca ggagaagagc gggcactgtg tcttgtggac
4920 gtgaagaaag tgaaacagtc cctggcccag tcccacctgc ctgcccagcc
cgacatctca 4980 cccaacattt ttgaagctgt caagggctgc cacttgtttg
gggcaggcaa gattgagaac 5040 gggctctgca tctgtgcagc catgcccagc
aaagtcgtca ttctccgcta caacgaaaac 5100 ctcagcaaat actgcatccg
gaaagagata gagacctcag agccctgcag ctgtatccac 5160 ttcaccaatt
acagtatcct cattggaacc aataaattct acgaaatcga catgaagcag 5220
tacacgctcg aggaattcct ggataagaat gaccattcct tggcacctgc tgtgtttgcc
5280 gcctcttcca acagcttccc tgtctcaatc gtgcaggtga acagcgcagg
gcagcgagag 5340 gagtacttgc tgtgtttcca cgaatttgga gtgttcgtgg
attcttacgg aagacgtagc 5400 cgcacagacg atctcaagtg gagtcgctta
cctttggcct ttgcctacag agaaccctat 5460 ctgtttgtga cccacttcaa
ctcactcgaa gtaattgaga tccaggcacg ctcctcagca 5520 gggacccctg
cccgagcgta cctggacatc ccgaacccgc gctacctggg ccctgccatt 5580
tcctcaggag cgatttactt ggcgtcctca taccaggata aattaagggt catttgctgc
5640 aagggaaacc tcgtgaagga gtccggcact gaacaccacc ggggcccgtc
cacctcccgc 5700 agcagcccca acaagcgagg cccacccacg tacaacgagc
acatcaccaa gcgcgtggcc 5760 tccagcccag cgccgcccga aggccccagc
cacccgcgag agccaagcac accccaccgc 5820 taccgcgagg ggcggaccga
gctgcgcagg gacaagtctc ctggccgccc cctggagcga 5880 gagaagtccc
ccggccggat gctcagcacg cggagagagc ggtcccccgg gaggctgttt 5940
gaagacagca gcaggggccg gctgcctgcg ggagccgtga ggaccccgct gtcccaggtg
6000 aacaagggaa gagggcagag tgcctctcaa gttttcacgg ttaacactgt
cacctattat 6060 gactggaata aaaagctgga caacctgcca gctaactggt
cagtcctgag gatcatccag 6120 ctgaatggag aaatccggca gcaggttgaa
aagtctgttc tgagaacaga ttattgctga 6180 gcagagttca tgtgacttct
agacgtggtg acttaaaaaa tggccttaag gctgcagagc 6240 cagccacctc
tgcttacaaa aagagtactt agtgcacatg actgtaagaa acaattgtaa 6300
aacctcatct agaaatcaga aagcttctaa tttctataga aatgacacct ccctggagcc
6360 gagagacaat ctgttgttga ttttgaagga caggcaagac caacactgta
tttagttcca 6420 tagccaggcc tcaacaggga caagtggctg gccttaaaaa
cacacagatg actggaaatg 6480 atgtgtggcc tcagtccctg tttcccagaa
ttttactggc aaaggagtta gcattcattt 6540 ttggcttaag aaaaatcgag
aatgtaggtt taga 6574 2 2053 PRT Homo sapiens 2 Met Leu Lys Phe Lys
Tyr Gly Ala Arg Asn Pro Leu Asp Ala Gly Ala 1 5 10 15 Ala Glu Pro
Ile Ala Ser Arg Ala Ser Arg Leu Asn Leu Phe Phe Gln 20 25 30 Gly
Lys Pro Pro Phe Met Thr Gln Gln Gln Met Ser Pro Leu Ser Arg 35 40
45 Glu Gly Ile Leu Asp Ala Leu Phe Val Leu Phe Glu Glu Cys Ser Gln
50 55 60 Pro Ala Leu Met Lys Ile Lys His Val Ser Asn Phe Val Arg
Lys Tyr 65 70 75 80 Ser Asp Thr Ile Ala Glu Leu Gln Glu Leu Gln Pro
Ser Ala Lys Asp 85 90 95 Phe Glu Val Arg Ser Leu Val Gly Cys Gly
His Phe Ala Glu Val Gln 100 105 110 Val Val Arg Glu Lys Ala Thr Gly
Asp Ile Tyr Ala Met Lys Val Met 115 120 125 Lys Lys Lys Ala Leu Leu
Ala Gln Glu Gln Val Ser Phe Phe Glu Glu 130 135 140 Glu Arg Asn Ile
Leu Ser Arg Ser Thr Ser Pro Trp Ile Pro Gln Leu 145 150 155 160 Gln
Tyr Ala Phe Gln Asp Lys Asn His Leu Tyr Leu Met Glu Glu Tyr 165 170
175 Gln Pro Gly Gly Asp Leu Leu Ser Leu Leu Asn Arg Tyr Glu Asp Gln
180 185 190 Leu Asp Glu Asn Leu Ile Gln Phe Tyr Leu Ala Glu Leu Ile
Leu Ala 195 200 205 Val His Ser Val His Leu Met Gly Tyr Val His Arg
Asp Ile Lys Pro 210 215 220 Glu Asn Ile Leu Val Asp Arg Thr Gly His
Ile Lys Leu Val Asp Phe 225 230 235 240 Gly Ser Ala Ala Lys Met Asn
Ser Asn Lys Met Val Asn Ala Lys Leu 245 250 255 Pro Ile Gly Thr Pro
Asp Tyr Met Ala Pro Glu Val Leu Thr Val Met 260 265 270 Asn Gly Asp
Gly Lys Gly Thr Tyr Gly Leu Asp Cys Asp Trp Trp Ser 275 280 285 Val
Gly Val Ile Ala Tyr Glu Met Ile Tyr Gly Arg Ser Pro Phe Ala 290 295
300 Glu Gly Thr Ser Ala Arg Thr Phe Asn Asn Ile Met Asn Phe Gln Arg
305 310 315 320 Phe Leu Lys Phe Pro Asp Asp Pro Lys Val Ser Ser Asp
Phe Leu Asp 325 330 335 Leu Ile Gln Ser Leu Leu Cys Gly Gln Lys Glu
Arg Leu Lys Phe Glu 340 345 350 Gly Leu Cys Cys His Pro Phe Phe Ser
Lys Ile Asp Trp Asn Asn Ile 355 360 365 Arg Asn Ser Pro Pro Pro Phe
Val Pro Thr Leu Lys Ser Asp Asp Asp 370 375 380 Thr Ser Asn Phe Asp
Glu Pro Glu Lys Asn Ser Trp Val Ser Ser Ser 385 390 395 400 Pro Cys
Gln Leu Ser Pro Ser Gly Phe Ser Gly Glu Glu Leu Pro Phe 405 410 415
Val Gly Phe Ser Tyr Ser Lys Ala Leu Gly Ile Leu Gly Arg Ser Glu 420
425 430 Ser Val Val Ser Gly Leu Asp Ser Pro Ala Lys Thr Ser Ser Met
Glu 435 440 445 Lys Lys Leu Leu Ile Lys Ser Lys Glu Leu Gln Asp Ser
Gln Asp Lys 450 455 460 Cys His Lys Met Glu Gln Glu Met Thr Arg Leu
His Arg Arg Val Ser 465 470 475 480 Glu Val Glu Ala Val Leu Ser Gln
Lys Glu Val Glu Leu Lys Ala Ser 485 490 495 Glu Thr Gln Arg Ser Leu
Leu Glu Gln Asp Leu Ala Thr Tyr Ile Thr 500 505 510 Glu Cys Ser Ser
Leu Lys Arg Ser Leu Glu Gln Ala Arg Met Glu Val 515 520 525 Ser Gln
Glu Asp Asp Lys Ala Leu Gln Leu Leu His Asp Ile Arg Glu 530 535 540
Gln Ser Arg Lys Leu Gln Glu Ile Lys Glu Gln Glu Tyr Gln Ala Gln 545
550 555 560 Val Glu Glu Met Arg Leu Met Met Asn Gln Leu Glu Glu Asp
Leu Val 565 570 575 Ser Ala Arg Arg Arg Ser Asp Leu Tyr Glu Ser Glu
Leu Arg Glu Ser 580 585 590 Arg Leu Ala Ala Glu Glu Phe Lys Arg Lys
Ala Thr Glu Cys Gln His 595 600 605 Lys Leu Leu Lys Ala Lys Asp Gln
Gly Lys Pro Glu Val Gly Glu Tyr 610 615 620 Ala Lys Leu Glu Lys Ile
Asn Ala Glu Gln Gln Leu Lys Ile Gln Glu 625 630 635 640 Leu Gln Glu
Lys Leu Glu Lys Ala Ala Lys Glu Arg Ala Glu Arg Glu 645 650 655 Leu
Glu Lys Leu Gln Asn Arg Glu Asp Ser Ser Glu Gly Ile Arg Lys 660 665
670 Lys Leu Val Glu Ala Glu Glu Arg Arg His Ser Leu Glu Asn Lys Val
675 680 685 Lys Arg Leu Glu Thr Met Glu Arg Arg Glu Asn Arg Leu Lys
Asp Asp 690 695 700 Ile Gln Thr Lys Ser Gln Gln Ile Gln Gln Met Ala
Asp Lys Ile Leu 705 710 715 720 Glu Leu Glu Glu Lys His Arg Glu Ala
Gln Val Ser Ala Gln His Leu 725 730 735 Glu Val His Leu Lys Gln Lys
Glu Gln His Tyr Glu Glu Lys Ile Lys 740 745 750 Val Leu Asp Asn Gln
Ile Lys Lys Asp Leu Ala Asp Lys Glu Thr Leu 755 760 765 Glu Asn Met
Met Gln Arg His Glu Glu Glu Ala His Glu Lys Gly Lys 770 775 780 Ile
Leu Ser Glu Gln Lys Ala Met Ile Asn Ala Met Asp Ser Lys Ile 785 790
795 800 Arg Ser Leu Glu Gln Arg Ile Val Glu Leu Ser Glu Ala Asn Lys
Leu 805 810 815 Ala Ala Asn Ser Ser Leu Phe Thr Gln Arg Asn Met Lys
Ala Gln Glu 820 825 830 Glu Met Ile Ser Glu Leu Arg Gln Gln Lys Phe
Tyr Leu Glu Thr Gln 835 840 845 Ala Gly Lys Leu Glu Ala Gln Asn Arg
Lys Leu Glu Glu Gln Leu Glu 850 855 860 Lys Ile Ser His Gln Asp His
Ser Asp Lys Asn Arg Leu Leu Glu Leu 865 870 875 880 Glu Thr Arg Leu
Arg Glu Val Ser Leu Glu His Glu Glu Gln Lys Leu 885 890 895 Glu Leu
Lys Arg Gln Leu Thr Glu Leu Gln Leu Ser Leu Gln Glu Arg 900 905 910
Glu Ser Gln Leu Thr Ala Leu Gln Ala Ala Arg Ala Ala Leu Glu Ser 915
