U.S. patent application number 11/636665 was filed with the patent office on 2007-05-03 for novel nucleic acid sequences encoding melanoma associated antigen molecules, aminotransferase molecles, atpase molecules, acyltransferase molecules, pyridoxal-phosphate dependant enzyme molecules and uses therefor.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Rajasekhar Bandaru, Maria Alexandra Glucksmann, Rachel E. Meyers, Laura A. Rudolph-Owen.
Application Number | 20070099230 11/636665 |
Document ID | / |
Family ID | 27585468 |
Filed Date | 2007-05-03 |
United States Patent
Application |
20070099230 |
Kind Code |
A1 |
Bandaru; Rajasekhar ; et
al. |
May 3, 2007 |
Novel nucleic acid sequences encoding melanoma associated antigen
molecules, aminotransferase molecles, ATPase molecules,
acyltransferase molecules, pyridoxal-phosphate dependant enzyme
molecules and uses therefor
Abstract
The invention provides isolated nucleic acids molecules that
encode novel polypeptides. The invention also provides antisense
nucleic acid molecules, recombinant expression vectors containing
the nucleic acid molecules of the invention, host cells into which
the expression vectors have been introduced, and nonhuman
transgenic animals in which a sequence of the invention has been
introduced or disrupted. The invention still further provides
isolated proteins, fusion proteins, antigenic peptides and
antibodies. Diagnostic methods utilizing compositions of the
invention are also provided.
Inventors: |
Bandaru; Rajasekhar;
(Watertown, MA) ; Glucksmann; Maria Alexandra;
(Lexington, MA) ; Meyers; Rachel E.; (Newton,
MA) ; Rudolph-Owen; Laura A.; (Jamaica Plain,
MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
40 Landsdowne Street
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
27585468 |
Appl. No.: |
11/636665 |
Filed: |
December 8, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11245400 |
Oct 6, 2005 |
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11636665 |
Dec 8, 2006 |
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10164966 |
Jun 7, 2002 |
7078205 |
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11245400 |
Oct 6, 2005 |
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10034864 |
Dec 27, 2001 |
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10164966 |
Jun 7, 2002 |
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09996194 |
Nov 28, 2001 |
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10164966 |
Jun 7, 2002 |
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09908928 |
Jul 19, 2001 |
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10164966 |
Jun 7, 2002 |
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09908180 |
Jul 18, 2001 |
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10164966 |
Jun 7, 2002 |
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09887389 |
Jun 22, 2001 |
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10164966 |
Jun 7, 2002 |
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09789300 |
Feb 20, 2001 |
6458576 |
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10164966 |
Jun 7, 2002 |
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60258517 |
Dec 28, 2000 |
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60250348 |
Nov 30, 2000 |
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60250073 |
Nov 30, 2000 |
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60253878 |
Nov 29, 2000 |
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60250338 |
Nov 30, 2000 |
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60220465 |
Jul 20, 2000 |
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60219740 |
Jul 20, 2000 |
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60214138 |
Jun 26, 2000 |
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60183208 |
Feb 17, 2000 |
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Current U.S.
Class: |
435/6.11 ;
435/320.1; 435/325; 435/6.18; 435/69.1; 435/7.23; 530/350;
536/23.5 |
Current CPC
Class: |
C07K 2319/00 20130101;
A01K 2217/05 20130101; C12N 9/1096 20130101; A01K 2217/075
20130101; C07K 14/47 20130101; C12N 9/14 20130101; C12N 9/1029
20130101; A61K 38/00 20130101; A61K 39/00 20130101; G01N 33/5743
20130101; C07K 14/4748 20130101; A61K 48/00 20130101; C12N 9/90
20130101 |
Class at
Publication: |
435/006 ;
536/023.5; 435/007.23; 435/069.1; 435/320.1; 435/325; 530/350 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/574 20060101 G01N033/574; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C07K 14/82 20060101
C07K014/82 |
Claims
1. An isolated 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, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
29, 31, 36, 38, 40, or 42; b) a nucleic acid molecule comprising a
fragment of at least 300 nucleotides of the nucleotide sequence of
SEQ ID NO:1, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29,
31, 36, 38, 40, or 42; c) a nucleic acid molecule which encodes a
polypeptide comprising the amino acid sequence of SEQ ID NO:2, 7,
11, 15, 19, 23, 27, 30, 37, or 41; d) a nucleic acid molecule which
encodes a fragment of a polypeptide comprising the amino acid
sequence of SEQ ID NO:2, 7, 11, 15, 19, 23, 27, 30, 37, or 41,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:2, 7, 11, 15, 19, 23, 27, 30, 37, or 41; and 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, 7, 11, 15, 19, 23, 27, 30, 37, or 41, wherein the nucleic
acid molecule hybridizes to a nucleic acid molecule comprising SEQ
ID NO:1, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29, 31,
36, 38, 40, or 42, or a complement thereof, under stringent
conditions.
2. The isolated nucleic acid molecule of claim 1, which is selected
from the group consisting of: a) a nucleic acid comprising the
nucleotide sequence of SEQ ID NO:1, 3, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 29, 31, 36, 38, 40, or 42; and b) a nucleic
acid molecule which encodes a polypeptide comprising the amino acid
sequence of SEQ ID NO:2, 7, 11, 15, 19, 23, 27, 30, 37, or 41.
3. The nucleic acid molecule of claim 1 further comprising vector
nucleic acid sequences.
4. The nucleic acid molecule of claim 1 further comprising nucleic
acid sequences encoding a heterologous polypeptide.
5. A non-human host cell containing the nucleic acid molecule of
claim 1.
6. The host cell of claim 5 which is a mammalian host cell.
7. An isolated polypeptide selected from the group consisting of:
a) a polypeptide which is encoded by a nucleic acid molecule
comprising a nucleotide sequence which is at least 60% identical to
a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1,
3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29, 31, 36, 38,
40, or 42, or a complement thereof; b) a naturally occurring
allelic variant of a polypeptide comprising the amino acid sequence
of SEQ ID NO:2, 7, 11, 15, 19, 23, 27, 30, 37, or 41, wherein the
polypeptide is encoded by a nucleic acid molecule which hybridizes
to a nucleic acid molecule comprising SEQ ID NO:1, 3, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 29, 31, 36, 38, 40, or 42, or a
complement thereof under stringent conditions; and c) a fragment of
a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 7,
11, 15, 19, 23, 27, 30, 37, or 41, wherein the fragment comprises
at least 15 contiguous amino acids of SEQ ID NO:2, 7, 11, 15, 19,
23, 27, 30, 37, or 41.
8. The polypeptide of claim 7 further comprising heterologous amino
acid sequences.
9. An antibody which selectively binds to a polypeptide of claim
7.
10. 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, 7, 11, 15, 19, 23, 27, 30, 37, or 41; b) a
polypeptide comprising a fragment of the amino acid sequence of SEQ
ID NO:2, 7, 11, 15, 19, 23, 27, 30, 37, or 41, wherein the fragment
comprises at least 15 contiguous amino acids of SEQ ID NO:2, 7, 11,
15, 19, 23, 27, 30, 37, or 41; and c) a naturally occurring allelic
variant of a polypeptide comprising the amino acid sequence of SEQ
ID NO:2, 7, 11, 15, 19, 23, 27, 30, 37, or 41, wherein the
polypeptide is encoded by a nucleic acid molecule which hybridizes
to a nucleic acid molecule comprising SEQ ID NO:1, 3, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 29, 31, 36, 38, 40, or 42;
comprising culturing the host cell of claim 5 under conditions in
which the nucleic acid molecule is expressed.
11. A method for detecting the presence of a polypeptide of claim 7
in a sample, comprising: a) contacting the sample with a compound
which selectively binds to a polypeptide of claim 7; and b)
determining whether the compound binds to the polypeptide in the
sample.
12. The method of claim 11, wherein the compound which binds to the
polypeptide is an antibody.
13. A kit comprising a compound which selectively binds to a
polypeptide of claim 7 and instructions for use.
14. A method for detecting the presence of a nucleic acid molecule
of claim 1 in a sample, comprising the steps of: a) contacting the
sample with a nucleic acid probe or primer which selectively
hybridizes to the nucleic acid molecule; and b) determining whether
the nucleic acid probe or primer binds to a nucleic acid molecule
in the sample.
15. The method of claim 14, wherein the sample comprises mRNA
molecules and is contacted with a nucleic acid probe.
16. A kit comprising a compound which selectively hybridizes to a
nucleic acid molecule of claim 1 and instructions for use.
17. A method for identifying a compound which binds to a
polypeptide of claim 7 comprising the steps of: a) contacting a
polypeptide, or a cell expressing a polypeptide of claim 7 with a
test compound; and b) determining whether the polypeptide binds to
the test compound.
18. The method of claim 17, wherein the binding of the test
compound to the polypeptide is detected by a method selected from
the group consisting of: a) detection of binding by direct
detecting of test compound/polypeptide binding; and, b) detection
of binding using a competition binding assay.
19. A method for modulating the activity of a polypeptide of claim
7 comprising contacting a polypeptide or a cell expressing a
polypeptide of claim 7 with a compound which binds to the
polypeptide in a sufficient concentration to modulate the activity
of the polypeptide.
20. A method for identifying a compound which modulates the
activity of a polypeptide of claim 7, comprising: a) contacting a
polypeptide of claim 7 with a test compound; and b) determining the
effect of the test compound on the activity of the polypeptide to
thereby identify a compound which modulates the activity of the
polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/245,400, filed Oct. 6, 2005 (allowed), which is a
divisional of U.S. patent application Ser. No. 10/164,966, filed
Jun. 7, 2002 (U.S. Pat. No. 7,078,205), which is: [0002] a
continuation-in-part of Ser. No. 10/034,864, filed Dec. 27, 2001,
which claims the benefit of U.S. Provisional Application No.
60/258,517, filed Dec. 28, 2000; [0003] and a continuation-in-part
of Ser. No. 09/996,194, filed Nov. 28, 2001, which claims the
benefit of U.S. Provisional Application No. 60/250,348, filed Nov.
30, 2000, U.S. Provisional Application No. 60/250,073, filed Nov.
30, 2000, U.S. Provisional Application No. 60/253,878, filed Nov.
29, 2000, and U.S. Provisional Application No. 60/250,338, filed
Nov. 20, 2000; [0004] and a continuation-in-part of Ser. No.
09/908,928, filed Jul. 19, 2001, which claims the benefit of U.S.
Provisional Application No. 60/220,465, filed Jul. 20, 2000; [0005]
and a continuation-in-part of Ser. No. 09/908,180, filed Jul. 18,
2001, which claims the benefit of U.S. Provisional Application No.
60/219,740, filed Jul. 20, 2000; [0006] and a continuation-in-part
of Ser. No. 09/887,389, filed Jun. 22, 2001, which claims the
benefit of U.S. Provisional Application No. 60/214,138, filed Jun.
26, 2000; [0007] and a continuation-in-part of Ser. No. 09/789,300,
filed Feb. 20, 2001, which claims the benefit of U.S. Provisional
Application No. 60/183,208, filed Feb. 17, 2000; all of which are
hereby incorporated herein in their entirety by reference.
[0008] The contents of the Sequence Listing are submitted herewith
on compact disc in duplicate. Each duplicate disc has a copy of the
file "sequence listing.txt" which is incorporated herein by this
reference. This file is 188 kilobytes and is a copy of the sequence
listing filed in U.S. patent application Ser. No. 10/164,966, filed
Jun. 7, 2002. This file was copied onto compact disc on Dec. 5,
2006.
Field of the Invention
[0009] The invention relates to novel nucleic acid sequences and
polypeptides. Also provided are vectors, host cells, and
recombinant methods for making and using the novel molecules.
TABLE-US-00001 TABLE OF CONTENTS Chapter 1 6 22406, A Novel Human
Pyridoxal-Phosphate Dependent Enzyme Family Member and Uses
Therefor i) 5 SEQ ID NOS: 1-5 ii) FIGS. 1-8 iii)
Continuation-In-Part of 09/789,300, filed Feb. 20, 2001, which
claims the benefit of U.S. Provisional Application No. 60/183,208,
filed Feb. 17, 2000 Chapter 2 6 32447, A Novel Human
Acyltransferase and Uses Thereof i) 5 SEQ ID NOS: 6-9 ii) FIGS.
9-10 iii) Continuation-In-Part of 09/887,389, filed Jun. 22, 2001,
which claims the benefit of U.S. Provisional Application No.
60/214,138, filed Jun. 26, 2000 Chapter 3 6 7716, A Novel Human
ATPase and Uses Therefor i) 5 SEQ ID NOS: 10-13 ii) FIGS. 11-17
iii) Continuation-In-Part of 09/908,180, filed Jul. 18, 2001, which
claims the benefit of U.S. Provisional Application No. 60/219,740,
filed Jul. 20, 2000 Chapter 4 6 25233, A Novel Human
Aminotransferase and Uses Therefor i) 5 SEQ ID NOS: 14-17 ii) FIGS.
18-24 iii) Continuation-In-Part of 09/908,928, filed Jul. 19, 2001,
which claims the benefit of U.S. Provisional Application No.
60/220,465, filed Jul. 20, 2000 Chapter 5 6 8035, 84242, 55304,
52999, AND 21999, Novel Human Proteins and Methods of Use Thereof
i) 5 SEQ ID NOS: 18-39 ii) FIGS. 25-33 iii) Continuation-In-Part of
09/996,194, filed Nov. 28, 2001, which claims the benefit of U.S.
Provisional Application No. 60/250,073, filed Nov. 30, 2000, and
U.S. Provisional Application No. 60/253,878, filed Nov. 29, 2000,
and U.S. Provisional Application No. 60/250,338, filed Nov. 30,
2000 Chapter 6 6 52020, A Novel Human Melanoma Associated Antigen
and Uses Therefor i) 5 SEQ ID NOS: 40-43 ii) FIGS. 34-35 iii)
Continuation-In-Part of 10/034,864, filed Dec. 27, 2001, which
claims the benefit of U.S. Provisional Application No. 60/258,517,
filed Dec. 28, 2000 Chapter 7 6 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, and 52020 Combined
Specification
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 depicts a hydropathy plot of human 22406. Relative
hydrophobic residues are shown above the dashed horizontal line,
and relative hydrophilic residues are below the dashed horizontal
line. The cysteine residues (cys) and N glycosylation site (Ngly)
are indicated by short vertical lines just below the hydropathy
trace. The numbers corresponding to the amino acid sequence (shown
in SEQ ID NO:2) of human 22406 are indicated. Polypeptides of the
invention include fragments which include: all or a part of a
hydrophobic sequence (a sequence above the dashed line); or all or
part of a hydrophilic fragment (a sequence below the dashed line).
Other fragments include a cysteine residue or an N-glycosylation
site. Predicted transmembrane domains (TM) are also depicted.
[0011] FIG. 2 shows an analysis of the 22406 amino acid sequence:
.alpha..beta.turn and coil regions; hydrophilicity; amphipathic
regions; flexible regions; antigenic index; and surface probability
plot.
[0012] FIG. 3 shows transmembrane segments predicted by MEMSAT and
Prosite matches for the 22406 open reading frame for amino acids
corresponding to specific functional sites. For the cAMP-and
cGMP-dependent protein kinase phosphorylation site, the actual
modified residue is the last amino acid. For the protein kinase C
phosphorylation sites, the actual modified residue is the first
amino acid. For the casein kinase II phosphorylation sites, the
actual modified residue is the first amino acid. For the
N-myristoylation site, the actual modified residue is the first
amino acid. In addition, Prosite matches the protein of the
invention to a serine/threonine dehydratase pyridoxal-phosphate
attachment site at about amino acids 47-60.
[0013] FIG. 4 shows expression of the 22406 protein in various
normal human tissues.
[0014] FIG. 5 shows the PSORT prediction of protein
localization.
[0015] FIGS. 6A-6B depict an alignment of the pyridoxal-phosphate
dependent enzyme family member domain (PALP) of human 22406 with a
consensus amino acid sequence derived from a hidden Markov model.
The upper sequence is the consensus amino acid sequence (SEQ ID
NO:4), while the lower amino acid sequence corresponds to amino
acids 19 to 315 of SEQ ID NO:2.
[0016] FIG. 7 displays the ProDom matches for 22406.
[0017] FIGS. 8A-8B display the expression levels of 22406 in
various tissues determined by quantitative PCR. The highest level
of expression is observed in brain cortex. The tissue types are as
follows from left to right: Aorta/Normal, Fetal Heart/Normal,
Heart/Normal, Heart/CHF, Vein/Normal, SMC/Aortic, Nerve/Normal,
Spinal Cord/Normal, Brain Cord/Normal, Brain Cortex/Normal, Brain
Hypothalmus/Normal, Glial Cells (Astrocytes), Glioblastoma,
Breast/Normal, Breast/Tumor, Ovary/Normal, Ovary/Tumor,
Pancreas/Normal, Prostate/Normal, Prostate/Tumor, Colon/Normal,
Colon/Tumor, Colon/IBD, Kidney/Normal, Liver/Normal,
Liver/Fibrosis, Fetal Liver/Normal, Lung/Normal, Lung/COPD,
Spleen/Normal, Tonsil/Normal, Lymph Node/Normal, Thymus/Normal,
Epithelial Cells (Prostate), Endothelial Cells (Aortic), Skeletal
Muscle/Normal, Fibroblasts (Dermal), Skin/Normal, Adipose/Normal,
Osteoblasts (Primary), Osteoblasts (Undiff), Osteoblasts (Diff),
Osteoclasts, NTC.
[0018] FIG. 9 depicts a hydropathy plot of human acyltransferase.
Relative hydrophobic residues are shown above the dashed horizontal
line, and relative hydrophilic residues are below the dashed
horizontal line. The cysteine residues (cys) and N glycosylation
site (Ngly) are indicated by short vertical lines just below the
hydropathy trace. The numbers corresponding to the amino acid
sequence of human acyltransferase are indicated. Polypeptides of
the invention include fragments which include: all or a part of a
hydrophobic sequence (a sequence above the dashed line); or all or
part of a hydrophilic fragment (a sequence below the dashed line).
Other fragments include a cysteine residue or an N-glycosylation
site.
[0019] FIG. 10 depicts an alignment of the acyltransferase domain
of human acyltransferase with a consensus amino acid sequence
derived from a hidden Markov model. The upper sequence is the
consensus amino acid sequence (SEQ ID NO:9), while the lower amino
acid sequence corresponds to amino acids 131 to 317 of SEQ ID
NO:7.
[0020] FIG. 11 depicts a hydropathy plot of human 7716. Relative
hydrophobic residues are shown above the dashed horizontal line,
and relative hydrophilic residues are below the dashed horizontal
line. The cysteine residues (cys) and N glycosylation site (Ngly)
are indicated by short vertical lines just below the hydropathy
trace. The numbers corresponding to the amino acid sequence of
human 7716 (shown in SEQ ID NO:11) are indicated. Polypeptides of
the invention include fragments which include: all or a part of a
hydrophobic sequence (a sequence above the dashed line); or all or
part of a hydrophilic fragment (a sequence below the dashed line).
Other fragments include a cysteine residue or as N-glycosylation
site.
[0021] FIG. 12 depicts an alignment of two ATPase domains of human
7716 with a consensus amino acid sequences derived from a hidden
Markov model. The upper sequence is the consensus amino acid
sequence (SEQ ID NO:13), while the lower amino acid sequence
corresponds to amino acids 236-421 of SEQ ID NO:11 for the domain 1
alignment and amino acids 500-705 of SEQ ID NO:11 for the domain 2
alignment.
[0022] FIG. 13 shows the expression of 7716 in clinical samples
from normal human breast tissue (columns 1-4) and human breast
tumor tissue (columns 5-9). Expression levels for 7716 were
determined by quantitative RT-PCR (Reverse Transcriptase Polymerase
Chain Reaction; Taqman.RTM. brand PCR kit, Applied Biosystems). The
quantitative RT-PCR reactions were performed according to the kit
manufacturer's instructions.
[0023] FIG. 14 shows the expression of 7716 in clinical samples
from normal human ovary tissue (columns 1-3) and human ovary tumor
tissue (columns 4-11). Expression levels for 7716 were determined
as described in the legend for FIG. 13.
[0024] FIG. 15 shows the expression of 7716 in clinical samples
from normal human lung tissue (columns 1-4), and human lung tumor
tissue (columns 5-12). Expression levels for 7716 were determined
as described in the legend for FIG. 13.
[0025] FIG. 16 shows the expression of 7716 in clinical samples
from normal human colon tissue (columns 1-4), human colon tumor
tissue (columns 5-10), human colon cancer liver metastases tissue
(columns 11-14); and normal human liver tissue (columns 15 and 16).
Expression levels of 7716 were determined as described in the
legend for FIG. 13.
[0026] FIG. 17 shows the expression of 7716 in clinical samples
from normal human brain tissue (columns 1-4), normal human
astrocytes (column 5), human brain tumor tissue (columns 6-10);
arresting human microvascular endothelial cells (column 11),
proliferating human microvascular endothelial cells (column 12),
placenta (column 13), fetal adrenal tissue (columns 14 and 15), and
fetal liver (columns 16 and 17). Expression levels of 7716 were
determined as described in the legend for FIG. 13.
[0027] FIG. 18 depicts a hydropathy plot of human 25233. Relative
hydrophobic residues are shown above the dashed horizontal line,
and relative hydrophilic residues are below the dashed horizontal
line. The cysteine residues (cys) and N glycosylation site (Ngly)
are indicated by short vertical lines just below the hydropathy
trace. The numbers corresponding to the amino acid sequence of
human 25233 (shown in SEQ ID NO:15) are indicated. Polypeptides of
the invention include fragments which include: all or a part of a
hydrophobic sequence (a sequence above the dashed line); or all or
part of a hydrophilic fragment (a sequence below the dashed line).
Other fragments include a cysteine residue or an N-glycosylation
site.
[0028] FIG. 19 depicts an alignment of the aminotransferase domain
of human 25233 with a consensus amino acid sequence derived from a
hidden Markov model. The upper sequence is the consensus amino acid
sequence (SEQ ID NO:17), while the lower amino acid sequence
corresponds to amino acids 83 to 517 of SEQ ID NO:15.
[0029] FIG. 20 shows an analysis of the 25233 open reading frame
for amino acid residues corresponding to specific functional sites.
A transmembrane region is predicted at amino acid residues 181-199
of SEQ ID NO:15. A glycosaminoglycan attachment site is found from
about amino acid residues 200-203 and 482-485 of SEQ ID NO:15; a
protein kinase C phosphorylation site from about amino acid
residues 42-44, 168-170, 278-280, 315-317, 339-341, and 372-374 of
SEQ ID NO:15; a casein kinase II phosphorylation site from about
amino acid residues 42-45, 126-129, 168-171, 224-227, and 329-332
of SEQ ID NO:15; and a N-myristoylation site from about amino acid
residues 144-149, 172-177, 186-191, 201-206, 270-275, 437-442, and
481-486 of SEQ ID NO:15.
[0030] FIGS. 21A-21B show the expression of 25233 in the following
human tissues and cell lines: Artery Normal (Column 1);Vein Normal
(Column 2); Aortic SMC (Smooth Muscle Cell) EARLY (Column 3);
Coronary SMC (Column 4); Static HUVEC (Human Umbilical Vein
Endothelial Cells) (Column 5); Shear HUVEC (Column 6); Heart Normal
(Column 7); Heart CHF (Congestive Heart Failure) (Column 8); Kidney
(Column 9); Skeletal Muscle (Column 10); Adipose Normal (Column
11); Pancreas (Column 12); Primary Osteoblasts (Column 13);
Osteoclasts (Differentiated); (Column 14); Skin Normal (Column 15);
Spinal Cord Normal (Column 16); Brain Cortex Normal (Column 17);
Brain Hypothalamus Normal (Column 18); Nerve (Column 19); DRG
(Dorsal Root Ganglion); (Column 20); Glial Cells (Astrocytes);
(Column 21); Glioblastoma (Column 22); Breast Normal (Column 23);
Breast Tumor (Column 24); Ovary Normal (Column 25); Ovary Tumor
(Column 26); Prostate Normal (Column 27); Prostate Tumor (Column
28); Epithelial Cells (Prostate); (Column 29); Colon Normal (Column
30); Colon Tumor (Column 31); Lung Normal (Column 32); Lung Tumor
(Column 33); Lung COPD (Chronic Obstructive Pulmonary Disease)
(Column 34); Colon IBD (Inflammatory Bowel Disease) (Column 35);
Liver Normal (Column 36); Liver Fibrosis (Column 37); Dermal
Cells-Fibroblasts (Column 38); Spleen Normal (Column 39); Tonsil
Normal (Column 40); Lymph Node (Column 41); Small Intestine (Column
42); Skin-Decubitus (Column 43); Synovium (Column 44); BM-MNC (Bone
Marrow Mononuclear Cells); (Column 45); and Activated PBMC
(Peripheral Blood Mononuclear Cells) (Column 46). Expression levels
were determined by reverse transcriptase (RT) quantitative PCR
(Taqman.RTM. brand quantitative PCR kit, Applied Biosystems). The
quantitative PCR reactions were performed according to the kit
manufacturer's instructions.
[0031] FIGS. 22A-22D show the expression of 25233 in human tissues
and cell lines, as follows. FIG. 22A: Breast N (Normal) (Columns
1-4); Breast T (Tumor) (Columns 5-12). FIG. 22B: Ovary N (Columns
1-4); Ovary T (Columns 5-12). FIG. 22C: Lung N (Columns 1-4); Lung
T (Columns 5-12). FIG. 22D depicts a time course study of 25233
expression levels in the human cancer cell line H460 with and
without p16: 24 hr H460-P16 (Column 1); 48 hr H460-P16 (Column 2);
72 hr H460-P16 (Column 3); 96 hr H460-P16 (Column 4); 48 hr H460
+P16 (Column 5); 72 hr H460 +P16 (Column 6); And 96 hr H460+P16
(Column 7). Expression levels were determined as in FIG. 21.
[0032] FIGS. 23A-23B show the expression of 25233 in human tissues
and cell lines, as follows. FIG. 23A: Colon N (Normal) (Columns
1-4); Colon T (Tumor) (Columns 5-11); Liver M (Metastases) (Columns
12-15); Liver N (Columns 16-17). FIG. 23B: Brain N (Columns 1-4);
Astrocytes (Column 5); Brain T (Columns 6-10); HMVEC-Arresting
(Column 11); HMVEC-Proliferating (Column 12); Placenta (Column 13);
Fetal Adrenal (Columns 14-15); Fetal Liver (Columns 16-17).
Expression levels were determined as in FIG. 21.
[0033] FIG. 24 shows the expression of 25233 in the following cell
lines: Breast cancer cell lines MCF-7 (column 1); ZR75 (column 2);
T47D (column 3); MDA 231 (column 4); and MDA 435 (column 5). Colon
cancer cell lines DLD-1 (column 6); SW 480 (column 7); SW 620
(column 8); HCT 116 (column 9); HT 29 (column 10); and Colo 205
(column 11). Lung cancer cell lines NCIH 125 (column 12); NCIH 67
(column 13); NCIH 322 (column 14); NCIH 460 (column 15); and A549
(column 16). NHBE (Normal human bronchial epithelial cells) (column
17). Expression levels were determined as in FIG. 21.
[0034] FIG. 25 depicts a hydropathy plot of human 8035. Relative
hydrophobic residues are shown above the dashed horizontal line,
and relative hydrophilic residues are below the dashed horizontal
line. The cysteine residues (cys) and N glycosylation site (Ngly)
are indicated by short vertical lines just below the hydropathy
trace. The numbers corresponding to the amino acid sequence (shown
in SEQ ID NO:19) of human 8035 are indicated. Polypeptides of the
invention include fragments that include: all or a part of a
hydrophobic sequence (a sequence above the dashed line); or all or
part of a hydrophilic fragment (a sequence below the dashed line).
Other fragments include a cysteine residue or an N-glycosylation
site.
[0035] FIG. 26 depicts a hydropathy plot of human 84242. Relative
hydrophobic residues are shown above the dashed horizontal line,
and relative hydrophilic residues are below the dashed horizontal
line. The cysteine residues (cys) and N glycosylation site (Ngly)
are indicated by short vertical lines just below the hydropathy
trace. The numbers corresponding to the amino acid sequence (shown
in SEQ ID NO:23) of human 84242 are indicated. Polypeptides of the
invention include fragments that include: all or a part of a
hydrophobic sequence (a sequence above the dashed line); or all or
part of a hydrophilic fragment (a sequence below the dashed line).
Other fragments include a cysteine residue or an N-glycosylation
site.
[0036] FIG. 27 depicts an alignment of the RING finger protein
domain (C3HC4 type) of human 8035 with a consensus amino acid
sequence derived from a hidden Markov model. The upper sequence is
the consensus amino acid sequence (SEQ ID NO:21), while the lower
amino acid sequence corresponds to amino acids 380 to 421 of SEQ ID
NO:19.
[0037] FIG. 28 depicts an alignment of the IBR (In Between RING
Fingers) protein domain of human 84242 with a consensus amino acid
sequence derived from a hidden Markov model. The upper sequence is
the consensus amino acid sequence (SEQ ID NO:25), while the lower
amino acid sequence corresponds to amino acids 2 to 67 of SEQ ID
NO:23.
[0038] FIG. 29 depicts a hydropathy plot of human 55304. Relative
hydrophobic residues are shown above the dashed horizontal line,
and relative hydrophilic residues are below the dashed horizontal
line. The cysteine residues (cys) and N glycosylation site (Ngly)
are indicated by short vertical lines just below the hydropathy
trace. The numbers corresponding to the amino acid sequence (shown
in SEQ ID NO:27) of human 55304 are indicated. Polypeptides of the
invention include fragments which include: all or a part of a
hydrophobic sequence (a sequence above the dashed line); or all or
part of a hydrophilic fragment (a sequence below the dashed line).
Other fragments include a cysteine residue or an N-glycosylation
site.
[0039] FIG. 30 depicts a hydropathy plot of human 52999. Relative
hydrophobic residues are shown above the dashed horizontal line,
and relative hydrophilic residues are below the dashed horizontal
line. The cysteine residues (cys) and N glycosylation site (Ngly)
are indicated by short vertical lines just below the hydropathy
trace. The numbers corresponding to the amino acid sequence (shown
in SEQ ID NO:30) of human 52999 are indicated. Polypeptides of the
invention include fragments which include: all or a part of a
hydrophobic sequence (a sequence above the dashed line); or all or
part of a hydrophilic fragment (a sequence below the dashed line).
Other fragments include a cysteine residue or an N-glycosylation
site.
[0040] FIG. 31 depicts an alignment of portions of the
metallopeptidase domain of human 52999 with consensus amino acid
sequences derived from hidden Markov models. The upper sequences
are the consensus amino acid sequences for the Peptidase_M8 family
of zinc metallopeptidases and the lower amino acid sequences
correspond to amino acids of human 52999. The upper sequence of
domain 1 of 4 is SEQ ID NO:32 and the lower amino acid sequence
corresponds to amino acids 180 to 192 of SEQ ID NO:30. The upper
sequence of domain 2 of 4 is SEQ ID NO:33 and the lower amino acid
sequence corresponds to amino acids 230 to 290 of SEQ ID NO:30. The
upper sequence of domain 3 of 4 is SEQ ID NO:34 and the lower amino
acid sequence corresponds to amino acids 354 to 409 of SEQ ID
NO:30. The upper sequence of domain 4 of 4 is SEQ ID NO:35 and the
lower amino acid sequence corresponds to amino acids 520 to 554 of
SEQ ID NO:30.
[0041] FIG. 32 depicts a hydropathy plot of human
ADP-ribosyltransferase. Relative hydrophobic residues are shown
above the dashed horizontal line, and relative hydrophilic residues
are below the dashed horizontal line. The cysteine residues (cys)
and N glycosylation site (Ngly) are indicated by short vertical
lines just below the hydropathy trace. The numbers corresponding to
the amino acid sequence (shown in SEQ ID NO:37) of human
ADP-ribosyltransferase are indicated. Polypeptides of the invention
include fragments which include: all or a part of a hydrophobic
sequence (a sequence above the dashed line); or all or part of a
hydrophilic fragment (a sequence below the dashed line). Other
fragments include a cysteine residue or as N-glycosylation
site.
[0042] FIG. 33 depicts an alignment of the human
ADP-ribosyltransferase polypeptiode with a consensus amino acid
sequence derived from a hidden Markov model. The upper sequence is
the consensus amino acid sequence (SEQ ID NO:39), while the lower
amino acid sequence corresponds to amino acid 3 to amino acid 271
of SEQ ID NO:37.
[0043] FIG. 34 depicts a hydropathy plot of human 52020. Relative
hydrophobic residues are shown above the dashed horizontal line,
and relative hydrophilic residues are below the dashed horizontal
line. The cysteine residues (cys) and N glycosylation site (Ngly)
are indicated by short vertical lines just below the hydropathy
trace. The numbers corresponding to the amino acid sequence (shown
in SEQ ID NO:41) of human 52020 are indicated. Polypeptides of the
invention include fragments which include: all or a part of a
hydrophobic sequence (a sequence above the dashed line); or all or
part of a hydrophilic fragment (a sequence below the dashed line).
Other fragments include a cysteine residue or as N-glycosylation
site.
[0044] FIG. 35 depicts an alignment of the MAGE domain of human
52020 with a consensus amino acid sequence derived from a hidden
Markov model. The upper sequence is the consensus amino acid
sequence (SEQ ID NO:43), while the lower amino acid sequence
corresponds to amino acids 1 to 208 of SEQ ID NO:41.
CHAPTER 1
22406, A Novel Human Pyridoxal-phosphate Dependent Enzyme Family
Member and Uses Therefor
Background of the Invention
[0045] The pyridoxal-phosphate dependent family of enzymes require
the co-factor, pyridoxal-5'-phosphate (pyridoxal-phosphate), for
catalytic activity. Pyridoxal-phosphate dependent enzymes (B6
enzymes) catalyze manifold reactions in the metabolism of amino
acids. L- and D-serine dehydratase, threonine dehydratase, and
serine racemase are a few of the members of this family of enzymes.
In all of the members of the family, the pyridoxal-phosphate group
is attached to a lysine residue. The sequence around this residue
is sufficiently conserved to allow the derivation of a pattern
specific to pyridoxal-phosphate dependent enzymes.
[0046] The pyridoxal-phosphate dependent family member, serine
racemase, has been shown to catalyze the direct racemization of
L-serine to D-serine with a requirement for pyridoxal 5'-phosphate
(Wolosker et al. (1999) PNAS 96:721-725). The properties of this
enzyme resemble those of bacterial racemases, suggesting that the
biosynthetic pathway for D-amino acids is conserved from bacteria
to mammalian brain.
[0047] It has been demonstrated that D-serine is the endogenous
ligand for the glycine site of the glutamate N-methyl-D-aspartate
(NMDA) receptor (Mothet et al. (2000) PNAS 97:4926-4931). The amino
acid D-serine is synthesized and stored in glia rather than in
neurons. Released glutamate acts on receptors on the protoplasmic
astrocytes closely opposed to the synapse to release D-serine,
which co-activates post-synaptic NMDA receptors together with
glutamate. As D-serine is formed by serine racemase, inhibitors of
this enzyme can be expected to reduce NMDA neurotransmission.
[0048] D-serine has been shown to modify behavioral changes
associated with learning, memory, convulsion, anxiety,
psychotomimetic induced abnormal behavior, cerebellar ataxia, and
neurodengeneration. Inhibitors of serine racemase can be expected
to quell anxiety and epilepsy and to prevent damage from stroke and
certain neurodegenerative conditions including Alzheimer's disease.
On the other hand, stimulating serine racemase might improve
schizophrenia symptoms, which are partly caused by depressed NMDA
receptor function.
[0049] Accordingly, members of the pyridoxal-phosphate dependent
enzyme class are a major target for drug action and development.
Therefore, it is valuable to the field of pharmaceutical
development to identify and characterize previously unknown serine
racemases. The present invention advances the state of the art by
providing a previously unidentified human pyridoxal-phosphate
dependent serine racemace.
Summary of the Invention
[0050] The present invention is based, in part, on the discovery of
a novel human pyridoxal-phosphate dependent enzyme family member,
referred to herein as "22406". The nucleotide sequence of a cDNA
encoding 22406 is shown in SEQ ID NO:1, and the amino acid sequence
of a 22406 polypeptide is shown in SEQ ID NO:2. In addition, the
nucleotide sequence of the coding region is depicted in SEQ ID
NO:3.
[0051] Accordingly, in one aspect the invention features a nucleic
acid molecule which encodes a 22406 protein or polypeptide, e.g., a
biologically active portion of the 22406 protein. In a preferred
embodiment, the isolated nucleic acid molecule encodes a
polypeptide having the amino acid sequence of SEQ ID NO:2. In other
embodiments, the invention provides an isolated 22406 nucleic acid
molecule having the nucleotide sequence shown in SEQ ID NO:1 or SEQ
ID NO:3. 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 or SEQ ID NO:3. In other embodiments, the invention
provides a nucleic acid molecule which hybridizes under stringent
hybridization conditions to a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, wherein the
nucleic acid encodes a full length 22406 protein or an active
fragment thereof.
[0052] In a related aspect, the invention further provides nucleic
acid constructs which include a 22406 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 22406 nucleic acid molecules of the
invention e.g., vectors and host cells suitable for producing 22406
nucleic acid molecules and polypeptides.
[0053] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of 22406-encoding nucleic acids.
[0054] In still another related aspect, isolated nucleic acid
molecules that are antisense to a 22406 encoding nucleic acid
molecule are provided.
[0055] In another aspect, the invention features 22406
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 22406-mediated or -related
disorders. In another embodiment, the invention provides 22406
polypeptides having a 22406 activity. Preferred polypeptides are
22406 proteins including at least one pyridoxal-phosphate dependent
enzyme family member domain, and, preferably, having a 22406
activity, e.g., a 22406 activity as described herein.
[0056] In other embodiments, the invention provides 22406
polypeptides, e.g., a 22406 polypeptide having the amino acid
sequence shown in SEQ ID NO:2; an amino acid sequence that is
substantially identical to the amino acid sequence shown in SEQ ID
NO:2; or an amino acid sequence encoded by a nucleic acid molecule
having a nucleotide sequence which hybridizes under stringent
hybridization conditions to a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, wherein the
nucleic acid encodes a full length 22406 protein or an active
fragment thereof.
[0057] In a related aspect, the invention further provides nucleic
acid constructs which include a 22406 nucleic acid molecule
described herein.
[0058] In a related aspect, the invention provides 22406
polypeptides or fragments operatively linked to non-22406
polypeptides to form fusion proteins.
[0059] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically bind 22406 polypeptides.
[0060] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the 22406 polypeptides or nucleic acids.
[0061] In still another aspect, the invention provides a process
for modulating 22406 polypeptide or nucleic acid expression or
activity, e.g. using the screened compounds. In certain
embodiments, the methods involve treatment of conditions related to
aberrant activity or expression of the 22406 polypeptides or
nucleic acids, such as conditions involving neurological
disorders.
[0062] The invention also provides assays for determining the
activity of or the presence or absence of 22406 polypeptides or
nucleic acid molecules in a biological sample, including for
disease diagnosis.
[0063] In further aspect the invention provides assays for
determining the presence or absence of a genetic alteration in a
22406 polypeptide or nucleic acid molecule, including for disease
diagnosis.
[0064] Other features and advantages of the invention will be
apparent from the following detailed description, Chapter 7,
Examples and claims.
Detailed Description of the Invention
[0065] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0066] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
Human 22406
[0067] The human 22406 sequence (SEQ ID NO:1), which is
approximately 1770 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
1020 nucleotides (nucleotides 69-1088 of SEQ ID NO:1; SEQ ID NO:3),
not including the terminal codon. The coding sequence encodes a 340
amino acid protein (SEQ ID NO:2). Chromosome mapping localized the
gene to human chromosome 17 between D17S849 and D17S796
(0.6-14cM).
[0068] Human 22406 contains a predicted pyridoxal-phosphate
dependent enzyme family member domain (PALP) (PFAM Accession
PF00291) located at about amino acid residues 19-315 of SEQ ID NO:2
(FIG. 6). The annotation "S_T_dehydratase" in the PFAM alignment of
FIG. 6 reflects a change in nomenclature of the Pfam identifier for
this class of enzyme domain. Human 22406 is also predicted to
contain two transmembrane domains which extend from about amino
acid residues 176-197 and 308-326 of SEQ ID NO:2 (FIG. 3).
[0069] The results of a BLASTX search reveal that the amino acid
sequence of 22406 shares about 90% sequence identity and about 96%
sequence similarity with a murine serine racemase (Accession No.
AF148321). Similar results of a BLASTN search reveal that the
nucleotide sequence of 22406 shares about 88% sequence identity
with this murine serine racemase (Accession No. AAF08701). This
serine racemase is a member of the pyridoxal-phosphate dependent
family of enzymes with the Pfam identifier, PALP, (Wolosker et al.
(1999) PNAS 96:13409-13414).
[0070] Members of the pyridoxal-phosphate dependent enzymes
frequently have the pyridoxal-phosphate group attached via a lysine
residue. The sequence around this residue is sufficiently conserved
to allow the derivation of a pattern specific to
pyridoxal-phosphate dependent enzymes. This pyridoxal-phosphate
attachment site consensus pattern (SEQ ID NO:5) is as follows:
[DESH]-x(4,5)-[STVG]-x-[AS]-[FYI]-K-[DLIFSA]-[RVMF]-[GA]-[LIVMGA]
[0071] In this sequence the "x" can represent any amino acid and
the brackets indicate that any of the amino acids contained within
are allowed at that position. The "K" is the lysine
pyridoxal-phosphate attachment site. The 22406 polypeptide contains
such a consensus pattern at amino acid residues 47-60 (FIG. 3). The
annotation "Dehydratase_Ser_Thr" rather than "PALP" in this figure
reflects the fact that the Pfam identifier for this class of enzyme
domain has been recently been updated from Dehydratase_Ser_Thr to
PALP.
[0072] For general information regarding PFAM identifiers, PS
prefix and PF prefix domain identification numbers, refer to
Sonnhammer et al. (1997) Protein 28:405-420 and the Pfam website
maintained in several locations, e.g. by the Sanger Institute
(sanger.ac.uk/Software/Pfam).
[0073] The 22406 protein contains a significant number of
structural characteristics in common with members of the
pyridoxal-phosphate dependent enzyme family. The term "family" when
referring to the protein and nucleic acid molecules of the
invention means two or more proteins or nucleic acid molecules
having a common structural domain or motif and having sufficient
amino acid or nucleotide sequence homology as defined herein. Such
family members can be naturally or non-naturally occurring and can
be from either the same or different species. For example, a family
can contain a first protein of human origin as well as other
distinct proteins of human origin, or alternatively, can contain
homologues of non-human origin, e.g., rat or mouse proteins.
Members of a family can also have common functional
characteristics.
[0074] As used herein, the term "pyridoxal-phosphate dependent
enzyme family member" refers to a protein or polypeptide which is
capable of metabolism of amino acids. As referred to herein,
pyridoxal-phosphate dependent family members preferably include a
catalytic domain of about 100-340 amino acid residues in length,
preferably about 200-320 amino acid residues in length, or more
preferably about 250-310 amino acid residues in length. A
pyridoxal-phosphate dependent enzyme family member typically
includes at least one of block of homology known as a
pyridoxal-phosphate attachment site characterized by the following
motif and described above:
[DESH]-x(4,5)-[STVG]-x-[AS]-[FYI]-K-[DLIFSA]-[RVMF]-[GA]-[LIVMGA]
(SEQ ID NO:5). Specificity of a pyridoxal-phosphate dependent
enzyme family member for catalysis of a particular amino acid
metabolic reaction is determined by sequence identity to such a
particular sub-class of pyridoxal-phosphate dependent enzyme family
members.
[0075] For example, the 22406 nucleotide and amino acid sequences
of the invention contain high sequence identity to the serine
racemase class of pyridoxal-phosphate dependent enzymes as
described above and found in Wolosker et al. (1999) PNAS
96:13409-13414, herein incorporated by reference in its entirety.
Based on this sequence similarity, the 22406 molecules of the
present invention are predicted to have similar biological
activities as pyridoxal-phosphate dependent serine racemase enzyme
family members.
[0076] Typically, pyridoxal-phosphate dependent enzyme family
members play a role in diverse cellular processes. For example, the
metabolism of amino acids involves specific reactions catalyzed by
various pyridoxal-phosphate dependent enzyme family members. The
pyridoxal-phosphate dependent serine racemase enzymes catalyze the
formation of D-serine from L-serine. This reaction is important as
D-serine is the endogenous ligand for the glycine site of the
glutamate N-methyl-D-aspartate (NMDA) receptor (Mothet et al.
(2000) PNAS 97:4926-4931). In the brain D-serine co-activates
post-synaptic NMDA receptors together with glutamate. NMDA receptor
function has been shown to be a mediator of behavioral changes
associated with a variety of neurological disorders. Thus, the
molecules of the present invention may be involved in one or more
of: 1) catalyzation of the formation of D-serine from L-serine; 2)
the activation of NMDA receptors; 3) learning; 4) memory; 5)
convulsion; 6) anxiety; 7) psychotomimetic induced abnormal
behavior; 8) cerebellar ataxia; and 9) neurodengeneration.
[0077] A 22406 polypeptide can include a "pyridoxal-phosphate
dependent enzyme family member domain" or regions homologous with
an "pyridoxal-phosphate dependent enzyme family member domain".
[0078] As used herein, the term "pyridoxal-phosphate dependent
enzyme family member domain" includes an amino acid sequence of
about 100-340 amino acid residues in length and having a bit score
for the alignment of the sequence to the pyridoxal-phosphate
dependent enzyme family member domain (HMM) of at least 8.
Preferably, an pyridoxal-phosphate dependent enzyme family member
domain includes at least about 200-320 amino acids, more preferably
about 250-310 amino acid residues, or about 290-300 amino acids and
has a bit score for the alignment of the sequence to the
pyridoxal-phosphate dependent enzyme family member domain (HMM) of
at least 16 or greater. The pyridoxal-phosphate dependent enzyme
family member domain (HMM) has been assigned the PFAM Accession
PF00291 (found at the Pfam website, pfam.wustl.edu). An alignment
of the pyridoxal-phosphate dependent enzyme family member domain
(amino acids 19 to 315 of SEQ ID NO:2) of human 22406 with a
consensus amino acid sequence derived from a hidden Markov model is
depicted in FIG. 6.
[0079] In a preferred embodiment 22406 polypeptide or protein has a
"pyridoxal-phosphate dependent enzyme family member domain (PALP)"
or a region which includes at least about 200-320, more preferably
about 250-310 or 290-300 amino acid residues and has at least about
60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with a "PALP
domain," e.g., the pyridoxal-phosphate dependent enzyme family
member domain of human 22406 (e.g., amino acid residues 19-315 of
SEQ ID NO:2).
[0080] To identify the presence of an "pyridoxal-phosphate
dependent enzyme family member" domain in a 22406 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
(sanger.ac.uk/Software/Pfam). 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.
[0081] In one embodiment, a 22406 protein includes 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 that spans a phospholipid membrane. More
preferably, a transmembrane domain includes about at least 18, 20,
22, 24, 25, 30, 35 or 40 amino acid residues and spans a
phospholipid 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,
the Pfam website at 7tm.sub.--1 (pfam.wustl.edu) and Zagotta W. N.
et al. (1996) Annual Rev. Neuronsci. 19:235-63, the contents of
which are incorporated herein by reference.
[0082] In one embodiment, a 22406 polypeptide or protein has at
least one transmembrane domain or a region which includes at least
18, 20, 22, 24, 25, 30, 35 or 40 amino acid residues and has at
least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a
"transmembrane domain," e.g., at least one transmembrane domain of
human 22406 (e.g., amino acid residues 176-197 and 308-326 of SEQ
ID NO:2).
[0083] In one embodiment, a 22406 protein includes at least one
"non-transmembrane domain." As used herein, "non-transmembrane
domains" are domains that reside outside of the membrane. When
referring to plasma membranes, non-transmembrane domains include
extracellular domains (i.e., outside of the cell) and intracellular
domains (i.e., within the cell). When referring to membrane-bound
proteins found in intracellular organelles (e.g., mitochondria,
endoplasmic reticulum, peroxisomes and microsomes),
non-transmembrane domains include those domains of the protein that
reside in the cytosol (i.e., the cytoplasm), the lumen of the
organelle, or the matrix or the intermembrane space (the latter two
relate specifically to mitochondria organelles). The C-terminal
amino acid residue of a non-transmembrane domain is adjacent to an
N-terminal amino acid residue of a transmembrane domain in a
naturally-occurring 22406 protein, or 22406-like protein.
[0084] In one embodiment a 22406 polypeptide or protein has at
least one "non-transmembrane domain" or a region which includes at
least about 1-175 acid residues, and has at least about 60%, 70%
80% 90% 95%, 99% or 100% homology with a "non-transmembrane
domain", e.g., a non-transmembrane domain of human 22406 (e.g.,
residues 1-175, 198-307, and 327-340 of SEQ ID NO:2). Preferably, a
non-transmembrane domain is capable of catalytic activity (e.g.,
catalyzing a serine racemazation reaction).
[0085] As the 22406 polypeptides of the invention may modulate
22406-mediated activities, they may be useful as of for developing
novel diagnostic and therapeutic agents for 22406-mediated or
related disorders, as described below.
[0086] As used herein, a "22406 activity", "biological activity of
22406" or "functional activity of 22406", refers to an activity
exerted by a 22406 protein, polypeptide or nucleic acid molecule on
e.g., a 22406-responsive cell or on a 22406 substrate, e.g., an
amino acid substrate, as determined in vivo or in vitro. In one
embodiment, a 22406 activity is a direct activity, such as an
association with a 22406 target molecule. A "target molecule" or
"binding partner" is a molecule with which a 22406 protein binds or
interacts in nature, e.g., an amino acid such as L-serine or
D-serine. A 22406 activity can also be an indirect activity, e.g.,
a cellular signaling activity mediated by interaction of the
D-serine product of 22406 catalysis with a D-serine "receptor",
"target molecule" or "binding partner". A D-serine "receptor",
"target molecule" or "binding partner" is herein defined as a
molecule with which D-serine binds or interacts in nature, and
these terms are herein used interchangeably. For example, the 22406
proteins of the present invention can have one or more of the
following activities: 1) catalyzation of the formation of D-serine
from L-serine; 2) activation of the NMDA receptor; 3) mediation of
learning; 4) mediation of memory; 5) mediation of convulsion; 6)
mediation of anxiety; 7) mediation of psychotomimetic induced
abnormal behavior; 8) mediation of cerebellar ataxia; 9) mediation
of neurodengeneration and 10) the ability to modulate,
competitively or non-competitively, any of 1-10. "Modulate" is
herein defined as increasing or decreasing an activity or process
by any mechanism, including but not limited to, inhibition or
antagonism by competitive or non-competitive binding.
[0087] Accordingly, 22406 protein may mediate various disorders,
particularly brain disorders, including but not limited to,
behavioral changes associated with learning, memory, convulsion,
anxiety, psychotomimetic induced abnormal behavior, cerebellar
ataxia, and neurodengeneration. Inhibitors of 22406 protein can be
expected to quell anxiety and epilepsy and to prevent damage from
stroke and certain neurodegenerative conditions including
Alzheimer's disease. On the other hand, stimulating 22406 protein
might improve schizophrenia symptoms, which are partly caused by
depressed NMDA receptor function.
[0088] In addition, 22406 protein can be expected to be involved in
various disorders of the tissues in which it is expressed. FIGS. 4
and 8 show expression of the 22406 protein in various normal human
tissues with highest expression in brain, heart, liver, skeletal
muscle, lymph node, prostate, dermal fibroblast, testes, and
thymus. Significant expression is also found in various other
tissues. In addition to the tissues shown in the Figures,
expression has also been observed in adrenal gland, bone,
endothelial cells, total fetal tissue, hypothalamus, keratinocytes,
natural killer cells, osteoblasts, pituitary, skin, spinal cord,
T-cells, colon to liver metastases and lymphoma.
[0089] Expression was also observed in two separate lung tumor cDNA
libraries while libraries of normal lung and bronchial epithelia
sequenced to equal depths yielded no sequences for the 22406
protein. Additionally, PCR analysis on panels containing normal and
tumor lung cDNAs showed that the gene may be expressed at higher
levels in lung tumor samples. Expression was also observed in
colonic tumor cDNA libraries.
[0090] Thus, 22406 can be also be expected to be involved in
disorders including heart disorders, liver disorders, lung
disorders, prostrate disorders, colon disorders, skeletal muscle
disorders, dermal fibroblast disorders, lymph node disorders, and
blood vessel disorders.
[0091] 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, Varicalla-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
degeneration, multiple system atrophy, including striatonigral
degeneration, 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.
[0092] Disorders involving the heart, include but are not limited
to, heart failure, including but not limited to, cardiac
hypertrophy, left-sided heart failure, and right-sided heart
failure; ischemic heart disease, including but not limited to
angina pectoris, myocardial infarction, chronic ischemic heart
disease, and sudden cardiac death; hypertensive heart disease,
including but not limited to, systemic (left-sided) hypertensive
heart disease and pulmonary (right-sided) hypertensive heart
disease; valvular heart disease, including but not limited to,
valvular degeneration caused by calcification, such as calcific
aortic stenosis, calcification of a congenitally bicuspid aortic
valve, and mitral annular calcification, and myxomatous
degeneration of the mitral valve (mitral valve prolapse), rheumatic
fever and rheumatic heart disease, infective endocarditis, and
noninfected vegetations, such as nonbacterial thrombotic
endocarditis and endocarditis of systemic lupus erythematosus
(Libman-Sacks disease), carcinoid heart disease, and complications
of artificial valves; myocardial disease, including but not limited
to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, and myocarditis; pericardial disease, including but
not limited to, pericardial effusion and hemopericardium and
pericarditis, including acute pericarditis and healed pericarditis,
and rheumatoid heart disease; neoplastic heart disease, including
but not limited to, primary cardiac tumors, such as myxoma, lipoma,
papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac
effects of noncardiac neoplasms; congenital heart disease,
including but not limited to, left-to-right shunts--late cyanosis,
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosus, and atrioventricular septal defect,
right-to-left shunts--early cyanosis, such as tetralogy of fallot,
transposition of great arteries, truncus arteriosus, tricuspid
atresia, and total anomalous pulmonary venous connection,
obstructive congenital anomalies, such as coarctation of aorta,
pulmonary stenosis and atresia, and aortic stenosis and atresia,
and disorders involving cardiac transplantation.
[0093] 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.
[0094] Disorders involving the liver include, but are not limited
to, hepatic injury; jaundice and cholestasis, such as bilirubin and
bile formation; hepatic failure and cirrhosis, such as cirrhosis,
portal hypertension, including ascites, portosystemic shunts, and
splenomegaly; infectious disorders, such as viral hepatitis,
including hepatitis A-E infection and infection by other hepatitis
viruses, clinicopathologic syndromes, such as the carrier state,
asymptomatic infection, acute viral hepatitis, chronic viral
hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and
toxin-induced liver disease, such as alcoholic liver disease;
inborn errors of metabolism and pediatric liver disease, such as
hemochromatosis, Wilson disease, .alpha..sub.1-antitrypsin
deficiency, and neonatal hepatitis; intrahepatic biliary tract
disease, such as secondary biliary cirrhosis, primary biliary
cirrhosis, primary sclerosing cholangitis, and anomalies of the
biliary tree; circulatory disorders, such as impaired blood flow
into the liver, including hepatic artery compromise and portal vein
obstruction and thrombosis, impaired blood flow through the liver,
including passive congestion and centrilobular necrosis and
peliosis hepatis, hepatic vein outflow obstruction, including
hepatic vein thrombosis (Budd-Chiari syndrome) and veno-occlusive
disease; hepatic disease associated with pregnancy, such as
preeclampsia and eclampsia, acute fatty liver of pregnancy, and
intrehepatic cholestasis of pregnancy; hepatic complications of
organ or bone marrow transplantation, such as drug toxicity after
bone marrow transplantation, graft-versus-host disease and liver
rejection, and nonimmunologic damage to liver allografts; tumors
and tumorous conditions, such as nodular hyperplasias, adenomas,
and malignant tumors, including primary carcinoma of the liver and
metastatic tumors.
[0095] The terms "cancer" or "neoplasms" include malignancies of
the various organ systems, such as 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.
[0096] 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 lung, prostate, and colon. The term 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.
[0097] 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.
[0098] Disorders involving the prostate include, but are not
limited to, inflammations, benign enlargement, for example, nodular
hyperplasia (benign prostatic hypertrophy or hyperplasia), and
tumors such as carcinoma.
[0099] Disorders involving precursor T-cell neoplasms include
precursor T lymphoblastic leukemia/lymphoma. Disorders involving
peripheral T-cell and natural killer cell neoplasms include T-cell
chronic lymphocytic leukemia, large granular lymphocytic leukemia,
mycosis fungoides and Sezary syndrome, peripheral T-cell lymphoma,
unspecified, angioimmunoblastic T-cell lymphoma, angiocentric
lymphoma (NK/T-cell lymphoma.sup.4a), intestinal T-cell lymphoma,
adult T-cell leukemia/lymphoma, and anaplastic large cell
lymphoma.
[0100] Disorders involving the skeletal muscle include tumors such
as rhabdomyosarcoma.
CHAPTER 2
32447, A Novel Human Acyltransferase and Uses Thereof
Background of the Invention
[0101] Glycerophospholipids, which include phopholipids and
triacylglycerol, are ubiquitous and critically important molecules.
Phospholipids are the predominant component of biomembranes, and
determine such properties as membrane permeability and the activity
of membrane proteins. Triacylglycerol is the major storage form of
energy in animals. In the de novo biosynthesis of
glycerophospholipids in most tissues, glycerol-3-phosphate is
esterified with a fatty acyl-CoA in the sn-1 position by
glycerol-3-phosphate acyltransferase to form
1-acylglycerol-3-phsophate (lysophosphatidic acid).
Lysophosphatidic acid is esterified in the sn-2 position with a
fatty acyl-CoA by 1-acylglycerol-3-phophate acyltransferase to form
1,2-diacylglycerol-3-phosphate (phosphatidic acid) (Dircks et al.
(1999) Progress in Lipid Research 38:461-479). Phosphatidic acid
can be converted to CDP-diacylglycerol and ultimately to
phosphatidylinositol, phosphatidylglycerol, and cardiolipin.
[0102] Glycerol-3-phophate acyltransferase (E.C. 2.3.1.15) is the
first committed and presumed to be a rate-limiting step in
glycerophospholipid biosynthesis. It catalyzes the esterification
of glycerol-3-phosphate in the sn-1 position with a fatty acyl-CoA
to form 1-acylglycerol-3-phosphate (lysophosphatidic acid). Two
isoforms of the enzyme have been detected, a mitochondrial form and
an endoplasmic reticulum isoform. The two forms of the enzyme can
be differentiated by their differential sensitivity to the
sulfhydryl modifying agent N-ethylmaleimide (NEM) (Haldar, D. et
al. (1979) J. Biol. Chem. 254(11):4502-9).
[0103] Glycerol-3-phosphate acyltransferase is found in most
tissues including liver, adipose, heart, lung, kidney, adrenal,
muscle, lactating mammary, intestinal mucosa, brain and in various
cultured cell lines. In most tissues the mitochondrial isoform
comprises 10% of the total activity. The mitochondrial isoform is
the isoform under nutritional and hormonal regulation that occurs
in lipogenic tissues such as liver and adipose tissue. The
mitochondrial isoform of the enzyme is regulated by hormonal and
nutritional fluctuations while the endoplasmic reticulum isoform is
unaffected (Dircks et al. (1999) Progress in Lipid Research
38:461-479).
[0104] Lysophophatidic acid is catalyzed in the sn-2 position by
1-acylglycerol-3-phosphate acyltransferase (E.C. 2.3.1.51), also
called lysophosphatidic acid acyltransferase to form
[0105] 1,2-diacylglycerol-phosphate (phosphatidic acid). This
enzyme has been cloned from many organisms, including several
species of bacteria and plants, yeast, human, and mouse. It has
been demonstrated that this acyltransferase increases several fold
when preadipocytes differentiate into adipocytes (Coleman et al.
(1978) J. Biol. Chem. 253:7256-61).
[0106] The final acylation step in triacylglycerol biosynthesis is
the esterification of fatty acyl-CoA in the sn-3 position by
diacylglycerol acyltransferase (E.C. 2.3.1.20) which occurs after
dephosphorylation of phosphatidic acid by phosphatide
phosphohydrolase. This acyltransferase is the only enzyme which is
specific to triacylglycerol synthesis and may play an important
role in triacylglycerol synthesis. This enzyme is localized in the
endoplasmic reticulum membrane. At present, very little information
is reported about this acyltransferase or its regulation.
[0107] Accordingly, acyltransferases are a major target for drug
action and development. Thus, it is valuable to the field of
pharmaceutical development to identify and characterize novel
acyltransferases and tissues and disorders in which these enzymes
are differentially expressed. The present invention advances the
state of the art by providing novel human acyltransferase molecules
and the uses thereof.
Summary of the Invention
[0108] The present invention is based, in part, on the discovery of
a novel human enzyme, referred to herein as "acyltransferase". The
nucleotide sequence of a cDNA encoding acyltransferase is shown in
SEQ ID NO:6, and the amino acid sequence of an acyltransferase
polypeptide is shown in SEQ ID NO:7 Accordingly, in one aspect, the
invention features a nucleic acid molecule which encodes an
acyltransferase protein or polypeptide, e.g., a biologically active
portion of the acyltransferase protein. In a preferred embodiment,
the isolated nucleic acid molecule encodes a polypeptide having the
amino acid sequence of SEQ ID NO:7. In other embodiments, the
invention provides an isolated acyltransferase nucleic acid
molecule having the nucleotide sequence shown in SEQ ID NO:6. 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:6. In other embodiments, the invention provides a nucleic
acid molecule which hybridizes under stringent hybridization
conditions to a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:6, wherein the nucleic acid encodes a full
length acyltransferase protein or an active fragment thereof.
[0109] In a related aspect, the invention further provides nucleic
acid constructs which include an acyltransferase 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 acyltransferase nucleic acid molecules of
the invention e.g., vectors and host cells suitable for producing
acyltransferase nucleic acid molecules and polypeptides.
[0110] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of acyltransferase-encoding nucleic acids.
[0111] In still another related aspect, isolated nucleic acid
molecules that are antisense to an acyltransferase encoding nucleic
acid molecule are provided.
[0112] In another aspect, the invention features, acyltransferase
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 acyltransferase-mediated
or -related disorders. In another embodiment, the invention
provides acyltransferase polypeptides having acyltransferase
activity. Preferred polypeptides are proteins including at least
domain or active site involved in the transfer of an acyl group
from acyl-CoA onto a substrate (e.g., transfer of an acyl group
onto sn-glycerol-3-phosphate from Acyl-CoA to yield
1-acylglycerol-3-phosphate(lysophosphatidate)).
[0113] In other embodiments, the invention provides acyltransferase
polypeptides, e.g., an acyltransferase polypeptide having the amino
acid sequence shown in SEQ ID NO:7; an amino acid sequence that is
substantially identical to the amino acid sequence shown in SEQ ID
NO:7; or an amino acid sequence encoded by a nucleic acid molecule
having a nucleotide sequence which hybridizes under stringent
hybridization conditions to a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:6, wherein the nucleic acid
encodes a full length acyltransferase protein or an active fragment
thereof.
[0114] In a related aspect, the invention further provides nucleic
acid constructs which include an acyltransferase nucleic acid
molecule described herein.
[0115] In a related aspect, the invention provides acyltransferase
polypeptides or fragments operatively linked to non-acyltransferase
polypeptides to form fusion proteins.
[0116] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically bind acyltransferase polypeptides.
[0117] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the acyltransferase polypeptides or nucleic acids.
[0118] In still another aspect, the invention provides a process
for modulating acyltransferase polypeptide or nucleic acid
expression or activity, e.g. using the screened compounds. In
certain embodiments, the methods involve treatment of conditions
related to aberrant activity or expression of the acyltransferase
polypeptides or nucleic acids, such as conditions involving
aberrant or deficient cellular proliferation or
differentiation.
[0119] The invention also provides assays for determining the
activity of or the presence or absence of acyltransferase
polypeptides or nucleic acid molecules in a biological sample,
including for disease diagnosis.
[0120] In further aspect the invention provides assays for
determining the presence or absence of a genetic alteration in an
acyltransferase polypeptide or nucleic acid molecule, including for
disease diagnosis.
Detailed Description of the Invention
[0121] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0122] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
Human Acyltransferase
[0123] The human acyltransferase sequence (SEQ ID NO:6), which is
approximately 2299 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
1632 nucleotides (SEQ ID NO:6) not including the terminal codon.
The coding sequence encodes a 544 amino acid protein (SEQ ID
NO:7).
[0124] This mature protein form is approximately 544 amino acid
residues in length. Human acyltransferase contains the following
regions or other structural features: MEMSAT predicted
transmembrane domains which extend from about amino acid residue 7
to 24, aa 58 to 81, and aa 140 to 164; a predicted signal peptide
from aa 1-20; and presumed mature peptide transmembrane segments
numbered from about aa 38 to 61, 90 to 108, and 120 to 144 of the
cleaved polypeptide, that is, numbered from the N-terminus of the
polypeptide resulting from the cleavage of the aa 1-20 signal
sequence.
[0125] The acyltransferase protein (SEQ ID NO:7) also includes the
following domains: cAMP- and cGMP-dependent protein kinase
phosphorylation sites at aa 104 to 107, aa 195 to 198, aa 204 to
207; N-glycosylation site at aa 225 to 228; protein kinase C
phosphorylation sites at aa 52 to 54, aa 194 to 196, aa 224 to 226,
aa 231 to 233, aa 350 to 352, aa 373 to 375, 526 to 528, and aa 539
to 541; casein kinase II phosphorylation sites at aa 134 to 137, aa
148 to 151, aa 165 to 168, aa 188 to 191, aa 198 to 201, aa 208 to
211, aa 279 to 282, aa 322 to 325, aa 427 to 430, aa 449 to 452, aa
482 to 485, aa 502 to 505, and 537 to 540; N-myristoylation sites
at aa 16 to 21, aa 221 to 226, aa 315 to 320, aa 376 to 381; and an
EF-hand calcium binding domain at aa 404 to 416, and aa 441 to
453.
[0126] For general information regarding PFAM identifiers, PS
prefix and PF prefix domain identification numbers, refer to
Sonnhammer et al. (1997) Protein 28:405-420 and the Pfam website
maintained in several locations, e.g. by the Sanger Institute
(sanger.ac.uk/Software/Pfam).
[0127] As used herein, the term "acyltransferase" refers to a
protein or polypeptide which is capable of transferring an "acyl
group" from Acyl-CoA onto a substrate. All three known and
described acyltransferases (glycerol-3-phosphate acyltransferase
(GPAT); 1-acylglycerol 3-phosphate acyltransferase(AGPAT); and
diacylglycerol acyltransferase) all act to transfer acyl groups.
Assays for acyltransferases have been previously described (Dircks
et al. (1999) Progress in Lipid Research 38:461-479).
[0128] Typically, acyltransferases play a role in diverse cellular
processes. For example, the glycerophospholipids are predominant
components of biomembranes and triacylglycerol is a major storage
form of energy in animals.
[0129] As used herein, the term "acyltransferase domain" includes
an amino acid sequence of about 80-300 amino acid residues in
length. Preferably, an acyltransferase domain includes at least
about 100-250 amino acids, more preferably about 130-200 amino acid
residues, or about 160-200 amino acids. The acyltransferase domain
(HMM) has been assigned the PFAM Accession PF01553 (at the Pfam
website, pfam.wustl.edu). An alignment of the acyltransferase
domain (amino acids 131 to 317 of SEQ ID NO:7) of human
acyltransferase with a consensus amino acid sequence derived from a
hidden Markov model is depicted in FIG. 10.
[0130] In a preferred embodiment acyltransferase polypeptide or
protein has an "acyltransferase domain" or a region which includes
at least about 100-250 more preferably about 130-200 or 160-200
amino acid residues and has at least about 60%, 70%, 80%, 90%, 95%,
99%, or 100% sequence identity with an "acyltransferase domain,"
e.g., the acyltransferase domain of human acyltransferase (e.g.,
amino acid residues 131-317 of SEQ ID NO:7).
[0131] To identify the presence of an acyltransferase domain in an
acyltransferase 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 (sanger.ac.uk/Software/Pfam). 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. 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.
[0132] In one embodiment, an acyltransferase protein includes at
least one transmembrane domain. As used herein, the term
"transmembrane domain" includes an amino acid sequence of at least
15 amino acid residues in length that spans a phospholipid
membrane. More preferably, a transmembrane domain includes about at
least 18, 20, 22, 24, or 25 amino acid residues and spans a
phospholipid 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,
the Pfam website at 7tm.sub.--1 (pfam.wustl.edu) and Zagotta W. N.
et al., (1996) Annual Rev. Neuronsci. 19: 235-63, the contents of
which are incorporated herein by reference.
[0133] In a preferred embodiment, an acyltransferase polypeptide or
protein has at least one transmembrane domain or a region which
includes at least 16, 18, 20, 22, 24, or 25 amino acid residues and
has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with
a "transmembrane domain," e.g., at least one transmembrane domain
of human acyltransferase (e.g., amino acid residues 7 to 24, 58 to
81, and 140 to 164 of SEQ ID NO:7).
[0134] In another embodiment, an acyltransferase protein includes
at least one "non-transmembrane domain." As used herein,
"non-transmembrane domains" are domains that reside outside of the
membrane. When referring to plasma membranes, non-transmembrane
domains include extracellular domains (i.e., outside of the cell)
and intracellular domains (i.e., within the cell). When referring
to membrane-bound proteins found in intracellular organelles (e.g.,
mitochondria, endoplasmic reticulum, peroxisomes and microsomes),
non-transmembrane domains include those domains of the protein that
reside in the cytosol (i.e., the cytoplasm), the lumen of the
organelle, or the matrix or the intermembrane space (the latter two
relate specifically to mitochondria organelles). The C-terminal
amino acid residue of a non-transmembrane domain is adjacent to an
N-terminal amino acid residue of a transmembrane domain in a
naturally occurring acyltransferase protein.
[0135] In a preferred embodiment, an acyltransferase protein has a
"non-transmembrane domain" or a region which includes at least
about 1-7, about 1-34, about 1-59, and about 1-381 amino acid
residues, and has at least about 60%, 70% 80% 90% 95%, 99% or 100%
sequence identity with a "non-transmembrane domain", e.g., a
non-transmembrane domain of human 32447 (e.g., residues 1-6, 25-57,
82-139, and 165-544 of SEQ ID NO:7). Preferably, a
non-transmembrane domain is capable of catalytic activity (e.g.,
acyltransferase activity).
[0136] A non-transmembrane domain located at the N-terminus of an
acyltransferase protein or polypeptide is referred to herein as an
"N-terminal non-transmembrane domain." As used herein, an
"N-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-350, preferably about 30-325, more
preferably about 50-320, or even more preferably about 80-310 amino
acid residues in length and is located outside the boundaries of a
membrane. For example, an N-terminal non-transmembrane domain is
located at about amino acid residues 1-6 of SEQ ID NO:7.
[0137] Similarly, a non-transmembrane domain located at the
C-terminus of an acyltransferase protein or polypeptide is referred
to herein as a "C-terminal non-transmembrane domain." As used
herein, a "C-terminal non-transmembrane domain" includes an amino
acid sequence having about 1-300, preferably about 15-290,
preferably about 20-270, more preferably about 25-255 amino acid
residues in length and is located outside the boundaries of a
membrane. For example, an C-terminal non-transmembrane domain is
located at about amino acid residues 165-544 of SEQ ID NO:7.
[0138] An acyltransferase polypeptide or protein can further
include a signal sequence. As used herein, a "signal sequence"
refers to a peptide of about 20-80 amino acid residues in length
which occurs at the N-terminus of secretory and integral membrane
proteins and which contains a majority of hydrophobic amino acid
residues. For example, a signal sequence contains at least about
12-25 amino acid residues, preferably about 30-70 amino acid
residues, more preferably about 20 amino acid residues, and has at
least about 40-70%, preferably about 50-65%, and more preferably
about 55-60% hydrophobic amino acid residues (e.g., alanine,
valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan,
or proline). Such a "signal sequence", also referred to in the art
as a "signal peptide", serves to direct a protein containing such a
sequence to a lipid bilayer. For example, in one embodiment, an
acyltransferase protein contains a signal sequence of about amino
acids 1-20 of SEQ ID NO:7. The "signal sequence" is cleaved during
processing of the mature protein. The mature acyltransferase
protein corresponds to amino acids 21-544 of SEQ ID NO:7.
[0139] As the acyltransferase polypeptides of the invention may
modulate acyltransferase-mediated activities, they may be useful
for developing novel diagnostic and therapeutic agents for
acyltransferase-mediated or related disorders, and for treatment of
those disorders, as described below.
[0140] As used herein, a "acyltransferase activity", "biological
activity of acyltransferase" or "functional activity of
acyltransferase", refers to an activity exerted by an
acyltransferase protein, polypeptide or nucleic acid molecule on
e.g., an acyltransferase-responsive cell or on an acyltransferase
substrates, e.g., glycerol-3-phosphate and fatty acyl CoA, or as
determined in vivo or in vitro. In one embodiment, an
acyltransferase activity is a direct activity, such as an
association with an acyltransferase target molecule. A "target
molecule" or "binding partner" is a molecule with which an
acyltransferase protein binds or interacts in nature.
[0141] Accordingly, acyltransferase protein may mediate various
disorders, including cellular proliferative and/or differentiative
disorders, brain disorders, heart disorders, blood vessel
disorders, and platelet disorders.
[0142] Examples of 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.
[0143] As used herein, the terms "cancer", "hyperproliferative" and
"neoplastic" refer to cells having the capacity for autonomous
growth, i.e., an abnormal state or condition characterized by
rapidly proliferating cell growth. Hyperproliferative and
neoplastic disease states may be categorized as pathologic, i.e.,
characterizing or constituting a disease state, or may be
categorized as non-pathologic, i.e., a deviation from normal but
not associated with a disease state. 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.
"Pathologic hyperproliferative" cells occur in disease states
characterized by malignant tumor growth. Examples of non-pathologic
hyperproliferative cells include proliferation of cells associated
with wound repair.
[0144] The terms "cancer" or "neoplasms" include malignancies of
the various organ systems, such as 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.
[0145] 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 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.
[0146] The term "sarcoma" is art recognized and refers to malignant
tumors of mesenchymal derivation.
[0147] The acyltransferase nucleic acid and protein of the
invention can be used to treat and/or diagnose a variety of
proliferative disorders. E.g., such 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, L., (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.
[0148] 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, Varicalla-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
degeneration, 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.
