U.S. patent application number 10/377072 was filed with the patent office on 2004-08-12 for novel 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 and 49933 molecules and uses therefor.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Chun, Miyoung, Curtis, Rory A. J., Glucksmann, Maria Alexandra, Logan, Thomas Joseph, Meyers, Rachel E., Rudolph-Owen, Laura A., Tsai, Fong-Ying, Williamson, Mark J..
Application Number | 20040157221 10/377072 |
Document ID | / |
Family ID | 46299020 |
Filed Date | 2004-08-12 |
United States Patent
Application |
20040157221 |
Kind Code |
A9 |
Curtis, Rory A. J. ; et
al. |
August 12, 2004 |
Novel 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 and 49933 molecules and uses therefor
Abstract
The invention provides isolated nucleic acids molecules,
designated 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 and 49933 nucleic acid molecules. The invention
also provides antisense nucleic acid molecules, recombinant
expression vectors containing 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 nucleic acid
molecules, host cells into which the expression vectors have been
introduced, and nonhuman transgenic animals in which a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 gene has been introduced or disrupted. The invention
still further provides isolated 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 proteins, fusion
proteins, antigenic peptides and anti-25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
antibodies. Diagnostic and therapeutic methods utilizing
compositions of the invention are also provided.
Inventors: |
Curtis, Rory A. J.;
(Ashland, MA) ; Logan, Thomas Joseph;
(Springfield, PA) ; Glucksmann, Maria Alexandra;
(Lexington, MA) ; Meyers, Rachel E.; (Newton,
MA) ; Williamson, Mark J.; (Saugus, MA) ;
Rudolph-Owen, Laura A.; (Medford, MA) ; Chun,
Miyoung; (Belmont, MA) ; Tsai, Fong-Ying;
(Newton, MA) |
Correspondence
Address: |
Jean M. Silveri
75 Sidney Street
Cambridge
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 0009501 A1 |
January 15, 2004 |
|
|
Family ID: |
46299020 |
Appl. No.: |
10/377072 |
Filed: |
February 27, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10377072 |
Feb 27, 2003 |
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09895860 |
Jun 29, 2001 |
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10377072 |
Feb 27, 2003 |
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09723806 |
Nov 28, 2000 |
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6686185 |
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10377072 |
Feb 27, 2003 |
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09843297 |
Apr 25, 2001 |
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6569667 |
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10377072 |
Feb 27, 2003 |
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09861801 |
May 21, 2001 |
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10377072 |
Feb 27, 2003 |
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09816494 |
Mar 23, 2001 |
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6664089 |
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09816494 |
Mar 23, 2001 |
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09815419 |
Mar 22, 2001 |
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10377072 |
Feb 27, 2003 |
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09888911 |
Jun 25, 2001 |
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10377072 |
Feb 27, 2003 |
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09908664 |
Jul 17, 2001 |
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10377072 |
Feb 27, 2003 |
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09935291 |
Aug 21, 2001 |
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60215370 |
Jun 29, 2000 |
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60187455 |
Mar 7, 2000 |
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60199801 |
Apr 26, 2000 |
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60205508 |
May 19, 2000 |
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60191858 |
Mar 24, 2000 |
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60213688 |
Jun 23, 2000 |
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60218675 |
Jul 17, 2000 |
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60250932 |
Nov 30, 2000 |
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60226504 |
Aug 21, 2000 |
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Current U.S.
Class: |
435/6.12 ;
435/183; 435/320.1; 435/325; 435/6.1; 435/69.1; 530/350;
536/23.2 |
Current CPC
Class: |
C12N 9/18 20130101; C12Y
207/01001 20130101; C07K 14/465 20130101; C12N 9/20 20130101; C12N
2310/3513 20130101; C12N 9/00 20130101; C12N 9/88 20130101; C07K
14/43545 20130101; C07K 14/43581 20130101; C07K 2319/00 20130101;
C12Y 301/04003 20130101; A01K 2217/05 20130101; A61K 38/00
20130101; A61P 43/00 20180101; C07K 14/463 20130101; C07K 14/47
20130101; C07K 14/705 20130101; C07K 14/395 20130101; C07K 14/43595
20130101; C12N 9/0071 20130101; C12N 9/0083 20130101; C12N 9/1205
20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/183; 435/320.1; 435/325; 530/350; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/00; C12P 021/02; C12N 005/06; C07K 014/47 |
Claims
What is claimed is:
1. An isolated 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 nucleic acid molecule selected
from the group consisting of: a) a nucleic acid molecule comprising
a nucleotide sequence which is at least 60% identical to the
nucleotide sequence of SEQ ID NO: 1, 3, 7, 9, 16, 18, 20, 22, 25,
27, 28, 30, 35, 37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72, or
the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC Accession Number ______; b) a nucleic acid molecule
comprising a fragment of at least 15 nucleotides of the nucleotide
sequence of SEQ ID NO: 1, 3, 7, 9, 16, 18, 20, 22, 25, 27, 28, 30,
35, 37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC
Accession Number ______; c) a nucleic acid molecule which encodes a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 8,
17, 21, 26, 29, 36, 40, 43, 64, 68 or 71, or the amino acid
sequence encoded by the cDNA insert of the plasmid deposited with
the ATCC Accession Number ______; d) a nucleic acid molecule which
encodes a fragment of a polypeptide comprising the amino acid
sequence of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or
71, or the amino acid sequence encoded by the cDNA insert of the
plasmid deposited with the ATCC Accession Number ______, wherein
the fragment comprises at least 15 contiguous amino acids of SEQ ID
NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71, or the amino
acid sequence encoded by the cDNA insert of the plasmid deposited
with the ATCC Accession Number ______; e) a nucleic acid molecule
which encodes a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 8,
17, 21, 26, 29, 36, 40, 43, 64, 68 or 71, or the amino acid
sequence encoded by the cDNA insert of the plasmid deposited with
the ATCC Accession Number ______, wherein the nucleic acid molecule
hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1, 3,
7, 9, 16, 18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41, 42, 44, 63,
65, 67, 69, 70 or 72, or a complement thereof, under stringent
conditions; f) a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO: 1, 3, 7, 9, 16, 18, 20, 22, 25, 27, 28, 30,
35, 37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC
Accession Number ______; and g) a nucleic acid molecule which
encodes a polypeptide comprising the amino acid sequence of SEQ ID
NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71, or the amino
acid sequence encoded by the cDNA insert of the plasmid deposited
with the ATCC Accession Number ______.
2. The isolated nucleic acid molecule of claim 1, which is the
nucleotide sequence SEQ ID NO: 1, 7, 16, 20, 25, 28, 35, 39, 42,
63, 67 or 70.
3. A host cell which contains the nucleic acid molecule of claim
1.
4. An isolated 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 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
60% identical to a nucleic acid comprising the nucleotide sequence
of SEQ ID NO: 1, 3, 7, 9, 16, 18, 20, 22, 25, 27, 28, 30, 35, 37,
39, 41, 42, 44, 63, 65, 67, 69, 70 or 72, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC
Accession Number ______, or a complement thereof; b) a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64,
68 or 71, or the amino acid sequence encoded by the cDNA insert of
the plasmid deposited with the ATCC Accession Number ______,
wherein the polypeptide is encoded by a nucleic acid molecule which
hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1, 3,
7, 9, 16, 18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41, 42, 44, 63,
65, 67, 69, 70 or 72, or a complement thereof under stringent
conditions; c) a fragment of a polypeptide comprising the amino
acid sequence of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64,
68 or 71, or the amino acid sequence encoded by the cDNA insert of
the plasmid deposited with the ATCC Accession Number ______,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71; and
d) the amino acid sequence of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36,
40, 43, 64, 68 or 71.
5. An antibody which selectively binds to a polypeptide of claim
4.
6. The polypeptide of claim 4, further comprising heterologous
amino acid sequences.
7. A method for producing a polypeptide selected from the group
consisting of: a) a polypeptide comprising the amino acid sequence
of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71, or
the amino acid sequence encoded by the cDNA insert of the plasmid
deposited with the ATCC Accession Number ______; b) a polypeptide
comprising a fragment of the amino acid sequence of SEQ ID NO: 2,
8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71, or the amino acid
sequence encoded by the cDNA insert of the plasmid deposited with
the ATCC Accession Number ______, wherein the fragment comprises at
least 15 contiguous amino acids of SEQ ID NO: 2, 8, 17, 21, 26, 29,
36, 40, 43, 64, 68 or 71, or the amino acid sequence encoded by the
cDNA insert of the plasmid deposited with the ATCC Accession Number
______; c) a naturally occurring allelic variant of a polypeptide
comprising the amino acid sequence of SEQ ID NO: 2, 8, 17, 21, 26,
29, 36, 40, 43, 64, 68 or 71, or the amino acid sequence encoded by
the cDNA insert of the plasmid deposited with the ATCC Accession
Number ______, wherein the polypeptide is encoded by a nucleic acid
molecule which hybridizes to a nucleic acid molecule comprising SEQ
ID NO: 1, 3, 7, 9, 16, 18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41,
42, 44, 63, 65, 67, 69, 70 or 72; and d) the amino acid sequence of
SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or71;
comprising culturing the host cell of claim 3 under conditions in
which the nucleic acid molecule is expressed.
8. A method for detecting the presence of a nucleic acid molecule
of claim 1 or a polypeptide encoded by the nucleic acid molecule in
a sample, comprising: a) contacting the sample with a compound
which selectively hybridizes to the nucleic acid molecule of claim
1 or binds to the polypeptide encoded by the nucleic acid molecule;
and b) determining whether the compound hybridizes to the nucleic
acid or binds to the polypeptide in the sample.
9. A kit comprising a compound which selectively hybridizes to a
nucleic acid molecule of claim 1 or binds to a polypeptide encoded
by the nucleic acid molecule and instructions for use.
10. A method for identifying a compound which binds to a
polypeptide or modulates the activity of the polypeptide of claim 4
comprising the steps of: a) contacting a polypeptide, or a cell
expressing a polypeptide of claim 4 with a test compound; and b)
determining whether the polypeptide binds to the test compound or
determining the effect of the test compound on the activity of the
polypeptide.
11. A method for modulating the activity of a polypeptide of claim
4 comprising contacting the polypeptide or a cell expressing the
polypeptide with a compound which binds to the polypeptide in a
sufficient concentration to modulate the activity of the
polypeptide.
12. A method for identifying a compound capable of treating a
disorder characterized by aberrant 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 activity,
comprising assaying the ability of the compound to modulate 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 nucleic acid expression or 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
polypeptide activity, thereby identifying a compound capable of
treating a disorder characterized by aberrant 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
activity.
13. A method of identifying a nucleic acid molecule associated with
a disorder characterized by aberrant 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 activity,
comprising: a) contacting a sample from a subject with a disorder
characterized by aberrant 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 activity, comprising
nucleic acid molecules with a hybridization probe comprising at
least 25 contiguous nucleotides of SEQ ID NO: 1, 7, 16, 20, 25, 28,
35, 39, 42, 63, 67 or 70 defined in claim 2; and b) detecting the
presence of a nucleic acid molecule in the sample that hybridizes
to the probe, thereby identifying a nucleic acid molecule
associated with a disorder characterized by aberrant 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 activity.
14. A method of identifying a polypeptide associated with a
disorder characterized by aberrant 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 activity,
comprising: a) contacting a sample comprising polypeptides with a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 polypeptide defined in claim 4; and b)
detecting the presence of a polypeptide in the sample that binds to
the 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 binding partner, thereby identifying the
polypeptide associated with a disorder characterized by aberrant
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 activity.
15. A method of identifying a subject having a disorder
characterized by aberrant 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 activity, comprising: a)
contacting a sample obtained from the subject comprising nucleic
acid molecules with a hybridization probe comprising at least 25
contiguous nucleotides of SEQ ID NO: 1, 7, 16, 20, 25, 28, 35, 39,
42, 63, 67 or70 defined in claim 2; and b) detecting the presence
of a nucleic acid molecule in the sample that hybridizes to the
probe, thereby identifying a subject having a disorder
characterized by aberrant 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 activity.
16. A method for treating a subject having a disorder characterized
by aberrant 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 activity, or a subject at risk of
developing a disorder characterized by aberrant 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 activity, comprising administering to the subject a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 modulator of the nucleic acid molecule defined in
claim 1 or the polypeptide encoded by the nucleic acid molecule or
contacting a cell with a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 modulator.
17. The method defined in claim 16 wherein said disorder is a
cellular proliferative and/or differentiative disorder, an
angiogenic disorder, a brain disorder, a neurological disorder, a
blood vessel disorder, a breast disorder, a colon disorder, a
kidney disorder, a lung disorder, an ovarian disorder, a prostate
disorder, a hematopoeitic disorder, a pancreatic disorder, a
skeletal muscle disorder, a skin disorder, a hormonal disorder, an
immune e.g., inflammatory, disorder, a cardiovascular disorder, a
lipid homeostasis disorder, an endothelial cell disorder, a liver
disorder, a disorder of the small intestine, a pain disorder, a
viral disease, a metabolic disorder, bone metabolism disorders or
platelet disorders.
18. The method of claim 16, wherein the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 modulator
is a) a small molecule; b) peptide; c) phosphopeptide; d)
anti-25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 antibody; e) a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 8,
17, 21, 26, 29, 36, 40, 43, 64, 68 or 71, or a fragment thereof; f)
a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 polypeptide comprising an amino acid sequence
which is at least 90 percent identical to the amino acid sequence
of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71,
wherein the percent identity is calculated using the ALIGN program
for comparing amino acid sequences, a PAM120 weight residue table,
a gap length penalty of 12, and a gap penalty of 4; or g) an
isolated naturally occurring allelic variant of a polypeptide
consisting of the amino acid sequence of SEQ ID NO: 2, 8, 17, 21,
26, 29, 36, 40, 43, 64, 68 or 71, wherein the polypeptide is
encoded by a nucleic acid molecule which hybridizes to a complement
of a nucleic acid molecule consisting of SEQ ID NO: 1, 7, 16, 20,
25, 28, 35, 39, 42, 63, 67 or 70 at 6.times. SSC at 45.degree. C.,
followed by one or more washes in 0.2.times. SSC, 0.1% SDS at
65.degree. C.
19. The method of claim 16, wherein the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 modulator
is a) an antisense 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 nucleic acid molecule; b) is a
ribozyme; c) the nucleotide sequence of SEQ ID NO: 1, 7, 16, 20,
25, 28, 35, 39, 42, 63, 67 or 70 or a fragment thereof; d) a
nucleic acid molecule encoding a polypeptide comprising an amino
acid sequence which is at least 90 percent identical to the amino
acid sequence of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64,
68 or 71, wherein the percent identity is calculated using the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4;
e) a nucleic acid molecule encoding a naturally occurring allelic
variant of a polypeptide comprising the amino acid sequence of SEQ
ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71, wherein the
nucleic acid molecule which hybridizes to a complement of a nucleic
acid molecule consisting of SEQ ID NO: 1, 7, 16, 20, 25, 28, 35,
39, 42, 63, 67 or 70 at 6.times. SSC at 45.degree. C., followed by
one or more washes in 0.2.times. SSC, 0.1% SDS at 65.degree. C.; or
f) a gene therapy vector.
Description
RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
patent application Ser. No. 09/895,860, filed Jun. 29, 2001
(pending), which claims the benefit of U.S. Provisional Application
Serial No. 60/215,370, filed Jun. 29, 2000. The present application
is also a continuation-in-part of U.S. patent application Ser. No.
09/723,806, filed Nov. 28, 2000 (pending), which claims the benefit
of U.S. Provisional Application Serial No. 60/187,455, filed Mar.
7, 2000. The present application is also a continuation-in-part of
U.S. patent application Ser. No. 09/843,297, filed Apr. 25, 2001
(pending), which claims the benefit of U.S. Provisional Application
Serial No. 60/199,801, filed Apr. 26, 2000. The present application
is also a continuation-in-part of U.S. patent application Ser. No.
09/861,801, filed May 21, 2001 (pending), which claims the benefit
of U.S. Provisional Application Serial No. 60/205,508, filed May
19, 2000. The present application is also a continuation-in-part of
U.S. patent application Ser. No. 09/816,494, filed Mar. 23, 2001
(pending), which claims the benefit of U.S Utility application Ser.
No. 09/815,419, filed Mar. 22, 2001 (pending), which claims the
benefit of U.S. Provisional Application Serial No. 60/191,858,
filed Mar. 24, 2000. The present application is also a
continuation-in-part of U.S. patent application Ser. No.
09/888,911, filed Jun. 25, 2001 (pending), which claims the benefit
of U.S. Provisional Application Serial No. 60/213,688, filed Jun.
23, 2000. The present application is also a continuation-in-part of
U.S. patent application Ser. No. 09/908,664, filed Jul. 17, 2001
(pending), which claims the benefit of U.S. Provisional Application
Serial No. 60/218,675, filed Jul. 17, 2000. The present application
is also a continuation-in-part of U.S. patent application Ser. No.
09/935,291, filed Aug. 21, 2001 (pending), which claims the benefit
of U.S. Provisional Application Serial No. 60/250,932, filed Nov.
30, 2000 and of U.S. provisional application Ser. No. 60/226,504,
filed Aug. 21, 2000. The entire contents of each of the
above-referenced patent applications are incorporated herein by
this reference.
BACKGROUND OF THE INVENTION
[0002] The enormous variety of biochemical reactions that comprise
life are nearly all mediated by a series of biological catalysts
known as enzymes. Enzymes are proteins which possess specific
properties that enable them to catalyze a series of reactions,
allowing metabolic pathways to degrade and to reconstruct products
needed to maintain organisms. By the binding of substrates through
geometrically and physically complementary reactions, enzymes are
stereospecific in binding substrates as well as in catalyzing
reactions. The stringency for this stereospecificity varies as some
enzymes are more specific to the identity of their substrates,
while others are capable of binding multiple substrates and can
catalyze numerous types of reactions.
[0003] Examples of enzymes include, for example, carboxylases,
fatty acid desaturases, serine/threonine dehydratases, hexokinases,
peptidyl tRNA hydrolases, dual specificity phosphatases,
phospholipases and transporters. Such enzymes have the ability to,
for example: (1) hydrolyze an ester linkage and/or liberate the
free acid form of a substrate, e.g., hydrolysis of a triglyceride
and/or liberation of free fatty acid(s) and glycerol; (2) catalyze
the formation of a double bond, preferably, at positions up to 9
carbons from the carboxyl end of a molecule, e.g., a fatty acid,
such as a polyunsaturated fatty acid; (3) catalyze the
phosphorylation of a sugar, e.g., an aldohexoses and a ketohexoses
(e.g., glucose, mannose, fructose, sorbitol and glucosamine); (4)
catalyze sugar metabolism; (5) transfer a phosphate from a
phosphate donor (e.g., ATP) to a sugar, e.g., an aldohexoses and a
ketohexoses (e.g., glucose, mannose, fructose, sorbitol and
glucosamine) to form a phosphorylated sugar, e.g.,
glucose-6-phosphate; (6) catalyze the removal of a phosphate group
attached to a tyrosine residue in a protein target, e.g., a growth
factor receptor; (7) catalyze the removal of a phosphate group
attached to a serine or threonine residue in a protein e.g., a
growth factor receptor; (8) hydrolyze covalent bond between peptide
and tRNA within peptidyl-tRNAs; (9) catalyze the hydrolysis of
phosphatidyl-inositol-4,5-bisphosphate (PIP2) producing
diacylglycerol and inositol 1,4,5-trisphosphate; (10) transport of
a substrate or target molecule (e.g., a Ca.sup.2+ ion) from one
side of a biological membrane to the other; and (11) be
phosphorylated or dephosphorylated. Accordingly, there exists a
need to identify additional human enzymes, for example, for use as
disease markers and as targets for identifying various therapeutic
modulators.
SUMMARY OF THE INVENTION
[0004] The present invention is based, at least in part, on the
discovery of novel nucleic acid molecules and proteins encoded by
such nucleic acid molecules, referred to herein as "25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933". The 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 nucleic acid and protein molecules of
the present invention are useful as modulating agents in regulating
a variety of cellular processes, e.g., including but not limited to
cell proliferation, differentiation, growth and division. In
particular, these nucleic acid molecules will be advantageous in
the regulation of any cellular function, uncontrolled proliferation
and differentiation, such as in cases of cancer. Accordingly, in
one aspect, this invention provides isolated nucleic acid molecules
encoding 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 proteins or biologically active
portions thereof, as well as nucleic acid fragments suitable as
primers or hybridization probes for the detection of 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933-encoding nucleic acids.
[0005] The nucleotide sequence of the cDNA encoding 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933, and the amino acid sequence of 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
polypeptides are depicted in Table 1.
1TABLE 1 Sequences of the invention ATCC Gene cDNA Protein Coding
Region Accession Name SEQ ID NO: SEQ ID NO: SEQ ID NO: Number 25869
1 2 3 25934 7 8 9 26335 16 17 18 50365 20 21 22 21117 25 26 27
38692 28 29 30 46508 35 36 37 16816 39 40 41 16839 42 43 44 49937
63 64 65 49931 67 68 69 49933 70 71 72
[0006] Accordingly, in one aspect, the invention features a nucleic
acid molecule which encodes a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein or
polypeptide, e.g., a biologically active portion of the 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein. In a preferred embodiment, the isolated
nucleic acid molecule encodes a polypeptide having the amino acid
sequence of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or
71. In other embodiments, the invention provides isolated 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 nucleic acid molecules having the nucleotide
sequence shown in SEQ ID NO: 1, 3, 7, 9, 16, 18, 20, 22, 25, 27,
28, 30, 35, 37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72 or the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC Accession Number ______. In still other embodiments, the
invention provides nucleic acid molecules that are substantially
identical (e.g., naturally occurring allelic variants) to the
nucleotide sequence shown in SEQ ID NO: 1, 3, 7, 9, 16, 18, 20, 22,
25, 27, 28, 30, 35, 37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72 or
the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC Accession Number ______. In other embodiments, the
invention provides a nucleic acid molecule which hybridizes under a
stringent hybridization condition as described herein to a nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO: 1,
3, 7, 9, 16, 18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41, 42, 44,
63, 65, 67, 69, 70 or 72 or the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC Accession Number ______,
wherein the nucleic acid encodes a full length 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
protein or an active fragment thereof.
[0007] In a related aspect, the invention further provides nucleic
acid constructs which include a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 nucleic acid molecules
of the invention e.g., vectors and host cells suitable for
producing polypeptides.
[0008] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933-encoding nucleic acids.
[0009] In still another related aspect, isolated nucleic acid
molecules that are antisense to a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 encoding
nucleic acid molecule are provided.
[0010] In another aspect, the invention features 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 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 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933-associated disorders. In another embodiment, the invention
provides 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 polypeptides having a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 activity.
[0011] In other embodiments, the invention provides 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 polypeptides, e.g., a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 polypeptide
having the amino acid sequence shown in SEQ ID NO: 2, 8, 17, 21,
26, 29, 36, 40, 43, 64, 68 or 71, or the amino acid sequence
encoded by the cDNA insert of the plasmid deposited with ATCC
Accession Number ______; an amino acid sequence that is
substantially identical to the amino acid sequence shown in SEQ ID
NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71, or the amino
acid sequence encoded by the cDNA insert of the plasmid deposited
with ATCC Accession Number ______; or an amino acid sequence
encoded by a nucleic acid molecule having a nucleotide sequence
which hybridizes under a stringent hybridization condition as
described herein to a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO: 1, 3, 7, 9, 16, 18, 20, 22, 25,
27, 28, 30, 35, 37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72, or
the nucleotide sequence of the insert of the plasmid deposited with
ATCC Accession Number ______, wherein the nucleic acid encodes a
full length 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 protein or an active fragment
thereof.
[0012] In a related aspect, the invention further provides nucleic
acid constructs which include a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 nucleic acid
molecule described herein.
[0013] In a related aspect, the invention provides 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 polypeptides or fragments operatively linked to non-25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 polypeptides to form fusion proteins.
[0014] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically or selectively bind 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
polypeptides.
[0015] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 polypeptides or nucleic acids.
[0016] In still another aspect, the invention provides a process
for modulating 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 polypeptide or nucleic acid
expression or activity, e.g., using the compounds identified in the
screens described herein. In certain embodiments, the methods
involve treatment of conditions related to aberrant activity or
expression of the 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 polypeptides or nucleic acids,
such as conditions or disorders involving aberrant or deficient
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 expression. Examples of such disorders
include, but are not limited to cellular proliferative and/or
differentiative disorders, angiogenic disorders, brain disorders,
neurological disorders, blood vessel disorders, breast disorders,
colon disorders, kidney disorders, lung disorders, ovarian
disorders, prostate disorders, hematopoeitic disorders, pancreatic
disorders, skeletal muscle disorders, skin disorders, hormonal
disorders, immune e.g., inflammatory, disorders, cardiovascular
disorders, lipid homeostasis disorders, endothelial cell disorders,
liver disorders, disorders of the small intestine, pain disorders,
viral diseases, metabolic disorders, bone metabolism disorders or
platelet disorders.
[0017] The invention also provides assays for determining the
activity of or the presence or absence of 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
polypeptides or nucleic acid molecules in a biological sample,
including for disease diagnosis.
[0018] In a further aspect, the invention provides assays for
determining the presence or absence of a genetic alteration in a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 polypeptide or nucleic acid molecule,
including for disease diagnosis.
[0019] In another aspect, the invention features 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.
At least one address of the plurality has a capture probe that
recognizes a 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 molecule. In one embodiment,
the capture probe is a nucleic acid, e.g., a probe complementary to
a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 nucleic acid sequence. In another embodiment,
the capture probe is a polypeptide, e.g., an antibody specific for
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 polypeptides. Also featured is a method of
analyzing a sample by contacting the sample to the aforementioned
array and detecting binding of the sample to the array.
[0020] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Human 25869 (COE-1)
[0022] The present invention is based, at least in part, on the
discovery of novel carboxylesterase family members, referred to
herein as "25869", "Carboxylesterase" or "COE-1" nucleic acid and
protein molecules. These novel molecules are capable of hydrolyzing
ester-, thioester-, and amide-containing compounds (e.g.,
triglycerides) to their respective free acids, are upregulated in
marmoset monkeys treated with cholestyramine, a cholesterol and/or
lipid lowering drug, and play a role in or function in a variety of
cellular processes, including lipid homeostasis, hydrolysis of
endogenous and/or exogenous compounds; detoxification and/or
activation of drugs, pro-drugs, toxins, and/or carcinogens; intra-
or inter-cellular signaling; gene expression; and/or cellular
growth and/or differentiation.
[0023] Carboxylesterases comprise a family of enzymes which
catalyze the hydrolysis of a variety of ester-, thioester-, and
amide-containing chemicals, as well as drugs (including prodrugs)
to their respective free acids. Carboxylesterases catalyze the
hydrolysis of endogenous compounds such as short- and long-chain
acyl-glycerols, long-chain acylcarnitine, and long-chain acyl-CoA
esters (Satoh, T. and Hosokawa, M. (1998) Annu. Rev. Pharmacol.
Toxicol. 38:257-88). The general enzymatic reaction that
carboxylesterases catalyze is:
a carboxylic ester+H.sub.2O<=>an alcohol+a carboxylic
anion
[0024] The hydrolytic activity of carboxylesterases is dependent on
the presence of a "catalytic triad" of amino acid residues that are
non-contiguous in the primary sequence but adjacent in the tertiary
structure (Ollis, D. L. et al. (1992) Protein Eng. 5:197-211;
Cygler, M. et al. (1993) Protein Sci. 2:366-382). In
carboxylesterases, the catalytic triad residues are Ser/His/Glu
(Satoh and Hosokawa (1998) supra; Oakeshott, J. G. et al. (1999)
Bioessays 21:1031-1042).
[0025] The first step of the hydrolysis reaction catalyzed by
carboxylesterases liberates the alcohol moiety of the substrate and
forms a covalent linkage between the remaining acid moiety of the
substrate and the serine residue of the catalytic triad. The second
step cleaves this linkage and liberates the acid moiety of the
substrate, largely through the action of the histidine residue of
the catalytic triad (Oakeshott et al. (1999) supra).
[0026] While many well-studied carboxylesterases have catalytic
triads, several members of the carboxyl/cholinesterase multigene
family have been reported to lack a functional catalytic triad
(Hortsch, M. et al. (1990) Development 110:1327-1340; Auld, V. J.
(1995) Cell 81:757-767; Ichtchenko, K. et al. (1995) Cell
81:435-443; Oakeshott, J. G. et al. (1995) Trends. Ecol. Evol.
10:103-110) and, thus, to lack carboxylesterase activity. However,
some of these enzymes have ligand-binding functions involved in
signal transduction.
[0027] Carboxylesterases are responsible for the hydrolysis of many
exogenous compounds, resulting in both the inactivation of drugs
and the activation of pro-drugs (Satoh, T. (1987) Reviews in
Biochem. Toxicol. 8:155-81; Heymann, E. (1980) Enzymatic Basis of
Detoxification 2:291-323; Heymann, E. (1982) Metabolic Basis of
Detoxification 1:229-45; Leinweber, F. -J. (1987) Drug. Metab. Rev.
18:379-439). Human liver and plasma carboxylesterase converts
lovastatin to its active form (Tang, B. K. and Kalow, W. (1995)
Eur. J. Clin. Pharmacol. 47:449-51) and converts an inactive
prodrug form of prostaglandin F2.alpha. to its active metabolite
(Cheng-Bennett, A. et al. (1994) Br. J. Ophthalmol. 78:560-67). A
significant number of drugs and endogenous compounds are substrates
of carboxylesterases, including dipivefrin hydrochloride (Nakamura,
M. et al. (1993) Ophthalmic Res. 25:46-51), carbonates (McCracken,
N. W. et al. (1993) Biochem. Pharmacol. 45:31-36; Huang, T. L. et
al. (1993) Pharmacol. Res. 10:639-48), cocaine (Dean, R. A. (1995)
J Pharmacol. Exp. Ther. (1995) 275:965-71; Brzezinski, M. R. et al.
(1994) Biochem. Pharmacol. 48:1747-55), salicylates (White, K. N.
et al. (1994) Biochem. Soc. Trans. 22:220S), capsaicin (Park, Y. H.
and Lee, S. S. (1994) Biochemn. Mol. Biol. Int. 34:351-60),
palmitoyl-coenzyme A (Hosokawa, M. et al. (1987) Mol. Pharmacol.
31:579-84; Hosokawa, M. et al. (1990) Arch. Biochem. Biophys.
277:219-27; Tsujita, T. and Okuda, H. (1993) J. Lipid. Res.
34:1773-81; Mentlein, R. et al. (1984) Arch. Biochem. Biophys.
234:612-21), haloperidol (Nambu, K. et al. (1987) Biochem.
Pharmacol. 36:1715-22), imidapril (Yamada, Y. et al. (1992)
Arzneimittel Forsch. 42:507-12), pyrrolizidine alkaloids (Dueker,
S. R. et al. (1992) Toxicol. Appl. Pharmacol. 117:116-21; Dueker,
S. R. et al. (1995) Arch. Toxicol. 69:725-28; Dueker, S. R. et al.
(1992) Drug. Metab. Dispos. 20:275-80), and steroids (Lund-Pero, M.
et al. (1994) Clin. Chim. Acta. 224:9-20).
[0028] The novel COE-1 molecules of the instant invention are
members of the lipase subfamily of carboxylesterases and show
significant homology to mouse and rat lipases. Analysis of the
activity of a recently characterized hepatic microsomal lipase,
ES-10, showed that increased expression of ES-10 in hepatic cell
lines resulted in an increase in the rate of depletion of
intracellular triacylglycerol stores, indicating that ES-10 is
capable of hydrolysis of stored triacylglycerol. In addition,
hepatocytes expressing ES-10 exhibited increased levels of apo-B
100 in the VLDL fraction (Lehner, R. and Vance, D. E. (1999)
Biochem. J. 343:1-10; Robbi, M. et al. (1990) Biochem. J.
269:451-458).
[0029] Because the COE-1 molecules of the instant invention show
significant homology to ES-10, COE-1 may function as an
intracellular lipase, the activity of which may contribute to the
mobilization of intracellular triacylglycerol stores, which can be
used for lipoprotein assembly. Accordingly, modulation of COE-1
activity may result in the modulation of serum lipoprotein and/or
triglyceride levels. For example, inhibition of COE-1 activity may
have positive effects on serum lipoprotein and triglyceride
profiles. In view of the foregoing activities, the COE-1 molecules
of the present invention provide novel diagnostic targets and
therapeutic agents to control carboxylesterase-associated
disorders.
[0030] As used herein, a "carboxylesterase-associated disorder"
includes a disorder, disease or condition which is caused or
characterized by a misregulation (e.g., downregulation or
upregulation) of carboxylesterase activity.
Carboxylesterase-associated disorders can detrimentally affect
cellular functions such as lipid homeostasis; cellular
proliferation, growth, differentiation, or migration; inter- or
intra-cellular communication; tissue function, such as cardiac
function or musculoskeletal function; systemic responses in an
organism, such as nervous system responses, hormonal responses
(e.g., insulin response), or immune responses; and protection of
cells from toxic compounds (e.g., carcinogens, toxins, or
mutagens).
[0031] In a preferred embodiment, a carboxylesterase-associated
disorder is a "lipid homeostasis disorder". Other examples of
carboxylesterase-associated disorders include cardiovascular
disorders, neurological (CNS) disorders, cellular proliferation,
growth, differentiation, or migration disorders, hormonal
disorders, immune disorders and disorders affecting tissues in
which COE-1 protein is expressed, e.g., the kidney, colon, liver,
brain, small intestine, and skeletal muscle, as assessed by TaqMan
analysis.
[0032] Members of the COE-1 family of proteins, for example,
include at least one "carboxylesterase domain" in the protein or
corresponding nucleic acid molecule. As used herein, the term
"carboxylesterase domain" includes a protein domain having at least
about 440-600 amino acid residues and a bit score of at least 440
when compared against a carboxylesterase Hidden Markov Model (HMM),
e.g., PFAM Accession Number PF00135. Preferably, a carboxylesterase
domain includes a protein having an amino acid sequence of about
460-580, 480-560, 500-540, or more preferably about 519 amino acid
residues, and a bit score of at least 470, 480, 490, 500, or more
preferably, 516.6. To identify the presence of a carboxylesterase
domain in a COE-1 protein, and make the determination that a
protein of interest has a particular profile, the amino acid
sequence of the protein is searched against a database of known
protein domains (e.g., the HMM database). The carboxylesterase
domain (HMM) has been assigned the PFAM Accession number PF00135
(see the PFAM website, available online through Washington
University in St. Louis). A search was performed against the HMM
database resulting in the identification of a carboxylesterase
domain in the amino acid sequence of human COE-1 at about residues
5-523 of SEQ ID NO: 2.
[0033] A description of the Pfam database can be found in Sonhammer
et al. (1997) Proteins 28:405-420, and a detailed description of
HMMs can be found, for example, in Gribskov et al.(1990) Methods
Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci.
USA 84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531;
and Stultz et al.(1993) Protein Sci. 2:305-314, the contents of
which are incorporated herein by reference.
[0034] In another embodiment, a COE-1 family member of the present
invention is identified based on the presence of a "catalytic
triad" in the protein or corresponding nucleic acid molecule. As
used herein, the term "catalytic triad" includes a group of three
amino acid residues which are non-contiguous in the primary
sequence but which are adjacent in the tertiary structure of a
protein and which actively participate in an enzymatic reaction
catalyzed by a carboxylesterase. In the carboxylesterases of the
present invention, the catalytic triad residues are typically
serine, histidine, and glutamic acid (Satoh and Hosokawa (1998)
supra; Oakeshott et al. (1999) supra). The amino acid residues of
the catalytic triad may also be referred to herein as the
"catalytic residues" (e.g., the "catalytic serine" or "catalytic
histidine") or the "active site residues" (e.g., the "active site
serine" or "active site histidine"). In a preferred embodiment, the
serine residue of a catalytic triad of the COE-1 molecules of the
present invention is contained within a catalytic serine motif, as
defined herein. In a further preferred embodiment, the serine
residue of a catalytic triad of the COE-1 molecules of the present
invention is contained within a carboxylesterases type-B serine
active site. An alignment of the COE-1 amino acid sequence (SEQ ID
NO: 2), using the GAP program in the GCG software package (Blosum
62 matrix) and a gap weight of 12 and a length weight of 4, with a
mouse liver carboxylesterase precursor (GenBank Accession No.
Q63880; SEQ ID NO: 4), identified two of the catalytic triad
residues as serine.sup.205 and histidine.sup.426 of human
COE-1.
[0035] In another embodiment, a COE-1 family member of the present
invention is identified based on the presence of a "catalytic
serine motif" in the protein or corresponding nucleic acid
molecule. As used herein, a "catalytic serine motif" includes a
group of five amino acid residues having the consensus sequence
G-X-S-X-G (SEQ ID NO: 6), wherein X indicates any amino acid
residue. Catalytic serine motifs are found in all known lipases
(Lehner, R. and Vance, D. E. (1999) Biochem. J. 343:1-10). In a
preferred embodiment, a catalytic serine motif includes a catalytic
serine, as defined herein. In a further preferred embodiment, a
catalytic serine motif is contained-within a carboxylesterases
type-B serine active site. An alignment of the COE-1 amino acid
sequence (SEQ ID NO: 2), using the GAP program in the GCG software
package (Blosum 62 matrix) and a gap weight of 12 and a length
weight of 4, with a mouse liver carboxylesterase precursor (GenBank
Accession No. Q63880; SEQ ID NO: 4), identified a catalytic serine
motif at about residues 203-207 of human COE-1 (SEQ ID NO: 2).
[0036] In another embodiment, a COE-1 protein of the present
invention is identified based on the presence of a
"carboxylesterases type-B serine active site" in the protein or
corresponding nucleic acid molecule. A carboxylesterases type-B
serine active site functions as part of the catalytic active site
of a carboxylesterase. The carboxylesterases type-B serine active
site has been assigned ProSite Accession Number PS00122. To
identify the presence of a carboxylesterases type-B serine active
site in a COE-1 protein, and to make the determination that a
protein of interest has a particular profile, the amino acid
sequence of the protein may be searched against a database of known
protein domains (e.g., the ProSite database) using the default
parameters (available online through the Swiss Institute of
Bioinformatics (SIB)). In a preferred embodiment, a
carboxylesterases type-B serine active site comprises a catalytic
serine motif, which further comprises a catalytic serine. A search
was performed against the ProSite database resulting in the
identification of a carboxylesterases type-B serine active site in
the amino acid sequence of human COE-1 (SEQ ID NO: 2) at about
residues 192-207.
[0037] In another embodiment, a COE-1 family member of the present
invention is identified based on the presence of an "ER retention
signal" in the protein or corresponding nucleic acid molecule. As
used herein, an "ER retention signal" includes a group of four
amino acid residues located at the C-terminus of a polypeptide
sequence which targets a protein to the lumen of the endoplasmic
reticulum. Based on homology to other proteins known to have ER
retention signals, an ER retention signal was identified in the
amino acid sequence of human COE-1 (SEQ ID NO: 2) at about residues
544-547.
[0038] Isolated proteins of the present invention, preferably COE-1
proteins, have an amino acid sequence sufficiently homologous to
the amino acid sequence of SEQ ID NO: 2, or are encoded by a
nucleotide sequence sufficiently homologous to SEQ ID NO: 1 or 3.
As used herein, the term "sufficiently homologous" refers to a
first amino acid or nucleotide sequence which contains a sufficient
or minimum number of identical or equivalent (e.g., an amino acid
residue which has a similar side chain) amino acid residues or
nucleotides to a second amino acid or nucleotide sequence such that
the first and second amino acid or nucleotide sequences share
common structural domains or motifs and/or a common functional
activity. For example, amino acid or nucleotide sequences which
share common structural domains having at least 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 85%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%,
99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or
more homology or identity across the amino acid sequences of the
domains and contain at least one and preferably two structural
domains or motifs, are defined herein as sufficiently homologous.
Furthermore, amino acid or nucleotide sequences which share at
least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 88%, 89%, 90%,
95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,
99.7%, 99.8%, 99.9% or more homology or identity and share a common
functional activity are defined herein as sufficiently
homologous.
[0039] In a preferred embodiment, a COE-1 protein includes at least
one or more of the following domains, motifs, and/or amino acid
residues: a carboxylesterase domain, a catalytic triad, a catalytic
serine, a catalytic histidine, a catalytic serine motif, a
carboxylesterases type-B serine active site, and/or an ER retention
signal, and has an amino acid sequence at least about 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 85%, 88%, 89%, 90%, 95%, 96%, 97%,
98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,
99.9% or more homologous or identical to the amino acid sequence of
SEQ ID NO: 2, or the amino acid sequence encoded by the DNA insert
of the plasmid deposited with ATCC as Accession Number ______. In
yet another preferred embodiment, a COE-1 protein includes at least
one or more of the following domains, motifs, and/or amino acid
residues: a carboxylesterase domain, a catalytic triad, a catalytic
serine, a catalytic histidine, a catalytic serine motif, a
carboxylesterases type-B serine active site, and/or an ER retention
signal, and is encoded by a nucleic acid molecule having a
nucleotide sequence which hybridizes under stringent hybridization
conditions to a complement of a nucleic acid molecule comprising
the nucleotide sequence of SEQ ID NO: 1 or 3. In another preferred
embodiment, a COE-1 protein includes at least one or more of the
following domains, motifs, and/or amino acid residues: a
carboxylesterase domain, a catalytic triad, a catalytic serine, a
catalytic histidine, a catalytic serine motif, a carboxylesterases
type-B serine active site, and/or an ER retention signal, and has a
COE-1 activity.
[0040] As used interchangeably herein, a "COE-1 activity",
"biological activity of COE-1" or "functional activity of COE-1",
includes an activity exerted or mediated by a COE-1 protein,
polypeptide or nucleic acid molecule on a COE-1 responsive cell or
on a COE-1 substrate, as determined in vivo or in vitro, according
to standard techniques. In one embodiment, a COE-1 activity is a
direct activity, such as an association with a COE-1 target
molecule. As used herein, a "target molecule" or "binding partner"
is a molecule with which a COE-1 protein binds or interacts in
nature, such that COE-1-mediated function is achieved. A COE-1
target molecule can be a non-COE-1 molecule or a COE-1 protein or
polypeptide of the present invention. In an exemplary embodiment, a
COE-1 target molecule is a COE-1 substrate or ligand, e.g., a
triglyceride. A COE-1 activity can also be an indirect activity,
such as a cellular signaling activity mediated by interaction of
the COE-1 protein with a COE-1 substrate or ligand.
[0041] In a preferred embodiment, a COE-1 activity is at least one
of the following activities: (i) interaction with a COE-1 substrate
or target molecule (e.g., a triglyceride); (ii) conversion of a
COE-1 substrate or target molecule to a product (e.g., hydrolysis
of an ester linkage and/or liberation of the free acid form of the
substrate, e.g., hydrolysis of a triglyceride and/or liberation of
free fatty acid(s) and glycerol); (iii) modulation of lipolysis;
(iv) modulation of lipid uptake by a cell (e.g., a liver cell); (v)
modulation of lipid synthesis and/or secretion; (vi) modulation of
intracellular lipid release and/or turnover; (vii) modulation of
intracellular lipid and/or triglyceride mass; (viii) modulation of
secreted lipid and/or triglyceride mass; (ix) modulation of serum
lipid, lipoprotein, and/or triglyceride levels; (x) modulation of
lipid homeostasis; (xi) direct or indirect modulation of
lipoprotein assembly; (xii) interaction with and/or hydrolysis of a
second non-COE-1 protein; (xiii) activation/deactivation of a COE-1
substrate or target molecule (e.g., activation/deactivation of a
carcinogen); (xiv) metabolism and/or detoxification of a drug; (xv)
modulation of cellular signaling and/or gene transcription (e.g.,
either directly or indirectly); and/or (xvi) modulation of cellular
proliferation and/or differentiation.
[0042] Isolation of the Human 25869 or COE-1 cDNA
[0043] The invention is based, at least in part, on the discovery
of genes encoding novel members of the carboxylesterase family. The
entire sequence of human clone Fbh25869 was determined and found to
contain an open reading frame termed human "25869" or "COE-1."
[0044] The nucleotide sequence encoding the human COE-1 is set
forth as SEQ ID NO: 1. The human COE-1 gene, which is approximately
2087 nucleotides in length, encodes a protein having a molecular
weight of approximately 60.2 kD and which is approximately 547
amino acid residues in length as set forth as SEQ ID NO: 2. The
coding region (open reading frame) of SEQ ID NO: 1 is set forth as
SEQ ID NO: 3. Clone Fbh25869, comprising the coding region of human
COE-1, was deposited with the American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on
______, and assigned Accession Number ______. This deposit will be
maintained under the terms of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure. This deposit were made merely as a
convenience for those of skill in the art and is not an admission
that a deposit is required under 35 U.S.C. .sctn. 112.
[0045] Analysis of the Human 25869 or COE-1 Molecules
[0046] The amino acid sequence of human COE-1 was analyzed using
the program PSORT (available online) to predict the localization of
the proteins within the cell. This program assesses the presence of
different targeting and localization of amino acid sequences within
the query sequence. The results of the analyses show that human
COE-1 may be localized to the cytoplasm, to the nucleus, or to the
mitochondria.
[0047] Searches of the amino acid sequence of human COE-1 were
performed against the HMM database. These searches resulted in the
identification of a "carboxylesterase domain" at about residues
5-523 of SEQ ID NO: 2 (score=516.6).
[0048] Searches of the amino acid sequence of human COE-1 were
further performed against the Prosite database. These searches
resulted in the identification in the amino acid sequence of human
COE-1 of a potential N-glycosylation site and a number of potential
protein kinase C phosphorylation sites, casein kinase II
phosphorylation sites, and N-myristoylation sites. These searches
further resulted in the identification of a carboxylesterases
type-B serine active site at about residues 192-207 of SEQ ID NO:
2.
[0049] A "catalytic triad" was also identified in the human COE-1.
An alignment of human COE-1 (SEQ ID NO: 2) with a mouse liver
carboxylesterase precursor (GenBank Accession No. Q63880; SEQ ID
NO: 4), identified two of the catalytic triad amino acids, namely
Ser.sup.205 and His.sup.426 within SEQ ID NO: 2.
[0050] A "catalytic serine motif" was also identified in the human
COE-1. An alignment of human COE-1 (SEQ ID NO: 2), using the GAP
program in the GCG software package (Blosum 62 matrix) and a gap
weight of 12 and a length weight of 4, with a mouse liver
carboxylesterase precursor (GenBank Accession No. Q63880; SEQ ID
NO: 4), identified a catalytic serine motif at about residues
203-207 of human COE-1 (SEQ ID NO: 2).
[0051] An "ER retention signal" was also identified in the human
COE-1. Based on homology to other proteins known to have ER
retention signals, an ER retention signal was identified in the
amino acid sequence of human COE-1 (SEQ ID NO: 2) at about residues
544-547.
[0052] Global alignments (using the GAP program in the GCG software
package (Blosum 62 matrix) and a gap weight of 12 and a length
weight of 4) of the human COE-1 amino acid sequence (SEQ ID NO: 2)
revealed that human COE-1 has a 67.3% identity with the amino acid
sequence of a mouse liver carboxylesterase precursor (SEQ ID NO: 4;
GenBank Accession No. Q63880) and a 46% identity with the amino
acid sequence of rat liver carboxylesterase 10 precursor (also
referred to as ES-10; SEQ ID NO: 5; GenBank Accession No.
P16303).
[0053] Tissue Distribution of COE-1 mRNA
[0054] This example describes the tissue distribution of human
COE-1 mRNA, as may be determined using in situ hybridization
analysis. For in situ analysis, various tissues, e.g., tissues
obtained from brain, are first frozen on dry ice.
Ten-micrometer-thick sections of the tissues are postfixed with 4%
formaldehyde in DEPC-treated 1.times. phosphate-buffered saline at
room temperature for 10 minutes before being rinsed twice in DEPC
1.times. phosphate-buffered saline and once in 0.1 M
triethanolamine-HCl (pH 8.0). Following incubation in 0.25% acetic
anhydride-0.1 M triethanolamine-HCl for 10 minutes, sections are
rinsed in DEPC 2.times. SSC (1.times. SSC is 0.15 M NaCl plus 0.015
M sodium citrate). Tissue is then dehydrated through a series of
ethanol washes, incubated in 100% chloroform for 5 minutes, and
then rinsed in 100% ethanol for 1 minute and 95% ethanol for 1
minute and allowed to air dry.
[0055] Hybridizations are performed with .sup.35S-radiolabeled
(5.times.10.sup.7 cpm/ml) cRNA probes. Probes are incubated in the
presence of a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5),
1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05%
yeast total RNA type .times.1, 1.times. Denhardt's solution, 50%
formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodium
dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hours at
55.degree. C.
[0056] After hybridization, slides are washed with 2.times. SSC.
Sections are then sequentially incubated at 37.degree. C. in TNE (a
solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM
EDTA), for 10 minutes, in TNE with 10 .mu.g of RNase A per ml for
30 minutes, and finally in TNE for 10 minutes. Slides are then
rinsed with 2.times. SSC at room temperature, washed with 2.times.
SSC at 50.degree. C. for 1 hour, washed with 0.2.times. SSC at
55.degree. C. for 1 hour, and 0.2.times. SSC at 60.degree. C. for 1
hour. Sections are then dehydrated rapidly through serial
ethanol-0.3 M sodium acetate concentrations before being air dried
and exposed to Kodak Biomax MR scientific imaging film for 24 hours
and subsequently dipped in NB-2 photoemulsion and exposed at
4.degree. C. for 7 days before being developed and counter
stained.
[0057] Analysis of Human COE-1 Expression using the TaqMan
Procedure
[0058] TaqMan analysis on a panel of: (1) normal artery; (2) normal
vein; (3) aortic smooth muscle cells--early; (4) coronary smooth
muscle cells; (5) human umbilical vein endothelial cells
(HUVECs)--static; (6) human umbilical vein endothelial cells
(HUVECs)--shear; (7) normal heart; (8) heart--congestive heart
failure (CHF); (9) kidney; (10) skeletal muscle; (11) normal
adipose tissue; (12) pancreas; (13) primary osteoblasts; (14)
differentiated osteoclasts; (15) normal skin; (16) normal spinal
cord; (17) normal brain cortex; (18) brain--hypothalamus; (19)
nerve; (20) dorsal root ganglion (DRG); (21) glial cells
(astrocytes); (22) glioblastoma; (23) normal breast; (24) breast
tumor; (25) normal ovary; (26) ovary tumor; (27) normal prostate;
(28) prostate tumor; (29) epithelial cells (prostate); (30) normal
colon; (31) colon tumor; (32) normal lung; (33) lung tumor; (34)
lung--chronic obstructive pulmonary disease (COPD); (35)
colon--inflammatory bowel disease (IBD); (36) normal liver; (37)
liver--fibrosis; (38) dermal cells--fibroblasts; (39) normal
spleen; (40) normal tonsil; (41) lymph node; (42) small intestine;
(43) skin--decubitus; (44) synovium; (45) bone marrow mononuclear
cells (BM-MNC); and (46) activated peripheral blood mononuclear
cells (PBMCs), revealed that human 25869 or COE-1 was highly
expressed in the kidney, colon, liver, brain, small intestine, and
skeletal muscle. Expression of human COE-1 was also upregulated in
marmoset monkeys treated with cholestyramine, a drug which lowers
cholesterol and/or lipids in the blood.
[0059] Analysis of Human 25869 or COE-1 Activity
[0060] The activity, e.g., the lipase activity, of COE-1 molecules
of the present invention may be determined by the use of any of the
following assays. All of the following assays are performed as
described in Lehner, R. and Vance, D. E. (1999) Biochem. J.
343:1-10, the contents of which are incorporated herein by
reference.
[0061] Materials
[0062] Oleic acid, essentially fatty acid-free bovine serum albumin
(BSA), p-nitrophenyl fatty acyl esters and Protein A-Sepharose CL
4B are purchased from Sigma (St. Louis, Mo., U.S.A.).
[9,10-3H]Triolein (28 mCi/mmol) is from Dupont NEN (Boston, Mass.,
U.S.A.). [1,3-.sup.3H]glycerol (2.6 Ci/mmol), [U-.sup.14C]glycerol
(149 mCi/mmol), [9,10-.sup.3H]oleic acid (10 Ci/mmol),
L-[4,5-.sup.3H]leucine (57 Ci/mmol) and ECL Western blotting
reagents are obtained from Amersham Canada (Oakville, Ontario,
Canada). Dulbecco's modified Eagle's medium (DMEM), sodium
pyruvate, penicillin/streptomycin, fetal bovine and horse sera and
Geneticin (G-418 sulfate) are from Gibco BRL (Life Technologies
Inc., Grand Island, N.Y., U.S.A.). Triascin C is purchased from
Biomol Research Laboratories, Inc. (Plymouth Meeting, Pa., U.S.A.).
All other chemicals and solvents are of reagent or better quality
and are obtained from any number of suppliers known to those of
skill in the art. Sheep anti-[human apolipoprotein (apo)B] IgG is
from Boehringer-Mannheim.
[0063] Cell Culture
[0064] Primary hepatocytes are isolated from male Sprague-Dawley
rats (body weight 125-150 g), fed ad libitum, by collagenase
perfusion of the liver. The cells are cultured in DMEM containing
15% (v/v) fetal bovine serum, as described in Yao, Z. and Vance, D.
E. (1988) J. Biol. Chem. 263:449-509. HepG2 cells, obtained from
A.T.C.C., are cultured in minimal Eagle's medium containing 0.1 mM
nonessential amino acids, 1 mM sodium pyruvate, penicillin (10
units/ml), streptomycin (100 .mu.g/ml) and 10% fetal bovine serum.
McArdle RH7777 cells obtained from A.T.C.C., are cultured in DMEM
containing pyruvate, antibiotics, 10% fetal bovine serum, and 10%
(v/v) horse serum. All cultures are maintained in 100 mm dishes
(Corning) at 37.degree. C. in humidified air (89-91% saturation)
containing 5% CO.sub.2.
[0065] Generation of Stable Cell Lines Expressing COE-1 cDNA
[0066] Cells, e.g., McArdle RH7777 cells, are transfected with 10
.mu.g of pBK-CMV plasmid vector (Stratagene) with or without COE-1
cDNA insert using a calcium precipitation procedure, as described
in Chen, C. and Okayama, H. (1987) Mol. Cell. Biol. 7:2745-2752.
Cells stably expressing either the empty vector or the vector
containing the COE-1 cDNA are selected for resistance to the
antibiotic G-418 (0.08% w/v). Transfected McArdle RH7777 cells are
grown in DMEM supplemented with 10% horse serum and 10% fetal
bovine serum, penicillin/streptomycin (40 units/ml) and 0.02% (w/v)
G-418. Cells are maintained at 37.degree. C. in humidified air
containing 5% Co.sub.2.
[0067] Preparation of Microsomal Membranes
[0068] Cells, e.g., McArdle RH7777 cells, from ten 100 mm diameter
culture dishes (approximately 50 mg of protein) are harvested into
5 ml of 10 mM Tris/HCl, pH 7.4, containing 250 mM sucrose and 5 mM
EDTA. Cells are homogenized with a Polytron, and the microsomal
membranes are isolated by ultracentrifugation from a
post-mitochondrial supernatant, as described in Lehner, R. and
Kuksis, A. (1993) J. Biol. Chem. 268:8781-8786.
[0069] Lipase Assay
[0070] Lipolytic activities in microsomal membranes (50 .mu.g of
protein) isolated from `mock` transfected (empty vector) and COE-1
cDNA transfected cells (e.g., McArdle RH7777 cells) are assessed
using either radiolabeled triolein or a chromogenic substrate
(p-nitrophenyl laurate) as described in Lehner, R. and Verger, R.
(1997) Biochemistry 36:1861-1868.
[0071] Immunoblot Analysis
[0072] Cells, e.g., McArdle RH7777 cells (non-transfected, `mock`
transfected, and COE-1 cDNA transfected) are harvested in
phosphate-buffered saline (PBS) and disrupted by brief sonication.
Cell homogenates (35 .mu.g of protein) are electrophoresed on an
SDS/12% (w/v) polyacrylamide gel, transferred to a nitrocellulose
membrane, and the expression of COE-1 is analyzed by blotting with
anti-COE-1 antibodies using standard methods.
[0073] Lipid Uptake by Transfected Cells
[0074] Cells, e.g., McArdle RH7777 cells (at approximately 80%
confluency in 60 mm diameter culture dishes) are incubated with 2
ml of serum-free DMEM containing 0.5% BSA and 100 .mu.Ci
[.sup.3H]oleic acid for 1 hour. The medium is aspirated, the cells
are washed with DMEM/0.5% BSA, and subsequently the cells are
incubated with 5 ml of DMEM/0.5% BAS for 2 hours. The medium is
removed, diluted with DMEM/0.5% BSA and 5 ml aliquots are added to
the cells, e.g., McArdle RH7777 cells, stably transfected with
either pBK-CMV vector or with pBK-CMV containing COE-1 cDNA.
Aliquots of medium (1 ml) are taken after 2, 4, and 6 hours of
incubation. At the end of the incubation period, the cells are
washed with ice-cold PBS, harvested in the same buffer, and
dispersed by brief sonication. Cellular and medium lipids are
extracted as described in Folch, J. et al. ((1957) J. Biol. Chem.
226:449-509) in the presence of non-labeled lipid carriers. The
lipids are applied to TLC plates and developed to one-third the
height with chloroform/methanol/acetic acid/water (25:15:4:2, by
volume) to separate glycerophospholipids, followed by development
in heptane/isopropyl ether/acetic acid (60:40:4, by volume) to
separate neutral lipids. Lipids are made visible by exposure to
iodine; bands corresponding to various lipid classes are scraped,
and the associated radioactivity is determined by scintillation
counting.
[0075] Effect of Triascin C on Glycerolipid Synthesis and
Secretion
[0076] Cells, e.g., McArdle RH7777 cells, stably transfected with
pBK-CMV or pBK-CMV containing COE-1 cDNA are grown to approximately
70% confluency in 60 mm diameter dishes and incubated for 24 hours
with serum-free DMEM containing 0.1 mM oleic acid complexed with
BSA (0.13% final concentration). The medium is aspirated and the
cells are incubated for 1 hour with DMEM containing 0.1 mM oleic
acid/BSA and various concentrations of triascin C in DMSO. The
final concentration of DMSO is 0.4%. After a 1 hour
incubation.+-.triascin C, 10 .mu.Ci/dish of [.sup.3H]glycerol is
added. Cells and medium are collected after 2 hours, lipids are
extracted, and the radioactivity associated with
phosphatidylcholine and triacylglycerol is analyzed as described
above.
[0077] Intracellular Lipid Turnover in Transfected Cells
[0078] Cells, e.g., McArdle RH7777 cells, stably transfected with
pBK-CMV or pBK-CMV containing COE-1 cDNA (grown to approximately
70% confluency) are incubated with serum-free DMEM containing 0.1
mM oleic acid complexed with BSA (0.13% final concentration) and 20
.mu.Ci [.sup.3H]glycerol for 15 hours. After 13 hours, triascin C
(20 .mu.M final concentration) is added. After an additional 2
hours of labeling, medium is removed, cells are washed and
incubated for 6 hours.+-.triascin C (chase). Cells are harvested,
lipids are extracted and separated by TLC, and the amount of
radioactivity in phosphatidylcholine and triacylglycerol is
determined.
[0079] Utilization of Intracellular Triacylglycerol Stores for
Lipoprotein Assembly and Secretion
[0080] Contribution of [.sup.14C]Glycerol-Labeled Intracellular
Triacylglycerol Pools in the Presence of Exogenous Oleate:
Pulse-Chase Protocol
[0081] Cells are incubated for 2 hours with 0.375 mM oleic
acid/0.5% BSA to increase the triacylglycerol stores. Cells are
then washed and incubated for 1 hour with DMEM followed by 2 hours
with 0.375 mM oleic acid/0.5% BSA and 0.25 .mu.Ci
[.sup.14C]glycerol. Radioactivity in cellular and medium
phosphatidylcholine and triacylglycerol is analyzed following lipid
extraction and TLC as described above.
[0082] Secretion of [.sup.3H]Glycerol-Prelabeled Intracellular
Triacylglycerol Pools in the Absence of Oleate
[0083] Cells are incubated for 16 hours with serum-free DMEM
containing 0.375 mM oleic acid/0.5% BSA and 0.25 .mu.Ci [.sup.14
C]glycerol, washed and incubated with DMEM in the absence of
extracellular oleate for up to 8 hours. Radioactivity in cellular
medium lipids is analyzed.
[0084] Lipid Secretion from Transfected Cells
[0085] Cells, e.g., McArdle RH7777 cells, at 60-70% confluency are
washed with DMEM and incubated in the absence of serum for 2 hours.
One set of dishes is incubated for 4 hours with serum-free DMEM
containing 0.5% BSA and 10 .mu.Ci [.sup.3H]glycerol. The other set
of dishes is incubated for 4 hours with serum-free DMEM containing
0.375 mM oleic acid/0.5% BSA and 10 .mu.Ci [.sup.3H]glycerol. At
the end of a 4 hour pulse, a set of oleate-treated cells is washed
with DMEM and incubated for various times with serum-free DMEM
containing 0.5% BSA (chase). Medium and cells are collected, and
lipids are extracted in the presence of non-labeled lipid carriers.
Lipids are separated by TLC and made visible by exposure to iodine,
and the radioactivity associated with phosphatidylcholine and
triacylglycerol is determined.
[0086] Triacylglycerol and Phosphatidylcholine Mass Secretion from
Transfected Cells
[0087] The experimental design for determination of the mass of
triacylglycerol and phosphatidylcholine secreted is identical to
that described above, except that cells are grown in 100 mm
diameter dishes, and medium from three dishes is combined. Lipids
are extracted by TLC, and the mass of phosphatidylcholine is
determined as described in Chalvardjian, A. and Rudnicki, E. (1970)
Anal. Biochem. 36:225-226. The mass of triacylglycerol is
determined according to the methods of Snyder, F. and Stephens, N.
((1959) Biochim. Biophys. Acta 34:244-245) using trioleoylglycerol
as an authentic standard.
[0088] Determination of Intracellular and Secreted Triacylglycerol
Mass
[0089] Cells (e.g., freshly prepared primary rat hepatocytes seeded
at (5-6).times.10.sup.6 cells/60 mm diameter dish; McArdle RH7777
cells at 80% confluency; or HepG2 cells) are incubated for 12 hours
in their respective growth media supplemented with 0.375 mM
oleate/0.5% BSA. Cells are then washed with DMEM and incubated for
2 hours with DMEM containing 0.375 mM oleate/0.5% BSA. The media
are then collected and triacylglycerol mass is analyzed
(triacylglycerol secretion at 2 hours of supplementation with
oleate). Cells are washed and incubated for 2 hours with DMEM in
the absence of oleate. Medium is removed, the cells washed, and
fresh DMEM replenished. The procedure is repeated every 2 hours.
Media and cell lipids are extracted and triacylglycerol mass
determined as described above.
[0090] ApoB Secretion
[0091] Cells stably transfected with either pBK-CMV or pBK-CMV
containing COE-1 cDNA grown to approximately 70% confluency in 60
mm diameter dishes are incubated for 16 hours with serum-free DMEM.
Cells are then incubated for 2 hours with DMEM containing 0.375 mM
oleic acid/0.5% BSA, washed for 1 hour with leucine-free
medium.+-.oleic acid/BSA and incubated for 2 hours with 2 ml of
either leucine-free DMEM containing 0.5% BSA and 250 .mu.Ci of
[.sup.3H]leucine, or leucine-free DMEM containing 0.375 mM oleic
acid/0.5% BSA and 250 .mu.Ci [.sup.3 H]leucine. Medium is removed
and briefly centrifuged to remove cellular debris. A 0.1 ml aliquot
of 10.times. immunoprecipitation buffer (1.5 M NaCl, 0.5 M
Tris/HCl, pH 7.4, 50 mM EDTA, 5% (v/v) Triton X-100, 1% (w/v) SDS;
Wu, X. et al. (1996) J. Lipid Res. 37:1198-1206) is added to the
culture medium (0.9 ml containing 1 mM final concentration of
benzamidine). Anti (human apoB) IgG (10 .mu.l) is then added. The
mixture is incubated for 12 hours at 4.degree. C., then 50 .mu.l of
Protein A-Sepharose is added, and the mixture is incubated for 3
more hours. The beads are pelleted by brief centrifugation, washed
three times with an excess of immunoprecipitation buffer, then 0.1
.mu.g of rat VLDL protein is added, followed by electrophoresis
sample buffer (125 mM Tris/HCl, pH 6.8, 4% SDS, 20% (v/v) glycerol,
10% (v/v) .beta.-mercaptoethanol, 0.02% Bromophenol Blue; Wu et al.
(1996) supra). Samples are boiled and electrophoresed through 5%
(w/v) polyacrylamide gels containing 0.1% SDS. Gels are silver
stained (Rabilloud, T. et al. (1988) Electrophoresis 9:288-291),
and bands corresponding to apoB48 and apoB100 are excised,
dissolved at 60.degree. C. in 0.2 ml of 60% (v/v) perchloric acid
followed by 0.4 ml of 30% (v/v) hydrogen peroxide (Mahin, D. T. and
Lofberg, R. T. (1966) Anal. Biochem. 16:500-509), and the
radioactivity associated with apoB48 and apoB100 is determined
using Hionic-Fluor scintillation cocktail (Packard Instrument Co.,
Meriden, Conn., U.S.A.).
[0092] For detection of apoB in VLDL, density centrifugation is
performed according to Chung et al. ((1980) J. Lipid Res.
21:284-291). To 1.2 ml of medium (containing 1 mM benzamidine) are
added 100 .mu.l of freshly prepared rat plasma and 0.7 grams KBr (4
M final concentration of KBr, density 1.3 g/ml). The mixture is
placed into 5.5 ml Quick-Seal centrifuge tubes (Beckman), carefully
overlayed with 0.9% NaCl, and centrifuged for 45 minutes at 41,600
g (65,000 revolutions/minute; VTi 65.2 rotor). Fractions of 0.5 ml
are collected from the bottom of the tubes, and the densities are
determined. Top fractions containing VLDL have a density of
<1.013 g/ml. Fractions are adjusted to 0.9 ml with water, and
apoB is immunoprecipitated and analyzed as described above.
[0093] Other Methods
[0094] 7.5 mM oleic acid/10% (w/v) BSA stock solution is prepared
by dissolving fatty acid-free BSA in DMEM. The solution is heated
to 56.degree. C., added to 0.106 grams oleic acid, stirred until
the solution clarifies, and sterilized by filtration.
[0095] Protein concentration is determined using the Bio-Rad
Protein Assay kit using BSA as a protein standard.
[0096] Human 25934
[0097] The human 25934 sequence (SEQ ID NO: 7), which is
approximately 1512 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
990 nucleotides (nucleotides 342-1334 of SEQ ID NO: 7; SEQ ID NO:
9). The coding sequence encodes a 330 amino acid protein (SEQ ID
NO: 8).
[0098] Human 25934 contains the following regions or other
structural features: a desaturase domain located at about amino
acid residues 51 to 295 of SEQ ID NO: 8; two transmembrane regions
at about amino acids 50-93 and 194-235 of SEQ ID NO: 8; three
cytoplasmic domains at about amino acids 1-49, 94-193, and 236-330
of SEQ ID NO: 8; one predicted N-glycosylation site (PS00001) at
about amino acids 233 to 236 of SEQ ID NO: 8; one predicted cAmp
and cGMP dependent protein kinase phosphorylation site (PS00004) at
about amino acids 311 to 314 of SEQ ID NO: 8; four predicted
Protein Kinase C phosphorylation sites (PS00005) at about amino
acids 98 to 100, 101 to 103, 255 to 257 and 308 to 310 of SEQ ID
NO: 8; two predicted Casein Kinase II phosphorylation sites
(PS00006) located at about amino acids 138 to 141 and 283-286 of
SEQ ID NO: 8; four predicted N-myristoylation sites (PS00008) from
about amino acids 23 to 28, 40 to 45, 59 to 64, and 88 to 93 of SEQ
ID NO: 8; one predicted amidation site (PS00009) from about amino
acid 170 to 173 of SEQ ID NO: 8; and one predicted fatty acid
desaturase family 1 signature (PS00476) from about amino acid 268
to 282 of SEQ ID NO: 8.
[0099] For general information regarding PFAM identifiers, PS
prefix and PF prefix domain identification numbers, refer to
Sonnhammer et al. (1997) Protein 28:405-420.
[0100] A plasmid containing the nucleotide sequence encoding human
25934 was deposited with American Type Culture Collection (ATCC),
10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and
assigned Accession Number ______. This deposit will be maintained
under the terms of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purposes of
Patent Procedure. This deposit was made merely as a convenience for
those of skill in the art and is not an admission that a deposit is
required under 35 U.S.C. .sctn. 112.
[0101] The 25934 protein contains a significant number of
structural characteristics in common with members of the desaturase
family.
[0102] Based on sequence homology, 25934 polypeptide is predicted
to be a member of the desaturase family of enzymes, specifically
the stearoyl-Co desaturase family (SCD family, EC 1.14.99.5).
[0103] Fatty acid desaturases are critical regulatory enzymes of
unsaturated fatty acid biosynthesis and catalyze the conversion of
a single bond between two carbon atoms (C--C) to a double bond
(C.dbd.C) in a fatty acyl chain. The resultant double bond is often
referred to as an unsaturated bond. Eukaryotic fatty acid
desaturases, typically, are iron-containing enzymes that catalyze
the NAD-(P)H and O.sub.2-dependent introduction of double bonds
into methylene-interrupted fatty acid chains. Examination of the
deduced amino acid sequence from mammals, fungi, insects, higher
plants and cyanobacteria has revealed three regions of conserved
primary sequence containing HX(3 or 4)H, HX(2 or 3), and HX(2 or
3)HH. This motif is also present in the bacterial membrane enzymes
alkaline hydroxylase (omega-hydroxylase) and xylene
monooxygenase.
[0104] There are three types of eukaryotic fatty acid desaturases,
acyl-CoA, acyl-ACP, and acyl-lipid desaturases (Ntambi et al.,
Biochem. and Biophys. Res. Com. 266:1-4, 1999). In plants and
cyanobacteria, acyl-lipid desaturases catalyzing most desaturation
reactions and introduce unsaturated bonds into fatty acids that are
in a lipid-bound form. Acyl-ACP desaturases are present in the
plastids of plant cells and insert a double bond into fatty acids
that are bound to acyl carrier protein (ACP). In animals, yeast and
fungal cells, Acyl-CoA introduce unsaturated bonds into fatty acids
that are bound to coenzyme A (CoA). A gene cloned from this family
is stearoyl-CoA desaturase and this gene has been identified in
many organisms including mice, rats, humans, yeast, ovines, and
hamsters.
[0105] Fatty acid desaturases can introduce an unsaturated bond at
a specific position in a fatty acyl chain, for example, at the -6,
-9, or -12 position. Desaturases are typically integral membrane
proteins induced in the endoplasmic reticulum by dietary
manipulations and then rapidly degraded (Ozols, J. (1997) MBC Vol.
8 (11): 2281-2290). Unsaturated fatty acids can be formed from a
variety of fatty acids including palmitate and stearate resulting
in the formation of unsaturated fatty acids palmitoleate (16:1),
and oleate (18:1).
[0106] In mammals, the rate limiting step in the biosynthesis of
monounsaturated fatty acids is the insertion of an unsaturated bond
by stearoyl-CoA desaturase (SCD) in the -9 position of the fatty
acid. SCD preferentially catalyzes the synthesis of oleic acid.
Oleate enriched low density lipoprotein (LDL) exhibits increased
affinity for the vessel wall, and is therefore pro-atherogenic
(Rudel, L. L. et al. (1997) J. Clin. Invest. 1:100(1):74-83). SCD
involvement in generating atherogenic LDL variants and in
regulating triglyceride synthesis is further supported by the
finding that polyunsaturated fatty acids (PUFA), which protect
against atherosclerosis, negatively regulate the expression of the
SCD gene (Rudel, L L et al. (1995) Atheroscler. Thromb. Vasc. Biol.
15(12):2101-10; Ntambi, J M (1999) J. Lipid Res. 40(9):1549-58).
Moreover, a mouse deficient for SCD exhibits significant reduction
in triglycerides (Miyazaki, M. et al. (2000) J. Biol. Chem, in
press).
[0107] SCD enzymes are structurally and functionally homologous to
one another, and can convert a single bond to a double bond in a
fatty acyl chain. SCD enzymes utilize oxygen and electrons from
cytochrome b.sub.5 for catalysis. Similar to other enzymes such as
ribonucleotide reductases and methane monoxygenases, stearoyl-CoA
desaturases can have a conserved iron binding motif which includes
eight histidines (Shanklin et al. (1997) Proc. Natl. Acad. Sci. USA
94:2981-2986), "H--X(3-4)-H--X(7-41)-H--
-X(2-3)-H--H--X(61-189)-H--X(2-3)-H--H (SEQ ID NO: 11)." The eight
histidine residues common to desaturase family members are
typically divided among three regions of the protein: region II
(H--X(3-4)-H); region Ib (the first H-X(2-3)-H--H sequence); and
region II (the second H--X(2-3)-H--H sequence) (Shanklin et al.
(1994) Biochemistry 33:12787-94).
[0108] SCDs typically contain two or three long hydrophobic domains
termed "transmembrane regions," each of which is capable of
spanning the membrane two times (Shanklin et al. (1994)
Biochemistry 33:12787-94). Because a transmembrane region is
capable of traversing the membrane twice, amino acid residues
flanking a transmembrane region reside on the same side of the
membrane (Stukey et al. (1990) J. Biol. Chem. 265:20144-49). Thus,
when region I (regions Ia and Ib) and region II are divided by a
transmembrane region in a desaturase family member, the regions
will typically reside on the same side of the membrane, e.g., the
cytoplasmic face of the endoplasmic reticulum membrane.
[0109] A 25934 polypeptides include a "desaturase domain" or
regions homologous with a "desaturase domain". As used herein, the
term "desaturase domain" includes an amino acid sequence of about
25 to 600 amino acid residues in length and having a bit score for
the alignment of the sequence to the fatty acid desaturase domain
(HMM) of at least 50. Preferably, a desaturase domain includes at
least about 50-500 amino acids, more preferably about 100-400 amino
acid residues, or about 200-250 amino acids and has a bit score for
the alignment of the sequence to the desaturase domain (HMM) of at
least 60, 80, 100, 150, 200, 250, 300, 450, 500 or greater.
[0110] In a preferred embodiment, 25934 polypeptide or protein has
a "desaturase domain" or a region which includes at least about
50-500 amino acids, more preferably about 100-400 amino acid
residues, or about 200-250 amino acid residues and has at least
about 70% 80% 90% 95%, 99%, or 100% homology with a "desaturase
domain," e.g., the desaturase domain of human 25934 (e.g., residues
51-295 of SEQ ID NO: 8). Preferably, the desaturase domain of a
25934 polypeptide includes at least one, two, three, four, five,
six, seven and preferably eight conserved histidines. Preferably,
the histidines form an eight-histidine motif, which binds two iron
atoms in the catalytic center. For example, a 25934 polypeptide
contains histidine residues at about amino acids 94, 99, 131, 134,
135, 272, 275, and 276 of SEQ ID NO: 8.
[0111] To identify the presence of a "desaturase" domain in a 25934
protein sequence, and make the determination that a polypeptide or
protein of interest has a particular profile, the amino acid
sequence of the protein can be searched against a database of HMMs
(e.g., the Pfam database, release 2.1) using the default
parameters. For example, the hmmsf program, which is available as
part of the HMMR package of search programs, is a family specific
default program for MILPAT0063 and a score of 15 is the default
threshold score for determining a hit. Alternatively, the threshold
score for determining a hit can be lowered (e.g., to 8 bits). A
description of the Pfam database can be found in Sonhammer et al.
(1997) Proteins 28(3):405-420 and a detailed description of HMMs
can be found, for example, in Gribskov et al.(1990) Meth. Enzymol.
183:146-159; Gribskov et al.(1987) Proc. Natl. Acad. Sci. USA
84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; and
Stultz et al.(1993) Protein Sci. 2:305-314, the contents of which
are incorporated herein by reference. A search was performed
against the HMM database resulting in the identification of a
"desaturase" domain in the amino acid sequence of human 25934 at
about residues 51-295 of SEQ ID NO: 8 (the identified desaturase
consensus domain is set forth in SEQ ID NO: 10).
[0112] A 25934 family member includes a desaturase domain and
optionally also a fatty acid desaturase family 1 signature, i.e., a
motif that matches the ProSite motif PS00476,
"G-E-X--[FY]--H--N--[FY]--H--H--X--F--- P--X-D-Y (SEQ ID NO: 12),"
e.g., this motif is found at about residues 268 to 282 of SEQ ID
NO: 8.
[0113] In one embodiment, a 25934 protein includes at least one,
preferably two, transmembrane regions. As used herein, the term
"transmembrane region" includes an amino acid sequence of about 20
amino acid residues in length that spans a phospholipid membrane,
e.g., an endoplasmic reticulum membrane, twice. More preferably, a
transmembrane region includes about at least 22, 24, 25, 30, 35,
40, 45, 50, 55, 60, 65, or 70 amino acid residues and spans a
phospholipid membrane twice. Transmembrane regions are rich in
hydrophobic residues, and typically have an a-helical structure. In
a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or
more of the amino acids of a transmembrane domain are hydrophobic,
e.g., leucines, isoleucines, tyrosines, or tryptophans.
Transmembrane regions are described in, for example,
pfam.wustl.edu/cgi-bin/getdesc?name=7 tm-1, and Zagotta W. N. et
al, (1996) Annual Rev. Neuronsci. 19: 235-63, the contents of which
are incorporated herein by reference.
[0114] In a preferred embodiment, a 25934 polypeptide or protein
has at least one transmembrane region or a region which includes at
least 20, 22, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino
acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or
100% homology with a "transmembrane region," e.g., at least one
transmembrane region of human 25934 (e.g., amino acid residues
50-93 or 194-235 of SEQ ID NO: 8).
[0115] In one embodiment, a 25934 protein includes at least one
cytoplasmic domain. When located at the N-terminal domain the
cytoplasmic domain is referred to herein as an "N-terminal
cytoplasmic domain". As used herein, an "N-terminal cytoplasmic
domain" includes an amino acid sequence having about 1-200,
preferably about 10-100, preferably about 20-90, more preferably
about 30-80, more preferably about 35-70, more preferably about
40-60, or even more preferably about 45-55 amino acid residues in
length and is located in the cytoplasm of a cell. The C-terminal
amino acid residue of a "N-terminal cytoplasmic domain" is adjacent
to an N-terminal amino acid residue of a transmembrane region in a
25934 protein. For example, an N-terminal cytoplasmic domain is
located at about amino acid residues 1-49 of SEQ ID NO: 8.
[0116] In a preferred embodiment, a 25934 polypeptide or protein
has at least one N-terminal cytoplasmic domain or a region which
includes at least about 5, preferably about 40-60, or even more
preferably about 45-55 amino acid residues and has at least about
60%, 70% 80% 90% 95%, 99%, or 100% homology with an "N-terminal
cytoplasmic domain," e.g., at least one N-terminal cytoplasmic
domain of human 25934 (e.g., residues 1-49 of SEQ ID NO: 8).
[0117] In another embodiment, a 25934 protein includes a
"cytoplasmic loop" in the sequence of the protein. As used herein,
a "cytoplasmic loop" includes an amino acid sequence having a
length of at least about 10, preferably about 20-250, preferably
about 30-150, more preferably about 80-120 amino acid residues and
is located within the cytoplasm of a cell. Accordingly, the
N-terminal amino acid residue of a "cytoplasmic loop" is adjacent
to a C-terminal amino acid residue of a transmembrane region and
the C-terminal residue of a "cytoplasmic loop" is adjacent to a
N-terminal amino acid residue of a transmembrane region in a 25934
protein. For example, a cytoplasmic loop is found at about amino
acid residues 94-193 of SEQ ID NO: 8.
[0118] In a preferred embodiment, a 25934 polypeptide or protein
has a cytoplasmic loop or a region which includes at least about
10, preferably about 20-250, preferably about 30-150, more
preferably about 80-120 amino acid residues and has at least about
60%, 70% 80% 90% 95%, 99%, or 100% homology with an "cytoplasmic
loop," e.g., the cytoplasmic loop of human 25934 (e.g., residues
94-193 of SEQ ID NO: 8).
[0119] In another embodiment, a 25934 protein includes a
"C-terminal cytoplasmic domain", also referred to herein as a
C-terminal cytoplasmic tail, in the sequence of the protein. As
used herein, a "C-terminal cytoplasmic domain" includes an amino
acid sequence having a length of at least about 30, preferably
about 50-150, preferably about 60-200, more preferably about 80-110
amino acid residues and is located within the cytoplasm of a cell.
Accordingly, the N-terminal amino acid residue of a "C-terminal
cytoplasmic domain" is adjacent to a C-terminal amino acid residue
of a transmembrane region in a 25934 protein. For example, a
C-terminal cytoplasmic domain is found at about amino acid residues
236-330 of SEQ ID NO: 8.
[0120] In a preferred embodiment, a 25934 polypeptide or protein
has a C-terminal cytoplasmic domain or a region which includes at
least about 30, preferably about 50-150, preferably about 60-200,
more preferably about 80-110 amino acid residues and has at least
about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an
"C-terminal cytoplasmic domain," e.g., the C-terminal cytoplasmic
domain of human 25934 (e.g., residues 236-330 of SEQ ID NO: 8).
[0121] 25934 polypeptides of the invention include 25934 fragments
which include: all or part of a hydrophobic sequence e.g., all or
part of the sequence from about residue 71 to about residue 91 of
SEQ ID NO: 8; all or part of a hydrophilic fragment; or other
fragments that include a cysteine or a glycosylation site.
[0122] A multiple sequence alignment of the 25934 amino acid
sequence with the human (SEQ ID NO: 13), rat (SEQ ID NO: 14), and
chicken (SEQ ID NO: 15) delta-9 desaturase proteins revealed 63.6%,
58.4%, and 58.4% identity between the 25934 amino acid sequence and
the chicken, human and rat amino acid sequences, respectively.
[0123] As the 25934 polypeptides of the invention may modulate
25934-mediated activities, they may be useful as of for developing
novel diagnostic and therapeutic agents for 25934-mediated or
related disorders, as described below.
[0124] As used herein, a "25934 activity", "biological activity of
25934" or "functional activity of 25934", refers to an activity
exerted by a 25934 protein, polypeptide or nucleic acid molecule on
e.g., a 25934-responsive cell or on a 25934 substrate, e.g., a
protein substrate, as determined in vivo or in vitro. In one
embodiment, a 25934 activity is a direct activity, such as an
association with a 25934 target molecule. A "target molecule" or
"binding partner" is a molecule with which a 25934 protein binds or
interacts in nature. In an exemplary embodiment, the binding
partner is a fatty acid, e.g., myristic, palmitic or stearic acid.
The 25934 proteins of the present invention can have one or more of
the following activities: (1) catalyzing the formation of a double
bond, preferably, at positions up to 9 carbons from the carboxyl
end of a molecule, e.g., a fatty acid, such as a polyunsaturated
fatty acid; (2) modulating the synthesis of monounsaturated fatty
acids, e.g., modulating the synthesis of a fatty acid synthesized
in an animal, e.g., oleic acid, palmitoyl- and stearoyl-CoA; (3)
modulating the desaturation of a fatty acid, e.g., a
polyunsaturated fatty acids; (4) modulating cellular lipid
composition, e.g., modulating the ratio of saturated and
unsaturated fatty acids; (5) modulating the energy state of
adipocytes; (6) modulating membrane fluidity; (7) modulating lipid
storage; (8) modulating proliferation and/or differentiation; (9)
modulating lipoprotein (e.g., LDL) composition and/or
concentration; (10) regulating triglyceride synthesis; (11)
altering the HDL/LDL ration; or (12) modulating fatty acid
metabolism.
[0125] Based on the above-described sequence similarities, the
25934 molecules of the present invention are predicted to have
similar biological activities as other desaturase family members,
and in particular, stearoyl CoA desaturases (SCD). For example, the
25934 polypeptide or a domain therein, e.g., desaturase domain, may
function to catalyze the conversion of a single bond between two
carbon atoms (C--C) to a double bond (C.dbd.C) in a fatty acid
chain. This modification is expected to occur at the n9 position of
the fatty acid. Desaturases are predicted to contribute to an
unfavorable LDL content state, e.g., by increasing LDL-oleate,
which is atherogenic (Rudel, L L. et al. (1997) J. Clin Invest.
1:100(1):74-83) as well as by playing a role in triglyceride
metabolism and/or biosynthesis. As shown by TaqMan analysis, Niacin
treatment in the marmoset model results in significant repression
of 25934 in the liver. Niacin has been shown to alter the
composition of LDL and HDL to a favorable state, to cause a
significant reduction in triglycerides, and to increase HDL
concentration (Goldberg, A. (2000) Am. J. Cardiol. 85(9): 1100-5.
Moreover, a mouse deficient for SCD exhibits significant reduction
in triglycerides (Miyazaki, M. et al. (2000) J. Biol. Chem.
275(39):30132-8). Accordingly, the 25934 molecules can act as novel
diagnostic targets and therapeutic agents for controlling disorders
associated with abnormal or aberrant desaturase activity and/or
triglyceride levels. In particular, it is predicted that targeting
the inhibition of 25934 nucleic acids and polypeptides will results
in the favorable modification, and possible reduction, of LDL
content and/or reduction of triglycerides. Thus, the 25934
molecules can act as novel targets for treating and/or diagnosing
fatty acid metabolic disorders (e.g., desaturation of fatty acids)
such as obesity and/or diabetes and more generally, cardiovascular
disorders.
[0126] The term "cardiovascular disorders" or "disease" includes
heart disorders, as well as disorders of the blood vessels of the
circulation system caused by, e.g., abnormally high concentrations
of lipids in the blood vessels.
[0127] In some embodiments, the therapeutic and prophylactic uses
of the compositions of the invention, further include the
administration of cholesterol lowering agents as a combination drug
therapies. The term "combination therapy" as used herein refers to
the administration to a subject (concurrently or sequentially) of
two or more cholesterol lowering agents. Current combination
therapy therapies using combinations of niacin and statins are
being used with positive results to treat hyperlipidemia (Guyton, J
R. (1999) Curr Cardiol Rep. 1(3):244-250; Otto, C. et al. (1999)
Internist (Berl) 40(12):1338-45). Other useful drug combinations
include those derived by addition of fish oil, bile acid binding
resins, or stanol esters, as well as nonstatin combinations susn as
niacin-resin or fibrate-niacin (Guyton, J R. (1999) supra). For
examples of dosages and administration schedules of the cholesterol
lowering agents, the teachings of Guyton, J R. (1999) supra, Otto,
C. et al. (1999) supra, Guyton, J R et al. (1998) Am J Cardiol
82(12A):82U-86U; Guyton, J R et al. (1998) Am J Cardiol.
82(6):737-43; Vega, G L et al. (1998) Am J. Cardiol.
81(4A):36B-42B; Schectman, G. (1996) Ann Intern Med.
125(12):990-1000; Nakamura, H. et al. (1993) Nippon Rinsho
51(8):2101-7; Goldberg, A. et al. (2000) Am J Cardiol 85(9):1100-5;
Morgan, J M et al. (1996) J Cardiovasc. Pharmac. Ther.
1(3):195-202; Stein, E A et al. (1996) J Cardiovasc Pharmacol Ther
1(2):107-116; and Goldberg, A C (1998) Am J Cardiol
82(12A):35U-41U, are expressly incorporated by reference.
[0128] The 25934 molecules can also be used to treat, diagnose or
prevent lipid disorders. Examples of lipid disorders include those
disorders which affect fatty acid metabolism. Fatty acids are
synthesized from acetyl-CoA, which is derived from carbohydrate,
protein and other non-lipid sources, and the pathway produces
saturated fatty acids, predominantly palmitic acid (10:0). In
mammals, the fatty acids may be elongated and desaturated.
Desaturation is catalyzed by desaturases which function by
inserting one or more double bonds at positions up to 9 carbons
from the carboxyl end of a fatty acid molecule.
[0129] The degree of fatty acid desaturation in cell membrane
lipids determines membrane fluidity. The activity of the desaturase
enzyme is critical for maintaining the ratio of saturated and
unsaturated fatty acids in cell membranes. Alterations in this
ratio can, e.g., alter the physical properties of membranes.
Moreover, alterations in the ratio of fatty acids have been
implicated in a range of diseases including diabetes, obesity,
hypertension, cancer, developmental disorders, immune disorders and
neurological and the above-described heart diseases. For example,
tumor tissue and virus-transformed cells have a higher content of
unsaturated fatty acids, especially oleic acid. Such shifts
increase the metabolic rates of many lipid-dependent enzymes and
are associated with a higher capacity for cell division.
[0130] As assessed by TaqMan analysis, the 25934 mRNA is found in
the brain, ovary, kidney and liver, therefore the molecules of the
invention can be used to develop novel agents or compounds to
treat, prevent and/or diagnose disorders involving aberrant
activities of those cells. For example, the molecules of the
invention can be used to treat, present and/or diagnose
neurological disorders, brain disorders, ovarian disorders, kidney
disorders and liver disorders, as described below.
[0131] Identification and Characterization of Human 25934 cDNA
[0132] The human 25934 sequence (SEQ ID NO: 7), which is
approximately 1512 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
990 nucleotides (nucleotides 342 to 1334 of SEQ ID NO: 7; SEQ ID
NO: 9). The coding sequence encodes a 330 amino acid protein (SEQ
ID NO: 8).
[0133] Tissue Distribution of 25934 mRNA
[0134] Endogenous human 25934 gene expression was determined using
the Perkin-Elmer/ABI 7700 Sequence Detection System which employs
TaqMan technology. To determine the level of 25934 in various human
tissues a primer/probe set was designed using Primer Express
(Perkin-Elmer) software and primary cDNA sequence information.
Total RNA was prepared from a series of human tissues using an
RNeasy kit from Qiagen. First strand cDNA was prepared from 1 Tg
total RNA using an oligo-dT primer and Superscript II reverse
transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng
total RNA was used per TaqMan reaction.
[0135] 25934 mRNA levels were analyzed in a variety of samples
isolated from the human fetal heart, spinal cord, brain (cortex,
hypothalamus, glial cells), ovary, kidney, liver, endothelial cells
and smooth muscle cells (SMC). The highest relative 25934 mRNA
expression, i.e., greater than 200 relative units, was observed in
spinal cord, brain and ovary. High level mRNA expression, i.e.,
greater than 100 relative units was observed in the kidney,
endothelial cells and human umbilical vein endothelial cells
(HUVEC). Expression in liver (a target organ for 25934) was
positive but lower relative to other tissues
[0136] The relative 25934 mRNA expression levels were determined
using an expanded TaqMan panel of human liver tissues and then this
panel was used to compare the expression of 25934 mRNA and stearoyl
CoA desaturase (SCD) mRNA. Expression of 25934 mRNA in human liver
is equivalent to the relative expression of the known SCD gene.
[0137] Further TaqMan analyses also demonstrate an inhibition of
25934 mRNA expression in the marmoset animal model. Niacin
treatment in the marmoset model results in significant repression
of 25934 in the liver.
[0138] Human 26335 (DHY)
[0139] The present invention is based, at least in part, on the
discovery of novel molecules, referred to herein as "26335",
"dehydratase" or "DHY" nucleic acid and protein molecules, which
are novel members of a family of enzymes possessing dehydratase
activity. These novel molecules are capable of deaminating serine
or threonine to pyruvate or 2-oxobutyrate, respectively, by
catalyzing a two-step reaction of dehydration of the amino acid,
followed by hydrolysis of the resulting imine. These novel
molecules may thus play a role in or function in a variety of
cellular processes, e.g., cellular proliferation, growth,
differentiation, migration, and inter- or intra-cellular
communication.
[0140] The biosynthesis and metabolism of amino acids is of
critical importance in many metabolic and catabolic pathways in
cells, and is fundamental to the production of cellular proteins. A
wide array of enzymes facilitate the synthesis, interconversion,
and degradation of amino acids, including transaminases, oxidases,
reductases, dehydrogenases, and kinases, among many others. One
such family of enzymes, the serine and threonine dehydratases,
catalyze the irreversible deamination of serine or threonine to
pyruvate or 2-oxobutyrate, respectively.
[0141] The reaction mechanism for these enzymes has been
characterized (Snell and Di Mari (1970) The Enzymes (Boyer, P. D.,
ed.), Academic Press: 3.sup.rd ed. Vol. 2: 335-370; and Ogawa et
al. (1989) Biochim. Biophys. Acta 996: 139-141). First, a Schiff
base is formed between a pyridoxal-5'phosphate cofactor and a
specific lysine residue which is strictly conserved within the
serine and threonine dehydratase family. A new Schiff base is
subsequently formed between the cofactor and the hydroxyamino acid
by transimination, catalyzing the removal of the .alpha.-proton
through stabilization of the resulting carbanion by the planar
.pi.-system of the prosthetic group. The hydroxyl group is
eliminated, and the resultant enamine is freed by a second
transimination. A tautomerization step results in the formation of
a ketimine, which hydrolyses to the 2-oxoacid and ammonia (Gabowski
et al. (1993) Trends in Biological Sciences 18: 297-300). A
subclass of the serine dehydratases found in anaerobic bacteria
substitutes an iron-sulfur cofactor for pyridoxal-5'-phosphate, and
exhibits an altered reaction mechanism with similarities to the
mechanism of aconitase (Hofmeister et al. (1993) Eur J Biochem
215(2):341-9). Threonine dehydratases, in general, are able to
deaminate either threonine or serine, while the serine dehydratases
have been found to be specific for the deamination of serine
(Grabowski et al. (1992) Eur. J. Biochem. 199:89-94; and Alfoldi et
al. (1968) J. Bacteriol. 96:1512-1518).
[0142] Members of the serine and threonine dehydratase family are
found in nearly all organisms, from bacteria to yeast to mammals.
Alignments of the amino acid sequences of family members from
disparate organisms have revealed two conserved regions, termed C1
and C2. The conserved C1 domain is located approximately 50 amino
acid residues from the N-terminus of the enzyme, and includes the
consensus sequence (G)S(F)K(I)RG (Datta et al. (1987) Proc. Natl.
Acad. Sci. USA 84: 393-397). This region of the protein has been
shown to bind the cofactor, pyridoxal-5'-phosphate, at the
conserved lysine residue (Schlitz and Schmitt (1981) FEBS Lett.
134:57-62). Conserved region C2 is located in the central region of
the amino acid sequences of these enzymes, and is predicted to have
a beta sheet-coil-beta sheet structure (Datta et al., supra). C2 is
rich in glycine, and is thought to be involved in the catalytic
activity of the enzymes (Marceau et al. (1988) J. Biol. Chem. 263:
16926-16933).
[0143] As used herein, the term "dehydratase" includes a molecule
which is involved in the metabolism and catabolism of biochemical
molecules necessary for energy metabolism, for intra- or
intercellular signaling, and for metabolism or catabolism of
metabolically important biomolecules. Typically, dehydratases are
involved in the deamination of amino acids, e.g., serine or
theronine. Examples of dehydratases include serine and threonine
dehydratases. Thus, the DHY molecules of the present invention
provide novel diagnostic targets and therapeutic agents to control
dehydratase-associated disorders.
[0144] As used herein, a "dehydratase-associated disorder" includes
a disorder, disease or condition which is caused or characterized
by a misregulation (e.g., downregulation or upregulation) of
dehydratase activity. Misregulation of dehydratase activity can
result in the overproduction or lack of production of one or more
amino acids or biologically important metabolic precursor molecules
(e.g., pyruvate or 2-oxobutyrate), and, by extension, aberrant
metabolite, energy molecules, and/or protein production in the cell
as a whole. Proteins produced by the cell not only include those
involved in normal cellular functioning (e.g., enzymes, receptors,
chaperonins, and transcription factors), but also important
signaling molecules (e.g., growth factors, cytokines, and
neuropeptides). Dehydratase-associated disorders, therefore, can
detrimentally affect cellular functions such as cellular
proliferation, growth, differentiation, or migration, inter- or
intra-cellular communication; and tissue function, such as cardiac
function or musculoskeletal function. Examples of
dehydratase-associated disorders include cellular proliferation,
growth, differentiation, or migration disorders, CNS disorders,
cardiovascular disorders and disorders affecting tissues in which
DHY protein is expressed.
[0145] As used herein, a "dehydratase-mediated activity" includes
an activity which involves the deamination of one or more amino
acids, e.g., threonine or serine. Dehydratase-mediated activities
include the production of biochemical molecules necessary for
energy metabolism, for intra- or intercellular signaling (e.g., the
production of growth factors), and for metabolism or catabolism of
metabolically important biomolecules (e.g., isoleucine and protein
production).
[0146] The family of DHY proteins comprises at least one
"transmembrane domain". As used herein, the term "transmembrane
domain" includes an amino acid sequence of about 15 amino acid
residues in length which spans the plasma membrane. More
preferably, a transmembrane domain includes about at least 20, 25,
30, 35, 40, or 45 amino acid residues and spans the plasma
membrane. Transmembrane domains are rich in hydrophobic residues,
and typically have an alpha-helical structure. In a preferred
embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the
amino acids of a transmembrane domain are hydrophobic, e.g.,
leucines, isoleucines, tyrosines, or tryptophans. Transmembrane
domains are described in, for example, Zagotta W. N. et al., (1996)
Annual Rev. Neurosci. 19: 235-263, the contents of which are
incorporated herein by reference. Amino acid residues 67-83,
167-187, 270-288, and 295-311 of the native DHY protein are
predicted to comprise transmembrane domains. Accordingly, DHY
proteins having at least 50-60% homology, preferably about 60-70%,
more preferably about 70-80%, or about 80-90% homology with a
transmembrane domain of human DHY are within the scope of the
invention.
[0147] In another embodiment, a DHY molecule of the present
invention is identified based on the presence of a
"serine/threonine dehydratase pyridoxal-phosphate attachment site"
in the protein or corresponding nucleic acid molecule. As used
herein, the term "serine/threonine dehydratase pyridoxal-phosphate
attachment site" includes a protein domain having an amino acid
sequence of about 10-20 amino acid residues. Preferably, a
serine/threonine dehydratase pyridoxal-phosphate attachment site
has about 14 residues and the following consensus sequence:
[DESH]-x(4,5)-[STVG]-x-[AS]-[FYI]-K-[DLIFSA]-[RVMF]-[GA]-[LIVMGA]
(SEQ ID NO: 19) (Datta et al. (1987) Proc. Natl. Acad. Sci. USA
84:393-397; and Ogawa et al. (1989) Biochim. Biophys. Acta
996:139-141). To identify the presence of a serine/threonine
dehydratase pyridoxal-phosphate attachment site in a DHY protein,
and make the determination that a protein of interest has a
particular profile, the amino acid sequence of the protein may be
searched against a database of known protein domains (e.g., the
ProSite database). The serine/threonine dehydratase
pyridoxal-phosphate attachment site has been assigned ProSite
accession number PS00165. A search was performed against the
ProSite database resulting in the identification of a
serine/threonine dehydratase pyridoxal-phosphate attachment site in
the amino acid sequence of human DHY (SEQ ID NO: 17) at about
residues 39-52 of SEQ ID NO: 17.
[0148] In another embodiment, a DHY molecule of the present
invention is identified based on the presence of a
"serine/threonine dehydratase domain" in the protein or
corresponding nucleic acid molecule. As used herein, the term
"serine/threonine dehydratase domain" includes a protein domain
having an amino acid sequence of about 200-400 amino acid residues
and a bit score of about 50, 60, 70, 80, 90, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, or 220 or more. Preferably,
a serine/threonine dehydratase domain includes at least about
250-350, or more preferably about 301 amino acid residues, and a
bit score of at least 229. To identify the presence of a
serine/threonine dehydratase domain in a DHY protein, and make the
determination that a protein of interest has a particular profile,
the amino acid sequence of the protein may be searched against a
database of known protein domains (e.g., the HMM database). A
search was performed against the HMM database resulting in the
identification of a serine/threonine dehydratase domain in the
amino acid sequence of human DHY (SEQ ID NO: 17) at about residues
11-311 of SEQ ID NO: 17.
[0149] In another embodiment, a DHY molecule of the present
invention is identified based on the presence of a "pyridoxal
phosphate-dependent lyase synthase domain" in the protein or
corresponding nucleic acid molecule. As used herein, the term
"pyridoxal phosphate-dependent lyase synthase domain" includes a
protein domain having an amino acid sequence of about 200-300 amino
acid residues and having a bit score for the alignment of the
sequence to the pyridoxal phosphate-dependent lyase synthase domain
of at least 10, 20, 30, 40, 50, 60, 70, 80 or higher. Preferably, a
pyridoxal phosphate-dependent lyase synthase domain includes at
least about 240-275, or more preferably about 265 amino acid
residues, and has a bit score for the alignment of the sequence to
the pyridoxal phosphate-dependent lyase synthase domain of at least
88. The pyridoxal phosphate-dependent lyase synthase domain has
been assigned ProDom entry 206. To identify the presence of a
pyridoxal phosphate-dependent lyase synthase domain in a DHY
protein, and to make the determination that a protein of interest
has a particular profile, the amino acid sequence of the protein
may be searched against a database of known protein domains (e.g.,
the ProDom database) using the default parameters. A search was
performed against the ProDom database resulting in the
identification of a pyridoxal phosphate-dependent lyase synthase
domain in the amino acid sequence of human DHY (SEQ ID NO: 17) at
about residues 18-282 of SEQ ID NO: 17.
[0150] In a preferred embodiment, the DHY molecules of the
invention include at least one or more of the following domains: a
transmembrane domain, a serine/threonine dehydratase
pyridoxal-phosphate attachment site, a serine/threonine dehydratase
domain, and/or a pyridoxal phosphate-dependent lyase synthase
domain.
[0151] Isolated proteins of the present invention, preferably DHY
proteins, have an amino acid sequence sufficiently identical to the
amino acid sequence of SEQ ID NO: 17, or are encoded by a
nucleotide sequence sufficiently identical to SEQ ID NO: 16 or 18.
As used herein, the term "sufficiently identical" refers to a first
amino acid or nucleotide sequence which contains a sufficient or
minimum number of identical or equivalent (e.g., an amino acid
residue which has a similar side chain) amino acid residues or
nucleotides to a second amino acid or nucleotide sequence such that
the first and second amino acid or nucleotide sequences share
common structural domains or motifs and/or a common functional
activity. For example, amino acid or nucleotide sequences which
share common structural domains have at least 30%, 40%, or 50%
homology, preferably 60% homology, more preferably 70%-80%, and
even more preferably 90-95% homology across the amino acid
sequences of the domains and contain at least one and preferably
two structural domains or motifs, are defined herein as
sufficiently identical. Furthermore, amino acid or nucleotide
sequences which share at least 30%, 40%, or 50%, preferably 60%,
more preferably 70-80%, or 90-95% homology and share a common
functional activity are defined herein as sufficiently
identical.
[0152] As used interchangeably herein, an "DHY activity",
"biological activity of DHY" or "functional activity of DHY",
refers to an activity exerted by a DHY protein, polypeptide or
nucleic acid molecule on a DHY responsive cell or tissue, or on a
DHY protein substrate, as determined in vivo, or in vitro,
according to standard techniques. In one embodiment, a DHY activity
is a direct activity, such as an association with a DHY-target
molecule. As used herein, a "target molecule" or "binding partner"
is a molecule with which a DHY protein binds or interacts in
nature, such that DHY-mediated function is achieved. A DHY target
molecule can be a non-DHY molecule or a DHY protein or polypeptide
of the present invention (e.g., pyridoxal-5'-phosphate). In an
exemplary embodiment, a DHY target molecule is a DHY ligand (e.g.,
serine or threonine). Alternatively, a DHY activity is an indirect
activity, such as a cellular signaling activity mediated by
interaction of the DHY protein with a DHY ligand. The biological
activities of DHY are described herein. For example, the DHY
proteins of the present invention can have one or more of the
following activities: 1) modulate metabolism and catabolism of
biochemical molecules necessary for energy production or storage
(e.g., amino acids, such as serine or threonine); 2) modulate
intra- or intercellular signaling; 3) modulate metabolism or
catabolism of metabolically important biomolecules; 4) modulate
cellular growth and differentiation; 5) modulate cellular
proliferation; and 6) modulate production of growth factors and
cytokines.
[0153] Accordingly, another embodiment of the invention features
isolated DHY proteins and polypeptides having a DHY activity. Other
preferred proteins are DHY proteins having one or more of the
following domains: a transmembrane domain, a serine/threonine
dehydratase pyridoxal-phosphate attachment site, a serine/threonine
dehydratase domain, and/or a pyridoxal phosphate-dependent lyase
synthase domain and, preferably, a DHY activity.
[0154] Additional preferred proteins have one or more of the
following domains: a transmembrane domain, a serine/threonine
dehydratase pyridoxal-phosphate attachment site, a serine/threonine
dehydratase domain, and/or a pyridoxal phosphate-dependent lyase
synthase domain, and are, preferably, 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 SEQ ID NO: 16 or 18.
[0155] Isolation of the 26335 or DHY cDNA
[0156] The invention is based, at least in part, on the discovery
of a human gene encoding a novel protein, referred to herein as
DHY. The entire sequence of human clone Fbh26335 was determined and
found to contain an open reading frame termed human "26335" or
"DHY", set forth in SEQ ID NO: 16 and SEQ ID NO: 18. The human DHY
gene, which is approximately 1327 nucleotides in length, encodes a
protein having a molecular weight of approximately 36.2 kD, which
is approximately 329 amino acid residues in length and which is set
forth in SEQ ID NO: 17. The coding region (open reading frame) of
SEQ ID NO: 16, is set forth as SEQ ID NO: 18.
[0157] Analysis of the Human DHY Molecule
[0158] The amino acid sequence of human DHY was analyzed using the
program PSORT to predict the localization of the protein within the
cell. This program assesses the presence of different targeting and
localization amino acid sequences within the query sequence. The
results of the analysis show that human DHY (SEQ ID NO: 17) may be
localized to the cytoplasm, to the mitochondrion, to golgi, to the
endoplasmic reticulum, extracellular to the cell or to the cell
wall, to vacuoles, to the nucleus, or to secretory vesicles.
[0159] A search of the amino acid sequence of DHY was performed
against the MEMSAT database. This search resulted in the
identification of four transmembrane domains in the amino acid
sequence of human 26335 or DHY (SEQ ID NO: 17) at about residues
67-83, 167-187, 270-288, and 295-311.
[0160] A search of the amino acid sequence of DHY was also
performed against the ProSite database. This search resulted in the
identification of a "serine/threonine dehydratase
pyridoxal-phosphate attachment site" in the amino acid sequence of
DHY (SEQ ID NO: 17) at about residues 39-52.
[0161] A search of the amino acid sequence of DHY was also
performed against the HMM database. This search resulted in the
identification of a "serine/threonine dehydratase domain" in the
amino acid sequence of DHY (SEQ ID NO: 17) at about residues 11-311
(score=229.5).
[0162] A search of the amino acid sequence of DHY was also
performed against the ProDom database. This search resulted in the
identification of a "pyridoxal phosphate-dependent lyase synthase
domain" in the amino acid sequence of human DHY (SEQ ID NO: 17) at
about residues 18-282 (score=88).
[0163] Tissue Distribution of Human 26335 or DHY mRNA using
TaqMan.TM. Analysis
[0164] Highest expression of DHY mRNA was detected in HMVECL,
U937/A10P10, bronchial epithelium, astrocytes, primary osteoblasts,
keratinocytes, bronchial epithelium mix (BEA8-2B), congestive heart
failure (CHF) heart tissue, the pituitary gland, fetal kidney
tissue, fetal liver tissue, mesangial, T24Ctl, T24 (treated),
adrenal gland tissue, Burkitt's Lymphoma tissue, mammary
epithelium, WT LNCap+casodex, A549 IL-1, SCC25 CDDP-tongue squamous
cell carcinoma tissue, testes, K563 (red blood cell line), A459
control (random-primed), liver tissue, prostate tissue, normal
colon tissue, HMC-1 (mast cell line), normal megakarocytes, colon
to liver metastasis (CHT128), colon to liver metastasis (CHT133),
normal breast tissue, PTH osteo, lung squamous cell carcinoma
tissue PIT299, and d8 dendritic cells.