920 925 Gln Leu Arg Gln Ala Lys Thr Glu Leu Glu Glu Thr Thr Ala Glu
Ala 930 935 940 Glu Glu Glu Ile Gln Ala Leu Thr Ala His Arg Asp Glu
Ile Gln Arg 945 950 955 960 Lys Phe Asp Ala Leu Arg Asn Ser Cys Thr
Val Ile Thr Asp Leu Glu 965 970 975 Glu Gln Leu Asn Gln Leu Thr Glu
Asp Asn Ala Glu Leu Asn Asn Gln 980 985 990 Asn Phe Tyr Leu Ser Lys
Gln Leu Asp Glu Ala Ser Gly Ala Asn Asp 995 1000 1005 Glu Ile Val
Gln Leu Arg Ser Glu Val Asp His Leu Arg Arg Glu Ile 1010 1015 1020
Thr Glu Arg Glu Met Gln Leu Thr Ser Gln Lys Gln Thr Met Glu Ala
1025 1030 1035 1040 Leu Lys Thr Thr Cys Thr Met Leu Glu Glu Gln Val
Met Asp Leu Glu 1045 1050 1055 Ala Leu Asn Asp Glu Leu Leu Glu Lys
Glu Arg Gln Trp Glu Ala Trp 1060 1065 1070 Arg Ser Val Leu Gly Asp
Glu Lys Ser Gln Phe Glu Cys Arg Val Arg 1075 1080 1085 Glu Leu Gln
Arg Met Leu Asp Thr Glu Lys Gln Ser Arg Ala Arg Ala 1090 1095 1100
Asp Gln Arg Ile Thr Glu Ser Arg Gln Val Val Glu Leu Ala Val Lys
1105 1110 1115 1120 Glu His Lys Ala Glu Ile Leu Ala Leu Gln Gln Ala
Leu Lys Glu Gln 1125 1130 1135 Lys Leu Lys Ala Glu Ser Leu Ser Asp
Lys Leu Asn Asp Leu Glu Lys 1140 1145 1150 Lys His Ala Met Leu Glu
Met Asn Ala Arg Ser Leu Gln Gln Lys Leu 1155 1160 1165 Glu Thr Glu
Arg Glu Leu Lys Gln Arg Leu Leu Glu Glu Gln Ala Lys 1170 1175 1180
Leu Gln Gln Gln Met Asp Leu Gln Lys Asn His Ile Phe Arg Leu Thr
1185 1190 1195 1200 Gln Gly Leu Gln Glu Ala Leu Asp Arg Ala Asp Leu
Leu Lys Thr Glu 1205 1210 1215 Arg Ser Asp Leu Glu Tyr Gln Leu Glu
Asn Ile Gln Val Leu Tyr Ser 1220 1225 1230 His Glu Lys Val Lys Met
Glu Gly Thr Ile Ser Gln Gln Thr Lys Leu 1235 1240 1245 Ile Asp Phe
Leu Gln Ala Lys Met Asp Gln Pro Ala Lys Lys Lys Lys 1250 1255 1260
Gly Leu Phe Ser Arg Arg Lys Glu Asp Pro Ala Leu Pro Thr Gln Val
1265 1270 1275 1280 Pro Leu Gln Tyr Asn Glu Leu Lys Leu Ala Leu Glu
Lys Glu Lys Ala 1285 1290 1295 Arg Cys Ala Glu Leu Glu Glu Ala Leu
Gln Lys Thr Arg Ile Glu Leu 1300 1305 1310 Arg Ser Ala Arg Glu Glu
Ala Ala His Arg Lys Ala Thr Asp His Pro 1315 1320 1325 His Pro Ser
Thr Pro Ala Thr Ala Arg Gln Gln Ile Ala Met Ser Ala 1330 1335 1340
Ile Val Arg Ser Pro Glu His Gln Pro Ser Ala Met Ser Leu Leu Ala
1345 1350 1355 1360 Pro Pro Ser Ser Arg Arg
Lys Glu Ser Ser Thr Pro Glu Glu Phe Ser 1365 1370 1375 Arg Arg Leu
Lys Glu Arg Met His His Asn Ile Pro His Arg Phe Asn 1380 1385 1390
Val Gly Leu Asn Met Arg Ala Thr Lys Cys Ala Val Cys Leu Asp Thr
1395 1400 1405 Val His Phe Gly Arg Gln Ala Ser Lys Cys Leu Glu Cys
Gln Val Met 1410 1415 1420 Cys His Pro Lys Cys Ser Thr Cys Leu Pro
Ala Thr Cys Gly Leu Pro 1425 1430 1435 1440 Ala Glu Tyr Ala Thr His
Phe Thr Glu Ala Phe Cys Arg Asp Lys Met 1445 1450 1455 Asn Ser Pro
Gly Leu Gln Thr Lys Glu Pro Ser Ser Ser Leu His Leu 1460 1465 1470
Glu Gly Trp Met Lys Val Pro Arg Asn Asn Lys Arg Gly Gln Gln Gly
1475 1480 1485 Trp Asp Arg Lys Tyr Ile Val Leu Glu Gly Ser Lys Val
Leu Ile Tyr 1490 1495 1500 Asp Asn Glu Ala Arg Glu Ala Gly Gln Arg
Pro Val Glu Glu Phe Glu 1505 1510 1515 1520 Leu Cys Leu Pro Asp Gly
Asp Val Ser Ile His Gly Ala Val Gly Ala 1525 1530 1535 Ser Glu Leu
Ala Asn Thr Ala Lys Ala Glu Lys Ala Glu Ala Asp Ala 1540 1545 1550
Lys Leu Leu Gly Asn Ser Leu Leu Lys Leu Glu Gly Asp Asp Arg Leu
1555 1560 1565 Asp Met Asn Cys Thr Leu Pro Phe Ser Asp Gln Val Val
Leu Val Gly 1570 1575 1580 Thr Glu Glu Gly Leu Tyr Ala Leu Asn Val
Leu Lys Asn Ser Leu Thr 1585 1590 1595 1600 His Val Pro Gly Ile Gly
Ala Val Phe Gln Ile Tyr Ile Ile Lys Asp 1605 1610 1615 Leu Glu Lys
Leu Leu Met Ile Ala Gly Glu Glu Arg Ala Leu Cys Leu 1620 1625 1630
Val Asp Val Lys Lys Val Lys Gln Ser Leu Ala Gln Ser His Leu Pro
1635 1640 1645 Ala Gln Pro Asp Ile Ser Pro Asn Ile Phe Glu Ala Val
Lys Gly Cys 1650 1655 1660 His Leu Phe Gly Ala Gly Lys Ile Glu Asn
Gly Leu Cys Ile Cys Ala 1665 1670 1675 1680 Ala Met Pro Ser Lys Val
Val Ile Leu Arg Tyr Asn Glu Asn Leu Ser 1685 1690 1695 Lys Tyr Cys
Ile Arg Lys Glu Ile Glu Thr Ser Glu Pro Cys Ser Cys 1700 1705 1710
Ile His Phe Thr Asn Tyr Ser Ile Leu Ile Gly Thr Asn Lys Phe Tyr
1715 1720 1725 Glu Ile Asp Met Lys Gln Tyr Thr Leu Glu Glu Phe Leu
Asp Lys Asn 1730 1735 1740 Asp His Ser Leu Ala Pro Ala Val Phe Ala
Ala Ser Ser Asn Ser Phe 1745 1750 1755 1760 Pro Val Ser Ile Val Gln
Val Asn Ser Ala Gly Gln Arg Glu Glu Tyr 1765 1770 1775 Leu Leu Cys
Phe His Glu Phe Gly Val Phe Val Asp Ser Tyr Gly Arg 1780 1785 1790
Arg Ser Arg Thr Asp Asp Leu Lys Trp Ser Arg Leu Pro Leu Ala Phe
1795 1800 1805 Ala Tyr Arg Glu Pro Tyr Leu Phe Val Thr His Phe Asn
Ser Leu Glu 1810 1815 1820 Val Ile Glu Ile Gln Ala Arg Ser Ser Ala
Gly Thr Pro Ala Arg Ala 1825 1830 1835 1840 Tyr Leu Asp Ile Pro Asn
Pro Arg Tyr Leu Gly Pro Ala Ile Ser Ser 1845 1850 1855 Gly Ala Ile
Tyr Leu Ala Ser Ser Tyr Gln Asp Lys Leu Arg Val Ile 1860 1865 1870
Cys Cys Lys Gly Asn Leu Val Lys Glu Ser Gly Thr Glu His His Arg
1875 1880 1885 Gly Pro Ser Thr Ser Arg Ser Ser Pro Asn Lys Arg Gly
Pro Pro Thr 1890 1895 1900 Tyr Asn Glu His Ile Thr Lys Arg Val Ala
Ser Ser Pro Ala Pro Pro 1905 1910 1915 1920 Glu Gly Pro Ser His Pro
Arg Glu Pro Ser Thr Pro His Arg Tyr Arg 1925 1930 1935 Glu Gly Arg
Thr Glu Leu Arg Arg Asp Lys Ser Pro Gly Arg Pro Leu 1940 1945 1950
Glu Arg Glu Lys Ser Pro Gly Arg Met Leu Ser Thr Arg Arg Glu Arg
1955 1960 1965 Ser Pro Gly Arg Leu Phe Glu Asp Ser Ser Arg Gly Arg
Leu Pro Ala 1970 1975 1980 Gly Ala Val Arg Thr Pro Leu Ser Gln Val
Asn Lys Gly Arg Gly Gln 1985 1990 1995 2000 Ser Ala Ser Gln Val Phe
Thr Val Asn Thr Val Thr Tyr Tyr Asp Trp 2005 2010 2015 Asn Lys Lys
Leu Asp Asn Leu Pro Ala Asn Trp Ser Val Leu Arg Ile 2020 2025 2030
Ile Gln Leu Asn Gly Glu Ile Arg Gln Gln Val Glu Lys Ser Val Leu
2035 2040 2045 Arg Thr Asp Tyr Cys 2050 3 6159 DNA Homo sapiens 3
atgttgaagt tcaaatatgg agcgcggaat cctttggatg ctggtgctgc tgaacccatt
60 gccagccggg cctccaggct gaatctgttc ttccagggga aaccaccctt
tatgactcaa 120 cagcagatgt ctcctctttc ccgagaaggg atattagatg
ccctctttgt tctctttgaa 180 gaatgcagtc agcctgctct gatgaagatt
aagcacgtga gcaactttgt ccggaagtat 240 tccgacacca tagctgagtt
acaggagctc cagccttcgg caaaggactt cgaagtcaga 300 agtcttgtag
gttgtggtca ctttgctgaa gtgcaggtgg taagagagaa agcaaccggg 360
gacatctatg ctatgaaagt gatgaagaag aaggctttat tggcccagga gcaggtttca
420 ttttttgagg aagagcggaa catattatct cgaagcacaa gcccgtggat
cccccaatta 480 cagtatgcct ttcaggacaa aaatcacctt tatctgatgg
aggaatatca gcctggaggg 540 gacttgctgt cacttttgaa tagatatgag
gaccagttag atgaaaacct gatacagttt 600 tacctagctg agctgatttt
ggctgttcac agcgttcatc tgatgggata cgtgcatcga 660 gacatcaagc