[0149] Disorders involving the heart, include but are not limited
to, heart failure, including but not limited to, cardiac
hypertrophy, left-sided heart failure, and right-sided heart
failure; ischemic heart disease, including but not limited to
angina pectoris, myocardial infarction, chronic ischemic heart
disease, and sudden cardiac death; hypertensive heart disease,
including but not limited to, systemic (left-sided) hypertensive
heart disease and pulmonary (right-sided) hypertensive heart
disease; valvular heart disease, including but not limited to,
valvular degeneration caused by calcification, such as calcific
aortic stenosis, calcification of a congenitally bicuspid aortic
valve, and mitral annular calcification, and myxomatous
degeneration of the mitral valve (mitral valve prolapse), rheumatic
fever and rheumatic heart disease, infective endocarditis, and
noninfected vegetations, such as nonbacterial thrombotic
endocarditis and endocarditis of systemic lupus erythematosus
(Libman-Sacks disease), carcinoid heart disease, and complications
of artificial valves; myocardial disease, including but not limited
to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, and myocarditis; pericardial disease, including but
not limited to, pericardial effusion and hemopericardium and
pericarditis, including acute pericarditis and healed pericarditis,
and rheumatoid heart disease; neoplastic heart disease, including
but not limited to, primary cardiac tumors, such as myxoma, lipoma,
papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac
effects of noncardiac neoplasms; congenital heart disease,
including but not limited to, left-to-right shunts--late cyanosis,
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosus, and atrioventricular septal defect,
right-to-left shunts--early cyanosis, such as tetralogy of fallot,
transposition of great arteries, truncus arteriosus, tricuspid
atresia, and total anomalous pulmonary venous connection,
obstructive congenital anomalies, such as coarctation of aorta,
pulmonary stenosis and atresia, and aortic stenosis and atresia,
and disorders involving cardiac transplantation.
[0150] 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.
[0151] By "variants" is intended proteins or polypeptides having an
amino acid sequence that is at least about 60%, 65%, preferably
about 75%, 85%, 95%, 96%, 97%, 98%, or 99% identical to the amino
acid sequence of SEQ ID NO:7. Variants also include polypeptides
encoded by a nucleic acid molecule that hybridizes to the nucleic
acid molecule of SEQ ID NO:6 or 3, or a complement thereof, under
stringent conditions. In another embodiment, a variant of an
isolated polypeptide of the present invention differs, by at least
1, but less than 5, 10, 20, 50, or 100 amino acid residues from the
sequence shown in SEQ ID NO:7. If alignment is needed for this
comparison the sequences should be aligned for maximum identity.
"Looped" out sequences from deletions or insertions, or mismatches,
are considered differences. Such variants generally retain the
functional activity of the acyltransferase proteins of the
invention. Variants include polypeptides that differ in amino acid
sequence due to natural allelic variation or mutagenesis.
CHAPTER 3
7716, A Novel Human ATPase and Uses Therefor
Background of the Invention
[0152] The AAA (ATPases Associated with a variety of cellular
Activities) family of proteins is a group of proteins linked by
common ancestry (reviewed by Beyer (1997) Protein Sci.
6:2043-2058). This Mg.sup.2+-dependent ATPase family is
characterized by the presence of one (type I) or two (type II)
copies of the AAA cassette, a conserved region of 220-250 amino
acids that includes the Walker signature sequences of P-loop
ATPases as well as other regions of similarity unique to AAA
ATPases (reviewed by Patel et al. (1998) Trends Cell Biol.
8:65-71). AAA ATPases play essential roles in a number of cellular
processes including the biogenesis of organelles, the regulation of
proteasome function, and the quality control and regulated
degradation of membrane proteins.
[0153] One of the best-characterized AAA ATPases is the NSF
(N-ethylmaleimide-sensitive factor), which is involved in the
docking/fusion step of all cellular fusion events, ranging from the
secretory pathway to synaptic transmission. Another example of an
AAA protein involved in membrane fusion events is Cdc48, which is
required for the ATP-dependent fusion of endoplasmic reticular
membranes. cdc48-1 was originally identified as a yeast cell-cycle
mutant that arrests in mitosis with an undivided nucleus. At least
two mammalian orthologs of Cdc48 have been identified, and have
been demonstrated to participate in Golgi membrane fusion events
(reviewed by Patel et al. (1998) Trends Cell Biol. 8:65-71). The
AAA ATPases PEX1 and PEX6 are required for peroxisome biogenesis,
and mutations in these genes are the most common cause of the
lethal neurologic disorders Zellweger syndrome, neonatal
adrenoleukodystrophy, and infantile Refsum disease (Dodt et al.
(1995) Nat. Genet. 9:115-124).
[0154] AAA ATPases also play a role in the 26S proteasome-mediated
proteolytic degradation of proteins via the ubiquitin pathway. The
regulatory component of the 26 S proteasome, which is responsible
for the recognition, binding, and unfolding of ubiquinated
molecules, contains at least six AAA ATPases (Sug1p, Sug2p, Yta1p,
Yta2p, Yta3p, and Yta5p). Evidence suggests that these subunits are
functionally distinct, with proteolysis of different cellular
substrates requiring specific AAA ATPase subunit activities
(reviewed by Patel et al. (1998) Trends Cell Biol. 8:65-71).
[0155] The proteolytic degradation of mitochondrial membrane
proteins also requires the activity of several integral membrane
AAA ATPases. These AAA ATPases combine proteolytic and
chaperone-like activities to form a membrane-integrated quality
control system (reviewed by Langer (2000) Trends Biochem. Sci.
25:247-251). AAA ATPase membrane proteases involved in the
degradation of membrane proteins include Yta10p (Afg3p), Yta12p
(Rca1p), and Yme1p.
[0156] The critical role that AAA ATPases play in numerous
biological processes and diseases make them important targets for
therapeutic intervention. It is consequently important to identify
novel genes encoding members of this enzyme family.
Summary of the Invention
[0157] The present invention is based, in part, on the discovery of
a novel human ATPase, referred to herein as "7716". The nucleotide
sequence of a cDNA encoding 7716 is shown in SEQ ID NO:10, and the
amino acid sequence of a 7716 polypeptide is shown in SEQ ID NO:11.
In addition, the nucleotide sequence of the coding region is
depicted in SEQ ID NO:12.
[0158] Accordingly, in one aspect the invention features a nucleic
acid molecule which encodes a 7716 protein or polypeptide, e.g., a
biologically active portion of the 7716 protein. In a preferred
embodiment, the isolated nucleic acid molecule encodes a
polypeptide having the amino acid sequence of SEQ ID NO:11. In
other embodiments, the invention provides an isolated 7716 nucleic
acid molecule having the nucleotide sequence shown in SEQ ID NO:10
or SEQ ID NO:12. 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:10 or SEQ ID NO:12. In other embodiments, the
invention provides a nucleic acid molecule which hybridizes under
stringent hybridization conditions to a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:10 or SEQ ID NO:12,
wherein the nucleic acid encodes a full length 7716 protein or an
active fragment thereof.
[0159] In a related aspect, the invention further provides nucleic
acid constructs which include a 7716 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 7716 nucleic acid molecules of the
invention e.g., vectors and host cells suitable for producing 7716
nucleic acid molecules and polypeptides.
[0160] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of 7716-encoding nucleic acids.
[0161] In still another related aspect, isolated nucleic acid
molecules that are antisense to a 7716 encoding nucleic acid
molecule are provided.
[0162] In another aspect, the invention features 7716 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 7716-mediated or -related disorders. In
another embodiment, the invention provides 7716 polypeptides having
a 7716 activity. Preferred polypeptides are 7716 proteins including
at least one ATPase domain, and, preferably, having a 7716
activity, e.g., a 7716 activity as described herein.
[0163] In other embodiments, the invention provides 7716
polypeptides, e.g., a 7716 polypeptide having the amino acid
sequence shown in SEQ ID NO:11; an amino acid sequence that is
substantially identical to the amino acid sequence shown in SEQ ID
NO:11; or an amino acid sequence encoded by a nucleic acid molecule
having a nucleotide sequence which hybridizes under stringent
hybridization conditions to a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:10 or SEQ ID NO:12, wherein the
nucleic acid encodes a full length 7716 protein or an active
fragment thereof.
[0164] In a related aspect, the invention further provides nucleic
acid constructs which include a 7716 nucleic acid molecule
described herein.
[0165] In a related aspect, the invention provides 7716
polypeptides or fragments operatively linked to non-7716
polypeptides to form fusion proteins.
[0166] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically bind 7716 polypeptides.
[0167] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the 7716 polypeptides or nucleic acids.
[0168] In still another aspect, the invention provides a process
for modulating 7716 polypeptide or nucleic acid expression or
activity, e.g. using the screened compounds. In certain
embodiments, the methods involve treatment of conditions related to
aberrant activity or expression of the 7716 polypeptides or nucleic
acids, such as conditions involving aberrant or deficient cellular
proliferation or differentiation.
[0169] The invention also provides assays for determining the
activity of or the presence or absence of 7716 polypeptides or
nucleic acid molecules in a biological sample, including for
disease diagnosis.
[0170] In further aspect the invention provides assays for
determining the presence or absence of a genetic alteration in a
7716 polypeptide or nucleic acid molecule, including for disease
diagnosis.
[0171] Other features and advantages of the invention will be
apparent from the following detailed description, Chapter 7,
Examples and claims.
Detailed Description of the Invention
[0172] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0173] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
Human 7716
[0174] The human 7716 sequence (SEQ ID NO:10), which is
approximately 2547 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
2259 nucleotides (nucleotides 63-2324 of SEQ ID NO:10; SEQ ID
NO:12). The coding sequence encodes a 753 amino acid protein (SEQ
ID NO:11).
[0175] Human 7716 contains the following regions or other
structural features: a predicted AAA ATPase domain (PFAM Accession
PF00004) located at about amino acid residues 236 to 421 of SEQ ID
NO:11, a second AAA ATPase domain located at about amino acid
residues 500 to 705 of SEQ ID NO:11; and two predicted
transmembrane domains, the first which extends from about amino
acid residues 144 to 166 of SEQ ID NO:11, and the second which
extends from about amino acid residues 225 to 246 of SEQ ID
NO:11.
[0176] The 7716 protein also includes the following domains: two
Prosite AAA-protein family signatures (amino acids 338-356 and
622-640; Prosite Accession No. PS00674), an ATP-binding
protease/ATP-dependent cell division protein domain (amino acids
236-705), a cell division protein domain derived from SwissProt
Accession No. 083350 (amino acids 233-713), a putative ATP/GTP
binding protein domain derived from SwissProt Accession No. 086711,
and a PEX6-like domain. For general information regarding PFAM
identifiers, PS prefix and PF prefix domain identification numbers,
refer to Sonnhammer et al. (1997) Protein 28:405-420 and the Pfam
website maintained in several locations, e.g. by the Sanger
Institute (sanger.ac.uk/Software/Pfam).
[0177] The 7716 protein contains a significant number of structural
characteristics in common with members of the ATPase family. The
term "family" when referring to the protein and nucleic acid
molecules of the invention means two or more proteins or nucleic
acid molecules having a common structural domain or motif and
having sufficient amino acid or nucleotide sequence homology as
defined herein. Such family members can be naturally or
non-naturally occurring and can be from either the same or
different species. For example, a family can contain a first
protein of human origin as well as other distinct proteins of human
origin, or alternatively, can contain homologues of non-human
origin, e.g., rat or mouse proteins. Members of a family can also
have common functional characteristics.
[0178] As used herein, the term "ATPase" refers to a protein or
polypeptide which is capable of catalyzing an ATP hydrolysis
reaction. As referred to herein, ATPases preferably include a
catalytic domain of about 220-250 amino acid residues in length. An
AAA ATPase domain typically includes at least three blocks of amino
acid sequence similarity commonly found in members of the AAA
ATPase family: a Walker A domain (found at about amino acids
241-248 and 506-513 of SEQ ID NO:11); a Walker B domain (found at
about amino acids 300-307 and 560-568 of SEQ ID NO:11); and an SRH
domain, a highly conserved domain among AAA ATPases which is
believed to be involved in ATP hydrolysis (found at about amino
acids 338-366 and 622-650 of SEQ ID NO:11). Based on these sequence
similarities, the 7716 molecules of the present invention are
predicted to have similar biological activities as AAA ATPase
family members.
[0179] Typically, AAA ATPases play a role in diverse cellular
processes. For example, AAA ATPases are required for organelle
biogenesis, and thus for cell division. Mutations in the genes
encoding the peroxisome biogenesis AAA ATPases PEX 1 and PEX 2 are
responsible for the majority of neurodegenerative diseases broadly
classified as Zellwegger syndrome. AAA ATPases are also involved in
other membrane fusion events, including secretion and synaptic
transmission. AAA ATPases also play a role in the regulation of
proteolysis by the 26S proteasome, and in the regulated degradation
of membrane proteins. Thus, the molecules of the present invention
may be involved in one or more of: 1) catalyzing the hydrolysis of
ATP; 2) the modulation of organelle biogenesis; 3) the regulation
of cell division; 4) the regulation of membrane fusion; 5) the
modulation of synaptic transmission; 6) the modulation of
secretion; 7) the regulation of proteolysis by the 26S proteasome;
or 8) the degradation of membrane proteins.
[0180] A 7716 polypeptide can include an "ATPase domain" or regions
homologous with an "ATPase domain." As used herein, the term
"ATPase domain" includes an amino acid sequence of about 80-250
amino acid residues in length and having a bit score for the
alignment of the sequence to the ATPase domain (HMM) of at least 8.
Preferably, an ATPase domain includes at least about 140-250 amino
acids, more preferably about 160-220 amino acid residues, or about
180-210 amino acids and has a bit score for the alignment of the
sequence to the ATPase domain (HMM) of at least 16 or greater. The
AAA ATPase domain (HMM) has been assigned the PFAM Accession
PF00004 (see the Pfam website, wustl.edu/Pfam). An alignment of the
AAA ATPase domain 1 (amino acids 236 to 421 of SEQ ID NO:11) and
domain 2 (amino acids 500 to 705 of SEQ ID NO:11) of human 7716
with a consensus amino acid sequence derived from a hidden Markov
model is depicted in FIG. 12.
[0181] In one embodiment, 7716 polypeptide or protein has a "ATPase
domain" or a region which includes at least about 140-250 more
preferably about 160-220 or 180-210 amino acid residues and has at
least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with an
"ATPase domain," e.g., the ATPase domains of human 7716 (e.g.,
amino acid residues 236-421 or 500-705 of SEQ ID NO:11).
[0182] To identify the presence of an "ATPase" domain in a 7716
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
(sanger.ac.uk/
[0183] Software/Pfam). 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.
[0184] In one embodiment, a 7716 protein includes 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 that spans a phospholipid membrane. More
preferably, a transmembrane domain includes about at least 18, 20,
22, 24, 25, 30, 35 or 40 amino acid residues and spans a
phospholipid 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,
the Pfam website at 7tm.sub.--1 (pfam.wustl.edu) and Zagotta W. N.
et al. (1996) Annual Rev. Neuronsci. 19:235-63, the contents of
which are incorporated herein by reference.
[0185] In a preferred embodiment, a 7716 polypeptide or protein has
at least one transmembrane domain or a region which includes at
least 18, 20, 22, 24, 25, 30, 35 or 40 amino acid residues and has
at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a
"transmembrane domain," e.g., at least one transmembrane domain of
human 7716 (e.g., amino acid residues 144-166 or 225-246 of SEQ ID
NO:11).
[0186] In another embodiment, a 7716 protein includes at least one
"non-transmembrane domain." As used herein, "non-transmembrane
domains" are domains that reside outside of the membrane. When
referring to plasma membranes, non-transmembrane domains include
extracellular domains (i.e., outside of the cell) and intracellular
domains (i.e., within the cell). When referring to membrane-bound
proteins found in intracellular organelles (e.g., mitochondria,
endoplasmic reticulum, peroxisomes and microsomes),
non-transmembrane domains include those domains of the protein that
reside in the cytosol (i.e., the cytoplasm), the lumen of the
organelle, or the matrix or the intermembrane space (the latter two
relate specifically to mitochondria organelles). The C-terminal
amino acid residue of a non-transmembrane domain is adjacent to an
N-terminal amino acid residue of a transmembrane domain in a
naturally-occurring 7716, or 7716-like protein.
[0187] In a preferred embodiment, a 7716 polypeptide or protein has
a "non-transmembrane domain" or a region which includes at least
about 1-350, preferably about 200-320, more preferably about
230-300, and even more preferably about 240-280 amino acid
residues, and has at least about 60%, 70% 80% 90% 95%, 99% or 100%
homology with a "non-transmembrane domain", e.g., a
non-transmembrane domain of human 7716 (e.g., residues 1-143,
167-224, and 247-753 of SEQ ID NO:11). Preferably, a
non-transmembrane domain is capable of catalytic activity (e.g.,
catalyzing an ATP hydrolysis reaction).
[0188] A non-transmembrane domain located at the N-terminus of a
7716 protein or polypeptide is referred to herein as an "N-terminal
non-transmembrane domain." As used herein, an "N-terminal
non-transmembrane domain" includes an amino acid sequence having
about 1-350, preferably about 30-325, more preferably about 50-320,
or even more preferably about 80-310 amino acid residues in length
and is located outside the boundaries of a membrane. For example,
an N-terminal non-transmembrane domain is located at about amino
acid residues 1-143 of SEQ ID NO:11.
[0189] Similarly, a non-transmembrane domain located at the
C-terminus of a 7716 protein or polypeptide is referred to herein
as a "C-terminal non-transmembrane domain." As used herein, an
"C-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-300, preferably about 15-290, preferably
about 20-270, more preferably about 25-255 amino acid residues in
length and is located outside the boundaries of a membrane. For
example, an C-terminal non-transmembrane domain is located at about
amino acid residues 247-753 of SEQ ID NO:11.
[0190] 7716 expression in human normal breast and breast tumor
tissue, human normal ovary and ovary tumor tissue, human normal
lung and lung tumor tissue, human normal colon, colon tumor, colon
cancer liver metastases, and normal liver tissue, and human
angiogenic tissue is shown in FIGS. 13, 14, 15, 16, and 17,
respectively. The methods of the invention are particularly
relevant to the tissues and disorders in which 7716 is
expressed.
[0191] As the 7716 polypeptides of the invention may modulate
7716-mediated activities, they may be useful as of for developing
novel diagnostic and therapeutic agents for 7716-mediated or
related disorders, as described below.
[0192] As used herein, a "7716 activity", "biological activity of
7716" or "functional activity of 7716", refers to an activity
exerted by a 7716 protein, polypeptide or nucleic acid molecule on
e.g., a 7716-responsive cell or on a 7716 substrate, e.g., a lipid
or protein substrate, as determined in vivo or in vitro. In one
embodiment, a 7716 activity is a direct activity, such as an
association with a 7716 target molecule. A "target molecule" or
"binding partner" is a molecule with which a 7716 protein binds or
interacts in nature, e.g., an ATP molecule which the 7716 protein
hydrolyzes. A 7716 activity can also be an indirect activity, e.g.,
a cellular signaling activity mediated by interaction of the 7716
protein with a 7716 ligand. For example, the 7716 proteins of the
present invention can have one or more of the following activities:
1) catalyzing the hydrolysis of ATP; 2) the modulation of organelle
biogenesis; 3) the regulation of cell division; 4) the regulation
of membrane fusion; 5) the modulation of synaptic transmission; 6)
the modulation of secretion; 7) the regulation of proteolysis by
the 26S proteasome; 8) the degradation of membrane proteins or 9)
the ability to antagonize or inhibit, competitively or
non-competitively, any of 1-8.
[0193] Accordingly, 7716 protein may mediate various disorders,
including cellular proliferative and/or differentiative disorders,
brain disorders, lung disorders, colon disorders, breast disorders,
ovary disorders, and liver disorders.
[0194] Examples of 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.
[0195] As used herein, the terms "cancer", "hyperproliferative" and
"neoplastic" refer to cells having the capacity for autonomous
growth, i.e., an abnormal state or condition characterized by
rapidly proliferating cell growth. Hyperproliferative and
neoplastic disease states may be categorized as pathologic, i.e.,
characterizing or constituting a disease state, or may be
categorized as non-pathologic, i.e., a deviation from normal but
not associated with a disease state. 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.
"Pathologic hyperproliferative" cells occur in disease states
characterized by malignant tumor growth. Examples of non-pathologic
hyperproliferative cells include proliferation of cells associated
with wound repair.
[0196] The terms "cancer" or "neoplasms" include malignancies of
the various organ systems, such as 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.
[0197] 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 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.
[0198] The term "sarcoma" is art recognized and refers to malignant
tumors of mesenchymal derivation.
[0199] The 7716 nucleic acid and protein of the invention can be
used to treat and/or diagnose a variety of proliferative disorders.
E.g., such 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, L. (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.
[0200] 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, Varicalla-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
degeneration, multiple system atrophy, including striatonigral
degeneration, 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.
[0201] Disorders involving 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.
[0202] 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.
[0203] 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.
[0204] 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
[0205] Disorders involving the liver include, but are not limited
to, hepatic injury; jaundice and cholestasis, such as bilirubin and
bile formation; hepatic failure and cirrhosis, such as cirrhosis,
portal hypertension, including ascites, portosystemic shunts, and
splenomegaly; infectious disorders, such as viral hepatitis,
including hepatitis A-E infection and infection by other hepatitis
viruses, clinicopathologic syndromes, such as the carrier state,
asymptomatic infection, acute viral hepatitis, chronic viral
hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and
toxin-induced liver disease, such as alcoholic liver disease;
inborn errors of metabolism and pediatric liver disease, such as
hemochromatosis, Wilson disease, .alpha..sub.1-antitrypsin
deficiency, and neonatal hepatitis; intrahepatic biliary tract
disease, such as secondary biliary cirrhosis, primary biliary
cirrhosis, primary sclerosing cholangitis, and anomalies of the
biliary tree; circulatory disorders, such as impaired blood flow
into the liver, including hepatic artery compromise and portal vein
obstruction and thrombosis, impaired blood flow through the liver,
including passive congestion and centrilobular necrosis and
peliosis hepatis, hepatic vein outflow obstruction, including
hepatic vein thrombosis (Budd-Chiari syndrome) and veno-occlusive
disease; hepatic disease associated with pregnancy, such as
preeclampsia and eclampsia, acute fatty liver of pregnancy, and
intrehepatic cholestasis of pregnancy; hepatic complications of
organ or bone marrow transplantation, such as drug toxicity after
bone marrow transplantation, graft-versus-host disease and liver
rejection, and nonimmunologic damage to liver allografts; tumors
and tumorous conditions, such as nodular hyperplasias, adenomas,
and malignant tumors, including primary carcinoma of the liver and
metastatic tumors.
CHAPTER 4
25233, A Novel Human Aminotransferase and Uses Therefor
Background of the Invention
[0206] Vitamin B.sub.6, in the form of its biologically active
phosphorylated derivatives pyridoxal-5'-phosphate (PLP) and
pyridoxamine-5'-phosphate, represents one of nature's most
versatile cofactors (Schneider et al. 2000, Structure 8:R1-R6).
Vitamin B.sub.6 dependent enzymes have a major role in the
metabolism of amino acids, and are found in various pathways
ranging from the interconversion of alpha-amino acids to the
biosynthesis of antibiotic compounds. In the resting enzyme, the
aldehyde group of PLP is covalently linked to a lysine residue at
the active site of the enzyme. Upon binding of the amino acid
substrate, the lysine residue is exchanged for the amino group of
the substrate forming a Schiff-base complex with PLP. In the next
step of the reaction, one of the bonds to the C alpha atom of the
aldimine is broken forming an enzyme-linked intermediate. This
process is facilitated by the electrophilic properties of the
cofactor. The multitude of reactions catalyzed by PLP-dependent
enzymes thus have several steps in common and variations arise from
enzymatic control of the different routes to the central
intermediate.
[0207] Grishin et al. (1995) Protein Sci. 4:1291-1304 have
classified the PLP-dependent enzymes into five different fold types
on the basis of amino acid sequence comparisons, predicted
secondary structure elements and available three-dimensional
structural information. These five structural classes are the
aspartate aminotransferase family (class-I), the tryptophan
synthase beta family (class-II), the alanine racemase family
(class-III), the D-amino acid family (class-IV), and the glycogen
phosphorylase family (class-V). See Schneider et al. (2000)
Structure 8:R1-R6.
[0208] The alpha-amino groups of the 20 L-amino acids commonly
found in proteins are removed during the oxidative degradation of
the amino acids (U.S. Pat. No. 6,013,509, herein incorporated by
reference). The removal of the alpha-amino groups, the first step
in the catabolism of most of the L-amino acids, is promoted by
aminotransferases (or transaminases). Cells contain several
different aminotransferases.
[0209] The measurement of alanine aminotransferase and aspartate
aminotransferase levels in blood serum is an important diagnostic
procedure in medicine. For instance, these enzymes are used as
indicators of heart damage and to monitor tissue recovery following
the damage (See U.S. Pat. No. 6,013,509, above). Increased serum
levels of the class-I aminotransferase, aspartate aminotransferase,
are indicative of muscle, cardiac, kidney, pancreatic, red blood
cell, or hepatic injury (see, for example, U.S. Pat. No. 5,834,226,
herein incorporated by reference). Generally speaking, aspartate
aminotransferase is elevated in diseases affecting tissues rich in
aspartate aminotransferase. See U.S. Pat. No. 5,834,226, above.
Examples of such conditions include acute myocardial infarction,
pulmonary emulsion, acute pancreatitis, viral and toxic hepatitis,
and acute cirrhosis. See U.S. Pat. No. 5,834,226, above. Elevated
aspartate aminotransferase levels have also been correlated with
various cancers and with active periodontal disease.
[0210] Accordingly, it is valuable to the field of pharmaceutical
development to identify and characterize previously unknown
aminotransferases. The present invention advances the state of the
art by providing a previously unidentified human
aminotransferase.
Summary of the Invention
[0211] The present invention is based, in part, on the discovery of
a novel human aminotransferase, referred to herein as "25233". The
nucleotide sequence of a cDNA encoding 25233 is shown as SEQ ID
NO:14. The amino acid sequence of a 25233 polypeptide is shown as
SEQ ID NO:15. In addition, the nucleotide sequence of the coding
region of 25233 (nucleotides 94-1665 of SEQ ID NO:14) is SEQ ID
NO:16.
[0212] Accordingly, in one aspect the invention features a nucleic
acid molecule which encodes a 25233 protein or polypeptide, e.g., a
biologically active portion of the 25233 protein. In a preferred
embodiment, the isolated nucleic acid molecule encodes a
polypeptide having the amino acid sequence of SEQ ID NO:15. In
other embodiments, the invention provides an isolated 25233 nucleic
acid molecule having the nucleotide sequence shown in SEQ ID NO:14,
SEQ ID NO:16. 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:14, SEQ ID NO:16. In other embodiments, the
invention provides a nucleic acid molecule which hybridizes under
stringent hybridization conditions to a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:16,
wherein the nucleic acid encodes a full length 25233 protein or an
active fragment thereof.
[0213] In a related aspect, the invention further provides nucleic
acid constructs which include a 25233 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 25233 nucleic acid molecules of the
invention; e.g., vectors and host cells suitable for producing
25233 nucleic acid molecules and polypeptides.
[0214] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of 25233-encoding nucleic acids.
[0215] In still another related aspect, isolated nucleic acid
molecules that are antisense to a 25233 encoding nucleic acid
molecule are provided.
[0216] In another aspect, the invention features 25233
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 25233-mediated or -related
disorders. In another embodiment, the invention provides 25233
polypeptides having a 25233 activity. Preferred polypeptides are
25233 proteins including at least one aminotransferase domain, and,
preferably, having a 25233 activity; e.g., a 25233 activity as
described herein.
[0217] In other embodiments, the invention provides 25233
polypeptides, e.g., a 25233 polypeptide having the amino acid
sequence shown in SEQ ID NO:15; an amino acid sequence that is
substantially identical to the amino acid sequence shown in SEQ ID
NO:15; or an amino acid sequence encoded by a nucleic acid molecule
having a nucleotide sequence which hybridizes under stringent
hybridization conditions to a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:14, SEQ ID NO:16, wherein the
nucleic acid encodes a full length 25233 protein or an active
fragment thereof.
[0218] In a related aspect, the invention further provides nucleic
acid constructs which include a 25233 nucleic acid molecule
described herein.
[0219] In a related aspect, the invention provides 25233
polypeptides or fragments operatively linked to non-25233
polypeptides to form fusion proteins.
[0220] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically bind 25233 polypeptides.
[0221] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the 25233 polypeptides or nucleic acids.
[0222] In still another aspect, the invention provides a process
for modulating 25233 polypeptide or nucleic acid expression or
activity, e.g. using the screened compounds. In certain
embodiments, the methods involve treatment of conditions related to
aberrant activity or expression of the 25233 polypeptides or
nucleic acids, such as conditions involving aberrant or deficient
cellular proliferation or differentiation.
[0223] The invention also provides assays for determining the
activity of or the presence or absence of 25233 polypeptides or
nucleic acid molecules in a biological sample, including for
disease diagnosis.
[0224] In further aspect the invention provides assays for
determining the presence or absence of a genetic alteration in a
25233 polypeptide or nucleic acid molecule, including for disease
diagnosis.
[0225] Other features and advantages of the invention will be
apparent from the following detailed description, Chapter 7,
Examples and the claims.
Detailed Description of the Invention
[0226] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0227] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
[0228] The human 25233 sequence (SEQ ID NO:14), which is
approximately 2127 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
1569 nucleotides (nucleotides 94-1662 of SEQ ID NO:14; nucleotides
1-1569 of SEQ ID NO:16), not including the terminal codon. The
coding sequence encodes a 523 amino acid protein (SEQ ID
NO:15).
[0229] This mature protein form is approximately 523 amino acid
residues in length (from about amino acid 1 to amino acid 523 of
SEQ ID NO:15). Human 25233 contains the following regions or other
structural features: a predicted aminotransferase domain (PFAM
Accession PF00155) located at about amino acid residues 83-517 of
SEQ ID NO:15; and a predicted transmembrane domain which extends
from about amino acid residue 181-199 of SEQ ID NO:15.
[0230] The 25233 protein also includes an ICE-like protease
(caspase) p20 domain from about amino acid 138-149 of SEQ ID
NO:15.
[0231] For general information regarding PFAM identifiers, PS
prefix and PF prefix domain identification numbers, refer to
Sonnhammer et al. (1997) Protein 28:405-420 and the Pfam website
maintained in several locations, e.g. by the Sanger Institute
(sanger.ac.uk/Software/Pfam).
[0232] The 25233 protein contains a significant number of
structural characteristics in common with members of the
aminotransferase class-I family. The term "family" when referring
to the protein and nucleic acid molecules of the invention means
two or more proteins or nucleic acid molecules having a common
structural domain or motif and having sufficient amino acid or
nucleotide sequence homology as defined herein. Such family members
can be naturally or non-naturally occurring and can be from either
the same or different species. For example, a family can contain a
first protein of human origin as well as other distinct proteins of
human origin, or alternatively, can contain homologues of non-human
origin, e.g., rat or mouse proteins. Members of a family can also
have common functional characteristics.
[0233] As used herein, the term "aminotransferase" refers to a
protein or polypeptide that is capable of catalyzing a
transamination, decarboxylation, deamination, racemization, or
aldol cleavage reaction. Aminotransferases are
pyridoxal-5'-phosphate (PLP) dependent enzymes and can have a
specificity for various amino acid or related substrates.
Typically, aminotransferases have a major role in the metabolism of
amino acids, and are found in various pathways ranging from the
interconversion of alpha-amino acids to the biosynthesis of
antibiotic compounds. These PLP-dependent enzymes have been grouped
into five different fold types, designated classes I-V, on the
basis of amino acid sequence comparisons, predicted secondary
structure elements and available three-dimensional structural
information. These five structural classes are the aspartate
aminotransferase family (class-I), the tryptophan synthase beta
family (class-II), the alanine racemase family (class-III), the
D-amino acid family (class-IV), and the glycogen phosphorylase
family (class-V). The 25233 protein of the present invention is
homologous to the class I aminotransferase family members.
[0234] The metabolism of amino acids involves specific reversible
transfer of nitrogenous groups catalyzed by aminotransferases.
Thus, the molecules of the present invention may be involved in one
or more of: 1) the transfer of an amino groups; 2) catalysis of
certain amino acids; 3) the modulation of the metabolism of various
amino acids; 4) the modulation of tumor cell growth and invasion;
5) cellular homeostasis.
[0235] A 25233 polypeptide can include an "aminotransferase domain"
or regions homologous with an "aminotransferase domain".
[0236] As used herein, the term "aminotransferase domain" includes
an amino acid sequence of about 400-450 amino acid residues in
length and having a bit score for the alignment of the sequence to
the aminotransferase domain (HMM) of at least 8. Preferably, an
aminotransferase domain includes at least about 410-445 amino
acids, more preferably about 420-440 amino acid residues, or about
430-435 amino acids and has a bit score for the alignment of the
sequence to the aminotransferase domain (HMM) of at least 10 or
greater. The aminotransferase domain (HMM) has been assigned the
PFAM Accession PF00155 (see the Pfam website, pfam.wustl.edu). An
alignment of the aminotransferase domain (amino acids 83-517 of SEQ
ID NO:15) of human aminotransferase with a consensus amino acid
sequence derived from a hidden Markov model is depicted in FIG.
19.
[0237] In a preferred embodiment 25233-like polypeptide or protein
has an "aminotransferase domain" or a region which includes at
least about 200-450 more preferably about 230-440 or 260-420 amino
acid residues and has at least about 60%, 70%, 80%, 90%, 95%, 99%,
or 100% sequence identity with an "aminotransferase domain," e.g.,
the aminotransferase domain of human 25233-like polypeptide (e.g.,
amino acid residues 83-517 of SEQ ID NO:15).
[0238] To identify the presence of an "aminotransferase" domain in
a 25233-like 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 (sanger.ac.uk/Software/Pfam). 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.
[0239] In one embodiment, an 25233-like protein includes 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 that spans a phospholipid membrane. More
preferably, a transmembrane domain includes about at least 15, 18,
20, 22, 24, or 25 amino acid residues and spans a phospholipid
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, the Pfam website at
7tm.sub.--1 (pfam.wustl.edu) and Zagotta W. N. et al. (1996) Annual
Rev. Neuronsci. 19:235-63, the contents of which are incorporated
herein by reference.
[0240] In a preferred embodiment, a 25233-like polypeptide or
protein has at least one transmembrane domain or a region which
includes at least 15, 18, 20, 22, 24, or 25 amino acid residues and
has at least about 60%, 70% 80% 90% 95%, 99%, or 100% sequence
identity with a "transmembrane domain," e.g., at least one
transmembrane domain of human 25233-like polypeptide (e.g., amino
acid residues 181-199 of SEQ ID NO:15).
[0241] In another embodiment, an 25233-like protein includes at
least one "non-transmembrane domain." As used herein,
"non-transmembrane domains" are domains that reside outside of the
membrane. When referring to plasma membranes, non-transmembrane
domains include extracellular domains (i.e., outside of the cell)
and intracellular domains (i.e., within the cell). When referring
to membrane-bound proteins found in intracellular organelles (e.g.,
mitochondria, endoplasmic reticulum, peroxisomes and microsomes),
non-transmembrane domains include those domains of the protein that
reside in the cytosol (i.e., the cytoplasm), the lumen of the
organelle, or the matrix or the intermembrane space (the latter two
relate specifically to mitochondria organelles). The C-terminal
amino acid residue of a non-transmembrane domain is adjacent to an
N-terminal amino acid residue of a transmembrane domain in a
naturally occurring 25233-like protein.
[0242] In a preferred embodiment, an 25233-like polypeptide or
protein has a "non-transmembrane domain" or a region which includes
at least about 1-320, preferably about 200-310, more preferably
about 230-300, and even more preferably about 240-280 amino acid
residues, and has at least about 60%, 70% 80% 90% 95%, 99% or 100%
sequence identity with a "non-transmembrane domain", e.g., a
non-transmembrane domain of human 25233-like polypeptide (e.g.,
residues 200-523 of SEQ ID NO:15). Preferably, a non-transmembrane
domain is capable of catalytic activity (e.g., transamination).
[0243] A non-transmembrane domain located at the N-terminus of a
25233-like protein or polypeptide is referred to herein as an
"N-terminal non-transmembrane domain." As used herein, an
"N-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-180, preferably about 30-160, more
preferably about 50-140, or even more preferably about 80-120 amino
acid residues in length and is located outside the boundaries of a
membrane. For example, an N-terminal non-transmembrane domain is
located at about amino acid residues 1-180 of SEQ ID NO:15.
[0244] Similarly, a non-transmembrane domain located at the
C-terminus of a 25233-like protein or polypeptide is referred to
herein as a "C-terminal non-transmembrane domain." As used herein,
an "C-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-323, preferably about 200-310, more
preferably about 230-300, and even more preferably about 240-280
amino acid residues in length and is located outside the boundaries
of a membrane. For example, an C-terminal non-transmembrane domain
is located at about amino acid residues 200-523 of SEQ ID
NO:15.
[0245] As the 25233 polypeptides of the invention may modulate
25233-mediated activities, they may be useful for developing novel
diagnostic and therapeutic agents for 25233-mediated or related
disorders, as described below.
[0246] As used herein, a "25233 activity", "biological activity of
25233" or "functional activity of 25233", refers to an activity
exerted by a 25233 protein, polypeptide or nucleic acid molecule on
e.g., a 25233-responsive cell or on a 25233 substrate, e.g., an
amino acid or related substrate, as determined in vivo or in vitro.
In one embodiment, a 25233 activity is a direct activity, such as
an association with a 25233 binding partner. A "binding partner" is
a molecule with which a 25233 protein binds or interacts in nature,
e.g., an amino acid that the 25233 protein transaminates. A 25233
activity can also be an indirect activity, e.g., a cellular
signaling activity mediated by interaction of the 25233 protein
with a 25233 binding partner. For example, the molecules of the
present invention may be involved in one or more of: 1) the
transfer of an amino groups; 2) catalysis of certain amino acids;
3) the modulation of the metabolism of various amino acids; 4) the
modulation of tumor cell growth and invasion; 5) cellular
homeostasis.
[0247] As used herein, the terms "cancer", "hyperproliferative" and
"neoplastic" refer to cells having the capacity for autonomous
growth, i.e., an abnormal state or condition characterized by
rapidly proliferating cell growth. Hyperproliferative and
neoplastic disease states may be categorized as pathologic, i.e.,
characterizing or constituting a disease state, or may be
categorized as non-pathologic, i.e., a deviation from normal but
not associated with a disease state. 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.
"Pathologic hyperproliferative" cells occur in disease states
characterized by malignant tumor growth. Examples of non-pathologic
hyperproliferative cells include proliferation of cells associated
with wound repair.
[0248] The terms "cancer" or "neoplasms" include malignancies of
the various organ systems, such as 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.
[0249] 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 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.
[0250] The term "sarcoma" is art recognized and refers to malignant
tumors of mesenchymal derivation.
[0251] Human 25233 showed elevated levels of expression in tissues
and cells including, but not limited to, pancreas, immune cells,
brain cortex, glial cells, liver, epithelial cells, breast tumor,
lung tumor, and colon tumor. See FIGS. 21 and 22A-D. Thus, the
25233 nucleic acid and protein of the invention can be used to
treat and/or diagnose a variety of proliferative disorders. E.g.,
such 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, L. (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.
[0252] Disorders involving 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.
[0253] 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.
[0254] 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, Varicalla-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
degeneration, multiple system atrophy, including striatonigral
degeneration, 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.
[0255] Disorders involving T-cells include, but are not limited to,
cell-mediated hypersensitivity, such as delayed type
hypersensitivity and T-cell-mediated cytotoxicity, and transplant
rejection; autoimmune diseases, such as systemic lupus
erythematosus, Sjogren syndrome, systemic sclerosis, inflammatory
myopathies, mixed connective tissue disease, and polyarteritis
nodosa and other vasculitides; immunologic deficiency syndromes,
including but not limited to, primary immunodeficiencies, such as
thymic hypoplasia, severe combined immunodeficiency diseases, and
AIDS; leukopenia; reactive (inflammatory) proliferations of white
cells, including but not limited to, leukocytosis, acute
nonspecific lymphadenitis, and chronic nonspecific lymphadenitis;
neoplastic proliferations of white cells, including but not limited
to lymphoid neoplasms, such as precursor T-cell neoplasms, such as
acute lymphoblastic leukemia/lymphoma, peripheral T-cell and
natural killer cell neoplasms that include peripheral T-cell
lymphoma, unspecified, adult T-cell leukemia/lymphoma, mycosis
fungoides and Sezary syndrome, and Hodgkin disease.
[0256] 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.
[0257] Disorders involving B-cells include, but are not limited to
precursor B-cell neoplasms, such as lymphoblastic
leukemia/lymphoma. Peripheral B-cell neoplasms include, but are not
limited to, chronic lymphocytic leukemia/small lymphocytic
lymphoma, follicular lymphoma, diffuse large B-cell lymphoma,
Burkitt lymphoma, plasma cell neoplasms, multiple myeloma, and
related entities, lymphoplasmacytic lymphoma (Waldenstr om
macroglobulinemia), mantle cell lymphoma, marginal zone lymphoma
(MALToma), and hairy cell leukemia.
[0258] 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.
[0259] Disorders in the male breast include, but are not limited
to, gynecomastia and carcinoma.
[0260] Disorders involving the prostate include, but are not
limited to, inflammations, benign enlargement, for example, nodular
hyperplasia (benign prostatic hypertrophy or hyperplasia), and
tumors such as carcinoma.
[0261] Disorders involving the heart, include but are not limited
to, heart failure, including but not limited to, cardiac
hypertrophy, left-sided heart failure, and right-sided heart
failure; ischemic heart disease, including but not limited to
angina pectoris, myocardial infarction, chronic ischemic heart
disease, and sudden cardiac death; hypertensive heart disease,
including but not limited to, systemic (left-sided) hypertensive
heart disease and pulmonary (right-sided) hypertensive heart
disease; valvular heart disease, including but not limited to,
valvular degeneration caused by calcification, such as calcific
aortic stenosis, calcification of a congenitally bicuspid aortic
valve, and mitral annular calcification, and myxomatous
degeneration of the mitral valve (mitral valve prolapse), rheumatic
fever and rheumatic heart disease, infective endocarditis, and
noninfected vegetations, such as nonbacterial thrombotic
endocarditis and endocarditis of systemic lupus erythematosus
(Libman-Sacks disease), carcinoid heart disease, and complications
of artificial valves; myocardial disease, including but not limited
to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, and myocarditis; pericardial disease, including but
not limited to, pericardial effusion and hemopericardium and
pericarditis, including acute pericarditis and healed pericarditis,
and rheumatoid heart disease; neoplastic heart disease, including
but not limited to, primary cardiac tumors, such as myxoma, lipoma,
papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac
effects of noncardiac neoplasms; congenital heart disease,
including but not limited to, left-to-right shunts--late cyanosis,
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosus, and atrioventricular septal defect,
right-to-left shunts--early cyanosis, such as tetralogy of fallot,
transposition of great arteries, truncus arteriosus, tricuspid
atresia, and total anomalous pulmonary venous connection,
obstructive congenital anomalies, such as coarctation of aorta,
pulmonary stenosis and atresia, and aortic stenosis and atresia,
and disorders involving cardiac transplantation.
[0262] Disorders involving red cells include, but are not limited
to, anemias, such as hemolytic anemias, including hereditary
spherocytosis, hemolytic disease due to erythrocyte enzyme defects:
glucose-6-phosphate dehydrogenase deficiency, sickle cell disease,
thalassemia syndromes, paroxysmal nocturnal hemoglobinuria,
immunohemolytic anemia, and hemolytic anemia resulting from trauma
to red cells; and anemias of diminished erythropoiesis, including
megaloblastic anemias, such as anemias of vitamin B12 deficiency:
pernicious anemia, and anemia of folate deficiency, iron deficiency
anemia, anemia of chronic disease, aplastic anemia, pure red cell
aplasia, and other forms of marrow failure.
[0263] 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.
[0264] 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.
[0265] Disorders involving the liver include, but are not limited
to, hepatic injury; jaundice and cholestasis, such as bilirubin and
bile formation; hepatic failure and cirrhosis, such as cirrhosis,
portal hypertension, including ascites, portosystemic shunts, and
splenomegaly; infectious disorders, such as viral hepatitis,
including hepatitis A-E infection and infection by other hepatitis
viruses, clinicopathologic syndromes, such as the carrier state,
asymptomatic infection, acute viral hepatitis, chronic viral
hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and
toxin-induced liver disease, such as alcoholic liver disease;
inborn errors of metabolism and pediatric liver disease, such as
hemochromatosis, Wilson disease, .alpha..sub.1-antitrypsin
deficiency, and neonatal hepatitis; intrahepatic biliary tract
disease, such as secondary biliary cirrhosis, primary biliary
cirrhosis, primary sclerosing cholangitis, and anomalies of the
biliary tree; circulatory disorders, such as impaired blood flow
into the liver, including hepatic artery compromise and portal vein
obstruction and thrombosis, impaired blood flow through the liver,
including passive congestion and centrilobular necrosis and
peliosis hepatis, hepatic vein outflow obstruction, including
hepatic vein thrombosis (Budd-Chiari syndrome) and veno-occlusive
disease; hepatic disease associated with pregnancy, such as
preeclampsia and eclampsia, acute fatty liver of pregnancy, and
intrehepatic cholestasis of pregnancy; hepatic complications of
organ or bone marrow transplantation, such as drug toxicity after
bone marrow transplantation, graft-versus-host disease and liver
rejection, and nonimmunologic damage to liver allografts; tumors
and tumorous conditions, such as nodular hyperplasias, adenomas,
and malignant tumors, including primary carcinoma of the liver and
metastatic tumors.
CHAPTER 5
8035, 84242, 55304, 52999, and 21999, Novel Human Proteins and
Methods of Use Thereof
Background of the Invention
[0266] RING Finger Proteins. Targeted protein proteolysis is
increasingly understood to be an important general mechanism by
which cells regulate protein levels and, consequently, their
functions at specific times. In eukaryotic cells, the main
mechanism for such control involves the specific covalent
modification by polyubiquitin, which labels target proteins for
proteolysis and subsequent degradation. There are many known
examples of such events, and ubiquitination is now recognized as a
major mechanism for cellular regulation (for review see: Freemont,
P.S. (2000) Current Biology 10:R84-R87; Joazeiro and Weissman
(2000) Cell 102:549-552; Jackson et al. (2000) Trends in Cell
Biology 10:429-439).
[0267] Protein ubiquitination begins with the formation of a
thiol-ester linkage between ubiquitin and the ubiquitin activating
enzyme (E1). Ubiquitin is then transferred to a ubiquitin
conjugating enzyme (E2), again through a thiol-ester linkage. The
ubiquitin ligases (E3's), which are primarily responsible for
providing specificity to the ubiquitin conjugation, interact with
both E2 and substrate to promote ubiquitination. The E3 enzymes are
thought to be the least conserved component of the ubiquitination
pathway.
[0268] Recent studies indicate that E3's can be divided into two
distinct protein classes; those containing a HECT domain and those
containing a RING finger domain. The RING finger class of E3
ubiquitin ligases can be further grouped into the SCF, VBC and
anaphase-promoting complexes, and single-polypeptide RING finger E3
enzymes. The RING finger motif and its variants have been found in
more than 200 eukaryotic proteins, but interestingly not in any
prokaryotic protein. Perhaps the most famous RING finger protein is
BRCA1, the product of a breast cancer-associated gene. Point
mutations within the RING finger domain of BRCA1 predispose females
having the mutations to breast cancer. Other well-known family
members include the protooncogene products Cbl, BMI-1, and PML; the
immunoglobulin gene recombination enzyme RAG1; the Rbx1 component
of the von Hippel Lindau (VHL) tumor suppressor complex; and the
p53 regulator MDM2, to name but a few.
[0269] RING finger proteins play pivotal roles in diverse cellular
processes and are implicated in contributing to disease. The
biological roles of RING finger proteins include regulation of
cellular proliferation, apoptosis, the cell cycle, cellular
signaling, transcription, DNA repair, degradation from the
endoplasmic reticulum (ER), and photomorphogenesis. In addition,
RING mutations in the RING finger protein, Parkin, are associated
with autosomal juvenile parkinsonism.
[0270] The tumor suppressor BRCA1 provides an example where loss of
RING finger function is associated with dysregulated growth and
malignancy, in the form of familial breast and ovarian cancer.
Another example where loss of RING finger function is associated
with malignancy is in the case of VHL disease. The RING finger
protein Rbx1 is a component of the E3 complex that includes the VHL
tumor suppressor protein, and VHL mutations that prevent assembly
of this E3 are associated with the malignancies of VHL disease,
perhaps due to the stabilization of proteins such as hypoxia
inducing factor 1 alpha.
[0271] The influence of RING finger E3 ubiquitin ligases on the
balance between cellular proliferation and apoptosis is
demonstrated by the following examples. First, Mdm2 is a RING
finger E3 ubiquitin ligase that functions as a regulator of the
tumor suppressor, p53. The regulation of p53 by Mdm2 has been
demonstrated to depend on the RING finger domain of Mdm2, thus,
implicating this RING finger E3 ubiquitin ligase as a critical
regulator of cellular proliferation. Second, the E3 ubiquitin
ligase activity of a group of RING finger containing proteins known
as Inhibitors of Apoptosis (IAP's) has been demonstrated to be the
activity responsible for IAP auto-ubiquitination, degradation, and
progression toward cell death in response to apoptotic stimuli
(Yang et al. (2000) Science 288:874-877). Thus, these E3 ubiquitin
ligases play a crucial role in the regulation of apoptosis.
[0272] One example of the clearly established role of RING finger
proteins in the regulation of the cell cycle is that mitotic
cyclins are targeted for degradation by ubiquitination mediated by
the APC (or cyclosome) that includes the small RING finger protein,
Apc11p.
[0273] The RING finger E3 ubiquitin ligases' role in the secretory
pathway is through regulation of the disposal of membrane proteins
from the endoplasmic reticulum (ER). One example of the impact of
this key role in the secretory pathway is the RING finger E3
facilitation of disposal of a membrane protein from the ER
contributing to the pathogenesis of AIDS. Beta TrCP, a RING finger
protein that targets beta-catenin and I.sub.kappaB.sub.alpha for
ubiquitination, also targets Vpu-bound CD4 for degradation,
resulting in an increase in the amount of HIV Env protein available
for virus production.
[0274] Accordingly, RING finger proteins are a major target for
drug action and development. Therefore, it is valuable to the field
of pharmaceutical development to identify and characterize
previously unknown RING finger proteins. The present invention
advances the state of the art by providing two previously
unidentified human RING finger proteins.
[0275] Aminopeptidases. Aminopeptidases are a group of
widely-distributed exopeptidases that catalyze the hydrolysis of
amino acid residues from the amino-terminus of peptide substrates.
Members of this enzyme family are found throughout the animal and
plant kingdoms, and are found subcellularly in organelles, in the
cytoplasm, and as membrane components. Aminopeptidases function in
many cellular processes, including protein maturation, the
regulation of hormone levels (including vasopressin and
noradrenaline levels), the regulation of the renin-angiotensin
system, and in cell-cycle control (including B cell precursor cell
cycle control).
[0276] In eukaryotes, aminopeptidases are associated with removal
of the initiator methionine. This enzyme family is also involved in
the metabolism of secreted regulatory molecules, such as hormones
and neurotransmitters, and modulation of cell-cell interactions. In
mammalian cells and tissues, these enzymes play a role in the
terminal stages of protein degradation, and in cell-cycle control.
Aminopeptidase also have a role in protein turnover and selective
elimination of obsolete or defective proteins.
[0277] Industrial uses of this enzyme family include modification
of amino termini in recombinantly expressed proteins. See A. Taylor
(1993) TIBS 18: 1993:167-172.
[0278] Many aminopeptidases are metalloenzymes, requiring divalent
cations for proteolytic activity. Most aminopeptidase metal binding
sites coordinate Zn.sup.2+ or Co.sup.2+. However, the metal binding
sites of certain aminopeptidases can readily bind Mn.sup.2+ and
Mg.sup.2+. Sites involved in Zn.sup.2+ coordination include the
"His His Glu" and "Asp Glu Lys" motifs.
[0279] Several aminopeptidase inhibitors have been identified.
These inhibitors include bestatin (which has been shown to bind to
the aminopeptidase active site), boronic and phosphonic acids,
.alpha.-methylleucine and isoamylthioamide. See A. Taylor (1993)
TIBS 18: 1993:167-172; Burley et al. (1992) J. Mol. Biol.
224:113-140; Taylor et al. (1993) Biochemistry 32:784-790.
[0280] Aminopeptidases play a role in the pathogenesis of a number
of disorders including hypertension, cancer, cataracts, and
leukemia, and inhibitors of these enzymes are currently being
evaluated as potential therapeutics for many of these disorders.
Aminopeptidase activity is also believed to contribute to the aging
process. Accordingly, aminopeptidases are a major target for drug
action and development. Therefore, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown aminopeptidases.
[0281] Metallopeptidases. Proteases function in carcinogenesis by
inactivating or activating regulators of the cell cycle,
differentiation, programmed cell death, or other processes
affecting cancer development and/or progression. Consistent with
the model involving protease activity and tumor progression,
certain protease inhibitors have been shown to be effective
inhibitors of carcinogenesis both in vitro and in vivo.
[0282] Metallopeptidases are a group of widely distributed
proteases that depend on bound Ca.sup.2+ or Zn.sup.2+ for activity;
however, certain metallopeptidases can readily utilize Mn.sup.2+
and Mg.sup.2+. The biological functions of metallopeptidases
include protein maturation and protein degradation, such as the
degradation of extracellular matrix proteins. As such,
metallopeptidases have been shown to have a role in tumor growth,
metastasis, and angiogenesis.
[0283] Accordingly, metallopeptidases are a major target for drug
action and development. Therefore, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown metallopeptidases. The present invention advances the state
of the art by providing a previously unidentified human
metallopeptidase.
[0284] ADP-ribosyltransferases. Mono (ADP-ribosyl) transferase (EC
2.4.2.31) catalyzes the transfer of the ADP-ribose moiety of
nicotinamide adenine dinucleotide (NAD) to an acceptor amino acid
in proteins. In vertebrates, there is a family of such enzymes that
transfer an NAD group to arginine. At least five distinct forms of
this enzyme have been identified so far. Some of the forms are
attached to the membrane by a GPI anchor while others seem to be
secreted. These proteins are typically about 250 to 300 amino acid
residues.
[0285] Mono-ADP ribosylation is a post-translational modification
of proteins in which the ADP-ribose moiety of NAD is transferred to
an acceptor protein and is responsible for the toxicity of some
bacterial toxins, e.g., cholera toxin and pertussis toxin.
ADP-ribosyltransferase activity has been detected in viruses,
bacteria, and eukaryotic cells. For example, cholera toxin
ADP-ribosylates an arginine in the .alpha.-subunit of the
stimulatory heterotrimeric guanine nucleotide-binding (G) protein,
resulting in the activation of adenylyl cyclase and an increase in
intracellular cyclic AMP. Eukaryotic ADP-ribosyltransferase
activity has been detected in several tissues, and cDNAs have been
cloned from rabbit, human skeletal muscle, chicken
polymorphonuclear granulocytes, and nucleoblasts, and mouse
lymphoma cells. Studies have shown that when the transferase cDNAs
are transfected into mammalian cells, the skeletal muscle and mouse
lymphocyte enzymes are extracellular glycosylphosphatidylinositol
(GPI)-anchored proteins. Consistent with its extracellular
location, the GPI-linked muscle transferase ADP-ribosylates
integrin .alpha.-7 on cultured myotubes (Zolkiewska et al. (1993)
J. Biol. Chem. 268:25273-25276). Also, inhibitor studies suggest
that the muscle transferase may participate in the regulation of
myogenesis (Kharadia, S. V. et al. (1992) Exp. Cell Res.
201:33-42). The muscle and lymphocyte ADP-ribosyltransferases
catalyze the ADP-ribosylation of arginine, agmatine, and other
simple guanidino compounds (Zolkiewska,A. et al. (1992) Proc. Natl.
Acad. Sci. U.S.A. 89:11352-11356).
Summary of the Invention
[0286] The present invention is based, in part, on the discovery of
novel human RING finger proteins, referred to herein as "8035 and
84242". The nucleotide sequences of the cDNA's encoding 8035 and
84242 are shown in SEQ ID NO:18 and SEQ ID NO:22, respectively, and
the amino acid sequences of the 8035 and 84242 polypeptides are
shown in SEQ ID NO:19 and SEQ ID NO:23, respectively. In addition,
the nucleotide sequences of the coding regions of these cDNA's are
depicted in SEQ ID NO:20 and SEQ ID NO:24, respectively.
[0287] Accordingly, in one aspect the invention features nucleic
acid molecules that encode the 8035 and 84242 proteins or
polypeptides, e.g., biologically active portions of the 8035 and
84242 proteins. In a preferred embodiment, the isolated nucleic
acid molecules encode polypeptides having the amino acid sequence
of SEQ ID NO:19 or SEQ ID NO:23. In other embodiments, the
invention provides isolated 8035 and 84242 nucleic acid molecules
having the nucleotide sequences shown in SEQ ID NO:18, SEQ ID
NO:20, SEQ ID NO:22 or SEQ ID NO:24. In still other embodiments,
the invention provides nucleic acid molecules that are
substantially identical (e.g., naturally occurring allelic
variants) to the nucleotide sequences shown in SEQ ID NO:18, SEQ ID
NO:20, SEQ ID NO:22 or SEQ ID NO:24. In other embodiments, the
invention provides nucleic acid molecules that hybridize under
stringent hybridization conditions to a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:18, SEQ ID NO:20,
SEQ ID NO:22 or SEQ ID NO:24, wherein the nucleic acid encodes a
full length 8035 or 84242 protein or an active fragment
thereof.
[0288] In a related aspect, the invention further provides nucleic
acid constructs that include a 8035 or 84242 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 8035 and 84242 nucleic acid molecules of
the invention e.g., vectors and host cells suitable for producing
8035 and 84242 nucleic acid molecules and polypeptides.
[0289] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of 8035 and 84242-encoding nucleic acids.
[0290] In still another related aspect, isolated nucleic acid
molecules that are antisense to an 8035 or an 84242 encoding
nucleic acid molecule are provided.
[0291] In another aspect, the invention features 8035 and 84242
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 8035 and 84242-mediated or
-related disorders. In another embodiment, the invention provides
8035 and 84242 polypeptides having 8035 or 84242 activity,
respectively. Preferred polypeptides are 8035 proteins including at
least one RING finger protein domain (C3HC4 type) and, preferably,
having an 8035 activity, e.g., an 8035 activity as described
herein; and 84242 proteins including at least one IBR (In Between
RING Finger) domain and, preferably, having an 84242 activity,
e.g., an 84242 activity as described herein.
[0292] In other embodiments, the invention provides 8035 and 84242
polypeptides, e.g., an 8035 or 84242 polypeptide having the amino
acid sequence shown in SEQ ID NO:19 or SEQ ID NO:23, respectively;
an amino acid sequence that is substantially identical to the amino
acid sequence shown in SEQ ID NO:19 or SEQ ID NO:23; or an amino
acid sequence encoded by a nucleic acid molecule having a
nucleotide sequence that hybridizes under stringent hybridization
conditions to a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID
NO:24, wherein the nucleic acids encode a full length 8035 or 84242
protein or an active fragment thereof.
[0293] In a related aspect, the invention further provides nucleic
acid constructs that include an 8035 or 84242 nucleic acid molecule
described herein.
[0294] In a related aspect, the invention provides 8035 and 84242
polypeptides or fragments operatively linked to non-8035 and
non-84242 polypeptides to form fusion proteins.
[0295] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically bind 8035 or 84242 polypeptides.
[0296] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the 8035 and 84242 polypeptides or nucleic acids.
[0297] In still another aspect, the invention provides a process
for modulating 8035 and 84242 polypeptide or nucleic acid
expression or activity, e.g., using the screened compounds. In
certain embodiments, the methods involve treatment of conditions
related to aberrant activity or expression of the 8035 and 84242
polypeptides or nucleic acids, such as conditions involving
aberrant regulation of cellular proliferation and/or
differentiation.
[0298] The invention also provides assays for determining the
activity of or the presence or absence of 8035 and 84242
polypeptides or nucleic acid molecules in a biological sample,
including for disease diagnosis.
[0299] In further aspect the invention provides assays for
determining the presence or absence of a genetic alteration in a
8035 or 84242 polypeptide or nucleic acid molecule, including for
disease diagnosis.
[0300] The present invention is also based, in part, on the
discovery of a novel human aminopeptidase, referred to herein as
"55304". The nucleotide sequence of a cDNA encoding 55304 is shown
in SEQ ID NO:26, and the amino acid sequence of a 55304 polypeptide
is shown in SEQ ID NO:27. In addition, the nucleotide sequence of
the coding region is depicted in SEQ ID NO:28.
[0301] Accordingly, in one aspect the invention features a nucleic
acid molecule which encodes a 55304 protein or polypeptide, e.g., a
biologically active portion of the 55304 protein. In a preferred
embodiment, the isolated nucleic acid molecule encodes a
polypeptide having the amino acid sequence of SEQ ID NO:27. In
other embodiments, the invention provides an isolated 55304 nucleic
acid molecule having the nucleotide sequence shown in SEQ ID NO:26
or SEQ ID NO:28. 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:26 or SEQ ID NO:28. In other embodiments, the
invention provides a nucleic acid molecule which hybridizes under
stringent hybridization conditions to a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:26 or SEQ ID NO:28,
wherein the nucleic acid encodes a full length 55304 protein or an
active fragment thereof.
[0302] In a related aspect, the invention further provides nucleic
acid constructs which include a 55304 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 55304 nucleic acid molecules of the
invention e.g., vectors and host cells suitable for producing 55304
nucleic acid molecules and polypeptides.
[0303] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of 55304-encoding nucleic acids.
[0304] In still another related aspect, isolated nucleic acid
molecules that are antisense to a 55304 encoding nucleic acid
molecule are provided.
[0305] In another aspect, the invention features 55304
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 55304-mediated or -related
disorders. In another embodiment, the invention provides 55304
polypeptides having a 55304 activity. Preferred polypeptides are
55304 proteins including at least one aminopeptidase domain, and,
preferably, having a 55304 activity, e.g., a 55304 activity as
described herein.
[0306] In other embodiments, the invention provides 55304
polypeptides, e.g., a 55304 polypeptide having the amino acid
sequence shown in SEQ ID NO:27; an amino acid sequence that is
substantially identical to the amino acid sequence shown in SEQ ID
NO:27; or an amino acid sequence encoded by a nucleic acid molecule
having a nucleotide sequence which hybridizes under stringent
hybridization conditions to a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:26 or SEQ ID NO:28, wherein the
nucleic acid encodes a full length 55304 protein or an active
fragment thereof.
[0307] In a related aspect, the invention further provides nucleic
acid constructs which include a 55304 nucleic acid molecule
described herein.
[0308] In a related aspect, the invention provides 55304
polypeptides or fragments operatively linked to non-55304
polypeptides to form fusion proteins.
[0309] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically bind 55304 polypeptides.
[0310] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the 55304 polypeptides or nucleic acids.
[0311] In still another aspect, the invention provides a process
for modulating 55304 polypeptide or nucleic acid expression or
activity, e.g. using the screened compounds. In certain
embodiments, the methods involve treatment of conditions related to
aberrant activity or expression of the 55304 polypeptides or
nucleic acids, such as conditions involving aberrant or deficient
cellular proliferation or differentiation.
[0312] The invention also provides assays for determining the
activity of or the presence or absence of 55304 polypeptides or
nucleic acid molecules in a biological sample, including for
disease diagnosis.
[0313] In further aspect the invention provides assays for
determining the presence or absence of a genetic alteration in a
55304 polypeptide or nucleic acid molecule, including for disease
diagnosis.
[0314] The present invention is based, in part, on the discovery of
a novel human metallopeptidase, referred to herein as "52999". The
nucleotide sequence of a cDNA encoding 52999 is shown in SEQ ID
NO:29, and the amino acid sequence of a 52999 polypeptide is shown
in SEQ ID NO:30. In addition, the nucleotide sequence of the coding
region is depicted in SEQ ID NO:3 1.
[0315] Accordingly, in one aspect the invention features a nucleic
acid molecule which encodes a 52999 protein or polypeptide, e.g., a
biologically active portion of the 52999 protein. In a preferred
embodiment, the isolated nucleic acid molecule encodes a
polypeptide having the amino acid sequence of SEQ ID NO:30. In
other embodiments, the invention provides an isolated 52999 nucleic
acid molecule having the nucleotide sequence shown in SEQ ID NO:29
SEQ ID NO:31. 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:29 or SEQ ID NO:31. In other embodiments, the
invention provides a nucleic acid molecule which hybridizes under
stringent hybridization conditions to a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:29 or SEQ ID NO:31,
wherein the nucleic acid encodes a full length 52999 protein or an
active fragment thereof.
[0316] In a related aspect, the invention further provides nucleic
acid constructs which include a 52999 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 52999 nucleic acid molecules of the
invention e.g., vectors and host cells suitable for producing 52999
nucleic acid molecules and polypeptides.
[0317] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of 52999-encoding nucleic acids.
[0318] In still another related aspect, isolated nucleic acid
molecules that are antisense to a 52999 encoding nucleic acid
molecule are provided.
[0319] In another aspect, the invention features 52999
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 52999-mediated or -related
disorders. In another embodiment, the invention provides 52999
polypeptides having a 52999 activity. Preferred polypeptides are
52999 proteins including at least one metallopeptidase domain, and,
preferably, having a 52999 activity, e.g., a 52999 activity as
described herein.
[0320] In other embodiments, the invention provides 52999
polypeptides, e.g., a 52999 polypeptide having the amino acid
sequence shown in SEQ ID NO:30; an amino acid sequence that is
substantially identical to the amino acid sequence shown in SEQ ID
NO:30; or an amino acid sequence encoded by a nucleic acid molecule
having a nucleotide sequence which hybridizes under stringent
hybridization conditions to a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:29 or SEQ ID NO:31, wherein the
nucleic acid encodes a full length 52999 protein or an active
fragment thereof.
[0321] In a related aspect, the invention further provides nucleic
acid constructs which include a 52999 nucleic acid molecule
described herein.
[0322] In a related aspect, the invention provides 52999
polypeptides or fragments operatively linked to non-52999
polypeptides to form fusion proteins.
[0323] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically bind 52999 polypeptides.
[0324] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the 52999 polypeptides or nucleic acids.
[0325] In still another aspect, the invention provides a process
for modulating 52999 polypeptide or nucleic acid expression or
activity, e.g. using the screened compounds. In certain
embodiments, the methods involve treatment of conditions related to
aberrant activity or expression of the 52999 polypeptides or
nucleic acids, such as inflammatory conditions and conditions
involving aberrant or deficient cellular proliferation or
differentiation.
[0326] The invention also provides assays for determining the
activity of or the presence or absence of 52999 polypeptides or
nucleic acid molecules in a biological sample, including for
disease diagnosis.
[0327] In further aspect the invention provides assays for
determining the presence or absence of a genetic alteration in a
52999 polypeptide or nucleic acid molecule, including for disease
diagnosis.
[0328] The present invention is based, in part, on the discovery of
a novel human ribosyltransferase referred to herein as mono-ADP
ribosyltransferase. The nucleotide sequence of a cDNA encoding
ADP-ribosyltransferase is shown in SEQ ID NO:36, and the amino acid
sequence of a ADP-ribosyltransferase polypeptide is shown in SEQ ID
NO:37.
[0329] Accordingly, in one aspect the invention features a nucleic
acid molecule which encodes a ADP-ribosyltransferase protein or
polypeptide, e.g., a biologically active portion of the
ADP-ribosyltransferase protein. In a preferred embodiment, the
isolated nucleic acid molecule encodes a polypeptide having the
amino acid sequence of SEQ ID NO:37. In other embodiments, the
invention provides an isolated ADP-ribosyltransferase nucleic acid
molecule having the nucleotide sequence shown in SEQ ID NO:36. 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:36. In other embodiments, the invention provides a nucleic
acid molecule which hybridizes under stringent hybridization
conditions to a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:36, wherein the nucleic acid encodes a full
length ADP-ribosyltransferase protein or an active fragment
thereof.
[0330] In a related aspect, the invention further provides nucleic
acid constructs which include a ADP-ribosyltransferase 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 ADP-ribosyltransferase nucleic acid
molecules of the invention e.g., vectors and host cells suitable
for producing ADP-ribosyltransferase nucleic acid molecules and
polypeptides.
[0331] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of ADP-ribosyltransferase-encoding nucleic acids.
[0332] In still another related aspect, isolated nucleic acid
molecules that are antisense to a ADP-ribosyltransferase encoding
nucleic acid molecule are provided.
[0333] In another aspect, the invention features
ADP-ribosyltransferase 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
ADP-ribosyltransferase-mediated or -related disorders. 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, 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 a disease. A therapeutic agent
includes, but is not limited to, small molecules, peptides,
antibodies, ribozymes and antisense oligonucleotides.
[0334] In another embodiment, the invention provides
ADP-ribosyltransferase polypeptides having a ADP-ribosyltransferase
activity. Preferred polypeptides are ADP-ribosyltransferase
proteins including at least one transferase domain, and,
preferably, having a ADP-ribosyltransferase activity, e.g., a
ADP-ribosyltransferase activity described herein.
[0335] In other embodiments, the invention provides
ADP-ribosyltransferase polypeptides, e.g., a ADP-ribosyltransferase
polypeptide having the amino acid sequence shown in SEQ ID NO:37;
an amino acid sequence that is substantially identical to the amino
acid sequence shown in SEQ ID NO:37; or an amino acid sequence
encoded by a nucleic acid molecule having a nucleotide sequence
which hybridizes under stringent hybridization conditions to a
nucleic acid molecule comprising the nucleotide sequence of SEQ ID
NO:36, wherein the nucleic acid encodes a full length
ADP-ribosyltransferase protein or an active fragment thereof.
[0336] In a related aspect, the invention further provides nucleic
acid constructs which include a ADP-ribosyltransferase nucleic acid
molecule described herein.
[0337] In a related aspect, the invention provides
ADP-ribosyltransferase polypeptides or fragments operatively linked
to non-ADP-ribosyltransferase polypeptides to form fusion
proteins.
[0338] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically bind ADP-ribosyltransferase
polypeptides.
[0339] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the ADP-ribosyltransferase polypeptides or nucleic acids.
[0340] In still another aspect, the invention provides a process
for modulating ADP-ribosyltransferase polypeptide or nucleic acid
expression or activity, e.g. using the screened compounds. In
certain embodiments, the methods involve treatment of conditions
related to aberrant activity or expression of the
ADP-ribosyltransferase polypeptides or nucleic acids, such as
conditions involving aberrant or deficient cellular
proliferation/differentiation or aberrant metabolic function.
[0341] The invention also provides assays for determining the
activity of or the presence or absence of ADP-ribosyltransferase
polypeptides or nucleic acid molecules in a biological sample,
including for disease diagnosis.
[0342] In further aspect the invention provides assays for
determining the presence or absence of a genetic alteration in a
ADP-ribosyltransferase polypeptide or nucleic acid molecule,
including for disease diagnosis.
[0343] Other features and advantages of the invention will be
apparent from the following detailed description, Chapter 7,
Examples and the claims.
Detailed Description of the Invention
[0344] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0345] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
Human 8035 and 84242
[0346] The human 8035 and 84242 sequences (SEQ ID NO:18 and SEQ ID
NO:22, respectively), that are approximately 2876 and 2810
nucleotides long including untranslated regions, respectively,
contain predicted methionine-initiated coding sequences of about
1302 and 1212 nucleotides (nucleotides 613-1914 of SEQ ID NO:18;
SEQ ID NO:20, and nucleotides 744-1955 of SEQ ID NO:22; SEQ ID
NO:24, respectively). The coding sequences encode a 433 and 403
amino acid protein (SEQ ID NO:19 and SEQ ID NO:23,
respectively).
[0347] Human 8035 contains a predicted RING finger protein domain
(C3HC4 type) (PFAM Accession PF00097) located at about amino acid
residues 380-421 of SEQ ID NO:19; and potential transmembrane
domains are recognized from about amino acid residue 26-43, 50-69,
78-94, 136-152, 162-178, 185-203, and 221-245 of SEQ ID NO:19.
[0348] Human 84242 contains a predicted IBR (In Between RING
Fingers) domain (PFAM Accession PF01485) located at about amino
acid residues 2-67 of SEQ ID NO:23; and potential transmembrane
domains are recognized from about amino acid residue 174-195,
221-245, and 329-345 of SEQ ID NO:23.
[0349] For general information regarding PFAM identifiers, PS
prefix and PF prefix domain identification numbers, refer to
Sonnhammer et al. (1997) Protein 28:405-420 and the Pfam website
maintained in several locations, e.g. by the Sanger Institute
(sanger.ac.uk/Software/Pfam).
[0350] As stated above the 8035 proteins share significant
structural characteristics with members of the C3HCH type RING
finger protein family and the 84242 protein contains another
cysteine-rich domain termed, IBR (In Between RING Fingers). The IBR
domain has a C6HC consensus pattern that defines this structure as
the forth family member of the zinc-binding RING, LIM, and LAP/PHD
fingers (van der Reijden et al. (1999) Protein Sci. 8:1557-1561).
The term "family" when referring to the protein and nucleic acid
molecules of the invention means two or more proteins or nucleic
acid molecules having a common structural domain or motif and
having sufficient amino acid or nucleotide sequence homology as
defined herein. Such family members can be naturally or
non-naturally occurring and can be from either the same or
different species. For example, a family can contain a first
protein of human origin as well as other distinct proteins of human
origin, or alternatively, can contain homologues of non-human
origin, e.g., rat or mouse proteins. Members of a family can also
have common functional characteristics.
[0351] A number of eukaryotic and viral proteins contain a C3HCH
type RING finger domain. This conserved cysteine-rich RING domain
binds two atoms of zinc, and is likely involved in mediating
protein-protein interactions. The 3 dimensional structure of the
zinc ligation system is unique to the RING domain and is referred
to as the "cross-brace" motif.
[0352] As such, the 84242 polypeptides of the present invention can
be expected to possess similar biological activities as the 8035
polypeptides of the invention and other RING finger protein family
members.
[0353] Typically, RING finger family proteins play a role in
diverse cellular processes. For example, proteins currently known
to include the C3HC4 domain have a role in the mediation of such
functions as recombination, particularly the rearrangement of
immunoglobulin and T-cell receptor genes; the regulation of gene
expression, particularly in various tumor cells, and as a trans-
activator and/or -repressor of the expression of many viral and
cellular promoters including the interleukin-2 receptor alpha
chain; the maintenance of the segment-specific repression of
homeotic selector genes and as a DNA-binding protein involved in X
chromosome dosage compensation; developmental regulation,
particularly male germ cell development and the regulation of
photomorphogenesis; cellular differentiation, particularly
differentiation of acute leukemia cells; the stabilization of
protein-protein interactions, particularly the stabilization of the
complex between the CDK7 kinase and cyclin H; peroxisome
biogenesis, particularly in Zellweger syndrome, an autosomal
recessive disorder associated with peroxisomal deficiencies; the
postranscriptional regulation of genes, particularly in VSG
expression sites; the regulation of adenylate cyclase activity; and
the regulation of DNA repair.