[0165] Lesser expression was also detected in HUVECL, HL60/S,
prostate epithelium, coronary smooth muscle cells, fetal lung
tissue, fetal thymus tissue, congestive heart failure (CHF) heart
tissue, prostate smooth muscle tissue, thyroid tissue, LPS 24 hour
osteoblasts, uterine smooth muscle tissue (treated), bronchial
smooth muscle tissue, umbilical smooth muscle tissue (treated),
A2780 WT, fetal liver tissue, fetal skin, fetal adrenal gland
tissue, midterm placental tissue, lung carcinoma tissue, embryonic
keritinocytes, testes, skin, adipose, placental tissue
(random-primed), kidney tissue (random-primed), HPK (random
primed), salivary gland, heart tissue, the thymus, stomach tissue,
spleen tissue, small intestine tissue, normal breast epithelia,
normal ovarian epithelia, colon carcinoma tissue, ovarian ascites,
serum starved embryonic lung tissue, lung squamous cell carcinoma
tissue, brain subcortical white matter, normal prostate tissue
(ziplox), HUVEC L (umbilical endothelium).
[0166] No expression was detected in U937/A10p50, CaCo, Hela cells,
HL60/Adr, fetal brain tissue, melanocytes, cerebellum, aortic
endothelial cells, prostate fibroblast tissue, mammary gland
tissue, natural killer cells, LPS 1 hr. osteoblasts, LPS 6 hr.
osteoblasts, WT LNCap+ testosterone, A2780ADR, fetal spleen tissue,
the esophagus, p65 con +/+, p65 IL-1 +/+, pulmonary artery smooth
muscle tissue, erythroleukemia cells, SCC25 WT-tongue squamous cell
carcinoma tissue, fetal hypothalamus, T cells (CD3 treated), T
cells (CD3 IL-4/IL-10 treated), T cells (CD3 IFNg/TFNa treated),
trachea tissue, ME180 IL-1 cervical carcinoma tissue, ME 180
control, MCP-1 mast cell line, HPKII, lung tissue (random primed),
heart tissue (random primed), fetal brain tissue (random primed),
testes (random primed), RAJI (Burkitt's lymphoma B cell), ST 486
(lymphoma B cell), HL60 (acute promyelocytic leukemia), umbilical
cord smooth muscle tissue (treated, random primed), uterine smooth
muscle (treated, random primed), mammary gland tissue (random
primed), small intestine tissue (random primed), fetal liver tissue
(random primed), skeletal liver tissue (random primed), stomach
tissue (random primed), spleen tissue (random primed), liver tissue
(random primed), brain tissue (random primed), uterine tissue,
uterine tissue (random primed), thymus tissue (random primed), 9
week fetus, lung tissue, skeletal muscle, retinal pigmentosa
epithelial tissue, retinal tissue, bone marrow, Th-1 induced T
cell, Th-2 induced T cell, colon carcinoma tissue (NDR 109), colon
carcinoma tissue (NDR82), fetal dorsal spinal cord tissue, lung
adenocarcinoma tissue (PIT245), megakaryocytes, BMCD34+, IBD colon
tissue, cervical cancer tissue, spinal cord, dorsal root ganglia,
and ovarian epithelium tumor tissue.
[0167] Human 50365
[0168] The human 50365 sequence (SEQ ID NO: 20, as recited below),
which is approximately 3669 nucleotides long including untranslated
regions, contains a predicted methionine-initiated coding sequence
of about 2754 nucleotides, including the termination codon. The
coding sequence encodes a 917 amino acid protein (SEQ ID NO: 21, as
recited below).
[0169] Human 50365 contains the following regions or other
structural features: two hexokinase domains located at about amino
acids 16 to 463 and 464 to 910 of SEQ ID NO: 21, the latter of
which includes a predicted hexokinase signature domain (PFAM
Accession Number PS00378) from about amino acid residue 597 to
about amino acid residue 622 of SEQ ID NO: 21; two N-glycosylation
sites (PS00001) from about amino acid 208 to about 211, and from
about amino acid 655 to about 658, of SEQ ID NO: 21; one
glycosaminoglycan attachment site (PS00002) from about amino acid
896 to about 899 of SEQ ID NO: 21; one cAMP- and cGMP-dependent
protein kinase site (PS00004) from about amino acid 500 to 503 of
SEQ ID NO: 21; twelve protein kinase C phosphorylation sites
(PS00005) from about amino acid 172 to 174, 379 to 381, 449 to 451,
508 to 510, 523 to 525, 547 to 549, 551 to 553, 772 to 774, 791 to
793, 826 to 828, 877 to 879, and 896 to 898, of SEQ ID NO: 21;
thirteen casein kinase II sites (PS00006) from about amino acid 35
to 38, 114 to 117, 161 to 164, 243 to 246, 275 to 278, 364 to 367,
569 to 572, 625 to 628, 722 to 725, 726 to 729, 787 to 790, 810 to
813, and 877 to 880, of SEQ ID NO: 21; two tyrosine kinase
phosphorylation sites (PS00007) from about amino acid 20 to 27, and
490 to 497, of SEQ ID NO: 21; twenty-five N-myristylation sites
(PS00008) from about amino acid 74 to 79, 151 to 156, 166 to 171,
179 to 184, 212 to 217, 227 to 232, 233 to 238, 299 to 304, 317 to
322, 348 to 353, 360 to 365, 411 to 416, 448 to 453, 518 to 523,
589 to 594, 613 to 618, 659 to 664, 674 to 679, 680 to 685, 746 to
751, 779 to 784, 807 to 812, 834 to 839, 858 to 863, and 895 to
900, of SEQ ID NO: 21; and two amidation sites (PS00009) from amino
acid 100 to 103, and amino acid 547 to 550 of SEQ ID NO: 21.
[0170] Human 50365 is predicted to be a soluble, cytoplasmic
polypeptide.
[0171] For general information regarding PFAM identifiers, PS
prefix and PF prefix domain identification numbers, refer to
Sonnhammer et al. (1997) Protein 28:405-420.
[0172] The 50365 protein contains a significant number of
structural characteristics in common with members of the hexokinase
family. Hexokinases are a family of sugar phosphorylating enzymes
which carry out the phosphorylation of hexoses, for example,
glucose, mannose, fructose, sorbitol and glucosamine, at the
6'-position. The phosphoryl donor can be MgATP, ITP, or dATP. Both
.alpha.- and .beta.-D-hexoses can be phosphorylated, although with
different kinetic constants.
[0173] Four major isoenzymes are found in vertebrates: types I, II,
III, and IV. The liver hexokinase isoenzyme (type IV) is also
misleadingly known as glucokinase, and is expressed only in the
liver and pancreatic .beta.-cells. This isoenzyme has an important
role in modulating insulin secretion. Structurally, the enzymes
typically include a small N-terminal hydrophobic region, followed
by two similar hexokinase domains of about 450 residues each. The
second such region has catalytic activity, while the first has a
regulatory role.
[0174] Hexokinases are present in nearly all cells. These enzymes
have been identified as important for normal glycolytic activity.
Irregularities in their function can lead to disorders such as
diabetes and hemolytic anemia arising from hexokinase
deficiency.
[0175] A 50365 polypeptide can include a "hexokinase domain" or
regions homologous with a "hexokinase domain". Type I, II, and III
mammalian hexokinase polypeptides typically include two hexokinase
domains. Each domain can form a structural unit that includes
features of an .alpha./.beta. sandwich. Each domain can include
amino acids with regulatory and/or catalytic functions, e.g.,
including a pocket for ATP and hexose substrates.
[0176] As used herein, the term "hexokinase domain" includes an
amino acid sequence of about 300 to about 600 amino acid residues
in length and having a bit score for the alignment of the sequence
to the hexokinase domain (HMM) of at least 300. Preferably, a
hexokinase domain includes at least about 350 to about 500 amino
acids, more preferably about 400 to about 490 amino acid residues
and has a bit score for the alignment of the sequence to the
hexokinase domain (HMM) of at least 500, 600, 700, 800 or greater.
The hexokinase domain (HMM) has been assigned the PFAM Accession
PF00349.
[0177] In a preferred embodiment 50365 polypeptide or protein has a
"hexokinase domain" or a region which includes at least about 500
to about 1200, more preferably about 550 to about 1100 or about 600
to about 1000 amino acid residues and has at least about 60%, 70%
80% 90% 95%, 99%, or 100% homology with a "hexokinase domain,"
e.g., the hexokinase domain of human 50365 (e.g., residues 597 to
622 of SEQ ID NO: 21).
[0178] Preferably, the hexokinase domain includes a "hexokinase
signature domain". This term refers to a protein domain having an
amino acid sequence of at least about 15 to about 30 more
preferably about 20 to about 30 or about 24 to about 28 amino acid
residues which includes the following amino acid sequence:
"L-G-F-T-F--S--F--P--C-x-Q-x-S--I-x-x-G-x-- L-I-x-W-T-K-G-F" (SEQ
ID NO: 24). Preferably, a 50365 polypeptide or protein has a
"hexokinase signature domain" or a region which includes and has at
least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a
"hexokinase signature domain," e.g., the hexokinase signature
domain of human 50365 (e.g., residues 597 to 622 of SEQ ID NO:
21).
[0179] To identify the presence of a "hexokinase" domain in a 50365
protein sequence, and make the determination that a polypeptide or
protein of interest has a particular profile, the amino acid
sequence of the protein can be searched against the Pfam database
of HMMs (e.g., the Pfam database, release 2.1) using the default
parameters. For example, the hmmsf program, which is available as
part of the HMMER package of search programs, is a family specific
default program for MILPAT0063 and a score of 15 is the default
threshold score for determining a hit. Alternatively, the threshold
score for determining a hit can be lowered (e.g., to 8 bits). A
description of the Pfam database can be found in Sonhammer et al.
(1997) Proteins 28(3):405-420 and a detailed description of HMMs
can be found, for example, in Gribskov et al.(1990) Meth. Enzymol.
183:146-159; Gribskov et al.(1987) Proc. Natl. Acad. Sci. USA
84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; and
Stultz et al.(1993) Protein Sci. 2:305-314, the contents of which
are incorporated herein by reference. A search was performed
against the HMM database resulting in the identification of two
"hexokinase domains" in the amino acid sequence of human 50365 at
about residues 16 to 463 and 464 to 910 of SEQ ID NO: 21, the
identified hexokinase domain consensus sequence is set forth in SEQ
ID NO: 23.
[0180] A 50365 family member can include at least one hexokinase
domain. Furthermore, a 50365 family member can include at least
one, preferably two N-glycosylation sites (PS00001); at least one
glycosaminoglycan attachment site (PS00002); at least one cAMP- and
cGMP-dependent protein kinase site (PS00004); at least one, two,
three, four, five, six, seven, eight, nine, ten, eleven, or
preferably twelve protein kinase C phosphorylation sites (PS00005);
at least one, two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, or preferably thirteen predicted casein kinase II
phosphorylation sites (PS00006); at least one, or preferably two
tyrosine kinase phosphorylation sites (PS00007); at least one, two,
three, four, five, six, seven, eight, nine, ten, eleven, twelve,
thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, twenty,
twenty-three, or preferably twenty-five predicted N-myristylation
sites (PS00008); and at least one, preferably two amidation sites
(PS00009).
[0181] 50365 polypeptides of the invention include fragments which
include: all or part of a hydrophobic sequence, e.g., the sequence
of from about amino acid residue 365 to about amino acid residue
380, or from about amino acid residue 645 to about amino acid
residue 655, of SEQ ID NO: 21; all or part of a hydrophilic
sequence, e.g., the sequence of from about amino acid residue 98 to
about amino acid residue 120, or from about amino acid residue 715
to about amino acid residue 745 of SEQ ID NO: 21.
[0182] As the 50365 polypeptides of the invention may modulate
50365-mediated activities, they may be useful as of for developing
novel diagnostic and therapeutic agents for 50365-mediated or
related disorders, as described below.
[0183] As used herein, a "50365 activity", "biological activity of
50365" or "functional activity of 50365", refers to an activity
exerted by a 50365 protein, polypeptide or nucleic acid molecule on
e.g., a 50365-responsive cell or on a 50365 substrate, e.g., a
protein substrate, as determined in vivo or in vitro. In one
embodiment, a 50365 activity is a direct activity, such as an
association with a 50365 target molecule. A "target molecule" or
"binding partner" is a molecule with which a 50365 protein binds or
interacts in nature. In an exemplary embodiment, is a 50365
substrate, e.g., an aldohexose or ketohexose (e.g., glucose,
mannose, fructose, sorbitol and glucosamine), or a
phosphate-containing molecule, e.g., ITP, dATP, or MgATP as
phosphoryl donor.
[0184] A 50365 activity can also be an indirect activity, e.g., a
cellular signaling activity mediated by interaction of the 50365
protein with a 50365 substrate. For example, the 50365 proteins of
the present invention can have one or more of the following
activities: (1) it can catalyze the phosphorylation of a sugar,
e.g., an aldohexoses and a ketohexoses (e.g., glucose, mannose,
fructose, sorbitol and glucosamine); (2) it can catalyze sugar
metabolism; (3) it can transfer a phosphate from a phosphate donor
(e.g., ATP) to a sugar, e.g., an aldohexoses and a ketohexoses
(e.g., glucose, mannose, fructose, sorbitol and glucosamine) to
form a phosphorylated sugar, e.g., glucose-6-phosphate; (4) it can
modulate glycolytic activities in a cell or tissue, e.g., a tissue
in which a 50365 protein is expressed, e.g., muscle tissue and
colon; or (5) it can modulate sugar metabolism; and/or (6) it can
modulate cellular proliferation and/or differentiation.
[0185] Based on its structural features, the 50365 molecules of the
present invention can have similar biological activities as
hexokinase family members.
[0186] Expression of 50365 mRNA is modulated in a number of
cancerous tissue samples. For example, 50365 mRNA is elevated in a
number of colon tumors and colonic liver metastases (see, e.g.,
section entitled "Tissue Distribution of 50365 mRNA by TaqMan
Analysis" below). Thus, the 50365 molecules can act as novel
diagnostic targets and therapeutic agents for controlling disorders
of neoplasia, e.g., cancer, a cell differentiative disorder, or a
cell proliferative disorder as well as colon and lung disorders.
50365 molecules can also act as indicators and an agent for
metabolic disorders, e.g., disorders of sugar metabolism and
glycolysis.
[0187] Identification and Characterization of Human 50365 cDNA
[0188] The human 50365 nucleic acid sequence is recited as
follows:
2 CCACGCGTCCGGCCTGGACTGGAAGCGTGCAACACTCCAGAGTCG (SEQ ID NO:20)
TAGGAGTGAACACTGCACAGGAATCTCTGCCCATCTCAGGAGAAACCAAACTTG
GGGAAAATGTTTGCGGTCCACTTGATGGCATTTTACTTCAGCAAGCTGAAGGAG
GACCAGATCAAGAAGGTGGACAGGTTCCTGTATCACATGCGGCTCTCCGATGAC
ACCCTTTTGGACATCATGAGGCGGTTCCGGGCTGAGATGGAGAAGGGCCTGGCA
AAGGACACCAACCCCACGGCTGCAGTGAAGATGTTGCCCACCTTCGTCAGGGCC
ATTCCCGATGGTTCCGAAAATGGGGAGTTCCTTTCCCTGGATCTCGGAGGGTCC
AAGTTCCGAGTGCTGAAGGTGCAAGTCGCTGAAGAGGGGAAGCGACACGTGCA
GATGGAGAGTCAGTTCTACCCAACGCCCAATGAAATCATCCGCGGGAACGGCAT
AGAGCTGTTTGAATATGTAGCTGACTGTCTGGCAGATTTCATGAAGACCAAAGA
TTTAAAGCATAAGAAATTGCCCCTTGGCCTAACTTTTTCTTTCCCCTGTCGACAG
ACTAAACTGGAAGAGGGTGTCCTACTTTCGTGGACAAAAAAGTTTAAGGCACGA
GGAGTTCAGGACACGGATGTGGTGAGCCGTCTGACCAAAGCCATGAGAAGACA
CAAGGACATGGACGTGGACATCCTGGCCCTGGTCAATGACACCGTGGGGACCAT
GATGACCTGTGCCTATGACGACCCCTACTGCGAAGTTGGTGTCATCATCGGAAC
TGGCACCAATGCGTGTTACATGGAGGACATGAGCAACATTGACCTGGTGGAGG
GCGACGAGGGCAGGATGTGCATCAACACAGAGTGGGGGGCCTTCGGGGACGAC
GGGGCCCTGGAGGACATTCGCACTGAGTTCGACAGGGAGCTGGACCTCGGCTCT
CTCAACCCAGGAAAGCAACTGTTCGAGAAGATGATCAGTGGCCTGTACCTGGGG
GAGCTTGTCAGGCTTATCTTGCTGAAGATGGCCAAGGCTGGCCTCCTGTTTGGTG
GTGAGAAATCTTCTGCTCTCCACACTAAGGGCAAGATCGAAACACGGCACGTGG
CTGCCATGGAGAAGTATAAAGAAGGCCTTGCTAATACAAGAGAGATCCTGGTG
GACCTGGGTCTGGAACCGTCTGAGGCTGACTGCATTGCCGTCCAGCATGTCTGT
ACCATCGTCTCCTTCCGCTCGGCCAATCTCTGTGCAGCAGCTCTGGCGGCCATCC
TGACACGCCTCCGGGAGAACAAGAAGGTGGAACGGCTCCGGACCACAGTGGGC
ATGGACGGCACCCTCTACAAGATACACCCTCAGTACCCAAAACGCCTGCACAAG
GTGGTGAGGAAACTGGTCCCAAGCTGTGATGTCCGCTTCCTCCTGTCAGAGAGT
GGCAGCACCAAGGGGGCCGCCATGGTGACCGCGGTGGCCTCCCGCGTGCAGGC
CCAGCGGAAGCAGATCGACAGGGTGCTGGCTTTGTTCCAGCTGACCCGAGAGCA
GCTCGTGGACGTGCAGGCCAAGATGCGGGCTGAGCTGGAGTATGGGCTGAAGA
AGAAGAGCCACGGGCTGGCCACGGTCAGGATGCTGCCCACCTACGTCTGCGGG
CTGCCGGACGGCACAGAGAAAGGAAAGTTTCTCGCCCTGGATCTTGGGGGAAC
CAACTTCCGGGTCCTCCTGGTGAAGATCAGAAGTGGACGGAGGTCAGTGCGAAT
GTACAACAAGATCTTCGCCATCCCCCTGGAGATCATGCAGGGCACTGGTGAGGA
GCTCTTTGATCACATTGTGCAGTGCATCGCCGACTTCCTGGACTACATGGGCCTC
AAGGGAGCCTCCCTACCTTTGGGCTTCACATTCTCATTTCCCTGCAGGCAGATGA
GCATTGACAAGGGAACACTCATAGGGTGGACCAAAGGTTTCAAGGCCACTGAC
TGTGAAGGGGAGGACGTGGTGGACATGCTCAGGGAAGCCATCAAGAGGAGAAA
CGAGTTTGACCTGGACATTGTTGCAGTCGTGAATGATACAGTGGGGACCATGAT
GACCTGTGGCTATGAAGATCCTAATTGTGAGATTGGCCTGATTGCAGGAACAGG
CAGCAACATGTGCTACATGGAGGACATGAGGAACATCGAGATGGTGGAGGGGG
GTGAAGGGAAGATGTGCATCAATACAGAGTGGGGAGGATTTGGAGACAATGGC
TGCATAGATGACATCCGGACCCGATACGACACGGAGGTGGATGAGGGGTCCTT
GAATCCTGGCAAGCAGAGATACGAGAAAATGACCAGTGGGATGTACTTGGGGG
AGATTGTGCGGCAGATCCTGATCGACCTGACCAAGCAGGGTCTCCTCTTCCGAG
GGCAGATTTCAGAGCGTCTCCGGACCAGGGGCATCTTCGAAACCAAGTTCCTGT
CCCAGATCGAAAGCGATCGGCTGGCCCTTCTCCAGGTCAGGAGGATTCTGCAGC
AGCTGGGCCTGGACAGCACGTGTGAGGACAGCATCGTGGTGAAGGAGGTGTGC
GGAGCCGTGTCCCGGCGGGCGGCCCAGCTCTGCGGTGCTGGCCTGGCCGCTATA
GTGGAAAAAAGGAGAGAAGACCAGGGGCTAGAGCACCTGAGGATCACTGTGGG
TGTGGACGGCACCCTGTACAAGCTGCACCCTCACTTTTCTAGAATATTGCAGGA
AACTGTGAAGGAACTAGCCCCTCGATGTGATGTGACATTCATGCTGTCAGAAGA
TGGCAGTGGAAAAGGGGCAGCACTGATCACTGCTGTGGCCAAGAGGTTACAGC
AGGCACAGAAGGAGAACTAGGAACCCCTGGGATTGGACCTGATGCATCTTGGA
TACTGAACAGCTTTTCCTCTGGCAGATCAGTTGGTCAGAGACCAATGGGCACCC
TCCTGGCTGACCTCACCTTCTGGATGGCCGAAAGAGAACCCCAGGTTCTCGGGT
ACTCTTAGTATCTTGTACTGGATTTGCAGTGACATTACATGACATCTCTATTTGG
TATATTTGGGCCAAAATGGGCCAACTTATGAAATCAAAGTGTCTGTCCTGAGAG
ATCCCCTTTCAACACATTGTTCAGGTGAGGCTTGAGCTGTCAATTCTCTATGGCT
TTCAGTCTTGTGGCTGCGGGACTTGGAAATATATAGAATCTGCCCATGTGGCTG
GCAGGCTGTTTCCCCATTGGGATGCTTAAGCCATCTCTTATAGGGGATTGGACCC
TGTACTTGTGGATGAACATTGGAGAGCAAGAGGAACTCACGTTATGAACTAGGG
GGATCTCATCTAACTTGTCCTTAACTTGCCATGTTGACTTCAAACCTGTTAAGAG
AACAAAGACTTTGAAGTATCCAGCCCCAGGGTGCAGAGAGGTTGATTGCCAGG
GAGCACTGCAGGAATCATTGCATGCTTAAAGCGAGTTATGTCAGCACCCTGTAG
GATTTTGTTCCTTATTAAGTGTGTGCCATGTGGTGGGGTGCTGTCTGGGGCATCT
GTTTTTCATTTTGCCTGTGGTTTGTGTTGCAGSTGTTGATAGTTGTTTTAAGGATT
GTTAGGTATAGGAAATCCAGTAAATTAATAAAAAAATTTTGATTTTCCAATAAA
AAAAAAAAAAAAAAA.
[0189] The human 50365 sequence (SEQ ID NO: 20) is approximately
3669 nucleotides long. The nucleic acid sequence includes an
initiation codon (ATG) and a termination codon (TAA) which are
underscored and bolded above. The region between and inclusive of
the initiation codon and the termination codon is a
methionine-initiated coding sequence of about 2754 nucleotides (SEQ
ID NO: 22), including the termination codon. The coding sequence
encodes a 917 amino acid protein (SEQ ID NO: 21), which is recited
as follows:
3 MFAVHLMAFYFSKLKEDQIKKVDRFLYHMRLSDDTLLDIMRRFRAE (SEQ ID NO:21)
MEKGLAKDTNPTAAVKMLPTFVRAIPDGSENGEFLSLDLGGSKFRVLKVQVAEEG
KRHVQMESQFYPTPNEIIRGNGWLFEYVADCLADFMKTKDLKHKKLPLGLTFSFPC
RQTKLEEGVLLSWTKKFKARGVQDTDVVSRLTKAMRRHKDMDVDILALVNDTVG
TMMTCAYDDPYCEVGVIIGTGTNACYMEDMSNIDLVEGDEGRMCINTEWGAFGD
DGALEDRTEFDRELDLGSLNPGKQLFEKMISGLYLGELVRLILLKMAKAGLLFGGE
KSSALHTKGKTETRHVAAMEKYKEGLANTREILVDLGLEPSEADCIAVQHVCTIVSF
RSANLCAAALAAILTRLRENKKVERLRTTVGMDGTLYKIHPQYPKRLHKVVRKLVP
SCDVRFLLSESGSTKGAAMVTAVASRVQAQRKQTDRVLALFQLTREQLVDVQAKM
RAELEYGLKKKSHGLATVRMLPTYVCGLPDGTEKGKFLALDLGGTNFRVLLVKIRS
GRRSVRMYNKIEAIPLEIMQGTGEELFDHIVQCIADFLDYMGLKGASLPLGFTFSFPC
RQMSDKGTLIGWTKGFKATDCEGEDVVDMLREAIKRRNEFDLDIVAVVNPTVGT
MMTCGYEDPNCEIGLIAGTGSNMCYMEDMRNIEMVEGGEGKMCINTEWGGFGDN
GCIDDIRTRYDTEVDEGSLNPGKQRYEKMTSGMYLGEIVRQILIPLTKQGLLFRGQIS
ERLRTRGIEETKFLSQIESDRLALLQVRRWQQLGLDSTCEDSIVVKEVCGAVSRRAA
QLCGAGLAAIVEKRREDQGLEHLRITVGVDGTLYKLHPHFSRILQETVKELAPRCDV
TFMLSEDGSGKGAALITAVAKRLQQAQKEN.
[0190] Tissue Distribution of 50365 mRNA by TaqMan Analysis
[0191] Endogenous human 50365 gene expression was determined using
the Perkin-Elmer/ABI7700 Sequence Detection System which employs
TaqMan technology.
[0192] To determine the level of 50365 in various human tissues a
primer/probe set was designed. Total RNA was prepared from a series
of human tissues using an RNeasy kit from Qiagen. First strand cDNA
was prepared from 1 .mu.g total RNA using an oligo-dT primer and
Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained
from approximately 50 ng total RNA was used per TaqMan reaction.
Tissues tested include the human tissues and several cell lines
shown in Tables 2 and 3 below. 50365 mRNA was detected in colon and
liver tissue, and upregulated in colonic liver metastases (Table
2). In addition, 50365 mRNA was also detectable in adenomas and
adenocarcinomas. 50365 expression was also found to a lesser extent
in some lung tumor and ovary tumor tissues (Table 3).
4 TABLE 2 Tissue Type Expression CHT 410 Colon Normal 0.32 CHT 425
Colon Normal 0.41 CHT 371 Colon Normal 1.06 PIT 281 Colon Normal
0.00 NDR 211 Colon Normal 0.22 CHT 122 Adenomas 0.21 CHT 887
Adenomas 1.65 CHT 414 Colonic Adenocarcinoma-B 0.47 CHT 841 Colonic
Adenocarcinoma-B 0.05 CHT 890 Colonic Adenocarcinoma-B 0.58 CHT 910
Colonic Adenocarcinoma-B 3.85 CHT 377 Colonic Adenocarcinoma-B 0.00
CHT 520 Colonic Adenocarcinoma-C 0.80 CHT 596 Colonic
Adenocarcinoma-C 0.77 CHT 907 Colonic Adenocarcinoma-C 2.41 CHT 372
Colonic Adenocarcinoma-C 2.09 NDR 210 Colonic Adenocarcinoma-C 0.95
CHT 1365 Colonic Adenocarcinoma-C 2.54 CLN 740 Liver Normal 0.00
CLN 741 Liver Normal 0.00 NDR 165 Liver Normal 0.00 NDR 150 Liver
Normal 0.14 PIT 236 Liver Normal 0.00 CHT 1878 Liver Normal 0.00
CHT 119 Colon Liver Metastasis 7.52 CHT 131 Colon Liver Metastasis
0.77 CHT 218 Colon Liver Metastasis 5.45 CHT 739 Colon Liver
Metastasis 10.53 CHT 755 Colon Liver Metastasis 3.64 CHT 215 Colon
Abdominal Metastasis 0.24 PIT 337 Colon Normal 0.29 CHT 807 Colonic
Adenocarcinoma-B 61.64 CHT 382 Colonic Adenocarcinoma-B 57.11 CHT
077 Colon Liver Metastasis 180.49
[0193] The mRNA expression data for 50365 mRNA tabulated in Table 2
indicate that 50365 expression is upregulated in some adenomas and
adenocarcinomas, and in most colonic liver metastases (see
"Relative Expression" values). Relative expression in Table 2 is
relative to expression of .beta.2-macroglobulin.
5 TABLE 3 Tissue Type Expression PIT 400 Breast Normal 0.00 PIT 372
Breast Normal 0.00 CHT 559 Breast Normal 0.00 MDA 236-Breast Tumor:
PD-IDC (ILC?) 0.00 MDA 304 Breast Tumor: MD-IDC 0.00 CHT 2002
Breast Tumor: IDC 0.00 CHT 562 Breast Tumor: IDC 0.00 NDR 138
Breast Tumor ILC (LG) 0.00 CHT 1841 Lymph node (Breast Metastasis)
0.00 PIT 58 Lung (Breast Metastasis) 0.00 CHT 620 Ovary Normal 0.00
PIT 208 Ovary Normal 0.00 CLN 012 Ovary Tumor 0.00 CLN 07 Ovary
Tumor 0.05 CLN 17 Ovary Tumor 1.38 MDA 25 Ovary Tumor 0.00 MDA 216
Ovary Tumor 0.00 PIT 298 Lung Normal 0.00 MDA 185 Lung Normal 0.00
CLN 930 Lung Normal 0.00 MPI 215 Lung Tumor--SmC 0.00 MDA 259 Lung
Tumor-PDNSCCL 0.00 CHT 832 Lung Tumor-PDNSCCL 0.97 MDA 262 Lung
Tumor-Small Cell Carcinoma 0.00 CHT 793 Lung Tumor-Adenocarcinoma
0.03 CHT 331 Lung Tumor-Adenocarcinoma 0.00 CHT 405 Colon Normal
0.16 CHT 523 Colon Normal 0.65 CHT 371 Colon Normal 2.38 CHT 382
Colon Tumor: MD 0.88 CHT 528 Colon Tumor: MD 7.84 CLN 609 Colon
Tumor 2.21 NDR 210 Colon Tumor: MD-PD 0.84 CHT 340 Colon-Liver
Metastasis 3.23 NDR 100 Colon-Liver Metastasis 1.11 PIT 260 Liver
Normal (female) 1.17 CHT 1653 Cervix Squamous CC 0.00 CHT 569
Cervix Squamous CC 0.00 A24 HMVEC-Arr 0.00 C48 HMVEC-Prol 0.00
Pooled Hemangiomas 0.00 HCT116N22 Normal Ox.ygenation 0.97
HCT116H22 Hypoxic 0.00
[0194] 50365 mRNA was analyzed by TaqMan in a number of cell lines
derived from normal and tumor cells (Table 3). Relative expression
in Table 3 is relative to expression of .beta.2-macroglobulin.
Elevated 50365 mRNA expression levels were detected in some colon
cell lines, e.g., normal colon, colon tumor; colonic liver
metastases; some lung cell lines, e.g., lung tumor-PDNSCCL (poorly
differentiated non-small cell carcinoma of the lung), lung
tumor-adenocarcinoma; and an ovary tumor cell line. 50365 mRNA was
also detected under normal oxygenation conditions.
[0195] Human 21117
[0196] The human 21117 sequence (SEQ ID NO: 25), which is
approximately 3544 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
1998 nucleotides (nucleotides 589 to 2586 of SEQ ID NO: 25; SEQ ID
NO: 27). The coding sequence encodes a 665 amino acid protein (SEQ
ID NO: 26).
[0197] Human 21117 contains the following regions or other
structural features: a dual specificity phosphatase catalytic
domain (PF00782) located from about amino acid residue 158 to 297
of SEQ ID NO: 26; a rhodanese-like domain (PF00581) located from
about amino acid residue 11 to 131 of SEQ ID NO: 26; and one
tyrosine specific protein phosphatase active site (PS00383) at
amino acids 242 to 254 of SEQ ID NO: 26.
[0198] The 21117 protein additionally includes: six predicted
N-glycosylation sites (PS00001) at amino acids 38 to 41, 49 to 52,
190 to 193, 212 to 215, 300 to 303, and 640 to 643 of SEQ ID NO:
26; two predicted cAMP and cGMP-dependent protein kinase
phosphorylation sites (PS00004) at amino acids 277 to 280 and 624
to 627 of SEQ ID NO: 26; twelve predicted Protein Kinase C sites
(PS00005) at about amino acids 12 to 14, 23 to 25, 72 to 74, 82 to
84, 393 to 395, 439 to 441, 473 to 475, 481 to 483, 486 to 488, 596
to 598, 604 to 606, and 609 to 611 of SEQ ID NO: 26; thirteen
casein kinase II phosphorylation sites (PS00006) at amino acids 21
to 24, 91 to 94, 214 to 217, 266 to 269, 369 to 372, 421 to 424,434
to 437,458 to 461,508 to 511, 589 to 592,612 to 615,617 to 620, and
642 to 645 of SEQ ID NO: 26; and seven predicted N-myristoylation
sites (PS00008) from about amino acid 134 to 139, 247 to 252, 329
to 334, 382 to 387, 520 to 525, 574 to 579, and 650 to 655 of SEQ
ID NO: 26.
[0199] 21117 polypeptides of the invention include 21117 fragments
that include: all or part of a hydrophobic sequence e.g., all or
part of the sequence from about residue 91 to about residue 106 of
SEQ ID NO: 26; and/or all or part of a hydrophilic sequence e.g.,
all or part of the sequence from about residue 592 to about residue
633 of SEQ ID NO: 26. Other fragments include a cysteine residue or
a glycosylation site.
[0200] Human 38692
[0201] The human 38692 sequence (SEQ ID NO: 28), which is
approximately 1114 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
672 nucleotides (nucleotides 89 to 760 of SEQ ID NO: 28; SEQ ID NO:
30). The coding sequence encodes a 223 amino acid protein (SEQ ID
NO: 29).
[0202] Human 38692 contains the following regions or other
structural features: a dual specificity phosphatase catalytic
domain (PF00782) located from about amino acid residue 28 to 173 of
SEQ ID NO: 29; one predicted Protein Kinase C phosphorylation site
(PS00005) at about amino acids 201 to 203 of SEQ ID NO: 29; one
predicted casein kinase II phosphorylation site (PS0006) at amino
acids 205 to 208 of SEQ ID NO: 29; two predicted N-myristoylation
sites (PS00008) from about amino acid 123 to 128 and 197 to 202 of
SEQ ID NO: 29; and two tyrosine kinase phosphorylation sites
(PS00007) at amino acids 15 to 23 and 142 to 149 of SEQ ID NO:
29.
[0203] 38692 polypeptides of the invention include 38692 fragments
that include: all or part of a hydrophobic sequence, e.g., all or
part of the sequence from about residue 31 to about residue 41 of
SEQ ID NO: 29; and/or all or part of a hydrophilic fragment e.g.,
all or part of the sequence from about residue 200 to about residue
212 of SEQ ID NO: 29. Other fragments include cysteine
residues.
[0204] 21117 and 38692 Dual Specificity Phosphatase Proteins
[0205] The intracellular phosphorylation of proteins is critical
for a plethora of regulatory and signalling pathways in eukaryotic
cells. Phosphorylation events can govern a wide range of cellular
processes, including cell proliferation, differentiation,
transcription, and morphology. An essential component of these
signalling pathways is the ability of the cell to desensitize,
recycle, and counteract phosphorylation signals. The cell primarily
utilizes enzymes, termed phosphatases, which remove the phosphate
on tyrosine, serine, and threonine side chains. The protein
phosphatases are divided into three groups according to catalytic
function: (1) protein phosphatases that dephosphorylate serine and
threonine residues; (2) protein phosphatases which dephosphorylate
tyrosine residues; and (3) dual specificity protein phosphatases
which dephosphorylate serine, threonine and tyrosine residues.
[0206] Serine/threonine protein phosphatases are associated with
the regulation of cholesterol biosynthesis, glycogen metabolism,
muscle contractility, calcium ion channels, protein synthesis,
regulation of the G2 to M transition of the cell cycle, regulation
of glycolysis (6-phosphofructo-2-kinase and pyruvate kinase),
glycogenolysis (phosphorylase kinase subunit), gluconeogenesis
(fructose-2,6-bisphosphat- ase and pyruvate kinase), amino-acid
degradation (phenylalanine hydroxylase), lipid metabolism
(acetyl-CoA carboxylase), catecholamine synthesis (tyrosine
hydroxylase) and protein synthesis (elongation factor 2).
[0207] Protein tyrosine phosphatases (PTPs) are a family of
intracellular and integral membrane phosphatases that
dephosphorylate tyrosine residues in proteins. PTPs have been
identified in mammals, Drosophila and Schiz. pombe and are
implicated in the control of normal and neoplastic growth and
proliferation. They have also been found encoded by plasmids in
bacteria of the genus Yersinia, where they are implicated in
pathogenicity.
[0208] Dual specificity phosphatases hydrolyze phosphotyrosine,
phosphothreonine, and phosphoserine residues (for a review, see,
e.g., Fauman and Saper (1996) Trends in Biochem. 21:412). This
class of proteins is exemplified by the VH1 or vaccinia virus late
H1 gene protein, whose catalytic activity is required for vaccinia
virus replication. A human homolog of VH1, VHR, has also been
identified. VH1-like dual specificity phosphatase can also include
the phosphatases PAC-1 and CL100/MKP-1, hVH-2/MKP-2, hVH-3, MKP-3,
MKP-X, MKP-4, hVH-5, and M3/6 proteins. The PAC-1 and CL100
proteins hydrolyze phosphothreonine and phosphotyrosine residues on
phosphorylated MAP (mitogen activated protein) kinases. In order to
modulate signalling events, the activity and expression of dual
specificity phosphatases can be finely regulated. For example, the
PAC-1 and CL100 phosphatase can be induced by growth factors
(Keyse, S (1995) Biochim. Biophys. Acta1265:152-160).
[0209] The 21117 and 38692 proteins contain a significant number of
structural characteristics in common with members of the dual
specificity phosphatase family.
[0210] Dual specificity phosphatase proteins are characterized by a
common fold. Examples of members of the dual specificity
phosphatase family include MAP kinase phosphatase-1 (MKP-1), which
dephosphorylates MAP kinase on both threonine and tyrosine residues
and a human, vaccinia H1-related phosphatase (VHR), which also
removes the phosphate from phosphothreonine and phosphotyrosine
residues. Dual specificity phosphatases are exemplified by the VH1
or vaccinia virus late H1 gene protein, which hydrolyzes both
phosphotyrosine, phosphothreonine, and phosphoserine. VH1 catalytic
activity is required for viral replication. A human homolog of VH1,
VHR, has been identified. The three dimensional structure of this
family is based on models from x-ray crystallographic data of
protein tyrosine phosphatases, and human VHR. The VHR structure
includes a core domain consisting of a five-stranded mixed
.beta.-sheet and six .alpha.-helices. This structure closely
superimposes on the structure of phosphotyrosine protein
phosphatases. However, dual specificity phosphatases lack the KNRY
motif, and the N-terminal structures of tyrosine protein
phosphatases which endow these enzymes with a deep active site
specific for aryl phosphates. Thus, dual specificity phosphatases
have a shallower active site relative to tyrosine protein
phosphatases and can accommodate phosphoserine and phosphothreonine
substrates. Even so dual specificity phosphatases can have a
greater than 50-fold faster rate of phosphatase activity for
phosphotyrosine substrates than phosphothreonine or phosphoserine
substrates.
[0211] Similar to the broader class of phosphatases, dual
specificity phosphatases have a highly conserved active site
including three catalytic residues, a cysteine, an arginine, and an
aspartic acid. The active site cysteine and arginine are found in
the "C--X.sub.5--R" motif of the tyrosine phosphatase signature
(Prosite PS00383). This motif forms a binding pocket for three of
the phosphate oxyanions. The cysteine acts as a nucleophile to
accept the PO.sub.3 group. The reaction transiently generates a
phospho-cysteine intermediate before the phosphate is transferred
to water. The active site arginine stabilizes the transition-state
by hydrogen bonding to phosphate oxygens. In addition the histidine
preceding the active site cysteine and the serine or threonine
following the active site arginine are responsible for lowering the
pK.sub.a of the cysteine to stabilize a negative charge on the
cysteine. The active site aspartic acid accelerates the reaction by
donating a protein to generate an uncharged hydroxyl (for a review,
see Fauman and Saper (1996) Trends in Biochem. 21:412). A
C--X.sub.5--R motif is found in the 21117 protein at about amino
acids 242 to 254 of SEQ ID NO: 26.
[0212] The 21117 and 38692 proteins of the present invention show
significant homology to members of the dual specificity phosphatase
family. Dual specificity phosphatases are known to play critical
roles in growth factor signaling. For example, vaccinia H1-related
(VHR)-like phosphatases are known to dephosphorylate growth factor
receptors and thereby eliminate their signaling. MAP-kinase
phosphatases terminate MAP-kinase activity, thus leading to
inhibition of growth factor-mediated mitogenic signaling. Thus,
dual specificity phosphatases play a key role in inhibiting
proliferation and stimulating the differentiation of cells. As the
21117 and 38692 proteins show homology to dual specificity
phosphatases, these proteins are likely to be involved in
modulating (e.g., inhibiting) the proliferation and (e.g.,
stimulating) the differentiation of the cells in which they are
expressed, e.g., hematopoietic cells such as eythroid cells,
myeloid cells, monocytes, or megakaryocytes. Accordingly, the 21117
and 38692 molecules of the invention may be useful for developing
novel diagnostic and therapeutic agents for 21117 and
38692-mediated or related disorders, as described below.
[0213] A 21117 or 38692 polypeptide of the invention can include a
"dual specificity phosphatase catalytic domain" or regions
homologous with a "dual specificity phosphatase catalytic domain".
As used herein, the term "dual specificity phosphatase catalytic
domain" refers to an amino acid sequence having about 50 to 250,
preferably about 100 to 200, more preferably about 120 to 160 amino
acid residues and having a bit score for the alignment of the
sequence to the dual specificity phosphatase domain (HMM) of at
least 50, preferably 100, more preferably 120, 200, or more. The
dual specificity phosphatase catalytic domain (HMM) has been
assigned the PFAM Accession Number PF00782.
[0214] A dual specificity phosphatase domain preferably includes
the conserved active site residues cysteine and arginine in a
C--X.sub.5--R motif found at about amino acids 242 to 254 of SEQ ID
NO: 26 (the 21117 protein). Preferably, a dual specificity
phosphatase domain includes a conserved general amino acid, e.g.,
aspartic acid. For example, a 21117 protein has an aspartic acid
located at about residue 213 of SEQ ID NO: 26 and a 38692 protein
has an aspartic acid located at about residue 89 of SEQ ID NO: 29.
Typically, dual specificity phosphatases are able to
dephosphorylate tyrosine residues and serine/threonine
residues.
[0215] In a preferred embodiment, a 21117 or 38692 polypeptide or
protein has a "dual specificity phosphatase catalytic domain" or a
region that includes at least about 50 to 250, preferably about 100
to 200, more preferably about 120 to 160, and even more preferably
about 130 to 150 amino acid residues and has at least about 70% 80%
90% 95%, 99%, or 100% homology with a "dual specificity phosphatase
catalytic domain," e.g., the dual specificity phosphatase catalytic
domain of human 21117 (e.g., residues 158 to 297 of SEQ ID NO: 26)
or 38692 (e.g., residues 28 to 173 of SEQ ID NO: 29).
[0216] To identify the presence of a "dual specificity phosphatase
catalytic domain" in a 21117 or 38692 protein sequence and to make
the determination that a polypeptide or protein of interest has a
particular profile, the amino acid sequence of the protein can be
searched against a database of HMMs (e.g., the Pfam database,
release 2.1) using default parameters. For example, the hmmsf
program, which is available as part of the HMMER package of search
programs, is a family specific default program for MILPAT0063 and a
score of 15 is the default threshold score for determining a hit.
Alternatively, the threshold score for determining a hit can be
lowered (e.g., to 8 bits). A description of the Pfam database can
be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a
detailed description of HMMs can be found, for example, in Gribskov
et al.(1990) Meth. Enzymol. 183:146-159; Gribskov et al.(1987)
Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al.(1994) J. Mol.
Biol. 235:1501-1531; and Stultz et al.(1993) Protein Sci.
2:305-314, the contents of which are incorporated herein by
reference. A search was performed against the HMM database
resulting in the identification of a "dual specificity phosphatase
catalytic domain" e.g., the dual specificity phosphatase catalytic
domain of human 21117 (amino acids 158 to 297 of SEQ ID NO: 26) or
human 38692 (amino acids 28 to 173 of SEQ ID NO: 29). The
identified dual specificity phosphatase catalytic domain consensus
sequences of human 21117 and of human 38692 are set forth in SEQ ID
NO: 31 and 32, respectively.
[0217] Human 21117 also contains a "rhodanese-like" domain
(PF00581) from about amino acid 11 to 131 of SEQ ID NO: 26. The
rhodanese-like domain is occasionally found in a single copy in
phosphatases, such as Cdc25 phosphatase, a dual-specificity
phosphatase. Rhodanese is about 300 amino acids in length and has a
conserved domain at the N-terminus and at the C-terminus. A
cysteine residue is part of the active site of the enzyme. In a
preferred embodiment, a 21117 polypeptide or protein has a
"rhodanese-like domain" or a region that includes at least about 80
to 300 amino acids, preferably about 100 to 150 amino acid
residues, and has at least about 70% 80% 90% 95%, 99%, or 100%
homology with a "rhodanese-like domain," e.g., the rhodanese-like
domain of human 21117 (e.g., residues 11 to 131 of SEQ ID NO: 26).
A search was performed against the HMM database resulting in the
identification of two "rhodanese-like domains" in human 21117
(amino acids 11 to 131 and amino acids 12 to 134 of SEQ ID NO: 26).
The identified rhodanese-like domain consensus sequences of human
21117 are set forth in SEQ ID NO: 33 and 34.
[0218] As used herein, a "21117 or 38692 activity", "biological
activity of 21117 or 38692" or "functional activity of 21117 or
38692", refers to an activity exerted by a 21117 or 38692 protein,
polypeptide or nucleic acid molecule on e.g., a 21117 or
38692-responsive cell or on a 21117 or 38692 substrate, e.g., a
protein substrate, as determined in vivo or in vitro. In one
embodiment, an 21117 or 38692 activity is a direct activity, such
as an association with a 21117 or 38692 target molecule. A "target
molecule" or "binding partner" is a molecule with which a 21117 or
38692 protein binds or interacts in nature. A 21117 or 38692
activity can also be an indirect activity, e.g., a cellular
signaling activity mediated by interaction of the 21117 or 38692
protein with an 21117 or 38692 receptor.
[0219] Based on the above-described sequence similarities, the
21117 or 38692 molecules of the present invention are predicted to
have similar biological activities as dual specificity phosphatase
family members, probably of the VHR-type. Since VHR-phosphatases
inhibit growth factor signaling by dephosphorylating, e.g., growth
factor receptors, the 21117 or 38692 molecules of the invention are
predicted to have one or more of the following activities: (1)
catalyze the removal of a phosphate group attached to a tyrosine
residue in a protein target, e.g., a growth factor receptor; (2)
catalyze the removal of a phosphate group attached to a serine or
threonine residue in a protein e.g., a growth factor receptor; (3)
modulate growth factor activity; (4) modulate an intracellular
signaling pathway, e.g., a MAP kinase or ERK kinase pathway; (5)
modulate (e.g., stimulate) cell differentiation, e.g.,
differentiation of a 38692- or a 21117-expressing cell, e.g., a
breast, colon, lung, or adipose cell, a bone cell, an endothelial
cell, a liver cell, or a hematopoietic cell (e.g., a myeloid
(neutrophil) cell, a monocyte, an erythroid cell, a bone marrow
cell, a CD34-expressing cell, a megakaryocyte); (6) stimulate
hematopoiesis; (7) modulate cell proliferation, e.g., proliferation
of a 38692- or a 21117-expressing cell, e.g., a breast, colon,
lung, or adipose cell, a bone cell, an endothelial cell, a liver
cell, or a hematopoietic cell (e.g., a myeloid (neutrophil) cell, a
monocyte, an erythroid cell, a bone marrow cell, a CD34-expressing
cell, a megakaryocyte); (8) inactivate cell surface growth factor
receptors, e.g., tyrosine kinase receptors; or (9) modulate
apoptosis, of a cell, e.g., a cancer cell, e.g., a leukemic
cell.
[0220] As assessed by TaqMan analysis described herein, 38692 mRNA
is expressed in hematopoietic cells, and in particular, in
erythroid cell lineages, therefore the molecules of the invention
can be used to develop novel agents or compounds to treat and/or
diagnose disorders involving aberrant activities of those cells
e.g., hematopoietic, and in particular, erythroid disorders, as
described below. For example, 38692 polypeptide is expressed in
megakaryocytes, fetal liver CD34+ cells, erythroid progenitor cells
(e.g., bone marrow glycophorin A positive cells (BM GPA+)), and
Bone Marrow Glycophorin A (BM GPA) low CD71+.
[0221] As used herein, a "CD34-positive cell" refers to a cell that
expresses detectable levels of the CD34 antigen, preferably human
CD34 antigen. The sequence for human CD34 is provided in SwissProt
Accession Number P28906. The CD34 antigen is typically present on
immature hematopoietic precursor cells and hematopoietic
colony-forming cells in the bone marrow, including unipotent
(CFU-GM, BFU-E) and pluripotent progenitors (CFU-GEMM, CFU-Mix and
CFU-blast). The CD34 is also expressed on stromal cell precursors.
Terminal deoxynucleotidyl transferase (TdT)-positive B- and
T-lymphoid precursors in normal bone also are CD34+. The CD34
antigen is typically present on early myeloid cells that express
the CD33 antigen, but lack the CD14 and CD15 antigens and on early
erythroid cells that express the CD71 antigen and dimly express the
CD45 antigen. The CD34 antigen is also found on capillary
endothelial cells and approximately 1% of human thymocytes. Normal
peripheral blood lymphocytes, monocytes, granulocytes and platelets
do not express the CD34 antigen. CD34 antigen density is highest on
early haematopoietic progenitor cells and decreases as the cells
mature. The antigen is undetectably on fully differentiated
haematopoietic cells. Approximately 60% of acute B-lymphoid
leukemia's and acute myeloid leukemia express the CD34 antigen. The
antigen is not expressed on chronic lymphoid leukemia (B or T
lineage) or lymphomas.
[0222] As the 38692 polypeptides of the invention may modulate
38692-mediated activities, they may be useful as of for developing
novel diagnostic and therapeutic agents for 38692-mediated or
related disorders, e.g., hematopoietic related disorders, or
erythroid-associated disorders. As assessed by TaqMan analysis,
38692 is expressed at high levels in fetal liver, HepG2.2.15-A
liver cells, and Hep3B hypoxia cells, therefore the molecules of
the invention can be used to develop novel agents or compounds to
treat and/or diagnose liver related disorders.
[0223] Further TaqMan analyses have demonstrated that 21117 mRNA is
expressed in normal breast, normal colon, normal adipose tissue,
prostate tumor and lung chronic obstructive pulmonary disorder
(COPD) tissue. Thus, diagnostic and therapeutic methods of using
the 21117 molecules of the invention to treat/diagnose breast,
colon, adipose, prostate, and lung disorders are also contemplated
by the present invention.
[0224] Identification and Characterization of Human 38692 and 21117
cDNA
[0225] The human 21117 sequence (SEQ ID NO: 25), which is
approximately 3544 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
1998 nucleotides (nucleotides 589 to 2586 of SEQ ID NO: 25; SEQ ID
NO: 27). The coding sequence encodes a 666 amino acid protein (SEQ
ID NO: 26).
[0226] The human 38692 sequence (SEQ ID NO: 28), which is
approximately 1114 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
672 nucleotides (nucleotides 89 to 760 of SEQ ID NO: 28; SEQ ID NO:
30). The coding sequence encodes a 224 amino acid protein (SEQ ID
NO: 29).
[0227] Tissue Distribution of 38692 or 21117 mRNA
[0228] Endogenous human 21117 and 38692 gene expression was
determined using the Perkin-Elmer/ABI 7700 Sequence Detection
System which employs TaqMan technology.
[0229] To determine the level of 21117 mRNA in various human
tissues a primer/probe set was designed using Primer Express
(Perkin-Elmer) software and primary cDNA sequence information.
Total RNA was prepared from a series of tissues using an RNeasy kit
from Qiagen. First strand cDNA was prepared from 1 .mu.g total RNA
using an oligo-dT primer and Superscript II reverse transcriptase
(Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was
used per TaqMan reaction.
[0230] 21117 mRNA levels were analyzed in a variety of tissue
samples, both normal and diseased, including (1) Aorta/normal; (2)
Fetal heart/normal; (3) Heart normal; (4) Heart/Coronary heart
failure (CHF); (5) Vein/Normal; (6) SMC (Aortic); (7) Spinal
cord/Normal; (8) Brain cortex/Normal; (9) Brain
hypothalamus/Normal; (10) Glial cells (Astrocytes); (11)
Brain/Glioblastoma; (12) Breast/Normal; (13) Breast tumor/IDC; (14)
ovary/Normal; (15) ovary/Tumor; (16) Pancreas; (17)
Prostate/Normal; (18) Prostate/Tumr; (19) Colon/normal; (20)
Colon/tumor; (21) Colon/IBD (inflammatory bowel disease); (22)
Kidney/normal; (23) Liver/normal; (24) Liver fibrosis; (25) Fetal
Liver/normal; (26) Lung/normal; (27) Lung/tumor; (28) Lung/chronic
obstructive pulmonary disease (COPD); (29) Spleen/normal; (30)
Tonsil/normal; (31) Lymph node/normal; (32) Thymus/normal; (33)
Epithelial Cells (prostate); (34) Endothelial Cells (aortic); (35)
Skeletal Muscle/Normal; (36) Fibroblasts (Dermal); (37)
Skin/normal; (38) Adipose/Normal; (39) Osteoblasts(primary); (40)
Osteoblasts (undifferentiated); (41) Osteoblasts (differentiated);
(42) Osteoclasts; (43) Aortic Smooth Muscle Cells (SMC) Early; (44)
Aortic SMC Late; (45) Osteoclasts (undiff); (46) shear human
umbilical vein endothelial cells (HUVEC). High relative levels of
expression were detected in normal colon, normal breast, chronic
obstructive pulmonary disease lung tissue, normal adipose tissue,
and undifferentiated osteoblasts.
[0231] 38692 expression was determined by TaqMan assays on mRNA
derived from various tissues and cell lines, including (1) Lung;
(2) Kidney; (3) Spleen; (4) Fetal Liver; (5) Granulocytes; (6) NHDF
resting; (7) NHDF/TGF-treated for 48 hr; (8) NHLF/CTN-treated for
48 hr; (9) NHLF/TGF-treated for 48 hr; (10) NC Heps; (11) Passage
Stellates; (12) Liver Pool; (13) LF/CHT 339; (14) LF/NDR 191; (15)
LF/NDR 079; (16) Lymph Nodes NDR 173; (17) Tonsils; (18) TH1 24 hr;
(19) CD4; (20) CD14 Resting; (21) CD19; (22) CD3 Resting; (23) bone
marrow mononuclear cells (BM MNC) LP26; (24) MnPB CD34+; (25) adult
bone marrow (ABM) CD34+; (26) Cord Blood CD34+; (27) Erythroid;
(28) Megakaryocytes LP16; (29) Neutrophils d14; (30) NBM
CD15+/CD14-/34+; (31) mBM CD15+/CD11b-; (32) BM/glycophorin A
(GPA); (33) Hepatocyte (Hep)G2-A; (34) HepG2.2.15-A; (35) HBV-Liver
MAI-1; (36) HL60; (37) leukemia cell line K562; (38) Molt 4; (39)
liver cell line Hep3B Nor; (40)Hep3B Hypoxia, as well as various
hematopoietic cell lines, including, (1) Lung; (2) Colon; (3)
Heart; (4) Spleen; (5) Kidney; (6) Liver NDR 200; (7) Fetal Liver;
(8) Skeletal Muscle; (9) m BM (bone marrow) mononuclear cells
(MNC); (10) mBM MNC LP7; (11) mBM CD34+ LP92; (12) mobilized
peripheral blood (mPB) CD34+ LF41; (13) mPB CD34+LF48; (14) adult
bone marrow (ABM) CD34+ LP91; (15) ABM CD34+ LP29; (16) Cord Blood
CD34+ LF109; (17) Fetal Liver CD34+ LP93; (18) Fetal Liver CD34+
LP45; (19) Bone Marrow Glycophorin A positive (BM GPA+) LP85; (20)
BM GPA+LP34-1; (21) BM GPA low CD71+LF38; (22) BM GPA low CD71+
LP85-2; (23) mobilized peripheral blood (mPB) CD41+/CD14-LP94; (24)
BM CD41+/CD14-LP78; (25) mBM CD15+LP15; (26) mBM CD15+/CD11b-LP7-4;
(27) mBM CD15+/CD11b+LP15-2; (28) BM CD15+/CD11b-LF80-4; (29) BM
CD15+/CD11b-LP23-2; (30) BM CD15+/CD34-LP27-2; (31) BM
CD15+/CD34-LP41-1; (32) Erythrocyte (Ery) d6 LP25-1; (33) Ery d6
LP31-1; (34) Ery d10 LP24-4; (35) Ery d12 LF24-8; (36) Ery d12
LF24-9; (37) Ery dl4 GPA+LP31-4; (38) Ery d14 CD36+LP31-7; (39)
Megakaryocyte (Meg) 24 hr LF23-2; (40) Meg 44 hr LF6-2; (41) Meg d7
LP31-2; (42) Meg d12 LF26; (43) Meg d14 LP31-5; (44) Neutrophil d4
LF30; (45) Neutrophil d6 LF26; (46) Neutrophil d6 LP27; (47)
Neutrophil d7 LP31-3; (48) Neutrophil d12 LP27; (49) Neutrophil d12
LP26B; (50) Neutroph d14 LP31-6. In some samples, mnRNA expression
was detected at the indicated times in culture (e.g., 24 hrs., 48
hrs., days in culture).
[0232] High relative levels of 38692 expression were found in
hepatic tissues (e.g., fetal liver cells, HepG2.2.15-A liver cells,
fetal liver CD34+ cells), and in hematopoietic cells such as K562
cells, Bone Marrow Glycophorin A (BM GPA) low CD71+ LF38; and BM
GPA low CD71+ LP85-2.
[0233] Human 46508
[0234] The human 46508 sequence (SEQ ID NO: 35), which is
approximately 1182 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
684 nucleotides, including the termination codon. The coding
sequence encodes a 227 amino acid protein (SEQ ID NO: 36).
[0235] Human 46508 contains the following regions or other
structural features: a peptidyl-tRNA hydrolase domain (PFAM
Accession PF01195) located at about amino acid residues 44 to 221
of SEQ ID NO: 36; two Protein Kinase C sites (PS00005) at about
amino acids 13 to 15, and 150 to 152 of SEQ ID NO: 36; two Casein
Kinase II sites (PS00006) located at about amino acids 125 to 128,
and 194 to 197 of SEQ ID NO: 36; seven N-myristoylation sites
(PS00008) located at about amino acids 4 to 9, 17 to 22, 23 to 28,
53 to 58, 74 to 79, 149 to 154, and 156 to 161 of SEQ ID NO: 36;
one amidation site (PS00009) located at about amino acid 40 to 43
of SEQ ID NO: 36; and one glycosaminoglycan attachment site
(PS00002) located at about amino acids 3 to 6 of SEQ ID NO: 36.
[0236] For general information regarding PFAM identifiers, PS
prefix and PF prefix domain identification numbers, refer to
Sonnhammer et al. (1997) Protein 28:405-420.
[0237] The 46508 protein contains a significant number of
structural characteristics in common with members of the
peptidyl-tRNA hydrolase family.
[0238] Peptidyl-tRNA hydrolases are a family of important enzymes
which hydrolyze ester linkages between the peptide moiety and the
tRNA of peptidyl-tRNAs (Kossel, H (1969) Biochim. Biophys. Acta
204:191-202; Garcia-Villegas, M. R. (1991) EMBO J. 10:3549-3555).
The esterase activity of peptidyl-tRNA hydrolases cleaves the
covalent bond between the nascent peptide and the tRNA. Such
cleavage results in the recycling of tRNA. The peptidyl-tRNA
hydrolase from E. coli is well characterized, and homologous
proteins are found in many eubacterial species. In E. coli, the
gene encoding peptidyl-tRNA hydrolase is essential. Further, the
required level of peptidyl-tRNA hydrolase activity for viability is
escalated under conditions that increase premature translational
termination such as exposure to antibiotics (Menninger and Coleman
(1993) Antimicrob. Agents Chemother. 37:2027-2029.) and reduced
when tRNAs particularly prone to dissociate from the ribosome are
supplied in excess (Heurgue-Hamard et al. (1996) EMBO J.
15:2826-2833).
[0239] The x-ray crystal structure of E. coli peptidyl-tRNA
hydrolase was determined at high resolution (Schmitt et al. (1997)
EMBO J. 16:4760-4769). The monomeric protein contains single
monomeric .alpha./.beta. globular domain of seven .beta.-strands
and six .alpha.-helices. The peptidyl-tRNA hydrolase enzyme
structure has structural similarity to an aminopeptidase from
Aeromonas proteolytica (GenPept:640150)(Chevrier, B. et al. (1994)
Structure 2:283-291) and to a lesser extent to bovine purine
nucleoside phosphorylase (GenPept:2624420) (Koellner, G. et al.
(1997) J. Mol. Biol. 265:202-216.). Genetic data and structural
analysis indicate that three residues, asparagine 10, histidine 20,
and aspartic acid 93 in the E. coli enzyme are critical residues
for catalysis. In addition, asparagine 68 and asparagine 114 of the
E. coli enzyme are poised to make favorable electrostatic contacts
with the peptide region of the peptidyl-tRNA substrate whereas
arginine 133 of the E. coli sequence may contact the tRNA portion
of the substrate. The amino acid identities of these positions are
conserved in alignments of eubacterial peptidyl-tRNA
transferases.
[0240] The 46508 polypeptide (SEQ ID NO: 36) has the three
conserved residues important for catalysis, namely: an asparagine
at position 51, a histidine at position 59, and an aspartic acid at
position 134 of SEQ ID NO: 36. In addition, conserved asparagines
and a conserved arginine, residues 68, 114, and 133 of the E. coli
peptidyl-tRNA hydrolase, respectively, contribute to the
specificity of substrate recognition. The 46508 polypeptide (SEQ ID
NO: 36) also has these three conserved residues, namely, an
asparagine at position 109, an asparagine at position 155, and an
arginine at position 173 of SEQ ID NO: 36.
[0241] Cells which utilize the translation machinery more intensely
than quiescent cells, e.g. rapidly growing cells, environmentally
stressed cells, and virally infected cells, are likely to produce
more peptidyl-tRNA substrates. Further, because of the increased
translation activity, such cells also require larger tRNA pools
than quiescent cells either in their cytoplasm or mitochondria, or
both. Accordingly the activity of peptidyl-tRNA hydrolase enzymes
may be required by such cells. Thus, inhibition of 46508 activity
might be a successful route to treatment of a variety of disorders,
including but not limited to, cell proliferation, cell
differentiation, viral infection, and metabolism.
[0242] A 46508 polypeptide can include a "peptidyl-tRNA hydrolase
domain" or regions homologous with a "peptidyl-tRNA hydrolase
domain". A 46508 polypeptide can optionally further include at
least one glycosaminoglycan attachment site; at least one,
preferably two, protein kinase C phosphorylation sites; at least
one, preferable two, casein kinase II phosphorylation sites; at
least one, two, three, four, five, six, preferably seven,
N-myristoylation sites; and at least one amidation site.
[0243] As used herein, the term "peptidyl-tRNA hydrolase domain"
includes an amino acid sequence of about 160 to 240 amino acid
residues in length and having a bit score for the alignment of the
sequence to the peptidyl-tRNA hydrolase domain profile (Pfam HMM)
of at least 80. Preferably, the peptidyl-tRNA hydrolase domain has
an amino acid sequence of about 170 to about 200 amino acids, more
preferable about 170 to 190 amino acids, or about 177 amino acids,
and has a bit score for the alignment of the sequence to the
peptidyl-tRNA hydrolase domain (HMM) of at least 100, preferably of
at least 120, more preferably of at least 130 or greater.
Preferably, the peptidyl-tRNA hydrolase domain further includes the
following highly conserved residues :one, preferably two, more
preferably three asparagine residues, a histidine residue, an
aspartic acid, and an arginine corresponding respectively to
asparagine 51, asparagine 109, asparagine 155, histidine 59,
aspartic acid 134, and arginine 173 of SEQ ID NO: 36. The
peptidyl-tRNA hydrolase domain (HMM) has been assigned the PFAM
Accession.
[0244] In a preferred embodiment 46508 polypeptide or protein has a
"peptidyl-tRNA hydrolase domain" or a region which includes at
least about 120 to about 200 amino acids, more preferably about 160
to 190, 170 to 180, or about 177 amino acid residues and has at
least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a
"peptidyl-tRNA hydrolase domain," e.g., the peptidyl-tRNA hydrolase
domain of human 46508 (e.g., residues 44 to 221 of SEQ ID NO:
36).
[0245] To identify the presence of a "peptidyl-tRNA hydrolase"
domain in a 46508 protein sequence, and make the determination that
a polypeptide or protein of interest has a particular profile, the
amino acid sequence of the protein can be searched against the Pfam
database of HMMs (e.g., the Pfam database, release 2.1) using the
default parameters. For example, the hmmsf program, which is
available as part of the HMMER package of search programs, is a
family specific default program for MILPAT0063 and a score of 15 is
the default threshold score for determining a hit. Alternatively,
the threshold score for determining a hit can be lowered (e.g., to
8 bits). A description of the Pfam database can be found in
Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed
description of HMMs can be found, for example, in Gribskov et
al.(1990) Meth. Enzymol. 183:146-159; Gribskov et al.(1987) Proc.
Natl. Acad. Sci. USA 84:4355-4358; Krogh et al.(1994) J. Mol. Biol.
235:1501-1531; and Stultz et al.(1993) Protein Sci. 2:305-314, the
contents of which are incorporated herein by reference. A search
was performed against the HMM database resulting in the
identification of a "peptidyl-tRNA hydrolase" domain in the amino
acid sequence of human 46508 at about residues 44 to about 221 of
SEQ ID NO: 36. The identified peptidyl-tRNA hydrolase domain
consensus sequence is set forth as SEQ ID NO: 38.
[0246] A 46508 family member can include at least one peptidyl-tRNA
hydrolase domain or regions homologous with a peptidyl-tRNA
hydrolase domain. Furthermore, a 46508 family member can include at
least one, preferably two, protein kinase C phosphorylation sites
(PS00005); at least one, preferable two, casein kinase II
phosphorylation sites (PS00006); at least one, two, three, four,
five, six, preferably seven, N-myristoylation sites; and at least
one amidation site.
[0247] 46508 polypeptides of the invention include fragments which
include: all or part of a hydrophobic sequence, e.g., the sequence
from about amino acid 60 to 70, from about 86 to 102, and from
about 189 to 195 of SEQ ID NO: 36; all or part of a hydrophilic
sequence, e.g., the sequence of from about amino acid 77 to 85,
from about 217 to 224 of SEQ ID NO: 36, a sequence which includes a
Cys, or a glycosylation site, of SEQ ID NO: 36.
[0248] As the 46508 polypeptides of the invention may modulate
46508-mediated activities, they may be useful as of for developing
novel diagnostic and therapeutic agents for 46508-mediated or
related disorders, as described below.
[0249] As used herein, a "46508 activity", "biological activity of
46508" or "functional activity of 46508", refers to an activity
exerted by a 46508 protein, polypeptide or nucleic acid molecule.
For example, a 46508 activity can be an activity exerted by 46508
in a physiological milieu on, e.g., a 46508-responsive cell or on a
46508 substrate, e.g., a protein substrate. A 46508 activity can be
determined in vivo or in vitro. In one embodiment, a 46508 activity
is a direct activity, such as an association with a 46508 target
molecule. A "target molecule" or "binding partner" is a molecule
with which a 46508 protein binds or interacts in nature. In an
other embodiment, 46508 activity can also be an indirect activity,
e.g. a cellular signaling activity mediated by interaction of the
46508 protein with a second protein or with a nucleic acid.
[0250] The features of the 46508 molecules of the present invention
can provide similar biological activities as peptidyl-tRNA
hydrolase family members. For example, the 46508 proteins of the
present invention can have one or more of the following activities:
(1) ability to bind tRNA; (2) ability to bind peptide fragments;
(3) ability to bind peptidyl-tRNAs; (4) ability to hydrolyze
covalent bond between peptide and tRNA within peptidyl-tRNAs; or
(5) ability to modulate translational efficiency. The 46508
polypeptide may perform one or more of these properties in the
milieu of the cell cytoplasm and/or of the cell mitochondria.
[0251] As shown below, increased 46508 mRNA expression is detected
in a variety of malignant and non-malignant tissues, including
cardiovascular tissues (e.g., endothelial cells, coronary smooth
muscle cells), pancreas, neural tissues (e.g., brain, hypothalamus,
DRG), skin, immune, e.g., erythroid cells, as well as a number of
primary and metastatic tumors, e.g., ovarian, breast, prostate and
lung tumors. Thus, the 46508 molecules can act as novel diagnostic
targets and therapeutic agents for controlling disorders of
involving aberrant activity of those cells, e.g., cell
proliferative disorders (e.g., cancer), cardiovascular disorders,
neurological disorders, pain disorders, pancreatic disorders,
breast disorders, colon disorders, ovarian disorders, lung
disorders, skin and immune, e.g., erythroid, disorders.
[0252] High transcriptional expression of 46508 was observed in
tumor samples compared to normal organ control samples. For
example, high expression was observed in 5/5 primary ovarian tumor
samples, 4/4 primary colon tumor samples, 2/2 colon to liver
metastases, and 3/6 primary lung tumor samples. Additionally, high
expression was observed in proliferating HMVEC cells when compared
to arrested HMVEC cells. Therefore, 46508 may mediate or be
involved in cellular proliferative and/or differentiative
disorders.
[0253] Moderate expression of 46508 was observed in normal heart
tissue samples, and in both normal and tumor breast tissue samples,
and thus 46508 may mediate disorders involving the heart, e.g.
cardiovascular disorders; and it may mediate disorders of the
breast, e.g, breast disorders. High to moderate expression of 46508
was also observed in normal pancreas tissue, in the skin and in the
normal brain cortex and hypothalamus, therefore, 46508 may mediate
disorders involving the pancreas, e.g. pancreatic disorders; it may
mediate disorders involving the skin, e.g., skin disorders; and it
may mediate disorders involving the brain cortex or hypothalamus,
e.g. disorders of the brain.
[0254] 46508 mRNA expression was also detected in the dorsal root
ganglia (DRG). Therefore, 46508-associated disorders can
detrimentally affect regulation and modulation of the pain
response; and vasoconstriction, inflammatory response and pain
therefrom. Examples of such disorders in which the 46508 molecules
of the invention may be directly or indirectly involved include
pain, pain syndromes, and inflammatory disorders, including
inflammatory pain.
[0255] Normal and tumorous samples of ovarian tissue also showed
expression of 46508 mRNA. Various data indicates that 46508 is
highly expressed in several ovarian cell lines, including
SKOV3/Var, A2780, MDA 2774 and ES-2. Thus 46508 may mediate
diseases involving the ovary.
[0256] Moderate expression of 46508 mRNA was also noted in normal
colon tissue, in normal and tumor prostate samples, in fibrotic
liver tissue samples, in both normal and lung tumor samples and
high expression was noted in colon tumor samples. Thus 46508 may
mediate diseases involving the colon, e.g. colon disorders; it may
mediate diseases involving the prostate, e.g. prostate disorders;
it may mediate diseases involving the liver, e.g. liver disorders;
and it may mediate diseases involving the lung, e.g. lung
disorders;
[0257] Identification and Characterization of Human 46508 cDNA
[0258] The human 46508 sequence (SEQ ID NO: 35), which is
approximately 1180 nucleotides long, including untranslated
regions, contains a predicted methionine-initiated coding sequence
of about 684 nucleotides, including the termination codon
(nucleotides indicated as "coding" of SEQ ID NO: 35; SEQ ID NO:
37). The coding sequence encodes a 227 amino acid protein (SEQ ID
NO: 36).
[0259] Tissue Distribution of 46508 mRNA by TaqMan Analysis
[0260] Endogenous human 46508 gene expression was determined using
the Perkin-Elmer/ABI 7700 Sequence Detection System which employs
TaqMan technology.
[0261] To determine the level of 46508 in various human tissues a
primer/probe set was designed. Total RNA was prepared from a series
of human tissues using an RNeasy kit from Qiagen. First strand cDNA
was prepared from 1 .mu.g total RNA using an oligo-dT primer and
Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained
from approximately 50 ng total RNA was used per TaqMan reaction.
Tissues tested include the human tissues and several cell lines
shown in Tables 4, 5 and 6.
[0262] Table 4 below shows expression of 46508 mRNA in various
normal and diseased tissues, detected using TaqMan analysis. The
highest transcriptional expression of 46508 was noted in HUVEC cell
line, with moderate to high expression found in normal pancreas,
brain, hypothalamus, skeletal muscle, DRG (dorsal root ganglion)
and skin. Moderate expression also noted in normal and tumor pairs
of breast, ovarian, prostate, colon and lung tissue along with
fibrotic liver, normal heart and diseased heart (CHF, congestive
heart failure) samples.
6TABLE 4 Tissue Distribution of 46508 mRNA by TaqMan Analysis
Tissue Type Expression Artery normal 8.6685 Aorta diseased 5.2992
Vein normal 1.7725 Coronary SMC 17.1577 HUVEC 49.0365 Hemangioma
5.0834 Heart normal 8.2009 Heart CHF 7.1393 Kidney 9.4204 Skeletal
Muscle 16.6308 Adipose normal 3.9608 Pancreas 20.3335 primary
osteoblasts 2.7431 Osteoclasts (diff) 0.8955 Skin normal 13.0031
Spinal cord normal 5.1365 Brain Cortex normal 24.2647 Brain
Hypothalamus normal 25.2951 Nerve 7.8942 DRG (Dorsal Root Ganglion)
16.5159 Breast normal 6.5016 Breast tumor 11.4382 Ovary normal
10.0965 Ovary Tumor 6.7542 Prostate Normal 10.273 Prostate Tumor
11.0485 Salivary glands 2.1822 Colon normal 3.5327 Colon Tumor
14.9885 Lung normal 4.9273 Lung tumor 7.3655 Lung COPD 5.0658 Colon
IBD 3.14 Liver normal 9.3229 Liver fibrosis 8.6385 Spleen normal
2.4129 Tonsil normal 2.83 Lymph node normal 5.3176 Small intestine
normal 2.0573 Macrophages 1.3526 Synovium 2.3388 BM-MNC 0.2563
Activated PBMC 1.5538 Neutrophils 1.5755 Megakaryocytes 1.57
Erythroid 15.3566 positive control 21.1969
[0263] Table 5 below also shows expression of 46508 mRNA in various
normal and diseased tissues, detected using TaqMan analysis. Table
5 shows the high transcriptional expression of 46508 in tumor
samples compared to normal organ matched controls. High
transcriptional expression was noted in 5/5 primary ovarian tumors,
4/4 primary colon tumors, 2/2 colon to liver metastases, 3/6
primary lung tumors and proliferating HMVEC cells when compared to
arrested HMVEC cells.
7TABLE 5 Expression of 46508 mRNA in Normal and Cancerous Tissues
Tissue Type Expression PIT 400 Breast Normal 28.36 PIT 372 Breast
Normal 36.40 CHT 1228 Breast Normal 8.88 MDA 304 Breast Tumor:
MD-IDC 8.34 CHT 2002 Breast Tumor: IDC 3.55 MDA 236-Breast Tumor:
PD-IDC 3.21 CHT 562 Breast Tumor: IDC 13.51 NDR 138 Breast Tumor
ILC (LG) 25.30 CHT 1841 Lymph node (Breast met) 7.19 PIT 58 Lung
(Breast met) 12.22 CHT 620 Ovary Normal 14.83 CHT 619 Ovary Normal
7.09 CLN 012 Ovary Tumor 40.53 CLN 07 Ovary Tumor 28.26 CLN 17
Ovary Tumor 94.40 MDA 25 Ovary Tumor 80.21 CLN 08 Ovary Tumor 28.07
PIT 298 Lung Normal 2.14 MDA 185 Lung Normal 6.05 CLN 930 Lung
Normal 12.13 MPI 215 Lung Tumor--SmC 9.69 MDA 259 Lung
Tumor-PDNSCCL 13.94 CHT 832 Lung Tumor-PDNSCCL 8.64 MDA 262 Lung
Tumor-SCC 68.87 CHT 793 Lung Tumor-ACA 20.83 CHT 331 Lung Tumor-ACA
8.14 CHT 405 Colon Normal 2.50 CHT 1685 Colon Normal 2.10 CHT 371
Colon Normal 0.95 CHT 382 Colon Tumor: MD 69.11 CHT 528 Colon
Tumor: MD 54.79 CLN 609 Colon Tumor 15.25 NDR 210 Colon Tumor:
MD-PD 121.16 CHT 340 Colon-Liver Met 15.25 CHT 1637 Colon-Liver Met
10.82 PIT 260 Liver N (female) 1.46 CHT 1653 Cervix Squamous CC
19.51 CHT 569 Cervix Squamous CC 1.31 A24 HMVEC-Arrested 11.56 C48
HMVEC-Proliferating 31.80 Pooled Hemangiomas 1.16 HCT116N22
Normoxic 76.42 HCT116H22 Hypoxic 68.39
[0264] Table 6 indicates that 46508 mRNA is highly expressed in
several ovarian cell lines including SKOV3/Var, A2780, MDA2774 and
ES-2. The table allows comparisons between two normal ovarian
surface epithelium samples (MDA 127 Normal Ovary and MDA 224 Normal
Ovary) and two ovarian ascites (MDA 124 Ovarian Ascites and MDA 126
Ovarian Ascites) samples. Expression of 46508 mRNA is upregulated
in one of the ascites samples. The table also shows an experiment
where the ovarian cancer cell line, HEY, was serum starved for 24
hours. Time points were taken at 0, 1, 3, 6, 9 and 12 hours after
the addition of 10% serum (HEY 0 hr, HEY 1 hr, HEY 3 hr, HEY 6 hr,
HEY 9 hr, and HEY 12 hr, respectively). Since cMyc protein is
highly upregulated at 1 hour after addition of serum and
phosphorylated at 6 hours, the experiment is a good model for
identifying targets that are downstream of cMyc. These data
indicate that 46508 mRNA may be regulated in a manner similar to
cMyc since the expression increases from 1 to 9 hours after the
addition of serum.
[0265] Also shown are data involving the ovarian cancer cell lines
SKOV3 and SKOV3/Variant. These cell lines were grown in three
different cellular environments: on plastic, in soft agar, and as a
subcutaneous tumor in nude mice (all cells grown in 10% serum). The
plastic sample was used as the "control" in each experiment. The
SKOV3/Var cell line is a variant of the parental cell line SKOV3
which is resistant to cisplatin. These data indicate that 46508
mRNA is upregulated in environments that may be more similar to the
tumor in vivo (the soft agar and subcutaneous tumor) compared to
growth on plastic.
8TABLE 6 Expression in Various Ovarian Cells Tissue Type Expression
SKOV-3 No GF 40.67 SKOV-3 EGF '15 41.52 SKOV-3 EGF '30 46.23 SKOV-3
EGF '60 38.88 SKOV-3 Hrg '15 36.02 SKOV-3 Hrg '30 41.67 SKOV-3 Hrg
'60 48.87 SKOV-3 Serum '30 54.98 SKOV-3 var No GF 151.25 SKOV-3 var
EGF '15 140.63 SKOV-3 var EGF '30 126.74 SKOV-3 var EGF '60 125.43
SKOV-3 var Hrg '15 141.61 SKOV-3 var Hrg '30 170.76 SKOV-3 var Hrg
'60 140.15 SKOV-3 var Serum '30 190.78 HEY Plastic 50.94 HEY Soft
Agar 22.96 SKOV-3 35.65 SKOV-3 var 122.00 A2780 159.87 A2780-ADR
60.79 OVCAR-3 54.98 OVCAR-4 59.75 MDA 2774 123.28 DOV13 36.52
Caov-3 18.14 ES-2 101.18 HEY 0 hr 55.17 HEY 1 hr 62.50 HEY 3 hr
74.84 HEY 6 hr 69.59 HEY 9 hr 77.75 HEY 12 hr 68.63 SKOV-3 SubQ
Tumor 18.01 SKOV-3 Variant Plastic 152.30 SKOV-3 Var SubQ Tumor
9.39 MDA 127 Normal Ovary 11.44 MDA 224 Normal Ovary 17.10 MDA 124
Ovarian Ascites 18.84 MDA 126 Ovarian Ascites 41.67 HEY 63.81
SKOV-3 Plastic 72.80
[0266] Human 16816
[0267] The human 16816 sequence (SEQ ID NO: 39), which is
approximately 2629 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
2289 nucleotides (nucleotides 257-2545 of SEQ ID NO: 39; SEQ ID NO:
41), including the terminal codon. The coding sequence encodes a
762 amino acid protein (SEQ ID NO: 40).
[0268] This mature protein form is approximately 762 amino acid
residues in length (from about amino acid 1 to amino acid 762 of
SEQ ID NO: 40). Human 16816 contains the following regions or other
structural features: one predicted phosphatidylinositol-specific
phospholipase C domain X (PFAM Accession Number PF00388) located at
about amino acid residues 291-436 of SEQ ID NO: 40; one predicted
phosphatidylinositol-specific phospholipase C domain Y (PFAM
Accession Number PF00387) located at about amino acid residues
492-609 of SEQ ID NO: 40; two predicted EF hand domains (PFAM
Accession Number PF00036) located at about amino acid residues
138-166 and 174-202 of SEQ ID NO: 40; one predicted C2 domain (PFAM
Accession Number PF00168) located at about amino acid residues
291-436 and 492-609 of SEQ ID NO: 40; two cAMP- and cGMP-dependent
protein kinase phosphorylation sites (PS00004) located at about
amino acids 435-438 and 482-485 of SEQ ID NO: 40; nine predicted
protein kinase C phosphorylation sites (PS00005) located at about
amino acids 31-33, 56-58, 68-70, 203-205, 257-259, 355-357,
504-506, 666-668 and 741-743 of SEQ ID NO: 40; seventeen predicted
casein kinase II phosphorylation sites (PS00006) located at about
amino 11-14, 62-65, 80-83, 100-103, 108-111, 127-130, 155-158,
223-226, 318-321, 410-413, 422-425, 438-441, 463-466, 467-470,
522-525, 649-652 and 710-713 of SEQ ID NO: 40; four predicted
N-myristoylation sites (PS00008) located at about amino acids
188-193, 219-224, 414-419 and 684-689 of SEQ ID NO: 40; two
predicted amidation sites (PS00009) located at about amino acids
96-99 and 433-436 of SEQ ID NO: 40; one RGD cell attachment
sequence (PS00016) located at about amino acids 145-147 of SEQ ID
NO: 40; and/or two EF hand calcium-binding domains (PS00018)
located at about amino acids 147-159 and 183-195 of SEQ ID NO:
40.
[0269] In one embodiment, a 16816 family member can include at
least one phosphatidylinositol-specific phospholipase C domain X
(PFAM Accession Number PF00388); at least one
phosphatidylinositol-specific phospholipase C domain Y (PFAM
Accession Number PF00387); at least one preferably two EF hand
domains (PFAM Accession Number PF00036 or PS00018); at least one
predicted C2 domain (PFAM Accession Number PF00168). Furthermore, a
16816 family member can include at least one and preferably two
cAMP- and cGMP-dependent protein kinase phosphorylation sites; at
least one, two, three, four, five, six, seven, eight, and
preferably nine protein kinase C phosphorylation sites (PS00005);
at least one, two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, fifteen, sixteen and preferably
seventeen casein kinase II phosphorylation sites (PS00006); at
least one, two, three, and preferably four N-myristolyation sites
(PS00008); at least one and preferably two predicted amidation
sites; or at least one RGD cell attachment sequence.
[0270] 16816 polypeptides of the invention include fragments which
include: all or part of a hydrophobic sequence, e.g., the sequence
from about amino acid 485 to 500, from about 650 to 660, and from
about 685 to 700 of SEQ ID NO: 40; all or part of a hydrophilic
sequence, e.g., the sequence from about amino acid 125 to 150, from
about 465 to 480, and from about 665 to 680 of SEQ ID NO: 40; a
sequence which includes a Cys, or a glycosylation site.
[0271] Human 16839
[0272] The human 16839 sequence (SEQ ID NO: 42), which is
approximately 2171 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
1827 nucleotides (nucleotides 232-2058 of SEQ ID NO: 42; SEQ ID NO:
44), including the terminal codon. The coding sequence encodes a
608 amino acid protein (SEQ ID NO: 43).
[0273] This mature protein form is approximately 608 amino acid
residues in length (from about amino acid 1 to amino acid 608 of
SEQ ID NO: 43). Human 16839 contains the following regions or other
structural features: one EF hand domain (PFAM Accession Number
PF00036) located at about amino acids 39 to 67 of SEQ ID NO: 43;
one phosphatidylinositol-specific phospholipase C domain X (PFAM
Accession Number PF00388) located at about amino acids 156 to 300
of SEQ ID NO: 43; one phosphatidylinositol-specifi- c phospholipase
C domain Y (PFAM Accession Number PF00387) located at about amino
acids 348 to 465 of SEQ ID NO: 43; one C2 domain (PFAM Accession
Number PF00168) located at about amino acids 484 to 572 of SEQ ID
NO: 43; two N-glycosylation sites (PS00001) located at about amino
acids 376-379 and 537-540 of SEQ ID NO: 43; three cAMP- and
cGMP-dependent protein kinase phosphorylation sites (PS00004)
located at about amino acids 310-313, 337-340 and 385-388 of SEQ ID
NO: 43; ten predicted protein kinase C phosphorylation sites
(PS00005) located at about amino acids 24-26, 68-70, 220-222,
303-305, 313-315, 340-342, 399-401, 485-487, 501-503 and 533-535 of
SEQ ID NO: 43; eight predicted casein kinase II phosphorylation
sites (PS00006) located at about amino 56-59, 68-71, 79-82,
267-270, 303-306, 356-359, 378-381 and 411-414 of SEQ ID NO: 43;
and three predicted N-myristoylation sites (PS00008) located at
about amino acids 16-21, 479-484 and 560-565 of SEQ ID NO: 43.
[0274] In one embodiment, a 16839 family member can include at
least one EF hand domain (PFAM Accession Number PF00036); at least
one phosphatidylinositol-specific phospholipase C domain X (PFAM
Accession Number PF00388); at least one
phosphatidylinositol-specific phospholipase C domain Y (PFAM
Accession Number PF00387); at least one C2 domain (PFAM Accession
Number PF00168). Furthermore, a 16839 family member can include at
least one N-glycosylation site (PS00001); at least one, two and
preferably three cAMP- and cGMP-dependent protein kinase
phosphorylation sites; at least one, two, three, four, five, six,
seven, eight, nine and preferably ten protein kinase C
phosphorylation sites (PS00005); at least one, two, three, four,
five, six, seven, and preferably eight casein kinase II
phosphorylation sites (PS00006); at least one, two, and preferably
three N-myristolyation sites (PS00008).
[0275] 16839 polypeptides of the invention include fragments which
include: all or part of a hydrophobic sequence, e.g., the sequence
from about amino acid 340 to 350, from about 480 to 490, and from
about 540 to 560 of SEQ ID NO: 43; all or part of a hydrophilic
sequence, e.g., the sequence from about amino acid 300 to 325, from
about 360 to 390, and from about 405 to 420 of SEQ ID NO: 43; a
sequence which includes a Cys, or a glycosylation site.
[0276] 16816 and 16839 Phospholipase C Proteins
[0277] The 16816 and 16839 protein contain a significant number of
structural characteristics in common with members of the
phospholipase C family.
[0278] Phospholipase C (PLC) belongs to a family of enzymes, also
known as disulfide isomerases, which play an important role in
mediating signal transduction pathways. Many extracellular
signaling molecules including hormones, growth factors,
neurotransmitters, and immunoglobulins bind to their respective
cell surface receptors and activate PLCs. Activated PLCs then
catalyze the hydrolysis of phosphatidyl-inositol-4,5-bisphosphate
(PIP2), a component of the plasma membrane, to produce
diacylglycerol and inositol 1,4,5-trisphosphate (IP3).
[0279] In their respective biochemical pathways, IP3 and
diacylglycerol serve as second messengers and trigger a series of
intracellular responses. IP3 induces the release of calcium from
internal cellular storage, and diacylglycerol activates protein
kinase C (PKC). Both pathways are part of transmembrane signal
transduction mechanisms, which regulate numerous cellular
processes, including secretion, neural activity, metabolism, and
proliferation.
[0280] PLC molecules have been found in a broad spectrum of
organisms including bacteria, simple eukaryotes, plants and animals
(Munnik et al., Biochim. Biophys. Acta. 1389:222-272, (1998)).
Several distinct isoforms of PLC have been identified in animals
and are categorized as PLC-beta, PLC-gamma, and PLC-delta. Subtypes
are designated by adding Arabic numbers after the Greek letters,
e.g., PLC-beta-1. PLCs have a molecular mass of 62-68 kDa, and
their amino acid sequences show two regions of significant
similarity.
[0281] The present invention is based, at least in part, on the
discovery of novel molecules, referred to herein as "phospholipase
C" or "16816" or "16839" nucleic acid and polypeptide molecules,
which play a role in or function in modulating signal transduction
pathways.
[0282] PLC molecules have been found in a broad spectrum of
organisms including bacteria, simple eukaryotes, plants and
animals. Members of a family can also have common functional
characteristics. Members of the PLC family share one or more common
domains such as a pleckstrin homology domain, an EF hand domain, a
phosphatidylinositol-specific phospholipase domain X (PLC-X)
domain, a phosphatidylinositol-specific phospholipase domain Y
(PLC-Y) domain or a C2 domain. Members of this family can also have
common functional characteristics, e.g., the ability to hydrolyze
phosphatidylinositols.
[0283] A 16816 polypeptide can include a "pleckstrin homology (PH)
domain" or regions homologous with a "PH domain". As used herein,
the term "PH domain" refers to a protein domain having an amino
acid sequence of about 10 to 200, preferably about 50 to 150, more
preferably about 108 amino acid residues. By "PH domain" is meant a
domain that can function as a recognition site for a
phosphatidylinositol, e.g., a 3, 4, 5-trisphosphate (PIP3) or
another kinase ligand product, and can function as a means to
localize PLC to the cytoplasmic face of the plasma membrane.
[0284] As used herein, the term "PH domain" includes an amino acid
sequence of about 108 amino acid residues in length and having a
bit score for the alignment of the sequence to the PH domain (HMM)
of at least 10. Preferably, a PH domain includes at least about
10-200 amino acids, more preferably about 50-150 amino acid
residues, or about 75-110 amino acids and has a bit score for the
alignment of the sequence to the PH domain (HMM) of at least 20,
30, or greater.
[0285] In a preferred embodiment a 16816 polypeptide or protein has
a "PH domain" or a region which includes at least about 10-200
amino acids, more preferably about 50-150 amino acid residues, or
about 107 amino acid residues and has at least about 60%, 70% 80%
90% 95%, 99%, or 100% homology with a "PH domain," e.g., the PH
domain of human 16816 (e.g., residues 17-124 of SEQ ID NO: 40). The
identified PH domain consensus sequence is set forth as SEQ ID NO:
45.
[0286] A 16816 or 16839 polypeptide can also include an "EF hand
domain" or regions homologous with an "EF hand domain". As used
herein, the term "EF hand domain" refers to a protein domain having
an amino acid sequence of about 5 to 50, preferably about 5 to 40,
more preferably about 28-29 amino acid residues. By "EF hand
domain" is meant a type of calcium-binding domain that consists of
a twelve residue loop flanked on both sides by a twelve residue
alpha-helical domain. In an EF-hand loop the calcium ion is
coordinated in a pentagonal bipyramidal configuration. The six
residues involved in the binding are in positions 1, 3, 5, 7, 9 and
12; these residues are denoted by X, Y, Z, -Y, -X and -Z. The
invariant Glu or Asp at position 12 provides two oxygens for
liganding Ca (bidentate ligand). Preferably, the EF hand domain
includes the following amino acid consensus sequence having Prosite
signatures as PS00018, or sequences homologous thereto.
D-x-[DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]-{GP}--
[LIVMC]-[DENQSTAGC]-x(2)-[DE]-[LIVMFYW] (SEQ ID NO: 62). In the
above conserved motif, and other motifs described herein, the
standard IUPAC one-letter code for the amino acids is used. Each
element in the pattern is separated by a dash (-); square brackets
([ ])indicate the particular residues that are accepted at that
position; x indicates that any residue is accepted at that
position; and numbers in parentheses (()) indicate the number of
residues represented by the accompanying amino acid. The EF hand
domains are located in mostly hydrophilic regions of the molecule
of human 16816 or 16839 polypeptide and which corresponds to about
amino acids 138-166 and 172-202 of SEQ ID NO: 40; or amino acids
39-67 of SEQ ID NO: 43. The EF hand domain (HMM) has been assigned
the PFAM Accession Number PF00036.
[0287] The "EF hand domain" includes an amino acid sequence of
about 28 amino acid residues in length and can have a bit score for
the alignment of the sequence to the EF hand (HMM) of at least 5.
Preferably, an EF hand domain includes at least about 5-50 amino
acids, or at least about 5-40, or about 28 amino acids and has a
bit score for the alignment of the sequence to the EF hand (HMM) of
at least 5, 10, 15, 20, or greater.
[0288] In a preferred embodiment, the 16816 or 16839 polypeptide or
protein has an "EF hand domain" or a region which includes at least
about 5-50, more preferably about 5-40 or 28-29 amino acid residues
and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology
with an "EF hand domain," e.g., the EF hand domain of human 16816
or 16839 (e.g., residues 138-166 and 172-202 of SEQ ID NO: 40; or
amino acids 39-67 of SEQ ID NO: 43). The identified EF hand domain
consensus sequences within 16816 are set forth as SEQ ID NO: 46 and
47, and the identified EF hand domain consensus sequence within
16839 is set forth as SEQ ID) NO: 56.
[0289] A 16816 or 16839 polypeptide can also include a
"phosphatidylinositol-specific phospholipase C domain X (referred
to herein as "PLC-X domain")" or regions homologous with a "PLC-X
domain". As used herein, the term "PLC-X domain" refers to a
protein domain having an amino acid sequence of about 8 to 200,
preferably about 15 to 170, more preferably about 145 amino acid
residues. By "PLC-X domain" is meant a subdomain that composes the
catalytic site of the phospholipase, e.g., PLC-X subdomain can fold
together with another subdomain, e.g.,
phosphatidylinositol-specific phospholipase C domain Y such that a
functioning catalytic site that hydrolyzes a phosphatidylinositol
is formed, e.g., phosphatidylinositol 4,5-bisphosphate, is
formed.
[0290] The "PLC-X domain" includes an amino acid sequence of about
145 amino acid residues in length and can have a bit score for the
alignment of the sequence to the phosphatidylinositol-specific
phospholipase-C domain X (HMM) of at least 50. Preferably, a PLC-X
domain includes at least about 15-170 amino acids, or at least
about 20-150, or about 145 amino acids and has a bit score for the
alignment of the sequence to the phosphatidylinositol-specific
phospholipase-C domain X (HMM) of at least 60, 70, 80, 90, 100,
150, 200, 250, or greater.
[0291] In a preferred embodiment, the 16816 or 16839 polypeptide or
protein has a "PLC-X" or a region which includes at least about
8-200, more preferably about 15-170 or 20-150 amino acid residues
and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology
with a "PLC-X domain," e.g., the PLC-X domain of human 16816 or
16839 (e.g., residues 291-436 of SEQ ID NO: 40; or residues 156-300
of SEQ ID NO: 43). The identified PLC-X domain consensus sequence
of 16816 is set forth in SEQ ID NO: 48 and the identified PLC-X
domain consensus sequence of 16839 is set forth in SEQ ID NO:
57.
[0292] A 16816 or 16839 polypeptide can include a
"phosphatidylinositol-sp- ecific phospholipase C domain Y (referred
to herein as PLC-Y domain)" or regions homologous with a "PLC-Y
domain". As used herein, the term "PLC-Y domain" refers to a
protein domain having an amino acid sequence of about 8 to 200,
preferably about 15 to 170, more preferably about 117 amino acid
residues. By "PLC-Y domain" is meant a subdomain that composes the
catalytic site of the phospholipase, e.g., the subdomain can fold
together with another subdomain, e.g., PLC-X domain such that a
functioning catalytic site that hydrolyzes a phosphatidylinositol,
e.g., phosphatidylinositol 4,5-bisphosphate, is formed.
[0293] The "PLC-Y domain" includes an amino acid sequence of about
117 amino acid residues in length and can have a bit score for the
alignment of the sequence to the PLC-Y domain (HMM) of at least 50.
Preferably, a PLC-Y domain includes at least about 15-170 amino
acids, or at least about 20-150, or about 117 amino acids and has a
bit score for the alignment of the sequence to the PLC-Y domain
(HMM) of at least 60, 70, 80, 90, 100, 110, 120, 140, 160, 180, or
greater.
[0294] In a preferred embodiment 16816 or 16839 polypeptide or
protein has a "PLC-Y domain" or a region which includes at least
about 8-200, more preferably about 15-170 or 20-150 amino acid
residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100%
homology with a "PLC-Y domain" e.g., PLC-Y domain of human 16816 or
16839 (e.g., residues 492-609 of SEQ ID NO: 40; or residues 348-465
of SEQ ID NO: 43). The identified PLC-Y domain consensus sequence
of 16816 is set forth in SEQ ID NO: 49 and the identified PLC-Y
domain consensus sequence of 16839 is set forth in SEQ ID NO:
58.
[0295] A 16816 or 16839 polypeptide can include a "calcium (Ca2+)
binding domain (referred to as C2 domain") or regions homologous
with a "C2 domain". As used herein, the term "C2" refers to a
protein domain having an amino acid sequence of about 8 to 200,
preferably about 15 to 170, more preferably about 20 to 100, or
still more preferably about 90 amino acid residues. By "C2 domain"
is meant a domain that can mediate interaction with calcium or
phospholipids.
[0296] The "C2 domain" includes an amino acid sequence of about 90
amino acid residues in length and can have a bit score for the
alignment of the sequence to the C2 domain (HMM) of at least 50.
Preferably, a C2 includes at least about 8-200, or at least about
15-170, or at least 20-100, or about 90 amino acids and has a bit
score for the alignment of the sequence to the C2 domain (HMM) of
at least 60, 70, 80, 85, or greater.
[0297] In a preferred embodiment, a 16816 or 16839 polypeptide or
protein has a "C2" or a region which includes at least about
10-200, more preferably about 15-170 or 20-100 amino acid residues
and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology
with a "C2," e.g., the C2 domain of human 16816 or 16839 (e.g.,
residues 629-719 of SEQ ID NO: 40; or residues 484-572 of SEQ ID
NO: 43). The identified C2 domain consensus sequence of 16816 is
set forth in SEQ ID NO: 50 and the identified C2 domain consensus
sequence of 16839 is set forth in SEQ ID NO: 59.
[0298] To identify the presence of a "PH domain," "EF hand," "PLC-X
domain," "PLC-Y domain," or a "C2 domain" in a 16816 or 16839
protein sequence, and make the determination that a polypeptide or
protein of interest has a particular profile, the amino acid
sequence of the protein can be searched against a database of HMMs
(e.g., the Pfam database, release 2.1) using the default
parameters. For example, the hmmsf program, which is available as
part of the HMMER package of search programs, is a family specific
default program for MILPAT0063 and a score of 15 is the default
threshold score for determining a hit. Alternatively, the threshold
score for determining a hit can be lowered (e.g., to 8 bits). A
description of the Pfam database can be found in Sonhammer et al.
(1997) Proteins 28(3):405-420 and a detailed description of HMMs
can be found, for example, in Gribskov et al. (1990) Meth. Enzymol.
183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA
84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and
Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which
are incorporated herein by reference.
[0299] A search was performed against the HMM database resulting in
the identification of a "PH domain" in the amino acid sequence of
human 16816 at about residues 17-124 of SEQ ID NO: 40. The
identified PH domain consensus sequence of 16816 is set forth in
SEQ ID NO: 45. This search also resulted in the identification of
"EF domain(s)" in the amino acid sequence of human 16816 or 16839
at about residues 138-166 and 172-202 of SEQ ID NO: 40 or amino
acids 39-67 of SEQ ID NO: 43 respectively. The identified EF domain
consensus sequences of 16816 are set forth in SEQ ID NOS: 46 and 47
and the identified EF domain consensus sequence of 16839 is set
forth in SEQ ID NO: 56. This search also resulted in the
identification of a "C2 domain" in the amino acid sequence of human
16816 or 16839 at about residues 629-719 of SEQ ID NO: 40 and
residues 484-572 of SEQ ID NO: 43, respectively. The identified C2
domain consensus sequence of 16816 is set forth in SEQ ID NO: 50
and the identified C2 domain consensus sequence of 16839 is set
forth in SEQ ID NO: 59. This search also resulted in the
identification of a "PLC-Y domain" in the amino acid sequence of
human 16816 or 16839 at about residues 492-609 of SEQ ID NO: 40 and
residues 348-465 of SEQ ID NO: 43. The identified PLC-Y domain
consensus sequence of 16816 is set forth in SEQ ID NO: 49 and the
identified PLC-Y domain consensus sequence of 16839 is set forth in
SEQ ID NO: 58. This search additionally resulted in the
identification of a "PLC-X domain" in the amino acid sequence of
human 16816 or 16839 at about residues 291-436 of SEQ ID NO: 40 and
residues 156-300 of SEQ ID NO: 43. The identified PLC-X domain
consensus sequence of 16816 is set forth in SEQ ID NO: 48 and the
identified PLC-X domain consensus sequence of 16839 is set forth in
SEQ ID NO: 57.
[0300] To identify the presence of a "phospholipase C" domain in a
16816 or 16839 protein sequence, and make the determination that a
polypeptide or protein of interest has a particular profile, the
amino acid sequence of the protein can be searched against a
database of domains, e.g., the ProDom database (Corpet et al.
(1999), Nucl. Acids Res. 27:263-267). The ProDom protein domain
database consists of an automatic compilation of homologous
domains. Current versions of ProDom are built using recursive
PSI-BLAST searches (Altschul S F et al. (1997) Nucleic Acids Res.
25:3389-3402; Gouzy et al. (1999) Computers and Chemistry
23:333-340) of the SWISS-PROT 38 and TREMBL protein databases. The
database automatically generates a consensus sequence for each
domain. A BLAST search was performed against the HMM database
resulting in the identification of a "phospholipase C" domain in
the amino acid sequence of human 16816 or 16839.
[0301] The phospholipase C domain is homologous to ProDom family
PD001214 ("phospholipase phosphodiesterase hydrolase
phosphoinositide-specific 1-phosphatidylinositol-45-bisphosphate
degradation transducer lipid beta" SEQ ID NO: 51, ProDomain Release
2001.1). An alignment of the phospholipase C domain (amino acids
275 to 436 of SEQ ID NO: 40) of human 16816 with a consensus amino
acid sequence (SEQ ID NO: 51) derived from a hidden Markov model
shows that the consensus sequence for SEQ ID NO: 51 is 54%
identical over amino acids 275 to 436 of SEQ ID NO: 40.
[0302] The phospholipase C domain is also homologous to ProDom
family PD186804 ("phospholipase C delta calcium-binding PLC-III
hydrolase phosphodiesterase lipid PLC-delta-1
1-phosphatidylinositol-45-bisphosphat- e" SEQ ID NO: 52, ProDomain
Release 2001.1. An alignment of the phospholipase C domain (amino
acids 1 to 191 of SEQ ID NO: 40) of human 16816 with a consensus
amino acid sequence (SEQ ID NO: 52) derived from a hidden Markov
model shows that the consensus sequence for SEQ ID NO: 52 is 44%
identical over amino acids 1 to 191 of SEQ ID NO: 40.
[0303] The phospholipase C domain is also homologous to ProDom
family PD001202 ("phospholipase phosphodiesterase hydrolase
phosphoinositide-specific 1-phosphatidylinositol-45-bisphosphate
degradation lipid transducer beta" SEQ ID NOS: 53 and 61, ProDomain
Release 2001.1. An alignment of the phospholipase C domain (amino
acids 491 to 608 of SEQ ID NO: 40) of human 16816 with a consensus
amino acid sequence (SEQ ID NO: 53) derived from a hidden Markov
model demonstrates that the consensus sequence for SEQ ID NO: 53 is
57% identical over amino acids 491 to 608 of SEQ ID NO: 40. An
alignment of the phospholipase C domain (amino acids 350 to 473 of
SEQ ID NO: 43) of human 16839 with a consensus amino acid sequence
(SEQ ID NO: 61) derived from a hidden Markov model demonstrates
that the consensus sequence for SEQ ID NO: 61 is 47% identical over
amino acids 350 to 473 of SEQ ID NO: 43.
[0304] The phospholipase C domain is also homologous to ProDom
family PD033204 ("C phospholipase delta-4 delta4 phospholipase" SEQ
ID NO: 54, ProDomain Release 2001.1. An alignment of the
phospholipase C domain (amino acids 722 to 761 of SEQ ID NO: 40) of
human 16816 with a consensus amino acid sequence (SEQ ID NO: 54)
derived from a hidden Markov model demonstrates that the consensus
sequence for SEQ ID NO: 54 is 70% identical over amino acids 722 to
761 of SEQ ID NO: 40.
[0305] The phospholipase C domain is also homologous to ProDom
family PD270355 ("1-phosphatidylinositol-4 phosphodiesterase-like
bisphosphate" SEQ ID NO: 55, ProDomain Release 2001.1. An alignment
of the phospholipase C domain (amino acids 562 to 621 of SEQ ID NO:
40) of human 16816 with a consensus amino acid sequence (SEQ ID NO:
55) derived from a hidden Markov model demonstrates that the
consensus sequence for SEQ ID NO: 55 is 46% identical over amino
acids 562 to 621 of SEQ ID NO: 40.
[0306] The phospholipase C domain is also homologous to ProDom
family PD001214 ("phospholipase phosphodiesterase hydrolase
phosphoinositide-specific 1-phosphatidylinositol-45-bisphosphate
degradation transducer lipid beta" SEQ ID NO: 60, ProDomain Release
2001.1. An alignment of the phospholipase C domain (amino acids 140
to 324 of SEQ ID NO: 43) of human 16839 with a consensus amino acid
sequence (SEQ ID NO: 60) derived from a hidden Markov model shows
that the consensus sequence for SEQ ID NO: 60 is 48% identical over
amino acids 140 to 324 of SEQ ID NO: 43.
[0307] As the 16816 or 16839 polypeptide of the invention may
modulate 16816 or 16839-mediated activity, they may be useful as of
for developing novel diagnostic and therapeutic agents for 16816 or
16839-mediated or related disorders, as described below. As used
herein, a "16816 or 16839 activity", "biological activity of 16816
or 16839" or "functional activity of 16816 or 16839", refers to an
activity exerted by a 16816 or 16839 protein, polypeptide or
nucleic acid molecule on e.g., a 16816 or 16839-responsive cell or
on a 16816 or 16839 substrate, e.g., a protein substrate, as
determined in vivo or in vitro. In one embodiment, a 16816 or 16839
activity is a direct activity, such as an association with a 16816
or 16839 target molecule. A "target molecule" or "binding partner"
is a molecule with which a 16816 or 16839 protein binds or
interacts with in nature. In an exemplary embodiment, it is a
receptor, e.g., a tyrosine kinase receptor. In another embodiment,
16816 or 16839 can associate with a second messenger molecule such
as a specialized adaptor molecule; with inositol phosphates and
inositol lipids; membrane proteins; or with a guanine nucleotide
binding-regulatory protein (G-protein). A 16816 or 16839 activity
can also be an indirect activity, e.g., a cellular signaling
activity mediated by interaction of the 16816 or 16839 protein with
a receptor or another signaling molecule. For example, the 16816 or
16839 proteins of the present invention can have one or more of the
following activities: (1) transduction of transmembrane signals;
(2) lipid-metabolizing activity, e.g., 16816 or 16839 can catalyze
the hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (PIP2)
producing diacylglycerol and inositol 1,4,5-trisphosphate; (3) the
regulation of transmission of signals from cellular receptors such
as hormones such as serotonin, growth factors such as
platelet-derived growth factor (PDGF), fibroblast growth factor
(FGF), and nerve growth factor (NGF), neurotransmitters and
immunoglobulins; (4) modulation of cell proliferation; (5)
modulation of cell differentiation; (6) modulation of cell
migration; (7) modulation of fertilization; and (8) modulation of
hypertension.