ctgagaacat tctcgttgac cgcacaggac acatcaagct ggtggatttt 720
ggatctgccg cgaaaatgaa ttcaaacaag atggtgaatg ccaaactccc gattgggacc
780 ccagattaca tggctcctga agtgctgact gtgatgaacg gggatggaaa
aggcacctac 840 ggcctggact gtgactggtg gtcagtgggc gtgattgcct
atgagatgat ttatgggaga 900 tcccccttcg cagagggaac ctctgccaga
accttcaata acattatgaa tttccagcgg 960 tttttgaaat ttccagatga
ccccaaagtg agcagtgact ttcttgatct gattcaaagc 1020 ttgttgtgcg
gccagaaaga gagactgaag tttgaaggtc tttgctgcca tcctttcttc 1080
tctaaaattg actggaacaa cattcgtaac tctcctcccc ccttcgttcc caccctcaag
1140 tctgacgatg acacctccaa ttttgatgaa ccagagaaga attcgtgggt
ttcatcctct 1200 ccgtgccagc tgagcccctc aggcttctcg ggtgaagaac
tgccgtttgt ggggttttcg 1260 tacagcaagg cactggggat tcttggtaga
tctgagtctg ttgtgtcggg tctggactcc 1320 cctgccaaga ctagctccat
ggaaaagaaa cttctcatca aaagcaaaga gctacaagac 1380 tctcaggaca
agtgtcacaa gatggagcag gaaatgaccc ggttacatcg gagagtgtca 1440
gaggtggagg ctgtgcttag tcagaaggag gtggagctga aggcctctga gactcagaga
1500 tccctcctgg agcaggacct tgctacctac atcacagaat gcagtagctt
aaagcgaagt 1560 ttggagcaag cacggatgga ggtgtcccag gaggatgaca
aagcactgca gcttctccat 1620 gatatcagag agcagagccg gaagctccaa
gaaatcaaag agcaggagta ccaggctcaa 1680 gtggaagaaa tgaggttgat
gatgaatcag ttggaagagg atcttgtctc agcaagaaga 1740 cggagtgatc
tctacgaatc tgagctgaga gagtctcggc ttgctgctga agaattcaag 1800
cggaaagcga cagaatgtca gcataaactg ttgaaggcta aggatcaagg gaagcctgaa
1860 gtgggagaat atgcgaaact ggagaagatc aatgctgagc agcagctcaa
aattcaggag 1920 ctccaagaga aactggagaa ggctgcaaag gagcgagccg
agagggagct ggagaagctg 1980 cagaaccgag aggattcttc tgaaggcatc
agaaagaagc tggtggaagc tgaggaacgc 2040 cgccattctc tggagaacaa
ggtaaagaga ctagagacca tggagcgtag agaaaacaga 2100 ctgaaggatg
acatccagac aaaatcccaa cagatccagc agatggctga taaaattctg 2160
gagctcgaag agaaacatcg ggaggcccaa gtctcagccc agcacctaga agtgcacctg
2220 aaacagaaag agcagcacta tgaggaaaag attaaagtgt tggacaatca
gataaagaaa 2280 gacctggctg acaaggagac actggagaac atgatgcaga
gacacgagga ggaggcccat 2340 gagaagggca aaattctcag cgaacagaag
gcgatgatca atgctatgga ttccaagatc 2400 agatccctgg aacagaggat
tgtggaactg tctgaagcca ataaacttgc agcaaatagc 2460 agtcttttta
cccaaaggaa catgaaggcc caagaagaga tgatttctga actcaggcaa 2520
cagaaatttt acctggagac acaggctggg aagttggagg cccagaaccg aaaactggag
2580 gagcagctgg agaagatcag ccaccaagac cacagtgaca agaatcggct
gctggaactg 2640 gagacaagat tgcgggaggt cagtctagag cacgaggagc
agaaactgga gctcaagcgc 2700 cagctcacag agctacagct ctccctgcag
gagcgcgagt cacagttgac agccctgcag 2760 gctgcacggg cggccctgga
gagccagctt cgccaggcga agacagagct ggaagagacc 2820 acagcagaag
ctgaagagga gatccaggca ctcacggcac atagagatga aatccagcgc 2880
aaatttgatg ctcttcgtaa cagctgtact gtaatcacag acctggagga gcagctaaac
2940 cagctgaccg aggacaacgc tgaactcaac aaccaaaact tctacttgtc
caaacaactc 3000 gatgaggctt ctggcgccaa cgacgagatt gtacaactgc
gaagtgaagt ggaccatctc 3060 cgccgggaga tcacggaacg agagatgcag
cttaccagcc agaagcaaac gatggaggct 3120 ctgaagacca cgtgcaccat
gctggaggaa caggtcatgg atttggaggc cctaaacgat 3180 gagctgctag
aaaaagagcg gcagtgggag gcctggagga gcgtcctggg tgatgagaaa 3240
tcccagtttg agtgtcgggt tcgagagctg cagagaatgc tggacaccga gaaacagagc
3300 agggcgagag ccgatcagcg gatcaccgag tctcgccagg tggtggagct
ggcagtgaag 3360 gagcacaagg ctgagattct cgctctgcag caggctctca
aagagcagaa gctgaaggcc 3420 gagagcctct ctgacaagct caatgacctg
gagaagaagc atgctatgct tgaaatgaat 3480 gcccgaagct tacagcagaa
gctggagact gaacgagagc tcaaacagag gcttctggaa 3540 gagcaagcca
aattacagca gcagatggac ctgcagaaaa atcacatttt ccgtctgact 3600
caaggactgc aagaagctct agatcgggct gatctactga agacagaaag aagtgacttg
3660 gagtatcagc tggaaaacat tcaggttctc tattctcatg aaaaggtgaa
aatggaaggc 3720 actatttctc aacaaaccaa actcattgat tttctgcaag
ccaaaatgga ccaacctgct 3780 aaaaagaaaa agggtttatt tagtcgacgg
aaagaggacc ctgctttacc cacacaggtt 3840 cctctgcagt acaatgagct
gaagctggcc ctggagaagg agaaagctcg ctgtgcagag 3900 ctagaggaag
cccttcagaa gacccgcatc gagctccggt ccgcccggga ggaagctgcc 3960
caccgcaaag caacggacca cccacaccca tccacgccag ccaccgcgag gcagcagatc
4020 gccatgtccg ccatcgtgcg gtcgccagag caccagccca gtgccatgag
cctgctggcc 4080 ccgccatcca gccgcagaaa ggagtcttca actccagagg
aatttagtcg gcgtcttaag 4140 gaacgcatgc accacaatat tcctcaccga
ttcaacgtag gactgaacat gcgagccaca 4200 aagtgtgctg tgtgtctgga
taccgtgcac tttggacgcc aggcatccaa atgtctcgaa 4260 tgtcaggtga
tgtgtcaccc caagtgctcc acgtgcttgc cagccacctg cggcttgcct 4320
gctgaatatg ccacacactt caccgaggcc ttctgccgtg acaaaatgaa ctccccaggt
4380 ctccagacca aggagcccag cagcagcttg cacctggaag ggtggatgaa
ggtgcccagg 4440 aataacaaac gaggacagca aggctgggac aggaagtaca
ttgtcctgga gggatcaaaa 4500 gtcctcattt atgacaatga agccagagaa
gctggacaga ggccggtgga agaatttgag 4560 ctgtgccttc ccgacgggga
tgtatctatt catggtgccg ttggtgcttc cgaactcgca 4620 aatacagcca
aagcagaaaa agcagaagct gatgctaaac tgcttggaaa ctccctgctg 4680
aaactggaag gtgatgaccg tctagacatg aactgcacgc tgcccttcag tgaccaggtg
4740 gtgttggtgg gcaccgagga agggctctac gccctgaatg tcttgaaaaa
ctccctaacc 4800 catgtcccag gaattggagc agtcttccaa atttatatta
tcaaggacct ggagaagcta 4860 ctcatgatag caggagaaga gcgggcactg
tgtcttgtgg acgtgaagaa agtgaaacag 4920 tccctggccc agtcccacct
gcctgcccag cccgacatct cacccaacat ttttgaagct 4980 gtcaagggct
gccacttgtt tggggcaggc aagattgaga acgggctctg catctgtgca 5040
gccatgccca gcaaagtcgt cattctccgc tacaacgaaa acctcagcaa atactgcatc
5100 cggaaagaga tagagacctc agagccctgc agctgtatcc acttcaccaa
ttacagtatc 5160 ctcattggaa ccaataaatt ctacgaaatc gacatgaagc
agtacacgct cgaggaattc 5220 ctggataaga atgaccattc cttggcacct
gctgtgtttg ccgcctcttc caacagcttc 5280 cctgtctcaa tcgtgcaggt
gaacagcgca gggcagcgag aggagtactt gctgtgtttc 5340 cacgaatttg
gagtgttcgt ggattcttac ggaagacgta gccgcacaga cgatctcaag 5400
tggagtcgct tacctttggc ctttgcctac agagaaccct atctgtttgt gacccacttc
5460 aactcactcg aagtaattga gatccaggca cgctcctcag cagggacccc
tgcccgagcg 5520 tacctggaca tcccgaaccc gcgctacctg ggccctgcca
tttcctcagg agcgatttac 5580 ttggcgtcct cataccagga taaattaagg
gtcatttgct gcaagggaaa cctcgtgaag 5640 gagtccggca ctgaacacca
ccggggcccg tccacctccc gcagcagccc caacaagcga 5700 ggcccaccca
cgtacaacga gcacatcacc aagcgcgtgg cctccagccc agcgccgccc 5760
gaaggcccca gccacccgcg agagccaagc acaccccacc gctaccgcga ggggcggacc
5820 gagctgcgca gggacaagtc tcctggccgc cccctggagc gagagaagtc
ccccggccgg 5880 atgctcagca cgcggagaga gcggtccccc gggaggctgt
ttgaagacag cagcaggggc 5940 cggctgcctg cgggagccgt gaggaccccg
ctgtcccagg tgaacaaggg aagagggcag 6000 agtgcctctc aagttttcac
ggttaacact gtcacctatt atgactggaa taaaaagctg 6060 gacaacctgc
cagctaactg gtcagtcctg aggatcatcc agctgaatgg agaaatccgg 6120