[0354] Genetic mutations, recombinations and chromosomal
translocations in RING finger protein family members have been
implicated in diseases such as cancer, particularly mammalian
breast and ovarian cancer, systemic lupus erythematosus, acute
promyelocytic leukemia (APL), VHL disease, primary Sjogren's
syndrome, Zellweger syndrome, and autosomal juvenile parkinsonism.
In addition, RING finger protein family members have been shown to
contribute to the pathogenesis of certain viral diseases including
those caused by HSV and HIV.
[0355] Thus, the molecules of the present invention may be involved
in one or more of: 1) regulation of recombination; 2) regulation of
gene expression; 3) developmental regulation; 4) regulation of
cellular proliferation and differentiation; 5) regulation of tumor
cell growth; 6) stabilization of protein-protein interactions; 7)
postranscriptional regulation of genes; 8) regulation of adenylate
cyclase activity; 9) regulation of the cell cycle; 10) regulation
of X chromosome dosage compensation; 11) regulation of DNA repair;
12) regulation of viral pathogenesis; 13) regulation of protein
degradation from the ER; and 14) regulation of apoptosis.
[0356] As used herein, the term "RING finger protein domain"
includes an amino acid sequence of about 30-60 amino acid residues
in length and having a bit score for the alignment of the sequence
to the RING finger protein domain (HMM) of at least 8. Preferably,
a RING finger protein domain has a bit score for the alignment of
the sequence to the RING finger protein domain (HMM) of at least 16
or greater. The RING finger protein domain (HMM) has been assigned
the PFAM Accession PF00097 (pfam.wustl.edu/). An alignment of the
RING finger protein domain (amino acids 380-421 of SEQ ID NO:19) of
human 8035 with a consensus amino acid sequence derived from a
hidden Markov model is depicted in FIG. 27.
[0357] Herein, the term "RING finger protein family member" may
also include a polypeptide that possess an IBR domain as described
above. An IBR domain includes an amino acid sequence of about 45-70
amino acid residues in length and having a bit score for the
alignment of the sequence to the IBR protein domain (HMM) of at
least 8. Preferably, an IBR protein domain has a bit score for the
alignment of the sequence to the IBR domain (HMM) of at least 16 or
greater. The IBR domain (HMM) has been assigned the PFAM Accession
PF01485 (pfam.wustl.edu/). An alignment of the IBR protein domain
(amino acids 2-67 of SEQ ID NO:23) of human 84242 with a consensus
amino acid sequence derived from a hidden Markov model is depicted
in FIG. 28.
[0358] In a preferred embodiment an 8035 polypeptide or protein has
at least one RING finger domain or region that includes at least
about 30-60 amino acid residues with at least about 60%, 70%, 80%,
90%, 95%, 99%, or 100% homology with a RING finger domain, e.g.,
the RING finger protein domains of human 8035 (e.g., amino acid
residues 380-421 of SEQ ID NO:19).
[0359] In another preferred embodiment an 84242 polypeptide or
protein has at least one RING finger domain as described above as
well as an IBR domain or region that includes at least about 45-70
amino acid residues with at least about 60%, 70%, 80%, 90%, 95%,
99%, or 100% homology with an IBR domain, e.g., the IBR protein
domain of human 84242 (e.g., amino acid residues 2-67 of SEQ ID
NO:23).
[0360] To identify the presence of a RING finger domain and/or an
IBR domain in a 8035 or 84242 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
(sanger.ac.uk/Software/Pfam). 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.
[0361] As the 8035 and 84242 polypeptides of the invention may
modulate 8035 and 84242-mediated activities, they may be useful for
developing novel diagnostic and therapeutic agents for 8035 and
84242-mediated or related disorders, as described below.
[0362] As used herein, an "8035 or 84242 activity", "biological
activity of 8035 or 84242" or "functional activity of 8035 or
84242", refers to an activity exerted by an 8035 or 84242 protein,
polypeptide or nucleic acid molecule on e.g., an 8035 or
84242-responsive cell or on an 8035 or 84242 substrate, e.g., a
protein substrate, as determined in vivo or in vitro. In one
embodiment, an 8035 or 84242 activity is a direct activity, such as
an association with an 8035 or 84242 target molecule. A 8035 or
84242 "target molecule" or "binding partner" or "ligand" or
"substrate" is a molecule with which an 8035 or 84242 protein binds
or interacts in nature, e.g., an E2 polypeptide or other protein
substrate that an 8035 or 84242 protein binds to facilitate protein
ubiquitination and protein degradation.
[0363] An 8035 or 84242 activity can also be an indirect activity,
e.g., a cellular signaling activity mediated by interaction of the
8035 or 84242 protein with an 8035 or 84242 ligand. For example,
the 8035 and 84242 proteins of the present invention can have one
or more of the following activities: 1) regulation of
recombination, including the rearrangement of immunoglobulin and
T-cell receptor genes; 2) regulation of gene expression such as by
the transactivation and/or repression of the expression of various
promoters; 3) developmental regulation including regulation of male
germ cell development, and maintenance of the segment-specific
repression of homeotic selector genes; 4) regulation of cellular
proliferation and differentiation; 5) regulation of tumor cell
growth; 6) stabilization of protein-protein interactions such as
stabilization of the complex between certain cyclin regulated
kinases; 7) postranscriptional regulation of genes including VSG
genes; 8) regulation of adenylate cyclase activity; 9) regulation
of the cell cycle such as regulation of mitosis; 10) regulation of
X chromosome dosage compensation; 11) regulation of DNA repair; 12)
regulation of viral pathogenesis; 13) regulation of protein
degradation from the ER; 14) regulation of photomorphogenesis; 15)
regulation of peroxisome biogenesis; and 16) regulation of
apoptosis.
[0364] Accordingly, 8035 and 84242 protein may be mediate various
disorders, particularly cellular proliferative and/or
differentiative disorders. Indeed, genetic mutations,
recombinations and chromosomal translocations in RING finger
protein family members have been implicated in diseases such as
cancer, particularly mammalian breast and ovarian cancer; systemic
lupus erythematosus; acute promyelocytic leukemia (APL); VHL
disease; primary Sjogren's syndrome; Zellweger syndrome; and
autosomal juvenile parkinsonism. In addition, RING finger protein
family members have been shown to contribute to the pathogenesis of
certain viral diseases including those caused by HSV and HIV.
[0365] Examples of 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.
[0366] As used herein, the terms "cancer", "hyperproliferative" and
"neoplastic" refer to cells having the capacity for autonomous
growth, i.e., an abnormal state or condition characterized by
rapidly proliferating cell growth. Hyperproliferative and
neoplastic disease states may be categorized as pathologic, i.e.,
characterizing or constituting a disease state, or may be
categorized as non-pathologic, i.e., a deviation from normal but
not associated with a disease state. 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.
"Pathologic hyperproliferative" cells occur in disease states
characterized by malignant tumor growth. Examples of non-pathologic
hyperproliferative cells include proliferation of cells associated
with wound repair.
[0367] The terms "cancer" or "neoplasms" include malignancies of
the various organ systems, such as 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.
[0368] 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 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.
[0369] The term "sarcoma" is art recognized and refers to malignant
tumors of mesenchymal derivation.
[0370] The 8035 and 84242 nucleic acid and protein of the invention
can be used to treat and/or diagnose a variety of proliferative
disorders. E.g., such 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,
L. (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.
[0371] 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, Varicalla-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
degeneration, multiple system atrophy, including striatonigral
degeneration, 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.
Human 55304
[0372] The human 55304 sequence (SEQ ID NO:26), which is
approximately 5502 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
2043 nucleotides (nucleotides 803-2845 of SEQ ID NO:26; SEQ ID
NO:28). The coding sequence encodes a 680 amino acid protein (SEQ
ID NO:27).
[0373] Human 55304 shows a high degree of sequence conservation
with known aminopeptidases at 16 key residues (amino acid numbers
318, 336, 344, 346, 363, 374, 397, 399, 410, 411, 412, 425, 448,
450, 489, 508 of SEQ ID NO:27). These amino acids are conserved
between the 55340 polypeptide and the corresponding residues from a
leucyl aminopeptidase from Vibrio proteolyticus (SwissProt
Accession No. Q01693), an aminopeptidase from Streptomyces griseus
(SwissProt Accession No. P80561), and aminopeptidase Y from
Saccharomyces cerevisiae (SwissProt Accession No. P37302).
[0374] The 55304 polypeptide contains 8 putative transmembrane
domains. These domains are located at amino acids 192-208, 227-251,
264-286, 302-318, 326-343, 356-379, 397-421, and 428-448 of the
55304 amino acid sequences shown in SEQ ID NO:27.
[0375] The 55304 protein also contains the following ProDom domain
matches: protein aminopeptidase/T1F15.12/HSP26-TIF32 hydrolase
(amino acids 1-69 of SEQ ID NO:27) and YBS.sub.--4/HSP26-TIF32
hydrolase/aminopeptidase zinc metalloprotease (amino acids 83-206
of SEQ ID NO:27).
[0376] The 55304 protein contains structural characteristics in
common with members of the aminopeptidase family.
[0377] As used herein, the term "aminopeptidase" refers to a
protein or polypeptide which is capable of catalyzing the removal
of an amino acid from the amino terminus of a peptide substrate.
Aminopeptidases can have a specificity for specific amino acids.
For example, the removal of the amino-terminal methionine from
proteins and peptides is catalyzed by the methionine aminopeptidase
class of aminopeptidases.
[0378] As referred to herein, aminopeptidases preferably include a
catalytic domain of about 100-250 amino acid residues in length,
preferably about 130-210 amino acid residues in length, or more
preferably about 180-200 amino acid residues in length. An
aminopeptidase domain typically includes conserved residues (i.e.
identical residues or conservatively substituted residues as
defined elsewhere herein) in at least 8, at least 9, at least 10,
at least 11, at least 12, at least 13, at least 14, at least 15, or
at 16 sites in the amino acid sequence of the protein. These sites
are located at amino acids 318, 336, 344, 346, 363, 374, 397, 399,
410, 411, 412, 425, 448, 450, 489, 508 of SEQ ID NO:27.
[0379] Typically, aminopeptidases play a role in diverse cellular
processes. For example, aminopeptidases function in protein
maturation, in the terminal degradation of polypeptides, in hormone
level regulation, in the regulation of the renin-angiotensin
system, and in cell cycle control.
[0380] Thus, the molecules of the present invention may be involved
in one or more of: 1) the removal of an amino acid from the amino
terminus of a peptide substrate; 2) protein maturation; 3) the
terminal degradation of proteins; 4) the modulation of hormone
levels; 5) the regulation of the cell cycle; or 6) the regulation
of the renin-angiotensin system.
[0381] In a preferred embodiment 55304 polypeptide or protein has
an "aminopeptidase domain" or a region which includes at least
about 100-250 more preferably about 130-200 or 160-200 amino acid
residues and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or
100% homology with an "aminopeptidase domain," e.g., the
aminopeptidase domain of human 55304 (e.g., amino acid residues
318-508, particularly amino acid residues 318, 336, 344, 346, 363,
374, 397, 399, 410, 411, 412, 425, 448, 450, 489, and 508 of SEQ ID
NO:27).
[0382] In one embodiment, a 55304 protein includes 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 that spans a phospholipid membrane. More
preferably, a transmembrane domain includes about at least 18, 20,
22, 24, 25, 30, 35 or 40 amino acid residues and spans a
phospholipid 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,
the Pfam website at 7tm.sub.--1 (pfam.wustl.edu) and Zagotta W. N.
et al. (1996) Annual Rev. Neuronsci. 19:235-63, the contents of
which are incorporated herein by reference.
[0383] In a preferred embodiment, a 55304 polypeptide or protein
has at least one transmembrane domain or a region which includes at
least 18, 20, 22, 24, 25, 30, 35 or 40 amino acid residues and has
at least about 60%, 70% 80% 90% 95%, 99%, or 100% sequence identity
with a "transmembrane domain," e.g., at least one transmembrane
domain of human 55304 (e.g., amino acid residues 192-208, 227-251,
264-286, 302-318, 326-343, 356-379, 397-421, or 428-448 of SEQ ID
NO:27).
[0384] In another embodiment, a 55304 protein includes at least one
"non-transmembrane domain." As used herein, "non-transmembrane
domains" are domains that reside outside of the membrane. When
referring to plasma membranes, non-transmembrane domains include
extracellular domains (i.e., outside of the cell) and intracellular
domains (i.e., within the cell). When referring to membrane-bound
proteins found in intracellular organelles (e.g., mitochondria,
endoplasmic reticulum, peroxisomes and microsomes),
non-transmembrane domains include those domains of the protein that
reside in the cytosol (i.e., the cytoplasm), the lumen of the
organelle, or the matrix or the intermembrane space (the latter two
relate specifically to mitochondria organelles). The C-terminal
amino acid residue of a non-transmembrane domain is adjacent to an
N-terminal amino acid residue of a transmembrane domain in a
naturally-occurring 55304, or 55304-like protein.
[0385] In a preferred embodiment, a 55304 polypeptide or protein
has a "non-transmembrane domain" or a region which includes at
least about 1-250, preferably about 1-2311, more preferably about
5-231 amino acid residues, and has at least about 60%, 70% 80% 90%
95%, 99% or 100% sequence identity with a "non-transmembrane
domain", e.g., a non-transmembrane domain of human 55304 (e.g.,
residues 1-191, 209-226, 252-263, 287-301, 319-325, 344-355,
380-396, 422-427, or 449-680 of SEQ ID NO:27). Preferably, a
non-transmembrane domain is capable of catalytic activity (e.g.,
catalyzing the removal of an amino terminal amino acid from a
peptide substrate).
[0386] A non-transmembrane domain located at the N-terminus of a
55304 protein or polypeptide is referred to herein as an
"N-terminal non-transmembrane domain." As used herein, an
"N-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-350, preferably about 50-325, more
preferably about 80-320, or even more preferably about 120-191
amino acid residues in length and is located outside the boundaries
of a membrane. For example, an N-terminal non-transmembrane domain
is located at about amino acid residues 1-191 of SEQ ID NO:27.
[0387] Similarly, a non-transmembrane domain located at the
C-terminus of a 55304 protein or polypeptide is referred to herein
as a "C-terminal non-transmembrane domain." As used herein, an
"C-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-300, preferably about 15-290, preferably
about 20-270, more preferably about 25-231 amino acid residues in
length and is located outside the boundaries of a membrane. For
example, a C-terminal non-transmembrane domain is located at about
amino acid residues 680-449 of SEQ ID NO:27.
[0388] As the 55304 polypeptides of the invention may modulate
55304-mediated activities, they may be useful as of for developing
novel diagnostic and therapeutic agents for 55304-mediated or
related disorders, as described below.
[0389] As used herein, a "55304 activity", "biological activity of
55304" or "functional activity of 55304", refers to an activity
exerted by a 55304 protein, polypeptide or nucleic acid molecule on
e.g., a 55304-responsive cell or on a 55304 substrate, e.g., a
protein substrate, as determined in vivo or in vitro. In one
embodiment, a 55304 activity is a direct activity, such as an
association with a 55304 target molecule. A "target molecule" or is
a molecule with which a 55304 protein binds or interacts in nature,
e.g., a peptide substrate from which 55304 removes an amino acid. A
55304 activity can also be an indirect activity, e.g., a cellular
signaling activity mediated by the product of 55304 proteolysis.
For example, the 55304 proteins of the present invention can have
one or more of the following activities: 1) the removal of an amino
acid from the amino terminus of a peptide substrate 2) protein
maturation 3) the terminal degradation of proteins; 4) the
modulation of hormone levels; 5) the regulation of the cell cycle;
or 6) the regulation of the renin-angiotensin system.
[0390] Accordingly, 55304 protein may be mediate various disorders,
including cellular proliferative and/or differentiative disorders,
hypertensive disorders, hormonal disorders, and disorders related
to protein maturation and degradation.
[0391] The 55304 nucleic acid and protein of the invention can be
used to treat and/or diagnose a variety of proliferative disorders
(see above for examples of such disorders).
[0392] Disorders involving the heart, include but are not limited
to, heart failure, including but not limited to, cardiac
hypertrophy, left-sided heart failure, and right-sided heart
failure; ischemic heart disease, including but not limited to
angina pectoris, myocardial infarction, chronic ischemic heart
disease, and sudden cardiac death; hypertensive heart disease,
including but not limited to, systemic (left-sided) hypertensive
heart disease and pulmonary (right-sided) hypertensive heart
disease; valvular heart disease, including but not limited to,
valvular degeneration caused by calcification, such as calcific
aortic stenosis, calcification of a congenitally bicuspid aortic
valve, and mitral annular calcification, and myxomatous
degeneration of the mitral valve (mitral valve prolapse), rheumatic
fever and rheumatic heart disease, infective endocarditis, and
noninfected vegetations, such as nonbacterial thrombotic
endocarditis and endocarditis of systemic lupus erythematosus
(Libman-Sacks disease), carcinoid heart disease, and complications
of artificial valves; myocardial disease, including but not limited
to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, and myocarditis; pericardial disease, including but
not limited to, pericardial effusion and hemopericardium and
pericarditis, including acute pericarditis and healed pericarditis,
and rheumatoid heart disease; neoplastic heart disease, including
but not limited to, primary cardiac tumors, such as myxoma, lipoma,
papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac
effects of noncardiac neoplasms; congenital heart disease,
including but not limited to, left-to-right shunts--late cyanosis,
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosus, and atrioventricular septal defect,
right-to-left shunts--early cyanosis, such as tetralogy of fallot,
transposition of great arteries, truncus arteriosus, tricuspid
atresia, and total anomalous pulmonary venous connection,
obstructive congenital anomalies, such as coarctation of aorta,
pulmonary stenosis and atresia, and aortic stenosis and atresia,
and disorders involving cardiac transplantation.
Human 52999
[0393] The present invention provides the human 52999 sequence (SEQ
ID NO:29), which is approximately 2566 nucleotides long including
untranslated regions, contains a predicted methionine-initiated
coding sequence of about 2277 nucleotides (nucleotides 194 to 2470
of SEQ ID NO:29; SEQ ID NO:31). The coding sequence encodes a 758
amino acid protein (SEQ ID NO:30).
[0394] The protein form of 52999 after cleavage of the predicted
signal sequence is approximately 739 amino acid residues in length
(from about amino acid 20 to amino acid 758 of SEQ ID NO:30). Human
52999 (SEQ ID NO:30) contains regions of high homology located at
about amino acid residues 180 to 192, 230 to 290, 354 to 409, and
520 to 554 of SEQ ID NO:30 that are consistent with 52999 belonging
to the Peptidase_M8 family of zinc metallopeptidases (PFAM
Accession PF01457; FIG. 31).
[0395] The majority of zinc-dependent metallopeptidases (with the
notable exception of the carboxypeptidases) such as the
Peptidase_M8 family share a common pattern of primary structure in
the part of their sequence involved in the binding of zinc, and can
be grouped together as a superfamily, known as the metzincins, on
the basis of this sequence similarity. From the tertiary structure
of thermolysin, the position of the residues acting as zinc ligands
and those involved in the catalytic activity are known. Two of the
zinc ligands are histidines which are very close together in the
sequence; C-terminal to the first histidine is a glutamic acid
residue which acts as a nucleophile and promotes the attack of a
water molecule on the carbonyl carbon of the substrate. A signature
pattern which includes the two histidine and the glutamic acid
residues is sufficient to detect this superfamily of proteins
(Rawlings and Barrett (1995) Methods Enzymol. 248:183-228).
[0396] The 52999 protein includes such a zinc metallopeptidase
zinc-binding signature sequence (ATLHELLHAL) from amino acids
272-281 of SEQ ID NO:30 (ProSite PS0014/PDOC00129), consistent with
the catalytic HEXXH zinc-binding motif of the zinc
metallopeptidases.
[0397] For general information regarding PFAM identifiers, PS
prefix and PF prefix domain identification numbers, refer to
Sonnhammer et al. (1997) Protein 28:405-420 and the Pfam website
maintained in several locations, e.g. by the Sanger Institute
(sanger.ac.uk/Software/Pfam).
[0398] The 52999 protein also contains predicted transmembrane
domains that extend from about amino acid 632-649 and 706-722 of
SEQ ID NO:30.
[0399] As used herein, the term "metallopeptidase" refers to a
protein or polypeptide that is capable of catalyzing the cleavage
of a polypeptide bond through hydrolysis (i.e., possessing
polypeptide hydrolytic activity) and contains at least one
co-factor selected from the group consisting of Zn.sup.2+,
Mn.sup.2+, Mg.sup.2+, and Ca.sup.2+. Metallopeptidases can have a
specificity for various polypeptide substrates including a
preference for hydrophobic residues at P1 and P1' and basic
residues at P2 and P3'. Based on the sequence similarities
described above, the 52999 molecules of the present invention are
predicted to have similar biological activities as metallopeptidase
family members.
[0400] The 52999 protein contains a significant number of
structural characteristics in common with members of the
metallopeptidase family as described above.
[0401] As the biological functions of metallopeptidases include
protein maturation and protein degradation, they typically play a
role in diverse cellular processes. In particular,
metallopeptidases have been shown to have a role in tumor growth,
metastasis, and angiogenesis; in inflammatory disorders including,
but not limited to osteoarthritis and rheumatoid arthritis,
multiple sclerosis, Crohn disease, psoriasis, periodontal disease,
and asthma; in macular degeneration; in restenosis; and in
Alzheimer's disease.
[0402] A 52999 polypeptide can include a "metallopeptidase
zinc-binding motif" or regions homologous with the "Peptidase_M8
family of metallopeptidases".
[0403] As used herein, the term "Peptidase_M8 family of
metallopeptidases" includes an amino acid sequence having a bit
score for the alignment of the sequence to the Peptidase_M8 family
domain (HMM) of at least 8. Preferably, a Peptidase_M8 family
domain has a bit score for the alignment of the sequence to the
metallopeptidase domain (HMM) of at least 16 or greater. The
Peptidase_M8 family (HMM) has been assigned the PFAM Accession
PF01457 (pfam.wustl.edu/). An alignment of the Peptidase_M8 family
domain of human 52999 (amino acids 180 to 192, 230 to 290, 354 to
409, and 520 to 554 of SEQ ID NO:30) with the consensus amino acid
sequences derived from a hidden Markov model is depicted in FIG.
31.
[0404] In a preferred embodiment 52999 polypeptide or protein has
regions with at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100%
homology with the Peptidase_M8 family of metallopeptidases (e.g.,
amino acid residues 180 to 192, 230 to 290, 354 to 409, and 520 to
554 of SEQ ID NO:30).
[0405] To identify the presence of a Peptidase_M8 metallopeptidase
region of homology in a 52999 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
(sanger.ac.uk/Software/Pfam). 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.
[0406] In one embodiment, a 52999 protein includes at least one
transmembrane domain. As used herein, the term "transmembrane
domain" includes an amino acid sequence of at least about 15 amino
acid residues in length that spans a phospholipid 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, the Pfam website at
7tm.sub.--1 (pfam.wustl.edu) and Zagotta W. N. et al. (1996) Annual
Rev. Neuronsci. 19:235-63, the contents of which are incorporated
herein by reference.
[0407] In a preferred embodiment, a 52999 polypeptide or protein
has at least one transmembrane domain or a region which includes at
least 15, 16, 17, 18, 20, 22, 24, 25, 30, 35 or 40 amino acid
residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100%
homology with a "transmembrane domain," e.g., at least one
transmembrane domain of human 52999 (e.g., amino acid residues
632-649 and 706-722 of SEQ ID NO:30).
[0408] In another embodiment, a 52999 protein includes at least one
non-transmembrane domain. As used herein, "non-transmembrane
domains" are domains that reside outside of the membrane. When
referring to plasma membranes, non-transmembrane domains include
extracellular domains (i.e., outside of the cell) and intracellular
domains (i.e., within the cell). When referring to membrane-bound
proteins found in intracellular organelles (e.g., mitochondria,
endoplasmic reticulum, peroxisomes and microsomes),
non-transmembrane domains include those domains of the protein that
reside in the cytosol (i.e., the cytoplasm), the lumen of the
organelle, or the matrix or the intermembrane space (the latter two
relate specifically to mitochondria organelles). The C-terminal
amino acid residue of a non-transmembrane domain is adjacent to an
N-terminal amino acid residue of a transmembrane domain in a
naturally-occurring 52999, or 52999-like protein.
[0409] In a preferred embodiment, a 52999 polypeptide or protein
has two non-transmembrane domains wherein the larger of the
non-transmembrane domains includes an amino acid sequence having at
least about 100-300, 300-500, or 500-600 or more amino acid
residues in length, and has at least about 60%, 70% 80% 90% 95%,
99% or 100% homology with the larger of the two non-transmembrane
domains of human 52999 (e.g., residues 21-612 of SEQ ID NO:30).
Preferably, the non-transmembrane domain is capable of polypeptide
hydrolytic activity.
[0410] A non-transmembrane domain located at the N-terminus of a
52999 protein or polypeptide is referred to herein as an
"N-terminal non-transmembrane domain." As used herein, an
"N-terminal non-transmembrane domain" includes an amino acid
sequence having at least about 1-300, 300-500, or 500-600 or more
amino acid residues in length, and is located outside the
boundaries of a membrane. For example, an N-terminal
non-transmembrane domain is located at about amino acid residues
21-612 of SEQ ID NO:30.
[0411] Similarly, a non-transmembrane domain located at the
C-terminus of a 52999 protein or polypeptide is referred to herein
as a "C-terminal non-transmembrane domain." As used herein, an
"C-terminal non-transmembrane domain" includes an amino acid
sequence having at least about 1-15, 15-25, or 25-36 or more amino
acid residues in length and is located outside the boundaries of a
membrane. For example, a C-terminal non-transmembrane domain is
located at about amino acid residues 723-758 of SEQ ID NO:30.
[0412] A 52999 molecule can further include a signal sequence. As
used herein, a "signal sequence" refers to a peptide of about 20-80
amino acid residues in length which occurs at the N-terminus of
secretory and integral membrane proteins and which contains a
majority of hydrophobic amino acid residues. For example, a signal
sequence contains at least about 20-25, 25-50, or 50-80 amino acid
residues and has at least about 40-90%, hydrophobic amino acid
residues (e.g., alanine, valine, leucine, isoleucine,
phenylalanine, tyrosine, tryptophan, or proline). Such a signal
sequence, also referred to in the art as a "signal peptide", serves
to direct a protein containing such a sequence to a lipid bilayer.
For example, in one embodiment, a 52999 protein contains a signal
sequence of about amino acids 1-20 of SEQ ID NO:30. The signal
sequence is cleaved during processing of the metallopeptidase. The
processed 52999 protein corresponds to amino acids 21 to 758 of SEQ
ID NO:30.
[0413] As the 52999 polypeptides of the invention may modulate
52999-mediated activities, they may be useful for developing novel
diagnostic and therapeutic agents for 52999-mediated or related
disorders, as described below.
[0414] As used herein, a "52999 activity", "biological activity of
52999" or "functional activity of 52999", refers to an activity
exerted by a 52999 protein, polypeptide or nucleic acid molecule on
e.g., a 52999-responsive cell or on a 52999 polypeptide substrate,
as determined in vivo or in vitro. In one embodiment, a 52999
activity is a direct activity, such as an association with a 52999
target molecule. A "target molecule" or "binding partner" or
"ligand" or "substrate" is a molecule with which a 52999 protein
binds or interacts in nature, e.g., a polypeptide that a 52999
protein cleaves. A 52999 activity can also be an indirect activity,
e.g., a cellular signaling activity mediated by interaction of the
52999 protein with a 52999 ligand. For example, the 52999 proteins
of the present invention can have one or more of the following
activities: 1) cleavage of a protein precursor to maturation; 2)
cleavage of a proenzyme to its active state; 3) catalysis of
protein degradation; 4) catalysis of the degradation of
extracellular matrix proteins; 5) modulation of tumor cell growth
and invasion; and 6) modulation of angiogenesis.
[0415] Accordingly, 52999 protein may be mediate various disorders,
including cellular proliferative and/or differentiative disorders;
inflammatory disorders including, but not limited to osteoarthritis
and rheumatoid arthritis, multiple sclerosis, Crohn disease,
psoriasis, periodontal disease, and asthma; macular degeneration;
restenosis; and Alzheimer's disease (see above for examples of such
disorders).
Human 21999
[0416] The human ADP-ribosyltransferase sequence (SEQ ID NO:36),
which is approximately 1485 nucleotides long including untranslated
regions, contains a predicted methionine-initiated coding sequence
of about 879 nucleotides. The coding sequence encodes a 292 amino
acid protein (SEQ ID NO:37).
[0417] As the ADP-ribosyltransferase polypeptides of the invention
may modulate ADP-ribosyltransferase-mediated activities, they may
be useful as of for developing novel diagnostic and therapeutic
agents for ADP-ribosyltransferase-mediated or related disorders, as
described below.
[0418] As used herein, a "ADP-ribosyltransferase activity",
"biological activity of ADP-ribosyltransferase" or "functional
activity of ADP-ribosyltransferase", refers to an activity exerted
by a ADP-ribosyltransferase protein, polypeptide or nucleic acid
molecule on e.g., a ADP-ribosyltransferase-responsive cell or on a
ADP-ribosyltransferase substrate, e.g., an ADP-ribose moiety
substrate, as determined in vivo or in vitro. In one embodiment, a
ADP-ribosyltransferase activity is a direct activity, such as an
association with a ADP-ribosyltransferase target molecule. A
"target molecule" or "binding partner" is a molecule with which a
ADP-ribosyltransferase protein binds or interacts in nature, e.g.,
an ADP-ribose moiety of NAD.
[0419] ADP-ribosyltransferase protein can be detected in a variety
of cell types, including viruses, bacteria and eukaryotic cells.
ADP-ribosylation of target proteins by bacterial toxin transferases
such as cholera, diptheria and pertussis toxins alters critical
pathways. For example, cholera toxin ADP-ribosylates an arginine in
the .A-inverted.-subunit of the stimulatory heterotrimeric guanine
nucleotide-binding (G) protein, resulting in the activation of
adenylyl cyclase and in increase in intracellular cAMP. Eukaryotic
ADP-ribosyltransferase activity has been detected in several
tissues including human skeletal muscle. In fact, inhibitor studies
suggest that the muscle transferase may participate
[0420] in the regulation of myogenesis (Kharadia, S. V. et al.
(1992) Exp. Cell. Res. 201: 33-42).
[0421] Accordingly, the ADP-ribosyltransferase may be involved in
various cellular metabolic and proliferative/differentiative
disorders. Examples of cellular proliferative and/or
[0422] differentiative disorders include cancer, e.g., carcinoma,
sarcoma, metastatic disorders
[0423] or hematopoietic neoplastic disorder.
[0424] The ADP-ribosyltransferase nucleic acid and protein of the
invention can be used to treat and/or diagnose a variety of
proliferative disorders (see above, for examples).
[0425] 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.
CHAPTER 6
52020, A Novel Human Melanoma Associated Antigen and Uses Therefor
cl Background of the Invention
[0426] The immune system has the ability to mount responses that
can destroy tumor cells. Among the various elements of the immune
system, cytotoxic T lymphocytes (CTL) are highly effective in
mediating the rejection of established tumors. CTL's recognize
antigenic determinants produced from any protein synthesized within
the cell, while antibodies recognize and bind only integral cell
surface molecules.
[0427] The anti-tumor activity of tumor-specific CTL is the result
of a series of complex molecular events. After cellular processing
of proteins in the cytoplasm of the tumor cells, small peptides are
transported to the endoplasmic reticulum, where they bind to newly
synthesized major histocompatibility gene complex (MHC) class I
molecules or HLA's. HLA/peptide complexes are then exported to the
surface of the tumor cell where they are recognized by
antigen-specific Class I-restricted CTL's. In addition to lysing
the tumor cell, the CTL's may also secrete lymphokines such as
tumor necrosis factor (TNF), and gamma-interferon (.gamma.-IFN),
which also contribute to the overall anti-tumor effect.
[0428] A family of genes referred to as the "MAGE" family after the
melanoma associated antigen encoding gene, MAGE-1, has been
discovered which are processed into peptides and expressed on tumor
cell surfaces as HLA/peptide complexes. The MAGE peptides are
recognized by specific CTL's leading to lysis of the tumor cells
from which they are expressed. The genes code for "tumor rejection
antigen precursors" and the peptides derived therefrom are referred
to as "tumor rejection antigens" (see Traversari et al. (1992)
Immunogenetics 35:145; van der Bruggen et al (1991), Science
254:1643; and U.S. Pat. No. 5,342,774; all of which are herein
incorporated by reference).
[0429] In fact, the MAGE-1 encoded human melanoma specific antigen,
MZ2-E, is recognized by CTL's derived from a cancer patient (Van
der Bruggen et al. (1991) Science 254:1643-1647). The MAGE-1 gene
is expressed by various melanoma cell lines as well as several
other types of tumor cells, but is not expressed in a panel of
normal tissues. Eleven additional members of the MAGE family, map
to the q28 region of chromosome X and have between 64% and 85%
identity in amino acid sequence to MAGE-1 (Chen et al. (1994) Proc.
Natl. Acad. Sci. 91:1004-1008); De Plaen et al. (1994)
Immunogenetics 40:360-369; Wang et al. (1994) Cytogenet. Cell
Genet. 67:116-119). These genes on the q28 region of chromosome X
are referred to as the MAGE-A family genes (MAGE-A1 to A12)
(Duffour et al. (1999) Eur. J. Immunol. 29:3329-3337).
[0430] The MAGE-A family of genes have been found to be expressed
at a high level in a number of tumors of various histologic types
including those from colorectal, lung, ovarian, breast, colon,
lung, liver, thyroid, and skin cancers (Mori et al. (1996) Ann.
Surg. 183-188; Sakata M. (1996) Kurume Med. J. 43:55-61; Yamada et
al. (1995) Int. J. Cancer 64:388-393; Zukut R. et al. (1993) Cancer
Res. 53:5-8; Zakut R. et al. (1990) Cancer Res. 53:5-8). In
addition, significantly increased levels of MAGE-A4 have been
detected in the sera of patients with hepatitis C virus
(HCV)-associated cancer and HCV- associated liver cirrhosis
indicating (Tsuzurahara et al. (1997) Jpn. J. Cancer Res.
88:915-918.) Examination of a large panel of healthy tissues
revealed expression of MAGE genes only in testis and placenta (De
Plaen et al., supra).
[0431] For example, the MAGE-A1 gene is expressed in approximately
40% of melanomas and in some other tumors and the MAGE-A2 and
MAGE-A3 genes are expressed in approximately 80-90% of the melanoma
lines that have been examined. Activation of MAGE-A1 in cancer
cells may be due to demethylation of the promoter sequence.
Treatment with the demethylating agent 5-aza-2'-deoxycytidine
activated MAGE-A1 expression not only in tumor cell lines, but also
in primary fibroblasts (De Smet C. et al. (1996) Proc. Natl. Acad.
Sci. 93:7149-7153).
[0432] Another family of tumor rejection antigen precursor genes
has been identified on the Xp arm of the X chromosome. These genes
are referred to as the MAGE-B family of genes (MAGE-B1 to B4). The
MAGE-B1 and MAGE-B2 genes are similarly expressed in tumors of
various histological origins, silent in normal tissues with the
exception of testis, and activated by a demethylation process
(Lurquin et al. (1997) Genomics 46:397-408). In addition, studies
by McCurdy et al. indicate that MAGE Xp-2 (MAGE-B2) is the target
of autoantibodies in systemic Lupus Erythematosus (SLE), suggesting
that this protein may also have a role in autoimmune and
inflammatory disorders (McCurdy et al. (1998) Molec. Genet. Metab.
63:3-13). A gene designated MAGE-C1 has also been identified.
[0433] The identification of tumor specific antigens, such as those
encoded by MAGE genes, and the corresponding T cell epitopes have
provided novel peptide-based vaccines useful in treating cancer
patients. For example, a nonapeptide fragment of MAGE-A1 stimulates
CTL's that respond to antigen MZ2-E (Traversari et al. (1992) J.
Exp. Med. 176:1453-1457). Cells that present the nonapeptide,
EADPT-GHSY, were used to immunize MAGE-A positive melanoma patients
(Hu et al. (1996) Cancer Res. 56:2479-2483). The immunization
increased the frequency of autologous melanoma-reactive CTL
precursors in the circulation. In combination with interleukin-2
the MAGE-A1 nonapeptide immunization led to a significant expansion
of the peptide-specific and autologous melanoma-reactive CTL
response (Hu et al., supra). In addition, a tumor rejection antigen
derived from MAGE-A3 tumor rejection antigen precursors is
presented by HLA-A1 molecules, and tumor rejection antigens derived
from MAGE-A2 complex with MHC class I molecule HLA-A2.
[0434] More recently it has been demonstrated that an anti-MAGE-A4
tumor rejection antigen CTL clone can lyse HLA-A2 tumor cells
expressing MAGE-A4 tumor rejection antigen precursors (Duffour et
al., supra). These data are especially important as MAGE-A4 is
expressed in 51% of lung carcinomas and 63% of esophageal
carcinomas, whereas about 50% of Caucasians and Asians express
HLA-A2. These results indicate that MAGE proteins other than
MAGE-A1 are likely to be valuable for cancer immunotherapy.
[0435] In addition to their value as anticancer agents, there is
evidence that MAGE proteins may also have use in the treatment of
neurodegenerative conditions. For example, MAGE genes are related
to the necdin gene, and the small potential transmembrane domain of
the MAGE proteins shows a particularly high degree of conservation
with the transmembrane domain of the necdin protein. It has been
postulated that this region associates with the transmembrane
domain of another protein (De Plaen et al, supra).
[0436] Necdin is a nuclear protein, first identified in neuronally
differentiated embryonal carcinoma cells and in the brain of adult
mice (Maruyama et al. (1991) Biochem. Biophys. Res. Commun.
178:291-296). Necdin is expressed in virtually all postmitotic
neurons in the central nervous system at all stages of development
(Uetsuki et al. (1996), J. Biol. Chem. 271:918-924). However,
necdin is not expressed in proliferative neuron-like cells
originating from tumors, and ectopic expression of necdin in NIH3T3
cells suppresses cell growth without affecting cell viability
(Aizawa et al., (1992) Dev. Brain Res. 63:265-274); Hayashi et al.,
(1995) Biochem. Biophys. Res. Commun. 213:317-324). Therefore,
necdin is likely to affect the transition in developing neurons
from proliferative to non-proliferative states (Uetsuki et al.,
supra). Furthermore, necdin has been shown to interact with viral
transforming proteins such as SV40 large T antigen and adenovirus
E1A, and with the transcription factor E2F1. Necdin can also
functionally replace the Rb as a growth suppressor in Rb deficient
osteosarcoma cells, suggesting that necdin is a neuron-specific
growth suppressor with a function similar to that of Rb (Taniura et
al. (1998) J. Biol. Chem. 273:720-728). Therefore, MAGE proteins
may also function to suppress growth in neuronal cells and, thus,
be involved in the pathophysiology of neurodegenerative
conditions.
[0437] It is well established that members of the MAGE protein
family play critical roles in a variety of important cellular
processes including the regulation of cellular growth and
differentiation; T-cell activation; CTL effector cell function; and
elicitation of auto-antibodies. As a result of these roles, the
MAGE proteins are involved in such important diseases and disorders
as cancers, tissue repair, neurodegenerative disorders, autoimmune
disorders, and inflammatory disorders.
[0438] Accordingly, the discovery of polynucleotides encoding
MAGE-like proteins, and the proteins themselves, provides a means
to investigate MAGE-mediated disorders, and provides new
compositions useful in the diagnosis and/or treatment of cancers,
neurodegenerative disorders, autoimmune disorders such as SLE, and
inflammatory disorders.
Brief Summary of the Invention
[0439] The present invention is based, in part, on the discovery of
a novel human MAGE-like polypeptide, referred to herein as "52020".
The nucleotide sequence of a cDNA encoding 52020 is shown in SEQ ID
NO:40, and the amino acid sequence of a 52020 polypeptide is shown
in SEQ ID NO:41. In addition, the nucleotide sequence of the coding
region is depicted in SEQ ID NO:42.
[0440] Accordingly, in one aspect the invention features a nucleic
acid molecule which encodes a 52020 protein or polypeptide, e.g., a
biologically active portion of the 52020 protein. In a preferred
embodiment, the isolated nucleic acid molecule encodes a
polypeptide having the amino acid sequence of SEQ ID NO:41. In
other embodiments, the invention provides an isolated 52020 nucleic
acid molecule having the nucleotide sequence shown in SEQ ID NO:40
or SEQ ID NO:42. 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:40 or SEQ ID NO:42. In other embodiments, the
invention provides a nucleic acid molecule which hybridizes under
stringent hybridization conditions to a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:40 or SEQ ID NO:42,
wherein the nucleic acid encodes a full length 52020 protein or an
active fragment thereof.
[0441] In a related aspect, the invention further provides nucleic
acid constructs which include a 52020 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 52020 nucleic acid molecules of the
invention e.g., vectors and host cells suitable for producing 52020
nucleic acid molecules and polypeptides.
[0442] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of 52020-encoding nucleic acids.
[0443] In still another related aspect, isolated nucleic acid
molecules that are antisense to a 52020 encoding nucleic acid
molecule are provided.
[0444] In another aspect, the invention features 52020
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 52020-mediated or -related
disorders. In another embodiment, the invention provides 52020
polypeptides having a 52020 activity. Preferred polypeptides are
52020 proteins including at least one MAGE domain, and, preferably,
having a 52020 activity, e.g., a 52020 activity as described
herein.
[0445] In other embodiments, the invention provides 52020
polypeptides, e.g., a 52020 polypeptide having the amino acid
sequence shown in SEQ ID NO:41; an amino acid sequence that is
substantially identical to the amino acid sequence shown in SEQ ID
NO:41; or an amino acid sequence encoded by a nucleic acid molecule
having a nucleotide sequence which hybridizes under stringent
hybridization conditions to a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:40 or SEQ ID NO:42, wherein the
nucleic acid encodes a full length 52020 protein or an active
fragment thereof.
[0446] In a related aspect, the invention further provides nucleic
acid constructs which include a 52020 nucleic acid molecule
described herein.
[0447] In a related aspect, the invention provides 52020
polypeptides or fragments operatively linked to non-52020
polypeptides to form fusion proteins.
[0448] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically bind 52020 polypeptides.
[0449] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the 52020 polypeptides or nucleic acids.
[0450] In still another aspect, the invention provides a process
for modulating 52020 polypeptide or nucleic acid expression or
activity, e.g. using the screened compounds. In certain
embodiments, the methods involve treatment of conditions related to
aberrant activity or expression of the 52020 polypeptides or
nucleic acids, such as conditions involving aberrant: cellular
proliferation or differentiation, T-cell activation, CTL effector
function, or elicitation of autoantibodies.
[0451] The invention also provides assays for determining the
activity of or the presence or absence of 52020 polypeptides or
nucleic acid molecules in a biological sample, including for
disease diagnosis.
[0452] In further aspect the invention provides assays for
determining the presence or absence of a genetic alteration in a
52020 polypeptide or nucleic acid molecule, including for disease
diagnosis.
[0453] Other features and advantages of the invention will be
apparent from the following detailed description, Chapter 7,
Examples and the claims.
Detailed Description of the Invention
[0454] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0455] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
Human 52020
[0456] The human 52020 sequence (SEQ ID NO:40), which is
approximately 2183 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
912 nucleotides (nucleotides 782 to 1693 of SEQ ID NO:40; SEQ ID
NO:42), not including the terminal codon. The coding sequence
encodes a 304 amino acid protein (SEQ ID NO:41).
[0457] Human 52020 contains the following regions or other
structural features: a predicted MAGE domain (PFAM Accession
PF01454) located at about amino acid residues 1-208 of SEQ ID
NO:41; and a predicted transmembrane domain which extends from
about amino acid residue 172-188 of SEQ ID NO:41.
[0458] For general information regarding PFAM identifiers, PS
prefix and PF prefix domain identification numbers, refer to
Sonnhammer et al. (1997) Protein 28:405-420 and the Pfam website
maintained in several locations, e.g. by the Sanger Institute
(sanger.ac.uk/Software/Pfam).
[0459] The 52020 protein contains a significant number of
structural characteristics in common with members of the MAGE
family. The term "family" when referring to the protein and nucleic
acid molecules of the invention means two or more proteins or
nucleic acid molecules having a common structural domain or motif
and having sufficient amino acid or nucleotide sequence homology as
defined herein. Such family members can be naturally or
non-naturally occurring and can be from either the same or
different species. For example, a family can contain a first
protein of human origin as well as other distinct proteins of human
origin, or alternatively, can contain homologues of non-human
origin, e.g., rat or mouse proteins. Members of a family can also
have common functional characteristics.
[0460] As used herein, the term "MAGE" refers to a protein or
polypeptide that is capable of regulating cellular growth and
differentiation; activating T-cells; effecting CTL cell function;
and/or eliciting auto-antibodies. MAGE-like proteins can be divided
into several subfamilies based upon their homology to a MAGE family
consensus sequence that is depicted in FIG. 35. These classes
consist of the MAGE-A proteins (MAGE-A1 through -A12), MAGE-B
proteins (MAGE-B1 through -B4), and the MAGE-C1 protein. The 52020
MAGE-like protein of the invention shows the most homology to the
MAGE-A4 and MAGE-B1 proteins.
[0461] Prodom analysis indicated amino acids from about 101 to
about 206 of SEQ ID NO:41 have about 45% sequence identity to the
consensus sequence of Prodom class PD003141. Polypeptides belong to
this Prodom classification include, for example,
Melanoma-associated Antigen B2 (MAGE-B2 Antigen) from Homo sapiens;
MAGE-B1 from Homo sapiens; MAGE-B4 antigen from Homo sapiens;
MAGE-B3 antigen from Homo sapiens; Melanoma-associated antigen
MAGE-like protein from Homo sapiens. See, for example, Lurquin et
al. (1997) Genomics 46:397-408; and Muscatelli et al. (1995) Proc.
Natl. Acad. Sci. U.S.A. 92:4987-4991.
[0462] Prodom analysis further indicated amino acids from about
207-275 of SEQ ID NO:41 have about 59% sequence identity to the
consensus sequence of Prodom class PD003293. Polypeptides belonging
to this Prodom classification include, for example, MAGE-12,
MAGE-1, MAGE-11, MAGE-10 antigen from Homo sapiens; Melanoma
Antigen Related Sequence 2 (SMAGE-2) from Mus musculus; and Necdin
from Mus musculus. The MAGE antigens from human belonging to this
Prodom class have been implicated in tumor transformation or
various aspects of tumor regression. See, for example, de Smet et
al. (1994) Immunogenetics 39:121-129; Ding et al. (1994) Biochem.
Biophys. Res. Commun. 202:549-555; and Gaugler et al. (1994) J.
Exp. Med. 179:921-930; and Schultz-Thater et al. (1994) Int. J.
Cancer 59:435-439. Based on these sequence similarities, the 52020
molecules of the present invention are predicted to have similar
biological activities as MAGE family members.
[0463] MAGE proteins play a role in diverse cellular processes. For
example, MAGE proteins are processed into peptides and expressed on
tumor cell surfaces as HLA/peptide complexes. The MAGE peptides are
recognized by specific CTL's leading to lysis of the tumor cells
from which they are expressed. In addition, to their role as tumor
specific antigens, MAGE proteins may also function as cell growth
suppressors. MAGE proteins may also function to suppress growth in
neuronal cells and, thus, be involved in the pathophysiology of
neurodegenerative conditions. Furthermore, MAGE-B2 is the target of
autoantibodies in SLE, suggesting that MAGE proteins function to
elicit autoantibodies and, thus, have a role in autoimmune and
inflammatory disorders.
[0464] Thus, the molecules of the present invention may be involved
in one or more of: 1) the regulation of cellular growth and
differentiation; 2) tissue repair; 3) T-cell activation; 4) CTL
effector cell function; and 5) elicitation of autoantibodies.
[0465] In one embodiment of the invention the 52020 MAGE-like
protein, or fragments thereof, are used as vaccines for treating
various cancerous conditions. There is ample evidence that points
to the presentation of MAGE tumor rejection antigens on tumor
cells, followed by the development of an immune response and
deletion of the tumor cells expressing the tumor rejection antigens
(see U.S. Pat. Nos. 5,342,774; 6,063,900; 6,034,214; and 6,019,987
all of which are herein incorporated in their entirety by
reference). The evidence in the art shows that when various MAGE
tumor rejection antigens are administered to cells, a CTL response
is mounted and presenting cells are lysed. This is behavior
characteristic of vaccines, and hence MAGE tumor rejection antigen
precursors's, or the resulting tumor rejection antigens may be
used, either alone or in pharmaceutically appropriate compositions,
as vaccines.
[0466] In another embodiment of the invention, specific anti-52020
CTL clones, or antibodies to a 52020 tumor rejection antigen are
used to diagnose or monitor cancerous conditions as is described in
more detail infra (see U.S. Pat. Nos. 5,908,778 and 5,763,165
herein incorporated by reference), by monitoring the CTL's in a
sample from a subject, binding of antibodies to tumor rejection
antigens, or the activity of anti-tumor rejection antigen CTL's in
connection with subject samples. Similarly, the expression of
nucleic acid molecules encoding tumor rejection antigen
precursors's can be monitored via amplification (e.g., polymerase
chain reaction (PCR)), anti-sense hybridization, probe
technologies, and so forth and described in more detail infra.
Various subject samples, including body fluids (e.g., blood, serum,
and other exudates), tissues and tumors may be so assayed.
[0467] Expression of the 52020 MAGE-like proteins of the invention
may distinguish cells of particular tumors from normal cells.
[0468] In another embodiment, antagonists or inhibitors of 52020
MAGE-like protein may be administered to a subject to treat or
prevent neurodegenerative conditions. Such conditions include, but
are not limited to, those brought on by ischemia, epilepsy,
convulsions, AIDS-related dementia, Alzheimer's disease,
schizophrenia, Alzheimer's and Parkinson's disease, amyotrophic
lateral sclerosis, and lathyrism.
[0469] In another embodiment, antibodies which specifically bind
52020 MAGE-like protein may be used for the diagnosis of conditions
or diseases characterized by expression of 52020 MAGE-like protein,
or in assays to monitor patients being treated with 52020 MAGE-like
protein agonists, antagonists or inhibitors. The antibodies useful
for diagnostic purposes may be prepared in the same manner as those
described above for therapeutics and in more detail infra.
Diagnostic assays for 52020 MAGE-like protein include methods which
utilize the antibody and a label to detect 52020 MAGE-like protein
in human body fluids or extracts of cells or tissues. The
antibodies may be used with or without modification, and may be
labeled by joining them, either covalently or non-covalently, with
a reporter molecule. A wide variety of reporter molecules which are
known in the art may be used (described in more detail infra).
[0470] In another embodiment of the invention, the polynucleotides
encoding 52020 MAGE-like protein may be used for diagnostic
purposes. The polynucleotides which may be used include
oligonucleotide sequences, antisense RNA and DNA molecules, and
PNA's. The polynucleotides may be used to detect and quantify gene
expression in biopsied tissues in which expression of 52020
MAGE-like may be correlated with disease (see U.S. Pat. No.
6,140,050 herein incorporated by reference). The diagnostic assay
may be used to distinguish between absence, presence, and excess
expression of 52020 MAGE-like, and to monitor regulation of 52020
MAGE-like levels.
[0471] A 52020 polypeptide can include a "MAGE domain" or regions
homologous with an "MAGE domain".
[0472] As used herein, the term "MAGE domain" includes an amino
acid sequence of about 80-208 amino acid residues in length and
having a bit score for the alignment of the sequence to the MAGE
domain (HMM) of at least 8. Preferably, a MAGE domain includes at
least about 208 amino acids and has a bit score for the alignment
of the sequence to the MAGE domain (HMM) of at least 16 or greater.
The MAGE domain (HMM) has been assigned the PFAM Accession PF01454
(pfam.wustl.edu/). An alignment of the MAGE domain (amino acids 1
to 208 of SEQ ID NO:41) of human 52020 with a consensus amino acid
sequence derived from a hidden Markov model is depicted in FIG. 35
and results in a bit score of 127.
[0473] In a preferred embodiment 52020 polypeptide or protein has a
"MAGE domain" or a region which includes at least about 80-208 more
preferably about 208 amino acid residues and has at least about
60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with a "MAGE
domain," e.g., the MAGE domain of human 52020 (e.g., amino acid
residues 1-208 of SEQ ID NO:41).
[0474] To identify the presence of an "MAGE" domain in a 52020
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
(sanger.ac.uk/Software/Pfam). 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.
[0475] In one embodiment, a 52020 protein includes 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 that spans a phospholipid membrane. More
preferably, a transmembrane domain includes about at least 17, 18,
20, 22, 24, 25, 30, 35 or 40 amino acid residues and spans a
phospholipid 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,
the Pfam website at 7tm.sub.--1 (pfam.wustl.edu) and Zagotta W. N.
et al. (1996) Annual Rev. Neuronsci. 19:235-63, the contents of
which are incorporated herein by reference.
[0476] In a preferred embodiment, a 52020 polypeptide or protein
has at least one transmembrane domain or a region which includes at
least 17, 18, 20, 22, 24, 25, 30, 35 or 40 amino acid residues and
has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with
a "transmembrane domain," e.g., at least one transmembrane domain
of human 52020 (e.g., amino acid residues 172-188 of SEQ ID
NO:41).
[0477] In another embodiment, a 52020 protein includes at least one
"non-transmembrane domain." As used herein, "non-transmembrane
domains" are domains that reside outside of the membrane. When
referring to plasma membranes, non-transmembrane domains include
extracellular domains (i.e., outside of the cell) and intracellular
domains (i.e., within the cell). When referring to membrane-bound
proteins found in intracellular organelles (e.g., mitochondria,
endoplasmic reticulum, peroxisomes and microsomes),
non-transmembrane domains include those domains of the protein that
reside in the cytosol (i.e., the cytoplasm), the lumen of the
organelle, or the matrix or the intermembrane space (the latter two
relate specifically to mitochondria organelles). The C-terminal
amino acid residue of a non-transmembrane domain is adjacent to an
N-terminal amino acid residue of a transmembrane domain in a
naturally-occurring 52020, or 52020-like protein.
[0478] In a preferred embodiment, a 52020 polypeptide or protein
has a "non-transmembrane domain" or a region which includes at
least about 1-200, preferably about 100-180, more preferably about
125-175, and even more preferably about 140-171 amino acid
residues, and has at least about 60%, 70% 80% 90% 95%, 99% or 100%
homology with a "non-transmembrane domain", e.g., a
non-transmembrane domain of human 52020 (e.g., residues 1-171 and
188-304 of SEQ ID NO:41). Preferably, a non-transmembrane domain is
capable of catalytic activity (e.g., catalyzing an acylation
reaction).
[0479] A non-transmembrane domain located at the N-terminus of a
52020 protein or polypeptide is referred to herein as an
"N-terminal non-transmembrane domain." As used herein, an
"N-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-200 and preferably about 30-200 amino acid
residues and is located outside the boundaries of a membrane. For
example, an N-terminal non-transmembrane domain is located at about
amino acid residues 1-171 of SEQ ID NO:41.
[0480] Similarly, a non-transmembrane domain located at the
C-terminus of a 52020 protein or polypeptide is referred to herein
as a "C-terminal non-transmembrane domain." As used herein, an
"C-terminal non-transmembrane domain" includes an amino acid
sequence having about 1-150, preferably about 15-150, preferably
about 20-150, more preferably about 30-150 amino acid residues in
length and is located outside the boundaries of a membrane. For
example, a C-terminal non-transmembrane domain is located at about
amino acid residues 189-304 of SEQ ID NO:41.
[0481] As the 52020 polypeptides of the invention may modulate
52020-mediated activities, they may be useful as of for developing
novel diagnostic and therapeutic agents for 52020-mediated or
related disorders, as described below.
[0482] As used herein, a "52020 activity", "biological activity of
52020" or "functional activity of 52020", refers to an activity
exerted by a 52020 protein, polypeptide or nucleic acid molecule on
e.g., a 52020-responsive cell or on a 52020 substrate, e.g., a CTL
or protein substrate, as determined in vivo or in vitro. In one
embodiment, a 52020 activity is a direct activity, such as an
association with a 52020 target molecule. A "target molecule" or
"binding partner" is a molecule or cell with which a 52020 protein
binds or interacts in nature. A 52020 activity can also be an
indirect activity, e.g., a cellular signaling activity mediated by
interaction of the 52020 protein with a 52020 ligand. For example,
the 52020 proteins of the present invention can have one or more of
the following activities: 1) regulation of cellular growth and
differentiation; 2) tissue repair; 3) T-cell activation; 4) CTL
effector cell function; and 5) elicitation of autoantibodies.
[0483] Accordingly, 52020 protein may mediate various disorders,
including cancers, tissue repair, neurodegenerative disorders,
autoimmune disorders, and inflammatory disorders.
[0484] Examples of cellular proliferative and/or differentiative
disorders include cancer, e.g., carcinoma, sarcoma, and metastatic
disorders. A metastatic tumor can arise from a multitude of primary
tumor types, including but not limited to those of colon, lung,
breast, ovary, epithelium, and liver origin.
[0485] As used herein, the terms "cancer", "hyperproliferative" and
"neoplastic" refer to cells having the capacity for autonomous
growth, i.e., an abnormal state or condition characterized by
rapidly proliferating cell growth. Hyperproliferative and
neoplastic disease states may be categorized as pathologic, i.e.,
characterizing or constituting a disease state, or may be
categorized as non-pathologic, i.e., a deviation from normal but
not associated with a disease state. 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.
"Pathologic hyperproliferative" cells occur in disease states
characterized by malignant tumor growth. Examples of non-pathologic
hyperproliferative cells include proliferation of cells associated
with wound repair.
[0486] The terms "cancer" or "neoplasms" include malignancies of
the various organ systems, such as 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, non-small cell carcinoma of the
lung, cancer of the small intestine and cancer of the
esophagus.
[0487] 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 lung, breast, head and neck, esophagus, epithelium
(especially melanoma), colon, and ovary. The term 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.
[0488] The term "sarcoma" is art recognized and refers to malignant
tumors of mesenchymal derivation.
[0489] The 52020 molecules can be used to treat and/or diagnose
neurodegenerative disorders such as, but not limited to, those
brought on by ischemia, epilepsy, convulsions, AIDS-related
dementia, Alzheimer's disease, schizophrenia, Alzheimer's and
Parkinson's disease, amyotrophic lateral sclerosis, and
lathyrism.
[0490] The 52020 molecules be used to treat and/or diagnose
inflammatory disorders including, but not limited to osteoarthritis
and rheumatoid arthritis, multiple sclerosis, Crohn disease,
psoriasis, periodontal disease, and asthma; macular degeneration;
restenosis; and Alzheimer's disease.
[0491] Similarly, aberrant expression and/or activity of 52020
molecules may mediate 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.
CHAPTER 7
22406, Acyltransferase (32447), 7716, 25233, 8035, 84242, 55304,
52999, 21999, and 52020 Combined Specification
[0492] The 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, and 52020 protein, fragments thereof, and derivatives
and other variants of the sequence in SEQ ID NO:2, SEQ ID NO:7, SEQ
ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27,
SEQ ID NO:30, SEQ ID NO:37 and SEQ ID NO:41, respectively, are
collectively referred to as "polypeptides or proteins of the
invention" or "22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, and 21999, 52020 polypeptides or proteins". Nucleic
acid molecules encoding such polypeptides or proteins are
collectively referred to as "nucleic acids of the invention" or
"22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999 and 52020 nucleic acids." 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999 and 52020 molecules refer
to 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999 and 52020 nucleic acids, polypeptides, and antibodies.
[0493] 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.
[0494] 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.
[0495] As used herein, the term "hybridizes under stringent
conditions" describes conditions for hybridization and washing.
Stringent conditions are known to those skilled in the art and can
be found in Current Protocols in Molecular Biology John Wiley &
Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are
described in that reference and either can be used. A preferred,
example of stringent hybridization conditions are hybridization in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50.degree. C. Another example of stringent hybridization conditions
are hybridization in 6.times. sodium chloride/sodium citrate (SSC)
at about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 55.degree. C. A further example of
stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
60.degree. C. Preferably, stringent hybridization conditions are
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 65.degree. C. Particularly preferred
stringency conditions (and the conditions that should be used if
the practitioner is uncertain about what conditions should be
applied to determine if a molecule is within a hybridization
limitation of the invention) are 0.5M 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. Preferably, an isolated nucleic acid molecule
that hybridizes under stringent conditions to an
acyltransferase-like sequence of the invention corresponds to a
naturally-occurring nucleic acid molecule.
[0496] Preferably, an isolated nucleic acid molecule of the
invention that hybridizes under stringent conditions to the
sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,
SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:38, SEQ
ID NO:40, or SEQ ID NO:42 corresponds to a naturally-occurring
nucleic acid molecule.
[0497] 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).
[0498] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules that include an open reading frame
encoding a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 protein, preferably a mammalian 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein, and can further include non-coding regulatory
sequences, and introns.
[0499] 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 a preparation of 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein having less than about 30%, 20%, 10% and more
preferably 5% (by dry weight), of non-22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 protein,
respectively, (also referred to herein as a "contaminating
protein"), or of chemical precursors or non-22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 chemicals,
respectively. When the 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 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.
[0500] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of 22406, acyltransferase,
7716, 25233, or 52020 or of 8035, 84242, 55304, 52999, or 21999
(e.g., the sequence of SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27,
SEQ ID NO:30, or SEQ ID NO:37) 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 22406
polypeptides of the present invention, e.g., those present in the
pyridoxal-phosphate attachment site, are predicted to be
particularly unamenable to alteration. Amino acid residues that are
conserved among the acyltransferase polypeptides of the present
invention, e.g., those present in the fatty acid synthase domain,
are predicted to be particularly unamenable to alteration. Amino
acid residues that are conserved among the 7716 polypeptides of the
present invention, e.g., those present in the ATPase domain, are
predicted to be particularly unamenable to alteration. Amino acid
residues that are conserved among the 25233 polypeptides of the
present invention, e.g., those present in the aminotransferase
domain, are predicted to be particularly unamenable to alteration.
Amino acid residues that are conserved among the 84242 or 8035
polypeptides of the present invention, e.g., those present in the
RING or IBR protein domains, are predicted to be particularly
unamenable to alteration. Similarly, amino acid residues that are
conserved among the 55304 polypeptides of the present invention,
e.g., those present in the aminopeptidase domain, are predicted to
be particularly unamenable to alteration. Amino acid residues that
are conserved among the 52999 polypeptides of the present
invention, in particular those present in the metal-binding active
site domain, are also not predicted to be amenable to alteration.
Amino acid residues that are conserved among the 21999 polypeptides
of the present invention, e.g., those present in the transferase
domain, are predicted to be particularly unamenable to alteration,
as well. Amino acid residues that are conserved among the 52020
polypeptides of the present invention, e.g., those present in the
MAGE domain, are predicted to be particularly unamenable to
alteration.
[0501] 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened for 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 biological activity to identify mutants that retain activity.
Following mutagenesis of the sequence of SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14,
SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID
NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ
ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42, the encoded
protein can be expressed recombinantly and the activity of the
protein can be determined.
[0502] As used herein, a "biologically active portion" of a 22406
protein includes a fragment of a 22406 protein which participates
in an interaction between a 22406 molecule and a non-22406
molecule. Biologically active portions of a 22406 protein include
peptides comprising amino acid sequences sufficiently homologous to
or derived from the amino acid sequence of the 22406 protein, e.g.,
the amino acid sequence shown in SEQ ID NO:2, which include less
amino acids than the full length 22406 proteins, and exhibit at
least one activity of a 22406 protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the 22406 protein, e.g., pyridoxal-phosphate dependent
enzyme family member activity. A biologically active portion of a
22406 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 22406 protein can be used as targets for developing
agents which modulate a 22406 mediated activity, e.g.,
pyridoxal-phosphate dependent enzyme family member activity.
[0503] As used herein, a "biologically active portion" of an
acyltransferase protein includes a fragment of an acyltransferase
protein which participates in an interaction between an
acyltransferase molecule and a non-acyltransferase molecule.
Biologically active portions of an acyltransferase protein include
peptides comprising amino acid sequences sufficiently homologous to
or derived from the amino acid sequence of the acyltransferase
protein, e.g., the amino acid sequence shown in SEQ ID NO:7, which
include less amino acids than the full length acyltransferase
proteins, and exhibit at least one activity of an acyltransferase
protein. Typically, biologically active portions comprise a domain
or motif with at least one activity of the acyltransferase protein,
e.g., transfer of an acyl group. A biologically active portion of
an acyltransferase protein can be a polypeptide which is, for
example, 10, 25, 50, 100, 200 or more amino acids in length.
Alternatively, a fragment of a polypeptide of the present invention
comprises an amino acid sequence consisting of amino acid residues
1-20, 20-40, 40-60, 60-80, 80-100, 100-120, 120-140, 140-160,
160-180, 180-200, 200-220, 220-240, 240-260, 260-280, 280-300,
300-320, 320-340, 340-360, 360-380, 380-400, 400-420, 420-440,
440-460, 460-480, 480-500, 500-520, 520-540 of SEQ ID NO:7.
Biologically active portions of an acyltransferase protein can be
used as targets for developing agents which modulate an
acyltransferase mediated activity, e.g., fatty acid synthase
activity.
[0504] As used herein, a "biologically active portion" of a 7716
protein includes a fragment of a 7716 protein which participates in
an interaction between a 7716 molecule and a non-7716 molecule.
Biologically active portions of a 7716 protein include peptides
comprising amino acid sequences sufficiently homologous to or
derived from the amino acid sequence of the 7716 protein, e.g., the
amino acid sequence shown in SEQ ID NO:11, which include less amino
acids than the full length 7716 proteins, and exhibit at least one
activity of a 7716 protein. Typically, biologically active portions
comprise a domain or motif with at least one activity of the 7716
protein, e.g., ATPase activity. A biologically active portion of a
7716 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 7716 protein can be used as targets for developing
agents which modulate a 7716 mediated activity, e.g., ATPase
activity.
[0505] As used herein, a "biologically active portion" of a 25233
protein includes a fragment of a 25233 protein which participates
in an interaction between a 25233 molecule and a non-25233
molecule. Biologically active portions of a 25233 protein include
peptides comprising amino acid sequences sufficiently homologous to
or derived from the amino acid sequence of the 25233 protein, e.g.,
the amino acid sequence shown in SEQ ID NO:15, which include less
amino acids than the full length 25233 proteins, and exhibit at
least one activity of a 25233 protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the 25233 protein, e.g., aminotransferase activity. A
biologically active portion of a 25233 protein can be a polypeptide
which is, for example, 10, 25, 50, 100, 200 or more amino acids in
length. Alternatively, a fragment of a polypeptide of the present
invention comprises an amino acid sequence consisting of amino acid
residues 1-20, 20-40, 40-60, 60-80, 80-100, 100-120, 120-140,
140-160, 160-180, 180-200, 200-220, 220-240, 240-260, 260-280,
280-300, 300-320, 320-340, 340-360, 360-380, 380-400, 400-420,
420-440, 440-460, 460-480, 480-500, 500-520, or 520-523 of SEQ ID
NO:15. Biologically active portions of a 25233 protein can be used
as targets for developing agents which modulate a 25233 mediated
activity, e.g., aminotransferase activity.
[0506] As used herein, a "biologically active portion" of an 8035,
84242, 55304, 52999, or 21999 protein includes a fragment of an
8035, 84242, 55304, 52999, or 21999 protein that participates in an
interaction between an 8035, 84242, 55304, 52999, or 21999 molecule
and a non-8035, non-84242, non-55304, non-52999, or non-21999
molecule. Biologically active portions of 8035, 84242, 55304,
52999, or 21999 proteins include peptides comprising amino acid
sequences sufficiently homologous to or derived from the amino acid
sequence of the 8035, 84242, 55304, 52999, or 21999 protein, e.g.,
the amino acid sequence shown in SEQ ID NO:19, SEQ ID NO:23, SEQ ID
NO:27, SEQ ID NO:30, and SEQ ID NO:37, respectively, that include
less amino acids than the full length 8035, 84242, 55304, 52999, or
21999 protein, and exhibit at least one activity of an 8035, 84242,
55304, 52999, or 21999 protein. Typically, biologically active
portions comprise a domain or motif with at least one activity of
the 8035, 84242, 55304, 52999, or 21999 protein, e.g., in the case
of 8035, or 84242, RING finger protein activity; in the case of
55304, aminopeptidase protein activity; in the case of 52999,
metallopeptidase protein activity; and in the case of 21999,
ribosyltransferase activity. A biologically active portion of an
8035, 84242, 55304, 52999, or 21999 protein can be a polypeptide
that is, for example, 10, 25, 50, 100, 200 or more amino acids in
length. Biologically active portions of an 8035, 84242, 55304,
52999, or 21999 protein can be used as targets for developing
agents that modulate an 8035, 84242, 55304, 52999, or 21999
mediated activity, e.g., in the case of 8035 or 84242, RING finger
protein activity; in the case of 55304, aminopeptidase protein
activity; in the case of 52999, metallopeptidase protein activity;
and in the case of 21999, ribosyltransferase activity.
[0507] As used herein, a "biologically active portion" of a 52020
protein includes a fragment of a 52020 protein which participates
in an interaction between a 52020 molecule and a non-52020
molecule. Biologically active portions of a 52020 protein include
peptides comprising amino acid sequences sufficiently homologous to
or derived from the amino acid sequence of the 52020 protein, e.g.,
the amino acid sequence shown in SEQ ID NO:41, which include less
amino acids than the full length 52020 proteins, and exhibit at
least one activity of a 52020 protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the 52020 protein, e.g., MAGE activity as described
herein. A biologically active portion of a 52020 protein can be a
polypeptide which is, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 25, 50, 100, 200 or more amino acids in length.
Biologically active portions of a 52020 protein can be used as
targets for developing agents which modulate a 52020 mediated
activity, e.g., MAGE activity.
[0508] Calculations of homology or sequence identity between
sequences (the terms are used interchangeably herein) are performed
as follows.
[0509] To determine the percent identity of two amino acid
sequences, or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, 90%, 100% of the length
of the reference sequence (e.g., when aligning a second sequence to
the 22406 amino acid sequence of SEQ ID NO:2 having 340 amino acid
residues, at least 102, preferably at least 136, more preferably at
least 170, even more preferably at least 204, and even more
preferably at least 238, 272, 306 or 340 amino acid residues are
aligned; e.g., when aligning a second sequence to the 7716 amino
acid sequence of SEQ ID NO:11 having 226 amino acid residues, at
least 302, preferably at least 377, more preferably at least 452,
even more preferably at least 528, and even more preferably at
least 603, 679 or 753 amino acid residues are aligned; e.g., when
aligning a second sequence to the 8035 amino acid sequence of SEQ
ID NO:19 having 130 amino acid residues, at least 173, preferably
at least 217, more preferably at least 260, even more preferably at
least 303, and even more preferably at least 346, 390 or 433 amino
acid residues are aligned; when aligning a second sequence to the
84242 amino acid sequence of SEQ ID NO:23 having 121 amino acid
residues, at least 161, preferably at least 202, more preferably at
least 242, even more preferably at least 282, and even more
preferably at least 322, 363 or 403 amino acid residues are
aligned; when aligning a second sequence to the 55304 amino acid
sequence of SEQ ID NO:27 having 204 amino acid residues, at least
272, preferably at least, more preferably at least 340, even more
preferably at least 408, and even more preferably at least 544, 612
or 680 amino acid residues are aligned; when aligning a second
sequence to the 52999 amino acid sequence of SEQ ID NO:30 having
227 amino acid residues, at least 303, preferably at least 379,
more preferably at least 455, even more preferably at least 530,
and even more preferably at least 606, 682 or 758 amino acid
residues are aligned; and, when aligning a second sequence to the
ADP-ribosyltransferase amino acid sequence of SEQ ID NO:37 having
88 amino acid residues, at least 117, preferably at least 146, more
preferably at least 177, even more preferably at least 204, and
even more preferably at least 234, 263 or 292 amino acid residues
are aligned; e.g., when aligning a second sequence to the 52020
amino acid sequence of SEQ ID NO:41 having 91 amino acid residues,
at least 122, preferably at least 152, more preferably at least
182, even more preferably at least 213, and even more preferably at
least 243, 24 or 304 amino acid residues are aligned). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0510] 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 (available at the bioinformatics page of the
website maintained by Accelrys, Inc., San Diego, Calif., USA),
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) is using a Blossum 62 scoring matrix with a gap open
penalty of 12, a gap extend penalty of 4, and a frameshift gap
penalty of 5.
[0511] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of E. Meyers and W.
Miller (1989) CABIOS 4:11-17 which has been incorporated into the
ALIGN program (version 2.0), using a PAM 120 weight residue table,
a gap length penalty of 12 and a gap penalty of 4.
[0512] 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 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 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(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used (accessible at the website
maintained by National Center for Biotechnology Information,
Bethesda, Md., USA).
[0513] "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.
[0514] "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.
[0515] 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.
[0516] Various aspects of the invention are described in further
detail below.
Isolated Nucleic Acid Molecules
[0517] In one aspect, the invention provides, isolated or purified,
nucleic acid molecules that encode a 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 polypeptide
described herein, e.g., a full length 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 protein or a
fragment thereof, e.g., a biologically active portion of 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein. Also included are nucleic acid fragments suitable
for use as a hybridization probes, that can be used, e.g., to
identify a nucleic acid molecule encoding a polypeptide of the
invention, 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 mRNA, and fragments suitable for use as
primers, e.g., PCR primers for the amplification or mutation of
nucleic acid molecules.
[0518] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO:1, or
a portion of any of these nucleotide sequences. In one embodiment,
the nucleic acid molecule includes sequences encoding the human
22406 protein (i.e., "the coding region", from nucleotides 69-1088
of SEQ ID NO:1, not including the terminal codon), as well as 5'
untranslated sequences (nucleotides 1-68 of SEQ ID NO:1).
Alternatively, the nucleic acid molecule can include only the
coding region of SEQ ID NO:1 (e.g., nucleotides 69-1088 of SEQ ID
NO:1, corresponding to SEQ ID NO:3) and, e.g., no flanking
sequences which normally accompany the subject sequence. In another
embodiment, the nucleic acid molecule encodes a sequence
corresponding to the mature protein of SEQ ID NO:2.
[0519] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO:6, or
a portion of any of these nucleotide sequences. In one embodiment,
the nucleic acid molecule includes sequences encoding the human
acyltransferase protein (i.e., "the coding region", from
nucleotides of SEQ ID NO:6, not including the terminal codon), as
well as 5' untranslated sequences (nucleotides of SEQ ID NO:6).