[0308] Based on the above-described sequence similarities, the
16816 or 16839 molecules of the present invention are predicted to
have similar biological activities as members of the PLC family.
Members of the PLC family play a very important role in
transmembrane signal transduction. Extracellular signaling
molecules including hormones, growth factors, neurotransmitters,
and immunoglobulins bind to their respective cell surface receptors
and activate phospholipase-C. PLC molecules have many functions
including: glycogenolysis in liver cells, histamine secretion by
mast cells, serotonin release by blood platelets, aggregation by
blood platelets, insulin release by pancreatic islet cells,
epinephrine secretion by adrenal chromaffin cells, and smooth
muscle contraction. In general, biological systems that are
activated by receptor tyrosine kinase cause the activation of
phospholipase-C. The role of an activated PLC is to catalyze the
hydrolysis of phosphatidyl-inositol-4,5-bisphospha- te (PIP2), a
minor component of the plasma membrane, to produce diacylglycerol
and inositol 1,4,5-trisphosphate (IP3). Inositol trisphosphate
releases calcium from intracellular stores and increases the influx
of calcium from the extracellular fluid. The calcium ions directly
regulate target enzymes and indirectly affect other enzymes by
functioning as a second messenger and interacting with
calcium-binding proteins, such as troponin C and calmodulin. For
example, calcium ions regulate muscle contraction, glycogen
breakdown and exocytosis. Diacylglycerol, a product of the
hydrolysis by PLCs, acts as a second messenger by activating
protein kinase C. Activated protein kinase C phosphorylates a great
number of intracellular proteins at the serine and threonine
residues and modulates different signaling pathways. For example,
the phosphorylation of glycogen synthase by protein kinase C stops
the synthesis of glycogen. Moreover, protein kinase C controls cell
division and proliferation. Both pathways are part of transmembrane
signal transduction mechanisms, which regulate cellular processes,
which include secretion, neural activity, metabolism,
differentiation and proliferation.
[0309] Both 16816 and 16839 proteins are homologous to the
phospholipase C molecule, PLC1. Stimulation of 16816 or 16839
activity is desirable in situations in which 16816 or 16839 is
abnormally downregulated and/or in which increased 16816 or 16839
activity is likely to have a beneficial effect. For example,
research on chromosome 20 q has associated this gene locus with
tumor suppressor activity. Deletions and mutations of the 20 q
chromosome have been associated with myelodysplasia and
myeloproliferative disorders. PLC1 is one of the genes present at
this locus, and has been found to deleted in these cases.
(Asimakopoulos et al. (1994) Blood 84(9):3086-94). As such, 16816
and 16839 may play a role in preventing or treating myeloid
disorders.
[0310] Likewise, inhibition of 16816 or 16839 activity is desirable
in situations in which 16816 or 16839 is abnormally upregulated
and/or in which decreased 16816 or 16839 activity is likely to have
a beneficial effect. It has been shown that PLC1 overexpression is
associated with hepatocellular carcinoma. One antibody, k-PLC1, was
shown to react with PLC1 (Wiedmann et al. (1987) Hepatology
7(3):543-50). As such, inhibitors such as 16816- or 16839-specific
antibodies may be useful to reduce the quantity of PLC1 in such
situations, and potentially decrease the severity and/or occurrence
of hepatocellular carcinoma. Thus, the 16816 or 16839 molecules can
act as novel diagnostic targets and therapeutic agents for
controlling disorders caused by abnormal or aberrant PLC activity.
Evidence indicates that a high percentage of primary human mammary
carcinomas concomitantly show abnormally high levels of PLC-gamma-1
(Kassis et al., Clin Cancer Res., August; 5(8):2251-60, 1999).
Likewise, studies on spontaneous hypertensive rats have suggested
that one of the main causes for the hypertension is an abnormal
activation of PLC-delta-1 resulting from point mutations in the X
and Y regions of the PLC amino acid sequence (Sanada et al.,
Hypertension 33(4):1036-42, 1999). Therefore, the 16816 or 16839
molecules can act as novel diagnostic targets and therapeutic
agents for controlling one or more of cellular proliferative and/or
differentiative disorders, disorders associated with bone
metabolism, immune disorders, hematopoietic disorders,
cardiovascular disorders, liver disorders, viral diseases, pain or
metabolic disorders.
[0311] As the 16816 or 16839 polypeptides of the invention may
modulate 16816 or 16839-mediated activities, they may be useful for
developing novel diagnostic and therapeutic agents for 16816 or
16839-mediated or related disorders, as described below.
[0312] Accordingly, 16816 or 16839 protein may mediate various
disorders, including cellular proliferative and/or differentiative
disorders, brain disorders, heart disorders, blood vessel
disorders, and platelet disorders.
[0313] Identification and Characterization of Human 16816 or 16839
cDNAs
[0314] The human 16816 or 16839 sequence (SEQ ID NO: 39 or SEQ ID
NO: 42), which is approximately 2629 or 2171 nucleotides long,
respectively, including untranslated regions, contains a predicted
methionine-initiated coding sequence of about 2289 or 1827
nucleotides, respectively (nucleotides 257-2545 of SEQ ID NO: 39;
SEQ ID NO: 41; or nucleotides 232-2058 of SEQ ID NO: 42; SEQ ID NO:
44). The coding sequence encodes a 762 or 608 amino acid protein
(SEQ ID NO: 40 or SEQ ID NO: 43), respectively.
[0315] Gene Expression Analysis
[0316] Total RNA was prepared from various human tissues by a
single step extraction method using RNA STAT-60 according to the
manufacturer's instructions (TelTest, Inc). Each RNA preparation
was treated with DNase I (Ambion) at 37.degree. C. for 1 hour.
DNAse I treatment was determined to be complete if the sample
required at least 38 PCR amplification cycles to reach a threshold
level of fluorescence using .beta.-2 microglobulin as an internal
amplicon reference. The integrity of the RNA samples following
DNase I treatment was confirmed by agarose gel electrophoresis and
ethidium bromide staining. After phenol extraction cDNA was
prepared from the sample using the SUPERSCRIPT.TM. Choice System
following the manufacturer's instructions (GibcoBRL). A negative
control of RNA without reverse transcriptase was mock reverse
transcribed for each RNA sample.
[0317] Human 16816 or 16839 expression was measured by TaqMan.RTM.
quantitative PCR (Perkin Elmer Applied Biosystems) in cDNA prepared
from a variety of normal and diseased (e.g., cancerous) human
tissues or cell lines. Probes were designed by PrimerExpress
software (PE Biosystems) based on the sequence of the human 16816
or 16839 gene. Each human 16816 or 16839 gene probe was labeled
using FAM (6-carboxyfluorescein), and the .beta.2-microglobulin
reference probe was labeled with a different fluorescent dye, VIC.
The differential labeling of the target gene and internal reference
gene thus enabled measurement in same well. Forward and reverse
primers and the probes for both .beta.2-microglobulin and target
gene were added to the TaqMan.RTM. Universal PCR Master Mix (PE
Applied Biosystems). Although the final concentration of primer and
probe could vary, each was internally consistent within a given
experiment. A typical experiment contained 200 nM of forward and
reverse primers plus 100 nM probe for .beta.-2 microglobulin and
600 nM forward and reverse primers plus 200 nM probe for the target
gene. TaqMan matrix experiments were carried out on an ABI PRISM
7700 Sequence Detection System (PE Applied Biosystems). The thermal
cycler conditions were as follows: hold for 2 min at 50.degree. C.
and 10 min at 95.degree. C., followed by two-step PCR for 40 cycles
of 95.degree. C. for 15 sec followed by 60.degree. C. for 1
min.
[0318] The following method was used to quantitatively calculate
human 16816 or 16839 gene expression in the various tissues
relative to .beta.-2 microglobulin expression in the same tissue.
The threshold cycle (Ct) value is defined as the cycle at which a
statistically significant increase in fluorescence is detected. A
lower Ct value is indicative of a higher mRNA concentration. The Ct
value of the human 16816 or 16839 gene is normalized by subtracting
the Ct value of the .beta.-2 microglobulin gene to obtain a
.DELTA.Ct value using the following formula: .DELTA.Ct=Ct.sub.human
59914 and 59921-Ct.sub..beta.-2 microglobulin. Expression is then
calibrated against a cDNA sample showing a comparatively low level
of expression of the human 16816 or 16839 gene. The .DELTA.Ct value
for the calibrator sample is then subtracted from .DELTA.Ct for
each tissue sample according to the following formula:
.DELTA..DELTA.Ct=.DELTA.Ct-.sub.sample-.DELTA.Ct-.sub.calibrator.
Relative expression is then calculated using the arithmetic formula
given by 2-.DELTA..DELTA.Ct. Expression of the target human 16816
or 16839 gene in each of the tissues tested is then graphically
represented as discussed in more detail below.
[0319] TaqMan real-time quantitative RT-PCR is used to detect the
presence of RNA transcript corresponding to human 16816 relative to
a no template control in a panel of human tissues or cells. It is
found that the highest expression of 16816 orthologs are expressed
in skeletal muscle tissue as shown in Table 7.
9TABLE 7 Tissue Type mean 16816 mean .beta.eta 2 .differential.Ct
expression Aorta/normal 38.87 23.105 15.765 0 Fetal heart/normal
33.43 20.82 12.61 0.159960132 Heart normal 34.62 18.985 15.635
0.019651514 Heart/CHF 35.64 20.47 15.17 0 Vein/Normal 33.81 19.15
14.66 0.038627826 Spinal cord/Normal 33.175 19.43 13.745
0.072835434 Brain cortex/Normal 30.42 21.2 9.22 1.676885618 Brain
hypothalamus/ 30.415 20.155 10.26 0.815515546 Normal Glial cells
(Astro- 33.495 21.58 11.915 0.258956968 cytes) Brain/Glioblastoma
29.77 18.41 11.36 0.380451455 Breast/Normal 34.145 19.195 14.95
0.031593778 Breast tumor/IDC 33.215 18.82 14.395 0.046416588
OVARY/Normal 35.47 21.23 14.24 0 OVARY/Tumor 37.815 19.785 18.03 0
Pancreas 35.73 18.125 17.605 0 Prostate/Normal 30.895 19.255 11.64
0.313336401 Prostate/Tumor 31.695 18.255 13.44 0.089982252
Colon/normal 31.035 17.45 13.585 0.081378093 Colon/tumor 36.88
18.84 18.04 0 Colon/IBD 35.185 18.445 16.74 0 Kidney/normal 30.255
21.005 9.25 1.642375811 Liver/normal 36.48 19.18 17.3 0 Liver
fibrosis 35.16 20.015 15.145 0 Fetal Liver/normal 37.02 22.14 14.88
0 Lung/normal 33.93 18.02 15.91 0.016241 Lung/tumor 34.845 18.955
15.89 0.016467716 Lung/COPD 33.345 18.13 15.215 0.026292302
Spleen/normal 39.23 20.19 19.04 0 Tonsil/normal 35.72 17.955 17.765
0 Lymph node/normal 34.55 18.485 16.065 0.014586568 Thymus/normal
33.475 19.675 13.8 0.070110984 Epithelial Cells 31.825 20.57 11.255
0.409173406 (prostate) Endothelial Cells 32.815 20.775 12.04
0.237464587 (aortic) Skeletal Muscle/ 24.19 19.34 4.85 34.674046
Normal Fibroblasts (Dermal) 31.89 18.915 12.975 0.124204064
Skin/normal 34.805 20.945 13.86 0.067254951 Adipose/Normal 32.94
18.88 14.06 0.058548835 Osteoblasts (pri- 32.595 20.175 12.42
0.182476715 mary) Osteoblasts (Undiff) 31.05 19.025 12.025
0.239946435 Osteoblasts (Diff) 30.815 18.315 12.5 0.172633492
Osteoclasts 34.17 17.725 16.445 0.011208867 Aortic SMC Early 34.705
20.23 14.475 0.043912768 Aortic SMC Late 34.18 22.97 11.21
0.42213732 shear HUVEC 31.315 20.955 10.36 0.76090291 static HUVEC
31.465 20.875 10.59 0.64877237
[0320] TaqMan real-time quantitative RT-PCR is used to detect the
presence of RNA transcript corresponding to human 16839 relative to
a no template control in a panel of human tissues or cells. It is
found that 16839 orthologs are expressed in teste as shown in the
Table 8 and DRG as shown in Table 9.
10TABLE 8 Tissue Type 16839.20 .beta.2.803 .differential.Ct
Expression Adrenal Gland 40.00 19.21 20.80 0.00 Brain 40.00 21.01
18.99 0.00 Heart 40.00 18.97 21.04 0.00 Kidney 39.90 18.98 20.92
0.00 Liver 40.00 18.61 21.40 0.00 Lung 40.00 17.12 22.88 0.00
Mammary Gland 40.00 17.94 22.07 0.00 Pancreas 40.00 20.50 19.50
0.00 Placenta 40.00 17.83 22.18 0.00 Prostate 40.00 17.20 22.80
0.00 Salivary Gland 40.00 18.48 21.52 0.00 Muscle 40.00 20.56 19.45
0.00 Sm. Intestine 40.00 18.82 21.19 0.00 Spleen 40.00 16.64 23.36
0.00 Stomach 40.00 18.52 21.48 0.00 Teste 26.97 20.05 6.92 1.69
Thymus 40.00 17.66 22.35 0.00 Trachea 40.00 18.93 21.07 0.00 Uterus
40.00 18.80 21.20 0.00 Spinal Cord 40.00 18.94 21.07 0.00 DRG 40.00
19.50 20.51 0.00 Skin 39.10 18.44 20.66 0.00
[0321]
11TABLE 9 Tissue Type Mean .beta. 2 Mean
.differential..differential. Ct Expression Artery normal 40 19.38
20.62 0 Vein normal 39.38 18.47 20.91 0 Aortic SMC EARLY 38.88
19.23 19.64 0 Coronary SMC 39.92 20.55 19.38 0 Static HUVEC 39.06
18.66 20.4 0 Heart normal 40 17.21 22.79 0 Heart CHF 39.26 17.18
22.07 0 Kidney 34.14 17.21 16.93 0.008 Skeletal Muscle 40 20.26
19.74 0 Adipose normal 40 18.7 21.31 0 Pancreas 39.41 19.23 20.18 0
primary osteoblasts 37.65 17.54 20.11 0 Osteoclasts (diff) 35.13
15.82 19.31 0 Skin normal 39.13 19.16 19.98 0 Spinal cord normal 40
18.59 21.41 0 Brain Cortex normal 38.95 18.59 20.36 0 Brain
Hypothalamus normal 40 19.43 20.57 0 Nerve 40 22.34 17.66 0 DRG
(Dorsal Root Ganglion) 34.58 19.82 14.76 0.0362 Glial Cells
(Astrocytes) 38.84 20.34 18.5 0 Glioblastoma 38.91 16.52 22.39 0
Breast normal 39.6 18.93 20.67 0 Breast tumor 39.98 17.11 22.88 0
Ovary normal 38.79 18.03 20.76 0 Ovary Tumor 40 18.23 21.77 0
Prostate Normal 38.15 18.3 19.85 0 Prostate Tumor 38.36 16.03 22.33
0 Epithelial Cells (Prostate) 39.45 19.29 20.16 0 Colon normal
39.44 16.6 22.84 0 Colon Tumor 38.73 16.22 22.51 0 Lung normal
38.99 16.12 22.87 0 Lung tumor 37.2 16.58 20.63 0 Lung COPD 38.73
16.72 22 0 Colon IBD 38.3 15.77 22.54 0 Liver normal 39.88 17.98
21.91 0 Liver fibrosis 37.62 19.28 18.34 0 Dermal Cells-fibroblasts
37.84 17.66 20.18 0 Spleen normal 37.22 18.06 19.16 0 Tonsil normal
37.55 15.44 22.11 0 Lymph node 39.7 17.23 22.47 0 Resting PBMC 40
18.8 21.2 0 Skin-Decubitus 36.45 18.82 17.64 0 Synovium 37.98 17.66
20.32 0 BM-MNC 38.51 14.83 23.68 0 (Bone marrow mononuclear cells)
Activated PBMC 37.06 13.93 23.13 0 Shear HUVEC 38 17.99 20.01
0.0009
[0322] Expression of 16839 was also detected in a panel of tissues
and liver cell lines as shown in Table 10.
12TABLE 10 Tissue Type Mean 18S Mean .differential..differential.
Ct Expression PIT 278/Heart 33.06 13.77 19.29 0.0016 PIT 351/Kidney
30.36 12.56 17.81 0.0044 PIT 915/Skeletal Muscle 38.28 11.87 26.41
0 NDR 63/Liver 33.23 11.69 21.54 0.0003 NDR 242/Liver 34.26 12.63
21.63 0.0003 PIT 260/Liver 33.58 11.77 21.81 0.0003 CHT 756/Liver
31.77 11.65 20.13 0.0009 MPI 155/Liver 34.91 14.68 20.23 0.0008 MPI
146/Liver 30.91 11.95 18.97 0.002 CHT 902/Liver 35.72 12.49 23.22 0
PIT 45/Liver 33.97 12.14 21.84 0.0003 PIT 292/Liver 35.46 13.65
21.82 0 CLN 784/Liver 32.56 11.76 20.8 0.0005 NDR 752/Liver 31.09
11.04 20.06 0.0009 CHT 1679/Liver 35.38 11.7 23.68 0 CHT 1420/Liver
33.06 11.15 21.91 0.0003 CHT 339/Liver 35.68 11.23 24.45 0 CHT
1237/Liver 33.41 11.02 22.4 0.0002
[0323] Expression of 16839 was also detected in a panel of breast
tumor cell lines as shown in Table 11.
13TABLE 11 Tissue Type Mean 16839.4 .beta. 2 Mean
.differential..differential. Ct Expression MCF10MS 35.1 18.84 16.25
0 MCF10A 33.88 18.81 15.07 0.03 MCF10AT.cl1 34.2 18.88 15.32 0.02
MCF10AT.cl3 35.19 18.22 16.98 0 MCF10AT1 36.74 19.19 17.56 0
MCF10AT3B 36.23 18.93 17.3 0 MCF10CA1a.cl1 36.94 16.34 20.59 0
MCF10AT3B Agar 37.76 25.07 12.68 0 MCF10CA1a.cl1 37.93 23.13 14.8 0
Agar MCF10A.m25 Plastic 32.39 23.05 9.35 1.54 MCF10CA Agar 33.63
20.71 12.93 0.13 MCF10CA Plastic 35.98 20.56 15.43 0 MCF3B Agar
37.34 21 16.34 0 MCF3B Plastic 32.67 20.54 12.14 0.22 MCF10A EGF 0
hr 28.23 16.57 11.66 0.31 MCF10A EGF 0.5 hr 28.84 16.61 12.23 0.21
MCF10A EGF 1 hr 28.68 16.79 11.9 0.26 MCF10A EGF 2 hr 29.54 16.88
12.66 0.15 MCF10A EGF 4 hr 31.21 16.97 14.24 0.05 MCF10A EGF 8 hr
31.16 16.65 14.51 0.04 MCF10A IGF1A 0 hr 30.54 20.41 10.13 0.90
MCF10A IGF1A 30.89 21.36 9.52 1.36 0.5 hr MCF10A IGF1A 1 hr 29.7
20.84 8.86 2.15 MCF10A IGF1A 3 hr 30.14 21.09 9.05 1.89 MCF10A
IGF1A 24 hr 28.53 20.43 8.1 3.66 MCF10AT3B.cl5 36.57 20.54 16.03 0
Plastic MCF10AT3B.cl6 38.18 20.7 17.48 0 Plastic MCF10AT3B.cl3
39.13 20.8 18.34 0 Plastic MCF10AT3B.cl1 35.92 21.07 14.85 0
Plastic MCF10AT3B.cl4 38.1 20.95 17.15 0 Plastic MCF10AT3B.cl2
36.49 20.75 15.74 0 Plastic MCF10AT3B.cl5 39.22 22.68 16.54 0 Agar
MCF10AT3B.cl6 35.7 23.13 12.57 0 Agar MCF-7 37.46 22.25 15.22 0
ZR--75 37.54 21.25 16.3 0 T47D 37.19 20.72 16.47 0 MDA-231 33.55
19.7 13.85 0.07 MDA-435 34.73 19.39 15.35 0.02 SkBr3 37.16 19.86
17.3 0 Hs578Bst 36.52 18.93 17.59 0 Hs578T 31.36 19 12.35 0.19
[0324] Expression of 16839 was also detected in an oncology phase
panel as shown in Table 12 and shows highest relative expression in
lung tumor (CHT 832 lung T-PDNCSCCL) and upregulation of 16839 was
found in {fraction (3/7)} lung tumor tissue or cell samples.
14TABLE 12 Tissue Type Mean 16839.4 .beta. 2 Mean
.differential..differential. Ct Expression PIT 400 Breast N 35.4
19.18 16.23 0 PIT 372 Breast N 35.38 19.66 15.72 0 CHT 558 Breast N
36.38 19.02 17.36 0 CLN 168 Breast T: 38.45 19.84 18.62 0 IDC MDA
304 Breast T: 37.04 17.84 19.2 0 MD-IDC NDR 58 Breast T: IDC 38.83
17.84 20.99 0 NDR 05 Breast T: IDC 35.22 20.57 14.65 0 CHT 562
Breast T: 34.08 18.71 15.37 0.02 IDC NDR 12 Breast T 35.52 21.39
14.13 0 PIT 208 Ovary N 37.06 18.56 18.5 0 CHT 620 Ovary N 36.05
19.2 16.85 0 CLN 03 Ovary T 40 19.83 20.17 0 CLN 17 Ovary T 39.49
20 19.5 0 MDA 25 Ovary T 38.42 21.65 16.78 0 MDA 216 Ovary T 38.04
20.11 17.93 0 CLN 012 Ovary T 39.27 21.3 17.98 0 MDA 185 Lung N 40
19.43 20.57 0 CLN 930 Lung N 37.99 20.29 17.7 0 MDA 183 Lung N
39.65 18.11 21.54 0 MPI 215 Lung T-- 31.34 18.62 12.72 0.15 SmC MDA
259 Lung T- 37.42 19.93 17.49 0 PDNSCCL CHT 832 Lung T- 28.18 18.86
9.32 1.56 PDNSCCL MDA 253 Lung T- 33.39 18.05 15.35 0.02 PDNSCC MDA
262 Lung T- 38.79 22.28 16.51 0 SCC CHT 211 Lung T-AC 39.1 19.29
19.82 0 CHT 793 Lung T- 35.74 18.55 17.19 0 ACA CHT 396 Colon N
39.82 17.7 22.13 0 CHT 523 Colon N 39.99 18.52 21.47 0 CHT 452
Colon N 40 17.5 22.5 0 CHT 382 Colon T: 38.42 17.97 20.45 0 MD CHT
528 Colon T: 34.85 17.54 17.32 0.01 MD CLN 609 Colon T 38.1 18.82
19.29 0 CHT 372 Colon T: 37.83 19.24 18.59 0 MD-PD CHT 340
Colon-Liver 37.93 19.99 17.94 0 Met NDR 100 Colon-Liver 36.52 18.26
18.27 0 Met PIT 260 Liver N 35.31 16.93 18.37 0 (female) ONC 102
Hemangio- 36.36 19.04 17.32 0 ma A24 HMVEC-Arr 35.31 18.75 16.55 0
C48 HMVEC-Prol 35.12 19.18 15.94 0
[0325] Human 49937, 49931 and 49933
[0326] The present invention is based, at least in part, on the
discovery of novel calcium transporter family members, referred to
interchangeably herein as "P-type ATPase", "E1-E2 ATPase", "human
E1-E2 ATPase", or "HEAT" nucleic acid and protein molecules (e.g.,
HEAT-1 (49937), HEAT-2 (49931) and HEAT-3(49933)). These novel
molecules are members of the E1-E2 ATPase superfamily and are
highly expressed in human vessels, endothelial cells, and vascular
smooth muscle cells, e.g., coronary vascular smooth muscle
cells.
[0327] The E1-E2 ATPase family is a large superfamily of cation
transport enzymes that contains at least 80 members found in
diverse organisms such as bacteria, archaea, and eukaryotes
(Palmgren, M. G. and Axelsen, K. B. (1998) Biochim. Biophys. Acta.
1365:37-45). These enzymes are involved in ATP hydrolysis-dependent
transmembrane movement of a variety of inorganic cations (e.g.,
H.sup.+, Na.sup.+, K.sup.+, Ca.sup.2+, Cu.sup.2+, Cd.sup.+, and
Mg.sup.2+ ions) across a concentration gradient, whereby the enzyme
converts the free energy of ATP hydrolysis into electrochemical ion
gradients. E1-E2 ATPases are also known as "P-type" ATPases,
referring to the existence of a covalent high-energy
phosphoryl-enzyme intermediate in the chemical reaction pathway of
these transporters. The superfamily contains four major groups:
Ca.sup.2+ transporting ATPases; Na.sup.+/K.sup.+- and gastric
H.sup.+/K.sup.+ transporting ATPases; plasma membrane H.sup.+
transporting ATPases of plants, fungi, and lower eukaryotes; and
all bacterial P-type ATPases (Kuhlbrandt et al. (1998) Curr. Opin.
Struct. Biol. 8:510-516).
[0328] The E1-E2 ATPases are involved in ATP hydrolysis-dependent
transmembrane movement of inorganic cations (e.g., Ca.sup.2+ ions)
across a concentration gradient. E1-E2 ATPases are phosphorylated
at a highly conserved DKTG sequence. Phosphorylation at this site
is thought to control the enzyme's substrate affinity. Most E1-E2
ATPases contain ten alpha-helical transmembrane domains, although
additional domains may be present. Members of the E1-E2 ATPase
superfamily are able to generate electrochemical ion gradients
which enable a variety of processes in the cell such as absorption,
secretion, transmembrane signaling, nerve impulse transmission,
excitation/contraction coupling, and growth and differentiation
(Scarborough (1999) Curr. Opin. Cell Biol. 11:517-522).
[0329] As indicated in herein, the HEAT molecules of the present
invention, e.g., HEAT-2, are up-regulated during shear,
proliferation, and tube formation of endothelial cells and, thus,
are believed to be involved in angiogenesis. Calcium ions are
involved in the regulation of many cellular activities. In vascular
smooth muscle cells, transient increases in intracellular calcium
levels mediate contraction. Thus, maintenance of a low steady-state
level of calcium is critical to maintaining proper cell function.
Additionally, since the main determinant of the
contraction-relaxation cycle of smooth muscle is calcium, calcium
concentration is an important factor in the regulation of vascular
tone. The normal concentration of calcium in the cell is in the
submicromolar range, while the concentration in the extracellular
compartment is in the millimolar range. In order to maintain
intracellular calcium concentration in the submicromolar range,
several mechanisms are operative in most cells. In smooth muscle
cells, these regulatory mechanisms include calcium extrusion via
Ca.sup.2+-transporting E1-E2 ATPases at the plasma membrane and at
the sarcoplasmic/endoplasmic reticulum.
[0330] Thus, as the HEAT molecules of the present invention are
Ca.sup.2+-transporting E1-E2 ATPases, and are highly expressed in
vessels, endothelial cells, and vascular smooth muscle cells, these
molecules are believed to be involved in vasotone regulation of
vascular smooth muscle cells, e.g., coronary vascular smooth muscle
cells. For example, activation of a HEAT molecule of the invention,
e.g., HEAT-3, may result in decreased cytosolic calcium
concentrations, thus reducing vascular tone. Inhibition of a HEAT
molecule of the invention, e.g., HEAT-3, may result in decreased
intracellular calcium store, which may subsequently lower the
calcium release by vasopressor stimulation, thereby reducing
vascular smooth muscle tone.
[0331] Accordingly, the HEAT molecules of the present invention
provide novel diagnostic targets and therapeutic agents for
cardivascular disorders. The HEAT molecules of the present
invention further provide novel diagnostic targets and therapeutic
agents for cellular proliferation, growth, or differentiation
disorders. Additional disorders that may be treated using the
molecules of the present invention include disorders affecting
tissues in which HEAT protein is expressed (e.g., vessels,
endothelial cells, and vascular smooth muscle cells).
[0332] The family of HEAT proteins of the present invention
comprises at least one "transmembrane domain," preferably at least
2, 3, or 4 transmembrane domains, more preferably 5, 6, 7, 8, or 9
transmembrane domains, even more preferably 10 or 11 transmembrane
domains, and most preferably, 12 transmembrane domains. As used
herein, the term "transmembrane domain" includes an amino acid
sequence of about 15 amino acid residues in length which spans the
plasma membrane. More preferably, a transmembrane domain includes
about at least 20, 25, 30, 35, 40, or 45 amino acid residues and
spans the plasma membrane. Transmembrane domains are rich in
hydrophobic residues, and typically have an alpha-helical
structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%,
90%, 95% or more of the amino acids of a transmembrane domain are
hydrophobic, e.g., leucines, isoleucines, tyrosines, or
tryptophans. Transmembrane domains are described in, for example,
Zagotta, W. N. et al. (1996) Annu. Rev. Neurosci. 19:235-263, the
contents of which are incorporated herein by reference. Amino acid
residues 8-25, 47-65, 231-253, 256-276, 428-448, 464-484, 936-954,
963-987, 994-1015, 1049-1065, 1079-1102, and 1118-1134 of the human
HEAT-1 protein (SEQ ID NO: 64) are predicted to comprise
transmembrane domains. Amino acid residues 29-50, 211-227, 234-253,
294-317, 410-434, 449-469, 941-960, 968-985, 1000-1020, 1076-1092,
1105-1129, 1144-1160 of the human HEAT-2 protein (SEQ ID NO: 68)
are predicted to comprise transmembrane domains. Amino acid
residues 65-89, 99-116, 242-258, 265-281, 445-464, 493-509,
990-1007, 1015-1031, 1049-1073, 1103-1119, 1134-1151, 1171-1187 of
the human HEAT-3 protein (SEQ ID NO: 71) are also predicted to
comprise transmembrane domains.
[0333] In another embodiment, members of the HEAT family of
proteins include at least one "E1-E2 ATPase domain" in the protein
or corresponding nucleic acid molecule. As used herein, the term
"E1-E2 ATPase" domain includes a protein domain having at least
about 70-110 amino acid residues and a bit score of at least 30
when compared against an E1-E2 ATPase Hidden Markov Model (HMM),
e.g., PFAM Accession Number PF00122. Preferably, an E1-E2 ATPase
domain includes a protein having an amino acid sequence of about
80-100, or more preferably about 87, 89, or 90 amino acid residues,
and a bit score of at least 35, 40, 50, or more preferably, 37.0,
51.4, or 53.4. To identify the presence of an E1-E2 ATPase domain
in a HEAT protein, and make the determination that a protein of
interest has a particular profile, the amino acid sequence of the
protein is searched against a database of known protein motifs
and/or domains (e.g., the HMM database). The E1-E2 ATPase domain
(HMM) has been assigned the PFAM Accession number PF00122 (see the
PFAM website, available online through Washington University in
Saint Louis). A search was performed against the HMM database
resulting in the identification of an E1-E2 ATPase domain in the
amino acid sequence of human HEAT-1 at about residues 299-387 of
SEQ ID NO: 64. A search was also performed against the HMM database
resulting in the identification of an E1-E2 ATPase domain in the
amino acid sequence of human HEAT-2 at about residues 278-365 of
SEQ ID NO: 68. A search was further performed against the HMM
database resulting in the identification of an E1-E2 ATPase domain
in the amino acid sequence of human HEAT-3 at about residues
302-392 of SEQ ID NO: 71.
[0334] A description of the Pfam database can be found in Sonhammer
et al. (1997) Proteins 28:405-420, and a detailed description of
HMMs can be found, for example, in Gribskov et al. (1990) Methods
Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci.
USA 84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531;
and Stultz et al.(1993) Protein Sci. 2:305-314, the contents of
which are incorporated herein by reference.
[0335] Preferably an E1-E2 ATPase domain is at least about 70-110
amino acid residues and has an "E1-E2 ATPase activity", for
example, the ability to interact with a HEAT substrate or target
molecule (e.g., ATP or a cation such as Ca.sup.2+); to transport a
HEAT substrate or target molecule (e.g., a cation such as
Ca.sup.2+) from one side of a biological membrane to the other; to
adopt an E1 conformation or an E2 conformation; to convert a HEAT
substrate or target molecule to a product (e.g., to hydrolyze ATP);
to interact with a second non-HEAT protein; to modulate intra- or
inter-cellular signaling and/or gene transcription (e.g., either
directly or indirectly); to modulate vascular smooth muscle tone;
to modulate cellular growth and/or proliferation; and/or to
modulate angiogenesis. Accordingly, identifying the presence of an
"E1-E2 ATPase domain" can include isolating a fragment of a HEAT
molecule (e.g., a HEAT polypeptide) and assaying for the ability of
the fragment to exhibit one of the aforementioned E1-E2 ATPase
domain activities.
[0336] In another embodiment, a HEAT molecule of the present
invention may also be identified based on its ability to adopt an
E1 conformation or an E2 conformation. As used herein, an "E1
conformation" of a HEAT protein includes a 3-dimensional
conformation of a HEAT protein which does not exhibit HEAT activity
(e.g., the ability to transport Ca.sup.2+), as defined herein. An
E1 conformation of a HEAT protein usually occurs when the HEAT
protein is unphosphorylated. As used herein, an "E2 conformation"
of a HEAT protein includes a 3-dimensional conformation of a HEAT
protein which exhibits HEAT activity (e.g., the ability to
transport c Ca.sup.2+), as defined herein. An E2 conformation of a
HEAT protein usually occurs when the HEAT protein is
phosphorylated.
[0337] In another embodiment, a HEAT protein of the present
invention is identified based on the presence of an "E1-E2 ATPases
phosphorylation site" in the protein or corresponding nucleic acid
molecule. An E1-E2 ATPases phosphorylation site functions in
accepting a phosphate moiety and has the following consensus
sequence: D-K-T-G-T-[LIVM]-[TI] (SEQ ID NO: 73), wherein D is
phosphorylated. The use of amino acids in brackets indicates that
the amino acid at the indicated position may be any one of the
amino acids within the brackets, e.g., [TI] indicates any of one of
either T (threonine) or I (isoleucine). The E1-E2 ATPases
phosphorylation site has been assigned ProSite Accession Number
PS00154. To identify the presence of an E1-E2 ATPases
phosphorylation site in a HEAT protein, and to make the
determination that a protein of interest has a particular profile,
the amino acid sequence of the protein may be searched against a
database of known protein domains (e.g., the ProSite database)
using the default parameters (available online through the Swiss
Institute for Bioinformatics). A search was performed against the
ProSite database resulting in the identification of an E1-E2
ATPases phosphorylation site in the amino acid sequence of human
HEAT-1 (SEQ ID NO: 64) at about residues 513-519. A similar search
resulted in the identification of an E1-E2 phosphorylation site in
the amino acid sequence of human HEAT-2 (SEQ ID NO: 68) at about
residues 498-504 and in the amino acid sequence of human HEAT-3
(SEQ ID NO: 71) at about residues 533-539.
[0338] Preferably an E1-E2 ATPase phosphorylation site has a
"phosphorylation site activity," for example, the ability to be
phosphorylated; to be dephosphorylated; to regulate the E1-E2
conformational change of the HEAT protein in which it is contained;
to regulate transport of Ca.sup.2+ across a biological membrane by
the HEAT protein in which it is contained; and/or to regulate the
activity (as defined herein) of the HEAT protein in which it is
contained. Accordingly, identifying the presence of an "E1-E2
ATPases phosphorylation site" can include isolating a fragment of a
HEAT molecule (e.g., a HEAT polypeptide) and assaying for the
ability of the fragment to exhibit one of the aforementioned
phosphorylation site activities.
[0339] The family of HEAT proteins of the present invention also
comprises at least one "large extramembrane domain" in the protein
or corresponding nucleic acid molecule. As used herein, a "large
extramembrane domain" includes a domain having greater than 20
amino acid residues that is found between transmembrane domains,
preferably on the cytoplasmic side of the plasma membrane, and does
not span or traverse the plasma membrane. A large extramembrane
domain preferably includes at least one, two, three, four or more
motifs or consensus sequences characteristic of P-type or E1-E2
ATPases, i.e., includes one, two, three, four, or more "P-type
ATPase consensus sequences or motifs". As used herein, the phrase
"P-type ATPase consensus sequences or motifs" includes any
consensus sequence or motif known in the art to be characteristic
of P-type ATPases, including, but not limited to, the P-type ATPase
sequence 1 motif (as defined herein), the P-type ATPase sequence 2
motif (as defined herein), the P-type ATPase sequence 3 motif (as
defined herein), and the E1-E2 ATPases phosphorylation site (as
defined herein).
[0340] In one embodiment, the family of HEAT proteins of the
present invention comprises at least one "N-terminal" large
extramembrane domain in the protein or corresponding nucleic acid
molecule. As used herein, an "N-terminal" large extramembrane
domain is found in the N-terminal 1/3.sup.rd of the protein,
preferably between the fourth and fifth transmembrane domains of a
HEAT protein, and includes about 50-270, 50-250, 60-230, 70-210,
80-190, 90-170, or preferably, 92, 151, or 163 amino acid residues.
In a preferred embodiment, an N-terminal large extramembrane domain
includes at least one P-type ATPase sequence 1 motif (as described
herein). An N-terminal large extramembrane domain was identified in
the amino acid sequence of human HEAT-1 at about residues 277-427
of SEQ ID NO: 64. An N-terminal large extramembrane domain was also
identified in the amino acid sequence of human HEAT-2 at about
residues 318-409 of SEQ ID NO: 68 and in the amino acid sequence of
human HEAT-3 at about residues 282-444 of SEQ ID NO: 71.
[0341] The family of HEAT proteins of the present invention also
comprises at least one "C-terminal" large extramembrane domain in
the protein or corresponding nucleic acid molecule. As used herein,
a "C-terminal" large extramembrane domain is found in the
C-terminal 2/3.sup.rds of the protein, preferably between the sixth
and seventh transmembrane domains of a HEAT protein and includes
about 340-590, 360-570, 380-550, 400-530, 420-510, 440-490, or
preferably, 451, 471, or 480 amino acid residues. In a preferred
embodiment, a C-terminal large extramembrane domain includes at
least one or more of the following motifs: a P-type ATPase sequence
2 motif (as described herein), a P-type ATPase sequence 3 motif (as
defined herein), and/or an E1-E2 ATPases phosphorylation site (as
defined herein). A C-terminal large extramembrane domain was
identified in the amino acid sequence of human HEAT-1 at about
residues 485-935 of SEQ ID NO: 64, in the amino acid sequence of
human HEAT-2 at about residues 470-940 of SEQ ID NO: 68, and in the
amino acid sequence of human HEAT-3 at about residues 510-989 of
SEQ ID NO: 71.
[0342] In another embodiment, a HEAT protein of the present
invention includes at least one "P-type ATPase sequence 1 motif" in
the protein or corresponding nucleic acid molecule. As used herein,
a "P-type ATPase sequence 1 motif" is a conserved sequence motif
diagnostic for P-type ATPases (Tang, X. et al. (1996) Science
272:1495-1497; Fagan, M. J. and Saier, M. H. (1994) J. Mol. Evol.
38:57). A P-type ATPase sequence 1 motif is involved in the
coupling of ATP hydrolysis with transport (e.g., transport of
Ca.sup.2+). The consensus sequence for a P-type ATPase sequence 1
motif is [DNS]-[QENR]-[SA]-[LIVSAN]-[LIV]-[TSN]-G-E-[SN] (SEQ ID
NO: 75). The use of amino acids in brackets indicates that the
amino acid at the indicated position may be any one of the amino
acids within the brackets, e.g., [SA] indicates any of one of
either S (serine) or A (alanine). In a preferred embodiment, a
P-type ATPase sequence 1 motif is contained within an N-terminal
large extramembrane domain. In another preferred embodiment, a
P-type ATPase sequence 1 motif in the HEAT proteins of the present
invention has at least 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid
resides which match the consensus sequence for a P-type ATPase
sequence 1 motif. A P-type ATPase sequence 1 motif was identified
in the amino acid sequence of human HEAT-1 at about residues
341-349 of SEQ ID NO: 64, in the amino acid sequence of human
HEAT-2 at about residues 318-326 of SEQ ID NO: 68, and in the amino
acid sequence of human HEAT-3 at about residues 348-356 of SEQ ID
NO: 71.
[0343] In another embodiment, a HEAT protein of the present
invention includes at least one "P-type ATPase sequence 2 motif" in
the protein or corresponding nucleic acid molecule. As used herein,
a "P-type ATPase sequence 2 motif" is a conserved sequence motif
diagnostic for P-type ATPases (Tang, X. et al. (1996) Science
272:1495-1497; Fagan, M. J. and Saier, M. H. (1994) J. Mol. Evol.
38:57). Preferably, a P-type ATPase sequence 2 motif overlaps with
and/or includes an E1-E2 ATPases phosphorylation site (as defined
herein). The consensus sequence for a P-type ATPase sequence 2
motif is [LIV]-[CAML]-[STFL]-D-K-T-G-T-[LI]-T (SEQ ID NO: 76). The
use of amino acids in brackets indicates that the amino acid at the
indicated position may be any one of the amino acids within the
brackets, e.g., [LI] indicates any of one of either L (leucine) or
I (isoleucine). In a preferred embodiment, a P-type ATPase sequence
2 motif is contained within a C-terminal large extramembrane
domain. In another preferred embodiment, a P-type ATPase sequence 2
motif in the HEAT proteins of the present invention has at least 1,
2, 3, 4, 5, 6, 7, 8, 9 or more amino acid resides which match the
consensus sequence for a P-type ATPase sequence 2 motif. A P-type
ATPase sequence 2 motif was identified in the amino acid sequence
of human HEAT-1 at about residues 510-519 of SEQ ID NO: 64, in the
amino acid sequence of human HEAT-2 at about residues 495-504 of
SEQ ID NO: 68, and in the amino acid sequence of human HEAT-3 at
about residues 530-539 of SEQ ID NO: 71.
[0344] In yet another embodiment, a HEAT protein of the present
invention includes at least one "P-type ATPase sequence 3 motif" in
the protein or corresponding nucleic acid molecule. As used herein,
a "P-type ATPase sequence 3 motif" is a conserved sequence motif
diagnostic for P-type ATPases (Tang, X. et al. (1996) Science
272:1495-1497; Fagan, M. J. and Saier, M. H. (1994) J. Mol. Evol.
38:57). A P-type ATPase sequence 3 motif is involved in ATP
binding. The consensus sequence for a P-type ATPase sequence 3
motif is [TIV]-G-D-G-X-N-D-[ASG]-P-[ASV]-L (SEQ ID NO: 77). X
indicates that the amino acid at the indicated position may be any
amino acid (i.e., is not conserved). The use of amino acids in
brackets indicates that the amino acid at the indicated position
may be any one of the amino acids within the brackets, e.g., [TIV]
indicates any of one of either T (threonine), I (isoleucine), or V
(valine). In a preferred embodiment, a P-type ATPase sequence 3
motif is contained within a C-terminal large extramembrane domain.
In another preferred embodiment, a P-type ATPase sequence 3 motif
in the HEAT proteins of the present invention has at least 1, 2, 3,
4, 5, 6, 7, 8 or more amino acid resides (including the amino acid
at the position indicated by "X") which match the consensus
sequence for a P-type ATPase sequence 3 motif. A P-type ATPase
sequence 3 motif was identified in the amino acid sequence of human
HEAT-1 at about residues 876-886 of SEQ ID NO: 64, in the amino
acid sequence of human HEAT-2 at about residues 881-891 of SEQ ID
NO: 68, and in the amino acid sequence of human HEAT-3 at about
residues 862-872 of SEQ ID NO: 71.
[0345] Isolated HEAT proteins of the present invention have an
amino acid sequence sufficiently homologous to the amino acid
sequence of SEQ ID NOS: 64, 68, or 71, or are encoded by a
nucleotide sequence sufficiently homologous to SEQ ID NOS: 63, 65,
67, 69, 70, or 72. As used herein, the term "sufficiently
homologous" refers to a first amino acid or nucleotide sequence
which contains a sufficient or minimum number of identical or
equivalent (e.g., an amino acid residue which has a similar side
chain) amino acid residues or nucleotides to a second amino acid or
nucleotide sequence such that the first and second amino acid or
nucleotide sequences share common structural domains or motifs
and/or a common functional activity. For example, amino acid or
nucleotide sequences which share common structural domains having
at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more homology or identity
across the amino acid sequences of the domains and contain at least
one and preferably two structural domains or motifs, are defined
herein as sufficiently homologous. Furthermore, amino acid or
nucleotide sequences which share at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or
more homology or identity and share a common functional activity
are defined herein as sufficiently homologous.
[0346] In a preferred embodiment, a HEAT protein includes at least
one or more of the following domains or motifs: a transmembrane
domain, an E1-E2 ATPase domain, an E1-E2 ATPases phosphorylation
site, an N-terminal large extramembrane domain, a C-terminal large
extramembrane domain, a P-type ATPase sequence 1 motif, a P-type
ATPase sequence 2 motif, and/or a P-type ATPase sequence 3 motif,
and has an amino acid sequence at least about 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%
or more homologous or identical to the amino acid sequence of SEQ
ID NOS: 64, 68, or 71. In yet another preferred embodiment, a HEAT
protein includes at least one or more of the following domains or
motifs: a transmembrane domain, an E1-E2 ATPase domain, an E1-E2
ATPases phosphorylation site, an N-terminal large extramembrane
domain, a C-terminal large extramembrane domain, a P-type ATPase
sequence 1 motif, a P-type ATPase sequence 2 motif, and/or a P-type
ATPase sequence 3 motif, and is encoded by a nucleic acid molecule
having a nucleotide sequence which hybridizes under stringent
hybridization conditions to a complement of a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NOS: 63, 65, 67, 69,
70, or 72. In another preferred embodiment, a HEAT protein includes
at least one or more of the following domains or motifs: a
transmembrane domain, an E1-E2 ATPase domain, an E1-E2 ATPases
phosphorylation site, an N-terminal large extramembrane domain, a
C-terminal large extramembrane domain, a P-type ATPase sequence 1
motif, a P-type ATPase sequence 2 motif, and/or a P-type ATPase
sequence 3 motif, and has a HEAT activity.
[0347] As used interchangeably herein, a "HEAT activity",
"biological activity of HEAT" or "functional activity of HEAT",
includes an activity exerted or mediated by a HEAT protein,
polypeptide or nucleic acid molecule on a HEAT responsive cell or
on a HEAT substrate, as determined in vivo or in vitro, according
to standard techniques. In one embodiment, a HEAT activity is a
direct activity, such as an association with a HEAT target
molecule. As used herein, a "target molecule" or "binding partner"
is a molecule with which a HEAT protein binds or interacts in
nature, such that HEAT-mediated function is achieved. A HEAT target
molecule can be a non-HEAT molecule or a HEAT protein or
polypeptide of the present invention. In an exemplary embodiment, a
HEAT target molecule is a HEAT substrate (e.g., a Ca.sup.2+ ion;
ATP; or a non-HEAT protein). A HEAT activity can also be an
indirect activity, such as a cellular signaling activity mediated
by interaction of the HEAT protein with a HEAT substrate (e.g.,
regulation of vascular smooth muscle tone, cellular growth and/or
proliferation, and/or angiogenesis).
[0348] In a preferred embodiment, a HEAT activity is at least one
of the following activities: (i) interaction with a HEAT substrate
or target molecule (e.g., a Ca.sup.2+ ion; ATP; or a non-HEAT
protein); (ii) transport of a HEAT substrate or target molecule
(e.g., a Ca.sup.2+ ion) from one side of a biological membrane to
the other; (iii) the ability to be phosphorylated or
dephosphorylated; (iv) adoption of an E1 conformation or an E2
conformation; (v) conversion of a HEAT substrate or target molecule
to a product (e.g., hydrolysis of ATP to ADP and free phosphate);
(vi) interaction with a second non-HEAT protein; (vii) modulation
of intra- or inter-cellular signaling and/or gene transcription
(e.g., either directly or indirectly); (viii) modulation of
vascular smooth muscle tone; (ix) modulation of cellular growth
and/or proliferation; and/or (x) modulation of angiogenesis.
[0349] Isolation of the Human HEAT cDNAs
[0350] The invention is based, at least in part, on the discovery
of genes encoding novel members of the E1-E2 ATPase family. The
entire sequence of human clones Fbh49937, Fbh49931, and Fbh49933
were determined and found to contain open reading frames termed
human "HEAT-1," human "HEAT-2," and human "HEAT-3,"
respectively.
[0351] The nucleotide sequence encoding the human HEAT-1 gene,
which is approximately 4055 nucleotides in length, is set forth as
SEQ ID NO: 63. The protein encoded by this nucleic acid molecule
has a molecular weight of approximately 129.8 kD, it comprises
about 1180 amino acids and it has the amino acid sequence set forth
as SEQ ID NO: 64. The coding region (open reading frame) of SEQ ID
NO: 63 is set forth as SEQ ID NO: 65.
[0352] The nucleotide sequence encoding the human HEAT-2 gene,
which is approximately 7249 nucleotides in length, is set forth as
SEQ ID NO: 67. The protein encoded by this nucleic acid molecule
has a molecular weight of approximately 138.2 kD, it comprises
about 1256 amino acids and it has the amino acid sequence set forth
as SEQ ID NO: 68. The coding region (open reading frame) of SEQ ID
NO: 67 is set forth as SEQ ID NO: 69.
[0353] The nucleotide sequence encoding the human HEAT-3 gene,
which is approximately 3919 nucleotides in length, is set forth as
SEQ ID NO: 70. The protein encoded by this nucleic acid molecule
has a molecular weight of approximately 132.5 kD, it comprises
about 1204 amino acids and it has the amino acid sequence set forth
as SEQ ID NO: 71. The coding region (open reading frame) of SEQ ID
NO: 70 is set forth as SEQ ID NO: 72.
[0354] Analysis of the Human HEAT Molecules
[0355] The amino acid sequences of human HEAT-1, HEAT-2, and HEAT-3
were analyzed using the program PSORT (available online; see Nakai,
K. and Kanehisa, M. (1992) Genomics 14:897-911) to predict the
localization of the proteins within the cell. This program assesses
the presence of different targeting and localization of amino acid
sequences within the query sequence. The results of the analyses
show that human HEAT-1 may be localized to the endoplasmic
reticulum, mitochondria, secretory vesicles, or vacuoles. The
results of these analyses further show that human HEAT-2 may be
localized to the endoplasmic reticulum or the mitochondria and that
human HEAT-3 may be localized to endoplasmic reticulum, the
mitochondria, or vacuoles.
[0356] Analyses of the amino acid sequence of human HEAT-1, HEAT-2,
and HEAT-3 were performed using MEMSAT. These analyses resulted in
the identification of twelve possible transmembrane domains in the
amino acid sequence of human HEAT-2 at residues 29-50, 211-227,
234-253, 294-317, 410-434, 449-469, 941-960, 968-985, 1000-1020,
1076-1092, 1105-1129, and 1144-1160 of SEQ ID NO: 68. These
analyses further resulted in the identification of twelve possible
transmembrane domains in the amino acid sequence of human HEAT-3 at
residues 65-89, 99-116, 242-258, 265-281, 445-464, 493-509,
990-1007, 1015-1031, 1049-1073, 1049-1073, 1103-1119, 1134-1151,
and 1171-1187 of SEQ ID NO: 71. Further analysis of the amino acid
sequence of SEQ ID NO: 71 (e.g., a Clustal W (1.74) multiple
sequence alignment, for example, of the human HEAT-3 amino acid
sequence with a known C. elegans cation-transporting ATPase
(YE56elegans; SEQ ID NO: 74; GenBank Accession No. P90747)) clearly
identifies the twelve transmembrane domains of the HEAT-3 gene. The
analysis of human HEAT-1 predicted twelve possible transmembrane
domains in the amino acid sequence of human HEAT-1 (SEQ ID NO: 64)
at about residues 8-25, 47-65, 256-276, 428-448, 464-484, 900-920,
936-954, 963-987, 994-1015, 1049-1065, 1079-1102, and 1118-1134.
The potential transmembrane domain at about residues 900-920 has a
notably low score of only 0.4 by MEMSAT analysis. Further analysis
of the amino acid sequence of SEQ ID NO: 64 (e.g., alignment with,
for example, a known C. elegans E1-E2 ATPase cation transporter
(SEQ ID NO: 66)) resulted in the identification of a twelfth
transmembrane domain at about amino acid residues 231-253 of SEQ ID
NO: 64. Accordingly, the human HEAT-1 protein of SEQ ID NO: 64 is
predicted to have at least twelve transmembrane domains, for
example, at about residues 8-25, 47-65, 231-253, 256-276, 428-448,
464-484, 936-954, 963-987, 994-1015, 1049-1065, 1079-1102, and
1118-1134.
[0357] Searches of the amino acid sequences of human HEAT-1,
HEAT-2, and HEAT-3 were also performed against the HMM database.
These searches resulted in the identification of an "E1-E2 ATPase"
domain in the amino acid sequence of HEAT-1 at about residues
299-387 (score=51.4) of SEQ ID NO: 64. These searches also resulted
in the identification of an "E1-E2 ATPase" domain in the amino acid
sequence of human HEAT-2 at about residues 278-365 (score=53.4) of
SEQ ID NO: 68. These searches further resulted in the
identification of an "E1-E2 ATPase" domain in the amino acid
sequence of human HEAT-3 at about residues 302-392 (score=37.0) of
SEQ ID NO: 71.
[0358] Searches of the amino acid sequence of human HEAT-1 were
performed against the Prosite database. These searches resulted in
the identification of an "E1-E2 ATPases phosphorylation site" at
about residues 513-519 of SEQ ID NO: 64. These searches also
resulted in the identification in the amino acid sequence of human
HEAT-1 of a number of potential N-glycosylation sites, cAMP- and
cGMP-dependent protein kinase phosphorylation sites, protein kinase
C phosphorylation sites, casein kinase II phosphorylation sites,
and N-myristoylation sites.
[0359] Searches of the amino acid sequence of human HEAT-2 were
also performed against the Prosite database. These searches
resulted in the identification of an "E1-E2 ATPases phosphorylation
site" at about residues 498-504 of SEQ ID NO: 68. These searches
also resulted in the identification in the amino acid sequence of
human HEAT-2 of a number of potential N-glycosylation sites, cAMP-
and cGMP-dependent protein kinase phosphorylation sites, protein
kinase C phosphorylation sites, casein kinase II phosphorylation
sites, tyrosine phosphorylation sites, and N-myristoylation
sites.
[0360] Searches of the amino acid sequence of human HEAT-3 were
further performed against the Prosite database. These searches
resulted in the identification of an "E1-E2 ATPases phosphorylation
site" at about residues 533-539 of SEQ ID NO: 71. These searches
also resulted in the identification in the amino acid sequence of
human HEAT-3 of a number of potential N-glycosylation sites, cAMP-
and cGMP-dependent protein kinase phosphorylation sites, protein
kinase C phosphorylation sites, casein kinase II phosphorylation
sites, and N-myristoylation sites.
[0361] The amino acid sequence of human HEAT-2 was used as a
database query using the BLASTP program. This search established
that human HEAT-2 has the highest homology to a putative yeast
Ca.sup.2+-transporting ATPase (high score=798,
probability=2.9e-87).
[0362] Table 13 depicts an alignment of a region important in
calcium binding from HEAT-1, HEAT-2, HEAT-3 with similar sequences
from a number of E1-E2 ATPases of various substrate specificities
from a number of different organisms. This region includes the
sixth transmembrane domain from each of HEAT-1, HEAT-2, and HEAT-3,
as well as a number of amino acid residues adjacent to the sixth
transmembrane domain. Amino acid residues determined to be
important for calcium binding by mutagenesis of a SERCA
calcium-transporting E1-E2 ATPase are indicated ("SERCA
mutagenesis"). Amino acid residues in this region that are critical
for calcium binding are indicated in bold. Substrate specificities
are as follows: Type V (calcium), Ca.sup.2+ (calcium), Cu.sup.2+
(copper), Na.sup.+/K.sup.+ (sodium/potassium), and PL
(phospholipid).
15TABLE 13 SEQ GenBank ID Substrate Gene Name Acc No. Sequence NO:
Specificity SERCA IPEGLPA 78 mutagenesis Fbh49937FL
DLVTVVVPPALPAAMTVCTLYAQSRLRR 79 (HEAT-1) Fbh49931FL
DIITITVPPALPAAMTAGIVYAQRRLKK 80 (HEAT-2) Fbh49933FL
LILTSVVPPELPIELSLAVNTSLIALAK 81 (HEAT-3) ATC9_Yeast_ Q12697
DIITIVVPPALPATLTIGTNFALSRLKEK 82 Type V yor291 ATC6_Yeast_ P39986
LIITTSVVPPELPMELTMAVNSSLAALAK 83 Type V SPF1_ye1031 w ATCY_SCHPO_
O14022 VLTILVPPALPATLSVGIANSIARLSRA 84 Type V sp_0140220
Gp_7324471_ AAF5962 DLVTIVVPPALPAVMGIGIFYAQRRLRQK 85 Type V CE 2
Sp_Q21286_Y Q21286 DIITIVVPPALPAANSVGIINANSRLKKK 86 Type V
BF7_CAEEL YH2M_CAEEL_ Q27533 DIITITVPPALPAANSVGIINAQL- RLKKK 87
Type V sp_q27533 YE56_CAEEL_ CAB0568 LILTSVIPPELPIELSLAVNSSLMALQKL
88 Type V CAB05683 3 ATP2A1_H_AA AAB5311
ALAVAAIPEGLPAVITTCLALGTRRMAKK 89 B53112 2 Ca.sup.2+ Rabbit P04191
ALAVAAIPEGLPAVITTCLALGTRRMAKK 90 SERCA1 Ca.sup.2+ ATCB_Chick_
P13585 ALAVAAIPEGLPAVITTCLALGTRRMAKK 91 SERCA1 Ca.sup.2+
ATC2_FELCA_ Q00779 ALAVAAIPEGLPAVITTCLALGTRRMAKK 92 SERCA2
Ca.sup.2+ SERCA AAB8229 ALAVAAIPEGLPAVITTCLALGTRRMAKK 93
Procambarus 1 Ca.sup.2+ clarkii hSERCA2 P16615
ALAVAAIPEGLPAVITTCLALGTRRMAKK 94 Ca.sup.2+ hSERCA3 Q93084
ALAVAAIPEGLPAVITTCLALGTRRMARK 95 Ca.sup.2+ ATCB_DROME_ P22700
AVAVAAIPEGLPAVITTCLALGTRRMAKK 96 P22700_1584 Ca.sup.2+ 16
ATC1_Yeast_ P13586 SLAVAAIPEGLPIIVTVTLALGVLRMAKR 97 PMR1 Ca.sup.2+
Y_PMC1 P38929 TVIVVAVPEGLPLAVTLALAFATTRMTKD 98 Ca.sup.2+ hPMCA1
P20020 TVLVVAVPEGLPLAVTISLAYSVKKMMKD 99 Ca.sup.2+ hPMCA2 Q01814
TVLVVAVPEGLPLAVTISLAYSVKKMMKD 100 Ca.sup.2+ Rat PMCA1
TVLVVAVPEGLPLAVTISLAYSVKKMMKD 101 Ca.sup.2+ PMCA3_H Q16720
TVLVVAVPEGLPLAVTISLAYSVKKMMKD 102 Ca.sup.2+ PMCA4_H P23634
TVLVVAVPEGLPLAVTISLAYSVKKMMKD 103 Ca.sup.2+ Pcalp_Yeast P38360
TVLIVSCPCVIGLAVPIVFVIASGVAAKR 104 Cu.sup.2+ AT7A_Human Q04656
TVLCIACPCSLGLATPTAVMVGTGVAAQN 105 Cu.sup.2+ AT7B_Human P35670
TVLCIACPCSLGLATPTAVMVGTGVAAQN 106 Cu.sup.2+ ATNA_DROME_ P13607
GIIVANVPEGLLATVTVCLTLTAKRMASK 107 P13607_7326 Na.sup.+/K.sup.+ 56
ATNA_HYDAT P35317 GIIVANVPEGLLATVTVCLTLTAKKMAKK 108
Na.sup.+/K.sup.+ ATN1_BUFMA P30714 GIIVANVPEGLLATVTVCLTLTAKRMARK
109 Na.sup.+/K.sup.+ ATN1 Human P05023
GIIVANVPEGLLATVTVCLTLTAKRMARK 110 Na.sup.+/K.sup.+ ATN2 Human
P50993 GIIVANVPEGLLATVTVCLTLTAKRMARK 111 Na.sup.+/K.sup.+ ATN3
Human P13637 GIIVANVPEGLLATVTVCLTVTAKRMARK 112 Na.sup.+/K.sup.+
ATPP2_H_AAD AAD3470 ILFNNLIPISLLVTLEVVKFTQAYFINWD 113 PL 34706 6
DRS2_ATC4_y P39524 ILFSNLVPISLFVTVELIKYYQAFMIGSD 114 PL east
[0363] Tissue Expression Analysis of HEAT-1, HEAT-2, and HEAT-3
mRNA Using Transcriptional Profiling and Taqman Analysis
[0364] This example describes the tissue distribution of human
HEAT-2 mRNA, as determined using transcriptional profiling analysis
and the TaqMan.TM. procedure. For transcriptional profiling
analysis, an array of several thousand cDNA clones are spotted onto
a nylon membrane and probed with a complex probe prepared by
radiolabeling cDNA made from mRNA from, for example, normal tissue,
and another, separate probe made from mRNA from another tissue, for
example, diseased tissue. Expression levels of each gene in the
first (e.g., normal) and the second (e.g., diseased) tissue are
then compared. Transcriptional profiling thus allows assessment of
the expression level of several thousand genes in an mRNA sample at
the same time.
[0365] Endothelial Cell Paradigms
[0366] To induce tube formation, human microvascular endothelial
cells isolated from the lung (HMVECs) were plated on Matrigel to
induce capillary-like tube formation. At 5 hours, the cells were
actively forming tubes, and RNA was harvested. Additional RNA
samples were prepared from cells 25 hours after plating on Matrigel
when tube formation was complete, and from actively proliferating
and confluent HMVECs grown on plastic.
[0367] Cells were also treated with laminar shear stress (LSS) of 7
dyn/cm.sup.2 for 24-30 hours, LSS plus one or six additional hours
of 12 dyn/cm.sup.2 ("1 h up" or "6 h up"), or LSS plus one or six
additional hours of 2 dyn/cm.sup.2 ("1 h down" or "6 h down").
[0368] HEAT-1
[0369] The expression levels of human HEAT-1 mRNA in various human
and monkey cell types and tissues was first determined using the
TaqMan procedure. The tisues and cells tested correspond to (1)
normal artery; (2) normal vein; (3) aortic smooth muscle cells
(early); (4) coronary smooth muscle cells; (5) umbilical vein
endothelial cells (static); (6) umbilical vein endothelial cells
(shear); (7) normal heart; (8) heart (congestive heart failure);
(9) kidney; (10) skeletal muscle; (11) normal adipose tissue; (12)
pancreas; (13) primary osteoblasts; (14) differentiated
osteoclasts; (15) normal skin; (16) normal spinal cord; (17) normal
brain cortex; (18) normal brain hypothalamus; (19) nerve; (20)
dorsal root ganglion; (21) glial cells (astrocytes); (22)
glioblastoma; (23) normal breast; (24) breast tumor; (25) normal
ovary; (26) ovarian tumor; (27) normal prostate; (28) prostate
tumor; (29) epithelial cells (prostate); (30) normal colon; (31)
colon tumor; (32) normal lung; (33) lung tumor; (34) lung (chronic
obstructive pulmonary disease); (35) colon (inflammatory bowel
disease); (36) normal liver; (37) liver fibrosis; (38) dermal cells
(fibroblasts); (39) normal spleen; (40) normal tonsil; (41) lymph
node; (42) small intestine; (43) skin (decubitus); (44) synovium;
(45) bone marrow mononuclear cells; and (46) activated peripheral
blood mononuclear cells. As assessed by this TaqMan analysis,
HEAT-1 is highly expressed in coronary artery vascular smooth
muscle cells, prostate epithelial cells, pancreas, and brain
(including cortex, hypothalamus, dorsal root ganglion cells, and
glial cells/astrocytes).
[0370] The expression levels of human HEAT-1 mRNA in various human
vascular rich organs was then determined using the TaqMan procedur,
the samples tested include (1) confluent microvascular endothelial
cells; (2) aortic smooth muscle cells; (3) fetal heart; (4) normal
heart atrium; (5) normal heart atrium; (6) normal heart ventricle;
(7) normal heart ventricle; (8) normal heart ventricle; (9) normal
heart ventricle; (10) normal heart ventricle; (11) diseased heart
ventricle; (12) diseased heart ventricle; (13) diseased heart
ventricle; (14) normal kidney; (15) normal kidney; (16) normal
kidney; (17) normal kidney; (18) normal kidney; (19) hypertensive
kidney; (20) hypertensive kidney; (21) hypertensive kidney; (22)
hypertensive kidney; (23) skeletal muscle; (24) skeletal muscle;
(25) liver; (26) liver; (27) normal fetal adrenal gland; (28) Wilms
tumor; (29) Wilms tumor; (30) normal spinal cord; and (31) diseased
cartilage. As assessed by this TaqMan panel, HEAT-1 is highly
expressed in Wilms' tumor, normal spinal cord, and microvascular
endothelial cells.
[0371] In another TaqMan experiment, the expression levels of human
HEAT-1 mRNA in various human and monkey vessels, namely (1) human
aortic smooth muscle cells; (2) human microvascular endothelial
cells; (3) human adipose tissue; (4) human normal carotid artery;
(5) human normal carotid artery; (6) human normal muscular artery;
(7) human diseased iliac artery; (8) human diseased tibial artery;
(9) human diseased aorta; (10) human normal saphenous vein; (11)
human normal saphenous vein; (12) human normal saphenous vein; (13)
human normal saphenous vein; (14) human diseased saphenous vein;
(15) human normal vein; (16) human normal vein; (17) human normal
vein; (18) monkey normal coronary artery; (19) monkey normal
coronary artery; (20) monkey normal coronary artery; (21) monkey
normal coronary artery; (22) monkey normal vein; and (23) no
transcriptase control, was determined. This TaqMan panel
demonstrated that HEAT-1 is highly expressed in vessels such as
arteries and veins.
[0372] The expression levels of human HEAT-1 mRNA in various human
coronary vascular cell types, namely (1) aortic smooth muscle
cells; (2) aortic smooth muscle cells; (3) aortic smooth muscle
cells; (4) coronary smooth muscle cells; (5) coronary smooth muscle
cells; (6) coronary smooth muscle cells; (7) coronary smooth muscle
cells; (8) macrophages; (9) macrophages treated with IFN.alpha.;
(10) macrophages treated with CD40; (11) macrophages treated with
LPS; (12) umbilical vein endothelial cells; (13) microvascular
endothelial cells; (14) aortic endothelial cells; (15) aortic
endothelial cells; (16) cortex renal epithelium; (17) renal
proximal tubule epithelium; (18) mesangial cells; (19) skeletal
muscle; (20) skeletal muscle; and (21) lung fibroblasts was also
determined using the Taqman procedure. This TaqMan panel
demonstrated that HEAT-1 is highly expressed in coronary and
vascular smooth muscle cells, as compared to other cell types.
[0373] The expression levels of human HEAT-1 mRNA in various human
endothelial cell paradigms was determined using the TaqMan
procedure. These experiments demonstrated that human HEAT-1 is
upregulated during shear stress of endothelial cells and that human
HEAT-1 is upregulated during proliferation and tube formation of
endothelial cells. These data strongly link human HEAT-1 to a role
in angiogenesis.
[0374] HEAT-2
[0375] The expression levels of human HEAT-2 mRNA in various human
and monkey cell types and tissues was first determined using
transcriptional profiling. The samples tested include (1) human
aortic smooth muscle cells; (2) human coronary artery smooth muscle
cells; (3) human umbilical vein endothelial cells; (4) human
microvascular endothelial cells (lung); (5) monkey aorta; (6)
monkey vein; (7) monkey heart; and (8) monkey liver. As shown in
these experiments, HEAT-2 is highly expressed in coronary artery
vascular smooth muscle cells, as compared to other tissues such as
aortic vascular smooth muscle cells, umbilical vein endothelial
cells, microvascular endothelial cells, heart, liver, aorta, and
vein. The expression levels of human HEAT-2 mRNA in various human
cell types and tissues was then confirmed in a second experiment
using the TaqMan procedure using a panel consisting of the
following tissues and samples: (1) normal aorta; (2) normal fetal
heart; (3) normal heart; (4) heart (congestive heart failure); (5)
normal vein; (6) aortic smooth muscle cells; (7) normal spinal
cord; (8) brain (normal cortex); (9) brain (hypothalamus); (10)
glial cells (astrocytes); (11) brain (glioblastoma); (12) normal
breast; (13) breast tumor (infiltrating ductal carcinoma); (14)
normal ovary; (15) ovarian tumor; (16) pancreas; (17) normal
prostate; (18) prostate tumor; (19) normal colon; (20) colon tumor;
(21) colon (inflammatory bowel disease); (22) normal kidney; (23)
normal liver; (24) fibrotic liver; (25) normal fetal liver; (26)
normal lung; (27) lung tumor; (28) lung (chronic obstructive
pulmonary disease); (29) normal spleen; (30) normal tonsil; (31)
normal lymph node; (32) normal thymus; (33) epithelial cells (from
prostate); (34) aortic endothelial cells; (35) skeletal muscle;
(36) dermal fibroblasts; (37) normal skin; (38) normal adipose
tissue; (39) primary osteoblasts; (40) undifferentiated
osteoblasts; (41) differentiated osteoblasts; (42) osteoclasts;
(43) aortic smooth muscle cells (early); (44) aortic smooth muscle
cells (late); (45) human umbilical vein endothelial cells (shear);
and (46) human umbilical vein endothelial cells (static).
[0376] The expression levels of human HEAT-2 mRNA in various human
vascular rich organs, namely (1) normal human heart; (2) normal
human heart; (3) normal human heart; (4) normal human heart; (5)
normal human heart; (6) normal human heart; (7) normal human heart;
(8) normal human heart; (9) diseased human heart; (10) diseased
human right ventricle; (11) diseased human left ventricle; (12)
normal monkey heart; (13) normal monkey heart; (14) normal monkey
heart; (15) normal human kidney; (16) normal human kidney; (17)
normal human kidney; (18) normal human kidney; (19) normal human
kidney; (20) human hypertensive kidney; (21) human hypertensive
kidney; (22) human hypertensive kidney; (23) human hypertensive
kidney; (24) human hypertensive kidney; (25) human liver; (26)
human liver; (27) human liver; (28) human skeletal muscle; (29)
human skeletal muscle; and (30) human skeletal muscle, was then
determined using the TaqMan procedure. These experiments
demonstrated that HEAT-2 is highly expressed in the heart.
[0377] In another experiment, the expression levels of human HEAT-2
mRNA in various human and monkey vessels, namely (1) human adipose
tissue; (2) human normal artery; (3) human normal artery; (4) human
carotid artery; (5) human carotid artery; (6) human normal artery;
(7) human diseased artery; (8) human diseased artery; (9) human
diseased artery; (10) human normal vein; (11) human normal vein;
(12) human vein; (13) human vein; (14) human normal vein; (15)
human varicose vein; (16) confluent human microvascular endothelial
cells; (17) human aortic smooth muscle cells; (18) monkey aorta;
(19) monkey aorta; (20) monkey aorta; (21) monkey artery; (22)
monkey artery; (23) monkey renal artery; (24) monkey renal artery;
(25) monkey renal artery; (26) monkey renal artery; (27) monkey
renal artery; (28) monkey coronary artery; (29) monkey coronary
artery; (30) monkey coronary artery; (31) monkey coronary artery;
(32) monkey coronary artery; (33) monkey coronary artery; and (34)
monkey coronary artery, was determined using the TaqMan procedure
and in situ hybridization. These experiments demonstrated that
HEAT-2 is highly expressed in vessels such as arteries and
veins.
[0378] The expression levels of human HEAT-2 mRNA in various human
coronary vascular cell types, namely (1) aortic smooth muscle
cells; (2) aortic smooth muscle cells; (3) aortic smooth muscle
cells; (4) aortic smooth muscle cells; (5) coronary smooth muscle
cells; (6) coronary smooth muscle cells; (7) coronary smooth muscle
cells; (8) coronary smooth muscle cells; (9) macrophages; (10)
macrophages treated with IFN.gamma.; (11) macrophages treated with
CD40; (12) macrophages treated with LPS; (13) umbilical vein
endothelial cells; (14) microvascular endothelial cells; (15)
aortic endothelial cells; (16) coronary artery endothelial cells;
(17) coronary artery endothelial cells; (18) cortex renal
epithelium; (19) renal proximal tubule epithelium; (20) mesangial
cells; (21) skeletal muscle; (22) skeletal muscle; and (23) lung
fibroblasts, was also determined using the TaqMan procedure. As
determined by these experiments, HEAT-2 is highly expressed in
coronary vascular smooth muscle cells, as compared to other cell
types.
[0379] The expression levels of human HEAT-2 mRNA in various human
endothelial cell paradigms was determined using the TaqMan
procedure. The samples tested include (1) umbilical vein
endothelial cells (static); (2) umbilical vein endothelial cells
(shear regulated); (3) umbilical vein endothelial cells
(proliferating); (4) umbilical vein endothelial cells (confluent);
(5) umbilical vein endothelial cells (without growth factor
treatment); (6) umbilical vein endothelial cells (Interleukin-1
stimulated); (7) microvascular endothelial cells (proliferating);
(8) microvascular endothelial cells (confluent); (9) microvascular
endothelial cells (proliferating); (10) microvascular endothelial
cells (confluent); (11) microvascular endothelial cells
(proliferating); (12) microvascular endothelial cells (confluent);
(13) microvascular endothelial cells (without growth factor
treatment); (14) coronary microvascular endothelial cells
(proliferating); (15) coronary microvascular endothelial cells
(confluent); (16) microvascular endothelial cells (5% serum plus
growth factors); (17) microvascular endothelial cells (5% serum
without growth factors); (18) microvascular endothelial cells (hEGF
treated); (19) microvascular endothelial cells (VEGF treated); (20)
microvascular endothelial cells (bFGF treated); (21) microvascular
endothelial cells (IGF treated); (22) 293 cells; (23) umbilical
vein endothelial cells (static 25 h); (24) umbilical vein
endothelial cells (laminar shear stress); (25) umbilical vein
endothelial cells (laminar shear stress+1 h up); (26) umbilical
vein endothelial cells (laminar shear stress+1 h down); (27)
umbilical vein endothelial cells (static 30 h); (28) umbilical vein
endothelial cells (laminar shear stress); (29) umbilical vein
endothelial cells (laminar shear stress+6 h up); (30) umbilical
vein endothelial cells (static 30 h); (31) umbilical vein
endothelial cells (laminar shear stress); and (32) umbilical vein
endothelial cells (laminar shear stress+6 h down). These
experiments demonstrate that HEAT-2 is upregulated during shear and
proliferation of endothelial cells. These data strongly link HEAT-2
to a role in angiogenesis.
[0380] Human HEAT-2 was also shown to be upregulated during tube
formation of endothelial cells. The expression level of human
HEAT-2 is 5-fold higher in the 5-hour Matrigel sample than in any
other sample, indicating that expression is significantly induced
during the process of capillary-like tube formation. There is also
significantly higher expression in proliferating HMVECs than in
confluent HMVECs grown on plastic. These results indicate a
pro-angiogenic function for human HEAT-2.
[0381] Human HEAT-2 mRNA expression was also detected by in situ
hybridization analysis in human endothelial cells and myocytes in
the heart and in endothelial cells and inflammatory cells in ApoE
knockout mouse diseased aortic roots.
[0382] HEAT-3
[0383] The expression levels of human HEAT-3 mRNA in various human
and monkey cell types and tissues, namely (1) normal aorta; (2)
normal fetal heart; (3) normal heart; (4) heart (congestive heart
failure); (5) normal vein; (6) normal spinal cord; (7) normal brain
cortex; (8) normal brain hypothalamus; (9) glial cells
(astrocytes); (10) glioblastoma (brain); (11) normal breast; (12)
breast tumor (infiltrating ductal carcinoma); (13) normal ovary;
(14) ovarian tumor; (15) pancreas; (16) normal prostate; (17)
prostate tumor; (18) normal colon; (19) colon tumor; (20) colon
(inflammatory bowel disease); (21) normal kidney; (22) normal
liver; (23) liver fibrosis; (24) normal fetal liver; (25) normal
lung; (26) lung tumor; (27) lung (chronic obstructive pulmonary
disease); (28) normal spleen; (29) normal tonsil; (30) normal lymph
node; (31) normal thymus; (32) epithelial cells (prostate); (33)
endothelial cells (aortic); (34) normal skeletal muscle; (35)
fibroblasts (dermal); (36) normal skin; (37) normal adipose tissue;
(38) primary osteoblasts; (39) undifferentiated osteoblasts; (40)
differentiated osteoblasts; (41) osteoclasts; (42) aortic smooth
muscle cells (early); (43) aortic smooth muscle cells (late); (44)
umbilical vein endothelial cells (laminar shear stress); (45)
umbilical vein endothelial cells (static); and (46)
undifferentiated osteoclasts, was first determined using the TaqMan
procedure. These results demonstrate that HEAT-3 is highly
expressed in coronary artery vascular smooth muscle cells, prostate
epithelial cells, pancreas, and brain (including cortex,
hypothalamus, and glial cells/astrocytes).
[0384] The expression levels of human HEAT-3 mRNA in various human
vascular rich organs, namely (1) normal heart; (2) normal heart;
(3) normal heart; (4) normal heart; (5) normal heart; (6) normal
heart; (7) normal heart; (8) normal heart; (9) diseased heart; (10)
diseased right ventricle; (11) normal fetal heart; (12) normal
kidney; (13) normal kidney; (14) normal kidney; (15) normal kidney;
(16) normal kidney; (17) hypertensive kidney; (18) hypertensive
kidney; (19) hypertensive kidney; (20) hypertensive kidney; (21)
hypertensive kidney; (22) skeletal muscle; (23) skeletal muscle;
(24) skeletal muscle; (25) liver; (26) liver; (27) normal monkey
heart; (28) normal monkey heart; (29) normal monkey heart; (30)
normal monkey heart; (31) smooth muscle cells (SMC); (32) confluent
human microvascular endothelial cells (HMVECs); (33) M human
umbilical vein endothelial cells (HUVECs); (34) human umbilical
vein endothelial cells (HUVECs)-vehicle; (35) M human amniotic
endothelial cells (HAECs); and (36) human amniotic endothelial
cells (HAECs)-vehicle, was then determined using the TaqMan
procedure. These experiments demonstrated that HEAT-3 is expressed
in the heart, kidney, and skeletal muscle.
[0385] In another experiment, the expression levels of human HEAT-3
mRNA in various vessels, namely (1) aortic smooth muscle cells; (2)
microvascular endothelial cells; (3) adipose tissue; (4) normal
artery; (5) normal artery; (6) normal artery; (7) diseased artery;
(8) diseased artery; (9) diseased aorta; (10) normal vein; (11)
normal vein; (12) normal vein; (13) normal vein; (14) diseased
vein; (15) normal vein; (16) normal vein; (17) normal vein; (18) LC
smooth muscle cells; (19) LC smooth muscle cells; (20) aortic
smooth muscle cells; (21) human microvascular endothelial cells;
(22) normal human carotid artery; (23) normal human carotid artery;
(24) normal human muscular artery; (25) human diseased iliac
artery; (26) human diseased tibial artery; (27) human diseased
aorta; (28) human normal saphenous vein; (29) human normal
saphenous vein; (30) human normal saphenous vein; (31) human normal
saphenous vein; (32) human diseased saphenous vein; (33) human
normal vein; and (34) human normal saphenous vein, was determined
using the TaqMan procedure. These experiments demonstrated that
HEAT-3 is highly expressed in vessels such as arteries and
veins.
[0386] The expression levels of human HEAT-3 mRNA in various human
coronary vascular cell types, namely (1) aortic smooth muscle
cells; (2) aortic smooth muscle cells; (3) coronary smooth muscle
cells; (4) coronary smooth muscle cells; (5) coronary smooth muscle
cells; (6) coronary smooth muscle cells; (7) macrophages; (8)
macrophages treated with IFN.gamma.; (9) macrophages treated with
CD40; (10) macrophages treated with LPS; (11) microvascular
endothelial cells; (12) aortic endothelial cells; (13) coronary
artery endothelial cells; (14) cortex renal epithelium; (15) renal
proximal tubule epithelium; (16) mesangial cells; and (17) skeletal
muscle, was also determined using the TaqMan procedure. These
experiments demonstrate that HEAT-3 is highly expressed in coronary
and aortic vascular smooth muscle cells, as well as in renal
proximal tubule epithelium, as compared to other cell types.
[0387] Tissue Distribution of HEAT mRNA Using in situ Analysis
[0388] Using in situ hybridization analysis, HEAT-2 mRNA was found
to be expressed in human endothelial cells and myocytes in the
heart and in endothelial cells and inflammatory cells in ApoE
knockout mice diseased aortic roots.
[0389] Assesment of Microvessel Contraction
[0390] The following describes the assessment of microvessel
contraction using rat microvessels, as described in, for example,
Bischoff, A. et al. (2000) Br. J. Pharmacol. 130:1871-1877.
Microvessels (e.g., mesenteric or renal microvessels such as
interlobar arteries) are prepared from rats (e.g., adult Wistar
rats) as described in Chen et al. (1996) Naunyn-Schmiedeberg's
Arch. Pharmacol. 353:314-323 and Chen et al. (1997) J. Auton.
Pharmacol. 17:137-146. Rats are killed by either decapitation or an
overdose of thiobutabarbitone. The vessels are mounted on 40 .mu.m
diameter stainless steel wires in a myograph chamber for isometric
recording of tension development. The vessels are then bathed in
Krebs-Henseleit buffer of the following composition: 119 mM NaCl,
25 mM NaHCO.sub.3, 4.7 mM KCl, 1.18 mM KH.sub.2PO.sub.4, 1.17 mM
MgSO.sub.4, 2.5 mM CaCl.sub.2, 0.026 mM EDTA, and 5.5 mM D-glucose.
The buffer temperature is maintained at 37.degree. C., and the
chamber is gassed with 5% CO2/95% O2 to maintain a pH of 7.4.
Additionally, 5 .mu.M cocaine and 1 .mu.M (.+-.)-propranolol may be
added to block neuronal catecholoamine uptake and
.beta.-adrenoceptor activation by high noradrenaline concentration.
Following equilibration, the vessels are challenged several times
with 125 mM KCl and 10 .mu.M noradrenaline. The vessels are then
treated with 100 .mu.M carbachol; vessels with a relaxation
response of at least 50% indicate a functionally intact
epithelium.
[0391] Assesment of Intracellular Free Calcium Concentrations in
Cultured Rat Aortic Smooth Muscle Cells
[0392] The following describes the assessment of intracellular free
calcium concentrations in cultured rat aortic smooth muscle cells,
as described in, for example, Bischoff, A. et al. (2000) Br. J.
Pharmacol. 130:1871-1877. Vascular smooth muscle cells are prepared
from rat thoracic aorta according to Rosskoph et al. (1995) Cell
Physiol. Biochem. 5:276-285). Briefly, freshly prepared aortae are
incubated for 30 minutes at room temperature with 125 U/ml
collagenase I in Hank's balanced salt solution (HBSS) of the
following composition: 118 mM NaCl, 5 mM KCl, 1 mM CaCl.sub.2, 1 mM
MgCl.sub.2, 5 mM D-glucose, and 15 mM HEPES pH 7.4. Thereafter,
remaining connective tissue and endothelium are removed, the aortae
are cut into small pieces and incubated for 4-6 hours at 37.degree.
C. in DMEM/F12 medium with 100 U/ml penicillin, 100 .mu.g/ml
streptomycin, and 250 ng/ml amphotericin B. Treatment with
collagenase (125 U/ml) and elastase (0.5 mg/ml) in HBSS without
Ca.sup.2+ and Mg.sup.2+ follow for 2 hours at 37.degree. C . The
reaction is stopped by addition of DMEM/F12 medium containing 20%
fetal calf serum and penicillin, streptomycin, and amphotericin B,
and the cells are plated onto 60-mm cell culture plates. The cells
are used between passage 3 and 6. The Ca.sup.2+ concentration
measurements are performed as described in Meyer zu Heringdorf et
al. (1996) Naunyn-Schmiedeberg's Arch. Pharmacol. 354:397-403.
Briefly, the cells are loaded with 1 .mu.M fura2/AM for 1 hour at
room temperature in HBSS, washed with HBSS, and used for
fluorescence measurements within the next hour. Ca.sup.2+
concentrations are measured in a continuously stirred cell
suspension at room temperature in a Hitachi F2000
spectrofluorometer as described in Meyer zu Heringdorf et al.
(1996) supra.
[0393] Calcium Transport Assay
[0394] The following describes the assessment of calcium transport
by HEAT molecules in cultured COS-1 cells, as described in, for
example, Maruyama, K. and MacLennan, D. H. (1988) Proc. Natl. Acad.
Sci. USA 85:3314-3318.
[0395] Cell Culture and DNA Transfection
[0396] COS-1 or HEK-293 cells are maintained in Dulbecco's modified
Eagle's medium (DMEM) with 0.1 mM .alpha.-MEM nonessential amino
acids, 4 mM L-glutamine, 100 units of pennicillin per ml, 100 .mu.g
of streptomycin per ml, and 10% fetal calf serum under 5%
CO.sub.2/95% air at 37.degree. C. Transfection of HEAT-containing
DNA is carried out by the DEAE dextran-chloroquine shock method
(Sompayrac, L. M. and Danna, K. J. (1981) Proc. Natl. Acad. Sci.
USA 78:7575-7578; Gorman, C. (1985) in DNA Cloning: A Practical
Approach, ed. Gover, D. M. (IRL, Washington, D.C.), Vol. 2, pp.
143-190) with 25 .mu.g of cesium chloride gradient-purified DNA and
1.5 mg of DEAE dextran per 10 cm Petri dish. Cells are then
incubated for 3 hours at 37.degree. C. in 6 ml of DMEM containing
300 .mu.g of chloroquine, washed, and cultured in DMEM for 48 or 72
hours. Control cells are treated in the same way with vector DNA or
with no added DNA.
[0397] Isolation of Microsomal Fraction
[0398] For isolation of a microsomal fraction (Resh, M. D. and
Erikson, R. L. (1985) J. Cell Biol. 100:409-417; Yamada, S. and
Ikemoto, N. (1980) J. Biol. Chem. 255:3108-3119), cells from five
10 cm Petri dishes are washed twice with 5 ml of a solution of
0.137 M NaCl/2.7 mM KCl/8 mM Na.sub.2HPO.sub.4/1.5 mM
KH.sub.2PO.sub.4 (PBS), harvested in a solution of 5 mM EDTA in PBS
and washed with 5 ml of PBS. The cells are swollen at 0.degree. C.
for 10 minutes in 2 ml of a hypotonic solution of 10 mM Tris-HCl,
pH 7.5/0.5 mM MgCl.sub.2, and then phenylmethylsolfonyl fluoride
and Trasylol are added to 0.1 mM and 100 units/ml, respectively.
The cells are homogenized with 30 strokes in a glass Dounce
homogenizer, and the homogenate is diluted with an equal volume of
a solution of 0.5 M sucrose/6 mM 2-mercaptoenthanol, 40 .mu.M
CaCl.sub.2/300 mM KCl/10 mM Tris-HCl, pH 7.5. The suspension is
centrifuged at 10,000.times. g for 20 minutes to pellet nuclei and
mitochondria. The supernatant is brought to a concentration of 0.6
M KCl by the addition of 0.9 ml of a 2.5 M solution. The suspension
is centrifuged at 100,000.times. g for 60 minutes to sediment the
microsomal fraction. The pellet is suspended in a solution
containing 0.25 M sucrose, 0.15 M KCl, 3 mM 2-mercaptoethanol, 20
.mu.M CaCl.sub.2, 10 mM Tris-HCl (pH 7.5), and centrifuged again at
100,000.times. g for 60 minutes. The final pellet, containing
approximately 100 .mu.g of protein, is suspended in the same
solution at a protein concentration of 1 mg/ml.
[0399] Ca.sup.2+ Transport Assay
[0400] Ca.sup.2+ transport activity is assayed in a reaction
mixture containing 20 .mu.M Mops-KOH (pH 6.8), 100 mM KCl, 5 mM
CaCl.sub.2, 5 mM ATP, 0.45 mM CaCl.sub.2 (containing .sup.45Ca at a
specific activity of 10.sup.6 cpm/.mu.mol), 0.5 mM EGTA, and 5 mM
potassium oxalate. The uptake reaction is initiated by the addition
of 10 .mu.g of microsomal protein to 1 ml of reaction mixture at
room temperature. At different time points, 0.15 ml samples are
filtered through a 0.3 .mu.m Millipore filter and washed with 10 ml
of 0.15 M KCl. Radioactivity on the filter is measured by liquid
scintillation counting. For the measurement of Ca.sup.2+ ion
dependency, free Ca.sup.2+ concentration is calculated by the
computer program of Fabiato and Fabiato ((1979) J. Physiol.
(London) 75:463-505). For the measurement of ATP dependency, an ATP
regenerating system consisting of 2.5 mM phosphoenolpyruvate and 50
.mu.g of pyruvate kinase per ml is used.
[0401] Measurement of Phosphorylated HEAT Intermediate
[0402] Microsomal protein (5 .mu.g) is added to 0.1 ml of a
solution of 20 mM Mops, pH 6.8/100 mM KCl/5 mM MgCl.sub.2/0.5 mM
EGTA in the presence or absence of 0.5 mM CaCl.sub.2. The reaction,
at ice temperature, is started by the addition of 5 .mu.M ATP
(10.sup.6 cpm/nmol) and stopped after 5 seconds by the addition of
0.6 ml of a mixture of 5% trichloroacetic acid and 5 mM potassium
phosphate. Incorporation of .sup.32P is determined either by
collecting the protein on a filter for scintillation counting or by
separating the protein in acidic NaDodSO.sub.4/polyacrylamide gels
for autoradiography Sarkadi, B. et al. (1986) J. Biol. Chem.
261:9552-9557).
[0403] Analysis of HEAT-3 Activity
[0404] The full-length HEAT-3 was inserted into the multiple
cloning site in the pCDNA3 vector. The DNA for the clone was
amplified and transfected into HEK-293 cells using calcium
phosphate precipitation. After 72 hours, the cells were harvested,
microsomal fractions isolated, and .sup.45Ca-uptake measured as a
function of calcium concentration using a filter assay.
[0405] Two different HEAT-3 fusion proteins were generated. One
HEAT-3 fusion protein was created by inserting the 3X Flag epitope
at the 3' end of the HEAT-3 gene. Another HEAT-3 fusion protein was
created by inserting the green fluorescent protein (GFP) at the 3'
end of the HEAT-3 gene. Fluorescence of this protein could be
observed with the naked eye and by confocal microscopy. Measurement
of expression using Western blotting with an anti-GFP antibody
showed that HEAT-3 is well expressed in the microsomal
fraction.
[0406] Confocal microscopy showed that the expression pattern of
HEAT-3 is similar to SERCA1, indicating that HEAT-3 is targeted to
and localized in the endoplasmic reticulum of HEK-293 cells.
[0407] Ca.sup.2+ uptake experiments (as described above) were
performed using both the Flag and GFP fusion proteins. An increase
in Ca.sup.2+ uptake of HEAT-3 was shown over GFP vector alone. Five
independent experiments were performed to confirm the increase of
calcium uptake with HEAT-3 as compared to vector alone. The Flag
fusion protein showed an increase of calcium uptake as compared to
Flag vector alone. In these experiments, the Vmax for HEAT-3 was
lower (about 20.times.) than the Vmax for SERCA1 under the same
conditions. The KCa for HEAT-3 was about 6.00 pCa units, as
compared with about 6.38 pCa units for SERCA1. In the presence of
ATP, there is 2-3 fold more calcium uptake compared to uptake in
the absence of ATP, indicating that the calcium uptake by HEAT-3 is
ATP dependent.
[0408] Definitions
[0409] The 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 protein, fragments thereof, and
derivatives and other variants of the sequence in SEQ ID NOS: 2, 8,
17, 21, 26, 29, 36, 40, 43, 64, 68 or 71 thereof are collectively
referred to as "polypeptides or proteins of the invention" or
"25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 polypeptides or proteins". Nucleic acid
molecules encoding such polypeptides or proteins are collectively
referred to as "nucleic acids of the invention" or "25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 nucleic acids."
[0410] 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.
[0411] The term "isolated or purified nucleic acid molecule"
includes nucleic acid molecules which are separated from other
nucleic acid molecules which are present in the natural source of
the nucleic acid. For example, with regards to genomic DNA, the
term "isolated" includes nucleic acid molecules which are separated
from the chromosome with which the genomic DNA is naturally
associated. Preferably, an "isolated" nucleic acid is free of
sequences which 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 kb, 4 kb, 3 kb, 2 kb, 1 kb,
0.5 kb or 0.1 kb 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.
[0412] As used herein, the term "hybridizes under low stringency,
medium stringency, high stringency, or very high stringency
conditions" describes conditions for hybridization and washing.
Guidance for performing hybridization reactions can be found in
Current Protocols in Molecular Biology (1989) John Wiley &
Sons, N.Y., 6.3.1-6.3.6, which is incorporated by reference.
Aqueous and nonaqueous methods are described in that reference and
either can be used. Specific hybridization conditions referred to
herein are as follows: 1) low stringency hybridization conditions
in 6.times. sodium chloride/sodium citrate (SSC) at about
45.degree. C., followed by two washes in 0.2.times. SSC, 0.1% SDS
at least at 50.degree. C. (the temperature of the washes can be
increased to 55.degree. C. for low stringency conditions); 2)
medium stringency hybridization conditions in 6.times. SSC at about
45.degree. C., followed by one or more washes in 0.2.times. SSC,
0.1% SDS at 60.degree. C.; 3) high stringency hybridization
conditions in 6.times. SSC at about 45.degree. C., followed by one
or more washes in 0.2.times. SSC, 0.1% SDS at 65.degree. C.; and
preferably 4) very high stringency hybridization conditions are
0.5M sodium phosphate, 7% SDS at 65.degree. C., followed by one or
more washes at 0.2.times. SSC, 1% SDS at 65.degree. C. Very high
stringency conditions (4) are the preferred conditions and the ones
that should be used unless otherwise specified.
[0413] 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).
[0414] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules which include an open reading frame
encoding a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 protein, preferably a mammalian 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein, and can further include non-coding
regulatory sequences, and introns.
[0415] 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 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein having less than about 30%, 20%, 10% and more
preferably 5% (by dry weight), of non-25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein
(also referred to herein as a "contaminating protein"), or of
chemical precursors or non-25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 chemicals. When
the 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 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.
[0416] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
(e.g., the sequence of SEQ ID NOS: 1, 3, 7, 9, 16, 18, 20, 22, 25,
27, 28, 30, 35, 37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72)
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 conserved domains, are predicted to be
particularly unamenable to alteration.
[0417] 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), nonpolar 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 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
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 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 coding sequence, such as by saturation mutagenesis,
and the resultant mutants can be screened for 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
biological activity to identify mutants that retain activity.
Following mutagenesis of SEQ ID NOS: 1, 3, 7, 9, 16, 18, 20, 22,
25, 27, 28, 30, 35, 37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72,
the encoded protein can be expressed recombinantly and the activity
of the protein can be determined.
[0418] As used herein, a "biologically active portion" of a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein includes a fragment of a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein which participates in an interaction between a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 molecule and a non-25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 molecule.
Biologically active portions of a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein
include peptides comprising amino acid sequences sufficiently
homologous to or derived from the amino acid sequence of the 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein, e.g., the amino acid sequence shown in SEQ
ID NOS: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71, which
include fewer amino acids than the full length 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
protein, and exhibit at least one activity of a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein. Typically, biologically active portions comprise a
domain or motif with at least one activity of the 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein. A biologically active portion of a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein can be a polypeptide which is, for example, 10, 25,
50, 100, 200 or more amino acids in length. Biologically active
portions of a 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 protein can be used as targets
for developing agents which modulate a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 mediated
activity.
[0419] The term "family" when referring to the protein and nucleic
acid molecules of the invention is intended to mean 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., rat or mouse proteins. Members of a family can also
have common functional characteristics.
[0420] Calculations of homology or sequence identity (the terms
"homology" and "identity" are used interchangeably herein) between
sequences are performed as follows:
[0421] 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. 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.
[0422] 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 and Wunsch (1970) J. Mol. Biol. 48:444-453 algorithm
which has been incorporated into the GAP program in the GCG
software package using either a Blossum 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 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 Blossum 62 scoring
matrix with a gap penalty of 12, a gap extend penalty of 4, and a
frameshift gap penalty of 5.
[0423] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of Meyers and
Miller ((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.
[0424] 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. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein molecules of the
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and
Gapped BLAST programs, the default parameters of the respective
programs (e.g., XBLAST and NBLAST) can be used.
[0425] Particular 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 polypeptides of the present
invention have an amino acid sequence substantially identical to
the amino acid sequence of SEQ ID NOS: 2, 8, 17, 21, 26, 29, 36,
40, 43, 64, 68 or 71. In the context of an amino acid sequence, the
term "substantially identical" is used herein to refer to a first
amino acid that contains a sufficient or minimum number of amino
acid residues that are i) identical to, or ii) conservative
substitutions of aligned amino acid residues in a second amino acid
sequence such that the first and second amino acid sequences can
have a common structural domain and/or common functional activity.
For example, amino acid sequences that contain a common structural
domain having at least about 60%, or 65% identity, likely 75%
identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NOS: 2, 8, 17, 21, 26, 29, 36, 40,
43, 64, 68 or 71 are termed substantially identical.
[0426] In the context of nucleotide sequence, the term
"substantially identical" is used herein to refer to a first
nucleic acid sequence that contains a sufficient or minimum number
of nucleotides that are identical to aligned nucleotides in a
second nucleic acid sequence such that the first and second
nucleotide sequences encode a polypeptide having common functional
activity, or encode a common structural polypeptide domain or a
common functional polypeptide activity. For example, nucleotide
sequences having at least about 60%, or 65% identity, likely 75%
identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NOS: 1, 3, 7, 9, 16, 18, 20, 22, 25,
27, 28, 30, 35, 37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72 are
termed substantially identical.
[0427] "Misexpression 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.
[0428] "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.
[0429] 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.
[0430] As used herein, cellular proliferative and/or
differentiative disorders include cancer, e.g., carcinoma, sarcoma,
metastatic disorders or hematopoietic neoplastic disorders, e.g.,
leukemias. A metastatic tumor can arise from a multitude of primary
tumor types, including but not limited to those of prostate, colon,
lung, breast and liver origin.
[0431] As used herein, the term "cancer" (also used interchangeably
with the terms, "hyperproliferative" and "neoplastic") refers to
cells having the capacity for autonomous growth, i.e., an abnormal
state or condition characterized by rapidly proliferating cell
growth. Cancerous disease states may be categorized as pathologic,
i.e., characterizing or constituting a disease state, e.g.,
malignant tumor growth, or may be categorized as non-pathologic,
i.e., a deviation from normal but not associated with a disease
state, e.g., cell proliferation associated with wound repair. The
term is meant to include all types of cancerous growths or
oncogenic processes, metastatic tissues or malignantly transformed
cells, tissues, or organs, irrespective of histopathologic type or
stage of invasiveness. The term "cancer" includes malignancies of
the various organ systems, such as those affecting lung, breast,
thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as
well as adenocarcinomas which include malignancies such as most
colon cancers, renal-cell carcinoma, prostate cancer and/or
testicular tumors, non-small cell carcinoma of the lung, cancer of
the small intestine and cancer of the esophagus. The term
"carcinoma" is art recognized and refers to malignancies of
epithelial or endocrine tissues including respiratory system
carcinomas, gastrointestinal system carcinomas, genitourinary
system carcinomas, testicular carcinomas, breast carcinomas,
prostatic carcinomas, endocrine system carcinomas, and melanomas.
Exemplary carcinomas include those forming from tissue of the
cervix, lung, prostate, breast, head and neck, colon and ovary. The
term "carcinoma" also includes carcinosarcomas, e.g., which include
malignant tumors composed of carcinomatous and sarcomatous tissues.
An "adenocarcinoma" refers to a carcinoma derived from glandular
tissue or in which the tumor cells form recognizable glandular
structures. The term "sarcoma" is art recognized and refers to
malignant tumors of mesenchymal derivation.
[0432] Examples of cellular proliferative and/or differentiative
disorders of the lung include, but are not limited to, tumors such
as bronchogenic carcinoma, including paraneoplastic syndromes,
bronchioloalveolar carcinoma, neuroendocrine tumors, such as
bronchial carcinoid, miscellaneous tumors, metastatic tumors, and
pleural tumors, including solitary fibrous tumors (pleural fibroma)
and malignant mesothelioma.
[0433] Examples of cellular proliferative and/or differentiative
disorders of the breast include, but are not limited to,
proliferative breast disease including, e.g., epithelial
hyperplasia, sclerosing adenosis, and small duct papillomas;
tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor,
and sarcomas, and epithelial tumors such as large duct papilloma;
carcinoma of the breast including in situ (noninvasive) carcinoma
that includes ductal carcinoma in situ (including Paget's disease)
and lobular carcinoma in situ, and invasive (infiltrating)
carcinoma including, but not limited to, invasive ductal carcinoma,
invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)
carcinoma, tubular carcinoma, and invasive papillary carcinoma, and
miscellaneous malignant neoplasms. Disorders in the male breast
include, but are not limited to, gynecomastia and carcinoma.
[0434] Examples of cellular proliferative and/or differentiative
disorders involving the colon include, but are not limited to,
tumors of the colon, such as non-neoplastic polyps, adenomas,
familial syndromes, colorectal carcinogenesis, colorectal
carcinoma, and carcinold tumors.
[0435] Examples of cancers or neoplastic conditions, in addition to
the ones described above, include, but are not limited to, a
fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
gastric cancer, esophageal cancer, rectal cancer, pancreatic
cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of
the head and neck, skin cancer, brain cancer, squamous cell
carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's
tumor, cervical cancer, testicular cancer, small cell lung
carcinoma, non-small cell lung carcinoma, bladder carcinoma,
epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma,
acoustic neuroma, oligodendroglioma, meningioma, melanoma,
neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi
sarcoma.
[0436] Other disorders related to angiogenesis and which may,
therefore, be treated using the molecules described herein, include
diabetic retinopathy, neovascularization (e.g., intraocular
neovascularization), psoriasis, endometriosis, Grave's disease,
ischemic disease, chronic inflammatory diseases, macular
degeneration, neovascular glaucoma, retinal fibroplasia, uveitis,
eye diseases associated with choroidal neovascularization and iris
neovascularization, hereditary hemorrhagic telangiectasia,
fibrodysplasia ossificans progressiva, idiopathic pulmonary
fibrosis, autosomal dominant polycystic kidney disease, synovitis,
familial exudative vitreoretinopathy (FEVR), Alagille syndrome,
Knobloch syndrome, disseminated lymphangiomatosis, toxic epidermal
necrolysis, Von Hippel Lindau disease (VHL), microbial-related
dysplastic and neoplastic angiomatous proliferative processes
(e.g., verruga peruana (VP)), Proteus syndrome (PS), Castleman's
disease, and Klippel-Trenaunay-Weber syndrome.
[0437] Proliferative disorders include hematopoietic neoplastic
disorders. As used herein, the term "hematopoietic neoplastic
disorders" includes diseases involving hyperplastic/neoplastic
cells of hematopoietic origin, e.g., arising from myeloid, lymphoid
or erythroid lineages, or precursor cells thereof. Preferably, the
diseases arise from poorly differentiated acute leukemias, e.g.,
erythroblastic leukemia and acute megakaryoblastic leukemia.
Additional exemplary myeloid disorders include, but are not limited
to, acute promyeloid leukemia (APML), acute myelogenous leukemia
(AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus
(1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid
malignancies include, but are not limited to acute lymphoblastic
leukemia (ALL) which includes B-lineage ALL and T-lineage ALL,
chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),
hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
Additional forms of malignant lymphomas include, but are not
limited to non-Hodgkin lymphoma and variants thereof, peripheral T
cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous
T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF),
Hodgkin's disease and Reed-Sternberg disease.
[0438] As used herein, disorders involving the brain include, but
are not limited to, disorders involving neurons, and disorders
involving glia, such as astrocytes, oligodendrocytes, ependymal
cells, and microglia; cerebral edema, raised intracranial pressure
and herniation, and hydrocephalus; malformations and developmental
diseases, such as neural tube defects, forebrain anomalies,
posterior fossa anomalies, and syringomyelia and hydromyelia;
perinatal brain injury; cerebrovascular diseases, such as those
related to hypoxia, ischemia, and infarction, including
hypotension, hypoperfusion, and low-flow states--global cerebral
ischemia and focal cerebral ischemia--infarction from obstruction
of local blood supply, intracranial hemorrhage, including
intracerebral (intraparenchymal) hemorrhage, subarachnoid
hemorrhage and ruptured berry aneurysms, and vascular
malformations, hypertensive cerebrovascular disease, including
lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-borne
(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes
simplex virus Type 2, Varicella-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including HIV-1 meningoencephalitis (subacute
encephalitis), vacuolar myelopathy, AIDS-associated myopathy,
peripheral neuropathy, and AIDS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyelinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyelitis and acute
necrotizing hemorrhagic encephalomyelitis, and other diseases with
demyelination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer disease and Pick
disease, degenerative diseases of basal ganglia and brain stem,
including Parkinsonism, idiopathic Parkinson disease (paralysis
agitans), progressive supranuclear palsy, corticobasal degenration,
multiple system atrophy, including striatonigral degenration,
Shy-Drager syndrome, and olivopontocerebellar atrophy, and
Huntington disease; spinocerebellar degenerations, including
spinocerebellar ataxias, including Friedreich ataxia, and
ataxia-telanglectasia, degenerative diseases affecting motor
neurons, including amyotrophic lateral sclerosis (motor neuron
disease), bulbospinal atrophy (Kennedy syndrome), and spinal
muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin B.sub.1) deficiency and vitamin B.sub.12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycemia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastoma multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain
stem glioma, oligodendroglioma, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), and neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type 1
neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease.