cagcaggttg aaaagtctgt tctgagaaca gattattgc 6159 4 2055 PRT Mus
musculus 4 Met Leu Lys Phe Lys Tyr Gly Val Arg Asn Pro Pro Glu Ala
Ser Ala 1 5 10 15 Ser Glu Pro Ile Ala Ser Arg Ala Ser Arg Leu Asn
Leu Phe Phe Gln 20 25 30 Gly Lys Pro Pro Leu Met Thr Gln Gln Gln
Met Ser Ala Leu Ser Arg 35 40 45 Glu Gly Met Leu Asp Ala Leu Phe
Ala Leu Phe Glu Glu Cys Ser Gln 50 55 60 Pro Ala Leu Met Lys Met
Lys His Val Ser Ser Phe Val Gln Lys Tyr 65 70 75 80 Ser Asp Thr Ile
Ala Glu Leu Arg Glu Leu Gln Pro Ser Ala Arg Asp 85 90 95 Phe Glu
Val Arg Ser Leu Val Gly Cys Gly His Phe Ala Glu Val Gln 100 105 110
Val Val Arg Glu Lys Ala Thr Gly Asp Val Tyr Ala Met Lys Ile Met 115
120 125 Lys Lys Lys Ala Leu Leu Ala Gln Glu Gln Val Ser Phe Phe Glu
Glu 130 135 140 Glu Arg Asn Ile Leu Ser Arg Ser Thr Ser Pro Trp Ile
Pro Gln Leu 145 150 155 160 Gln Tyr Ala Phe Gln Asp Lys Asn Asn Leu
Tyr Leu Val Met Glu Tyr 165 170 175 Gln Pro Gly Gly Asp Phe Leu Ser
Leu Leu Asn Arg Tyr Glu Asp Gln 180 185 190 Leu Asp Glu Ser Met Ile
Gln Phe Tyr Leu Ala Glu Leu Ile Leu Ala 195 200 205 Val His Ser Val
His Gln Met Gly Tyr Val His Arg Asp Ile Lys Pro 210 215 220 Glu Asn
Ile Leu Ile Asp Arg Thr Gly Glu Ile Lys Leu Val Asp Phe 225 230 235
240 Gly Ser Ala Ala Lys Met Asn Ser Asn Lys Val Asp Ala Lys Leu Pro
245 250 255 Ile Gly Thr Pro Asp Tyr Met Ala Pro Glu Val Leu Thr Val
Met Asn 260 265 270 Glu Asp Arg Arg Gly Thr Tyr Gly Leu Asp Cys Asp
Trp Trp Ser Val 275 280 285 Gly Val Val Ala Tyr Glu Met Val Tyr Gly
Lys Thr Pro Phe Thr Glu 290 295 300 Gly Thr Ser Ala Arg Thr Phe Asn
Asn Ile Met Asn Phe Gln Arg Phe 305 310 315 320 Leu Lys Phe Pro Asp
Asp Pro Lys Val Ser Ser Glu Leu Leu Asp Leu 325 330 335 Leu Gln Ser
Leu Leu Cys Val Gln Lys Glu Arg Leu Lys Phe Glu Gly 340 345 350 Leu
Cys Cys His Pro Phe Phe Ala Arg Thr Asp Trp Asn Asn Ile Arg 355 360
365 Asn Ser Pro Pro Pro Phe Val Pro Thr Leu Lys Ser Asp Asp Asp Thr
370 375 380 Ser Asn Phe Asp Glu Pro Glu Lys Asn Ser Trp Ala Phe Ile
Leu Cys 385 390 395 400 Val Pro Ala Glu Pro Leu Ala Phe Ser Gly Glu
Glu Leu Pro Phe Val 405 410 415 Gly Phe Ser Tyr Ser Lys Ala Leu Gly
Tyr Leu Gly Arg Ser Glu Ser 420 425 430 Val Val Ser Ser Leu Asp Ser
Pro Ala Lys Val Ser Ser Met Glu Lys 435 440 445 Lys Leu Leu Ile Lys
Ser Lys Glu Leu Gln Asp Ser Gln Asp Lys Cys 450 455 460 His Lys Met
Glu Gln Glu Met Thr Arg Leu His Arg Arg Val Ser Glu 465 470 475 480
Val Glu Ala Val Leu Ser Gln Lys Glu Val Glu Leu Lys Ala Ser Glu 485
490 495 Thr Gln Arg Ser Leu Leu Glu Gln Asp Leu Ala Thr Tyr Ile Thr
Glu 500 505 510 Cys Ser Ser Leu Lys Arg Ser Leu Glu Gln Ala Arg Met
Glu Val Ser 515 520 525 Gln Glu Asp Asp Lys Ala Leu Gln Leu Leu His
Asp Ile Arg Glu Gln 530 535 540 Ser Arg Lys Leu Gln Glu Ile Lys Glu
Gln Glu Tyr Gln Ala Gln Val 545 550 555 560 Glu Glu Met Arg Leu Met
Met Asn Gln Leu Glu Glu Asp Leu Val Ser 565 570 575 Ala Arg Arg Arg
Ser Asp Leu Tyr Glu Ser Glu Leu Arg Glu Ser Arg 580 585 590 Leu Ala
Ala Glu Glu Phe Lys Arg Lys Ala Asn Glu Cys Gln His Lys 595 600 605
Leu Met Lys Ala Lys Asp Gln Gly Lys Pro Glu Val Gly Glu Tyr Ser 610
615 620 Lys Leu Glu Lys Ile Asn Ala Glu Gln Gln Leu Lys Ile Gln Glu
Leu 625 630 635 640 Gln Glu Lys Leu Glu Lys Ala Val Lys Ala Ser Thr
Glu Ala Thr Glu 645 650 655 Leu Leu Gln Asn Ile Arg Gln Ala Lys Glu
Arg Ala Glu Arg Glu Leu 660 665 670 Glu Lys Leu His Asn Arg Glu Asp
Ser Ser Glu Gly Ile Lys Lys Lys 675 680 685 Leu Val Glu Ala Glu Glu
Arg Arg His Ser Leu Glu Asn Lys Val Lys 690 695 700 Arg Leu Glu Thr
Met Glu Arg Arg Glu Asn Arg Leu Lys Asp Asp Ile 705 710 715 720 Gln
Thr Lys Ser Glu Gln Ile Gln Gln Met Ala Asp Lys Ile Leu Glu 725
730
735 Leu Glu Glu Lys His Arg Glu Ala Gln Val Ser Ala Gln His Leu Glu
740 745 750 Val His Leu Lys Gln Lys Glu Gln His Tyr Glu Glu Lys Ile
Lys Val 755 760 765 Leu Asp Asn Gln Ile Lys Lys Asp Leu Ala Asp Lys
Glu Ser Leu Glu 770 775 780 Asn Met Met Gln Arg His Glu Glu Glu Ala
His Glu Lys Gly Lys Ile 785 790 795 800 Leu Ser Glu Gln Lys Ala Met
Ile Asn Ala Met Asp Ser Lys Ile Arg 805 810 815 Ser Leu Glu Gln Arg
Ile Val Glu Leu Ser Glu Ala Asn Lys Leu Ala 820 825 830 Ala Asn Ser
Ser Leu Phe Thr Gln Arg Asn Met Lys Ala Gln Glu Glu 835 840 845 Met
Ile Ser Glu Leu Arg Gln Gln Lys Phe Tyr Leu Glu Thr Gln Ala 850 855
860 Gly Lys Leu Glu Ala Gln Asn Arg Lys Leu Glu Glu Gln Leu Glu Lys
865 870 875 880 Ile Ser His Gln Asp His Ser Asp Lys Ser Arg Leu Leu
Glu Leu Glu 885 890 895 Thr Arg Leu Arg Glu Val Ser Leu Glu His Glu
Glu Gln Lys Leu Glu 900 905 910 Leu Lys Arg Gln Leu Thr Glu Leu Gln
Leu Ser Leu Gln Glu Arg Glu 915 920 925 Ser Gln Leu Thr Ala Leu Gln
Ala Ala Arg Ala Ala Leu Glu Ser Gln 930 935 940 Leu Arg Gln Ala Lys
Thr Glu Leu Glu Glu Thr Thr Ala Glu Ala Glu 945 950 955 960 Glu Glu
Ile Gln Ala Leu Thr Ala His Arg Asp Glu Ile Gln Arg Lys 965 970 975
Phe Asp Ala Leu Arg Asn Ser Cys Thr Val Ile Thr Asp Leu Glu Glu 980
985 990 Gln Leu Asn Gln Leu Thr Glu Asp Asn Ala Glu Leu Asn Asn Gln
Asn 995 1000 1005 Phe Tyr Leu Ser Lys Gln Leu Asp Glu Ala Ser Gly
Ala Asn Asp Glu 1010 1015 1020 Ile Val Gln Leu Arg Ser Glu Val Asp
His Leu Arg Arg Glu Ile Thr 1025 1030 1035 1040 Glu Arg Glu Met Gln
Leu Thr Ser Gln Lys Gln Thr Met Glu Ala Leu 1045 1050 1055 Lys Thr
Thr Cys Thr Met Leu Glu Glu Gln Val Leu Asp Leu Glu Ala 1060 1065
1070 Leu Asn Asp Glu Leu Leu Glu Lys Glu Arg Gln Trp Glu Ala Trp
Arg 1075 1080 1085 Ser Val Leu Gly Asp Glu Lys Ser Gln Phe Glu Cys
Arg Val Arg Glu 1090 1095 1100 Leu Gln Arg Met Leu Asp Thr Glu Lys
Gln Ser Arg Ala Arg Ala Asp 1105 1110 1115 1120 Gln Arg Ile Thr Glu
Ser Arg Gln Val Val Glu Leu Ala Val Lys Glu 1125 1130 1135 His Lys
Ala Glu Ile Leu Ala Leu Gln Gln Ala Leu Lys Glu Gln Lys 1140 1145
1150 Leu Lys Ala Glu Ser Leu Ser Asp Lys Leu Asn Asp Leu Glu Lys
Lys 1155 1160 1165 His Ala Met Leu Glu Met Asn Ala Arg Ser Leu Gln
Gln Lys Leu Glu 1170 1175 1180 Thr Glu Arg Glu Leu Lys Gln Arg Leu
Leu Glu Glu Gln Ala Lys Leu 1185 1190 1195 1200 Gln Gln Gln Met Asp
Leu Gln Lys Asn His Ile Phe Arg Leu Thr Gln 1205 1210 1215 Gly Leu
Gln Glu Ala Leu Asp Arg Ala Asp Leu Leu Lys Thr Glu Arg 1220 1225
1230 Ser Asp Leu Glu Tyr Gln Leu Glu Asn Ile Gln Val Leu Tyr Ser
His 1235 1240 1245 Glu Lys Val Lys Met Glu Gly Thr Ile Ser Gln Gln
Thr Lys Leu Ile 1250 1255 1260 Asp Phe Leu Gln Ala Lys Met Asp Gln
Pro Ala Lys Lys Lys Lys Val 1265 1270 1275 1280 Pro Leu Gln Tyr Asn
Glu Leu Lys Leu Ala Leu Glu Lys Glu Lys Ala 1285 1290 1295 Arg Cys
Ala Glu Leu Glu Glu Ala Leu Gln Lys Thr Arg Ile Glu Leu 1300 1305