Alternatively, the nucleic acid molecule can include only the
coding region of SEQ ID NO:6 (e.g., nucleotides of SEQ ID NO:6).
and e.g., no flanking sequences which normally accompany the
subject sequence. In another embodiment, the nucleic acid molecule
encodes a sequence corresponding to the mature protein of SEQ ID
NO:7.
[0520] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO:10,
SEQ ID NO:12, or a portion of any of these nucleotide sequences. In
one embodiment, the nucleic acid molecule includes sequences
encoding the human 7716 protein (i.e., "the coding region", from
nucleotides 63-2321 of SEQ ID NO:10, not including the terminal
codon), as well as 5' untranslated sequences (nucleotides 1-62 of
SEQ ID NO:10). Alternatively, the nucleic acid molecule can include
only the coding region of SEQ ID NO:10 (e.g., nucleotides 63-2321
of SEQ ID NO:10, corresponding to SEQ ID NO:12) and, e.g., no
flanking sequences which normally accompany the subject sequence.
In another embodiment, the nucleic acid molecule encodes a sequence
corresponding to the mature protein of SEQ ID NO:11.
[0521] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO:14,
or a portion of any of these nucleotide sequences. In one
embodiment, the nucleic acid molecule includes sequences encoding
the human 25233 protein (i.e., "the coding region", from
nucleotides 94 to 1662 of SEQ ID NO:14, not including the terminal
codon), as well as 5' untranslated sequences (nucleotides 1-93 of
SEQ ID NO:14). Alternatively, the nucleic acid molecule can include
only the coding region of SEQ ID NO:14 (e.g., nucleotides 94 to
1662 of SEQ ID NO:14, corresponding to SEQ ID NO:16) and, e.g., no
flanking sequences which normally accompany the subject sequence.
In another embodiment, the nucleic acid molecule encodes a sequence
corresponding to the mature protein of SEQ ID NO:15.
[0522] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO:18,
or a portion of any of these nucleotide sequences. In one
embodiment, an isolated nucleic acid molecule of the invention
includes the nucleotide sequence shown in SEQ ID NO:22, or a
portion of any of these nucleotide sequences. In one embodiment,
the nucleic acid molecule includes sequences encoding the human
8035 protein (i.e., "the coding region", from nucleotides 613-1914
of SEQ ID NO:18), as well as 5' untranslated sequences (nucleotides
1-612 of SEQ ID NO:18). In one embodiment, the nucleic acid
molecule includes sequences encoding the human 84242 protein (i.e.,
"the coding region", from nucleotides 744-1038 of SEQ ID NO:22), as
well as 5' untranslated sequences (nucleotides 1-743 of SEQ ID
NO:22). Alternatively, the nucleic acid molecule can include only
the coding region of SEQ ID NO:18 or SEQ ID NO:22 (e.g.,
nucleotides 613-1914 of SEQ ID NO:18, corresponding to SEQ ID
NO:20, and nucleotides 744-1955 of SEQ ID NO:22, corresponding to
SEQ ID NO:24, respectively) and, e.g., no flanking sequences which
normally accompany the subject sequence. In another embodiment, the
nucleic acid molecule encodes a sequence corresponding to the
mature protein of SEQ ID NO:19 or SEQ ID NO:23.
[0523] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO:26,
or a portion of any of these nucleotide sequences. In one
embodiment, the nucleic acid molecule includes sequences encoding
the human 55304 protein (i.e., "the coding region", from
nucleotides 803-2845 of SEQ ID NO:26), as well as 5' untranslated
sequences (nucleotides 1-802 of SEQ ID NO:26). Alternatively, the
nucleic acid molecule can include only the coding region of SEQ ID
NO:26 (e.g., nucleotides 803-2845 of SEQ ID NO:26, corresponding to
SEQ ID NO:28) and, e.g., no flanking sequences which normally
accompany the subject sequence. In another embodiment, the nucleic
acid molecule encodes a sequence corresponding to the mature
protein of SEQ ID NO:27.
[0524] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO:29,
or a portion of any of these nucleotide sequences. In one
embodiment, the nucleic acid molecule includes sequences encoding
the human 52999 protein (i.e., "the coding region", from
nucleotides 194-2470 of SEQ ID NO:29), as well as 5' untranslated
sequences (nucleotides 1-193 of SEQ ID NO:29). Alternatively, the
nucleic acid molecule can include only the coding region of SEQ ID
NO:29 (e.g., nucleotides 194-2470 of SEQ ID NO:29, corresponding to
SEQ ID NO:31) and, e.g., no flanking sequences which normally
accompany the subject sequence. In another embodiment, the nucleic
acid molecule encodes a sequence corresponding to the mature
protein of SEQ ID NO:30.
[0525] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO:36,
or a portion of any of these nucleotide sequences. In one
embodiment, the nucleic acid molecule includes sequences encoding
the human ADP-ribosyltransferase protein (i.e., "the coding
region", from nucleotides 255 to 1133 of SEQ ID NO:36), as well as
5' untranslated sequences of SEQ ID NO:36. Alternatively, the
nucleic acid molecule can include only the coding region of SEQ ID
NO:36 and, e.g., no flanking sequences which normally accompany the
subject sequence. In another embodiment, the nucleic acid molecule
encodes a sequence corresponding to the mature protein of SEQ ID
NO:37.
[0526] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO:40,
or a portion of any of these nucleotide sequences. In one
embodiment, the nucleic acid molecule includes sequences encoding
the human 52020 protein (i.e., "the coding region", from
nucleotides 782-1693 of SEQ ID NO:40, not including the terminal
codon), as well as 5' untranslated sequences (nucleotides 1-781 of
SEQ ID NO:40). Alternatively, the nucleic acid molecule can include
only the coding region of SEQ ID NO:40 (e.g., nucleotides 782-1693
of SEQ ID NO:40, corresponding to SEQ ID NO:42) and, e.g., no
flanking sequences which normally accompany the subject sequence.
In another embodiment, the nucleic acid molecule encodes a sequence
corresponding to the mature protein of SEQ ID NO:41.
[0527] 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, SEQ ID
NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31,
SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42, 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, SEQ
ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ
ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,
SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID
NO:31, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42,
such that it can hybridize to the nucleotide sequences shown in SEQ
ID NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ
ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,
SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or
SEQ ID NO:42, thereby forming a stable duplex.
[0528] In one embodiment, an isolated nucleic acid molecule of the
present invention includes a nucleotide sequence which is at least
about 95%, 96%, 97%, 98%, 99%, or more homologous to the nucleotide
sequence shown in SEQ ID NO:1 or SEQ ID NO:3. In the case of an
isolated nucleic acid molecule which is longer than or equivalent
in length to the reference sequence, e.g., SEQ ID NO:1, or SEQ ID
NO:3, the comparison is made with the full length of the reference
sequence. Where the isolated nucleic acid molecule is shorter than
the reference sequence, e.g., shorter than SEQ ID NO:1, or SEQ ID
NO:3, the comparison is made to a segment of the reference sequence
of the same length (excluding any loop required by the homology
calculation).
[0529] 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 nucleotide sequence
shown in SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ
ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24,
SEQ ID NO:29, or SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:40 or SEQ ID
NO:42. In the case of an isolated nucleic acid molecule which is
longer than or equivalent in length to the reference sequence,
e.g., SEQ ID NO:6, SEQ ID NO:10, or SEQ ID NO:12, SEQ ID NO:14 or
SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NO:20, and SEQ ID NO:22 or SEQ
ID NO:24, SEQ ID NO:29, or SEQ ID NO:31, SEQ ID NO:36, SEQ ID
NO:40, SEQ ID NO:42, the comparison is made with the full length of
the reference sequence. Where the isolated nucleic acid molecule is
shorter than the reference sequence, e.g., shorter than SEQ ID
NO:6, SEQ ID NO:10, or SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16,
SEQ ID NO:18 or SEQ ID NO:20, and SEQ ID NO:22 or SEQ ID NO:24, SEQ
ID NO:29, or SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:40, SEQ ID
NO:42, the comparison is made to a segment of the reference
sequence of the same length (excluding any loop required by the
homology calculation).
[0530] 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 nucleotide sequence
shown in SEQ ID NO:26 or SEQ ID NO:28. In the case of an isolated
nucleic acid molecule which is longer than or equivalent in length
to the reference sequence, e.g., SEQ ID NO:26, or SEQ ID NO:28, the
comparison is made with the full length of the reference sequence.
Where the isolated nucleic acid molecule is shorter than the
reference sequence, e.g., shorter than SEQ ID NO:26, or SEQ ID
NO:28, the comparison is made to a segment of the reference
sequence of the same length (excluding any gap required by the
alignment calculation).
22406 Nucleic Acid Fragments
[0531] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3.
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 22406 protein, e.g., an immunogenic or biologically
active portion of a 22406 protein. A fragment can comprise:
nucleotides 19-315 of SEQ ID NO:1, which encodes an
pyridoxal-phosphate dependent enzyme family member domain of human
22406. Alternatively, a fragment can comprise: nucleotides 47-60 of
SEQ ID NO:1, which encodes an pyridoxal-phosphate attachment site
of human 22406. The nucleotide sequence determined from the cloning
of the 22406 gene allows for the generation of probes and primers
designed for use in identifying and/or cloning other 22406 family
members, or fragments thereof, as well as 22406 homologues, or
fragments thereof, from other species.
[0532] 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 150 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.
[0533] 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, the nucleic
acid fragment can include an pyridoxal-phosphate dependent enzyme
family member domain. In a preferred embodiment the fragment is at
least, 50, 100, 200, 300, 400, 500, 600, 700, or 885 base pairs in
length.
[0534] 22406 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 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive
nucleotides of a sense or antisense sequence of SEQ ID NO:1 or SEQ
ID NO:3, or of a naturally occurring allelic variant or mutant of
SEQ ID NO:1 or SEQ ID NO:3.
[0535] In one embodiment the nucleic acid is a probe which is at
least 5, 10, 20 or 30, 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.
[0536] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid which encodes a 22406
pyridoxal-phosphate dependent enzyme family member domain (e.g.,
about amino acid residues 19-315 of SEQ ID NO:2).
[0537] 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 22406 sequence, e.g., a region described
herein. The primers should be at least 5, 10, 20, 30, or 50 base
pairs in length and less than 100, or less than 200, base pairs in
length. The primers should be identical, or differs by one base
from a sequence disclosed herein or from a naturally occurring
variant; e.g., primers suitable for amplifying all or a portion of
any of the following regions are provided: a 22406
pyridoxal-phosphate dependent enzyme family member domain (e.g.,
about amino acid residues 19-315 of SEQ ID NO:2).
[0538] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0539] A nucleic acid fragment encoding a "biologically active
portion of a 22406 polypeptide" can be prepared by isolating a
portion of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3,
which encodes a polypeptide having a 22406 biological activity
(e.g., the biological activities of the 22406 proteins as described
herein), expressing the encoded portion of the 22406 protein (e.g.,
by recombinant expression in vitro) and assessing the activity of
the encoded portion of the 22406 protein. For example, a nucleic
acid fragment encoding a biologically active portion of 22406
includes a pyridoxal-phosphate dependent enzyme family member
domain (e.g., about amino acid residues 19-315 of SEQ ID NO:2). A
nucleic acid fragment encoding a biologically active portion of a
22406 polypeptide, may comprise a nucleotide sequence which is
greater than 300-885 or more nucleotides in length.
[0540] In preferred embodiments, nucleic acids include a nucleotide
sequence which is about 100, 200, 300, 400, 500, 600, 700, 800,
900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 nucleotides in
length and hybridizes under stringent hybridization conditions to a
nucleic acid molecule of SEQ ID NO:1 or SEQ ID NO:3. A fragment of
a nucleotide sequence of the present invention comprises a
nucleotide sequence consisting of nucleotides 1-100, 100-200,
200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900,
900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500,
1500-1600, 1600-1700, 1700-1770 of SEQ ID NO:1.
Acyltransferase Nucleic Acid Fragments
[0541] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO:6. 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 an
acyltransferase protein, e.g., an immunogenic or biologically
active portion of an acyltransferase protein. A fragment can
comprise nucleotides of SEQ ID NO:6, which encode a domain or
active site of the native human acyltransferase. The nucleotide
sequence determined from the cloning of the acyltransferase gene
allows for the generation of probes and primers designed for use in
identifying and/or cloning other acyltransferase family members, or
fragments thereof, as well as acyltransferase homologues, or
fragments thereof, from other species.
[0542] 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 150 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.
[0543] 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, the nucleic
acid fragment can include an acyltransferase. In a preferred
embodiment the fragment is at least, 50, 100, 200, 300, 400, 500,
600, 700, 900, 1000, 1100, 1200, 1300, 1400, 1500, or 1600 base
pairs in length.
[0544] Acyltransferase 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:6 or SEQ ID NO:8, or of a naturally
occurring allelic variant or mutant of SEQ ID NO:6 or SEQ ID
NO:8.
[0545] 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.
[0546] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid which encodes an
acyltransferase domain.
[0547] 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 an acyltransferase sequence, e.g., a region
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 differs by one base
from a sequence disclosed herein or from a naturally occurring
variant. E.g., primers suitable for amplifying all or a portion of
any of the following regions are provided: an acyltransferase
domain (e.g., about amino acid residues of SEQ ID NO:7).
[0548] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0549] A nucleic acid fragment encoding a "biologically active
portion of an acyltransferase polypeptide" can be prepared by
isolating a portion of the nucleotide sequence of SEQ ID NO:6,
which encodes a polypeptide having an acyltransferase biological
activity (e.g., the biological activities of the acyltransferase
proteins as described herein), expressing the encoded portion of
the acyltransferase protein (e.g., by recombinant expression in
vitro) and assessing the activity of the encoded portion of the
acyltransferase protein. For example, a nucleic acid fragment
encoding a biologically active portion of acyltransferase includes
an fatty acid synthase domain (e.g., about amino acid residues of
SEQ ID NO:7). A nucleic acid fragment encoding a biologically
active portion of an acyltransferase polypeptide, may comprise a
nucleotide sequence which is greater than 300-1400 or more
nucleotides in length.
[0550] In preferred embodiments, nucleic acids include a nucleotide
sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000,
1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or 1900 nucleotides
in length and hybridizes under stringent hybridization conditions
to a nucleic acid molecule of SEQ ID NO:6.
7716 Nucleic Acid Fragments
[0551] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO:10 or SEQ ID
NO:12. 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 7716 protein, e.g., an immunogenic or
biologically active portion of a 7716 protein. A fragment can
comprise: nucleotides 706-1263 or 1498-2115 of SEQ ID NO:10, each
of which encodes an ATPase domain of human 7716. The nucleotide
sequence determined from the cloning of the 7716 gene allows for
the generation of probes and primers designed for use in
identifying and/or cloning other 7716 family members, or fragments
thereof, as well as 7716 homologues, or fragments thereof, from
other species.
[0552] 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 150 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.
[0553] 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, the nucleic
acid fragment can include an ATPase domain. In a preferred
embodiment the fragment is at least, 50, 100, 200, 300, 400, 500,
600, 700, or 900 base pairs in length.
[0554] 7716 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:10 or SEQ ID NO:12, or of a
naturally occurring allelic variant or mutant of SEQ ID NO:10 or
SEQ ID NO:12.
[0555] 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.
[0556] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid which encodes an ATPase domain
(e.g., about amino acid residues 236-421 or 500-705 of SEQ ID
NO:11).
[0557] 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 7716 sequence, e.g., a region 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 differs by one base from a
sequence disclosed herein or from a naturally occurring variant.
E.g., primers suitable for amplifying all or a portion of any of
the following regions are provided: an ATPase domain (e.g., about
amino acid residues 236-421 or 500-705 of SEQ ID NO:11).
[0558] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0559] A nucleic acid fragment encoding a "biologically active
portion of a 7716 polypeptide" can be prepared by isolating a
portion of the nucleotide sequence of SEQ ID NO:10 or SEQ ID NO:12,
which encodes a polypeptide having a 7716 biological activity
(e.g., the biological activities of the 7716 proteins as described
herein), expressing the encoded portion of the 7716 protein (e.g.,
by recombinant expression in vitro) and assessing the activity of
the encoded portion of the 7716 protein. For example, a nucleic
acid fragment encoding a biologically active portion of 7716
includes an ATPase domain (e.g., about amino acid residues 236-421
or 500-705 of SEQ ID NO:11). A nucleic acid fragment encoding a
biologically active portion of a 7716 polypeptide, may comprise a
nucleotide sequence which is greater than 300-1200 or more
nucleotides in length.
[0560] In preferred embodiments, nucleic acids include 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, or 2600 nucleotides in length and
hybridizes under stringent hybridization conditions to a nucleic
acid molecule of SEQ ID NO:10 or SEQ ID NO:12.
25233 Nucleic Acid Fragments
[0561] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO:14 or SEQ ID
NO:16. 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 25233 protein, e.g., an immunogenic or
biologically active portion of a 25233 protein. A fragment can
comprise: nucleotides 339-1644 of SEQ ID NO:14, which encodes an
aminotransferase domain of human 25233. The nucleotide sequence
determined from the cloning of the 25233 gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning other 25233 family members, or fragments thereof, as
well as 25233 homologues, or fragments thereof, from other
species.
[0562] 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 150 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.
[0563] 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, the nucleic
acid fragment can include an aminotransferase domain. In a
preferred embodiment the fragment is at least 15, 20, 50, 100, 200,
300, 400, 500, 600, 700, or 900 base pairs in length.
Alternatively, a nucleic acid molecules that is a fragment of a
25233-like nucleotide sequence of the present invention comprises a
nucleotide sequence consisting of nucleotides 1-100, 100-200,
200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900,
900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500,
1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000, 2000-2100,
or 2100-2127 of SEQ ID NO:14 or nucleotides 1-100, 100-200,
200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900,
900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, or
1500-1572 of SEQ ID NO:16.
[0564] 25233 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:14 or SEQ ID NO:16, or of a
naturally occurring allelic variant or mutant of SEQ ID NO:14 or
SEQ ID NO:16.
[0565] 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.
[0566] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid which encodes an
aminotransferase domain (e.g., about nucleotides 339-1644 of SEQ ID
NO:14 or nucleotides 247-1551 of SEQ ID NO:16).
[0567] 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 25233 sequence, e.g., a region 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 differs by one base from a
sequence disclosed herein or from a naturally occurring variant.
E.g., primers suitable for amplifying all or a portion of any of
the following regions are provided: a nucleic acid encoding an
aminotransferase domain (e.g., about nucleic acid residues 339-1644
of SEQ ID NO:14 or 247-1551 of SEQ ID NO:16).
[0568] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0569] A nucleic acid fragment encoding a "biologically active
portion of a 25233 polypeptide" can be prepared by isolating a
portion of the nucleotide sequence of SEQ ID NO:14 or SEQ ID NO:16,
which encodes a polypeptide having a 25233 biological activity
(e.g., the biological activities of the 25233 proteins as described
herein), expressing the encoded portion of the 25233 protein (e.g.,
by recombinant expression in vitro) and assessing the activity of
the encoded portion of the 25233 protein. For example, a nucleic
acid fragment encoding a biologically active portion of 25233
includes an aminotransferase domain (e.g., about nucleic acid
residues 339-1644 of SEQ ID NO:14 or 247-1551 of SEQ ID NO:16). A
nucleic acid fragment encoding a biologically active portion of a
25233 polypeptide, may comprise a nucleotide sequence which is
greater than 300-1200 or more nucleotides in length.
[0570] In preferred embodiments, nucleic acids include a nucleotide
sequence which is about 400, 600, 800, 1000, 1200, 1400, 1600,
1700, 1800, 1900, 2000, 2100, or 2127 nucleotides in length and
hybridizes under stringent hybridization conditions to a nucleic
acid molecule of SEQ ID NO:14, or SEQ ID NO:16.
8035 and 84242 Nucleic Acid Fragments
[0571] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO:18, SEQ ID NO:20,
SEQ ID NO:22 or SEQ ID NO:24. 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 an 8035 or 84242
protein, e.g., an immunogenic or biologically active portion of an
8035 or 84242 protein. A fragment can comprise nucleotides
1750-1875 of SEQ ID NO:18, that encodes a RING finger protein
domain of human 8035. A fragment can comprise nucleotides 837-1038
of SEQ ID NO:22, that encodes an IBR protein domain of human 84242.
The nucleotide sequences determined from the cloning of the 8035
and 84242 genes allow for the generation of probes and primers
designed for use in identifying and/or cloning other 8035 and 84242
family members, or fragments thereof, as well as 8035 and 84242
homologues, or fragments thereof, from other species.
[0572] In another embodiment, a nucleic acid includes a nucleotide
sequence that includes part, or all, of the coding regions and
extends into either (or both) the 5' or 3' noncoding regions. Other
embodiments include a fragment that includes a nucleotide sequence
encoding an amino acid fragment described herein. Nucleic acid
fragments can encode a specific domain or site described herein or
fragments thereof, or fragments comprising a specific domain or
site described herein that are at least 150 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 be construed as encompassing
those fragments that may have been disclosed prior to the
invention.
[0573] 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, the nucleic
acid fragment can include a RING finger protein domain and/or an
IBR protein domain. In a preferred embodiment the fragment is at
least, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700,
900, 1100, 1200 or 1300 base pairs in length.
[0574] 8035 and 84242 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:18, SEQ ID NO:20, of SEQ ID NO:22
or SEQ ID NO:24, or of a naturally occurring allelic variant or
mutant of SEQ ID NO:18, SEQ ID NO:20, of SEQ ID NO:22 or SEQ ID
NO:24.
[0575] In a preferred embodiment the nucleic acid is a probe that
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.
[0576] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid that encodes a RING finger
protein domain and/or an IBR protein domain (e.g., about amino acid
residues 380-421 of SEQ ID NO:19 and about amino acid residues 2-67
or 102-133 of SEQ ID NO:23).
[0577] 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 an 8035 or 84242 sequence, e.g., a region
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.
E.g., primers suitable for amplifying all or a portion of any of
the following regions are provided: a RING finger protein domain
(e.g., about amino acid residues 380-421 of SEQ ID NO:19; an IBR
protein domain (e.g., about amino acid residues 2-67 of SEQ ID
NO:23).
[0578] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0579] A nucleic acid fragment encoding a "biologically active
portion of an 8035 or 84242 polypeptide" can be prepared by
isolating a portion of the nucleotide sequence of SEQ ID NO:18, SEQ
ID NO:20, SEQ ID NO:22 or SEQ ID NO:24, that encodes a polypeptide
having an 8035 or 84242 biological activity (e.g., the biological
activities of the 8035 and 84242 proteins as described herein),
expressing the encoded portion of the 8035 or 84242 protein (e.g.,
by recombinant expression in vitro) and assessing the activity of
the encoded portion of the 8035 or 84242 protein. For example, a
nucleic acid fragment encoding a biologically active portion of
8035 includes an RING finger protein domain (e.g., about amino acid
residues 380-421 of SEQ ID NO:19). A nucleic acid fragment encoding
a biologically active portion of an 8035 polypeptide, may comprise
a nucleotide sequence which is greater than 125-1200 or more
nucleotides in length. For example, a nucleic acid fragment
encoding a biologically active portion of 84242 includes an IBR
domain and/or a RING finger protein domain (e.g., about amino acid
residues 2-67 and 102-133 of SEQ ID NO:23, respectively). A nucleic
acid fragment encoding a biologically active portion of an 84242
polypeptide, may comprise a nucleotide sequence which is greater
than 125-1200 or more nucleotides in length.
[0580] In preferred embodiments, nucleic acids include a nucleotide
sequence which is about 125, 150, 200, 300, 500, 600, 700, 800,
900, 1000, 1100, 1200, 1300, or 1302 nucleotides in length in the
case of 8035 and which is about 125, 150, 200, 300, 500, 600, 700,
800, 900, 1000, 1100, 1200, or 1212 nucleotides in length in the
case of 84242 and hybridizes under stringent hybridization
conditions to a nucleic acid molecule of SEQ ID NO:18, SEQ ID
NO:20, SEQ ID NO:22 or SEQ ID NO:24.
55304 Nucleic Acid Fragments
[0581] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO:26 or SEQ ID
NO:28. 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 55304 protein, e.g., an immunogenic or
biologically active portion of a 55304 protein. A fragment can
comprise: nucleotides 803-2845 of SEQ ID NO:26, which encodes an
aminopeptidase domain of human 55304. The nucleotide sequence
determined from the cloning of the 55304 gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning other 55304 family members, or fragments thereof, as
well as 55304 homologues, or fragments thereof, from other
species.
[0582] 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 150 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.
[0583] 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, the nucleic
acid fragment can include an Aminopeptidase domain. In a preferred
embodiment the fragment is at least, 50, 100, 200, 300,400, 500,
600, 700, 800, 900, 1000, or 1500 base pairs in length.
[0584] 55304 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:26 or SEQ ID NO:28, or of a
naturally occurring allelic variant or mutant of SEQ ID NO:26 or
SEQ ID NO:28.
[0585] 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.
[0586] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid which encodes an Aminopeptidase
domain (e.g., about amino acid residues 318-508 of SEQ ID
NO:27).
[0587] 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 55304 sequence, e.g., a region 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 differs by one base from a
sequence disclosed herein or from a naturally occurring variant.
E.g., primers suitable for amplifying all or a portion of any of
the following regions are provided: an Aminopeptidase domain (e.g.,
about amino acid residues 318-508 of SEQ ID NO:27).
[0588] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0589] A nucleic acid fragment encoding a "biologically active
portion of a 55304 polypeptide" can be prepared by isolating a
portion of the nucleotide sequence of SEQ ID NO:26 or SEQ ID NO:28,
which encodes a polypeptide having a 55304 biological activity
(e.g., the biological activities of the 55304 proteins as described
herein), expressing the encoded portion of the 55304 protein (e.g.,
by recombinant expression in vitro) and assessing the activity of
the encoded portion of the 55304 protein. For example, a nucleic
acid fragment encoding a biologically active portion of 55304
includes an aminopeptidase domain (e.g., about amino acid residues
318-508 of SEQ ID NO:27). A nucleic acid fragment encoding a
biologically active portion of a 55304 polypeptide, may comprise a
nucleotide sequence which is greater than 300-1200 or more
nucleotides in length.
[0590] In preferred embodiments, nucleic acids include a nucleotide
sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000,
1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or 2043
nucleotides in length and hybridizes under stringent hybridization
conditions to a nucleic acid molecule of SEQ ID NO:26 or SEQ ID
NO:28.
52999 Nucleic Acid Fragments
[0591] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO:29 or SEQ ID
NO:31. 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 52999 protein, e.g., an immunogenic or
biologically active portion of a 52999 protein. A fragment can
comprise all or a portion of the nucleotides from about nucleotide
253-2086 of SEQ ID NO:29, that encode a polypeptide hydrolytic
domain of human 52999. The nucleotide sequence determined from the
cloning of the 52999 gene allows for the generation of probes and
primers designed for use in identifying and/or cloning other 52999
family members, or fragments thereof, as well as 52999 homologues,
or fragments thereof, from other species.
[0592] 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 150 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.
[0593] A nucleic acid fragment can include a sequence corresponding
to a region or functional site described herein. A nucleic acid
fragment can also include one or more regions or functional sites
described herein. Thus, for example, a nucleic acid fragment can
include a polypeptide hydrolytic domain or a conserved region or
motif. In a preferred embodiment the fragment is at least 50, 100,
200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 or more base pairs
in length.
[0594] 52999 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:29 or SEQ ID NO:31, or of a
naturally occurring allelic variant or mutant of SEQ ID NO:29 or
SEQ ID NO:31.
[0595] 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.
[0596] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid which encodes a portion of an
endopepdidase domain (e.g., about amino acid residues 21-631 of SEQ
ID NO:30).
[0597] 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 52999 sequence, e.g., a region 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 differs by one base from a
sequence disclosed herein or from a naturally occurring variant.
E.g., primers suitable for amplifying all or a portion of any of a
polypeptide hydrolytic domain (e.g., about amino acid residues
21-631 of SEQ ID NO:30).
[0598] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0599] A nucleic acid fragment encoding a "biologically active
portion of a 52999 polypeptide" can be prepared by isolating a
portion of the nucleotide sequence of SEQ ID NO:29 or SEQ ID NO:31,
which encodes a polypeptide having a 52999 biological activity
(e.g., the biological activities of the 52999 proteins as described
herein), expressing the encoded portion of the 52999 protein (e.g.,
by recombinant expression in vitro) and assessing the activity of
the encoded portion of the 52999 protein. For example, a nucleic
acid fragment encoding a biologically active portion of 52999 may
include a polypeptide hydrolytic domain (e.g., about amino acid
residues 21-631 of SEQ ID NO:30). A nucleic acid fragment encoding
a biologically active portion of a 52999 polypeptide, may comprise
a nucleotide sequence that is 300-1800 or more nucleotides in
length.
[0600] In preferred embodiments, nucleic acids include a nucleotide
sequence that is about 300, 400, 500, 600, 700, 800, 900, 1000,
1100, 1200, 1300, 1400, 1600, 1800, 2000, 2200 or 2277 nucleotides
in length and hybridizes under stringent hybridization conditions
to a nucleic acid molecule of SEQ ID NO:29 or SEQ ID NO:31.
21999 Nucleic Acid Fragments
[0601] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO:36. 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
ADP-ribosyltransferase protein, e.g., an immunogenic or
biologically active portion of a ADP-ribosyltransferase protein. A
fragment can comprise nucleotide sequences which code for a portion
of the ADP-ribosyltransferase protein of SEQ ID NO:37 and retains
biological activity. The nucleotide sequence determined from the
cloning of the ADP-ribosyltransferase gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning other ADP-ribosyltransferase family members, or
fragments thereof, as well as ADP-ribosyltransferase homologues, or
fragments thereof, from other species.
[0602] 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 150 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.
[0603] 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. In a preferred embodiment the
fragment is at least, 50, 100, 200, 300, 400, 500, 600, 700, or 900
base pairs in length.
[0604] ADP-ribosyltransferase 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:36, or of
a naturally occurring allelic variant or mutant of SEQ ID
NO:36.
[0605] 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.
[0606] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid which encodes a fragment of SEQ
ID NO:37.
[0607] 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 ADP-ribosyltransferase sequence, e.g., a
region 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 differs by one
base from a sequence disclosed herein or from a naturally occurring
variant. E.g., primers suitable for amplifying all or a portion of
any of the region of SEQ ID NO:37 are provided.
[0608] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0609] A nucleic acid fragment encoding a "biologically active
portion of a ADP-ribosyltransferase polypeptide" can be prepared by
isolating a portion of the nucleotide sequence of SEQ ID NO:36,
which encodes a polypeptide having a ADP-ribosyltransferase
biological activity (e.g., the biological activities of the
ADP-ribosyltransferase proteins as described herein), expressing
the encoded portion of the ADP-ribosyltransferase protein (e.g., by
recombinant expression in vitro) and assessing the activity of the
encoded portion of the ADP-ribosyltransferase protein.
[0610] In preferred embodiments, nucleic acids include a nucleotide
sequence which is about 300, 400, 500, 600, 700, 800, or 879
nucleotides in length and hybridizes under stringent hybridization
conditions to a nucleic acid molecule of SEQ ID NO:36.
52020 Nucleic Acid Fragments
[0611] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO:40 or SEQ ID
NO:42. 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 52020 protein, e.g., an immunogenic or
biologically active portion of a 52020 protein, e.g., such as tumor
rejection antigen. A fragment can comprise: nucleotides 1-208 of
SEQ ID NO:40, which encodes an MAGE domain of human 52020. The
nucleotide sequence determined from the cloning of the 52020 gene
allows for the generation of probes and primers designed for use in
identifying and/or cloning other 52020 family members, or fragments
thereof, as well as 52020 homologues, or fragments thereof, from
other species.
[0612] 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 150 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.
[0613] 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, the nucleic
acid fragment can include an MAGE domain. In a preferred embodiment
the fragment is at least, 50, 100, 200, 300, 400, 500, 600, 700, or
900 base pairs in length.
[0614] 52020 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:40 or SEQ ID NO:42, or of a
naturally occurring allelic variant or mutant of SEQ ID NO:40 or
SEQ ID NO:42.
[0615] 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.
[0616] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid which encodes a MAGE domain
(e.g., about amino acid residues 1-208 of SEQ ID NO:41).
[0617] 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 52020 sequence, e.g., a region 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 differs by one base from a
sequence disclosed herein or from a naturally occurring variant.
E.g., primers suitable for amplifying all or a portion of any of
the following regions are provided: a MAGE domain (e.g., about
amino acid residues 1-208 of SEQ ID NO:41).
[0618] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0619] A nucleic acid fragment encoding a "biologically active
portion of a 52020 polypeptide" can be prepared by isolating a
portion of the nucleotide sequence of SEQ ID NO:40 or SEQ ID NO:42,
which encodes a polypeptide having a 52020 biological activity
(e.g., the biological activities of the 52020 proteins as described
herein), expressing the encoded portion of the 52020 protein (e.g.,
by recombinant expression in vitro) and assessing the activity of
the encoded portion of the 52020 protein. For example, a nucleic
acid fragment encoding a biologically active portion of 52020
includes a MAGE domain (e.g., about amino acid residues 1-208 of
SEQ ID NO:41). A nucleic acid fragment encoding a biologically
active portion of a 52020 polypeptide, may comprise a nucleotide
sequence which is about 300-900 or more nucleotides in length. In
another embodiment a nucleic acid fragment of a 52020
polynucleotide may encode a biologically active polypeptide that
functions as a tumor rejection antigen. This nucleic acid fragment
may comprise a nucleotide sequence of SEQ ID NO:42 consisting of 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or more
nucleic acid residues.
[0620] In other embodiments, the nucleic acid fragments may include
nucleotides 1-100, 100-200, 200-300, 300-400, 400-500, 500-600,
600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200,
1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800,
1800-1900, 1900-2000, 200-2100, or 2100-2183 of SEQ ID NO:40. In
other embodiments, nucleic acids include a nucleotide sequence that
is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,
1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2183 nucleotides
in length and hybridizes under stringent hybridization conditions
to a nucleic acid molecule of SEQ ID NO:40 or SEQ ID NO:42.
Nucleic Acid Variants
[0621] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO:1, SEQ
ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ
ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,
SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID
NO:31, SEQ ID NO:36, SEQ ID NO:40, or SEQ ID NO:42. Such
differences can be due to degeneracy of the genetic code (and
result in a nucleic acid which encodes the same 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
ADP-ribosyltransferase, or 52020 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 than that that is shown in SEQ ID NO:2, SEQ ID NO:7,
SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID
NO:27, SEQ ID NO:30, SEQ ID NO:37 or SEQ ID NO:41. 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.
[0622] Nucleic acids of the invention 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, preferably at least 10%, or 20% of the codons have
been altered such that the sequence is optimized for expression in
E. coli, yeast, human, insect, or CHO cells.
[0623] 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).
[0624] In a preferred embodiment, the nucleic acid differs from
that of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ
ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,
SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:40, or SEQ ID
NO:42, 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 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.
[0625] 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 at least about
95%, 96%, 97%, 98%, 99% or more identical to the amino acid
sequence shown in SEQ ID NO:2 or a fragment of this sequence. Such
nucleic acid molecules can readily be obtained as being able to
hybridize under stringent conditions, to the nucleotide sequence
shown in SEQ ID NO:3 or a fragment of this sequence. Nucleic acid
molecules corresponding to orthologs, homologs, and allelic
variants of the 22406 cDNAs of the invention can further be
isolated by mapping to the same chromosome or locus as the 22406
gene. Preferred variants include those that are correlated with
pyridoxal-phosphate dependent racemase activity.
[0626] Allelic variants of 22406, e.g., human 22406, include both
functional and non-functional proteins. Functional allelic variants
are naturally occurring amino acid sequence variants of the 22406
protein within a population that maintain the ability to form
D-serine from L-serine. Functional allelic variants will typically
contain only conservative substitution of one or more amino acids
of SEQ ID NO:2, or substitution, deletion or insertion of
non-critical residues in non-critical regions of the protein.
Non-functional allelic variants are naturally-occurring amino acid
sequence variants of the 22406, e.g., human 22406, protein within a
population that do not have the ability to form D-serine from
L-serine. Non-functional allelic variants will typically contain a
non-conservative substitution, a deletion, or insertion, or
premature truncation of the amino acid sequence of SEQ ID NO:2, or
a substitution, insertion, or deletion in critical residues or
critical regions of the protein.
[0627] 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 amino acid sequence shown in SEQ ID NO:7 or a
fragment of this sequence. Nucleic acid molecules corresponding to
orthologs, homologs, and allelic variants of the acyltransferase
cDNAs of the invention can further be isolated by mapping to the
same chromosome or locus as the acyltransferase gene. Preferred
variants include those that are correlated with fatty acid synthase
activity.
[0628] Allelic variants of acyltransferase, e.g., human
acyltransferase, include both functional and non-functional
proteins. Functional allelic variants are naturally occurring amino
acid sequence variants of the acyltransferase protein within a
population that maintain the ability to modulate the
phosphorylation state of itself or another protein or polypeptide.
Functional allelic variants will typically contain only
conservative substitution of one or more amino acids of SEQ ID
NO:7, 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 acyltransferase, e.g., human acyltransferase,
protein within a population that do not have the ability to attach
an acyl chain to a lipid precursor. 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:7, or a substitution, insertion, or deletion in critical
residues or critical regions of the protein.
[0629] 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 amino acid sequence shown in SEQ ID NO:11 or a
fragment of this sequence. Such nucleic acid molecules can readily
be obtained as being able to hybridize under stringent conditions,
to the nucleotide sequence shown in SEQ ID NO:12 or a fragment of
this sequence. Nucleic acid molecules corresponding to orthologs,
homologs, and allelic variants of the 7716 cDNAs of the invention
can further be isolated by mapping to the same chromosome or locus
as the 7716 gene. Preferred variants include those that are
correlated with ATPase activity.
[0630] Allelic variants of 7716, e.g., human 7716, include both
functional and non-functional proteins. Functional allelic variants
are naturally occurring amino acid sequence variants of the 7716
protein within a population that maintain the ability to hydrolyze
ATP. Functional allelic variants will typically contain only
conservative substitution of one or more amino acids of SEQ ID
NO:11, 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 7716, e.g., human 7716, protein within a population
that do not have the ability to hydrolyze ATP. 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:11, or a substitution,
insertion, or deletion in critical residues or critical regions of
the protein.
[0631] 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%, 65%, preferably about 75%, 85%, 95%, or 98% identical to
SEQ ID NO:15 or a fragment of this sequence. Such nucleic acid
molecules can readily be obtained as being able to hybridize under
stringent conditions, to the nucleotide sequence shown in SEQ ID
NO:16 or a fragment of this sequence. Nucleic acid molecules
corresponding to orthologs, homologs, and allelic variants of the
25233 cDNAs of the invention can further be isolated by mapping to
the same chromosome or locus as the 25233 gene. Preferred variants
include those that are correlated with aminotransferase
activity.
[0632] Allelic variants of 25233, e.g., human 25233, include both
functional and non-functional proteins. Functional allelic variants
are naturally occurring amino acid sequence variants of the 25233
protein within a population that maintain the ability to catalyze
transamination, decarboxylation, deamination, racemization, or
aldol cleavage reactions. Functional allelic variants will
typically contain only conservative substitution of one or more
amino acids of SEQ ID NO:15, 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 25233, e.g., human 25233, protein within a
population that do not have the ability to catalyze transamination,
decarboxylation, deamination, racemization, or aldol cleavage
reactions. 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:15, or
a substitution, insertion, or deletion in critical residues or
critical regions of the protein.
[0633] 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 amino acid sequence shown in SEQ ID NO:19 or SEQ
ID NO:23, or fragments of these sequences. Such nucleic acid
molecules can readily be obtained as being able to hybridize under
stringent conditions, to the nucleotide sequence shown in SEQ ID
NO:20 or SEQ ID NO:24, or fragments of these sequences. Nucleic
acid molecules corresponding to orthologs, homologs, and allelic
variants of the 8035 and 84242 cDNAs of the invention can further
be isolated by mapping to the same chromosome or locus as the 8035
or 84242 gene. Preferred variants include those that are correlated
with RING finger protein activity (E3 ubiquitin ligase activity),
e.g. variants that comprise nucleotide sequences encoding
polypeptides that share identity to the amino acid sequence shown
in SEQ ID NO:19 or SEQ ID NO:23 or a fragment of these sequences
retain RING finger protein activity (E3 ubiquitin ligase
activity).
[0634] Allelic variants of 8035 and 84242, e.g., human 8035 and
84242, include both functional and non-functional proteins.
Functional allelic variants are naturally occurring amino acid
sequence variants of the 8035 and 84242 proteins within a
population that maintain the ability to function as E3 ubiquitin
ligases. Functional allelic variants will typically contain only
conservative substitution of one or more amino acids of SEQ ID
NO:19 or SEQ ID NO:23 or substitution, deletion, or insertion of
non-critical residues in non-critical regions of these proteins.
Non-functional allelic variants are naturally-occurring amino acid
sequence variants of 8035 and 84242, e.g., human 8035 and 84242,
proteins within a population that do not have the ability function
as E3 ubiquitin ligases. 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:19 or SEQ ID NO:23, or a substitution, insertion, or
deletion in critical residues or critical regions of these
proteins.
[0635] 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 amino acid sequence shown in SEQ ID NO:27 or a
fragment of this sequence. Such nucleic acid molecules can readily
be obtained as being able to hybridize under stringent conditions,
to the nucleotide sequence shown in SEQ ID NO:28 or a fragment of
this sequence. Nucleic acid molecules corresponding to orthologs,
homologs, and allelic variants of the 55304 cDNAs of the invention
can further be isolated by mapping to the same chromosome or locus
as the 55304 gene. Preferred variants include those that are
correlated with aminopeptidase activity.
[0636] Allelic variants of 55304, e.g., human 55304, include both
functional and non-functional proteins. Functional allelic variants
are naturally occurring amino acid sequence variants of the 55304
protein within a population that maintain the ability to hydrolyse
the amino terminal amino acid from a peptide substrate. Functional
allelic variants will typically contain only conservative
substitution of one or more amino acids of SEQ ID NO:27, 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 55304, e.g., human 55304, protein within a population that do
not have the ability to remove the amino terminal amino acid from a
peptide substrate. 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:27,
or a substitution, insertion, or deletion in critical residues or
critical regions of the protein.
[0637] 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 amino acid sequence shown in SEQ ID NO:30 or a
fragment of this sequence. Such nucleic acid molecules can readily
be obtained as being able to hybridize under stringent conditions,
to the nucleotide sequence shown in SEQ ID NO:31 or a fragment of
this sequence. Nucleic acid molecules corresponding to orthologs,
homologs, and allelic variants of the 52999 cDNAs of the invention
can further be isolated by mapping to the same chromosome or locus
as the 52999 gene. Preferred variants include those that are
correlated with metallopeptidase activity, e.g. variants that
comprise nucleotide sequences encoding polypeptides that share
identity to the amino acid sequence shown in SEQ ID NO:30 or a
fragment of this sequence retain metallopeptidase activity.
[0638] Allelic variants of 52999, e.g., human 52999, include both
functional and non-functional proteins. Functional allelic variants
are naturally occurring amino acid sequence variants of the 52999
protein within a population that maintain polypeptide hydrolytic
activity. Functional allelic variants will typically contain only
conservative substitution of one or more amino acids of SEQ ID
NO:30, 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 52999, e.g., human 52999, protein within a
population that do not have the ability to catalyze the cleavage of
polypeptide bonds. 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:30,
or a substitution, insertion, or deletion in critical residues or
critical regions of the protein.
[0639] 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 amino acid sequence shown in SEQ ID NO:37 or a
fragment of this sequence. Such nucleic acid molecules can readily
be obtained as being able to hybridize under stringent conditions,
to the nucleotide sequence shown in SEQ ID NO:36 or a fragment of
this sequence. Nucleic acid molecules corresponding to orthologs,
homologs, and allelic variants of the ADP-ribosyltransferase cDNAs
of the invention can further be isolated by mapping to the same
chromosome or locus as the ADP-ribosyltransferase gene.
[0640] Allelic variants of ADP-ribosyltransferase, e.g., human
ADP-ribosyltransferase, include both functional and non-functional
proteins. Functional allelic variants are naturally occurring amino
acid sequence variants of the ADP-ribosyltransferase protein within
a population that maintain the ability to transfer an ADP-ribose
moiety to an acceptor protein. Functional allelic variants will
typically contain only conservative substitution of one or more
amino acids of SEQ ID NO:37, 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 ADP-ribosyltransferase, e.g., human
ADP-ribosyltransferase, protein within a population that do not
have the ability to transfer an ADP-ribose moiety to an acceptor
protein. 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:37, or
a substitution, insertion, or deletion in critical residues or
critical regions of the protein.
[0641] 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% or 75%, more typically at
least about 80% or 85%, and most typically at least about 90%, 95%,
96%, 97%, 98% or 94% or more identical to the amino acid sequence
shown in SEQ ID NO:41 or a fragment of this sequence. Such nucleic
acid molecules can readily be obtained as being able to hybridize
under stringent conditions, to the nucleotide sequence shown in SEQ
ID NO:42 or a fragment of this sequence. Nucleic acid molecules
corresponding to orthologs, homologs, and allelic variants of the
52020 cDNAs of the invention can further be isolated by mapping to
the same chromosome or locus as the 52020 gene. Preferred variants
include those that are correlated with MAGE activity.
[0642] Allelic variants of 52020, e.g., human 52020, include both
functional and non-functional proteins. Functional allelic variants
are naturally occurring amino acid sequence variants of the 52020
protein within a population that maintain the activity of 52020
MAGE-like protein as described herein and possess the amino acid
sequence conservation with SEQ ID NO:41 as described in the
previous paragraph. Functional allelic variants will typically
contain only conservative substitution of one or more amino acids
of SEQ ID NO:41, 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 52020, e.g., human 52020, protein within a
population that do not retain 52020 MAGE-like protein activity.
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:41, or
a substitution, insertion, or deletion in critical residues or
critical regions of the protein.
[0643] Moreover, nucleic acid molecules encoding other 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
ADP-ribosyltransferase, or 52020 family members and, thus, which
have a nucleotide sequence which differs from the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
ADP-ribosyltransferase, or 52020 sequences of SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31,
SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:42, are intended to be within
the scope of the invention.
Antisense Nucleic Acid Molecules, Ribozymes and Modified 22406,
Acyltransferase, 7716, 25233, 8035. 84242, 55304, 52999, 21999, or
52020 Nucleic Acid Molecules
[0644] In another aspect, the invention features, an isolated
nucleic acid molecule which is antisense to 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020. 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 coding strand, or to only a portion thereof (e.g., the coding
region of human 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 corresponding to SEQ ID NO:3, SEQ ID
NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ
ID NO:28, SEQ ID NO:31, SEQ ID NO:38, SEQ ID NO:42, respectively).
In another embodiment, the antisense nucleic acid molecule is
antisense to a "noncoding region" of the coding strand of a
nucleotide sequence encoding 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 (e.g., the 5' and 3'
untranslated regions).
[0645] An antisense nucleic acid can be designed such that it is
complementary to the entire coding region of 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 mRNA, but more preferably is an oligonucleotide which is
antisense to only a portion of the coding or noncoding region of
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 mRNA. For example, the antisense oligonucleotide
can be complementary to the region surrounding the translation
start site of 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 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.
[0646] 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).
[0647] 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein to thereby inhibit expression of the protein, e.g.,
by inhibiting transcription and/or translation. Alternatively,
antisense nucleic acid molecules can be modified to target selected
cells and then administered systemically. For systemic
administration, antisense molecules can be modified such that they
specifically bind to receptors or antigens expressed on a selected
cell surface, e.g., by linking the antisense nucleic acid molecules
to peptides or antibodies 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.
[0648] 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).
[0649] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. A ribozyme having specificity for a
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020-encoding nucleic acid can include one or more
sequences complementary to the nucleotide sequence of a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 cDNA disclosed herein (i.e., the sequence of SEQ ID NO:1, SEQ
ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ
ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,
SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID
NO:31, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42),
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 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020-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, 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 mRNA can be used
to select a catalytic RNA having a specific ribonuclease activity
from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.
W. (1993) Science 261:1411-1418.
[0650] 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 (e.g., the 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 promoter
and/or enhancers) to form triple helical structures that prevent
transcription of the 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 gene in target cells. See
generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;
Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher,
L. J. (1992) Bioassays 14(12):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.
[0651] The invention also provides detectably labeled
oligonucleotide primer and probe molecules. Typically, such labels
are chemiluminescent, fluorescent, radioactive, or
colorimetric.
[0652] A 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 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 B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1):
5-23). 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 B. et al. (1996)
supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci.
93:14670-675.
[0653] PNAs of 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 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 B. (1996) supra)); or as probes or primers for DNA
sequencing or hybridization (Hyrup B. et al. (1996) supra;
Perry-O'Keefe supra).
[0654] In other embodiments, the oligonucleotide may 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. W088/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. W089/10134). 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 may be conjugated to another molecule, (e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, or hybridization-triggered cleavage agent).
[0655] The invention also includes molecular beacon oligonucleotide
primer and probe molecules having at least one region which is
complementary to a 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 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.
Isolated 22406 Polypeptides
[0656] In another aspect, the invention features, an isolated 22406
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-22406 antibodies. 22406 protein can be isolated from
cells or tissue sources using standard protein purification
techniques. 22406 protein or fragments thereof can be produced by
recombinant DNA techniques or synthesized chemically.
[0657] 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 postranslational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same postranslational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of postranslational modifications, e.g., glycosylation or
cleavage, present when expressed in a native cell.
[0658] In a preferred embodiment, a 22406 polypeptide has one or
more of the following characteristics:
[0659] (i) it has the ability to form D-serine from L-serine;
[0660] (ii) it has a molecular weight, e.g., a deduced molecular
weight, amino acid composition or other physical characteristic of
the polypeptide of SEQ ID NO:2;
[0661] (iii) it has an overall sequence identity of at least 95%,
96%, 97%, 98%, or 99%, with a polypeptide of SEQ ID NO:2;
[0662] (iv) it has an pyridoxal-phosphate dependent enzyme family
member domain which preferably has an overall sequence identity of
about 70%, 80%, 90% or 95% with amino acid residues 19-315 of SEQ
ID NO:2;
[0663] (v) it has a pyridoxal-phosphate attachment site conserved
sequence as described herein; and
[0664] (vi) it has at least 70%, preferably 80%, and most
preferably 95% of the cysteines found amino acid sequence of the
native protein.
[0665] In a preferred embodiment the 22406 protein, or fragment
thereof, differs from the corresponding sequence in SEQ ID NO:2. 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 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. (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 pyridoxal-phosphate dependent enzyme family member domain. In
another preferred embodiment one or more differences are in
non-active site residues, e.g. outside of the pyridoxal-phosphate
dependent enzyme family member domain.
[0666] Other embodiments include a protein that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such 22406 proteins
differ in amino acid sequence from SEQ ID NO:2, yet retain
biological activity.
[0667] In one embodiment, a biologically active portion of a 22406
protein includes an pyridoxal-phosphate dependent enzyme family
member domain. In another embodiment, a biologically active portion
of a 22406 protein includes a pyridoxal-phosphate attachment site
domain. 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 22406 protein.
[0668] In a preferred embodiment, the 22406 protein has an amino
acid sequence shown in SEQ ID NO:2. In other embodiments, the 22406
protein is substantially identical to SEQ ID NO:2. In yet another
embodiment, the 22406 protein is substantially identical to SEQ ID
NO:2 and retains the functional activity of the protein of SEQ ID
NO:2, as described in detail above. Accordingly, in another
embodiment, the 22406 protein is a protein which includes an amino
acid sequence at least about 95%, 96%, 97%, 98%, 99%, or more
identical to SEQ ID NO:2.
Isolated Acyltransferase Polypeptides
[0669] In another aspect, the invention features, an isolated
acyltransferase 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-acyltransferase antibodies.
Acyltransferase protein can be isolated from cells or tissue
sources using standard protein purification techniques.
Acyltransferase protein or fragments thereof can be produced by
recombinant DNA techniques or synthesized chemically.
[0670] 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 postranslational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same postranslational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of postranslational modifications, e.g., glycosylation or
cleavage, present when expressed in a native cell.
[0671] In a preferred embodiment, an acyltransferase polypeptide
has one or more of the following characteristics:
[0672] (i) it is involved in transfer of an acyl group from Acyl-Co
onto a substrate;
[0673] (ii) it has a molecular weight, e.g., a deduced molecular
weight, amino acid composition or other physical characteristic of
the polypeptide of SEQ ID NO:7;
[0674] (iii) it has an overall sequence similarity of at least 50%,
preferably at least 60%, more preferably at least 70, 80, 90, or
95%, with a polypeptide of SEQ ID NO:7;
[0675] (iv) it has an acyltransferase active site which preferably
has an overall sequence similarity of about 70%, 80%, 90% or 95%
with amino acid residues 200-216 of SEQ ID NO:7;
[0676] In a preferred embodiment the acyltransferase protein, or
fragment thereof, differs from the corresponding sequence in SEQ ID
NO:7. 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:7 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:7. (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 fatty acid synthase domain. In another preferred embodiment one
or more differences are in non-active site residues, e.g. outside
of the fatty acid synthase domain.
[0677] Other embodiments include a protein that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such acyltransferase
proteins differ in amino acid sequence from SEQ ID NO:7, yet retain
biological activity.
[0678] In one embodiment, a biologically active portion of an
acyltransferase protein includes an fatty acid synthase domain. In
another embodiment, a biologically active portion of an
acyltransferase protein includes a domain. 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
acyltransferase protein.
[0679] In a preferred embodiment, the acyltransferase protein has
an amino acid sequence shown in SEQ ID NO:7. In other embodiments,
the acyltransferase protein is substantially identical to SEQ ID
NO:7. In yet another embodiment, the acyltransferase protein is
substantially identical to SEQ ID NO:7 and retains the functional
activity of the protein of SEQ ID NO:7, as described in detail
above. Accordingly, in another embodiment, the acyltransferase
protein is a protein which includes an amino acid sequence at least
about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical
to SEQ ID NO:7.
Isolated 7716 Polypeptides
[0680] In another aspect, the invention features, an isolated 7716
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-7716 antibodies. 7716 protein can be isolated from cells
or tissue sources using standard protein purification techniques.
7716 protein or fragments thereof can be produced by recombinant
DNA techniques or synthesized chemically.
[0681] 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 postranslational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same postranslational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of postranslational modifications, e.g., glycosylation or
cleavage, present when expressed in a native cell.
[0682] In a preferred embodiment, a 7716 polypeptide has one or
more of the following characteristics:
[0683] (i) it hydrolyzes ATP;
[0684] (ii) it has a molecular weight, e.g., a deduced molecular
weight, amino acid composition or other physical characteristic of
the polypeptide of SEQ ID NO:11;
[0685] (iii) it has an overall sequence identity of at least 50%,
preferably at least 60%, more preferably at least 70, 80, 90, or
95%, with a polypeptide of SEQ ID NO:11;
[0686] (iv) it has an ATPase domain which preferably has an overall
sequence identity of about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% with amino acid residues 236-421 or 500-705 of
SEQ ID NO:11;
[0687] (v) it has at least 70%, preferably 80%, and most preferably
95% of the cysteines found amino acid sequence of the native
protein.
[0688] In a preferred embodiment the 7716 protein, or fragment
thereof, differs from the corresponding sequence in SEQ ID NO:11.
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:11 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:11. (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 ATPase domain. In another preferred
embodiment one or more differences are in non-active site residues,
e.g. outside of the ATPase domain.
[0689] Other embodiments include a protein that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such 7716 proteins
differ in amino acid sequence from SEQ ID NO:11, yet retain
biological activity.
[0690] In one embodiment, a biologically active portion of a 7716
protein includes an ATPase domain. In another embodiment, a
biologically active portion of a 7716 protein includes a ATP
Binding Protease/ATP-dependent cell division protein domain.
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 7716 protein.
[0691] In a preferred embodiment, the 7716 protein has an amino
acid sequence shown in SEQ ID NO:11. In other embodiments, the 7716
protein is substantially identical to SEQ ID NO:11. In yet another
embodiment, the 7716 protein is substantially identical to SEQ ID
NO:11 and retains the functional activity of the protein of SEQ ID
NO:11, as described in detail above. Accordingly, in another
embodiment, the 7716 protein is a protein which includes an amino
acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to
SEQ ID NO:11.
Isolated 25233 Polypeptides
[0692] In another aspect, the invention features, an isolated 25233
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-25233 antibodies. 25233 protein can be isolated from
cells or tissue sources using standard protein purification
techniques. 25233 protein or fragments thereof can be produced by
recombinant DNA techniques or synthesized chemically.
[0693] 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 postranslational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same postranslational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of postranslational modifications, e.g., glycosylation or
cleavage, present when expressed in a native cell.
[0694] In a preferred embodiment, a 25233 polypeptide has one or
more of the following characteristics:
[0695] (i) it is capable of catalyzing a transamination,
decarboxylation, deamination, racemization, or aldol cleavage
reaction;
[0696] (ii) it has a molecular weight, e.g., a deduced molecular
weight, amino acid composition or other physical characteristic of
the polypeptide of SEQ ID NO:15;
[0697] (iii) it has an overall sequence identity of at least 50%,
preferably at least 60%, more preferably at least 70, 80, 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99%, with a polypeptide of SEQ ID
NO:15;
[0698] (iv) it has an aminotransferase domain which preferably has
an overall sequence identity of about 70%, 80%, 90% or 95% with
amino acid residues 83-517 of SEQ ID NO:15;
[0699] (v) it has at least 70%, preferably 80%, and most preferably
95% of the cysteines found in the amino acid sequence of the native
protein.
[0700] In a preferred embodiment the 25233 protein, or fragment
thereof, differs from the corresponding sequence in SEQ ID NO:15.
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:15 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:15. (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 aminotransferase domain. In another
preferred embodiment one or more differences are in non-active site
residues, e.g. outside of the aminotransferase domain.
[0701] Other embodiments include a protein that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such 25233 proteins
differ in amino acid sequence from SEQ ID NO:15, yet retain
biological activity.
[0702] In one embodiment, a biologically active portion of a 25233
protein includes an aminotransferase domain. 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
25233 protein.
[0703] In a preferred embodiment, the 25233 protein has an amino
acid sequence shown in SEQ ID NO:15. In other embodiments, the
25233 protein is substantially identical to SEQ ID NO:15. In yet
another embodiment, the 25233 protein is substantially identical to
SEQ ID NO:15 and retains the functional activity of the protein of
SEQ ID NO:15, as described in detail above. Accordingly, in another
embodiment, the 25233 protein is a protein which includes an amino
acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to
SEQ ID NO:15.
Isolated 8035 and 84242 Polypeptides
[0704] In another aspect, the invention features, an isolated 8035
or 84242 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-8035 or anti-84242 antibodies. 8035 and
84242 protein can be isolated from cells or tissue sources using
standard protein purification techniques. 8035 and 84242 protein or
fragments thereof can be produced by recombinant DNA techniques or
synthesized chemically.
[0705] 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 postranslational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same postranslational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of postranslational modifications, e.g., glycosylation or
cleavage, present when expressed in a native cell.
[0706] In a preferred embodiment, an 8035 or 84242 polypeptide has
one or more of the following characteristics:
[0707] (i) it functions as an E3 ubiquitin ligase;
[0708] (ii) it has a molecular weight, e.g., a deduced molecular
weight, amino acid composition or other physical characteristic of
the polypeptide of SEQ ID NO:19 or SEQ ID NO:23;
[0709] (iii) it has an overall sequence identity of at least 50%,
preferably at least 60%, more preferably at least 70, 80, 90, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, with a polypeptide of
SEQ ID NO:19 or SEQ ID NO:23;
[0710] (iv) it has at least one RING finger protein domain and/or
an IBR protein domain which preferably have an overall sequence
identity of about 70%, 80%, 90% or 95% with amino acid residues
380-421 of SEQ ID NO:19 (RING), amino acid residues 2-67 of SEQ ID
NO:23 (IBR), or amino acid residues 102-133 of SEQ ID NO:23
(RING);
[0711] (v) it has at least 70%, preferably 80%, and most preferably
95% of the cysteines found in the amino acid sequence of the native
proteins.
[0712] In a preferred embodiment the 8035 or 84242 protein, or
fragments thereof, differs from the corresponding sequence in SEQ
ID NO:19 or SEQ ID NO:23, respectively. In one embodiment it
differs by at least 1 but by less than 15, 10 or 5 amino acid
residues. In another it differs from the corresponding sequence in
SEQ ID NO:19 or SEQ ID NO:23 by at least 1 residue but less than
20%, 15%, 10% or 5% of the residues in it differ from the
corresponding sequence in SEQ ID NO:19 or SEQ ID NO:23. (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. RING finger
protein domain. In another preferred embodiment one or more
differences are in non-active site residues, e.g. outside of the
RING finger protein domain.
[0713] Other embodiments include proteins that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such 8035 and 84242
proteins differ in amino acid sequence from SEQ ID NO:19 and SEQ ID
NO:23, yet retain biological activity.
[0714] In one embodiment, a biologically active portion of an 8035
or 84242 protein includes a RING finger protein domain. In another
embodiment, a biologically active portion of an 8035 or 84242
protein includes an IBR protein domain and a RING finger protein
domain. Moreover, other biologically active portions, in which
other regions of the proteins are deleted, can be prepared by
recombinant techniques and evaluated for one or more of the
functional activities of a native 8035 or 84242 protein.
[0715] In a preferred embodiment, the 8035 or 84242 protein has an
amino acid sequence shown in SEQ ID NO:19 or SEQ ID NO:23,
respectively. In other embodiments, the 8035 or 84242 protein is
substantially identical to SEQ ID NO:19 or SEQ ID NO:23,
respectively. In yet another embodiment, the 8035 or 84242 protein
is substantially identical to SEQ ID NO:19 or SEQ ID NO:23 and
retains the functional activity of the protein of SEQ ID NO:19 or
SEQ ID NO:23, respectively, as described in detail above.
Accordingly, in another embodiment, the 8035 or 84242 protein is a
protein which includes an amino acid sequence at least about 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or more identical to SEQ ID NO:19 or SEQ ID NO:23,
respectively.
Isolated 55304 Polypeptides
[0716] In another aspect, the invention features, an isolated 55304
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-55304 antibodies. 55304 protein can be isolated from
cells or tissue sources using standard protein purification
techniques. 55304 protein or fragments thereof can be produced by
recombinant DNA techniques or synthesized chemically.
[0717] 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 postranslational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same postranslational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of postranslational modifications, e.g., glycosylation or
cleavage, present when expressed in a native cell.
[0718] In a preferred embodiment, a 55304 polypeptide has one or
more of the following characteristics:
[0719] (i) it catalyzes the removal of an amino terminal amino acid
from a peptide substrate;
[0720] (ii) it has a molecular weight, e.g., a deduced molecular
weight, amino acid composition or other physical characteristic of
the polypeptide of SEQ ID NO:27;
[0721] (iii) it has an overall sequence identity of at least 50%,
preferably at least 60%, more preferably at least 70, 80, 90, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, with a polypeptide of
SEQ ID NO:27 as determined using the preferred algorithm described
elsewhere herein;
[0722] (iv) it has an aminopeptidase domain which preferably has an
overall sequence identity of about 68%, 75%, 81%, 87.5% or 93% with
amino acid residues 318, 336, 344, 346, 363, 374, 397, 399, 410,
411, 412, 425, 448, 450, 489, and 508 of SEQ ID NO:27, i.e. 11, 12,
13, 14, or 15 of these amino acids are conserved between the 5304
protein and the corresponding residues of the amino acid sequence
set forth in SEQ ID NO:27;
[0723] (v) it has at least 70%, preferably 80%, and most preferably
95% of the cysteines found amino acid sequence of the native
protein.
[0724] In a preferred embodiment the 55304 protein, or fragment
thereof, differs from the corresponding sequence in SEQ ID NO:27.
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:27 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:27. (If this comparison
requires alignment the sequences should be aligned for maximum
homology, e.g. by the GAP algorithm described elsewhere herein.
Gapped 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 aminopeptidase domain. In another preferred embodiment one or
more differences are in non-active site residues, e.g. outside of
the aminopeptidase domain.
[0725] Other embodiments include a protein that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such 55304 proteins
differ in amino acid sequence from SEQ ID NO:27, yet retain
biological activity.
[0726] In one embodiment, a biologically active portion of a 55304
protein includes an Aminopeptidase domain. 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
55304 protein.
[0727] In a preferred embodiment, the 55304 protein has an amino
acid sequence shown in SEQ ID NO:27. In other embodiments, the
55304 protein is substantially identical to SEQ ID NO:27. In yet
another embodiment, the 55304 protein is substantially identical to
SEQ ID NO:27 and retains the functional activity of the protein of
SEQ ID NO:27, as described in detail above. Accordingly, in another
embodiment, the 55304 protein is a protein which includes an amino
acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to
SEQ ID NO:27.
Isolated 52999 Polypeptides
[0728] In another aspect, the invention features, an isolated 52999
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-52999 antibodies. 52999 protein can be isolated from
cells or tissue sources using standard protein purification
techniques. 52999 protein or fragments thereof can be produced by
recombinant DNA techniques or synthesized chemically.
[0729] 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 postranslational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same postranslational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of postranslational modifications, e.g., glycosylation or
cleavage, present when expressed in a native cell.
[0730] In a preferred embodiment, a 52999 polypeptide has one or
more of the following characteristics:
[0731] (i) it is capable of catalyzing the cleavage of a
polypeptide through hydrolysis;
[0732] (ii) it has a molecular weight, e.g., a deduced molecular
weight, amino acid composition or other physical characteristic of
the polypeptide of SEQ ID NO:30;
[0733] (iii) it has an overall sequence identity of at least 50%,
preferably at least 60%, more preferably at least 70, 80, 90, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, with a polypeptide of
SEQ ID NO:30;
[0734] (iv) it has a zinc-binding signature sequence that
preferably has an overall sequence identity of about 70%, 80%, 90%,
or 95% or more with amino acid residues 272-281 of SEQ ID
NO:30;
[0735] (v) it has at least 70%, preferably 80%, and most preferably
95% of the cysteines found amino acid sequence of the native
protein.
[0736] In a preferred embodiment the 52999 protein, or fragment
thereof, differs from the corresponding sequence in SEQ ID NO:30.
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:30 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:30. (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 metallopeptidase domain. In another
preferred embodiment one or more differences are in non-active site
residues, e.g. outside of the metallopeptidase domain.
[0737] Other embodiments include a protein that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such 52999 proteins
differ in amino acid sequence from SEQ ID NO:30, yet retain
biological activity.
[0738] In one embodiment, a biologically active portion of a 52999
protein includes a polypeptide hydrolytic domain. In another
embodiment, a biologically active portion of a 52999 protein
includes a portion of the polypeptide hydrolytic domain that
includes the zinc-binding signature sequence. Moreover, other
biologically active portions, in which other regions of the protein
are deleted, can be prepared by recombinant techniques and
evaluated for the functional activities of a native 52999
protein.
[0739] In a preferred embodiment, the 52999 protein has an amino
acid sequence shown in SEQ ID NO:30. In other embodiments, the
52999 protein is substantially identical to SEQ ID NO:30. In yet
another embodiment, the 52999 protein is substantially identical to
SEQ ID NO:30 and retains the functional activity of the protein of
SEQ ID NO:30, as described in detail above. Accordingly, in another
embodiment, the 52999 protein is a protein which includes an amino
acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to
SEQ ID NO:30.
Isolated 21999 Polypeptides
[0740] In another aspect, the invention features, an isolated
ADP-ribosyltransferase 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-ADP-ribosyltransferase antibodies.
ADP-ribosyltransferase protein can be isolated from cells or tissue
sources using standard protein purification techniques.
ADP-ribosyltransferase protein or fragments thereof can be produced
by recombinant DNA techniques or synthesized chemically.
[0741] 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 postranslational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same postranslational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of postranslational modifications, e.g., glycosylation or
cleavage, present when expressed in a native cell.
[0742] In a preferred embodiment, a ADP-ribosyltransferase
polypeptide has one or more of the following characteristics:
[0743] (i) can act to transfer an ADP-ribose moiety of NAD to an
acceptor protein;
[0744] (ii) it has a molecular weight, e.g., a deduced molecular
weight, amino acid composition or other physical characteristic of
the polypeptide of SEQ ID NO:37;
[0745] (iii) it has an overall sequence identity of at least 50%,
preferably at least 60%, more preferably at least 70, 80, 90, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, with a polypeptide of
SEQ ID NO:37.
[0746] In a preferred embodiment the ADP-ribosyltransferase
protein, or fragment thereof, differs from the corresponding
sequence in SEQ ID NO:37. 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:37 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:37. (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 transferase
domain. In another preferred embodiment one or more differences are
in non-active site residues, e.g. outside of the transferase
domain.
[0747] Other embodiments include a protein that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such
ADP-ribosyltransferase proteins differ in amino acid sequence from
SEQ ID NO:37, yet retain biological activity.
[0748] In one embodiment, a biologically active portion of a
ADP-ribosyltransferase protein includes a transferase domain.
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 ADP-ribosyltransferase protein.
[0749] In a preferred embodiment, the ADP-ribosyltransferase
protein has an amino acid sequence shown in SEQ ID NO:37. In other
embodiments, the ADP-ribosyltransferase protein is substantially
identical to SEQ ID NO:37. In yet another embodiment, the
ADP-ribosyltransferase protein is substantially identical to SEQ ID
NO:37 and retains the functional activity of the protein of SEQ ID
NO:37, as described in detail above. Accordingly, in another
embodiment, the ADP-ribosyltransferase protein is a protein which
includes an amino acid sequence at least about 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more
identical to SEQ ID NO:37.
Isolated 52020 Polypeptides
[0750] In another aspect, the invention features, an isolated 52020
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-52020 antibodies. 52020 protein can be isolated from
cells or tissue sources using standard protein purification
techniques. 52020 protein or fragments thereof can be produced by
recombinant DNA techniques or synthesized chemically.
[0751] 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 postranslational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same postranslational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of postranslational modifications, e.g., glycosylation or
cleavage, present when expressed in a native cell.
[0752] In a preferred embodiment, a 52020 polypeptide has one or
more of the following characteristics:
[0753] (i) it is capable of regulating cellular and
differentiation, tissue repair, activating T-cells, effective CTL
cell function, and eliciting auto-antibodies (ii) it has a
molecular weight, e.g., a deduced molecular weight, amino acid
composition or other physical characteristic of the polypeptide of
SEQ ID NO:41;
[0754] (iii) it has an overall sequence identity of at least 50%,
preferably at least 60%, more preferably at least 70%, 80%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, with a polypeptide
of SEQ ID NO:41;
[0755] (iv) it has a MAGE domain as defined by the consensus amino
acid sequence shown in FIG. 35 which preferably has an overall
sequence identity of about 60%, 70%, 80%, 90% or 95% with amino
acid residues 1-208 of SEQ ID NO:41;
[0756] (v) it has at least 70%, preferably 80%, and most preferably
95% of the cysteines found amino acid sequence of the native
protein.
[0757] In a preferred embodiment the 52020 protein, or fragment
thereof, differs from the corresponding sequence in SEQ ID NO:41.
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:41 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:41. (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 MAGE domain. In another preferred
embodiment one or more differences are in residues without
activity.
[0758] Other embodiments include a protein that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such 52020 proteins
differ in amino acid sequence from SEQ ID NO:41, yet retain
biological activity.
[0759] In one embodiment, a biologically active portion of a 52020
protein includes an MAGE domain. In another embodiment, a
biologically active portion of a 52020 protein includes amino acid
residues capable of being processed to function as tumor rejection
antigens. 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 52020 protein.
[0760] In a preferred embodiment, the 52020 protein has an amino
acid sequence shown in SEQ ID NO:41. In other embodiments, the
52020 protein is substantially identical to SEQ ID NO:41. In yet
another embodiment, the 52020 protein is substantially identical to
SEQ ID NO:41 and retains the functional activity of the protein of
SEQ ID NO:41, as described in detail herein supra. Accordingly, in
another embodiment, the 52020 protein is a protein which includes
an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical
to SEQ ID NO:41.
22406, Acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 Chimeric or Fusion Proteins
[0761] In another aspect, the invention provides 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 chimeric or fusion proteins. As used herein, a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 "chimeric protein" or "fusion protein" includes a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 polypeptide linked to a non-22406, non-acyltransferase,
non-7716, non-25233, non-8035, non-84242, non-55304, non-52999,
non-21999 or non-52020 polypeptide. A "non-22406,
non-acyltransferase, non-7716, non-25233, non-8035, non-84242,
non-55304, non-52999, non-21999 or non-52020 polypeptide" refers to
a polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous to the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein, respectively, e.g., a protein which is different
from the 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 protein and which is derived from the same
or a different organism. The 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 polypeptides of the
fusion proteins can correspond to all or a portion e.g., a fragment
described herein of a 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 amino acid sequence. In a
preferred embodiment, a 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 fusion protein includes at
least one biologically active portion of a 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 protein or
at least one (or two) biologically active portion of a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein. The non-22406, non-acyltransferase, non-7716,
non-25233, non-8035, non-84242, non-55304, non-52999, non-21999 or
non-52020 polypeptide can be fused to the N-terminus or C-terminus
of the 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 polypeptide.
[0762] The fusion protein can include a moiety which has a high
affinity for a ligand. For example, the fusion protein can be a
GST-22406, GST-acyltransferase, GST-7716, GST-25233, GST-8035,
GST-84242, GST-55304, GST-52999, GST-21999 or GST-52020 fusion
protein in which the 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 sequences are fused to the
C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020.
Alternatively, the fusion protein can be a 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 protein
containing a heterologous signal sequence at its N-terminus. In
certain host cells (e.g., mammalian host cells), expression and/or
secretion of 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 can be increased through use of a
heterologous signal sequence.
[0763] Fusion proteins can include all or a part of a serum
protein, e.g., an IgG constant region, or human serum albumin.
[0764] The 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 fusion proteins of the invention can be
incorporated into pharmaceutical compositions and administered to a
subject in vivo. The 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 fusion proteins can be used to
affect the bioavailability of a 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 substrate. 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 fusion proteins may be useful therapeutically for the
treatment of disorders caused by, for example, (i) aberrant
modification or mutation of a gene encoding a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein; (ii) misregulation of the 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 gene; and
(iii) aberrant post-translational modification of a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein. "Treatment" is herein 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" as defined herein, includes,
but is not limited to, small molecules, peptides, antibodies,
ribozymes and antisense oligonucleotides.
[0765] Moreover, the 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020-fusion proteins of the
invention can be used as immunogens to produce anti-22406,
anti-acyltransferase, anti-7716, anti-25233, anti-8035, anti-84242,
anti-55304, anti-52999, anti-21999 or anti-52020 antibodies,
respectively, in a subject, to purify 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 ligands and in
screening assays to identify molecules which inhibit the
interaction of 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 with a 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 substrate.
[0766] Expression vectors are commercially available that already
encode a fusion moiety (e.g., a GST polypeptide). A 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020-encoding nucleic acid can be cloned into such an expression
vector such that the fusion moiety is linked in-frame to the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein.