[0439] As used herein, neurological disorders include disorders of
the central nervous system (CNS) and the peripheral nervous system,
e.g., cognitive and neurodegenerative disorders, Examples of
neurological disorders include, but are not limited to, autonomic
function disorders such as hypertension and sleep disorders, and
neuropsychiatric disorders, such as depression, schizophrenia,
schizoaffective disorder, Korsakoff's psychosis, alcoholism,
anxiety disorders, or phobic disorders; learning or memory
disorders, e.g., amnesia or age-related memory loss, attention
deficit disorder, dysthymic disorder, major depressive disorder,
mania, obsessive-compulsive disorder, psychoactive substance use
disorders, anxiety, phobias, panic disorder, as well as bipolar
affective disorder, e.g., severe bipolar affective (mood) disorder
(BP-1), and bipolar affective neurological disorders, e.g.,
migraine and obesity. Such neurological disorders include, for
example, disorders involving neurons, and disorders involving glia,
such as astrocytes, oligodendrocytes, ependymal cells, and
microglia; cerebral edema, raised intracranial pressure and
herniation, and hydrocephalus; malformations and developmental
diseases, such as neural tube defects, forebrain anomalies,
posterior fossa anomalies, and syringomyelia and hydromyelia;
perinatal brain injury; cerebrovascular diseases, such as those
related to hypoxia, ischemia, and infarction, including
hypotension, hypoperfusion, and low-flow states--global cerebral
ischemia and focal cerebral ischemia--infarction from obstruction
of local blood supply, intracranial hemorrhage, including
intracerebral (intraparenchymal) hemorrhage, subarachnoid
hemorrhage and ruptured berry aneurysms, and vascular
malformations, hypertensive cerebrovascular disease, including
lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-borne
(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes
simplex virus Type 2, Varicella-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including HIV-1 meningoencephalitis (subacute
encephalitis), vacuolar myelopathy, AIDS-associated myopathy,
peripheral neuropathy, and AIDS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyelinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyelitis and acute
necrotizing hemorrhagic encephalomyelitis, and other diseases with
demyelination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer's disease and
Pick's disease, degenerative diseases of basal ganglia and brain
stem, including Parkinsonism, idiopathic Parkinson's disease
(paralysis agitans) and other Lewy diffuse body diseases,
progressive supranuclear palsy, corticobasal degenration, multiple
system atrophy, including striatonigral degenration, Shy-Drager
syndrome, and olivopontocerebellar atrophy, and Huntington's
disease, senile dementia, Gilles de la Tourette's syndrome,
epilepsy, and Jakob-Creutzfieldt disease; spinocerebellar
degenerations, including spinocerebellar ataxias, including
Friedreich ataxia, and ataxia-telanglectasia, degenerative diseases
affecting motor neurons, including amyotrophic lateral sclerosis
(motor neuron disease), bulbospinal atrophy (Kennedy syndrome), and
spinal muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin B.sub.1) deficiency and vitamin B.sub.12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycemia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastoma multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain
stem glioma, oligodendroglioma, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), and neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type 1
neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease. Further
CNS-related disorders include, for example, those listed in the
American Psychiatric Association's Diagnostic and Statistical
manual of Mental Disorders (DSM), the most current version of which
is incorporated herein by reference in its entirety.
[0440] As used herein, disorders involving blood vessels include,
but are not limited to, responses of vascular cell walls to injury,
such as endothelial dysfunction and endothelial activation and
intimal thickening; vascular diseases including, but not limited
to, congenital anomalies, such as arteriovenous fistula,
atherosclerosis, and hypertensive vascular disease, such as
hypertension; inflammatory disease--the vasculitides, such as giant
cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa
(classic), Kawasaki syndrome (mucocutaneous lymph node syndrome),
microscopic polyanglitis (microscopic polyarteritis,
hypersensitivity or leukocytoclastic anglitis), Wegener
granulomatosis, thromboanglitis obliterans (Buerger disease),
vasculitis associated with other disorders, and infectious
arteritis; Raynaud disease; aneurysms and dissection, such as
abdominal aortic aneurysms, syphilitic (luetic) aneurysms, and
aortic dissection (dissecting hematoma); disorders of veins and
lymphatics, such as varicose veins, thrombophlebitis and
phlebothrombosis, obstruction of superior vena cava (superior vena
cava syndrome), obstruction of inferior vena cava (inferior vena
cava syndrome), and lymphangitis and lymphedema; tumors, including
benign tumors and tumor-like conditions, such as hemangioma,
lymphangioma, glomus tumor (glomangioma), vascular ectasias, and
bacillary angiomatosis, and intermediate-grade (borderline
low-grade malignant) tumors, such as Kaposi sarcoma and
hemangloendothelioma, and malignant tumors, such as angiosarcoma
and hemangiopericytoma; and pathology of therapeutic interventions
in vascular disease, such as balloon angioplasty and related
techniques and vascular replacement, such as coronary artery bypass
graft surgery.
[0441] As used herein, disorders of the breast include, but are not
limited to, disorders of development; inflammations, including but
not limited to, acute mastitis, periductal mastitis, periductal
mastitis (recurrent subareolar abscess, squamous metaplasia of
lactiferous ducts), mammary duct ectasia, fat necrosis,
granulomatous mastitis, and pathologies associated with silicone
breast implants; fibrocystic changes; proliferative breast disease
including, but not limited to, epithelial hyperplasia, sclerosing
adenosis, and small duct papillomas; tumors including, but not
limited to, stromal tumors such as fibroadenoma, phyllodes tumor,
and sarcomas, and epithelial tumors such as large duct papilloma;
carcinoma of the breast including in situ (noninvasive) carcinoma
that includes ductal carcinoma in situ (including Paget's disease)
and lobular carcinoma in situ, and invasive (infiltrating)
carcinoma including, but not limited to, invasive ductal carcinoma,
no special type, invasive lobular carcinoma, medullary carcinoma,
colloid (mucinous) carcinoma, tubular carcinoma, and invasive
papillary carcinoma, and miscellaneous malignant neoplasms.
Disorders in the male breast include, but are not limited to,
gynecomastia and carcinoma.
[0442] As used herein, disorders involving the colon include, but
are not limited to, congenital anomalies, such as atresia and
stenosis, Meckel diverticulum, congenital aganglionic
megacolon-Hirschsprung disease; enterocolitis, such as diarrhea and
dysentery, infectious enterocolitis, including viral
gastroenteritis, bacterial enterocolitis, necrotizing
enterocolitis, antibiotic-associated colitis (pseudomembranous
colitis), and collagenous and lymphocytic colitis, miscellaneous
intestinal inflammatory disorders, including parasites and
protozoa, acquired immunodeficiency syndrome, transplantation,
drug-induced intestinal injury, radiation enterocolitis,
neutropenic colitis (typhlitis), and diversion colitis; idiopathic
inflammatory bowel disease, such as Crohn disease and ulcerative
colitis; tumors of the colon, such as non-neoplastic polyps,
adenomas, familial syndromes, colorectal carcinogenesis, colorectal
carcinoma, and carcinoid tumors.
[0443] As used herein, disorders involving the kidney include, but
are not limited to, congenital anomalies including, but not limited
to, cystic diseases of the kidney, that include but are not limited
to, cystic renal dysplasia, autosomal dominant (adult) polycystic
kidney disease, autosomal recessive (childhood) polycystic kidney
disease, and cystic diseases of renal medulla, which include, but
are not limited to, medullary sponge kidney, and
nephronophthisis-uremic medullary cystic disease complex, acquired
(dialysis-associated) cystic disease, such as simple cysts;
glomerular diseases including pathologies of glomerular injury that
include, but are not limited to, in situ immune complex deposition,
that includes, but is not limited to, anti-GBM nephritis, Heymann
nephritis, and antibodies against planted antigens, circulating
immune complex nephritis, antibodies to glomerular cells,
cell-mediated immunity in glomerulonephritis, activation of
alternative complement pathway, epithelial cell injury, and
pathologies involving mediators of glomerular injury including
cellular and soluble mediators, acute glomerulonephritis, such as
acute proliferative (poststreptococcal, postinfectious)
glomerulonephritis, including but not limited to, poststreptococcal
glomerulonephritis and nonstreptococcal acute glomerulonephritis,
rapidly progressive (crescentic) glomerulonephritis, nephrotic
syndrome, membranous glomerulonephritis (membranous nephropathy),
minimal change disease (lipoid nephrosis), focal segmental
glomerulosclerosis, membranoproliferative glomerulonephritis, IgA
nephropathy (Berger disease), focal proliferative and necrotizing
glomerulonephritis (focal glomerulonephritis), hereditary
nephritis, including but not limited to, Alport syndrome and thin
membrane disease (benign familial hematuria), chronic
glomerulonephritis, glomerular lesions associated with systemic
disease, including but not limited to, systemic lupus
erythematosus, Henoch-Schonlein purpura, bacterial endocarditis,
diabetic glomerulosclerosis, amyloidosis, fibrillary and
immunotactoid glomerulonephritis, and other systemic disorders;
diseases affecting tubules and interstitium, including acute
tubular necrosis and tubulointerstitial nephritis, including but
not limited to, pyelonephritis and urinary tract infection, acute
pyelonephritis, chronic pyelonephritis and reflux nephropathy, and
tubulointerstitial nephritis induced by drugs and toxins, including
but not limited to, acute drug-induced interstitial nephritis,
analgesic abuse nephropathy, nephropathy associated with
nonsteroidal anti-inflammatory drugs, and other tubulointerstitial
diseases including, but not limited to, urate nephropathy,
hypercalcemia and nephrocalcinosis, and multiple myeloma; diseases
of blood vessels including benign nephrosclerosis, malignant
hypertension and accelerated nephrosclerosis, renal artery
stenosis, and thrombotic microangiopathies including, but not
limited to, classic (childhood) hemolytic-uremic syndrome, adult
hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura,
idiopathic HUS/TTP, and other vascular disorders including, but not
limited to, atherosclerotic ischemic renal disease, atheroembolic
renal disease, sickle cell disease nephropathy, diffuse cortical
necrosis, and renal infarcts; urinary tract obstruction
(obstructive uropathy); urolithiasis (renal calculi, stones); and
tumors of the kidney including, but not limited to, benign tumors,
such as renal papillary adenoma, renal fibroma or hamartoma
(renomedullary interstitial cell tumor), angiomyolipoma, and
oncocytoma, and malignant tumors, including renal cell carcinoma
(hypernephroma, adenocarcinoma of kidney), which includes
urothelial carcinomas of renal pelvis.
[0444] Examples of disorders of the lung include, but are not
limited to, congenital anomalies; atelectasis; diseases of vascular
origin, such as pulmonary congestion and edema, including
hemodynamic pulmonary edema and edema caused by microvascular
injury, adult respiratory distress syndrome (diffuse alveolar
damage), pulmonary embolism, hemorrhage, and infarction, and
pulmonary hypertension and vascular sclerosis; chronic obstructive
pulmonary disease, such as emphysema, chronic bronchitis, bronchial
asthma, and bronchiectasis; diffuse interstitial (infiltrative,
restrictive) diseases, such as pneumoconioses, sarcoidosis,
idiopathic pulmonary fibrosis, desquamative interstitial
pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia
(pulmonary infiltration with eosinophilia), Bronchiolitis
obliterans-organizing pneumonia, diffuse pulmonary hemorrhage
syndromes, including Goodpasture syndrome, idiopathic pulmonary
hemosiderosis and other hemorrhagic syndromes, pulmonary
involvement in collagen vascular disorders, and pulmonary alveolar
proteinosis; complications of therapies, such as drug-induced lung
disease, radiation-induced lung disease, and lung transplantation;
tumors, such as bronchogenic carcinoma, including paraneoplastic
syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors,
such as bronchial carcinoid, miscellaneous tumors, and metastatic
tumors; pathologies of the pleura, including inflammatory pleural
effusions, noninflammatory pleural effusions, pneumothorax, and
pleural tumors, including solitary fibrous tumors (pleural fibroma)
and malignant mesothelioma.
[0445] As used herein, disorders involving the ovary include, for
example, polycystic ovarian disease, Stein-leventhal syndrome,
Pseudomyxoma peritonei and stromal hyperthecosis; ovarian tumors
such as, tumors of coelomic epithelium, serous tumors, mucinous
tumors, endometeriod tumors, clear cell adenocarcinoma,
cystadenofibroma, brenner tumor, surface epithelial tumors; germ
cell tumors such as mature (benign) teratomas, monodermal
teratomas, immature malignant teratomas, dysgerminoma, endodermal
sinus tumor, choriocarcinoma; sex cord-stomal tumors such as,
granulosa-theca cell tumors, thecoma-fibromas, androblastomas, hill
cell tumors, and gonadoblastoma; and metastatic tumors such as
Krukenberg tumors.
[0446] As used herein, "a prostate disorder" refers to an abnormal
condition occurring in the male pelvic region characterized by,
e.g., male sexual dysfunction and/or urinary symptoms. This
disorder may be manifested in the form of genitourinary
inflammation (e.g., inflammation of smooth muscle cells) as in
several common diseases of the prostate including prostatitis,
benign prostatic hyperplasia and cancer, e.g., adenocarcinoma or
carcinoma, of the prostate.
[0447] As used herein, the term "hematopoietic disorder" includes
neoplastic and non-neoplastic hematopoietic or immune disorders.
Examples of neoplastic immune disorders include, but are not
limited to, erythroid leukemias, or leukemias of erythroid
precursor cells, e.g., poorly differentiated acute leukemias such
as erythroblastic leukemia and acute megakaryoblastic leukemia;
acute promyeloid leukemia (APML), acute myelogenous leukemia (AML)
and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L.
(1991) Crit Rev. in Oncol./Hemotol. 11:267-97). In particular, AML
can include the uncontrolled proliferation of CD34+ cells such as
AML subtypes M1 and M2, myeloblastic leukemias with and without
maturation, and AML subtype M6, erythroleukemia (Di Guglielmo's
disease). Additional neoplastic disorders include a myelodysplastic
syndrome or preleukemic disorder, e.g., oligoblastic leukemia,
smoldering leukemia. Additional cancers of the erythroid lineage
include erythroblastosis, and other relevant diseases of the bone
marrow.
[0448] The term "leukemia" or "leukemic cancer" is intended to have
its clinical meaning, namely, a neoplastic disease in which white
corpuscle maturation is arrested at a primitive stage of cell
development. The disease is characterized by an increased number of
leukemic blast cells in the bone marrow, and by varying degrees of
failure to produce normal hematopoietic cells. The condition may be
either acute or chronic. Leukemias are further typically
categorized as being either lymphocytic i.e., being characterized
by cells which have properties in common with normal lymphocytes,
or myelocytic (or myelogenous), i.e., characterized by cells having
some characteristics of normal granulocytic cells. Acute
lymphocytic leukemia ("ALL") arises in lymphoid tissue, and
ordinarily first manifests its presence in bone marrow. Acute
myelocytic leukemia ("AML") arises from bone marrow hematopoietic
stem cells or their progeny. The term acute myelocytic leukemia
subsumes several subtypes of leukemia: myeloblastic leukemia,
promyelocytic leukemia, and myelomonocytic leukemia. In addition,
leukemias with erythroid or megakaryocytic properties are
considered myelogenous leukemias as well.
[0449] Examples of non-neoplastic hematopoieitic disorders or
diseases include, but are not limited to, autoimmune diseases
(including, for example, diabetes mellitus, arthritis (including
rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, psoriatic arthritis), multiple sclerosis,
encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,
autoimmune thyroiditis, dermatitis (including atopic dermatitis and
eczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's
disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Graves' disease, sarcoidosis,
primary biliary cirrhosis, uveitis posterior, and interstitial lung
fibrosis), graft-versus-host disease, cases of transplantation, and
allergy such as, atopic allergy.
[0450] As used herein, the term "erythroid associated disorders"
include disorders involving aberrant (increased or deficient)
erythroblast proliferation, e.g., an erythroleukemia, and aberrant
(increased or deficient) erythroblast differentiation, e.g., an
anemia. Erythrocyte-associated disorders include anemias such as,
for example, hemolytic anemias due to hereditary cell membrane
abnormalities, such as hereditary spherocytosis, hereditary
elliptocytosis, and hereditary pyropoikilocytosis; hemolytic
anemias due to acquired cell membrane defects, such as paroxysmal
nocturnal hemoglobinuria and spur cell anemia; hemolytic anemias
caused by antibody reactions, for example to the RBC antigens, or
antigens of the ABO system, Lewis system, Ii system, Rh system,
Kidd system, Duffy system, and Kell system; methemoglobinemia; a
failure of erythropoiesis, for example, as a result of aplastic
anemia, pure red cell aplasia, myelodysplastic syndromes,
sideroblastic anemias, and congenital dyserythropoietic anemia;
secondary anemia in nonhematolic disorders, for example, as a
result of chemotherapy, alcoholism, or liver disease; anemia of
chronic disease, such as chronic renal failure; and endocrine
deficiency diseases.
[0451] Agents that modulate polypeptide or nucleic acid activity or
expression of the molecules of the invention can be used to treat
anemias, in particular, anemias associated with cancer
chemotherapy, chronic renal failure, malignancies, adult and
juvenile rheumatoid arthritis, disorders of haemoglobin synthesis,
prematurity, and zidovudine treatment of HIV infection. A subject
receiving the treatment can be additionally treated with a second
agent, e.g., erythropoietin, to futher ameliorate the
condition.
[0452] As used herein, the term "erythropoietin" or "EPO" refers to
a glycoprotein produced in the kidney, which is the principal
hormone responsible for stimulating red blood cell production
(erythrogenesis). EPO stimulates the division and differentiation
of committed erythroid progenitors in the bone marrow. Normal
plasma erythropoietin levels range from 0.01 to 0.03 Units/mL, and
can increase up to 100 to 1,000-fold during hypoxia or anemia.
Graber and Krantz, Ann. Rev. Med. 29:51 (1978); Eschbach and
Adamson, Kidney Intl. 28:1 (1985). Recombinant human erythropoietin
(rHuEpo or epoetin alfa) is commercially available as EPOGEN.RTM.
(epoetin alfa, recombinant human erythropoietin) (Amgen Inc.,
Thousand Oaks, Calif.) and as PROCRIT.RTM. (epoetin alfa,
recombinant human erythropoietin) (Ortho Biotech Inc., Raritan,
N.J.).
[0453] Another example of an erythroid-associated disorder is
erythrocytosis. Erythrocytosis, a disorder of red blood cell
overproduction caused by excessive and/or ectopic erythropoietin
production, can be caused by cancers, e.g., a renal cell cancer, a
hepatocarcinoma, and a central nervous system cancer. Diseases
associated with erythrocytosis include polycythemias, e.g.,
polycythemia vera, secondary polycythemia, and relative
polycythemia.
[0454] As used herein, disorders involving the pancreas include
those of the exocrine pancreas such as congenital anomalies,
including but not limited to, ectopic pancreas; pancreatitis,
including but not limited to, acute pancreatitis; cysts, including
but not limited to, pseudocysts; tumors, including but not limited
to, cystic tumors and carcinoma of the pancreas; and disorders of
the endocrine pancreas such as, diabetes mellitus; islet cell
tumors, including but not limited to, insulinomas, gastrinomas, and
other rare islet cell tumors.
[0455] As used herein, skeletal muscle disorders include, but are
not limited to, muscular dystrophy (e.g., Duchenne muscular
dystrophy, Becker muscular dystrophy, Emery-Dreifuss muscular
dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral
muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular
dystrophy, distal muscular dystrophy, and congenital muscular
dystrophy), motor neuron diseases (e.g., amyotrophic lateral
sclerosis, infantile progressive spinal muscular atrophy,
intermediate spinal muscular atrophy, spinal bulbar muscular
atrophy, and adult spinal muscular atrophy), myopathies (e.g.,
inflammatory myopathies (e.g., dermatomyositis and polymyositis),
myotonia congenita, paramyotonia congenita, central core disease,
nemaline myopathy, myotubular myopathy, and periodic paralysis),
tumors such as rhabdomyosarcoma, and metabolic diseases of muscle
(e.g., phosphorylase deficiency, acid maltase deficiency,
phosphofructokinase deficiency, debrancher enzyme deficiency,
mitochondrial myopathy, carnitine deficiency, carnitine palmityl
transferase deficiency, phosphoglycerate kinase deficiency,
phosphoglycerate mutase deficiency, lactate dehydrogenase
deficiency, and myoadenylate deaminase deficiency).
[0456] Diseases of the skin, include but are not limited to,
disorders of pigmentation and melanocytes, including but not
limited to, vitiligo, freckle, melasma, lentigo, nevocellular
nevus, dysplastic nevi, and malignant melanoma; benign epithelial
tumors, including but not limited to, seborrheic keratoses,
acanthosis nigricans, fibroepithelial polyp, epithelial cyst,
keratoacanthoma, and adnexal (appendage) tumors; premalignant and
malignant epidermal tumors, including but not limited to, actinic
keratosis, squamous cell carcinoma, basal cell carcinoma, and
merkel cell carcinoma; tumors of the dermis, including but not
limited to, benign fibrous histiocytoma, dermatofibrosarcoma
protuberans, xanthomas, and dermal vascular tumors; tumors of
cellular immigrants to the skin, including but not limited to,
histiocytosis X, mycosis fungoides (cutaneous T-cell lymphoma), and
mastocytosis; disorders of epidermal maturation, including but not
limited to, ichthyosis; acute inflammatory dermatoses, including
but not limited to, urticaria, acute eczematous dermatitis, and
erythema multiforme; chronic inflammatory dermatoses, including but
not limited to, psoriasis, lichen planus, and lupus erythematosus;
blistering (bullous) diseases, including but not limited to,
pemphigus, bullous pemphigoid, dermatitis herpetiformis, and
noninflammatory blistering diseases: epidermolysis bullosa and
porphyria; disorders of epidermal appendages, including but not
limited to, acne vulgaris; panniculitis, including but not limited
to, erythema nodosum and erythema induratum; and infection and
infestation, such as verrucae, molluscum contagiosum, impetigo,
superficial fungal infections, and arthropod bites, stings, and
infestations.
[0457] As used herein, hormonal disorders and diseases include type
I and type II diabetes mellitus, pituitary disorders (e.g., growth
disorders), thyroid disorders (e.g., hypothyroidism or
hyperthyroidism), and reproductive or fertility disorders (e.g.,
disorders which affect the organs of the reproductive system, e.g.,
the prostate gland, the uterus, or the vagina; disorders which
involve an imbalance in the levels of a reproductive hormone in a
subject; disorders affecting the ability of a subject to reproduce;
and disorders affecting secondary sex characteristic development,
e.g., adrenal hyperplasia).
[0458] Examples of immune, e.g., inflammatory, (e.g. respiratory
inflammatory) disorders or diseases include, but are not limited
to, autoimmune diseases (including, for example, diabetes mellitus,
arthritis (including rheumatoid arthritis, juvenile rheumatoid
arthritis, osteoarthritis, psoriatic arthritis), multiple
sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus
erythematosis, autoimmune thyroiditis, dermatitis (including atopic
dermatitis and eczematous dermatitis), psoriasis, Sjogren's
Syndrome, inflammatory bowel disease, e.g. Crohn's disease and
ulcerative colitis, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, asthma, allergic asthma, chronic obstructive
pulmonary disease, cutaneous lupus erythematosus, scleroderma,
vaginitis, proctitis, drug eruptions, leprosy reversal reactions,
erythema nodosum leprosum, autoimmune uveitis, allergic
encephalomyelitis, acute necrotizing hemorrhagic encephalopathy,
idiopathic bilateral progressive sensorineural hearing loss,
aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia,
polychondritis, Wegener's granulomatosis, chronic active hepatitis,
Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves'
disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior,
and interstitial lung fibrosis), graft-versus-host disease, cases
of transplantation, and allergy such as, atopic allergy.
[0459] As used herein, disorders involving the heart, or
"cardiovascular disease" or a "cardiovascular disorder" includes a
disease or disorder which affects the cardiovascular system, e.g.,
the heart, the blood vessels, and/or the blood. A cardiovascular
disorder can be caused by an imbalance in arterial pressure, a
malfunction of the heart, or an occlusion of a blood vessel, e.g.,
by a thrombus. A cardiovascular disorder includes, but is not
limited to disorders such as arteriosclerosis, atherosclerosis,
cardiac hypertrophy, ischemia reperfusion injury, restenosis,
arterial inflammation, vascular wall remodeling, ventricular
remodeling, rapid ventricular pacing, coronary microembolism,
tachycardia, bradycardia, pressure overload, aortic bending,
coronary artery ligation, vascular heart disease, valvular disease,
including but not limited to, valvular degeneration caused by
calcification, rheumatic heart disease, endocarditis, or
complications of artificial valves; atrial fibrillation, long-QT
syndrome, congestive heart failure, sinus node dysfunction, angina,
heart failure, hypertension, atrial fibrillation, atrial flutter,
pericardial disease, including but not limited to, pericardial
effusion and pericarditis; cardiomyopathies, e.g., dilated
cardiomyopathy or idiopathic cardiomyopathy, myocardial infarction,
coronary artery disease, coronary artery spasm, ischemic disease,
arrhythmia, sudden cardiac death, and cardiovascular developmental
disorders (e.g., arteriovenous malformations, arteriovenous
fistulae, raynaud's syndrome, neurogenic thoracic outlet syndrome,
causalgia/reflex sympathetic dystrophy, hemangioma, aneurysm,
cavernous angioma, aortic valve stenosis, atrial septal defects,
atrioventricular canal, coarctation of the aorta, ebsteins anomaly,
hypoplastic left heart syndrome, interruption of the aortic arch,
mitral valve prolapse, ductus arteriosus, patent foramen ovale,
partial anomalous pulmonary venous return, pulmonary atresia with
ventricular septal defect, pulmonary atresia without ventricular
septal defect, persistance of the fetal circulation, pulmonary
valve stenosis, single ventricle, total anomalous pulmonary venous
return, transposition of the great vessels, tricuspid atresia,
truncus arteriosus, ventricular septal defects). A cardiovascular
disease or disorder also can include an endothelial cell
disorder.
[0460] As used herein, the term "atherosclerosis" is intended to
have its clinical meaning. This term refers to a cardiovascular
condition occurring as a result of narrowing down of the arterial
walls. The narrowing is due to the formation of plaques (raised
patches) or streaks in the inner lining of the arteries. These
plaques consist of foam cells of low-density lipoproteins,
oxidized-LDL, decaying muscle cells, fibrous tissue, clumps of
blood platelets, cholesterol, and sometimes calcium. They tend to
form in regions of turbulent blood flow and are found most often in
people with high concentrations of cholesterol in the bloodstream.
The number and thickness of plaques increase with age, causing loss
of the smooth lining of the blood vessels and encouraging the
formation of thrombi (blood clots). Sometimes fragments of thrombi
break off and form emboli, which travel through the bloodstream and
block smaller vessels. The blood supply is restricted to the heart,
eventually forming a blood clot leading to death. The major causes
of atherosclerosis are hypercholesterolemia (and low HDL),
hypoalphoproteinemia, and hyperlipidemia marked by high circulating
cholesterol and high lipids like LDL-cholesterol and triglycerides
in the blood. These lipids are deposited in the arterial walls,
obstructing the blood flow and forming atherosclerotic plaques
leading to death.
[0461] As used herein the term "hypercholesterolemia" is a
condition with elevated levels of circulating total cholesterol,
LDL-cholesterol and VLDL-cholesterol as per the guidelines of the
Expert Panel Report of the National Cholesterol Educational Program
(NCEP) of Detection, Evaluation of Treatment of high cholesterol in
adults (see, Arch. Int. Med. (1988)148, 36-39).
[0462] As used herein the term "hyperlipidemia" or "hyperlipemia"
is a condition where the blood lipid parameters are elevated in the
blood. This condition manifests an abnormally high concentration of
fats. The lipid fractions in the circulating blood are, total
cholesterol, low density lipoproteins, very low density
lipoproteins and triglycerides.
[0463] As used herein the term "lipoprotein" such as VLDL, LDL and
HDL, refers to a group of proteins found in the serum, plasma and
lymph and are important for lipid transport. The chemical
composition of each lipoprotein differs in that the HDL has a
higher proportion of protein versus lipid, whereas the VLDL has a
lower proportion of protein versus lipid.
[0464] As used herein, the term "triglyceride" means a lipid or
neutral fat consisting of glycerol combined with three fatty acid
molecules.
[0465] As used herein the term "xanthomatosis" is a disease
evidenced by a yellowish swelling or plaques in the skin resulting
from deposits of fat. The presence of xanthomas are usually
accompanied by raised blood cholesterol levels.
[0466] As used herein the term "apolipoprotein B" or "apoprotein B"
or "Apo B" refers to the protein component of the LDL cholesterol
transport proteins. Cholesterol synthesized de novo is transported
from the liver and intestine to peripheral tissues in the form of
lipoproteins. Most of the apolipoprotein B is secreted into the
circulatory system as VLDL.
[0467] As used herein the term "apolipoprotein A" or "apoprotein A"
or "Apo A" refers to the protein component of the HDL cholesterol
transport proteins.
[0468] "Procedural vascular trauma" includes the effects of
surgical/medical-mechanical interventions into mammalian
vasculature, but does not include vascular trauma due to the
organic vascular pathologies listed hereinabove, or to unintended
traumas, such as due to an accident. Thus, procedural vascular
traumas within the scope of the present treatment method include
(1) organ grafting or transplantation, such as transplantation and
grafting of heart, kidney, liver and the like, e.g., involving
vessel anastomosis; (2) vascular surgery, such as coronary bypass
surgery, biopsy, heart valve replacement, atheroectomy,
thrombectomy, and the like; (3) transcatheter vascular therapies
(TVT) including angioplasty, e.g., laser angioplasty and PTCA
procedures discussed hereinbelow, employing balloon catheters, or
indwelling catheters; (4) vascular grafting using natural or
synthetic materials, such as in saphenous vein coronary bypass
grafts, dacron and venous grafts used for peripheral arterial
reconstruction, etc.; (5) placement of a mechanical shunt, such as
a PTFE hemodialysis shunt used for arteriovenous communications;
and (6) placement of an intravascular stent, which may be metallic,
plastic or a biodegradable polymer. See U.S. patent application
Ser. No. 08/389,712, filed Feb. 15, 1995, which is incorporated by
reference herein. For a general discussion of implantable devices
and biomaterials from which they can be formed, see H. Kambic et
al., "Biomaterials in Artificial Organs", Chem. Eng. News, 30 (Apr.
14, 1986), the disclosure of which is incorporated by reference
herein.
[0469] As used herein, "cholesterol lowering agents," include
agents which are useful for lowering serum cholesterol such as for
example bile acid sequestering resins (e.g. colestipol
hydrochloride or cholestyramine), fish oil, stanol esters, an
ApoAII-lowering agent, a VLDL lowering agent, an ApoAI-stimulating
agent, fibric acid derivatives (e.g. clofibrate, fenofibrate, or
gemfibrozil), thiazolidenediones (e.g. troglitazone), or HMG-CoA
reductase inhibitors (e.g. statins, such as fluvastatin sodium,
lovastatin, pravastatin sodium, or simvastatin), as well as
nicotinic acid, niacin, or probucol.
[0470] "VLDL-lowering agent" includes an agent which decreases the
hepatic synthesis of triglyceride-rich lipoproteins or increases
the catabolism of triglyceride-rich lipoproteins, e.g., fibrates
such as gemfibrozil, or the statins, increases the expression of
the apoE-mediated clearance pathway, or improves insulin
sensitivity in diabetics, e.g., the thiazolidene diones.
[0471] As used herein, a "lipid homeostasis disorder" includes a
disorder, disease, or condition associated with, caused by, and/or
linked to abnormal regulation (e.g., upregulation or
downregulation) of lipid metabolism. Lipid homeostasis disorders
may be caused by or associated with aberrant lipolysis, aberrant
lipid uptake, aberrant lipid synthesis and/or secretion, aberrant
intracellular lipid release and/or turnover, aberrant intracellular
triglyceride release and/or turnover, aberrant intracellular lipid
and/or triglyceride mass, and/or aberrant secreted lipid and/or
triglyceride mass within or from a cell, e.g., a liver cell. Lipid
homeostasis disorders include, but are not limited to,
atherosclerosis, obesity, diabetes, insulin resistance,
hyperlipidemia, hypolipidemia, dyslipidemia, hypercholesterolemia,
hypocholesterolemia, triglyceride storage disease, cardiovascular
disease, coronary artery disease, hypertension, stroke, overweight,
anorexia, cachexia, hyperlipoproteinemia, hypolipoproteinemia,
Niemann Pick disease, hypertriglyceridemia, hypotriglyceridemia,
pancreatitis, diffuse idiopathic skeletal hyperostosis (DISH),
atherogenic lipoprotein phenotype (ALP), epilepsy, liver disease,
fatty liver, steatohepatitis, and polycystic ovarian syndrome.
[0472] Disorders which can be treated or diagnosed by methods
described herein include, but are not limited to, disorders
associated with an accumulation in the liver of fibrous tissue,
such as that resulting from an imbalance between production and
degradation of the extracellular matrix accompanied by the collapse
and condensation of preexisting fibers. The methods described
herein can be used to diagnose or treat hepatocellular necrosis or
injury induced by a wide variety of agents including processes
which disturb homeostasis, such as an inflammatory process, tissue
damage resulting from toxic injury or altered hepatic blood flow,
and infections (e.g., bacterial, viral and parasitic). For example,
the methods can be used for the early detection of hepatic injury,
such as portal hypertension or hepatic fibrosis. In addition, the
methods can be employed to detect liver fibrosis attributed to
inborn errors of metabolism, for example, fibrosis resulting from a
storage disorder such as Gaucher's disease (lipid abnormalities) or
a glycogen storage disease, A1-antitrypsin deficiency; a disorder
mediating the accumulation (e.g., storage) of an exogenous
substance, for example, hemochromatosis (iron-overload syndrome)
and copper storage diseases (Wilson's disease), disorders resulting
in the accumulation of a toxic metabolite (e.g., tyrosinemia,
fructosemia and galactosemia) and peroxisomal disorders (e.g.,
Zellweger syndrome). Additionally, the methods described herein can
be used for the early detection and treatment of liver injury
associated with the administration of various chemicals or drugs,
such as for example, methotrexate, isonizaid, oxyphenisatin,
methyldopa, chlorpromazine, tolbutamide or alcohol, or which
represents a hepatic manifestation of a vascular disorder such as
obstruction of either the intrahepatic or extrahepatic bile flow or
an alteration in hepatic circulation resulting, for example, from
chronic heart failure, veno-occlusive disease, portal vein
thrombosis or Budd-Chiari syndrome.
[0473] Disorders involving the small intestine include the
malabsorption syndromes such as, celiac sprue, tropical sprue
(postinfectious sprue), whipple disease, disaccharidase (lactase)
deficiency, abetalipoproteinemia, and tumors of the small intestine
including adenomas and adenocarcinoma.
[0474] Examples of pain conditions include, but are not limited to,
pain elicited during various forms of tissue injury, e.g.,
inflammation, infection, and ischemia; pain associated with
musculoskeletal disorders, e.g., Joint pain, or arthritis; tooth
pain; headaches, e.g., migrane; pain associated with surgery; pain
related to inflammation, e.g., irritable bowel syndrome; chest
pain; or hyperalgesia, e.g., excessive sensitivity to pain
(described in, for example, Fields (1987) Pain, New
York:McGraw-Hill). Other examples of pain disorders or pain
syndromes include, but are not limited to, complex regional pain
syndrome (CRPS), reflex sympathetic dystrophy (RSD), causalgia,
neuralgia, central pain and dysesthesia syndrome, carotidynia,
neurogenic pain, refractory cervicobrachial pain syndrome,
myofascial pain syndrome, craniomandibular pain dysfunction
syndrome, chronic idiopathic pain syndrome, Costen's
pain-dysfunction, acute chest pain syndrome, nonulcer dyspepsia,
interstitial cystitis, gynecologic pain syndrome, patellofemoral
pain syndrome, anterior knee pain syndrome, recurrent abdominal
pain in children, colic, low back pain syndrome, neuropathic pain,
phantom pain from amputation, phantom tooth pain, or pain asymbolia
(the inability to feel pain). Other examples of pain conditions
include pain induced by parturition, or post partum pain.
[0475] As used herein, an "endothelial cell disorder" includes a
disorder characterized by aberrant, unregulated, or unwanted
endothelial cell activity, e.g., proliferation, migration,
angiogenesis, or vascularization; or aberrant expression of cell
surface adhesion molecules or genes associated with angiogenesis,
e.g., TIE-2, FLT and FLK. Endothelial cell disorders include
tumorigenesis, tumor metastasis, psoriasis, diabetic retinopathy,
endometriosis, Grave's disease, ischemic disease (e.g.,
atherosclerosis), and chronic inflammatory diseases (e.g.,
rheumatoid arthritis).
[0476] Additionally, the molecules of the invention can play an
important role in the etiology of certain viral diseases, including
but not limited to Hepatitis B, Hepatitis C and Herpes Simplex
Virus (HSV). Modulators of the activity of the molecules of the
invention could be used to control viral diseases. The modulators
can be used in the treatment and/or diagnosis of viral infected
tissue or virus-associated tissue fibrosis, especially liver and
liver fibrosis. Also, such modulators can be used in the treatment
and/or diagnosis of virus-associated carcinoma, especially
hepatocellular cancer.
[0477] Additionally, molecules of the invention can play an
important role in the regulation of metabolism or pain disorders.
Diseases of metabolic imbalance include, but are not limited to,
obesity, anorexia nervosa, cachexia, lipid disorders, and diabetes.
Examples of pain disorders include, but are, not limited to, pain
response elicited during various forms of tissue injury, e.g.,
inflammation, infection, and ischemia, usually referred to as
hyperalgesia (described in, for example, Fields (1987) Pain, New
York:McGraw-Hill); pain associated with musculoskeletal disorders,
e.g., joint pain; tooth pain; headaches; pain associated with
surgery; pain related to irritable bowel syndrome; or chest
pain.
[0478] Aberrant expression and/or activity of the molecules of the
invention can mediate disorders associated with bone metabolism.
"Bone metabolism" refers to direct or indirect effects in the
formation or degeneration of bone structures, e.g., bone formation,
bone resorption, etc., which can ultimately affect the
concentrations in serum of calcium and phosphate. This term also
includes activities mediated by the molecules of the invention in
bone cells, e.g. osteoclasts and osteoblasts, that can in turn
result in bone formation and degeneration. For example, molecules
of the invention can support different activities of bone resorbing
osteoclasts such as the stimulation of differentiation of monocytes
and mononuclear phagocytes into osteoclasts. Accordingly, molecules
of the invention that modulate the production of bone cells can
influence bone formation and degeneration, and thus can be used to
treat bone disorders. Examples of such disorders include, but are
not limited to, osteoporosis, osteodystrophy, osteomalacia,
rickets, osteitis fibrosa cystica, renal osteodystrophy,
osteosclerosis, anti-convulsant treatment, osteopenia,
fibrogenesis-imperfecta ossium, secondary hyperparathyrodism,
hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive
jaundice, drug induced metabolism, medullary carcinoma, chronic
renal disease, rickets, sarcoidosis, glucocorticoid antagonism,
malabsorption syndrome, steatorrhea, tropical sprue, idiopathic
hypercalcemia and milk fever.
[0479] As used herein, platelet disorders include, but are not
limited to, thrombocytopenia, include idiopathic thrombocytopenic
purpura, including acute idiopathic thrombocytopenic purpura,
drug-induced thrombocytopenia, HIV-associated thrombocytopenia, and
thrombotic microangiopathies: thrombotic thrombocytopenic purpura
and hemolytic-uremic syndrome.
[0480] Various aspects of the invention are described in further
detail below.
[0481] Isolated Nucleic Acid Molecules
[0482] In one aspect, the invention provides, an isolated or
purified, nucleic acid molecule that encodes a 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
polypeptide described herein, e.g., a full length 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein or a fragment thereof, e.g., a biologically active
portion of 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 protein. Also included is a nucleic
acid fragment suitable for use as a hybridization probe, which can
be used, e.g., to identify a nucleic acid molecule encoding a
polypeptide of the invention, 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 mRNA, and
fragments suitable for use as primers, e.g., PCR primers for the
amplification or mutation of nucleic acid molecules.
[0483] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO: 1,
3, 7, 9, 16, 18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41, 42, 44,
63, 65, 67, 69, 70 or 72, or a portion of any of this nucleotide
sequence. In one embodiment, the nucleic acid molecule includes
sequences encoding the human 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein (i.e.,
"the coding region" of SEQ ID NO: 1, 7, 16, 20, 25, 28, 35, 39, 42,
63, 67 or 70, as shown in SEQ ID NO: 3, 9, 18, 22, 27, 30, 37, 41,
44, 65, 69 or 72, respectively), as well as 5' untranslated
sequences and 3' untranslated sequences. Alternatively, the nucleic
acid molecule can include only the coding region of SEQ ID NO: 1,
7, 16, 20, 25, 28, 35, 39, 42, 63, 67 or 70 (e.g., SEQ ID NO: 3, 9,
18, 22, 27, 30, 37, 41, 44, 65, 69 or 72) and, e.g., no flanking
sequences which normally accompany the subject sequence. In another
embodiment, the nucleic acid molecule encodes a sequence
corresponding to a fragment of the protein corresponding to
conserved domains identified within SEQ ID NO: 2, 8, 17, 21, 26,
29, 36, 40, 43, 64, 68 or 71.
[0484] 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 SEQ ID NO: 1, 3, 7,
9, 16, 18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41, 42, 44, 63, 65,
67, 69, 70 or 72, or a portion of any of these nucleotide
sequences. In other embodiments, the nucleic acid molecule of the
invention is sufficiently complementary to the nucleotide sequence
shown in SEQ ID NO: 1, 3, 7, 9, 16, 18, 20, 22, 25, 27, 28, 30, 35,
37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72 such that it can
hybridize to the nucleotide sequence shown in SEQ ID NO: 1, 3, 7,
9, 16, 18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41, 42, 44, 63, 65,
67, 69, 70 or 72, thereby forming a stable duplex.
[0485] In one embodiment, an isolated nucleic acid molecule of the
present 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%, 99%, or more homologous to the entire length of the
nucleotide sequence shown in SEQ ID NO: 1, 3, 7, 9, 16, 18, 20, 22,
25, 27, 28, 30, 35, 37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72,
or a portion, preferably of the same length, of any of these
nucleotide sequences.
[0486] 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 Nucleic Acid Fragments
[0487] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO: 1, 3, 7, 9, 16,
18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41, 42, 44, 63, 65, 67, 69,
70 or 72. For example, such a nucleic acid molecule can include a
fragment which can be used as a probe or primer or a fragment
encoding a portion of a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein, e.g., an
immunogenic or biologically active portion of a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein. A fragment can comprise those nucleotides of SEQ ID
NO: 1, 3, 7, 9, 16, 18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41, 42,
44, 63, 65, 67, 69, 70 or 72, which encode a domain of human 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933. The nucleotide sequence determined from the cloning
of the 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 gene allows for the generation of
probes and primers designed for use in identifying and/or cloning
other 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 family members, or fragments thereof,
as well as 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 homologs, or fragments thereof, from
other species.
[0488] 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' noncoding region. Other
embodiments include a fragment which 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 which are at
least 100 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.
[0489] 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. Thus, for example, a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,49937,49931
or 49933 nucleic acid fragment can include a sequence corresponding
to a domain, as described herein.
[0490] 25869,25934,26335, 50365,21117,38692, 46508, 16816,
16839,49937, 49931 or 49933 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 SEQ ID NO: 1, 3, 7,
9, 16, 18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41, 42, 44, 63, 65,
67, 69, 70 or 72, or of a naturally occurring allelic variant or
mutant of SEQ ID NO: 1, 3, 7, 9, 16, 18, 20, 22, 25, 27, 28, 30,
35, 37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72.
[0491] 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 less than in 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.
[0492] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid which encodes a domain
identified in the 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or49933 sequences.
[0493] 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 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 sequence, e.g., a
domain, region, site or other sequence described herein. 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 differ by one base from a sequence
disclosed herein or from a naturally occurring variant.
[0494] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0495] A nucleic acid fragment encoding a "biologically active
portion of a 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 polypeptide" can be prepared by
isolating a portion of the nucleotide sequence of SEQ ID NO: 1, 3,
7, 9, 16, 18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41, 42, 44, 63,
65, 67, 69, 70 or 72, which encodes a polypeptide having a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 biological activity (e.g., the biological activities
of the 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 proteins are described herein),
expressing the encoded portion of the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein
(e.g., by recombinant expression in vitro) and assessing the
activity of the encoded portion of the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein. A
nucleic acid fragment encoding a biologically active portion of a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 polypeptide, can comprise a nucleotide
sequence which is greater than 300 or more nucleotides in
length.
[0496] In preferred embodiments, a nucleic acid includes a
nucleotide sequence which is about 300, 400, 500, 600, 700, 800,
900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,
2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000,
3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100,
4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200,
5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300,
6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7249 or more
nucleotides in length and hybridizes under stringent hybridization
conditions to a nucleic acid molecule of SEQ ID NO: 1, 3, 7, 9, 16,
18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41, 42, 44, 63, 65, 67, 69,
70 or 72.
[0497] 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 Nucleic Acid Variants
[0498] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO: 1, 3,
7, 9, 16, 18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41, 42, 44, 63,
65, 67, 69, 70 or 72. Such differences can be due to degeneracy of
the genetic code (and result in a nucleic acid which encodes the
same 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 proteins as those encoded by the nucleotide
sequence disclosed herein. In another embodiment, an isolated
nucleic acid molecule of the invention has a nucleotide sequence
encoding a protein having an amino acid sequence which differs, by
at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues
that shown in SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68
or 71. 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.
[0499] Nucleic acids of the inventor can be chosen for having
codons, which are preferred, or non-preferred, for a particular
expression system. E.g., 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.
[0500] 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).
[0501] In a preferred embodiment, the nucleic acid differs from
that of SEQ ID NO: 1, 3, 7, 9, 16, 18, 20, 22, 25, 27, 28, 30, 35,
37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72, e.g., as follows: by
at least one but less than 10, 20, 30, or 40 nucleotides; at least
one but less than 1%, 5%, 10% or 20% of the nucleotides 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.
[0502] 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 SEQ ID NO: 2, 8, 17,
21, 26, 29, 36, 40, 43, 64, 68 or 71 or a fragment of this
sequence. Such nucleic acid molecules can readily be identified as
being able to hybridize under stringent conditions, to the
nucleotide sequence shown in SEQ ID NO: 2, 8, 17, 21, 26, 29, 36,
40, 43, 64, 68 or 71 or a fragment of the sequence. Nucleic acid
molecules corresponding to orthologs, homologs, and allelic
variants of the 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 cDNAs of the invention can
further be isolated by mapping to the same chromosome or locus as
the 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 gene.
[0503] Preferred variants include those that are correlated with
activities specific to the molecules of the invention, i.e.
carboxylase activity, fatty acid desaturase activity,
serine/threonine dehydratase activity, hexokinase activity,
peptidyl tRNA hydrolase activity, dual specificity phosphatase
activity, phospholipase activity, transporter activity, or
other.
[0504] Allelic variants of 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933, e.g., human
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933, include both functional and non-functional
proteins. Functional allelic variants are naturally occurring amino
acid sequence variants of the 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein within a
population that maintain the ability to (1) hydrolyze an ester
linkage and/or liberate the free acid form of a substrate, e.g.,
hydrolysis of a triglyceride and/or liberation of free fatty
acid(s) and glycerol; (2) catalyze the formation of a double bond,
preferably, at positions up to 9 carbons from the carboxyl end of a
molecule, e.g., a fatty acid, such as a polyunsaturated fatty acid;
(3) catalyze the phosphorylation of a sugar, e.g., an aldohexoses
and a ketohexoses (e.g., glucose, mannose, fructose, sorbitol and
glucosamine); (4) catalyze sugar metabolism; (5) transfer a
phosphate from a phosphate donor (e.g., ATP) to a sugar, e.g., an
aldohexoses and a ketohexoses (e.g., glucose, mannose, fructose,
sorbitol and glucosamine) to form a phosphorylated sugar, e.g.,
glucose-6-phosphate; (6) catalyze the removal of a phosphate group
attached to a tyrosine residue in a protein target, e.g., a growth
factor receptor; (7) catalyze the removal of a phosphate group
attached to a serine or threonine residue in a protein e.g., a
growth factor receptor; (8) hydrolyze covalent bond between peptide
and tRNA within peptidyl-tRNAs; (9) catalyze the hydrolysis of
phosphatidyl-inositol-4,5-bisphosphate (PIP2) producing
diacylglycerol and inositol 1,4,5-trisphosphate; (10) transport a
substrate or target molecule (e.g., a Ca.sup.2+ ion) from one side
of a biological membrane to the other; or (11) be phosphorylated or
dephosphorylated. Functional allelic variants will typically
contain only conservative substitution of one or more amino acids
of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71, 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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933, e.g., human 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933, protein
within a population that do not have the ability to (1) hydrolyze
an ester linkage and/or liberate the free acid form of a substrate,
e.g., hydrolysis of a triglyceride and/or liberation of free fatty
acid(s) and glycerol; (2) catalyze the formation of a double bond,
preferably, at positions up to 9 carbons from the carboxyl end of a
molecule, e.g., a fatty acid, such as a polyunsaturated fatty acid;
(3) catalyze the phosphorylation of a sugar, e.g., an aldohexoses
and a ketohexoses (e.g., glucose, mannose, fructose, sorbitol and
glucosamine); (4) catalyze sugar metabolism; (5) transfer a
phosphate from a phosphate donor (e.g., ATP) to a sugar, e.g., an
aldohexoses and a ketohexoses (e.g., glucose, mannose, fructose,
sorbitol and glucosamine) to form a phosphorylated sugar, e.g.,
glucose-6-phosphate; (6) catalyze the removal of a phosphate group
attached to a tyrosine residue in a protein target, e.g., a growth
factor receptor; (7) catalyze the removal of a phosphate group
attached to a serine or threonine residue in a protein e.g., a
growth factor receptor; (8) hydrolyze covalent bond between peptide
and tRNA within peptidyl-tRNAs; (9) catalyze the hydrolysis of
phosphatidyl-inositol-4,5-- bisphosphate (PIP2) producing
diacylglycerol and inositol 1,4,5-trisphosphate; (10) transport a
substrate or target molecule (e.g., a Ca.sup.2+ ion) from one side
of a biological membrane to the other; or (11) be phosphorylated or
dephosphorylated. 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,
8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71, or a substitution,
insertion, or deletion in critical residues or critical regions of
the protein.
[0505] Moreover, nucleic acid molecules encoding other 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 family members and, thus, which have a nucleotide
sequence which differs from the 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 sequences of SEQ
ID NO: 1, 3, 7, 9, 16, 18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41,
42, 44, 63, 65, 67, 69, 70 or 72 are intended to be within the
scope of the invention.
[0506] Antisense Nucleic Acid Molecules, Ribozymes and Modified
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 Nucleic Acid Molecules
[0507] In another aspect, the invention features, an isolated
nucleic acid molecule which is antisense to 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933. An
"antisense" nucleic acid can include a nucleotide sequence which 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 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
coding strand, or to only a portion thereof (e.g., the coding
region of human 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 corresponding to SEQ ID NO: 3,
9, 18, 22, 27, 30, 37, 41, 44, 65, 69 or 72, respectively). In
another embodiment, the antisense nucleic acid molecule is
antisense to a "noncoding region" of the coding strand of a
nucleotide sequence encoding 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 (e.g., the 5' and
3' untranslated regions).
[0508] An antisense nucleic acid can be designed such that it is
complementary to the entire coding region of 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
mRNA, but more preferably is an oligonucleotide which is antisense
to only a portion of the coding or noncoding region of 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 mRNA. For example, the antisense oligonucleotide can
be complementary to the region surrounding the translation start
site of 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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, 80, or more nucleotides
in length.
[0509] 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 subcloned 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).
[0510] 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 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 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 or selectively bind to receptors or antigens expressed
on a selected cell surface, e.g., by linking the antisense nucleic
acid molecules to peptides or antibodies which 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 II or pol III
promoter are preferred.
[0511] 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) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0512] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. A ribozyme having specificity for a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933-encoding nucleic acid can include one or more
sequences complementary to the nucleotide sequence of a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 cDNA disclosed herein (i.e., SEQ ID NO: 1, 3, 7, 9,
16, 18, 20, 22, 25, 27, 28, 30, 35, 37, 39, 41, 42, 44, 63, 65, 67,
69, 70 or 72), and a sequence having known catalytic sequence
responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or
Haselhoff and Gerlach (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 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No.
4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 mRNA can be used to select a catalytic RNA
having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel and Szostak (1993) Science
261:1411-1418.
[0513] 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 (e.g., the 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
promoter and/or enhancers) to form triple helical structures that
prevent transcription of the 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 gene in target
cells. See generally, Helene (1991) Anticancer Drug Des. 6:569-84;
Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992)
Bioassays 14:807-15. 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'-3', 3'-5' manner, such that they
base pair 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.
[0514] The invention also provides detectably labeled
oligonucleotide primer and probe molecules. Typically, such labels
are chemiluminescent, fluorescent, radioactive, or
colorimetric.
[0515] A 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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 (see
Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4:
5-23).
[0516] As used herein, the terms "peptide nucleic acid" or "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-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. 93:
14670-675.
[0517] PNAs of 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 nucleic acid molecules can be
used in therapeutic and diagnostic applications. For example, PNAs
can be used as antisense or antigene agents for sequence-specific
modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs
of 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 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
(Hyrup et al. (1996) supra)); or as probes or primers for DNA
sequencing or hybridization (Hyrup et al. (1996) supra;
Perry-O'Keefe supra).
[0518] 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 (see, 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 No. WO88/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In
addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (see, e.g., Krol et al.
(1988) Bio-Techniques 6:958-976) or intercalating agents. (see,
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).
[0519] The invention also includes molecular beacon oligonucleotide
primer and probe molecules having at least one region which is
complementary to a 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 nucleic acid of the invention,
two complementary regions one having a fluorophore and one a
quencher such that the molecular beacon is useful for quantitating
the presence of the 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 nucleic acid of the
invention in a sample. Molecular beacon nucleic acids are
described, for example, in Lizardi et al., U.S. Pat. No. 5,854,033;
Nazarenko et al., U.S. Pat. No. 5,866,336, and Livak et al., U.S.
Pat. No. 5,876,930.
[0520] Isolated 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 Polypeptides
[0521] In another aspect, the invention features, an isolated
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 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-25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 antibodies.
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein can be isolated from cells or tissue
sources using standard protein purification techniques. 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein or fragments thereof can be produced by
recombinant DNA techniques or synthesized chemically.
[0522] Polypeptides of the invention include those which 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 in a native cell.
[0523] In a preferred embodiment, a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
polypeptide has one or more of the following characteristics: it
has the ability to (1) hydrolyze an ester linkage and/or liberate
the free acid form of a substrate, e.g., hydrolysis of a
triglyceride and/or liberation of free fatty acid(s) and glycerol;
(2) catalyze the formation of a double bond, preferably, at
positions up to 9 carbons from the carboxyl end of a molecule,
e.g., a fatty acid, such as a polyunsaturated fatty acid; (3)
catalyze the phosphorylation of a sugar, e.g., an aldohexoses and a
ketohexoses (e.g., glucose, mannose, fructose, sorbitol and
glucosamine); (4) catalyze sugar metabolism; (5) transfer a
phosphate from a phosphate donor (e.g., ATP) to a sugar, e.g., an
aldohexoses and a ketohexoses (e.g., glucose, mannose, fructose,
sorbitol and glucosamine) to form a phosphorylated sugar, e.g.,
glucose-6-phosphate; (6) catalyze the removal of a phosphate group
attached to a tyrosine residue in a protein target, e.g., a growth
factor receptor; (7) catalyze the removal of a phosphate group
attached to a serine or threonine residue in a protein e.g., a
growth factor receptor; (8) hydrolyze covalent bond between peptide
and tRNA within peptidyl-tRNAs; (9) catalyze the hydrolysis of
phosphatidyl-inositol-4,5-bisphosphate (PIP2) producing
diacylglycerol and inositol 1,4,5-trisphosphate; (10) transport a
substrate or target molecule (e.g., a Ca.sup.2+ ion) from one side
of a biological membrane to the other; (11) be phosphorylated or
dephosphorylated; (12) it has a molecular weight, e.g., a deduced
molecular weight, preferably ignoring any contribution of post
translational modifications, amino acid composition or other
physical characteristic of a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 polypeptide,
e.g., a polypeptide of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43,
64, 68 or 71; (13) it has an overall sequence similarity of at
least 60%, preferably at least 70%, more preferably at least 80,
90, or 95%, with a polypeptide of SEQ ID NO: 2, 8, 17, 21, 26, 29,
36, 40, 43, 64, 68 or 71; (14) it is expressed in a multitude of
human tissues and cell lines (refer to section for each molecule of
the invention); and (15) it has specific domains which are
preferably about 70%, 80%, 90% or 95% identical to the identified
amino acid residues of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43,
64, 68 or 71 (refer to section for each molecule of the invention
for domain names and locations within amino acid sequence).
[0524] In a preferred embodiment the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein,
or fragment thereof, differs from the corresponding sequence in SEQ
ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71. In one
embodiment it differs by at least one but by less than 15, 10 or 5
amino acid residues. In another it differs from the corresponding
sequence in SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or
71 by at least one residue but less than 20%, 15%, 10% or 5% of the
residues in it differ from the corresponding sequence in SEQ ID NO:
2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71. (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 residue or a
conservative substitution. In a preferred embodiment the
differences are not in the identified or conserved domain(s) within
SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71. In
another embodiment one or more differences are in the cidentified
or conserved domain(s) within SEQ ID NO: 2, 8, 17, 21, 26, 29, 36,
40, 43, 64, 68 or71. 7
[0525] Other embodiments include a protein that contains one or
more changes in amino acid sequence, e.g., a change in an amino
acid residue which is not essential for activity. Such 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 proteins differ in amino acid sequence from SEQ ID
NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71, yet retain
biological activity.
[0526] 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, 8, 17, 21, 26, 29, 36, 40, 43,
64, 68 or 71.
[0527] A 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 protein or fragment is provided which
varies from the sequence of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36,
40, 43, 64, 68 or 71 in regions defined by amino acids that are not
within identified or conserved domains or regions by at least one
but by less than 15, 10 or 5 amino acid residues in the protein or
fragment but which does not differ from SEQ ID NO: 2, 8, 17, 21,
26, 29, 36, 40, 43, 64, 68 or 71 in regions defined by amino acids
that are within identified or conserved domains or regions. (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.
[0528] In one embodiment, a biologically active portion of a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein includes an identified domain (refer to
section for each molecule of the invention). 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
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein.
[0529] In a preferred embodiment, the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein
has an amino acid sequence shown in SEQ ID NO: 2, 8, 17, 21, 26,
29, 36, 40, 43, 64, 68 or 71. In other embodiments, the 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein is sufficiently or substantially identical
to SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or 71. In
yet another embodiment, the 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein is
sufficiently or substantially identical to SEQ ID NO: 2, 8, 17, 21,
26, 29, 36, 40, 43, 64, 68 or 71 and retains the functional
activity of the protein of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40,
43, 64, 68 or 71, as described in detail in the subsections
above.
[0530] 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 Chimeric or Fusion Proteins
[0531] In another aspect, the invention provides 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 chimeric or fusion proteins. As used herein, a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 "chimeric protein" or "fusion protein" includes a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 polypeptide linked to a non-25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
polypeptide. A "non-25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous to the 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein, e.g., a protein which is different from the 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein and which is derived from the same or a
different organism. The 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 polypeptide of the
fusion protein can correspond to all or a portion e.g., a fragment
described herein of a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 amino acid sequence. In
a preferred embodiment, a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 fusion protein includes
at least one (or two) biologically active portion of a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein. The non-25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 polypeptide can
be fused to the N-terminus or C-terminus of the 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 polypeptide.
[0532] The fusion protein can include a moiety which has a high
affinity for a ligand. For example, the fusion protein can be a
GST-25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 fusion protein in which the 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 sequences are fused to the C-terminus of the GST sequences.
Such fusion proteins can facilitate the purification of recombinant
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933. Alternatively, the fusion protein can be a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein containing a heterologous signal
sequence at its N-terminus. In certain host cells (e.g., mammalian
host cells), expression and/or secretion of 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 can
be increased through use of a heterologous signal sequence.
[0533] Fusion proteins can include all or a part of a serum
protein, e.g., a portion of an immunoglobulin (e.g., IgG, IgA, or
IgE), e.g., an Fc region and/or the hinge C1 and C2 sequences of an
immunoglobulin or human serum albumin.
[0534] The 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 fusion proteins of the invention can
be incorporated into pharmaceutical compositions and administered
to a subject in vivo. The 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 fusion proteins can be
used to affect the bioavailability of a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 substrate.
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 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 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
protein; (ii) mis-regulation of the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 gene; and
(iii) aberrant post-translational modification of a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein.
[0535] Moreover, the 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933-fusion proteins of the
invention can be used as immunogens to produce anti-25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 antibodies in a subject, to purify 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
ligands and in screening assays to identify molecules which inhibit
the interaction of 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 with a 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
substrate.
[0536] Expression vectors are commercially available that already
encode a fusion moiety (e.g., a GST polypeptide). A 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933-encoding nucleic acid can be cloned into such an expression
vector such that the fusion moiety is linked in-frame to the 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein.
[0537] Variants of 25869, 25934, 26335, 50365, 21117, 38692. 46508,
16816, 16839, 49937, 49931 or 49933 Proteins
[0538] In another aspect, the invention also features a variant of
a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 polypeptide, e.g., which functions as an
agonist (mimetics) or as an antagonist. Variants of the 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 proteins can be generated by mutagenesis, e.g.,
discrete point mutation, the insertion or deletion of sequences or
the truncation of a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein. An agonist of
the 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 proteins can retain substantially the same,
or a subset, of the biological activities of the naturally
occurring form of a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein. An antagonist
of a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein can inhibit one or more of the
activities of the naturally occurring form of the 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein by, for example, competitively modulating a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-mediated activity of a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein.
[0539] Variants of a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein can be
identified by screening combinatorial libraries of mutants, e.g.,
truncation mutants, of a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein for agonist or
antagonist activity.
[0540] Libraries of fragments e.g., N terminal, C terminal, or
internal fragments, of a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein coding sequence
can be used to generate a variegated population of fragments for
screening and subsequent selection of variants of a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein.
[0541] Variants in which a cysteine residues is added or deleted or
in which a residue which is glycosylated is added or deleted are
particularly preferred.
[0542] 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 are
known in the art. Recursive ensemble mutagenesis (REM), a new
technique which enhances the frequency of functional mutants in the
libraries, can be used in combination with the screening assays to
identify 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 variants (Arkin and Yourvan (1992)
Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993)
Protein Engineering 6:327-331).
[0543] Cell based assays can be exploited to analyze a variegated
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 library. For example, a library of expression
vectors can be transfected into a cell line, e.g., a cell line,
which ordinarily responds to 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 in a
substrate-dependent manner. The transfected cells are then
contacted with 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 and the effect of the
expression of the mutant on signaling by the 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
substrate can be detected, e.g., by measuring either carboxylase
activity, fatty acid desaturase activity, serine/threonine
dehydratase activity, hexokinase activity, peptidyl tRNA hydrolase
activity, dual specificity phosphatase activity, phospholipase
activity, transporter activity, or other activity disclosed herein.
Plasmid DNA can then be recovered from the cells which score for
inhibition, or alternatively, potentiation of signaling by the
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 substrate, and the individual clones further
characterized.
[0544] In another aspect, the invention features a method of making
a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 polypeptide, e.g., a peptide having a
non-wild type activity, e.g., an antagonist, agonist, or super
agonist of a naturally occurring 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 polypeptide,
e.g., a naturally occurring 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 polypeptide. The
method includes altering the sequence of a 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
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.
[0545] In another aspect, the invention features a method of making
a fragment or analog of a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 polypeptide a biological
activity of a naturally occurring 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
polypeptide. The method includes altering the sequence, e.g., by
substitution or deletion of one or more residues, of a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 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.
[0546] Anti-25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 Antibodies
[0547] In another aspect, the invention provides an anti-25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 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
scFV and dcFV fragments, Fab and F(ab').sub.2 fragments which can
be generated by treating the antibody with an enzyme such as papain
or pepsin, respectively.
[0548] 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.
[0549] A full-length 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein or, antigenic
peptide fragment of 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 can be used as an
immunogen or can be used to identify anti-25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
antibodies made with other immunogens, e.g., cells, membrane
preparations, and the like. The antigenic peptide of 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 should include at least 8 amino acid residues of the amino
acid sequence shown in SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43,
64, 68 or 71 and encompasses an epitope of 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933.
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.
[0550] Fragments of 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 which include
hydrophilic regions of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43,
64, 68 or 71 can be used to make, e.g., used as immunogens or used
to characterize the specificity of an antibody, antibodies against
hydrophilic regions of the 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein.
Similarly, fragments of 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 which include
hydrophobic regions of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43,
64, 68 or 71 can be used to make an antibody against a hydrophobic
region of the 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 protein; fragments of 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 which include residues within extra cellular
domain(s) of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40, 43, 64, 68 or
71 can be used to make an antibody against an extracellular or
non-cytoplasmic region of the 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein;
fragments of 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 which include residues within
intracellular regions of SEQ ID NO: 2, 8, 17, 21, 26, 29, 36, 40,
43, 64, 68 or 71 can be used to make an antibody against an
intracellular region of the 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein; a
fragment of 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 which include residues within
identified or conserved domains of SEQ ID NO: 2, 8, 17, 21, 26, 29,
36, 40, 43, 64, 68 or 71 can be used to make an antibody against
the identified or conserved domain of the 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
protein.
[0551] Antibodies reactive with, or specific or selective for, any
of these regions, or other regions or domains described herein are
provided.
[0552] Preferred epitopes encompassed by the antigenic peptide are
regions of 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 protein sequence can be used to
indicate the regions that have a particularly high probability of
being localized to the surface of the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein
and are thus likely to constitute surface residues useful for
targeting antibody production.
[0553] In a preferred embodiment the antibody can bind to the
extracellular portion of the 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein, e.g., it
can bind to a whole cell which expresses the 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
protein. In another embodiment, the antibody binds an intracellular
portion of the 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 protein.
[0554] In a preferred embodiment the antibody binds an epitope on
any domain or region on 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 proteins described
herein.
[0555] Additionally, chimeric, humanized, and completely human
antibodies are also within the scope of the invention. Chimeric,
humanized, but most preferably, completely human antibodies are
desirable for applications which include repeated administration,
e.g., therapeutic treatment of human patients, and some diagnostic
applications.
[0556] Chimeric and humanized monoclonal antibodies, comprising
both human and non-human portions, can be made using standard
recombinant DNA techniques. Such chimeric and humanized monoclonal
antibodies can be produced by recombinant DNA techniques known in
the art, for example using methods described in Robinson et al.
International Application No. PCT/US86/02269; Akira, et al.
European Patent Application 184,187; Taniguchi, European Patent
Application 171,496; Morrison et al. European Patent Application
173,494; Neuberger et al. PCT International Publication No. WO
86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.
European Patent Application 125,023; Better et al. (1988) Science
240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA
84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et
al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al.
(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature
314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.
80:1553-1559).
[0557] A humanized or complementarity determining region
(CDR)-grafted antibody will have at least one or two, but generally
all three recipient CDR's (of heavy and or light immuoglobulin
chains) replaced with a donor CDR. The antibody may be replaced
with at least a portion of a non-human CDR or only some of the
CDR's may be replaced with non-human CDR's. It is only necessary to
replace the number of CDR's required for binding of the humanized
antibody to a 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 or a fragment thereof.
Preferably, the donor will be a rodent antibody, e.g., a rat or
mouse antibody, and the recipient will be a human framework or a
human consensus framework. Typically, the immunoglobulin providing
the CDR's is called the "donor" and the immunoglobulin providing
the framework is called the "acceptor." In one embodiment, the
donor immunoglobulin is a non-human (e.g., rodent). The acceptor
framework is a naturally-occurring (e.g., a human) framework or a
consensus framework, or a sequence about 85% or higher, preferably
90%, 95%, 99% or higher identical thereto.
[0558] As used herein, the term "consensus sequence" refers to the
sequence formed from the most frequently occurring amino acids (or
nucleotides) in a family of related sequences (See e.g., Winnaker,
(1987) From Genes to Clones (Verlagsgesellschaft, Weinheim,
Germany). In a family of proteins, each position in the consensus
sequence is occupied by the amino acid occurring most frequently at
that position in the family. If two amino acids occur equally
frequently, either can be included in the consensus sequence. A
"consensus framework" refers to the framework region in the
consensus immunoglobulin sequence.
[0559] An antibody can be humanized by methods known in the art.
Humanized antibodies can be generated by replacing sequences of the
Fv variable region which are not directly involved in antigen
binding with equivalent sequences from human Fv variable regions.
General methods for generating humanized antibodies are provided by
Morrison (1985) Science 229:1202-1207, by Oi et al. (1986)
BioTechniques 4:214, and by Queen et al. U.S. Pat. Nos. 5,585,089,
5,693,761 and 5,693,762, the contents of all of which are hereby
incorporated by reference. Those methods include isolating,
manipulating, and expressing the nucleic acid sequences that encode
all or part of immunoglobulin Fv variable regions from at least one
of a heavy or light chain. Sources of such nucleic acid are well
known to those skilled in the art and, for example, may be obtained
from a hybridoma producing an antibody against a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 polypeptide or fragment thereof. The recombinant DNA encoding
the humanized antibody, or fragment thereof, can then be cloned
into an appropriate expression vector.
[0560] Humanized or CDR-grafted antibodies can be produced by
CDR-grafting or CDR substitution, wherein one, two, or all CDR's of
an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No.
5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.
(1988) Science 239:1534; Beidler et al. (1988) J. Immunol.
141:4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all
of which are hereby expressly incorporated by reference. Winter
describes a CDR-grafting method which may be used to prepare the
humanized antibodies of the present invention (UK Patent
Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat.
No. 5,225,539), the contents of which is expressly incorporated by
reference.
[0561] Also within the scope of the invention are humanized
antibodies in which specific amino acids have been substituted,
deleted or added. Preferred humanized antibodies have amino acid
substitutions in the framework region, such as to improve binding
to the antigen. For example, a humanized antibody will have
framework residues identical to the donor framework residue or to
another amino acid other than the recipient framework residue. To
generate such antibodies, a selected, small number of acceptor
framework residues of the humanized immunoglobulin chain can be
replaced by the corresponding donor amino acids. Preferred
locations of the substitutions include amino acid residues adjacent
to the CDR, or which are capable of interacting with a CDR (see
e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids
from the donor are described in U.S. Pat. No. 5,585,089, e.g.,
columns 12-16 of U.S. Pat. No. 5,585,089, the e.g., columns 12-16
of U.S. Pat. No. 5,585,089, the contents of which are hereby
incorporated by reference. Other techniques for humanizing
antibodies are described in Padlan et al. EP 519596 A1, published
on Dec. 23, 1992.
[0562] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Such antibodies can be
produced using transgenic mice that are incapable of expressing
endogenous immunoglobulin heavy and light chains genes, but which
can express human heavy and light chain genes. See, for example,
Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93); and U.S.
Pat. Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and
5,545,806. In addition, companies such as Abgenix, Inc. (Fremont,
Calif.) and Medarex, Inc. (Princeton, N.J.), can be engaged to
provide human antibodies directed against a selected antigen using
technology similar to that described above.
[0563] Completely human antibodies that recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a murine antibody, is used to guide the selection
of a completely human antibody recognizing the same epitope. This
technology is described by Jespers et al. (1994) Bio/Technology
12:899-903).
[0564] The anti-25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 antibody can be a single chain
antibody. A single-chain antibody (scFV) can be engineered as
described in, for example, Colcher et al. (1999) Ann. N Y Acad.
Sci. 880:263-80; and Reiter (1996) Clin. Cancer Res. 2:245-52. The
single chain antibody can be dimerized or multimerized to generate
multivalent antibodies having specificities for different epitopes
of the same target 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 protein.
[0565] In a preferred embodiment, the antibody has reduced or no
ability to bind an Fc receptor. For example, it is an isotype or
subtype, fragment or other mutant, which does not support binding
to an Fc receptor, e.g., it has a mutagenized or deleted Fc
receptor binding region.
[0566] An antibody (or fragment thereof) may be conjugated to a
therapeutic moiety such as a cytotoxin, a therapeutic agent or a
radioactive 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, puromycin, maytansinoids, e.g.,
maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat.
Nos. 5,475,092, 5,585,499, 5,846,545) 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, CC-1065,
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, vinblastine,
taxol and maytansinoids).
[0567] Radioactive ions include, but are not limited to iodine,
yttrium and praseodymium.
[0568] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic moiety is not to be
construed as limited to classical chemical therapeutic agents. For
example, the therapeutic moiety may be a protein or polypeptide
possessing a desired biological activity. Such proteins may
include, for example, a toxin such as abrin, ricin A, 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,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophase colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0569] 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.
[0570] An anti-25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 antibody (e.g., monoclonal
antibody) can be used to isolate 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 by standard
techniques, such as affinity chromatography or immunoprecipitation.
Moreover, an anti-25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 antibody can be used to detect
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein (e.g., in a cellular lysate or cell
supernatant) in order to evaluate the abundance and pattern of
expression of the protein. Anti-25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 antibodies can be
used diagnostically to monitor protein levels in tissue as part of
a clinical testing procedure, e.g., to 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 labelling). 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.
[0571] In preferred embodiments, an antibody can be made by
immunizing with a purified 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 antigen, or a
fragment thereof, e.g., a fragment described herein, a membrane
associated antigen, tissues, e.g., crude tissue preparations, whole
cells, preferably living cells, lysed cells, or cell fractions,
e.g., membrane fractions.
[0572] Antibodies which bind only a native 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
protein, only denatured or otherwise non-native 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein, or which bind both, are within the invention.
Antibodies with linear or conformational epitopes are within the
invention. Conformational epitopes sometimes can be identified by
identifying antibodies which bind to native but not denatured
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein.
[0573] Recombinant Expression Vectors, Host Cells and Genetically
Engineered Cells
[0574] 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.
[0575] A vector can include a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 nucleic acid in a
form suitable for expression of the nucleic acid in a host
cell.
[0576] 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 which
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.,
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 proteins, mutant forms of 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 proteins, fusion proteins, and the like).
[0577] The recombinant expression vectors of the invention can be
designed for expression of 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 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, Calif. Alternatively, the recombinant expression vector
can be transcribed and translated in vitro, for example using T7
promoter regulatory sequences and T7 polymerase.
[0578] 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 and Johnson
(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.
[0579] Purified fusion proteins can be used in 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
activity assays, (e.g., direct assays or competitive assays
described in detail below), or to generate antibodies specific or
selective for 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 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 which are subsequently transplanted into irradiated
recipients. The pathology of the subject recipient is then examined
after sufficient time has passed (e.g., six weeks).
[0580] To maximize recombinant protein expression in E. coli is to
express the protein in a host bacteria with an impaired capacity to
proteolytically cleave the recombinant protein (Gottesman (1990)
Gene Expression Technology: Methods in Enzymology 185, Academic
Press, San Diego, Calif. 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)
Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid
sequences of the invention can be carried out by standard DNA
synthesis techniques.
[0581] The 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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.
[0582] When used in mammalian cells, the expression vector's
control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40.
[0583] 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 and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (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 Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example, the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[0584] 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
et al., (1986) Reviews--Trends in Genetics 1:1.
[0585] Another aspect the invention provides a host cell which
includes a nucleic acid molecule described herein, e.g., a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 nucleic acid molecule within a recombinant
expression vector or a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 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 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 still included
within the scope of the term as used herein.
[0586] A host cell can be any prokaryotic or eukaryotic cell. For
example, a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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 CV-1 origin, SV-40 (COS)
cells). Other suitable host cells are known to those skilled in the
art.
[0587] 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.
[0588] A host cell of the invention can be used to produce (i.e.,
express) a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 protein. Accordingly, the invention
further provides methods for producing a 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
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 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein has been introduced) in a suitable medium such that a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein is produced. In another embodiment,
the method further includes isolating a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein
from the medium or the host cell.
[0589] In another aspect, the invention features, a cell or
purified preparation of cells which include a 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
transgene, or which otherwise misexpress 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933.
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 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 transgene, e.g., a heterologous form of a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933, e.g., a gene derived from humans (in the case of a non-human
cell). The 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 transgene can be misexpressed, e.g.,
overexpressed or underexpressed. In other preferred embodiments,
the cell or cells include a gene which misexpresses an endogenous
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933, e.g., a gene the expression of which is
disrupted, e.g., a knockout. Such cells can serve as a model for
studying disorders which are related to mutated or misexpressed
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 alleles or for use in drug screening.
[0590] In another aspect, the invention features, a human cell,
e.g., a hematopoietic stem cell, transformed with nucleic acid
which encodes a subject 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 polypeptide.
[0591] Also provided are cells, preferably human cells, e.g., human
hematopoietic or fibroblast cells, in which an endogenous 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 is under the control of a regulatory sequence that
does not normally control the expression of the endogenous 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 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 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
gene. For example, an endogenous 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 gene which is
"transcriptionally silent," e.g., not normally expressed, or
expressed only at very low levels, can be activated by inserting a
regulatory element which is capable of promoting the expression of
a normally expressed gene product in that cell. Techniques such as
targeted homologous recombinations, can be used to insert the
heterologous DNA as described in, e.g., Chappel, U.S. Pat. No.
5,272,071; WO 91/06667, published in May 16, 1991.
[0592] Transgenic Animals
[0593] The invention provides non-human transgenic animals. Such
animals are useful for studying the function and/or activity of a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein and for identifying and/or evaluating
modulators of 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 gene has been
altered by, 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.
[0594] 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 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 protein to particular cells. A
transgenic founder animal can be identified based upon the presence
of a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 transgene in its genome and/or expression of
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein can
further be bred to other transgenic animals carrying other
transgenes.
[0595] 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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.
[0596] For example, the following animal models may be used in the
methods of the invention: the hypertensive transgenic mouse model
that lacks fat and has lipoatrophic diabetes (Reitmann, M. L. et
al. (1999) Ann. N.Y. Acad. Sci. 192:289-96; Moitra J. et al. (1998)
Genes Dev. 12:3168-81); a VEGF transgenic animal model for
atherosclerosis and angiogenesis (Sueishi, K. et al. (1997) Ann. N.
Y. Acad. Sci. 811:311-324); guinea pigs, which are used as models
for cholesterol and lipoprotein metabolism, as well as early
atherosclerosis development (Fernandez, M. L. (2001) J. Nutr.
131(1):10-20); the macrophage scavenger receptor class A (SR-A)
transgenic mouse, which shows reduced atherosclerosis (De Winther,
M. P. et al. (2000) Int. J. Tissue React. 22(2-3):85-91); the St.
Thomas' Hospital rabbit strain, an animal model with genetically
elevated plasma levels of VLDL, IDL, and low-density lipoprotein
(LDL) (Nordestgaard, B. G. et al. (1992) Eur. J. Epidemiol. 8 Suppl
1:92-8); the Watanabe heritable hyperlipidemic (WHHL) rabbit, the
animal model for familial hypercholesterolemia (Buja, L. M. et al.
(1990) Eur. Heart J. 11 Suppl E:41-52); atherosclerosis induced in
Cynomolgus macaque monkeys by feeding cholesterol (Weingand, K. W.
(1989) Exp. Mol. Pathol. 50(1):1-15); atherosclerosis induced by
infection with Marek's disease herpesvirus in chickens (1999) Am.
Heart J. 138(5 Pt 2):S465-8); genetically selected lines of
Japanese quail, highly susceptible (SUS) and resistant (RES) to
atherosclerosis (1987) Atherosclerosis 68(1-2):77-8); the
atherosclerotic and hypertensive strain of male broad-breasted
white turkeys (BBWT) (Pagnan, A. (1980) Artery 6(4):320-7);
diet-induced, apoE deficiency-induced, or LDL receptor-deficiency
induced atherosclerosis in mice (Smith, J. D. (1997) J. Intern.
Med. 242(2):99-109); the JCR:LA-corpulent rat, an experimental
model for the obese-diabetic-dyslipidemic syndrome that mimics the
human condition and exhibits spontaneous development of
atherosclerosis and myocardial lesions (Brindley, D. N. (1995)
Metabolism 44(2 Suppl 2):23-7); marmosets or other animals treated
with cholestyramine or other cholesterol and/or lipid lowering
drugs; and numerous other animal models of atherosclerosis
(reviewed in Bocan, T. M. (1998) Curr. Pharm. Des. 4(1):37-52;
Fekete, S. (1993) Acta Vet. Hung. 41(1-2):3-9).
[0597] Further examples of animals that can be used include the
transgenic mouse described in U.S. Pat. No. 5,932,779 that contains
a mutation in an endogenous melanocortin-4-receptor (MC4-R) gene;
animals having mutations which lead to syndromes that include
obesity symptoms (described in, for example, Friedman, J. M. et al.
(1991) Mamm. Genome 1:130-144; Friedman, J. M. and Liebel, R. L.
(1992) Cell 69:217-220; Bray, G. A. (1992) Prog. Brain Res.
93:333-341; and Bray, G. A. (1989) Amer. J. Clin. Nutr. 5:891-902);
the animals described in Stubdal, H. et al. (2000) Mol. Cell Biol.
20(3):878-82 (the mouse tubby phenotype characterized by
maturity-onset obesity); the animals described in Abadie, J. M. et
al. (2000) Lipids 35(6):613-20 (the obese Zucker rat (ZR), a
genetic model of human youth-onset obesity and type 2 diabetes
mellitus); the animals described in Shaughnessy, S. et al. (2000)
Diabetes 49(6):904-11 (mice null for the adipocyte fatty acid
binding protein); or the animals described in Loskutoff, D. J. et
al. (2000) Ann. N.Y. Acad. Sci. 902:272-81 (the fat mouse). Other
examples of animals that may be used include non-recombinant,
non-genetic animal models of obesity such as, for example, rabbit,
mouse, or rat models in which the animal has been exposed to
long-term over-eating.
[0598] The invention also includes a population of cells from a
transgenic animal, as discussed, e.g., below.
[0599] Uses
[0600] The nucleic acid molecules, proteins, protein homologs, 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).
[0601] The isolated nucleic acid molecules of the invention can be
used, for example, to express a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein (e.g.,
via a recombinant expression vector in a host cell in gene therapy
applications), to detect a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 mRNA (e.g., in a
biological sample) or a genetic alteration in a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 gene, and to modulate 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 activity, as
described further below. The 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 proteins can be
used to treat disorders characterized by insufficient, aberrant or
excessive production of a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 substrate or production
of 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 inhibitors. In addition, the 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 proteins can be used to screen for naturally occurring 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 substrates, to screen for drugs or compounds which
modulate 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 activity, as well as to treat
disorders characterized by insufficient, aberrant or excessive
production of 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 protein or production of 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein forms which have decreased, aberrant or
unwanted activity compared to 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 wild type protein
(e.g., aberrant or deficient carboxylase actvity, fatty acid
desaturase activity, serine/threonine dehydratase activity,
hexokinase activity, peptidyl tRNA hydrolase actvity, dual
specificity phosphatase activity, phospholipase activity,
transporter actvity, or other activity disclosed herein). Moreover,
the anti-25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 antibodies of the invention can be
used to detect and isolate 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 proteins,
regulate the bioavailability of 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 proteins, and
modulate 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 activity.
[0602] A method of evaluating a compound for the ability to
interact with, e.g., bind, a subject 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
polypeptide is provided. The method includes: contacting the
compound with the subject 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 polypeptide; and
evaluating ability of the compound to interact with, e.g., to bind
or form a complex with the subject 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
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 which interact with subject 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
polypeptide. It can also be used to find natural or synthetic
inhibitors of subject 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 polypeptide. Screening
methods are discussed in more detail below.
[0603] Screening Assays:
[0604] The invention provides methods (also referred to herein as
"screening 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 to 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 proteins, have a stimulatory or
inhibitory effect on, for example, 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 expression
or 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 activity, or have a stimulatory or inhibitory
effect on, for example, the expression or activity of a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 substrate. Compounds thus identified can be used to
modulate the activity of target gene products (e.g., 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 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.
[0605] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein or polypeptide or a biologically
active portion thereof. In another embodiment, the invention
provides assays for screening candidate or test compounds which
bind to or modulate the activity of a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein or
polypeptide or a biologically active portion thereof.
[0606] 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; see, e.g., Zuckermann et al. (1994) J. Med. Chem.
37:2678-85); 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).
[0607] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909-13; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422-426; Zuckermann et al. (1994). J. Med.
Chem. 37:2678-85; 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 in Gallop et al.
(1994) J. Med. Chem. 37:1233-51.
[0608] 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 (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S.
Pat. No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol.
Biol. 222:301-310; Ladner supra.).
[0609] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein or biologically
active portion thereof is contacted with a test compound, and the
ability of the test compound to modulate 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
activity is determined. Determining the ability of the test
compound to modulate 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 activity can be
accomplished by monitoring, for example, carboxylase actvity, fatty
acid desaturase activity, serine/threonine dehydratase activity,
hexokinase activity, peptidyl tRNA hydrolase actvity, dual
specificity phosphatase activity, phospholipase activity,
transporter actvity, or other activity disclosed herein. The cell,
for example, can be of mammalian origin, e.g., human.
[0610] The ability of the test compound to modulate 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 binding to a compound, e.g., a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 substrate,
or to bind to 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 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 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 can
be determined by detecting the labeled compound, e.g., substrate,
in a complex. Alternatively, 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 could be coupled
with a radioisotope or enzymatic label to monitor the ability of a
test compound to modulate 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 binding to a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 substrate in a complex. For example, compounds
(e.g., 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 substrates) can be labeled with
.sup.125I, .sup.14C, .sup.35S or .sup.3H., either directly or
indirectly, and the radioisotope detected by direct counting of
radioemmission 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.
[0611] The ability of a compound (e.g., a 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
substrate) to interact with 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 without the labeling of either
the compound or the 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933. 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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933.
[0612] In yet another embodiment, a cell-free assay is provided in
which a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 protein or biologically active portion
thereof is contacted with a test compound and the ability of the
test compound to bind to the 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein or
biologically active portion thereof is evaluated. Preferred
biologically active portions of the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 proteins
to be used in assays of the present invention include fragments
which participate in interactions with non-25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
molecules, e.g., fragments with high surface probability
scores.
[0613] Soluble and/or membrane-bound forms of isolated proteins
(e.g., 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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
may 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).sub.n,
3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),
3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane
sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane
sulfonate.
[0614] 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.
[0615] The interaction between two molecules can also be detected,
e.g., using fluorescence energy transfer (FET) (see, for example,
Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al.,
U.S. Pat. No. 4,868,103). A fluorophore label on the first, `donor`
molecule is selected such that its 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).
[0616] In another embodiment, determining the ability of the 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein to bind to a target molecule can be
accomplished using real-time Biomolecular Interaction Analysis
(BIA) (see, e.g., Sjolander and Urbaniczky (1991) Anal. Chem.
63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol.
5:699-705). "Surface plasmon resonance" 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 surface plasmon resonance (SPR)), resulting in a
detectable signal which can be used as an indication of real-time
reactions between biological molecules.
[0617] 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.
[0618] It may be desirable to immobilize either 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933, an anti-25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 antibody or its target molecule
to facilitate separation of complexed from uncomplexed forms of one
or both of the proteins, as well as to accommodate automation of
the assay. Binding of a test compound to a 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
protein, or interaction of a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 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/25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose 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 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein, and the
mixture incubated under conditions conducive to complex formation
(e.g., at physiological conditions for salt and pH).
[0619] 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
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 binding or activity determined using standard
techniques.
[0620] Other techniques for immobilizing either a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein or a target, molecule on matrices include using
conjugation of biotin and streptavidin. Biotinylated 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein or target molecules can be prepared from biotin-NHS
(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).
[0621] 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, unreacted 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 or selective for the immobilized
component (the antibody, in turn, can be directly labeled or
indirectly labeled with, e.g., a labeled anti-Ig antibody).
[0622] In one embodiment, this assay is performed utilizing
antibodies reactive with 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein or target
molecules but which do not interfere with binding of the 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein to its target molecule. Such antibodies can
be derivatized to the wells of the plate, and unbound target or
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 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
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein or target molecule, as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein or target
molecule.
[0623] Alternatively, cell free assays can be conducted in a liquid
phase. In such an assay, the reaction products are separated from
unreacted components, by any of a number of standard techniques,
including but not limited to: differential centrifugation (see, for
example, Rivas and Minton (1993) Trends Biochem Sci 18:284-7);
chromatography (gel filtration chromatography, ion-exchange
chromatography); electrophoresis (see, e.g., Ausubel et al., eds.
(1999) Current Protocols in Molecular Biology, J. Wiley, New
York.); and immunoprecipitation (see, for example, Ausubel et al.,
eds. (1999) Current Protocols in Molecular Biology, J. Wiley, New
York). Such resins and chromatographic techniques are known to one
skilled in the art (see, e.g., Heegaard (1998) J Mol Recognit
11:141-8; Hage and Tweed (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.
[0624] In a preferred embodiment, the assay includes contacting the
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein or biologically active portion
thereof with a known compound which binds 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 protein, wherein determining
the ability of the test compound to interact with a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein includes determining the ability of the test compound
to preferentially bind to 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 or biologically active
portion thereof, or to modulate the activity of a target molecule,
as compared to the known compound.
[0625] 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 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 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 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein
through modulation of the activity of a downstream effector of a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 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.
[0626] 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.
[0627] 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.
[0628] 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.
[0629] 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 or selective for the species to be anchored can
be used to anchor the species to the solid surface.
[0630] 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,
unreacted 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 or selective 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.
[0631] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected; e.g., using an immobilized antibody specific or selective
for one of the binding components to anchor any complexes formed in
solution, and a labeled antibody specific or selective 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.
[0632] 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 (see, 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.
[0633] In yet another aspect, the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 proteins
can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, 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; and Brent WO94/10300),
to identify other proteins, which bind to or interact with 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 ("25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933-binding proteins" or "25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-bp") and are involved in 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 activity.
Such 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933-bps can be activators or inhibitors of
signals by the 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 proteins or 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 targets as, for example, downstream elements of a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-mediated signaling pathway.
[0634] 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 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 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: 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
protein can be the fused to the activator domain.) If the "bait"
and the "prey" proteins are able to interact, in vivo, forming a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933-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) which 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 which encodes the protein which interacts
with the 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 protein.
[0635] In another embodiment, modulators of 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
expression are identified. For example, a cell or cell free mixture
is contacted with a candidate compound and the expression of 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 mRNA or protein evaluated relative to the level of
expression of 25869, 25934, 26335, 50365, 21117, 38692,46508,
16816, 16839, 49937, 49931 or 49933 mRNA or protein in the absence
of the candidate compound. When expression of 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
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 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 mRNA or protein expression.
Alternatively, when expression of 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 mRNA or
protein is less (statistically significantly less) in the presence
of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
mRNA or protein expression. The level of 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
mRNA or protein expression can be determined by methods described
herein for detecting 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 mRNA or protein.
[0636] 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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein can be confirmed in vivo, e.g., in an
animal such as an animal model for aberrant or deficient
carboxylase actvity, fatty acid desaturase activity,
serine/threonine dehydratase activity, hexokinase activity,
peptidyl tRNA hydrolase actvity, dual specificity phosphatase
activity, phospholipase activity, transporter actvity, or other
activity disclosed herein.
[0637] 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 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 modulating agent, an
antisense 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 nucleic acid molecule, a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933-specific antibody, or a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933-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.
[0638] Detection Assays
[0639] 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 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 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.
[0640] Chromosome Mapping
[0641] The 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 nucleotide sequences or portions
thereof can be used to map the location of the 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
genes on a chromosome. This process is called chromosome mapping.
Chromosome mapping is useful in correlating the 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 sequences with genes associated with disease.
[0642] Briefly, 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 genes can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp in
length) from the 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 nucleotide sequences. 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 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
sequences will yield an amplified fragment.
[0643] 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.
(D'Eustachio et al. (1983) Science 220:919-924).
[0644] Other mapping strategies e.g., in situ hybridization
(described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA,
87:6223-27), pre-screening with labeled flow-sorted chromosomes,
and pre-selection by hybridization to chromosome specific cDNA
libraries can be used to map 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 to a chromosomal
location.
[0645] 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 this technique, see Verma et al. (1988)
Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,
New York).
[0646] 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 noncoding regions
of the genes actually are 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.
[0647] 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 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), described in, for
example, Egeland et al. (1987) Nature, 325:783-787.
[0648] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 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.
[0649] Tissue Typing
[0650] 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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).
[0651] 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 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 nucleotide sequences described herein can be used to
prepare two PCR primers from the 5' and 3' ends of the sequences.
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.
[0652] Allelic variation occurs to some degree in the coding
regions of these sequences, and to a greater degree in the
noncoding 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 noncoding regions,
fewer sequences are necessary to differentiate individuals. The
noncoding sequences of SEQ ID NO: 1, 7, 16, 20, 25, 28, 35, 39, 42,
63, 67 or 70 can provide positive individual identification with a
panel of perhaps 10 to 1,000 primers which each yield a noncoding
amplified sequence of 100 bases. If predicted coding sequences,
such as those in SEQ ID NO: 3, 9, 18, 22, 27, 30, 37, 41, 44, 65,
69 or 72 are used, a more appropriate number of primers for
positive individual identification would be 500-2,000.
[0653] If a panel of reagents from 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 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.
[0654] Use of Partial 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 Sequences in Forensic
Biology
[0655] 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.
[0656] 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 base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to
noncoding regions of SEQ ID NO: 1, 7, 16, 20, 25, 28, 35, 39, 42,
63, 67 or 70 (e.g., fragments derived from the noncoding regions of
SEQ ID NO: 1, 7, 16, 20, 25, 28, 35, 39, 42, 63, 67 or 70 having a
length of at least 20 bases, preferably at least 30 bases) are
particularly appropriate for this use.
[0657] The 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 nucleotide sequences described herein
can further be used to provide polynucleotide reagents, e.g.,
labeled or labelable probes which can be used in, for example, an
in situ hybridization technique, to identify a specific tissue.
This can be very useful in cases where a forensic pathologist is
presented with a tissue of unknown origin. Panels of such 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 probes can be used to identify tissue by species
and/or by organ type.
[0658] In a similar fashion, these reagents, e.g., 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 primers or probes can be used to screen tissue culture for
contamination (i.e. screen for the presence of a mixture of
different types of cells in a culture).
[0659] Predictive Medicine
[0660] 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.
[0661] Generally, the invention provides, a method of determining
if a subject is at risk for a disorder related to a lesion in or
the misexpression of a gene which encodes 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933.
Such disorders include, e.g., a disorder associated with the
misexpression of 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 gene; cellular proliferative
and/or differentiative disorders, angiogenic disorders, brain
disorders, neurological disorders, blood vessel disorders, breast
disorders, colon disorders, kidney disorders, lung disorders,
ovarian disorders, prostate disorders, hematopoeitic disorders,
pancreatic disorders, skeletal muscle disorders, skin disorders,
hormonal disorders, immune e.g., inflammatory, disorders,
cardiovascular disorders, lipid homeostasis disorders, endothelial
cell disorders, liver disorders, disorders of the small intestine,
pain disorders, viral diseases, metabolic disorders, bone
metabolism disorders or platelet disorders.
[0662] The method includes one or more of the following: detecting,
in a tissue of the subject, the presence or absence of a mutation
which affects the expression of the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 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; detecting, in a tissue of the subject, the presence
or absence of a mutation which alters the structure of the 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 gene; detecting, in a tissue of the subject, the
misexpression of the 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 gene, at the mRNA level,
e.g., detecting a non-wild type level of an mRNA; or detecting, in
a tissue of the subject, the misexpression of the gene, at the
protein level, e.g., detecting a non-wild type level of a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 polypeptide.
[0663] In preferred embodiments the method includes: ascertaining
the existence of at least one of: a deletion of one or more
nucleotides from the 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 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.
[0664] 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, 7, 16, 20, 25, 28, 35, 39,
42, 63, 67 or 70, or naturally occurring mutants thereof or 5' or
3' flanking sequences naturally associated with the 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 gene; (ii) exposing the probe/primer to nucleic acid of the
tissue; and detecting, by hybridization, e.g., in situ
hybridization, of the probe/primer to the nucleic acid, the
presence or absence of the genetic lesion.
[0665] In preferred embodiments detecting the misexpression
includes ascertaining the existence of at least one of: an
alteration in the level of a messenger RNA transcript of the 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 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 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or49933.
[0666] Methods of the invention can be used prenatally or to
determine if a subject's offspring will be at risk for a
disorder.
[0667] In preferred embodiments the method includes determining the
structure of a 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 gene, an abnormal structure
being indicative of risk for the disorder.
[0668] In preferred embodiments the method includes contacting a
sample from the subject with an antibody to the 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein or a nucleic acid, which hybridizes specifically with
the gene. These and other embodiments are discussed below.
[0669] Diagnostic and Prognostic Assays
[0670] The presence, level, or absence of 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 protein or nucleic acid (e.g., mRNA,
genomic DNA) that encodes 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein such that the
presence of 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or49933 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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 gene can be measured in a number of ways,
including, but not limited to: measuring the mRNA encoded by the
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 genes; measuring the amount of protein
encoded by the 25869, 25934, 26335, 50365, 21117,-38692, 46508,
16816, 16839, 49937, 49931 or 49933 genes; or measuring the
activity of the protein encoded by the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 genes.
[0671] The level of mRNA corresponding to the 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
gene in a cell can be determined both by in situ and by in vitro
formats.
[0672] 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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 nucleic acid, such as the nucleic acid
of SEQ ID NO: 1, 7, 16, 20, 25, 28, 35, 39, 42, 63, 67 or 70, 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 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 mRNA or genomic DNA. Other suitable probes for use in the
diagnostic assays are described herein.
[0673] 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
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 genes.
[0674] The level of mRNA in a sample that is encoded by one of
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 can be evaluated with nucleic acid
amplification, e.g., by rtPCR (Mullis (1987) 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
(Lizardi et al., 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 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 flanked by
the primers.
[0675] 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 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 gene being analyzed.
[0676] In another embodiment, the methods further contacting a
control sample with a compound or agent capable of detecting 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 mRNA, or genomic DNA, and comparing the presence of
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 mRNA or genomic DNA in the control sample
with the presence of 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 mRNA or genomic DNA in
the test sample.
[0677] A variety of methods can be used to determine the level of
protein encoded by 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933. 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.
[0678] The detection methods can be used to detect 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein in a biological sample in vitro as well as in vivo.
In vitro techniques for detection of 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein
include enzyme linked immunosorbent assays (ELISAs),
immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA),
radioimmunoassay (RIA), and Western blot analysis. In vivo
techniques for detection of 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein include
introducing into a subject a labeled anti-25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
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.
[0679] In another embodiment, the methods further include
contacting the control sample with a compound or agent capable of
detecting 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 protein, and comparing the presence of
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein in the control sample with the
presence of 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 protein in the test sample.
[0680] The invention also includes kits for detecting the presence
of 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 in a biological sample. For example, the kit
can include a compound or agent capable of detecting 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 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
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein or nucleic acid.
[0681] 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.
[0682] 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 which 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.
[0683] The diagnostic methods described herein can identify
subjects having, or at risk of developing, a disease or disorder
associated with misexpressed or aberrant or unwanted 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 expression or activity. As used herein, the term "unwanted"
includes an unwanted phenomenon involved in a biological response
such as pain or deregulated cell proliferation.
[0684] In one embodiment, a disease or disorder associated with
aberrant or unwanted 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 expression or activity
is identified. A test sample is obtained from a subject and 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein or nucleic acid (e.g., mRNA or genomic DNA)
is evaluated, wherein the level, e.g., the presence or absence, of
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein or nucleic acid is diagnostic for a
subject having or at risk of developing a disease or disorder
associated with aberrant or unwanted 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 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.
[0685] 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 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 expression or activity. For example, such methods can be used
to determine whether a subject can be effectively treated with an
agent for a cellular proliferative and/or differentiative
disorders, angiogenic disorders, brain disorders, neurological
disorders, blood vessel disorders, breast disorders, colon
disorders, kidney disorders, lung disorders, ovarian disorders,
prostate disorders, hematopoeitic disorders, pancreatic disorders,
skeletal muscle disorders, skin disorders, hormonal disorders,
immune e.g., inflammatory, disorders, cardiovascular disorders,
lipid homeostasis disorders, endothelial cell disorders, liver
disorders, disorders of the small intestine, pain disorders, viral
diseases, metabolic disorders, bone metabolism disorders or
platelet disorders.
[0686] The methods of the invention can also be used to detect
genetic alterations in a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 gene, thereby
determining if a subject with the altered gene is at risk for a
disorder characterized by misregulation in 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
protein activity or nucleic acid expression, such as a cellular
proliferative and/or differentiative disorders, angiogenic
disorders, brain disorders, neurological disorders, blood vessel
disorders, breast disorders, colon disorders, kidney disorders,
lung disorders, ovarian disorders, prostate disorders,
hematopoeitic disorders, pancreatic disorders, skeletal muscle
disorders, skin disorders, hormonal disorders, immune e.g.,
inflammatory, disorders, cardiovascular disorders, lipid
homeostasis disorders, endothelial cell disorders, liver disorders,
disorders of the small intestine, pain disorders, viral diseases,
metabolic disorders, bone metabolism disorders or platelet
disorders. 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 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933-protein,
or the mis-expression of the 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 gene; 2) an addition of one or
more nucleotides to a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 gene; 3) a substitution
of one or more nucleotides of a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 gene, 4) a
chromosomal rearrangement of a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 gene; 5) an
alteration in the level of a messenger RNA transcript of a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 gene, 6) aberrant modification of a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 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 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 gene, 8) a non-wild type
level of a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933-protein, 9) allelic loss of a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 gene, and 10) inappropriate post-translational
modification of a 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933-protein.
[0687] 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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933-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 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 gene under conditions such that
hybridization and amplification of the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 gene (if
present) occurs, 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 may be desirable to use as a
preliminary amplification step in conjunction with any of the
techniques used for detecting mutations described herein.
Alternatively, other amplification methods described herein or
known in the art can be used.
[0688] In another embodiment, mutations in a 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
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 (see, for example, 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.
[0689] In other embodiments, genetic mutations in 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 can be identified by hybridizing a sample and control nucleic
acids, e.g., DNA or RNA, two dimensional arrays, 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) Human Mutation 7:
244-255; Kozal et al. (1996) Nature Medicine 2: 753-759). For
example, genetic mutations in 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 can be identified
in two dimensional arrays containing light-generated DNA probes as
described in Cronin, M. T. 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.
[0690] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 gene and detect mutations by comparing the
sequence of the sample 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 with the corresponding
wild-type (control) sequence. Automated sequencing procedures can
be utilized when performing the diagnostic assays (Naeve et al.
(1995) Biotechniques 19:448-53), including sequencing by mass
spectrometry.
[0691] Other methods for detecting mutations in the 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 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)
Methods Enzymol. 217:286-295).
[0692] 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 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 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).
[0693] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
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, see also Cotton (1993) Mutat.
Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl.
9:73-79). Single-stranded DNA fragments of sample and control
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 nucleic acids will be denatured and allowed
to renature. 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).
[0694] 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 bp 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).
[0695] 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).
[0696] Alternatively, allele specific amplification technology
which 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) Nucleic
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 may 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 88:189-93). 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.
[0697] The methods described herein can be performed, for example,
by utilizing 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 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 gene.
[0698] Use of 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937,49931 or 49933 Molecules as Surrogate
Markers
[0699] The 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 molecules of the invention can be detected, and can be
correlated with one or more biological states in vivo. For example,
the 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 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 in the art include: Koomen
et al. (2000) J. Mass. Spectrom. 35: 258-264; and James (1994) AIDS
Treatment News Archive 209.
[0700] The 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 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-25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 antibodies can be employed in an immune-based detection
system for a 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 protein marker, or 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-specific radiolabeled probes can be used to detect a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 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 in the
art include: Matsuda et al. 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; and Nicolau (1999) Am. J.
Health-Syst. Pharm. 56 Suppl. 3: S16-S20.
[0701] The 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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 (see, 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., 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 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 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 DNA can correlate with a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 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.
[0702] Pharmaceutical Compositions
[0703] The nucleic acid and polypeptides, fragments thereof, as
well as anti-25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 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.
[0704] 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.
[0705] 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 polyetheylene 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 manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0706] 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 which 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.
[0707] 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, or corn
starch; a lubricant such as magnesium stearate or Sterotes; 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.
[0708] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0709] 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.
[0710] 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.
[0711] 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 with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0712] 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.
[0713] 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
which 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.
[0714] 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.
[0715] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg 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,
unconjugated or conjugated as described herein, can include a
single treatment or, preferably, can include a series of
treatments.
[0716] For antibodies, the preferred dosage is 0.1 mg/kg of body
weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act
in the brain, a dosage of 50 mg/kg to 100 mg/kg 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 lipidation
of antibodies is described by Cruikshank et al. ((1997) J. Acquired
Immune Deficiency Syndromes and Human Retrovirology 14:193).
[0717] The present invention encompasses agents which modulate
expression or activity. An agent can, 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 heteroorganic and organometallic 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.
[0718] 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 can, 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.
[0719] 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 (see 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.
[0720] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0721] Methods of Treatment:
[0722] 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 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 expression or activity.
As used herein, the term "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. A therapeutic agent includes, but is
not limited to, small molecules, peptides, antibodies, ribozymes
and antisense oligonucleotides.
[0723] 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 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 molecules
of the present invention or 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 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.
[0724] In one aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant or unwanted 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 expression or activity,
by administering to the subject a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 or an
agent which modulates 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 expression or at least
one 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 activity. Subjects at risk for a disease
which is caused or contributed to by aberrant or unwanted 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 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 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 aberrance, such that a disease or disorder is
prevented or, alternatively, delayed in its progression. Depending
on the type of 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 aberrance, for example, a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933, 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 agonist or 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 antagonist agent can be used for treating the subject. The
appropriate agent can be determined based on screening assays
described herein.
[0725] It is possible that some 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 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.
[0726] The 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 molecules can act as novel diagnostic
targets and therapeutic agents for controlling one or more of a
cellular proliferative and/or differentiative disorder, angiogenic
disorder, brain disorder, neurological disorder, blood vessel
disorder, breast disorder, colon disorder, kidney disorder, lung
disorder, ovarian disorder, prostate disorder, hematopoeitic
disorder, pancreatic disorder, skeletal muscle disorder, skin
disorder, hormonal disorder, immune e.g., inflammatory, disorder,
cardiovascular disorder, lipid homeostasis disorder, endothelial
cell disorder, liver disorder, disorder of the small intestine,
pain disorder, viral disease, metabolic disorder bone metabolism
disorders or platelet disorders, all of which are described
above.
[0727] As discussed, successful treatment of 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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, human, 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).
[0728] 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.
[0729] 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.
[0730] Another method by which nucleic acid molecules can be
utilized in treating or preventing a disease characterized by
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 expression is through the use of aptamer
molecules specific for 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein. Aptamers are
nucleic acid molecules having a tertiary structure which permits
them to specifically or selectively bind to protein ligands (see,
e.g., Osborne et al. (1997) Curr. Opin. Chem Biol. 1: 5-9; and
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 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 protein activity can be
specifically decreased without the introduction of drugs or other
molecules which can have pluripotent effects.
[0731] Antibodies can be generated that are both specific for
target gene product and that reduce target gene product activity.
Such antibodies can, therefore, by administered in instances
whereby negative modulatory techniques are appropriate for the
treatment of 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 disorders. For a description of
antibodies, see the Antibody section above.
[0732] In circumstances wherein injection of an animal or a human
subject with a 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 protein or epitope for
stimulating antibody production is harmful to the subject, it is
possible to generate an immune response against 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 through the use of anti-idiotypic antibodies (see, for
example, Herlyn (1999) Ann Med 31:66-78; and
Bhattacharya-Chatterjee and Foon (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
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein.
[0733] Vaccines directed to a disease characterized by 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 expression can also be generated in this
fashion.
[0734] 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 (see e.g., Marasco et al. (1993) Proc. Natl.
Acad. Sci. USA 90:7889-7893).
[0735] 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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 disorders. A therapeutically effective
dose refers to that amount of the compound sufficient to result in
amelioration of symptoms of the disorders. Toxicity and therapeutic
efficacy of such compounds can be determined by standard
pharmaceutical procedures as described above.
[0736] 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.
[0737] 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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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 which contains a repeated "negative image" of the compound
and is able to selectively rebind the molecule under biological
assay conditions. A detailed review of this technique can be seen
in Ansell et al (1996) Current Opinion in Biotechnology 7:89-94 and
in Shea (1994) Trends in Polymer Science 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. An example of the
use of such matrixes in this way can be seen 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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 can be readily monitored and used in
calculations of IC.sub.50.
[0738] 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 fiberoptic devices, in turn allowing the dose in a test
subject to be quickly optimized based on its individual IC.sub.50.
An rudimentary example of such a "biosensor" is discussed in Kriz
et al (1995) Analytical Chemistry 67:2142-2144.