1310 Arg Ser Ala Arg Glu Glu Ala Ala His Arg Lys Ala Thr Asp His
Pro 1315 1320 1325 His Pro Ser Thr Pro Ala Thr Ala Arg Gln Gln Ile
Ala Met Ser Ala 1330 1335 1340 Ile Val Arg Ser Pro Glu His Gln Pro
Ser Ala Met Ser Leu Leu Ala 1345 1350 1355 1360 Pro Pro Ser Ser Arg
Arg Lys Glu Ser Ser Thr Pro Glu Glu Phe Ser 1365 1370 1375 Arg Arg
Leu Lys Glu Arg Met His His Asn Ile Pro His Arg Phe Asn 1380 1385
1390 Val Gly Leu Asn Met Arg Ala Thr Lys Cys Ala Val Cys Leu Asp
Thr 1395 1400 1405 Val His Phe Gly Arg Gln Ala Ser Lys Cys Leu Glu
Cys Gln Val Met 1410 1415 1420 Cys His Pro Lys Cys Ser Thr Cys Leu
Pro Ala Thr Cys Gly Leu Pro 1425 1430 1435 1440 Ala Glu Tyr Ala Thr
His Phe Thr Glu Ala Phe Cys Arg Asp Lys Met 1445 1450 1455 Asn Ser
Pro Gly Leu Gln Ser Lys Glu Pro Gly Ser Ser Leu His Leu 1460 1465
1470 Glu Gly Trp Met Lys Val Pro Arg Asn Asn Lys Arg Gly Gln Gln
Gly 1475 1480 1485 Trp Asp Arg Lys Tyr Ile Val Leu Glu Gly Ser Lys
Val Leu Ile Tyr 1490 1495 1500 Asp Asn Glu Ala Arg Glu Ala Gly Gln
Arg Pro Val Glu Glu Phe Glu 1505 1510 1515 1520 Leu Cys Leu Pro Asp
Gly Asp Val Ser Ile His Gly Ala Val Gly Ala 1525 1530 1535 Ser Glu
Leu Ala Asn Thr Ala Lys Ala Asp Val Pro Tyr Ile Leu Lys 1540 1545
1550 Met Glu Ser His Pro His Thr Thr Cys Trp Pro Gly Arg Thr Leu
Tyr 1555 1560 1565 Leu Leu Ala Pro Ser Phe Pro Asp Lys Gln Arg Trp
Val Thr Ala Leu 1570 1575 1580 Glu Ser Val Val Ala Gly Gly Arg Val
Ser Arg Glu Lys Ala Glu Ala 1585 1590 1595 1600 Asp Ala Lys Leu Leu
Gly Asn Ser Leu Leu Lys Leu Glu Gly Asp Asp 1605 1610 1615 Arg Leu
Asp Met Asn Cys Thr Leu Pro Phe Ser Asp Gln Val Val Leu 1620 1625
1630 Val Gly Thr Glu Glu Gly Leu Tyr Ala Leu Asn Val Leu Lys Asn
Ser 1635 1640 1645 Leu Thr His Ile Pro Gly Ile Gly Ala Val Phe Gln
Ile Tyr Ile Ile 1650 1655 1660 Lys Asp Leu Glu Lys Leu Leu Met Ile
Ala Gly Glu Glu Arg Ala Leu 1665 1670 1675 1680 Cys Leu Val Asp Val
Lys Lys Val Lys Gln Ser Leu Ala Gln Ser His 1685 1690 1695 Leu Pro
Ala Gln Pro Asp Val Ser Pro Asn Ile Phe Glu Ala Val Lys 1700 1705
1710 Gly Cys His Leu Phe Ala Ala Gly Lys Ile Glu Asn Ser Leu Cys
Ile 1715 1720 1725 Cys Ala Ala Met Pro Ser Lys Val Val Ile Leu Arg
Tyr Asn Asp Asn 1730 1735 1740 Leu Ser Lys Tyr Cys Ile Arg Lys Glu
Ile Glu Thr Ser Glu Pro Cys 1745 1750 1755 1760 Ser Cys Ile His Phe
Thr Asn Tyr Ser Ile Leu Ile Gly Thr Asn Lys 1765 1770 1775 Phe Tyr
Glu Ile Asp Met Lys Gln Tyr Thr Leu Asp Glu Phe Leu Asp 1780 1785
1790 Lys Asn Asp His Ser Leu Ala Pro Ala Val Phe Ala Ser Ser Ser
Asn 1795 1800 1805 Ser Phe Pro Val Ser Ile Val Gln Ala Asn Ser Ala
Gly Gln Arg Glu 1810 1815 1820 Glu Tyr Leu Leu Cys Phe His Glu Phe
Gly Val Phe Val Asp Ser Tyr 1825 1830 1835 1840 Gly Arg Arg Ser Arg
Thr Asp Asp Leu Lys Trp Ser Arg Leu Pro Leu 1845 1850 1855 Ala Phe
Ala Tyr Arg Glu Pro Tyr Leu Phe Val Thr His Phe Asn Ser 1860 1865
1870 Leu Glu Val Ile Glu Ile Gln Ala Arg Ser Ser Leu Gly Ser Pro
Ala 1875 1880 1885 Arg Ala Tyr Leu Glu Ile Pro Asn Pro Arg Tyr Leu
Gly Pro Ala Ile 1890 1895 1900 Ser Ser Gly Ala Ile Tyr Leu Ala Ser
Ser Tyr Gln Asp Lys Leu Arg 1905 1910 1915 1920 Val Ile Cys Cys Lys
Gly Asn Leu Val Lys Glu Ser Gly Thr Glu Gln 1925 1930 1935 His Arg
Val Pro Ser Thr Ser Arg Ser Ser Pro Asn Lys Arg Gly Pro 1940 1945
1950 Pro Thr Tyr Asn Glu His Ile Thr Lys Arg Val Ala Ser Ser Pro
Ala 1955 1960 1965 Pro Pro Glu Gly Pro Ser His Pro Arg Glu Pro Ser
Thr Pro His Arg 1970 1975 1980 Tyr Arg Asp Arg Glu Gly Arg Thr Glu
Leu Arg Arg Asp Lys Ser Pro 1985 1990 1995 2000 Gly Arg Pro Leu Glu
Arg Glu Lys Ser Pro Gly Arg Met Leu Ser Thr 2005 2010 2015 Arg Arg
Glu Arg Ser Pro Gly Arg Leu Phe Glu Asp Ser Ser Arg Gly 2020 2025
2030 Arg Leu Pro Ala Gly Ala Val Arg Thr Pro Leu Ser Gln Val Asn
Lys 2035 2040 2045 Val Trp Asp Gln Ser Ser Val 2050 2055 5 1641 PRT
Mus musculus 5 Pro Phe Val Pro Thr Leu Lys Ser Asp Asp Asp Thr Ser
Asn Phe Asp 1 5 10 15 Glu Pro Glu Lys Asn Ser Trp Val Ser Ser Ser
Val Cys Gln Leu Ser 20 25 30 Pro Ser Gly Phe Ser Gly Glu Glu Leu
Pro Phe Val Gly Phe Ser Tyr 35 40 45 Ser Lys Ala Leu Gly Tyr Leu
Gly Arg Ser Glu Ser Val Val Ser Ser 50 55 60 Leu Asp Ser Pro Ala
Lys Val Ser Ser Met Glu Lys Lys Leu Leu Ile 65 70 75 80 Lys Ser Lys
Glu Leu Gln Asp Ser Gln Asp Lys Cys His Lys Met Glu 85 90 95 Gln
Glu Met Thr Arg Leu His Arg Arg Val Ser Glu Val Glu Ala Val 100 105
110 Leu Ser Gln Lys Glu Val Glu Leu Lys Ala Ser Glu Thr Gln Arg Ser
115 120 125 Leu Leu Glu Gln Asp Leu Ala Thr Tyr Ile Thr Glu Cys Ser
Ser Leu 130 135 140 Lys Arg Ser Leu Glu Gln Ala Arg Met Glu Val Ser
Gln Glu Asp Asp 145 150 155 160 Lys Ala Leu Gln Leu Leu His Asp Ile
Arg Glu Gln Ser Arg Lys Leu 165 170 175 Gln Glu Ile Lys Glu Gln Glu
Tyr Gln Ala Gln Val Glu Glu Met Arg 180 185 190 Leu Met Met Asn Gln
Leu Glu Glu Asp Leu Val Ser Ala Arg Arg Arg 195 200 205 Ser Asp Leu
Tyr Glu Ser Glu Leu Arg Glu Ser Arg Leu Ala Ala Glu 210 215 220 Glu
Phe Lys Arg Lys Ala Asn Glu Cys Gln His Lys Leu Met Lys Ala 225 230
235 240 Lys Asp Gln Gly Lys Pro Glu Val Gly Glu Tyr Ser Lys Leu Glu
Lys 245 250 255 Ile Asn Ala Glu Gln Gln Leu Lys Ile Gln Glu Leu Gln
Glu Lys Leu 260 265 270 Glu Lys Ala Val Lys Ala Ser Thr Glu Ala Thr
Glu Leu Leu Gln Asn 275 280 285 Ile Arg Gln Ala Lys Glu Arg Ala Glu
Arg Glu Leu Glu Lys Leu His 290 295 300 Asn Arg Glu Asp Ser Ser Glu
Gly Ile Lys Lys Lys Leu Val Glu Ala 305 310 315 320 Glu Glu Leu Glu
Glu Lys His Arg Glu Ala Gln Val Ser Ala Gln His 325 330 335 Leu Glu
Val His Leu Lys Gln Lys Glu Gln His Tyr Glu Glu Lys Ile 340 345 350
Lys Val Leu Asp Asn Gln Ile Lys Lys Asp Leu Ala Asp Lys Glu Ser 355
360 365 Leu Glu Asn Met Met Gln Arg His Glu Glu Glu Ala His Glu Lys
Gly 370 375 380 Lys Ile Leu Ser Glu Gln Lys Ala Met Ile Asn Ala Met
Asp Ser Lys 385 390 395 400 Ile Arg Ser Leu Glu Gln Arg Ile Val Glu
Leu Ser Glu Ala Asn Lys 405 410 415 Leu Ala Ala Asn Ser Ser Leu Phe
Thr Gln Arg Asn Met Lys Ala Gln 420 425 430 Glu Glu Met Ile Ser Glu
Leu Arg Gln Gln Lys Phe Tyr Leu Glu Thr 435 440 445 Gln Ala Gly Lys
Leu Glu Ala Gln Asn Arg Lys Leu Glu Glu Gln Leu 450 455 460 Glu Lys
Ile Ser His Gln Asp His Ser Asp Lys Ser Arg Leu Leu Glu 465 470 475
480 Leu Glu Thr Arg Leu Arg Glu Val Ser Leu Glu His Glu Glu Gln Lys
485 490 495 Leu Glu Leu Lys Arg Gln Leu Thr Glu Leu Gln Leu Ser Leu
Gln Glu 500 505 510 Arg Glu Ser Gln Leu Thr Ala Leu Gln Ala Ala Arg
Ala Ala Leu Glu 515 520 525 Ser Gln Leu Arg Gln Ala Lys Thr Glu Leu
Glu Glu Thr Thr Ala Glu 530 535 540 Ala Glu Glu Glu Ile Gln Ala Leu
Thr Ala His Arg Asp Glu Ile Gln 545 550 555 560 Arg Lys Phe Asp Ala