Variants of 22406, Acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 Proteins
[0767] In another aspect, the invention also features a variant of
a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 polypeptide, e.g., which functions as an agonist
(mimetics) or as an antagonist. Variants of the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 proteins can be generated by mutagenesis, e.g., discrete
point mutation, the insertion or deletion of sequences or the
truncation of a 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 protein. An agonist of the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 proteins can retain substantially the same, or a subset, of
the biological activities of the naturally occurring form of a
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 protein. An antagonist of a 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 protein can
inhibit one or more of the activities of the naturally occurring
form of the 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 protein by, for example,
competitively modulating a 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020-mediated activity of a
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 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 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020
protein.
[0768] Variants of a 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 protein can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of a 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 protein for agonist or antagonist
activity.
[0769] Libraries of fragments e.g., N terminal, C terminal, or
internal fragments, of a 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 protein coding sequence can be
used to generate a variegated population of fragments for screening
and subsequent selection of variants of a 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020
protein.
[0770] 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.
[0771] Methods for screening gene products of combinatorial
libraries made by point mutations or truncation, and for screening
cDNA libraries for gene products having a selected property.
Recursive ensemble mutagenesis (REM), a new technique which
enhances the frequency of functional mutants in the libraries, can
be used in combination with the screening assays to identify 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA
89:7811-7815; Delgrave et al. (1993) Protein Engineering
6(3):327-331).
[0772] Cell based assays can be exploited to analyze a variegated
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 library. For example, a library of expression
vectors can be transfected into a cell line, e.g., a cell line,
which ordinarily responds to 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 in a substrate-dependent
manner. The transfected cells are then contacted with 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 and the effect of the expression of the mutant on signaling
by the 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 substrate can be detected. For example,
22406-transfected cells are then contacted with D-serine and the
effect of the expression of the mutant on signaling by the D-serine
22406 substrate can be detected, e.g., by measuring serine racemase
activity; the effect of the expression of the mutant on signaling
by the acyltransferase substrate can be detected, e.g., by
measuring fatty acid synthase activity; effect of the expression of
the mutant on signaling by the 7716 substrate can be detected,
e.g., by measuring ATPase activity; the effect of the expression of
the mutant on signaling by the 25233 substrate can be detected,
e.g., by measuring aminotransferase activity; where 8035 or 84242
is tested, detection can be accomplished by measuring RING finger
protein-mediated activity; where 55304 is tested, detection can be
accomplished by measuring aminopeptidase activity; where 52999 is
tested, detection can be accomplished by measuring polypeptide
hydrolytic activity; where 21999 is tested, detection can be
accomplished by measuring transferase activity in the reaction
wherein NAD(+) and L-arginine are converted by the
ADP-ribosyltransferase enzyme to form the end-products nicotinamide
and N2-(ADP-D-ribosyl)-L-arginine; and the effect of the expression
of the mutant on signaling by the 52020 substrate can be detected,
e.g., by measuring MAGE activity. Plasmid DNA can then be recovered
from the cells which score for inhibition, or alternatively,
potentiation of signaling by the 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 substrate, and
the individual clones further characterized.
[0773] In another aspect, the invention features a method of making
a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 polypeptide, e.g., a peptide having a non-wild type
activity, e.g., an antagonist, agonist, or super agonist of a
naturally occurring 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 polypeptide, e.g., a naturally
occurring 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 polypeptide. The method includes: altering
the sequence of a 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 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.
[0774] In another aspect, the invention features a method of making
a fragment or analog of a 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 polypeptide having a
biological activity of a naturally occurring 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 polypeptide. The method includes: altering the sequence,
e.g., by substitution or deletion of one or more residues, of a
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 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.
Anti-22406, Anti-acyltransferase, Anti-7716, Anti-25233, Anti-8035,
Anti-84242, Anti-55304, Anti-52999, Anti-21999, and Anti-52020
Antibodies
[0775] In another aspect, the invention provides an anti-22406,
anti-acyltransferase, anti-7716, anti-25233, anti-8035, anti-84242,
anti-55304, anti-52999, anti-21999, and anti-52020 antibody. The
term "antibody" as used herein refers to an immunoglobulin molecule
or immunologically active portion thereof, i.e., an antigen-binding
portion. Examples of immunologically active portions of
immunoglobulin molecules include F(ab) and F(ab').sub.2 fragments
which can be generated by treating the antibody with an enzyme such
as pepsin.
[0776] 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.
[0777] A full-length 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 protein or, antigenic peptide
fragment of 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 can be used as an immunogen or can be
used to identify anti-22406, anti-acyltransferase, anti-7716,
anti-25233, anti-8035, anti-84242, anti-55304, anti-52999,
anti-21999 or anti-52020 antibodies made with other immunogens,
e.g., cells, membrane preparations, and the like. The antigenic
peptide of 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 should include at least 8 amino acid
residues of the amino acid sequence shown in SEQ ID NO:2, SEQ ID
NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ
ID NO:27, SEQ ID NO:30, SEQ ID NO:37 or SEQ ID NO:41 respectively,
and encompasses an epitope of 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020. 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.
[0778] Fragments of 22406 which include, e.g., residues 130-150 of
SEQ ID NO:2 of SEQ ID NO:5 can be used to make, e.g., used as
immunogens, or used to characterize the specificity of an antibody
or antibodies against what are believed to be hydrophilic regions
of the 22406 protein. Similarly, a fragment of 22406 which
includes, e.g., residues 175-200 of SEQ ID NO:2 can be used to make
an antibody against what is believed to be a hydrophobic region of
the 22406 protein; a fragment of 22406 which includes residues
45-62 of SEQ ID NO:2 can be used to make an antibody against the
pyridoxal-phosphate dependent enzyme family member region of the
22406 protein. Antibodies reactive with, or specific for, any of
these regions, or other regions or domains described herein are
provided.
[0779] Fragments of 7716 which include, e.g., residues 255-290 of
SEQ ID NO:11 can be used to make, e.g., used as immunogens, or used
to characterize the specificity of an antibody or antibodies
against what are believed to be hydrophilic regions of the 7716
protein. Similarly, a fragment of 7716 which includes, e.g.,
residues 385-410 of SEQ ID NO:11 can be used to make an antibody
against what is believed to be a hydrophobic region of the 7716
protein; a fragment of 7716 which includes residues 236-421 or
500-705 of SEQ ID NO:11 can be used to make an antibody against an
ATPase region of the 7716 protein. Antibodies reactive with, or
specific for, any of these regions, or other regions or domains
described herein are provided.
[0780] Fragments of 25233 which include, e.g., residues 122-137 or
residues 243-256 of SEQ ID NO:15 can be used to make, e.g., used as
immunogens, or used to characterize the specificity of an antibody
or antibodies against what are believed to be hydrophilic regions
of the 25233 protein. Similarly, a fragment of 25233 which
includes, e.g., residues 363-393 of SEQ ID NO:15 can be used to
make an antibody against what is believed to be a hydrophobic
region of the 25233 protein; a fragment of 25233 which includes
residues 83-517 of SEQ ID NO:15 can be used to make an antibody
against the aminotransferase region of the 25233 protein.
Antibodies reactive with, or specific for, any of these regions, or
other regions or domains described herein are provided.
[0781] Fragments of 8035 which include, e.g., residues 350-390 of
SEQ ID NO:19 can be used to make, e.g., used as immunogens, or used
to characterize the specificity of an antibody or antibodies
against what are believed to be hydrophilic regions of the 8035
protein. Similarly, a fragment of 8035 which includes, e.g.,
residues 200-230 of SEQ ID NO:19 can be used to make an antibody
against what is believed to be a hydrophobic region of the 8035
protein; a fragment of 8035 which includes residues 380-421 of SEQ
ID NO:19 can be used to make an antibody against the RING finger
protein region of the 8035 protein. Antibodies reactive with, or
specific for, any of these regions, or other regions or domains
described herein are provided.
[0782] Fragments of 84242 which include, e.g., residues 190-220 of
SEQ ID NO:23 can be used to make, e.g., used as immunogens, or used
to characterize the specificity of an antibody or antibodies
against what are believed to be hydrophilic regions of the 84242
protein. Similarly, a fragment of 84242 which includes, e.g.,
residues 115-150 of SEQ ID NO:23 can be used to make an antibody
against what is believed to be a hydrophobic region of the 84242
protein; a fragment of 84242 which includes residues 2-67 of SEQ ID
NO:23 can be used to make an antibody against the IBR protein
region of the 84242 protein. Antibodies reactive with, or specific
for, any of these regions, or other regions or domains described
herein are provided.
[0783] Fragments of 55304 which include, e.g., residues 650-670 of
SEQ ID NO:27 of SEQ ID NO:22 can be used to make, e.g., used as
immunogens, or used to characterize the specificity of an antibody
or antibodies against what are believed to be hydrophilic regions
of the 55304 protein. Similarly, a fragment of 55304 which
includes, e.g., residues 240-260 of SEQ ID NO:27 can be used to
make an antibody against what is believed to be a hydrophobic
region of the 55304 protein; a fragment of 55304 which includes
residues 318-508 of SEQ ID NO:27 can be used to make an antibody
against the aminopeptidase region of the 55304 protein.
[0784] Fragments of 52999 that include residues from about amino
acid 291-320 of SEQ ID NO:30 can be used to make, e.g., used as
immunogens, or characterize the specificity of an antibody or
antibodies against what are believed to be hydrophilic regions of
the 52999 protein. Similarly, a fragment of 52999 that includes
residues from about amino acid 321-345 of SEQ ID NO:30 can be used
to make an antibody against what is believed to be a hydrophobic
region of the 52999 protein; a fragment of 52999 that includes
residues from about amino acid 270-290 of SEQ ID NO:30 can be used
to make an antibody against the active site region of the 52999
protein.
[0785] Fragments of 21999 can be used to make immunogens, or used
to characterize the specificity of an antibody or antibodies
against what are believed to be hydrophilic regions of the
ADP-ribosyltransferase protein.
[0786] Fragments of 52020 which include, e.g., residues 1-30 of SEQ
ID NO:41 of SEQ ID NO:5 can be used to make, e.g., used as
immunogens, or used to characterize the specificity of an antibody
or antibodies against what are believed to be hydrophilic regions
of the 52020 protein. Similarly, a fragment of 52020 which
includes, e.g., residues 170-190 of SEQ ID NO:41 can be used to
make an antibody against what is believed to be a hydrophobic
region of the 52020 protein; a fragment of 52020 which includes
residues 1-208 of SEQ ID NO:41 can be used to make an antibody
against the MAGE consensus region of the 52020 protein. Antibodies
reactive with, or specific for, any of these regions, or other
regions or domains described herein are provided.
[0787] In a preferred embodiment the antibody fails to bind an Fc
receptor, e.g. it is a type which does not support Fc receptor
binding or has been modified, e.g., by deletion or other mutation,
such that is does not have a functional Fc receptor binding
region.
[0788] Preferred epitopes encompassed by the antigenic peptide are
regions of 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 that are located on the surface of the
proteins, e.g., hydrophilic regions, as well as regions with high
antigenicity. For example, an Emini surface probability analysis of
the human 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 protein sequence can be used to indicate the
regions that have a particularly high probability of being
localized to the surface of the 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 protein,
respectively, and are thus likely to constitute surface residues
useful for targeting antibody production.
[0789] In a preferred embodiment the antibody binds an epitope on
any domain or region on 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 proteins described herein.
[0790] Chimeric, humanized, but most preferably, completely human
antibodies are desirable for applications which include repeated
administration, e.g., therapeutic treatment (and some diagnostic
applications) of human patients.
[0791] The anti-22406, anti-acyltransferase, anti-7716, anti-25233,
anti-8035, anti-84242, anti-55304, anti-52999, anti-21999 or
anti-52020 antibody can be a single chain antibody. A single-chain
antibody (scFV) may be engineered (see, for example, Colcher, D. et
al. (1999, Jun. 30) Ann. NY Acad. Sci.880:263-80; and Reiter, Y.
(1996 Feb) Clin. Cancer Res.2(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
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 protein.
[0792] An anti-22406, anti-acyltransferase, anti-7716, anti-25233,
anti-8035, anti-84242, anti-55304, anti-52999, anti-21999 or
anti-52020 antibody (e.g., monoclonal antibody) can be used to
isolate 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 by standard techniques, such as affinity
chromatography or immunoprecipitation. Moreover, an anti-22406,
anti-acyltransferase, anti-7716, anti-25233, anti-8035, anti-84242,
anti-55304, anti-52999, anti-21999 or anti-52020 antibody can be
used to detect 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 protein (e.g., in a cellular lysate
or cell supernatant) in order to evaluate the abundance and pattern
of expression of the protein. Anti-22406, anti-acyltransferase,
anti-7716, anti-25233, anti-8035, anti-84242, anti-55304,
anti-52999, anti-21999 or anti-52020 antibodies can be used
diagnostically to monitor protein levels in tissue as part of a
clinical testing procedure, e.g., to, for example, determine the
efficacy of a given treatment regimen. Detection can be facilitated
by coupling (i.e., physically linking) the antibody to a detectable
substance (i.e., antibody labeling). Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
Recombinant Expression Vectors, Host Cells and Genetically
Engineered Cells
[0793] 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.
[0794] A vector can include a 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 nucleic acid in a form
suitable for expression of the nucleic acid in a host cell.
Preferably the recombinant expression vector includes one or more
regulatory sequences operatively linked to the nucleic acid
sequence to be expressed. The term "regulatory sequence" includes
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Regulatory sequences include those 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.,
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 proteins, mutant forms of 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 proteins,
fusion proteins, and the like).
[0795] The recombinant expression vectors of the invention can be
designed for expression of 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 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, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0796] 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, D. B. and
Johnson, K. S. (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.
[0797] Purified fusion proteins can be used in 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 activity assays, (e.g., direct assays or competitive assays
described in detail below), or to generate antibodies specific for
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 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 (6) weeks).
[0798] To maximize recombinant protein expression in E. coli is to
express the protein in host bacteria with an impaired capacity to
proteolytically cleave the recombinant protein (Gottesman, S., Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990) 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.
[0799] The 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 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.
[0800] 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.
[0801] 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).
[0802] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. Regulatory sequences
(e.g., viral promoters and/or enhancers) operatively linked to a
nucleic acid cloned in the antisense orientation can be chosen
which direct the constitutive, tissue specific or cell type
specific expression of antisense RNA in a variety of cell types.
The antisense expression vector can be in the form of a recombinant
plasmid, phagemid or attenuated virus. For a discussion of the
regulation of gene expression using antisense genes see Weintraub,
H. et al. (1986) Antisense RNA as a molecular tool for genetic
analysis, Reviews--Trends in Genetics, Vol. 1(1).
[0803] Another aspect the invention provides a host cell which
includes a nucleic acid molecule described herein, e.g., a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 nucleic acid molecule within a recombinant expression vector
or a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 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 rather also to the progeny or
potential progeny of such a cell. Because certain modifications may
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.
[0804] A host cell can be any prokaryotic or eukaryotic cell. For
example, a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 protein can be expressed in bacterial cells
such as E. coli, insect cells, yeast or mammalian cells (such as
Chinese hamster ovary cells (CHO) or COS cells). Other suitable
host cells are known to those skilled in the art.
[0805] 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.
[0806] A host cell of the invention can be used to produce (i.e.,
express) a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 protein. Accordingly, the invention further
provides methods for producing a 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 protein has been
introduced) in a suitable medium such that a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein is produced. In another embodiment, the method
further includes isolating a 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 protein from the medium
or the host cell.
[0807] In another aspect, the invention features, a cell or
purified preparation of cells which include a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 transgene, or which otherwise misexpress 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020. 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
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 transgene, e.g., a heterologous form of a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020, e.g., a gene derived from humans (in the case of a non-human
cell). The 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 transgene can be misexpressed, e.g.,
overexpressed or underexpressed. In other preferred embodiments,
the cell or cells include a gene which misexpress an endogenous
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020, 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 alleles or for use in drug screening.
[0808] In another aspect, the invention features, a human cell,
e.g., a brain cell, transformed with nucleic acid which encodes a
subject 22406 polypeptide.
[0809] In another aspect, the invention features, a human cell,
e.g., a hematopoietic stem cell, transformed with nucleic acid
which encodes a subject acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999 or 52020 polypeptide.
[0810] Also provided are cells or a purified preparation thereof,
e.g., human cells, in which an endogenous 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 is under
the control of a regulatory sequence that does not normally control
the expression of the endogenous 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 gene. For example, an endogenous 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 gene, e.g., a gene which is "transcriptionally silent," e.g.,
not normally expressed, or expressed only at very low levels, may
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 on May 16,
1991.
Transgenic Animals
[0811] The invention provides non-human transgenic animals. Such
animals are useful for studying the function and/or activity of a
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 protein and for identifying and/or evaluating
modulators of 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 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.
[0812] 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 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 protein to particular cells. A
transgenic founder animal can be identified based upon the presence
of a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 transgene in its genome and/or expression of
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 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 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 protein can
further be bred to other transgenic animals carrying other
transgenes.
[0813] 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 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.
[0814] The invention also includes a population of cells from a
transgenic animal, as discussed herein.
Uses
[0815] The nucleic acid molecules, proteins, protein homologues,
and antibodies described herein can be used in one or more of the
following methods: a) screening assays; b) predictive medicine
(e.g., diagnostic assays, prognostic assays, monitoring clinical
trials, and pharmacogenetics); and c) methods of treatment (e.g.,
therapeutic and prophylactic).
[0816] The isolated nucleic acid molecules of the invention can be
used, for example, to express a 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 protein (e.g.,
via a recombinant expression vector in a host cell in gene therapy
applications), to detect a 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 mRNA (e.g., in a
biological sample) or a genetic alteration in a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 gene, and to modulate 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 activity, as described
further below. The 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 proteins can be used to treat
disorders characterized by insufficient or excessive production of
a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 substrate or production of 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020
inhibitors.
[0817] In addition, the 22406 proteins can be used to screen for
naturally occurring 22406 substrates, to screen for drugs or
compounds which modulate 22406 activity, as well as to treat
disorders characterized by insufficient or excessive production of
22406 protein or production of 22406 protein forms which have
decreased, aberrant or unwanted activity compared to 22406
wild-type protein. Such disorders include those of the brain,
particularly those disorders associated with convulsion, anxiety,
and neurodengeneration.
[0818] In addition, the acyltransferase proteins can be used to
screen for naturally occurring acyltransferase substrates, to
screen for drugs or compounds which modulate acyltransferase
activity, as well as to treat disorders characterized by
insufficient or excessive production of acyltransferase protein or
production of acyltransferase protein forms which have decreased,
aberrant or unwanted activity compared to acyltransferase wild-type
protein. Such disorders include those characterized by aberrant
signaling or aberrant, e.g., hyperproliferative, cell growth.
[0819] In addition, the 7716 proteins can be used to screen for
naturally occurring 7716 substrates, to screen for drugs or
compounds which modulate 7716 activity, as well as to treat
disorders characterized by insufficient or excessive production of
7716 protein or production of 7716 protein forms which have
decreased, aberrant or unwanted activity compared to 7716 wild-type
protein. Such disorders include those characterized by aberrance in
organelle biogenesis, cell proliferation, membrane fusion,
degradation of membrane proteins, or degradation of ubiquitin
pathway target proteins.
[0820] In addition, the 25233 proteins can be used to screen for
naturally occurring 25233 substrates, to screen for drugs or
compounds which modulate 25233 activity, as well as to treat
disorders characterized by insufficient or excessive production of
25233 protein or production of 25233 protein forms which have
decreased, aberrant or unwanted activity compared to 25233
wild-type protein. Such disorders include those characterized by
increased serum levels of aminotransferase.
[0821] In addition, the 8035, 84242, 55304, 52999, or 21999
proteins can be used to screen for naturally occurring 8035, 84242,
55304, 52999, or 21999 substrates, to screen for drugs or compounds
which modulate 8035, 84242, 55304, 52999, or 21999 activity, as
well as to treat disorders characterized by insufficient or
excessive production of 8035, 84242, 55304, 52999, or 21999 protein
or production of 8035, 84242, 55304, 52999, or 21999 protein forms
which have decreased, aberrant or unwanted activity compared to
8035, 84242, 55304, 52999, or 21999 wild-type protein. In the case
of 8035 and 84242, such disorders include those characterized by
aberrant cellular proliferative and/or differentiative disorders.
In the case of 55304, such disorders include those characterized by
aberrant protein proteolysis or maturation or aberrant, e.g.
hyperproliferative, cell growth. In the case of 52999, such
disorders include those characterized by aberrant protein
processing or protein degradation. In the case of 21999, such
disorders include those characterized by aberrant cellular
metabolism or aberrant growth, e.g., hyperproliferative, cell
growth.
[0822] In addition, the 52020 proteins can be used to screen for
naturally occurring 52020 binding partners, to screen for drugs or
compounds which modulate 52020 activity, as well as to treat
disorders characterized by insufficient or excessive production of
52020 protein or production of 52020 protein forms which have
decreased, aberrant or unwanted activity compared to 52020
wild-type protein. Such disorders include those characterized by
aberrant cell growth.
[0823] Moreover, the anti-22406, anti-acyltransferase, anti-7716,
anti-25233, anti-8035, anti-84242, anti-55304, anti-52999,
anti-21999 or anti-52020 antibodies of the invention can be used to
detect and isolate 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999 or 52020 proteins, regulate the
bioavailability of 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999 or 52020 proteins, and modulate 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999 or
52020 activity, respectively.
[0824] A method of evaluating a compound for the ability to
interact with, e.g., bind, a subject 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 polypeptide is
provided. The method includes: contacting the compound with the
subject 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 polypeptide; and evaluating ability of the
compound to interact with, e.g., to bind or form a complex with the
subject 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 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
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 polypeptide. It can also be used to find natural or
synthetic inhibitors of subject 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 polypeptide.
Screening methods are discussed in more detail below.
Screening Assays:
[0825] 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 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 proteins, have a stimulatory or inhibitory
effect on, for example, 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 expression or 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 activity, or have a stimulatory or inhibitory effect on, for
example, the expression or activity of a 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 substrate.
Compounds thus identified can be used to modulate the activity of
target gene products (e.g., 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 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.
[0826] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 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 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 protein or polypeptide
or a biologically active portion thereof.
[0827] 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, R. N. 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, K.S. (1997) Anticancer Drug Des.
12:145).
[0828] 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; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0829] Libraries of compounds may 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 or spores (Ladner, U.S. Pat. No. 5,223,409), 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.).
[0830] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 protein or biologically active
portion thereof is contacted with a test compound, and the ability
of the test compound to modulate 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 activity is
determined. Determining the ability of the test compound to
modulate 22406 activity can be accomplished by monitoring, for
example, serine racemase activity. Determining the ability of the
test compound to modulate acyltransferase activity can be
accomplished by monitoring, for example, fatty acid synthase
activity. Determining the ability of the test compound to modulate
7716 activity can be accomplished by monitoring, for example,
ATPase activity. Determining the ability of the test compound to
modulate 25233 activity can be accomplished by monitoring, for
example, aminotransferase activity. Determining the ability of the
test compound to modulate 8035 or 84242 activity can be
accomplished by monitoring, for example, RING finger E3 ubiquitin
ligase protein activity. Determining the ability of the test
compound to modulate 55304 activity can be accomplished by
monitoring, for example, aminopeptidase activity. Determining the
ability of the test compound to modulate 52999 activity can be
accomplished by monitoring, for example, polypeptide hydrolytic
activity. Determining the ability of the test compound to modulate
21999 activity can be accomplished by monitoring, for example,
ADP-moiety transferase activity. Determining the ability of the
test compound to modulate 52020 activity can be accomplished by
monitoring, for example, MAGE activity. The cell, for example, can
be of mammalian origin, e.g., human. Cell homogenates, or
fractions, preferably membrane containing fractions, can also be
tested.
[0831] The ability of the test compound to modulate 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 binding to a compound, e.g., a 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 substrate, or to
bind to 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 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 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 can be
determined by detecting the labeled compound, e.g., substrate, in a
complex. Alternatively, 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 could be coupled with a
radioisotope or enzymatic label to monitor the ability of a test
compound to modulate 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 binding to a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 substrate in a complex. For example, compounds (e.g., 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 substrates) can be labeled with .sup.125I, .sup.35S,
.sup.14C, or .sup.3H, either directly or indirectly, and the
radioisotope detected by direct counting of 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.
[0832] The ability of a compound (e.g., a 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 substrate)
to interact with 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 without the labeling of either the
compound or the 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020. McConnell, H. M. 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 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020.
[0833] In yet another embodiment, a cell-free assay is provided in
which a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 protein or biologically active portion
thereof is contacted with a test compound and the ability of the
test compound to bind to the 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 protein or biologically
active portion thereof is evaluated. Preferred biologically active
portions of the 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 proteins to be used in assays of the
present invention include fragments which participate in
interactions with non-22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 molecules, e.g., fragments
with high surface probability scores.
[0834] Soluble and/or membrane-bound forms of isolated proteins
(e.g., 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 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.
[0835] 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.
[0836] In one embodiment, assays are performed where the ability of
an agent to block pyridoxal-phosphate dependent serine racemase
activity within a cell is evaluated.
[0837] In one embodiment, assays are performed where the ability of
an agent to block fatty acid synthase activity within a cell is
evaluated.
[0838] In one embodiment, assays are performed where the ability of
an agent to block ATPase activity within a cell is evaluated.
[0839] In one embodiment, assays are performed where the ability of
an agent to block aminotransferase activity within a cell is
evaluated.
[0840] In one embodiment related to 8035 and 84242, assays are
performed where the ability of an agent to block RING finger
protein E3 ubiquitin ligase activity within a cell is evaluated. In
another embodiment involving 55304, assays are performed where the
ability of an agent to block aminopeptidase activity within a cell
is evaluated. In yet another embodiment related to 52999, assays
are performed where the ability of an agent to block
metallopeptidase activity within a cell is evaluated. In another
embodiment related to 21999, an assay is a cell-based assay in
which a cell which expresses a ADP-ribosyltransferase protein or
biologically active portion thereof is contacted with a test
compound, and the ability of the test compound to modulate
ADP-ribosyltransferase activity is determined. Determining the
ability of the test compound to modulate ADP-ribosyltransferase
activity can be accomplished by monitoring, for example, ADP-moiety
transferase activity. The cell, for example, can be of mammalian
origin, e.g., human. Cell homogenates, or fractions, preferably
membrane containing fractions, can also be tested.
[0841] In one embodiment, assays are performed where the ability of
an agent to block MAGE activity within a cell is evaluated.
[0842] 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 may simply
utilize the natural fluorescent energy of tryptophan residues.
Labels are chosen that emit different wavelengths of light, such
that the `acceptor` molecule label may 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).
[0843] In another embodiment, determining the ability of the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein to bind to a target molecule can be accomplished
using real-time Biomolecular Interaction Analysis (BIA) (see, e.g.,
Sjolander, S. and Urbaniczky, C. (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.TM.). 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.
[0844] 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.
[0845] It may be desirable to immobilize either 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020, an anti-22406, anti-acyltransferase, anti-7716, anti-25233,
anti-8035, anti-84242, anti-55304, anti-52999, anti-21999 or
anti-52020 antibody, or its target molecule, respectively, 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 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 protein, or
interaction of a 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 protein with a target molecule,
respectively, 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/22406,
glutathione-S-transferase/acyltransferase,
glutathione-S-transferase/7716, glutathione-S-transferase/25233,
glutathione-S-transferase/8035, glutathione-S-transferase/84242,
glutathione-S-transferase/55304, glutathione-S-transferase/52999,
glutathione-S-transferase/21999 or glutathione-S-transferase/52020
fusion proteins or glutathione-S-transferase/target fusion proteins
can be adsorbed onto glutathione SEPHAROSE.TM. beads (Sigma
Chemical, St. Louis, Mo.) or glutathione derivatized microtiter
plates, which are then combined with the test compound or the test
compound and either the non-adsorbed target protein or 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein, respectively, and the mixture incubated under
conditions conducive to complex formation (e.g., at physiological
conditions for salt and pH). Following incubation, the beads or
microtiter plate wells are washed to remove any unbound components,
the matrix immobilized in the case of beads, complex determined
either directly or indirectly, for example, as described above.
Alternatively, the complexes can be dissociated from the matrix,
and the level of 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 binding or activity determined using
standard techniques.
[0846] Other techniques for immobilizing either a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein or a target molecule on matrices include using
conjugation of biotin and streptavidin. Biotinylated 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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).
[0847] 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 for the immobilized component (the
antibody, in turn, can be directly labeled or indirectly labeled
with, e.g., a labeled anti-Ig antibody).
[0848] In one embodiment, this assay is performed utilizing
antibodies reactive with 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 protein or target molecules
but which do not interfere with binding of the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein, respectively, to its target molecule. Such
antibodies can be derivatized to the wells of the plate, and
unbound target or 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 protein or target molecule, as well
as enzyme-linked assays which rely on detecting an enzymatic
activity associated with the 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 protein or target
molecule.
[0849] 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, G., and Minton, A. P. (1993 August) Trends Biochem
Sci 18(8):284-7); chromatography (gel filtration chromatography,
ion-exchange chromatography); electrophoresis (see, e.g., Ausubel,
F. et al. eds. Current Protocols in Molecular Biology 1999, J.
Wiley: New York.); and immunoprecipitation (see, for example,
Ausubel, F. et al. eds. Current Protocols in Molecular Biology
1999, J. Wiley: New York). Such resins and chromatographic
techniques are known to one skilled in the art (see, e.g.,
Heegaard, N. H. (1998 Winter) J Mol. Recognit.11(1-6):141-8; Hage,
D. S., and Tweed, S. A. (1997, Oct. 10) J. Chromatogr. B Biomed.
Sci. Appl .699(1-2):499-525). Further, fluorescence energy transfer
may also be conveniently utilized, as described herein, to detect
binding without further purification of the complex from
solution.
[0850] In a preferred embodiment, the assay includes contacting the
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 protein or biologically active portion thereof with
a known compound which binds 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020, respectively, 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 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 protein, wherein determining the
ability of the test compound to interact with a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein includes determining the ability of the test compound
to preferentially bind to 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 or biologically active
portion thereof, or to modulate the activity of a target molecule,
as compared to the known compound.
[0851] 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 protein
through modulation of the activity of a downstream effector of a
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 target molecule, respectively. 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.
[0852] To identify compounds that interfere with the interaction
between the target gene product and its cellular or extracellular
binding partner(s), e.g., a substrate, a reaction mixture
containing the target gene product and the binding partner is
prepared, under conditions and for a time sufficient, to allow the
two products to form complex. In order to test an inhibitory agent,
the reaction mixture is provided in the presence and absence of the
test compound. The test compound can be initially included in the
reaction mixture, or can be added at a time subsequent to the
addition of the target gene and its cellular or extracellular
binding partner. Control reaction mixtures are incubated without
the test compound or with a placebo. The formation of any complexes
between the target gene product and the cellular or extracellular
binding partner is then detected. The formation of a complex in the
control reaction, but not in the reaction mixture containing the
test compound, indicates that the compound interferes with the
interaction of the target gene product and the interactive binding
partner. Additionally, complex formation within reaction mixtures
containing the test compound and normal target gene product can
also be compared to complex formation within reaction mixtures
containing the test compound and mutant target gene product. This
comparison can be important in those cases wherein it is desirable
to identify compounds that disrupt interactions of mutant but not
normal target gene products.
[0853] 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.
[0854] In a heterogeneous assay system, either the target gene
product or the interactive cellular or extracellular binding
partner, is anchored onto a solid surface (e.g., a microtiter
plate), while the non-anchored species is labeled, either directly
or indirectly. The anchored species can be immobilized by
non-covalent or covalent attachments. Alternatively, an immobilized
antibody specific for the species to be anchored can be used to
anchor the species to the solid surface.
[0855] 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 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.
[0856] 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 for one of
the binding components to anchor any complexes formed in solution,
and a labeled antibody specific for the other partner to detect
anchored complexes. Again, depending upon the order of addition of
reactants to the liquid phase, test compounds that inhibit complex
or that disrupt preformed complexes can be identified.
[0857] 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.
[0858] In yet another aspect, the 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 ("22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020-binding proteins" or "22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020-bp") and are involved in 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 activity, respectively.
Such 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020-bps can be activators or inhibitors of
signals by the 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 proteins or 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 targets,
respectively, as, for example, downstream elements of a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020-mediated signaling pathway.
[0859] 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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: 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 protein can be fused to the
activator domain.) If the "bait" and the "prey" proteins are able
to interact, in vivo, forming a 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020-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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein.
[0860] In another embodiment, modulators of 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 expression
are identified. For example, a cell or cell free mixture is
contacted with a candidate compound and the expression of 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 mRNA or protein evaluated relative to the level of expression
of 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 mRNA or protein in the absence of the candidate
compound. When expression of 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 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
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 mRNA or protein expression, respectively.
Alternatively, when expression of 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 mRNA or protein
expression, respectively. The level of 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 mRNA or
protein expression can be determined by methods described herein
for detecting 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 mRNA or protein, respectively.
[0861] 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 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 protein can be confirmed in vivo, e.g., in
an animal.
[0862] 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 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 modulating agent, an
antisense 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 nucleic acid molecule, a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020-specific antibody, or a 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020-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.
Detection Assays
[0863] 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 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 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.
Chromosome Mapping
[0864] The 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 nucleotide sequences or portions thereof can
be used to map the location of the 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 genes,
respectively, on a chromosome. This process is called chromosome
mapping. Chromosome mapping is useful in correlating the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 sequences with genes associated with disease.
[0865] Briefly, 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp in length) from the
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 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 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 sequences will
yield an amplified fragment.
[0866] 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 P. et al. (1983) Science 220:919-924).
[0867] Other mapping strategies e.g., in situ hybridization
(described in Fan, Y. 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 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 to a chromosomal
location.
[0868] 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. Human
Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York
1988).
[0869] 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.
[0870] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland, J. et al. (1987) Nature 325:783-787.
[0871] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 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.
Tissue Typing
[0872] 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 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).
[0873] 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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.
[0874] 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, SEQ I) NO:6, SEQ ID NO:10, SEQ
ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29,
SEQ ID NO:36 and SEQ ID NO:40 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, SEQ ID
NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ
ID NO:28, SEQ ID NO:31, SEQ ID NO:38 or SEQ ID NO:42 are used, a
more appropriate number of primers for positive individual
identification would be 500-2,000.
[0875] If a panel of reagents from 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 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.
Use of Partial 22406, Acyltransferase, 7716, 25233, 8035, 84242.
55304, 52999, 21999 or 52020 Sequences in Forensic Biology
[0876] 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.
[0877] 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, SEQ ID NO:6, SEQ ID NO:10, SEQ ID
NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ
ID NO:36 or SEQ ID NO:40 (e.g., fragments derived from the
noncoding regions of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:10, SEQ ID
NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ
ID NO:36 or SEQ ID NO:40 having a length of at least 20 bases,
preferably at least 30 bases) are particularly appropriate for this
use.
[0878] The 22406 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, e.g., a
tissue containing pyridoxal-phosphate dependent serine racemase
activity. The acyltransferase 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,
e.g., a tissue containing fatty acid synthase activity. The 7716
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, e.g., a tissue containing
ATPase activity. The 25233 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,
e.g., a tissue containing aminotransferase activity. The 8035,
84242, 55304, 52999, or 21999 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,
e.g., a tissue containing RING finger E3 ubiquitin ligase protein
activity (8035 and 84242), aminopeptidase activity (55304),
metallopreptidase activity (52999), and ADP-ribosyltransferase
activity (21999). The 52020 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,
e.g., a tissue containing MAGE activity. This can be very useful in
cases where a forensic pathologist is presented with a tissue of
unknown origin. Panels of such 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 probes can be used to
identify tissue by species and/or by organ type.
[0879] In a similar fashion, these reagents, e.g., 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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).
Predictive Medicine
[0880] 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.
[0881] 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 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020.
[0882] Such disorders include, e.g., a disorder associated with the
misexpression of 22406, or a neurological disorder.
[0883] Such disorders include, e.g., a disorder associated with the
misexpression of acyltransferase, or fatty acid biosynthesis or
lipid metabolism related disorder.
[0884] Such disorders include, e.g., a disorder associated with the
misexpression of 7716, or lipid metabolism related disorder.
[0885] Such disorders include, e.g., a disorder associated with the
misexpression of 25233, or lipid metabolism related disorder.
[0886] Such disorders include, e.g., a disorder associated with the
misexpression of 8035 or 84242, those disorders resulting from
aberrant cellular proliferation and/or differentiation including
diseases such as cancer, acute promyelocytic leukemia (APL), VHL
disease, and systemic lupus erythematosus. In addition, RING finger
protein family members have been shown to contribute to the
pathogenesis of certain viral diseases including those caused by
HSV and HIV. Other such disorders include, e.g., a disorder
associated with the misexpression of 55304, 52999, 21999, or lipid
metabolism related disorder.
[0887] Such disorders include, e.g., 52020-mediated aberrant cell
growth.
[0888] The method includes one or more of the following:
[0889] detecting, in a tissue of the subject, the presence or
absence of a mutation which affects the expression of the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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;
[0890] detecting, in a tissue of the subject, the presence or
absence of a mutation which alters the structure of the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 gene;
[0891] detecting, in a tissue of the subject, the misexpression of
the 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 gene, at the mRNA level, e.g., detecting a non-wild
type level of an mRNA;
[0892] 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 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 polypeptide.
[0893] In preferred embodiments the method includes: ascertaining
the existence of at least one of: a deletion of one or more
nucleotides from the 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 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.
[0894] 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, SEQ ID NO:6, SEQ ID NO:10, SEQ
ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29,
SEQ ID NO:36, SEQ ID NO:40, naturally occurring mutants thereof or
5' or 3' flanking sequences naturally associated with the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 gene, respectively; (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.
[0895] 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020.
[0896] Methods of the invention can be used prenatally or to
determine if a subject's offspring will be at risk for a
disorder.
[0897] In preferred embodiments the method includes determining the
structure of a 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 gene, an abnormal structure being
indicative of risk for the disorder.
[0898] In preferred embodiments the method includes contacting a
sample form the subject with an antibody to the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein or a nucleic acid, which hybridizes specifically with
the gene. These and other embodiments are discussed below.
Diagnostic and Prognostic Assays
[0899] The presence, level, or absence of 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein or nucleic acid (e.g., mRNA, genomic DNA) that
encodes 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 protein such that the presence of 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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. The level of expression of the
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 gene can be measured in a number of ways,
including, but not limited to: measuring the mRNA encoded by the
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 genes; measuring the amount of protein encoded by
the 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 genes; or measuring the activity of the protein
encoded by the 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 genes.
[0900] The level of mRNA corresponding to the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 gene in a cell can be determined both by in situ and by in
vitro formats.
[0901] 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 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 nucleic acid, such as the nucleic
acid of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ
ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:36 or
SEQ ID NO:40, 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
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 mRNA or genomic DNA, respectively. Other suitable
probes for use in the diagnostic assays are described herein.
[0902] 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
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 genes.
[0903] The level of mRNA in a sample that is encoded by one of
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 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.
[0904] 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 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 gene being analyzed.
[0905] In another embodiment, the methods further contacting a
control sample with a compound or agent capable of detecting 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 mRNA, or genomic DNA, and comparing the presence of 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 mRNA or genomic DNA in the control sample with the presence
of 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 mRNA or genomic DNA, respectively, in the test
sample.
[0906] A variety of methods can be used to determine the level of
protein encoded by 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020. 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.
[0907] The detection methods can be used to detect 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein in a biological sample in vitro as well as in vivo.
In vitro techniques for detection of 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 protein include
enzyme linked immunosorbent assays (ELISAs), immunoprecipitations,
immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay
(RIA), and Western blot analysis. In vivo techniques for detection
of 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 protein include introducing into a subject a
labeled anti-22406, anti-acyltransferase, anti-7716, anti-25233,
anti-8035, anti-84242, anti-55304, anti-52999, anti-21999, or
anti-52020 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.
[0908] In another embodiment, the methods further include
contacting the control sample with a compound or agent capable of
detecting 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 protein, and comparing the presence of
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 protein in the control sample with the presence of
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 protein, respectively, in the test sample.
[0909] The invention also includes kits for detecting the presence
of 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 in a biological sample. For example, the kit can
include a compound or agent capable of detecting 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 protein or nucleic acid.
[0910] 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.
[0911] 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.
[0912] 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 expression or activity. As used herein, the term "unwanted"
includes an unwanted phenomenon involved in a biological response
such as, for example, in the case of 22406, neurodegeneration, in
the case of acyltranferase, pain or deregulated cell proliferation,
in the case of 7716, neurodegeneration or deregulated cell
proliferation, in the case of 25233, muscle, cardiac, kidney,
pancreatic, red blood cell, or hepatic injury, or cancer, or active
periodontal disease, in the case of 8035 and 84242, deregulated
cell proliferation and/or differentiation, in the case of 55304
deregulated cell proliferation or hypertension, in the case of
52999, inflammation or deregulated cell proliferation, in the case
of 21999, deregulated cell proliferation or depressed cellular
metabolism, or in the case of 52020, deregulated cell growth.
[0913] In one embodiment, a disease or disorder associated with
aberrant or unwanted 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 expression or activity is
identified. A test sample is obtained from a subject and 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein or nucleic acid (e.g., mRNA or genomic DNA) is
evaluated, wherein the level, e.g., the presence or absence, of
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 protein or nucleic acid is diagnostic for a subject
having or at risk of developing a disease or disorder associated
with aberrant or unwanted 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 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.
[0914] 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 22406 expression
or activity. For example, such methods can be used to determine
whether a subject can be effectively treated with an agent for a
neurodegenerative disorder.
[0915] 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 7716 expression or
activity. For example, such methods can be used to determine
whether a subject can be effectively treated with an agent for
neurodegeneration or a cellular growth related disorder.
[0916] 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 25233 expression
or activity. For example, such methods can be used to determine
whether a subject can be effectively treated with an agent for
muscle, cardiac, kidney, pancreatic, red blood cell, or hepatic
injury, or cancer, or active periodontal disease.
[0917] 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 acyltransferase,
8035, 84242, 55304, 52999, 21999, or 52020 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
growth related disorder.
[0918] The methods of the invention can also be used to detect
genetic alterations in a 22406 gene, thereby determining if a
subject with the altered gene is at risk for a disorder
characterized by misregulation in 22406 protein activity or nucleic
acid expression, such as a neurodegenerative disorder. The methods
of the invention can also be used to detect genetic alterations in
a 7716 gene, thereby determining if a subject with the altered gene
is at risk for a disorder characterized by misregulation in 7716
protein activity or nucleic acid expression, such as
neurodegeneration or a cellular growth related disorder. The
methods of the invention can also be used to detect genetic
alterations in a 25233 gene, thereby determining if a subject with
the altered gene is at risk for a disorder characterized by
misregulation in 25233 protein activity or nucleic acid expression,
such as those arising as a result of a metabolic defect. The
methods of the invention can also be used to detect genetic
alterations in a acyltransferase, 8035, 84242, 55304, 52999, 21999
or 52020 gene, thereby determining if a subject with the altered
gene is at risk for a disorder characterized by misregulation in
8035, 84242, 55304, 52999, 21999 or 52020 protein activity or
nucleic acid expression, such as a cellular growth related
disorder. 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 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020-protein, or the
misexpression of the 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 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
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 gene; 2) an addition of one or more nucleotides to
a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 gene; 3) a substitution of one or more nucleotides
of a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 gene, 4) a chromosomal rearrangement of a
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 gene; 5) an alteration in the level of a messenger
RNA transcript of a 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 gene, 6) aberrant modification
of a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 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 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 gene, 8) a
non-wild type level of a 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020-protein, 9) allelic loss of a
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 gene, and 10) inappropriate post-translational
modification of a 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020-protein.
[0919] 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 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020-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 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 gene under conditions such that
hybridization and amplification of the 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020-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.
[0920] Alternative amplification methods include: self sustained
sequence replication (Guatelli, J. C. et al. (1990) Proc. Natl.
Acad. Sci. USA 87:1874-1878), transcriptional amplification system
(Kwoh, D. Y. et al. (1989) Proc. Natl. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al. (1988)
Bio-Technology 6:1197), or other nucleic acid amplification
methods, followed by the detection of the amplified molecules using
techniques known to those of skill in the art.
[0921] In another embodiment, mutations in a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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.
[0922] In other embodiments, genetic mutations in 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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, M. T. et al. (1996) Human
Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine
2:753-759). For example, genetic mutations in 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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.
[0923] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 gene and detect mutations by comparing the sequence
of the sample 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 with the corresponding wild-type
(control) sequence. Automated sequencing procedures can be utilized
when performing the diagnostic assays (Naeve et al.(1995)
Biotechniques 19:448-453), including sequencing by mass
spectrometry.
[0924] Other methods for detecting mutations in the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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 230:1242-1246; Cotton
et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397-4401; Saleeba et
al. (1992) Methods Enzymol. 217:286-295).
[0925] 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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).
[0926] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 genes. For example, single strand conformation polymorphism
(SSCP) may be used to detect differences in electrophoretic
mobility between mutant and wild type nucleic acids (Orita et al.
(1989) Proc. Natl. Acad. Sci. USA: 86:2766-2770, 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 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 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 may be labeled or detected with
labeled probes. The sensitivity of the assay may 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).
[0927] 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 313:495-498). 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).
[0928] 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).
[0929] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may 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 6:1-7). It is anticipated
that in certain embodiments amplification may also be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci USA 88:189-193). 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.
[0930] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving a 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 gene.
Use of 22406, Acyltransferase, 7716, 25233, 8035, 84242. 55304,
52999, 21999 or 52020 Molecules as Surrogate Markers
[0931] The 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 molecules of the invention may be detected, and may be
correlated with one or more biological states in vivo. For example,
the 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 molecules of the invention may 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 may
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 may be made using cholesterol levels as a surrogate marker,
and an analysis of HIV infection may 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.
[0932] The 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 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 may 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 may be monitored by the
pharmacodynamic marker. Similarly, the presence or quantity of the
pharmacodynamic marker may 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 may be sufficient to activate multiple
rounds of marker (e.g., a 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 marker) transcription or
expression, the amplified marker may be in a quantity which is more
readily detectable than the drug itself. Also, the marker may be
more easily detected due to the nature of the marker itself; for
example, using the methods described herein, anti-22406,
anti-acyltransferase, anti-7716, anti-25233, anti-8035, anti-84242,
anti-55304, anti-52999, anti-21999 or anti-52020 antibodies may be
employed in an immune-based detection system for a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein marker, respectively, or 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020-specific
radiolabeled probes may be used to detect a 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 mRNA
marker, respectively. Furthermore, the use of a pharmacodynamic
marker may 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.
[0933] The 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 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(12): 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,
may be selected. For example, based on the presence or quantity of
RNA, or protein (e.g., 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 protein or RNA) for specific
tumor markers in a subject, a drug or course of treatment may 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 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 DNA may
correlate 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 drug response, respectively. The use of
pharmacogenomic markers therefore permits the application of the
most appropriate treatment for each subject without having to
administer the therapy.
Pharmaceutical Compositions
[0934] The nucleic acid and polypeptides, fragments thereof, as
well as anti-22406, anti-acyltransferase, anti-7716, anti-25233,
anti-8035, anti-84242, anti-55304, anti-52999, anti-21999, or
anti-52020 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.
[0935] 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.
[0936] 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.RTM. EL solubilizer (BASF; Florham
Park, 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 mannitol, 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.
[0937] 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.
[0938] 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.
[0939] 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.
[0940] 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.
[0941] 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.
[0942] 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.
[0943] 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.
[0944] 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 may 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.
[0945] 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 may 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 may 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 may
be measured, for example, by high performance liquid
chromatography.
[0946] 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 may influence the dosage and timing required to
effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of a protein, polypeptide, or antibody can include
a single treatment or, preferably, can include a series of
treatments.
[0947] 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).
[0948] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics (e.g., peptoids), amino
acids, amino acid analogs, polynucleotides, polynucleotide analogs,
nucleotides, nucleotide analogs, organic or inorganic compounds
(i.e, including 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.
[0949] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram. It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. When one or more of these small molecules is to be
administered to an animal (e.g., a human) in order to modulate
expression or activity of a polypeptide or nucleic acid of the
invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0950] An antibody (or fragment thereof) may be conjugated to a
therapeutic moiety such as a cytotoxin, a therapeutic agent or a
radioactive metal ion. A cytotoxin or cytotoxic agent includes any
agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0951] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug 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.
[0952] 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.
[0953] 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.
[0954] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
Methods of Treatment
[0955] 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 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 expression or activity.
[0956] 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 acyltransferase expression or activity.
"Treatment" is herein 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. With regards to both prophylactic and therapeutic
methods of treatment, such treatments may 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 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 molecules of the
present invention or 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 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.
[0957] In one aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant or unwanted 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 expression or activity, by
administering to the subject a 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 agent which modulates
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 expression or at least one 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 activity,
respectively. Subjects at risk for a disease which is caused or
contributed to by aberrant or unwanted 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 aberrance, such that a disease or disorder is prevented or,
alternatively, delayed in its progression. Depending on the type of
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 aberrance, for example, a 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 agonist, or
a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 antagonist agent can be used for treating the
subject. The appropriate agent can be determined based on screening
assays described herein.
[0958] It is possible that some 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 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.
[0959] As discussed, successful treatment of 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 disorders. Such
molecules can include, but are not limited to peptides,
phosphopeptides, small organic or inorganic molecules, or
antibodies (including, for example, polyclonal, monoclonal,
humanized, anti-idiotypic, chimeric or single chain antibodies, and
FAb, F(ab').sub.2 and FAb expression library fragments, scFV
molecules, and epitope-binding fragments thereof).
[0960] 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.
[0961] 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.
[0962] Another method by which nucleic acid molecules may be
utilized in treating or preventing a disease characterized by
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 expression is through the use of aptamer molecules
specific for 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 protein, respectively. Aptamers are
nucleic acid molecules having a tertiary structure which permits
them to specifically bind to protein ligands (see, e.g., Osborne,
et al. (1997) Curr. Opin. Chem. Biol. 1(1):5-9; and Patel, D. J.
(1997 June) Curr. Opin. Chem. Biol. 1(1):32-46). Since nucleic acid
molecules may in many cases be more conveniently introduced into
target cells than therapeutic protein molecules may be, aptamers
offer a method by which 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 protein activity may be
specifically decreased without the introduction of drugs or other
molecules which may have pluripotent effects.
[0963] Antibodies can be generated that are both specific for
target gene product and that reduce target gene product activity.
Such antibodies may, therefore, be administered in instances
whereby negative modulatory techniques are appropriate for the
treatment of 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 disorders. For a description of
antibodies, see the Antibody section above.
[0964] In circumstances wherein injection of an animal or a human
subject with a 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 protein or epitope for stimulating
antibody production is harmful to the subject, it is possible to
generate an immune response against 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 through the use
of anti-idiotypic antibodies (see, for example, Herlyn, D. (1999)
Ann. Med. 31(1):66-78; and Bhattacharya-Chatterjee, M., and Foon,
K. A. (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 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 protein. Vaccines
directed to a disease characterized by 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 expression
may also be generated in this fashion.
[0965] In instances where the target antigen is intracellular and
whole antibodies are used, internalizing antibodies may 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).
[0966] 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 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 disorders. A therapeutically effective dose
refers to that amount of the compound sufficient to result in
amelioration of symptoms of the disorders.
[0967] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
that exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects can be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0968] 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.
[0969] 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 may
utilize antibody mimics and/or "biosensors" that have been created
through molecular imprinting techniques. The compound which is able
to modulate 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 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, R. J. et al. (1996) Current Opinion in Biotechnology
7:89-94 and in Shea, K. J. (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, G. 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 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 can be
readily monitored and used in calculations of IC.sub.50.
[0970] 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.
A rudimentary example of such a "biosensor" is discussed in Kriz,
D. et al. (1995) Analytical Chemistry 67:2142-2144.
[0971] Another aspect of the invention pertains to methods of
modulating 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 expression or activity for therapeutic
purposes. Accordingly, in an exemplary embodiment, the modulatory
method of the invention involves contacting a cell with a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 agent that modulates one or more of the activities of 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein activity, respectively, associated with the cell. An
agent that modulates 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 protein activity can be an
agent as described herein, such as a nucleic acid or a protein, a
naturally-occurring target molecule of a 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 protein
(e.g., a 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 substrate or binding partner), a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 antibody, a 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 agonist or antagonist, a
peptidomimetic of a 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 agonist or antagonist, or
other small molecule.
[0972] In one embodiment, the agent stimulates one or more 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 activities. Examples of such stimulatory agents include
active 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 protein and a nucleic acid molecule encoding
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020, respectively. In another embodiment, the agent
inhibits one or more 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 activities. Examples of such
inhibitory agents include antisense 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 nucleic acid
molecules, anti-22406, anti-acyltransferase, anti-7716, anti-25233,
anti-8035, anti-84242, anti-55304, anti-52999, anti-21999, or
anti-52020 antibodies, and 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 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., upregulates
or downregulates) 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 expression or activity. In another
embodiment, the method involves administering a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 protein or nucleic acid molecule as therapy to compensate for
reduced, aberrant, or unwanted 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 expression or activity,
respectively.
[0973] Stimulation of 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 activity is desirable in
situations in which 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 is abnormally downregulated
and/or in which increased 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 activity is likely to
have a beneficial effect. For example, stimulation of 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 activity is desirable in situations in which 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 is downregulated and/or in which increased 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 activity is likely to have a beneficial effect. Likewise,
inhibition of 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 activity is desirable in situations
in which 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 is abnormally upregulated and/or in which
decreased 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 activity is likely to have a beneficial
effect.
[0974] The 22406 molecules can act as novel diagnostic targets and
therapeutic agents for controlling one or more of brain disorders,
including but not limited to, behavioral changes associated with
learning, memory, convulsion, anxiety, psychotomimetic induced
abnormal behavior, cerebellar ataxia, and neurodengeneration.
Inhibitors of 22406 protein can be expected to quell anxiety and
epilepsy and to prevent damage from stroke and certain
neurodegenerative conditions including Alzheimer's disease. On the
other hand, stimulating 22406 protein might improve schizophrenia
symptoms, which are partly caused by depressed NMDA receptor
function. In addition, 22406 protein can be expected to be involved
in various disorders of the tissues in which it is expressed,
including heart disorders, liver disorders, prostrate disorders,
skeletal muscle disorders, dermal fibroblast disorders, and blood
vessel disorders. All of the disorders described supra are
disorders that may be treated or diagnosed by methods described
herein.
[0975] The acyltransferase molecules can act as novel diagnostic
targets and therapeutic agents for controlling one or more of
cellular proliferative and/or differentiative disorders,
cardiovascular disorders, as described above, as well as disorders
associated with lipid metabolism, hematopoietic disorders, liver
disorders, viral diseases, pain or metabolic disorders.
[0976] Examples of hematopoietic disorders 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.
[0977] Disorders which may 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, Al-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 may
be useful 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.
[0978] Additionally, acyltransferase molecules may 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 acyltransferase activity 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, acyltransferase modulators can be used in the
treatment and/or diagnosis of virus-associated carcinoma,
especially hepatocellular cancer.
[0979] Additionally, acyltransferase may 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, H. L., (1987) Pain, New
York:McGraw-Hill); pain associated with muscoloskeletal disorders,
e.g., joint pain; tooth pain; headaches; pain associated with
surgery.
[0980] The 7716 molecules can act as novel diagnostic targets and
therapeutic agents for controlling one or more of cellular
proliferative and/or differentiative disorders, organelle
biogenesis disorders (including those resulting in
neurodegeneration, membrane fusion disorders (including disorders
relating to secretion or synaptic transmission) as well as
disorders associated with aberrant regulation of proteolysis of
targets of the 26S proteasome, and aberrant degradation of membrane
proteins.
[0981] Aberrant expression and/or activity of 7716 molecules may
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 may ultimately affect the concentrations in
serum of calcium and phosphate. This term also includes activities
mediated by 7716 molecules effects in bone cells, e.g. osteoclasts
and osteoblasts, that may in turn result in bone formation and
degeneration. For example, 7716 molecules may support different
activities of bone resorbing osteoclasts such as the stimulation of
differentiation of monocytes and mononuclear phagocytes into
osteoclasts. Accordingly, 7716 molecules that modulate the
production of bone cells can influence bone formation and
degeneration, and thus may 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.
[0982] Examples of hematopoietic disorders 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.
[0983] Disorders which may 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, Al-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 may
be useful 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.
[0984] Additionally, 7716 molecules may 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 7716 activity 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, 7716
modulators can be used in the treatment and/or diagnosis of
virus-associated carcinoma, especially hepatocellular cancer.
[0985] Additionally, 7716 may 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, H. L. (1987) Pain, New
York:McGraw-Hill); pain associated with muscoloskeletal disorders,
e.g., joint pain; tooth pain; headaches; pain associated with
surgery; pain related to irritable bowel syndrome; or chest
pain.
[0986] The 25233 molecules can act as novel diagnostic targets and
therapeutic agents for controlling one or more of muscle, cardiac,
kidney, pancreatic, red blood cell, or hepatic disorders, cancer,
or active periodontal disease. Examples of disorders which may 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 may be useful
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.
[0987] The 8035, 84242, 55304, 52999, or 21999 molecules can act as
novel diagnostic targets and therapeutic agents for controlling one
or more of cellular proliferative and/or differentiative disorders,
viral diseases, or metabolic disorders.
[0988] Aberrant expression and/or activity of 8035 and 84242
molecules may lead to disorders resulting from aberrant cellular
proliferation and/or differentiation including diseases such as
cancer, acute promyelocytic leukemia (APL), VHL disease, and
systemic lupus erythematosus. In addition, RING finger protein
family members such as 8035 and 84242 have been shown to contribute
to the pathogenesis of certain viral diseases including those
caused by HSV and HIV.
[0989] The 55304 molecules can act as novel diagnostic targets and
therapeutic agents for controlling one or more of cellular
proliferative and/or differentiative disorders, hypertensive
disorders, hormone disorders, and disorders associated with protein
maturation as described above, as well as disorders associated with
bone metabolism, hematopoietic disorders, liver disorders, viral
diseases, pain or metabolic disorders.
[0990] Aberrant expression and/or activity of 55304 molecules may
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 may ultimately affect the concentrations in
serum of calcium and phosphate. This term also includes activities
mediated by 55304 molecules effects in bone cells, e.g. osteoclasts
and osteoblasts, that may in turn result in bone formation and
degeneration. For example, 55304 molecules may support different
activities of bone resorbing osteoclasts such as the stimulation of
differentiation of monocytes and mononuclear phagocytes into
osteoclasts. Accordingly, 55304 molecules that modulate the
production of bone cells can influence bone formation and
degeneration, and thus may 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.
[0991] Examples of hematopoietic disorders 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.
[0992] Disorders which may 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, Al-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 may
be useful 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.
[0993] Additionally, 55304 molecules may 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 55304 activity 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, 55304
modulators can be used in the treatment and/or diagnosis of
virus-associated carcinoma, especially hepatocellular cancer.
[0994] Additionally, 55304 may 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, H. L. (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.
[0995] The 52999 molecules can act as novel diagnostic targets and
therapeutic agents for controlling one or more of cellular
proliferative and/or differentiative disorders; inflammatory
disorders including, but not limited to osteoarthritis and
rheumatoid arthritis, multiple sclerosis, Crohn disease, psoriasis,
periodontal disease, and asthma; macular degeneration; restenosis;
and Alzheimer's disease.
[0996] Similarly, aberrant expression and/or activity of 52999
molecules may mediate disorders associated with, for example,
hematopoietic disorders including, but 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.
[0997] The 21999 molecules can act as novel diagnostic targets and
therapeutic agents for controlling one or more of cellular
proliferative and/or differentiative disorders, cardiovascular
disorders, as described above, as well as disorders associated with
hematopoietic disorders, liver disorders, viral diseases, or
metabolic disorders. Examples of these disorders may be found
above.
[0998] The 52020 molecules can act as novel diagnostic targets and
therapeutic agents for controlling one or more of cancers, tissue
repair, neurodegenerative disorders, autoimmune disorders, and
inflammatory disorders as described herein supra.
Pharmacogenomics
[0999] The 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 molecules of the present invention, as well
as agents, or modulators which have a stimulatory or inhibitory
effect on 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020 activity (e.g., 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 gene
expression) as identified by a screening assay described herein can
be administered to individuals to treat (prophylactically or
therapeutically) 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 associated disorders (e.g., cellular
growth related disorders) associated with aberrant or unwanted
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 activity. 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) may 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 may
consider applying knowledge obtained in relevant pharmacogenomics
studies in determining whether to administer a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 molecule or a 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 modulator as well as tailoring
the dosage and/or therapeutic regimen of treatment with a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 molecule or 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 modulator.
[1000] 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, M. et al. (1996) Clin. Exp. Pharmacol.
Physiol. 23(10-11):983-985 and Linder, M. W. et al. (1997) Clin.
Chem. 43(2):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.
[1001] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high-resolution map can be generated from a
combination of some ten million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[1002] 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 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 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.
[1003] 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 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 molecule or 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 modulator
of the present invention) can give an indication whether gene
pathways related to toxicity have been turned on.
[1004] 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 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 molecule or 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 modulator, such as a modulator identified by one of the
exemplary screening assays described herein.
[1005] 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 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 genes of
the present invention, wherein these products may be associated
with resistance of the cells to a therapeutic agent. Specifically,
the activity of the proteins encoded by the 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 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., cancer cells,
will become sensitive to treatment with an agent that the
unmodified target cells were resistant to.
[1006] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 protein can be applied
in clinical trials. For example, the effectiveness of an agent
determined by a screening assay as described herein to increase
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 gene expression, protein levels, or upregulate
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 activity, can be monitored in clinical trials of
subjects exhibiting decreased 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 gene expression, protein
levels, or downregulated 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 activity, respectively.
Alternatively, the effectiveness of an agent determined by a
screening assay to decrease 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 gene expression, protein
levels, or downregulate 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 activity, can be monitored in
clinical trials of subjects exhibiting increased 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 gene expression, protein levels, or upregulated 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 activity, respectively. In such clinical trials, the
expression or activity of a 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 gene, and preferably,
other genes that have been implicated in, for example, a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020-associated disorder can be used as a "read out" or markers of
the phenotype of a particular cell.
Other Embodiments
[1007] In another aspect, the invention features, a method of
analyzing a plurality of capture probes. The method can be used,
e.g., to analyze gene expression. The method includes: providing a
two dimensional array having a plurality of addresses, each address
of the plurality being positionally distinguishable from each other
address of the plurality, and each address of the plurality having
a unique capture probe, e.g., a nucleic acid or peptide sequence;
contacting the array with a 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020, preferably purified,
nucleic acid, preferably purified, polypeptide, preferably
purified, or antibody, and thereby evaluating the plurality of
capture probes. Binding, e.g., in the case of a nucleic acid,
hybridization with a capture probe at an address of the plurality,
is detected, e.g., by signal generated from a label attached to the
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 nucleic acid, polypeptide, or antibody.
[1008] 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.
[1009] The method can include contacting the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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.
[1010] The plurality of capture probes can be a plurality of
nucleic acid probes each of which specifically hybridizes, with an
allele of 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020. 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. 22406 is
associated with pyridoxal-phosphate dependent serine racemase
activity, thus it is useful for disorders associated with the
brain. Acyltransferase is associated with fatty acid synthase
activity, thus it is useful for disorders associated with abnormal
lipid metabolism. 7716 is associated with ATPase activity, thus it
is useful for disorders associated with abnormal lipid metabolism.
25233 is associated with aminotransferase activity, thus it is
useful for disorders associated with abnormal lipid metabolism.
8035 and 84242 are associated with RING finger protein activity,
thus it is useful for disorders associated with abnormal cellular
proliferation and/or differentiation. 55304 is associated with
aminopeptidase activity, thus it is useful for disorders associated
with abnormal lipid metabolism. 52999 is associated with
metallopeptidase activity, thus it, too, is useful for disorders
associated with abnormal lipid metabolism. 52020 is associated with
MAGE activity, thus it is useful for disorders associated with
abnormal cell growth.
[1011] The method can be used to detect SNPs, as described
above.
[1012] In another aspect, the invention features, a method of
analyzing a plurality of probes. The method is useful, e.g., for
analyzing gene expression. The method includes: providing a two
dimensional array having a plurality of addresses, each address of
the plurality being positionally distinguishable from each other
address of the plurality having a unique capture probe, e.g.,
wherein the capture probes are from a cell or subject which express
or misexpress 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020 or from a cell or subject in which a
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 mediated response has been elicited, e.g., by
contact of the cell with 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 nucleic acid or protein, or
administration to the cell or subject 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 nucleic acid or
protein; contacting the array with one or more inquiry probe,
wherein an inquiry probe can be a nucleic acid, polypeptide, or
antibody (which is preferably other than 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 nucleic
acid, polypeptide, or antibody); providing a two dimensional array
having a plurality of addresses, each address of the plurality
being positionally distinguishable from each other address of the
plurality, and each address of the plurality having a unique
capture probe, e.g., wherein the capture probes are from a cell or
subject which does not express 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 (or does not express as
highly as in the case of the 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 positive plurality of
capture probes) or from a cell or subject which in which a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 mediated response has not been elicited (or has been elicited
to a lesser extent than in the first sample); contacting the array
with one or more inquiry probes (which is preferably other than a
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 nucleic acid, polypeptide, or antibody), and
thereby evaluating the plurality of capture probes. Binding, e.g.,
in the case of a nucleic acid, hybridization with a capture probe
at an address of the plurality, is detected, e.g., by signal
generated from a label attached to the nucleic acid, polypeptide,
or antibody.
[1013] In another aspect, the invention features, a method of
analyzing 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304,
52999, 21999, or 52020, e.g., analyzing structure, function, or
relatedness to other nucleic acid or amino acid sequences. The
method includes: providing a 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 nucleic acid or amino
acid sequence; comparing the 22406, acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716, 25233, 8035, 84242,
55304, 52999, 21999, or 52020.
[1014] Preferred databases include GenBank.TM.. The method can
include evaluating the sequence identity between a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 sequence and a database sequence. The method can be performed
by accessing the database at a second site, e.g., over the
internet.
[1015] In another aspect, the invention features, a set of
oligonucleotides, useful, e.g., for identifying SNP's, or
identifying specific alleles of 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020. 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
different labels, such that an oligonucleotides which hybridizes to
one allele provides a signal that is distinguishable from an
oligonucleotides which hybridizes to a second allele.
[1016] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are incorporated herein by
reference.
EXAMPLES
Example 1
Identification and Characterization of Human 22406 cDNAs
[1017] The human 22406 sequence (SEQ ID NO:1), which is
approximately 1770 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
1020 nucleotides (nucleotides 69-1088 of SEQ ID NO:1; SEQ ID NO:3).
The coding sequence encodes a 340 amino acid protein (SEQ ID
NO:2).
Example 2
Tissue Distribution of 22406 mRNA
[1018] Expression levels of 22406 in various tissue and cell types
were determined by quantitative RT-PCR (Reverse Transcriptase
Polymerase Chain Reaction; Taqman.RTM. brand PCR kit, Applied
Biosystems) (FIG. 8). The quantitative RT-PCR reactions were
performed according to the kit manufacturer's instructions.
[1019] 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 22406 cDNA (SEQ ID NO:1)
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.
[1020] FIGS. 4 and 8 show expression of the 22406 protein in
various human tissues. In FIG. 8 the tissue types are as follows
from left to right: Aorta/Normal, Fetal Heart/Normal, Heart/Normal,
Heart/CHF, Vein/Normal, SMC/Aortic, Nerve/Normal, Spinal
Cord/Normal, Brain Cord/Normal, Brain Cortex/Normal, Brain
Hypothalmus/Normal, Glial Cells (Astrocytes), Glioblastoma,
Breast/Normal, Breast/Tumor, Ovary/Normal, Ovary/Tumor,
Pancreas/Normal, Prostate/Normal, Prostate/Tumor, Colon/Normal,
Colon/Tumor, Colon/IBD, Kidney/Normal, Liver/Normal,
Liver/Fibrosis, Fetal Liver/Normal, Lung/Normal, Lung/COPD,
Spleen/Normal, Tonsil/Normal, Lymph Node/Normal, Thymus/Normal,
Epithelial Cells (Prostate), Endothelial Cells (Aortic), Skeletal
Muscle/Normal, Fibroblasts (Dermal), Skin/Normal, Adipose/Normal,
Osteoblasts (Primary), Osteoblasts (Undiff), Osteoblasts (Diff),
Osteoclasts, NTC.
Example 3
Identification and Characterization of Human Acyltransferase
cDNAs
[1021] The human acyltransferase sequence (SEQ ID NO:6), which is
approximately 2465 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
1632 nucleotides (from nucleotide position 495 to position 2126 of
SEQ ID NO:6). The coding sequence (SEQ ID NO:8) encodes a 554 amino
acid protein (SEQ ID NO:7).
Example 4
Tissue Distribution of Acyltransferase mRNA
[1022] 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 acyltransferase cDNA (SEQ
ID NO:6). 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 5
Identification and Characterization of Human 7716 cDNAs
[1023] The human 7716 sequence (SEQ ID NO:10), which is
approximately 2547 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
2259 nucleotides (nucleotides 63-2321 of SEQ ID NO:10; SEQ ID
NO:12). The coding sequence encodes a 753 amino acid protein (SEQ
ID NO:11).
Example 6
Tissue Distribution of 7716 mRNA
[1024] 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 7716 cDNA (SEQ ID NO:10)
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 7
Identification and Characterization of Human 25233 cDNAs
[1025] The human 25233 sequence (SEQ ID NO:14), which is
approximately 2127 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
1572 nucleotides (nucleotides 94-1665 of SEQ ID NO:14; nucleotides
1-1572 of SEQ ID NO:16). The coding sequence encodes a 523 amino
acid protein (SEQ ID NO:15).
Example 8
Distribution of Human 25233 in Various Cells and Tissues
[1026] Human 25233 showed elevated levels of expression in
pancreas, brain cortex, glial cells, breast tumor, epithelial
cells, and liver. See FIG. 21. Expression levels of 25233 were
observed to be higher in tumors of the breast, ovary and lung, as
compared with 25233 expression levels in normal breast, ovary, and
lung tissue. See FIGS. 22A-D. 25233 expression levels were somewhat
higher in tumors of the colon, as compared to normal colon tissue.
See FIGS. 23A-B. Generally, 25233 expression levels increased over
time in the human cancer cell line H460 with and without p16. See
FIG. 22D. On the other hand, TABLE-US-00002 TABLE 1 Expression of
25233 in breast cancer cell lines MCF10A and MCF3B at various
intervals after exposure to EGF (1-6 and 7-12, respectively).
Expression in tumorigenic and non-tumorigenic breast cancer cell
lines (13-22) are shown, as well. Relative Cell Line Expression 1
10 MCF10A EGF 0 hr 16.9 2 MCF10A EGF 0.5 hr 19.0 3 MCF10A EGF 1 hr
16.5 4 MCF10A EGF 2 hr 16.2 5 MCF10A EGF 4 hr 28.6 6 MCF10A EGF 8
hr 20.1 7 MCF3B EGF 0 hr 58.1 8 MCF3B EGF 0.5 hr 49.5 9 MCF3B EGF 1
hr 43.6 10 MCF3B EGF 2 hr 57.1 11 MCF3B EGE 4 hr 58.3 12 MCF3B EGF
8 hr 52.6 13 MCF10A-NT 24.4 14 MCF10AT.c11-NT 23.9 15
MCF10AT.c13-NT 60.2 16 MCF10MS-NT 19.6 17 MCF10CA1a.c11-T 8.2 18
MCF10AT1-T 62.7 19 MCF10AT3B-T 15.5 20 MCF10AT3B-agar 20.5 21
MCF10CA1a.cl1-agar 37.4 22 MCF10A-m25-plastic 58.9
25233 expression levels appeared steady over time in breast tissue
cell lines treated with EGF, as well. See Table 1. Expression
levels were determined by quantitative PCR (Taqman.RTM. brand
quantitative PCR kit, Applied Biosystems). The quantitative PCR
reactions were performed according to the kit manufacturer's
instructions.
[1027] In situ hybridization analysis was also carried out (data
not shown). In lung tumor and colon metastasis tissue, expression
of 25233 was observed in immune cells; 25233 expression was not
observed in the tumor tissue itself.
Example 9
Identification and Characterization of Human 8035 and 84242
cDNAs
[1028] The human 8035 sequence (SEQ ID NO:18), which is
approximately 2876 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
1299 nucleotides (nucleotides 613-1914 of SEQ ID NO:18; SEQ ID
NO:20). The coding sequence encodes a 433 amino acid protein (SEQ
ID NO:19).
[1029] The human 84242 sequence (SEQ ID NO:22), which is
approximately 2810 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
1209 nucleotides (nucleotides 744-1955 of SEQ ID NO:22; SEQ ID
NO:24). The coding sequence encodes a 403 amino acid protein (SEQ
ID NO:23).
Example 10
Distribution of Human 84242 in Various Cells and Tissues
[1030] Human 84242 showed elevated levels of expression in coronary
smooth muscle, human umbilical vein endothelia, heart, kidney,
skeletal muscle, brain, dorsal root ganglion, breast, prostate,
colon, lung, skin, bone marrow, blood, and erythroid cells and
tissues. See Table 2. Expression levels of 84242 were observed to
be higher in tumors of the breast, prostate, colon, and lung,
relative to normal tissue. See Table 2, rows 22, 26, 29, and 31.
TABLE-US-00003 TABLE 2 Expression of 84242 in various cells and
tissues. PHASE 1.5.1 EXPRESSION OF 84242(F1R1) RELATIVE TISSUE TYPE
EXPRESSION 1 Artery normal 9.2907 2 Aorta diseased 8.2294 3 Vein
normal 2.9399 4 Coronary SMC (Smooth Muscle Cell) 12.4734 5 HUVEC
(Human Umbilical Vein Endothelial 40.9498 Cells) 6 Hemangioma
3.5697 7 Heart normal 11.6381 8 Heart CHF (Congestive Heart
Failure) 12.6038 9 Kidney 11.5577 10 Skeletal Muscle 18.6459 11
Adipose normal 2.7717 12 Pancreas 4.8259 13 primary osteoblasts
9.1946 14 Osteoclasts (Differentiated) 2.9913 15 Skin normal 6.2584
16 Spinal cord normal 4.3043 17 Brain Cortex normal 50.942 18 Brain
Hypothalamus, normal 9.9575 19 Nerve 5.8393 20 DRG (Dorsal Root
Ganglion) 12.8241 21 Breast normal 4.6453 22 Breast tumor 10.1667
23 Ovary normal 8.5789 24 Ovary Tumor 3.0968 25 Prostate Normal
4.5027 26 Prostate Tumor 12.0904 27 Salivary glands 1.816 28 Colon
normal 6.9682 29 Colon Tumor 20.0535 30 Lung normal 4.4253 31 Lung
tumor 21.7175 32 Lung COPD (Chronic Obstructive Pulmonary 6.0872
Disease) 33 Colon IBD (Inflammatory Bowel Disease) 7.8125 34 Liver
normal 2.7241 35 Liver fibrosis 4.9102 36 Spleen normal 2.5241 37
Tonsil normal 8.7591 38 Lymph node, normal 4.4253 39 Small
intestine normal 6.5241 40 Skin-Decubitus 12.7355 41 Synovium
2.4129 42 BM-MNC (Bone Marrow-Mononuclear Cell) 15.0405 43
Activated PBMC (Peripheral Blood Mononuclear 15.2505 Cell) 44
Neutrophils 3.2848 45 Megakaryocytes 4.996 46 Erythroid 17.337
[1031] Expression levels were determined by quantitative PCR
(Taqman.RTM. brand quantitative PCR kit, Applied Biosystems). The
quantitative PCR reactions were performed according to the kit
manufacturer's instructions.