[0739] Another aspect of the invention pertains to methods of
modulating 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 expression or activity for therapeutic
purposes. Accordingly, in an exemplary embodiment, the modulatory
method of the invention involves contacting a cell with a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 or agent that modulates one or more of the
activities of 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 protein activity associated
with the cell. An agent that modulates 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein
activity can be an agent as described herein, such as a nucleic
acid or a protein, a naturally-occurring target molecule of a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 protein (e.g., a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 substrate
or receptor), a 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 antibody, a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 agonist or antagonist, a peptidomimetic of a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 agonist or antagonist, or other small molecule.
[0740] In one embodiment, the agent stimulates one or 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 activities. Examples of such stimulatory agents include
active 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 protein and a nucleic acid molecule
encoding 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933. In another embodiment, the agent
inhibits one or more 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 activities. Examples of
such inhibitory agents include antisense 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
nucleic acid molecules, anti-25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 antibodies, and
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 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
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 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) 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 expression or activity. In another
embodiment, the method involves administering a 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 protein or nucleic acid molecule as therapy to compensate for
reduced, aberrant, or unwanted 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 expression or
activity.
[0741] Stimulation of 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 activity is desirable in
situations in which 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 is abnormally
downregulated and/or in which increased 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 activity
is likely to have a beneficial effect. For example, stimulation of
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 activity is desirable in situations in which
a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 is downregulated and/or in which increased
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 activity is likely to have a beneficial
effect. Likewise, inhibition of 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 activity is
desirable in situations in which 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 is abnormally
upregulated and/or in which decreased 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 activity
is likely to have a beneficial effect.
[0742] Pharmacogenomics
[0743] The 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 molecules of the present invention, as
well as agents, or modulators which have a stimulatory or
inhibitory effect on 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 activity (e.g., 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 gene expression) as identified by a screening assay
described herein can be administered to individuals to treat
(prophylactically or therapeutically) 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933-associated
disorders (e.g., aberrant or deficient carboxylase actvity, fatty
acid desaturase activity, serine/threonine dehydratase activity,
hexokinase activity, peptidyl tRNA hydrolase actvity, dual
specificity phosphatase activity, phospholipase activity,
transporter actvity, or other activity disclosed herein) associated
with aberrant or unwanted 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 activity.
[0744] 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 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 molecule
or 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 modulator as well as tailoring the dosage
and/or therapeutic regimen of treatment with a 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
molecule or 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 modulator.
[0745] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum et al. (1996) Clin. Exp. Pharmacol. Physiol.
23:983-985 and 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
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0746] 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 can occur once per every 1000
bases of DNA. A SNP can be involved in a disease process, however,
the vast majority can 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.
[0747] 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 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 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.
[0748] Alternatively, a method termed the "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 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 molecule or 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 modulator of the present invention) can give an indication
whether gene pathways related to toxicity have been turned on.
[0749] 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 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 molecule or
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 modulator, such as a modulator identified by
one of the exemplary screening assays described herein.
[0750] 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 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
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 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 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., human cells, will become sensitive to treatment
with an agent to which the unmodified target cells were
resistant.
[0751] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 protein can be
applied in clinical trials. For example, the effectiveness of an
agent determined by a screening assay as described herein to
increase 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 gene expression, protein levels, or
upregulate 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 activity, can be monitored in clinical
trials of subjects exhibiting decreased 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 gene
expression, protein levels, or downregulated 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
activity. Alternatively, the effectiveness of an agent determined
by a screening assay to decrease 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 gene expression,
protein levels, or downregulate 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 activity, can be
monitored in clinical trials of subjects exhibiting increased
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 gene expression, protein levels, or
upregulated 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 activity. In such clinical trials, the
expression or activity of a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 gene, and
preferably, other genes that have been implicated in, for example,
a [FAMILYNAME]-associated or another 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or49933-associated
disorder can be used as a "read out" or markers of the phenotype of
a particular cell.
[0752] Other Embodiments
[0753] In another aspect, the invention features a method of
analyzing a plurality of capture probes. The method is useful,
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,
wherein the capture probes are from a cell or subject which
expresses 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 or from a cell or subject in which a
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 mediated response has been elicited;
contacting the array with a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 nucleic acid
(preferably purified), a 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 polypeptide (preferably
purified), or an anti-25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 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 a signal generated
from a label attached to the 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 nucleic acid,
polypeptide, or antibody.
[0754] 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.
[0755] The method can include contacting the 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
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 each 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.
[0756] The plurality of capture probes can be a plurality of
nucleic acid probes each of which specifically hybridizes, with an
allele of 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933. 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.
[0757] The method can be used to detect SNPs, as described
above.
[0758] In another aspect, the invention features, a method of
analyzing 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933, e.g., analyzing structure, function,
or relatedness to other nucleic acid or amino acid sequences. The
method includes: providing a 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 nucleic acid or
amino acid sequence; comparing the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933.
[0759] The method can include evaluating the sequence identity
between a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 sequence and a database sequence. The
method can be performed by accessing the database at a second site,
e.g., over the internet. Preferred databases include GenBank.TM.
and SwissProt.
[0760] In another aspect, the invention features, a set of
oligonucleotides, useful, e.g., for identifying SNP's, or
identifying specific alleles of 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933. 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 oligonucleotides of
the plurality identical in sequence with one another (except for
differences in length). The oligonucleotides can be provided with
differential labels, such that an oligonucleotide which hybridizes
to one allele provides a signal that is distinguishable from an
oligonucleotides which hybridizes to a second allele.
[0761] The sequences of 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 molecules are 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 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
molecule. 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 exist in nature or in purified form.
[0762] A 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 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 compact
disc and CD-ROM; electrical storage media such as RAM, ROM, EPROM,
EEPROM, and the like; and general hard disks and hybrids of these
categories such as magnetic/optical storage media. The medium is
adapted or configured for having thereon 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
sequence information of the present invention.
[0763] As used herein, the term "electronic apparatus" is intended
to include any suitable computing or processing apparatus of other
device configured or adapted for storing data or information.
Examples of electronic apparatus suitable for use with the present
invention include stand-alone computing apparatus; networks,
including a local area network (LAN), a wide area network (WAN)
Internet, Intranet, and Extranet; electronic appliances such as
personal digital assistants (PDAs), cellular phones, pagers, and
the like; and local and distributed processing systems.
[0764] As used herein, "recorded" refers to a process for storing
or encoding information on the electronic apparatus readable
medium. Those skilled in the art can readily adopt any of the
presently known methods for recording information on known media to
generate manufactures comprising the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 sequence
information.
[0765] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 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 and Microsoft Word, or represented in the form of an
ASCII file, stored in a database application, such as DB2, Sybase,
Oracle, or the like. 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.
[0766] By providing the 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 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 which match a particular target sequence
or target motif.
[0767] The present invention therefore provides a medium for
holding instructions for performing a method for determining
whether a subject has a protein kinase, methyltransferase, acyl-CoA
dehydrogenase, short chain dehyrdogenase, reductase,
acyltransferase, phosphatase, transferase, ATP-ase or
synthase-associated or another 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or49933-associated disease
or disorder or a pre-disposition to a protein kinase,
methyltransferase, acyl-CoA dehydrogenase, short chain
dehyrdogenase, reductase, acyltransferase, phosphatase,
transferase, ATP-ase or synthase-associated or another 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-associated disease or disorder, wherein the method
comprises the steps of determining 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 sequence
information associated with the subject and based on the 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 sequence information, determining whether the
subject has a protein kinase, methyltransferase, acyl-CoA
dehydrogenase, short chain dehyrdogenase, reductase,
acyltransferase, phosphatase, transferase, ATP-ase or synthase
-associated or another 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933-associated disease or
disorder and/or recommending a particular treatment for the
disease, disorder, or pre-disease condition.
[0768] The present invention further provides in an electronic
system and/or in a network, a method for determining whether a
subject has a protein kinase, methyltransferase, acyl-CoA
dehydrogenase, short chain dehyrdogenase, reductase,
acyltransferase, phosphatase, transferase, ATP-ase or
synthase-associated or another 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933-associated
disease or disorder or a pre-disposition to a disease associated
with 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933, wherein the method comprises the steps of
determining 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 sequence information associated with
the subject, and based on the 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 sequence
information, determining whether the subject has a protein kinase,
methyltransferase, acyl-CoA dehydrogenase, short chain
dehyrdogenase, reductase, acyltransferase, phosphatase,
transferase, ATP-ase or synthase-associated or another 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-associated disease or disorder or a pre-disposition
to a protein kinase, methyltransferase, acyl-CoA dehydrogenase,
short chain dehyrdogenase, reductase, acyltransferase, phosphatase,
transferase, ATP-ase or synthase-associated or another 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-associated disease or disorder, and/or recommending
a particular treatment for the disease, disorder, or pre-disease
condition. The method may further comprise the step of receiving
phenotypic information associated with the subject and/or acquiring
from a network phenotypic information associated with the
subject.
[0769] The present invention also provides in a network, a method
for determining whether a subject has a protein kinase,
methyltransferase, acyl-CoA dehydrogenase, short chain
dehyrdogenase, reductase, acyltransferase, phosphatase,
transferase, ATP-ase or synthase-associated or another 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-associated disease or disorder or a pre-disposition
to a protein kinase, methyltransferase, acyl-CoA dehydrogenase,
short chain dehyrdogenase, reductase, acyltransferase, phosphatase,
transferase, ATP-ase or synthase-associated or another 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-associated disease or disorder, said method
comprising the steps of receiving 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 sequence
information from the subject and/or information related thereto,
receiving phenotypic information associated with the subject,
acquiring information from the network corresponding to 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 and/or corresponding to a protein kinase,
methyltransferase, acyl-CoA dehydrogenase, short chain
dehyrdogenase, reductase, acyltransferase, phosphatase,
transferase, ATP-ase or synthase-associated or another 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-associated disease or disorder, and based on one or
more of the phenotypic information, the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
information (e.g., sequence information and/or information related
thereto), and the acquired information, determining whether the
subject has a protein kinase, methyltransferase, acyl-CoA
dehydrogenase, short chain dehyrdogenase, reductase,
acyltransferase, phosphatase, transferase, ATP-ase or
synthase-associated or another 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933-associated
disease or disorder or a pre-disposition to a protein kinase,
methyltransferase, acyl-CoA dehydrogenase, short chain
dehyrdogenase, reductase, acyltransferase, phosphatase,
transferase, ATP-ase or synthase-associated or another 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-associated disease or disorder. The method may
further comprise the step of recommending a particular treatment
for the disease, disorder, or pre-disease condition.
[0770] The present invention also provides a business method for
determining whether a subject has a protein kinase,
methyltransferase, acyl-CoA dehydrogenase, short chain
dehyrdogenase, reductase, acyltransferase, phosphatase,
transferase, ATP-ase or synthase-associated or another 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 -associated disease or disorder or a pre-disposition
to a protein kinase, methyltransferase, acyl-CoA dehydrogenase,
short chain dehyrdogenase, reductase, acyltransferase, phosphatase,
transferase, ATP-ase or synthase-associated or another 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-associated disease or disorder, said method
comprising the steps of receiving information related to 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 (e.g., sequence information and/or information
related thereto), receiving phenotypic information associated with
the subject, acquiring information from the network related to
25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 and/or related to a protein kinase,
methyltransferase, acyl-CoA dehydrogenase, short chain
dehyrdogenase, reductase, acyltransferase, phosphatase,
transferase, ATP-ase or synthase-associated or another 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-associated disease or disorder, and based on one or
more of the phenotypic information, the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
information, and the acquired information, determining whether the
subject has a protein kinase, methyltransferase, acyl-CoA
dehydrogenase, short chain dehyrdogenase, reductase,
acyltransferase, phosphatase, transferase, ATP-ase or
synthase-associated or another 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933-associated
disease or disorder or a pre-disposition to a protein kinase,
methyltransferase, acyl-CoA dehydrogenase, short chain
dehyrdogenase, reductase, acyltransferase, phosphatase,
transferase, ATP-ase or synthase-associated or another 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-associated disease or disorder. The method may
further comprise the step of recommending a particular treatment
for the disease, disorder, or pre-disease condition.
[0771] The invention also includes an array comprising a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 sequence of the present invention. The array can be
used to assay expression of one or more genes in the array. In one
embodiment, the array can be used to assay gene expression in a
tissue to ascertain tissue specificity of genes in the array. In
this manner, up to about 7600 genes can be simultaneously assayed
for expression, one of which can be 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933. This
allows a profile to be developed showing a battery of genes
specifically expressed in one or more tissues.
[0772] In addition to such qualitative information, the invention
allows the quantitation of gene expression. Thus, not only tissue
specificity, but also the level of expression of a battery of genes
in the tissue if ascertainable. Thus, genes can be grouped on the
basis of their tissue expression per se and level of expression in
that tissue. This is useful, for example, in ascertaining the
relationship of gene expression in that tissue. Thus, one tissue
can be perturbed and the effect on gene expression in a second
tissue can be determined. In this context, the effect of one cell
type on another cell type in response to a biological stimulus can
be determined. In this context, the effect of one cell type on
another cell type in response to a biological stimulus can be
determined. Such a determination is useful, for example, to know
the effect of cell-cell interaction at the level of gene
expression. If an agent is administered therapeutically to treat
one cell type but has an undesirable effect on another cell type,
the invention provides an assay to determine the molecular basis of
the undesirable effect and thus provides the opportunity to
co-administer a counteracting agent or otherwise treat the
undesired effect. Similarly, even within a single cell type,
undesirable biological effects can be determined at the molecular
level. Thus, the effects of an agent on expression of other than
the target gene can be ascertained and counteracted.
[0773] In another embodiment, the array can be used to monitor the
time course of expression of one or more genes in the array. This
can occur in various biological contexts, as disclosed herein, for
example development of a protein kinase, methyltransferase,
acyl-CoA dehydrogenase, short chain dehyrdogenase, reductase,
acyltransferase, phosphatase, transferase, ATP-ase or
synthase-associated or another 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933-associated
disease or disorder, progression of protein kinase,
methyltransferase, acyl-CoA dehydrogenase, short chain
dehyrdogenase, reductase, acyltransferase, phosphatase,
transferase, ATP-ase or synthase-associated or another 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-associated disease or disorder, and processes, such
a cellular transformation associated with the protein kinase,
methyltransferase, acyl-CoA dehydrogenase, short chain
dehyrdogenase, reductase, acyltransferase, phosphatase,
transferase, ATP-ase or synthase-associated or another 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933-associated disease or disorder.
[0774] The array is also useful for ascertaining the effect of the
expression of a gene on the expression of other genes in the same
cell or in different cells (e.g., acertaining the effect of 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 expression on the expression of other genes). This
provides, for example, for a selection of alternate molecular
targets for therapeutic intervention if the ultimate or downstream
target cannot be regulated.
[0775] The array is also useful for ascertaining differential
expression patterns of one or more genes in normal and abnormal
cells. This provides a battery of genes (e.g., including 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933) that could serve as a molecular target for
diagnosis or therapeutic intervention.
[0776] 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, may
be of shorter length.
[0777] 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 (NCBI).
[0778] Thus, the invention features a method of making a computer
readable record of a sequence of a 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 sequence
which 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 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.
[0779] In another aspect, the invention features a method of
analyzing a sequence. The method includes: providing a 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 sequence, or record, in computer readable form;
comparing a second sequence to the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 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 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 sequence includes a sequence being compared. In a preferred
embodiment the 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 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.
EXEMPLIFICATION
EXAMPLE 1
[0780] Tissue Distribution of 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 mRNA
[0781] 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 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
cDNA (SEQ ID NO: 1, 3, 7, 9, 16, 18, 20, 22, 25, 27, 28, 30, 35,
37, 39, 41, 42, 44, 63, 65, 67, 69, 70 or 72) or 25869, 25934,
26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or
49933 cDNA can be used. The DNA was radioactively labeled with
.sup.32P-dCTP using the Prime-It 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 hybridization solution (Clontech) and washed at high
stringency according to manufacturer's recommendations.
EXAMPLE 2
[0782] TaqMan.TM. Procedure
[0783] The Taqman.TM. procedure is a quantitative, real-time
PCR-based approach to detecting mRNA. The RT-PCR reaction exploits
the 5' nuclease activity of AmpliTaq Gold.TM. DNA Polymerase to
cleave a TaqMan.TM. probe during PCR. Briefly, cDNA was generated
from the samples of interest and served as the starting material
for PCR amplification. In addition to the 5' and 3' gene-specific
primers, a gene-specific oligonucleotide probe (complementary to
the region being amplified) was included in the reaction (i.e., the
Taqman.TM. probe). The TaqMan.TM. probe included an oligonucleotide
with a fluorescent reporter dye covalently linked to the 5' end of
the probe (such as FAM (6-carboxyfluorescein), TET
(6-carboxy-4,7,2',7'-tetrachlorofluorescein), JOE
(6-carboxy-4,5-dichloro- -2,7-dimethoxyfluorescein), or VIC) and a
quencher dye (TAMRA (6-carboxy-N,N,N',N'-tetramethylrhodamine) at
the 3' end of the probe.
[0784] During the PCR reaction, cleavage of the probe separated the
reporter dye and the quencher dye, resulting in increased
fluorescence of the reporter. Accumulation of PCR products was
detected directly by monitoring the increase in fluorescence of the
reporter dye. When the probe was intact, the proximity of the
reporter dye to the quencher dye resulted in suppression of the
reporter fluorescence. During PCR, if the target of interest was
present, the probe specifically annealed between the forward and
reverse primer sites. The 5'-3' nucleolytic activity of the
AmpliTaq.TM. Gold DNA Polymerase cleaved the probe between the
reporter and the quencher only if the probe hybridized to the
target. The probe fragments were then displaced from the target,
and polymerization of the strand continued. The 3' end of the probe
was blocked to prevent extension of the probe during PCR. This
process occurred in every cycle and did not interfere with the
exponential accumulation of product. RNA was prepared using the
trizol method and treated with DNase to remove contaminating
genomic DNA. cDNA was synthesized using standard techniques. Mock
cDNA synthesis in the absence of reverse transcriptase resulted in
samples with no detectable PCR amplification of the control GAPDH
or .beta.-actin gene confirming efficient removal of genomic DNA
contamination.
EXAMPLE 3
[0785] In Situ Hybridization Procedure
[0786] For in situ analysis, various tissues, e.g. tissues obtained
from brain, are first frozen on dry ice. Ten-micrometer-thick
sections of the tissues are postfixed with 4% formaldehyde in DEPC
treated 1.times. phosphate-buffered saline at room temperature for
10 minutes before being rinsed twice in DEPC 1.times.
phosphate-buffered saline and once in 0.1 M triethanolamine-HCl (pH
8.0). Following incubation in 0.25% acetic anhydride-0.1 M
triethanolamine-HCl for 10 minutes, sections are rinsed in DEPC
2.times. SSC (1.times. SSC is 0.15M NaCl plus 0.015M sodium
citrate). Tissue is then dehydrated through a series of ethanol
washes, incubated in 100% chloroform for 5 minutes, and then rinsed
in 100% ethanol for 1 minute and 95% ethanol for 1 minute and
allowed to air dry.
[0787] Hybridizations are performed with .sup.35S-radiolabeled
(5.times.10.sup.7 cpm/ml) cRNA probes. Probes are incubated in the
presence of a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5),
1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05%
yeast total RNA type X1, 1.times. Denhardt's solution, 50%
formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodium
dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hours at
55.degree. C.
[0788] After hybridization, slides are washed with 2.times.SSC.
Sections are then sequentially incubated at 37.degree. C. in TNE (a
solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM
EDTA), for 10 minutes, in TNE with 10 ug of RNase A per ml for 30
minutes, and finally in TNE for 10 minutes. Slides are then rinsed
with 2.times.SSC at room temperature, washed with 2.times. SSC at
50.degree. C. for 1 hour, washed with 0.2.times. SSC at 55.degree.
C. for 1 hour, and 0.2.times. SSC at 60.degree. C. for 1 hour.
Sections are then dehydrated rapidly through serial ethanol-0.3 M
sodium acetate concentrations before being air dried and exposed to
Kodak Biomax MR scientific imaging film for 24 hours and
subsequently dipped in NB-2 photoemulsion and exposed at 4.degree.
C. for 7 days before being developed and counter stained.
EXAMPLE 4
[0789] Recombinant Expression of 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 in Bacterial
Cells
[0790] In this example, 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 is expressed as a
recombinant glutathione-S-transferase (GST) fusion polypeptide in
E. coli and the fusion polypeptide is isolated and characterized.
Specifically, 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 is fused to GST and this fusion
polypeptide is expressed in E. coli, e.g., strain PEB199.
Expression of the GST-25869, -25934, -26335, -50365, -21117,
-38692, -46508, -16816, -16839, -49937, -49931 or -49933 fusion
protein in PEB199 is induced with IPTG. The recombinant fusion
polypeptide is purified from crude bacterial lysates of the induced
PEB 199 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 5
[0791] Expression of Recombinant 25869, 25934, 26335, 50365, 21117,
38692, 46508, 16816, 16839, 49937, 49931 or 49933 Protein in COS
Cells
[0792] To express the 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 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 25869,
25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839, 49937,
49931 or 49933 protein and an HA tag (Wilson et al. (1984) Cell
37:767) or a FLAG 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.
[0793] To construct the plasmid, the 25869, 25934, 26335, 50365,
21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117, 38692,
46508, 16816, 16839, 49937, 49931 or 49933 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 tag and the last 20
nucleotides of the 25869, 25934, 26335, 50365, 21117, 38692, 46508,
16816, 16839, 49937, 49931 or 49933 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 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816, 16839,
49937, 49931 or 49933 gene is inserted in the correct orientation.
The ligation mixture is transformed into E. coli cells (strains
HB101, DH5.alpha., 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.
[0794] COS cells are subsequently transfected with the 25869-,
25934-, 26335-, 50365-, 21117-, 38692-, 46508-, 16816-, 16839-,
49937-, 49931- or 49933-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, J., Fritsh, E. F., and Maniatis, T.
Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989. The expression of the 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
polypeptide is detected by radiolabelling (.sup.35S-methionine or
.sup.35S-cysteine available from NEN, Boston, Mass., can be used)
and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1988) 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 mM NaCl, 1%
NP-40, 0.1% SDS, 0.5% DOC, 50 mM 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.
[0795] Alternatively, DNA containing the 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
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 25869, 25934, 26335,
50365, 21117, 38692, 46508, 16816, 16839, 49937, 49931 or 49933
polypeptide is detected by radiolabelling and immunoprecipitation
using a 25869, 25934, 26335, 50365, 21117, 38692, 46508, 16816,
16839, 49937, 49931 or 49933 specific monoclonal antibody.
[0796] The contents of all references, patents and published patent
applications cited throughout this application are incorporated
herein by reference.
[0797] Equivalents
[0798] 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.
Sequence CWU 1
1
114 1 2087 DNA Homo Sapiens CDS (222)...(1865) 1 accacgcgtc
cgcagcttgg tcaacagagc tagaccctgt ctcaaacaca aaaaataaaa 60
acaaagaaat gggcaagtgg ctggccccaa ggcacaaggc cctagtggca ggcccggtct
120 ggcctggagt ggagtggtag tgactctcag gcaggcaggg aggaggaagt
tgggcgtcaa 180 cctaagacca ggctcaccgg cttgctggga aggttaccaa g atg
ctg tgt ggg ccc 236 Met Leu Cys Gly Pro 1 5 gaa gtt gct cag cct gaa
gta gac acc acc ctg ggt cgt gtg cga ggc 284 Glu Val Ala Gln Pro Glu
Val Asp Thr Thr Leu Gly Arg Val Arg Gly 10 15 20 cgg cag gtg ggc
gtg aag ggc aca gac cgc ctt gtg aat gtc ttt ctg 332 Arg Gln Val Gly
Val Lys Gly Thr Asp Arg Leu Val Asn Val Phe Leu 25 30 35 ggc att
cca ttt gcc cag ccg cca ctg ggc cct gac cgg ttc tca gcc 380 Gly Ile
Pro Phe Ala Gln Pro Pro Leu Gly Pro Asp Arg Phe Ser Ala 40 45 50
cca cac cca gca cag ccc tgg gag ggt gtg cgg gat gcc agc act gcg 428
Pro His Pro Ala Gln Pro Trp Glu Gly Val Arg Asp Ala Ser Thr Ala 55
60 65 ccc cca atg tgc cta caa gac gtg gag agc atg aac agc agc aga
ttt 476 Pro Pro Met Cys Leu Gln Asp Val Glu Ser Met Asn Ser Ser Arg
Phe 70 75 80 85 gtc ctc aac gga aaa cag cag atc ttc tcc gtt tca gag
gac tgc ctg 524 Val Leu Asn Gly Lys Gln Gln Ile Phe Ser Val Ser Glu
Asp Cys Leu 90 95 100 gtc ctc aac gtc tat agc cca gct gag gtc ccc
gca ggg tcc ggt agg 572 Val Leu Asn Val Tyr Ser Pro Ala Glu Val Pro
Ala Gly Ser Gly Arg 105 110 115 ccg gtc atg gta tgg gtc cat gga ggc
gct ctg ata act ggc gct gcc 620 Pro Val Met Val Trp Val His Gly Gly
Ala Leu Ile Thr Gly Ala Ala 120 125 130 acc tcc tac gat gga tca gct
ctg gct gcc tat ggg gat gtg gtc gtg 668 Thr Ser Tyr Asp Gly Ser Ala
Leu Ala Ala Tyr Gly Asp Val Val Val 135 140 145 gtt aca gtc cag tac
cgc ctt ggg gtc ctt ggc ttc ttc agc act gga 716 Val Thr Val Gln Tyr
Arg Leu Gly Val Leu Gly Phe Phe Ser Thr Gly 150 155 160 165 gat gag
cat gca cct ggc aac cag ggc ttc cta gat gtg gta gct gct 764 Asp Glu
His Ala Pro Gly Asn Gln Gly Phe Leu Asp Val Val Ala Ala 170 175 180
ttg cgc tgg gtg caa gaa aac atc gcc ccc ttc ggg ggt gac ctc aac 812
Leu Arg Trp Val Gln Glu Asn Ile Ala Pro Phe Gly Gly Asp Leu Asn 185
190 195 tgt gtc act gtc ttt ggt gga tct gcc ggt ggg agc atc atc tct
ggc 860 Cys Val Thr Val Phe Gly Gly Ser Ala Gly Gly Ser Ile Ile Ser
Gly 200 205 210 ctg gtc ctg tcc cca gtg gct gca ggg ctg ttc cac aga
gcc atc aca 908 Leu Val Leu Ser Pro Val Ala Ala Gly Leu Phe His Arg
Ala Ile Thr 215 220 225 cag agt ggg gtc atc acc acc cca ggg atc atc
gac tct cac cct tgg 956 Gln Ser Gly Val Ile Thr Thr Pro Gly Ile Ile
Asp Ser His Pro Trp 230 235 240 245 ccc cta gct cag aaa atc gca aac
acc ttg gcc tgc agc tcc agc tcc 1004 Pro Leu Ala Gln Lys Ile Ala
Asn Thr Leu Ala Cys Ser Ser Ser Ser 250 255 260 ccg gct gag atg gtg
cag tgc ctt cag cag aaa gaa gga gaa gag ctg 1052 Pro Ala Glu Met
Val Gln Cys Leu Gln Gln Lys Glu Gly Glu Glu Leu 265 270 275 gtc ctt
agc aag aag ctg aaa aat act atc tat cct ctc acc gtt gat 1100 Val
Leu Ser Lys Lys Leu Lys Asn Thr Ile Tyr Pro Leu Thr Val Asp 280 285
290 ggc act gtc ttc ccc aaa agc ccc aag gaa ctc ctg aag gag aag ccc
1148 Gly Thr Val Phe Pro Lys Ser Pro Lys Glu Leu Leu Lys Glu Lys
Pro 295 300 305 ttc cac tct gtg ccc ttc ctc atg ggt gtc aac aac cat
gag ttc agc 1196 Phe His Ser Val Pro Phe Leu Met Gly Val Asn Asn
His Glu Phe Ser 310 315 320 325 tgg ctc atc ccc agg ggc tgg ggt ctc
ctg gat aca atg gag cag atg 1244 Trp Leu Ile Pro Arg Gly Trp Gly
Leu Leu Asp Thr Met Glu Gln Met 330 335 340 agc cgg gag gac atg ctg
gcc atc tca aca ccc gtc ttg acc agt ctg 1292 Ser Arg Glu Asp Met
Leu Ala Ile Ser Thr Pro Val Leu Thr Ser Leu 345 350 355 gat gtg ccc
cct gag atg atg ccc acc gtc ata gat gaa tac cta gga 1340 Asp Val
Pro Pro Glu Met Met Pro Thr Val Ile Asp Glu Tyr Leu Gly 360 365 370
agc aac tcg gac gca caa gcc aaa tgc cag gcg ttc cag gaa ttc atg
1388 Ser Asn Ser Asp Ala Gln Ala Lys Cys Gln Ala Phe Gln Glu Phe
Met 375 380 385 ggt gac gta ttc atc aat gtt ccc acc gtc agt ttt tca
aga tac ctt 1436 Gly Asp Val Phe Ile Asn Val Pro Thr Val Ser Phe
Ser Arg Tyr Leu 390 395 400 405 cga gat tct gga agc cct gtc ttt ttc
tat gag ttc cag cat cga ccc 1484 Arg Asp Ser Gly Ser Pro Val Phe
Phe Tyr Glu Phe Gln His Arg Pro 410 415 420 agt tct ttt gcg aag atc
aaa cct gcc tgg gtg aag gct gat cat ggg 1532 Ser Ser Phe Ala Lys
Ile Lys Pro Ala Trp Val Lys Ala Asp His Gly 425 430 435 gcc gag ggt
gct ttt gtg ttc gga ggt ccc ttc ctc atg gac gag agc 1580 Ala Glu
Gly Ala Phe Val Phe Gly Gly Pro Phe Leu Met Asp Glu Ser 440 445 450
tcc cgc ctg gcc ttt cca gag gcc aca gag gag gag aag cag cta agc
1628 Ser Arg Leu Ala Phe Pro Glu Ala Thr Glu Glu Glu Lys Gln Leu
Ser 455 460 465 ctc acc atg atg gcc cag tgg acc cac ttt gcc cgg aca
ggg gac ccc 1676 Leu Thr Met Met Ala Gln Trp Thr His Phe Ala Arg
Thr Gly Asp Pro 470 475 480 485 aat agc aag gct ctg cct cct tgg ccc
caa ttc aac cag gcg gaa caa 1724 Asn Ser Lys Ala Leu Pro Pro Trp
Pro Gln Phe Asn Gln Ala Glu Gln 490 495 500 tat ctg gag atc aac cca
gtg cca cgg gcc gga cag aag ttc agg gag 1772 Tyr Leu Glu Ile Asn
Pro Val Pro Arg Ala Gly Gln Lys Phe Arg Glu 505 510 515 gcc tgg atg
cag ttc tgg tca gag acg ctc ccc agc aag ata caa cag 1820 Ala Trp
Met Gln Phe Trp Ser Glu Thr Leu Pro Ser Lys Ile Gln Gln 520 525 530
tgg cac cag aag cag aag aac agg aag gcc cag gag gac ctc tga 1865
Trp His Gln Lys Gln Lys Asn Arg Lys Ala Gln Glu Asp Leu * 535 540
545 ggccaggcct gaaccttctt ggctggggca aaccactctt caagtggtgg
cagagtccca 1925 gcacggcagc ccgcctctcc ccctgctgag actttaatct
ccaccagccc ttaaagtgtc 1985 ggccgctctg tgactggagt tatgctcttt
tgaaatgtca caaggccgcc tcccacctct 2045 ggggcattgt acaagttctt
ccctctcaaa aaaaaaaaaa aa 2087 2 547 PRT Homo Sapiens 2 Met Leu Cys
Gly Pro Glu Val Ala Gln Pro Glu Val Asp Thr Thr Leu 1 5 10 15 Gly
Arg Val Arg Gly Arg Gln Val Gly Val Lys Gly Thr Asp Arg Leu 20 25
30 Val Asn Val Phe Leu Gly Ile Pro Phe Ala Gln Pro Pro Leu Gly Pro
35 40 45 Asp Arg Phe Ser Ala Pro His Pro Ala Gln Pro Trp Glu Gly
Val Arg 50 55 60 Asp Ala Ser Thr Ala Pro Pro Met Cys Leu Gln Asp
Val Glu Ser Met 65 70 75 80 Asn Ser Ser Arg Phe Val Leu Asn Gly Lys
Gln Gln Ile Phe Ser Val 85 90 95 Ser Glu Asp Cys Leu Val Leu Asn
Val Tyr Ser Pro Ala Glu Val Pro 100 105 110 Ala Gly Ser Gly Arg Pro
Val Met Val Trp Val His Gly Gly Ala Leu 115 120 125 Ile Thr Gly Ala
Ala Thr Ser Tyr Asp Gly Ser Ala Leu Ala Ala Tyr 130 135 140 Gly Asp
Val Val Val Val Thr Val Gln Tyr Arg Leu Gly Val Leu Gly 145 150 155
160 Phe Phe Ser Thr Gly Asp Glu His Ala Pro Gly Asn Gln Gly Phe Leu
165 170 175 Asp Val Val Ala Ala Leu Arg Trp Val Gln Glu Asn Ile Ala
Pro Phe 180 185 190 Gly Gly Asp Leu Asn Cys Val Thr Val Phe Gly Gly
Ser Ala Gly Gly 195 200 205 Ser Ile Ile Ser Gly Leu Val Leu Ser Pro
Val Ala Ala Gly Leu Phe 210 215 220 His Arg Ala Ile Thr Gln Ser Gly
Val Ile Thr Thr Pro Gly Ile Ile 225 230 235 240 Asp Ser His Pro Trp
Pro Leu Ala Gln Lys Ile Ala Asn Thr Leu Ala 245 250 255 Cys Ser Ser
Ser Ser Pro Ala Glu Met Val Gln Cys Leu Gln Gln Lys 260 265 270 Glu
Gly Glu Glu Leu Val Leu Ser Lys Lys Leu Lys Asn Thr Ile Tyr 275 280
285 Pro Leu Thr Val Asp Gly Thr Val Phe Pro Lys Ser Pro Lys Glu Leu
290 295 300 Leu Lys Glu Lys Pro Phe His Ser Val Pro Phe Leu Met Gly
Val Asn 305 310 315 320 Asn His Glu Phe Ser Trp Leu Ile Pro Arg Gly
Trp Gly Leu Leu Asp 325 330 335 Thr Met Glu Gln Met Ser Arg Glu Asp
Met Leu Ala Ile Ser Thr Pro 340 345 350 Val Leu Thr Ser Leu Asp Val
Pro Pro Glu Met Met Pro Thr Val Ile 355 360 365 Asp Glu Tyr Leu Gly
Ser Asn Ser Asp Ala Gln Ala Lys Cys Gln Ala 370 375 380 Phe Gln Glu
Phe Met Gly Asp Val Phe Ile Asn Val Pro Thr Val Ser 385 390 395 400
Phe Ser Arg Tyr Leu Arg Asp Ser Gly Ser Pro Val Phe Phe Tyr Glu 405
410 415 Phe Gln His Arg Pro Ser Ser Phe Ala Lys Ile Lys Pro Ala Trp
Val 420 425 430 Lys Ala Asp His Gly Ala Glu Gly Ala Phe Val Phe Gly
Gly Pro Phe 435 440 445 Leu Met Asp Glu Ser Ser Arg Leu Ala Phe Pro
Glu Ala Thr Glu Glu 450 455 460 Glu Lys Gln Leu Ser Leu Thr Met Met
Ala Gln Trp Thr His Phe Ala 465 470 475 480 Arg Thr Gly Asp Pro Asn
Ser Lys Ala Leu Pro Pro Trp Pro Gln Phe 485 490 495 Asn Gln Ala Glu
Gln Tyr Leu Glu Ile Asn Pro Val Pro Arg Ala Gly 500 505 510 Gln Lys
Phe Arg Glu Ala Trp Met Gln Phe Trp Ser Glu Thr Leu Pro 515 520 525
Ser Lys Ile Gln Gln Trp His Gln Lys Gln Lys Asn Arg Lys Ala Gln 530
535 540 Glu Asp Leu 545 3 1644 DNA Homo Sapiens CDS (1)...(1644) 3
atg ctg tgt ggg ccc gaa gtt gct cag cct gaa gta gac acc acc ctg 48
Met Leu Cys Gly Pro Glu Val Ala Gln Pro Glu Val Asp Thr Thr Leu 1 5
10 15 ggt cgt gtg cga ggc cgg cag gtg ggc gtg aag ggc aca gac cgc
ctt 96 Gly Arg Val Arg Gly Arg Gln Val Gly Val Lys Gly Thr Asp Arg
Leu 20 25 30 gtg aat gtc ttt ctg ggc att cca ttt gcc cag ccg cca
ctg ggc cct 144 Val Asn Val Phe Leu Gly Ile Pro Phe Ala Gln Pro Pro
Leu Gly Pro 35 40 45 gac cgg ttc tca gcc cca cac cca gca cag ccc
tgg gag ggt gtg cgg 192 Asp Arg Phe Ser Ala Pro His Pro Ala Gln Pro
Trp Glu Gly Val Arg 50 55 60 gat gcc agc act gcg ccc cca atg tgc
cta caa gac gtg gag agc atg 240 Asp Ala Ser Thr Ala Pro Pro Met Cys
Leu Gln Asp Val Glu Ser Met 65 70 75 80 aac agc agc aga ttt gtc ctc
aac gga aaa cag cag atc ttc tcc gtt 288 Asn Ser Ser Arg Phe Val Leu
Asn Gly Lys Gln Gln Ile Phe Ser Val 85 90 95 tca gag gac tgc ctg
gtc ctc aac gtc tat agc cca gct gag gtc ccc 336 Ser Glu Asp Cys Leu
Val Leu Asn Val Tyr Ser Pro Ala Glu Val Pro 100 105 110 gca ggg tcc
ggt agg ccg gtc atg gta tgg gtc cat gga ggc gct ctg 384 Ala Gly Ser
Gly Arg Pro Val Met Val Trp Val His Gly Gly Ala Leu 115 120 125 ata
act ggc gct gcc acc tcc tac gat gga tca gct ctg gct gcc tat 432 Ile
Thr Gly Ala Ala Thr Ser Tyr Asp Gly Ser Ala Leu Ala Ala Tyr 130 135
140 ggg gat gtg gtc gtg gtt aca gtc cag tac cgc ctt ggg gtc ctt ggc
480 Gly Asp Val Val Val Val Thr Val Gln Tyr Arg Leu Gly Val Leu Gly
145 150 155 160 ttc ttc agc act gga gat gag cat gca cct ggc aac cag
ggc ttc cta 528 Phe Phe Ser Thr Gly Asp Glu His Ala Pro Gly Asn Gln
Gly Phe Leu 165 170 175 gat gtg gta gct gct ttg cgc tgg gtg caa gaa
aac atc gcc ccc ttc 576 Asp Val Val Ala Ala Leu Arg Trp Val Gln Glu
Asn Ile Ala Pro Phe 180 185 190 ggg ggt gac ctc aac tgt gtc act gtc
ttt ggt gga tct gcc ggt ggg 624 Gly Gly Asp Leu Asn Cys Val Thr Val
Phe Gly Gly Ser Ala Gly Gly 195 200 205 agc atc atc tct ggc ctg gtc
ctg tcc cca gtg gct gca ggg ctg ttc 672 Ser Ile Ile Ser Gly Leu Val
Leu Ser Pro Val Ala Ala Gly Leu Phe 210 215 220 cac aga gcc atc aca
cag agt ggg gtc atc acc acc cca ggg atc atc 720 His Arg Ala Ile Thr
Gln Ser Gly Val Ile Thr Thr Pro Gly Ile Ile 225 230 235 240 gac tct
cac cct tgg ccc cta gct cag aaa atc gca aac acc ttg gcc 768 Asp Ser
His Pro Trp Pro Leu Ala Gln Lys Ile Ala Asn Thr Leu Ala 245 250 255
tgc agc tcc agc tcc ccg gct gag atg gtg cag tgc ctt cag cag aaa 816
Cys Ser Ser Ser Ser Pro Ala Glu Met Val Gln Cys Leu Gln Gln Lys 260
265 270 gaa gga gaa gag ctg gtc ctt agc aag aag ctg aaa aat act atc
tat 864 Glu Gly Glu Glu Leu Val Leu Ser Lys Lys Leu Lys Asn Thr Ile
Tyr 275 280 285 cct ctc acc gtt gat ggc act gtc ttc ccc aaa agc ccc
aag gaa ctc 912 Pro Leu Thr Val Asp Gly Thr Val Phe Pro Lys Ser Pro
Lys Glu Leu 290 295 300 ctg aag gag aag ccc ttc cac tct gtg ccc ttc
ctc atg ggt gtc aac 960 Leu Lys Glu Lys Pro Phe His Ser Val Pro Phe
Leu Met Gly Val Asn 305 310 315 320 aac cat gag ttc agc tgg ctc atc
ccc agg ggc tgg ggt ctc ctg gat 1008 Asn His Glu Phe Ser Trp Leu
Ile Pro Arg Gly Trp Gly Leu Leu Asp 325 330 335 aca atg gag cag atg
agc cgg gag gac atg ctg gcc atc tca aca ccc 1056 Thr Met Glu Gln
Met Ser Arg Glu Asp Met Leu Ala Ile Ser Thr Pro 340 345 350 gtc ttg
acc agt ctg gat gtg ccc cct gag atg atg ccc acc gtc ata 1104 Val
Leu Thr Ser Leu Asp Val Pro Pro Glu Met Met Pro Thr Val Ile 355 360
365 gat gaa tac cta gga agc aac tcg gac gca caa gcc aaa tgc cag gcg
1152 Asp Glu Tyr Leu Gly Ser Asn Ser Asp Ala Gln Ala Lys Cys Gln
Ala 370 375 380 ttc cag gaa ttc atg ggt gac gta ttc atc aat gtt ccc
acc gtc agt 1200 Phe Gln Glu Phe Met Gly Asp Val Phe Ile Asn Val
Pro Thr Val Ser 385 390 395 400 ttt tca aga tac ctt cga gat tct gga
agc cct gtc ttt ttc tat gag 1248 Phe Ser Arg Tyr Leu Arg Asp Ser
Gly Ser Pro Val Phe Phe Tyr Glu 405 410 415 ttc cag cat cga ccc agt
tct ttt gcg aag atc aaa cct gcc tgg gtg 1296 Phe Gln His Arg Pro
Ser Ser Phe Ala Lys Ile Lys Pro Ala Trp Val 420 425 430 aag gct gat
cat ggg gcc gag ggt gct ttt gtg ttc gga ggt ccc ttc 1344 Lys Ala
Asp His Gly Ala Glu Gly Ala Phe Val Phe Gly Gly Pro Phe 435 440 445
ctc atg gac gag agc tcc cgc ctg gcc ttt cca gag gcc aca gag gag
1392 Leu Met Asp Glu Ser Ser Arg Leu Ala Phe Pro Glu Ala Thr Glu
Glu 450 455 460 gag aag cag cta agc ctc acc atg atg gcc cag tgg acc
cac ttt gcc 1440 Glu Lys Gln Leu Ser Leu Thr Met Met Ala Gln Trp
Thr His Phe Ala 465 470 475 480 cgg aca ggg gac ccc aat agc aag gct
ctg cct cct tgg ccc caa ttc 1488 Arg Thr Gly Asp Pro Asn Ser Lys
Ala Leu Pro Pro Trp Pro Gln Phe 485 490 495 aac cag gcg gaa caa tat
ctg gag atc aac cca gtg cca cgg gcc gga 1536 Asn Gln Ala Glu Gln
Tyr Leu Glu Ile Asn Pro Val Pro Arg Ala Gly 500 505 510 cag aag ttc
agg gag gcc tgg atg cag ttc tgg tca gag acg ctc ccc 1584 Gln Lys
Phe Arg Glu Ala Trp Met Gln Phe Trp Ser Glu Thr Leu Pro 515 520 525
agc aag ata caa cag tgg cac cag aag cag aag aac agg aag gcc cag
1632 Ser Lys Ile Gln Gln Trp His Gln Lys Gln Lys Asn Arg Lys Ala
Gln 530 535 540 gag gac ctc tga 1644 Glu Asp Leu * 545 4 554 PRT
Mus Musculus 4 Met Ala Cys Leu Leu Leu Ile Phe Pro Thr Thr Val Ile
Gly Pro Lys 1 5 10 15 Val Thr Gln Pro Glu Val Asp Thr Pro Leu Gly
Arg Val Arg Gly Arg 20
25 30 Gln Val Gly Val Lys Asp Thr Asp Arg Met Val Asn Val Phe Leu
Gly 35 40 45 Ile Pro Phe Ala Gln Ala Pro Leu Gly Pro Leu Arg Phe
Ser Ala Pro 50 55 60 Leu Pro Pro Gln Pro Trp Glu Gly Val Arg Asp
Ala Ser Ile Asn Pro 65 70 75 80 Pro Met Cys Leu Gln Asp Val Glu Arg
Met Ser Asn Ser Arg Phe Thr 85 90 95 Leu Asn Glu Lys Met Lys Ile
Phe Pro Ile Ser Glu Asp Cys Leu Thr 100 105 110 Leu Asn Ile Tyr Ser
Pro Thr Glu Ile Thr Ala Gly Asp Lys Arg Pro 115 120 125 Val Met Val
Trp Ile His Gly Gly Ser Leu Arg Val Gly Ser Ser Thr 130 135 140 Ser
His Asp Gly Ser Ala Leu Ala Ala Tyr Gly Asp Val Val Val Val 145 150
155 160 Thr Val Gln Tyr Arg Leu Gly Ile Phe Gly Phe Leu Ser Thr Gly
Asp 165 170 175 Lys His Met Pro Gly Asn Arg Gly Phe Leu Asp Val Val
Ala Ala Leu 180 185 190 Arg Trp Val Gln Gly Asn Ile Ala Pro Phe Gly
Gly Asp Pro Asn Cys 195 200 205 Val Thr Ile Phe Gly Asn Ser Ala Gly
Gly Ile Ile Val Ser Ser Leu 210 215 220 Leu Leu Ser Pro Met Ser Ala
Gly Leu Phe His Arg Ala Ile Ser Gln 225 230 235 240 Ser Gly Val Val
Ile Ser Lys Ile Leu Glu Asp Leu Asn Ala Trp Ser 245 250 255 Glu Ala
Gln Asn Phe Ala Asn Ser Val Ala Cys Gly Ser Ala Ser Pro 260 265 270
Ala Glu Leu Val Gln Cys Leu Leu Gln Lys Glu Gly Lys Asp Leu Ile 275
280 285 Thr Lys Lys Asn Val Asn Ile Ser Tyr Thr Val Asn Asp Ser Phe
Phe 290 295 300 Pro Gln Arg Pro Gln Lys Leu Leu Ala Asn Lys Gln Phe
Pro Thr Val 305 310 315 320 Pro Tyr Leu Leu Gly Val Thr Asn His Glu
Phe Gly Trp Leu Leu Leu 325 330 335 Lys Phe Trp Asn Ile Leu Asp Lys
Met Glu His Leu Ser Gln Glu Asp 340 345 350 Leu Leu Glu Asn Ser Arg
Pro Leu Leu Ala His Met Gln Leu Pro Pro 355 360 365 Glu Ile Met Pro
Thr Val Ile Asp Glu Tyr Leu Asp Asn Gly Ser Asp 370 375 380 Glu Ser
Ala Thr Arg Tyr Ala Leu Gln Glu Leu Leu Gly Asp Ile Thr 385 390 395
400 Leu Val Ile Pro Thr Leu Ile Phe Ser Lys Tyr Leu Gln Asp Ala Gly
405 410 415 Cys Pro Val Phe Leu Tyr Glu Phe Gln His Thr Pro Ser Ser
Phe Ala 420 425 430 Lys Phe Lys Pro Ala Trp Val Lys Ala Asp His Ser
Ser Glu Asn Ala 435 440 445 Phe Val Phe Gly Gly Pro Phe Leu Thr Asp
Glu Ser Ser Leu Leu Ala 450 455 460 Phe Pro Glu Ala Thr Glu Glu Glu
Lys Gln Leu Ser Leu Thr Met Met 465 470 475 480 Ala Gln Trp Ser Gln
Phe Ala Arg Thr Gly Asn Pro Asn Gly Lys Gly 485 490 495 Leu Pro Pro
Trp Pro Gln Leu Asn Gln Leu Glu Gln Tyr Leu Glu Ile 500 505 510 Gly
Leu Glu Pro Arg Thr Gly Val Lys Leu Lys Lys Gly Arg Leu Gln 515 520
525 Phe Trp Thr Glu Thr Leu Pro Arg Lys Ile Gln Glu Trp His Arg Glu
530 535 540 Gln Arg Ser Arg Lys Val Pro Glu Glu Leu 545 550 5 565
PRT Rattus Norvegicus 5 Met Arg Leu Tyr Pro Leu Val Trp Leu Phe Leu
Ala Ala Cys Thr Ala 1 5 10 15 Trp Gly Tyr Pro Ser Ser Pro Pro Val
Val Asn Thr Val Lys Gly Lys 20 25 30 Val Leu Gly Lys Tyr Val Asn
Leu Glu Gly Phe Ala Gln Pro Val Ala 35 40 45 Val Phe Leu Gly Ile
Pro Phe Ala Lys Pro Pro Leu Gly Ser Leu Arg 50 55 60 Phe Ala Pro
Pro Gln Pro Ala Glu Pro Trp Asn Phe Val Lys Asn Thr 65 70 75 80 Thr
Ser Tyr Pro Pro Met Cys Ser Gln Asp Ala Val Gly Gly Gln Val 85 90
95 Leu Ser Glu Leu Phe Thr Asn Arg Lys Glu Asn Ile Pro Leu Gln Phe
100 105 110 Ser Glu Asp Cys Leu Tyr Leu Asn Val Tyr Thr Pro Ala Asp
Leu Thr 115 120 125 Lys Asn Ser Arg Leu Pro Val Met Val Trp Ile His
Gly Gly Gly Leu 130 135 140 Val Val Gly Gly Ala Ser Thr Tyr Asp Gly
Gln Val Leu Ser Ala His 145 150 155 160 Glu Asn Val Val Val Val Thr
Ile Gln Tyr Arg Leu Gly Ile Trp Gly 165 170 175 Phe Phe Ser Thr Gly
Asp Glu His Ser Arg Gly Asn Trp Gly His Leu 180 185 190 Asp Gln Val
Ala Ala Leu His Trp Val Gln Asp Asn Ile Ala Asn Phe 195 200 205 Gly
Gly Asn Pro Gly Ser Val Thr Ile Phe Gly Glu Ser Ala Gly Gly 210 215
220 Phe Ser Val Ser Ala Leu Val Leu Ser Pro Leu Ala Lys Asn Leu Phe
225 230 235 240 His Arg Ala Ile Ser Glu Ser Gly Val Val Leu Thr Ser
Ala Leu Ile 245 250 255 Thr Thr Asp Ser Lys Pro Ile Ala Lys Leu Ile
Ala Thr Leu Ser Gly 260 265 270 Cys Lys Thr Thr Thr Ser Ala Val Met
Val His Cys Leu Arg Gln Lys 275 280 285 Thr Glu Asp Glu Leu Leu Glu
Thr Ser Leu Lys Leu Asn Leu Phe Lys 290 295 300 Leu Asp Leu Leu Gly
Asn Pro Lys Glu Ser Tyr Pro Phe Leu Pro Thr 305 310 315 320 Val Ile
Asp Gly Val Val Leu Pro Lys Thr Pro Glu Glu Ile Leu Ala 325 330 335
Glu Lys Ser Phe Asn Thr Val Pro Tyr Ile Val Gly Ile Asn Lys Gln 340
345 350 Glu Phe Gly Trp Ile Ile Pro Thr Leu Met Gly Tyr Pro Leu Ser
Glu 355 360 365 Gly Lys Leu Asp Gln Lys Thr Ala Lys Ser Leu Leu Trp
Lys Ser Tyr 370 375 380 Pro Thr Leu Lys Ile Ser Glu Lys Met Ile Pro
Val Val Ala Glu Lys 385 390 395 400 Tyr Phe Gly Gly Thr Asp Asp Pro
Ala Lys Arg Lys Asp Leu Phe Gln 405 410 415 Asp Leu Val Ala Asp Val
Ile Phe Gly Val Pro Ser Val Met Val Ser 420 425 430 Arg Ser His Arg
Asp Ala Gly Ala Pro Thr Phe Met Tyr Glu Phe Glu 435 440 445 Tyr Arg
Pro Ser Phe Val Ser Ala Met Arg Pro Lys Thr Val Ile Gly 450 455 460
Asp His Gly Asp Glu Leu Phe Ser Val Phe Gly Ser Pro Phe Leu Lys 465
470 475 480 Asp Gly Ala Ser Glu Glu Glu Thr Asn Leu Ser Lys Met Val
Met Lys 485 490 495 Tyr Trp Ala Asn Phe Ala Arg Asn Gly Ser Pro Asn
Gly Gly Gly Leu 500 505 510 Pro His Trp Pro Glu Tyr Asp Gln Lys Glu
Gly Tyr Leu Lys Ile Gly 515 520 525 Ala Ser Thr Gln Ala Ala Gln Arg
Leu Lys Asp Lys Glu Val Ala Phe 530 535 540 Trp Ser Glu Leu Arg Ala
Lys Glu Ala Ala Glu Glu Pro Ser His Trp 545 550 555 560 Lys His Val
Glu Leu 565 6 5 PRT Artificial Sequence Catalytic Serine Motif 6
Gly Xaa Ser Xaa Gly 1 5 7 1512 DNA Homo Sapiens CDS (342)...(1334)
7 ccacgcgtcc ggactagttc catttccaca gctcctcctc cccggccgcg cgcccctccc
60 gccccgcgcg cgcctcctct ttctcgcggc cgagttcagc ccgggcagcc
atatggggga 120 tacgccagca acagacgccg gccgccaaga tctgcatccc
taggccacgc taagaccctg 180 gggaagagcg caggagcccg ggagaagggc
tggaaggagg ggactggacg tgcggagaat 240 tcccccctaa aaggcagaag
cccccgcccc caccctcgag ctccgctcgg gcagagcgcc 300 tgcctgcctg
ccgctgctgc gggcgcccac ctcgcccagc c atg cca ggc ccg gcc 356 Met Pro
Gly Pro Ala 1 5 acc gac gcg ggg aag atc cct ttc tgc gac gcc aag gaa
gaa atc cgt 404 Thr Asp Ala Gly Lys Ile Pro Phe Cys Asp Ala Lys Glu
Glu Ile Arg 10 15 20 gcc ggg ctc gaa agc tct gag ggc ggc ggc ggc
ccg gag agg cca ggc 452 Ala Gly Leu Glu Ser Ser Glu Gly Gly Gly Gly
Pro Glu Arg Pro Gly 25 30 35 gcg cgc ggg cag cgg cag aac atc gtc
tgg agg aat gtc gtc ctg atg 500 Ala Arg Gly Gln Arg Gln Asn Ile Val
Trp Arg Asn Val Val Leu Met 40 45 50 agc ttg ctc cac ttg ggg gcc
gtg tac tcc ctg gtg ctc atc ccc aaa 548 Ser Leu Leu His Leu Gly Ala
Val Tyr Ser Leu Val Leu Ile Pro Lys 55 60 65 gcc aag cca ctc act
ctg ctc tgg gcc tac ttc tgc ttc ctc ctg gcc 596 Ala Lys Pro Leu Thr
Leu Leu Trp Ala Tyr Phe Cys Phe Leu Leu Ala 70 75 80 85 gct ctg ggt
gtg aca gct ggt gcc cat cgc ttg tgg agc cac agg tcc 644 Ala Leu Gly
Val Thr Ala Gly Ala His Arg Leu Trp Ser His Arg Ser 90 95 100 tac
cgg gcc aag ctg cct ctg agg ata ttt ctg gct gtc gcc aac tcc 692 Tyr
Arg Ala Lys Leu Pro Leu Arg Ile Phe Leu Ala Val Ala Asn Ser 105 110
115 atg gct ttc cag aat gac atc ttc gag tgg tcc agg gac cac cga gcc
740 Met Ala Phe Gln Asn Asp Ile Phe Glu Trp Ser Arg Asp His Arg Ala
120 125 130 cac cac aag tac tca gag acg gat gct gac ccc cac aat gcc
cgc cgg 788 His His Lys Tyr Ser Glu Thr Asp Ala Asp Pro His Asn Ala
Arg Arg 135 140 145 ggc ttc ttc ttc tcc cat att ggg tgg ctg ttt gtt
cgc aag cat cga 836 Gly Phe Phe Phe Ser His Ile Gly Trp Leu Phe Val
Arg Lys His Arg 150 155 160 165 gat gtt att gag aag ggg aga aag ctt
gac gtc act gac ctg ctt gct 884 Asp Val Ile Glu Lys Gly Arg Lys Leu
Asp Val Thr Asp Leu Leu Ala 170 175 180 gat cct gtg gtc cgg atc cag
aga aag tac tat aag atc tcc gtg gtg 932 Asp Pro Val Val Arg Ile Gln
Arg Lys Tyr Tyr Lys Ile Ser Val Val 185 190 195 ctc atg tgc ttt gtg
gtc ccc acg ctg gtg ccc tgg tac atc tgg gga 980 Leu Met Cys Phe Val
Val Pro Thr Leu Val Pro Trp Tyr Ile Trp Gly 200 205 210 gag agt ctg
tgg aat tcc tac ttc ttg gcc tct att ctc cgc tat acc 1028 Glu Ser
Leu Trp Asn Ser Tyr Phe Leu Ala Ser Ile Leu Arg Tyr Thr 215 220 225
atc tca ctc aac atc agc tgg ctg gtc aac agc gcc gcc cac atg tat
1076 Ile Ser Leu Asn Ile Ser Trp Leu Val Asn Ser Ala Ala His Met
Tyr 230 235 240 245 gga aac cgg ccc tat gac aag cac atc agc cct cgg
cag aac cca ctc 1124 Gly Asn Arg Pro Tyr Asp Lys His Ile Ser Pro
Arg Gln Asn Pro Leu 250 255 260 gtc gct ctg ggt gcc att ggt gaa ggc
ttc cat aat tac cat cac acc 1172 Val Ala Leu Gly Ala Ile Gly Glu
Gly Phe His Asn Tyr His His Thr 265 270 275 ttt ccc ttt gac tac tct
gcg agt gaa ttt ggc tta aat ttt aac cca 1220 Phe Pro Phe Asp Tyr
Ser Ala Ser Glu Phe Gly Leu Asn Phe Asn Pro 280 285 290 acc acc tgg
ttc att gat ttc atg tgc tgg ctg ggg ctg gcc act gac 1268 Thr Thr
Trp Phe Ile Asp Phe Met Cys Trp Leu Gly Leu Ala Thr Asp 295 300 305
cgc aaa cgg gca acc aag ccg atg atc gag gcc cgg aag gcc agg act
1316 Arg Lys Arg Ala Thr Lys Pro Met Ile Glu Ala Arg Lys Ala Arg
Thr 310 315 320 325 gga gac agc agt gct tga acttggaaca gccatcccac
atgtctgccg 1364 Gly Asp Ser Ser Ala * 330 ttgcaacctc ggttcatggc
tttggttaca atagctctct tgtacattgg atcgtgggag 1424 ggggcagagg
gtggggaagg aacgagtcaa tgtggtttgg gaatgttttt gtttatctca 1484
aaataatgtt gaaatacaat tatcaatg 1512 8 330 PRT Homo Sapiens 8 Met
Pro Gly Pro Ala Thr Asp Ala Gly Lys Ile Pro Phe Cys Asp Ala 1 5 10
15 Lys Glu Glu Ile Arg Ala Gly Leu Glu Ser Ser Glu Gly Gly Gly Gly
20 25 30 Pro Glu Arg Pro Gly Ala Arg Gly Gln Arg Gln Asn Ile Val
Trp Arg 35 40 45 Asn Val Val Leu Met Ser Leu Leu His Leu Gly Ala
Val Tyr Ser Leu 50 55 60 Val Leu Ile Pro Lys Ala Lys Pro Leu Thr
Leu Leu Trp Ala Tyr Phe 65 70 75 80 Cys Phe Leu Leu Ala Ala Leu Gly
Val Thr Ala Gly Ala His Arg Leu 85 90 95 Trp Ser His Arg Ser Tyr
Arg Ala Lys Leu Pro Leu Arg Ile Phe Leu 100 105 110 Ala Val Ala Asn
Ser Met Ala Phe Gln Asn Asp Ile Phe Glu Trp Ser 115 120 125 Arg Asp
His Arg Ala His His Lys Tyr Ser Glu Thr Asp Ala Asp Pro 130 135 140
His Asn Ala Arg Arg Gly Phe Phe Phe Ser His Ile Gly Trp Leu Phe 145
150 155 160 Val Arg Lys His Arg Asp Val Ile Glu Lys Gly Arg Lys Leu
Asp Val 165 170 175 Thr Asp Leu Leu Ala Asp Pro Val Val Arg Ile Gln
Arg Lys Tyr Tyr 180 185 190 Lys Ile Ser Val Val Leu Met Cys Phe Val
Val Pro Thr Leu Val Pro 195 200 205 Trp Tyr Ile Trp Gly Glu Ser Leu
Trp Asn Ser Tyr Phe Leu Ala Ser 210 215 220 Ile Leu Arg Tyr Thr Ile
Ser Leu Asn Ile Ser Trp Leu Val Asn Ser 225 230 235 240 Ala Ala His
Met Tyr Gly Asn Arg Pro Tyr Asp Lys His Ile Ser Pro 245 250 255 Arg
Gln Asn Pro Leu Val Ala Leu Gly Ala Ile Gly Glu Gly Phe His 260 265
270 Asn Tyr His His Thr Phe Pro Phe Asp Tyr Ser Ala Ser Glu Phe Gly
275 280 285 Leu Asn Phe Asn Pro Thr Thr Trp Phe Ile Asp Phe Met Cys
Trp Leu 290 295 300 Gly Leu Ala Thr Asp Arg Lys Arg Ala Thr Lys Pro
Met Ile Glu Ala 305 310 315 320 Arg Lys Ala Arg Thr Gly Asp Ser Ser
Ala 325 330 9 990 DNA Homo Sapiens CDS (1)...(990) 9 atg cca ggc
ccg gcc acc gac gcg ggg aag atc cct ttc tgc gac gcc 48 Met Pro Gly
Pro Ala Thr Asp Ala Gly Lys Ile Pro Phe Cys Asp Ala 1 5 10 15 aag
gaa gaa atc cgt gcc ggg ctc gaa agc tct gag ggc ggc ggc ggc 96 Lys
Glu Glu Ile Arg Ala Gly Leu Glu Ser Ser Glu Gly Gly Gly Gly 20 25
30 ccg gag agg cca ggc gcg cgc ggg cag cgg cag aac atc gtc tgg agg
144 Pro Glu Arg Pro Gly Ala Arg Gly Gln Arg Gln Asn Ile Val Trp Arg
35 40 45 aat gtc gtc ctg atg agc ttg ctc cac ttg ggg gcc gtg tac
tcc ctg 192 Asn Val Val Leu Met Ser Leu Leu His Leu Gly Ala Val Tyr
Ser Leu 50 55 60 gtg ctc atc ccc aaa gcc aag cca ctc act ctg ctc
tgg gcc tac ttc 240 Val Leu Ile Pro Lys Ala Lys Pro Leu Thr Leu Leu
Trp Ala Tyr Phe 65 70 75 80 tgc ttc ctc ctg gcc gct ctg ggt gtg aca
gct ggt gcc cat cgc ttg 288 Cys Phe Leu Leu Ala Ala Leu Gly Val Thr
Ala Gly Ala His Arg Leu 85 90 95 tgg agc cac agg tcc tac cgg gcc
aag ctg cct ctg agg ata ttt ctg 336 Trp Ser His Arg Ser Tyr Arg Ala
Lys Leu Pro Leu Arg Ile Phe Leu 100 105 110 gct gtc gcc aac tcc atg
gct ttc cag aat gac atc ttc gag tgg tcc 384 Ala Val Ala Asn Ser Met
Ala Phe Gln Asn Asp Ile Phe Glu Trp Ser 115 120 125 agg gac cac cga
gcc cac cac aag tac tca gag acg gat gct gac ccc 432 Arg Asp His Arg
Ala His His Lys Tyr Ser Glu Thr Asp Ala Asp Pro 130 135 140 cac aat
gcc cgc cgg ggc ttc ttc ttc tcc cat att ggg tgg ctg ttt 480 His Asn
Ala Arg Arg Gly Phe Phe Phe Ser His Ile Gly Trp Leu Phe 145 150 155
160 gtt cgc aag cat cga gat gtt att gag aag ggg aga aag ctt gac gtc
528 Val Arg Lys His Arg Asp Val Ile Glu Lys Gly Arg Lys Leu Asp Val
165 170 175 act gac ctg ctt gct gat cct gtg gtc cgg atc cag aga aag
tac tat 576 Thr Asp Leu Leu Ala Asp Pro Val Val Arg Ile Gln Arg Lys
Tyr Tyr 180 185 190 aag atc tcc gtg gtg ctc atg tgc ttt gtg gtc ccc
acg ctg gtg ccc 624 Lys Ile Ser Val Val Leu Met Cys Phe Val Val Pro
Thr Leu Val Pro 195 200 205 tgg tac atc tgg gga gag agt ctg tgg aat
tcc tac ttc ttg gcc tct 672 Trp Tyr Ile Trp Gly Glu Ser Leu Trp Asn
Ser Tyr Phe Leu Ala Ser 210 215 220 att ctc cgc tat acc
atc tca ctc aac atc agc tgg ctg gtc aac agc 720 Ile Leu Arg Tyr Thr
Ile Ser Leu Asn Ile Ser Trp Leu Val Asn Ser 225 230 235 240 gcc gcc
cac atg tat gga aac cgg ccc tat gac aag cac atc agc cct 768 Ala Ala
His Met Tyr Gly Asn Arg Pro Tyr Asp Lys His Ile Ser Pro 245 250 255
cgg cag aac cca ctc gtc gct ctg ggt gcc att ggt gaa ggc ttc cat 816
Arg Gln Asn Pro Leu Val Ala Leu Gly Ala Ile Gly Glu Gly Phe His 260
265 270 aat tac cat cac acc ttt ccc ttt gac tac tct gcg agt gaa ttt
ggc 864 Asn Tyr His His Thr Phe Pro Phe Asp Tyr Ser Ala Ser Glu Phe
Gly 275 280 285 tta aat ttt aac cca acc acc tgg ttc att gat ttc atg
tgc tgg ctg 912 Leu Asn Phe Asn Pro Thr Thr Trp Phe Ile Asp Phe Met
Cys Trp Leu 290 295 300 ggg ctg gcc act gac cgc aaa cgg gca acc aag
ccg atg atc gag gcc 960 Gly Leu Ala Thr Asp Arg Lys Arg Ala Thr Lys
Pro Met Ile Glu Ala 305 310 315 320 cgg aag gcc agg act gga gac agc
agt gct 990 Arg Lys Ala Arg Thr Gly Asp Ser Ser Ala 325 330 10 248
PRT Artificial Sequence Consensus Sequence 10 Ile Leu Leu Gly Ala
Leu His Leu Gly Ala Leu Tyr Leu Leu Ala Leu 1 5 10 15 Leu Pro Thr
Glu Leu Lys Trp Lys Thr Val Ile Val Ala Leu Leu Leu 20 25 30 Tyr
Val Ile Thr Gly Gly Leu Gly Ile Thr Ala Gly Tyr His Arg Leu 35 40
45 Trp Ser His Arg Ser Tyr Lys Ala Lys Leu Pro Leu Arg Ile Phe Leu
50 55 60 Ala Ile Phe Gly Thr Leu Ala Val Gln Gly Ser Ile Tyr Glu
Trp Ala 65 70 75 80 Arg Asp His Arg Ala His His Lys Tyr Ser Asp Thr
Asp Ala Asp Pro 85 90 95 His Asp Ala Asn Arg Gly Phe Phe Phe Ser
His Val Gly Trp Leu Leu 100 105 110 Val Lys Lys His Pro Ala Val Lys
Glu Lys Gly Lys Lys Leu Asp Leu 115 120 125 Ser Asp Leu Lys Ala Asp
Pro Val Val Arg Phe Gln His Arg Tyr Tyr 130 135 140 Ile Pro Leu Met
Val Leu Met Gly Phe Ile Leu Pro Thr Leu Val Pro 145 150 155 160 Gly
Tyr Leu Trp Gly Glu Thr Phe Trp Gly Gly Phe Val Trp Ala Gly 165 170
175 Phe Leu Arg Leu Val Phe Val Leu His Ala Thr Trp Cys Val Asn Ser
180 185 190 Ala Ala His Lys Phe Gly Tyr Arg Pro Tyr Asp Ser Arg Ile
Thr Pro 195 200 205 Arg Asn Asn Trp Leu Val Ala Leu Val Thr Phe Gly
Glu Gly Trp His 210 215 220 Asn Phe His His Thr Phe Pro Tyr Asp Tyr
Arg Asn Ala Glu Lys Trp 225 230 235 240 Lys Trp Glu Tyr Asp Leu Thr
Lys 245 11 248 PRT Artificial Sequence Conserved Iron Binding Motif
11 His Xaa Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa His 35 40 45 Xaa Xaa Xaa His His Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130
135 140 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 145 150 155 160 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215 220 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 225 230 235 240 Xaa
Xaa His Xaa Xaa Xaa His His 245 12 15 PRT Artificial Sequence Fatty
Acid Desaturase Family 1 Signature Sequence 12 Gly Glu Xaa Xaa His
Asn Xaa His His Xaa Phe Pro Xaa Asp Tyr 1 5 10 15 13 359 PRT Homo
Sapiens 13 Met Pro Ala His Leu Leu Gln Asp Asp Ile Ser Ser Ser Tyr
Thr Thr 1 5 10 15 Thr Thr Thr Ile Thr Ala Pro Pro Ser Arg Val Leu
Gln Asn Gly Gly 20 25 30 Asp Lys Leu Glu Thr Met Pro Leu Tyr Leu
Glu Asp Asp Ile Arg Pro 35 40 45 Asp Ile Lys Asp Asp Ile Tyr Asp
Pro Thr Tyr Lys Asp Lys Glu Gly 50 55 60 Pro Ser Pro Lys Val Glu
Tyr Val Trp Arg Asn Ile Ile Leu Met Ser 65 70 75 80 Leu Leu His Leu
Gly Ala Leu Tyr Gly Ile Thr Leu Ile Pro Thr Cys 85 90 95 Lys Phe
Tyr Thr Trp Leu Trp Gly Val Phe Tyr Tyr Phe Val Ser Ala 100 105 110
Leu Gly Ile Thr Ala Gly Ala His Arg Leu Trp Ser His Arg Ser Tyr 115
120 125 Lys Ala Arg Leu Pro Leu Arg Leu Phe Leu Ile Ile Ala Asn Thr
Met 130 135 140 Ala Phe Gln Asn Asp Val Tyr Glu Trp Ala Arg Asp His
Arg Ala His 145 150 155 160 His Lys Phe Ser Glu Thr His Ala Asp Pro
His Asn Ser Arg Arg Gly 165 170 175 Phe Phe Phe Ser His Val Gly Trp
Leu Leu Val Arg Lys His Pro Ala 180 185 190 Val Lys Glu Lys Gly Ser
Thr Leu Asp Leu Ser Asp Leu Glu Ala Glu 195 200 205 Lys Leu Val Met
Phe Gln Arg Arg Tyr Tyr Lys Pro Gly Leu Leu Leu 210 215 220 Met Cys
Phe Ile Leu Pro Thr Leu Val Pro Trp Tyr Phe Trp Gly Glu 225 230 235
240 Thr Phe Gln Asn Ser Val Phe Val Ala Thr Phe Leu Arg Tyr Ala Val
245 250 255 Val Leu Asn Ala Thr Trp Leu Val Asn Ser Ala Ala His Leu
Phe Gly 260 265 270 Tyr Arg Pro Tyr Asp Lys Asn Ile Ser Pro Arg Glu
Asn Ile Leu Val 275 280 285 Ser Leu Gly Ala Val Gly Glu Gly Phe His
Asn Tyr His His Ser Phe 290 295 300 Pro Tyr Asp Tyr Ser Ala Ser Glu
Tyr Arg Trp His Ile Asn Phe Thr 305 310 315 320 Thr Phe Phe Ile Asp
Cys Met Ala Ala Leu Gly Leu Ala Tyr Asp Arg 325 330 335 Lys Lys Val
Ser Lys Ala Ala Ile Leu Ala Arg Ile Lys Arg Thr Gly 340 345 350 Asp
Gly Asn Tyr Lys Ser Gly 355 14 358 PRT Rattus Norvegicus 14 Met Pro
Ala His Met Leu Gln Glu Ile Ser Ser Ser Tyr Thr Thr Thr 1 5 10 15
Thr Thr Ile Thr Glu Pro Pro Ser Gly Asn Leu Gln Asn Gly Arg Glu 20
25 30 Lys Met Lys Lys Val Pro Leu Tyr Leu Glu Glu Asp Ile Arg Pro
Glu 35 40 45 Met Arg Glu Asp Ile His Asp Pro Ser Tyr Gln Asp Glu
Glu Gly Pro 50 55 60 Pro Pro Lys Leu Glu Tyr Val Trp Arg Asn Ile
Ile Leu Met Ala Leu 65 70 75 80 Leu His Val Gly Ala Leu Tyr Gly Ile
Thr Leu Ile Pro Ser Ser Lys 85 90 95 Val Tyr Thr Leu Leu Trp Gly
Ile Phe Tyr Tyr Leu Ile Ser Ala Leu 100 105 110 Gly Ile Thr Ala Gly
Ala His Arg Leu Trp Ser His Arg Thr Tyr Lys 115 120 125 Ala Arg Leu
Pro Leu Arg Ile Phe Leu Ile Ile Ala Asn Thr Met Ala 130 135 140 Phe
Gln Asn Asp Val Tyr Glu Trp Ala Arg Asp His Arg Ala His His 145 150
155 160 Lys Phe Ser Glu Thr His Ala Asp Pro His Asn Ser Arg Arg Gly
Phe 165 170 175 Phe Phe Ser His Val Gly Trp Leu Leu Val Arg Lys His
Pro Ala Val 180 185 190 Lys Glu Lys Gly Gly Lys Leu Asp Met Ser Asp
Leu Lys Ala Glu Lys 195 200 205 Leu Val Met Phe Gln Arg Arg Tyr Tyr
Lys Pro Gly Leu Leu Leu Met 210 215 220 Cys Phe Ile Leu Pro Thr Leu
Val Pro Trp Tyr Cys Trp Gly Glu Thr 225 230 235 240 Phe Leu His Ser
Leu Phe Val Ser Thr Phe Leu Arg Tyr Thr Leu Val 245 250 255 Leu Asn
Ala Thr Trp Leu Val Asn Ser Ala Ala His Leu Tyr Gly Tyr 260 265 270
Arg Pro Tyr Asp Lys Asn Ile Gln Ser Arg Glu Asn Ile Leu Val Ser 275
280 285 Leu Gly Ser Val Gly Glu Gly Phe His Asn Tyr His His Ala Phe
Pro 290 295 300 Tyr Asp Tyr Ser Ala Ser Glu Tyr Arg Trp His Ile Asn
Phe Thr Thr 305 310 315 320 Phe Phe Ile Asp Cys Met Ala Ala Leu Gly
Leu Ala Tyr Asp Arg Lys 325 330 335 Lys Val Ser Lys Ala Ala Val Leu
Ala Arg Ile Lys Arg Thr Gly Asp 340 345 350 Gly Ser His Lys Ser Ser
355 15 357 PRT Gallus Gallus 15 Met Pro Ala His Leu Leu Gln Glu Glu
Glu Phe Ser Ser Ala Ser Ser 1 5 10 15 Thr Thr Thr Val Thr Ser Arg
Val Thr Lys Asn Gly Asn Val Ile Met 20 25 30 Glu Lys Asp Leu Leu
Asn His Asp Asp Val Ala Ala Glu Arg Gly Met 35 40 45 Val Asp Asp
Leu Phe Asp Glu Thr Tyr Arg Glu Lys Glu Gly Pro Lys 50 55 60 Pro
Pro Leu Arg Tyr Val Trp Arg Asn Ile Ile Leu Met Ser Leu Leu 65 70
75 80 His Leu Gly Ala Ile Ile Gly Leu Thr Leu Ile Pro Ser Ala Lys
Ile 85 90 95 Gln Thr Leu Ala Trp Ala Ile Leu Cys Phe Val Leu Ser
Ala Leu Gly 100 105 110 Ile Thr Ala Gly Ser His Arg Leu Trp Ser His
Arg Ser Tyr Lys Ala 115 120 125 Thr Leu Pro Leu Arg Ile Phe Leu Thr
Ile Ala Asn Ser Met Ala Phe 130 135 140 Gln Asn Asp Ile Tyr Glu Trp
Ala Arg Asp His Arg Val His His Lys 145 150 155 160 Phe Ser Glu Thr
His Ala Asp Pro His Asn Ala Met Arg Gly Tyr Phe 165 170 175 Phe Ser
His Met Ala Trp Leu Leu Val Arg Lys His Pro Asp Val Ile 180 185 190
Glu Lys Gly Gln Lys Leu Asp Leu Ser Asp Leu Lys Ala Asp Lys Val 195
200 205 Val Met Phe Gln Arg Arg Tyr Tyr Lys Pro Ser Val Val Leu Leu
Cys 210 215 220 Phe Thr Leu Pro Thr Leu Val Pro Trp Tyr Phe Trp Asp
Glu Ser Ile 225 230 235 240 Ile Ile Ser Phe Phe Ile Pro Ala Ile Leu
Arg Tyr Thr Leu Gly Leu 245 250 255 Asn Ala Thr Trp Leu Val Asn Ser
Ala Ala His Met Phe Gly Asn Arg 260 265 270 Pro Tyr Asp Gln Asn Ile
Asn Pro Arg Glu Asn Pro Leu Val Ser Val 275 280 285 Gly Ala Leu Gly
Glu Gly Phe His Asn Tyr His His Thr Phe Pro Tyr 290 295 300 Asp Tyr
Ser Thr Ser Glu Phe Gly Trp Arg Phe Asn Leu Thr Thr Ala 305 310 315
320 Phe Ile Asp Leu Met Cys Leu Leu Gly Leu Ala Ser Asp Arg Lys Lys
325 330 335 Val Ser Lys Glu Val Ile Leu Ala Arg Lys Met Arg Thr Gly
Asp Gly 340 345 350 Ser His Lys Ser Gly 355 16 1327 DNA Homo
Sapiens CDS (107)...(1096) 16 cacgcgtccg ggaaagagct ggttccctgg
caggctggag ggcaggagct ggggccacgc 60 tggtctggga tagttgggca
gggaggctgt ctacctggtc tccaga atg gac ggc 115 Met Asp Gly 1 cct gtg
gca gag cat gcc aag cag gag ccc ttt cac gtg gtc aca cct 163 Pro Val
Ala Glu His Ala Lys Gln Glu Pro Phe His Val Val Thr Pro 5 10 15 ctg
ttg gag agc tgg gcg ctg tcc cag gtg gcg ggc atg cct gtc ttc 211 Leu
Leu Glu Ser Trp Ala Leu Ser Gln Val Ala Gly Met Pro Val Phe 20 25
30 35 ctc aag tgt gag aat gtg cag ccc agc ggc tcc ttc aag att cgg
ggc 259 Leu Lys Cys Glu Asn Val Gln Pro Ser Gly Ser Phe Lys Ile Arg
Gly 40 45 50 att ggg cat ttc tgc cag gag atg gcc aag aag gga tgc
aga cac ctg 307 Ile Gly His Phe Cys Gln Glu Met Ala Lys Lys Gly Cys
Arg His Leu 55 60 65 gtg tgc tcc tca ggg ggt aat gcg ggc atc gct
gct gcc tat gct gct 355 Val Cys Ser Ser Gly Gly Asn Ala Gly Ile Ala
Ala Ala Tyr Ala Ala 70 75 80 agg aag ctg ggc att cct gcc acc atc
gtg ctc ccc gag agc acc tcc 403 Arg Lys Leu Gly Ile Pro Ala Thr Ile
Val Leu Pro Glu Ser Thr Ser 85 90 95 ctg cag gtg gtg cag agg ctg
cag gcg gag ggg gcc gag gtt cag ctg 451 Leu Gln Val Val Gln Arg Leu
Gln Ala Glu Gly Ala Glu Val Gln Leu 100 105 110 115 act gga aag gtc
tgg gac gag gcc aat ctg agg gcg caa gag ttg gcc 499 Thr Gly Lys Val
Trp Asp Glu Ala Asn Leu Arg Ala Gln Glu Leu Ala 120 125 130 aag agg
gac ggc tgg gag aat gtc ccc ccg ttt gac cac ccc cta ata 547 Lys Arg
Asp Gly Trp Glu Asn Val Pro Pro Phe Asp His Pro Leu Ile 135 140 145
tgg aaa ggc cac gcc agc ctg gtg cag gag ctg aaa gca gtg ctg agg 595
Trp Lys Gly His Ala Ser Leu Val Gln Glu Leu Lys Ala Val Leu Arg 150
155 160 acc cca cca ggt gcc ctg gtg ctg gca gtt ggg ggt ggg ggt ctc
ctg 643 Thr Pro Pro Gly Ala Leu Val Leu Ala Val Gly Gly Gly Gly Leu
Leu 165 170 175 gcc ggg gtg gtg gct ggc ctg ctg gag gtg ggc tgg cag
cat gta ccc 691 Ala Gly Val Val Ala Gly Leu Leu Glu Val Gly Trp Gln
His Val Pro 180 185 190 195 atc att gcc atg gag acc cat ggg gca cac
tgc ttc aat gcg gcc atc 739 Ile Ile Ala Met Glu Thr His Gly Ala His
Cys Phe Asn Ala Ala Ile 200 205 210 aca gcc ggc aag ctg gtc aca ctt
cca gac atc acc agt gtg gcc aag 787 Thr Ala Gly Lys Leu Val Thr Leu
Pro Asp Ile Thr Ser Val Ala Lys 215 220 225 agc ctg ggt gcc aag acg
gtg gcc gct cgg gcc ctg gag tgc atg cag 835 Ser Leu Gly Ala Lys Thr
Val Ala Ala Arg Ala Leu Glu Cys Met Gln 230 235 240 gtg tgc aag att
cac tct gaa gtg gtg gag gac acc gag gct gtg agc 883 Val Cys Lys Ile
His Ser Glu Val Val Glu Asp Thr Glu Ala Val Ser 245 250 255 gct gtg
cag cag ctc ctg gat gat gag cgt atg ctg gtg gag cct gcc 931 Ala Val
Gln Gln Leu Leu Asp Asp Glu Arg Met Leu Val Glu Pro Ala 260 265 270
275 tgt ggg gca gcc tta gca gcc atc tac tca ggc ctc ctg cgg agg ctc
979 Cys Gly Ala Ala Leu Ala Ala Ile Tyr Ser Gly Leu Leu Arg Arg Leu
280 285 290 cag gcc gag ggc tgc ctg ccc cct tcc ctg act tca gtt gtg
gta atc 1027 Gln Ala Glu Gly Cys Leu Pro Pro Ser Leu Thr Ser Val
Val Val Ile 295 300 305 gtg tgt gga ggc aac aac atc aac agc cga gag