Leu Arg Asn Ser Cys Thr Val Ile Thr Asp Leu 565 570 575 Glu Glu Gln
Leu Asn Gln Leu Thr Glu Asp Asn Ala Glu Leu Asn Asn 580 585 590 Gln
Asn Phe Tyr Leu Ser Lys Gln Leu Asp Glu Ala Ser Gly Ala Asn 595 600
605 Asp Glu Ile Val Gln Leu Arg Ser Glu Val Asp His Leu Arg Arg Glu
610 615 620 Ile Thr Glu Arg Glu Met Gln Leu Thr Ser Gln Lys Gln Thr
Met Glu 625 630 635 640 Ala Leu Lys Thr Thr Cys Thr Met Leu Glu Glu
Gln Val Leu Asp Leu 645 650 655 Glu Ala Leu Asn Asp Glu Leu Leu Glu
Lys Glu Arg Gln Trp Glu Ala 660 665 670 Trp Arg Ser Val Leu Gly Asp
Glu Lys Ser Gln Phe Glu Cys Arg Val 675 680 685 Arg Glu Leu Gln Arg
Met Leu Asp Thr Glu Lys Gln Ser Arg Ala Arg 690 695 700 Ala Asp Gln
Arg Ile Thr Glu Ser Arg Gln Val Val Glu Leu Ala Val 705 710 715 720
Lys Glu His Lys Ala Glu Ile Leu Ala Leu Gln Gln Ala Leu Lys Glu 725
730 735 Gln Lys Leu Lys Ala Glu Ser Leu Ser Asp Lys Leu Asn Asp Leu
Glu 740 745 750 Lys Lys His Ala Met Leu Glu Met Asn Ala Arg Ser Leu
Gln Gln Lys 755 760 765 Leu Glu Thr Glu Arg Glu Leu Lys Gln Arg Leu
Leu Glu Glu Gln Ala 770 775 780 Lys Leu Gln Gln Gln Met Asp Leu Gln
Lys Asn His Ile Phe Arg Leu 785 790 795 800 Thr Gln Gly Leu Gln Glu
Ala Leu Asp Arg Ala Asp Leu Leu Lys Thr 805 810 815 Glu Arg Ser Asp
Leu Glu Tyr Gln Leu Glu Asn Ile Gln Val Leu Tyr 820 825 830 Ser His
Glu Lys Val Lys Met Glu Gly Thr Ile Ser Gln Gln Thr Lys 835 840 845
Leu Ile Asp Phe Leu Gln Ala Lys Met Asp Gln Pro Ala Lys Lys Lys 850
855 860 Lys Val Pro Leu Gln Tyr Asn Glu Leu Lys Leu Ala Leu Glu Lys
Glu 865 870 875 880 Lys Ala Arg Cys Ala Glu Leu Glu Glu Ala Leu Gln
Lys Thr Arg Ile 885 890 895 Glu Leu Arg Ser Ala Arg Glu Glu Ala Ala
His Arg Lys Ala Thr Asp 900 905 910 His Pro His Pro Ser Thr Pro Ala
Thr Ala Arg Gln Gln Ile Ala Met 915 920 925 Ser Ala Ile Val Arg Ser
Pro Glu His Gln Pro Ser Ala Met Ser Leu 930 935 940 Leu Ala Pro Pro
Ser Ser Arg Arg Lys Glu Ser Ser Thr Pro Glu Glu 945 950 955 960 Phe
Ser Arg Arg Leu Lys Glu Arg Met His His Asn Ile Pro His Arg 965 970
975 Phe Asn Val Gly Leu Asn Met Arg Ala Thr Lys Cys Ala Val Cys Leu
980 985 990 Asp Thr Val His Phe Gly Arg Gln Ala Ser Lys Cys Leu Glu
Cys Gln 995 1000 1005 Val Met Cys His Pro Lys Cys Ser Thr Cys Leu
Pro Ala Thr Cys Gly 1010 1015 1020 Leu Pro Ala Glu Tyr Ala Thr His
Phe Thr Glu Ala Phe Cys Arg Asp 1025 1030 1035 1040 Lys Met Asn Ser
Pro Gly Leu Gln Ser Lys Glu Pro Gly Ser Ser Leu 1045 1050 1055 His
Leu Glu Gly Trp Met Lys Val Pro Arg Asn Asn Lys Arg Gly Gln 1060
1065 1070 Gln Gly Trp Asp Arg Lys Tyr Ile Val Leu Glu Gly Ser Lys
Val Leu 1075 1080 1085 Ile Tyr Asp Asn Glu Ala Arg Glu Ala Gly Gln
Arg Pro Val Glu Glu 1090 1095 1100 Phe Glu Leu Cys Leu
Pro Asp Gly Asp Val Ser Ile His Gly Ala Val 1105 1110 1115 1120 Gly
Ala Ser Glu Leu Ala Asn Thr Ala Lys Ala Asp Val Pro Tyr Ile 1125
1130 1135 Leu Lys Met Glu Ser His Pro His Thr Thr Cys Trp Pro Gly
Arg Thr 1140 1145 1150 Leu Tyr Leu Leu Ala Pro Ser Phe Pro Asp Lys
Gln Arg Trp Val Thr 1155 1160 1165 Ala Leu Glu Ser Val Val Ala Gly
Gly Arg Val Ser Arg Glu Lys Ala 1170 1175 1180 Glu Ala Asp Ala Lys
Leu Leu Gly Asn Ser Leu Leu Lys Leu Glu Gly 1185 1190 1195 1200 Asp
Asp Arg Leu Asp Met Asn Cys Thr Leu Pro Phe Ser Asp Gln Val 1205
1210 1215 Val Leu Val Gly Thr Glu Glu Gly Leu Tyr Ala Leu Asn Val
Leu Lys 1220 1225 1230 Asn Ser Leu Thr His Ile Pro Gly Ile Gly Ala
Val Phe Gln Ile Tyr 1235 1240 1245 Ile Ile Lys Asp Leu Glu Lys Leu
Leu Met Ile Ala Gly Glu Glu Arg 1250 1255 1260 Ala Leu Cys Leu Val
Asp Val Lys Lys Val Lys Gln Ser Leu Ala Gln 1265 1270 1275 1280 Ser
His Leu Pro Ala Gln Pro Asp Val Ser Pro Asn Ile Phe Glu Ala 1285
1290 1295 Val Lys Gly Cys His Leu Phe Ala Ala Gly Lys Ile Glu Asn
Ser Leu 1300 1305 1310 Cys Ile Cys Ala Ala Met Pro Ser Lys Val Val
Ile Leu Arg Tyr Asn 1315 1320 1325 Asp Asn Leu Ser Lys Tyr Cys Ile
Arg Lys Glu Ile Glu Thr Ser Glu 1330 1335 1340 Pro Cys Ser Cys Ile
His Phe Thr Asn Tyr Ser Ile Leu Ile Gly Thr 1345 1350 1355 1360 Asn
Lys Phe Tyr Glu Ile Asp Met Lys Gln Tyr Thr Leu Asp Glu Phe 1365
1370 1375 Leu Asp Lys Asn Asp His Ser Leu Ala Pro Ala Val Phe Ala
Ser Ser 1380 1385 1390 Ser Asn Ser Phe Pro Val Ser Ile Val Gln Ala
Asn Ser Ala Gly Gln 1395 1400 1405 Arg Glu Glu Tyr Leu Leu Cys Phe
His Glu Phe Gly Val Phe Val Asp 1410 1415 1420 Ser Tyr Gly Arg Arg
Ser Arg Thr Asp Asp Leu Lys Trp Ser Arg Leu 1425 1430 1435 1440 Pro
Leu Ala Phe Ala Tyr Arg Glu Pro Tyr Leu Phe Val Thr His Phe 1445
1450 1455 Asn Ser Leu Glu Val Ile Glu Ile Gln Ala Arg Ser Ser Leu
Gly Ser 1460 1465 1470 Pro Ala Arg Ala Tyr Leu Glu Ile Pro Asn Pro
Arg Tyr Leu Gly Pro 1475 1480 1485 Ala Ile Ser Ser Gly Ala Ile Tyr
Leu Ala Ser Ser Tyr Gln Asp Lys 1490 1495 1500 Leu Arg Val Ile Cys
Cys Lys Gly Asn Leu Val Lys Glu Ser Gly Thr 1505 1510 1515 1520 Glu
Gln His Arg Val Pro Ser Thr Ser Arg Ser Ser Pro Asn Lys Arg 1525
1530 1535 Gly Pro Pro Thr Tyr Asn Glu His Ile Thr Lys Arg Val Ala
Ser Ser 1540 1545 1550 Pro Ala Pro Pro Glu Gly Pro Ser His Pro Arg
Glu Pro Ser Thr Pro 1555 1560 1565 His Arg Tyr Arg Asp Arg Glu Gly
Arg Thr Glu Leu Arg Arg Asp Lys 1570 1575 1580 Ser Pro Gly Arg Pro
Leu Glu Arg Glu Lys Ser Pro Gly Arg Met Leu 1585 1590 1595 1600 Ser
Thr Arg Arg Glu Arg Ser Pro Gly Arg Leu Phe Glu Asp Ser Ser 1605
1610 1615 Arg Gly Arg Leu Pro Ala Gly Ala Val Arg Thr Pro Leu Ser
Gln Val 1620 1625 1630 Asn Lys Val Trp Asp Gln Ser Ser Val 1635
1640 6 1597 PRT Mus musculus 6 Met Leu Leu Gly Glu Glu Ala Met Met
Glu Gln Glu Met Thr Arg Leu 1 5 10 15 His Arg Arg Val Ser Glu Val
Glu Ala Val Leu Ser Gln Lys Glu Val 20 25 30 Glu Leu Lys Ala Ser
Glu Thr Gln Arg Ser Leu Leu Glu Gln Asp Leu 35 40 45 Ala Thr Tyr
Ile Thr Glu Cys Ser Ser Leu Lys Arg Ser Leu Glu Gln 50 55 60 Ala
Arg Met Glu Val Ser Gln Glu Asp Asp Lys Ala Leu Gln Leu Leu 65 70
75 80 His Asp Ile Arg Glu Gln Ser Arg Lys Leu Gln Glu Ile Lys Glu
Gln 85 90 95 Glu Tyr Gln Ala Gln Val Glu Glu Met Arg Leu Met Met
Asn Gln Leu 100 105 110 Glu Glu Asp Leu Val Ser Ala Arg Arg Arg Ser
Asp Leu Tyr Glu Ser 115 120 125 Glu Leu Arg Glu Ser Arg Leu Ala Ala
Glu Glu Phe Lys Arg Lys Ala 130 135 140 Asn Glu Cys Gln His Lys Leu
Met Lys Ala Lys Asp Gln Gly Lys Pro 145 150 155 160 Glu Val Gly Glu
Tyr Ser Lys Leu Glu Lys Ile Asn Ala Glu Gln Gln 165 170 175 Leu Lys
Ile Gln Glu Leu Gln Glu Lys Leu Glu Lys Ala Val Lys Ala 180 185 190
Ser Thr Glu Ala Thr Glu Leu Leu Gln Asn Ile Arg Gln Ala Lys Glu 195
200 205 Arg Ala Glu Arg Glu Leu Glu Lys Leu His Asn Arg Glu Asp Ser
Ser 210 215 220 Glu Gly Ile Lys Lys Lys Leu Val Glu Ala Glu Glu Arg
Arg His Ser 225 230 235 240 Leu Glu Asn Lys Val Lys Arg Leu Glu Thr