Example 11
Identification and Characterization of Human 55304 cDNAs
[1032] The human 55304 sequence (SEQ ID NO:26), which is
approximately 5502 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
2039 nucleotides (nucleotides 803-2845 of SEQ ID NO:26; SEQ ID
NO:28). The coding sequence encodes a 680 amino acid protein (SEQ
ID NO:27).
Example 12
Distribution of Human 55304 in Various Cells and Tissues
[1033] Human 555304 showed elevated levels of expression in brain,
kidney, testes, and epithelial cells of the prostate. See Tables 3
and 4. TABLE-US-00004 TABLE 3 Expression of 55304 in various cells
and tissues. 55304 HUMAN PANEL PHASE I RELATIVE TISSUE EXPRESSION 1
Adrenal Gland 0.95 2 Brain 2.66 3 Heart 0.32 4 Kidney 3.66 5 Liver
0.12 6 Lung 0.15 7 Mammary Gland 0.25 8 Placenta 1.18 9 Prostate
0.68 10 Salivary Gland 1.55 11 Muscle 0.74 12 Sm. Intestine 0.42 13
Spleen 0.08 14 Stomach 0.68 15 Testes 5 16 Thymus 0.34 17 Trachea
0.45 18 Uterus 0.16 19 Spinal Cord 1.19 20 Skin 0.20 21 DRG (Dorsal
Root Ganglion) 0.45
[1034] Expression levels of 55304 were observed to be higher in
tumors of the colon and lung, relative to normal tissue. See Table
4, rows 31 and 33. TABLE-US-00005 TABLE 4 Expression of 55304 in
various cells and tissues. PHASE 1.3.3 EXPRESSION OF 55304.1
RELATIVE TISSUE TYPE EXPRESSION 1 Artery normal 0 2 Vein normal 0 3
Aortic SMC (Smooth Muscle Cell) EARLY 1.1981 4 Coronary SMC 2.022 5
Static HUVEC (Human Umbilical Vein Endothelial 1.4497 Cell) 6 Shear
HUVEC 0.6465 7 Heart normal 0.2366 8 Heart CHF (Chronic Heart
Failure) 1.736 9 Kidney 38.8754 10 Skeletal Muscle 0.1922 11
Adipose normal 0 12 Pancreas 1.1613 13 Primary Osteoblasts 0.0619
14 Osteoclasts (differentiated) 0.0143 15 Skin normal 0.4192 16
Spinal cord normal 3.1619 17 Brain Cortex normal 22.5614 18 Brain
Hypothalamus normal 11.8415 19 Nerve 1.1063 20 DRG (Dorsal Root
Ganglion) 3.472 21 Glial Cells (Astrocytes) 3.2395 22 Glioblastoma
0.2814 23 Breast normal 0.0458 24 Breast tumor 0.3818 25 Ovary
normal 5.3546 26 Ovary Tumor 1.1735 27 Prostate Normal 0.5003 28
Prostate Tumor 0.1529 29 Epithelial Cells (Prostate) 40.2463 30
Colon normal 0.1002 31 Colon Tumor 8.6385 32 Lung normal 0 33 Lung
tumor 4.9444 34 Lung COPD (Chronic Obstructive Pulmonary 0.0954
Disorder) 35 Colon IBD (Inflammatory Bowel Disease) 0.0903 36 Liver
normal 0.309 37 Liver fibrosis 0.4733 38 Dermal Cells-fibroblasts
0.2416 39 Spleen normal 0.059 40 Tonsil normal 0.6159 41 Lymph node
0.4684 42 Small Intestine 0.1299 43 Skin-Decubitus 0.5108 44
Synovium 0.0287 45 BM-MNC (Bone Marrow Mononuclear Cell) 0.6095 46
Activated PBMC (Peripheral Blood Mononuclear 0.0125 Cell)
[1035] Expression levels were determined as described in Example
12.
Example 13
Identification and Characterization of Human 52999 cDNAs
[1036] The human 52999 sequence (SEQ ID NO:29), which is
approximately 2566 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
2277 nucleotides (nucleotides 194-2470 of SEQ ID NO:29; SEQ ID
NO:1). The coding sequence encodes a 758 amino acid protein (SEQ ID
NO:30).
Example 14
Identification and Characterization of 21999 Human
ADP-ribosyltransferase cDNAs
[1037] The human ADP-ribosyltransferase sequence (SEQ ID NO:36),
which is approximately 1485 nucleotides long including untranslated
regions, contains a predicted methionine-initiated coding sequence
of about 876 nucleotides (nucleotides 255-1133 of SEQ ID NO:36).
The coding sequence encodes a 292 amino acid protein (SEQ ID
NO:37).
Example 15
Distribution of Human 21999 in Various Cells and Tissues
[1038] Human 21999 showed elevated levels of expression in skeletal
muscle and ovary tissue. See Table 5. TABLE-US-00006 TABLE 5
Expression of 21999 in various cells and tissues. PHASE 1.6.5 OF
21999 RELATIVE TISSUE TYPE EXPRESSION 1 Artery normal 0 2 Aorta
diseased 0 3 Vein normal 0 4 Coronary SMC (Smooth Muscle Cell) 0 5
HUVEC (Human Umbilical Vein Endothelial Cell) 0 6 Hemangioma 0 7
Heart normal 0 8 Heart CHF (Chronic Heart Failure) 0.1044 9 Kidney
0 10 Skeletal Muscle 1.5218 11 Adipose normal 0 12 Pancreas 0 13
Primary Osteoblasts 0 14 Osteoclasts (differentiated) 0 15 Skin
normal 0 16 Spinal cord normal 0 17 Brain Cortex normal 0 18 Brain
Hypothalamus normal 0 19 Nerve 0 20 DRG (Dorsal Root Ganglion) 0 21
Breast normal 0.1482 22 Breast tumor 0 23 Ovary normal 1.2191 24
Ovary Tumor 0.0332 25 Prostate Normal 0.0641 26 Prostate Tumor 0 27
Salivary glands 0 28 Colon normal 0 29 Colon Tumor 0 30 Lung normal
0.0202 31 Lung tumor 0.871 32 Lung COPD (Chronic Obstructive
Pulmonary 0 Disease) 33 Colon IBD (Inflammatory Bowel Disease) 0 34
Liver normal 0 35 Liver fibrosis 0 36 Spleen normal 0 37 Tonsil
normal 0 38 Lymph node normal 0 39 Small intestine normal 0 40
Macrophages 0 41 Synovium 0 42 BM-MNC (Bone-Marrow Mononuclear
Cells) 0 43 Activated PBMC (Peripheral Blood Mononuclear 0 Cells)
44 Neutrophils 0 45 Megakaryocytes 0 46 Erythroid 0 47 Positive
Control 10.5253
[1039] Expression levels of 21999 were observed to be higher in
tumors of the lung and colon, as well as in metastatic liver,
relative to normal tissue. See Table 6, rows 22, 26, 30, 34, and
35. 21999 expression was also elevated in both normoxic and hypoxic
colon tumor cell lines (HCT116). Table 6, rows 42 and 43.
TABLE-US-00007 TABLE 6 Expression of 21999 in various cells and
tissues. 21999.1 ONCOLOGY PHASE II PANEL RELATIVE TISSUE TYPE
EXPRESSION 1 PIT 400 Breast Normal 0.00 2 PIT 372 Breast Normal
3.15 3 CHT 1228 Breast Normal 1.31 4 MDA 304 Breast Tumor: MD-IDC
(Moderately 0.00 Differentiated-Invasive Ductal Carcinoma) 5 CHT
2002 Breast Tumor: IDC 0.00 6 MDA 236-Breast Tumor: PD-IDC(ILC?)
(Poorly 0.00 Differentiated-IDC(Invasive Lobular Carcinoma?)) 7 CHT
562 Breast Tumor: IDC 0.00 8 NDR 138 Breast Tumor ILC (LG) 0.00 9
CHT 1841 Lymph node (Breast metastases) 0.00 10 PIT 58 Lung (Breast
metastases) 0.00 11 CHT 620 Ovary Normal 14.38 12 PIT 208 Ovary
Normal 6.39 13 CLN 012 Ovary Tumor 96.05 14 CLN 07 Ovary Tumor 0.07
15 CLN 17 Ovary Tumor 9.82 16 MDA 25 Ovary Tumor 0.35 17 CLN 08
Ovary Tumor 0.48 18 PIT 298 Lung Normal 0.05 19 MDA 185 Lung Normal
0.00 20 CLN 930 Lung Normal 0.57 21 MPI 215 Lung Tumor--SmC 0.00 22
MDA 259 Lung Tumor-PDNSCCL 32.13 23 CHT 832 Lung Tumor-PDNSCCL 0.00
24 MDA 262 Lung Tumor-SCC (Squamous Cell 1.01 Carcinoma) 25 CHT 793
Lung Tumor-ACA 0.00 26 CHT 331 Lung Tumor-ACA 14.63 27 CHT 405
Colon Normal 0.00 28 CHT 523 Colon Normal 0.20 29 CHT 371 Colon
Normal 0.00 30 CHT 382 Colon Tumor: MD 8.46 31 CHT 528 Colon Tumor:
MD 0.17 32 CLN 609 Colon Tumor 0.13 33 NDR 210 Colon Tumor: MD-PD
0.00 34 CHT 340 Colon-Liver Metastases 6.19 35 CHT 1637 Colon-Liver
Metastases 2.14 36 PIT 260 Liver Normal (female) 0.00 37 CHT 1653
Cervix Squamous CC 0.00 38 CHT 569 Cervix Squamous CC 0.00 39 A24
HMVEC (Human Microvessel Endothelial 0.00 Cell) - Arresting 40 C48
HMVEC-Proliferating 0.00 41 Pooled Hemangiomas 0.00 42 HCT116N22
Normoxic 51.30 43 HCT116H22 Hypoxic 47.37
[1040] Expression levels were determined as described in Example
12.
Example 16
Tissue Distribution of 8035, 84242, 55304, 52999, or 21999 mRNA
[1041] 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 8035, 84242, 55304, 52999,
or 21999 cDNA (SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID
NO:29, or SEQ ID NO:36) can be used. The DNA is 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 17
Identification and Characterization of Human 52020 cDNAs
[1042] The human 52020 sequence (SEQ ID NO:40), which is
approximately 2183 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
912 nucleotides, not including the stop codon (nucleotides782-1693
of SEQ ID NO:40; nucleotides 1-912 of SEQ ID NO:42). The coding
sequence encodes a 304 amino acid protein (SEQ ID NO:41).
Example 18
Tissue Distribution of 52020 mRNA
[1043] 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 52020 cDNA (SEQ ID NO:40)
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 19
Recombinant Expression of 22406, Acyltransferase, 7716. 25233,
8035, 84242, 55304 52999, 21999, or 52020 in Bacterial Cells
[1044] In this example, 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 is expressed as a recombinant
glutathione-S-transferase (GST) fusion polypeptide in E. coli and
the fusion polypeptide is isolated and characterized. Specifically,
22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 is fused to GST and this fusion polypeptide is
expressed in E. coli, e.g., strain PEB199. Expression of the
GST-22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 fusion protein in PEB 199 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 20
Expression of Recombinant 22406, Acyltransferase, 7716, 25233,
8035, 84242, 55304, 52999, 21999, or 52020 Protein in COS Cells
[1045] To express the 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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.
[1046] To construct the plasmid, the 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716, 25233, 8035,
84242, 55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 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 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 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.
[1047] COS cells are subsequently transfected with the 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020-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 22406, acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999,
21999, or 52020 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.
[1048] Alternatively, DNA containing the 22406, acyltransferase,
7716, 25233, 8035, 84242, 55304, 52999, 21999, or 52020 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 22406, acyltransferase, 7716,
25233, 8035, 84242, 55304, 52999, 21999, or 52020 polypeptide is
detected by radiolabelling and immunoprecipitation using a 22406,
acyltransferase, 7716, 25233, 8035, 84242, 55304, 52999, 21999, or
52020 specific monoclonal antibody.
Equivalents
[1049] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[1050] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
43 1 1770 DNA Homo sapiens CDS (69)...(1091) 1 cacgcgtccg
ggccggggag gcgcgcggag gctggagctg gaggcgcggc gccggtgagc 60 tgagaacc
atg tgt gct cag tat tgc atc tcc ttt gct gat gtt gaa aaa 110 Met Cys
Ala Gln Tyr Cys Ile Ser Phe Ala Asp Val Glu Lys 1 5 10 gct cat atc
aac att cga gat tct atc cac ctc aca cca gtg cta aca 158 Ala His Ile
Asn Ile Arg Asp Ser Ile His Leu Thr Pro Val Leu Thr 15 20 25 30 agc
tcc att ttg aat caa cta aca ggg cgc aat ctt ttc ttc aaa tgt 206 Ser
Ser Ile Leu Asn Gln Leu Thr Gly Arg Asn Leu Phe Phe Lys Cys 35 40
45 gaa ctc ttc cag aaa aca gga tct ttt aag att cgt ggt gct ctc aat
254 Glu Leu Phe Gln Lys Thr Gly Ser Phe Lys Ile Arg Gly Ala Leu Asn
50 55 60 gcc gtc aga agc ttg gtt cct gat gct tta gaa agg aag ccg
aaa gct 302 Ala Val Arg Ser Leu Val Pro Asp Ala Leu Glu Arg Lys Pro
Lys Ala 65 70 75 gtt gtt act cac agc agt gga aac cat ggc cag gct
ctc acc tat gct 350 Val Val Thr His Ser Ser Gly Asn His Gly Gln Ala
Leu Thr Tyr Ala 80 85 90 gcc aaa ttg gaa gga att cct gct tat att
gtg gtg ccc cag aca gct 398 Ala Lys Leu Glu Gly Ile Pro Ala Tyr Ile
Val Val Pro Gln Thr Ala 95 100 105 110 cca gac tgt aaa aaa ctt gca
ata caa gcc tac gga gcg tca att gta 446 Pro Asp Cys Lys Lys Leu Ala
Ile Gln Ala Tyr Gly Ala Ser Ile Val 115 120 125 tac tgt gaa cct agt
gat gag tcc aga gaa aat gtt gca aaa aga gtt 494 Tyr Cys Glu Pro Ser
Asp Glu Ser Arg Glu Asn Val Ala Lys Arg Val 130 135 140 aca gaa gaa
aca gaa ggc atc atg gta cat ccc aac cag gag cct gca 542 Thr Glu Glu
Thr Glu Gly Ile Met Val His Pro Asn Gln Glu Pro Ala 145 150 155 gtg
ata gct gga caa ggg aca att gcc ctg gaa gtg ctg aac cag gtt 590 Val
Ile Ala Gly Gln Gly Thr Ile Ala Leu Glu Val Leu Asn Gln Val 160 165
170 cct ttg gtg gat gca ctg gtg gta cct gta ggt gga gga gga atg ctt
638 Pro Leu Val Asp Ala Leu Val Val Pro Val Gly Gly Gly Gly Met Leu
175 180 185 190 gct gga ata gca att aca gtt aag gct ctg aaa cct agt
gtg aag gta 686 Ala Gly Ile Ala Ile Thr Val Lys Ala Leu Lys Pro Ser
Val Lys Val 195 200 205 tat gct gct gaa ccc tca aat gca gat gac tgc
tac cag tcc aag ctg 734 Tyr Ala Ala Glu Pro Ser Asn Ala Asp Asp Cys
Tyr Gln Ser Lys Leu 210 215 220 aag ggg aaa ctg atg ccc aat ctt tat
cct cca gaa acc ata gca gat 782 Lys Gly Lys Leu Met Pro Asn Leu Tyr
Pro Pro Glu Thr Ile Ala Asp 225 230 235 ggt gtc aaa tcc agc att ggc
ttg aac acc tgg cct att atc agg gac 830 Gly Val Lys Ser Ser Ile Gly
Leu Asn Thr Trp Pro Ile Ile Arg Asp 240 245 250 ctt gtg gat gat atc
ttc act gtc aca gag gat gaa att aag tgt gca 878 Leu Val Asp Asp Ile
Phe Thr Val Thr Glu Asp Glu Ile Lys Cys Ala 255 260 265 270 acc cag
ctg gtg tgg gag agg atg aaa cta ctc att gaa cct aca gct 926 Thr Gln
Leu Val Trp Glu Arg Met Lys Leu Leu Ile Glu Pro Thr Ala 275 280 285
ggt gtt gga gtg gct gct gtg ctg tct caa cat ttt caa act gtt tcc 974
Gly Val Gly Val Ala Ala Val Leu Ser Gln His Phe Gln Thr Val Ser 290
295 300 cca gaa gta aag aac att tgt att gtg ctc agt ggt gga aat gta
gac 1022 Pro Glu Val Lys Asn Ile Cys Ile Val Leu Ser Gly Gly Asn
Val Asp 305 310 315 tta acc tcc tcc ata act tgg gtg aag cag gct gaa
agg cca gct tct 1070 Leu Thr Ser Ser Ile Thr Trp Val Lys Gln Ala
Glu Arg Pro Ala Ser 320 325 330 tat cag tct gtt tct gtt taa
tttacagaaa aggaaatggt gggaattcag 1121 Tyr Gln Ser Val Ser Val * 335
340 tgtctttaga tactgaagac attttgtttc ctagtattgt caactcttag
ttatcagatt 1181 cttaatggag agtggctatt tcattaagat ttaatagttt
tttttggact aagtagtgga 1241 aaaactttta tacttaactg agacattttg
tcaaggctaa aaaaaagtct tgcaaaatgg 1301 ggcagtggac tgacaggctg
acatagaaaa taaactttgc ccaatcacaa cttgtgcctc 1361 ccatccctgg
agtactgact ggcaccggta agacagaatc tctttgaatc cattactcca 1421
tgcccccttg aggcactgtt gaagaaatct cacttttcag ccagggtact ggttctggta
1481 catatggatc ataagtccat ttggggaaga ctcgtttata caggttcatc
agtactgtgt 1541 cttgagattt tagcttccca tcaaagctgc atttcatgtg
gccatgggta cctagaaaga 1601 catcagaaca agtcggtcaa attaaaagta
gaaaatttta aagcaatgac ttccaaccca 1661 acagtcattt agcaacactg
cagaaatgca gacatggtct caaatcccgt gtttccttac 1721 ctaaaggttc
cttgatatgt cctctccggc ccccacttcg ttctcagtt 1770 2 340 PRT Homo
sapiens 2 Met Cys Ala Gln Tyr Cys Ile Ser Phe Ala Asp Val Glu Lys
Ala His 1 5 10 15 Ile Asn Ile Arg Asp Ser Ile His Leu Thr Pro Val
Leu Thr Ser Ser 20 25 30 Ile Leu Asn Gln Leu Thr Gly Arg Asn Leu
Phe Phe Lys Cys Glu Leu 35 40 45 Phe Gln Lys Thr Gly Ser Phe Lys
Ile Arg Gly Ala Leu Asn Ala Val 50 55 60 Arg Ser Leu Val Pro Asp
Ala Leu Glu Arg Lys Pro Lys Ala Val Val 65 70 75 80 Thr His Ser Ser
Gly Asn His Gly Gln Ala Leu Thr Tyr Ala Ala Lys 85 90 95 Leu Glu
Gly Ile Pro Ala Tyr Ile Val Val Pro Gln Thr Ala Pro Asp 100 105 110
Cys Lys Lys Leu Ala Ile Gln Ala Tyr Gly Ala Ser Ile Val Tyr Cys 115
120 125 Glu Pro Ser Asp Glu Ser Arg Glu Asn Val Ala Lys Arg Val Thr
Glu 130 135 140 Glu Thr Glu Gly Ile Met Val His Pro Asn Gln Glu Pro
Ala Val Ile 145 150 155 160 Ala Gly Gln Gly Thr Ile Ala Leu Glu Val
Leu Asn Gln Val Pro Leu 165 170 175 Val Asp Ala Leu Val Val Pro Val
Gly Gly Gly Gly Met Leu Ala Gly 180 185 190 Ile Ala Ile Thr Val Lys
Ala Leu Lys Pro Ser Val Lys Val Tyr Ala 195 200 205 Ala Glu Pro Ser
Asn Ala Asp Asp Cys Tyr Gln Ser Lys Leu Lys Gly 210 215 220 Lys Leu
Met Pro Asn Leu Tyr Pro Pro Glu Thr Ile Ala Asp Gly Val 225 230 235
240 Lys Ser Ser Ile Gly Leu Asn Thr Trp Pro Ile Ile Arg Asp Leu Val
245 250 255 Asp Asp Ile Phe Thr Val Thr Glu Asp Glu Ile Lys Cys Ala
Thr Gln 260 265 270 Leu Val Trp Glu Arg Met Lys Leu Leu Ile Glu Pro
Thr Ala Gly Val 275 280 285 Gly Val Ala Ala Val Leu Ser Gln His Phe
Gln Thr Val Ser Pro Glu 290 295 300 Val Lys Asn Ile Cys Ile Val Leu
Ser Gly Gly Asn Val Asp Leu Thr 305 310 315 320 Ser Ser Ile Thr Trp
Val Lys Gln Ala Glu Arg Pro Ala Ser Tyr Gln 325 330 335 Ser Val Ser
Val 340 3 1020 DNA Homo sapiens 3 atgtgtgctc agtattgcat ctcctttgct
gatgttgaaa aagctcatat caacattcga 60 gattctatcc acctcacacc
agtgctaaca agctccattt tgaatcaact aacagggcgc 120 aatcttttct
tcaaatgtga actcttccag aaaacaggat cttttaagat tcgtggtgct 180
ctcaatgccg tcagaagctt ggttcctgat gctttagaaa ggaagccgaa agctgttgtt
240 actcacagca gtggaaacca tggccaggct ctcacctatg ctgccaaatt
ggaaggaatt 300 cctgcttata ttgtggtgcc ccagacagct ccagactgta
aaaaacttgc aatacaagcc 360 tacggagcgt caattgtata ctgtgaacct
agtgatgagt ccagagaaaa tgttgcaaaa 420 agagttacag aagaaacaga
aggcatcatg gtacatccca accaggagcc tgcagtgata 480 gctggacaag
ggacaattgc cctggaagtg ctgaaccagg ttcctttggt ggatgcactg 540
gtggtacctg taggtggagg aggaatgctt gctggaatag caattacagt taaggctctg
600 aaacctagtg tgaaggtata tgctgctgaa ccctcaaatg cagatgactg
ctaccagtcc 660 aagctgaagg ggaaactgat gcccaatctt tatcctccag
aaaccatagc agatggtgtc 720 aaatccagca ttggcttgaa cacctggcct
attatcaggg accttgtgga tgatatcttc 780 actgtcacag aggatgaaat
taagtgtgca acccagctgg tgtgggagag gatgaaacta 840 ctcattgaac
ctacagctgg tgttggagtg gctgctgtgc tgtctcaaca ttttcaaact 900
gtttccccag aagtaaagaa catttgtatt gtgctcagtg gtggaaatgt agacttaacc
960 tcctccataa cttgggtgaa gcaggctgaa aggccagctt cttatcagtc
tgtttctgtt 1020 4 378 PRT Artificial Sequence Pyridoxal-Phosphate
Dependent Enzyme Family Domain Sequence 4 Val Thr Glu Leu Ile Gly
Asn Thr Pro Leu Val Arg Leu Asn Arg Leu 1 5 10 15 Ser Lys Glu Leu
Gly Glu Gly Leu Gly Ala Asn Ala Ala Val Glu Ile 20 25 30 Tyr Leu
Lys Leu Glu Asp Leu Asn Gly Pro Thr Gly Ser Phe Lys Asp 35 40 45
Arg Gly Leu Ala Leu Asn Met Ile Leu Leu Ala Glu Lys Leu Gly Lys 50
55 60 Lys Gly Gly Ile Val Pro Gly Thr Val Gln Val Glu Ser Lys Thr
Thr 65 70 75 80 Ile Ile Glu Pro Thr Ser Gly Asn Thr Gly Ile Ala Leu
Ala Leu Ala 85 90 95 Ala Ala Leu Leu Gly Leu Lys Cys Thr Ile Val
Met Pro Ala Thr Asp 100 105 110 Thr Ser Arg Glu Lys Arg Ala Gln Leu
Arg Ala Leu Gly Ala Glu Leu 115 120 125 Val Val Val Pro Val Ala Gly
Gly Gly Ser Asp Asp Leu Ala Asp Ala 130 135 140 Ile Ala Lys Ala Glu
Glu Leu Ala Glu Glu Asn Pro Glu Asn Ala Tyr 145 150 155 160 Leu Leu
Asn Gln Ala Ala Gly Pro Phe Asp Asn Pro Ala Asn Pro Glu 165 170 175
Ile Ala Gly Gln Lys Thr Ile Gly Pro Glu Ile Trp Glu Gln Leu Gly 180
185 190 Gly Lys Glu Ile Ser Leu Gly Arg Leu Pro Asp Ala Val Val Ala
Pro 195 200 205 Val Gly Gly Gly Gly Thr Ile Thr Gly Ile Ala Arg Tyr
Leu Lys Glu 210 215 220 Leu Asn Pro Asp Gly Lys Ile Asp Val Leu Glu
Leu Pro Val Lys Val 225 230 235 240 Ile Gly Val Glu Pro Glu Gly Ser
Ala Val Leu Ser Gly Ser Leu Lys 245 250 255 Ala Thr Leu Thr Leu Ala
Gly Lys Pro Gly Pro Leu His Gly Arg Asp 260 265 270 Ser Lys Tyr Leu
Leu Gln Asp Glu Pro Val Thr Leu Pro Glu Thr Lys 275 280 285 Ser Ile
Gly Ile Gly Leu Gly Val Pro Arg Val Gly Glu Phe Val Pro 290 295 300
Pro Ile Leu Asp Glu Leu Leu Asp Arg Arg Gln Gly Ile Asp Glu Val 305
310 315 320 Val Thr Val Thr Asp Glu Glu Ala Leu Glu Ala Ala Arg Leu
Leu Ala 325 330 335 Arg Glu Glu Gly Ile Leu Val Gly Pro Ser Ser Gly
Ala Ala Val Ala 340 345 350 Ala Ala Leu Lys Leu Ala Lys Glu Gly Lys
Lys Pro Leu Asn Lys Gly 355 360 365 Lys Thr Ile Val Val Ile Leu Ser
Gly Gly 370 375 5 14 PRT Artificial Sequence Pyridoxal-Phosphate
Attachment Site Consensus Sequence misc_feature 2, 3, 4, 5, 7 Xaa =
Any amino acid 5 Asp Xaa Xaa Xaa Xaa Ser Xaa Ala Phe Lys Asp Arg
Gly Leu 1 5 10 6 2299 DNA Homo sapiens CDS (495)...(2129) 6
agtggttgct caatggcatt actggatcca ggttaggatt atctgagaat acatttatct
60 gcattttacg gaaggcaacc gaggttcata gagatagtga tttggccagc
gtcacagkcc 120 cagtaaaagg gattgaaaat ccaggtccgt ccgaccctaa
agcagggaac tctgcctagt 180 gtctcgctgt ggaatgttag ggatcctggg
gtacccttca gggtcttggc tcggaaggaa 240 aacattcccc tccgagggga
tggactatat taccaagggg gtggagccag atgcctgagg 300 gggtgtggcc
agagcctggg gcgtgtcaca gccgaagggg cagggcggca gcagcaggcg 360
tctaagtaac ttcagcgcct gcgcagaggc tccccagcgt cgccctaggc tgggactcta
420 gtaggtcttc ggctcagttt tggctgcagc gcccgcgtag atcgcttcgg
ccgggttcta 480 cgcccggctc aact atg agc cgg tgc gcc cag gcg gcg gaa
gtg gcg gcc 530 Met Ser Arg Cys Ala Gln Ala Ala Glu Val Ala Ala 1 5
10 aca gtg cca ggt gcc ggc gtc ggg aac gtg ggg ctg cgg ccg ccc atg
578 Thr Val Pro Gly Ala Gly Val Gly Asn Val Gly Leu Arg Pro Pro Met
15 20 25 gtg ccc cgt cag gcg tcc ttc ttc ccg ccg ccg gtg ccg aac
ccc ttc 626 Val Pro Arg Gln Ala Ser Phe Phe Pro Pro Pro Val Pro Asn
Pro Phe 30 35 40 gtg cag cag acg cag atc ggc tcc gcg agg cgg gtc
cag att gtc ctt 674 Val Gln Gln Thr Gln Ile Gly Ser Ala Arg Arg Val
Gln Ile Val Leu 45 50 55 60 ctt ggg att atc ttg ctt cca att cgt gtc
tta ttg gtt gcg tta att 722 Leu Gly Ile Ile Leu Leu Pro Ile Arg Val
Leu Leu Val Ala Leu Ile 65 70 75 tta tta ctt gca tgg cca ttt gct
gca att tca aca gta tgc tgt cct 770 Leu Leu Leu Ala Trp Pro Phe Ala
Ala Ile Ser Thr Val Cys Cys Pro 80 85 90 gaa aag ctg acc cac cca
ata act ggt tgg agg agg aaa att act caa 818 Glu Lys Leu Thr His Pro
Ile Thr Gly Trp Arg Arg Lys Ile Thr Gln 95 100 105 aca gct ttg aaa
ttt ctg ggt cgt gct atg ttc ttt tca atg gga ttt 866 Thr Ala Leu Lys
Phe Leu Gly Arg Ala Met Phe Phe Ser Met Gly Phe 110 115 120 ata gtt
gct gta aaa gga aag att gca agt cct ttg gaa gca cca gtt 914 Ile Val
Ala Val Lys Gly Lys Ile Ala Ser Pro Leu Glu Ala Pro Val 125 130 135
140 ttt gtt gct gcc cct cat tca aca ttc ttt gat gga att gcc tgt gtt
962 Phe Val Ala Ala Pro His Ser Thr Phe Phe Asp Gly Ile Ala Cys Val
145 150 155 gta gct ggg tta cct tct ata gta tct cga aat gag aat gca
caa gtc 1010 Val Ala Gly Leu Pro Ser Ile Val Ser Arg Asn Glu Asn
Ala Gln Val 160 165 170 cct ctg att ggc aga ctg tta cgg gct gtg caa
cca gtt ttg gtg tcc 1058 Pro Leu Ile Gly Arg Leu Leu Arg Ala Val
Gln Pro Val Leu Val Ser 175 180 185 cgt gta gat ccg gat tcc cga aaa
aac aca ata aat gaa ata ata aag 1106 Arg Val Asp Pro Asp Ser Arg
Lys Asn Thr Ile Asn Glu Ile Ile Lys 190 195 200 cga aca aca tca gga
gga gaa tgg ccc cag ata cta gtt ttc cca gaa 1154 Arg Thr Thr Ser
Gly Gly Glu Trp Pro Gln Ile Leu Val Phe Pro Glu 205 210 215 220 ggt
act tgt act aat cgt tcc tgt ttg att act ttt aaa cca gga gcc 1202
Gly Thr Cys Thr Asn Arg Ser Cys Leu Ile Thr Phe Lys Pro Gly Ala 225
230 235 ttc att cca gga gtt cca gtg cag cca gtc ctc ctc aga tac cca
aac 1250 Phe Ile Pro Gly Val Pro Val Gln Pro Val Leu Leu Arg Tyr
Pro Asn 240 245 250 aag ctg gat act gtg acc tgg aca tgg caa gga tat
aca ttc att cag 1298 Lys Leu Asp Thr Val Thr Trp Thr Trp Gln Gly
Tyr Thr Phe Ile Gln 255 260 265 ctt tgt atg ctt act ttc tgc cag ctc
ttc aca aag gta gaa gtt gag 1346 Leu Cys Met Leu Thr Phe Cys Gln
Leu Phe Thr Lys Val Glu Val Glu 270 275 280 ttt atg cca gtt caa gta
cca aat gat gaa gaa aaa aat gat cct gtc 1394 Phe Met Pro Val Gln
Val Pro Asn Asp Glu Glu Lys Asn Asp Pro Val 285 290 295 300 ctt ttt
gcc aat aaa gtc cgg aat tta atg gca gaa gct ctg gga ata 1442 Leu
Phe Ala Asn Lys Val Arg Asn Leu Met Ala Glu Ala Leu Gly Ile 305 310
315 cca gta aca gat cat acc tat gaa gac tgc aga ttg atg att tca gca
1490 Pro Val Thr Asp His Thr Tyr Glu Asp Cys Arg Leu Met Ile Ser
Ala 320 325 330 gga cag cta aca ttg cct atg gaa gct ggg ctg gtg gaa
ttt act aaa 1538 Gly Gln Leu Thr Leu Pro Met Glu Ala Gly Leu Val
Glu Phe Thr Lys 335 340 345 att agc cga aaa ttg aaa tta gat tgg gat
ggt gtt cgt aag cat ttg 1586 Ile Ser Arg Lys Leu Lys Leu Asp Trp
Asp Gly Val Arg Lys His Leu 350 355 360 gat gaa tat gca tct att gcg
agt tcc tca aaa gga gga aga att gga 1634 Asp Glu Tyr Ala Ser Ile
Ala Ser Ser Ser Lys Gly Gly Arg Ile Gly 365 370 375 380 att gaa gaa
ttc gcc aag tat tta aag ttg cct gtt tca gat gtc ttg 1682 Ile Glu
Glu Phe Ala Lys Tyr Leu Lys Leu Pro Val Ser Asp Val Leu 385 390 395
aga caa ctt ttt gca ctc ttt gac agg aac cat gat ggc agc att gac
1730 Arg Gln Leu Phe Ala Leu Phe Asp Arg Asn His Asp Gly Ser Ile
Asp 400 405 410 ttc cga gag tat gtg att ggc ctg gct gtc ttg tgc aac
cct tcc aac 1778 Phe Arg Glu Tyr Val Ile Gly Leu Ala Val Leu Cys
Asn Pro Ser Asn 415 420 425 aca gag gag atc atc cag gtg gca ttt aag
ctg ttt gac gtt gat gag 1826 Thr Glu Glu Ile Ile Gln Val Ala Phe
Lys Leu Phe Asp Val Asp Glu 430 435 440 gat ggc tac ata acg gag gaa
gag ttc tcc acc att cta cag gct tcc 1874 Asp Gly Tyr Ile Thr Glu
Glu Glu Phe Ser Thr Ile Leu Gln Ala Ser 445 450 455 460 ctt gga gtg
cct gac ctt gat
gtt tct ggt ctc ttc aag gaa ata gcc 1922 Leu Gly Val Pro Asp Leu
Asp Val Ser Gly Leu Phe Lys Glu Ile Ala 465 470 475 caa ggg gac tca
att tcc tat gag gaa ttt aaa agt ttt gcc tta aag 1970 Gln Gly Asp
Ser Ile Ser Tyr Glu Glu Phe Lys Ser Phe Ala Leu Lys 480 485 490 cat
cca gaa tat gct aag ata ttt aca aca tac cta gac ctc cag acg 2018
His Pro Glu Tyr Ala Lys Ile Phe Thr Thr Tyr Leu Asp Leu Gln Thr 495
500 505 tgc cat gtg ttt tca tta cca aaa gaa gtc cag aca acc ccc tcc
acc 2066 Cys His Val Phe Ser Leu Pro Lys Glu Val Gln Thr Thr Pro
Ser Thr 510 515 520 gcc agt aat aaa gtc agc cct gaa aag cat gaa gag
agt acc tca gac 2114 Ala Ser Asn Lys Val Ser Pro Glu Lys His Glu
Glu Ser Thr Ser Asp 525 530 535 540 aaa aaa gat gac tga aagcagtatt
tccaataagg aaaacacagt agcttttgct 2169 Lys Lys Asp Asp * tgaaattgta
aaggcactta ttgataatac ttttaatgtg ttggtaatga tgtttaaaat 2229
tgaaagattt ttaaaataaa aatgatagat tttcttacta aaaaaaaaaa aaaaaaaaaa
2289 aaaaaaaaaa 2299 7 544 PRT Homo sapiens 7 Met Ser Arg Cys Ala
Gln Ala Ala Glu Val Ala Ala Thr Val Pro Gly 1 5 10 15 Ala Gly Val
Gly Asn Val Gly Leu Arg Pro Pro Met Val Pro Arg Gln 20 25 30 Ala
Ser Phe Phe Pro Pro Pro Val Pro Asn Pro Phe Val Gln Gln Thr 35 40
45 Gln Ile Gly Ser Ala Arg Arg Val Gln Ile Val Leu Leu Gly Ile Ile
50 55 60 Leu Leu Pro Ile Arg Val Leu Leu Val Ala Leu Ile Leu Leu
Leu Ala 65 70 75 80 Trp Pro Phe Ala Ala Ile Ser Thr Val Cys Cys Pro
Glu Lys Leu Thr 85 90 95 His Pro Ile Thr Gly Trp Arg Arg Lys Ile
Thr Gln Thr Ala Leu Lys 100 105 110 Phe Leu Gly Arg Ala Met Phe Phe
Ser Met Gly Phe Ile Val Ala Val 115 120 125 Lys Gly Lys Ile Ala Ser
Pro Leu Glu Ala Pro Val Phe Val Ala Ala 130 135 140 Pro His Ser Thr
Phe Phe Asp Gly Ile Ala Cys Val Val Ala Gly Leu 145 150 155 160 Pro
Ser Ile Val Ser Arg Asn Glu Asn Ala Gln Val Pro Leu Ile Gly 165 170
175 Arg Leu Leu Arg Ala Val Gln Pro Val Leu Val Ser Arg Val Asp Pro
180 185 190 Asp Ser Arg Lys Asn Thr Ile Asn Glu Ile Ile Lys Arg Thr
Thr Ser 195 200 205 Gly Gly Glu Trp Pro Gln Ile Leu Val Phe Pro Glu
Gly Thr Cys Thr 210 215 220 Asn Arg Ser Cys Leu Ile Thr Phe Lys Pro
Gly Ala Phe Ile Pro Gly 225 230 235 240 Val Pro Val Gln Pro Val Leu
Leu Arg Tyr Pro Asn Lys Leu Asp Thr 245 250 255 Val Thr Trp Thr Trp
Gln Gly Tyr Thr Phe Ile Gln Leu Cys Met Leu 260 265 270 Thr Phe Cys
Gln Leu Phe Thr Lys Val Glu Val Glu Phe Met Pro Val 275 280 285 Gln
Val Pro Asn Asp Glu Glu Lys Asn Asp Pro Val Leu Phe Ala Asn 290 295
300 Lys Val Arg Asn Leu Met Ala Glu Ala Leu Gly Ile Pro Val Thr Asp
305 310 315 320 His Thr Tyr Glu Asp Cys Arg Leu Met Ile Ser Ala Gly
Gln Leu Thr 325 330 335 Leu Pro Met Glu Ala Gly Leu Val Glu Phe Thr
Lys Ile Ser Arg Lys 340 345 350 Leu Lys Leu Asp Trp Asp Gly Val Arg
Lys His Leu Asp Glu Tyr Ala 355 360 365 Ser Ile Ala Ser Ser Ser Lys
Gly Gly Arg Ile Gly Ile Glu Glu Phe 370 375 380 Ala Lys Tyr Leu Lys
Leu Pro Val Ser Asp Val Leu Arg Gln Leu Phe 385 390 395 400 Ala Leu
Phe Asp Arg Asn His Asp Gly Ser Ile Asp Phe Arg Glu Tyr 405 410 415
Val Ile Gly Leu Ala Val Leu Cys Asn Pro Ser Asn Thr Glu Glu Ile 420
425 430 Ile Gln Val Ala Phe Lys Leu Phe Asp Val Asp Glu Asp Gly Tyr
Ile 435 440 445 Thr Glu Glu Glu Phe Ser Thr Ile Leu Gln Ala Ser Leu
Gly Val Pro 450 455 460 Asp Leu Asp Val Ser Gly Leu Phe Lys Glu Ile
Ala Gln Gly Asp Ser 465 470 475 480 Ile Ser Tyr Glu Glu Phe Lys Ser
Phe Ala Leu Lys His Pro Glu Tyr 485 490 495 Ala Lys Ile Phe Thr Thr
Tyr Leu Asp Leu Gln Thr Cys His Val Phe 500 505 510 Ser Leu Pro Lys
Glu Val Gln Thr Thr Pro Ser Thr Ala Ser Asn Lys 515 520 525 Val Ser
Pro Glu Lys His Glu Glu Ser Thr Ser Asp Lys Lys Asp Asp 530 535 540
8 1632 DNA Homo sapiens CDS (1)...(1632) 8 atg agc cgg tgc gcc cag
gcg gcg gaa gtg gcg gcc aca gtg cca ggt 48 Met Ser Arg Cys Ala Gln
Ala Ala Glu Val Ala Ala Thr Val Pro Gly 1 5 10 15 gcc ggc gtc ggg
aac gtg ggg ctg cgg ccg ccc atg gtg ccc cgt cag 96 Ala Gly Val Gly
Asn Val Gly Leu Arg Pro Pro Met Val Pro Arg Gln 20 25 30 gcg tcc
ttc ttc ccg ccg ccg gtg ccg aac ccc ttc gtg cag cag acg 144 Ala Ser
Phe Phe Pro Pro Pro Val Pro Asn Pro Phe Val Gln Gln Thr 35 40 45
cag atc ggc tcc gcg agg cgg gtc cag att gtc ctt ctt ggg att atc 192
Gln Ile Gly Ser Ala Arg Arg Val Gln Ile Val Leu Leu Gly Ile Ile 50
55 60 ttg ctt cca att cgt gtc tta ttg gtt gcg tta att tta tta ctt
gca 240 Leu Leu Pro Ile Arg Val Leu Leu Val Ala Leu Ile Leu Leu Leu
Ala 65 70 75 80 tgg cca ttt gct gca att tca aca gta tgc tgt cct gaa
aag ctg acc 288 Trp Pro Phe Ala Ala Ile Ser Thr Val Cys Cys Pro Glu
Lys Leu Thr 85 90 95 cac cca ata act ggt tgg agg agg aaa att act
caa aca gct ttg aaa 336 His Pro Ile Thr Gly Trp Arg Arg Lys Ile Thr
Gln Thr Ala Leu Lys 100 105 110 ttt ctg ggt cgt gct atg ttc ttt tca
atg gga ttt ata gtt gct gta 384 Phe Leu Gly Arg Ala Met Phe Phe Ser
Met Gly Phe Ile Val Ala Val 115 120 125 aaa gga aag att gca agt cct
ttg gaa gca cca gtt ttt gtt gct gcc 432 Lys Gly Lys Ile Ala Ser Pro
Leu Glu Ala Pro Val Phe Val Ala Ala 130 135 140 cct cat tca aca ttc
ttt gat gga att gcc tgt gtt gta gct ggg tta 480 Pro His Ser Thr Phe
Phe Asp Gly Ile Ala Cys Val Val Ala Gly Leu 145 150 155 160 cct tct
ata gta tct cga aat gag aat gca caa gtc cct ctg att ggc 528 Pro Ser
Ile Val Ser Arg Asn Glu Asn Ala Gln Val Pro Leu Ile Gly 165 170 175
aga ctg tta cgg gct gtg caa cca gtt ttg gtg tcc cgt gta gat ccg 576
Arg Leu Leu Arg Ala Val Gln Pro Val Leu Val Ser Arg Val Asp Pro 180
185 190 gat tcc cga aaa aac aca ata aat gaa ata ata aag cga aca aca
tca 624 Asp Ser Arg Lys Asn Thr Ile Asn Glu Ile Ile Lys Arg Thr Thr
Ser 195 200 205 gga gga gaa tgg ccc cag ata cta gtt ttc cca gaa ggt
act tgt act 672 Gly Gly Glu Trp Pro Gln Ile Leu Val Phe Pro Glu Gly
Thr Cys Thr 210 215 220 aat cgt tcc tgt ttg att act ttt aaa cca gga
gcc ttc att cca gga 720 Asn Arg Ser Cys Leu Ile Thr Phe Lys Pro Gly
Ala Phe Ile Pro Gly 225 230 235 240 gtt cca gtg cag cca gtc ctc ctc
aga tac cca aac aag ctg gat act 768 Val Pro Val Gln Pro Val Leu Leu
Arg Tyr Pro Asn Lys Leu Asp Thr 245 250 255 gtg acc tgg aca tgg caa
gga tat aca ttc att cag ctt tgt atg ctt 816 Val Thr Trp Thr Trp Gln
Gly Tyr Thr Phe Ile Gln Leu Cys Met Leu 260 265 270 act ttc tgc cag
ctc ttc aca aag gta gaa gtt gag ttt atg cca gtt 864 Thr Phe Cys Gln
Leu Phe Thr Lys Val Glu Val Glu Phe Met Pro Val 275 280 285 caa gta
cca aat gat gaa gaa aaa aat gat cct gtc ctt ttt gcc aat 912 Gln Val
Pro Asn Asp Glu Glu Lys Asn Asp Pro Val Leu Phe Ala Asn 290 295 300
aaa gtc cgg aat tta atg gca gaa gct ctg gga ata cca gta aca gat 960
Lys Val Arg Asn Leu Met Ala Glu Ala Leu Gly Ile Pro Val Thr Asp 305
310 315 320 cat acc tat gaa gac tgc aga ttg atg att tca gca gga cag
cta aca 1008 His Thr Tyr Glu Asp Cys Arg Leu Met Ile Ser Ala Gly
Gln Leu Thr 325 330 335 ttg cct atg gaa gct ggg ctg gtg gaa ttt act
aaa att agc cga aaa 1056 Leu Pro Met Glu Ala Gly Leu Val Glu Phe
Thr Lys Ile Ser Arg Lys 340 345 350 ttg aaa tta gat tgg gat ggt gtt
cgt aag cat ttg gat gaa tat gca 1104 Leu Lys Leu Asp Trp Asp Gly
Val Arg Lys His Leu Asp Glu Tyr Ala 355 360 365 tct att gcg agt tcc
tca aaa gga gga aga att gga att gaa gaa ttc 1152 Ser Ile Ala Ser
Ser Ser Lys Gly Gly Arg Ile Gly Ile Glu Glu Phe 370 375 380 gcc aag
tat tta aag ttg cct gtt tca gat gtc ttg aga caa ctt ttt 1200 Ala
Lys Tyr Leu Lys Leu Pro Val Ser Asp Val Leu Arg Gln Leu Phe 385 390
395 400 gca ctc ttt gac agg aac cat gat ggc agc att gac ttc cga gag
tat 1248 Ala Leu Phe Asp Arg Asn His Asp Gly Ser Ile Asp Phe Arg
Glu Tyr 405 410 415 gtg att ggc ctg gct gtc ttg tgc aac cct tcc aac
aca gag gag atc 1296 Val Ile Gly Leu Ala Val Leu Cys Asn Pro Ser
Asn Thr Glu Glu Ile 420 425 430 atc cag gtg gca ttt aag ctg ttt gac
gtt gat gag gat ggc tac ata 1344 Ile Gln Val Ala Phe Lys Leu Phe
Asp Val Asp Glu Asp Gly Tyr Ile 435 440 445 acg gag gaa gag ttc tcc
acc att cta cag gct tcc ctt gga gtg cct 1392 Thr Glu Glu Glu Phe
Ser Thr Ile Leu Gln Ala Ser Leu Gly Val Pro 450 455 460 gac ctt gat
gtt tct ggt ctc ttc aag gaa ata gcc caa ggg gac tca 1440 Asp Leu
Asp Val Ser Gly Leu Phe Lys Glu Ile Ala Gln Gly Asp Ser 465 470 475
480 att tcc tat gag gaa ttt aaa agt ttt gcc tta aag cat cca gaa tat
1488 Ile Ser Tyr Glu Glu Phe Lys Ser Phe Ala Leu Lys His Pro Glu
Tyr 485 490 495 gct aag ata ttt aca aca tac cta gac ctc cag acg tgc
cat gtg ttt 1536 Ala Lys Ile Phe Thr Thr Tyr Leu Asp Leu Gln Thr
Cys His Val Phe 500 505 510 tca tta cca aaa gaa gtc cag aca acc ccc
tcc acc gcc agt aat aaa 1584 Ser Leu Pro Lys Glu Val Gln Thr Thr
Pro Ser Thr Ala Ser Asn Lys 515 520 525 gtc agc cct gaa aag cat gaa
gag agt acc tca gac aaa aaa gat gac 1632 Val Ser Pro Glu Lys His
Glu Glu Ser Thr Ser Asp Lys Lys Asp Asp 530 535 540 9 195 PRT
Artificial Sequence Prodom consensus sequence for Acyltransferase 9
Leu Glu Asn Leu Pro Lys Lys Gly Pro Ala Ile Val Val Ser Asn His 1 5
10 15 Arg Ser Tyr Leu Asp Ile Leu Val Leu Ser Ala Ala Leu Pro Arg
Arg 20 25 30 Gly Pro Trp Leu Val Arg Arg Leu Val Phe Ile Ala Lys
Lys Glu Leu 35 40 45 Leu Lys Val Pro Leu Leu Phe Gly Trp Leu Met
Arg Leu Ala Gly Ala 50 55 60 Ile Phe Ile Asp Arg Asn Asn Arg Ala
Lys Asp Ala Leu Ala Ala Ala 65 70 75 80 Asp Glu Leu Val Arg Val Leu
Glu Leu Leu Arg Lys Gly Arg Ser Val 85 90 95 Leu Ile Phe Pro Glu
Gly Thr Arg Ser Arg Ser Gly Glu Leu Leu Pro 100 105 110 Pro Phe Lys
Lys Gly Ile Ala Ala Phe Arg Leu Ala Leu Lys Ala Gly 115 120 125 Val
Pro Ile Val Pro Val Val Ile Val Ser Gly Thr Glu Glu Leu Glu 130 135
140 Pro Lys Asn Glu Ala Gly Lys Leu Leu Arg Leu Ala Arg Lys Lys Gly
145 150 155 160 Pro Val Thr Val Arg Val Leu Pro Pro Ile Pro Leu Asp
Pro Glu Asp 165 170 175 Ile Lys Glu Leu Ala Glu Arg Leu Arg Asp Ile
Leu Val Gln Ala Leu 180 185 190 Glu Glu Leu 195 10 2547 DNA Homo
sapiens CDS (63)...(2324) 10 gctttttgtg ggccgggtgg gtttcctaat
ctggtttcgt ctgcttggtt catctgtgtg 60 cg atg gct ccg gac tcg gat ccc
ttc cct gaa ggg ccg ctc tta aag 107 Met Ala Pro Asp Ser Asp Pro Phe
Pro Glu Gly Pro Leu Leu Lys 1 5 10 15 ctg cta ccc tta gac gct aga
gac cgg ggc acc cag cgc tgc cgc ctg 155 Leu Leu Pro Leu Asp Ala Arg
Asp Arg Gly Thr Gln Arg Cys Arg Leu 20 25 30 ggc ccg gcc gcc ctc
cac gcc ctg ggc gcg cgc ttg ggc tcg gca gtg 203 Gly Pro Ala Ala Leu
His Ala Leu Gly Ala Arg Leu Gly Ser Ala Val 35 40 45 aag atc tcg
cta ccc gac ggc ggc tcc tgc ctc tgc act gcc tgg cct 251 Lys Ile Ser
Leu Pro Asp Gly Gly Ser Cys Leu Cys Thr Ala Trp Pro 50 55 60 cgg
cgg gac gga gcg gac ggc ttt gtg cag ctg gac ccg ctg tgc gcg 299 Arg
Arg Asp Gly Ala Asp Gly Phe Val Gln Leu Asp Pro Leu Cys Ala 65 70
75 agc ccc ggg gcg gcg gtc ggg gcg tcg aga tcc cgg agg agt ctc agc
347 Ser Pro Gly Ala Ala Val Gly Ala Ser Arg Ser Arg Arg Ser Leu Ser
80 85 90 95 ctg aat cgc ctc ctc cta gtg ccc tgt ccg ccc ctg cgg cgc
gtc gcc 395 Leu Asn Arg Leu Leu Leu Val Pro Cys Pro Pro Leu Arg Arg
Val Ala 100 105 110 gtg tgg ccg gtg ttg cga gag cgg gca ggc gcg ccc
ggt gcc cgg aat 443 Val Trp Pro Val Leu Arg Glu Arg Ala Gly Ala Pro
Gly Ala Arg Asn 115 120 125 aca gcc gcg gtg ctg gag gcg gca cag gag
ctg ctg aga aac cga ccg 491 Thr Ala Ala Val Leu Glu Ala Ala Gln Glu
Leu Leu Arg Asn Arg Pro 130 135 140 atc tcc ctg ggc cac gtg gtg gtc
gct ccg cca ggc gct cct ggc ctg 539 Ile Ser Leu Gly His Val Val Val
Ala Pro Pro Gly Ala Pro Gly Leu 145 150 155 gtg gct gcc ttg cac atc
gtc ggc ggg acg ccc agt ccc gat ccc gct 587 Val Ala Ala Leu His Ile
Val Gly Gly Thr Pro Ser Pro Asp Pro Ala 160 165 170 175 ggg ctg gtc
acc cct cgt acc cgc gtc agc ctt ggc ggg gag cct ccg 635 Gly Leu Val
Thr Pro Arg Thr Arg Val Ser Leu Gly Gly Glu Pro Pro 180 185 190 tcg
gaa gcc cag ccg cag ccc gag gtg ccc ctg gga ggt ctt tcg gag 683 Ser
Glu Ala Gln Pro Gln Pro Glu Val Pro Leu Gly Gly Leu Ser Glu 195 200
205 gcg gcc gac tcg ctg cgg gag ctc ctc cgc ctc ccg ctc cgc tac ccg
731 Ala Ala Asp Ser Leu Arg Glu Leu Leu Arg Leu Pro Leu Arg Tyr Pro
210 215 220 cgc gcc ctg acc gcg ctg ggc tta gcg gtg cct cgc ggg gtg
ctc ctg 779 Arg Ala Leu Thr Ala Leu Gly Leu Ala Val Pro Arg Gly Val
Leu Leu 225 230 235 gcg ggg ccc ccc gga gtg ggc aag acc cag ctg gtg
cag gcc gtg gcg 827 Ala Gly Pro Pro Gly Val Gly Lys Thr Gln Leu Val
Gln Ala Val Ala 240 245 250 255 cgc gag gcg ggc gcg gag ctg ctg gca
gtc agc gcc ccg gcg ctg cag 875 Arg Glu Ala Gly Ala Glu Leu Leu Ala
Val Ser Ala Pro Ala Leu Gln 260 265 270 ggt tcc cgg cct ggg gag acc
gag gag aac gtg cgg cgg gtc ttc cag 923 Gly Ser Arg Pro Gly Glu Thr
Glu Glu Asn Val Arg Arg Val Phe Gln 275 280 285 cgc gcc cgg gaa ctg
gcc agc cgc gga ccc agc ctc ctc ttc ctg gac 971 Arg Ala Arg Glu Leu
Ala Ser Arg Gly Pro Ser Leu Leu Phe Leu Asp 290 295 300 gag atg gac
gcc ttg tgt ccc cag cgg ggc agt cga gca ccc gag agc 1019 Glu Met
Asp Ala Leu Cys Pro Gln Arg Gly Ser Arg Ala Pro Glu Ser 305 310 315
cgc gta gtg gcc cag gtg ttg acg ctg ctg gac ggc gcc agt ggg gac
1067 Arg Val Val Ala Gln Val Leu Thr Leu Leu Asp Gly Ala Ser Gly
Asp 320 325 330 335 cgc gag gtc gtg gtt gtg gga gcc act aac cgg ccg
gac gct cta gac 1115 Arg Glu Val Val Val Val Gly Ala Thr Asn Arg
Pro Asp Ala Leu Asp 340 345 350 cca gcg ctg cgt aga ccc ggg aga ttt
gac cga gag gtg gtc att ggg 1163 Pro Ala Leu Arg Arg Pro Gly Arg
Phe Asp Arg Glu Val Val Ile Gly 355 360 365 act ccc aca ctt aaa caa
aga aag gaa att ctg caa gtg att acc tcg 1211 Thr Pro Thr Leu Lys
Gln Arg Lys Glu Ile Leu Gln Val Ile Thr Ser 370 375 380 aag atg ccc
atc tcc agt cat gtt gat ttg ggc ctt ctt gca gaa atg 1259 Lys Met
Pro
Ile Ser Ser His Val Asp Leu Gly Leu Leu Ala Glu Met 385 390 395 aca
gtt ggc tat gtt ggt gcc gac ctg aca gca ctc tgt agg gag gct 1307
Thr Val Gly Tyr Val Gly Ala Asp Leu Thr Ala Leu Cys Arg Glu Ala 400
405 410 415 gcc atg cat gcc ctc ctt cat agt gag aag aac cag gac aat
cct gtg 1355 Ala Met His Ala Leu Leu His Ser Glu Lys Asn Gln Asp
Asn Pro Val 420 425 430 att gat gaa ata gac ttc ctt gaa gct ttt aaa
aat att cag ccc tca 1403 Ile Asp Glu Ile Asp Phe Leu Glu Ala Phe
Lys Asn Ile Gln Pro Ser 435 440 445 tcg ttt cga agc gtc att gga tta
atg gat atc aag cct gtt gac tgg 1451 Ser Phe Arg Ser Val Ile Gly
Leu Met Asp Ile Lys Pro Val Asp Trp 450 455 460 gag gag att ggt ggc
ctt gaa gat gta aaa ctg aag tta aaa cag agc 1499 Glu Glu Ile Gly
Gly Leu Glu Asp Val Lys Leu Lys Leu Lys Gln Ser 465 470 475 att gag
tgg cct ctg aaa ttc cct tgg gaa ttt gtt aga atg ggc ctg 1547 Ile
Glu Trp Pro Leu Lys Phe Pro Trp Glu Phe Val Arg Met Gly Leu 480 485
490 495 aca caa cca aag gga gtt ctc ctc tat ggg ccc cct gga tgt gct
aaa 1595 Thr Gln Pro Lys Gly Val Leu Leu Tyr Gly Pro Pro Gly Cys
Ala Lys 500 505 510 acc act ctg gtg agg gcc ctg gcc aca agc tgt cac
tgc tct ttc gtt 1643 Thr Thr Leu Val Arg Ala Leu Ala Thr Ser Cys
His Cys Ser Phe Val 515 520 525 tca gtg agt gga gct gat ctg ttt tca
ccg ttt gtt gga gat tca gaa 1691 Ser Val Ser Gly Ala Asp Leu Phe
Ser Pro Phe Val Gly Asp Ser Glu 530 535 540 aaa gtg ttg tct cag ata
ttt cga caa gca aga gca agc act cca gca 1739 Lys Val Leu Ser Gln
Ile Phe Arg Gln Ala Arg Ala Ser Thr Pro Ala 545 550 555 att ttg ttt
ttg gat gaa att gat tca atc ttg gga gct cgc tca gcc 1787 Ile Leu
Phe Leu Asp Glu Ile Asp Ser Ile Leu Gly Ala Arg Ser Ala 560 565 570
575 agc aag aca gga tgt gat gtt caa gaa cga gtt ctt tct gtt ctc ctg
1835 Ser Lys Thr Gly Cys Asp Val Gln Glu Arg Val Leu Ser Val Leu
Leu 580 585 590 aat gaa tta gat ggt gtt gga ctt aag aca ata gag aga
aga gga agt 1883 Asn Glu Leu Asp Gly Val Gly Leu Lys Thr Ile Glu
Arg Arg Gly Ser 595 600 605 aaa tca agt caa cag gag ttt caa gaa gtt
ttt aac cga agt gtc atg 1931 Lys Ser Ser Gln Gln Glu Phe Gln Glu
Val Phe Asn Arg Ser Val Met 610 615 620 att att gca gca aca aat aga
cct gat gtg tta gat act gct ttg tta 1979 Ile Ile Ala Ala Thr Asn
Arg Pro Asp Val Leu Asp Thr Ala Leu Leu 625 630 635 cga cct gga aga
tta gat aag atc atc tat atc cca cct cca gat cac 2027 Arg Pro Gly
Arg Leu Asp Lys Ile Ile Tyr Ile Pro Pro Pro Asp His 640 645 650 655
aag ggc agg ctt tct att tta aaa gtc tgt aca aaa acc atg cca ata
2075 Lys Gly Arg Leu Ser Ile Leu Lys Val Cys Thr Lys Thr Met Pro
Ile 660 665 670 ggg cct gat gtc tcc tta gaa aac ctc gca gca gaa acc
tgt ttt ttt 2123 Gly Pro Asp Val Ser Leu Glu Asn Leu Ala Ala Glu
Thr Cys Phe Phe 675 680 685 tct gga gct gat ctt aga aac ctc tgc aca
gaa gct gct ttg ctg gct 2171 Ser Gly Ala Asp Leu Arg Asn Leu Cys
Thr Glu Ala Ala Leu Leu Ala 690 695 700 ctg caa gaa aat gga cta gac
gca act aca gtg aaa caa gag cac ttt 2219 Leu Gln Glu Asn Gly Leu
Asp Ala Thr Thr Val Lys Gln Glu His Phe 705 710 715 cta aaa tca ctt
aag act gta aaa ccg tcg tta agt tgc aag gac ttg 2267 Leu Lys Ser
Leu Lys Thr Val Lys Pro Ser Leu Ser Cys Lys Asp Leu 720 725 730 735
gct tta tat gaa aac tta ttt aag aaa gaa gga ttt tct aac gtg gaa
2315 Ala Leu Tyr Glu Asn Leu Phe Lys Lys Glu Gly Phe Ser Asn Val
Glu 740 745 750 ggt att taa aaatcacctt aaactcttgt tcagttcaca
ttaattgaaa 2364 Gly Ile * tgtgaacttg cctgtcgttt gcaacttcac
acttttagaa tttgtgttta tatttcctgt 2424 aagtgaataa ataaaacaaa
acaaaacaaa aaaaacttgt gcctgataag ctaaggctca 2484 tttattttta
aaaggcatat taaataaaat actgtaattt aggaagaaaa aaaaaaaaaa 2544 aaa
2547 11 753 PRT Homo sapiens 11 Met Ala Pro Asp Ser Asp Pro Phe Pro
Glu Gly Pro Leu Leu Lys Leu 1 5 10 15 Leu Pro Leu Asp Ala Arg Asp
Arg Gly Thr Gln Arg Cys Arg Leu Gly 20 25 30 Pro Ala Ala Leu His
Ala Leu Gly Ala Arg Leu Gly Ser Ala Val Lys 35 40 45 Ile Ser Leu
Pro Asp Gly Gly Ser Cys Leu Cys Thr Ala Trp Pro Arg 50 55 60 Arg
Asp Gly Ala Asp Gly Phe Val Gln Leu Asp Pro Leu Cys Ala Ser 65 70
75 80 Pro Gly Ala Ala Val Gly Ala Ser Arg Ser Arg Arg Ser Leu Ser
Leu 85 90 95 Asn Arg Leu Leu Leu Val Pro Cys Pro Pro Leu Arg Arg
Val Ala Val 100 105 110 Trp Pro Val Leu Arg Glu Arg Ala Gly Ala Pro
Gly Ala Arg Asn Thr 115 120 125 Ala Ala Val Leu Glu Ala Ala Gln Glu
Leu Leu Arg Asn Arg Pro Ile 130 135 140 Ser Leu Gly His Val Val Val
Ala Pro Pro Gly Ala Pro Gly Leu Val 145 150 155 160 Ala Ala Leu His
Ile Val Gly Gly Thr Pro Ser Pro Asp Pro Ala Gly 165 170 175 Leu Val
Thr Pro Arg Thr Arg Val Ser Leu Gly Gly Glu Pro Pro Ser 180 185 190
Glu Ala Gln Pro Gln Pro Glu Val Pro Leu Gly Gly Leu Ser Glu Ala 195
200 205 Ala Asp Ser Leu Arg Glu Leu Leu Arg Leu Pro Leu Arg Tyr Pro
Arg 210 215 220 Ala Leu Thr Ala Leu Gly Leu Ala Val Pro Arg Gly Val
Leu Leu Ala 225 230 235 240 Gly Pro Pro Gly Val Gly Lys Thr Gln Leu
Val Gln Ala Val Ala Arg 245 250 255 Glu Ala Gly Ala Glu Leu Leu Ala
Val Ser Ala Pro Ala Leu Gln Gly 260 265 270 Ser Arg Pro Gly Glu Thr
Glu Glu Asn Val Arg Arg Val Phe Gln Arg 275 280 285 Ala Arg Glu Leu
Ala Ser Arg Gly Pro Ser Leu Leu Phe Leu Asp Glu 290 295 300 Met Asp
Ala Leu Cys Pro Gln Arg Gly Ser Arg Ala Pro Glu Ser Arg 305 310 315
320 Val Val Ala Gln Val Leu Thr Leu Leu Asp Gly Ala Ser Gly Asp Arg
325 330 335 Glu Val Val Val Val Gly Ala Thr Asn Arg Pro Asp Ala Leu
Asp Pro 340 345 350 Ala Leu Arg Arg Pro Gly Arg Phe Asp Arg Glu Val
Val Ile Gly Thr 355 360 365 Pro Thr Leu Lys Gln Arg Lys Glu Ile Leu
Gln Val Ile Thr Ser Lys 370 375 380 Met Pro Ile Ser Ser His Val Asp
Leu Gly Leu Leu Ala Glu Met Thr 385 390 395 400 Val Gly Tyr Val Gly
Ala Asp Leu Thr Ala Leu Cys Arg Glu Ala Ala 405 410 415 Met His Ala
Leu Leu His Ser Glu Lys Asn Gln Asp Asn Pro Val Ile 420 425 430 Asp
Glu Ile Asp Phe Leu Glu Ala Phe Lys Asn Ile Gln Pro Ser Ser 435 440
445 Phe Arg Ser Val Ile Gly Leu Met Asp Ile Lys Pro Val Asp Trp Glu
450 455 460 Glu Ile Gly Gly Leu Glu Asp Val Lys Leu Lys Leu Lys Gln
Ser Ile 465 470 475 480 Glu Trp Pro Leu Lys Phe Pro Trp Glu Phe Val
Arg Met Gly Leu Thr 485 490 495 Gln Pro Lys Gly Val Leu Leu Tyr Gly
Pro Pro Gly Cys Ala Lys Thr 500 505 510 Thr Leu Val Arg Ala Leu Ala
Thr Ser Cys His Cys Ser Phe Val Ser 515 520 525 Val Ser Gly Ala Asp
Leu Phe Ser Pro Phe Val Gly Asp Ser Glu Lys 530 535 540 Val Leu Ser
Gln Ile Phe Arg Gln Ala Arg Ala Ser Thr Pro Ala Ile 545 550 555 560
Leu Phe Leu Asp Glu Ile Asp Ser Ile Leu Gly Ala Arg Ser Ala Ser 565
570 575 Lys Thr Gly Cys Asp Val Gln Glu Arg Val Leu Ser Val Leu Leu
Asn 580 585 590 Glu Leu Asp Gly Val Gly Leu Lys Thr Ile Glu Arg Arg
Gly Ser Lys 595 600 605 Ser Ser Gln Gln Glu Phe Gln Glu Val Phe Asn
Arg Ser Val Met Ile 610 615 620 Ile Ala Ala Thr Asn Arg Pro Asp Val
Leu Asp Thr Ala Leu Leu Arg 625 630 635 640 Pro Gly Arg Leu Asp Lys
Ile Ile Tyr Ile Pro Pro Pro Asp His Lys 645 650 655 Gly Arg Leu Ser
Ile Leu Lys Val Cys Thr Lys Thr Met Pro Ile Gly 660 665 670 Pro Asp
Val Ser Leu Glu Asn Leu Ala Ala Glu Thr Cys Phe Phe Ser 675 680 685
Gly Ala Asp Leu Arg Asn Leu Cys Thr Glu Ala Ala Leu Leu Ala Leu 690
695 700 Gln Glu Asn Gly Leu Asp Ala Thr Thr Val Lys Gln Glu His Phe
Leu 705 710 715 720 Lys Ser Leu Lys Thr Val Lys Pro Ser Leu Ser Cys
Lys Asp Leu Ala 725 730 735 Leu Tyr Glu Asn Leu Phe Lys Lys Glu Gly
Phe Ser Asn Val Glu Gly 740 745 750 Ile 12 2262 DNA Homo sapiens 12
atggctccgg actcggatcc cttccctgaa gggccgctct taaagctgct acccttagac
60 gctagagacc ggggcaccca gcgctgccgc ctgggcccgg ccgccctcca
cgccctgggc 120 gcgcgcttgg gctcggcagt gaagatctcg ctacccgacg
gcggctcctg cctctgcact 180 gcctggcctc ggcgggacgg agcggacggc
tttgtgcagc tggacccgct gtgcgcgagc 240 cccggggcgg cggtcggggc
gtcgagatcc cggaggagtc tcagcctgaa tcgcctcctc 300 ctagtgccct
gtccgcccct gcggcgcgtc gccgtgtggc cggtgttgcg agagcgggca 360
ggcgcgcccg gtgcccggaa tacagccgcg gtgctggagg cggcacagga gctgctgaga
420 aaccgaccga tctccctggg ccacgtggtg gtcgctccgc caggcgctcc
tggcctggtg 480 gctgccttgc acatcgtcgg cgggacgccc agtcccgatc
ccgctgggct ggtcacccct 540 cgtacccgcg tcagccttgg cggggagcct
ccgtcggaag cccagccgca gcccgaggtg 600 cccctgggag gtctttcgga
ggcggccgac tcgctgcggg agctcctccg cctcccgctc 660 cgctacccgc
gcgccctgac cgcgctgggc ttagcggtgc ctcgcggggt gctcctggcg 720
gggccccccg gagtgggcaa gacccagctg gtgcaggccg tggcgcgcga ggcgggcgcg
780 gagctgctgg cagtcagcgc cccggcgctg cagggttccc ggcctgggga
gaccgaggag 840 aacgtgcggc gggtcttcca gcgcgcccgg gaactggcca
gccgcggacc cagcctcctc 900 ttcctggacg agatggacgc cttgtgtccc
cagcggggca gtcgagcacc cgagagccgc 960 gtagtggccc aggtgttgac
gctgctggac ggcgccagtg gggaccgcga ggtcgtggtt 1020 gtgggagcca
ctaaccggcc ggacgctcta gacccagcgc tgcgtagacc cgggagattt 1080
gaccgagagg tggtcattgg gactcccaca cttaaacaaa gaaaggaaat tctgcaagtg
1140 attacctcga agatgcccat ctccagtcat gttgatttgg gccttcttgc
agaaatgaca 1200 gttggctatg ttggtgccga cctgacagca ctctgtaggg
aggctgccat gcatgccctc 1260 cttcatagtg agaagaacca ggacaatcct
gtgattgatg aaatagactt ccttgaagct 1320 tttaaaaata ttcagccctc
atcgtttcga agcgtcattg gattaatgga tatcaagcct 1380 gttgactggg
aggagattgg tggccttgaa gatgtaaaac tgaagttaaa acagagcatt 1440
gagtggcctc tgaaattccc ttgggaattt gttagaatgg gcctgacaca accaaaggga
1500 gttctcctct atgggccccc tggatgtgct aaaaccactc tggtgagggc
cctggccaca 1560 agctgtcact gctctttcgt ttcagtgagt ggagctgatc
tgttttcacc gtttgttgga 1620 gattcagaaa aagtgttgtc tcagatattt
cgacaagcaa gagcaagcac tccagcaatt 1680 ttgtttttgg atgaaattga
ttcaatcttg ggagctcgct cagccagcaa gacaggatgt 1740 gatgttcaag
aacgagttct ttctgttctc ctgaatgaat tagatggtgt tggacttaag 1800
acaatagaga gaagaggaag taaatcaagt caacaggagt ttcaagaagt ttttaaccga
1860 agtgtcatga ttattgcagc aacaaataga cctgatgtgt tagatactgc
tttgttacga 1920 cctggaagat tagataagat catctatatc ccacctccag
atcacaaggg caggctttct 1980 attttaaaag tctgtacaaa aaccatgcca
atagggcctg atgtctcctt agaaaacctc 2040 gcagcagaaa cctgtttttt
ttctggagct gatcttagaa acctctgcac agaagctgct 2100 ttgctggctc
tgcaagaaaa tggactagac gcaactacag tgaaacaaga gcactttcta 2160