ctg cag gcc ttg aaa 1075 Val Cys Gly Gly Asn Asn Ile Asn Ser Arg
Glu Leu Gln Ala Leu Lys 310 315 320 acc cac ctg ggc cag gtc tga
ggggtcccat cctggcccca aagacccctg 1126 Thr His Leu Gly Gln Val * 325
agaggcccat ggacagtcct gtgtctggat gaggaggact cagtgctggc agatggcagt
1186 ggaagctgcc ctgtgcaact gtgctggctg cctcctgaag gaagccctcc
tggactgctt 1246 cttttggctc tccgacaact ccggccaata aacactttct
gaattgagtt tgcgaataaa 1306 aaaaaaaaaa aaaaaaaaaa a 1327 17 329 PRT
Homo Sapiens 17 Met Asp Gly Pro Val Ala Glu His Ala Lys Gln Glu Pro
Phe His Val 1 5 10 15 Val Thr Pro Leu Leu Glu Ser Trp Ala Leu Ser
Gln Val Ala Gly Met 20 25 30 Pro Val Phe Leu Lys Cys Glu Asn Val
Gln Pro Ser Gly Ser Phe Lys
35 40 45 Ile Arg Gly Ile Gly His Phe Cys Gln Glu Met Ala Lys Lys
Gly Cys 50 55 60 Arg His Leu Val Cys Ser Ser Gly Gly Asn Ala Gly
Ile Ala Ala Ala 65 70 75 80 Tyr Ala Ala Arg Lys Leu Gly Ile Pro Ala
Thr Ile Val Leu Pro Glu 85 90 95 Ser Thr Ser Leu Gln Val Val Gln
Arg Leu Gln Ala Glu Gly Ala Glu 100 105 110 Val Gln Leu Thr Gly Lys
Val Trp Asp Glu Ala Asn Leu Arg Ala Gln 115 120 125 Glu Leu Ala Lys
Arg Asp Gly Trp Glu Asn Val Pro Pro Phe Asp His 130 135 140 Pro Leu
Ile Trp Lys Gly His Ala Ser Leu Val Gln Glu Leu Lys Ala 145 150 155
160 Val Leu Arg Thr Pro Pro Gly Ala Leu Val Leu Ala Val Gly Gly Gly
165 170 175 Gly Leu Leu Ala Gly Val Val Ala Gly Leu Leu Glu Val Gly
Trp Gln 180 185 190 His Val Pro Ile Ile Ala Met Glu Thr His Gly Ala
His Cys Phe Asn 195 200 205 Ala Ala Ile Thr Ala Gly Lys Leu Val Thr
Leu Pro Asp Ile Thr Ser 210 215 220 Val Ala Lys Ser Leu Gly Ala Lys
Thr Val Ala Ala Arg Ala Leu Glu 225 230 235 240 Cys Met Gln Val Cys
Lys Ile His Ser Glu Val Val Glu Asp Thr Glu 245 250 255 Ala Val Ser
Ala Val Gln Gln Leu Leu Asp Asp Glu Arg Met Leu Val 260 265 270 Glu
Pro Ala Cys Gly Ala Ala Leu Ala Ala Ile Tyr Ser Gly Leu Leu 275 280
285 Arg Arg Leu Gln Ala Glu Gly Cys Leu Pro Pro Ser Leu Thr Ser Val
290 295 300 Val Val Ile Val Cys Gly Gly Asn Asn Ile Asn Ser Arg Glu
Leu Gln 305 310 315 320 Ala Leu Lys Thr His Leu Gly Gln Val 325 18
990 DNA Homo Sapiens CDS (1)...(990) 18 atg gac ggc cct gtg gca gag
cat gcc aag cag gag ccc ttt cac gtg 48 Met Asp Gly Pro Val Ala Glu
His Ala Lys Gln Glu Pro Phe His Val 1 5 10 15 gtc aca cct ctg ttg
gag agc tgg gcg ctg tcc cag gtg gcg ggc atg 96 Val Thr Pro Leu Leu
Glu Ser Trp Ala Leu Ser Gln Val Ala Gly Met 20 25 30 cct gtc ttc
ctc aag tgt gag aat gtg cag ccc agc ggc tcc ttc aag 144 Pro Val Phe
Leu Lys Cys Glu Asn Val Gln Pro Ser Gly Ser Phe Lys 35 40 45 att
cgg ggc att ggg cat ttc tgc cag gag atg gcc aag aag gga tgc 192 Ile
Arg Gly Ile Gly His Phe Cys Gln Glu Met Ala Lys Lys Gly Cys 50 55
60 aga cac ctg gtg tgc tcc tca ggg ggt aat gcg ggc atc gct gct gcc
240 Arg His Leu Val Cys Ser Ser Gly Gly Asn Ala Gly Ile Ala Ala Ala
65 70 75 80 tat gct gct agg aag ctg ggc att cct gcc acc atc gtg ctc
ccc gag 288 Tyr Ala Ala Arg Lys Leu Gly Ile Pro Ala Thr Ile Val Leu
Pro Glu 85 90 95 agc acc tcc ctg cag gtg gtg cag agg ctg cag gcg
gag ggg gcc gag 336 Ser Thr Ser Leu Gln Val Val Gln Arg Leu Gln Ala
Glu Gly Ala Glu 100 105 110 gtt cag ctg act gga aag gtc tgg gac gag
gcc aat ctg agg gcg caa 384 Val Gln Leu Thr Gly Lys Val Trp Asp Glu
Ala Asn Leu Arg Ala Gln 115 120 125 gag ttg gcc aag agg gac ggc tgg
gag aat gtc ccc ccg ttt gac cac 432 Glu Leu Ala Lys Arg Asp Gly Trp
Glu Asn Val Pro Pro Phe Asp His 130 135 140 ccc cta ata tgg aaa ggc
cac gcc agc ctg gtg cag gag ctg aaa gca 480 Pro Leu Ile Trp Lys Gly
His Ala Ser Leu Val Gln Glu Leu Lys Ala 145 150 155 160 gtg ctg agg
acc cca cca ggt gcc ctg gtg ctg gca gtt ggg ggt ggg 528 Val Leu Arg
Thr Pro Pro Gly Ala Leu Val Leu Ala Val Gly Gly Gly 165 170 175 ggt
ctc ctg gcc ggg gtg gtg gct ggc ctg ctg gag gtg ggc tgg cag 576 Gly
Leu Leu Ala Gly Val Val Ala Gly Leu Leu Glu Val Gly Trp Gln 180 185
190 cat gta ccc atc att gcc atg gag acc cat ggg gca cac tgc ttc aat
624 His Val Pro Ile Ile Ala Met Glu Thr His Gly Ala His Cys Phe Asn
195 200 205 gcg gcc atc aca gcc ggc aag ctg gtc aca ctt cca gac atc
acc agt 672 Ala Ala Ile Thr Ala Gly Lys Leu Val Thr Leu Pro Asp Ile
Thr Ser 210 215 220 gtg gcc aag agc ctg ggt gcc aag acg gtg gcc gct
cgg gcc ctg gag 720 Val Ala Lys Ser Leu Gly Ala Lys Thr Val Ala Ala
Arg Ala Leu Glu 225 230 235 240 tgc atg cag gtg tgc aag att cac tct
gaa gtg gtg gag gac acc gag 768 Cys Met Gln Val Cys Lys Ile His Ser
Glu Val Val Glu Asp Thr Glu 245 250 255 gct gtg agc gct gtg cag cag
ctc ctg gat gat gag cgt atg ctg gtg 816 Ala Val Ser Ala Val Gln Gln
Leu Leu Asp Asp Glu Arg Met Leu Val 260 265 270 gag cct gcc tgt ggg
gca gcc tta gca gcc atc tac tca ggc ctc ctg 864 Glu Pro Ala Cys Gly
Ala Ala Leu Ala Ala Ile Tyr Ser Gly Leu Leu 275 280 285 cgg agg ctc
cag gcc gag ggc tgc ctg ccc cct tcc ctg act tca gtt 912 Arg Arg Leu
Gln Ala Glu Gly Cys Leu Pro Pro Ser Leu Thr Ser Val 290 295 300 gtg
gta atc gtg tgt gga ggc aac aac atc aac agc cga gag ctg cag 960 Val
Val Ile Val Cys Gly Gly Asn Asn Ile Asn Ser Arg Glu Leu Gln 305 310
315 320 gcc ttg aaa acc cac ctg ggc cag gtc tga 990 Ala Leu Lys Thr
His Leu Gly Gln Val * 325 19 15 PRT Artificial Sequence
Serine/Threonine Dehydratase Pyridoxal- Phosphate Attachment Site
Conserved Sequence 19 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys
Xaa Xaa Xaa Xaa 1 5 10 15 20 3669 DNA Homo Sapiens CDS
(106)...(2859) 20 ccacgcgtcc ggcctggact ggaagcgtgc aacactccag
agtcgtagga gtgaacactg 60 cacaggaatc tctgcccatc tcaggagaaa
ccaaacttgg ggaaa atg ttt gcg gtc 117 Met Phe Ala Val 1 cac ttg atg
gca ttt tac ttc agc aag ctg aag gag gac cag atc aag 165 His Leu Met
Ala Phe Tyr Phe Ser Lys Leu Lys Glu Asp Gln Ile Lys 5 10 15 20 aag
gtg gac agg ttc ctg tat cac atg cgg ctc tcc gat gac acc ctt 213 Lys
Val Asp Arg Phe Leu Tyr His Met Arg Leu Ser Asp Asp Thr Leu 25 30
35 ttg gac atc atg agg cgg ttc cgg gct gag atg gag aag ggc ctg gca
261 Leu Asp Ile Met Arg Arg Phe Arg Ala Glu Met Glu Lys Gly Leu Ala
40 45 50 aag gac acc aac ccc acg gct gca gtg aag atg ttg ccc acc
ttc gtc 309 Lys Asp Thr Asn Pro Thr Ala Ala Val Lys Met Leu Pro Thr
Phe Val 55 60 65 agg gcc att ccc gat ggt tcc gaa aat ggg gag ttc
ctt tcc ctg gat 357 Arg Ala Ile Pro Asp Gly Ser Glu Asn Gly Glu Phe
Leu Ser Leu Asp 70 75 80 ctc gga ggg tcc aag ttc cga gtg ctg aag
gtg caa gtc gct gaa gag 405 Leu Gly Gly Ser Lys Phe Arg Val Leu Lys
Val Gln Val Ala Glu Glu 85 90 95 100 ggg aag cga cac gtg cag atg
gag agt cag ttc tac cca acg ccc aat 453 Gly Lys Arg His Val Gln Met
Glu Ser Gln Phe Tyr Pro Thr Pro Asn 105 110 115 gaa atc atc cgc ggg
aac ggc ata gag ctg ttt gaa tat gta gct gac 501 Glu Ile Ile Arg Gly
Asn Gly Ile Glu Leu Phe Glu Tyr Val Ala Asp 120 125 130 tgt ctg gca
gat ttc atg aag acc aaa gat tta aag cat aag aaa ttg 549 Cys Leu Ala
Asp Phe Met Lys Thr Lys Asp Leu Lys His Lys Lys Leu 135 140 145 ccc
ctt ggc cta act ttt tct ttc ccc tgt cga cag act aaa ctg gaa 597 Pro
Leu Gly Leu Thr Phe Ser Phe Pro Cys Arg Gln Thr Lys Leu Glu 150 155
160 gag ggt gtc cta ctt tcg tgg aca aaa aag ttt aag gca cga gga gtt
645 Glu Gly Val Leu Leu Ser Trp Thr Lys Lys Phe Lys Ala Arg Gly Val
165 170 175 180 cag gac acg gat gtg gtg agc cgt ctg acc aaa gcc atg
aga aga cac 693 Gln Asp Thr Asp Val Val Ser Arg Leu Thr Lys Ala Met
Arg Arg His 185 190 195 aag gac atg gac gtg gac atc ctg gcc ctg gtc
aat gac acc gtg ggg 741 Lys Asp Met Asp Val Asp Ile Leu Ala Leu Val
Asn Asp Thr Val Gly 200 205 210 acc atg atg acc tgt gcc tat gac gac
ccc tac tgc gaa gtt ggt gtc 789 Thr Met Met Thr Cys Ala Tyr Asp Asp
Pro Tyr Cys Glu Val Gly Val 215 220 225 atc atc gga act ggc acc aat
gcg tgt tac atg gag gac atg agc aac 837 Ile Ile Gly Thr Gly Thr Asn
Ala Cys Tyr Met Glu Asp Met Ser Asn 230 235 240 att gac ctg gtg gag
ggc gac gag ggc agg atg tgc atc aac aca gag 885 Ile Asp Leu Val Glu
Gly Asp Glu Gly Arg Met Cys Ile Asn Thr Glu 245 250 255 260 tgg ggg
gcc ttc ggg gac gac ggg gcc ctg gag gac att cgc act gag 933 Trp Gly
Ala Phe Gly Asp Asp Gly Ala Leu Glu Asp Ile Arg Thr Glu 265 270 275
ttc gac agg gag ctg gac ctc ggc tct ctc aac cca gga aag caa ctg 981
Phe Asp Arg Glu Leu Asp Leu Gly Ser Leu Asn Pro Gly Lys Gln Leu 280
285 290 ttc gag aag atg atc agt ggc ctg tac ctg ggg gag ctt gtc agg
ctt 1029 Phe Glu Lys Met Ile Ser Gly Leu Tyr Leu Gly Glu Leu Val
Arg Leu 295 300 305 atc ttg ctg aag atg gcc aag gct ggc ctc ctg ttt
ggt ggt gag aaa 1077 Ile Leu Leu Lys Met Ala Lys Ala Gly Leu Leu
Phe Gly Gly Glu Lys 310 315 320 tct tct gct ctc cac act aag ggc aag
atc gaa aca cgg cac gtg gct 1125 Ser Ser Ala Leu His Thr Lys Gly
Lys Ile Glu Thr Arg His Val Ala 325 330 335 340 gcc atg gag aag tat
aaa gaa ggc ctt gct aat aca aga gag atc ctg 1173 Ala Met Glu Lys
Tyr Lys Glu Gly Leu Ala Asn Thr Arg Glu Ile Leu 345 350 355 gtg gac
ctg ggt ctg gaa ccg tct gag gct gac tgc att gcc gtc cag 1221 Val
Asp Leu Gly Leu Glu Pro Ser Glu Ala Asp Cys Ile Ala Val Gln 360 365
370 cat gtc tgt acc atc gtc tcc ttc cgc tcg gcc aat ctc tgt gca gca
1269 His Val Cys Thr Ile Val Ser Phe Arg Ser Ala Asn Leu Cys Ala
Ala 375 380 385 gct ctg gcg gcc atc ctg aca cgc ctc cgg gag aac aag
aag gtg gaa 1317 Ala Leu Ala Ala Ile Leu Thr Arg Leu Arg Glu Asn
Lys Lys Val Glu 390 395 400 cgg ctc cgg acc aca gtg ggc atg gac ggc
acc ctc tac aag ata cac 1365 Arg Leu Arg Thr Thr Val Gly Met Asp
Gly Thr Leu Tyr Lys Ile His 405 410 415 420 cct cag tac cca aaa cgc
ctg cac aag gtg gtg agg aaa ctg gtc cca 1413 Pro Gln Tyr Pro Lys
Arg Leu His Lys Val Val Arg Lys Leu Val Pro 425 430 435 agc tgt gat
gtc cgc ttc ctc ctg tca gag agt ggc agc acc aag ggg 1461 Ser Cys
Asp Val Arg Phe Leu Leu Ser Glu Ser Gly Ser Thr Lys Gly 440 445 450
gcc gcc atg gtg acc gcg gtg gcc tcc cgc gtg cag gcc cag cgg aag
1509 Ala Ala Met Val Thr Ala Val Ala Ser Arg Val Gln Ala Gln Arg
Lys 455 460 465 cag atc gac agg gtg ctg gct ttg ttc cag ctg acc cga
gag cag ctc 1557 Gln Ile Asp Arg Val Leu Ala Leu Phe Gln Leu Thr
Arg Glu Gln Leu 470 475 480 gtg gac gtg cag gcc aag atg cgg gct gag
ctg gag tat ggg ctg aag 1605 Val Asp Val Gln Ala Lys Met Arg Ala
Glu Leu Glu Tyr Gly Leu Lys 485 490 495 500 aag aag agc cac ggg ctg
gcc acg gtc agg atg ctg ccc acc tac gtc 1653 Lys Lys Ser His Gly
Leu Ala Thr Val Arg Met Leu Pro Thr Tyr Val 505 510 515 tgc ggg ctg
ccg gac ggc aca gag aaa gga aag ttt ctc gcc ctg gat 1701 Cys Gly
Leu Pro Asp Gly Thr Glu Lys Gly Lys Phe Leu Ala Leu Asp 520 525 530
ctt ggg gga acc aac ttc cgg gtc ctc ctg gtg aag atc aga agt gga
1749 Leu Gly Gly Thr Asn Phe Arg Val Leu Leu Val Lys Ile Arg Ser
Gly 535 540 545 cgg agg tca gtg cga atg tac aac aag atc ttc gcc atc
ccc ctg gag 1797 Arg Arg Ser Val Arg Met Tyr Asn Lys Ile Phe Ala
Ile Pro Leu Glu 550 555 560 atc atg cag ggc act ggt gag gag ctc ttt
gat cac att gtg cag tgc 1845 Ile Met Gln Gly Thr Gly Glu Glu Leu
Phe Asp His Ile Val Gln Cys 565 570 575 580 atc gcc gac ttc ctg gac
tac atg ggc ctc aag gga gcc tcc cta cct 1893 Ile Ala Asp Phe Leu
Asp Tyr Met Gly Leu Lys Gly Ala Ser Leu Pro 585 590 595 ttg ggc ttc
aca ttc tca ttt ccc tgc agg cag atg agc att gac aag 1941 Leu Gly
Phe Thr Phe Ser Phe Pro Cys Arg Gln Met Ser Ile Asp Lys 600 605 610
gga aca ctc ata ggg tgg acc aaa ggt ttc aag gcc act gac tgt gaa
1989 Gly Thr Leu Ile Gly Trp Thr Lys Gly Phe Lys Ala Thr Asp Cys
Glu 615 620 625 ggg gag gac gtg gtg gac atg ctc agg gaa gcc atc aag
agg aga aac 2037 Gly Glu Asp Val Val Asp Met Leu Arg Glu Ala Ile
Lys Arg Arg Asn 630 635 640 gag ttt gac ctg gac att gtt gca gtc gtg
aat gat aca gtg ggg acc 2085 Glu Phe Asp Leu Asp Ile Val Ala Val
Val Asn Asp Thr Val Gly Thr 645 650 655 660 atg atg acc tgt ggc tat
gaa gat cct aat tgt gag att ggc ctg att 2133 Met Met Thr Cys Gly
Tyr Glu Asp Pro Asn Cys Glu Ile Gly Leu Ile 665 670 675 gca gga aca
ggc agc aac atg tgc tac atg gag gac atg agg aac atc 2181 Ala Gly
Thr Gly Ser Asn Met Cys Tyr Met Glu Asp Met Arg Asn Ile 680 685 690
gag atg gtg gag ggg ggt gaa ggg aag atg tgc atc aat aca gag tgg
2229 Glu Met Val Glu Gly Gly Glu Gly Lys Met Cys Ile Asn Thr Glu
Trp 695 700 705 gga gga ttt gga gac aat ggc tgc ata gat gac atc cgg
acc cga tac 2277 Gly Gly Phe Gly Asp Asn Gly Cys Ile Asp Asp Ile
Arg Thr Arg Tyr 710 715 720 gac acg gag gtg gat gag ggg tcc ttg aat
cct ggc aag cag aga tac 2325 Asp Thr Glu Val Asp Glu Gly Ser Leu
Asn Pro Gly Lys Gln Arg Tyr 725 730 735 740 gag aaa atg acc agt ggg
atg tac ttg ggg gag att gtg cgg cag atc 2373 Glu Lys Met Thr Ser
Gly Met Tyr Leu Gly Glu Ile Val Arg Gln Ile 745 750 755 ctg atc gac
ctg acc aag cag ggt ctc ctc ttc cga ggg cag att tca 2421 Leu Ile
Asp Leu Thr Lys Gln Gly Leu Leu Phe Arg Gly Gln Ile Ser 760 765 770
gag cgt ctc cgg acc agg ggc atc ttc gaa acc aag ttc ctg tcc cag
2469 Glu Arg Leu Arg Thr Arg Gly Ile Phe Glu Thr Lys Phe Leu Ser
Gln 775 780 785 atc gaa agc gat cgg ctg gcc ctt ctc cag gtc agg agg
att ctg cag 2517 Ile Glu Ser Asp Arg Leu Ala Leu Leu Gln Val Arg
Arg Ile Leu Gln 790 795 800 cag ctg ggc ctg gac agc acg tgt gag gac
agc atc gtg gtg aag gag 2565 Gln Leu Gly Leu Asp Ser Thr Cys Glu
Asp Ser Ile Val Val Lys Glu 805 810 815 820 gtg tgc gga gcc gtg tcc
cgg cgg gcg gcc cag ctc tgc ggt gct ggc 2613 Val Cys Gly Ala Val
Ser Arg Arg Ala Ala Gln Leu Cys Gly Ala Gly 825 830 835 ctg gcc gct
ata gtg gaa aaa agg aga gaa gac cag ggg cta gag cac 2661 Leu Ala
Ala Ile Val Glu Lys Arg Arg Glu Asp Gln Gly Leu Glu His 840 845 850
ctg agg atc act gtg ggt gtg gac ggc acc ctg tac aag ctg cac cct
2709 Leu Arg Ile Thr Val Gly Val Asp Gly Thr Leu Tyr Lys Leu His
Pro 855 860 865 cac ttt tct aga ata ttg cag gaa act gtg aag gaa cta
gcc cct cga 2757 His Phe Ser Arg Ile Leu Gln Glu Thr Val Lys Glu
Leu Ala Pro Arg 870 875 880 tgt gat gtg aca ttc atg ctg tca gaa gat
ggc agt gga aaa ggg gca 2805 Cys Asp Val Thr Phe Met Leu Ser Glu
Asp Gly Ser Gly Lys Gly Ala 885 890 895 900 gca ctg atc act gct gtg
gcc aag agg tta cag cag gca cag aag gag 2853 Ala Leu Ile Thr Ala
Val Ala Lys Arg Leu Gln Gln Ala Gln Lys Glu 905 910 915 aac tag
gaacccctgg gattggacct gatgcatctt ggatactgaa cagcttttcc 2909 Asn *
tctggcagat cagttggtca gagaccaatg ggcaccctcc tggctgacct caccttctgg
2969 atggccgaaa gagaacccca ggttctcggg tactcttagt atcttgtact
ggatttgcag 3029 tgacattaca tgacatctct atttggtata tttgggccaa
aatgggccaa cttatgaaat 3089 caaagtgtct gtcctgagag atcccctttc
aacacattgt tcaggtgagg cttgagctgt 3149 caattctcta tggctttcag
tcttgtggct gcgggacttg gaaatatata gaatctgccc 3209 atgtggctgg
caggctgttt ccccattggg atgcttaagc catctcttat aggggattgg 3269
accctgtact tgtggatgaa cattggagag caagaggaac tcacgttatg aactaggggg
3329 atctcatcta acttgtcctt
aacttgccat gttgacttca aacctgttaa gagaacaaag 3389 actttgaagt
atccagcccc agggtgcaga gaggttgatt gccagggagc actgcaggaa 3449
tcattgcatg cttaaagcga gttatgtcag caccctgtag gattttgttc cttattaagt
3509 gtgtgccatg tggtggggtg ctgtctgggg catctgtttt tcattttgcc
tgtggtttgt 3569 gttgcagstg ttgatagttg ttttaaggat tgttaggtat
aggaaatcca gtaaattaat 3629 aaaaaaattt tgattttcca ataaaaaaaa
aaaaaaaaaa 3669 21 917 PRT Homo Sapiens 21 Met Phe Ala Val His Leu
Met Ala Phe Tyr Phe Ser Lys Leu Lys Glu 1 5 10 15 Asp Gln Ile Lys
Lys Val Asp Arg Phe Leu Tyr His Met Arg Leu Ser 20 25 30 Asp Asp
Thr Leu Leu Asp Ile Met Arg Arg Phe Arg Ala Glu Met Glu 35 40 45
Lys Gly Leu Ala Lys Asp Thr Asn Pro Thr Ala Ala Val Lys Met Leu 50
55 60 Pro Thr Phe Val Arg Ala Ile Pro Asp Gly Ser Glu Asn Gly Glu
Phe 65 70 75 80 Leu Ser Leu Asp Leu Gly Gly Ser Lys Phe Arg Val Leu
Lys Val Gln 85 90 95 Val Ala Glu Glu Gly Lys Arg His Val Gln Met
Glu Ser Gln Phe Tyr 100 105 110 Pro Thr Pro Asn Glu Ile Ile Arg Gly
Asn Gly Ile Glu Leu Phe Glu 115 120 125 Tyr Val Ala Asp Cys Leu Ala
Asp Phe Met Lys Thr Lys Asp Leu Lys 130 135 140 His Lys Lys Leu Pro
Leu Gly Leu Thr Phe Ser Phe Pro Cys Arg Gln 145 150 155 160 Thr Lys
Leu Glu Glu Gly Val Leu Leu Ser Trp Thr Lys Lys Phe Lys 165 170 175
Ala Arg Gly Val Gln Asp Thr Asp Val Val Ser Arg Leu Thr Lys Ala 180
185 190 Met Arg Arg His Lys Asp Met Asp Val Asp Ile Leu Ala Leu Val
Asn 195 200 205 Asp Thr Val Gly Thr Met Met Thr Cys Ala Tyr Asp Asp
Pro Tyr Cys 210 215 220 Glu Val Gly Val Ile Ile Gly Thr Gly Thr Asn
Ala Cys Tyr Met Glu 225 230 235 240 Asp Met Ser Asn Ile Asp Leu Val
Glu Gly Asp Glu Gly Arg Met Cys 245 250 255 Ile Asn Thr Glu Trp Gly
Ala Phe Gly Asp Asp Gly Ala Leu Glu Asp 260 265 270 Ile Arg Thr Glu
Phe Asp Arg Glu Leu Asp Leu Gly Ser Leu Asn Pro 275 280 285 Gly Lys
Gln Leu Phe Glu Lys Met Ile Ser Gly Leu Tyr Leu Gly Glu 290 295 300
Leu Val Arg Leu Ile Leu Leu Lys Met Ala Lys Ala Gly Leu Leu Phe 305
310 315 320 Gly Gly Glu Lys Ser Ser Ala Leu His Thr Lys Gly Lys Ile
Glu Thr 325 330 335 Arg His Val Ala Ala Met Glu Lys Tyr Lys Glu Gly
Leu Ala Asn Thr 340 345 350 Arg Glu Ile Leu Val Asp Leu Gly Leu Glu
Pro Ser Glu Ala Asp Cys 355 360 365 Ile Ala Val Gln His Val Cys Thr
Ile Val Ser Phe Arg Ser Ala Asn 370 375 380 Leu Cys Ala Ala Ala Leu
Ala Ala Ile Leu Thr Arg Leu Arg Glu Asn 385 390 395 400 Lys Lys Val
Glu Arg Leu Arg Thr Thr Val Gly Met Asp Gly Thr Leu 405 410 415 Tyr
Lys Ile His Pro Gln Tyr Pro Lys Arg Leu His Lys Val Val Arg 420 425
430 Lys Leu Val Pro Ser Cys Asp Val Arg Phe Leu Leu Ser Glu Ser Gly
435 440 445 Ser Thr Lys Gly Ala Ala Met Val Thr Ala Val Ala Ser Arg
Val Gln 450 455 460 Ala Gln Arg Lys Gln Ile Asp Arg Val Leu Ala Leu
Phe Gln Leu Thr 465 470 475 480 Arg Glu Gln Leu Val Asp Val Gln Ala
Lys Met Arg Ala Glu Leu Glu 485 490 495 Tyr Gly Leu Lys Lys Lys Ser
His Gly Leu Ala Thr Val Arg Met Leu 500 505 510 Pro Thr Tyr Val Cys
Gly Leu Pro Asp Gly Thr Glu Lys Gly Lys Phe 515 520 525 Leu Ala Leu
Asp Leu Gly Gly Thr Asn Phe Arg Val Leu Leu Val Lys 530 535 540 Ile
Arg Ser Gly Arg Arg Ser Val Arg Met Tyr Asn Lys Ile Phe Ala 545 550
555 560 Ile Pro Leu Glu Ile Met Gln Gly Thr Gly Glu Glu Leu Phe Asp
His 565 570 575 Ile Val Gln Cys Ile Ala Asp Phe Leu Asp Tyr Met Gly
Leu Lys Gly 580 585 590 Ala Ser Leu Pro Leu Gly Phe Thr Phe Ser Phe
Pro Cys Arg Gln Met 595 600 605 Ser Ile Asp Lys Gly Thr Leu Ile Gly
Trp Thr Lys Gly Phe Lys Ala 610 615 620 Thr Asp Cys Glu Gly Glu Asp
Val Val Asp Met Leu Arg Glu Ala Ile 625 630 635 640 Lys Arg Arg Asn
Glu Phe Asp Leu Asp Ile Val Ala Val Val Asn Asp 645 650 655 Thr Val
Gly Thr Met Met Thr Cys Gly Tyr Glu Asp Pro Asn Cys Glu 660 665 670
Ile Gly Leu Ile Ala Gly Thr Gly Ser Asn Met Cys Tyr Met Glu Asp 675
680 685 Met Arg Asn Ile Glu Met Val Glu Gly Gly Glu Gly Lys Met Cys
Ile 690 695 700 Asn Thr Glu Trp Gly Gly Phe Gly Asp Asn Gly Cys Ile
Asp Asp Ile 705 710 715 720 Arg Thr Arg Tyr Asp Thr Glu Val Asp Glu
Gly Ser Leu Asn Pro Gly 725 730 735 Lys Gln Arg Tyr Glu Lys Met Thr
Ser Gly Met Tyr Leu Gly Glu Ile 740 745 750 Val Arg Gln Ile Leu Ile
Asp Leu Thr Lys Gln Gly Leu Leu Phe Arg 755 760 765 Gly Gln Ile Ser
Glu Arg Leu Arg Thr Arg Gly Ile Phe Glu Thr Lys 770 775 780 Phe Leu
Ser Gln Ile Glu Ser Asp Arg Leu Ala Leu Leu Gln Val Arg 785 790 795
800 Arg Ile Leu Gln Gln Leu Gly Leu Asp Ser Thr Cys Glu Asp Ser Ile
805 810 815 Val Val Lys Glu Val Cys Gly Ala Val Ser Arg Arg Ala Ala
Gln Leu 820 825 830 Cys Gly Ala Gly Leu Ala Ala Ile Val Glu Lys Arg
Arg Glu Asp Gln 835 840 845 Gly Leu Glu His Leu Arg Ile Thr Val Gly
Val Asp Gly Thr Leu Tyr 850 855 860 Lys Leu His Pro His Phe Ser Arg
Ile Leu Gln Glu Thr Val Lys Glu 865 870 875 880 Leu Ala Pro Arg Cys
Asp Val Thr Phe Met Leu Ser Glu Asp Gly Ser 885 890 895 Gly Lys Gly
Ala Ala Leu Ile Thr Ala Val Ala Lys Arg Leu Gln Gln 900 905 910 Ala
Gln Lys Glu Asn 915 22 2754 DNA Homo Sapiens CDS (1)...(2754) 22
atg ttt gcg gtc cac ttg atg gca ttt tac ttc agc aag ctg aag gag 48
Met Phe Ala Val His Leu Met Ala Phe Tyr Phe Ser Lys Leu Lys Glu 1 5
10 15 gac cag atc aag aag gtg gac agg ttc ctg tat cac atg cgg ctc
tcc 96 Asp Gln Ile Lys Lys Val Asp Arg Phe Leu Tyr His Met Arg Leu
Ser 20 25 30 gat gac acc ctt ttg gac atc atg agg cgg ttc cgg gct
gag atg gag 144 Asp Asp Thr Leu Leu Asp Ile Met Arg Arg Phe Arg Ala
Glu Met Glu 35 40 45 aag ggc ctg gca aag gac acc aac ccc acg gct
gca gtg aag atg ttg 192 Lys Gly Leu Ala Lys Asp Thr Asn Pro Thr Ala
Ala Val Lys Met Leu 50 55 60 ccc acc ttc gtc agg gcc att ccc gat
ggt tcc gaa aat ggg gag ttc 240 Pro Thr Phe Val Arg Ala Ile Pro Asp
Gly Ser Glu Asn Gly Glu Phe 65 70 75 80 ctt tcc ctg gat ctc gga ggg
tcc aag ttc cga gtg ctg aag gtg caa 288 Leu Ser Leu Asp Leu Gly Gly
Ser Lys Phe Arg Val Leu Lys Val Gln 85 90 95 gtc gct gaa gag ggg
aag cga cac gtg cag atg gag agt cag ttc tac 336 Val Ala Glu Glu Gly
Lys Arg His Val Gln Met Glu Ser Gln Phe Tyr 100 105 110 cca acg ccc
aat gaa atc atc cgc ggg aac ggc ata gag ctg ttt gaa 384 Pro Thr Pro
Asn Glu Ile Ile Arg Gly Asn Gly Ile Glu Leu Phe Glu 115 120 125 tat
gta gct gac tgt ctg gca gat ttc atg aag acc aaa gat tta aag 432 Tyr
Val Ala Asp Cys Leu Ala Asp Phe Met Lys Thr Lys Asp Leu Lys 130 135
140 cat aag aaa ttg ccc ctt ggc cta act ttt tct ttc ccc tgt cga cag
480 His Lys Lys Leu Pro Leu Gly Leu Thr Phe Ser Phe Pro Cys Arg Gln
145 150 155 160 act aaa ctg gaa gag ggt gtc cta ctt tcg tgg aca aaa
aag ttt aag 528 Thr Lys Leu Glu Glu Gly Val Leu Leu Ser Trp Thr Lys
Lys Phe Lys 165 170 175 gca cga gga gtt cag gac acg gat gtg gtg agc
cgt ctg acc aaa gcc 576 Ala Arg Gly Val Gln Asp Thr Asp Val Val Ser
Arg Leu Thr Lys Ala 180 185 190 atg aga aga cac aag gac atg gac gtg
gac atc ctg gcc ctg gtc aat 624 Met Arg Arg His Lys Asp Met Asp Val
Asp Ile Leu Ala Leu Val Asn 195 200 205 gac acc gtg ggg acc atg atg
acc tgt gcc tat gac gac ccc tac tgc 672 Asp Thr Val Gly Thr Met Met
Thr Cys Ala Tyr Asp Asp Pro Tyr Cys 210 215 220 gaa gtt ggt gtc atc
atc gga act ggc acc aat gcg tgt tac atg gag 720 Glu Val Gly Val Ile
Ile Gly Thr Gly Thr Asn Ala Cys Tyr Met Glu 225 230 235 240 gac atg
agc aac att gac ctg gtg gag ggc gac gag ggc agg atg tgc 768 Asp Met
Ser Asn Ile Asp Leu Val Glu Gly Asp Glu Gly Arg Met Cys 245 250 255
atc aac aca gag tgg ggg gcc ttc ggg gac gac ggg gcc ctg gag gac 816
Ile Asn Thr Glu Trp Gly Ala Phe Gly Asp Asp Gly Ala Leu Glu Asp 260
265 270 att cgc act gag ttc gac agg gag ctg gac ctc ggc tct ctc aac
cca 864 Ile Arg Thr Glu Phe Asp Arg Glu Leu Asp Leu Gly Ser Leu Asn
Pro 275 280 285 gga aag caa ctg ttc gag aag atg atc agt ggc ctg tac
ctg ggg gag 912 Gly Lys Gln Leu Phe Glu Lys Met Ile Ser Gly Leu Tyr
Leu Gly Glu 290 295 300 ctt gtc agg ctt atc ttg ctg aag atg gcc aag
gct ggc ctc ctg ttt 960 Leu Val Arg Leu Ile Leu Leu Lys Met Ala Lys
Ala Gly Leu Leu Phe 305 310 315 320 ggt ggt gag aaa tct tct gct ctc
cac act aag ggc aag atc gaa aca 1008 Gly Gly Glu Lys Ser Ser Ala
Leu His Thr Lys Gly Lys Ile Glu Thr 325 330 335 cgg cac gtg gct gcc
atg gag aag tat aaa gaa ggc ctt gct aat aca 1056 Arg His Val Ala
Ala Met Glu Lys Tyr Lys Glu Gly Leu Ala Asn Thr 340 345 350 aga gag
atc ctg gtg gac ctg ggt ctg gaa ccg tct gag gct gac tgc 1104 Arg
Glu Ile Leu Val Asp Leu Gly Leu Glu Pro Ser Glu Ala Asp Cys 355 360
365 att gcc gtc cag cat gtc tgt acc atc gtc tcc ttc cgc tcg gcc aat
1152 Ile Ala Val Gln His Val Cys Thr Ile Val Ser Phe Arg Ser Ala
Asn 370 375 380 ctc tgt gca gca gct ctg gcg gcc atc ctg aca cgc ctc
cgg gag aac 1200 Leu Cys Ala Ala Ala Leu Ala Ala Ile Leu Thr Arg
Leu Arg Glu Asn 385 390 395 400 aag aag gtg gaa cgg ctc cgg acc aca
gtg ggc atg gac ggc acc ctc 1248 Lys Lys Val Glu Arg Leu Arg Thr
Thr Val Gly Met Asp Gly Thr Leu 405 410 415 tac aag ata cac cct cag
tac cca aaa cgc ctg cac aag gtg gtg agg 1296 Tyr Lys Ile His Pro
Gln Tyr Pro Lys Arg Leu His Lys Val Val Arg 420 425 430 aaa ctg gtc
cca agc tgt gat gtc cgc ttc ctc ctg tca gag agt ggc 1344 Lys Leu
Val Pro Ser Cys Asp Val Arg Phe Leu Leu Ser Glu Ser Gly 435 440 445
agc acc aag ggg gcc gcc atg gtg acc gcg gtg gcc tcc cgc gtg cag
1392 Ser Thr Lys Gly Ala Ala Met Val Thr Ala Val Ala Ser Arg Val
Gln 450 455 460 gcc cag cgg aag cag atc gac agg gtg ctg gct ttg ttc
cag ctg acc 1440 Ala Gln Arg Lys Gln Ile Asp Arg Val Leu Ala Leu
Phe Gln Leu Thr 465 470 475 480 cga gag cag ctc gtg gac gtg cag gcc
aag atg cgg gct gag ctg gag 1488 Arg Glu Gln Leu Val Asp Val Gln
Ala Lys Met Arg Ala Glu Leu Glu 485 490 495 tat ggg ctg aag aag aag
agc cac ggg ctg gcc acg gtc agg atg ctg 1536 Tyr Gly Leu Lys Lys
Lys Ser His Gly Leu Ala Thr Val Arg Met Leu 500 505 510 ccc acc tac
gtc tgc ggg ctg ccg gac ggc aca gag aaa gga aag ttt 1584 Pro Thr
Tyr Val Cys Gly Leu Pro Asp Gly Thr Glu Lys Gly Lys Phe 515 520 525
ctc gcc ctg gat ctt ggg gga acc aac ttc cgg gtc ctc ctg gtg aag
1632 Leu Ala Leu Asp Leu Gly Gly Thr Asn Phe Arg Val Leu Leu Val
Lys 530 535 540 atc aga agt gga cgg agg tca gtg cga atg tac aac aag
atc ttc gcc 1680 Ile Arg Ser Gly Arg Arg Ser Val Arg Met Tyr Asn
Lys Ile Phe Ala 545 550 555 560 atc ccc ctg gag atc atg cag ggc act
ggt gag gag ctc ttt gat cac 1728 Ile Pro Leu Glu Ile Met Gln Gly
Thr Gly Glu Glu Leu Phe Asp His 565 570 575 att gtg cag tgc atc gcc
gac ttc ctg gac tac atg ggc ctc aag gga 1776 Ile Val Gln Cys Ile
Ala Asp Phe Leu Asp Tyr Met Gly Leu Lys Gly 580 585 590 gcc tcc cta
cct ttg ggc ttc aca ttc tca ttt ccc tgc agg cag atg 1824 Ala Ser
Leu Pro Leu Gly Phe Thr Phe Ser Phe Pro Cys Arg Gln Met 595 600 605
agc att gac aag gga aca ctc ata ggg tgg acc aaa ggt ttc aag gcc
1872 Ser Ile Asp Lys Gly Thr Leu Ile Gly Trp Thr Lys Gly Phe Lys
Ala 610 615 620 act gac tgt gaa ggg gag gac gtg gtg gac atg ctc agg
gaa gcc atc 1920 Thr Asp Cys Glu Gly Glu Asp Val Val Asp Met Leu
Arg Glu Ala Ile 625 630 635 640 aag agg aga aac gag ttt gac ctg gac
att gtt gca gtc gtg aat gat 1968 Lys Arg Arg Asn Glu Phe Asp Leu
Asp Ile Val Ala Val Val Asn Asp 645 650 655 aca gtg ggg acc atg atg
acc tgt ggc tat gaa gat cct aat tgt gag 2016 Thr Val Gly Thr Met
Met Thr Cys Gly Tyr Glu Asp Pro Asn Cys Glu 660 665 670 att ggc ctg
att gca gga aca ggc agc aac atg tgc tac atg gag gac 2064 Ile Gly
Leu Ile Ala Gly Thr Gly Ser Asn Met Cys Tyr Met Glu Asp 675 680 685
atg agg aac atc gag atg gtg gag ggg ggt gaa ggg aag atg tgc atc
2112 Met Arg Asn Ile Glu Met Val Glu Gly Gly Glu Gly Lys Met Cys
Ile 690 695 700 aat aca gag tgg gga gga ttt gga gac aat ggc tgc ata
gat gac atc 2160 Asn Thr Glu Trp Gly Gly Phe Gly Asp Asn Gly Cys
Ile Asp Asp Ile 705 710 715 720 cgg acc cga tac gac acg gag gtg gat
gag ggg tcc ttg aat cct ggc 2208 Arg Thr Arg Tyr Asp Thr Glu Val
Asp Glu Gly Ser Leu Asn Pro Gly 725 730 735 aag cag aga tac gag aaa
atg acc agt ggg atg tac ttg ggg gag att 2256 Lys Gln Arg Tyr Glu
Lys Met Thr Ser Gly Met Tyr Leu Gly Glu Ile 740 745 750 gtg cgg cag
atc ctg atc gac ctg acc aag cag ggt ctc ctc ttc cga 2304 Val Arg
Gln Ile Leu Ile Asp Leu Thr Lys Gln Gly Leu Leu Phe Arg 755 760 765
ggg cag att tca gag cgt ctc cgg acc agg ggc atc ttc gaa acc aag
2352 Gly Gln Ile Ser Glu Arg Leu Arg Thr Arg Gly Ile Phe Glu Thr
Lys 770 775 780 ttc ctg tcc cag atc gaa agc gat cgg ctg gcc ctt ctc
cag gtc agg 2400 Phe Leu Ser Gln Ile Glu Ser Asp Arg Leu Ala Leu
Leu Gln Val Arg 785 790 795 800 agg att ctg cag cag ctg ggc ctg gac
agc acg tgt gag gac agc atc 2448 Arg Ile Leu Gln Gln Leu Gly Leu
Asp Ser Thr Cys Glu Asp Ser Ile 805 810 815 gtg gtg aag gag gtg tgc
gga gcc gtg tcc cgg cgg gcg gcc cag ctc 2496 Val Val Lys Glu Val
Cys Gly Ala Val Ser Arg Arg Ala Ala Gln Leu 820 825 830 tgc ggt gct
ggc ctg gcc gct ata gtg gaa aaa agg aga gaa gac cag 2544 Cys Gly
Ala Gly Leu Ala Ala Ile Val Glu Lys Arg Arg Glu Asp Gln 835 840 845
ggg cta gag cac ctg agg atc act gtg ggt gtg gac ggc acc ctg tac
2592 Gly Leu Glu His Leu Arg Ile Thr Val Gly Val Asp Gly Thr Leu
Tyr 850 855 860 aag ctg cac cct cac ttt tct aga ata ttg cag gaa act
gtg aag gaa 2640 Lys Leu His Pro His Phe Ser Arg Ile Leu Gln Glu
Thr Val Lys Glu 865 870 875 880 cta gcc cct cga tgt gat gtg aca ttc
atg ctg tca gaa gat ggc agt 2688 Leu Ala Pro Arg Cys Asp Val Thr
Phe Met Leu Ser Glu Asp Gly Ser 885 890 895 gga aaa ggg gca gca ctg
atc act gct gtg gcc aag agg tta cag cag 2736 Gly Lys Gly Ala Ala
Leu Ile Thr Ala Val Ala Lys Arg Leu Gln Gln 900 905
910 gca cag aag gag aac tag 2754 Ala Gln Lys Glu Asn * 915 23 482
PRT Artificial Sequence Consensus Sequence 23 Ala Asp Leu Leu Gln
Ala Val Glu Glu Leu Leu Asp Asp Phe Thr Val 1 5 10 15 Ser Thr Glu
Thr Leu Arg Glu Val Thr Lys Arg Phe Ile Lys Glu Met 20 25 30 Glu
Lys Gly Leu Ser Pro Pro Lys Glu Gly Gly Asn Thr Ala Ser Val 35 40
45 Val Lys Met Leu Pro Thr Phe Val Arg Ser Thr Pro Thr Gly Thr Glu
50 55 60 Lys Gly Asp Phe Leu Ala Leu Asp Leu Gly Gly Thr Asn Phe
Arg Val 65 70 75 80 Leu Leu Val Lys Leu Gly Gly Asn Gly Lys Gly Val
Glu Met Thr Gln 85 90 95 Ser Lys Tyr Arg Ile Pro Glu Glu Leu Met
Thr Gly Glu Asn Val Thr 100 105 110 Gly Glu Gln Leu Phe Asp Phe Ile
Ala Glu Cys Ile Lys Asp Phe Met 115 120 125 Asp Glu Gln Phe Pro Lys
Gly Lys Lys Glu Pro Leu Pro Leu Gly Phe 130 135 140 Thr Phe Ser Phe
Pro Cys Ser Gln Thr Ser Ile Asn Glu Gly Ile Leu 145 150 155 160 Ile
Arg Trp Thr Lys Gly Phe Lys Ile Gly Arg Ala Thr Asn Ser Gly 165 170
175 Val Glu Gly His Asp Val Val Gln Leu Leu Arg Glu Ala Ile Lys Arg
180 185 190 Arg Gly Ala Phe Pro Ile Asp Val Val Ala Val Val Asn Asp
Thr Val 195 200 205 Gly Thr Leu Met Ser Cys Ala Tyr Thr Lys Gly Arg
Gly Asp Pro Glu 210 215 220 Cys Glu Thr Val Ile Gly Leu Ile Val Gly
Thr Gly Thr Asn Ala Cys 225 230 235 240 Tyr Met Glu Glu Met Arg Asn
Ile Glu Lys Leu Glu Gly Lys Leu Lys 245 250 255 Asp Asp Ile Pro Asp
Glu Gly Arg Met Cys Ile Asn Met Glu Trp Gly 260 265 270 Ala Phe Gly
Asp Asn Gly His Leu Asp Leu Pro Arg Thr Lys Tyr Asp 275 280 285 Val
Val Ile Asp Glu Glu Ser Pro Asn Pro Gly Gln Gln Leu Phe Glu 290 295
300 Lys Met Ile Ser Gly Met Tyr Leu Gly Glu Ile Val Arg Leu Ile Leu
305 310 315 320 Leu Asp Leu Thr Lys Glu Gly Leu Leu Phe Lys Gly Gln
Asp Ser Pro 325 330 335 Lys Leu Lys Thr Arg Gly Ser Phe Glu Thr Ser
Val Leu Ser Arg Ile 340 345 350 Glu Ser Asp Pro Ser Glu Asn Leu Glu
Asp Val Arg Ala Ile Leu Gln 355 360 365 Thr Ala Leu Gly Leu Glu Thr
Thr Asp Glu Glu Arg Lys Leu Val Arg 370 375 380 Arg Val Cys Glu Ala
Val Ser Thr Arg Ala Ala Arg Leu Cys Ala Ala 385 390 395 400 Gly Leu
Ala Ala Ile Leu Lys Lys Ile Arg Glu Asn Arg Gly Arg Glu 405 410 415
Arg Leu Lys Val Thr Val Gly Val Asp Gly Ser Val Tyr Lys Leu Tyr 420
425 430 Pro Gly Phe Lys Glu Arg Leu Ala Glu Ala Leu Arg Asp Leu Leu
Pro 435 440 445 Asp Cys Glu Gly Ser Glu Glu Asp Lys Lys Val Ser Ile
Ile Pro Ala 450 455 460 Glu Asp Gly Ser Gly Lys Gly Ala Ala Leu Val
Ala Ala Val Ala Ala 465 470 475 480 Lys Leu 24 26 PRT Artificial
Sequence Hexokinase Signature Domain 24 Leu Gly Phe Thr Phe Ser Phe
Pro Cys Xaa Gln Xaa Ser Ile Xaa Xaa 1 5 10 15 Gly Xaa Leu Ile Xaa
Trp Thr Lys Gly Phe 20 25 25 3544 DNA Homo Sapiens CDS
(589)...(2586) 25 tcctataggg agtcgccccg cgtccgaaaa gattataagt
aaatactctg ctctttcaag 60 tgaaccaaac ctatcaaacc tgtttagaaa
ataaaccagg cagaataaaa tgatgcgaaa 120 tgttcatttt aaaaaacttc
aggatgggca caaacacaca gaagtgggaa atgaataaaa 180 gagtattgat
aaatttttga aaattgttga agctgagtaa tgggctttca gtccagtgta 240
aagctgttgg agcgcgggag caaaggtaaa gaatgatgta atgcgctggc tgctccaaag
300 catcttttgt tgtggaatgg ttattccagt catctcttta tgaatcaaat
gtgaggggct 360 gctttgtgga cggagtcctt tgcaagagca catcaacggg
aaagagaaag agacattcac 420 ttggagggct cttgctgaaa atgggtttaa
ctctcctttt gccagtcacc accagcctga 480 cctcatacac ttttagtaca
atggagtggc tgagcctttg agcacaccac cattacatca 540 tcgtggcaaa
ttaaagaagg aggtgggaaa agaggactta ttgttgtc atg gcc cat 597 Met Ala
His 1 gag atg att gga act caa att gtt act gag agg ttg gtg gct ctg
ctg 645 Glu Met Ile Gly Thr Gln Ile Val Thr Glu Arg Leu Val Ala Leu
Leu 5 10 15 gaa agt gga acg gaa aaa gtg ctg cta att gat agc cgg cca
ttt gtg 693 Glu Ser Gly Thr Glu Lys Val Leu Leu Ile Asp Ser Arg Pro
Phe Val 20 25 30 35 gaa tac aat aca tcc cac att ttg gaa gcc att aat
atc aac tgc tcc 741 Glu Tyr Asn Thr Ser His Ile Leu Glu Ala Ile Asn
Ile Asn Cys Ser 40 45 50 aag ctt atg aag cga agg ttg caa cag gac
aaa gtg tta att aca gag 789 Lys Leu Met Lys Arg Arg Leu Gln Gln Asp
Lys Val Leu Ile Thr Glu 55 60 65 ctc atc cag cat tca gcg aaa cat
aag gtt gac att gat tgc agt cag 837 Leu Ile Gln His Ser Ala Lys His
Lys Val Asp Ile Asp Cys Ser Gln 70 75 80 aag gtt gta gtt tac gat
caa agc tcc caa gat gtt gcc tct ctc tct 885 Lys Val Val Val Tyr Asp
Gln Ser Ser Gln Asp Val Ala Ser Leu Ser 85 90 95 tca gac tgt ttt
ctc act gta ctt ctg ggt aaa ctg gag aag agc ttc 933 Ser Asp Cys Phe
Leu Thr Val Leu Leu Gly Lys Leu Glu Lys Ser Phe 100 105 110 115 aac
tct gtt cac ctg ctt gca ggt ggg ttt gct gag ttc tct cgt tgt 981 Asn
Ser Val His Leu Leu Ala Gly Gly Phe Ala Glu Phe Ser Arg Cys 120 125
130 ttc cct ggc ctc tgt gaa gga aaa tcc act cta gtc cct acc tgc att
1029 Phe Pro Gly Leu Cys Glu Gly Lys Ser Thr Leu Val Pro Thr Cys
Ile 135 140 145 tct cag cct tgc tta cct gtt gcc aac att ggg cca acc
cga att ctt 1077 Ser Gln Pro Cys Leu Pro Val Ala Asn Ile Gly Pro
Thr Arg Ile Leu 150 155 160 ccc aat ctt tat ctt ggc tgc cag cga gat
gtc ctc aac aag gag ctg 1125 Pro Asn Leu Tyr Leu Gly Cys Gln Arg
Asp Val Leu Asn Lys Glu Leu 165 170 175 atg cag cag aat ggg att ggt
tat gtg tta aat gcc agc aat acc tgt 1173 Met Gln Gln Asn Gly Ile
Gly Tyr Val Leu Asn Ala Ser Asn Thr Cys 180 185 190 195 cca aag cct
gac ttt atc ccc gag tct cat ttc ctg cgt gtg cct gtg 1221 Pro Lys
Pro Asp Phe Ile Pro Glu Ser His Phe Leu Arg Val Pro Val 200 205 210
aat gac agc ttt tgt gag aaa att ttg ccg tgg ttg gac aaa tca gta
1269 Asn Asp Ser Phe Cys Glu Lys Ile Leu Pro Trp Leu Asp Lys Ser
Val 215 220 225 gat ttc att gag aaa gca aaa gcc tcc aat gga tgt gtt
cta gtg cac 1317 Asp Phe Ile Glu Lys Ala Lys Ala Ser Asn Gly Cys
Val Leu Val His 230 235 240 tgt tta gct ggg atc tcc cgc tcc gcc acc
atc gct atc gcc tac atc 1365 Cys Leu Ala Gly Ile Ser Arg Ser Ala
Thr Ile Ala Ile Ala Tyr Ile 245 250 255 atg aag agg atg gac atg tct
tta gat gaa gct tac aga ttt gtg aaa 1413 Met Lys Arg Met Asp Met
Ser Leu Asp Glu Ala Tyr Arg Phe Val Lys 260 265 270 275 gaa aaa aga
cct act ata tct cca aac ttc aat ttt ctg ggc caa ctc 1461 Glu Lys
Arg Pro Thr Ile Ser Pro Asn Phe Asn Phe Leu Gly Gln Leu 280 285 290
ctg gac tat gag aag aag att aag aac cag act gga gca tca ggg cca
1509 Leu Asp Tyr Glu Lys Lys Ile Lys Asn Gln Thr Gly Ala Ser Gly
Pro 295 300 305 aag agc aaa ctc aag ctg ctg cac ctg gag aag cca aat
gaa cct gtc 1557 Lys Ser Lys Leu Lys Leu Leu His Leu Glu Lys Pro
Asn Glu Pro Val 310 315 320 cct gct gtc tca gag ggt gga cag aaa agc
gag acg ccc ctc agt cca 1605 Pro Ala Val Ser Glu Gly Gly Gln Lys
Ser Glu Thr Pro Leu Ser Pro 325 330 335 ccc tgt gcc gac tct gct acc
tca gag gca gca gga caa agg ccc gtg 1653 Pro Cys Ala Asp Ser Ala
Thr Ser Glu Ala Ala Gly Gln Arg Pro Val 340 345 350 355 cat ccc gcc
agc gtg ccc agc gtg ccc agc gtg cag ccg tcg ctg tta 1701 His Pro
Ala Ser Val Pro Ser Val Pro Ser Val Gln Pro Ser Leu Leu 360 365 370
gag gac agc ccg ctg gta cag gcg ctc agt ggg ctg cac ctg tcc gca
1749 Glu Asp Ser Pro Leu Val Gln Ala Leu Ser Gly Leu His Leu Ser
Ala 375 380 385 gac agg ctg gaa gac agc aat aag ctc aag cgt tcc ttc
tct ctg gat 1797 Asp Arg Leu Glu Asp Ser Asn Lys Leu Lys Arg Ser
Phe Ser Leu Asp 390 395 400 atc aaa tca gtt tca tat tca gcc agc atg
gca gca tcc tta cat ggc 1845 Ile Lys Ser Val Ser Tyr Ser Ala Ser
Met Ala Ala Ser Leu His Gly 405 410 415 ttc tcc tca tca gaa gat gct
ttg gaa tac tac aaa cct tcc act act 1893 Phe Ser Ser Ser Glu Asp
Ala Leu Glu Tyr Tyr Lys Pro Ser Thr Thr 420 425 430 435 ctg gat ggg
acc aac aag cta tgc cag ttc tcc cct gtt cag gaa cta 1941 Leu Asp
Gly Thr Asn Lys Leu Cys Gln Phe Ser Pro Val Gln Glu Leu 440 445 450
tcg gag cag act ccc gaa acc agt cct gat aag gag gaa gcc agc atc
1989 Ser Glu Gln Thr Pro Glu Thr Ser Pro Asp Lys Glu Glu Ala Ser
Ile 455 460 465 ccc aag aag ctg cag acc gcc agg cct tca gac agc cag
agc aag cga 2037 Pro Lys Lys Leu Gln Thr Ala Arg Pro Ser Asp Ser
Gln Ser Lys Arg 470 475 480 ttg cat tcg gtc aga acc agc agc agt ggc
acc gcc cag agg tcc ctt 2085 Leu His Ser Val Arg Thr Ser Ser Ser
Gly Thr Ala Gln Arg Ser Leu 485 490 495 tta tct cca ctg cat cga agt
ggg agc gtg gag gac aat tac cac acc 2133 Leu Ser Pro Leu His Arg
Ser Gly Ser Val Glu Asp Asn Tyr His Thr 500 505 510 515 agc ttc ctt
ttc ggc ctt tcc acc agc cag cag cac ctc acg aag tct 2181 Ser Phe
Leu Phe Gly Leu Ser Thr Ser Gln Gln His Leu Thr Lys Ser 520 525 530
gct ggc ctg ggc ctt aag ggc tgg cac tcg gat atc ttg gcc ccc cag
2229 Ala Gly Leu Gly Leu Lys Gly Trp His Ser Asp Ile Leu Ala Pro
Gln 535 540 545 acc tct acc cct tcc ctg acc agc agc tgg tat ttt gcc
aca gag tcc 2277 Thr Ser Thr Pro Ser Leu Thr Ser Ser Trp Tyr Phe
Ala Thr Glu Ser 550 555 560 tca cac ttc tac tct gcc tca gcc atc tac
gga ggc agt gcc agt tac 2325 Ser His Phe Tyr Ser Ala Ser Ala Ile
Tyr Gly Gly Ser Ala Ser Tyr 565 570 575 tct gcc tac agc tgc agc cag
ctg ccc act tgc gga gac caa gtc tat 2373 Ser Ala Tyr Ser Cys Ser
Gln Leu Pro Thr Cys Gly Asp Gln Val Tyr 580 585 590 595 tct gtg cgc
agg cgg cag aag cca agt gac aga gct gac tcg cgg cgg 2421 Ser Val
Arg Arg Arg Gln Lys Pro Ser Asp Arg Ala Asp Ser Arg Arg 600 605 610
agc tgg cat gaa gag agc ccc ttt gaa aag cag ttt aaa cgc aga agc
2469 Ser Trp His Glu Glu Ser Pro Phe Glu Lys Gln Phe Lys Arg Arg
Ser 615 620 625 tgc caa atg gaa ttt gga gag agc atc atg tca gag aac
agg tca cgg 2517 Cys Gln Met Glu Phe Gly Glu Ser Ile Met Ser Glu
Asn Arg Ser Arg 630 635 640 gaa gag ctg ggg aaa gtg ggc agt cag tct
agc ttt tcg ggc agc atg 2565 Glu Glu Leu Gly Lys Val Gly Ser Gln
Ser Ser Phe Ser Gly Ser Met 645 650 655 gaa atc att gag gtc tcc tga
gaagaaagac acttgtgact tctatagaca 2616 Glu Ile Ile Glu Val Ser * 660
665 attttttttt cttgttcaca aaaaaattcc ctgtaaatct gaaatatata
tatgtacata 2676 catatatatt tttggaaaat ggagctatgg tgtaaaagca
acaggtggat caacccagtt 2736 gttactctct taacatctgc atttgagaga
tcagctaata cttctctcaa caaaaatgga 2796 agggcagatg ctagaatccc
ccctagacgg aggaaaacca ttttattcag tgaattacac 2856 atcctcttgt
tcttaaaaaa gcaagtgtct ttggtgttgg aggacaaaat cccctaccat 2916
tttcacgttg tgctactaag agatctcaaa tattagtctt tgtccggacc cttccatagt
2976 acaccttagc gctgagactg agccagcttg ggggtcaggt aggtagaccc
tgttagggac 3036 agagcctagt ggtaaatcca agagaaatga tcctatccaa
agctgattca caaacccacg 3096 ctcacctgac agccgaggga cacgagcatc
actctgctgg acggaccatt aggggccttg 3156 ccaaggtcta ccttagagca
aacccagtac ctcagacagg aaagtcgggg ctttgaccac 3216 taccatatct
ggtagcccat tttctaggca ttgtgaatag gtaggtagct agtcacactt 3276
ttcagaccaa ttcaaactgt ctatgcacaa aattcccgtg ggcctagatg gagataattt
3336 ttttttcttc tcagctttat gaagagaagg gaaactgtct aggattcagc
tgaaccacca 3396 ggaacctggc aacatcacga tttaagctaa ggttgggagg
ctaacgagtc tacctccctc 3456 tttgtaaatc aaagaattgt ttaaaatggg
attgtcaatc ctttaaataa agatgaactt 3516 ggtttcaaaa aaaaaaaaaa
aaaaaagg 3544 26 665 PRT Homo Sapiens 26 Met Ala His Glu Met Ile
Gly Thr Gln Ile Val Thr Glu Arg Leu Val 1 5 10 15 Ala Leu Leu Glu
Ser Gly Thr Glu Lys Val Leu Leu Ile Asp Ser Arg 20 25 30 Pro Phe
Val Glu Tyr Asn Thr Ser His Ile Leu Glu Ala Ile Asn Ile 35 40 45
Asn Cys Ser Lys Leu Met Lys Arg Arg Leu Gln Gln Asp Lys Val Leu 50
55 60 Ile Thr Glu Leu Ile Gln His Ser Ala Lys His Lys Val Asp Ile
Asp 65 70 75 80 Cys Ser Gln Lys Val Val Val Tyr Asp Gln Ser Ser Gln
Asp Val Ala 85 90 95 Ser Leu Ser Ser Asp Cys Phe Leu Thr Val Leu
Leu Gly Lys Leu Glu 100 105 110 Lys Ser Phe Asn Ser Val His Leu Leu
Ala Gly Gly Phe Ala Glu Phe 115 120 125 Ser Arg Cys Phe Pro Gly Leu
Cys Glu Gly Lys Ser Thr Leu Val Pro 130 135 140 Thr Cys Ile Ser Gln
Pro Cys Leu Pro Val Ala Asn Ile Gly Pro Thr 145 150 155 160 Arg Ile
Leu Pro Asn Leu Tyr Leu Gly Cys Gln Arg Asp Val Leu Asn 165 170 175
Lys Glu Leu Met Gln Gln Asn Gly Ile Gly Tyr Val Leu Asn Ala Ser 180
185 190 Asn Thr Cys Pro Lys Pro Asp Phe Ile Pro Glu Ser His Phe Leu
Arg 195 200 205 Val Pro Val Asn Asp Ser Phe Cys Glu Lys Ile Leu Pro
Trp Leu Asp 210 215 220 Lys Ser Val Asp Phe Ile Glu Lys Ala Lys Ala
Ser Asn Gly Cys Val 225 230 235 240 Leu Val His Cys Leu Ala Gly Ile
Ser Arg Ser Ala Thr Ile Ala Ile 245 250 255 Ala Tyr Ile Met Lys Arg
Met Asp Met Ser Leu Asp Glu Ala Tyr Arg 260 265 270 Phe Val Lys Glu
Lys Arg Pro Thr Ile Ser Pro Asn Phe Asn Phe Leu 275 280 285 Gly Gln
Leu Leu Asp Tyr Glu Lys Lys Ile Lys Asn Gln Thr Gly Ala 290 295 300
Ser Gly Pro Lys Ser Lys Leu Lys Leu Leu His Leu Glu Lys Pro Asn 305
310 315 320 Glu Pro Val Pro Ala Val Ser Glu Gly Gly Gln Lys Ser Glu
Thr Pro 325 330 335 Leu Ser Pro Pro Cys Ala Asp Ser Ala Thr Ser Glu
Ala Ala Gly Gln 340 345 350 Arg Pro Val His Pro Ala Ser Val Pro Ser
Val Pro Ser Val Gln Pro 355 360 365 Ser Leu Leu Glu Asp Ser Pro Leu
Val Gln Ala Leu Ser Gly Leu His 370 375 380 Leu Ser Ala Asp Arg Leu
Glu Asp Ser Asn Lys Leu Lys Arg Ser Phe 385 390 395 400 Ser Leu Asp
Ile Lys Ser Val Ser Tyr Ser Ala Ser Met Ala Ala Ser 405 410 415 Leu
His Gly Phe Ser Ser Ser Glu Asp Ala Leu Glu Tyr Tyr Lys Pro 420 425
430 Ser Thr Thr Leu Asp Gly Thr Asn Lys Leu Cys Gln Phe Ser Pro Val
435 440 445 Gln Glu Leu Ser Glu Gln Thr Pro Glu Thr Ser Pro Asp Lys
Glu Glu 450 455 460 Ala Ser Ile Pro Lys Lys Leu Gln Thr Ala Arg Pro
Ser Asp Ser Gln 465 470 475 480 Ser Lys Arg Leu His Ser Val Arg Thr
Ser Ser Ser Gly Thr Ala Gln 485 490 495 Arg Ser Leu Leu Ser Pro Leu
His Arg Ser Gly Ser Val Glu Asp Asn 500 505 510 Tyr His Thr Ser Phe
Leu Phe Gly Leu Ser Thr Ser Gln Gln His Leu 515 520 525 Thr Lys Ser
Ala Gly Leu Gly Leu Lys Gly Trp His Ser Asp Ile Leu 530 535 540 Ala
Pro Gln Thr Ser Thr Pro Ser Leu Thr Ser Ser Trp Tyr Phe Ala 545
550
555 560 Thr Glu Ser Ser His Phe Tyr Ser Ala Ser Ala Ile Tyr Gly Gly
Ser 565 570 575 Ala Ser Tyr Ser Ala Tyr Ser Cys Ser Gln Leu Pro Thr
Cys Gly Asp 580 585 590 Gln Val Tyr Ser Val Arg Arg Arg Gln Lys Pro
Ser Asp Arg Ala Asp 595 600 605 Ser Arg Arg Ser Trp His Glu Glu Ser
Pro Phe Glu Lys Gln Phe Lys 610 615 620 Arg Arg Ser Cys Gln Met Glu
Phe Gly Glu Ser Ile Met Ser Glu Asn 625 630 635 640 Arg Ser Arg Glu
Glu Leu Gly Lys Val Gly Ser Gln Ser Ser Phe Ser 645 650 655 Gly Ser
Met Glu Ile Ile Glu Val Ser 660 665 27 1998 DNA Homo Sapiens CDS
(1)...(1998) 27 atg gcc cat gag atg att gga act caa att gtt act gag
agg ttg gtg 48 Met Ala His Glu Met Ile Gly Thr Gln Ile Val Thr Glu
Arg Leu Val 1 5 10 15 gct ctg ctg gaa agt gga acg gaa aaa gtg ctg
cta att gat agc cgg 96 Ala Leu Leu Glu Ser Gly Thr Glu Lys Val Leu
Leu Ile Asp Ser Arg 20 25 30 cca ttt gtg gaa tac aat aca tcc cac
att ttg gaa gcc att aat atc 144 Pro Phe Val Glu Tyr Asn Thr Ser His
Ile Leu Glu Ala Ile Asn Ile 35 40 45 aac tgc tcc aag ctt atg aag
cga agg ttg caa cag gac aaa gtg tta 192 Asn Cys Ser Lys Leu Met Lys
Arg Arg Leu Gln Gln Asp Lys Val Leu 50 55 60 att aca gag ctc atc
cag cat tca gcg aaa cat aag gtt gac att gat 240 Ile Thr Glu Leu Ile
Gln His Ser Ala Lys His Lys Val Asp Ile Asp 65 70 75 80 tgc agt cag
aag gtt gta gtt tac gat caa agc tcc caa gat gtt gcc 288 Cys Ser Gln
Lys Val Val Val Tyr Asp Gln Ser Ser Gln Asp Val Ala 85 90 95 tct
ctc tct tca gac tgt ttt ctc act gta ctt ctg ggt aaa ctg gag 336 Ser
Leu Ser Ser Asp Cys Phe Leu Thr Val Leu Leu Gly Lys Leu Glu 100 105
110 aag agc ttc aac tct gtt cac ctg ctt gca ggt ggg ttt gct gag ttc
384 Lys Ser Phe Asn Ser Val His Leu Leu Ala Gly Gly Phe Ala Glu Phe
115 120 125 tct cgt tgt ttc cct ggc ctc tgt gaa gga aaa tcc act cta
gtc cct 432 Ser Arg Cys Phe Pro Gly Leu Cys Glu Gly Lys Ser Thr Leu
Val Pro 130 135 140 acc tgc att tct cag cct tgc tta cct gtt gcc aac
att ggg cca acc 480 Thr Cys Ile Ser Gln Pro Cys Leu Pro Val Ala Asn
Ile Gly Pro Thr 145 150 155 160 cga att ctt ccc aat ctt tat ctt ggc
tgc cag cga gat gtc ctc aac 528 Arg Ile Leu Pro Asn Leu Tyr Leu Gly
Cys Gln Arg Asp Val Leu Asn 165 170 175 aag gag ctg atg cag cag aat
ggg att ggt tat gtg tta aat gcc agc 576 Lys Glu Leu Met Gln Gln Asn
Gly Ile Gly Tyr Val Leu Asn Ala Ser 180 185 190 aat acc tgt cca aag
cct gac ttt atc ccc gag tct cat ttc ctg cgt 624 Asn Thr Cys Pro Lys
Pro Asp Phe Ile Pro Glu Ser His Phe Leu Arg 195 200 205 gtg cct gtg
aat gac agc ttt tgt gag aaa att ttg ccg tgg ttg gac 672 Val Pro Val
Asn Asp Ser Phe Cys Glu Lys Ile Leu Pro Trp Leu Asp 210 215 220 aaa
tca gta gat ttc att gag aaa gca aaa gcc tcc aat gga tgt gtt 720 Lys
Ser Val Asp Phe Ile Glu Lys Ala Lys Ala Ser Asn Gly Cys Val 225 230
235 240 cta gtg cac tgt tta gct ggg atc tcc cgc tcc gcc acc atc gct
atc 768 Leu Val His Cys Leu Ala Gly Ile Ser Arg Ser Ala Thr Ile Ala
Ile 245 250 255 gcc tac atc atg aag agg atg gac atg tct tta gat gaa
gct tac aga 816 Ala Tyr Ile Met Lys Arg Met Asp Met Ser Leu Asp Glu
Ala Tyr Arg 260 265 270 ttt gtg aaa gaa aaa aga cct act ata tct cca
aac ttc aat ttt ctg 864 Phe Val Lys Glu Lys Arg Pro Thr Ile Ser Pro
Asn Phe Asn Phe Leu 275 280 285 ggc caa ctc ctg gac tat gag aag aag
att aag aac cag act gga gca 912 Gly Gln Leu Leu Asp Tyr Glu Lys Lys
Ile Lys Asn Gln Thr Gly Ala 290 295 300 tca ggg cca aag agc aaa ctc
aag ctg ctg cac ctg gag aag cca aat 960 Ser Gly Pro Lys Ser Lys Leu
Lys Leu Leu His Leu Glu Lys Pro Asn 305 310 315 320 gaa cct gtc cct
gct gtc tca gag ggt gga cag aaa agc gag acg ccc 1008 Glu Pro Val
Pro Ala Val Ser Glu Gly Gly Gln Lys Ser Glu Thr Pro 325 330 335 ctc
agt cca ccc tgt gcc gac tct gct acc tca gag gca gca gga caa 1056
Leu Ser Pro Pro Cys Ala Asp Ser Ala Thr Ser Glu Ala Ala Gly Gln 340
345 350 agg ccc gtg cat ccc gcc agc gtg ccc agc gtg ccc agc gtg cag
ccg 1104 Arg Pro Val His Pro Ala Ser Val Pro Ser Val Pro Ser Val
Gln Pro 355 360 365 tcg ctg tta gag gac agc ccg ctg gta cag gcg ctc
agt ggg ctg cac 1152 Ser Leu Leu Glu Asp Ser Pro Leu Val Gln Ala
Leu Ser Gly Leu His 370 375 380 ctg tcc gca gac agg ctg gaa gac agc
aat aag ctc aag cgt tcc ttc 1200 Leu Ser Ala Asp Arg Leu Glu Asp
Ser Asn Lys Leu Lys Arg Ser Phe 385 390 395 400 tct ctg gat atc aaa
tca gtt tca tat tca gcc agc atg gca gca tcc 1248 Ser Leu Asp Ile
Lys Ser Val Ser Tyr Ser Ala Ser Met Ala Ala Ser 405 410 415 tta cat
ggc ttc tcc tca tca gaa gat gct ttg gaa tac tac aaa cct 1296 Leu
His Gly Phe Ser Ser Ser Glu Asp Ala Leu Glu Tyr Tyr Lys Pro 420 425
430 tcc act act ctg gat ggg acc aac aag cta tgc cag ttc tcc cct gtt
1344 Ser Thr Thr Leu Asp Gly Thr Asn Lys Leu Cys Gln Phe Ser Pro
Val 435 440 445 cag gaa cta tcg gag cag act ccc gaa acc agt cct gat
aag gag gaa 1392 Gln Glu Leu Ser Glu Gln Thr Pro Glu Thr Ser Pro
Asp Lys Glu Glu 450 455 460 gcc agc atc ccc aag aag ctg cag acc gcc
agg cct tca gac agc cag 1440 Ala Ser Ile Pro Lys Lys Leu Gln Thr
Ala Arg Pro Ser Asp Ser Gln 465 470 475 480 agc aag cga ttg cat tcg
gtc aga acc agc agc agt ggc acc gcc cag 1488 Ser Lys Arg Leu His
Ser Val Arg Thr Ser Ser Ser Gly Thr Ala Gln 485 490 495 agg tcc ctt
tta tct cca ctg cat cga agt ggg agc gtg gag gac aat 1536 Arg Ser
Leu Leu Ser Pro Leu His Arg Ser Gly Ser Val Glu Asp Asn 500 505 510
tac cac acc agc ttc ctt ttc ggc ctt tcc acc agc cag cag cac ctc
1584 Tyr His Thr Ser Phe Leu Phe Gly Leu Ser Thr Ser Gln Gln His
Leu 515 520 525 acg aag tct gct ggc ctg ggc ctt aag ggc tgg cac tcg
gat atc ttg 1632 Thr Lys Ser Ala Gly Leu Gly Leu Lys Gly Trp His
Ser Asp Ile Leu 530 535 540 gcc ccc cag acc tct acc cct tcc ctg acc
agc agc tgg tat ttt gcc 1680 Ala Pro Gln Thr Ser Thr Pro Ser Leu
Thr Ser Ser Trp Tyr Phe Ala 545 550 555 560 aca gag tcc tca cac ttc
tac tct gcc tca gcc atc tac gga ggc agt 1728 Thr Glu Ser Ser His
Phe Tyr Ser Ala Ser Ala Ile Tyr Gly Gly Ser 565 570 575 gcc agt tac
tct gcc tac agc tgc agc cag ctg ccc act tgc gga gac 1776 Ala Ser
Tyr Ser Ala Tyr Ser Cys Ser Gln Leu Pro Thr Cys Gly Asp 580 585 590
caa gtc tat tct gtg cgc agg cgg cag aag cca agt gac aga gct gac
1824 Gln Val Tyr Ser Val Arg Arg Arg Gln Lys Pro Ser Asp Arg Ala
Asp 595 600 605 tcg cgg cgg agc tgg cat gaa gag agc ccc ttt gaa aag
cag ttt aaa 1872 Ser Arg Arg Ser Trp His Glu Glu Ser Pro Phe Glu
Lys Gln Phe Lys 610 615 620 cgc aga agc tgc caa atg gaa ttt gga gag
agc atc atg tca gag aac 1920 Arg Arg Ser Cys Gln Met Glu Phe Gly
Glu Ser Ile Met Ser Glu Asn 625 630 635 640 agg tca cgg gaa gag ctg
ggg aaa gtg ggc agt cag tct agc ttt tcg 1968 Arg Ser Arg Glu Glu
Leu Gly Lys Val Gly Ser Gln Ser Ser Phe Ser 645 650 655 ggc agc atg
gaa atc att gag gtc tcc tga 1998 Gly Ser Met Glu Ile Ile Glu Val
Ser * 660 665 28 1114 DNA Homo Sapiens CDS (89)...(760) 28
ctatagggag tcgcccacgc gtccggggcg gtggcgcgct gacacctggc ggcggcggag
60 ggcgggcaga agcccgcggg ccagcacc atg gag gac gtg aag ctg gag ttc
112 Met Glu Asp Val Lys Leu Glu Phe 1 5 cct tcc ctt cca cag tgc aag
gaa gac gcc gag gag tgg acc tac cct 160 Pro Ser Leu Pro Gln Cys Lys
Glu Asp Ala Glu Glu Trp Thr Tyr Pro 10 15 20 atg aga cga gag atg
cag gaa att tta cct gga ttg ttc tta ggc cca 208 Met Arg Arg Glu Met
Gln Glu Ile Leu Pro Gly Leu Phe Leu Gly Pro 25 30 35 40 tat tca tct
gct atg aaa agc aag cta cct gta cta cag aaa cat gga 256 Tyr Ser Ser
Ala Met Lys Ser Lys Leu Pro Val Leu Gln Lys His Gly 45 50 55 ata
acc cat ata ata tgc ata cga caa aat att gaa gca aac ttt att 304 Ile
Thr His Ile Ile Cys Ile Arg Gln Asn Ile Glu Ala Asn Phe Ile 60 65
70 aaa cca aac ttt cag cag tta ttt aga tat tta gtc ctg gat att gca
352 Lys Pro Asn Phe Gln Gln Leu Phe Arg Tyr Leu Val Leu Asp Ile Ala
75 80 85 gat aat cca gtt gaa aat ata ata cgt ttt ttc cct atg act
aag gaa 400 Asp Asn Pro Val Glu Asn Ile Ile Arg Phe Phe Pro Met Thr
Lys Glu 90 95 100 ttt att gat ggg agc tta caa atg gga gga aaa gtt
ctt gtg cat gga 448 Phe Ile Asp Gly Ser Leu Gln Met Gly Gly Lys Val
Leu Val His Gly 105 110 115 120 aat gca ggg atc tcc aga agt gca gcc
ttt gtt att gca tac att atg 496 Asn Ala Gly Ile Ser Arg Ser Ala Ala
Phe Val Ile Ala Tyr Ile Met 125 130 135 gaa aca ttt gga atg aag tac
aga gat gct ttt gct tat gtt caa gaa 544 Glu Thr Phe Gly Met Lys Tyr
Arg Asp Ala Phe Ala Tyr Val Gln Glu 140 145 150 aga aga ttt tgt att
aat cct aat gct gga ttt gtc cat caa ctt cag 592 Arg Arg Phe Cys Ile
Asn Pro Asn Ala Gly Phe Val His Gln Leu Gln 155 160 165 gaa tat gaa
gcc atc tac cta gca aaa tta aca ata cag atg atg tca 640 Glu Tyr Glu
Ala Ile Tyr Leu Ala Lys Leu Thr Ile Gln Met Met Ser 170 175 180 cca
ctc cag ata gaa agg tca tta tct gtt cat tct ggt acc aca ggc 688 Pro
Leu Gln Ile Glu Arg Ser Leu Ser Val His Ser Gly Thr Thr Gly 185 190
195 200 agt ttg aag aga aca cat gaa gaa gag gat gat ttt gga acc atg
caa 736 Ser Leu Lys Arg Thr His Glu Glu Glu Asp Asp Phe Gly Thr Met
Gln 205 210 215 gtg gcg act gca cag aat ggc tga cttgaagagc
aacatcatag agtgtgaatt 790 Val Ala Thr Ala Gln Asn Gly * 220
tctatttggg aaggagaaaa tacaagagaa aattataatg taaaatggta aaaacataag
850 tagttttttt ttcaattaca tgttgcttcc agacatactt ctctgcaact
tgttgagcaa 910 cattttaaga tgttggactt ctgcaataga tgacactgat
ggttttactc ctttttttaa 970 aaacacatgc gcgcgcacac acacatgctt
tacaagtttt attataaacc aagaattttg 1030 gacttgcaaa gaggtattat
tgcaataatg cacttttcat acttgaaatt tatttgtatg 1090 atataaagtt
attactttaa acaa 1114 29 223 PRT Homo Sapiens 29 Met Glu Asp Val Lys
Leu Glu Phe Pro Ser Leu Pro Gln Cys Lys Glu 1 5 10 15 Asp Ala Glu
Glu Trp Thr Tyr Pro Met Arg Arg Glu Met Gln Glu Ile 20 25 30 Leu
Pro Gly Leu Phe Leu Gly Pro Tyr Ser Ser Ala Met Lys Ser Lys 35 40
45 Leu Pro Val Leu Gln Lys His Gly Ile Thr His Ile Ile Cys Ile Arg
50 55 60 Gln Asn Ile Glu Ala Asn Phe Ile Lys Pro Asn Phe Gln Gln
Leu Phe 65 70 75 80 Arg Tyr Leu Val Leu Asp Ile Ala Asp Asn Pro Val
Glu Asn Ile Ile 85 90 95 Arg Phe Phe Pro Met Thr Lys Glu Phe Ile
Asp Gly Ser Leu Gln Met 100 105 110 Gly Gly Lys Val Leu Val His Gly
Asn Ala Gly Ile Ser Arg Ser Ala 115 120 125 Ala Phe Val Ile Ala Tyr
Ile Met Glu Thr Phe Gly Met Lys Tyr Arg 130 135 140 Asp Ala Phe Ala
Tyr Val Gln Glu Arg Arg Phe Cys Ile Asn Pro Asn 145 150 155 160 Ala
Gly Phe Val His Gln Leu Gln Glu Tyr Glu Ala Ile Tyr Leu Ala 165 170
175 Lys Leu Thr Ile Gln Met Met Ser Pro Leu Gln Ile Glu Arg Ser Leu
180 185 190 Ser Val His Ser Gly Thr Thr Gly Ser Leu Lys Arg Thr His
Glu Glu 195 200 205 Glu Asp Asp Phe Gly Thr Met Gln Val Ala Thr Ala
Gln Asn Gly 210 215 220 30 672 DNA Homo Sapiens CDS (1)...(672) 30
atg gag gac gtg aag ctg gag ttc cct tcc ctt cca cag tgc aag gaa 48
Met Glu Asp Val Lys Leu Glu Phe Pro Ser Leu Pro Gln Cys Lys Glu 1 5
10 15 gac gcc gag gag tgg acc tac cct atg aga cga gag atg cag gaa
att 96 Asp Ala Glu Glu Trp Thr Tyr Pro Met Arg Arg Glu Met Gln Glu
Ile 20 25 30 tta cct gga ttg ttc tta ggc cca tat tca tct gct atg
aaa agc aag 144 Leu Pro Gly Leu Phe Leu Gly Pro Tyr Ser Ser Ala Met
Lys Ser Lys 35 40 45 cta cct gta cta cag aaa cat gga ata acc cat
ata ata tgc ata cga 192 Leu Pro Val Leu Gln Lys His Gly Ile Thr His
Ile Ile Cys Ile Arg 50 55 60 caa aat att gaa gca aac ttt att aaa
cca aac ttt cag cag tta ttt 240 Gln Asn Ile Glu Ala Asn Phe Ile Lys
Pro Asn Phe Gln Gln Leu Phe 65 70 75 80 aga tat tta gtc ctg gat att
gca gat aat cca gtt gaa aat ata ata 288 Arg Tyr Leu Val Leu Asp Ile
Ala Asp Asn Pro Val Glu Asn Ile Ile 85 90 95 cgt ttt ttc cct atg
act aag gaa ttt att gat ggg agc tta caa atg 336 Arg Phe Phe Pro Met
Thr Lys Glu Phe Ile Asp Gly Ser Leu Gln Met 100 105 110 gga gga aaa
gtt ctt gtg cat gga aat gca ggg atc tcc aga agt gca 384 Gly Gly Lys
Val Leu Val His Gly Asn Ala Gly Ile Ser Arg Ser Ala 115 120 125 gcc
ttt gtt att gca tac att atg gaa aca ttt gga atg aag tac aga 432 Ala
Phe Val Ile Ala Tyr Ile Met Glu Thr Phe Gly Met Lys Tyr Arg 130 135
140 gat gct ttt gct tat gtt caa gaa aga aga ttt tgt att aat cct aat
480 Asp Ala Phe Ala Tyr Val Gln Glu Arg Arg Phe Cys Ile Asn Pro Asn
145 150 155 160 gct gga ttt gtc cat caa ctt cag gaa tat gaa gcc atc
tac cta gca 528 Ala Gly Phe Val His Gln Leu Gln Glu Tyr Glu Ala Ile
Tyr Leu Ala 165 170 175 aaa tta aca ata cag atg atg tca cca ctc cag
ata gaa agg tca tta 576 Lys Leu Thr Ile Gln Met Met Ser Pro Leu Gln
Ile Glu Arg Ser Leu 180 185 190 tct gtt cat tct ggt acc aca ggc agt
ttg aag aga aca cat gaa gaa 624 Ser Val His Ser Gly Thr Thr Gly Ser
Leu Lys Arg Thr His Glu Glu 195 200 205 gag gat gat ttt gga acc atg
caa gtg gcg act gca cag aat ggc tga 672 Glu Asp Asp Phe Gly Thr Met
Gln Val Ala Thr Ala Gln Asn Gly * 210 215 220 31 173 PRT Artificial
Sequence Consensus Sequence 31 Gly Pro Ser Glu Ile Leu Pro His Leu
Tyr Leu Gly Ser Tyr Ser Thr 1 5 10 15 Ala Ser Glu Ala Asn Leu Ala
Leu Leu Lys Lys Leu Gly Ile Thr His 20 25 30 Val Ile Asn Val Thr
Glu Glu Val Pro Asn Pro Phe Glu Leu Asp Lys 35 40 45 Lys Asn Asp
Arg His Tyr Thr Asn Ala Tyr Ile Ser Lys Asn Ser Gly 50 55 60 Phe
Thr Tyr Leu Gln Ile Pro Asn Val Asp Asp His Ile Tyr Tyr His 65 70
75 80 Ile Ala Trp Asn His Glu Thr Lys Ile Ser Lys Tyr Phe Asp Glu
Ala 85 90 95 Val Asp Phe Ile Asp Asp Ala Arg Gln Lys Gly Gly Lys
Val Leu Val 100 105 110 His Cys Gln Ala Gly Ile Ser Arg Ser Ala Thr
Leu Ile Ile Ala Tyr 115 120 125 Leu Met Lys Thr Arg Asn Leu Ser Leu
Asn Glu Ala Tyr Asp Phe Val 130 135 140 Tyr Val Tyr His Ile Lys Glu
Arg Arg Cys Pro Ile Ile Ser Pro Asn 145 150 155 160 Phe Gly Phe Leu
Arg Gln Leu Ile Glu Tyr Glu Arg Lys 165 170 32 172 PRT Artificial
Sequence Consensus Sequence 32 Gly Pro Ser Glu Ile Leu Pro His Leu
Tyr Leu Gly Ser Tyr Ser Asp 1 5 10 15 Ala Ser Glu Ala Asn Leu Ala
Leu Leu Lys Lys Leu Gly Ile Thr His 20
25 30 Val Ile Asn Val Thr Glu Glu Val Pro Asn Asn Phe Glu Leu Lys
Lys 35 40 45 Lys Asn Asp Arg Tyr Tyr Thr Asn Glu Tyr Ile Ser Lys
Gly Ser Gly 50 55 60 Phe Thr Tyr Leu Gln Ile Pro Asn Val Asp Asp
Ile Tyr Tyr His Ile 65 70 75 80 Ala Trp Asn Thr Glu Thr Lys Ile Ser
Lys Tyr Leu Glu Glu Ala Val 85 90 95 Glu Phe Ile Glu Asp Ala Glu
Lys Lys Gly Gly Lys Val Leu Val His 100 105 110 Cys Gln Ala Gly Val
Ser Arg Ser Ala Thr Leu Val Ile Ala Tyr Leu 115 120 125 Met Lys Thr
Arg Asn Leu Ser Leu Arg Asp Ala Tyr Asp Phe Val Tyr 130 135 140 Val
Tyr His Ile Lys Glu Arg Arg Cys Pro Ile Ile Ser Pro Asn Phe 145 150
155 160 Gly Phe Leu Arg Gln Leu Ile Glu Tyr Glu Arg Lys 165 170 33
131 PRT Artificial Sequence Consensus Sequence 33 Thr Ala Gly Glu
Leu Lys Ala Leu Leu Glu Ser Ala Pro Lys Leu Ile 1 5 10 15 Leu Ile
Asp Val Arg Ser Pro Glu Phe Gly Glu Glu Tyr Glu Tyr Glu 20 25 30
Gly Gly His Ile Pro Gly Ala Val Asn Val Pro Glu Glu Glu Ile Glu 35
40 45 Ala Leu Leu Asp Arg Ser Gly Ile Leu Pro Asp Ile Glu Lys Leu
His 50 55 60 Leu Leu Lys Asp Pro Glu Glu Leu Ala Lys Leu Phe Gly
Glu Leu Gly 65 70 75 80 Ser Ser Lys Asp Lys Arg Val Ile Val Tyr Cys
Arg Ser Gly Arg Gly 85 90 95 Leu Leu Arg Asn Arg Arg Ser Ala Leu
Ala Ala Leu Leu Leu Lys Lys 100 105 110 Leu Gly Tyr Pro Glu Val Tyr
Ile Leu Lys Gly Gly Tyr Lys Glu Trp 115 120 125 Leu Ala Lys 130 34
94 PRT Artificial Sequence Consensus Sequence 34 Val Leu Glu Glu
Leu Lys Leu Leu Leu Asn Glu Asp Val Val Leu Leu 1 5 10 15 Asp Val
Arg Ser Pro Glu Glu Tyr Glu Gly Gly His Ile Pro Gly Ala 20 25 30
Val Asn Ile Pro Leu Ser Glu Leu Leu Asp Arg Leu Gly Leu Asp Lys 35
40 45 Asp Lys Pro Val Ile Val Tyr Cys Arg Ser Gly Val Arg Ser Ala
Ala 50 55 60 Lys Ala Ala Trp Leu Leu Arg Glu Leu Gly Phe Lys Asn
Val Tyr Leu 65 70 75 80 Leu Asp Gly Gly Tyr Lys Glu Trp Ser Ala Ala
Gly Pro Pro 85 90 35 1182 DNA Homo Sapiens CDS (326)...(1009) 35
tggaggtaga aaacttttat tagcksgtcc ggttgaggcc tacagcgggg aaaggacttg
60 ccagattttc gccgcaagtc agggccatag cggggggcat aacaaggcct
cccaaccgaa 120 ggtcaagcaa gagctccgag cgtcccacac aagtcccgaa
gggacactgt gacgccgcgc 180 tactgaaggc gcctgggttc ccggactcgg
ccaccgcctc gccgcttccg cccctcagaa 240 gcatggcggc cacgtagccc
ggcccggatt ggacgttggc ggtggacgcc aaacagttgg 300 caacacgatt
ggctgctgcg gggtg atg acg tca ggg ggc ggt gtc gga gtg 352 Met Thr
Ser Gly Gly Gly Val Gly Val 1 5 aat ggg ggc agc atg agg ccg ggc ggc
ttt ttg ggc gcc gga cag cgg 400 Asn Gly Gly Ser Met Arg Pro Gly Gly
Phe Leu Gly Ala Gly Gln Arg 10 15 20 25 ctg agt aga gcc atg agc cga
tgt gtt ttg gag cct cgc ccc ccg ggg 448 Leu Ser Arg Ala Met Ser Arg
Cys Val Leu Glu Pro Arg Pro Pro Gly 30 35 40 aag cgg tgg atg gtg
gct ggc ctg ggg aat ccc gga ctg ccc ggc acg 496 Lys Arg Trp Met Val
Ala Gly Leu Gly Asn Pro Gly Leu Pro Gly Thr 45 50 55 cga cac agc
gtg ggc atg gcg gtg ctg ggg cag ctg gcg cgg cgg ctg 544 Arg His Ser
Val Gly Met Ala Val Leu Gly Gln Leu Ala Arg Arg Leu 60 65 70 ggt
gtg gcg gag agt tgg acg cgc gac cgg cac tgt gcc gcc gac ctc 592 Gly
Val Ala Glu Ser Trp Thr Arg Asp Arg His Cys Ala Ala Asp Leu 75 80
85 gcc ctg gcc ccg ctg ggg gat gcc caa ctg gtc ctg ctc cgg cca cgg
640 Ala Leu Ala Pro Leu Gly Asp Ala Gln Leu Val Leu Leu Arg Pro Arg
90 95 100 105 cgg ctt atg aac gcc aac ggg cgc agc gtg gcc cgg gct
gcg gag ctg 688 Arg Leu Met Asn Ala Asn Gly Arg Ser Val Ala Arg Ala
Ala Glu Leu 110 115 120 ttt ggg ctg act gcc gag gaa gtc tac ctg gtg
cat gat gag ctg gac 736 Phe Gly Leu Thr Ala Glu Glu Val Tyr Leu Val
His Asp Glu Leu Asp 125 130 135 aag ccc ctg ggg aga ctg gct ctg aag
ctg ggg ggc agt gcc agg ggc 784 Lys Pro Leu Gly Arg Leu Ala Leu Lys
Leu Gly Gly Ser Ala Arg Gly 140 145 150 cac aat gga gtc cgt tcc tgc
att agc tgc ctc aac tcc aat gca atg 832 His Asn Gly Val Arg Ser Cys
Ile Ser Cys Leu Asn Ser Asn Ala Met 155 160 165 cca agg ctg cgg gtg
ggt atc ggg cgc ccg gcg cac cct gag gcg gtt 880 Pro Arg Leu Arg Val
Gly Ile Gly Arg Pro Ala His Pro Glu Ala Val 170 175 180 185 cag gcc
cat gtg ctg ggc tgc ttc tcc cct gct gag cag gag ctg ctg 928 Gln Ala
His Val Leu Gly Cys Phe Ser Pro Ala Glu Gln Glu Leu Leu 190 195 200
cct ctg ttg ctg gat cga gcc acc gac ctg atc ttg gac cac atc cgt 976
Pro Leu Leu Leu Asp Arg Ala Thr Asp Leu Ile Leu Asp His Ile Arg 205
210 215 gag cga agc cag ggg ccc tca ctg ggg ccg tga cactagtggc
catggctgcc 1029 Glu Arg Ser Gln Gly Pro Ser Leu Gly Pro * 220 225
tgcctgactg tagtgcccac caacccagcc actgccacag agctgccacg ccagccttgg
1089 tatctacttt ttatacaaat ctcctctaga ctgttccagg ctgcctgcgg
attaaagtgg 1149 gggtgactgt gaaaaaaaaa aaaaaaaaaa gga 1182 36 227
PRT Homo Sapiens 36 Met Thr Ser Gly Gly Gly Val Gly Val Asn Gly Gly
Ser Met Arg Pro 1 5 10 15 Gly Gly Phe Leu Gly Ala Gly Gln Arg Leu
Ser Arg Ala Met Ser Arg 20 25 30 Cys Val Leu Glu Pro Arg Pro Pro
Gly Lys Arg Trp Met Val Ala Gly 35 40 45 Leu Gly Asn Pro Gly Leu
Pro Gly Thr Arg His Ser Val Gly Met Ala 50 55 60 Val Leu Gly Gln
Leu Ala Arg Arg Leu Gly Val Ala Glu Ser Trp Thr 65 70 75 80 Arg Asp
Arg His Cys Ala Ala Asp Leu Ala Leu Ala Pro Leu Gly Asp 85 90 95
Ala Gln Leu Val Leu Leu Arg Pro Arg Arg Leu Met Asn Ala Asn Gly 100
105 110 Arg Ser Val Ala Arg Ala Ala Glu Leu Phe Gly Leu Thr Ala Glu
Glu 115 120 125 Val Tyr Leu Val His Asp Glu Leu Asp Lys Pro Leu Gly
Arg Leu Ala 130 135 140 Leu Lys Leu Gly Gly Ser Ala Arg Gly His Asn
Gly Val Arg Ser Cys 145 150 155 160 Ile Ser Cys Leu Asn Ser Asn Ala
Met Pro Arg Leu Arg Val Gly Ile 165 170 175 Gly Arg Pro Ala His Pro
Glu Ala Val Gln Ala His Val Leu Gly Cys 180 185 190 Phe Ser Pro Ala
Glu Gln Glu Leu Leu Pro Leu Leu Leu Asp Arg Ala 195 200 205 Thr Asp
Leu Ile Leu Asp His Ile Arg Glu Arg Ser Gln Gly Pro Ser 210 215 220
Leu Gly Pro 225 37 684 DNA Homo Sapiens CDS (1)...(684) 37 atg acg
tca ggg ggc ggt gtc gga gtg aat ggg ggc agc atg agg ccg 48 Met Thr
Ser Gly Gly Gly Val Gly Val Asn Gly Gly Ser Met Arg Pro 1 5 10 15
ggc ggc ttt ttg ggc gcc gga cag cgg ctg agt aga gcc atg agc cga 96
Gly Gly Phe Leu Gly Ala Gly Gln Arg Leu Ser Arg Ala Met Ser Arg 20
25 30 tgt gtt ttg gag cct cgc ccc ccg ggg aag cgg tgg atg gtg gct
ggc 144 Cys Val Leu Glu Pro Arg Pro Pro Gly Lys Arg Trp Met Val Ala
Gly 35 40 45 ctg ggg aat ccc gga ctg ccc ggc acg cga cac agc gtg
ggc atg gcg 192 Leu Gly Asn Pro Gly Leu Pro Gly Thr Arg His Ser Val
Gly Met Ala 50 55 60 gtg ctg ggg cag ctg gcg cgg cgg ctg ggt gtg
gcg gag agt tgg acg 240 Val Leu Gly Gln Leu Ala Arg Arg Leu Gly Val
Ala Glu Ser Trp Thr 65 70 75 80 cgc gac cgg cac tgt gcc gcc gac ctc
gcc ctg gcc ccg ctg ggg gat 288 Arg Asp Arg His Cys Ala Ala Asp Leu
Ala Leu Ala Pro Leu Gly Asp 85 90 95 gcc caa ctg gtc ctg ctc cgg
cca cgg cgg ctt atg aac gcc aac ggg 336 Ala Gln Leu Val Leu Leu Arg
Pro Arg Arg Leu Met Asn Ala Asn Gly 100 105 110 cgc agc gtg gcc cgg
gct gcg gag ctg ttt ggg ctg act gcc gag gaa 384 Arg Ser Val Ala Arg
Ala Ala Glu Leu Phe Gly Leu Thr Ala Glu Glu 115 120 125 gtc tac ctg
gtg cat gat gag ctg gac aag ccc ctg ggg aga ctg gct 432 Val Tyr Leu
Val His Asp Glu Leu Asp Lys Pro Leu Gly Arg Leu Ala 130 135 140 ctg
aag ctg ggg ggc agt gcc agg ggc cac aat gga gtc cgt tcc tgc 480 Leu
Lys Leu Gly Gly Ser Ala Arg Gly His Asn Gly Val Arg Ser Cys 145 150
155 160 att agc tgc ctc aac tcc aat gca atg cca agg ctg cgg gtg ggt
atc 528 Ile Ser Cys Leu Asn Ser Asn Ala Met Pro Arg Leu Arg Val Gly
Ile 165 170 175 ggg cgc ccg gcg cac cct gag gcg gtt cag gcc cat gtg
ctg ggc tgc 576 Gly Arg Pro Ala His Pro Glu Ala Val Gln Ala His Val
Leu Gly Cys 180 185 190 ttc tcc cct gct gag cag gag ctg ctg cct ctg
ttg ctg gat cga gcc 624 Phe Ser Pro Ala Glu Gln Glu Leu Leu Pro Leu
Leu Leu Asp Arg Ala 195 200 205 acc gac ctg atc ttg gac cac atc cgt
gag cga agc cag ggg ccc tca 672 Thr Asp Leu Ile Leu Asp His Ile Arg
Glu Arg Ser Gln Gly Pro Ser 210 215 220 ctg ggg ccg tga 684 Leu Gly
Pro * 225 38 193 PRT Artificial Sequence Consensus Sequence 38 Thr
Ile Lys Leu Ile Val Gly Leu Gly Asn Pro Gly Lys Gln Tyr Ala 1 5 10
15 Glu Thr Arg His Asn Ala Gly Phe Met Val Leu Asp Leu Leu Ala Ser
20 25 30 Arg Leu Gly Leu Ser Leu Arg Glu Glu Lys Arg Phe Phe Gly
Leu Gly 35 40 45 Gly Lys Val Leu Val Ser Gly Lys Lys His Cys Val
Ile Leu Leu Lys 50 55 60 Pro Arg Thr Tyr Met Asn Leu Ser Gly Lys
Ala Val Leu Ala Leu Ala 65 70 75 80 Ser Phe Tyr Lys Ile Lys Pro Glu
Glu Ile Leu Val Val His Asp Asp 85 90 95 Leu Asp Leu Pro Leu Gly
Lys Ile Arg Leu Lys Gln Gly Gly Gly Ala 100 105 110 Gly Arg Gly His
Asn Gly Leu Lys Ser Ile Ile Ser His Leu Gly Asn 115 120 125 Thr Asn
Asn Phe Asn Arg Leu Arg Ile Gly Ile Gly Arg Pro Asn Pro 130 135 140
Gly Ser Asn Asp Val Ala Glu Phe Val Leu Ser Lys Phe Ser Pro Ala 145
150 155 160 Glu Arg Pro Leu Leu Glu Lys Ala Leu Asp Lys Ala Ile Glu
Ala Leu 165 170 175 Glu Met Ile Ile Glu Gly His Gly Met Asn Lys Leu
Met Asn Arg Phe 180 185 190 Asn 39 2629 DNA Homo Sapiens CDS
(257)...(2545) 39 gcacgagaca cctctcccct tcttactgct tccctccggc
tataacttgc cagtcacagc 60 agccagctgc tgtagaagag gggaggaaac
aagccagtgc aaggggagca aaagagaaaa 120 ggagccaggc tgggcttcct
gatcccacag catcgcagag ctcgggaggc acagctcaca 180 gacacaggaa
acacaggact gctattctgc tctcctgccc acggtgatct ggtgccagct 240
ggtggaacag tgggtg atg gcg tcc ctg ctg caa gac cag ctg acc act gat
292 Met Ala Ser Leu Leu Gln Asp Gln Leu Thr Thr Asp 1 5 10 cag gac
ttg ctg ctg atg cag gaa ggc atg ccg atg cgc aag gtg agg 340 Gln Asp
Leu Leu Leu Met Gln Glu Gly Met Pro Met Arg Lys Val Arg 15 20 25
tcc aaa agc tgg aag aag cta aga tac ttc aga ctt cag aat gac ggc 388
Ser Lys Ser Trp Lys Lys Leu Arg Tyr Phe Arg Leu Gln Asn Asp Gly 30
35 40 atg aca gtc tgg cat gca cgg cag gcc agg ggc agt gcc aag ccc
agc 436 Met Thr Val Trp His Ala Arg Gln Ala Arg Gly Ser Ala Lys Pro
Ser 45 50 55 60 ttc tca atc tct gat gtg gag aca ata cgt aat ggc cat
gat tcc gag 484 Phe Ser Ile Ser Asp Val Glu Thr Ile Arg Asn Gly His
Asp Ser Glu 65 70 75 ttg ctg cgt agc ctg gca gag gag ctc ccc ctg
gag cag ggc ttc acc 532 Leu Leu Arg Ser Leu Ala Glu Glu Leu Pro Leu
Glu Gln Gly Phe Thr 80 85 90 att gtc ttc cat ggc cgc cgc tcc aac
ctg gac ctg atg gcc aac agt 580 Ile Val Phe His Gly Arg Arg Ser Asn
Leu Asp Leu Met Ala Asn Ser 95 100 105 gtt gag gag gcc cag ata tgg
atg cga ggg ctc cag ctg ttg gtg gat 628 Val Glu Glu Ala Gln Ile Trp
Met Arg Gly Leu Gln Leu Leu Val Asp 110 115 120 ctt gtc acc agc atg
gac cat cag gag cgc ctg gac caa tgg ctg agc 676 Leu Val Thr Ser Met
Asp His Gln Glu Arg Leu Asp Gln Trp Leu Ser 125 130 135 140 gat tgg
ttt caa cgt gga gac aaa aat cag gat ggt aag atg agt ttc 724 Asp Trp
Phe Gln Arg Gly Asp Lys Asn Gln Asp Gly Lys Met Ser Phe 145 150 155
caa gaa gtt cag cgg tta ttg cac cta atg aat gtg gaa atg gac caa 772
Gln Glu Val Gln Arg Leu Leu His Leu Met Asn Val Glu Met Asp Gln 160
165 170 gaa tat gcc ttc agt ctt ttt cag gca gca gac acg tcc cag tct
gga 820 Glu Tyr Ala Phe Ser Leu Phe Gln Ala Ala Asp Thr Ser Gln Ser
Gly 175 180 185 acc ctg gaa gga gaa gaa ttc gta cag ttc tat aag gca
ttg act aaa 868 Thr Leu Glu Gly Glu Glu Phe Val Gln Phe Tyr Lys Ala
Leu Thr Lys 190 195 200 cgt gct gag gtg cag gaa ctg ttt gaa agt ttt
tca gct gat ggg cag 916 Arg Ala Glu Val Gln Glu Leu Phe Glu Ser Phe
Ser Ala Asp Gly Gln 205 210 215 220 aag ctg act ctg ctg gaa ttt ttg
gat ttc ctc caa gag gag cag aag 964 Lys Leu Thr Leu Leu Glu Phe Leu
Asp Phe Leu Gln Glu Glu Gln Lys 225 230 235 gag aga gac tgc acc tct
gag ctt gct ctg gaa ctc att gac cgc tat 1012 Glu Arg Asp Cys Thr
Ser Glu Leu Ala Leu Glu Leu Ile Asp Arg Tyr 240 245 250 gaa cct tca
gac agt ggc aaa ctg cgg cat gtg ctg agt atg gat ggc 1060 Glu Pro
Ser Asp Ser Gly Lys Leu Arg His Val Leu Ser Met Asp Gly 255 260 265
ttc ctc agc tac ctc tgc tct aag gat gga gac atc ttc aac cca gcc
1108 Phe Leu Ser Tyr Leu Cys Ser Lys Asp Gly Asp Ile Phe Asn Pro
Ala 270 275 280 tgc ctc ccc atc tat cag gat atg act caa ccc ctg aac
cac tac ttc 1156 Cys Leu Pro Ile Tyr Gln Asp Met Thr Gln Pro Leu
Asn His Tyr Phe 285 290 295 300 atc tgc tct tct cat aac acc tac cta
gtg ggg gac cag ctt tgc ggc 1204 Ile Cys Ser Ser His Asn Thr Tyr
Leu Val Gly Asp Gln Leu Cys Gly 305 310 315 cag agc agc gtc gag gga
tat ata cgg gcc ctg aag cgg ggg tgc cgc 1252 Gln Ser Ser Val Glu
Gly Tyr Ile Arg Ala Leu Lys Arg Gly Cys Arg 320 325 330 tgc gtg gag
gtg gat gta tgg gat gga cct agc ggg gaa cct gtc gtt 1300 Cys Val
Glu Val Asp Val Trp Asp Gly Pro Ser Gly Glu Pro Val Val 335 340 345
tac cac gga cac acc ctg acc tcc cgc atc ctg ttc aaa gat gtc gtg
1348 Tyr His Gly His Thr Leu Thr Ser Arg Ile Leu Phe Lys Asp Val
Val 350 355 360 gcc aca gta gca cag tat gcc ttc cag aca tca gac tac
cca gtc atc 1396 Ala Thr Val Ala Gln Tyr Ala Phe Gln Thr Ser Asp
Tyr Pro Val Ile 365 370 375 380 ttg tcc ctg gag acc cac tgc agc tgg
gag cag cag cag acc atg gcc 1444 Leu Ser Leu Glu Thr His Cys Ser
Trp Glu Gln Gln Gln Thr Met Ala 385 390 395 cgt cat ctg act gag atc
ctg ggg gag cag ctg ctg agc acc acc ttg 1492 Arg His Leu Thr Glu
Ile Leu Gly Glu Gln Leu Leu Ser Thr Thr Leu 400 405 410 gat ggg gtg
ctg ccc act cag ctg ccc tcg cct gag gag ctt cgg agg 1540 Asp Gly
Val Leu Pro Thr Gln Leu Pro Ser Pro Glu Glu Leu Arg Arg 415 420 425
aag atc ctg gtg aag ggg aag aag tta aca ctt gag gaa gac ctg gaa
1588 Lys Ile Leu Val Lys Gly Lys Lys Leu Thr Leu Glu Glu Asp Leu
Glu 430 435 440 tat gag gaa gag gaa gca gaa cct gag ttg gaa gag tca
gaa ttg gcg 1636 Tyr Glu Glu Glu Glu Ala Glu Pro Glu Leu Glu Glu
Ser Glu Leu Ala 445 450 455 460 ctg gag tcc cag ttt gag act
gag cct gag ccc cag gag cag aac ctt 1684 Leu Glu Ser Gln Phe Glu
Thr Glu Pro Glu Pro Gln Glu Gln Asn Leu 465 470 475 cag aat aag gac
aaa aag aag aaa tcc aag ccc atc ttg tgt cca gcc 1732 Gln Asn Lys
Asp Lys Lys Lys Lys Ser Lys Pro Ile Leu Cys Pro Ala 480 485 490 ctc
tct tcc ctg gtt atc tac ttg aag tct gtc tca ttc cgc agc ttc 1780
Leu Ser Ser Leu Val Ile Tyr Leu Lys Ser Val Ser Phe Arg Ser Phe 495
500 505 aca cat tca aag gag cac tac cac ttc tac gag ata tca tct ttc
tct 1828 Thr His Ser Lys Glu His Tyr His Phe Tyr Glu Ile Ser Ser
Phe Ser 510 515 520 gaa acc aag gcc aag cgc ctc atc aag gag gct ggc
aat gag ttt gtg 1876 Glu Thr Lys Ala Lys Arg Leu Ile Lys Glu Ala
Gly Asn Glu Phe Val 525 530 535 540 cag cac aat act tgg cag tta agc
cgt gtg tat ccc agc ggc ctg agg 1924 Gln His Asn Thr Trp Gln Leu
Ser Arg Val Tyr Pro Ser Gly Leu Arg 545 550 555 aca gac tct tcc aac
tac aac ccc cag gaa ctc tgg aat gca ggc tgc 1972 Thr Asp Ser Ser
Asn Tyr Asn Pro Gln Glu Leu Trp Asn Ala Gly Cys 560 565 570 cag atg
gtg gcc atg aat atg cag act gca ggg ctt gaa atg gac atc 2020 Gln
Met Val Ala Met Asn Met Gln Thr Ala Gly Leu Glu Met Asp Ile 575 580
585 tgt gat ggg cat ttc cgc cag aat ggc ggc tgt ggc tat gtg ctg aag
2068 Cys Asp Gly His Phe Arg Gln Asn Gly Gly Cys Gly Tyr Val Leu
Lys 590 595 600 cca gac ttc ctg cgt gat atc cag agt tct ttc cac cct
gag aag ccc 2116 Pro Asp Phe Leu Arg Asp Ile Gln Ser Ser Phe His
Pro Glu Lys Pro 605 610 615 620 atc agc cct ttc aaa gcc cag act ctc
tta atc cag gtg atc agc ggt 2164 Ile Ser Pro Phe Lys Ala Gln Thr
Leu Leu Ile Gln Val Ile Ser Gly 625 630 635 cag caa ctc ccc aaa gtg
gac aag acc aaa gag ggg tcc att gtg gat 2212 Gln Gln Leu Pro Lys
Val Asp Lys Thr Lys Glu Gly Ser Ile Val Asp 640 645 650 cca ctg gtg
aaa gtg cag atc ttt ggc gtt cgt cta gac aca gca cgg 2260 Pro Leu
Val Lys Val Gln Ile Phe Gly Val Arg Leu Asp Thr Ala Arg 655 660 665
cag gag acc aac tat gtg gag aac aat ggt ttt aat cca tac tgg ggg
2308 Gln Glu Thr Asn Tyr Val Glu Asn Asn Gly Phe Asn Pro Tyr Trp
Gly 670 675 680 cag aca cta tgt ttc cgg gtg ctg gtg cct gaa ctt gcc
atg ctg cgt 2356 Gln Thr Leu Cys Phe Arg Val Leu Val Pro Glu Leu
Ala Met Leu Arg 685 690 695 700 ttt gtg gta atg gat tat gac tgg aaa
tcc cga aat gac ttt att ggt 2404 Phe Val Val Met Asp Tyr Asp Trp
Lys Ser Arg Asn Asp Phe Ile Gly 705 710 715 cag tac acc ctg cct tgg
acc tgc atg caa caa ggt tac cgc cac att 2452 Gln Tyr Thr Leu Pro
Trp Thr Cys Met Gln Gln Gly Tyr Arg His Ile 720 725 730 cac ctg ctg
tcc aaa gat ggc atc agc ctc cgc cca gct tcc atc ttt 2500 His Leu
Leu Ser Lys Asp Gly Ile Ser Leu Arg Pro Ala Ser Ile Phe 735 740 745
gtg tat atc tgc atc cag gaa ggc ctg gag ggg gat gag tcc tga 2545
Val Tyr Ile Cys Ile Gln Glu Gly Leu Glu Gly Asp Glu Ser * 750 755
760 ggtgggcatt tcacgggaag ggttggtgtg ctggctttag acggggagaa
acatctggaa 2605 ggatgctcga gggggggccc gggc 2629 40 762 PRT Homo
Sapiens 40 Met Ala Ser Leu Leu Gln Asp Gln Leu Thr Thr Asp Gln Asp
Leu Leu 1 5 10 15 Leu Met Gln Glu Gly Met Pro Met Arg Lys Val Arg
Ser Lys Ser Trp 20 25 30 Lys Lys Leu Arg Tyr Phe Arg Leu Gln Asn
Asp Gly Met Thr Val Trp 35 40 45 His Ala Arg Gln Ala Arg Gly Ser
Ala Lys Pro Ser Phe Ser Ile Ser 50 55 60 Asp Val Glu Thr Ile Arg
Asn Gly His Asp Ser Glu Leu Leu Arg Ser 65 70 75 80 Leu Ala Glu Glu
Leu Pro Leu Glu Gln Gly Phe Thr Ile Val Phe His 85 90 95 Gly Arg
Arg Ser Asn Leu Asp Leu Met Ala Asn Ser Val Glu Glu Ala 100 105 110
Gln Ile Trp Met Arg Gly Leu Gln Leu Leu Val Asp Leu Val Thr Ser 115
120 125 Met Asp His Gln Glu Arg Leu Asp Gln Trp Leu Ser Asp Trp Phe
Gln 130 135 140 Arg Gly Asp Lys Asn Gln Asp Gly Lys Met Ser Phe Gln