Met Glu Arg Arg Glu Asn 245 250 255 Arg Leu Lys Asp Asp Ile Gln Thr
Lys Ser Glu Gln Ile Gln Gln Met 260 265 270 Ala Asp Lys Ile Leu Glu
Leu Glu Glu Lys His Arg Glu Ala Gln Val 275 280 285 Ser Ala Gln His
Leu Glu Val His Leu Lys Gln Lys Glu Gln His Tyr 290 295 300 Glu Glu
Lys Ile Lys Val Leu Asp Asn Gln Ile Lys Lys Asp Leu Ala 305 310 315
320 Asp Lys Glu Ser Leu Glu Asn Met Met Gln Arg His Glu Glu Glu Ala
325 330 335 His Glu Lys Gly Lys Ile Leu Ser Glu Gln Lys Ala Met Ile
Asn Ala 340 345 350 Met Asp Ser Lys Ile Arg Ser Leu Glu Gln Arg Ile
Val Glu Leu Ser 355 360 365 Glu Ala Asn Lys Leu Ala Ala Asn Ser Ser
Leu Phe Thr Gln Arg Asn 370 375 380 Met Lys Ala Gln Glu Glu Met Ile
Ser Glu Leu Arg Gln Gln Lys Phe 385 390 395 400 Tyr Leu Glu Thr Gln
Ala Gly Lys Leu Glu Ala Gln Asn Arg Lys Leu 405 410 415 Glu Glu Gln
Leu Glu Lys Ile Ser His Gln Asp His Ser Asp Lys Ser 420 425 430 Arg
Leu Leu Glu Leu Glu Thr Arg Leu Arg Glu Val Ser Leu Glu His 435 440
445 Glu Glu Gln Lys Leu Glu Leu Lys Arg Gln Leu Thr Glu Leu Gln Leu
450 455 460 Ser Leu Gln Glu Arg Glu Ser Gln Leu Thr Ala Leu Gln Ala
Ala Arg 465 470 475 480 Ala Ala Leu Glu Ser Gln Leu Arg Gln Ala Lys
Thr Glu Leu Glu Glu 485 490 495 Thr Thr Ala Glu Ala Glu Glu Glu Ile
Gln Ala Leu Thr Ala His Arg 500 505 510 Asp Glu Ile Gln Arg Lys Phe
Asp Ala Leu Arg Asn Ser Cys Thr Val 515 520 525 Ile Thr Asp Leu Glu
Glu Gln Leu Asn Gln Leu Thr Glu Asp Asn Ala 530 535 540 Glu Leu Asn
Asn Gln Asn Phe Tyr Leu Ser Lys Gln Leu Asp Glu Ala 545 550 555 560
Ser Gly Ala Asn Asp Glu Ile Val Gln Leu Arg Ser Glu Val Asp His 565
570 575 Leu Arg Arg Glu Ile Thr Glu Arg Glu Met Gln Leu Thr Ser Gln
Lys 580 585 590 Gln Thr Met Glu Ala Leu Lys Thr Thr Cys Thr Met Leu
Glu Glu Gln 595 600 605 Val Leu Asp Leu Glu Ala Leu Asn Asp Glu Leu
Leu Glu Lys Glu Arg 610 615 620 Gln Trp Glu Ala Trp Arg Ser Val Leu
Gly Asp Glu Lys Ser Gln Phe 625 630 635 640 Glu Cys Arg Val Arg Glu
Leu Gln Arg Met Leu Asp Thr Glu Lys Gln 645 650 655 Ser Arg Ala Arg
Ala Asp Gln Arg Ile Thr Glu Ser Arg Gln Val Val 660 665 670 Glu Leu
Ala Val Lys Glu His Lys Ala Glu Ile Leu Ala Leu Gln Gln 675 680 685
Ala Leu Lys Glu Gln Lys Leu Lys Ala Glu Ser Leu Ser Asp Lys Leu 690
695 700 Asn Asp Leu Glu Lys Lys His Ala Met Leu Glu Met Asn Ala Arg
Ser 705 710 715 720 Leu Gln Gln Lys Leu Glu Thr Glu Arg Glu Leu Lys
Gln Arg Leu Leu 725 730 735 Glu Glu Gln Ala Lys Leu Gln Gln Gln Met
Asp Leu Gln Lys Asn His 740 745 750 Ile Phe Arg Leu Thr Gln Gly Leu
Gln Glu Ala Leu Asp Arg Ala Asp 755 760 765 Leu Leu Lys Thr Glu Arg
Ser Asp Leu Glu Tyr Gln Leu Glu Asn Ile 770 775 780 Gln Val Leu Tyr
Ser His Glu Lys Val Lys Met Glu Gly Thr Ile Ser 785 790 795 800 Gln
Gln Thr Lys Leu Ile Asp Phe Leu Gln Ala Lys Met Asp Gln Pro 805 810
815 Ala Lys Lys Lys Lys Val Pro Leu Gln Tyr Asn Glu Leu Lys Leu Ala
820 825 830 Leu Glu Lys Glu Lys Ala Arg Cys Ala Glu Leu Glu Glu Ala
Leu Gln 835 840 845 Lys Thr Arg Ile Glu Leu Arg Ser Ala Arg Glu Glu
Ala Ala His Arg 850 855 860 Lys Ala Thr Asp His Pro His Pro Ser Thr
Pro Ala Thr Ala Arg Gln 865 870 875 880 Gln Ile Ala Met Ser Ala Ile
Val Arg Ser Pro Glu His Gln Pro Ser 885 890 895 Ala Met Ser Leu Leu
Ala Pro Pro Ser Ser Arg Arg Lys Glu Ser Ser 900 905 910 Thr Pro Glu
Glu Phe Ser Arg Arg Leu Lys Glu Arg Met His His Asn 915 920 925 Ile
Pro His Arg Phe Asn Val Gly Leu Asn Met Arg Ala Thr Lys Cys 930 935
940 Ala Val Cys Leu Asp Thr Val His Phe Gly Arg Gln Ala Ser Lys Cys
945 950 955 960 Leu Glu Cys Gln Val Met Cys His Pro Lys Cys Ser Thr
Cys Leu Pro 965 970 975 Ala Thr Cys Gly Leu Pro Ala Glu Tyr Ala Thr
His Phe Thr Glu Ala 980 985 990 Phe Cys Arg Asp Lys Met Asn Ser Pro
Gly Leu Gln Ser Lys Glu Pro 995 1000 1005 Gly Ser Ser Leu His Leu
Glu Gly Trp Met Lys Val Pro Arg Asn Asn 1010 1015 1020 Lys Arg Gly
Gln Gln Gly Trp Asp Arg Lys Tyr Ile Val Leu Glu Gly 1025 1030 1035
1040 Ser Lys Val Leu Ile Tyr Asp Asn Glu Ala Arg Glu Ala Gly Gln
Arg 1045 1050 1055 Pro Val Glu Glu Phe Glu Leu Cys Leu Pro Asp Gly
Asp Val Ser Ile 1060 1065 1070 His Gly Ala Val Gly Ala Ser Glu Leu
Ala Asn Thr Ala Lys Ala Asp 1075 1080 1085 Val Pro Tyr Ile Leu Lys
Met Glu Ser His Pro His Thr Thr Cys Trp 1090 1095 1100 Pro Gly Arg
Thr Leu Tyr Leu Leu Ala Pro Ser Phe Pro Asp Lys Gln 1105 1110 1115
1120 Arg Trp Val Thr Ala Leu Glu Ser Val Val Ala Gly Gly Arg Val
Ser 1125 1130 1135 Arg Glu Lys Ala Glu Ala Asp Ala Lys Leu Leu Gly
Asn Ser Leu Leu 1140 1145 1150 Lys Leu Glu Gly Asp Asp Arg Leu Asp
Met Asn Cys Thr Leu Pro Phe 1155 1160 1165 Ser Asp Gln Val Val Leu
Val Gly Thr Glu Glu Gly Leu Tyr Ala Leu 1170 1175 1180 Asn Val Leu
Lys Asn Ser Leu Thr His Ile Pro Gly Ile Gly Ala Val 1185 1190 1195
1200 Phe Gln Ile Tyr Ile Ile Lys Asp Leu Glu Lys Leu Leu Met Ile
Ala 1205 1210 1215 Gly Glu Glu Arg Ala Leu Cys Leu Val Asp Val Lys
Lys Val Lys Gln 1220 1225 1230 Ser Leu Ala Gln Ser His Leu Pro Ala
Gln Pro Asp Val Ser Pro Asn 1235 1240 1245 Ile Phe Glu Ala Val Lys
Gly Cys His Leu Phe Ala Ala Gly Lys Ile 1250 1255 1260 Glu Asn Ser
Leu Cys Ile Cys Ala Ala Met Pro Ser Lys Val Val Ile 1265 1270 1275
1280 Leu Arg Tyr Asn Asp Asn Leu Ser Lys Tyr Cys Ile Arg Lys Glu
Ile 1285 1290 1295 Glu Thr Ser Glu Pro Cys Ser Cys Ile His Phe Thr
Asn Tyr Ser Ile 1300 1305 1310 Leu Ile Gly Thr Asn Lys Phe Tyr Glu
Ile Asp Met Lys Gln Tyr Thr 1315 1320 1325 Leu Asp Glu Phe Leu Asp
Lys Asn Asp His Ser Leu Ala Pro Ala Val 1330 1335 1340 Phe Ala Ser
Ser Ser Asn Ser Phe Pro Val Ser Ile Val Gln Ala Asn 1345 1350 1355
1360 Ser Ala Gly Gln Arg Glu Glu Tyr Leu Leu Cys Phe His Glu Phe
Gly 1365 1370 1375 Val Phe Val Asp Ser Tyr Gly Arg Arg Ser Arg Thr
Asp Asp Leu Lys 1380 1385 1390 Trp Ser Arg Leu Pro Leu Ala Phe Ala
Tyr Arg Glu Pro Tyr Leu Phe 1395 1400 1405 Val Thr His Phe Asn Ser
Leu Glu Val Ile Glu Ile Gln Ala Arg Ser 1410 1415 1420 Ser Leu Gly
Ser Pro Ala Arg Ala Tyr Leu Glu Ile Pro Asn Pro Arg 1425 1430 1435
1440 Tyr Leu Gly Pro Ala Ile Ser Ser Gly Ala Ile Tyr Leu Ala Ser
Ser 1445 1450 1455 Tyr Gln Asp Lys Leu Arg Val Ile Cys Cys Lys Gly
Asn Leu Val Lys 1460 1465 1470 Glu Ser Gly Thr Glu Gln His Arg Val
Pro Ser Thr Ser Arg Ser Ser 1475 1480 1485 Pro Asn Lys Arg Gly Pro
Pro Thr Tyr Asn Glu His Ile Thr Lys Arg 1490 1495 1500 Val Ala Ser
Ser Pro Ala Pro Pro Glu Gly Pro Ser His Pro Arg Glu 1505 1510 1515
1520 Pro Ser Thr Pro His Arg Tyr Arg Asp Arg Glu Gly Arg Thr Glu
Leu 1525 1530 1535 Arg Arg Asp Lys Ser Pro Gly Arg Pro Leu Glu Arg
Glu Lys Ser Pro 1540 1545 1550 Gly Arg Met Leu Ser Thr Arg Arg Glu