aaatcactta agactgtaaa accgtcgtta agttgcaagg acttggcttt atatgaaaac
2220 ttatttaaga aagaaggatt ttctaacgtg gaaggtattt aa 2262 13 216 PRT
Artificial Sequence Consensus for AAA ATPase domain 13 Gly Val Leu
Leu Tyr Gly Pro Pro Gly Thr Gly Lys Thr Leu Leu Ala 1 5 10 15 Lys
Ala Val Ala Lys Glu Leu Gly Val Pro Phe Ile Ser Ile Ser Gly 20 25
30 Ser Glu Leu Val Ser Lys Tyr Val Gly Glu Ser Glu Lys Arg Val Arg
35 40 45 Ala Leu Phe Glu Leu Ala Arg Lys Ser Leu Lys Lys Ala Ala
Pro Ser 50 55 60 Pro Ile Ile Phe Ile Asp Glu Ile Asp Ala Leu Ala
Pro Lys Arg Gly 65 70 75 80 Asp Glu Gly Asp Val Ser Glu Arg Val Val
Asn Gln Leu Leu Thr Glu 85 90 95 Met Asp Leu Glu Arg Ile Gly Phe
Glu Lys His Tyr Leu Arg Val Ser 100 105 110 Asp Val Val Asp Leu Ser
Gly Val Ile Val Ile Ala Ala Thr Asn Arg 115 120 125 Pro Asp Leu Leu
Asp Pro Ala Leu Leu Arg Pro Gly Arg Phe Asp Arg 130 135 140 Arg Ile
Glu Val Pro Leu Pro Asp Glu Glu Glu Arg Leu Glu Ile Leu 145 150 155
160 Lys Ile His Leu Lys Lys Met Pro Leu Ala Leu Cys Gln Glu Arg Ser
165 170 175 Glu Leu Ala Lys Asp Val Asp Leu Asp Glu Leu Ala Lys Glu
Leu Ala 180 185 190 Arg Arg Thr Pro Gly Phe Ser Gly Ala Asp Leu Ala
Ala Leu Cys Arg 195 200 205 Glu Ala Ala Leu Arg Ala Leu Arg 210 215
14 2127 DNA Homo sapiens CDS (94)...(1665) misc_feature 1895 n =
A,T,C or G 14 cgccggggcc gggtagctgc tccaggcgcg cgagctaacc
gagtgcggcg agggcctacc 60 aggggcgaca gggtttctct ccgcaagcgc gcg atg
cag cgg gcg gcg acg ctg 114 Met Gln Arg Ala Ala Thr Leu 1 5 gtc cgg
cgg ggc tgt ggt ccc cgg acc ccc agc tcc tgg ggc cgc agc 162 Val Arg
Arg Gly Cys Gly Pro Arg Thr Pro Ser Ser Trp Gly Arg Ser 10 15 20
cag agc agc gcg gcc gcc gag gcc tcg gcg gtg ctc aag gtg cgg ccc 210
Gln Ser Ser Ala Ala Ala Glu Ala Ser Ala Val Leu Lys Val Arg Pro 25
30 35 gag cgc agc cgg cgc gag cgc atc ctc acg ctg gag tcc atg aac
ccg 258 Glu Arg Ser Arg Arg Glu Arg Ile Leu Thr Leu Glu Ser Met Asn
Pro 40 45 50 55 cag gtg aag gcg gtg gag tac gcc gtg cgg gga ccc atc
gtg ctc aag 306 Gln Val Lys Ala Val Glu Tyr Ala Val Arg Gly Pro Ile
Val Leu Lys 60 65 70 gcc ggc gag atc gag ctc gag ctg cag cgg ggt
atc aaa aag cca ttc 354 Ala Gly Glu Ile Glu Leu Glu Leu Gln Arg Gly
Ile Lys Lys Pro Phe 75 80 85 aca gag gtc atc cga gcc aac atc ggg
gac gcc cag gct atg ggg cag 402 Thr Glu Val Ile Arg Ala Asn Ile Gly
Asp Ala Gln Ala Met Gly Gln 90 95 100 cag cca atc acc ttc ctc cgg
cag gtg atg gca cta tgc acc tac cca 450 Gln Pro Ile Thr Phe Leu Arg
Gln Val Met Ala Leu Cys Thr Tyr Pro 105 110 115 aac ctg ctg gac agc
ccc agc ttc cca gaa gat gct aag aaa cgt gcc 498 Asn Leu Leu Asp Ser
Pro Ser Phe Pro Glu Asp Ala Lys Lys Arg Ala 120 125 130 135 cgg cgg
atc ctg cag gct tgt ggc ggg aac agc ctg ggg tcc tac agt 546 Arg Arg
Ile Leu Gln Ala Cys Gly Gly Asn Ser Leu Gly Ser Tyr Ser 140 145 150
gct agc cag ggt gtc aac tgc atc cgt gaa gat gtg gct gcc tac atc 594
Ala Ser Gln Gly Val Asn Cys Ile Arg Glu Asp Val Ala Ala Tyr Ile 155
160 165 acc agg agg gat ggc ggt gtg cct gcg gac ccc gac aac atc tac
ctg 642 Thr Arg Arg Asp Gly Gly Val Pro Ala Asp Pro Asp Asn Ile Tyr
Leu 170 175 180 acc acg gga gct agt gac ggc att tct acg atc ctg aag
atc ctc gtc 690 Thr Thr Gly Ala Ser Asp Gly Ile Ser Thr Ile Leu Lys
Ile Leu Val 185 190 195 tcc ggg ggc ggc aag tca cgg aca ggt gtg atg
atc ccc atc cca caa 738 Ser Gly Gly Gly Lys Ser Arg Thr Gly Val Met
Ile Pro Ile Pro Gln 200 205 210 215 tat ccc ctc tat tca gct gtc atc
tct gag ctc gac gcc atc cag gtg 786 Tyr Pro Leu Tyr Ser Ala Val Ile
Ser Glu Leu Asp Ala Ile Gln Val 220 225 230 aat tac tac ctg gac gag
gag aac tgc tgg gcg ctg aat gtg aat gag 834 Asn Tyr Tyr Leu Asp Glu
Glu Asn Cys Trp Ala Leu Asn Val Asn Glu 235 240 245 ctc cgg cgg gcg
gtg cag gag gcc aaa gac cac tgt gat cct aag gtg 882 Leu Arg Arg Ala
Val Gln Glu Ala Lys Asp His Cys Asp Pro Lys Val 250 255 260 ctc tgc
ata
atc aac cct ggg aac ccc aca ggc cag gta caa agc aga 930 Leu Cys Ile
Ile Asn Pro Gly Asn Pro Thr Gly Gln Val Gln Ser Arg 265 270 275 aag
tgc ata gaa gat gtg atc cac ttt gcc tgg gaa gag aag ctc ttt 978 Lys
Cys Ile Glu Asp Val Ile His Phe Ala Trp Glu Glu Lys Leu Phe 280 285
290 295 ctc ctg gct gat gag gtg tac cag gac aac gtg tac tct cca gat
tgc 1026 Leu Leu Ala Asp Glu Val Tyr Gln Asp Asn Val Tyr Ser Pro
Asp Cys 300 305 310 aga ttc cac tcc ttc aag aag gtg ctg tac gag atg
ggg ccc gag tac 1074 Arg Phe His Ser Phe Lys Lys Val Leu Tyr Glu
Met Gly Pro Glu Tyr 315 320 325 tcc agc aac gtg gag ctc gcc tcc ttc
cac tcc acc tcc aag ggc tac 1122 Ser Ser Asn Val Glu Leu Ala Ser
Phe His Ser Thr Ser Lys Gly Tyr 330 335 340 atg ggc gag tgt ggt tac
aga gga ggc tac atg gag gtg atc aac ctg 1170 Met Gly Glu Cys Gly
Tyr Arg Gly Gly Tyr Met Glu Val Ile Asn Leu 345 350 355 cac cct gag
atc aag ggc cag ctg gtg aag ctg ctg tcg gtg cgc ctg 1218 His Pro
Glu Ile Lys Gly Gln Leu Val Lys Leu Leu Ser Val Arg Leu 360 365 370
375 tgc ccc cca gtg tct ggg cag gcc gcc atg gac att gtc gtg aac ccc
1266 Cys Pro Pro Val Ser Gly Gln Ala Ala Met Asp Ile Val Val Asn
Pro 380 385 390 ccg gtg gca gga gag gag tcc ttt gag caa ttc agc cga
gag aag gag 1314 Pro Val Ala Gly Glu Glu Ser Phe Glu Gln Phe Ser
Arg Glu Lys Glu 395 400 405 tcg gtc ctg ggt aat ctg gcc aaa aaa gca
aag ctg acg gaa gac ctg 1362 Ser Val Leu Gly Asn Leu Ala Lys Lys
Ala Lys Leu Thr Glu Asp Leu 410 415 420 ttt aac caa gtc cca gga att
cac tgc aac ccc ttg cag ggg gcc atg 1410 Phe Asn Gln Val Pro Gly
Ile His Cys Asn Pro Leu Gln Gly Ala Met 425 430 435 tac gcc ttc cct
cgg atc ttc att cct gcc aaa gct gtg gag gct gct 1458 Tyr Ala Phe
Pro Arg Ile Phe Ile Pro Ala Lys Ala Val Glu Ala Ala 440 445 450 455
cag gcc cat caa atg gct cca gac atg ttc tac tgc atg aag ctc ctg
1506 Gln Ala His Gln Met Ala Pro Asp Met Phe Tyr Cys Met Lys Leu
Leu 460 465 470 gag gag act ggc atc tgt gtc gtg ccc ggc agt ggc ttt
ggg cag agg 1554 Glu Glu Thr Gly Ile Cys Val Val Pro Gly Ser Gly
Phe Gly Gln Arg 475 480 485 gaa ggc act tac cac ttc agg atg act atc
ctc cct cca gtg gag aag 1602 Glu Gly Thr Tyr His Phe Arg Met Thr
Ile Leu Pro Pro Val Glu Lys 490 495 500 ctg aaa acg gtg ctg cag aag
gtg aaa gac ttc cac atc aac ttc ctg 1650 Leu Lys Thr Val Leu Gln
Lys Val Lys Asp Phe His Ile Asn Phe Leu 505 510 515 gag aag tac gcg
tga ggacgcctga gccccagcgg gagacctgtc cttggctctt 1705 Glu Lys Tyr
Ala * 520 cctcccaatg cccgtcaggc tgaactcgcc tcccccgtga ctytgcctcg
ggcctcgcag 1765 aggccgctgg tcacttygtc atcattttgc ccctggagac
gtctttcttt gtgccttgat 1825 gttgagagcg cctctctttt gagcaaacaa
gcattctata tgcaaccaga gtagagggga 1885 cctgctcagn caggtgtgac
cagggttctc tgaatctgtt attgtttttg cttctggaaa 1945 gttcatttgg
ggtttacaac aactaggatg tgttgggtga gatgtttcag atctggagaa 2005
atgagcaggt gtcgggaaat gtgtgactta accgtggtga gggctggaaa tccaaactcc
2065 cccccatgat ctgtgaaata aagcccttag cggtgaaaaa aaaaaaaaaa
aaaaaaaarr 2125 cg 2127 15 523 PRT Homo sapiens 15 Met Gln Arg Ala
Ala Thr Leu Val Arg Arg Gly Cys Gly Pro Arg Thr 1 5 10 15 Pro Ser
Ser Trp Gly Arg Ser Gln Ser Ser Ala Ala Ala Glu Ala Ser 20 25 30
Ala Val Leu Lys Val Arg Pro Glu Arg Ser Arg Arg Glu Arg Ile Leu 35
40 45 Thr Leu Glu Ser Met Asn Pro Gln Val Lys Ala Val Glu Tyr Ala
Val 50 55 60 Arg Gly Pro Ile Val Leu Lys Ala Gly Glu Ile Glu Leu
Glu Leu Gln 65 70 75 80 Arg Gly Ile Lys Lys Pro Phe Thr Glu Val Ile
Arg Ala Asn Ile Gly 85 90 95 Asp Ala Gln Ala Met Gly Gln Gln Pro
Ile Thr Phe Leu Arg Gln Val 100 105 110 Met Ala Leu Cys Thr Tyr Pro
Asn Leu Leu Asp Ser Pro Ser Phe Pro 115 120 125 Glu Asp Ala Lys Lys
Arg Ala Arg Arg Ile Leu Gln Ala Cys Gly Gly 130 135 140 Asn Ser Leu
Gly Ser Tyr Ser Ala Ser Gln Gly Val Asn Cys Ile Arg 145 150 155 160
Glu Asp Val Ala Ala Tyr Ile Thr Arg Arg Asp Gly Gly Val Pro Ala 165
170 175 Asp Pro Asp Asn Ile Tyr Leu Thr Thr Gly Ala Ser Asp Gly Ile
Ser 180 185 190 Thr Ile Leu Lys Ile Leu Val Ser Gly Gly Gly Lys Ser
Arg Thr Gly 195 200 205 Val Met Ile Pro Ile Pro Gln Tyr Pro Leu Tyr
Ser Ala Val Ile Ser 210 215 220 Glu Leu Asp Ala Ile Gln Val Asn Tyr
Tyr Leu Asp Glu Glu Asn Cys 225 230 235 240 Trp Ala Leu Asn Val Asn
Glu Leu Arg Arg Ala Val Gln Glu Ala Lys 245 250 255 Asp His Cys Asp
Pro Lys Val Leu Cys Ile Ile Asn Pro Gly Asn Pro 260 265 270 Thr Gly
Gln Val Gln Ser Arg Lys Cys Ile Glu Asp Val Ile His Phe 275 280 285
Ala Trp Glu Glu Lys Leu Phe Leu Leu Ala Asp Glu Val Tyr Gln Asp 290
295 300 Asn Val Tyr Ser Pro Asp Cys Arg Phe His Ser Phe Lys Lys Val
Leu 305 310 315 320 Tyr Glu Met Gly Pro Glu Tyr Ser Ser Asn Val Glu
Leu Ala Ser Phe 325 330 335 His Ser Thr Ser Lys Gly Tyr Met Gly Glu
Cys Gly Tyr Arg Gly Gly 340 345 350 Tyr Met Glu Val Ile Asn Leu His
Pro Glu Ile Lys Gly Gln Leu Val 355 360 365 Lys Leu Leu Ser Val Arg
Leu Cys Pro Pro Val Ser Gly Gln Ala Ala 370 375 380 Met Asp Ile Val
Val Asn Pro Pro Val Ala Gly Glu Glu Ser Phe Glu 385 390 395 400 Gln
Phe Ser Arg Glu Lys Glu Ser Val Leu Gly Asn Leu Ala Lys Lys 405 410
415 Ala Lys Leu Thr Glu Asp Leu Phe Asn Gln Val Pro Gly Ile His Cys
420 425 430 Asn Pro Leu Gln Gly Ala Met Tyr Ala Phe Pro Arg Ile Phe
Ile Pro 435 440 445 Ala Lys Ala Val Glu Ala Ala Gln Ala His Gln Met
Ala Pro Asp Met 450 455 460 Phe Tyr Cys Met Lys Leu Leu Glu Glu Thr
Gly Ile Cys Val Val Pro 465 470 475 480 Gly Ser Gly Phe Gly Gln Arg
Glu Gly Thr Tyr His Phe Arg Met Thr 485 490 495 Ile Leu Pro Pro Val
Glu Lys Leu Lys Thr Val Leu Gln Lys Val Lys 500 505 510 Asp Phe His
Ile Asn Phe Leu Glu Lys Tyr Ala 515 520 16 1572 DNA Homo sapiens
CDS (1)...(1572) 16 atg cag cgg gcg gcg acg ctg gtc cgg cgg ggc tgt
ggt ccc cgg acc 48 Met Gln Arg Ala Ala Thr Leu Val Arg Arg Gly Cys
Gly Pro Arg Thr 1 5 10 15 ccc agc tcc tgg ggc cgc agc cag agc agc
gcg gcc gcc gag gcc tcg 96 Pro Ser Ser Trp Gly Arg Ser Gln Ser Ser
Ala Ala Ala Glu Ala Ser 20 25 30 gcg gtg ctc aag gtg cgg ccc gag
cgc agc cgg cgc gag cgc atc ctc 144 Ala Val Leu Lys Val Arg Pro Glu
Arg Ser Arg Arg Glu Arg Ile Leu 35 40 45 acg ctg gag tcc atg aac
ccg cag gtg aag gcg gtg gag tac gcc gtg 192 Thr Leu Glu Ser Met Asn
Pro Gln Val Lys Ala Val Glu Tyr Ala Val 50 55 60 cgg gga ccc atc
gtg ctc aag gcc ggc gag atc gag ctc gag ctg cag 240 Arg Gly Pro Ile
Val Leu Lys Ala Gly Glu Ile Glu Leu Glu Leu Gln 65 70 75 80 cgg ggt
atc aaa aag cca ttc aca gag gtc atc cga gcc aac atc ggg 288 Arg Gly
Ile Lys Lys Pro Phe Thr Glu Val Ile Arg Ala Asn Ile Gly 85 90 95
gac gcc cag gct atg ggg cag cag cca atc acc ttc ctc cgg cag gtg 336
Asp Ala Gln Ala Met Gly Gln Gln Pro Ile Thr Phe Leu Arg Gln Val 100
105 110 atg gca cta tgc acc tac cca aac ctg ctg gac agc ccc agc ttc
cca 384 Met Ala Leu Cys Thr Tyr Pro Asn Leu Leu Asp Ser Pro Ser Phe
Pro 115 120 125 gaa gat gct aag aaa cgt gcc cgg cgg atc ctg cag gct
tgt ggc ggg 432 Glu Asp Ala Lys Lys Arg Ala Arg Arg Ile Leu Gln Ala
Cys Gly Gly 130 135 140 aac agc ctg ggg tcc tac agt gct agc cag ggt
gtc aac tgc atc cgt 480 Asn Ser Leu Gly Ser Tyr Ser Ala Ser Gln Gly
Val Asn Cys Ile Arg 145 150 155 160 gaa gat gtg gct gcc tac atc acc
agg agg gat ggc ggt gtg cct gcg 528 Glu Asp Val Ala Ala Tyr Ile Thr
Arg Arg Asp Gly Gly Val Pro Ala 165 170 175 gac ccc gac aac atc tac
ctg acc acg gga gct agt gac ggc att tct 576 Asp Pro Asp Asn Ile Tyr
Leu Thr Thr Gly Ala Ser Asp Gly Ile Ser 180 185 190 acg atc ctg aag
atc ctc gtc tcc ggg ggc ggc aag tca cgg aca ggt 624 Thr Ile Leu Lys
Ile Leu Val Ser Gly Gly Gly Lys Ser Arg Thr Gly 195 200 205 gtg atg
atc ccc atc cca caa tat ccc ctc tat tca gct gtc atc tct 672 Val Met
Ile Pro Ile Pro Gln Tyr Pro Leu Tyr Ser Ala Val Ile Ser 210 215 220
gag ctc gac gcc atc cag gtg aat tac tac ctg gac gag gag aac tgc 720
Glu Leu Asp Ala Ile Gln Val Asn Tyr Tyr Leu Asp Glu Glu Asn Cys 225
230 235 240 tgg gcg ctg aat gtg aat gag ctc cgg cgg gcg gtg cag gag
gcc aaa 768 Trp Ala Leu Asn Val Asn Glu Leu Arg Arg Ala Val Gln Glu
Ala Lys 245 250 255 gac cac tgt gat cct aag gtg ctc tgc ata atc aac
cct ggg aac ccc 816 Asp His Cys Asp Pro Lys Val Leu Cys Ile Ile Asn
Pro Gly Asn Pro 260 265 270 aca ggc cag gta caa agc aga aag tgc ata
gaa gat gtg atc cac ttt 864 Thr Gly Gln Val Gln Ser Arg Lys Cys Ile
Glu Asp Val Ile His Phe 275 280 285 gcc tgg gaa gag aag ctc ttt ctc
ctg gct gat gag gtg tac cag gac 912 Ala Trp Glu Glu Lys Leu Phe Leu
Leu Ala Asp Glu Val Tyr Gln Asp 290 295 300 aac gtg tac tct cca gat
tgc aga ttc cac tcc ttc aag aag gtg ctg 960 Asn Val Tyr Ser Pro Asp
Cys Arg Phe His Ser Phe Lys Lys Val Leu 305 310 315 320 tac gag atg
ggg ccc gag tac tcc agc aac gtg gag ctc gcc tcc ttc 1008 Tyr Glu
Met Gly Pro Glu Tyr Ser Ser Asn Val Glu Leu Ala Ser Phe 325 330 335
cac tcc acc tcc aag ggc tac atg ggc gag tgt ggt tac aga gga ggc
1056 His Ser Thr Ser Lys Gly Tyr Met Gly Glu Cys Gly Tyr Arg Gly
Gly 340 345 350 tac atg gag gtg atc aac ctg cac cct gag atc aag ggc
cag ctg gtg 1104 Tyr Met Glu Val Ile Asn Leu His Pro Glu Ile Lys
Gly Gln Leu Val 355 360 365 aag ctg ctg tcg gtg cgc ctg tgc ccc cca
gtg tct ggg cag gcc gcc 1152 Lys Leu Leu Ser Val Arg Leu Cys Pro
Pro Val Ser Gly Gln Ala Ala 370 375 380 atg gac att gtc gtg aac ccc
ccg gtg gca gga gag gag tcc ttt gag 1200 Met Asp Ile Val Val Asn
Pro Pro Val Ala Gly Glu Glu Ser Phe Glu 385 390 395 400 caa ttc agc
cga gag aag gag tcg gtc ctg ggt aat ctg gcc aaa aaa 1248 Gln Phe
Ser Arg Glu Lys Glu Ser Val Leu Gly Asn Leu Ala Lys Lys 405 410 415
gca aag ctg acg gaa gac ctg ttt aac caa gtc cca gga att cac tgc
1296 Ala Lys Leu Thr Glu Asp Leu Phe Asn Gln Val Pro Gly Ile His
Cys 420 425 430 aac ccc ttg cag ggg gcc atg tac gcc ttc cct cgg atc
ttc att cct 1344 Asn Pro Leu Gln Gly Ala Met Tyr Ala Phe Pro Arg
Ile Phe Ile Pro 435 440 445 gcc aaa gct gtg gag gct gct cag gcc cat
caa atg gct cca gac atg 1392 Ala Lys Ala Val Glu Ala Ala Gln Ala
His Gln Met Ala Pro Asp Met 450 455 460 ttc tac tgc atg aag ctc ctg
gag gag act ggc atc tgt gtc gtg ccc 1440 Phe Tyr Cys Met Lys Leu
Leu Glu Glu Thr Gly Ile Cys Val Val Pro 465 470 475 480 ggc agt ggc
ttt ggg cag agg gaa ggc act tac cac ttc agg atg act 1488 Gly Ser
Gly Phe Gly Gln Arg Glu Gly Thr Tyr His Phe Arg Met Thr 485 490 495
atc ctc cct cca gtg gag aag ctg aaa acg gtg ctg cag aag gtg aaa
1536 Ile Leu Pro Pro Val Glu Lys Leu Lys Thr Val Leu Gln Lys Val
Lys 500 505 510 gac ttc cac atc aac ttc ctg gag aag tac gcg tga
1572 Asp Phe His Ile Asn Phe Leu Glu Lys Tyr Ala * 515 520 17 447
PRT Artificial Sequence Pfam Aminotransferase family model 17 Leu
Ser Ser Met Ala Ala Asn Val Ser His Gly Pro Gly Asp Pro Ile 1 5 10
15 Leu Gly Val Trp Glu Ala Phe Lys Glu Asp Pro Arg Pro Gly Lys Asp
20 25 30 Asn Pro Asn Gly Val Ile Gly Val Gly Ala Tyr Glu Pro Gln
Leu Gly 35 40 45 Lys Asp Leu Val Leu Pro Ala Val Lys Lys Ala Glu
Lys Arg Leu Ala 50 55 60 Leu Asp Arg Glu Gly Asn Ile Glu Phe Arg
Glu Ile Lys Glu Tyr Leu 65 70 75 80 Pro Ile His Gly Leu Pro Glu Phe
Arg Glu Ala Ile Ala Lys Leu Leu 85 90 95 Phe Gly Ala Arg Ser Pro
Lys Leu Lys Phe Lys Arg Val Arg Val Val 100 105 110 Gln Thr Leu Gly
Gly Thr Gly Ala Leu Arg Leu Ala Ala Asp Phe Leu 115 120 125 Ala Asn
Pro Gly Asp Gly Ser Arg Gly Arg Glu Val Leu Val Pro Thr 130 135 140
Pro Thr Trp Pro Asn Tyr Lys Arg Asp Ile Phe Trp Ala Ala Gly Val 145
150 155 160 Glu Val Ile Val Pro Tyr His Tyr Tyr Lys Asp Glu Asn Asn
Phe Gly 165 170 175 Leu Asp Phe Glu Ala Leu Glu Ala Ala Ile Glu Lys
Ala Pro Glu Lys 180 185 190 Asn Ile Lys Thr Lys Val Leu Leu His Asn
Asn Pro His Asn Pro Thr 195 200 205 Gly Thr Asp Pro Thr Arg Glu Gln
Leu Lys Lys Ile Ala Asp Val Val 210 215 220 Lys Glu Lys Asn Ile Leu
Leu Leu Ser Asp Glu Ala Tyr Gln Gly Phe 225 230 235 240 Val Phe Gly
Ser Leu Asp Glu Asp Ala Ala Ser Val Ala Glu Phe Ala 245 250 255 Glu
Glu Val Lys Glu Glu Met Glu Cys Asn Gly Asp Glu Leu Leu Val 260 265
270 Val Gln Ser Phe Ser Lys Asn Phe Gly Leu Tyr Gly Trp Arg Val Gly
275 280 285 Ala Ile Tyr Val Val Asn Pro Arg Ile Gly Asp Ala Val Ile
Ser Ala 290 295 300 Ala Ala Lys Met Ser Ser Ala Gly Arg Val Ser Ser
Gln Leu Gln Ala 305 310 315 320 Leu Ala Arg Ala Met Tyr Ser Asn Pro
Asp Phe Pro Pro Asp His Gly 325 330 335 Ala Glu Ile Val Ala Arg Ile
Leu Glu Arg Arg Asp Leu Phe Thr Ser 340 345 350 Trp Leu Glu Glu Val
Lys Gly Met Ala Cys Arg Ile Pro Asn Gly Arg 355 360 365 Leu Tyr Leu
Trp Met Asp Leu Arg Lys Leu Leu Lys Glu Glu Asp Asp 370 375 380 Trp
Ser His Ile Ile Glu Gln Glu Gly Met Phe Ser Phe Thr Trp Leu 385 390
395 400 Leu Asn Glu Glu Gln Val Asn Val Ser Pro Gly Ser Glu Phe His
Ile 405 410 415 Tyr Glu Pro Gly Trp Gly Arg Ile Ser Leu Ala Gly Leu
Ser Glu Ala 420 425 430 Asn Val Glu Glu Ala Ala Glu Arg Ile Arg Ala
Phe Val Lys Arg 435 440 445 18 2876 DNA Homo sapiens CDS
(613)...(1914) 18 gtcaccacgc gtccgcggac gcgcgtccgg cgcccagcgg
agtaggggct gcgcttgggg 60 tttgctgaag ctggctgcct ctcccactcc
ccttttgggt gcaaagcgcc gctagcggga 120 agacgggggc cgggcgggga
caggggcacc tgcgtagctg gactgagagc ctgcgcccag 180 cttacatcga
ccccacccgg ccccggcccg acccgacgcg acccgatccg atccgatccc 240
attccatccg ttcctcgtct cctcccggtc tgacccgttg cccggccgtg gttcgccaca
300 ccaggcatcc aaagctgagg tcgctcctac ggcctgggct cgccttcgct
ttagagatgt 360 ttggcctctt ccctcccaaa cagcccatct tcaaaacctg
gactcttgga ctggcacctg 420 gccacctttc cctctaccaa gactccactt
ccgtcttacc cacttcttcc tcagattctt 480 ggtaccccct gggttggaga
ctgctcattt tccttccaaa ttaatcccag accccctaaa 540 atattgacaa
ccttgacaac cccccaaccg aggagccaga ctttgttttg gactaacttc 600
catagccctg tc atg gag gca gtg tac ctg gta gtg aat ggg ttg ggc ctg
651
Met Glu Ala Val Tyr Leu Val Val Asn Gly Leu Gly Leu 1 5 10 gtg ctg
gac gtg ctg acc ttg gtg ttg gac ctc aac ttc ctg ctg gtg 699 Val Leu
Asp Val Leu Thr Leu Val Leu Asp Leu Asn Phe Leu Leu Val 15 20 25
tcc tcc ctc ctg gct tcc ctg gcc tgg ctc ctg gcc ttc gtc tac aac 747
Ser Ser Leu Leu Ala Ser Leu Ala Trp Leu Leu Ala Phe Val Tyr Asn 30
35 40 45 ctg ccg cac acg gta ctg act agt ctt ctg cac ttg ggc cgc
gga gtc 795 Leu Pro His Thr Val Leu Thr Ser Leu Leu His Leu Gly Arg
Gly Val 50 55 60 ttg ctt tca ttg ctg gcc ttg atc gaa gcc gtg gtc
cgg ttc aca tgt 843 Leu Leu Ser Leu Leu Ala Leu Ile Glu Ala Val Val
Arg Phe Thr Cys 65 70 75 ggg ggc ttg cag gcc ttg tgt act ctg ctg
tat agc tgc tgc tct ggc 891 Gly Gly Leu Gln Ala Leu Cys Thr Leu Leu
Tyr Ser Cys Cys Ser Gly 80 85 90 cta gag agc cta aag ctc ctg ggg
cac ctg gcc tct cat ggg gca ctg 939 Leu Glu Ser Leu Lys Leu Leu Gly
His Leu Ala Ser His Gly Ala Leu 95 100 105 cgg agc agg gag ata ctg
cac cgg ggc gtc ctc aat gtg gtc tcc agt 987 Arg Ser Arg Glu Ile Leu
His Arg Gly Val Leu Asn Val Val Ser Ser 110 115 120 125 ggc cat gct
ttg ctg cgc cag gcc tgt gac atc tgt gcc att gcc atg 1035 Gly His
Ala Leu Leu Arg Gln Ala Cys Asp Ile Cys Ala Ile Ala Met 130 135 140
agc ctg gtg gct tat gtg atc aac agc ctg gtc aac atc tgc ctc atc
1083 Ser Leu Val Ala Tyr Val Ile Asn Ser Leu Val Asn Ile Cys Leu
Ile 145 150 155 ggc act cag aac ctc ttt tcc ctg gtg ctg gcc ctg tgg
gat gca gtg 1131 Gly Thr Gln Asn Leu Phe Ser Leu Val Leu Ala Leu
Trp Asp Ala Val 160 165 170 acc ggg cct ctg tgg agg atg aca gac gta
gtg gct gcc ttc cta gcc 1179 Thr Gly Pro Leu Trp Arg Met Thr Asp
Val Val Ala Ala Phe Leu Ala 175 180 185 cac att tcc agc agt gct gtg
gcc atg gcc atc ctc ctt tgg aca ccc 1227 His Ile Ser Ser Ser Ala
Val Ala Met Ala Ile Leu Leu Trp Thr Pro 190 195 200 205 tgc caa cta
gcc ctg gag ctg ctg gcc tca gct gcc cgc ctc ctg gcc 1275 Cys Gln
Leu Ala Leu Glu Leu Leu Ala Ser Ala Ala Arg Leu Leu Ala 210 215 220
agc ttt gtg ctt gtc aat ctc act ggc ttg gtg ttg cta gct tgt gtg
1323 Ser Phe Val Leu Val Asn Leu Thr Gly Leu Val Leu Leu Ala Cys
Val 225 230 235 ctg gca gtg acg gtg act gtg ttg cat ccg gac ttc acc
ctg agg ctg 1371 Leu Ala Val Thr Val Thr Val Leu His Pro Asp Phe
Thr Leu Arg Leu 240 245 250 gct acc cag gca ctc agc cag ctc cat gcc
cgg cca tcc tac cac cgt 1419 Ala Thr Gln Ala Leu Ser Gln Leu His
Ala Arg Pro Ser Tyr His Arg 255 260 265 ctt cga gag gat gtc atg cgg
ctc tct cgc cta gca ctg ggc tca gag 1467 Leu Arg Glu Asp Val Met
Arg Leu Ser Arg Leu Ala Leu Gly Ser Glu 270 275 280 285 gcc tgg cgc
cga gtc tgg agc cgc agt ctg cag ctg gcg agt tgg cca 1515 Ala Trp
Arg Arg Val Trp Ser Arg Ser Leu Gln Leu Ala Ser Trp Pro 290 295 300
aac cgg gga ggg gca cct gga gct ccc cag ggt gac cct atg agg gta
1563 Asn Arg Gly Gly Ala Pro Gly Ala Pro Gln Gly Asp Pro Met Arg
Val 305 310 315 ttc tca gtt agg acc cgg aga cag gac act ctt cct gaa
gcg ggg cgc 1611 Phe Ser Val Arg Thr Arg Arg Gln Asp Thr Leu Pro
Glu Ala Gly Arg 320 325 330 aga tca gag gca gaa gag gag gag gcc agg
acc atc aga gtg aca cct 1659 Arg Ser Glu Ala Glu Glu Glu Glu Ala
Arg Thr Ile Arg Val Thr Pro 335 340 345 gtc agg ggc cga gag agg ctc
aat gag gag gag cct cca ggt ggg caa 1707 Val Arg Gly Arg Glu Arg
Leu Asn Glu Glu Glu Pro Pro Gly Gly Gln 350 355 360 365 gac cct tgg
aaa ttg ctg aag gag caa gag gag cgg aag aag tgt gtc 1755 Asp Pro
Trp Lys Leu Leu Lys Glu Gln Glu Glu Arg Lys Lys Cys Val 370 375 380
atc tgc cag gac cag agc aag aca gtg ttg ctc ctg ccc tgc cgg cat
1803 Ile Cys Gln Asp Gln Ser Lys Thr Val Leu Leu Leu Pro Cys Arg
His 385 390 395 ctg tgc ctg tgc cag gcc tgc act gaa atc ctg atg cgc
cac ccc gtc 1851 Leu Cys Leu Cys Gln Ala Cys Thr Glu Ile Leu Met
Arg His Pro Val 400 405 410 tac cac cgc aat tgc ccg ctc tgc cgc cgg
ggc atc ctg cag acc ctc 1899 Tyr His Arg Asn Cys Pro Leu Cys Arg
Arg Gly Ile Leu Gln Thr Leu 415 420 425 aat gtc tac ctc tga
agcctccttc cctgcctgcc cacccctcca tgctccacgc 1954 Asn Val Tyr Leu *
430 aggcactcac gctaggacag cattaacacc tcatctccgg gtcctggtct
gaatcccctc 2014 ctacccctgt ggccatcctg ccatacatcc aggacattga
gttggaagac tatgatctgg 2074 gtgggggcag gataacatgg cttctcttta
cccagtgggt cccttcgatg ctgagggtgg 2134 tgagtatgtc actatgcaag
ggccctgaga ctatttgctg tgggctctcc tccagcctgc 2194 ccagggccca
cccagatgcc tctggggtta cccctgtctg cttctggttt ttctgttgga 2254
gatctatagg tccttttcct gcctccttca catttcctcc ccagcttttg cggccacaac
2314 acatcagtgt catttgggtg ttttggcaac tcaggggcct tcggatgatc
ttaaaccttt 2374 gtgttcagcc agagcccctg tgccctggta ggcgttgggg
ttagtatctc tcgggtgccc 2434 tcagagccac ctctgcctgt gatcgtctga
tgaggctccc tcccaacctg atccaaaagc 2494 cagtctcagg agtttacccc
tgggatgggg gatgcatctg cacctgactt tggggccacg 2554 tgccctgtgg
caccccagct cactgggagt ctcaggaggg ataaccggat ttctgctctt 2614
tcccctgtca ctcccacatc acacagaaaa atggcattcc tctctgtctc tccctggcat
2674 ggagagggca gactgtgcac atttcactag ggtccaaata cagaagggcc
cagggcccag 2734 gggcttgcag cttcgtgagg ggtctctggc ccagtttcca
atgaataaag ttctcttgac 2794 agctcaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaggc cgcaagctta tcaatttcga 2854 cctatactgg gtcgtattac aa 2876
19 433 PRT Homo sapiens 19 Met Glu Ala Val Tyr Leu Val Val Asn Gly
Leu Gly Leu Val Leu Asp 1 5 10 15 Val Leu Thr Leu Val Leu Asp Leu
Asn Phe Leu Leu Val Ser Ser Leu 20 25 30 Leu Ala Ser Leu Ala Trp
Leu Leu Ala Phe Val Tyr Asn Leu Pro His 35 40 45 Thr Val Leu Thr
Ser Leu Leu His Leu Gly Arg Gly Val Leu Leu Ser 50 55 60 Leu Leu
Ala Leu Ile Glu Ala Val Val Arg Phe Thr Cys Gly Gly Leu 65 70 75 80
Gln Ala Leu Cys Thr Leu Leu Tyr Ser Cys Cys Ser Gly Leu Glu Ser 85
90 95 Leu Lys Leu Leu Gly His Leu Ala Ser His Gly Ala Leu Arg Ser
Arg 100 105 110 Glu Ile Leu His Arg Gly Val Leu Asn Val Val Ser Ser
Gly His Ala 115 120 125 Leu Leu Arg Gln Ala Cys Asp Ile Cys Ala Ile
Ala Met Ser Leu Val 130 135 140 Ala Tyr Val Ile Asn Ser Leu Val Asn
Ile Cys Leu Ile Gly Thr Gln 145 150 155 160 Asn Leu Phe Ser Leu Val
Leu Ala Leu Trp Asp Ala Val Thr Gly Pro 165 170 175 Leu Trp Arg Met
Thr Asp Val Val Ala Ala Phe Leu Ala His Ile Ser 180 185 190 Ser Ser
Ala Val Ala Met Ala Ile Leu Leu Trp Thr Pro Cys Gln Leu 195 200 205
Ala Leu Glu Leu Leu Ala Ser Ala Ala Arg Leu Leu Ala Ser Phe Val 210
215 220 Leu Val Asn Leu Thr Gly Leu Val Leu Leu Ala Cys Val Leu Ala
Val 225 230 235 240 Thr Val Thr Val Leu His Pro Asp Phe Thr Leu Arg
Leu Ala Thr Gln 245 250 255 Ala Leu Ser Gln Leu His Ala Arg Pro Ser
Tyr His Arg Leu Arg Glu 260 265 270 Asp Val Met Arg Leu Ser Arg Leu
Ala Leu Gly Ser Glu Ala Trp Arg 275 280 285 Arg Val Trp Ser Arg Ser
Leu Gln Leu Ala Ser Trp Pro Asn Arg Gly 290 295 300 Gly Ala Pro Gly
Ala Pro Gln Gly Asp Pro Met Arg Val Phe Ser Val 305 310 315 320 Arg
Thr Arg Arg Gln Asp Thr Leu Pro Glu Ala Gly Arg Arg Ser Glu 325 330
335 Ala Glu Glu Glu Glu Ala Arg Thr Ile Arg Val Thr Pro Val Arg Gly
340 345 350 Arg Glu Arg Leu Asn Glu Glu Glu Pro Pro Gly Gly Gln Asp
Pro Trp 355 360 365 Lys Leu Leu Lys Glu Gln Glu Glu Arg Lys Lys Cys
Val Ile Cys Gln 370 375 380 Asp Gln Ser Lys Thr Val Leu Leu Leu Pro
Cys Arg His Leu Cys Leu 385 390 395 400 Cys Gln Ala Cys Thr Glu Ile
Leu Met Arg His Pro Val Tyr His Arg 405 410 415 Asn Cys Pro Leu Cys
Arg Arg Gly Ile Leu Gln Thr Leu Asn Val Tyr 420 425 430 Leu 20 1302
DNA Homo sapiens CDS (1)...(1302) 20 atg gag gca gtg tac ctg gta
gtg aat ggg ttg ggc ctg gtg ctg gac 48 Met Glu Ala Val Tyr Leu Val
Val Asn Gly Leu Gly Leu Val Leu Asp 1 5 10 15 gtg ctg acc ttg gtg
ttg gac ctc aac ttc ctg ctg gtg tcc tcc ctc 96 Val Leu Thr Leu Val
Leu Asp Leu Asn Phe Leu Leu Val Ser Ser Leu 20 25 30 ctg gct tcc
ctg gcc tgg ctc ctg gcc ttc gtc tac aac ctg ccg cac 144 Leu Ala Ser
Leu Ala Trp Leu Leu Ala Phe Val Tyr Asn Leu Pro His 35 40 45 acg
gta ctg act agt ctt ctg cac ttg ggc cgc gga gtc ttg ctt tca 192 Thr
Val Leu Thr Ser Leu Leu His Leu Gly Arg Gly Val Leu Leu Ser 50 55
60 ttg ctg gcc ttg atc gaa gcc gtg gtc cgg ttc aca tgt ggg ggc ttg
240 Leu Leu Ala Leu Ile Glu Ala Val Val Arg Phe Thr Cys Gly Gly Leu
65 70 75 80 cag gcc ttg tgt act ctg ctg tat agc tgc tgc tct ggc cta
gag agc 288 Gln Ala Leu Cys Thr Leu Leu Tyr Ser Cys Cys Ser Gly Leu
Glu Ser 85 90 95 cta aag ctc ctg ggg cac ctg gcc tct cat ggg gca
ctg cgg agc agg 336 Leu Lys Leu Leu Gly His Leu Ala Ser His Gly Ala
Leu Arg Ser Arg 100 105 110 gag ata ctg cac cgg ggc gtc ctc aat gtg
gtc tcc agt ggc cat gct 384 Glu Ile Leu His Arg Gly Val Leu Asn Val
Val Ser Ser Gly His Ala 115 120 125 ttg ctg cgc cag gcc tgt gac atc
tgt gcc att gcc atg agc ctg gtg 432 Leu Leu Arg Gln Ala Cys Asp Ile
Cys Ala Ile Ala Met Ser Leu Val 130 135 140 gct tat gtg atc aac agc
ctg gtc aac atc tgc ctc atc ggc act cag 480 Ala Tyr Val Ile Asn Ser
Leu Val Asn Ile Cys Leu Ile Gly Thr Gln 145 150 155 160 aac ctc ttt
tcc ctg gtg ctg gcc ctg tgg gat gca gtg acc ggg cct 528 Asn Leu Phe
Ser Leu Val Leu Ala Leu Trp Asp Ala Val Thr Gly Pro 165 170 175 ctg
tgg agg atg aca gac gta gtg gct gcc ttc cta gcc cac att tcc 576 Leu
Trp Arg Met Thr Asp Val Val Ala Ala Phe Leu Ala His Ile Ser 180 185
190 agc agt gct gtg gcc atg gcc atc ctc ctt tgg aca ccc tgc caa cta
624 Ser Ser Ala Val Ala Met Ala Ile Leu Leu Trp Thr Pro Cys Gln Leu
195 200 205 gcc ctg gag ctg ctg gcc tca gct gcc cgc ctc ctg gcc agc
ttt gtg 672 Ala Leu Glu Leu Leu Ala Ser Ala Ala Arg Leu Leu Ala Ser
Phe Val 210 215 220 ctt gtc aat ctc act ggc ttg gtg ttg cta gct tgt
gtg ctg gca gtg 720 Leu Val Asn Leu Thr Gly Leu Val Leu Leu Ala Cys
Val Leu Ala Val 225 230 235 240 acg gtg act gtg ttg cat ccg gac ttc
acc ctg agg ctg gct acc cag 768 Thr Val Thr Val Leu His Pro Asp Phe
Thr Leu Arg Leu Ala Thr Gln 245 250 255 gca ctc agc cag ctc cat gcc
cgg cca tcc tac cac cgt ctt cga gag 816 Ala Leu Ser Gln Leu His Ala
Arg Pro Ser Tyr His Arg Leu Arg Glu 260 265 270 gat gtc atg cgg ctc
tct cgc cta gca ctg ggc tca gag gcc tgg cgc 864 Asp Val Met Arg Leu
Ser Arg Leu Ala Leu Gly Ser Glu Ala Trp Arg 275 280 285 cga gtc tgg
agc cgc agt ctg cag ctg gcg agt tgg cca aac cgg gga 912 Arg Val Trp
Ser Arg Ser Leu Gln Leu Ala Ser Trp Pro Asn Arg Gly 290 295 300 ggg
gca cct gga gct ccc cag ggt gac cct atg agg gta ttc tca gtt 960 Gly
Ala Pro Gly Ala Pro Gln Gly Asp Pro Met Arg Val Phe Ser Val 305 310
315 320 agg acc cgg aga cag gac act ctt cct gaa gcg ggg cgc aga tca
gag 1008 Arg Thr Arg Arg Gln Asp Thr Leu Pro Glu Ala Gly Arg Arg
Ser Glu 325 330 335 gca gaa gag gag gag gcc agg acc atc aga gtg aca
cct gtc agg ggc 1056 Ala Glu Glu Glu Glu Ala Arg Thr Ile Arg Val
Thr Pro Val Arg Gly 340 345 350 cga gag agg ctc aat gag gag gag cct
cca ggt ggg caa gac cct tgg 1104 Arg Glu Arg Leu Asn Glu Glu Glu
Pro Pro Gly Gly Gln Asp Pro Trp 355 360 365 aaa ttg ctg aag gag caa
gag gag cgg aag aag tgt gtc atc tgc cag 1152 Lys Leu Leu Lys Glu
Gln Glu Glu Arg Lys Lys Cys Val Ile Cys Gln 370 375 380 gac cag agc
aag aca gtg ttg ctc ctg ccc tgc cgg cat ctg tgc ctg 1200 Asp Gln
Ser Lys Thr Val Leu Leu Leu Pro Cys Arg His Leu Cys Leu 385 390 395
400 tgc cag gcc tgc act gaa atc ctg atg cgc cac ccc gtc tac cac cgc
1248 Cys Gln Ala Cys Thr Glu Ile Leu Met Arg His Pro Val Tyr His
Arg 405 410 415 aat tgc ccg ctc tgc cgc cgg ggc atc ctg cag acc ctc
aat gtc tac 1296 Asn Cys Pro Leu Cys Arg Arg Gly Ile Leu Gln Thr
Leu Asn Val Tyr 420 425 430 ctc tga 1302 Leu * 21 54 PRT Artificial
Sequence Pfam consensus sequence 21 Cys Pro Ile Cys Leu Thr Thr Phe
Asp Leu Asp Glu Pro Lys Pro Phe 1 5 10 15 Lys Glu Pro Val Leu Leu
Pro Cys Gly His Ser Phe Cys Ser Lys Cys 20 25 30 Ile Val Glu Leu
Leu Arg Leu Ser Gln Asn Ser Lys Asn Asn Ser Val 35 40 45 Tyr Lys
Cys Pro Leu Cys 50 22 2810 DNA Homo sapiens CDS (744)...(1955)
misc_feature 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354,
355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,
368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,
381, 382, 383, 384, 385, 386, 387, 388, 389, 390 n = A,T,C or G
misc_feature 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401,
402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,
415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427,
428, 429, 430, 431, 432, 433, 434, 435, 436, 437 n = A,T,C or G
misc_feature 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448,
449, 450, 451 n = A,T,C or G 22 cgcctgcgca gggcagcggc ccgcggggcg
gaggctttat aatcacttcg tcgttgccgc 60 tcggcttcta tcgccgggag
ggcggttgag gcggtggtgg cggcgtcggc ggcggccggc 120 gctggctgag
gggcgctgag gcgggagctg tggcgctggg cgcccctggc tcctcggcct 180
ctgccggcca tgggctccga gaaggactcc gagtcgccgc gctccacatc gctacatgcg
240 gccgcacccg accctaagtg ccgcagcggc ggccggcgcc ggcgcctcac
cttgcacagc 300 gtcttttttg cctcggcccg cggccgccgc gcccgggcca
agcnnnnnnn nnnnnnnnnn 360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 420 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn ncgcaggccg agccgccgcc ccggcggcgg 480 cggagcctgg
gttcgacgat gaggaggcgg cggagggcgg tggcccgggc gcggaggagg 540
tggagtgtcc gctgtgcctg gtgcggctgc cgcctgagcg ggccccgcgc ctcctcagct
600 gtccgcaccg ctcgtgccgg gactgcctcc gccactacct gcgcctggag
ataagcgaga 660 gcagggtgcc catcagctgc cccgagtgca gcgagcgact
caacccgcac gacatccgct 720 tgctgctcgc cgacccgccg ctt atg cac aag tac
gag gag ttc atg ctg cgc 773 Met His Lys Tyr Glu Glu Phe Met Leu Arg
1 5 10 cgc tac cta gcc tcg gac ccc gac tgc cgc tgg tgc ccg gcc ccg
gac 821 Arg Tyr Leu Ala Ser Asp Pro Asp Cys Arg Trp Cys Pro Ala Pro
Asp 15 20 25 tgc ggt tat gct gtt att gcc tat ggc tgt gcc agc tgc
ccg aag cta 869 Cys Gly Tyr Ala Val Ile Ala Tyr Gly Cys Ala Ser Cys
Pro Lys Leu 30 35 40 act tgt gag agg gaa ggt tgc cag act gag ttc
tgc tac cac tgc aag 917 Thr Cys Glu Arg Glu Gly Cys Gln Thr Glu Phe
Cys Tyr His Cys Lys 45 50 55 cag ata tgg cat cca aat cag aca tgc
gat atg gcc cgt caa cag agg 965 Gln Ile Trp His Pro Asn Gln Thr Cys
Asp Met Ala Arg Gln Gln Arg 60 65 70 gcc cag act tta cga gtt cgg
acc aaa cac act tca ggt ctc agt tat 1013 Ala Gln Thr Leu Arg Val
Arg Thr Lys His Thr Ser Gly Leu Ser Tyr 75 80 85 90 ggg caa gaa tct
gga cca gat gac atc aag cca tgc cca cga tgc agt 1061 Gly Gln Glu
Ser Gly Pro Asp Asp Ile Lys Pro Cys Pro Arg Cys
Ser 95 100 105 gca tac att atc aag atg aat gat gga agc tgt aat cac
atg acc tgt 1109 Ala Tyr Ile Ile Lys Met Asn Asp Gly Ser Cys Asn
His Met Thr Cys 110 115 120 gca gtg tgt ggc tgt gaa ttc tgt tgg ctt
tgt atg aaa gag atc tca 1157 Ala Val Cys Gly Cys Glu Phe Cys Trp
Leu Cys Met Lys Glu Ile Ser 125 130 135 gac ttg cat tac ctc agc ccc
tct ggc tgt aca ttc tgg ggc aag aag 1205 Asp Leu His Tyr Leu Ser
Pro Ser Gly Cys Thr Phe Trp Gly Lys Lys 140 145 150 cca tgg agc cgt
aag aag aaa att ctt tgg cag ctg ggc acg ttg att 1253 Pro Trp Ser
Arg Lys Lys Lys Ile Leu Trp Gln Leu Gly Thr Leu Ile 155 160 165 170
ggt gct cca gtg ggg att tct ctc att gat ggc att gcc att cct gcc
1301 Gly Ala Pro Val Gly Ile Ser Leu Ile Asp Gly Ile Ala Ile Pro
Ala 175 180 185 atg gtc att ggc att cct gtt tat gtt gga agg aag att
cac agc agg 1349 Met Val Ile Gly Ile Pro Val Tyr Val Gly Arg Lys
Ile His Ser Arg 190 195 200 tat gag gga agg aaa acc tcc aaa cac aag
agg aat ttg gct atc act 1397 Tyr Glu Gly Arg Lys Thr Ser Lys His
Lys Arg Asn Leu Ala Ile Thr 205 210 215 gga gga gtg act ttg tcg gtc
att gca tcc cca gtt att gct gca gtt 1445 Gly Gly Val Thr Leu Ser
Val Ile Ala Ser Pro Val Ile Ala Ala Val 220 225 230 agt gtt ggt att
ggt gtc ccc att atg ctg gca tat gtt tat ggg gtt 1493 Ser Val Gly
Ile Gly Val Pro Ile Met Leu Ala Tyr Val Tyr Gly Val 235 240 245 250
gtg ccc att tct ctt tgt cgt gga ggt ggc tgt gga gtt agc aca gcc
1541 Val Pro Ile Ser Leu Cys Arg Gly Gly Gly Cys Gly Val Ser Thr
Ala 255 260 265 aac gga aaa gga gtg aaa att gaa ttt gat gaa gat gat
ggt cca atc 1589 Asn Gly Lys Gly Val Lys Ile Glu Phe Asp Glu Asp
Asp Gly Pro Ile 270 275 280 aca gtg gca gat gcc tgg aga gcc ctc aag
aat ccc agc att ggg gaa 1637 Thr Val Ala Asp Ala Trp Arg Ala Leu
Lys Asn Pro Ser Ile Gly Glu 285 290 295 agc agc att gaa ggc ctg act
agt gta ttg agc act agt gga agc cct 1685 Ser Ser Ile Glu Gly Leu
Thr Ser Val Leu Ser Thr Ser Gly Ser Pro 300 305 310 aca gat gga ctt
agt gtt atg caa ggt cct tac agc gaa acg gcc agc 1733 Thr Asp Gly
Leu Ser Val Met Gln Gly Pro Tyr Ser Glu Thr Ala Ser 315 320 325 330
ttt gca gcc ctc tca ggg ggc acg ctg agt ggc ggc att ctc tcc agt
1781 Phe Ala Ala Leu Ser Gly Gly Thr Leu Ser Gly Gly Ile Leu Ser
Ser 335 340 345 ggc aag gga aaa tat agc agg tta gaa gtt caa gcc gat
gtc caa aag 1829 Gly Lys Gly Lys Tyr Ser Arg Leu Glu Val Gln Ala
Asp Val Gln Lys 350 355 360 gaa att ttc ccc aaa gac aca gcc agt ctt
ggt gca att agt gac aac 1877 Glu Ile Phe Pro Lys Asp Thr Ala Ser
Leu Gly Ala Ile Ser Asp Asn 365 370 375 gca agc act cgt gct atg gcc
ggt tcc ata atc agt tcc tac aac cca 1925 Ala Ser Thr Arg Ala Met
Ala Gly Ser Ile Ile Ser Ser Tyr Asn Pro 380 385 390 cag gac agg ttt
agc atg atc cat gca tga ctcagcaaag tggattttgt 1975 Gln Asp Arg Phe
Ser Met Ile His Ala * 395 400 ctccacagag aatgcaacaa tatggaaatc
caagtggaca ttgaagccaa accaagccac 2035 tatcagctgg tgagtggaag
cagcacggag gactcgctcc atgttcatgc tcagatggca 2095 gagaatgaag
aagaaggtag tggtggcgga ggcagtgaag aggatccccc ctgcagacac 2155
caaagctgtg aacagaaaga ctgcctggcc agcaaacctt gggacatcag cctggcccag
2215 cctgaaagca tccgcagtga cctagagagt tctgatgcac agtcagacga
tgtgccagac 2275 atcacctcag atgagtgtgg ctccccccgc tcccatactg
cagcctgccc ctcgaccccc 2335 agagcccaag gtgcaccgag cccaagtgcc
catatgaacc tctctgccct agccgaggga 2395 caaactgtct tgaagccaga
aggtggagaa gccagagtat gaagtggaat gaatgctcct 2455 gttctgagaa
gcacacttgt aactgcatct tttggaattt tttttttttt ttttccaagg 2515
ggtagagatt tatgtatttt atttcacaga ttctctggtc acaggttttt gcccagggaa
2575 attctgagaa attcacaatt tcttaccaga taaaacatga aaagtttgcc
gttagttccc 2635 ctcccctccc ctccctcttt ttagttttaa tttattggtt
aaactgatgg cagcaatcca 2695 tgaggtgtgt caaagagtgt acatatgtat
gtgtgtatat tgaatgctaa acatattact 2755 gaaagacaca ttttaataaa
gatttctgtc ataattcaaa aaaaaaaaaa aaaaa 2810 23 403 PRT Homo sapiens
23 Met His Lys Tyr Glu Glu Phe Met Leu Arg Arg Tyr Leu Ala Ser Asp
1 5 10 15 Pro Asp Cys Arg Trp Cys Pro Ala Pro Asp Cys Gly Tyr Ala
Val Ile 20 25 30 Ala Tyr Gly Cys Ala Ser Cys Pro Lys Leu Thr Cys
Glu Arg Glu Gly 35 40 45 Cys Gln Thr Glu Phe Cys Tyr His Cys Lys
Gln Ile Trp His Pro Asn 50 55 60 Gln Thr Cys Asp Met Ala Arg Gln
Gln Arg Ala Gln Thr Leu Arg Val 65 70 75 80 Arg Thr Lys His Thr Ser
Gly Leu Ser Tyr Gly Gln Glu Ser Gly Pro 85 90 95 Asp Asp Ile Lys
Pro Cys Pro Arg Cys Ser Ala Tyr Ile Ile Lys Met 100 105 110 Asn Asp
Gly Ser Cys Asn His Met Thr Cys Ala Val Cys Gly Cys Glu 115 120 125
Phe Cys Trp Leu Cys Met Lys Glu Ile Ser Asp Leu His Tyr Leu Ser 130
135 140 Pro Ser Gly Cys Thr Phe Trp Gly Lys Lys Pro Trp Ser Arg Lys
Lys 145 150 155 160 Lys Ile Leu Trp Gln Leu Gly Thr Leu Ile Gly Ala
Pro Val Gly Ile 165 170 175 Ser Leu Ile Asp Gly Ile Ala Ile Pro Ala
Met Val Ile Gly Ile Pro 180 185 190 Val Tyr Val Gly Arg Lys Ile His
Ser Arg Tyr Glu Gly Arg Lys Thr 195 200 205 Ser Lys His Lys Arg Asn
Leu Ala Ile Thr Gly Gly Val Thr Leu Ser 210 215 220 Val Ile Ala Ser
Pro Val Ile Ala Ala Val Ser Val Gly Ile Gly Val 225 230 235 240 Pro
Ile Met Leu Ala Tyr Val Tyr Gly Val Val Pro Ile Ser Leu Cys 245 250
255 Arg Gly Gly Gly Cys Gly Val Ser Thr Ala Asn Gly Lys Gly Val Lys
260 265 270 Ile Glu Phe Asp Glu Asp Asp Gly Pro Ile Thr Val Ala Asp
Ala Trp 275 280 285 Arg Ala Leu Lys Asn Pro Ser Ile Gly Glu Ser Ser
Ile Glu Gly Leu 290 295 300 Thr Ser Val Leu Ser Thr Ser Gly Ser Pro
Thr Asp Gly Leu Ser Val 305 310 315 320 Met Gln Gly Pro Tyr Ser Glu
Thr Ala Ser Phe Ala Ala Leu Ser Gly 325 330 335 Gly Thr Leu Ser Gly
Gly Ile Leu Ser Ser Gly Lys Gly Lys Tyr Ser 340 345 350 Arg Leu Glu
Val Gln Ala Asp Val Gln Lys Glu Ile Phe Pro Lys Asp 355 360 365 Thr
Ala Ser Leu Gly Ala Ile Ser Asp Asn Ala Ser Thr Arg Ala Met 370 375
380 Ala Gly Ser Ile Ile Ser Ser Tyr Asn Pro Gln Asp Arg Phe Ser Met
385 390 395 400 Ile His Ala 24 1212 DNA Homo sapiens CDS
(1)...(1212) 24 atg cac aag tac gag gag ttc atg ctg cgc cgc tac cta
gcc tcg gac 48 Met His Lys Tyr Glu Glu Phe Met Leu Arg Arg Tyr Leu
Ala Ser Asp 1 5 10 15 ccc gac tgc cgc tgg tgc ccg gcc ccg gac tgc
ggt tat gct gtt att 96 Pro Asp Cys Arg Trp Cys Pro Ala Pro Asp Cys
Gly Tyr Ala Val Ile 20 25 30 gcc tat ggc tgt gcc agc tgc ccg aag
cta act tgt gag agg gaa ggt 144 Ala Tyr Gly Cys Ala Ser Cys Pro Lys
Leu Thr Cys Glu Arg Glu Gly 35 40 45 tgc cag act gag ttc tgc tac
cac tgc aag cag ata tgg cat cca aat 192 Cys Gln Thr Glu Phe Cys Tyr
His Cys Lys Gln Ile Trp His Pro Asn 50 55 60 cag aca tgc gat atg
gcc cgt caa cag agg gcc cag act tta cga gtt 240 Gln Thr Cys Asp Met
Ala Arg Gln Gln Arg Ala Gln Thr Leu Arg Val 65 70 75 80 cgg acc aaa
cac act tca ggt ctc agt tat ggg caa gaa tct gga cca 288 Arg Thr Lys
His Thr Ser Gly Leu Ser Tyr Gly Gln Glu Ser Gly Pro 85 90 95 gat
gac atc aag cca tgc cca cga tgc agt gca tac att atc aag atg 336 Asp
Asp Ile Lys Pro Cys Pro Arg Cys Ser Ala Tyr Ile Ile Lys Met 100 105
110 aat gat gga agc tgt aat cac atg acc tgt gca gtg tgt ggc tgt gaa
384 Asn Asp Gly Ser Cys Asn His Met Thr Cys Ala Val Cys Gly Cys Glu
115 120 125 ttc tgt tgg ctt tgt atg aaa gag atc tca gac ttg cat tac
ctc agc 432 Phe Cys Trp Leu Cys Met Lys Glu Ile Ser Asp Leu His Tyr
Leu Ser 130 135 140 ccc tct ggc tgt aca ttc tgg ggc aag aag cca tgg
agc cgt aag aag 480 Pro Ser Gly Cys Thr Phe Trp Gly Lys Lys Pro Trp
Ser Arg Lys Lys 145 150 155 160 aaa att ctt tgg cag ctg ggc acg ttg
att ggt gct cca gtg ggg att 528 Lys Ile Leu Trp Gln Leu Gly Thr Leu
Ile Gly Ala Pro Val Gly Ile 165 170 175 tct ctc att gat ggc att gcc
att cct gcc atg gtc att ggc att cct 576 Ser Leu Ile Asp Gly Ile Ala
Ile Pro Ala Met Val Ile Gly Ile Pro 180 185 190 gtt tat gtt gga agg
aag att cac agc agg tat gag gga agg aaa acc 624 Val Tyr Val Gly Arg
Lys Ile His Ser Arg Tyr Glu Gly Arg Lys Thr 195 200 205 tcc aaa cac
aag agg aat ttg gct atc act gga gga gtg act ttg tcg 672 Ser Lys His
Lys Arg Asn Leu Ala Ile Thr Gly Gly Val Thr Leu Ser 210 215 220 gtc
att gca tcc cca gtt att gct gca gtt agt gtt ggt att ggt gtc 720 Val
Ile Ala Ser Pro Val Ile Ala Ala Val Ser Val Gly Ile Gly Val 225 230
235 240 ccc att atg ctg gca tat gtt tat ggg gtt gtg ccc att tct ctt
tgt 768 Pro Ile Met Leu Ala Tyr Val Tyr Gly Val Val Pro Ile Ser Leu
Cys 245 250 255 cgt gga ggt ggc tgt gga gtt agc aca gcc aac gga aaa
gga gtg aaa 816 Arg Gly Gly Gly Cys Gly Val Ser Thr Ala Asn Gly Lys
Gly Val Lys 260 265 270 att gaa ttt gat gaa gat gat ggt cca atc aca
gtg gca gat gcc tgg 864 Ile Glu Phe Asp Glu Asp Asp Gly Pro Ile Thr
Val Ala Asp Ala Trp 275 280 285 aga gcc ctc aag aat ccc agc att ggg
gaa agc agc att gaa ggc ctg 912 Arg Ala Leu Lys Asn Pro Ser Ile Gly
Glu Ser Ser Ile Glu Gly Leu 290 295 300 act agt gta ttg agc act agt
gga agc cct aca gat gga ctt agt gtt 960 Thr Ser Val Leu Ser Thr Ser
Gly Ser Pro Thr Asp Gly Leu Ser Val 305 310 315 320 atg caa ggt cct
tac agc gaa acg gcc agc ttt gca gcc ctc tca ggg 1008 Met Gln Gly
Pro Tyr Ser Glu Thr Ala Ser Phe Ala Ala Leu Ser Gly 325 330 335 ggc
acg ctg agt ggc ggc att ctc tcc agt ggc aag gga aaa tat agc 1056
Gly Thr Leu Ser Gly Gly Ile Leu Ser Ser Gly Lys Gly Lys Tyr Ser 340
345 350 agg tta gaa gtt caa gcc gat gtc caa aag gaa att ttc ccc aaa
gac 1104 Arg Leu Glu Val Gln Ala Asp Val Gln Lys Glu Ile Phe Pro
Lys Asp 355 360 365 aca gcc agt ctt ggt gca att agt gac aac gca agc
act cgt gct atg 1152 Thr Ala Ser Leu Gly Ala Ile Ser Asp Asn Ala
Ser Thr Arg Ala Met 370 375 380 gcc ggt tcc ata atc agt tcc tac aac
cca cag gac agg ttt agc atg 1200 Ala Gly Ser Ile Ile Ser Ser Tyr
Asn Pro Gln Asp Arg Phe Ser Met 385 390 395 400 atc cat gca tga
1212 Ile His Ala * 25 72 PRT Artificial Sequence Pfam consensus
sequence 25 Glu Lys Tyr Glu Lys Phe Met Val Arg Ser Tyr Val Glu Lys
Asn Pro 1 5 10 15 Asp Leu Lys Trp Cys Pro Gly Pro Asp Cys Ser Tyr
Ala Val Arg Leu 20 25 30 Thr Glu Val Ser Ser Ser Thr Glu Leu Ala
Glu Pro Pro Arg Val Glu 35 40 45 Cys Lys Lys Pro Ala Cys Gly Thr
Ser Phe Cys Phe Lys Cys Gly Ala 50 55 60 Glu Trp His Ala Pro Val
Ser Cys 65 70 26 5502 DNA Homo sapiens CDS (803)...(2845) 26
ttaaactccc atgtgtgagg agtgtgcctc cctgygccct ctcagctctg aggctggycg
60 tctttcgggg tgttcctttt ggcaaatata cactgtaatc ttgagtctaa
atttatatgt 120 tgaaatgcta ccttttttaa agtaagaagc taaataaaat
tattttacta tcagtatcag 180 taaaaaaaaa aaaaaaaggg cggccgcgcc
accgccggag agggaggccc gagcgcagga 240 gcctctggtg gatgggtgca
gcggcggcgg gaggacgcgg aagaggagcc ccgggggtag 300 cggcggcgcg
agcaggggcg cggggaccgg gctgtctgag gtgcgcgccg cgctggggct 360
cgcgctctac ctgatcgcgc tgcggacgct ggtgcagctc tcgctgcagc agctcgtgct
420 acgcggggcc gctggacacc gcggggagtt cgacgcgctc caagccaggg
attatcttga 480 acacataacc tccattggcc ccaggactac aggaagtcca
gaaaatgaaa ttctgaccgt 540 gcactacctt ttggaacaga ttaaactgat
tgaagtgcaa agcaacagcc ttcataagat 600 ttcagtagat gtacaacggc
ccacaggctc ttttagcatt gatttcttgg gaggttttac 660 aagctattat
gacaacatca ccaatgttgt ggtaaagctg gaacccagag atggagccca 720
gcatgctgtc ttggctaatt gtcattttga ctcagtagca aactcaccag gtgccagtga
780 tgatgcagtt agctgctcag tg atg ctg gaa gtc ctt cgc gtc ttg tca
aca 832 Met Leu Glu Val Leu Arg Val Leu Ser Thr 1 5 10 tct tca gaa
gcc ttg cat cat gct gtc ata ttt ctc ttt aat ggt gct 880 Ser Ser Glu
Ala Leu His His Ala Val Ile Phe Leu Phe Asn Gly Ala 15 20 25 gag
gaa aat gtc ttg caa gcc agt cat ggt ttc att act cag cac ccc 928 Glu
Glu Asn Val Leu Gln Ala Ser His Gly Phe Ile Thr Gln His Pro 30 35
40 tgg gct agc ttg att cgt gca ttc att aac cta gag gca gca ggt gta
976 Trp Ala Ser Leu Ile Arg Ala Phe Ile Asn Leu Glu Ala Ala Gly Val
45 50 55 gga ggg aaa gaa ctt gta ttc caa aca ggt cct gaa aat cct
tgg ttg 1024 Gly Gly Lys Glu Leu Val Phe Gln Thr Gly Pro Glu Asn
Pro Trp Leu 60 65 70 gtt caa gct tat gtt tca gca gct aaa cac cct
ttt gct tct gtg gtg 1072 Val Gln Ala Tyr Val Ser Ala Ala Lys His
Pro Phe Ala Ser Val Val 75 80 85 90 gct cag gag gtt ttt cag agt gga
atc att cct tca gat act gac ttt 1120 Ala Gln Glu Val Phe Gln Ser
Gly Ile Ile Pro Ser Asp Thr Asp Phe 95 100 105 cgt atc tac agg gat
ttt ggg aac att cca gga ata gac tta gct ttt 1168 Arg Ile Tyr Arg
Asp Phe Gly Asn Ile Pro Gly Ile Asp Leu Ala Phe 110 115 120 att gag
aat gga tac att tat cac acc aag tat gac aca gcg gac aga 1216 Ile
Glu Asn Gly Tyr Ile Tyr His Thr Lys Tyr Asp Thr Ala Asp Arg 125 130
135 att cta aca gat tcc att cag aga gca ggt gac aac att tta gca gtt
1264 Ile Leu Thr Asp Ser Ile Gln Arg Ala Gly Asp Asn Ile Leu Ala
Val 140 145 150 ctt aag cat cta gct aca tct gat atg ctg gct gct gct
tct aag tat 1312 Leu Lys His Leu Ala Thr Ser Asp Met Leu Ala Ala
Ala Ser Lys Tyr 155 160 165 170 cga cat gga aac atg gtc ttc ttt gat
gtg ctg ggc ctg ttt gtc att 1360 Arg His Gly Asn Met Val Phe Phe
Asp Val Leu Gly Leu Phe Val Ile 175 180 185 gcc tac ccc tct cgt att
ggc tca atc ata aac tac atg gtg gta atg 1408 Ala Tyr Pro Ser Arg
Ile Gly Ser Ile Ile Asn Tyr Met Val Val Met 190 195 200 ggt gtt gtt
ttg tac ctg ggc aaa aaa ttt ttg cag ccc aaa cat aag 1456 Gly Val
Val Leu Tyr Leu Gly Lys Lys Phe Leu Gln Pro Lys His Lys 205 210 215
act ggt aac tac aag aag gac ttc ttg tgt gga ctt ggc atc act ttg
1504 Thr Gly Asn Tyr Lys Lys Asp Phe Leu Cys Gly Leu Gly Ile Thr
Leu 220 225 230 atc agc tgg ttc act agc ctt gtt acc gtt ctc att ata
gca gtg ttc 1552 Ile Ser Trp Phe Thr Ser Leu Val Thr Val Leu Ile
Ile Ala Val Phe 235 240 245 250 atc tct ctt att gga cag tct ctc tca
tgg tat aac cac ttc tat gtc 1600 Ile Ser Leu Ile Gly Gln Ser Leu
Ser Trp Tyr Asn His Phe Tyr Val 255 260 265 tcc gtt tgt ctg tat gga
act gca act gta gcc aaa ata ata ctt ata 1648 Ser Val Cys Leu Tyr
Gly Thr Ala Thr Val Ala Lys Ile Ile Leu Ile 270 275 280 cat act ctt
gcg aaa aga ttt tat tac atg aat gcc agt gcc cag tat 1696 His Thr
Leu Ala Lys Arg Phe Tyr Tyr Met Asn Ala Ser Ala Gln Tyr 285 290 295
ctg gga gaa gta ttt ttt gac att tcg ctg ttt gtc cat tgc tgt ttt
1744 Leu Gly Glu Val Phe Phe Asp Ile Ser Leu Phe Val His Cys Cys
Phe 300 305 310 ctt gtt acc ctc act tac caa gga ctt tgc tcg gcg ttt
att agt gct 1792 Leu Val Thr Leu Thr Tyr Gln Gly Leu Cys Ser Ala
Phe Ile Ser Ala 315 320 325
330 gtc tgg gta gca ttc cca ttg ctc aca aag ctc tgt gtg cat aag gac
1840 Val Trp Val Ala Phe Pro Leu Leu Thr Lys Leu Cys Val His Lys
Asp 335 340 345 ttc aag cag cat ggt gcc caa gga aaa ttt att gct ttt
tac ctt ttg 1888 Phe Lys Gln His Gly Ala Gln Gly Lys Phe Ile Ala
Phe Tyr Leu Leu 350 355 360 ggg atg ttt att cct tat ctt tat gca ttg
tac ctc atc tgg gca gta 1936 Gly Met Phe Ile Pro Tyr Leu Tyr Ala
Leu Tyr Leu Ile Trp Ala Val 365 370 375 ttt gag atg ttt acc cct atc
ctc ggg aga agt ggt tct gaa atc cca 1984 Phe Glu Met Phe Thr Pro
Ile Leu Gly Arg Ser Gly Ser Glu Ile Pro 380 385 390 cct gat gtt gtg
ctg gca tcc att ttg gct ggc tgt aca atg att ctc 2032 Pro Asp Val
Val Leu Ala Ser Ile Leu Ala Gly Cys Thr Met Ile Leu 395 400 405 410
tcg tcc tat ttt att aac ttc atc tac ctt gcc aag agc aca aaa aaa
2080 Ser Ser Tyr Phe Ile Asn Phe Ile Tyr Leu Ala Lys Ser Thr Lys
Lys 415 420 425 acc atg cta act tta act ttg gta tgt gca att aca ttc
ctc ctt gtt 2128 Thr Met Leu Thr Leu Thr Leu Val Cys Ala Ile Thr
Phe Leu Leu Val 430 435 440 tgc agt gga aca ttt ttt cca tat agc tcc
aat cct gct aat ccg aag 2176 Cys Ser Gly Thr Phe Phe Pro Tyr Ser
Ser Asn Pro Ala Asn Pro Lys 445 450 455 cca aag aga gtg ttt ctt cag
cat atg act aga aca ttc cat gac ttg 2224 Pro Lys Arg Val Phe Leu
Gln His Met Thr Arg Thr Phe His Asp Leu 460 465 470 gaa gga aat gca
gtt aaa cgg gac tct gga ata tgg atc aat ggg ttt 2272 Glu Gly Asn
Ala Val Lys Arg Asp Ser Gly Ile Trp Ile Asn Gly Phe 475 480 485 490
gat tat act gga att tct cac ata acc cct cac att cct gag atc aat
2320 Asp Tyr Thr Gly Ile Ser His Ile Thr Pro His Ile Pro Glu Ile
Asn 495 500 505 gat agt atc cga gct cac tgt gag gag aat gca cct ctt
tgt ggt ttt 2368 Asp Ser Ile Arg Ala His Cys Glu Glu Asn Ala Pro
Leu Cys Gly Phe 510 515 520 cct tgg tat ctt cca gtg cac ttt ctg atc
agg aaa aac tgg tat ctt 2416 Pro Trp Tyr Leu Pro Val His Phe Leu
Ile Arg Lys Asn Trp Tyr Leu 525 530 535 cct gcc cca gaa gtt tct cca
aga aat cct cct cat ttc cga ctc ata 2464 Pro Ala Pro Glu Val Ser
Pro Arg Asn Pro Pro His Phe Arg Leu Ile 540 545 550 tcc aaa gaa cag
aca cct tgg gat tct ata aaa ttg act ttt gaa gca 2512 Ser Lys Glu
Gln Thr Pro Trp Asp Ser Ile Lys Leu Thr Phe Glu Ala 555 560 565 570