Glu Val Gln 145 150 155 160 Arg Leu Leu His Leu Met Asn Val Glu Met
Asp Gln Glu Tyr Ala Phe 165 170 175 Ser Leu Phe Gln Ala Ala Asp Thr
Ser Gln Ser Gly Thr Leu Glu Gly 180 185 190 Glu Glu Phe Val Gln Phe
Tyr Lys Ala Leu Thr Lys Arg Ala Glu Val 195 200 205 Gln Glu Leu Phe
Glu Ser Phe Ser Ala Asp Gly Gln Lys Leu Thr Leu 210 215 220 Leu Glu
Phe Leu Asp Phe Leu Gln Glu Glu Gln Lys Glu Arg Asp Cys 225 230 235
240 Thr Ser Glu Leu Ala Leu Glu Leu Ile Asp Arg Tyr Glu Pro Ser Asp
245 250 255 Ser Gly Lys Leu Arg His Val Leu Ser Met Asp Gly Phe Leu
Ser Tyr 260 265 270 Leu Cys Ser Lys Asp Gly Asp Ile Phe Asn Pro Ala
Cys Leu Pro Ile 275 280 285 Tyr Gln Asp Met Thr Gln Pro Leu Asn His
Tyr Phe Ile Cys Ser Ser 290 295 300 His Asn Thr Tyr Leu Val Gly Asp
Gln Leu Cys Gly Gln Ser Ser Val 305 310 315 320 Glu Gly Tyr Ile Arg
Ala Leu Lys Arg Gly Cys Arg Cys Val Glu Val 325 330 335 Asp Val Trp
Asp Gly Pro Ser Gly Glu Pro Val Val Tyr His Gly His 340 345 350 Thr
Leu Thr Ser Arg Ile Leu Phe Lys Asp Val Val Ala Thr Val Ala 355 360
365 Gln Tyr Ala Phe Gln Thr Ser Asp Tyr Pro Val Ile Leu Ser Leu Glu
370 375 380 Thr His Cys Ser Trp Glu Gln Gln Gln Thr Met Ala Arg His
Leu Thr 385 390 395 400 Glu Ile Leu Gly Glu Gln Leu Leu Ser Thr Thr
Leu Asp Gly Val Leu 405 410 415 Pro Thr Gln Leu Pro Ser Pro Glu Glu
Leu Arg Arg Lys Ile Leu Val 420 425 430 Lys Gly Lys Lys Leu Thr Leu
Glu Glu Asp Leu Glu Tyr Glu Glu Glu 435 440 445 Glu Ala Glu Pro Glu
Leu Glu Glu Ser Glu Leu Ala Leu Glu Ser Gln 450 455 460 Phe Glu Thr
Glu Pro Glu Pro Gln Glu Gln Asn Leu Gln Asn Lys Asp 465 470 475 480
Lys Lys Lys Lys Ser Lys Pro Ile Leu Cys Pro Ala Leu Ser Ser Leu 485
490 495 Val Ile Tyr Leu Lys Ser Val Ser Phe Arg Ser Phe Thr His Ser
Lys 500 505 510 Glu His Tyr His Phe Tyr Glu Ile Ser Ser Phe Ser Glu
Thr Lys Ala 515 520 525 Lys Arg Leu Ile Lys Glu Ala Gly Asn Glu Phe
Val Gln His Asn Thr 530 535 540 Trp Gln Leu Ser Arg Val Tyr Pro Ser
Gly Leu Arg Thr Asp Ser Ser 545 550 555 560 Asn Tyr Asn Pro Gln Glu
Leu Trp Asn Ala Gly Cys Gln Met Val Ala 565 570 575 Met Asn Met Gln
Thr Ala Gly Leu Glu Met Asp Ile Cys Asp Gly His 580 585 590 Phe Arg
Gln Asn Gly Gly Cys Gly Tyr Val Leu Lys Pro Asp Phe Leu 595 600 605
Arg Asp Ile Gln Ser Ser Phe His Pro Glu Lys Pro Ile Ser Pro Phe 610
615 620 Lys Ala Gln Thr Leu Leu Ile Gln Val Ile Ser Gly Gln Gln Leu
Pro 625 630 635 640 Lys Val Asp Lys Thr Lys Glu Gly Ser Ile Val Asp
Pro Leu Val Lys 645 650 655 Val Gln Ile Phe Gly Val Arg Leu Asp Thr
Ala Arg Gln Glu Thr Asn 660 665 670 Tyr Val Glu Asn Asn Gly Phe Asn
Pro Tyr Trp Gly Gln Thr Leu Cys 675 680 685 Phe Arg Val Leu Val Pro
Glu Leu Ala Met Leu Arg Phe Val Val Met 690 695 700 Asp Tyr Asp Trp
Lys Ser Arg Asn Asp Phe Ile Gly Gln Tyr Thr Leu 705 710 715 720 Pro
Trp Thr Cys Met Gln Gln Gly Tyr Arg His Ile His Leu Leu Ser 725 730
735 Lys Asp Gly Ile Ser Leu Arg Pro Ala Ser Ile Phe Val Tyr Ile Cys
740 745 750 Ile Gln Glu Gly Leu Glu Gly Asp Glu Ser 755 760 41 2289
DNA Homo Sapiens CDS (1)...(2289) 41 atg gcg tcc ctg ctg caa gac
cag ctg acc act gat cag gac ttg ctg 48 Met Ala Ser Leu Leu Gln Asp
Gln Leu Thr Thr Asp Gln Asp Leu Leu 1 5 10 15 ctg atg cag gaa ggc
atg ccg atg cgc aag gtg agg tcc aaa agc tgg 96 Leu Met Gln Glu Gly
Met Pro Met Arg Lys Val Arg Ser Lys Ser Trp 20 25 30 aag aag cta
aga tac ttc aga ctt cag aat gac ggc atg aca gtc tgg 144 Lys Lys Leu
Arg Tyr Phe Arg Leu Gln Asn Asp Gly Met Thr Val Trp 35 40 45 cat
gca cgg cag gcc agg ggc agt gcc aag ccc agc ttc tca atc tct 192 His
Ala Arg Gln Ala Arg Gly Ser Ala Lys Pro Ser Phe Ser Ile Ser 50 55
60 gat gtg gag aca ata cgt aat ggc cat gat tcc gag ttg ctg cgt agc
240 Asp Val Glu Thr Ile Arg Asn Gly His Asp Ser Glu Leu Leu Arg Ser
65 70 75 80 ctg gca gag gag ctc ccc ctg gag cag ggc ttc acc att gtc
ttc cat 288 Leu Ala Glu Glu Leu Pro Leu Glu Gln Gly Phe Thr Ile Val
Phe His 85 90 95 ggc cgc cgc tcc aac ctg gac ctg atg gcc aac agt
gtt gag gag gcc 336 Gly Arg Arg Ser Asn Leu Asp Leu Met Ala Asn Ser
Val Glu Glu Ala 100 105 110 cag ata tgg atg cga ggg ctc cag ctg ttg
gtg gat ctt gtc acc agc 384 Gln Ile Trp Met Arg Gly Leu Gln Leu Leu
Val Asp Leu Val Thr Ser 115 120 125 atg gac cat cag gag cgc ctg gac
caa tgg ctg agc gat tgg ttt caa 432 Met Asp His Gln Glu Arg Leu Asp
Gln Trp Leu Ser Asp Trp Phe Gln 130 135 140 cgt gga gac aaa aat cag
gat ggt aag atg agt ttc caa gaa gtt cag 480 Arg Gly Asp Lys Asn Gln
Asp Gly Lys Met Ser Phe Gln Glu Val Gln 145 150 155 160 cgg tta ttg
cac cta atg aat gtg gaa atg gac caa gaa tat gcc ttc 528 Arg Leu Leu
His Leu Met Asn Val Glu Met Asp Gln Glu Tyr Ala Phe 165 170 175 agt
ctt ttt cag gca gca gac acg tcc cag tct gga acc ctg gaa gga 576 Ser
Leu Phe Gln Ala Ala Asp Thr Ser Gln Ser Gly Thr Leu Glu Gly 180 185
190 gaa gaa ttc gta cag ttc tat aag gca ttg act aaa cgt gct gag gtg
624 Glu Glu Phe Val Gln Phe Tyr Lys Ala Leu Thr Lys Arg Ala Glu Val
195 200 205 cag gaa ctg ttt gaa agt ttt tca gct gat ggg cag aag ctg
act ctg 672 Gln Glu Leu Phe Glu Ser Phe Ser Ala Asp Gly Gln Lys Leu
Thr Leu 210 215 220 ctg gaa ttt ttg gat ttc ctc caa gag gag cag aag
gag aga gac tgc 720 Leu Glu Phe Leu Asp Phe Leu Gln Glu Glu Gln Lys
Glu Arg Asp Cys 225 230 235 240 acc tct gag ctt gct ctg gaa ctc att
gac cgc tat gaa cct tca gac 768 Thr Ser Glu Leu Ala Leu Glu Leu Ile
Asp Arg Tyr Glu Pro Ser Asp 245 250 255 agt ggc aaa ctg cgg cat gtg
ctg agt atg gat ggc ttc ctc agc tac 816 Ser Gly Lys Leu Arg His Val
Leu Ser Met Asp Gly Phe Leu Ser Tyr 260 265 270 ctc tgc tct aag gat
gga gac atc ttc aac cca gcc tgc ctc ccc atc 864 Leu Cys Ser Lys Asp
Gly Asp Ile Phe Asn Pro Ala Cys Leu Pro Ile 275 280 285 tat cag gat
atg act caa ccc ctg aac cac tac ttc atc tgc tct tct 912 Tyr Gln Asp
Met Thr Gln Pro Leu Asn His Tyr Phe Ile Cys Ser Ser 290 295 300 cat
aac acc tac cta gtg ggg gac cag ctt tgc ggc cag agc agc gtc 960 His
Asn Thr Tyr Leu Val Gly Asp Gln Leu Cys Gly Gln Ser Ser Val 305 310
315 320 gag gga tat ata cgg gcc ctg aag cgg ggg tgc cgc tgc gtg gag
gtg 1008 Glu Gly Tyr Ile Arg Ala Leu Lys Arg Gly Cys Arg Cys Val
Glu Val 325 330 335 gat gta tgg gat gga cct agc ggg gaa cct gtc gtt
tac cac gga cac 1056 Asp Val Trp Asp Gly Pro Ser Gly Glu Pro Val
Val Tyr His Gly His 340 345 350 acc ctg acc tcc cgc atc ctg ttc aaa
gat gtc gtg gcc aca gta gca 1104 Thr Leu Thr Ser Arg Ile Leu Phe
Lys Asp Val Val Ala Thr Val Ala 355 360 365 cag tat gcc ttc cag aca
tca gac tac cca gtc atc ttg tcc ctg gag 1152 Gln Tyr Ala Phe Gln
Thr Ser Asp Tyr Pro Val Ile Leu Ser Leu Glu 370 375 380 acc cac tgc
agc tgg gag cag cag cag acc atg gcc cgt cat ctg act 1200 Thr His
Cys Ser Trp Glu Gln Gln Gln Thr Met Ala Arg His Leu Thr 385 390 395
400 gag atc ctg ggg gag cag ctg ctg agc acc acc ttg gat ggg gtg ctg
1248 Glu Ile Leu Gly Glu Gln Leu Leu Ser Thr Thr Leu Asp Gly Val
Leu 405 410 415 ccc act cag ctg ccc tcg cct gag gag ctt cgg agg aag
atc ctg gtg 1296 Pro Thr Gln Leu Pro Ser Pro Glu Glu Leu Arg Arg
Lys Ile Leu Val 420 425 430 aag ggg aag aag tta aca ctt gag gaa gac
ctg gaa tat gag gaa gag 1344 Lys Gly Lys Lys Leu Thr Leu Glu Glu
Asp Leu Glu Tyr Glu Glu Glu 435 440 445 gaa gca gaa cct gag ttg gaa
gag tca gaa ttg gcg ctg gag tcc cag 1392 Glu Ala Glu Pro Glu Leu
Glu Glu Ser Glu Leu Ala Leu Glu Ser Gln 450 455 460 ttt gag act gag
cct gag ccc cag gag cag aac ctt cag aat aag gac 1440 Phe Glu Thr
Glu Pro Glu Pro Gln Glu Gln Asn Leu Gln Asn Lys Asp 465 470 475 480
aaa aag aag aaa tcc aag ccc atc ttg tgt cca gcc ctc tct tcc ctg
1488 Lys Lys Lys Lys Ser Lys Pro Ile Leu Cys Pro Ala Leu Ser Ser
Leu 485 490 495 gtt atc tac ttg aag tct gtc tca ttc cgc agc ttc aca
cat tca aag 1536 Val Ile Tyr Leu Lys Ser Val Ser Phe Arg Ser Phe
Thr His Ser Lys 500 505 510 gag cac tac cac ttc tac gag ata tca tct
ttc tct gaa acc aag gcc 1584 Glu His Tyr His Phe Tyr Glu Ile Ser
Ser Phe Ser Glu Thr Lys Ala 515 520 525 aag cgc ctc atc aag gag gct
ggc aat gag ttt gtg cag cac aat act 1632 Lys Arg Leu Ile Lys Glu
Ala Gly Asn Glu Phe Val Gln His Asn Thr 530 535 540 tgg cag tta agc
cgt gtg tat ccc agc ggc ctg agg aca gac tct tcc 1680 Trp Gln Leu
Ser Arg Val Tyr Pro Ser Gly Leu Arg Thr Asp Ser Ser 545 550 555 560
aac tac aac ccc cag gaa ctc tgg aat gca ggc tgc cag atg gtg gcc
1728 Asn Tyr Asn Pro Gln Glu Leu Trp Asn Ala Gly Cys Gln Met Val
Ala 565 570 575 atg aat atg cag act gca ggg ctt gaa atg gac atc tgt
gat ggg cat 1776 Met Asn Met Gln Thr Ala Gly Leu Glu Met Asp Ile
Cys Asp Gly His 580 585 590 ttc cgc cag aat ggc ggc tgt ggc tat gtg
ctg aag cca gac ttc ctg 1824 Phe Arg Gln Asn Gly Gly Cys Gly Tyr
Val Leu Lys Pro Asp Phe Leu 595 600 605 cgt gat atc cag agt tct ttc
cac cct gag aag ccc atc agc cct ttc 1872 Arg Asp Ile Gln Ser Ser
Phe His Pro Glu Lys Pro Ile Ser Pro Phe 610 615 620 aaa gcc cag act
ctc tta atc cag gtg atc agc ggt cag caa ctc ccc 1920 Lys Ala Gln
Thr Leu Leu Ile Gln Val Ile Ser Gly Gln Gln Leu Pro 625 630 635 640
aaa gtg gac aag acc aaa gag ggg tcc att gtg gat cca ctg gtg aaa
1968 Lys Val Asp Lys Thr Lys Glu Gly Ser Ile Val Asp Pro Leu Val
Lys 645 650 655 gtg cag atc ttt ggc gtt cgt cta gac aca gca cgg cag
gag acc aac 2016 Val Gln Ile Phe Gly Val Arg Leu Asp Thr Ala Arg
Gln Glu Thr Asn 660 665 670 tat gtg gag aac aat ggt ttt aat cca tac
tgg ggg cag aca cta tgt 2064 Tyr Val Glu Asn Asn Gly Phe Asn Pro
Tyr Trp Gly Gln Thr Leu Cys 675 680 685 ttc cgg gtg ctg gtg cct gaa
ctt gcc atg ctg cgt ttt gtg gta atg 2112 Phe Arg Val Leu Val Pro
Glu Leu Ala Met Leu Arg Phe Val Val Met 690 695 700 gat tat gac tgg
aaa tcc cga aat gac ttt att ggt cag tac acc ctg 2160 Asp Tyr Asp
Trp Lys Ser Arg Asn Asp Phe Ile Gly Gln Tyr Thr Leu 705 710 715 720
cct tgg acc tgc atg caa caa ggt tac cgc cac att cac ctg ctg tcc
2208 Pro Trp Thr Cys Met Gln Gln Gly Tyr Arg
His Ile His Leu Leu Ser 725 730 735 aaa gat ggc atc agc ctc cgc cca
gct tcc atc ttt gtg tat atc tgc 2256 Lys Asp Gly Ile Ser Leu Arg
Pro Ala Ser Ile Phe Val Tyr Ile Cys 740 745 750 atc cag gaa ggc ctg
gag ggg gat gag tcc tga 2289 Ile Gln Glu Gly Leu Glu Gly Asp Glu
Ser * 755 760 42 2171 DNA Homo Sapiens CDS (232)...(2058) 42
ccacgcgtcc gcccacgcgt ccgcagccaa ccagctatac ctcttttgaa gattttaaga
60 acttagcctc ctgaacagtc ttcttcgaaa gtgaaaagtg gtaacagctg
atgagtatca 120 agaaattatt ttccgcaaag gggcagagtt aattgtattt
ggaacccatg acagcaccta 180 ctggggaaag acttctaagt gaggagaaac
ggctctacag gtcatgaaac t atg gaa 237 Met Glu 1 atg aga tgg ttt ttg
tca aag att cag gat gac ttc aga ggt gga aaa 285 Met Arg Trp Phe Leu
Ser Lys Ile Gln Asp Asp Phe Arg Gly Gly Lys 5 10 15 att aac cta gaa
aaa act cag agg tta ctt gaa aaa tta gat att cgg 333 Ile Asn Leu Glu
Lys Thr Gln Arg Leu Leu Glu Lys Leu Asp Ile Arg 20 25 30 tgc agt
tat att cat gtg aaa cag att ttt aag gac aat gac agg ctg 381 Cys Ser
Tyr Ile His Val Lys Gln Ile Phe Lys Asp Asn Asp Arg Leu 35 40 45 50
aaa caa gga aga atc acc ata gaa gaa ttt aga gca att tat cga att 429
Lys Gln Gly Arg Ile Thr Ile Glu Glu Phe Arg Ala Ile Tyr Arg Ile 55
60 65 atc acg cac aga gaa gaa att att gag att ttc aac aca tat tct
gaa 477 Ile Thr His Arg Glu Glu Ile Ile Glu Ile Phe Asn Thr Tyr Ser
Glu 70 75 80 aac cgg aaa att ctt tta gca agt aat ctg gct caa ttt
ctg aca caa 525 Asn Arg Lys Ile Leu Leu Ala Ser Asn Leu Ala Gln Phe
Leu Thr Gln 85 90 95 gaa caa tat gca gct gag atg agt aaa gct att
gct ttt gag atc att 573 Glu Gln Tyr Ala Ala Glu Met Ser Lys Ala Ile
Ala Phe Glu Ile Ile 100 105 110 cag aaa tac gag cct atc gaa gaa gtt
agg aaa gca cac caa atg tca 621 Gln Lys Tyr Glu Pro Ile Glu Glu Val
Arg Lys Ala His Gln Met Ser 115 120 125 130 tta gaa ggt ttt aca aga
tac atg gat tca cgt gaa tgt cta ctg ttt 669 Leu Glu Gly Phe Thr Arg
Tyr Met Asp Ser Arg Glu Cys Leu Leu Phe 135 140 145 aaa aat gaa tgt
aga aaa gtt tat caa gat atg act cat cca tta aat 717 Lys Asn Glu Cys
Arg Lys Val Tyr Gln Asp Met Thr His Pro Leu Asn 150 155 160 gat tat
ttt att tca tct tca cat aac aca tat ttg gta tct gat caa 765 Asp Tyr
Phe Ile Ser Ser Ser His Asn Thr Tyr Leu Val Ser Asp Gln 165 170 175
tta ttg gga cca agt gac ctt tgg gga tat gca agt gcc ctt gtg aaa 813
Leu Leu Gly Pro Ser Asp Leu Trp Gly Tyr Ala Ser Ala Leu Val Lys 180
185 190 gga tgc cgt tgt ttg gag att gac tgc tgg gat gga gca caa aat
gaa 861 Gly Cys Arg Cys Leu Glu Ile Asp Cys Trp Asp Gly Ala Gln Asn
Glu 195 200 205 210 cct gtt gta tat cat ggc tac aca ctc aca agc aaa
ctt ctg ttt aaa 909 Pro Val Val Tyr His Gly Tyr Thr Leu Thr Ser Lys
Leu Leu Phe Lys 215 220 225 act gtt atc caa gct ata cac aag tat gca
ttc atg aca tct gac tac 957 Thr Val Ile Gln Ala Ile His Lys Tyr Ala
Phe Met Thr Ser Asp Tyr 230 235 240 cca gtg gtg ctc tct tta gaa aat
cac tgc tcc act gcc caa caa gaa 1005 Pro Val Val Leu Ser Leu Glu
Asn His Cys Ser Thr Ala Gln Gln Glu 245 250 255 gta atg gca gac aat
ttg cag gct act ttt gga gag tcc ttg ctt tct 1053 Val Met Ala Asp
Asn Leu Gln Ala Thr Phe Gly Glu Ser Leu Leu Ser 260 265 270 gat atg
ctt gat gat ttt cct gat act cta cca tca cca gag gca cta 1101 Asp
Met Leu Asp Asp Phe Pro Asp Thr Leu Pro Ser Pro Glu Ala Leu 275 280
285 290 aaa ttc aaa ata tta gtt aaa aat aag aaa ata gga acc tta aag
gaa 1149 Lys Phe Lys Ile Leu Val Lys Asn Lys Lys Ile Gly Thr Leu
Lys Glu 295 300 305 acc cat gaa aga aaa ggt tct gat aag cat gga gac
aat caa gac aag 1197 Thr His Glu Arg Lys Gly Ser Asp Lys His Gly
Asp Asn Gln Asp Lys 310 315 320 gaa aca ggg gta aaa aag tta cct gga
gta atg ctt ttc aag aaa aag 1245 Glu Thr Gly Val Lys Lys Leu Pro
Gly Val Met Leu Phe Lys Lys Lys 325 330 335 aag acc agg aag cta aaa
att gct ctg gcc tta tct gat ctt gtc att 1293 Lys Thr Arg Lys Leu
Lys Ile Ala Leu Ala Leu Ser Asp Leu Val Ile 340 345 350 tat acg aaa
gct gag aaa ttc aaa agc ttt caa cat tca aga tta tat 1341 Tyr Thr
Lys Ala Glu Lys Phe Lys Ser Phe Gln His Ser Arg Leu Tyr 355 360 365
370 cag caa ttt aat gaa aat aat tct att ggg gag aca caa gcc cga aaa
1389 Gln Gln Phe Asn Glu Asn Asn Ser Ile Gly Glu Thr Gln Ala Arg
Lys 375 380 385 ctt tca aaa ttg cga gtc cat gag ttt att ttt cac acc
agg aag ttc 1437 Leu Ser Lys Leu Arg Val His Glu Phe Ile Phe His
Thr Arg Lys Phe 390 395 400 att acc aga ata tat ccc aaa gca aca aga
gca gac tct tct aat ttt 1485 Ile Thr Arg Ile Tyr Pro Lys Ala Thr
Arg Ala Asp Ser Ser Asn Phe 405 410 415 aat ccc caa gaa ttt tgg aat
ata ggt tgt caa atg gtg gct tta aat 1533 Asn Pro Gln Glu Phe Trp
Asn Ile Gly Cys Gln Met Val Ala Leu Asn 420 425 430 ttc cag acc cct
ggt ctg ccc atg gat ctg caa aat ggg aaa ttt ttg 1581 Phe Gln Thr
Pro Gly Leu Pro Met Asp Leu Gln Asn Gly Lys Phe Leu 435 440 445 450
gat aat ggt ggt tct gga tat att ttg aaa cca cat ttc tta aga gag
1629 Asp Asn Gly Gly Ser Gly Tyr Ile Leu Lys Pro His Phe Leu Arg
Glu 455 460 465 agt aaa tca tac ttt aac cca agt aac ata aaa gag ggt
atg cca att 1677 Ser Lys Ser Tyr Phe Asn Pro Ser Asn Ile Lys Glu
Gly Met Pro Ile 470 475 480 aca ctt aca ata agg ctc atc agt ggt atc
cag ttg cct ctt act cat 1725 Thr Leu Thr Ile Arg Leu Ile Ser Gly
Ile Gln Leu Pro Leu Thr His 485 490 495 tca tca tct aac aaa ggt gat
tca tta gta att ata gaa gtt ttt ggt 1773 Ser Ser Ser Asn Lys Gly
Asp Ser Leu Val Ile Ile Glu Val Phe Gly 500 505 510 gtt cca aat gat
caa atg aag cag cag act cgt gta att aaa aaa aat 1821 Val Pro Asn
Asp Gln Met Lys Gln Gln Thr Arg Val Ile Lys Lys Asn 515 520 525 530
gct ttt agt cca aga tgg aat gaa aca ttc aca ttt att att cat gtc
1869 Ala Phe Ser Pro Arg Trp Asn Glu Thr Phe Thr Phe Ile Ile His
Val 535 540 545 cca gaa ttg gca ttg ata cgt ttt gtt gtt gaa ggt caa
ggt tta ata 1917 Pro Glu Leu Ala Leu Ile Arg Phe Val Val Glu Gly
Gln Gly Leu Ile 550 555 560 gca gga aat gaa ttt ctt ggg caa tat act
ttg cca ctt cta tgc atg 1965 Ala Gly Asn Glu Phe Leu Gly Gln Tyr
Thr Leu Pro Leu Leu Cys Met 565 570 575 aac aaa ggt tat cgt cgt att
cct ctg ttt tcc aga atg ggt gag agc 2013 Asn Lys Gly Tyr Arg Arg
Ile Pro Leu Phe Ser Arg Met Gly Glu Ser 580 585 590 ctt gag cct gct
tca ctg ttt gtt tat gtt tgg tac gtc aga taa 2058 Leu Glu Pro Ala
Ser Leu Phe Val Tyr Val Trp Tyr Val Arg * 595 600 605 cagctaatga
taaatgacat atcattagct atgcatcgca ataaaacagc caaaatgaaa 2118
aaaaaaaaaa aaaaaaaaaa attggcggcc gcaagcttat tccctttagt aag 2171 43
608 PRT Homo Sapiens 43 Met Glu Met Arg Trp Phe Leu Ser Lys Ile Gln
Asp Asp Phe Arg Gly 1 5 10 15 Gly Lys Ile Asn Leu Glu Lys Thr Gln
Arg Leu Leu Glu Lys Leu Asp 20 25 30 Ile Arg Cys Ser Tyr Ile His
Val Lys Gln Ile Phe Lys Asp Asn Asp 35 40 45 Arg Leu Lys Gln Gly
Arg Ile Thr Ile Glu Glu Phe Arg Ala Ile Tyr 50 55 60 Arg Ile Ile
Thr His Arg Glu Glu Ile Ile Glu Ile Phe Asn Thr Tyr 65 70 75 80 Ser
Glu Asn Arg Lys Ile Leu Leu Ala Ser Asn Leu Ala Gln Phe Leu 85 90
95 Thr Gln Glu Gln Tyr Ala Ala Glu Met Ser Lys Ala Ile Ala Phe Glu
100 105 110 Ile Ile Gln Lys Tyr Glu Pro Ile Glu Glu Val Arg Lys Ala
His Gln 115 120 125 Met Ser Leu Glu Gly Phe Thr Arg Tyr Met Asp Ser
Arg Glu Cys Leu 130 135 140 Leu Phe Lys Asn Glu Cys Arg Lys Val Tyr
Gln Asp Met Thr His Pro 145 150 155 160 Leu Asn Asp Tyr Phe Ile Ser
Ser Ser His Asn Thr Tyr Leu Val Ser 165 170 175 Asp Gln Leu Leu Gly
Pro Ser Asp Leu Trp Gly Tyr Ala Ser Ala Leu 180 185 190 Val Lys Gly
Cys Arg Cys Leu Glu Ile Asp Cys Trp Asp Gly Ala Gln 195 200 205 Asn
Glu Pro Val Val Tyr His Gly Tyr Thr Leu Thr Ser Lys Leu Leu 210 215
220 Phe Lys Thr Val Ile Gln Ala Ile His Lys Tyr Ala Phe Met Thr Ser
225 230 235 240 Asp Tyr Pro Val Val Leu Ser Leu Glu Asn His Cys Ser
Thr Ala Gln 245 250 255 Gln Glu Val Met Ala Asp Asn Leu Gln Ala Thr
Phe Gly Glu Ser Leu 260 265 270 Leu Ser Asp Met Leu Asp Asp Phe Pro
Asp Thr Leu Pro Ser Pro Glu 275 280 285 Ala Leu Lys Phe Lys Ile Leu
Val Lys Asn Lys Lys Ile Gly Thr Leu 290 295 300 Lys Glu Thr His Glu
Arg Lys Gly Ser Asp Lys His Gly Asp Asn Gln 305 310 315 320 Asp Lys
Glu Thr Gly Val Lys Lys Leu Pro Gly Val Met Leu Phe Lys 325 330 335
Lys Lys Lys Thr Arg Lys Leu Lys Ile Ala Leu Ala Leu Ser Asp Leu 340
345 350 Val Ile Tyr Thr Lys Ala Glu Lys Phe Lys Ser Phe Gln His Ser
Arg 355 360 365 Leu Tyr Gln Gln Phe Asn Glu Asn Asn Ser Ile Gly Glu
Thr Gln Ala 370 375 380 Arg Lys Leu Ser Lys Leu Arg Val His Glu Phe
Ile Phe His Thr Arg 385 390 395 400 Lys Phe Ile Thr Arg Ile Tyr Pro
Lys Ala Thr Arg Ala Asp Ser Ser 405 410 415 Asn Phe Asn Pro Gln Glu
Phe Trp Asn Ile Gly Cys Gln Met Val Ala 420 425 430 Leu Asn Phe Gln
Thr Pro Gly Leu Pro Met Asp Leu Gln Asn Gly Lys 435 440 445 Phe Leu
Asp Asn Gly Gly Ser Gly Tyr Ile Leu Lys Pro His Phe Leu 450 455 460
Arg Glu Ser Lys Ser Tyr Phe Asn Pro Ser Asn Ile Lys Glu Gly Met 465
470 475 480 Pro Ile Thr Leu Thr Ile Arg Leu Ile Ser Gly Ile Gln Leu
Pro Leu 485 490 495 Thr His Ser Ser Ser Asn Lys Gly Asp Ser Leu Val
Ile Ile Glu Val 500 505 510 Phe Gly Val Pro Asn Asp Gln Met Lys Gln
Gln Thr Arg Val Ile Lys 515 520 525 Lys Asn Ala Phe Ser Pro Arg Trp
Asn Glu Thr Phe Thr Phe Ile Ile 530 535 540 His Val Pro Glu Leu Ala
Leu Ile Arg Phe Val Val Glu Gly Gln Gly 545 550 555 560 Leu Ile Ala
Gly Asn Glu Phe Leu Gly Gln Tyr Thr Leu Pro Leu Leu 565 570 575 Cys
Met Asn Lys Gly Tyr Arg Arg Ile Pro Leu Phe Ser Arg Met Gly 580 585
590 Glu Ser Leu Glu Pro Ala Ser Leu Phe Val Tyr Val Trp Tyr Val Arg
595 600 605 44 1827 DNA Homo Sapiens CDS (1)...(1827) 44 atg gaa
atg aga tgg ttt ttg tca aag att cag gat gac ttc aga ggt 48 Met Glu
Met Arg Trp Phe Leu Ser Lys Ile Gln Asp Asp Phe Arg Gly 1 5 10 15
gga aaa att aac cta gaa aaa act cag agg tta ctt gaa aaa tta gat 96
Gly Lys Ile Asn Leu Glu Lys Thr Gln Arg Leu Leu Glu Lys Leu Asp 20
25 30 att cgg tgc agt tat att cat gtg aaa cag att ttt aag gac aat
gac 144 Ile Arg Cys Ser Tyr Ile His Val Lys Gln Ile Phe Lys Asp Asn
Asp 35 40 45 agg ctg aaa caa gga aga atc acc ata gaa gaa ttt aga
gca att tat 192 Arg Leu Lys Gln Gly Arg Ile Thr Ile Glu Glu Phe Arg
Ala Ile Tyr 50 55 60 cga att atc acg cac aga gaa gaa att att gag
att ttc aac aca tat 240 Arg Ile Ile Thr His Arg Glu Glu Ile Ile Glu
Ile Phe Asn Thr Tyr 65 70 75 80 tct gaa aac cgg aaa att ctt tta gca
agt aat ctg gct caa ttt ctg 288 Ser Glu Asn Arg Lys Ile Leu Leu Ala
Ser Asn Leu Ala Gln Phe Leu 85 90 95 aca caa gaa caa tat gca gct
gag atg agt aaa gct att gct ttt gag 336 Thr Gln Glu Gln Tyr Ala Ala
Glu Met Ser Lys Ala Ile Ala Phe Glu 100 105 110 atc att cag aaa tac
gag cct atc gaa gaa gtt agg aaa gca cac caa 384 Ile Ile Gln Lys Tyr
Glu Pro Ile Glu Glu Val Arg Lys Ala His Gln 115 120 125 atg tca tta
gaa ggt ttt aca aga tac atg gat tca cgt gaa tgt cta 432 Met Ser Leu
Glu Gly Phe Thr Arg Tyr Met Asp Ser Arg Glu Cys Leu 130 135 140 ctg
ttt aaa aat gaa tgt aga aaa gtt tat caa gat atg act cat cca 480 Leu
Phe Lys Asn Glu Cys Arg Lys Val Tyr Gln Asp Met Thr His Pro 145 150
155 160 tta aat gat tat ttt att tca tct tca cat aac aca tat ttg gta
tct 528 Leu Asn Asp Tyr Phe Ile Ser Ser Ser His Asn Thr Tyr Leu Val
Ser 165 170 175 gat caa tta ttg gga cca agt gac ctt tgg gga tat gca
agt gcc ctt 576 Asp Gln Leu Leu Gly Pro Ser Asp Leu Trp Gly Tyr Ala
Ser Ala Leu 180 185 190 gtg aaa gga tgc cgt tgt ttg gag att gac tgc
tgg gat gga gca caa 624 Val Lys Gly Cys Arg Cys Leu Glu Ile Asp Cys
Trp Asp Gly Ala Gln 195 200 205 aat gaa cct gtt gta tat cat ggc tac
aca ctc aca agc aaa ctt ctg 672 Asn Glu Pro Val Val Tyr His Gly Tyr
Thr Leu Thr Ser Lys Leu Leu 210 215 220 ttt aaa act gtt atc caa gct
ata cac aag tat gca ttc atg aca tct 720 Phe Lys Thr Val Ile Gln Ala
Ile His Lys Tyr Ala Phe Met Thr Ser 225 230 235 240 gac tac cca gtg
gtg ctc tct tta gaa aat cac tgc tcc act gcc caa 768 Asp Tyr Pro Val
Val Leu Ser Leu Glu Asn His Cys Ser Thr Ala Gln 245 250 255 caa gaa
gta atg gca gac aat ttg cag gct act ttt gga gag tcc ttg 816 Gln Glu
Val Met Ala Asp Asn Leu Gln Ala Thr Phe Gly Glu Ser Leu 260 265 270
ctt tct gat atg ctt gat gat ttt cct gat act cta cca tca cca gag 864
Leu Ser Asp Met Leu Asp Asp Phe Pro Asp Thr Leu Pro Ser Pro Glu 275
280 285 gca cta aaa ttc aaa ata tta gtt aaa aat aag aaa ata gga acc
tta 912 Ala Leu Lys Phe Lys Ile Leu Val Lys Asn Lys Lys Ile Gly Thr
Leu 290 295 300 aag gaa acc cat gaa aga aaa ggt tct gat aag cat gga
gac aat caa 960 Lys Glu Thr His Glu Arg Lys Gly Ser Asp Lys His Gly
Asp Asn Gln 305 310 315 320 gac aag gaa aca ggg gta aaa aag tta cct
gga gta atg ctt ttc aag 1008 Asp Lys Glu Thr Gly Val Lys Lys Leu
Pro Gly Val Met Leu Phe Lys 325 330 335 aaa aag aag acc agg aag cta
aaa att gct ctg gcc tta tct gat ctt 1056 Lys Lys Lys Thr Arg Lys
Leu Lys Ile Ala Leu Ala Leu Ser Asp Leu 340 345 350 gtc att tat acg
aaa gct gag aaa ttc aaa agc ttt caa cat tca aga 1104 Val Ile Tyr
Thr Lys Ala Glu Lys Phe Lys Ser Phe Gln His Ser Arg 355 360 365 tta
tat cag caa ttt aat gaa aat aat tct att ggg gag aca caa gcc 1152
Leu Tyr Gln Gln Phe Asn Glu Asn Asn Ser Ile Gly Glu Thr Gln Ala 370
375 380 cga aaa ctt tca aaa ttg cga gtc cat gag ttt att ttt cac acc
agg 1200 Arg Lys Leu Ser Lys Leu Arg Val His Glu Phe Ile Phe His
Thr Arg 385 390 395 400 aag ttc att acc aga ata tat ccc aaa gca aca
aga gca gac tct tct 1248 Lys Phe Ile Thr Arg Ile Tyr Pro Lys Ala
Thr Arg Ala Asp Ser Ser 405 410 415 aat ttt aat ccc caa gaa ttt tgg
aat ata ggt tgt caa atg gtg gct 1296 Asn Phe Asn Pro Gln Glu Phe
Trp Asn Ile Gly Cys Gln Met Val Ala 420 425 430 tta aat ttc cag acc
cct ggt ctg ccc atg gat ctg caa aat ggg aaa 1344 Leu Asn Phe Gln
Thr Pro Gly Leu Pro Met Asp Leu Gln Asn Gly Lys
435 440 445 ttt ttg gat aat ggt ggt tct gga tat att ttg aaa cca cat
ttc tta 1392 Phe Leu Asp Asn Gly Gly Ser Gly Tyr Ile Leu Lys Pro
His Phe Leu 450 455 460 aga gag agt aaa tca tac ttt aac cca agt aac
ata aaa gag ggt atg 1440 Arg Glu Ser Lys Ser Tyr Phe Asn Pro Ser
Asn Ile Lys Glu Gly Met 465 470 475 480 cca att aca ctt aca ata agg
ctc atc agt ggt atc cag ttg cct ctt 1488 Pro Ile Thr Leu Thr Ile
Arg Leu Ile Ser Gly Ile Gln Leu Pro Leu 485 490 495 act cat tca tca
tct aac aaa ggt gat tca tta gta att ata gaa gtt 1536 Thr His Ser
Ser Ser Asn Lys Gly Asp Ser Leu Val Ile Ile Glu Val 500 505 510 ttt
ggt gtt cca aat gat caa atg aag cag cag act cgt gta att aaa 1584
Phe Gly Val Pro Asn Asp Gln Met Lys Gln Gln Thr Arg Val Ile Lys 515
520 525 aaa aat gct ttt agt cca aga tgg aat gaa aca ttc aca ttt att
att 1632 Lys Asn Ala Phe Ser Pro Arg Trp Asn Glu Thr Phe Thr Phe
Ile Ile 530 535 540 cat gtc cca gaa ttg gca ttg ata cgt ttt gtt gtt
gaa ggt caa ggt 1680 His Val Pro Glu Leu Ala Leu Ile Arg Phe Val
Val Glu Gly Gln Gly 545 550 555 560 tta ata gca gga aat gaa ttt ctt
ggg caa tat act ttg cca ctt cta 1728 Leu Ile Ala Gly Asn Glu Phe
Leu Gly Gln Tyr Thr Leu Pro Leu Leu 565 570 575 tgc atg aac aaa ggt
tat cgt cgt att cct ctg ttt tcc aga atg ggt 1776 Cys Met Asn Lys
Gly Tyr Arg Arg Ile Pro Leu Phe Ser Arg Met Gly 580 585 590 gag agc
ctt gag cct gct tca ctg ttt gtt tat gtt tgg tac gtc aga 1824 Glu
Ser Leu Glu Pro Ala Ser Leu Phe Val Tyr Val Trp Tyr Val Arg 595 600
605 taa 1827 * 45 85 PRT Artificial Sequence Consensus Amino Acid
Sequence 45 Val Ile Lys Glu Gly Trp Leu Leu Lys Lys Ser Lys Ser Trp
Lys Lys 1 5 10 15 Arg Tyr Phe Val Leu Phe Asn Asn Val Leu Leu Tyr
Tyr Lys Asp Ser 20 25 30 Lys Lys Lys Pro Lys Gly Ser Ile Pro Leu
Ser Gly Cys Gln Val Glu 35 40 45 Lys Pro Asp Lys Asn Cys Phe Glu
Ile Arg Thr Asp Arg Thr Leu Leu 50 55 60 Leu Gln Ala Glu Ser Glu
Glu Glu Arg Lys Glu Trp Val Lys Ala Ile 65 70 75 80 Gln Ser Ala Ile
Arg 85 46 29 PRT Artificial Sequence Consensus Amino Acid Sequence
46 Glu Leu Lys Glu Ala Phe Lys Glu Phe Asp Lys Asp Gly Asp Gly Lys
1 5 10 15 Ile Ser Phe Glu Glu Phe Lys Ala Ala Leu Lys Lys Leu 20 25
47 29 PRT Artificial Sequence Consensus Amino Acid Sequence 47 Glu
Leu Lys Glu Ala Phe Lys Glu Phe Asp Lys Asp Gly Asp Gly Lys 1 5 10
15 Ile Ser Phe Glu Glu Phe Lys Ala Ala Leu Lys Lys Leu 20 25 48 153
PRT Artificial Sequence Consensus Amino Acid Sequence 48 Asp Met
Ser Ile Pro Leu Ser His Tyr Phe Ile Ser Ser Ser His Asn 1 5 10 15
Thr Tyr Leu Thr Gly Lys Gln Leu Trp Gly Lys Ser Ser Val Glu Ser 20
25 30 Tyr Arg Gln Gln Leu Asp Ala Gly Cys Arg Cys Val Glu Leu Asp
Cys 35 40 45 Trp Asp Gly Lys Pro Asp Asp Glu Pro Ile Ile Tyr His
Gly His Thr 50 55 60 Leu Thr Leu Glu Ile Lys Leu Lys Asp Val Leu
Glu Ala Ile Lys Asp 65 70 75 80 Phe Ala Phe Lys Pro Thr Ser Pro Tyr
Pro Val Ile Leu Ser Leu Glu 85 90 95 Asn His Cys Asn Ser Asp Asp
Gln Gln Arg Lys Met Ala Lys Tyr Phe 100 105 110 Lys Glu Ile Phe Gly
Asp Met Leu Leu Thr Lys Pro Thr Leu Asp Ser 115 120 125 Leu Thr Thr
Glu Pro Gly Leu Pro Leu Pro Ser Leu Lys Asp Leu Arg 130 135 140 Gly
Lys Ile Leu Leu Lys Asn Lys Lys 145 150 49 128 PRT Artificial
Sequence Consensus Amino Acid Sequence 49 Glu Leu Ser Asn Leu Val
Asn Tyr Ile Gln Ser Ile Lys Phe Arg Ser 1 5 10 15 Phe Glu Leu Ser
Gly Glu Glu Lys Asn Thr Ser Tyr Glu Ile Ser Ser 20 25 30 Phe Ser
Glu Arg Lys Val Lys Ala Lys Lys Leu Leu Lys Glu Ser Pro 35 40 45
Val Glu Phe Val Lys Tyr Asn Lys Arg Gln Leu Ser Arg Val Tyr Pro 50
55 60 Lys Gly Thr Arg Val Asp Ser Ser Asn Phe Met Pro Gln Val Phe
Trp 65 70 75 80 Asn Ala Gly Cys Gln Met Val Ala Leu Asn Phe Gln Thr
Ser Asp Leu 85 90 95 Pro Met Gln Ile Asn Asp Gly Met Phe Glu Tyr
Asn Gly Gly Gln Pro 100 105 110 Asp Gly Ser Phe Lys Ser Gly Tyr Leu
Leu Lys Pro Glu Phe Leu Arg 115 120 125 50 95 PRT Artificial
Sequence Consensus Amino Acid Sequence 50 Leu Thr Val Thr Val Ile
Glu Ala Arg Asn Leu Pro Lys Met Asp Lys 1 5 10 15 Val Asn Gly Arg
Leu Ser Asp Pro Tyr Val Lys Val Ser Leu Leu Gly 20 25 30 Asp Lys
Lys Asp Leu Lys Lys Phe Lys Thr Lys Val Val Lys Lys Thr 35 40 45
Asn Gly Leu Asn Pro Val Trp Asn Glu Glu Thr Phe Val Phe Glu Lys 50
55 60 Val Pro Leu Pro Glu Leu Ala Ser Lys Thr Leu Arg Phe Ala Val
Tyr 65 70 75 80 Asp Glu Asp Arg Phe Ser Arg Asp Asp Phe Ile Gly Gln
Val Thr 85 90 95 51 170 PRT Artificial Sequence Consensus Amino
Acid Sequence 51 Ser Pro Asp Cys Asn Val Phe Asp Pro Glu His Lys
Gln Val His Gln 1 5 10 15 Asp Met Asn Gln Pro Leu Ser His Tyr Phe
Ile Asn Ser Ser His Asn 20 25 30 Thr Tyr Leu Thr Gly Asn Gln Leu
Ser Ser Gly Glu Ser Ser Val Glu 35 40 45 Met Tyr Arg Gln Ala Leu
Leu Lys Gly Cys Arg Cys Ile Glu Leu Asp 50 55 60 Cys Trp Asp Gly
Lys Asp Gly Asp Pro Glu Pro Ile Ile Thr His Gly 65 70 75 80 His Thr
Met Thr Thr Glu Ile Ser Phe Lys Asp Cys Leu Glu Ala Ile 85 90 95
Lys Glu His Ala Phe Val Thr Ser Glu Tyr Pro Val Ile Leu Ser Leu 100
105 110 Glu Asn His Cys Asp Ser Thr Pro Gln Gln Gln Ala Lys Met Ala
Glu 115 120 125 Tyr Cys Lys Glu Val Phe Gly Asp Met Leu Phe Thr Glu
Pro Leu Glu 130 135 140 Glu Ser Pro Leu Glu Pro Gly Lys Glu Leu Pro
Ser Pro Glu Glu Leu 145 150 155 160 Lys Arg Lys Ile Leu Ile Lys Asn
Lys Lys 165 170 52 202 PRT Artificial Sequence Consensus Amino Acid
Sequence 52 Met Ala Ser Gln Ile Lys Lys Ile Ser Ser Thr Asn Asp Cys
Leu Gln 1 5 10 15 Phe Met Gln Lys Gly Ser Glu Leu Lys Lys Val Arg
Ser Asn Ser Trp 20 25 30 Lys Tyr Asn Arg Tyr Phe Thr Leu Asp Asp
Asp Met Gln Thr Leu Trp 35 40 45 Trp Glu Pro His Trp Phe Ser Lys
Lys Asp Ser Glu Lys Pro Lys Phe 50 55 60 Asp Ile Ser Asp Ile Lys
Glu Ile Arg Met Gly Lys Asn Thr Glu Thr 65 70 75 80 Phe Arg Asn Asn
Gly Lys Glu Phe Gln Ile Gln Glu Pro Glu Asp Cys 85 90 95 Cys Phe
Ser Ile Ile Phe Gly Glu Asn Tyr Phe His Glu Ser Leu Asp 100 105 110
Leu Val Ala Asn Ser Ala Asp Val Ala Asn Ile Trp Val Ser Gly Leu 115
120 125 Arg Tyr Leu Val Asp Tyr Ala Lys His Met Leu Asp Asn Tyr Gln
Glu 130 135 140 Gln Leu Asp Gln Trp Leu Arg Glu Trp Phe Gln Gln Ala
Asp Arg Asn 145 150 155 160 Lys Asp Ser Arg Met Ser Phe Arg Glu Ala
Gln Asn Leu Leu Lys Leu 165 170 175 Met Asn Val Gln Met Asp Glu Glu
Tyr Ala Phe Ser Ile Phe Arg Glu 180 185 190 Cys Asp Phe Ser Gln Ser
Asn Thr Leu Asp 195 200 53 119 PRT Artificial Sequence Consensus
Amino Acid Sequence 53 Pro Glu Leu Ser Asp Leu Val Asn Tyr Cys Gln
Pro Val Lys Phe Lys 1 5 10 15 Gly Phe Glu Met Ala Glu Glu Lys Asn
Thr Tyr Tyr His Met Ser Ser 20 25 30 Phe Ser Glu Asn Lys Ala Glu
Lys Leu Val Asn Lys Glu His Pro Lys 35 40 45 Glu Phe Val Arg Tyr
Asn Gln Arg Asn Leu Leu Arg Val Tyr Pro Lys 50 55 60 Gly Thr Arg
Ile Asp Ser Ser Asn Tyr Asn Pro Met Val Phe Trp Asn 65 70 75 80 His
Gly Cys Gln Met Val Ala Leu Asn Phe Gln Thr His Gly Arg Ser 85 90
95 Met Trp Leu Asn Gln Gly Met Phe Arg Ala Asn Gly Gly Cys Gly Tyr
100 105 110 Val Leu Lys Pro Asp Phe Leu 115 54 40 PRT Artificial
Sequence Consensus Amino Acid Sequence 54 Trp Thr Cys Met Lys His
Gly Tyr Arg His Val Ser Leu Leu Ser Lys 1 5 10 15 Asp Gly Thr Ser
Leu His Pro Ala Ser His Phe Val Tyr Thr Cys Met 20 25 30 Gln Glu
Asp Leu Asp Met Asp Glu 35 40 55 63 PRT Artificial Sequence
Consensus Amino Acid Sequence 55 Tyr Lys Pro Gln Arg Ala Trp Met
His Gly Ala Gln Met Ile Ala Leu 1 5 10 15 Ser Arg Gln Asp Asp Lys
Glu Lys Leu Trp Leu Met Gln Gly Met Phe 20 25 30 Arg Ala Asn Gly
Gly Cys Gly Tyr Val Lys Lys Pro Asn Phe Leu Leu 35 40 45 Asn Ala
Gly Ser Ser Gly Val Phe Tyr Pro Thr Glu Asn Pro Val 50 55 60 56 29
PRT Artificial Sequence Consensus Amino Acid Sequence 56 Glu Leu
Lys Glu Ala Phe Lys Glu Phe Asp Lys Asp Gly Asp Gly Lys 1 5 10 15
Ile Ser Phe Glu Glu Phe Lys Ala Ala Leu Lys Lys Leu 20 25 57 153
PRT Artificial Sequence Consensus Amino Acid Sequence 57 Asp Met
Ser Ile Pro Leu Ser His Tyr Phe Ile Ser Ser Ser His Asn 1 5 10 15
Thr Tyr Leu Thr Gly Lys Gln Leu Trp Gly Lys Ser Ser Val Glu Ser 20
25 30 Tyr Arg Gln Gln Leu Asp Ala Gly Cys Arg Cys Val Glu Leu Asp
Cys 35 40 45 Trp Asp Gly Lys Pro Asp Asp Glu Pro Ile Ile Tyr His
Gly His Thr 50 55 60 Leu Thr Leu Glu Ile Lys Leu Lys Asp Val Leu
Glu Ala Ile Lys Asp 65 70 75 80 Phe Ala Phe Lys Pro Thr Ser Pro Tyr
Pro Val Ile Leu Ser Leu Glu 85 90 95 Asn His Cys Asn Ser Asp Asp
Gln Gln Arg Lys Met Ala Lys Tyr Phe 100 105 110 Lys Glu Ile Phe Gly
Asp Met Leu Leu Thr Lys Pro Thr Leu Asp Ser 115 120 125 Leu Thr Thr
Glu Pro Gly Leu Pro Leu Pro Ser Leu Lys Asp Leu Arg 130 135 140 Gly
Lys Ile Leu Leu Lys Asn Lys Lys 145 150 58 128 PRT Artificial
Sequence Consensus Amino Acid Sequence 58 Glu Leu Ser Asn Leu Val
Asn Tyr Ile Gln Ser Ile Lys Phe Arg Ser 1 5 10 15 Phe Glu Leu Ser
Gly Glu Glu Lys Asn Thr Ser Tyr Glu Ile Ser Ser 20 25 30 Phe Ser
Glu Arg Lys Val Lys Ala Lys Lys Leu Leu Lys Glu Ser Pro 35 40 45
Val Glu Phe Val Lys Tyr Asn Lys Arg Gln Leu Ser Arg Val Tyr Pro 50
55 60 Lys Gly Thr Arg Val Asp Ser Ser Asn Phe Met Pro Gln Val Phe
Trp 65 70 75 80 Asn Ala Gly Cys Gln Met Val Ala Leu Asn Phe Gln Thr
Ser Asp Leu 85 90 95 Pro Met Gln Ile Asn Asp Gly Met Phe Glu Tyr
Asn Gly Gly Gln Pro 100 105 110 Asp Gly Ser Phe Lys Ser Gly Tyr Leu
Leu Lys Pro Glu Phe Leu Arg 115 120 125 59 95 PRT Artificial
Sequence Consensus Amino Acid Sequence 59 Leu Thr Val Thr Val Ile
Glu Ala Arg Asn Leu Pro Lys Met Asp Lys 1 5 10 15 Val Asn Gly Arg
Leu Ser Asp Pro Tyr Val Lys Val Ser Leu Leu Gly 20 25 30 Asp Lys
Lys Asp Leu Lys Lys Phe Lys Thr Lys Val Val Lys Lys Thr 35 40 45
Asn Gly Leu Asn Pro Val Trp Asn Glu Glu Thr Phe Val Phe Glu Lys 50
55 60 Val Pro Leu Pro Glu Leu Ala Ser Lys Thr Leu Arg Phe Ala Val
Tyr 65 70 75 80 Asp Glu Asp Arg Phe Ser Arg Asp Asp Phe Ile Gly Gln
Val Thr 85 90 95 60 191 PRT Artificial Sequence Consensus Amino
Acid Sequence 60 Ser Pro Asp Cys Asn Val Phe Asp Pro Glu His Lys
Gln Val His Gln 1 5 10 15 Asp Met Asn Gln Pro Leu Ser His Tyr Phe
Ile Asn Ser Ser His Asn 20 25 30 Thr Tyr Leu Thr Gly Asn Gln Leu
Ser Ser Gly Glu Ser Ser Val Glu 35 40 45 Met Tyr Arg Gln Ala Leu
Leu Lys Gly Cys Arg Cys Ile Glu Leu Asp 50 55 60 Cys Trp Asp Gly
Lys Asp Gly Asp Pro Glu Pro Ile Ile Thr His Gly 65 70 75 80 His Thr
Met Thr Thr Glu Ile Ser Phe Lys Asp Cys Leu Glu Ala Ile 85 90 95
Lys Glu His Ala Phe Val Thr Ser Glu Tyr Pro Val Ile Leu Ser Leu 100
105 110 Glu Asn His Cys Asp Ser Thr Pro Gln Gln Gln Ala Lys Met Ala
Glu 115 120 125 Tyr Cys Lys Glu Val Phe Gly Asp Met Leu Phe Thr Glu
Pro Leu Glu 130 135 140 Glu Ser Pro Leu Glu Pro Gly Lys Glu Leu Pro
Ser Pro Glu Glu Leu 145 150 155 160 Lys Arg Lys Ile Leu Ile Lys Asn
Lys Lys Leu Lys Glu His Ser Glu 165 170 175 Glu Lys Glu Ser Glu Glu
Lys Lys Thr Asp Glu Glu Thr Glu Ser 180 185 190 61 128 PRT
Artificial Sequence Consensus Amino Acid Sequence 61 Ser Asp Leu
Val Asn Tyr Cys Gln Pro Val Lys Phe Lys Gly Phe Glu 1 5 10 15 Met
Ala Glu Glu Lys Asn Thr Tyr Tyr His Met Ser Ser Phe Ser Glu 20 25
30 Asn Lys Ala Glu Lys Leu Val Asn Lys Glu His Pro Lys Glu Phe Val
35 40 45 Arg Tyr Asn Gln Arg Asn Leu Leu Arg Val Tyr Pro Lys Gly
Thr Arg 50 55 60 Ile Asp Ser Ser Asn Tyr Asn Pro Met Val Phe Trp
Asn His Gly Cys 65 70 75 80 Gln Met Val Ala Leu Asn Phe Gln Thr His
Gly Arg Ser Met Trp Leu 85 90 95 Asn Gln Gly Met Phe Arg Ala Asn
Gly Gly Cys Gly Tyr Val Leu Lys 100 105 110 Pro Asp Phe Leu Leu Lys
Ala Arg Pro Asn Asp Glu Val Phe Asp Pro 115 120 125 62 51 PRT
Artificial Sequence Consensus Amino Acid Sequence 62 Asp Xaa Asp
Asn Ser Ile Leu Val Phe Tyr Trp Asp Glu Asn Ser Thr 1 5 10 15 Gly
Asp Asn Gln Gly His Arg Lys Gly Pro Leu Ile Val Met Cys Asp 20 25
30 Glu Asn Gln Ser Thr Ala Gly Cys Xaa Xaa Asp Glu Leu Ile Val Met
35 40 45 Phe Tyr Trp 50 63 4055 DNA Homo Sapiens CDS (210)...(3752)
63 tactataggg agtcgaccca cgcgtccggc cgcgccgagc ctggtggccc
aggtgccccg 60 cccgcgtcag ccctgctcca gccccgcgct agcccagcgc
ccctcgcccc gggccgtccg 120 gaccgcgccc ccgcccaggg ccttgcgcac
gccggggccc aggccgaggg ccgcagcgcc 180 ggggccggcg atgagcgcga
ggagccggc atg agc gca gac agc agc cct ctc 233 Met Ser Ala Asp Ser
Ser Pro Leu 1 5 gtg ggc agc acg ccc acc ggt tat ggg acc ctg acg ata
ggg aca tca 281 Val Gly Ser Thr Pro Thr Gly Tyr Gly Thr Leu Thr Ile
Gly Thr Ser 10 15 20 ata gat ccc ctc agc tcc tca gtt tca tcc gtg
agg ctc agc ggc tac 329 Ile Asp Pro Leu Ser Ser Ser Val Ser Ser Val
Arg Leu Ser Gly Tyr 25 30 35 40 tgt ggc agt cca tgg agg gtc atc ggc
tat cac gtc gtg gtc tgg atg 377 Cys Gly Ser Pro Trp Arg Val Ile Gly
Tyr His Val Val Val Trp Met 45 50 55 atg gct ggg atc
cct ttg ctg ctc ttc cgt tgg aag ccc ctg tgg ggg 425 Met Ala Gly Ile
Pro Leu Leu Leu Phe Arg Trp Lys Pro Leu Trp Gly 60 65 70 gtg cgg
ctg cgg ctc cgg ccc tgc aac ctg gcc cac gcc gaa aca ctc 473 Val Arg
Leu Arg Leu Arg Pro Cys Asn Leu Ala His Ala Glu Thr Leu 75 80 85
gtt atc gaa ata aga gac aaa gag gat agt tcc tgg cag ctc ttc act 521
Val Ile Glu Ile Arg Asp Lys Glu Asp Ser Ser Trp Gln Leu Phe Thr 90
95 100 gtc cag gtg cag act gag gcc atc ggc gag ggc agc ctg gag ccg
tcc 569 Val Gln Val Gln Thr Glu Ala Ile Gly Glu Gly Ser Leu Glu Pro
Ser 105 110 115 120 cca cag tcc cag gca gag gat ggc cgg agc cag gcg
gca gtt ggg gcg 617 Pro Gln Ser Gln Ala Glu Asp Gly Arg Ser Gln Ala
Ala Val Gly Ala 125 130 135 gta cca gag ggt gcc tgg aag gat acg gcc
cag ctc cac aag agc gag 665 Val Pro Glu Gly Ala Trp Lys Asp Thr Ala
Gln Leu His Lys Ser Glu 140 145 150 gag gcg gtg agt gtc gga cag aag
cgg gtg ctg cgg tat tac ctc ttc 713 Glu Ala Val Ser Val Gly Gln Lys
Arg Val Leu Arg Tyr Tyr Leu Phe 155 160 165 cag ggc cag cgc tat atc
tgg atc gag acc cag caa gcc ttc tac cag 761 Gln Gly Gln Arg Tyr Ile
Trp Ile Glu Thr Gln Gln Ala Phe Tyr Gln 170 175 180 gtc agc ctc ctg
gac cat ggc cgc tct tgt gac gac gtc cac cgc tcc 809 Val Ser Leu Leu
Asp His Gly Arg Ser Cys Asp Asp Val His Arg Ser 185 190 195 200 cgc
cat ggc ctc agc ctc cag gac caa atg gtg agg aag gcc att tac 857 Arg
His Gly Leu Ser Leu Gln Asp Gln Met Val Arg Lys Ala Ile Tyr 205 210
215 ggc ccc aac gtg atc agc ata ccg gtc aag tcc tac ccc cag ctg ctg
905 Gly Pro Asn Val Ile Ser Ile Pro Val Lys Ser Tyr Pro Gln Leu Leu
220 225 230 gtg gac gag gca ctg aac ccc tac tat ggg ttc cag gcc ttc
agc atc 953 Val Asp Glu Ala Leu Asn Pro Tyr Tyr Gly Phe Gln Ala Phe
Ser Ile 235 240 245 gcg ctg tgg ctg gct gac cac tac tac tgg tac gcc
ctg tgc atc ttc 1001 Ala Leu Trp Leu Ala Asp His Tyr Tyr Trp Tyr
Ala Leu Cys Ile Phe 250 255 260 ctc att tcc tcc atc tcc atc tgc ctg
tcg ctg tac aag acc aga aag 1049 Leu Ile Ser Ser Ile Ser Ile Cys
Leu Ser Leu Tyr Lys Thr Arg Lys 265 270 275 280 caa agc cag act cta
agg gac atg gtc aag ttg tcc atg cgg gtg tgc 1097 Gln Ser Gln Thr
Leu Arg Asp Met Val Lys Leu Ser Met Arg Val Cys 285 290 295 gtg tgc
cgg cca ggg gga gag gaa gag tgg gtg gac tcc agt gag cta 1145 Val
Cys Arg Pro Gly Gly Glu Glu Glu Trp Val Asp Ser Ser Glu Leu 300 305
310 gtg ccc gga gac tgc ctg gtg ctg ccc cag gag ggt ggg ctg atg ccc
1193 Val Pro Gly Asp Cys Leu Val Leu Pro Gln Glu Gly Gly Leu Met
Pro 315 320 325 tgt gat gcc gcc ctg gtg gcc ggc gag tgc atg gtg aat
gag agc tct 1241 Cys Asp Ala Ala Leu Val Ala Gly Glu Cys Met Val
Asn Glu Ser Ser 330 335 340 ctg aca gga gag agc att cca gtg ctg aag
acg gca ctg ccg gag ggg 1289 Leu Thr Gly Glu Ser Ile Pro Val Leu
Lys Thr Ala Leu Pro Glu Gly 345 350 355 360 ctg ggg ccc tac tgt gca
gag aca cac cgg cgg cac aca ctc ttc tgc 1337 Leu Gly Pro Tyr Cys
Ala Glu Thr His Arg Arg His Thr Leu Phe Cys 365 370 375 ggg acc ctc
atc ttg cag gcc cgg gcc tat gtg gga ccg cac gtc ctg 1385 Gly Thr
Leu Ile Leu Gln Ala Arg Ala Tyr Val Gly Pro His Val Leu 380 385 390
gca gtg gtg acc cgc aca ggg ttc tgc acg gca aaa ggg ggc ctg gtg
1433 Ala Val Val Thr Arg Thr Gly Phe Cys Thr Ala Lys Gly Gly Leu
Val 395 400 405 agc tcc atc ttg cac ccc cgg ccc atc aac ttc aag ttc
tat aaa cac 1481 Ser Ser Ile Leu His Pro Arg Pro Ile Asn Phe Lys
Phe Tyr Lys His 410 415 420 agc atg aag ttt gtg gct gcc ctc tct gtc
ctg gct ctc ctc ggc acc 1529 Ser Met Lys Phe Val Ala Ala Leu Ser
Val Leu Ala Leu Leu Gly Thr 425 430 435 440 atc tac agc atc ttc atc
ctc tac cga aac cgg gtg cct ctg aat gag 1577 Ile Tyr Ser Ile Phe
Ile Leu Tyr Arg Asn Arg Val Pro Leu Asn Glu 445 450 455 att gta atc
cgg gct ctc gac ctg gtg acc gtg gtg gtg cca cct gcc 1625 Ile Val
Ile Arg Ala Leu Asp Leu Val Thr Val Val Val Pro Pro Ala 460 465 470
ctg cct gct gcc atg act gtg tgc acg ctc tac gcc cag agc cga ctg
1673 Leu Pro Ala Ala Met Thr Val Cys Thr Leu Tyr Ala Gln Ser Arg
Leu 475 480 485 cgg aga cag ggc att ttc tgc atc cac cca ctg cgc atc
aac ctg ggg 1721 Arg Arg Gln Gly Ile Phe Cys Ile His Pro Leu Arg
Ile Asn Leu Gly 490 495 500 ggc aag ctg cag ctg gtg tgt ttc gac aag
acg ggc acc ctc act gag 1769 Gly Lys Leu Gln Leu Val Cys Phe Asp
Lys Thr Gly Thr Leu Thr Glu 505 510 515 520 gac ggc tta gac gtg atg
ggg gtg gtg ccc ctg aag ggg cag gca ttc 1817 Asp Gly Leu Asp Val
Met Gly Val Val Pro Leu Lys Gly Gln Ala Phe 525 530 535 ctg ccc ctg
gtc cca gag cct cgc cgc ctg cct gtg ggg ccc ctg ctc 1865 Leu Pro
Leu Val Pro Glu Pro Arg Arg Leu Pro Val Gly Pro Leu Leu 540 545 550
cga gca ctg gcc acc tgc cat gcc ctc agc cgg ctc cag gac acc ccc
1913 Arg Ala Leu Ala Thr Cys His Ala Leu Ser Arg Leu Gln Asp Thr
Pro 555 560 565 gtg ggc gac ccc atg gac ttg aag atg gtg gag tct act
ggc tgg gtc 1961 Val Gly Asp Pro Met Asp Leu Lys Met Val Glu Ser
Thr Gly Trp Val 570 575 580 ctg gag gaa gag ccg gct gca gac tca gca
ttt ggg acc cag gtc ttg 2009 Leu Glu Glu Glu Pro Ala Ala Asp Ser
Ala Phe Gly Thr Gln Val Leu 585 590 595 600 gca gtg atg aga cct cca
ctt tgg gag ccc cag ctg cag gca atg gag 2057 Ala Val Met Arg Pro
Pro Leu Trp Glu Pro Gln Leu Gln Ala Met Glu 605 610 615 gag ccc ccg
gtg cca gtc agc gtc ctc cac cgc ttc ccc ttc tct tcg 2105 Glu Pro
Pro Val Pro Val Ser Val Leu His Arg Phe Pro Phe Ser Ser 620 625 630
gct ctg cag cgc atg agt gtg gtg gtg gcg tgg cca ggg gcc act cag
2153 Ala Leu Gln Arg Met Ser Val Val Val Ala Trp Pro Gly Ala Thr
Gln 635 640 645 ccc gag gcc tac gtc aaa ggc tcc ccg gag ctg gtg gca
ggg ctc tgc 2201 Pro Glu Ala Tyr Val Lys Gly Ser Pro Glu Leu Val
Ala Gly Leu Cys 650 655 660 aac ccc gag aca gtg ccc acc gac ttc gcc
cag atg ctg cag agc tat 2249 Asn Pro Glu Thr Val Pro Thr Asp Phe
Ala Gln Met Leu Gln Ser Tyr 665 670 675 680 aca gct gct ggc tac cgt
gtc gtg gcc ctg gcc agc aag cca ctg ccc 2297 Thr Ala Ala Gly Tyr
Arg Val Val Ala Leu Ala Ser Lys Pro Leu Pro 685 690 695 act gtg ccc
agc ctg gag gca gcc cag caa ctg acg agg gac act gtg 2345 Thr Val
Pro Ser Leu Glu Ala Ala Gln Gln Leu Thr Arg Asp Thr Val 700 705 710
gaa gga gac ctg agc ctc ctg ggg ctg ctg gtc atg agg aac cta ctg
2393 Glu Gly Asp Leu Ser Leu Leu Gly Leu Leu Val Met Arg Asn Leu
Leu 715 720 725 aag ccg cag aca acg cca gtt atc cag gct ctg cga agg
acc cgc atc 2441 Lys Pro Gln Thr Thr Pro Val Ile Gln Ala Leu Arg
Arg Thr Arg Ile 730 735 740 cgc gcc gtc atg gtg aca ggg gac aac ctg
cag aca gcg gtg act gtg 2489 Arg Ala Val Met Val Thr Gly Asp Asn
Leu Gln Thr Ala Val Thr Val 745 750 755 760 gcc cgg ggc tgt ggc atg
gtg gcc ccc cag gag cat ctg atc atc gtc 2537 Ala Arg Gly Cys Gly
Met Val Ala Pro Gln Glu His Leu Ile Ile Val 765 770 775 cac gcc acc
cac cct gag cgg ggt cag cct gcc tct ctc gag ttc ctg 2585 His Ala
Thr His Pro Glu Arg Gly Gln Pro Ala Ser Leu Glu Phe Leu 780 785 790
ccg atg gag tcc ccc aca gcc gtg aat ggc gtt aag gat cct gac cag
2633 Pro Met Glu Ser Pro Thr Ala Val Asn Gly Val Lys Asp Pro Asp
Gln 795 800 805 gct gca agc tac acc gtg gag cca gac ccc cga tcc agg
cac ctg gcc 2681 Ala Ala Ser Tyr Thr Val Glu Pro Asp Pro Arg Ser
Arg His Leu Ala 810 815 820 ctc agc ggg ccc acc ttt ggt atc att gtg
aag cac ttc ccc aag ctg 2729 Leu Ser Gly Pro Thr Phe Gly Ile Ile
Val Lys His Phe Pro Lys Leu 825 830 835 840 ctg ccc aag gtc ctg gtc
cag ggc act gtc ttt gcc cgc atg gcc cct 2777 Leu Pro Lys Val Leu
Val Gln Gly Thr Val Phe Ala Arg Met Ala Pro 845 850 855 gag cag aag
aca gag ctg gtg tgc gag cta cag aag ctt cag tac tgc 2825 Glu Gln
Lys Thr Glu Leu Val Cys Glu Leu Gln Lys Leu Gln Tyr Cys 860 865 870
gtg ggc atg tgc gga gac ggt gcc aat gac tgt ggg gcc ctg aag gcg
2873 Val Gly Met Cys Gly Asp Gly Ala Asn Asp Cys Gly Ala Leu Lys
Ala 875 880 885 gct gat gtc ggc atc tcg ctg tcc cag gca gaa gcc tca
gtg gtc tca 2921 Ala Asp Val Gly Ile Ser Leu Ser Gln Ala Glu Ala
Ser Val Val Ser 890 895 900 ccc ttc acc tcg agc atg gcc agt att gag
tgc gtg ccc atg gtc atc 2969 Pro Phe Thr Ser Ser Met Ala Ser Ile
Glu Cys Val Pro Met Val Ile 905 910 915 920 agg gag ggg cgc tgt tcc
ctt gac act tcg ttc agc gtc ttc aag tac 3017 Arg Glu Gly Arg Cys
Ser Leu Asp Thr Ser Phe Ser Val Phe Lys Tyr 925 930 935 atg gct ctg
tac agc ctg acc cag ttc atc tcc gtc ctg atc ctc tac 3065 Met Ala
Leu Tyr Ser Leu Thr Gln Phe Ile Ser Val Leu Ile Leu Tyr 940 945 950
acg atc aac acc aac ctg ggt gac ctg cag ttc ctg gcc atc gac ctg
3113 Thr Ile Asn Thr Asn Leu Gly Asp Leu Gln Phe Leu Ala Ile Asp
Leu 955 960 965 gtc atc acc acc aca gtg gca gtg ctc atg agc cgc acg
ggg cca gcg 3161 Val Ile Thr Thr Thr Val Ala Val Leu Met Ser Arg
Thr Gly Pro Ala 970 975 980 ctg gtc ctg gga cgg gta cgg cca ccg ggg
gcg ctg ctc agc gtg ccc 3209 Leu Val Leu Gly Arg Val Arg Pro Pro
Gly Ala Leu Leu Ser Val Pro 985 990 995 1000 gtg ctc agc agc ctg
ctg ctg cag atg gtc ctg gtg acc ggc gtg cag 3257 Val Leu Ser Ser
Leu Leu Leu Gln Met Val Leu Val Thr Gly Val Gln 1005 1010 1015 cta
ggg ggc tac ttc ctg acc ctg gcc cag cca tgg ttc gtg cct ctg 3305
Leu Gly Gly Tyr Phe Leu Thr Leu Ala Gln Pro Trp Phe Val Pro Leu
1020 1025 1030 aac agg aca gtg gcc gca cca gac aac ctg ccc aac tac
gag aac acc 3353 Asn Arg Thr Val Ala Ala Pro Asp Asn Leu Pro Asn
Tyr Glu Asn Thr 1035 1040 1045 gtg gtc ttc tct ctg tcc agc ttc cag
tac ctc atc ctg gct gca gcc 3401 Val Val Phe Ser Leu Ser Ser Phe
Gln Tyr Leu Ile Leu Ala Ala Ala 1050 1055 1060 gtg tcc aag ggg gcg
ccc ttc cgc cgg ccg ctc tac acc aat gtg ccc 3449 Val Ser Lys Gly
Ala Pro Phe Arg Arg Pro Leu Tyr Thr Asn Val Pro 1065 1070 1075 1080
ttc ctg gtg gcc ctg gcg ctc ctg agc tcc gtc ctg gtg ggc ctt gtc
3497 Phe Leu Val Ala Leu Ala Leu Leu Ser Ser Val Leu Val Gly Leu
Val 1085 1090 1095 ctg gtc ccc ggc ctc ctg cag ggg ccg ctg gcg ctg
agg aac atc act 3545 Leu Val Pro Gly Leu Leu Gln Gly Pro Leu Ala
Leu Arg Asn Ile Thr 1100 1105 1110 gac acc ggc ttc aag ctg ctg ctg
ctg ggt ctg gtc acc ctc aac ttc 3593 Asp Thr Gly Phe Lys Leu Leu
Leu Leu Gly Leu Val Thr Leu Asn Phe 1115 1120 1125 gtg ggg gcc ttc
atg ctg gag agc gtg cta gac cag tgc ctc ccc gcc 3641 Val Gly Ala
Phe Met Leu Glu Ser Val Leu Asp Gln Cys Leu Pro Ala 1130 1135 1140
tgc ctg cgc cgc ctc cgg ccc aag cgg gcc tcc aag aag cgc ttc aag
3689 Cys Leu Arg Arg Leu Arg Pro Lys Arg Ala Ser Lys Lys Arg Phe
Lys 1145 1150 1155 1160 cag ctg gaa cga gag ctg gcc gag cag ccc tgg
cca ccg ctg ccc gcc 3737 Gln Leu Glu Arg Glu Leu Ala Glu Gln Pro
Trp Pro Pro Leu Pro Ala 1165 1170 1175 ggc ccc ctg agg tag
tgcaggccca cgggcacccc agacactgga actccctgcc 3792 Gly Pro Leu Arg *
1180 tctgagccac caactggacc cctctccagc aacaccaccg ccaccacctc
ccacatccct 3852 gaggttggcg actgtctaca ctcctccccc gagaccaccc
ccaccctggg gaagcgttga 3912 ctactgtccc ctaccttgga ccatcccgcg
taggggtggc agcccccagc tcccctcagt 3972 gctgctgtca gtgtagcaaa
taaagtcatg atattttcct ggcaaaaaaa aaaaaaaaaa 4032 aaaaaaaaaa
aaaaaaaaaa aaa 4055 64 1180 PRT Homo Sapiens 64 Met Ser Ala Asp Ser
Ser Pro Leu Val Gly Ser Thr Pro Thr Gly Tyr 1 5 10 15 Gly Thr Leu
Thr Ile Gly Thr Ser Ile Asp Pro Leu Ser Ser Ser Val 20 25 30 Ser
Ser Val Arg Leu Ser Gly Tyr Cys Gly Ser Pro Trp Arg Val Ile 35 40
45 Gly Tyr His Val Val Val Trp Met Met Ala Gly Ile Pro Leu Leu Leu
50 55 60 Phe Arg Trp Lys Pro Leu Trp Gly Val Arg Leu Arg Leu Arg
Pro Cys 65 70 75 80 Asn Leu Ala His Ala Glu Thr Leu Val Ile Glu Ile
Arg Asp Lys Glu 85 90 95 Asp Ser Ser Trp Gln Leu Phe Thr Val Gln
Val Gln Thr Glu Ala Ile 100 105 110 Gly Glu Gly Ser Leu Glu Pro Ser
Pro Gln Ser Gln Ala Glu Asp Gly 115 120 125 Arg Ser Gln Ala Ala Val
Gly Ala Val Pro Glu Gly Ala Trp Lys Asp 130 135 140 Thr Ala Gln Leu
His Lys Ser Glu Glu Ala Val Ser Val Gly Gln Lys 145 150 155 160 Arg
Val Leu Arg Tyr Tyr Leu Phe Gln Gly Gln Arg Tyr Ile Trp Ile 165 170
175 Glu Thr Gln Gln Ala Phe Tyr Gln Val Ser Leu Leu Asp His Gly Arg
180 185 190 Ser Cys Asp Asp Val His Arg Ser Arg His Gly Leu Ser Leu
Gln Asp 195 200 205 Gln Met Val Arg Lys Ala Ile Tyr Gly Pro Asn Val
Ile Ser Ile Pro 210 215 220 Val Lys Ser Tyr Pro Gln Leu Leu Val Asp
Glu Ala Leu Asn Pro Tyr 225 230 235 240 Tyr Gly Phe Gln Ala Phe Ser
Ile Ala Leu Trp Leu Ala Asp His Tyr 245 250 255 Tyr Trp Tyr Ala Leu
Cys Ile Phe Leu Ile Ser Ser Ile Ser Ile Cys 260 265 270 Leu Ser Leu
Tyr Lys Thr Arg Lys Gln Ser Gln Thr Leu Arg Asp Met 275 280 285 Val
Lys Leu Ser Met Arg Val Cys Val Cys Arg Pro Gly Gly Glu Glu 290 295
300 Glu Trp Val Asp Ser Ser Glu Leu Val Pro Gly Asp Cys Leu Val Leu
305 310 315 320 Pro Gln Glu Gly Gly Leu Met Pro Cys Asp Ala Ala Leu
Val Ala Gly 325 330 335 Glu Cys Met Val Asn Glu Ser Ser Leu Thr Gly
Glu Ser Ile Pro Val 340 345 350 Leu Lys Thr Ala Leu Pro Glu Gly Leu
Gly Pro Tyr Cys Ala Glu Thr 355 360 365 His Arg Arg His Thr Leu Phe
Cys Gly Thr Leu Ile Leu Gln Ala Arg 370 375 380 Ala Tyr Val Gly Pro
His Val Leu Ala Val Val Thr Arg Thr Gly Phe 385 390 395 400 Cys Thr
Ala Lys Gly Gly Leu Val Ser Ser Ile Leu His Pro Arg Pro 405 410 415
Ile Asn Phe Lys Phe Tyr Lys His Ser Met Lys Phe Val Ala Ala Leu 420
425 430 Ser Val Leu Ala Leu Leu Gly Thr Ile Tyr Ser Ile Phe Ile Leu
Tyr 435 440 445 Arg Asn Arg Val Pro Leu Asn Glu Ile Val Ile Arg Ala
Leu Asp Leu 450 455 460 Val Thr Val Val Val Pro Pro Ala Leu Pro Ala
Ala Met Thr Val Cys 465 470 475 480 Thr Leu Tyr Ala Gln Ser Arg Leu
Arg Arg Gln Gly Ile Phe Cys Ile 485 490 495 His Pro Leu Arg Ile Asn
Leu Gly Gly Lys Leu Gln Leu Val Cys Phe 500 505 510 Asp Lys Thr Gly
Thr Leu Thr Glu Asp Gly Leu Asp Val Met Gly Val 515 520 525 Val Pro
Leu Lys Gly Gln Ala Phe Leu Pro Leu Val Pro Glu Pro Arg 530 535 540
Arg Leu Pro Val Gly Pro Leu Leu Arg Ala Leu Ala Thr Cys His Ala 545
550 555 560 Leu Ser Arg Leu Gln Asp Thr Pro Val Gly Asp Pro Met Asp
Leu Lys 565 570 575 Met Val Glu Ser
Thr Gly Trp Val Leu Glu Glu Glu Pro Ala Ala Asp 580 585 590 Ser Ala
Phe Gly Thr Gln Val Leu Ala Val Met Arg Pro Pro Leu Trp 595 600 605
Glu Pro Gln Leu Gln Ala Met Glu Glu Pro Pro Val Pro Val Ser Val 610
615 620 Leu His Arg Phe Pro Phe Ser Ser Ala Leu Gln Arg Met Ser Val
Val 625 630 635 640 Val Ala Trp Pro Gly Ala Thr Gln Pro Glu Ala Tyr
Val Lys Gly Ser 645 650 655 Pro Glu Leu Val Ala Gly Leu Cys Asn Pro
Glu Thr Val Pro Thr Asp 660 665 670 Phe Ala Gln Met Leu Gln Ser Tyr
Thr Ala Ala Gly Tyr Arg Val Val 675 680 685 Ala Leu Ala Ser Lys Pro
Leu Pro Thr Val Pro Ser Leu Glu Ala Ala 690 695 700 Gln Gln Leu Thr
Arg Asp Thr Val Glu Gly Asp Leu Ser Leu Leu Gly 705 710 715 720 Leu
Leu Val Met Arg Asn Leu Leu Lys Pro Gln Thr Thr Pro Val Ile 725 730
735 Gln Ala Leu Arg Arg Thr Arg Ile Arg Ala Val Met Val Thr Gly Asp
740 745 750 Asn Leu Gln Thr Ala Val Thr Val Ala Arg Gly Cys Gly Met
Val Ala 755 760 765 Pro Gln Glu His Leu Ile Ile Val His Ala Thr His
Pro Glu Arg Gly 770 775 780 Gln Pro Ala Ser Leu Glu Phe Leu Pro Met
Glu Ser Pro Thr Ala Val 785 790 795 800 Asn Gly Val Lys Asp Pro Asp
Gln Ala Ala Ser Tyr Thr Val Glu Pro 805 810 815 Asp Pro Arg Ser Arg
His Leu Ala Leu Ser Gly Pro Thr Phe Gly Ile 820 825 830 Ile Val Lys
His Phe Pro Lys Leu Leu Pro Lys Val Leu Val Gln Gly 835 840 845 Thr
Val Phe Ala Arg Met Ala Pro Glu Gln Lys Thr Glu Leu Val Cys 850 855
860 Glu Leu Gln Lys Leu Gln Tyr Cys Val Gly Met Cys Gly Asp Gly Ala
865 870 875 880 Asn Asp Cys Gly Ala Leu Lys Ala Ala Asp Val Gly Ile
Ser Leu Ser 885 890 895 Gln Ala Glu Ala Ser Val Val Ser Pro Phe Thr
Ser Ser Met Ala Ser 900 905 910 Ile Glu Cys Val Pro Met Val Ile Arg
Glu Gly Arg Cys Ser Leu Asp 915 920 925 Thr Ser Phe Ser Val Phe Lys
Tyr Met Ala Leu Tyr Ser Leu Thr Gln 930 935 940 Phe Ile Ser Val Leu
Ile Leu Tyr Thr Ile Asn Thr Asn Leu Gly Asp 945 950 955 960 Leu Gln
Phe Leu Ala Ile Asp Leu Val Ile Thr Thr Thr Val Ala Val 965 970 975
Leu Met Ser Arg Thr Gly Pro Ala Leu Val Leu Gly Arg Val Arg Pro 980
985 990 Pro Gly Ala Leu Leu Ser Val Pro Val Leu Ser Ser Leu Leu Leu
Gln 995 1000 1005 Met Val Leu Val Thr Gly Val Gln Leu Gly Gly Tyr
Phe Leu Thr Leu 1010 1015 1020 Ala Gln Pro Trp Phe Val Pro Leu Asn
Arg Thr Val Ala Ala Pro Asp 1025 1030 1035 1040 Asn Leu Pro Asn Tyr
Glu Asn Thr Val Val Phe Ser Leu Ser Ser Phe 1045 1050 1055 Gln Tyr
Leu Ile Leu Ala Ala Ala Val Ser Lys Gly Ala Pro Phe Arg 1060 1065
1070 Arg Pro Leu Tyr Thr Asn Val Pro Phe Leu Val Ala Leu Ala Leu
Leu 1075 1080 1085 Ser Ser Val Leu Val Gly Leu Val Leu Val Pro Gly
Leu Leu Gln Gly 1090 1095 1100 Pro Leu Ala Leu Arg Asn Ile Thr Asp
Thr Gly Phe Lys Leu Leu Leu 1105 1110 1115 1120 Leu Gly Leu Val Thr
Leu Asn Phe Val Gly Ala Phe Met Leu Glu Ser 1125 1130 1135 Val Leu
Asp Gln Cys Leu Pro Ala Cys Leu Arg Arg Leu Arg Pro Lys 1140 1145
1150 Arg Ala Ser Lys Lys Arg Phe Lys Gln Leu Glu Arg Glu Leu Ala
Glu 1155 1160 1165 Gln Pro Trp Pro Pro Leu Pro Ala Gly Pro Leu Arg
1170 1175 1180 65 3540 DNA Homo Sapiens CDS (1)...(3540) 65 atg agc
gca gac agc agc cct ctc gtg ggc agc acg ccc acc ggt tat 48 Met Ser
Ala Asp Ser Ser Pro Leu Val Gly Ser Thr Pro Thr Gly Tyr 1 5 10 15
ggg acc ctg acg ata ggg aca tca ata gat ccc ctc agc tcc tca gtt 96
Gly Thr Leu Thr Ile Gly Thr Ser Ile Asp Pro Leu Ser Ser Ser Val 20
25 30 tca tcc gtg agg ctc agc ggc tac tgt ggc agt cca tgg agg gtc
atc 144 Ser Ser Val Arg Leu Ser Gly Tyr Cys Gly Ser Pro Trp Arg Val
Ile 35 40 45 ggc tat cac gtc gtg gtc tgg atg atg gct ggg atc cct
ttg ctg ctc 192 Gly Tyr His Val Val Val Trp Met Met Ala Gly Ile Pro
Leu Leu Leu 50 55 60 ttc cgt tgg aag ccc ctg tgg ggg gtg cgg ctg
cgg ctc cgg ccc tgc 240 Phe Arg Trp Lys Pro Leu Trp Gly Val Arg Leu
Arg Leu Arg Pro Cys 65 70 75 80 aac ctg gcc cac gcc gaa aca ctc gtt
atc gaa ata aga gac aaa gag 288 Asn Leu Ala His Ala Glu Thr Leu Val
Ile Glu Ile Arg Asp Lys Glu 85 90 95 gat agt tcc tgg cag ctc ttc
act gtc cag gtg cag act gag gcc atc 336 Asp Ser Ser Trp Gln Leu Phe
Thr Val Gln Val Gln Thr Glu Ala Ile 100 105 110 ggc gag ggc agc ctg
gag ccg tcc cca cag tcc cag gca gag gat ggc 384 Gly Glu Gly Ser Leu
Glu Pro Ser Pro Gln Ser Gln Ala Glu Asp Gly 115 120 125 cgg agc cag
gcg gca gtt ggg gcg gta cca gag ggt gcc tgg aag gat 432 Arg Ser Gln
Ala Ala Val Gly Ala Val Pro Glu Gly Ala Trp Lys Asp 130 135 140 acg
gcc cag ctc cac aag agc gag gag gcg gtg agt gtc gga cag aag 480 Thr
Ala Gln Leu His Lys Ser Glu Glu Ala Val Ser Val Gly Gln Lys 145 150
155 160 cgg gtg ctg cgg tat tac ctc ttc cag ggc cag cgc tat atc tgg
atc 528 Arg Val Leu Arg Tyr Tyr Leu Phe Gln Gly Gln Arg Tyr Ile Trp
Ile 165 170 175 gag acc cag caa gcc ttc tac cag gtc agc ctc ctg gac
cat ggc cgc 576 Glu Thr Gln Gln Ala Phe Tyr Gln Val Ser Leu Leu Asp
His Gly Arg 180 185 190 tct tgt gac gac gtc cac cgc tcc cgc cat ggc
ctc agc ctc cag gac 624 Ser Cys Asp Asp Val His Arg Ser Arg His Gly
Leu Ser Leu Gln Asp 195 200 205 caa atg gtg agg aag gcc att tac ggc
ccc aac gtg atc agc ata ccg 672 Gln Met Val Arg Lys Ala Ile Tyr Gly
Pro Asn Val Ile Ser Ile Pro 210 215 220 gtc aag tcc tac ccc cag ctg
ctg gtg gac gag gca ctg aac ccc tac 720 Val Lys Ser Tyr Pro Gln Leu
Leu Val Asp Glu Ala Leu Asn Pro Tyr 225 230 235 240 tat ggg ttc cag
gcc ttc agc atc gcg ctg tgg ctg gct gac cac tac 768 Tyr Gly Phe Gln
Ala Phe Ser Ile Ala Leu Trp Leu Ala Asp His Tyr 245 250 255 tac tgg
tac gcc ctg tgc atc ttc ctc att tcc tcc atc tcc atc tgc 816 Tyr Trp
Tyr Ala Leu Cys Ile Phe Leu Ile Ser Ser Ile Ser Ile Cys 260 265 270
ctg tcg ctg tac aag acc aga aag caa agc cag act cta agg gac atg 864
Leu Ser Leu Tyr Lys Thr Arg Lys Gln Ser Gln Thr Leu Arg Asp Met 275
280 285 gtc aag ttg tcc atg cgg gtg tgc gtg tgc cgg cca ggg gga gag
gaa 912 Val Lys Leu Ser Met Arg Val Cys Val Cys Arg Pro Gly Gly Glu
Glu 290 295 300 gag tgg gtg gac tcc agt gag cta gtg ccc gga gac tgc
ctg gtg ctg 960 Glu Trp Val Asp Ser Ser Glu Leu Val Pro Gly Asp Cys
Leu Val Leu 305 310 315 320 ccc cag gag ggt ggg ctg atg ccc tgt gat
gcc gcc ctg gtg gcc ggc 1008 Pro Gln Glu Gly Gly Leu Met Pro Cys
Asp Ala Ala Leu Val Ala Gly 325 330 335 gag tgc atg gtg aat gag agc
tct ctg aca gga gag agc att cca gtg 1056 Glu Cys Met Val Asn Glu
Ser Ser Leu Thr Gly Glu Ser Ile Pro Val 340 345 350 ctg aag acg gca
ctg ccg gag ggg ctg ggg ccc tac tgt gca gag aca 1104 Leu Lys Thr
Ala Leu Pro Glu Gly Leu Gly Pro Tyr Cys Ala Glu Thr 355 360 365 cac
cgg cgg cac aca ctc ttc tgc ggg acc ctc atc ttg cag gcc cgg 1152
His Arg Arg His Thr Leu Phe Cys Gly Thr Leu Ile Leu Gln Ala Arg 370
375 380 gcc tat gtg gga ccg cac gtc ctg gca gtg gtg acc cgc aca ggg
ttc 1200 Ala Tyr Val Gly Pro His Val Leu Ala Val Val Thr Arg Thr
Gly Phe 385 390 395 400 tgc acg gca aaa ggg ggc ctg gtg agc tcc atc
ttg cac ccc cgg ccc 1248 Cys Thr Ala Lys Gly Gly Leu Val Ser Ser
Ile Leu His Pro Arg Pro 405 410 415 atc aac ttc aag ttc tat aaa cac
agc atg aag ttt gtg gct gcc ctc 1296 Ile Asn Phe Lys Phe Tyr Lys
His Ser Met Lys Phe Val Ala Ala Leu 420 425 430 tct gtc ctg gct ctc
ctc ggc acc atc tac agc atc ttc atc ctc tac 1344 Ser Val Leu Ala
Leu Leu Gly Thr Ile Tyr Ser Ile Phe Ile Leu Tyr 435 440 445 cga aac
cgg gtg cct ctg aat gag att gta atc cgg gct ctc gac ctg 1392 Arg
Asn Arg Val Pro Leu Asn Glu Ile Val Ile Arg Ala Leu Asp Leu 450 455
460 gtg acc gtg gtg gtg cca cct gcc ctg cct gct gcc atg act gtg tgc
1440 Val Thr Val Val Val Pro Pro Ala Leu Pro Ala Ala Met Thr Val
Cys 465 470 475 480 acg ctc tac gcc cag agc cga ctg cgg aga cag ggc
att ttc tgc atc 1488 Thr Leu Tyr Ala Gln Ser Arg Leu Arg Arg Gln
Gly Ile Phe Cys Ile 485 490 495 cac cca ctg cgc atc aac ctg ggg ggc
aag ctg cag ctg gtg tgt ttc 1536 His Pro Leu Arg Ile Asn Leu Gly
Gly Lys Leu Gln Leu Val Cys Phe 500 505 510 gac aag acg ggc acc ctc
act gag gac ggc tta gac gtg atg ggg gtg 1584 Asp Lys Thr Gly Thr
Leu Thr Glu Asp Gly Leu Asp Val Met Gly Val 515 520 525 gtg ccc ctg
aag ggg cag gca ttc ctg ccc ctg gtc cca gag cct cgc 1632 Val Pro
Leu Lys Gly Gln Ala Phe Leu Pro Leu Val Pro Glu Pro Arg 530 535 540
cgc ctg cct gtg ggg ccc ctg ctc cga gca ctg gcc acc tgc cat gcc
1680 Arg Leu Pro Val Gly Pro Leu Leu Arg Ala Leu Ala Thr Cys His
Ala 545 550 555 560 ctc agc cgg ctc cag gac acc ccc gtg ggc gac ccc
atg gac ttg aag 1728 Leu Ser Arg Leu Gln Asp Thr Pro Val Gly Asp
Pro Met Asp Leu Lys 565 570 575 atg gtg gag tct act ggc tgg gtc ctg
gag gaa gag ccg gct gca gac 1776 Met Val Glu Ser Thr Gly Trp Val
Leu Glu Glu Glu Pro Ala Ala Asp 580 585 590 tca gca ttt ggg acc cag
gtc ttg gca gtg atg aga cct cca ctt tgg 1824 Ser Ala Phe Gly Thr
Gln Val Leu Ala Val Met Arg Pro Pro Leu Trp 595 600 605 gag ccc cag
ctg cag gca atg gag gag ccc ccg gtg cca gtc agc gtc 1872 Glu Pro
Gln Leu Gln Ala Met Glu Glu Pro Pro Val Pro Val Ser Val 610 615 620
ctc cac cgc ttc ccc ttc tct tcg gct ctg cag cgc atg agt gtg gtg
1920 Leu His Arg Phe Pro Phe Ser Ser Ala Leu Gln Arg Met Ser Val
Val 625 630 635 640 gtg gcg tgg cca ggg gcc act cag ccc gag gcc tac
gtc aaa ggc tcc 1968 Val Ala Trp Pro Gly Ala Thr Gln Pro Glu Ala
Tyr Val Lys Gly Ser 645 650 655 ccg gag ctg gtg gca ggg ctc tgc aac
ccc gag aca gtg ccc acc gac 2016 Pro Glu Leu Val Ala Gly Leu Cys
Asn Pro Glu Thr Val Pro Thr Asp 660 665 670 ttc gcc cag atg ctg cag
agc tat aca gct gct ggc tac cgt gtc gtg 2064 Phe Ala Gln Met Leu
Gln Ser Tyr Thr Ala Ala Gly Tyr Arg Val Val 675 680 685 gcc ctg gcc
agc aag cca ctg ccc act gtg ccc agc ctg gag gca gcc 2112 Ala Leu
Ala Ser Lys Pro Leu Pro Thr Val Pro Ser Leu Glu Ala Ala 690 695 700
cag caa ctg acg agg gac act gtg gaa gga gac ctg agc ctc ctg ggg
2160 Gln Gln Leu Thr Arg Asp Thr Val Glu Gly Asp Leu Ser Leu Leu
Gly 705 710 715 720 ctg ctg gtc atg agg aac cta ctg aag ccg cag aca
acg cca gtt atc 2208 Leu Leu Val Met Arg Asn Leu Leu Lys Pro Gln
Thr Thr Pro Val Ile 725 730 735 cag gct ctg cga agg acc cgc atc cgc
gcc gtc atg gtg aca ggg gac 2256 Gln Ala Leu Arg Arg Thr Arg Ile
Arg Ala Val Met Val Thr Gly Asp 740 745 750 aac ctg cag aca gcg gtg
act gtg gcc cgg ggc tgt ggc atg gtg gcc 2304 Asn Leu Gln Thr Ala
Val Thr Val Ala Arg Gly Cys Gly Met Val Ala 755 760 765 ccc cag gag
cat ctg atc atc gtc cac gcc acc cac cct gag cgg ggt 2352 Pro Gln
Glu His Leu Ile Ile Val His Ala Thr His Pro Glu Arg Gly 770 775 780
cag cct gcc tct ctc gag ttc ctg ccg atg gag tcc ccc aca gcc gtg
2400 Gln Pro Ala Ser Leu Glu Phe Leu Pro Met Glu Ser Pro Thr Ala
Val 785 790 795 800 aat ggc gtt aag gat cct gac cag gct gca agc tac
acc gtg gag cca 2448 Asn Gly Val Lys Asp Pro Asp Gln Ala Ala Ser
Tyr Thr Val Glu Pro 805 810 815 gac ccc cga tcc agg cac ctg gcc ctc
agc ggg ccc acc ttt ggt atc 2496 Asp Pro Arg Ser Arg His Leu Ala
Leu Ser Gly Pro Thr Phe Gly Ile 820 825 830 att gtg aag cac ttc ccc
aag ctg ctg ccc aag gtc ctg gtc cag ggc 2544 Ile Val Lys His Phe
Pro Lys Leu Leu Pro Lys Val Leu Val Gln Gly 835 840 845 act gtc ttt
gcc cgc atg gcc cct gag cag aag aca gag ctg gtg tgc 2592 Thr Val
Phe Ala Arg Met Ala Pro Glu Gln Lys Thr Glu Leu Val Cys 850 855 860
gag cta cag aag ctt cag tac tgc gtg ggc atg tgc gga gac ggt gcc
2640 Glu Leu Gln Lys Leu Gln Tyr Cys Val Gly Met Cys Gly Asp Gly
Ala 865 870 875 880 aat gac tgt ggg gcc ctg aag gcg gct gat gtc ggc
atc tcg ctg tcc 2688 Asn Asp Cys Gly Ala Leu Lys Ala Ala Asp Val
Gly Ile Ser Leu Ser 885 890 895 cag gca gaa gcc tca gtg gtc tca ccc
ttc acc tcg agc atg gcc agt 2736 Gln Ala Glu Ala Ser Val Val Ser
Pro Phe Thr Ser Ser Met Ala Ser 900 905 910 att gag tgc gtg ccc atg
gtc atc agg gag ggg cgc tgt tcc ctt gac 2784 Ile Glu Cys Val Pro
Met Val Ile Arg Glu Gly Arg Cys Ser Leu Asp 915 920 925 act tcg ttc
agc gtc ttc aag tac atg gct ctg tac agc ctg acc cag 2832 Thr Ser
Phe Ser Val Phe Lys Tyr Met Ala Leu Tyr Ser Leu Thr Gln 930 935 940
ttc atc tcc gtc ctg atc ctc tac acg atc aac acc aac ctg ggt gac
2880 Phe Ile Ser Val Leu Ile Leu Tyr Thr Ile Asn Thr Asn Leu Gly
Asp 945 950 955 960 ctg cag ttc ctg gcc atc gac ctg gtc atc acc acc
aca gtg gca gtg 2928 Leu Gln Phe Leu Ala Ile Asp Leu Val Ile Thr
Thr Thr Val Ala Val 965 970 975 ctc atg agc cgc acg ggg cca gcg ctg
gtc ctg gga cgg gta cgg cca 2976 Leu Met Ser Arg Thr Gly Pro Ala
Leu Val Leu Gly Arg Val Arg Pro 980 985 990 ccg ggg gcg ctg ctc agc
gtg ccc gtg ctc agc agc ctg ctg ctg cag 3024 Pro Gly Ala Leu Leu
Ser Val Pro Val Leu Ser Ser Leu Leu Leu Gln 995 1000 1005 atg gtc
ctg gtg acc ggc gtg cag cta ggg ggc tac ttc ctg acc ctg 3072 Met
Val Leu Val Thr Gly Val Gln Leu Gly Gly Tyr Phe Leu Thr Leu 1010
1015 1020 gcc cag cca tgg ttc gtg cct ctg aac agg aca gtg gcc gca
cca gac 3120 Ala Gln Pro Trp Phe Val Pro Leu Asn Arg Thr Val Ala
Ala Pro Asp 1025 1030 1035 1040 aac ctg ccc aac tac gag aac acc gtg
gtc ttc tct ctg tcc agc ttc 3168 Asn Leu Pro Asn Tyr Glu Asn Thr
Val Val Phe Ser Leu Ser Ser Phe 1045 1050 1055 cag tac ctc atc ctg
gct gca gcc gtg tcc aag ggg gcg ccc ttc cgc 3216 Gln Tyr Leu Ile
Leu Ala Ala Ala Val Ser Lys Gly Ala Pro Phe Arg 1060 1065 1070 cgg
ccg ctc tac acc aat gtg ccc ttc ctg gtg gcc ctg gcg ctc ctg 3264
Arg Pro Leu Tyr Thr Asn Val Pro Phe Leu Val Ala Leu Ala Leu Leu
1075 1080 1085 agc tcc gtc ctg gtg ggc ctt gtc ctg gtc ccc ggc ctc
ctg cag ggg 3312 Ser Ser Val Leu Val Gly Leu Val Leu Val Pro Gly
Leu Leu Gln Gly 1090 1095 1100 ccg ctg gcg ctg agg aac atc act gac
acc ggc ttc aag ctg ctg ctg 3360 Pro Leu Ala Leu Arg Asn Ile Thr
Asp Thr Gly Phe Lys Leu Leu Leu 1105 1110 1115 1120 ctg ggt ctg gtc
acc ctc aac ttc gtg ggg gcc ttc atg ctg gag agc 3408 Leu Gly Leu
Val Thr Leu Asn Phe Val Gly Ala Phe Met Leu Glu Ser 1125 1130 1135
gtg cta gac cag tgc ctc ccc gcc tgc ctg cgc
cgc ctc cgg ccc aag 3456 Val Leu Asp Gln Cys Leu Pro Ala Cys Leu
Arg Arg Leu Arg Pro Lys 1140 1145 1150 cgg gcc tcc aag aag cgc ttc
aag cag ctg gaa cga gag ctg gcc gag 3504 Arg Ala Ser Lys Lys Arg
Phe Lys Gln Leu Glu Arg Glu Leu Ala Glu 1155 1160 1165 cag ccc tgg
cca ccg ctg ccc gcc ggc ccc ctg agg 3540 Gln Pro Trp Pro Pro Leu
Pro Ala Gly Pro Leu Arg 1170 1175 1180 66 1187 PRT Caenorhabditis
elegans 66 Met Thr Leu Glu Ser Gly Asp His Thr Leu Thr Leu Phe Ala
Tyr Arg 1 5 10 15 Thr Gly Pro Phe Arg Thr Ile Leu Phe Tyr Ala Leu
Thr Val Leu Thr 20 25 30 Leu Gly Ile Phe Arg Leu Ile Leu His Trp
Lys Gln Lys Trp Asp Val 35 40 45 Lys Met Arg Met Val Pro Cys Thr
Phe Glu Ala Ala Glu Tyr Ile Tyr 50 55 60 Ile Ile Asp Asn His Asn
Val Ser Glu Leu Gln Pro Val Leu Arg Lys 65 70 75 80 Ser Asn Ala Thr
Ile Pro Thr Glu Asn Gly Glu Met Arg Lys Val Pro 85 90 95 Glu Leu
Arg Trp Phe Val Tyr Arg Lys Leu Glu Tyr Val Trp Ile Asp 100 105 110
Asp Leu Asn Ser Asp Glu Ser Val Asp Glu Ile Ser Asp Asn Asp Asn 115
120 125 Cys Trp Lys Thr Ser Phe Glu Ile Ala Asn Arg Ile Pro Cys Arg
Ser 130 135 140 Leu Leu Ala Val Ser Glu Ser Asn Phe Gly Leu Thr Leu
Ser Glu Ile 145 150 155 160 Ser Arg Arg Leu Glu Phe Tyr Gly Arg Asn
Glu Ile Val Val Gln Leu 165 170 175 Arg Pro Ile Leu Tyr Leu Leu Val
Met Glu Val Ile Thr Pro Phe Tyr 180 185 190 Val Phe Gln Ile Phe Ser
Val Thr Val Trp Tyr Asn Asp Glu Tyr Ala 195 200 205 Tyr Tyr Ala Ser
Leu Ile Val Ile Leu Ser Leu Gly Ser Ile Val Met 210 215 220 Asp Val
Tyr Gln Ile Arg Thr Gln Glu Ile Arg Leu Arg Ser Met Val 225 230 235
240 His Ser Thr Glu Ser Val Glu Val Ile Arg Glu Gly Thr Glu Met Thr
245 250 255 Ile Gly Ser Asp Gln Leu Val Pro Gly Asp Ile Leu Leu Ile
Pro Pro 260 265 270 His Gly Cys Leu Met Gln Cys Asp Ser Val Leu Met
Asn Gly Thr Val 275 280 285 Ile Val Asn Glu Ser Val Leu Thr Gly Glu
Ser Val Pro Ile Thr Lys 290 295 300 Val Ala Leu Thr Asp Glu Thr Asn
Asp Ser Val Phe Asn Ile Glu Lys 305 310 315 320 Asn Ser Lys Asn Val
Leu Phe Cys Gly Thr Gln Val Leu Gln Thr Arg 325 330 335 Phe Tyr Arg
Gly Lys Lys Val Lys Ala Ile Val Leu Arg Thr Ala Tyr 340 345 350 Ser
Thr Leu Lys Gly Gln Leu Val Arg Ser Ile Met Tyr Pro Lys Pro 355 360
365 Val Asp Phe Arg Phe Thr Lys Asp Leu Phe Lys Phe Ile Leu Phe Leu
370 375 380 Ala Cys Ile Ser Gly Cys Gly Phe Ile Tyr Thr Ile Ile Val
Met Ile 385 390 395 400 Met Arg Gly Asn Thr Leu Arg Arg Ile Ile Val
Arg Ser Leu Asp Ile 405 410 415 Ile Thr Ile Thr Val Pro Pro Ala Leu
Pro Ala Ala Met Ser Val Gly 420 425 430 Ile Ile Asn Ala Gln Leu Arg
Leu Lys Lys Lys Glu Ile Phe Cys Ile 435 440 445 Ser Pro Ser Thr Ile
Asn Thr Cys Gly Ala Ile Asn Val Val Cys Phe 450 455 460 Asp Lys Thr
Gly Thr Leu Thr Glu Asp Gly Leu Asp Phe His Val Val 465 470 475 480
Arg Pro Val Met Ser Ala Val Asn Gln Glu Ile Gln Lys Val Lys Leu 485
490 495 Glu Lys Ser Asn Arg Thr Glu Phe Met Gly Glu Met Thr Glu Leu
Thr 500 505 510 Ser Arg Asn Gly Leu Pro Phe Asp Gly Asp Leu Val Lys
Ala Ile Ala 515 520 525 Thr Cys His Ser Leu Thr Arg Ile Asn Gly Val
Leu His Gly Asp Pro 530 535 540 Leu Asp Leu Ile Leu Phe Gln Lys Thr
Gly Trp Thr Met Glu Glu Gly 545 550 555 560 Ile Glu Gly Asp Ile Glu
Glu Glu Thr Gln Arg Phe Asp Asn Val Gln 565 570 575 Pro Ser Ile Ile
Lys Pro Thr Asp Asp Lys Ser Ala Glu Tyr Ser Val 580 585 590 Ile Arg
Gln Phe Thr Phe Ser Ser Ser Leu Gln Arg Met Ser Val Ile 595 600 605
Val Phe Asp Pro Arg Glu Asp Arg Pro Asp Asn Met Met Leu Tyr Ser 610
615 620 Lys Gly Ser Pro Glu Met Ile Leu Ser Leu Cys Asp Pro Asn Thr
Val 625 630 635 640 Pro Glu Asp Tyr Leu Leu Gln Val Asn Ser Tyr Ala
Gln His Gly Phe 645 650 655 Arg Leu Ile Ala Val Ala Arg Arg Pro Leu
Asp Leu Asn Phe Asn Lys 660 665 670 Ala Ser Lys Val Lys Arg Asp Ala
Val Glu Cys Asp Leu Glu Met Leu 675 680 685 Gly Leu Ile Val Met Glu
Asn Arg Val Lys Pro Val Thr Leu Gly Val 690 695 700 Ile Asn Gln Leu
Asn Arg Ala Asn Ile Arg Thr Val Met Val Thr Gly 705 710 715 720 Asp
Asn Leu Leu Thr Gly Leu Ser Val Ala Arg Glu Cys Gly Ile Ile 725 730
735 Arg Pro Ser Lys Arg Ala Phe Leu Val Glu His Val Pro Gly Glu Leu
740 745 750 Asp Glu Tyr Gly Arg Thr Lys Ile Phe Val Lys Gln Ser Val
Ser Ser 755 760 765 Ser Asp Glu Val Ile Glu Asp Asp Ala Ser Val Ser
Ile Ser Met Cys 770 775 780 Ser Ser Thr Trp Lys Gly Ser Ser Glu Gly
Asp Gly Phe Ser Pro Thr 785 790 795 800 Asn Thr Glu Val Glu Thr Pro
Asn Pro Val Thr Ala Asp Ser Leu Gly 805 810 815 His Leu Ile Ala Ser
Ser Tyr His Leu Ala Ile Ser Gly Pro Thr Phe 820 825 830 Ala Val Ile
Val His Glu Tyr Pro Glu Leu Val Asp Gln Leu Cys Ser 835 840 845 Val
Cys Asp Val Phe Ala Arg Met Ala Pro Asp Gln Lys Gln Ser Leu 850 855
860 Val Glu Gln Leu Gln Gln Ile Asp Tyr Thr Val Ala Met Cys Gly Asp
865 870 875 880 Gly Ala Asn Asp Cys Ala Ala Leu Lys Ala Ala His Ala
Gly Ile Ser 885 890 895 Leu Ser Asp Ala Glu Ala Ser Ile Ala Ala Pro
Phe Thr Ser Lys Val 900 905 910 Pro Asp Ile Arg Cys Val Pro Thr Val
Ile Ser Glu Gly Arg Ala Ala 915 920 925 Leu Val Thr Ser Phe Gly Ile
Phe Lys Tyr Met Ala Gly Tyr Ser Leu 930 935 940 Thr Gln Phe Val Thr
Val Met His Leu Tyr Trp Ile Ser Asn Ile Leu 945 950 955 960 Thr Asp
Gly Gln Phe Met Tyr Ile Asp Met Phe Leu Ile Thr Met Phe 965 970 975
Ala Leu Leu Phe Gly Asn Thr Pro Ala Phe Tyr Arg Leu Ala His Thr 980
985 990 Pro Pro Pro Thr Arg Leu Leu Ser Ile Ala Ser Met Thr Ser Val
Val 995 1000 1005 Gly Gln Leu Ile Ile Ile Gly Val Val Gln Phe Ile
Val Phe Phe Ser 1010 1015 1020 Thr Ser Gln Gln Pro Trp Phe Thr Pro
Tyr Gln Pro Pro Val Asp Asp 1025 1030 1035 1040 Glu Val Glu Asp Lys
Arg Ser Met Gln Gly Thr Ala Leu Phe Cys Val 1045 1050 1055 Ser Met
Phe Gln Tyr Ile Ile Leu Ala Leu Val Tyr Ser Lys Gly Pro 1060 1065
1070 Pro Phe Arg Gly Asn Leu Trp Ser Asn Lys Pro Ile Tyr Lys Lys
Lys 1075 1080 1085 Arg Ser Ile Glu Ala Ile Ile Asp Tyr Val Pro Thr
Thr Asn Ser Asp 1090 1095 1100 His Ile Arg Arg Pro Ser Ile Asn Gly
Val Thr Ser Ser Arg Thr Glu 1105 1110 1115 1120 Ser Thr Leu Leu Ser
Ala Glu Gly Gln Gln Leu His Met Thr Thr Ser 1125 1130 1135 Lys Asn
Gly Lys Gly Gly Glu Asn Pro His Ser Ser Ala Leu Phe Glu 1140 1145
1150 Arg Leu Ile Ser Arg Ile Gly Gly Glu Pro Thr Trp Leu Thr Asn
Pro 1155 1160 1165 Ile Pro Pro His Ser Leu Ser Glu Pro Glu Glu Pro
Glu Lys Leu Glu 1170 1175 1180 Arg Thr Tyr 1185 67 7249 DNA Homo
Sapiens CDS (225)...(3995) 67 cacgcgtccg ggctgggctg aggcgaggcg
gcggcggcga cagcggcggc cgggtccccc 60 gcggcccctg gggctggtcc
ggccgcgagg gaggccgcgg aggaggcggc gcggcggcgg 120 ccagtgagcg
gccccgatct gacagacatc cctgaatctt ggtgtttgga cataggagtg 180
atcttccatt gtttgaagca ctggaccttt aatccactgt aggt atg gac agg gaa
236 Met Asp Arg Glu 1 gaa agg aag acc atc aat cag ggt caa gaa gat
gaa atg gag att tat 284 Glu Arg Lys Thr Ile Asn Gln Gly Gln Glu Asp
Glu Met Glu Ile Tyr 5 10 15 20 ggt tac aat ttg agt cgc tgg aag ctt
gcc ata gtt tct tta gga gtg 332 Gly Tyr Asn Leu Ser Arg Trp Lys Leu
Ala Ile Val Ser Leu Gly Val 25 30 35 att tgc tct gat ggg ttt ctc
ctc ctc ctc ctc tat tgg atg cct gag 380 Ile Cys Ser Asp Gly Phe Leu
Leu Leu Leu Leu Tyr Trp Met Pro Glu 40 45 50 tgg cgg gtg aaa gcg
acc tgt gtc aga gct gca att aaa gac tgt gaa 428 Trp Arg Val Lys Ala
Thr Cys Val Arg Ala Ala Ile Lys Asp Cys Glu 55 60 65 gta gtg ctg
ctg agg act act gat gaa ttc aaa atg tgg ttt tgt gca 476 Val Val Leu
Leu Arg Thr Thr Asp Glu Phe Lys Met Trp Phe Cys Ala 70 75 80 aaa
att cgc gtt ctt tct ttg gaa act tac cca gtt tca agt cca aaa 524 Lys
Ile Arg Val Leu Ser Leu Glu Thr Tyr Pro Val Ser Ser Pro Lys 85 90
95 100 tct atg tct aat aag ctt tca aat ggc cat gca gtt tgt tta att
gag 572 Ser Met Ser Asn Lys Leu Ser Asn Gly His Ala Val Cys Leu Ile
Glu 105 110 115 aat ccc act gaa gaa aat agg cac agg atc agt aaa tat
tca cag act 620 Asn Pro Thr Glu Glu Asn Arg His Arg Ile Ser Lys Tyr
Ser Gln Thr 120 125 130 gaa tca caa cag att cgt tat ttc acc cac cat
agt gta aaa tat ttc 668 Glu Ser Gln Gln Ile Arg Tyr Phe Thr His His
Ser Val Lys Tyr Phe 135 140 145 tgg aat gat acc att cac aat ttt gat
ttc tta aag gga ctg gat gaa 716 Trp Asn Asp Thr Ile His Asn Phe Asp
Phe Leu Lys Gly Leu Asp Glu 150 155 160 ggt gtt tct tgt acg tca att
tat gaa aag cat agt gca gga ctg aca 764 Gly Val Ser Cys Thr Ser Ile
Tyr Glu Lys His Ser Ala Gly Leu Thr 165 170 175 180 aag ggg atg cat
gcc tac aga aaa ctg ctt tat gga gta aat gaa att 812 Lys Gly Met His
Ala Tyr Arg Lys Leu Leu Tyr Gly Val Asn Glu Ile 185 190 195 gct gta
aaa gtg cct tct gtt ttt aag ctt cta att aaa gag gtt ctc 860 Ala Val
Lys Val Pro Ser Val Phe Lys Leu Leu Ile Lys Glu Val Leu 200 205 210
aac cca ttt tac att ttc cag ctg ttc agt gtt ata ctg tgg agc act 908
Asn Pro Phe Tyr Ile Phe Gln Leu Phe Ser Val Ile Leu Trp Ser Thr 215
220 225 gat gaa tac tat tac tat gct cta gct att gtg gtt atg tcc ata
gta 956 Asp Glu Tyr Tyr Tyr Tyr Ala Leu Ala Ile Val Val Met Ser Ile
Val 230 235 240 tca atc gta agc tca cta tat tcc att aga aag caa tat
gtt atg ttg 1004 Ser Ile Val Ser Ser Leu Tyr Ser Ile Arg Lys Gln
Tyr Val Met Leu 245 250 255 260 cat gac atg gtg gca act cat agt acc
gta aga gtt tca gtt tgt aga 1052 His Asp Met Val Ala Thr His Ser
Thr Val Arg Val Ser Val Cys Arg 265 270 275 gta aat gaa gaa ata gaa