Arg Ser Pro Gly Arg Leu Phe 1555 1560 1565 Glu Asp Ser Ser Arg Gly
Arg Leu Pro Ala Gly Ala Val Arg Thr Pro 1570 1575 1580 Leu Ser Gln
Val Asn Lys Val Trp Asp Gln Ser Ser Val 1585 1590 1595 7 1286 PRT
Homo sapiens 7 Val Leu Asp Asn Gln Ile Lys Lys Asp Leu Ala Asp Lys
Glu Thr Leu 1 5 10 15 Glu Asn Met Met Gln Arg His Glu Glu Glu Ala
His Glu Lys Gly Lys 20 25 30 Ile Leu Ser Glu Gln Lys Ala Met Ile
Asn Ala Met Asp Ser Lys Ile 35 40 45 Arg Ser Leu Glu Gln Arg Ile
Val Glu Leu Ser Glu Ala Asn Lys Leu 50 55 60 Ala Ala Asn Ser Ser
Leu Phe Thr Gln Arg Asn Met Lys Ala Gln Glu 65 70 75 80 Glu Met Ile
Ser Glu Leu Arg Gln Gln Lys Phe Tyr Leu Glu Thr Gln 85 90 95 Ala
Gly Lys Leu Glu Ala Gln Asn Arg Lys Leu Glu Glu Gln Leu Glu 100 105
110 Lys Ile Ser His Gln Asp His Ser Asp Lys Asn Arg Leu Leu Glu Leu
115 120 125 Glu Thr Arg Leu Arg Glu Val Ser Leu Glu His Glu Glu Gln
Lys Leu 130 135 140 Glu Leu Lys Arg Gln Leu Thr Glu Leu Gln Leu Ser
Leu Gln Glu Arg 145 150 155 160 Glu Ser Gln Leu Thr Ala Leu Gln Ala
Ala Arg Ala Ala Leu Glu Ser 165 170 175 Gln Leu Arg Gln Ala Lys Thr
Glu Leu Glu Glu Thr Thr Ala Glu Ala 180 185 190 Glu Glu Glu Ile Gln
Ala Leu Thr Ala His Arg Asp Glu Ile Gln Arg 195 200 205 Lys Phe Asp
Ala Leu Arg Asn Ser Cys Thr Val Ile Thr Asp Leu Glu 210 215 220 Glu
Gln Leu Asn Gln Leu Thr Glu Asp Asn Ala Glu Leu Asn Asn Gln 225 230
235 240 Asn Phe Tyr Leu Ser Lys Gln Leu Asp Glu Ala Ser Gly Ala Asn
Asp 245 250 255 Glu Ile Val Gln Leu Arg Ser Glu Val Asp His Leu Arg
Arg Glu Ile 260 265 270 Thr Glu Arg Glu Met Gln Leu Thr Ser Gln Lys
Gln Thr Met Glu Ala 275 280 285 Leu Lys Thr
Thr Cys Thr Met Leu Glu Glu Gln Val Met Asp Leu Glu 290 295 300 Ala
Leu Asn Asp Glu Leu Leu Glu Lys Glu Arg Gln Trp Glu Ala Trp 305 310
315 320 Arg Ser Val Leu Gly Asp Glu Lys Ser Gln Phe Glu Cys Arg Val
Arg 325 330 335 Glu Leu Gln Arg Met Leu Asp Thr Glu Lys Gln Ser Arg
Ala Arg Ala 340 345 350 Asp Gln Arg Ile Thr Glu Ser Arg Gln Val Val
Glu Leu Ala Val Lys 355 360 365 Glu His Lys Ala Glu Ile Leu Ala Leu
Gln Gln Ala Leu Lys Glu Gln 370 375 380 Lys Leu Lys Ala Glu Ser Leu
Ser Asp Lys Leu Asn Asp Leu Glu Lys 385 390 395 400 Lys His Ala Met
Leu Glu Met Asn Ala Arg Ser Leu Gln Gln Lys Leu 405 410 415 Glu Thr
Glu Arg Glu Leu Lys Gln Arg Leu Leu Glu Glu Gln Ala Lys 420 425 430
Leu Gln Gln Gln Met Asp Leu Gln Lys Asn His Ile Phe Arg Leu Thr 435
440 445 Gln Gly Leu Gln Glu Ala Leu Asp Arg Ala Asp Leu Leu Lys Thr
Glu 450 455 460 Arg Ser Asp Leu Glu Tyr Gln Leu Glu Asn Ile Gln Val
Leu Tyr Ser 465 470 475 480 His Glu Lys Val Lys Met Glu Gly Thr Ile
Ser Gln Gln Thr Lys Leu 485 490 495 Ile Asp Phe Leu Gln Ala Lys Met
Asp Gln Pro Ala Lys Lys Lys Lys 500 505 510 Val Pro Leu Gln Tyr Asn
Glu Leu Lys Leu Ala Leu Glu Lys Glu Lys 515 520 525 Ala Arg Cys Ala
Glu Leu Glu Glu Ala Leu Gln Lys Thr Arg Ile Glu 530 535 540 Leu Arg
Ser Ala Arg Glu Glu Ala Ala His Arg Lys Ala Thr Asp His 545 550 555
560 Pro His Pro Ser Thr Pro Ala Thr Ala Arg Gln Gln Ile Ala Met Ser
565 570 575 Ala Ile Val Arg Ser Pro Glu His Gln Pro Ser Ala Met Ser
Leu Leu 580 585 590 Ala Pro Pro Ser Ser Arg Arg Lys Glu Ser Ser Thr
Pro Glu Glu Phe 595 600 605 Ser Arg Arg Leu Lys Glu Arg Met His His
Asn Ile Pro His Arg Phe 610 615 620 Asn Val Gly Leu Asn Met Arg Ala
Thr Lys Cys Ala Val Cys Leu Asp 625 630 635 640 Thr Val His Phe Gly
Arg Gln Ala Ser Lys Cys Leu Glu Cys Gln Val 645 650 655 Met Cys His
Pro Lys Cys Ser Thr Cys Leu Pro Ala Thr Cys Gly Leu 660 665 670 Pro
Ala Glu Tyr Ala Thr His Phe Thr Glu Ala Phe Cys Arg Asp Lys 675 680
685 Met Asn Ser Pro Gly Leu Gln Thr Lys Glu Pro Ser Ser Ser Leu His
690 695 700 Leu Glu Gly Trp Met Lys Val Pro Arg Asn Asn Lys Arg Gly
Gln Gln 705 710 715 720 Gly Trp Asp Arg Lys Tyr Ile Val Leu Glu Gly
Ser Lys Val Leu Ile 725 730 735 Tyr Asp Asn Glu Ala Arg Glu Ala Gly
Gln Arg Pro Val Glu Glu Phe 740 745 750 Glu Leu Cys Leu Pro Asp Gly
Asp Val Ser Ile His Gly Ala Val Gly 755 760 765 Ala Ser Glu Leu Ala
Asn Thr Ala Lys Ala Asp Val Pro Tyr Ile Leu 770 775 780 Lys Met Glu
Ser His Pro His Thr Thr Cys Trp Pro Gly Arg Thr Leu 785 790 795 800
Tyr Leu Leu Ala Pro Ser Phe Pro Asp Lys Gln Arg Trp Val Thr Ala 805
810 815 Leu Glu Ser Val Val Ala Gly Gly Arg Val Ser Arg Glu Lys Ala
Glu 820 825 830 Ala Asp Ala Lys Leu Leu Gly Asn Ser Leu Leu Lys Leu
Glu Gly Asp 835 840 845 Asp Arg Leu Asp Met Asn Cys Thr Leu Pro Phe
Ser Asp Gln Val Val 850 855 860 Leu Val Gly Thr Glu Glu Gly Leu Tyr
Ala Leu Asn Val Leu Lys Asn 865 870 875 880 Ser Leu Thr His Val Pro
Gly Ile Gly Ala Val Phe Gln Ile Tyr Ile 885 890 895 Ile Lys Asp Leu
Glu Lys Leu Leu Met Ile Ala Gly Glu Glu Arg Ala 900 905 910 Leu Cys
Leu Val Asp Val Lys Lys Val Lys Gln Ser Leu Ala Gln Ser 915 920 925
His Leu Pro Ala Gln Pro Asp Ile Ser Pro Asn Ile Phe Glu Ala Val 930
935 940 Lys Gly Cys His Leu Phe Gly Ala Gly Lys Ile Glu Asn Gly Leu
Cys 945 950 955 960 Ile Cys Ala Ala Met Pro Ser Lys Val Val Ile Leu
Arg Tyr Asn Glu 965 970 975 Asn Leu Ser Lys Tyr Cys Ile Arg Lys Glu
Ile Glu Thr Ser Glu Pro 980 985 990 Cys Ser Cys Ile His Phe Thr Asn
Tyr Ser Ile Leu Ile Gly Thr Asn 995 1000 1005 Lys Phe Tyr Glu Ile
Asp Met Lys Gln Tyr Thr Leu Glu Glu Phe Leu 1010 1015 1020 Asp Lys
Asn Asp His Ser Leu Ala Pro Ala Val Phe Ala Ala Ser Ser 1025 1030
1035 1040 Asn Ser Phe Pro Val Ser Ile Val Gln Val Asn Ser Ala Gly
Gln Arg 1045 1050 1055 Glu Glu Tyr Leu Leu Cys Phe His Glu Phe Gly
Val Phe Val Asp Ser 1060 1065 1070 Tyr Gly Arg Arg Ser Arg Thr Asp
Asp Leu Lys Trp Ser Arg Leu Pro 1075 1080 1085 Leu Ala Phe Ala Tyr
Arg Glu Pro Tyr Leu Phe Val Thr His Phe Asn 1090 1095 1100 Ser Leu
Glu Val Ile Glu Ile Gln Ala Arg Ser Ser Ala Gly Thr Pro 1105 1110
1115 1120 Ala Arg Ala Tyr Leu Asp Ile Pro Asn Pro Arg Tyr Leu Gly
Pro Ala 1125 1130 1135 Ile Ser Ser Gly Ala Ile Tyr Leu Ala Ser Ser
Tyr Gln Asp Lys Leu 1140 1145 1150 Arg Val Ile Cys Cys Lys Gly Asn
Leu Val Lys Glu Ser Gly Thr Glu 1155 1160 1165 His His Arg Gly Pro
Ser Thr Ser Arg Ser Ser Pro Asn Lys Arg Gly 1170 1175 1180 Pro Pro
Thr Tyr Asn Glu His Ile Thr Lys Arg Val Ala Ser Ser Pro 1185 1190
1195 1200 Ala Pro Pro Glu Gly Pro Ser His Pro Arg Glu Pro Ser Thr
Pro His 1205 1210 1215 Arg Tyr Arg Glu Gly Arg Thr Glu Leu Arg Arg
Asp Lys Ser Pro Gly 1220 1225 1230 Arg Pro Leu Glu Arg Glu Lys Ser
Pro Gly Arg Met Leu Ser Thr Arg 1235 1240 1245 Arg Glu Arg Ser Pro
Gly Arg Leu Phe Glu Asp Ser Ser Arg Gly Arg 1250 1255 1260 Leu Pro
Ala Gly Ala Val Arg Thr Pro Leu Ser Gln Val Asn Lys Val 1265 1270
1275 1280 Trp Asp Gln Ser Ser Val 1285
* * * * *
References