gaa atc ttt tct acc gac ctt gtg cca gga 1100 Val Asn Glu Glu Ile
Glu Glu Ile Phe Ser Thr Asp Leu Val Pro Gly 280 285 290 gat gtc atg
gtc att cca tta aat ggg aca ata atg cct tgt gat gct 1148 Asp Val
Met Val Ile Pro Leu Asn Gly Thr Ile Met Pro Cys Asp Ala 295 300 305
gtg ctt att aat ggt acc tgc att gta aac gaa agc atg tta aca gga
1196 Val Leu Ile Asn Gly Thr Cys Ile Val Asn Glu Ser Met Leu Thr
Gly 310 315 320 gaa agt gtt cca gtg aca aag act aat ttg cca aat cct
tca gtg gat 1244 Glu Ser Val Pro Val Thr Lys Thr Asn Leu Pro Asn
Pro Ser Val Asp 325 330 335 340 gtg aaa gga ata gga gat gaa tta tat
aat cca gaa aca cat aaa cga 1292 Val Lys Gly Ile Gly Asp Glu Leu
Tyr Asn Pro Glu Thr His Lys Arg 345 350 355 cat act ttg ttt tgt ggg
aca act gtt att cag act cgt ttc tac act 1340 His Thr Leu Phe Cys
Gly Thr Thr Val Ile Gln Thr Arg Phe Tyr Thr 360 365 370 gga gaa ctc
gtc aaa gcc ata gtt gtt aga aca gga ttt agt act tcc 1388 Gly Glu
Leu Val Lys Ala Ile Val Val Arg Thr Gly Phe Ser Thr Ser 375 380 385
aaa gga cag ctt gtt cgt tcc ata ttg tat ccc aaa cca act gat ttt
1436 Lys Gly Gln Leu Val Arg Ser Ile Leu Tyr Pro Lys Pro Thr Asp
Phe 390 395 400 aaa ctc tac aga gat gcc tac ttg ttt cta cta tgt ctt
gtg gca gtt 1484 Lys Leu Tyr Arg Asp Ala Tyr Leu Phe Leu Leu Cys
Leu Val Ala Val 405 410 415 420 gct ggc att ggg ttt atc tac act att
att aat agc att tta aat gag 1532 Ala Gly Ile Gly Phe Ile Tyr Thr
Ile Ile Asn Ser Ile Leu Asn Glu 425 430 435 gta caa gtt ggg gtc ata
att atc gag tct ctt gat att atc aca att 1580 Val Gln Val Gly Val
Ile Ile Ile Glu Ser Leu Asp Ile Ile Thr Ile 440 445 450 act gtg ccc
cct gca ctt cct gct gca atg act gct ggt att gtg tat 1628 Thr Val
Pro Pro Ala Leu Pro Ala Ala Met Thr Ala Gly Ile Val Tyr 455 460 465
gct cag aga aga ctg aaa aaa atc ggt att ttc tgt atc agt cct caa
1676 Ala Gln Arg Arg Leu Lys Lys Ile Gly Ile Phe Cys Ile Ser Pro
Gln 470 475 480 aga ata aat att tgt gga cag ctc aat ctt gtt tgc ttt
gac aag act 1724 Arg Ile Asn Ile Cys Gly Gln Leu Asn Leu Val Cys
Phe Asp Lys Thr 485 490 495 500 gga act cta act gaa gat ggt tta gat
ctt tgg ggg att caa cga gtg 1772 Gly Thr Leu Thr Glu Asp Gly Leu
Asp Leu Trp Gly Ile Gln Arg Val 505 510 515 gaa aat gca cga ttt ctt
tca cca gaa gaa aat gtg tgc aat gag atg 1820 Glu Asn Ala Arg Phe
Leu Ser Pro Glu Glu Asn Val Cys Asn Glu Met 520 525 530 ttg gta aaa
tcc cag ttt gtt gct tgt atg gct act tgt cat tca ctt 1868 Leu Val
Lys Ser Gln Phe Val Ala Cys Met Ala Thr Cys His Ser Leu 535 540 545
aca aaa att gaa gga gtg ctc tct ggt gat cca ctt gat ctg aaa atg
1916 Thr Lys Ile Glu Gly Val Leu Ser Gly Asp Pro Leu Asp Leu Lys
Met 550 555 560 ttt gag gct att gga tgg att ctg gaa gaa gca act gaa
gaa gaa aca 1964 Phe Glu Ala Ile Gly Trp Ile Leu Glu Glu Ala Thr
Glu Glu Glu Thr 565 570 575 580 gca ctt cat aat cga att atg ccc aca
gtg gtt cgt cct ccc aaa caa 2012 Ala Leu His Asn Arg Ile Met Pro
Thr Val Val Arg Pro Pro Lys Gln 585 590 595 ctg ctt cct gaa tct acc
cct gca gga aac caa gaa atg gag ctg ttt 2060 Leu Leu Pro Glu Ser
Thr Pro Ala Gly Asn Gln Glu Met Glu Leu Phe 600 605 610 gaa ctt cca
gct act tat gag ata gga att gtt cgc cag ttc cca ttt 2108 Glu Leu
Pro Ala Thr Tyr Glu Ile Gly Ile Val Arg Gln Phe Pro Phe 615 620 625
tct tct gct ttg caa cgt atg agt gtg gtt gcc agg gtg ctg ggg gat
2156 Ser Ser Ala Leu Gln Arg Met Ser Val Val Ala Arg Val Leu Gly
Asp 630 635 640 agg aaa atg gac gcc tac atg aaa gga gcg ccc gag gcc
att gcc ggt 2204 Arg Lys Met Asp Ala Tyr Met Lys Gly Ala Pro Glu
Ala Ile Ala Gly 645 650 655 660 ctc tgt aaa cct gaa aca gtt cct gtc
gat ttt caa aac gtt ttg gaa 2252 Leu Cys Lys Pro Glu Thr Val Pro
Val Asp Phe Gln Asn Val Leu Glu 665 670 675 gac ttc act aaa cag ggc
ttc cgt gtg att gct ctt gca cac aga aaa 2300 Asp Phe Thr Lys Gln
Gly Phe Arg Val Ile Ala Leu Ala His Arg Lys 680 685 690 ttg gag tca
aaa ctg aca tgg cat aaa gta cag aat att agc aga gat 2348 Leu Glu
Ser Lys Leu Thr Trp His Lys Val Gln Asn Ile Ser
Arg Asp 695 700 705 gca att gag aac aac atg gat ttt atg gga tta att
ata atg cag aac 2396 Ala Ile Glu Asn Asn Met Asp Phe Met Gly Leu
Ile Ile Met Gln Asn 710 715 720 aaa tta aag caa gaa acc cct gca gta
ctt gaa gat ttg cat aaa gcc 2444 Lys Leu Lys Gln Glu Thr Pro Ala
Val Leu Glu Asp Leu His Lys Ala 725 730 735 740 aac att cgc acc gtc
atg gtc aca ggt gac agt atg ttg act gct gtc 2492 Asn Ile Arg Thr
Val Met Val Thr Gly Asp Ser Met Leu Thr Ala Val 745 750 755 tct gtg
gcc aga gat tgt gga atg att cta cct cag gat aaa gtg att 2540 Ser
Val Ala Arg Asp Cys Gly Met Ile Leu Pro Gln Asp Lys Val Ile 760 765
770 att gct gaa gca tta cct cca aag gat ggg aaa gtt gcc aaa ata aat
2588 Ile Ala Glu Ala Leu Pro Pro Lys Asp Gly Lys Val Ala Lys Ile
Asn 775 780 785 tgg cat tat gca gac tcc ctc acg cag tgc agt cat cca
tca gca att 2636 Trp His Tyr Ala Asp Ser Leu Thr Gln Cys Ser His
Pro Ser Ala Ile 790 795 800 gac cca gag gct att ccg gtt aaa ttg gtc
cat gat agc tta gag gat 2684 Asp Pro Glu Ala Ile Pro Val Lys Leu
Val His Asp Ser Leu Glu Asp 805 810 815 820 ctt caa atg act cgt tat
cat ttt gca atg aat gga aaa tca ttc tca 2732 Leu Gln Met Thr Arg
Tyr His Phe Ala Met Asn Gly Lys Ser Phe Ser 825 830 835 gtg ata ctg
gag cat ttt caa gac ctt gtt cct aag ttg atg ttg cat 2780 Val Ile
Leu Glu His Phe Gln Asp Leu Val Pro Lys Leu Met Leu His 840 845 850
ggc acc gtg ttt gcc cgt atg gca cct gat cag aag aca cag ttg ata
2828 Gly Thr Val Phe Ala Arg Met Ala Pro Asp Gln Lys Thr Gln Leu
Ile 855 860 865 gaa gca ttg caa aat gtt gat tat ttt gtt ggg atg tgt
ggt gat ggc 2876 Glu Ala Leu Gln Asn Val Asp Tyr Phe Val Gly Met
Cys Gly Asp Gly 870 875 880 gca aat gat tgt ggt gct ttg aag agg gca
cac gga ggc att tcc tta 2924 Ala Asn Asp Cys Gly Ala Leu Lys Arg
Ala His Gly Gly Ile Ser Leu 885 890 895 900 tcg gag ctc gaa gct tca
gtg gca tct ccc ttt acc tct aag act cct 2972 Ser Glu Leu Glu Ala
Ser Val Ala Ser Pro Phe Thr Ser Lys Thr Pro 905 910 915 agt att tcc
tgt gtg cca aac ctt atc agg gaa ggc cgt gct gct tta 3020 Ser Ile
Ser Cys Val Pro Asn Leu Ile Arg Glu Gly Arg Ala Ala Leu 920 925 930
ata act tcc ttc tgt gtg ttt aaa ttc atg gca ttg tac agc att atc
3068 Ile Thr Ser Phe Cys Val Phe Lys Phe Met Ala Leu Tyr Ser Ile
Ile 935 940 945 cag tac ttc agt gtt act ctg ctg tat tct atc tta agt
aac cta gga 3116 Gln Tyr Phe Ser Val Thr Leu Leu Tyr Ser Ile Leu
Ser Asn Leu Gly 950 955 960 gac ttc cag ttt ctc ttc att gat ctg gca
atc att ttg gta gtg gta 3164 Asp Phe Gln Phe Leu Phe Ile Asp Leu
Ala Ile Ile Leu Val Val Val 965 970 975 980 ttt aca atg agt tta aat
cct gcc tgg aaa gaa ctt gtg gca caa aga 3212 Phe Thr Met Ser Leu
Asn Pro Ala Trp Lys Glu Leu Val Ala Gln Arg 985 990 995 cca cct tcg
ggt ctt ata tct ggg gcc ctt ctc ttc tcc gtt ttg tct 3260 Pro Pro
Ser Gly Leu Ile Ser Gly Ala Leu Leu Phe Ser Val Leu Ser 1000 1005
1010 cag att atc atc tgc att gga ttt caa tct ttg ggt ttt ttt tgg
gtc 3308 Gln Ile Ile Ile Cys Ile Gly Phe Gln Ser Leu Gly Phe Phe
Trp Val 1015 1020 1025 aaa cag caa cct tgg tat gaa gtg tgg cat cca
aaa tca gat gct tgt 3356 Lys Gln Gln Pro Trp Tyr Glu Val Trp His
Pro Lys Ser Asp Ala Cys 1030 1035 1040 aat aca aca gga agc ggg ttt
tgg aat tct tca cac gta gac aat gaa 3404 Asn Thr Thr Gly Ser Gly
Phe Trp Asn Ser Ser His Val Asp Asn Glu 1045 1050 1055 1060 acc gaa
ctt gat gaa cat aat ata caa aat tat gaa aat acc aca gtg 3452 Thr
Glu Leu Asp Glu His Asn Ile Gln Asn Tyr Glu Asn Thr Thr Val 1065
1070 1075 ttt ttt att tcc agt ttt cag tac ctc ata gtg gca att gcc
ttt tca 3500 Phe Phe Ile Ser Ser Phe Gln Tyr Leu Ile Val Ala Ile
Ala Phe Ser 1080 1085 1090 aaa gga aaa ccc ttc agg caa cct tgc tac
aaa aat tat ttt ttt gtt 3548 Lys Gly Lys Pro Phe Arg Gln Pro Cys
Tyr Lys Asn Tyr Phe Phe Val 1095 1100 1105 ttt tct gtg att ttt tta
tat att ttt ata tta ttc atc atg ttg tat 3596 Phe Ser Val Ile Phe
Leu Tyr Ile Phe Ile Leu Phe Ile Met Leu Tyr 1110 1115 1120 cca gtt
gcc tct gtt gac cag gtt ctt cag ata gtg tgt gta cca tat 3644 Pro
Val Ala Ser Val Asp Gln Val Leu Gln Ile Val Cys Val Pro Tyr 1125
1130 1135 1140 cag tgg cgt gta act atg ctc atc att gtt ctt gtc aat
gcc ttt gtg 3692 Gln Trp Arg Val Thr Met Leu Ile Ile Val Leu Val
Asn Ala Phe Val 1145 1150 1155 tct atc aca gtg gag aac ttc ttc ctt
gac atg gtc ctt tgg aaa gtt 3740 Ser Ile Thr Val Glu Asn Phe Phe
Leu Asp Met Val Leu Trp Lys Val 1160 1165 1170 gtg ttc aac cga gac
aaa caa gga gag tat cgg ttc agc acc aca cag 3788 Val Phe Asn Arg
Asp Lys Gln Gly Glu Tyr Arg Phe Ser Thr Thr Gln 1175 1180 1185 cca
ccg cag gag tca gtg gat cgg tgg gga aaa tgc tgc tta ccc tgg 3836
Pro Pro Gln Glu Ser Val Asp Arg Trp Gly Lys Cys Cys Leu Pro Trp
1190 1195 1200 gcc ctg ggc tgt aga aag aag aca cca aag gca aag tac
atg tat ctg 3884 Ala Leu Gly Cys Arg Lys Lys Thr Pro Lys Ala Lys
Tyr Met Tyr Leu 1205 1210 1215 1220 gcg cag gag ctc ttg gtt gat cca
gaa tgg cca cca aaa cct cag aca 3932 Ala Gln Glu Leu Leu Val Asp
Pro Glu Trp Pro Pro Lys Pro Gln Thr 1225 1230 1235 acc aca gaa gct
aaa gct tta gtt aag gag aat gga tca tgt caa atc 3980 Thr Thr Glu
Ala Lys Ala Leu Val Lys Glu Asn Gly Ser Cys Gln Ile 1240 1245 1250
atc acc ata aca tag cagtgaatca gtctcagtgg tattgctgat agcagtattc
4035 Ile Thr Ile Thr * 1255 aggaatatgt gattttagga gtttctgatc
ctgtgtgtca gaatggcact agttcagttt 4095 atgtcccttc tgatatagta
gcttatttga cagctttgct cttccttaaa ataaaaacag 4155 aaaaatatat
cgtcctaaca gttaaattaa caatcaatcc ataaagtcct atatcttcat 4215
tcagcaaccc aaatattaca tacatttcca gaattttctt gattgttact ttcagtgata
4275 ttctttatat tgggtacagg agaagtttgg tgtttggtag gtttttcaac
attagttttt 4335 gagactagtt tacctcttca catttatgct cacaaccctc
ttgttagaaa agtctgtgtt 4395 tatatacagg ctgtaagttt gtgattgata
aaaagaagat gagtgttaat tagcctccag 4455 tgaaaatata ctgaaagcct
gttttcattt gattccaatg tttcttccaa agaattctgt 4515 ataaacatat
gccaattccc tatgatggtc tagagttagg aatgagtgtt tatggtgttg 4575
cttatagaac aactcaggta atctccattt ctggttttat attttctgta caaactgcct
4635 gggttttatt tttctaatca gcaaggtgct tcactgcctt cttgagacgc
ctctcaaagc 4695 tcttaaatgg ctcctgtgct atgtgtggtg ttggcagtct
aatttgcttc tgttaaatgt 4755 tgtagaacct ttttcactag gaaataagat
tcatttcttt cggcagtaga tgtagattca 4815 tcttttaacg tttcttcaaa
tttgtttctg tcaggctttg tgttatttta aatggttttt 4875 taaaattttc
ttctatgttt tcaattacct aaagacatag gataatagtt ttttttaagt 4935
tagaatttta cctcataaaa ttttttgagg tttgatgtat gtctctgtct tatcaataat
4995 gaggcttaaa aaatactgga tttgaatggc tgccgttttt tcaaagcaat
atgaatttga 5055 tgagtttgtt ttatgccatt aggtggcgcc agaggtcaga
acatgtctat tttgaattgg 5115 atcgttacaa atgagcatat ttgatgcgga
aatttctggg agaaaaaaaa ttgaggaaat 5175 aaagttaaaa aattgacatt
cattgagcca aaagagatgt ggagaaacat ttttcacctt 5235 tctgtttggc
ctgattaaca tttaaattct tgccaaaatt aaggtaactt ttaaaaaaca 5295
ccttttatag gtggatccag cagtctggca acgcccacag ttaccacaac acagaaaact
5355 gatcgtgcta taaaatggac gctaaactat gaaaacagtg tgacattgtt
ctctgttctt 5415 ccagagccag taacatgctt gctcgtgctt tctacttcta
gctgatcatt cttttcccaa 5475 catatattta caaattacca aattttacct
agaattttag gaccaaatgg ttctcactct 5535 ttatgctgca aagacctgga
tgatgtttgg taactataga aaaatagaaa ttacactcag 5595 gatcactgtt
actgctattg ccactgatga ttcctgcaaa aatataatcg aagttttcca 5655
tcaaatgtat aatatgctat taatacacat tagatgataa cagttgttcc atgaatgatt
5715 ctatgaagct atgcatctta gacctcttga gctgtgaatt agcactattt
tctatagtta 5775 cttattctct ggatcatttt ataatttcca tattaatttc
aaatatgctc gtgttattct 5835 tcagtgattt ccacaattgt gcattttatt
ctttggttta agtactgaag catataatga 5895 aagtaattgc taagtagcag
cttaaaaatt caattatccg attgtattta acatctttaa 5955 gagcatgatc
ataaagagct atttttgaca cccccccccc acttttttaa catttagagt 6015
tagtaagggt tttatatctc ttctgtccat attgttttca aaggaatgag gtgtttaggt
6075 ggctggaaaa gcatttgtag gaagttagat ttgaatatag acaaggtggg
ttattcacgt 6135 tgagaatgtt atttgaagaa tgcctgtgaa gccaggtgtg
ggttctactc agtgccatag 6195 atagactgag tcttctctcg taggttacca
ttacatagta attttgattc tgaattacac 6255 attaaattat ttgagtttat
acagacctaa attttaaaat ctgtacatat attattttga 6315 tgtattaaga
tgaatattgc tgatttaaat tttatttatg cacatactta aaggacagaa 6375
atgtctggga aagtaattgt taaataatga tatgtaactt tttaactttt taaataaata
6435 acaagatttt taatgtgtgt ctccctcagg gttgtttaaa gttttttttc
tccctcaagt 6495 ataaatagtg gtaactatat gttttgtatc ttctagcacc
aactgctgta aagcaatgct 6555 gcaaataatg cttgaataca agtggctaag
ccaacaacag aataaatact tttatagtag 6615 ttttataatc ctgaaattcg
aaagctttcc caattgcact tgcatctaaa caaaactgtt 6675 gcagttttta
ctctatttat tttgttcccc atgtttatga aagtcctgca cagtttcaaa 6735
ggcatggtaa ataatatatc aatgtttatg tagtctgtta cagaaacagc tatagataac
6795 attatccagt gaagagcaaa attcaagctt tagaaaatat tcatgcatgc
aattttgaca 6855 tatctaaaaa taggtttttg tatatttatg gtgggaggtg
gttgggaact tttaacaaaa 6915 tggggtgtta atttttgtac agtctgtggg
catttacaca tttttaatgt attaaaattt 6975 ggtaattatg tgtacattaa
attaataaaa gttacttcta gttatgattt gtgaattccc 7035 taagaccttg
gattttttta agtaacttta tatcagaaat gatactgcat ctttatattt 7095
ttaaaattgt attgctgctc aagaatggta ccctcttgtc aaaaaggcat acattcataa
7155 ttgtacattc agcattgtaa ataatcttat gaaacctttt ttgattgaag
ctattcaaaa 7215 taaaaatttt aatgaatgaa aaaaaaaaaa aaaa 7249 68 1256
PRT Homo Sapiens 68 Met Asp Arg Glu Glu Arg Lys Thr Ile Asn Gln Gly
Gln Glu Asp Glu 1 5 10 15 Met Glu Ile Tyr Gly Tyr Asn Leu Ser Arg
Trp Lys Leu Ala Ile Val 20 25 30 Ser Leu Gly Val Ile Cys Ser Asp
Gly Phe Leu Leu Leu Leu Leu Tyr 35 40 45 Trp Met Pro Glu Trp Arg
Val Lys Ala Thr Cys Val Arg Ala Ala Ile 50 55 60 Lys Asp Cys Glu
Val Val Leu Leu Arg Thr Thr Asp Glu Phe Lys Met 65 70 75 80 Trp Phe
Cys Ala Lys Ile Arg Val Leu Ser Leu Glu Thr Tyr Pro Val 85 90 95
Ser Ser Pro Lys Ser Met Ser Asn Lys Leu Ser Asn Gly His Ala Val 100
105 110 Cys Leu Ile Glu Asn Pro Thr Glu Glu Asn Arg His Arg Ile Ser
Lys 115 120 125 Tyr Ser Gln Thr Glu Ser Gln Gln Ile Arg Tyr Phe Thr
His His Ser 130 135 140 Val Lys Tyr Phe Trp Asn Asp Thr Ile His Asn
Phe Asp Phe Leu Lys 145 150 155 160 Gly Leu Asp Glu Gly Val Ser Cys
Thr Ser Ile Tyr Glu Lys His Ser 165 170 175 Ala Gly Leu Thr Lys Gly
Met His Ala Tyr Arg Lys Leu Leu Tyr Gly 180 185 190 Val Asn Glu Ile
Ala Val Lys Val Pro Ser Val Phe Lys Leu Leu Ile 195 200 205 Lys Glu
Val Leu Asn Pro Phe Tyr Ile Phe Gln Leu Phe Ser Val Ile 210 215 220
Leu Trp Ser Thr Asp Glu Tyr Tyr Tyr Tyr Ala Leu Ala Ile Val Val 225
230 235 240 Met Ser Ile Val Ser Ile Val Ser Ser Leu Tyr Ser Ile Arg
Lys Gln 245 250 255 Tyr Val Met Leu His Asp Met Val Ala Thr His Ser
Thr Val Arg Val 260 265 270 Ser Val Cys Arg Val Asn Glu Glu Ile Glu
Glu Ile Phe Ser Thr Asp 275 280 285 Leu Val Pro Gly Asp Val Met Val
Ile Pro Leu Asn Gly Thr Ile Met 290 295 300 Pro Cys Asp Ala Val Leu
Ile Asn Gly Thr Cys Ile Val Asn Glu Ser 305 310 315 320 Met Leu Thr
Gly Glu Ser Val Pro Val Thr Lys Thr Asn Leu Pro Asn 325 330 335 Pro
Ser Val Asp Val Lys Gly Ile Gly Asp Glu Leu Tyr Asn Pro Glu 340 345
350 Thr His Lys Arg His Thr Leu Phe Cys Gly Thr Thr Val Ile Gln Thr
355 360 365 Arg Phe Tyr Thr Gly Glu Leu Val Lys Ala Ile Val Val Arg
Thr Gly 370 375 380 Phe Ser Thr Ser Lys Gly Gln Leu Val Arg Ser Ile
Leu Tyr Pro Lys 385 390 395 400 Pro Thr Asp Phe Lys Leu Tyr Arg Asp
Ala Tyr Leu Phe Leu Leu Cys 405 410 415 Leu Val Ala Val Ala Gly Ile
Gly Phe Ile Tyr Thr Ile Ile Asn Ser 420 425 430 Ile Leu Asn Glu Val
Gln Val Gly Val Ile Ile Ile Glu Ser Leu Asp 435 440 445 Ile Ile Thr
Ile Thr Val Pro Pro Ala Leu Pro Ala Ala Met Thr Ala 450 455 460 Gly
Ile Val Tyr Ala Gln Arg Arg Leu Lys Lys Ile Gly Ile Phe Cys 465 470
475 480 Ile Ser Pro Gln Arg Ile Asn Ile Cys Gly Gln Leu Asn Leu Val
Cys 485 490 495 Phe Asp Lys Thr Gly Thr Leu Thr Glu Asp Gly Leu Asp
Leu Trp Gly 500 505 510 Ile Gln Arg Val Glu Asn Ala Arg Phe Leu Ser
Pro Glu Glu Asn Val 515 520 525 Cys Asn Glu Met Leu Val Lys Ser Gln
Phe Val Ala Cys Met Ala Thr 530 535 540 Cys His Ser Leu Thr Lys Ile
Glu Gly Val Leu Ser Gly Asp Pro Leu 545 550 555 560 Asp Leu Lys Met
Phe Glu Ala Ile Gly Trp Ile Leu Glu Glu Ala Thr 565 570 575 Glu Glu
Glu Thr Ala Leu His Asn Arg Ile Met Pro Thr Val Val Arg 580 585 590
Pro Pro Lys Gln Leu Leu Pro Glu Ser Thr Pro Ala Gly Asn Gln Glu 595
600 605 Met Glu Leu Phe Glu Leu Pro Ala Thr Tyr Glu Ile Gly Ile Val
Arg 610 615 620 Gln Phe Pro Phe Ser Ser Ala Leu Gln Arg Met Ser Val
Val Ala Arg 625 630 635 640 Val Leu Gly Asp Arg Lys Met Asp Ala Tyr
Met Lys Gly Ala Pro Glu 645 650 655 Ala Ile Ala Gly Leu Cys Lys Pro
Glu Thr Val Pro Val Asp Phe Gln 660 665 670 Asn Val Leu Glu Asp Phe
Thr Lys Gln Gly Phe Arg Val Ile Ala Leu 675 680 685 Ala His Arg Lys
Leu Glu Ser Lys Leu Thr Trp His Lys Val Gln Asn 690 695 700 Ile Ser
Arg Asp Ala Ile Glu Asn Asn Met Asp Phe Met Gly Leu Ile 705 710 715
720 Ile Met Gln Asn Lys Leu Lys Gln Glu Thr Pro Ala Val Leu Glu Asp
725 730 735 Leu His Lys Ala Asn Ile Arg Thr Val Met Val Thr Gly Asp
Ser Met 740 745 750 Leu Thr Ala Val Ser Val Ala Arg Asp Cys Gly Met
Ile Leu Pro Gln 755 760 765 Asp Lys Val Ile Ile Ala Glu Ala Leu Pro
Pro Lys Asp Gly Lys Val 770 775 780 Ala Lys Ile Asn Trp His Tyr Ala
Asp Ser Leu Thr Gln Cys Ser His 785 790 795 800 Pro Ser Ala Ile Asp
Pro Glu Ala Ile Pro Val Lys Leu Val His Asp 805 810 815 Ser Leu Glu
Asp Leu Gln Met Thr Arg Tyr His Phe Ala Met Asn Gly 820 825 830 Lys
Ser Phe Ser Val Ile Leu Glu His Phe Gln Asp Leu Val Pro Lys 835 840
845 Leu Met Leu His Gly Thr Val Phe Ala Arg Met Ala Pro Asp Gln Lys
850 855 860 Thr Gln Leu Ile Glu Ala Leu Gln Asn Val Asp Tyr Phe Val
Gly Met 865 870 875 880 Cys Gly Asp Gly Ala Asn Asp Cys Gly Ala Leu
Lys Arg Ala His Gly 885 890 895 Gly Ile Ser Leu Ser Glu Leu Glu Ala
Ser Val Ala Ser Pro Phe Thr 900 905 910 Ser Lys Thr Pro Ser Ile Ser
Cys Val Pro Asn Leu Ile Arg Glu Gly 915 920 925 Arg Ala Ala Leu Ile
Thr Ser Phe Cys Val Phe Lys Phe Met Ala Leu 930 935 940 Tyr Ser Ile
Ile Gln Tyr Phe Ser Val Thr Leu Leu Tyr Ser Ile Leu 945 950 955 960
Ser Asn Leu Gly Asp Phe Gln Phe Leu Phe Ile Asp Leu Ala Ile Ile 965
970 975 Leu Val Val Val Phe Thr Met Ser Leu Asn Pro Ala Trp Lys Glu
Leu 980 985 990 Val Ala Gln Arg Pro Pro Ser Gly Leu Ile Ser Gly Ala
Leu Leu Phe 995 1000 1005 Ser Val Leu Ser Gln Ile Ile Ile Cys Ile
Gly Phe Gln Ser Leu Gly 1010
1015 1020 Phe Phe Trp Val Lys Gln Gln Pro Trp Tyr Glu Val Trp His
Pro Lys 1025 1030 1035 1040 Ser Asp Ala Cys Asn Thr Thr Gly Ser Gly
Phe Trp Asn Ser Ser His 1045 1050 1055 Val Asp Asn Glu Thr Glu Leu
Asp Glu His Asn Ile Gln Asn Tyr Glu 1060 1065 1070 Asn Thr Thr Val
Phe Phe Ile Ser Ser Phe Gln Tyr Leu Ile Val Ala 1075 1080 1085 Ile
Ala Phe Ser Lys Gly Lys Pro Phe Arg Gln Pro Cys Tyr Lys Asn 1090
1095 1100 Tyr Phe Phe Val Phe Ser Val Ile Phe Leu Tyr Ile Phe Ile
Leu Phe 1105 1110 1115 1120 Ile Met Leu Tyr Pro Val Ala Ser Val Asp
Gln Val Leu Gln Ile Val 1125 1130 1135 Cys Val Pro Tyr Gln Trp Arg
Val Thr Met Leu Ile Ile Val Leu Val 1140 1145 1150 Asn Ala Phe Val
Ser Ile Thr Val Glu Asn Phe Phe Leu Asp Met Val 1155 1160 1165 Leu
Trp Lys Val Val Phe Asn Arg Asp Lys Gln Gly Glu Tyr Arg Phe 1170
1175 1180 Ser Thr Thr Gln Pro Pro Gln Glu Ser Val Asp Arg Trp Gly
Lys Cys 1185 1190 1195 1200 Cys Leu Pro Trp Ala Leu Gly Cys Arg Lys
Lys Thr Pro Lys Ala Lys 1205 1210 1215 Tyr Met Tyr Leu Ala Gln Glu
Leu Leu Val Asp Pro Glu Trp Pro Pro 1220 1225 1230 Lys Pro Gln Thr
Thr Thr Glu Ala Lys Ala Leu Val Lys Glu Asn Gly 1235 1240 1245 Ser
Cys Gln Ile Ile Thr Ile Thr 1250 1255 69 3768 DNA Homo Sapiens CDS
(1)...(3768) 69 atg gac agg gaa gaa agg aag acc atc aat cag ggt caa
gaa gat gaa 48 Met Asp Arg Glu Glu Arg Lys Thr Ile Asn Gln Gly Gln
Glu Asp Glu 1 5 10 15 atg gag att tat ggt tac aat ttg agt cgc tgg
aag ctt gcc ata gtt 96 Met Glu Ile Tyr Gly Tyr Asn Leu Ser Arg Trp
Lys Leu Ala Ile Val 20 25 30 tct tta gga gtg att tgc tct gat ggg
ttt ctc ctc ctc ctc ctc tat 144 Ser Leu Gly Val Ile Cys Ser Asp Gly
Phe Leu Leu Leu Leu Leu Tyr 35 40 45 tgg atg cct gag tgg cgg gtg
aaa gcg acc tgt gtc aga gct gca att 192 Trp Met Pro Glu Trp Arg Val
Lys Ala Thr Cys Val Arg Ala Ala Ile 50 55 60 aaa gac tgt gaa gta
gtg ctg ctg agg act act gat gaa ttc aaa atg 240 Lys Asp Cys Glu Val
Val Leu Leu Arg Thr Thr Asp Glu Phe Lys Met 65 70 75 80 tgg ttt tgt
gca aaa att cgc gtt ctt tct ttg gaa act tac cca gtt 288 Trp Phe Cys
Ala Lys Ile Arg Val Leu Ser Leu Glu Thr Tyr Pro Val 85 90 95 tca
agt cca aaa tct atg tct aat aag ctt tca aat ggc cat gca gtt 336 Ser
Ser Pro Lys Ser Met Ser Asn Lys Leu Ser Asn Gly His Ala Val 100 105
110 tgt tta att gag aat ccc act gaa gaa aat agg cac agg atc agt aaa
384 Cys Leu Ile Glu Asn Pro Thr Glu Glu Asn Arg His Arg Ile Ser Lys
115 120 125 tat tca cag act gaa tca caa cag att cgt tat ttc acc cac
cat agt 432 Tyr Ser Gln Thr Glu Ser Gln Gln Ile Arg Tyr Phe Thr His
His Ser 130 135 140 gta aaa tat ttc tgg aat gat acc att cac aat ttt
gat ttc tta aag 480 Val Lys Tyr Phe Trp Asn Asp Thr Ile His Asn Phe
Asp Phe Leu Lys 145 150 155 160 gga ctg gat gaa ggt gtt tct tgt acg
tca att tat gaa aag cat agt 528 Gly Leu Asp Glu Gly Val Ser Cys Thr
Ser Ile Tyr Glu Lys His Ser 165 170 175 gca gga ctg aca aag ggg atg
cat gcc tac aga aaa ctg ctt tat gga 576 Ala Gly Leu Thr Lys Gly Met
His Ala Tyr Arg Lys Leu Leu Tyr Gly 180 185 190 gta aat gaa att gct
gta aaa gtg cct tct gtt ttt aag ctt cta att 624 Val Asn Glu Ile Ala
Val Lys Val Pro Ser Val Phe Lys Leu Leu Ile 195 200 205 aaa gag gtt
ctc aac cca ttt tac att ttc cag ctg ttc agt gtt ata 672 Lys Glu Val
Leu Asn Pro Phe Tyr Ile Phe Gln Leu Phe Ser Val Ile 210 215 220 ctg
tgg agc act gat gaa tac tat tac tat gct cta gct att gtg gtt 720 Leu
Trp Ser Thr Asp Glu Tyr Tyr Tyr Tyr Ala Leu Ala Ile Val Val 225 230
235 240 atg tcc ata gta tca atc gta agc tca cta tat tcc att aga aag
caa 768 Met Ser Ile Val Ser Ile Val Ser Ser Leu Tyr Ser Ile Arg Lys
Gln 245 250 255 tat gtt atg ttg cat gac atg gtg gca act cat agt acc
gta aga gtt 816 Tyr Val Met Leu His Asp Met Val Ala Thr His Ser Thr
Val Arg Val 260 265 270 tca gtt tgt aga gta aat gaa gaa ata gaa gaa
atc ttt tct acc gac 864 Ser Val Cys Arg Val Asn Glu Glu Ile Glu Glu
Ile Phe Ser Thr Asp 275 280 285 ctt gtg cca gga gat gtc atg gtc att
cca tta aat ggg aca ata atg 912 Leu Val Pro Gly Asp Val Met Val Ile
Pro Leu Asn Gly Thr Ile Met 290 295 300 cct tgt gat gct gtg ctt att
aat ggt acc tgc att gta aac gaa agc 960 Pro Cys Asp Ala Val Leu Ile
Asn Gly Thr Cys Ile Val Asn Glu Ser 305 310 315 320 atg tta aca gga
gaa agt gtt cca gtg aca aag act aat ttg cca aat 1008 Met Leu Thr
Gly Glu Ser Val Pro Val Thr Lys Thr Asn Leu Pro Asn 325 330 335 cct
tca gtg gat gtg aaa gga ata gga gat gaa tta tat aat cca gaa 1056
Pro Ser Val Asp Val Lys Gly Ile Gly Asp Glu Leu Tyr Asn Pro Glu 340
345 350 aca cat aaa cga cat act ttg ttt tgt ggg aca act gtt att cag
act 1104 Thr His Lys Arg His Thr Leu Phe Cys Gly Thr Thr Val Ile
Gln Thr 355 360 365 cgt ttc tac act gga gaa ctc gtc aaa gcc ata gtt
gtt aga aca gga 1152 Arg Phe Tyr Thr Gly Glu Leu Val Lys Ala Ile
Val Val Arg Thr Gly 370 375 380 ttt agt act tcc aaa gga cag ctt gtt
cgt tcc ata ttg tat ccc aaa 1200 Phe Ser Thr Ser Lys Gly Gln Leu
Val Arg Ser Ile Leu Tyr Pro Lys 385 390 395 400 cca act gat ttt aaa
ctc tac aga gat gcc tac ttg ttt cta cta tgt 1248 Pro Thr Asp Phe
Lys Leu Tyr Arg Asp Ala Tyr Leu Phe Leu Leu Cys 405 410 415 ctt gtg
gca gtt gct ggc att ggg ttt atc tac act att att aat agc 1296 Leu
Val Ala Val Ala Gly Ile Gly Phe Ile Tyr Thr Ile Ile Asn Ser 420 425
430 att tta aat gag gta caa gtt ggg gtc ata att atc gag tct ctt gat
1344 Ile Leu Asn Glu Val Gln Val Gly Val Ile Ile Ile Glu Ser Leu
Asp 435 440 445 att atc aca att act gtg ccc cct gca ctt cct gct gca
atg act gct 1392 Ile Ile Thr Ile Thr Val Pro Pro Ala Leu Pro Ala
Ala Met Thr Ala 450 455 460 ggt att gtg tat gct cag aga aga ctg aaa
aaa atc ggt att ttc tgt 1440 Gly Ile Val Tyr Ala Gln Arg Arg Leu
Lys Lys Ile Gly Ile Phe Cys 465 470 475 480 atc agt cct caa aga ata
aat att tgt gga cag ctc aat ctt gtt tgc 1488 Ile Ser Pro Gln Arg
Ile Asn Ile Cys Gly Gln Leu Asn Leu Val Cys 485 490 495 ttt gac aag
act gga act cta act gaa gat ggt tta gat ctt tgg ggg 1536 Phe Asp
Lys Thr Gly Thr Leu Thr Glu Asp Gly Leu Asp Leu Trp Gly 500 505 510
att caa cga gtg gaa aat gca cga ttt ctt tca cca gaa gaa aat gtg
1584 Ile Gln Arg Val Glu Asn Ala Arg Phe Leu Ser Pro Glu Glu Asn
Val 515 520 525 tgc aat gag atg ttg gta aaa tcc cag ttt gtt gct tgt
atg gct act 1632 Cys Asn Glu Met Leu Val Lys Ser Gln Phe Val Ala
Cys Met Ala Thr 530 535 540 tgt cat tca ctt aca aaa att gaa gga gtg
ctc tct ggt gat cca ctt 1680 Cys His Ser Leu Thr Lys Ile Glu Gly
Val Leu Ser Gly Asp Pro Leu 545 550 555 560 gat ctg aaa atg ttt gag
gct att gga tgg att ctg gaa gaa gca act 1728 Asp Leu Lys Met Phe
Glu Ala Ile Gly Trp Ile Leu Glu Glu Ala Thr 565 570 575 gaa gaa gaa
aca gca ctt cat aat cga att atg ccc aca gtg gtt cgt 1776 Glu Glu
Glu Thr Ala Leu His Asn Arg Ile Met Pro Thr Val Val Arg 580 585 590
cct ccc aaa caa ctg ctt cct gaa tct acc cct gca gga aac caa gaa
1824 Pro Pro Lys Gln Leu Leu Pro Glu Ser Thr Pro Ala Gly Asn Gln
Glu 595 600 605 atg gag ctg ttt gaa ctt cca gct act tat gag ata gga
att gtt cgc 1872 Met Glu Leu Phe Glu Leu Pro Ala Thr Tyr Glu Ile
Gly Ile Val Arg 610 615 620 cag ttc cca ttt tct tct gct ttg caa cgt
atg agt gtg gtt gcc agg 1920 Gln Phe Pro Phe Ser Ser Ala Leu Gln
Arg Met Ser Val Val Ala Arg 625 630 635 640 gtg ctg ggg gat agg aaa
atg gac gcc tac atg aaa gga gcg ccc gag 1968 Val Leu Gly Asp Arg
Lys Met Asp Ala Tyr Met Lys Gly Ala Pro Glu 645 650 655 gcc att gcc
ggt ctc tgt aaa cct gaa aca gtt cct gtc gat ttt caa 2016 Ala Ile
Ala Gly Leu Cys Lys Pro Glu Thr Val Pro Val Asp Phe Gln 660 665 670
aac gtt ttg gaa gac ttc act aaa cag ggc ttc cgt gtg att gct ctt
2064 Asn Val Leu Glu Asp Phe Thr Lys Gln Gly Phe Arg Val Ile Ala
Leu 675 680 685 gca cac aga aaa ttg gag tca aaa ctg aca tgg cat aaa
gta cag aat 2112 Ala His Arg Lys Leu Glu Ser Lys Leu Thr Trp His
Lys Val Gln Asn 690 695 700 att agc aga gat gca att gag aac aac atg
gat ttt atg gga tta att 2160 Ile Ser Arg Asp Ala Ile Glu Asn Asn
Met Asp Phe Met Gly Leu Ile 705 710 715 720 ata atg cag aac aaa tta
aag caa gaa acc cct gca gta ctt gaa gat 2208 Ile Met Gln Asn Lys
Leu Lys Gln Glu Thr Pro Ala Val Leu Glu Asp 725 730 735 ttg cat aaa
gcc aac att cgc acc gtc atg gtc aca ggt gac agt atg 2256 Leu His
Lys Ala Asn Ile Arg Thr Val Met Val Thr Gly Asp Ser Met 740 745 750
ttg act gct gtc tct gtg gcc aga gat tgt gga atg att cta cct cag
2304 Leu Thr Ala Val Ser Val Ala Arg Asp Cys Gly Met Ile Leu Pro
Gln 755 760 765 gat aaa gtg att att gct gaa gca tta cct cca aag gat
ggg aaa gtt 2352 Asp Lys Val Ile Ile Ala Glu Ala Leu Pro Pro Lys
Asp Gly Lys Val 770 775 780 gcc aaa ata aat tgg cat tat gca gac tcc
ctc acg cag tgc agt cat 2400 Ala Lys Ile Asn Trp His Tyr Ala Asp
Ser Leu Thr Gln Cys Ser His 785 790 795 800 cca tca gca att gac cca
gag gct att ccg gtt aaa ttg gtc cat gat 2448 Pro Ser Ala Ile Asp
Pro Glu Ala Ile Pro Val Lys Leu Val His Asp 805 810 815 agc tta gag
gat ctt caa atg act cgt tat cat ttt gca atg aat gga 2496 Ser Leu
Glu Asp Leu Gln Met Thr Arg Tyr His Phe Ala Met Asn Gly 820 825 830
aaa tca ttc tca gtg ata ctg gag cat ttt caa gac ctt gtt cct aag
2544 Lys Ser Phe Ser Val Ile Leu Glu His Phe Gln Asp Leu Val Pro
Lys 835 840 845 ttg atg ttg cat ggc acc gtg ttt gcc cgt atg gca cct
gat cag aag 2592 Leu Met Leu His Gly Thr Val Phe Ala Arg Met Ala
Pro Asp Gln Lys 850 855 860 aca cag ttg ata gaa gca ttg caa aat gtt
gat tat ttt gtt ggg atg 2640 Thr Gln Leu Ile Glu Ala Leu Gln Asn
Val Asp Tyr Phe Val Gly Met 865 870 875 880 tgt ggt gat ggc gca aat
gat tgt ggt gct ttg aag agg gca cac gga 2688 Cys Gly Asp Gly Ala
Asn Asp Cys Gly Ala Leu Lys Arg Ala His Gly 885 890 895 ggc att tcc
tta tcg gag ctc gaa gct tca gtg gca tct ccc ttt acc 2736 Gly Ile
Ser Leu Ser Glu Leu Glu Ala Ser Val Ala Ser Pro Phe Thr 900 905 910
tct aag act cct agt att tcc tgt gtg cca aac ctt atc agg gaa ggc
2784 Ser Lys Thr Pro Ser Ile Ser Cys Val Pro Asn Leu Ile Arg Glu
Gly 915 920 925 cgt gct gct tta ata act tcc ttc tgt gtg ttt aaa ttc
atg gca ttg 2832 Arg Ala Ala Leu Ile Thr Ser Phe Cys Val Phe Lys
Phe Met Ala Leu 930 935 940 tac agc att atc cag tac ttc agt gtt act
ctg ctg tat tct atc tta 2880 Tyr Ser Ile Ile Gln Tyr Phe Ser Val
Thr Leu Leu Tyr Ser Ile Leu 945 950 955 960 agt aac cta gga gac ttc
cag ttt ctc ttc att gat ctg gca atc att 2928 Ser Asn Leu Gly Asp
Phe Gln Phe Leu Phe Ile Asp Leu Ala Ile Ile 965 970 975 ttg gta gtg
gta ttt aca atg agt tta aat cct gcc tgg aaa gaa ctt 2976 Leu Val
Val Val Phe Thr Met Ser Leu Asn Pro Ala Trp Lys Glu Leu 980 985 990
gtg gca caa aga cca cct tcg ggt ctt ata tct ggg gcc ctt ctc ttc
3024 Val Ala Gln Arg Pro Pro Ser Gly Leu Ile Ser Gly Ala Leu Leu
Phe 995 1000 1005 tcc gtt ttg tct cag att atc atc tgc att gga ttt
caa tct ttg ggt 3072 Ser Val Leu Ser Gln Ile Ile Ile Cys Ile Gly
Phe Gln Ser Leu Gly 1010 1015 1020 ttt ttt tgg gtc aaa cag caa cct
tgg tat gaa gtg tgg cat cca aaa 3120 Phe Phe Trp Val Lys Gln Gln
Pro Trp Tyr Glu Val Trp His Pro Lys 1025 1030 1035 1040 tca gat gct
tgt aat aca aca gga agc ggg ttt tgg aat tct tca cac 3168 Ser Asp
Ala Cys Asn Thr Thr Gly Ser Gly Phe Trp Asn Ser Ser His 1045 1050
1055 gta gac aat gaa acc gaa ctt gat gaa cat aat ata caa aat tat
gaa 3216 Val Asp Asn Glu Thr Glu Leu Asp Glu His Asn Ile Gln Asn
Tyr Glu 1060 1065 1070 aat acc aca gtg ttt ttt att tcc agt ttt cag
tac ctc ata gtg gca 3264 Asn Thr Thr Val Phe Phe Ile Ser Ser Phe
Gln Tyr Leu Ile Val Ala 1075 1080 1085 att gcc ttt tca aaa gga aaa
ccc ttc agg caa cct tgc tac aaa aat 3312 Ile Ala Phe Ser Lys Gly
Lys Pro Phe Arg Gln Pro Cys Tyr Lys Asn 1090 1095 1100 tat ttt ttt
gtt ttt tct gtg att ttt tta tat att ttt ata tta ttc 3360 Tyr Phe
Phe Val Phe Ser Val Ile Phe Leu Tyr Ile Phe Ile Leu Phe 1105 1110
1115 1120 atc atg ttg tat cca gtt gcc tct gtt gac cag gtt ctt cag
ata gtg 3408 Ile Met Leu Tyr Pro Val Ala Ser Val Asp Gln Val Leu
Gln Ile Val 1125 1130 1135 tgt gta cca tat cag tgg cgt gta act atg
ctc atc att gtt ctt gtc 3456 Cys Val Pro Tyr Gln Trp Arg Val Thr
Met Leu Ile Ile Val Leu Val 1140 1145 1150 aat gcc ttt gtg tct atc
aca gtg gag aac ttc ttc ctt gac atg gtc 3504 Asn Ala Phe Val Ser
Ile Thr Val Glu Asn Phe Phe Leu Asp Met Val 1155 1160 1165 ctt tgg
aaa gtt gtg ttc aac cga gac aaa caa gga gag tat cgg ttc 3552 Leu
Trp Lys Val Val Phe Asn Arg Asp Lys Gln Gly Glu Tyr Arg Phe 1170
1175 1180 agc acc aca cag cca ccg cag gag tca gtg gat cgg tgg gga
aaa tgc 3600 Ser Thr Thr Gln Pro Pro Gln Glu Ser Val Asp Arg Trp
Gly Lys Cys 1185 1190 1195 1200 tgc tta ccc tgg gcc ctg ggc tgt aga
aag aag aca cca aag gca aag 3648 Cys Leu Pro Trp Ala Leu Gly Cys
Arg Lys Lys Thr Pro Lys Ala Lys 1205 1210 1215 tac atg tat ctg gcg
cag gag ctc ttg gtt gat cca gaa tgg cca cca 3696 Tyr Met Tyr Leu
Ala Gln Glu Leu Leu Val Asp Pro Glu Trp Pro Pro 1220 1225 1230 aaa
cct cag aca acc aca gaa gct aaa gct tta gtt aag gag aat gga 3744
Lys Pro Gln Thr Thr Thr Glu Ala Lys Ala Leu Val Lys Glu Asn Gly
1235 1240 1245 tca tgt caa atc atc acc ata aca 3768 Ser Cys Gln Ile
Ile Thr Ile Thr 1250 1255 70 3919 DNA Homo Sapiens CDS
(68)...(3682) 70 ttaccggaag taaaacttcg gaagtgaggc gttcctctgc
ccggaagtga gcgccgcgct 60 aggaaag atg gcg gca gcg gcg gcg gtg ggc
aac gcg gtg ccc tgc ggg 109 Met Ala Ala Ala Ala Ala Val Gly Asn Ala
Val Pro Cys Gly 1 5 10 gcc cgg cct tgc ggg gtc cgg cct gac ggg cag
ccc aag ccc ggg ccg 157 Ala Arg Pro Cys Gly Val Arg Pro Asp Gly Gln
Pro Lys Pro Gly Pro 15 20 25 30 cag ccg cgc gcg ctc ctt gcc gcc ggg
ccg gcg ctc ata gcg aac ggt 205 Gln Pro Arg Ala Leu Leu Ala Ala Gly
Pro Ala Leu Ile Ala Asn Gly 35 40 45 gac gag ctg gtg gct gcc gtg
tgg ccg tac cgg cgg ttg gcg ctg ttg 253 Asp Glu Leu Val Ala Ala Val
Trp Pro Tyr Arg Arg Leu Ala Leu Leu 50 55 60 cgg cgc ctc acg gtg
ctg cca ttc gcc ggg ctg ctt tac ccg gcc tgg 301 Arg Arg Leu Thr Val
Leu Pro Phe Ala Gly Leu Leu Tyr Pro Ala Trp 65 70 75 ttg ggt gcc
gca gcc gct ggc tgc tgg ggc tgg ggc agc agt tgg gtg 349 Leu Gly Ala
Ala Ala Ala Gly Cys Trp Gly Trp Gly Ser Ser Trp Val 80 85
90 cag atc ccc gaa gct gcg ctg ctc gtg ctt gcc acc atc tgc ctc gcg
397 Gln Ile Pro Glu Ala Ala Leu Leu Val Leu Ala Thr Ile Cys Leu Ala
95 100 105 110 cac gcg ctc act gtc ctc tcg ggg cat tgg tct gtg cac
gcg cat tgc 445 His Ala Leu Thr Val Leu Ser Gly His Trp Ser Val His
Ala His Cys 115 120 125 gcg ctc acc tgc acc ccg gag tac gac ccc agc
aaa gcg acc ttt gtg 493 Ala Leu Thr Cys Thr Pro Glu Tyr Asp Pro Ser
Lys Ala Thr Phe Val 130 135 140 aag gtg gcg cca acc ccc aac aat ggc
tcc acg gag ctc gtg gcc ctg 541 Lys Val Ala Pro Thr Pro Asn Asn Gly
Ser Thr Glu Leu Val Ala Leu 145 150 155 cac cgc aat gag ggc gaa gac
ggg ctt gag gtg ctg tcc ttc gaa ttc 589 His Arg Asn Glu Gly Glu Asp
Gly Leu Glu Val Leu Ser Phe Glu Phe 160 165 170 cag aag atc aag tat
tcc tac gat gcc ctg gag aag aag cag ttt ctc 637 Gln Lys Ile Lys Tyr
Ser Tyr Asp Ala Leu Glu Lys Lys Gln Phe Leu 175 180 185 190 ccc gtg
gcc ttt cct gtg gga aac gcc ttc tca tac tat cag agc aac 685 Pro Val
Ala Phe Pro Val Gly Asn Ala Phe Ser Tyr Tyr Gln Ser Asn 195 200 205
aga ggc ttc cag gaa gac tca gag atc cga gca gct gag aag aaa ttt 733
Arg Gly Phe Gln Glu Asp Ser Glu Ile Arg Ala Ala Glu Lys Lys Phe 210
215 220 ggg agc aac aag gcc gag atg gtg gtg cct gac ttc tcg gag ctt
ttc 781 Gly Ser Asn Lys Ala Glu Met Val Val Pro Asp Phe Ser Glu Leu
Phe 225 230 235 aag gag aga gcc aca gcc ccc ttc ttt gta ttt cag gtg
ttc tgt gtg 829 Lys Glu Arg Ala Thr Ala Pro Phe Phe Val Phe Gln Val
Phe Cys Val 240 245 250 ggg ctc tgg tgc ctg gat gag tac tgg tac tac
agc gtc ttt acg cta 877 Gly Leu Trp Cys Leu Asp Glu Tyr Trp Tyr Tyr
Ser Val Phe Thr Leu 255 260 265 270 tcc atg ctg gtg gcg ttc gag gcc
tcg ctg gtg cag cag cag atg cgg 925 Ser Met Leu Val Ala Phe Glu Ala
Ser Leu Val Gln Gln Gln Met Arg 275 280 285 aac atg tcg gag atc cgg
aag atg ggc aac aag ccc cac atg atc cag 973 Asn Met Ser Glu Ile Arg
Lys Met Gly Asn Lys Pro His Met Ile Gln 290 295 300 gtc tac cga agc
cgc aag tgg agg ccc att gcc agt gat gag atc gta 1021 Val Tyr Arg
Ser Arg Lys Trp Arg Pro Ile Ala Ser Asp Glu Ile Val 305 310 315 cca
ggg gac atc gtc tcc atc ggc cgc tcc cca cag gag aac ctg gtg 1069
Pro Gly Asp Ile Val Ser Ile Gly Arg Ser Pro Gln Glu Asn Leu Val 320
325 330 cca tgt gac gtg ctt ctg ctg cga ggc cgc tgc atc gta gac gag
gcc 1117 Pro Cys Asp Val Leu Leu Leu Arg Gly Arg Cys Ile Val Asp
Glu Ala 335 340 345 350 atg ctc acg ggg gag tcc gtg cca cag atg aag
gag ccc atc gaa gac 1165 Met Leu Thr Gly Glu Ser Val Pro Gln Met
Lys Glu Pro Ile Glu Asp 355 360 365 ctc agc cca gac cgg gtg ctg gac
ctc cag gct gat tcc cgg ctg cac 1213 Leu Ser Pro Asp Arg Val Leu
Asp Leu Gln Ala Asp Ser Arg Leu His 370 375 380 gtc atc ttc ggg ggc
acc aag gtg gtg cag cac atc ccc cca cag aaa 1261 Val Ile Phe Gly
Gly Thr Lys Val Val Gln His Ile Pro Pro Gln Lys 385 390 395 gcc acc
acg ggc ctg aag ccg gtt gac agc ggg tgc gtg gcc tac gtc 1309 Ala
Thr Thr Gly Leu Lys Pro Val Asp Ser Gly Cys Val Ala Tyr Val 400 405
410 ctg cgg acc gga ttc aac aca tct cag ggc aag ctg ctg cgc acc atc
1357 Leu Arg Thr Gly Phe Asn Thr Ser Gln Gly Lys Leu Leu Arg Thr
Ile 415 420 425 430 ctc ttc ggg gtc aag agg gtg act gcg aac aac ctg
gag acc ttc atc 1405 Leu Phe Gly Val Lys Arg Val Thr Ala Asn Asn
Leu Glu Thr Phe Ile 435 440 445 ttc atc ctc ttc ctc ctg gtg ttt gcc
atc gct gca gct gcc tat gta 1453 Phe Ile Leu Phe Leu Leu Val Phe
Ala Ile Ala Ala Ala Ala Tyr Val 450 455 460 tgg att gaa ggt acc aag
gac ccc agc cgg aac cgc tac aag ctg ttt 1501 Trp Ile Glu Gly Thr
Lys Asp Pro Ser Arg Asn Arg Tyr Lys Leu Phe 465 470 475 ctg gag tgc
acc ctg atc ctc acc tcg gtc gtg cct cct gag ctg ccc 1549 Leu Glu
Cys Thr Leu Ile Leu Thr Ser Val Val Pro Pro Glu Leu Pro 480 485 490
atc gag ctg tcc ctg gcc gtc aac acc tcc ctc atc gcc ctg gcc aag
1597 Ile Glu Leu Ser Leu Ala Val Asn Thr Ser Leu Ile Ala Leu Ala
Lys 495 500 505 510 ctc tac atg tac tgc aca gag ccc ttc cgg atc ccc
ttt gct ggc aag 1645 Leu Tyr Met Tyr Cys Thr Glu Pro Phe Arg Ile
Pro Phe Ala Gly Lys 515 520 525 gtc gag gtg tgc tgc ttt gac aag acg
ggg acg ttg acc agt gac agc 1693 Val Glu Val Cys Cys Phe Asp Lys
Thr Gly Thr Leu Thr Ser Asp Ser 530 535 540 ctg gtg gtg cgc ggt gtg
gcc ggg ctg aga gac ggg aag gag gtg acc 1741 Leu Val Val Arg Gly
Val Ala Gly Leu Arg Asp Gly Lys Glu Val Thr 545 550 555 cca gtg tcc
agc atc cct gta gaa aca cac cgg gcc ctg gcc tcg tgc 1789 Pro Val
Ser Ser Ile Pro Val Glu Thr His Arg Ala Leu Ala Ser Cys 560 565 570
cac tcg ctc atg cag ctg gac gac ggc acc ctc gtg ggt gac cct cta
1837 His Ser Leu Met Gln Leu Asp Asp Gly Thr Leu Val Gly Asp Pro
Leu 575 580 585 590 gag aag gcc atg ctg acg gcc gtg gac tgg acg ctg
acc aaa gat gag 1885 Glu Lys Ala Met Leu Thr Ala Val Asp Trp Thr
Leu Thr Lys Asp Glu 595 600 605 aaa gta ttc ccc cga agt att aaa act
cag ggg ctg aaa att cac cag 1933 Lys Val Phe Pro Arg Ser Ile Lys
Thr Gln Gly Leu Lys Ile His Gln 610 615 620 cgc ttt cat ttt gcc agt
gcc ctg aag cga atg tcc gtg ctt gcc tcg 1981 Arg Phe His Phe Ala
Ser Ala Leu Lys Arg Met Ser Val Leu Ala Ser 625 630 635 tat gag aag
ctg ggc tcc acc gac ctc tgc tac atc gcg gcc gtg aag 2029 Tyr Glu
Lys Leu Gly Ser Thr Asp Leu Cys Tyr Ile Ala Ala Val Lys 640 645 650
ggg gcc ccc gaa act ctg cac tcc atg ttc tcc cag tgc ccg ccc gac
2077 Gly Ala Pro Glu Thr Leu His Ser Met Phe Ser Gln Cys Pro Pro
Asp 655 660 665 670 tac cac cac atc cac acc gag atc tcc cgg gaa gga
gcc cgc gtc ctg 2125 Tyr His His Ile His Thr Glu Ile Ser Arg Glu
Gly Ala Arg Val Leu 675 680 685 gcg ctg ggg tac aag gag ctg gga cac
ctc act cac cag cag gcc cgg 2173 Ala Leu Gly Tyr Lys Glu Leu Gly
His Leu Thr His Gln Gln Ala Arg 690 695 700 gag gtc aag cgg gag gcc
ctg gag tgc agc ctc aag ttc gtc ggc ttc 2221 Glu Val Lys Arg Glu
Ala Leu Glu Cys Ser Leu Lys Phe Val Gly Phe 705 710 715 att gtg gtc
tcc tgc ccg ctc aag gct gac tcc aag gcc gtg atc cgg 2269 Ile Val
Val Ser Cys Pro Leu Lys Ala Asp Ser Lys Ala Val Ile Arg 720 725 730
gag atc cag aat gcg tcc cac cgg gtg gtc atg atc acg gga gac aac
2317 Glu Ile Gln Asn Ala Ser His Arg Val Val Met Ile Thr Gly Asp
Asn 735 740 745 750 ccg ctc act gca tgc cac gtg gcc cag gag ctg cac
ttc att gaa aag 2365 Pro Leu Thr Ala Cys His Val Ala Gln Glu Leu
His Phe Ile Glu Lys 755 760 765 gcc cac acg ctg atc ctg cag cct ccc
tcc gag aaa ggc cgg cag tgc 2413 Ala His Thr Leu Ile Leu Gln Pro
Pro Ser Glu Lys Gly Arg Gln Cys 770 775 780 gag tgg cgc tcc att gac
ggc agc atc gtg ctg ccc ctg gcc cgg ggc 2461 Glu Trp Arg Ser Ile
Asp Gly Ser Ile Val Leu Pro Leu Ala Arg Gly 785 790 795 tcc cca aag
gca ctg gcc ctg gag tac gca ctg tgc ctc aca ggc gac 2509 Ser Pro
Lys Ala Leu Ala Leu Glu Tyr Ala Leu Cys Leu Thr Gly Asp 800 805 810
ggc ttg gcc cac ctg cag gcc acc gac ccc cag cag ctg ctc cgc ctc
2557 Gly Leu Ala His Leu Gln Ala Thr Asp Pro Gln Gln Leu Leu Arg
Leu 815 820 825 830 atc ccc cat gtg cag gtg ttc gcc cgt gtg gct ccc
aag cag aag gag 2605 Ile Pro His Val Gln Val Phe Ala Arg Val Ala
Pro Lys Gln Lys Glu 835 840 845 ttt gtc atc acc agc ctg aag gag ctg
ggc tac gtg acc ctc atg tgt 2653 Phe Val Ile Thr Ser Leu Lys Glu
Leu Gly Tyr Val Thr Leu Met Cys 850 855 860 ggg gat ggc acc aac gac
gtg ggc gcc ctg aag cat gct gac gtg ggt 2701 Gly Asp Gly Thr Asn
Asp Val Gly Ala Leu Lys His Ala Asp Val Gly 865 870 875 gtg gcg ctc
ttg gcc aat gcc cct gag cgg gtt gtc gag cgg cga cgg 2749 Val Ala
Leu Leu Ala Asn Ala Pro Glu Arg Val Val Glu Arg Arg Arg 880 885 890
cgg ccc cgg gac agc cca acc ctg agc aac agt ggc atc aga gcc acc
2797 Arg Pro Arg Asp Ser Pro Thr Leu Ser Asn Ser Gly Ile Arg Ala
Thr 895 900 905 910 tcc agg aca gcc aag cag cgg tcg ggg ctc cct ccc
tcc gag gag cag 2845 Ser Arg Thr Ala Lys Gln Arg Ser Gly Leu Pro
Pro Ser Glu Glu Gln 915 920 925 cca acc tcc cag agg gac cgc ctg agc
cag gtg ctg cga gac ctc gag 2893 Pro Thr Ser Gln Arg Asp Arg Leu
Ser Gln Val Leu Arg Asp Leu Glu 930 935 940 gac gag agt acg ccc att
gtg aaa ctg ggg gat gcc agc atc gca gca 2941 Asp Glu Ser Thr Pro
Ile Val Lys Leu Gly Asp Ala Ser Ile Ala Ala 945 950 955 ccc ttc acc
tcc aag ctc tca tcc atc cag tgc atc tgc cac gtg atc 2989 Pro Phe
Thr Ser Lys Leu Ser Ser Ile Gln Cys Ile Cys His Val Ile 960 965 970
aag cag ggc cgc tgc acg ctg gtg acc acg cta cag atg ttc aag atc
3037 Lys Gln Gly Arg Cys Thr Leu Val Thr Thr Leu Gln Met Phe Lys
Ile 975 980 985 990 ctg gcg ctc aat gcc ctc atc ctg gcc tac agc cag
agc gtc ctc tac 3085 Leu Ala Leu Asn Ala Leu Ile Leu Ala Tyr Ser
Gln Ser Val Leu Tyr 995 1000 1005 ctg gag gga gtc aag ttc agt gac
ttc cag gcc acc cta cag ggg ctg 3133 Leu Glu Gly Val Lys Phe Ser
Asp Phe Gln Ala Thr Leu Gln Gly Leu 1010 1015 1020 ctg ctg gcc ggc
tgc ttc ctc ttc atc tcc cgt tcc aag ccc ctc aag 3181 Leu Leu Ala
Gly Cys Phe Leu Phe Ile Ser Arg Ser Lys Pro Leu Lys 1025 1030 1035
acc ctc tcc cga gaa cgg ccc ctg ccc aac atc ttc aac ctg tac acc
3229 Thr Leu Ser Arg Glu Arg Pro Leu Pro Asn Ile Phe Asn Leu Tyr
Thr 1040 1045 1050 atc ctc acc gtc atg ctc cag ttc ttt gtg cac ttc
ctg agc ctt gtc 3277 Ile Leu Thr Val Met Leu Gln Phe Phe Val His
Phe Leu Ser Leu Val 1055 1060 1065 1070 tac ctg tac cgt gag gcc cag
gcc cgg agc ccc gag aag cag gag cag 3325 Tyr Leu Tyr Arg Glu Ala
Gln Ala Arg Ser Pro Glu Lys Gln Glu Gln 1075 1080 1085 ttc gtg gac
ttg tac aag gag ttt gag cca agc ctg gtc aac agc acc 3373 Phe Val
Asp Leu Tyr Lys Glu Phe Glu Pro Ser Leu Val Asn Ser Thr 1090 1095
1100 gtc tac atc atg gcc atg gcc atg cag atg gcc acc ttc gcc atc
aat 3421 Val Tyr Ile Met Ala Met Ala Met Gln Met Ala Thr Phe Ala
Ile Asn 1105 1110 1115 tac aaa ggc ccg ccc ttc atg gag agc ctg ccc
gag aac aag ccc ctg 3469 Tyr Lys Gly Pro Pro Phe Met Glu Ser Leu
Pro Glu Asn Lys Pro Leu 1120 1125 1130 gtg tgg agt ctg gca gtt tca
ctc ctg gcc atc att ggc ctg ctc ctc 3517 Val Trp Ser Leu Ala Val
Ser Leu Leu Ala Ile Ile Gly Leu Leu Leu 1135 1140 1145 1150 ggc tcc
tcg ccc gac ttc aac agc cag ttt ggc ctc gtg gac atc cct 3565 Gly
Ser Ser Pro Asp Phe Asn Ser Gln Phe Gly Leu Val Asp Ile Pro 1155
1160 1165 gtg gag ttc aag ctg gtc att gcc cag gtc ctg ctc ctg gac
ttc tgc 3613 Val Glu Phe Lys Leu Val Ile Ala Gln Val Leu Leu Leu
Asp Phe Cys 1170 1175 1180 ctg gcg ctc ctg gcc gac cgc gtc ctg cag
ttc ttc ctg ggg acc ccg 3661 Leu Ala Leu Leu Ala Asp Arg Val Leu
Gln Phe Phe Leu Gly Thr Pro 1185 1190 1195 aag ctg aaa gtg cct tcc
tga gatggcagtg ctggtaccca ctgcccaccc 3712 Lys Leu Lys Val Pro Ser *
1200 tggctgccgc tgggcgggaa ccccaacagg gccccgggag ggaaccctgc
ccccaacccc 3772 ccacagcaag gctgtacagt ctcgcccttg gaagactgag
ctgggacccc cacagccatc 3832 cgctggcttg gccagcagaa ccagccccaa
gccagcacct ttggtaaata aagcagcatc 3892 tgagatttta aaaaaaaaaa aaaaaaa
3919 71 1204 PRT Homo Sapiens 71 Met Ala Ala Ala Ala Ala Val Gly
Asn Ala Val Pro Cys Gly Ala Arg 1 5 10 15 Pro Cys Gly Val Arg Pro
Asp Gly Gln Pro Lys Pro Gly Pro Gln Pro 20 25 30 Arg Ala Leu Leu
Ala Ala Gly Pro Ala Leu Ile Ala Asn Gly Asp Glu 35 40 45 Leu Val
Ala Ala Val Trp Pro Tyr Arg Arg Leu Ala Leu Leu Arg Arg 50 55 60
Leu Thr Val Leu Pro Phe Ala Gly Leu Leu Tyr Pro Ala Trp Leu Gly 65
70 75 80 Ala Ala Ala Ala Gly Cys Trp Gly Trp Gly Ser Ser Trp Val
Gln Ile 85 90 95 Pro Glu Ala Ala Leu Leu Val Leu Ala Thr Ile Cys
Leu Ala His Ala 100 105 110 Leu Thr Val Leu Ser Gly His Trp Ser Val
His Ala His Cys Ala Leu 115 120 125 Thr Cys Thr Pro Glu Tyr Asp Pro
Ser Lys Ala Thr Phe Val Lys Val 130 135 140 Ala Pro Thr Pro Asn Asn
Gly Ser Thr Glu Leu Val Ala Leu His Arg 145 150 155 160 Asn Glu Gly
Glu Asp Gly Leu Glu Val Leu Ser Phe Glu Phe Gln Lys 165 170 175 Ile
Lys Tyr Ser Tyr Asp Ala Leu Glu Lys Lys Gln Phe Leu Pro Val 180 185
190 Ala Phe Pro Val Gly Asn Ala Phe Ser Tyr Tyr Gln Ser Asn Arg Gly
195 200 205 Phe Gln Glu Asp Ser Glu Ile Arg Ala Ala Glu Lys Lys Phe
Gly Ser 210 215 220 Asn Lys Ala Glu Met Val Val Pro Asp Phe Ser Glu
Leu Phe Lys Glu 225 230 235 240 Arg Ala Thr Ala Pro Phe Phe Val Phe
Gln Val Phe Cys Val Gly Leu 245 250 255 Trp Cys Leu Asp Glu Tyr Trp
Tyr Tyr Ser Val Phe Thr Leu Ser Met 260 265 270 Leu Val Ala Phe Glu
Ala Ser Leu Val Gln Gln Gln Met Arg Asn Met 275 280 285 Ser Glu Ile
Arg Lys Met Gly Asn Lys Pro His Met Ile Gln Val Tyr 290 295 300 Arg
Ser Arg Lys Trp Arg Pro Ile Ala Ser Asp Glu Ile Val Pro Gly 305 310
315 320 Asp Ile Val Ser Ile Gly Arg Ser Pro Gln Glu Asn Leu Val Pro
Cys 325 330 335 Asp Val Leu Leu Leu Arg Gly Arg Cys Ile Val Asp Glu
Ala Met Leu 340 345 350 Thr Gly Glu Ser Val Pro Gln Met Lys Glu Pro
Ile Glu Asp Leu Ser 355 360 365 Pro Asp Arg Val Leu Asp Leu Gln Ala
Asp Ser Arg Leu His Val Ile 370 375 380 Phe Gly Gly Thr Lys Val Val
Gln His Ile Pro Pro Gln Lys Ala Thr 385 390 395 400 Thr Gly Leu Lys
Pro Val Asp Ser Gly Cys Val Ala Tyr Val Leu Arg 405 410 415 Thr Gly
Phe Asn Thr Ser Gln Gly Lys Leu Leu Arg Thr Ile Leu Phe 420 425 430
Gly Val Lys Arg Val Thr Ala Asn Asn Leu Glu Thr Phe Ile Phe Ile 435
440 445 Leu Phe Leu Leu Val Phe Ala Ile Ala Ala Ala Ala Tyr Val Trp
Ile 450 455 460 Glu Gly Thr Lys Asp Pro Ser Arg Asn Arg Tyr Lys Leu
Phe Leu Glu 465 470 475 480 Cys Thr Leu Ile Leu Thr Ser Val Val Pro
Pro Glu Leu Pro Ile Glu 485 490 495 Leu Ser Leu Ala Val Asn Thr Ser
Leu Ile Ala Leu Ala Lys Leu Tyr 500 505 510 Met Tyr Cys Thr Glu Pro
Phe Arg Ile Pro Phe Ala Gly Lys Val Glu 515 520 525 Val Cys Cys Phe
Asp Lys Thr Gly Thr Leu Thr Ser Asp Ser Leu Val 530 535 540 Val Arg
Gly Val Ala Gly Leu Arg Asp Gly Lys Glu Val Thr Pro Val 545 550 555
560 Ser Ser Ile Pro Val Glu Thr His Arg Ala Leu Ala Ser Cys His Ser
565 570 575 Leu Met Gln Leu Asp Asp Gly Thr Leu Val Gly Asp Pro Leu
Glu Lys 580 585 590 Ala Met Leu Thr Ala Val Asp Trp Thr Leu Thr Lys
Asp Glu Lys Val 595 600 605
Phe Pro Arg Ser Ile Lys Thr Gln Gly Leu Lys Ile His Gln Arg Phe 610
615 620 His Phe Ala Ser Ala Leu Lys Arg Met Ser Val Leu Ala Ser Tyr
Glu 625 630 635 640 Lys Leu Gly Ser Thr Asp Leu Cys Tyr Ile Ala Ala
Val Lys Gly Ala 645 650 655 Pro Glu Thr Leu His Ser Met Phe Ser Gln
Cys Pro Pro Asp Tyr His 660 665 670 His Ile His Thr Glu Ile Ser Arg
Glu Gly Ala Arg Val Leu Ala Leu 675 680 685 Gly Tyr Lys Glu Leu Gly
His Leu Thr His Gln Gln Ala Arg Glu Val 690 695 700 Lys Arg Glu Ala
Leu Glu Cys Ser Leu Lys Phe Val Gly Phe Ile Val 705 710 715 720 Val
Ser Cys Pro Leu Lys Ala Asp Ser Lys Ala Val Ile Arg Glu Ile 725 730
735 Gln Asn Ala Ser His Arg Val Val Met Ile Thr Gly Asp Asn Pro Leu
740 745 750 Thr Ala Cys His Val Ala Gln Glu Leu His Phe Ile Glu Lys
Ala His 755 760 765 Thr Leu Ile Leu Gln Pro Pro Ser Glu Lys Gly Arg
Gln Cys Glu Trp 770 775 780 Arg Ser Ile Asp Gly Ser Ile Val Leu Pro
Leu Ala Arg Gly Ser Pro 785 790 795 800 Lys Ala Leu Ala Leu Glu Tyr
Ala Leu Cys Leu Thr Gly Asp Gly Leu 805 810 815 Ala His Leu Gln Ala
Thr Asp Pro Gln Gln Leu Leu Arg Leu Ile Pro 820 825 830 His Val Gln
Val Phe Ala Arg Val Ala Pro Lys Gln Lys Glu Phe Val 835 840 845 Ile
Thr Ser Leu Lys Glu Leu Gly Tyr Val Thr Leu Met Cys Gly Asp 850 855
860 Gly Thr Asn Asp Val Gly Ala Leu Lys His Ala Asp Val Gly Val Ala
865 870 875 880 Leu Leu Ala Asn Ala Pro Glu Arg Val Val Glu Arg Arg
Arg Arg Pro 885 890 895 Arg Asp Ser Pro Thr Leu Ser Asn Ser Gly Ile
Arg Ala Thr Ser Arg 900 905 910 Thr Ala Lys Gln Arg Ser Gly Leu Pro
Pro Ser Glu Glu Gln Pro Thr 915 920 925 Ser Gln Arg Asp Arg Leu Ser
Gln Val Leu Arg Asp Leu Glu Asp Glu 930 935 940 Ser Thr Pro Ile Val
Lys Leu Gly Asp Ala Ser Ile Ala Ala Pro Phe 945 950 955 960 Thr Ser
Lys Leu Ser Ser Ile Gln Cys Ile Cys His Val Ile Lys Gln 965 970 975
Gly Arg Cys Thr Leu Val Thr Thr Leu Gln Met Phe Lys Ile Leu Ala 980
985 990 Leu Asn Ala Leu Ile Leu Ala Tyr Ser Gln Ser Val Leu Tyr Leu
Glu 995 1000 1005 Gly Val Lys Phe Ser Asp Phe Gln Ala Thr Leu Gln
Gly Leu Leu Leu 1010 1015 1020 Ala Gly Cys Phe Leu Phe Ile Ser Arg
Ser Lys Pro Leu Lys Thr Leu 1025 1030 1035 1040 Ser Arg Glu Arg Pro
Leu Pro Asn Ile Phe Asn Leu Tyr Thr Ile Leu 1045 1050 1055 Thr Val
Met Leu Gln Phe Phe Val His Phe Leu Ser Leu Val Tyr Leu 1060 1065
1070 Tyr Arg Glu Ala Gln Ala Arg Ser Pro Glu Lys Gln Glu Gln Phe
Val 1075 1080 1085 Asp Leu Tyr Lys Glu Phe Glu Pro Ser Leu Val Asn
Ser Thr Val Tyr 1090 1095 1100 Ile Met Ala Met Ala Met Gln Met Ala
Thr Phe Ala Ile Asn Tyr Lys 1105 1110 1115 1120 Gly Pro Pro Phe Met
Glu Ser Leu Pro Glu Asn Lys Pro Leu Val Trp 1125 1130 1135 Ser Leu
Ala Val Ser Leu Leu Ala Ile Ile Gly Leu Leu Leu Gly Ser 1140 1145
1150 Ser Pro Asp Phe Asn Ser Gln Phe Gly Leu Val Asp Ile Pro Val
Glu 1155 1160 1165 Phe Lys Leu Val Ile Ala Gln Val Leu Leu Leu Asp
Phe Cys Leu Ala 1170 1175 1180 Leu Leu Ala Asp Arg Val Leu Gln Phe
Phe Leu Gly Thr Pro Lys Leu 1185 1190 1195 1200 Lys Val Pro Ser 72
3612 DNA Homo Sapiens CDS (1)...(3612) 72 atg gcg gca gcg gcg gcg
gtg ggc aac gcg gtg ccc tgc ggg gcc cgg 48 Met Ala Ala Ala Ala Ala
Val Gly Asn Ala Val Pro Cys Gly Ala Arg 1 5 10 15 cct tgc ggg gtc
cgg cct gac ggg cag ccc aag ccc ggg ccg cag ccg 96 Pro Cys Gly Val
Arg Pro Asp Gly Gln Pro Lys Pro Gly Pro Gln Pro 20 25 30 cgc gcg
ctc ctt gcc gcc ggg ccg gcg ctc ata gcg aac ggt gac gag 144 Arg Ala
Leu Leu Ala Ala Gly Pro Ala Leu Ile Ala Asn Gly Asp Glu 35 40 45
ctg gtg gct gcc gtg tgg ccg tac cgg cgg ttg gcg ctg ttg cgg cgc 192
Leu Val Ala Ala Val Trp Pro Tyr Arg Arg Leu Ala Leu Leu Arg Arg 50
55 60 ctc acg gtg ctg cca ttc gcc ggg ctg ctt tac ccg gcc tgg ttg
ggt 240 Leu Thr Val Leu Pro Phe Ala Gly Leu Leu Tyr Pro Ala Trp Leu
Gly 65 70 75 80 gcc gca gcc gct ggc tgc tgg ggc tgg ggc agc agt tgg
gtg cag atc 288 Ala Ala Ala Ala Gly Cys Trp Gly Trp Gly Ser Ser Trp
Val Gln Ile 85 90 95 ccc gaa gct gcg ctg ctc gtg ctt gcc acc atc
tgc ctc gcg cac gcg 336 Pro Glu Ala Ala Leu Leu Val Leu Ala Thr Ile
Cys Leu Ala His Ala 100 105 110 ctc act gtc ctc tcg ggg cat tgg tct
gtg cac gcg cat tgc gcg ctc 384 Leu Thr Val Leu Ser Gly His Trp Ser
Val His Ala His Cys Ala Leu 115 120 125 acc tgc acc ccg gag tac gac
ccc agc aaa gcg acc ttt gtg aag gtg 432 Thr Cys Thr Pro Glu Tyr Asp
Pro Ser Lys Ala Thr Phe Val Lys Val 130 135 140 gcg cca acc ccc aac
aat ggc tcc acg gag ctc gtg gcc ctg cac cgc 480 Ala Pro Thr Pro Asn
Asn Gly Ser Thr Glu Leu Val Ala Leu His Arg 145 150 155 160 aat gag
ggc gaa gac ggg ctt gag gtg ctg tcc ttc gaa ttc cag aag 528 Asn Glu
Gly Glu Asp Gly Leu Glu Val Leu Ser Phe Glu Phe Gln Lys 165 170 175
atc aag tat tcc tac gat gcc ctg gag aag aag cag ttt ctc ccc gtg 576
Ile Lys Tyr Ser Tyr Asp Ala Leu Glu Lys Lys Gln Phe Leu Pro Val 180
185 190 gcc ttt cct gtg gga aac gcc ttc tca tac tat cag agc aac aga
ggc 624 Ala Phe Pro Val Gly Asn Ala Phe Ser Tyr Tyr Gln Ser Asn Arg
Gly 195 200 205 ttc cag gaa gac tca gag atc cga gca gct gag aag aaa
ttt ggg agc 672 Phe Gln Glu Asp Ser Glu Ile Arg Ala Ala Glu Lys Lys
Phe Gly Ser 210 215 220 aac aag gcc gag atg gtg gtg cct gac ttc tcg
gag ctt ttc aag gag 720 Asn Lys Ala Glu Met Val Val Pro Asp Phe Ser
Glu Leu Phe Lys Glu 225 230 235 240 aga gcc aca gcc ccc ttc ttt gta
ttt cag gtg ttc tgt gtg ggg ctc 768 Arg Ala Thr Ala Pro Phe Phe Val
Phe Gln Val Phe Cys Val Gly Leu 245 250 255 tgg tgc ctg gat gag tac
tgg tac tac agc gtc ttt acg cta tcc atg 816 Trp Cys Leu Asp Glu Tyr
Trp Tyr Tyr Ser Val Phe Thr Leu Ser Met 260 265 270 ctg gtg gcg ttc
gag gcc tcg ctg gtg cag cag cag atg cgg aac atg 864 Leu Val Ala Phe
Glu Ala Ser Leu Val Gln Gln Gln Met Arg Asn Met 275 280 285 tcg gag
atc cgg aag atg ggc aac aag ccc cac atg atc cag gtc tac 912 Ser Glu
Ile Arg Lys Met Gly Asn Lys Pro His Met Ile Gln Val Tyr 290 295 300
cga agc cgc aag tgg agg ccc att gcc agt gat gag atc gta cca ggg 960
Arg Ser Arg Lys Trp Arg Pro Ile Ala Ser Asp Glu Ile Val Pro Gly 305
310 315 320 gac atc gtc tcc atc ggc cgc tcc cca cag gag aac ctg gtg
cca tgt 1008 Asp Ile Val Ser Ile Gly Arg Ser Pro Gln Glu Asn Leu
Val Pro Cys 325 330 335 gac gtg ctt ctg ctg cga ggc cgc tgc atc gta
gac gag gcc atg ctc 1056 Asp Val Leu Leu Leu Arg Gly Arg Cys Ile
Val Asp Glu Ala Met Leu 340 345 350 acg ggg gag tcc gtg cca cag atg
aag gag ccc atc gaa gac ctc agc 1104 Thr Gly Glu Ser Val Pro Gln
Met Lys Glu Pro Ile Glu Asp Leu Ser 355 360 365 cca gac cgg gtg ctg
gac ctc cag gct gat tcc cgg ctg cac gtc atc 1152 Pro Asp Arg Val
Leu Asp Leu Gln Ala Asp Ser Arg Leu His Val Ile 370 375 380 ttc ggg
ggc acc aag gtg gtg cag cac atc ccc cca cag aaa gcc acc 1200 Phe
Gly Gly Thr Lys Val Val Gln His Ile Pro Pro Gln Lys Ala Thr 385 390
395 400 acg ggc ctg aag ccg gtt gac agc ggg tgc gtg gcc tac gtc ctg
cgg 1248 Thr Gly Leu Lys Pro Val Asp Ser Gly Cys Val Ala Tyr Val
Leu Arg 405 410 415 acc gga ttc aac aca tct cag ggc aag ctg ctg cgc
acc atc ctc ttc 1296 Thr Gly Phe Asn Thr Ser Gln Gly Lys Leu Leu
Arg Thr Ile Leu Phe 420 425 430 ggg gtc aag agg gtg act gcg aac aac
ctg gag acc ttc atc ttc atc 1344 Gly Val Lys Arg Val Thr Ala Asn
Asn Leu Glu Thr Phe Ile Phe Ile 435 440 445 ctc ttc ctc ctg gtg ttt
gcc atc gct gca gct gcc tat gta tgg att 1392 Leu Phe Leu Leu Val
Phe Ala Ile Ala Ala Ala Ala Tyr Val Trp Ile 450 455 460 gaa ggt acc
aag gac ccc agc cgg aac cgc tac aag ctg ttt ctg gag 1440 Glu Gly
Thr Lys Asp Pro Ser Arg Asn Arg Tyr Lys Leu Phe Leu Glu 465 470 475
480 tgc acc ctg atc ctc acc tcg gtc gtg cct cct gag ctg ccc atc gag
1488 Cys Thr Leu Ile Leu Thr Ser Val Val Pro Pro Glu Leu Pro Ile
Glu 485 490 495 ctg tcc ctg gcc gtc aac acc tcc ctc atc gcc ctg gcc
aag ctc tac 1536 Leu Ser Leu Ala Val Asn Thr Ser Leu Ile Ala Leu
Ala Lys Leu Tyr 500 505 510 atg tac tgc aca gag ccc ttc cgg atc ccc
ttt gct ggc aag gtc gag 1584 Met Tyr Cys Thr Glu Pro Phe Arg Ile
Pro Phe Ala Gly Lys Val Glu 515 520 525 gtg tgc tgc ttt gac aag acg
ggg acg ttg acc agt gac agc ctg gtg 1632 Val Cys Cys Phe Asp Lys
Thr Gly Thr Leu Thr Ser Asp Ser Leu Val 530 535 540 gtg cgc ggt gtg
gcc ggg ctg aga gac ggg aag gag gtg acc cca gtg 1680 Val Arg Gly
Val Ala Gly Leu Arg Asp Gly Lys Glu Val Thr Pro Val 545 550 555 560
tcc agc atc cct gta gaa aca cac cgg gcc ctg gcc tcg tgc cac tcg
1728 Ser Ser Ile Pro Val Glu Thr His Arg Ala Leu Ala Ser Cys His
Ser 565 570 575 ctc atg cag ctg gac gac ggc acc ctc gtg ggt gac cct
cta gag aag 1776 Leu Met Gln Leu Asp Asp Gly Thr Leu Val Gly Asp
Pro Leu Glu Lys 580 585 590 gcc atg ctg acg gcc gtg gac tgg acg ctg
acc aaa gat gag aaa gta 1824 Ala Met Leu Thr Ala Val Asp Trp Thr
Leu Thr Lys Asp Glu Lys Val 595 600 605 ttc ccc cga agt att aaa act
cag ggg ctg aaa att cac cag cgc ttt 1872 Phe Pro Arg Ser Ile Lys
Thr Gln Gly Leu Lys Ile His Gln Arg Phe 610 615 620 cat ttt gcc agt
gcc ctg aag cga atg tcc gtg ctt gcc tcg tat gag 1920 His Phe Ala
Ser Ala Leu Lys Arg Met Ser Val Leu Ala Ser Tyr Glu 625 630 635 640
aag ctg ggc tcc acc gac ctc tgc tac atc gcg gcc gtg aag ggg gcc
1968 Lys Leu Gly Ser Thr Asp Leu Cys Tyr Ile Ala Ala Val Lys Gly
Ala 645 650 655 ccc gaa act ctg cac tcc atg ttc tcc cag tgc ccg ccc
gac tac cac 2016 Pro Glu Thr Leu His Ser Met Phe Ser Gln Cys Pro
Pro Asp Tyr His 660 665 670 cac atc cac acc gag atc tcc cgg gaa gga
gcc cgc gtc ctg gcg ctg 2064 His Ile His Thr Glu Ile Ser Arg Glu
Gly Ala Arg Val Leu Ala Leu 675 680 685 ggg tac aag gag ctg gga cac
ctc act cac cag cag gcc cgg gag gtc 2112 Gly Tyr Lys Glu Leu Gly
His Leu Thr His Gln Gln Ala Arg Glu Val 690 695 700 aag cgg gag gcc
ctg gag tgc agc ctc aag ttc gtc ggc ttc att gtg 2160 Lys Arg Glu
Ala Leu Glu Cys Ser Leu Lys Phe Val Gly Phe Ile Val 705 710 715 720
gtc tcc tgc ccg ctc aag gct gac tcc aag gcc gtg atc cgg gag atc
2208 Val Ser Cys Pro Leu Lys Ala Asp Ser Lys Ala Val Ile Arg Glu
Ile 725 730 735 cag aat gcg tcc cac cgg gtg gtc atg atc acg gga gac
aac ccg ctc 2256 Gln Asn Ala Ser His Arg Val Val Met Ile Thr Gly
Asp Asn Pro Leu 740 745 750 act gca tgc cac gtg gcc cag gag ctg cac
ttc att gaa aag gcc cac 2304 Thr Ala Cys His Val Ala Gln Glu Leu
His Phe Ile Glu Lys Ala His 755 760 765 acg ctg atc ctg cag cct ccc
tcc gag aaa ggc cgg cag tgc gag tgg 2352 Thr Leu Ile Leu Gln Pro
Pro Ser Glu Lys Gly Arg Gln Cys Glu Trp 770 775 780 cgc tcc att gac
ggc agc atc gtg ctg ccc ctg gcc cgg ggc tcc cca 2400 Arg Ser Ile
Asp Gly Ser Ile Val Leu Pro Leu Ala Arg Gly Ser Pro 785 790 795 800
aag gca ctg gcc ctg gag tac gca ctg tgc ctc aca ggc gac ggc ttg
2448 Lys Ala Leu Ala Leu Glu Tyr Ala Leu Cys Leu Thr Gly Asp Gly
Leu 805 810 815 gcc cac ctg cag gcc acc gac ccc cag cag ctg ctc cgc
ctc atc ccc 2496 Ala His Leu Gln Ala Thr Asp Pro Gln Gln Leu Leu
Arg Leu Ile Pro 820 825 830 cat gtg cag gtg ttc gcc cgt gtg gct ccc
aag cag aag gag ttt gtc 2544 His Val Gln Val Phe Ala Arg Val Ala
Pro Lys Gln Lys Glu Phe Val 835 840 845 atc acc agc ctg aag gag ctg
ggc tac gtg acc ctc atg tgt ggg gat 2592 Ile Thr Ser Leu Lys Glu
Leu Gly Tyr Val Thr Leu Met Cys Gly Asp 850 855 860 ggc acc aac gac
gtg ggc gcc ctg aag cat gct gac gtg ggt gtg gcg 2640 Gly Thr Asn
Asp Val Gly Ala Leu Lys His Ala Asp Val Gly Val Ala 865 870 875 880
ctc ttg gcc aat gcc cct gag cgg gtt gtc gag cgg cga cgg cgg ccc
2688 Leu Leu Ala Asn Ala Pro Glu Arg Val Val Glu Arg Arg Arg Arg
Pro 885 890 895 cgg gac agc cca acc ctg agc aac agt ggc atc aga gcc
acc tcc agg 2736 Arg Asp Ser Pro Thr Leu Ser Asn Ser Gly Ile Arg
Ala Thr Ser Arg 900 905 910 aca gcc aag cag cgg tcg ggg ctc cct ccc
tcc gag gag cag cca acc 2784 Thr Ala Lys Gln Arg Ser Gly Leu Pro
Pro Ser Glu Glu Gln Pro Thr 915 920 925 tcc cag agg gac cgc ctg agc
cag gtg ctg cga gac ctc gag gac gag 2832 Ser Gln Arg Asp Arg Leu
Ser Gln Val Leu Arg Asp Leu Glu Asp Glu 930 935 940 agt acg ccc att
gtg aaa ctg ggg gat gcc agc atc gca gca ccc ttc 2880 Ser Thr Pro
Ile Val Lys Leu Gly Asp Ala Ser Ile Ala Ala Pro Phe 945 950 955 960
acc tcc aag ctc tca tcc atc cag tgc atc tgc cac gtg atc aag cag
2928 Thr Ser Lys Leu Ser Ser Ile Gln Cys Ile Cys His Val Ile Lys
Gln 965 970 975 ggc cgc tgc acg ctg gtg acc acg cta cag atg ttc aag
atc ctg gcg 2976 Gly Arg Cys Thr Leu Val Thr Thr Leu Gln Met Phe
Lys Ile Leu Ala 980 985 990 ctc aat gcc ctc atc ctg gcc tac agc cag
agc gtc ctc tac ctg gag 3024 Leu Asn Ala Leu Ile Leu Ala Tyr Ser
Gln Ser Val Leu Tyr Leu Glu 995 1000 1005 gga gtc aag ttc agt gac
ttc cag gcc acc cta cag ggg ctg ctg ctg 3072 Gly Val Lys Phe Ser
Asp Phe Gln Ala Thr Leu Gln Gly Leu Leu Leu 1010 1015 1020 gcc ggc
tgc ttc ctc ttc atc tcc cgt tcc aag ccc ctc aag acc ctc 3120 Ala
Gly Cys Phe Leu Phe Ile Ser Arg Ser Lys Pro Leu Lys Thr Leu 1025
1030 1035 1040 tcc cga gaa cgg ccc ctg ccc aac atc ttc aac ctg tac
acc atc ctc 3168 Ser Arg Glu Arg Pro Leu Pro Asn Ile Phe Asn Leu
Tyr Thr Ile Leu 1045 1050 1055 acc gtc atg ctc cag ttc ttt gtg cac
ttc ctg agc ctt gtc tac ctg 3216 Thr Val Met Leu Gln Phe Phe Val
His Phe Leu Ser Leu Val Tyr Leu 1060 1065 1070 tac cgt gag gcc cag
gcc cgg agc ccc gag aag cag gag cag ttc gtg 3264 Tyr Arg Glu Ala
Gln Ala Arg Ser Pro Glu Lys Gln Glu Gln Phe Val 1075 1080 1085 gac
ttg tac aag gag ttt gag cca agc ctg gtc aac agc acc gtc tac 3312
Asp Leu Tyr Lys Glu Phe Glu Pro Ser Leu Val Asn Ser Thr Val Tyr
1090 1095 1100 atc atg gcc atg gcc atg cag atg gcc acc ttc gcc atc
aat tac aaa 3360 Ile Met Ala Met Ala Met Gln Met Ala Thr Phe Ala
Ile Asn Tyr Lys 1105 1110 1115 1120 ggc ccg ccc ttc atg gag agc ctg
ccc gag aac aag ccc ctg gtg tgg 3408 Gly Pro Pro Phe Met Glu Ser
Leu Pro Glu Asn Lys Pro Leu Val Trp 1125 1130 1135 agt ctg gca gtt
tca ctc ctg gcc atc att ggc ctg ctc ctc ggc tcc 3456 Ser Leu Ala
Val Ser Leu Leu Ala Ile Ile Gly
Leu Leu Leu Gly Ser 1140 1145 1150 tcg ccc gac ttc aac agc cag ttt
ggc ctc gtg gac atc cct gtg gag 3504 Ser Pro Asp Phe Asn Ser Gln
Phe Gly Leu Val Asp Ile Pro Val Glu 1155 1160 1165 ttc aag ctg gtc
att gcc cag gtc ctg ctc ctg gac ttc tgc ctg gcg 3552 Phe Lys Leu
Val Ile Ala Gln Val Leu Leu Leu Asp Phe Cys Leu Ala 1170 1175 1180
ctc ctg gcc gac cgc gtc ctg cag ttc ttc ctg ggg acc ccg aag ctg
3600 Leu Leu Ala Asp Arg Val Leu Gln Phe Phe Leu Gly Thr Pro Lys
Leu 1185 1190 1195 1200 aaa gtg cct tcc 3612 Lys Val Pro Ser 73 7
PRT Artificial Sequence Consensus Amino Acid Sequence 73 Asp Lys
Thr Gly Thr Xaa Xaa 1 5 74 1157 PRT Caenorhabditis elegans 74 Met
Gly Val Asp Gln Leu Val Glu Thr Ile Ile Pro Tyr Asn Leu Arg 1 5 10
15 Ser Ile Ala Thr His Leu Tyr Val Pro Pro Phe Thr Ile Ile Thr Ala
20 25 30 Ile Trp Thr Tyr Val Trp Leu Asn Ile Phe Gly Tyr Glu Glu
Tyr Tyr 35 40 45 Glu Leu Gly Met Leu Gly Tyr Ala Ala Ile Phe Val
Ile Leu Ala Leu 50 55 60 Val Leu Leu Phe Cys His Trp Met Met Pro
Val Arg Cys Phe Leu Met 65 70 75 80 Cys Ser Lys Gln Glu Asp Val Arg
Ile Ala Ser His Val Cys Val Ile 85 90 95 Pro Thr Gln Asn Asn Gly
Trp Pro Glu Leu Val Lys Leu Met Arg Thr 100 105 110 Thr Arg Asp Lys
Gln Thr Lys Leu Trp Phe Glu Phe Gln Arg Val His 115 120 125 Tyr Thr
Trp Asp Glu Glu Ser Arg Glu Phe Gln Thr Lys Thr Leu Asp 130 135 140
Thr Ala Lys Pro Met Val Phe Phe Gln Lys Ser His Gly Phe Glu Val 145
150 155 160 Glu Glu His Val Lys Asp Ala Lys Tyr Leu Leu Gly Asp Asn
Lys Thr 165 170 175 Glu Met Ile Val Pro Gln Phe Leu Glu Met Phe Ile
Glu Arg Ala Thr 180 185 190 Ala Pro Phe Phe Val Phe Gln Val Phe Cys
Val Gly Leu Trp Cys Leu 195 200 205 Glu Asp Met Trp Tyr Tyr Ser Leu
Phe Thr Leu Phe Met Leu Met Thr 210 215 220 Phe Glu Ala Thr Leu Val
Lys Gln Gln Met Lys Asn Met Ser Glu Ile 225 230 235 240 Arg Asn Met
Gly Asn Lys Thr Tyr Met Ile Asn Val Leu Arg Gly Lys 245 250 255 Lys
Trp Gln Lys Ile Lys Ile Glu Glu Leu Val Ala Gly Asp Ile Val 260 265
270 Ser Ile Gly Arg Gly Ala Glu Glu Glu Cys Val Pro Cys Asp Leu Leu
275 280 285 Leu Leu Arg Gly Pro Cys Ile Val Asp Glu Ser Met Leu Thr
Gly Glu 290 295 300 Ser Val Pro Gln Met Lys Glu Pro Ile Glu Asp Val
Glu Lys Asp Lys 305 310 315 320 Ile Phe Asp Ile Glu Thr Asp Ser Arg
Leu His Val Ile Phe Gly Gly 325 330 335 Thr Lys Ile Val Gln His Thr
Ala Pro Gly Lys Ala Ala Glu Gly Met 340 345 350 Val Lys Ser Pro Asp
Gly Asn Cys Ile Cys Tyr Val Ile Arg Thr Gly 355 360 365 Phe Asn Thr
Ser Gln Gly Lys Leu Leu Arg Thr Ile Met Phe Gly Val 370 375 380 Lys
Lys Ala Thr Ala Asn Asn Leu Glu Thr Phe Cys Phe Ile Leu Phe 385 390
395 400 Leu Leu Ile Phe Ala Ile Ala Ala Ala Ala Tyr Leu Trp Ile Lys
Gly 405 410 415 Ser Val Asp Glu Thr Arg Ser Lys Tyr Lys Leu Phe Leu
Glu Cys Thr 420 425 430 Leu Ile Leu Thr Ser Val Ile Pro Pro Glu Leu
Pro Ile Glu Leu Ser 435 440 445 Leu Ala Val Asn Ser Ser Leu Met Ala
Leu Gln Lys Leu Gly Ile Phe 450 455 460 Cys Thr Glu Pro Phe Arg Ile
Pro Phe Ala Gly Lys Val Asp Ile Cys 465 470 475 480 Cys Phe Asp Lys
Thr Gly Thr Leu Thr Thr Asp Asn Leu Val Val Glu 485 490 495 Gly Val
Ala Leu Asn Asn Gln Lys Glu Gly Met Ile Arg Asn Ala Glu 500 505 510
Asp Leu Pro His Glu Ser Leu Gln Val Leu Ala Ser Cys His Ser Leu 515
520 525 Val Arg Phe Glu Glu Asp Leu Val Gly Asp Pro Leu Glu Lys Ala
Cys 530 535 540 Leu Ser Trp Cys Gly Trp Asn Leu Thr Lys Gly Asp Ala
Val Met Pro 545 550 555 560 Pro Lys Thr Ala Ala Lys Gly Ile Ser Gly
Ile Lys Ile Phe His Arg 565 570 575 Tyr His Phe Ser Ser Ala Met Lys
Arg Met Thr Val Val Ala Gly Tyr 580 585 590 Gln Ser Pro Gly Thr Ser
Asp Thr Thr Phe Ile Val Ala Val Lys Gly 595 600 605 Ala Pro Glu Val
Leu Arg Asn Met Tyr Ala Asp Leu Pro Ser Asp Tyr 610 615 620 Asp Glu
Thr Tyr Thr Arg Leu Thr Arg Gln Gly Ser Arg Val Leu Ala 625 630 635
640 Met Gly Ile Arg Lys Leu Gly Glu Thr Arg Val Gly Glu Leu Arg Asp
645 650 655 Lys Lys Arg Glu Asn Phe Glu Asn Asp Leu Ala Phe Ala Gly
Phe Val 660 665 670 Val Ile Ser Cys Pro Leu Lys Ser Asp Thr Lys Thr
Met Ile Arg Glu 675 680 685 Ile Met Asp Ser Ser His Val Val Ala Met
Ile Thr Gly Asp Asn Pro 690 695 700 Leu Thr Ala Cys His Val Ser Lys
Val Leu Lys Phe Thr Lys Lys Ser 705 710 715 720 Leu Pro Thr Leu Val
Leu Asp Glu Pro Ala Asp Gly Val Asp Trp Met 725 730 735 Trp Lys Ser
Val Asp Gly Thr Ile Glu Leu Pro Leu Lys Pro Glu Thr 740 745 750 Lys
Asn Lys Met Glu Arg Lys Ala Phe Phe Asn Ser His Glu Phe Cys 755 760
765 Leu Thr Gly Ser Ala Phe His His Leu Val His Asn Glu His Thr Phe
770 775 780 Leu Arg Glu Leu Ile Leu His Val Lys Val Phe Ala Arg Met
Ala Pro 785 790 795 800 Lys Gln Lys Glu Arg Ile Ile Asn Glu Leu Lys
Ser Leu Gly Lys Val 805 810 815 Thr Leu Met Cys Gly Asp Gly Thr Asn
Asp Val Gly Ala Leu Lys His 820 825 830 Ala Asn Val Gly Val Ala Leu
Leu Thr Asn Pro Tyr Asp Ala Glu Lys 835 840 845 Ala Ala Glu Lys Glu
Lys Glu Lys Lys Ala Lys Ile Glu Glu Ala Arg 850 855 860 Ser Leu Val
Arg Ser Gly Ala Gln Leu Pro Gln Arg Pro Gly Ala Pro 865 870 875 880
Gly Ala Pro Pro Ala Ala Asn Ala Ala Arg Pro Arg Leu Asp Asn Leu 885
890 895 Met Lys Glu Leu Glu Glu Glu Glu Lys Ala Gln Val Ile Lys Leu
Gly 900 905 910 Asp Ala Ser Ile Ala Ala Pro Phe Thr Ser Lys Tyr Thr
Ser Ile Ala 915 920 925 Ser Ile Cys His Val Ile Lys Gln Gly Arg Cys
Thr Leu Val Thr Thr 930 935 940 Leu Gln Met Phe Lys Ile Leu Ala Leu
Asn Ala Leu Val Ser Ala Tyr 945 950 955 960 Ser Leu Ser Ala Leu Tyr
Leu Asp Gly Val Lys Phe Ser Asp Thr Gln 965 970 975 Ala Thr Ile Gln
Gly Leu Leu Leu Ala Ala Cys Phe Leu Phe Ile Ser 980 985 990 Lys Ser
Lys Pro Leu Lys Thr Leu Ser Arg Gln Arg Pro Met Ala Asn 995 1000
1005 Ile Phe Asn Ala Tyr Thr Leu Leu Thr Val Thr Leu Gln Phe Ile
Val 1010 1015 1020 His Phe Ser Cys Leu Leu Tyr Ile Val Gly Leu Ala
His Glu Ala Asn 1025 1030 1035 1040 Thr Glu Lys Ala Pro Val Asp Leu
Glu Ala Lys Phe Thr Pro Asn Ile 1045 1050 1055 Leu Asn Thr Thr Val
Tyr Ile Ile Ser Met Ala Leu Gln Val Cys Thr 1060 1065 1070 Phe Ala
Val Asn Tyr Arg Gly Arg Pro Phe Met Glu Ser Leu Phe Glu 1075 1080
1085 Asn Lys Ala Met Leu Tyr Ser Ile Met Phe Ser Gly Gly Ala Val
Phe 1090 1095 1100 Thr Leu Ala Ser Gly Gln Ala Thr Asp Leu Met Ile
Gln Phe Glu Leu 1105 1110 1115 1120 Val Val Leu Pro Glu Ala Leu Arg
Asn Ala Leu Leu Met Cys Val Thr 1125 1130 1135 Ala Asp Leu Val Ile
Cys Tyr Ile Ile Asp Arg Gly Leu Asn Phe Leu 1140 1145 1150 Leu Gly
Asp Met Phe 1155 75 9 PRT Artificial Sequence Consensus Amino Acid
Sequence 75 Xaa Xaa Xaa Xaa Xaa Xaa Gly Glu Xaa 1 5 76 10 PRT
Artificial Sequence Consensus Amino Acid Sequence 76 Xaa Xaa Xaa
Asp Lys Thr Gly Thr Xaa Thr 1 5 10 77 11 PRT Artificial Sequence
Consensus Amino Acid Sequence 77 Xaa Gly Asp Gly Xaa Asn Asp Xaa
Pro Xaa Leu 1 5 10 78 7 PRT Artificial Sequence Amino Acid Residues
Important for Calcium Transport 78 Ile Pro Glu Gly Leu Pro Ala 1 5
79 28 PRT Homo Sapiens 79 Asp Leu Val Thr Val Val Val Pro Pro Ala
Leu Pro Ala Ala Met Thr 1 5 10 15 Val Cys Thr Leu Tyr Ala Gln Ser
Arg Leu Arg Arg 20 25 80 28 PRT Homo Sapiens 80 Asp Ile Ile Thr Ile
Thr Val Pro Pro Ala Leu Pro Ala Ala Met Thr 1 5 10 15 Ala Gly Ile
Val Tyr Ala Gln Arg Arg Leu Lys Lys 20 25 81 28 PRT Homo Sapiens 81
Leu Ile Leu Thr Ser Val Val Pro Pro Glu Leu Pro Ile Glu Leu Ser 1 5
10 15 Leu Ala Val Asn Thr Ser Leu Ile Ala Leu Ala Lys 20 25 82 29
PRT Saccharomyces cerevisiae 82 Asp Ile Ile Thr Ile Val Val Pro Pro
Ala Leu Pro Ala Thr Leu Thr 1 5 10 15 Ile Gly Thr Asn Phe Ala Leu
Ser Arg Leu Lys Glu Lys 20 25 83 28 PRT Saccharomyces cerevisiae 83
Leu Ile Ile Thr Ser Val Val Pro Pro Glu Leu Pro Met Glu Leu Thr 1 5
10 15 Met Ala Val Asn Ser Ser Leu Ala Ala Leu Ala Lys 20 25 84 28
PRT Schizosaccharomyces pombe 84 Val Leu Thr Ile Leu Val Pro Pro
Ala Leu Pro Ala Thr Leu Ser Val 1 5 10 15 Gly Ile Ala Asn Ser Ile
Ala Arg Leu Ser Arg Ala 20 25 85 29 PRT Caenorhabditis elegans 85
Asp Leu Val Thr Ile Val Val Pro Pro Ala Leu Pro Ala Val Met Gly 1 5
10 15 Ile Gly Ile Phe Tyr Ala Gln Arg Arg Leu Arg Gln Lys 20 25 86
29 PRT Caenorhabditis elegans 86 Asp Ile Ile Thr Ile Val Val Pro
Pro Ala Leu Pro Ala Ala Met Ser 1 5 10 15 Val Gly Ile Ile Asn Ala
Asn Ser Arg Leu Lys Lys Lys 20 25 87 29 PRT Caenorhabditis elegans
87 Asp Ile Ile Thr Ile Thr Val Pro Pro Ala Leu Pro Ala Ala Met Ser
1 5 10 15 Val Gly Ile Ile Asn Ala Gln Leu Arg Leu Lys Lys Lys 20 25
88 29 PRT Caenorhabditis elegans 88 Leu Ile Leu Thr Ser Val Ile Pro
Pro Glu Leu Pro Ile Glu Leu Ser 1 5 10 15 Leu Ala Val Asn Ser Ser
Leu Met Ala Leu Gln Lys Leu 20 25 89 29 PRT Homo Sapiens 89 Ala Leu
Ala Val Ala Ala Ile Pro Glu Gly Leu Pro Ala Val Ile Thr 1 5 10 15
Thr Cys Leu Ala Leu Gly Thr Arg Arg Met Ala Lys Lys 20 25 90 29 PRT
Oryctolagus cuniculus 90 Ala Leu Ala Val Ala Ala Ile Pro Glu Gly
Leu Pro Ala Val Ile Thr 1 5 10 15 Thr Cys Leu Ala Leu Gly Thr Arg
Arg Met Ala Lys Lys 20 25 91 29 PRT Gallus gallus 91 Ala Leu Ala
Val Ala Ala Ile Pro Glu Gly Leu Pro Ala Val Ile Thr 1 5 10 15 Thr
Cys Leu Ala Leu Gly Thr Arg Arg Met Ala Lys Lys 20 25 92 29 PRT
Felis catus 92 Ala Leu Ala Val Ala Ala Ile Pro Glu Gly Leu Pro Ala
Val Ile Thr 1 5 10 15 Thr Cys Leu Ala Leu Gly Thr Arg Arg Met Ala
Lys Lys 20 25 93 29 PRT Procambarus clarkii 93 Ala Leu Ala Val Ala
Ala Ile Pro Glu Gly Leu Pro Ala Val Ile Thr 1 5 10 15 Thr Cys Leu
Ala Leu Gly Thr Arg Arg Met Ala Lys Lys 20 25 94 29 PRT Homo
Sapiens 94 Ala Leu Ala Val Ala Ala Ile Pro Glu Gly Leu Pro Ala Val
Ile Thr 1 5 10 15 Thr Cys Leu Ala Leu Gly Thr Arg Arg Met Ala Lys
Lys 20 25 95 29 PRT Homo Sapiens 95 Ala Leu Ala Val Ala Ala Ile Pro
Glu Gly Leu Pro Ala Val Ile Thr 1 5 10 15 Thr Cys Leu Ala Leu Gly
Thr Arg Arg Met Ala Arg Lys 20 25 96 29 PRT Drosophila melanogaster
96 Ala Val Ala Val Ala Ala Ile Pro Glu Gly Leu Pro Ala Val Ile Thr
1 5 10 15 Thr Cys Leu Ala Leu Gly Thr Arg Arg Met Ala Lys Lys 20 25
97 29 PRT Saccharomyces cerevisiae 97 Ser Leu Ala Val Ala Ala Ile
Pro Glu Gly Leu Pro Ile Ile Val Thr 1 5 10 15 Val Thr Leu Ala Leu
Gly Val Leu Arg Met Ala Lys Arg 20 25 98 29 PRT Saccharomyces
cerevisiae 98 Thr Val Ile Val Val Ala Val Pro Glu Gly Leu Pro Leu
Ala Val Thr 1 5 10 15 Leu Ala Leu Ala Phe Ala Thr Thr Arg Met Thr
Lys Asp 20 25 99 29 PRT Homo Sapiens 99 Thr Val Leu Val Val Ala Val
Pro Glu Gly Leu Pro Leu Ala Val Thr 1 5 10 15 Ile Ser Leu Ala Tyr
Ser Val Lys Lys Met Met Lys Asp 20 25 100 29 PRT Homo Sapiens 100
Thr Val Leu Val Val Ala Val Pro Glu Gly Leu Pro Leu Ala Val Thr 1 5
10 15 Ile Ser Leu Ala Tyr Ser Val Lys Lys Met Met Lys Asp 20 25 101
29 PRT Rattus norvegicus 101 Thr Val Leu Val Val Ala Val Pro Glu
Gly Leu Pro Leu Ala Val Thr 1 5 10 15 Ile Ser Leu Ala Tyr Ser Val
Lys Lys Met Met Lys Asp 20 25 102 29 PRT Homo Sapiens 102 Thr Val
Leu Val Val Ala Val Pro Glu Gly Leu Pro Leu Ala Val Thr 1 5 10 15
Ile Ser Leu Ala Tyr Ser Val Lys Lys Met Met Lys Asp 20 25 103 29
PRT Homo Sapiens 103 Thr Val Leu Val Val Ala Val Pro Glu Gly Leu
Pro Leu Ala Val Thr 1 5 10 15 Ile Ser Leu Ala Tyr Ser Val Lys Lys
Met Met Lys Asp 20 25 104 29 PRT Saccharomyces cerevisiae 104 Thr
Val Leu Ile Val Ser Cys Pro Cys Val Ile Gly Leu Ala Val Pro 1 5 10
15 Ile Val Phe Val Ile Ala Ser Gly Val Ala Ala Lys Arg 20 25 105 29
PRT Homo Sapiens 105 Thr Val Leu Cys Ile Ala Cys Pro Cys Ser Leu
Gly Leu Ala Thr Pro 1 5 10 15 Thr Ala Val Met Val Gly Thr Gly Val
Gly Ala Gln Asn 20 25 106 29 PRT Homo Sapiens 106 Thr Val Leu Cys
Ile Ala Cys Pro Cys Ser Leu Gly Leu Ala Thr Pro 1 5 10 15 Thr Ala
Val Met Val Gly Thr Gly Val Ala Ala Gln Asn 20 25 107 29 PRT
Drosophila melanogaster 107 Gly Ile Ile Val Ala Asn Val Pro Glu Gly
Leu Leu Ala Thr Val Thr 1 5 10 15 Val Cys Leu Thr Leu Thr Ala Lys
Arg Met Ala Ser Lys 20 25 108 29 PRT Hydra vulgaris 108 Gly Ile Ile
Val Ala Asn Val Pro Glu Gly Leu Leu Ala Thr Val Thr 1 5 10 15 Val
Cys Leu Thr Leu Thr Ala Lys Lys Met Ala Lys Lys 20 25 109 29 PRT
Bufo marinus 109 Gly Ile Ile Val Ala Asn Val Pro Glu Gly Leu Leu
Ala Thr Val Thr 1 5 10 15 Val Cys Leu Thr Leu Thr Ala Lys Arg Met
Ala Arg Lys 20 25 110 29 PRT Homo Sapiens 110 Gly Ile Ile Val Ala
Asn Val Pro Glu Gly Leu Leu Ala Thr Val Thr 1 5 10 15 Val Cys Leu
Thr Leu Thr Ala Lys Arg Met Ala Arg Lys 20 25 111 29 PRT Homo
Sapiens 111 Gly Ile Ile Val Ala Asn Val Pro Glu Gly Leu Leu Ala Thr
Val Thr 1 5 10 15 Val Cys Leu Thr Leu Thr Ala Lys Arg Met Ala Arg
Lys 20 25 112 29 PRT Homo Sapiens 112 Gly Ile Ile Val Ala Asn Val
Pro Glu Gly Leu Leu Ala Thr Val Thr 1 5 10 15 Val Cys Leu Thr
Val
Thr Ala Lys Arg Met Ala Arg Lys 20 25 113 29 PRT Homo Sapiens 113
Ile Leu Phe Asn Asn Leu Ile Pro Ile Ser Leu Leu Val Thr Leu Glu 1 5
10 15 Val Val Lys Phe Thr Gln Ala Tyr Phe Ile Asn Trp Asp 20 25 114
29 PRT Saccharomyces cerevisiae 114 Ile Leu Phe Ser Asn Leu Val Pro
Ile Ser Leu Phe Val Thr Val Glu 1 5 10 15 Leu Ile Lys Tyr Tyr Gln
Ala Phe Met Ile Gly Ser Asp 20 25
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