aca gga cca agc cat atg tcc ttc tat gtt cga gcc cac aaa ggg tca
2560 Thr Gly Pro Ser His Met Ser Phe Tyr Val Arg Ala His Lys Gly
Ser 575 580 585 aca ctt tct cag tgg tct ctt ggc aat ggc acc cca gtc
aca agt aaa 2608 Thr Leu Ser Gln Trp Ser Leu Gly Asn Gly Thr Pro
Val Thr Ser Lys 590 595 600 gga gga gac tac ttt gtc ttt tac tcc cat
gga ctc cag gcc tct gca 2656 Gly Gly Asp Tyr Phe Val Phe Tyr Ser
His Gly Leu Gln Ala Ser Ala 605 610 615 tgg cag ttc tgg ata gaa gtg
cag gtt tca gaa gaa cat cct gaa gga 2704 Trp Gln Phe Trp Ile Glu
Val Gln Val Ser Glu Glu His Pro Glu Gly 620 625 630 atg gtc acc gtg
gcc att gct gcc cac tat ctg tct ggg gaa gac aag 2752 Met Val Thr
Val Ala Ile Ala Ala His Tyr Leu Ser Gly Glu Asp Lys 635 640 645 650
aga tcc cct caa ctg gat gct ctg aag gaa aag ttc cca gat tgg aca
2800 Arg Ser Pro Gln Leu Asp Ala Leu Lys Glu Lys Phe Pro Asp Trp
Thr 655 660 665 ttt ccc tct gcc tgg gtg tgc acc tac gat ctc ttt gta
ttt taa 2845 Phe Pro Ser Ala Trp Val Cys Thr Tyr Asp Leu Phe Val
Phe * 670 675 680 tcttgtggat gagctctaag tacatgccca gtggatactc
catgtgacat ggtttctccc 2905 tatgttacgt ggatgtttgt aacgtaagtc
aatgaatttt aatgatcata tgttcaaaga 2965 gctttctggg ttaacgcttt
tcagggccaa gcactataag ggtttagctg tggcgcagtg 3025 atgcatggcc
tgttgacact tgaaaatgcc agtcttttgg cacttcagca catgtgggta 3085
ctgccactac acacacgtca ttttatatga ccttaaggac aaagccaaca atccacttca
3145 atagctgccc ctttaggatc aagaaagatg tacactgtca gagcattgtt
aatgagacaa 3205 aagttgtttc caatttaagc cccaaaacca tttgttgtat
tagtggatgg tgggtaaaat 3265 atcattcact gaggtaatga ttccccttga
gaatataact ctgtgtaggt cactggaaag 3325 tgattgccat agggctggga
gagaagcatt gcactcttga ggctgtagcc tgtgtcaagc 3385 tgtttcttca
ggcagcctct caaatgtgct ttgtctctct gtgctgaggc ctggaccctg 3445
tgctgagctg gtgactcact gtcctgacaa gtggacacac agatgcactg ctgtgctgct
3505 ttcctgaggt ggttttctat gcctgttttc ctctgaaaca tgtctgttac
ccctctccat 3565 cttaccaagt tgaaaagggg aatatttggc cacatacccc
tctggttttc gtaggttctt 3625 ttggttcaga atattgtttg tgccagtaca
tgaccttaac ttccttcctc agagcactga 3685 gctgccatct gggctattct
ggggtagaag gaaggctggg agtggtggga attttataaa 3745 tatttattct
cttttctttg tttcatagga gtcttgtgtt atacaaggtt agtccttcat 3805
ggtataatct tactgatgca ctgggcctat ctttttgttt tccagccagt tgaatagatt
3865 agtttttctc agtaacttac tatccagcag actggctttc ctgagacttg
aggttgtggc 3925 ttatactgga atgagaccac tgtacgtgta ggtggttcag
atcctgcgta atggcagcat 3985 gaggacttaa aaggtggttt tcattttgaa
gatggctatg tagcttgtaa ggtgtatcac 4045 agcagtacct ctcatggctt
tttggttcca gcagtgaggg cattggtgag atcaatggta 4105 aactgtgcaa
gctttctttt tatcattagg aaatgtgaaa cgttggacaa attttgagtt 4165
ttaacaagga caaaaagttg aaagaaaagg cacagttaac aaaaaagggt ggctagattt
4225 atcttgggtg atggaggaaa tgagagagga atgctcttga aaggtggtct
gtggatctgt 4285 ctgaatagaa agagcacagt aagtatgcat tgccggagaa
aacgtccttg aagctgcttg 4345 tctcatgtgt atgatgtgct ttttaaatca
tgcccctcgt tgcctgccta atctgtgact 4405 ccctaaaaac taactgggcc
catgtagatg gggctgcaac cagagctgaa taacatgtta 4465 ggctcacaca
tgcatcagca ctgcacactg gaatcattgc tcttcctgga ctttgtagaa 4525
atcagtctca agtgcttcaa gagtctggct cctgctactt ttatctgtca ggtagcacat
4585 aaggtttgca gggtttatat tttgtataga atcacagttg tggagaaaaa
gtaataattt 4645 ctcaatgaat tttaaaaatg ggcctatttt ctatccccgt
ggttcatctg atataattag 4705 tgttccctgt gaattccccc cctctatggg
aaggatgcct ttactcttta tcagtaataa 4765 attatgactg ttttcatatt
gccttagggt tatttccctg tgtaaaccat tgtcttttgt 4825 tttggttttc
tttagcatta tgaagctttg gtattgtaca aggtcagtag taagatgctc 4885
actagtctca gggcttgtgt aatattctgg gaggtcattt aaatgccaga aatggtcaag
4945 caattataca cagtatttat gactctgtta agcataccgt ttgtctgtca
cattagtaga 5005 ttctgagatt aaaaaaaatt tttaaagagt gatcatttaa
ataatttcta aaagggtctt 5065 ttcaagctct aacaaagtca ctaacaaatg
cattattttc tacagaatta gatgttagta 5125 gtacagtact gcatattcag
ggaaaaagtg tgaggaattg atttcaaaat agttcgttct 5185 tgtgtttgac
ctaagaatga ttgtcgcatg aagtgtttgt ttttacagtt tagcatatat 5245
aaacaaacat gataggattc cttaagatgt taccacccag ggggccacaa gccagcctgc
5305 tgtctcagga agctgtagaa ggagtgtttg tcaatttctt gtcactggtt
tgctgactta 5365 ctgaggatta attgttgcct tacaatgtta ctgaaataaa
ctgtttaata aaaaaaaaaa 5425 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaacaaa 5485 aaaaaaaggg cggccgc 5502 27
680 PRT Homo sapiens 27 Met Leu Glu Val Leu Arg Val Leu Ser Thr Ser
Ser Glu Ala Leu His 1 5 10 15 His Ala Val Ile Phe Leu Phe Asn Gly
Ala Glu Glu Asn Val Leu Gln 20 25 30 Ala Ser His Gly Phe Ile Thr
Gln His Pro Trp Ala Ser Leu Ile Arg 35 40 45 Ala Phe Ile Asn Leu
Glu Ala Ala Gly Val Gly Gly Lys Glu Leu Val 50 55 60 Phe Gln Thr
Gly Pro Glu Asn Pro Trp Leu Val Gln Ala Tyr Val Ser 65 70 75 80 Ala
Ala Lys His Pro Phe Ala Ser Val Val Ala Gln Glu Val Phe Gln 85 90
95 Ser Gly Ile Ile Pro Ser Asp Thr Asp Phe Arg Ile Tyr Arg Asp Phe
100 105 110 Gly Asn Ile Pro Gly Ile Asp Leu Ala Phe Ile Glu Asn Gly
Tyr Ile 115 120 125 Tyr His Thr Lys Tyr Asp Thr Ala Asp Arg Ile Leu
Thr Asp Ser Ile 130 135 140 Gln Arg Ala Gly Asp Asn Ile Leu Ala Val
Leu Lys His Leu Ala Thr 145 150 155 160 Ser Asp Met Leu Ala Ala Ala
Ser Lys Tyr Arg His Gly Asn Met Val 165 170 175 Phe Phe Asp Val Leu
Gly Leu Phe Val Ile Ala Tyr Pro Ser Arg Ile 180 185 190 Gly Ser Ile
Ile Asn Tyr Met Val Val Met Gly Val Val Leu Tyr Leu 195 200 205 Gly
Lys Lys Phe Leu Gln Pro Lys His Lys Thr Gly Asn Tyr Lys Lys 210 215
220 Asp Phe Leu Cys Gly Leu Gly Ile Thr Leu Ile Ser Trp Phe Thr Ser
225 230 235 240 Leu Val Thr Val Leu Ile Ile Ala Val Phe Ile Ser Leu
Ile Gly Gln 245 250 255 Ser Leu Ser Trp Tyr Asn His Phe Tyr Val Ser
Val Cys Leu Tyr Gly 260 265 270 Thr Ala Thr Val Ala Lys Ile Ile Leu
Ile His Thr Leu Ala Lys Arg 275 280 285 Phe Tyr Tyr Met Asn Ala Ser
Ala Gln Tyr Leu Gly Glu Val Phe Phe 290 295 300 Asp Ile Ser Leu Phe
Val His Cys Cys Phe Leu Val Thr Leu Thr Tyr 305 310 315 320 Gln Gly
Leu Cys Ser Ala Phe Ile Ser Ala Val Trp Val Ala Phe Pro 325 330 335
Leu Leu Thr Lys Leu Cys Val His Lys Asp Phe Lys Gln His Gly Ala 340
345 350 Gln Gly Lys Phe Ile Ala Phe Tyr Leu Leu Gly Met Phe Ile Pro
Tyr 355 360 365 Leu Tyr Ala Leu Tyr Leu Ile Trp Ala Val Phe Glu Met
Phe Thr Pro 370 375 380 Ile Leu Gly Arg Ser Gly Ser Glu Ile Pro Pro
Asp Val Val Leu Ala 385 390 395 400 Ser Ile Leu Ala Gly Cys Thr Met
Ile Leu Ser Ser Tyr Phe Ile Asn 405 410 415 Phe Ile Tyr Leu Ala Lys
Ser Thr Lys Lys Thr Met Leu Thr Leu Thr 420 425 430 Leu Val Cys Ala
Ile Thr Phe Leu Leu Val Cys Ser Gly Thr Phe Phe 435 440 445 Pro Tyr
Ser Ser Asn Pro Ala Asn Pro Lys Pro Lys Arg Val Phe Leu 450 455 460
Gln His Met Thr Arg Thr Phe His Asp Leu Glu Gly Asn Ala Val Lys 465
470 475 480 Arg Asp Ser Gly Ile Trp Ile Asn Gly Phe Asp Tyr Thr Gly
Ile Ser 485 490 495 His Ile Thr Pro His Ile Pro Glu Ile Asn Asp Ser
Ile Arg Ala His 500 505 510 Cys Glu Glu Asn Ala Pro Leu Cys Gly Phe
Pro Trp Tyr Leu Pro Val 515 520 525 His Phe Leu Ile Arg Lys Asn Trp
Tyr Leu Pro Ala Pro Glu Val Ser 530 535 540 Pro Arg Asn Pro Pro His
Phe Arg Leu Ile Ser Lys Glu Gln Thr Pro 545 550 555 560 Trp Asp Ser
Ile Lys Leu Thr Phe Glu Ala Thr Gly Pro Ser His Met 565 570 575 Ser
Phe Tyr Val Arg Ala His Lys Gly Ser Thr Leu Ser Gln Trp Ser 580 585
590 Leu Gly Asn Gly Thr Pro Val Thr Ser Lys Gly Gly Asp Tyr Phe Val
595 600 605 Phe Tyr Ser His Gly Leu Gln Ala Ser Ala Trp Gln Phe Trp
Ile Glu 610 615 620 Val Gln Val Ser Glu Glu His Pro Glu Gly Met Val
Thr Val Ala Ile 625 630 635 640 Ala Ala His Tyr Leu Ser Gly Glu Asp
Lys Arg Ser Pro Gln Leu Asp 645 650 655 Ala Leu Lys Glu Lys Phe Pro
Asp Trp Thr Phe Pro Ser Ala Trp Val 660 665 670 Cys Thr Tyr Asp Leu
Phe Val Phe 675 680 28 2043 DNA Homo sapiens CDS (1)...(2043) 28
atg ctg gaa gtc ctt cgc gtc ttg tca aca tct tca gaa gcc ttg cat 48
Met Leu Glu Val Leu Arg Val Leu Ser Thr Ser Ser Glu Ala Leu His 1 5
10 15 cat gct gtc ata ttt ctc ttt aat ggt gct gag gaa aat gtc ttg
caa 96 His Ala Val Ile Phe Leu Phe Asn Gly Ala Glu Glu Asn Val Leu
Gln 20 25 30 gcc agt cat ggt ttc att act cag cac ccc tgg gct agc
ttg att cgt 144 Ala Ser His Gly Phe Ile Thr Gln His Pro Trp Ala Ser
Leu Ile Arg 35 40 45 gca ttc att aac cta gag gca gca ggt gta gga
ggg aaa gaa ctt gta 192 Ala Phe Ile Asn Leu Glu Ala Ala Gly Val Gly
Gly Lys Glu Leu Val 50 55 60 ttc caa aca ggt cct gaa aat cct tgg
ttg gtt caa gct tat gtt tca 240 Phe Gln Thr Gly Pro Glu Asn Pro Trp
Leu Val Gln Ala Tyr Val Ser 65 70 75 80 gca gct aaa cac cct ttt gct
tct gtg gtg gct cag gag gtt ttt cag 288 Ala Ala Lys His Pro Phe Ala
Ser Val Val Ala Gln Glu Val Phe Gln 85 90 95 agt gga atc att cct
tca gat act gac ttt cgt atc tac agg gat ttt 336 Ser Gly Ile Ile Pro
Ser Asp Thr Asp Phe Arg Ile Tyr Arg Asp Phe 100 105 110 ggg aac att
cca gga ata gac tta gct ttt att gag aat gga tac att 384 Gly Asn Ile
Pro Gly Ile Asp Leu Ala Phe Ile Glu Asn Gly Tyr Ile 115 120 125 tat
cac acc aag tat gac aca gcg gac aga att cta aca gat tcc att 432 Tyr
His Thr Lys Tyr Asp Thr Ala Asp Arg Ile Leu Thr Asp Ser Ile 130 135
140 cag aga gca ggt gac aac att tta gca gtt ctt aag cat cta gct aca
480 Gln Arg Ala Gly Asp Asn Ile Leu Ala Val Leu Lys His Leu Ala Thr
145 150 155 160 tct gat atg ctg gct gct gct tct aag tat cga cat gga
aac atg gtc 528 Ser Asp Met Leu Ala Ala Ala Ser Lys Tyr Arg His Gly
Asn Met Val 165 170 175 ttc ttt gat gtg ctg ggc ctg ttt gtc att gcc
tac ccc tct cgt att 576 Phe Phe Asp Val Leu Gly Leu Phe Val Ile Ala
Tyr Pro Ser Arg Ile 180 185 190 ggc tca atc ata aac tac atg gtg gta
atg ggt gtt gtt ttg tac ctg 624 Gly Ser Ile Ile Asn Tyr Met Val Val
Met Gly Val Val Leu Tyr Leu 195 200 205 ggc aaa aaa ttt ttg cag ccc
aaa cat aag act ggt aac tac aag aag 672 Gly Lys Lys Phe Leu Gln Pro
Lys His Lys Thr Gly Asn Tyr Lys Lys 210 215 220 gac ttc ttg tgt gga
ctt ggc atc act ttg atc agc tgg ttc act agc 720 Asp Phe Leu Cys Gly
Leu Gly Ile Thr Leu Ile Ser Trp Phe Thr Ser 225 230 235 240 ctt gtt
acc gtt ctc att ata gca gtg ttc atc tct ctt att gga cag 768 Leu Val
Thr Val Leu Ile Ile Ala Val Phe Ile Ser Leu Ile Gly Gln 245 250 255
tct ctc tca tgg tat aac cac ttc tat gtc tcc gtt tgt ctg tat gga 816
Ser Leu Ser Trp Tyr Asn His Phe Tyr Val Ser Val Cys Leu Tyr Gly 260
265 270 act gca act gta gcc aaa ata ata ctt ata cat act ctt gcg aaa
aga 864 Thr Ala Thr Val Ala Lys Ile Ile Leu Ile His Thr Leu Ala Lys
Arg 275 280 285 ttt tat tac atg aat gcc agt gcc cag tat ctg gga gaa
gta ttt ttt 912 Phe Tyr Tyr Met Asn Ala Ser Ala Gln Tyr Leu Gly Glu
Val Phe Phe 290 295 300 gac att tcg ctg ttt gtc cat tgc tgt ttt ctt
gtt acc ctc act tac 960 Asp Ile Ser Leu Phe Val His Cys Cys Phe Leu
Val Thr Leu Thr Tyr 305 310 315 320 caa gga ctt tgc tcg gcg ttt att
agt gct gtc tgg gta gca ttc cca 1008 Gln Gly Leu Cys Ser Ala Phe
Ile Ser Ala Val Trp Val Ala Phe Pro 325 330 335 ttg ctc aca aag ctc
tgt gtg cat aag gac ttc aag cag cat ggt gcc 1056 Leu Leu Thr Lys
Leu Cys Val His Lys Asp Phe Lys Gln His Gly Ala 340 345 350 caa gga
aaa ttt att gct ttt tac ctt ttg ggg atg ttt att cct tat 1104 Gln
Gly Lys Phe Ile Ala Phe Tyr Leu Leu Gly Met Phe Ile Pro Tyr 355 360
365 ctt tat gca ttg tac ctc atc tgg gca gta ttt gag atg ttt acc cct
1152 Leu Tyr Ala Leu Tyr Leu Ile Trp Ala Val Phe Glu Met Phe Thr
Pro 370 375 380 atc ctc ggg aga agt ggt tct gaa atc cca cct gat gtt
gtg ctg gca 1200 Ile Leu Gly Arg Ser Gly Ser Glu Ile Pro Pro Asp
Val Val Leu Ala 385 390 395 400 tcc att ttg gct ggc tgt aca atg att
ctc tcg tcc tat ttt att aac 1248 Ser Ile Leu Ala Gly Cys Thr Met
Ile Leu Ser Ser Tyr Phe Ile Asn 405 410 415 ttc atc tac ctt gcc aag
agc aca aaa aaa acc atg cta act tta act 1296 Phe Ile Tyr Leu Ala
Lys Ser Thr Lys Lys Thr Met Leu Thr Leu Thr 420 425 430 ttg gta tgt
gca att aca ttc ctc ctt gtt tgc agt gga aca ttt ttt 1344 Leu Val
Cys Ala Ile Thr Phe Leu Leu Val Cys Ser Gly Thr Phe Phe 435 440 445
cca tat agc tcc aat cct gct aat ccg aag cca aag aga gtg ttt ctt
1392 Pro Tyr Ser Ser Asn Pro Ala Asn Pro Lys Pro Lys Arg Val Phe
Leu 450 455 460 cag cat atg act aga
aca ttc cat gac ttg gaa gga aat gca gtt aaa 1440 Gln His Met Thr
Arg Thr Phe His Asp Leu Glu Gly Asn Ala Val Lys 465 470 475 480 cgg
gac tct gga ata tgg atc aat ggg ttt gat tat act gga att tct 1488
Arg Asp Ser Gly Ile Trp Ile Asn Gly Phe Asp Tyr Thr Gly Ile Ser 485
490 495 cac ata acc cct cac att cct gag atc aat gat agt atc cga gct
cac 1536 His Ile Thr Pro His Ile Pro Glu Ile Asn Asp Ser Ile Arg
Ala His 500 505 510 tgt gag gag aat gca cct ctt tgt ggt ttt cct tgg
tat ctt cca gtg 1584 Cys Glu Glu Asn Ala Pro Leu Cys Gly Phe Pro
Trp Tyr Leu Pro Val 515 520 525 cac ttt ctg atc agg aaa aac tgg tat
ctt cct gcc cca gaa gtt tct 1632 His Phe Leu Ile Arg Lys Asn Trp
Tyr Leu Pro Ala Pro Glu Val Ser 530 535 540 cca aga aat cct cct cat
ttc cga ctc ata tcc aaa gaa cag aca cct 1680 Pro Arg Asn Pro Pro
His Phe Arg Leu Ile Ser Lys Glu Gln Thr Pro 545 550 555 560 tgg gat
tct ata aaa ttg act ttt gaa gca aca gga cca agc cat atg 1728 Trp
Asp Ser Ile Lys Leu Thr Phe Glu Ala Thr Gly Pro Ser His Met 565 570
575 tcc ttc tat gtt cga gcc cac aaa ggg tca aca ctt tct cag tgg tct
1776 Ser Phe Tyr Val Arg Ala His Lys Gly Ser Thr Leu Ser Gln Trp
Ser 580 585 590 ctt ggc aat ggc acc cca gtc aca agt aaa gga gga gac
tac ttt gtc 1824 Leu Gly Asn Gly Thr Pro Val Thr Ser Lys Gly Gly
Asp Tyr Phe Val 595 600 605 ttt tac tcc cat gga ctc cag gcc tct gca
tgg cag ttc tgg ata gaa 1872 Phe Tyr Ser His Gly Leu Gln Ala Ser
Ala Trp Gln Phe Trp Ile Glu 610 615 620 gtg cag gtt tca gaa gaa cat
cct gaa gga atg gtc acc gtg gcc att 1920 Val Gln Val Ser Glu Glu
His Pro Glu Gly Met Val Thr Val Ala Ile 625 630 635 640 gct gcc cac
tat ctg tct ggg gaa gac aag aga tcc cct caa ctg gat 1968 Ala Ala
His Tyr Leu Ser Gly Glu Asp Lys Arg Ser Pro Gln Leu Asp 645 650 655
gct ctg aag gaa aag ttc cca gat tgg aca ttt ccc tct gcc tgg gtg
2016 Ala Leu Lys Glu Lys Phe Pro Asp Trp Thr Phe Pro Ser Ala Trp
Val 660 665 670 tgc acc tac gat ctc ttt gta ttt taa 2043 Cys Thr
Tyr Asp Leu Phe Val Phe * 675 680 29 2566 DNA Homo sapiens CDS
(194)...(2470) 29 raagggacta gtcctgcaag tttaaacgaa tttcactcct
cttactacac tatagggctc 60 gagcggccgc ccgggcaggt ggggggcata
ggtgaaaaat agtaaagaga ggaaagccag 120 aaacagtgag ccagggaaca
aggggacgga agacaggatc caggctgtat ctacggagag 180 acacagaagg gag atg
ctg ctg ctg ccg ctg ctg ctg ctg ctg ccg cca 229 Met Leu Leu Leu Pro
Leu Leu Leu Leu Leu Pro Pro 1 5 10 cta gtc ctc agg gtt gct gca agc
cga tgt cta cat gat gag aca cag 277 Leu Val Leu Arg Val Ala Ala Ser
Arg Cys Leu His Asp Glu Thr Gln 15 20 25 aag tct gtg agc ctt ctc
agg ccc cct ttc tcc caa ctc ccc tca aaa 325 Lys Ser Val Ser Leu Leu
Arg Pro Pro Phe Ser Gln Leu Pro Ser Lys 30 35 40 tct cgc tct tcc
tcc ctc acc ctc cct agc tcc cgt gat cct caa ccc 373 Ser Arg Ser Ser
Ser Leu Thr Leu Pro Ser Ser Arg Asp Pro Gln Pro 45 50 55 60 cta cga
atc caa agc tgc tat cta gga gat cat ata tca gat gga gct 421 Leu Arg
Ile Gln Ser Cys Tyr Leu Gly Asp His Ile Ser Asp Gly Ala 65 70 75
tgg gat cct gag gga gaa ggg atg aga ggg gga tcc cga gcc ctg gcc 469
Trp Asp Pro Glu Gly Glu Gly Met Arg Gly Gly Ser Arg Ala Leu Ala 80
85 90 gca gtg aga gag gcc act cag cga atc cag gct gtt cta gca gtc
cct 517 Ala Val Arg Glu Ala Thr Gln Arg Ile Gln Ala Val Leu Ala Val
Pro 95 100 105 cca gtg caa gga ccc ctg ctt ctg agt cga gac cct gca
cag tat tgc 565 Pro Val Gln Gly Pro Leu Leu Leu Ser Arg Asp Pro Ala
Gln Tyr Cys 110 115 120 cac gct gtc tgg gga gac cca gat agc cca aac
tac cac agg tgc agc 613 His Ala Val Trp Gly Asp Pro Asp Ser Pro Asn
Tyr His Arg Cys Ser 125 130 135 140 ctc ttg aac cca gga tac aaa gga
gag agt tgc ctg ggg gca aag att 661 Leu Leu Asn Pro Gly Tyr Lys Gly
Glu Ser Cys Leu Gly Ala Lys Ile 145 150 155 cct gac acc cat ctt cgc
ggt tat gcc ttg tgg ccg gag cag ggt ccc 709 Pro Asp Thr His Leu Arg
Gly Tyr Ala Leu Trp Pro Glu Gln Gly Pro 160 165 170 cca caa ctg gtc
cag cca gat ggg cct ggg gtc caa aac act gat ttt 757 Pro Gln Leu Val
Gln Pro Asp Gly Pro Gly Val Gln Asn Thr Asp Phe 175 180 185 ctc ctg
tat gtg cga gtt gct cac act tcc aag tgc cac caa gag aca 805 Leu Leu
Tyr Val Arg Val Ala His Thr Ser Lys Cys His Gln Glu Thr 190 195 200
gtc tca ctc tgt tgc cca ggc tgg agt aca gcg gcc caa tca cag ctc 853
Val Ser Leu Cys Cys Pro Gly Trp Ser Thr Ala Ala Gln Ser Gln Leu 205
210 215 220 acc gca gcc ttg acc tcc tgg gct cag ccc tct gtc ata gcc
tat gct 901 Thr Ala Ala Leu Thr Ser Trp Ala Gln Pro Ser Val Ile Ala
Tyr Ala 225 230 235 gcc tgc tgc cag ctg gac tca gaa gac agg ccc ctc
gct ggt acc att 949 Ala Cys Cys Gln Leu Asp Ser Glu Asp Arg Pro Leu
Ala Gly Thr Ile 240 245 250 gtc tac tgt gcc caa cat ctc acc agc ccc
agc ctc agc cac agt gac 997 Val Tyr Cys Ala Gln His Leu Thr Ser Pro
Ser Leu Ser His Ser Asp 255 260 265 atc gtc atg gcc aca tta cat gaa
ttg ctc cat gcc ttg ggt ttc tct 1045 Ile Val Met Ala Thr Leu His
Glu Leu Leu His Ala Leu Gly Phe Ser 270 275 280 gga cag ctc ttc aag
aaa tgg cga gac tgc ccc tca gga ttc agt gtt 1093 Gly Gln Leu Phe
Lys Lys Trp Arg Asp Cys Pro Ser Gly Phe Ser Val 285 290 295 300 aga
gag aac tgt tct aca agg caa caa gtg aca agg caa gat gag tgg 1141
Arg Glu Asn Cys Ser Thr Arg Gln Gln Val Thr Arg Gln Asp Glu Trp 305
310 315 gga caa ctg ctt ctc acc acc cca gct gtt agc ctc agc ctg gcc
aaa 1189 Gly Gln Leu Leu Leu Thr Thr Pro Ala Val Ser Leu Ser Leu
Ala Lys 320 325 330 cac ttg gga gtg tcg ggg gct tcc ctg ggt gtt ccc
ttg gaa gaa gag 1237 His Leu Gly Val Ser Gly Ala Ser Leu Gly Val
Pro Leu Glu Glu Glu 335 340 345 gag ggc ctt ctg tcc tcg cac tgg gag
gcc aga cta ctc cag ggt tct 1285 Glu Gly Leu Leu Ser Ser His Trp
Glu Ala Arg Leu Leu Gln Gly Ser 350 355 360 tta atg act gct acc ttt
gat gga gcc cag cgc act cga ctc gac cca 1333 Leu Met Thr Ala Thr
Phe Asp Gly Ala Gln Arg Thr Arg Leu Asp Pro 365 370 375 380 atc acc
ctc gct gcc ttc aaa gac tca ggc tgg tac cag gtc aac cac 1381 Ile
Thr Leu Ala Ala Phe Lys Asp Ser Gly Trp Tyr Gln Val Asn His 385 390
395 agc gct gca gag gag ctg ttg tgg ggc cag gga tct ggc cca gaa ttt
1429 Ser Ala Ala Glu Glu Leu Leu Trp Gly Gln Gly Ser Gly Pro Glu
Phe 400 405 410 ggc ttg gtg acc aca tgt ggg act ggc tcc tca gac ttc
ttc tgt act 1477 Gly Leu Val Thr Thr Cys Gly Thr Gly Ser Ser Asp
Phe Phe Cys Thr 415 420 425 ggc agt ggg ctg ggc tgc cac tac ctg cac
ctg gac aag gga agc tgc 1525 Gly Ser Gly Leu Gly Cys His Tyr Leu
His Leu Asp Lys Gly Ser Cys 430 435 440 tcc tca gac ccc atg ctg gaa
ggc tgc cgc atg tac aag ccc tta gcc 1573 Ser Ser Asp Pro Met Leu
Glu Gly Cys Arg Met Tyr Lys Pro Leu Ala 445 450 455 460 aat ggg agt
gaa tgc tgg aag aag gaa aac gga ttc cct gct ggg gtg 1621 Asn Gly
Ser Glu Cys Trp Lys Lys Glu Asn Gly Phe Pro Ala Gly Val 465 470 475
gat aat ccc cat ggg gag atc tac cat ccc cag agc cgt tgc ttc ttt
1669 Asp Asn Pro His Gly Glu Ile Tyr His Pro Gln Ser Arg Cys Phe
Phe 480 485 490 gcc aac ctc act tca cag ctg ctc cct ggg gat aag ccc
agg cat cct 1717 Ala Asn Leu Thr Ser Gln Leu Leu Pro Gly Asp Lys
Pro Arg His Pro 495 500 505 tct ctt acc cca cac ctc aag gaa gca gag
ctc atg ggc cgc tgc tac 1765 Ser Leu Thr Pro His Leu Lys Glu Ala
Glu Leu Met Gly Arg Cys Tyr 510 515 520 tta cat caa tgc aca ggg agg
gga gct tac aag gtg cag gtg gag ggc 1813 Leu His Gln Cys Thr Gly
Arg Gly Ala Tyr Lys Val Gln Val Glu Gly 525 530 535 540 tcg cct tgg
gtc cca tgc ctt cct gga aag gtt ata cag ata cct ggg 1861 Ser Pro
Trp Val Pro Cys Leu Pro Gly Lys Val Ile Gln Ile Pro Gly 545 550 555
tac tat ggt ctt ctc ttc tgt ccc cgg ggt cgg ctg tgt cag act aat
1909 Tyr Tyr Gly Leu Leu Phe Cys Pro Arg Gly Arg Leu Cys Gln Thr
Asn 560 565 570 gaa ggt atc aat gct gtt act tcc cca cct gtg agt ctt
tca acc cca 1957 Glu Gly Ile Asn Ala Val Thr Ser Pro Pro Val Ser
Leu Ser Thr Pro 575 580 585 gat cca cta ttc cag ctc tct tta gaa tta
gct ggg cct cca gga cac 2005 Asp Pro Leu Phe Gln Leu Ser Leu Glu
Leu Ala Gly Pro Pro Gly His 590 595 600 tct ctg ggg aag gaa cag caa
gaa ggg cta gct gaa gca gta ctg gag 2053 Ser Leu Gly Lys Glu Gln
Gln Glu Gly Leu Ala Glu Ala Val Leu Glu 605 610 615 620 gct ttg gcg
agc aga ggc ggc act ggc agg tgc tat ttc cat ggc cca 2101 Ala Leu
Ala Ser Arg Gly Gly Thr Gly Arg Cys Tyr Phe His Gly Pro 625 630 635
tca att acc act agc ttg gtg ttt act gtg cat atg tgg aag tcc cct
2149 Ser Ile Thr Thr Ser Leu Val Phe Thr Val His Met Trp Lys Ser
Pro 640 645 650 ggc tgc caa ggg cct tca gtt gct aca ctg cac aag gcc
ctg act ctg 2197 Gly Cys Gln Gly Pro Ser Val Ala Thr Leu His Lys
Ala Leu Thr Leu 655 660 665 act ctc cag aaa aaa ccc cta gaa gtg tat
cat gga gga gcc aac ttt 2245 Thr Leu Gln Lys Lys Pro Leu Glu Val
Tyr His Gly Gly Ala Asn Phe 670 675 680 acc aca caa ccc agc aag ttg
ctg gtt act tca gac cat aat ccc tcc 2293 Thr Thr Gln Pro Ser Lys
Leu Leu Val Thr Ser Asp His Asn Pro Ser 685 690 695 700 atg acc cac
cta agg ctg tcc atg gga ctc tgc cta atg ctg cta atc 2341 Met Thr
His Leu Arg Leu Ser Met Gly Leu Cys Leu Met Leu Leu Ile 705 710 715
ctg gtg ggt gta atg gga acc aca gcc tac cag aaa aga gcc act ctt
2389 Leu Val Gly Val Met Gly Thr Thr Ala Tyr Gln Lys Arg Ala Thr
Leu 720 725 730 cct gtg aga cca tct gcc tct tac cat tca cca gag ctc
cac agc aca 2437 Pro Val Arg Pro Ser Ala Ser Tyr His Ser Pro Glu
Leu His Ser Thr 735 740 745 agg gtc cca gtt aga gga ata agg gag gtg
tga tgttgcccag aacatgacag 2490 Arg Val Pro Val Arg Gly Ile Arg Glu
Val * 750 755 ggggtaagga agagaataat ttcttgtgag acgactggat
ggaaaatcta ttgggtatac 2550 ttaatttcta ctttct 2566 30 758 PRT Homo
sapiens 30 Met Leu Leu Leu Pro Leu Leu Leu Leu Leu Pro Pro Leu Val
Leu Arg 1 5 10 15 Val Ala Ala Ser Arg Cys Leu His Asp Glu Thr Gln
Lys Ser Val Ser 20 25 30 Leu Leu Arg Pro Pro Phe Ser Gln Leu Pro
Ser Lys Ser Arg Ser Ser 35 40 45 Ser Leu Thr Leu Pro Ser Ser Arg
Asp Pro Gln Pro Leu Arg Ile Gln 50 55 60 Ser Cys Tyr Leu Gly Asp
His Ile Ser Asp Gly Ala Trp Asp Pro Glu 65 70 75 80 Gly Glu Gly Met
Arg Gly Gly Ser Arg Ala Leu Ala Ala Val Arg Glu 85 90 95 Ala Thr
Gln Arg Ile Gln Ala Val Leu Ala Val Pro Pro Val Gln Gly 100 105 110
Pro Leu Leu Leu Ser Arg Asp Pro Ala Gln Tyr Cys His Ala Val Trp 115
120 125 Gly Asp Pro Asp Ser Pro Asn Tyr His Arg Cys Ser Leu Leu Asn
Pro 130 135 140 Gly Tyr Lys Gly Glu Ser Cys Leu Gly Ala Lys Ile Pro
Asp Thr His 145 150 155 160 Leu Arg Gly Tyr Ala Leu Trp Pro Glu Gln
Gly Pro Pro Gln Leu Val 165 170 175 Gln Pro Asp Gly Pro Gly Val Gln
Asn Thr Asp Phe Leu Leu Tyr Val 180 185 190 Arg Val Ala His Thr Ser
Lys Cys His Gln Glu Thr Val Ser Leu Cys 195 200 205 Cys Pro Gly Trp
Ser Thr Ala Ala Gln Ser Gln Leu Thr Ala Ala Leu 210 215 220 Thr Ser
Trp Ala Gln Pro Ser Val Ile Ala Tyr Ala Ala Cys Cys Gln 225 230 235
240 Leu Asp Ser Glu Asp Arg Pro Leu Ala Gly Thr Ile Val Tyr Cys Ala
245 250 255 Gln His Leu Thr Ser Pro Ser Leu Ser His Ser Asp Ile Val
Met Ala 260 265 270 Thr Leu His Glu Leu Leu His Ala Leu Gly Phe Ser
Gly Gln Leu Phe 275 280 285 Lys Lys Trp Arg Asp Cys Pro Ser Gly Phe
Ser Val Arg Glu Asn Cys 290 295 300 Ser Thr Arg Gln Gln Val Thr Arg
Gln Asp Glu Trp Gly Gln Leu Leu 305 310 315 320 Leu Thr Thr Pro Ala
Val Ser Leu Ser Leu Ala Lys His Leu Gly Val 325 330 335 Ser Gly Ala
Ser Leu Gly Val Pro Leu Glu Glu Glu Glu Gly Leu Leu 340 345 350 Ser
Ser His Trp Glu Ala Arg Leu Leu Gln Gly Ser Leu Met Thr Ala 355 360
365 Thr Phe Asp Gly Ala Gln Arg Thr Arg Leu Asp Pro Ile Thr Leu Ala
370 375 380 Ala Phe Lys Asp Ser Gly Trp Tyr Gln Val Asn His Ser Ala
Ala Glu 385 390 395 400 Glu Leu Leu Trp Gly Gln Gly Ser Gly Pro Glu
Phe Gly Leu Val Thr 405 410 415 Thr Cys Gly Thr Gly Ser Ser Asp Phe
Phe Cys Thr Gly Ser Gly Leu 420 425 430 Gly Cys His Tyr Leu His Leu
Asp Lys Gly Ser Cys Ser Ser Asp Pro 435 440 445 Met Leu Glu Gly Cys
Arg Met Tyr Lys Pro Leu Ala Asn Gly Ser Glu 450 455 460 Cys Trp Lys
Lys Glu Asn Gly Phe Pro Ala Gly Val Asp Asn Pro His 465 470 475 480
Gly Glu Ile Tyr His Pro Gln Ser Arg Cys Phe Phe Ala Asn Leu Thr 485
490 495 Ser Gln Leu Leu Pro Gly Asp Lys Pro Arg His Pro Ser Leu Thr
Pro 500 505 510 His Leu Lys Glu Ala Glu Leu Met Gly Arg Cys Tyr Leu
His Gln Cys 515 520 525 Thr Gly Arg Gly Ala Tyr Lys Val Gln Val Glu
Gly Ser Pro Trp Val 530 535 540 Pro Cys Leu Pro Gly Lys Val Ile Gln
Ile Pro Gly Tyr Tyr Gly Leu 545 550 555 560 Leu Phe Cys Pro Arg Gly
Arg Leu Cys Gln Thr Asn Glu Gly Ile Asn 565 570 575 Ala Val Thr Ser
Pro Pro Val Ser Leu Ser Thr Pro Asp Pro Leu Phe 580 585 590 Gln Leu
Ser Leu Glu Leu Ala Gly Pro Pro Gly His Ser Leu Gly Lys 595 600 605
Glu Gln Gln Glu Gly Leu Ala Glu Ala Val Leu Glu Ala Leu Ala Ser 610
615 620 Arg Gly Gly Thr Gly Arg Cys Tyr Phe His Gly Pro Ser Ile Thr
Thr 625 630 635 640 Ser Leu Val Phe Thr Val His Met Trp Lys Ser Pro
Gly Cys Gln Gly 645 650 655 Pro Ser Val Ala Thr Leu His Lys Ala Leu
Thr Leu Thr Leu Gln Lys 660 665 670 Lys Pro Leu Glu Val Tyr His Gly
Gly Ala Asn Phe Thr Thr Gln Pro 675 680 685 Ser Lys Leu Leu Val Thr
Ser Asp His Asn Pro Ser Met Thr His Leu 690 695 700 Arg Leu Ser Met
Gly Leu Cys Leu Met Leu Leu Ile Leu Val Gly Val 705 710 715 720 Met
Gly Thr Thr Ala Tyr Gln Lys Arg Ala Thr Leu Pro Val Arg Pro 725 730
735 Ser Ala Ser Tyr His Ser Pro Glu Leu His Ser Thr Arg Val Pro Val
740 745 750 Arg Gly Ile Arg Glu Val 755 31 2277 DNA Homo sapiens
CDS (1)...(2277) 31 atg ctg ctg ctg ccg ctg ctg ctg ctg ctg ccg cca
cta gtc ctc agg 48 Met Leu Leu Leu Pro Leu Leu Leu Leu Leu Pro Pro
Leu Val Leu Arg 1 5 10 15 gtt gct gca agc cga tgt cta cat gat gag
aca cag aag tct gtg agc 96 Val Ala Ala Ser Arg Cys Leu His Asp Glu
Thr Gln Lys Ser Val
Ser 20 25 30 ctt ctc agg ccc cct ttc tcc caa ctc ccc tca aaa tct
cgc tct tcc 144 Leu Leu Arg Pro Pro Phe Ser Gln Leu Pro Ser Lys Ser
Arg Ser Ser 35 40 45 tcc ctc acc ctc cct agc tcc cgt gat cct caa
ccc cta cga atc caa 192 Ser Leu Thr Leu Pro Ser Ser Arg Asp Pro Gln
Pro Leu Arg Ile Gln 50 55 60 agc tgc tat cta gga gat cat ata tca
gat gga gct tgg gat cct gag 240 Ser Cys Tyr Leu Gly Asp His Ile Ser
Asp Gly Ala Trp Asp Pro Glu 65 70 75 80 gga gaa ggg atg aga ggg gga
tcc cga gcc ctg gcc gca gtg aga gag 288 Gly Glu Gly Met Arg Gly Gly
Ser Arg Ala Leu Ala Ala Val Arg Glu 85 90 95 gcc act cag cga atc
cag gct gtt cta gca gtc cct cca gtg caa gga 336 Ala Thr Gln Arg Ile
Gln Ala Val Leu Ala Val Pro Pro Val Gln Gly 100 105 110 ccc ctg ctt
ctg agt cga gac cct gca cag tat tgc cac gct gtc tgg 384 Pro Leu Leu
Leu Ser Arg Asp Pro Ala Gln Tyr Cys His Ala Val Trp 115 120 125 gga
gac cca gat agc cca aac tac cac agg tgc agc ctc ttg aac cca 432 Gly
Asp Pro Asp Ser Pro Asn Tyr His Arg Cys Ser Leu Leu Asn Pro 130 135
140 gga tac aaa gga gag agt tgc ctg ggg gca aag att cct gac acc cat
480 Gly Tyr Lys Gly Glu Ser Cys Leu Gly Ala Lys Ile Pro Asp Thr His
145 150 155 160 ctt cgc ggt tat gcc ttg tgg ccg gag cag ggt ccc cca
caa ctg gtc 528 Leu Arg Gly Tyr Ala Leu Trp Pro Glu Gln Gly Pro Pro
Gln Leu Val 165 170 175 cag cca gat ggg cct ggg gtc caa aac act gat
ttt ctc ctg tat gtg 576 Gln Pro Asp Gly Pro Gly Val Gln Asn Thr Asp
Phe Leu Leu Tyr Val 180 185 190 cga gtt gct cac act tcc aag tgc cac
caa gag aca gtc tca ctc tgt 624 Arg Val Ala His Thr Ser Lys Cys His
Gln Glu Thr Val Ser Leu Cys 195 200 205 tgc cca ggc tgg agt aca gcg
gcc caa tca cag ctc acc gca gcc ttg 672 Cys Pro Gly Trp Ser Thr Ala
Ala Gln Ser Gln Leu Thr Ala Ala Leu 210 215 220 acc tcc tgg gct cag
ccc tct gtc ata gcc tat gct gcc tgc tgc cag 720 Thr Ser Trp Ala Gln
Pro Ser Val Ile Ala Tyr Ala Ala Cys Cys Gln 225 230 235 240 ctg gac
tca gaa gac agg ccc ctc gct ggt acc att gtc tac tgt gcc 768 Leu Asp
Ser Glu Asp Arg Pro Leu Ala Gly Thr Ile Val Tyr Cys Ala 245 250 255
caa cat ctc acc agc ccc agc ctc agc cac agt gac atc gtc atg gcc 816
Gln His Leu Thr Ser Pro Ser Leu Ser His Ser Asp Ile Val Met Ala 260
265 270 aca tta cat gaa ttg ctc cat gcc ttg ggt ttc tct gga cag ctc
ttc 864 Thr Leu His Glu Leu Leu His Ala Leu Gly Phe Ser Gly Gln Leu
Phe 275 280 285 aag aaa tgg cga gac tgc ccc tca gga ttc agt gtt aga
gag aac tgt 912 Lys Lys Trp Arg Asp Cys Pro Ser Gly Phe Ser Val Arg
Glu Asn Cys 290 295 300 tct aca agg caa caa gtg aca agg caa gat gag
tgg gga caa ctg ctt 960 Ser Thr Arg Gln Gln Val Thr Arg Gln Asp Glu
Trp Gly Gln Leu Leu 305 310 315 320 ctc acc acc cca gct gtt agc ctc
agc ctg gcc aaa cac ttg gga gtg 1008 Leu Thr Thr Pro Ala Val Ser
Leu Ser Leu Ala Lys His Leu Gly Val 325 330 335 tcg ggg gct tcc ctg
ggt gtt ccc ttg gaa gaa gag gag ggc ctt ctg 1056 Ser Gly Ala Ser
Leu Gly Val Pro Leu Glu Glu Glu Glu Gly Leu Leu 340 345 350 tcc tcg
cac tgg gag gcc aga cta ctc cag ggt tct tta atg act gct 1104 Ser
Ser His Trp Glu Ala Arg Leu Leu Gln Gly Ser Leu Met Thr Ala 355 360
365 acc ttt gat gga gcc cag cgc act cga ctc gac cca atc acc ctc gct
1152 Thr Phe Asp Gly Ala Gln Arg Thr Arg Leu Asp Pro Ile Thr Leu
Ala 370 375 380 gcc ttc aaa gac tca ggc tgg tac cag gtc aac cac agc
gct gca gag 1200 Ala Phe Lys Asp Ser Gly Trp Tyr Gln Val Asn His
Ser Ala Ala Glu 385 390 395 400 gag ctg ttg tgg ggc cag gga tct ggc
cca gaa ttt ggc ttg gtg acc 1248 Glu Leu Leu Trp Gly Gln Gly Ser
Gly Pro Glu Phe Gly Leu Val Thr 405 410 415 aca tgt ggg act ggc tcc
tca gac ttc ttc tgt act ggc agt ggg ctg 1296 Thr Cys Gly Thr Gly
Ser Ser Asp Phe Phe Cys Thr Gly Ser Gly Leu 420 425 430 ggc tgc cac
tac ctg cac ctg gac aag gga agc tgc tcc tca gac ccc 1344 Gly Cys
His Tyr Leu His Leu Asp Lys Gly Ser Cys Ser Ser Asp Pro 435 440 445
atg ctg gaa ggc tgc cgc atg tac aag ccc tta gcc aat ggg agt gaa
1392 Met Leu Glu Gly Cys Arg Met Tyr Lys Pro Leu Ala Asn Gly Ser
Glu 450 455 460 tgc tgg aag aag gaa aac gga ttc cct gct ggg gtg gat
aat ccc cat 1440 Cys Trp Lys Lys Glu Asn Gly Phe Pro Ala Gly Val
Asp Asn Pro His 465 470 475 480 ggg gag atc tac cat ccc cag agc cgt
tgc ttc ttt gcc aac ctc act 1488 Gly Glu Ile Tyr His Pro Gln Ser
Arg Cys Phe Phe Ala Asn Leu Thr 485 490 495 tca cag ctg ctc cct ggg
gat aag ccc agg cat cct tct ctt acc cca 1536 Ser Gln Leu Leu Pro
Gly Asp Lys Pro Arg His Pro Ser Leu Thr Pro 500 505 510 cac ctc aag
gaa gca gag ctc atg ggc cgc tgc tac tta cat caa tgc 1584 His Leu
Lys Glu Ala Glu Leu Met Gly Arg Cys Tyr Leu His Gln Cys 515 520 525
aca ggg agg gga gct tac aag gtg cag gtg gag ggc tcg cct tgg gtc
1632 Thr Gly Arg Gly Ala Tyr Lys Val Gln Val Glu Gly Ser Pro Trp
Val 530 535 540 cca tgc ctt cct gga aag gtt ata cag ata cct ggg tac
tat ggt ctt 1680 Pro Cys Leu Pro Gly Lys Val Ile Gln Ile Pro Gly
Tyr Tyr Gly Leu 545 550 555 560 ctc ttc tgt ccc cgg ggt cgg ctg tgt
cag act aat gaa ggt atc aat 1728 Leu Phe Cys Pro Arg Gly Arg Leu
Cys Gln Thr Asn Glu Gly Ile Asn 565 570 575 gct gtt act tcc cca cct
gtg agt ctt tca acc cca gat cca cta ttc 1776 Ala Val Thr Ser Pro
Pro Val Ser Leu Ser Thr Pro Asp Pro Leu Phe 580 585 590 cag ctc tct
tta gaa tta gct ggg cct cca gga cac tct ctg ggg aag 1824 Gln Leu
Ser Leu Glu Leu Ala Gly Pro Pro Gly His Ser Leu Gly Lys 595 600 605
gaa cag caa gaa ggg cta gct gaa gca gta ctg gag gct ttg gcg agc
1872 Glu Gln Gln Glu Gly Leu Ala Glu Ala Val Leu Glu Ala Leu Ala
Ser 610 615 620 aga ggc ggc act ggc agg tgc tat ttc cat ggc cca tca
att acc act 1920 Arg Gly Gly Thr Gly Arg Cys Tyr Phe His Gly Pro
Ser Ile Thr Thr 625 630 635 640 agc ttg gtg ttt act gtg cat atg tgg
aag tcc cct ggc tgc caa ggg 1968 Ser Leu Val Phe Thr Val His Met
Trp Lys Ser Pro Gly Cys Gln Gly 645 650 655 cct tca gtt gct aca ctg
cac aag gcc ctg act ctg act ctc cag aaa 2016 Pro Ser Val Ala Thr
Leu His Lys Ala Leu Thr Leu Thr Leu Gln Lys 660 665 670 aaa ccc cta
gaa gtg tat cat gga gga gcc aac ttt acc aca caa ccc 2064 Lys Pro
Leu Glu Val Tyr His Gly Gly Ala Asn Phe Thr Thr Gln Pro 675 680 685
agc aag ttg ctg gtt act tca gac cat aat ccc tcc atg acc cac cta
2112 Ser Lys Leu Leu Val Thr Ser Asp His Asn Pro Ser Met Thr His
Leu 690 695 700 agg ctg tcc atg gga ctc tgc cta atg ctg cta atc ctg
gtg ggt gta 2160 Arg Leu Ser Met Gly Leu Cys Leu Met Leu Leu Ile
Leu Val Gly Val 705 710 715 720 atg gga acc aca gcc tac cag aaa aga
gcc act ctt cct gtg aga cca 2208 Met Gly Thr Thr Ala Tyr Gln Lys
Arg Ala Thr Leu Pro Val Arg Pro 725 730 735 tct gcc tct tac cat tca
cca gag ctc cac agc aca agg gtc cca gtt 2256 Ser Ala Ser Tyr His
Ser Pro Glu Leu His Ser Thr Arg Val Pro Val 740 745 750 aga gga ata
agg gag gtg tga 2277 Arg Gly Ile Arg Glu Val * 755 32 13 PRT
Artificial Sequence Pfam consensus sequence 32 Gly Glu Gly Val Ser
Asn Thr Asp Phe Val Leu Tyr Val 1 5 10 33 65 PRT Artificial
Sequence Pfam consensus sequence 33 Pro Gly Val Leu Ala Trp Ala Thr
Thr Cys Gln Val Phe Ser Asp Phe 1 5 10 15 Gly Arg Pro Ala Val Gly
Val Ile Asn Ile Pro Ala Ala Asn Ile Thr 20 25 30 Ser Arg Asn His
Tyr Asp Gln Leu Val Thr Arg Val Val Thr His Glu 35 40 45 Ile Ala
His Ala Leu Gly Phe Ser Val Gly Leu Tyr Thr Phe Phe Glu 50 55 60
Glu 65 34 55 PRT Artificial Sequence Pfam consensus sequence 34 Ser
His Trp Lys Lys Arg Asn Ala Lys Asp Glu Leu Met Ala Gly Ala 1 5 10
15 Ala Gly Ser Asp Ala Gly Tyr Tyr Ser Ala Leu Thr Met Ala Val Phe
20 25 30 Glu Asp Leu Gly Phe Tyr Lys Ala Asp Phe Ser Lys Ala Glu
Asp Met 35 40 45 Pro Trp Gly Lys Asn Ala Gly 50 55 35 37 PRT
Artificial Sequence Pfam consensus sequence 35 Ala Ala Leu Cys Ala
Asn Val Lys Cys Asp Thr Ala Thr Arg Thr Tyr 1 5 10 15 Ser Val Gln
Val Tyr Gly Ser Ser Gly Tyr Tyr Pro Cys Thr Pro Gly 20 25 30 Leu
Arg Val Glu Leu 35 36 1485 DNA Homo sapiens CDS (255)...(1133) 36
acaggctgta tcttctcaaa atttcattga ttgggctcaa tgaagtcacc tgcaacatrg
60 tagtagagta gggctccctt ttcacacgct ttttggaagg cttcttcaag
tcacattttc 120 cgttcggtct tcctttgccc tgtgtttgcr gtcatcatgt
gaggggctac ctatgttcag 180 cccaggcaac ccacagggag agagggcaga
gcggggagat ggcccctggt gagcactgag 240 gctccccttc aagg atg gcg ctg
gcg gct ttg atg atc gcc ctc ggc agc 290 Met Ala Leu Ala Ala Leu Met
Ile Ala Leu Gly Ser 1 5 10 ctc ggc ctc cac acc tgg cag gcc cag gct
gtt ccc acc atc ctg ccc 338 Leu Gly Leu His Thr Trp Gln Ala Gln Ala
Val Pro Thr Ile Leu Pro 15 20 25 ctg ggc ctg gct cca gac acc ttt
gac gat acc tat gtg ggt tgt gca 386 Leu Gly Leu Ala Pro Asp Thr Phe
Asp Asp Thr Tyr Val Gly Cys Ala 30 35 40 gag gag atg gag gag aag
gca gcc ccc ctg cta aag gag gaa atg gcc 434 Glu Glu Met Glu Glu Lys
Ala Ala Pro Leu Leu Lys Glu Glu Met Ala 45 50 55 60 cac cat gcc ctg
ctg cgg gaa tcc tgg gag gca gcc cag gag acc tgg 482 His His Ala Leu
Leu Arg Glu Ser Trp Glu Ala Ala Gln Glu Thr Trp 65 70 75 gag gac
aag cgt cga ggg ctt acc ttg ccc cct ggc ttc aaa gcc cag 530 Glu Asp
Lys Arg Arg Gly Leu Thr Leu Pro Pro Gly Phe Lys Ala Gln 80 85 90
aat gga ata gcc att atg gtc tac acc aac tca tcg aac acc ttg tac 578
Asn Gly Ile Ala Ile Met Val Tyr Thr Asn Ser Ser Asn Thr Leu Tyr 95
100 105 tgg gag ttg aat cag gcc gtg cgg acg ggc gga ggc tcc cgg gag
ctc 626 Trp Glu Leu Asn Gln Ala Val Arg Thr Gly Gly Gly Ser Arg Glu
Leu 110 115 120 tac atg agg cac ttt ccc ttc aag gcc ctg cat ttc tac
ctg atc cgg 674 Tyr Met Arg His Phe Pro Phe Lys Ala Leu His Phe Tyr
Leu Ile Arg 125 130 135 140 gcc ctg cag ctg ctg cga ggc agt ggg ggc
tgc agc agg gga cct ggg 722 Ala Leu Gln Leu Leu Arg Gly Ser Gly Gly
Cys Ser Arg Gly Pro Gly 145 150 155 gag gtg gtg ttc cga ggt gtg ggc
agc ctt cgc ttt gaa ccc aag agg 770 Glu Val Val Phe Arg Gly Val Gly
Ser Leu Arg Phe Glu Pro Lys Arg 160 165 170 ctg ggg gac tct gtc cgc
ttg ggc cag ttt gcc tcc agc tcc ctg gat 818 Leu Gly Asp Ser Val Arg
Leu Gly Gln Phe Ala Ser Ser Ser Leu Asp 175 180 185 aag gca gtg gcc
cac aga ttt ggt aat gcc acc ctc ttc tct cta aca 866 Lys Ala Val Ala
His Arg Phe Gly Asn Ala Thr Leu Phe Ser Leu Thr 190 195 200 act tgc
ttt ggg gcc cct ata cag gcc ttc tct gtc ttt ccc aag gag 914 Thr Cys
Phe Gly Ala Pro Ile Gln Ala Phe Ser Val Phe Pro Lys Glu 205 210 215
220 cgc gag gtg ctg att ccc ccc cat gaa gtc ttt ttg gtt acc aga ttc
962 Arg Glu Val Leu Ile Pro Pro His Glu Val Phe Leu Val Thr Arg Phe
225 230 235 tct cag gat gga gcc cag agc ttg gtg act ctc tgg agc tat
aat cag 1010 Ser Gln Asp Gly Ala Gln Ser Leu Val Thr Leu Trp Ser
Tyr Asn Gln 240 245 250 acc tgt agc cat ttt aac tgc gcc tat ctg ggt
ggg gag aag agg cgg 1058 Thr Cys Ser His Phe Asn Cys Ala Tyr Leu
Gly Gly Glu Lys Arg Arg 255 260 265 ggc tgt gtg tct gcg cca gga gcc
ctg gga acg ggt gac ctt cat atg 1106 Gly Cys Val Ser Ala Pro Gly
Ala Leu Gly Thr Gly Asp Leu His Met 270 275 280 acg aag agg cac ctc
cag cag cct tga gaagcaagaa catggttccg 1153 Thr Lys Arg His Leu Gln
Gln Pro * 285 290 gacccagccc tagcagcctt ctccccaacc aggatgttgg
cctggggagg ccacagcagg 1213 gctgagggaa ctctgctatg tgatggggac
ttcctgggac aagcaaggaa agtactgagg 1273 cagccacttg attgaacggt
gttgcaatgt ggagacatgg agttttattg aggtagctac 1333 gtgattaaat
ggtattgcag tgtggadaaa dgrramwwmm wgggacaagc aaggaaagta 1393
ctgaggcagc cacttgattg aacggtgttg caatgtggag acatggagtt ttattgaggt
1453 agctacgtga ttaaatggta ttgcagtgtg ga 1485 37 292 PRT Homo
sapiens 37 Met Ala Leu Ala Ala Leu Met Ile Ala Leu Gly Ser Leu Gly
Leu His 1 5 10 15 Thr Trp Gln Ala Gln Ala Val Pro Thr Ile Leu Pro
Leu Gly Leu Ala 20 25 30 Pro Asp Thr Phe Asp Asp Thr Tyr Val Gly
Cys Ala Glu Glu Met Glu 35 40 45 Glu Lys Ala Ala Pro Leu Leu Lys
Glu Glu Met Ala His His Ala Leu 50 55 60 Leu Arg Glu Ser Trp Glu
Ala Ala Gln Glu Thr Trp Glu Asp Lys Arg 65 70 75 80 Arg Gly Leu Thr
Leu Pro Pro Gly Phe Lys Ala Gln Asn Gly Ile Ala 85 90 95 Ile Met
Val Tyr Thr Asn Ser Ser Asn Thr Leu Tyr Trp Glu Leu Asn 100 105 110
Gln Ala Val Arg Thr Gly Gly Gly Ser Arg Glu Leu Tyr Met Arg His 115
120 125 Phe Pro Phe Lys Ala Leu His Phe Tyr Leu Ile Arg Ala Leu Gln
Leu 130 135 140 Leu Arg Gly Ser Gly Gly Cys Ser Arg Gly Pro Gly Glu
Val Val Phe 145 150 155 160 Arg Gly Val Gly Ser Leu Arg Phe Glu Pro
Lys Arg Leu Gly Asp Ser 165 170 175 Val Arg Leu Gly Gln Phe Ala Ser
Ser Ser Leu Asp Lys Ala Val Ala 180 185 190 His Arg Phe Gly Asn Ala
Thr Leu Phe Ser Leu Thr Thr Cys Phe Gly 195 200 205 Ala Pro Ile Gln
Ala Phe Ser Val Phe Pro Lys Glu Arg Glu Val Leu 210 215 220 Ile Pro
Pro His Glu Val Phe Leu Val Thr Arg Phe Ser Gln Asp Gly 225 230 235
240 Ala Gln Ser Leu Val Thr Leu Trp Ser Tyr Asn Gln Thr Cys Ser His
245 250 255 Phe Asn Cys Ala Tyr Leu Gly Gly Glu Lys Arg Arg Gly Cys
Val Ser 260 265 270 Ala Pro Gly Ala Leu Gly Thr Gly Asp Leu His Met
Thr Lys Arg His 275 280 285 Leu Gln Gln Pro 290 38 879 DNA Homo
sapiens CDS (1)...(879) 38 atg gcg ctg gcg gct ttg atg atc gcc ctc
ggc agc ctc ggc ctc cac 48 Met Ala Leu Ala Ala Leu Met Ile Ala Leu
Gly Ser Leu Gly Leu His 1 5 10 15 acc tgg cag gcc cag gct gtt ccc
acc atc ctg ccc ctg ggc ctg gct 96 Thr Trp Gln Ala Gln Ala Val Pro
Thr Ile Leu Pro Leu Gly Leu Ala 20 25 30 cca gac acc ttt gac gat
acc tat gtg ggt tgt gca gag gag atg gag 144 Pro Asp Thr Phe Asp Asp
Thr Tyr Val Gly Cys Ala Glu Glu Met Glu 35 40 45 gag aag gca gcc
ccc ctg cta aag gag gaa atg gcc cac cat gcc ctg 192 Glu Lys Ala Ala
Pro Leu Leu Lys Glu Glu Met Ala His His Ala Leu 50 55 60 ctg cgg
gaa tcc tgg gag gca gcc cag gag acc tgg gag gac aag cgt 240 Leu Arg
Glu Ser Trp Glu Ala Ala Gln Glu Thr Trp Glu Asp Lys Arg 65 70 75 80
cga ggg ctt acc ttg ccc cct ggc ttc aaa gcc cag aat gga ata gcc 288
Arg Gly Leu Thr Leu Pro Pro Gly Phe Lys Ala Gln Asn Gly Ile Ala 85
90 95 att atg gtc tac acc aac tca tcg aac acc ttg tac tgg gag ttg
aat 336 Ile Met Val Tyr Thr Asn Ser Ser Asn Thr Leu Tyr Trp Glu Leu
Asn 100 105 110 cag gcc gtg cgg
acg ggc gga ggc tcc cgg gag ctc tac atg agg cac 384 Gln Ala Val Arg
Thr Gly Gly Gly Ser Arg Glu Leu Tyr Met Arg His 115 120 125 ttt ccc
ttc aag gcc ctg cat ttc tac ctg atc cgg gcc ctg cag ctg 432 Phe Pro
Phe Lys Ala Leu His Phe Tyr Leu Ile Arg Ala Leu Gln Leu 130 135 140
ctg cga ggc agt ggg ggc tgc agc agg gga cct ggg gag gtg gtg ttc 480
Leu Arg Gly Ser Gly Gly Cys Ser Arg Gly Pro Gly Glu Val Val Phe 145
150 155 160 cga ggt gtg ggc agc ctt cgc ttt gaa ccc aag agg ctg ggg
gac tct 528 Arg Gly Val Gly Ser Leu Arg Phe Glu Pro Lys Arg Leu Gly
Asp Ser 165 170 175 gtc cgc ttg ggc cag ttt gcc tcc agc tcc ctg gat
aag gca gtg gcc 576 Val Arg Leu Gly Gln Phe Ala Ser Ser Ser Leu Asp
Lys Ala Val Ala 180 185 190 cac aga ttt ggt aat gcc acc ctc ttc tct
cta aca act tgc ttt ggg 624 His Arg Phe Gly Asn Ala Thr Leu Phe Ser
Leu Thr Thr Cys Phe Gly 195 200 205 gcc cct ata cag gcc ttc tct gtc
ttt ccc aag gag cgc gag gtg ctg 672 Ala Pro Ile Gln Ala Phe Ser Val
Phe Pro Lys Glu Arg Glu Val Leu 210 215 220 att ccc ccc cat gaa gtc
ttt ttg gtt acc aga ttc tct cag gat gga 720 Ile Pro Pro His Glu Val
Phe Leu Val Thr Arg Phe Ser Gln Asp Gly 225 230 235 240 gcc cag agc
ttg gtg act ctc tgg agc tat aat cag acc tgt agc cat 768 Ala Gln Ser
Leu Val Thr Leu Trp Ser Tyr Asn Gln Thr Cys Ser His 245 250 255 ttt
aac tgc gcc tat ctg ggt ggg gag aag agg cgg ggc tgt gtg tct 816 Phe
Asn Cys Ala Tyr Leu Gly Gly Glu Lys Arg Arg Gly Cys Val Ser 260 265
270 gcg cca gga gcc ctg gga acg ggt gac ctt cat atg acg aag agg cac
864 Ala Pro Gly Ala Leu Gly Thr Gly Asp Leu His Met Thr Lys Arg His
275 280 285 ctc cag cag cct tga 879 Leu Gln Gln Pro * 290 39 296
PRT Artificial Sequence Pfam consensus sequence 39 Met Pro Ala Leu
His Phe Val Leu Leu Leu Ser Val Gly Leu Leu Leu 1 5 10 15 Ser Thr
Gln Ala Leu Ser Ser Ala Ile Gln Gln Lys Asp Gly Leu Val 20 25 30
Lys Glu Leu Val Leu Asp Met Ala Pro Asn Ser Phe Asp Asp Gln Tyr 35
40 45 Leu Gly Cys Val Asp Arg Met Glu Ala Lys Tyr Leu Pro Gln Leu
Leu 50 55 60 Lys Glu Glu Phe Ala Ala Asn Glu Val Leu Ala Val Gly
Trp Glu Ser 65 70 75 80 Ala Lys Ala Lys Trp Gln Glu Arg Lys Ala Arg
Gly Ser Val Trp Gly 85 90 95 Ser Leu Pro Tyr Pro Ser Pro Pro Met
Gly Phe Lys Asp Glu His Gly 100 105 110 Ile Ala Leu Leu Ala Tyr Thr
Ala Ser Ser Gln Glu Gln Thr Pro Leu 115 120 125 Tyr Arg Glu Phe Asn
Glu Ala Val Arg Glu Ala Gly Arg Ser Arg Glu 130 135 140 Asp Tyr Leu
His His Phe His Phe Lys Ala Leu His Phe Tyr Leu Thr 145 150 155 160
Arg Ala Leu Gln Leu Leu Arg Ser Ser Gly Gly Cys Gln Pro Gly Pro 165
170 175 Cys His Val Val Tyr Arg Gly Val Arg Gly Leu Arg Phe Arg Pro
Gln 180 185 190 Gly Gly Gly Ala Ser Val Arg Phe Gly Gln Phe Thr Ser
Ser Ser Leu 195 200 205 Lys Lys Lys Val Ala Gln Ser Ser Glu Phe Phe
Phe Gly Gln Asp Thr 210 215 220 Phe Phe Ser Ile Lys Thr Cys Leu Gly
Val Pro Ile Lys Ala Phe Ser 225 230 235 240 Phe Phe Pro Ser Glu Glu
Glu Val Leu Ile Pro Pro Phe Glu Val Phe 245 250 255 Gln Val Ile Asn
Thr Ser Arg Pro Thr Ala Gly Ser Ala Ile Ile Leu 260 265 270 Leu Ser
Ser Lys Gly Lys Cys Ser Thr Tyr Asn Cys Glu Tyr Leu Lys 275 280 285
Gly Lys Lys Thr Glu Asn Cys Ile 290 295 40 2183 DNA Homo sapiens
CDS (782)...(1696) misc_feature 2134, 2147, 2153 n = A,T,C or G 40
gccgagctcg aattgcggcc gcatgcatgc ataagcttgc tcgagtctag attttttttt
60 tttttttttt gaggttgatc aaaaaccttt attctggtcc tcatcattca
ggaagccact 120 tatgcagaac actttcttca tctctgctct aagcagcagc
agctgcatcc aggcttgata 180 tggaagacag acagaaatag tggaagacct
tgcagagaag agctaattga tgagatctct 240 gcttatcccg gcataggtga
aggcagaaca ggaagagtgt gtgtaagtgc aaagactccg 300 ttcttcaaag
ctgaggcctg tgcagagggg tgaactgaag gcttcagatg aaggctcaca 360
aaactgcccc gctcggcaat caatctaaag taccttccag aaaagcaaat tcatccacag
420 cttctattgc attatccaag tctctcctct gaagggtttt cctttttccc
tgctgagcgc 480 aacagtaggc atcttttgca atggtctcca caaacagttc
cgcggctcgt gccagaatga 540 agatggcttc ctgtcccgct agcgtcacgt
cgggatctgc cttcaccaag gccttcactc 600 gcgccagagg caacctcgag
agacgagccc caggcacact cgttggggcc tggggctgcg 660 aggccgctgc
ctccccagca ggtacctcct cctctcgggg cgtcccgctt cctgccgccg 720
ccgccgcgtc gacgcggctg cttgcctacc ggagtgtgcg ccggcacctg ccgccggaga
780 c atg ttg caa aaa ccg agg aac cgg ggc cgc tct ggc ggc cag gcc
gag 829 Met Leu Gln Lys Pro Arg Asn Arg Gly Arg Ser Gly Gly Gln Ala
Glu 1 5 10 15 agg gac aga gac tgg agc cat agc gga aac ccc ggg gct
tcg cgg gcc 877 Arg Asp Arg Asp Trp Ser His Ser Gly Asn Pro Gly Ala
Ser Arg Ala 20 25 30 ggg gaa gac gcc cgg gtt ctc aga gac ggc ttt
gcc gag gag gcc ccg 925 Gly Glu Asp Ala Arg Val Leu Arg Asp Gly Phe
Ala Glu Glu Ala Pro 35 40 45 agc acg tcc cgc ggg ccg ggc ggc tcg
cag ggg tcg cag ggc ccc tcg 973 Ser Thr Ser Arg Gly Pro Gly Gly Ser
Gln Gly Ser Gln Gly Pro Ser 50 55 60 cct cag ggc gcc cgc cgg gcc
cag gcc gcc ccc gcc gtg ggg ccc agg 1021 Pro Gln Gly Ala Arg Arg
Ala Gln Ala Ala Pro Ala Val Gly Pro Arg 65 70 75 80 agc cag aag cag
ctg gag ctg aaa gtg tcc gag ctg gtg cag ttc ttg 1069 Ser Gln Lys
Gln Leu Glu Leu Lys Val Ser Glu Leu Val Gln Phe Leu 85 90 95 ctg
att aaa gac cag aag aag att ccg atc aag cgg gcc gac ata ctg 1117
Leu Ile Lys Asp Gln Lys Lys Ile Pro Ile Lys Arg Ala Asp Ile Leu 100
105 110 aag cac gtc atc ggg gac tac aag gac atc ttc ccc gac ctc ttc
aaa 1165 Lys His Val Ile Gly Asp Tyr Lys Asp Ile Phe Pro Asp Leu
Phe Lys 115 120 125 cgg gcc gcc gag cgc ctc cag tac gtc ttc ggg tat
aag ctg gtg gaa 1213 Arg Ala Ala Glu Arg Leu Gln Tyr Val Phe Gly
Tyr Lys Leu Val Glu 130 135 140 ctt gaa ccc aag agc aac act tac atc
ctc atc aac acc ctg gag cct 1261 Leu Glu Pro Lys Ser Asn Thr Tyr
Ile Leu Ile Asn Thr Leu Glu Pro 145 150 155 160 gtg gag gag gat gcc
gag atg agg ggt gac caa ggc acg ccc act acg 1309 Val Glu Glu Asp
Ala Glu Met Arg Gly Asp Gln Gly Thr Pro Thr Thr 165 170 175 ggc ctc
ctg atg atc gtc tta ggg ctc atc ttt atg aag ggc aac acc 1357 Gly
Leu Leu Met Ile Val Leu Gly Leu Ile Phe Met Lys Gly Asn Thr 180 185
190 atc aag gaa act gaa gcc tgg gac ttt ctg cgg cgc tta ggg gtc tac
1405 Ile Lys Glu Thr Glu Ala Trp Asp Phe Leu Arg Arg Leu Gly Val
Tyr 195 200 205 ccc acc aag aag cat tta att ttc gga gat cca aag aaa
ctc att act 1453 Pro Thr Lys Lys His Leu Ile Phe Gly Asp Pro Lys
Lys Leu Ile Thr 210 215 220 gag gac ttt gtg cga cag cgt tac ctg gaa
tac cgg cgg ata ccc cac 1501 Glu Asp Phe Val Arg Gln Arg Tyr Leu
Glu Tyr Arg Arg Ile Pro His 225 230 235 240 acc gac ccc gtc gac tac
gaa ttc cag tgg ggc ccg cga acc aac ctg 1549 Thr Asp Pro Val Asp
Tyr Glu Phe Gln Trp Gly Pro Arg Thr Asn Leu 245 250 255 gaa acc agc
aag atg aaa gtt ctt aag ttt gtg gcc aag gtc cat aat 1597 Glu Thr
Ser Lys Met Lys Val Leu Lys Phe Val Ala Lys Val His Asn 260 265 270
caa gac ccc aag gac tgg cca gcg cag tac tgt gag gct ttg gca gat
1645 Gln Asp Pro Lys Asp Trp Pro Ala Gln Tyr Cys Glu Ala Leu Ala
Asp 275 280 285 gag gag aac agg gcc aga cct cag cct agt ggc cca gct
cca tcc tct 1693 Glu Glu Asn Arg Ala Arg Pro Gln Pro Ser Gly Pro
Ala Pro Ser Ser 290 295 300 tga aaggtggatt cagagggacc cccgggacaa
gggtctgaga cccaaaggca 1746 * cagtttagag gattggggga agggagaacg
aacccaggga gcatattgct gtaaacgctt 1806 caatgtgtgt agctttagga
tgtgtttgca aagttttgtt tttttaatgt tgtgttattt 1866 tgctccagat
tttcatctat aaacaaagga gcatttgttt tgattttact ctttttggta 1926
taaaaaattt tgctagctta rtaaaacgaa ttggaaaact tgmytatkat ctggaacaga
1986 taatgcaaga aggracmcwt aagtaagttg yttttggtgc caagaaaata
aaaaagctat 2046 tatcaaggtc tcctaactac cccagttttg taagggaaaa
ataaaaaagt ttttattaaa 2106 atttaaaaaa aaaaaaaaar gggccggncg
cttagactta ntcttanaga aaaaaacctt 2166 cccacacctt ccccctg 2183 41
304 PRT Homo sapiens 41 Met Leu Gln Lys Pro Arg Asn Arg Gly Arg Ser
Gly Gly Gln Ala Glu 1 5 10 15 Arg Asp Arg Asp Trp Ser His Ser Gly
Asn Pro Gly Ala Ser Arg Ala 20 25 30 Gly Glu Asp Ala Arg Val Leu
Arg Asp Gly Phe Ala Glu Glu Ala Pro 35 40 45 Ser Thr Ser Arg Gly
Pro Gly Gly Ser Gln Gly Ser Gln Gly Pro Ser 50 55 60 Pro Gln Gly
Ala Arg Arg Ala Gln Ala Ala Pro Ala Val Gly Pro Arg 65 70 75 80 Ser
Gln Lys Gln Leu Glu Leu Lys Val Ser Glu Leu Val Gln Phe Leu 85 90
95 Leu Ile Lys Asp Gln Lys Lys Ile Pro Ile Lys Arg Ala Asp Ile Leu
100 105 110 Lys His Val Ile Gly Asp Tyr Lys Asp Ile Phe Pro Asp Leu
Phe Lys 115 120 125 Arg Ala Ala Glu Arg Leu Gln Tyr Val Phe Gly Tyr
Lys Leu Val Glu 130 135 140 Leu Glu Pro Lys Ser Asn Thr Tyr Ile Leu
Ile Asn Thr Leu Glu Pro 145 150 155 160 Val Glu Glu Asp Ala Glu Met
Arg Gly Asp Gln Gly Thr Pro Thr Thr 165 170 175 Gly Leu Leu Met Ile
Val Leu Gly Leu Ile Phe Met Lys Gly Asn Thr 180 185 190 Ile Lys Glu
Thr Glu Ala Trp Asp Phe Leu Arg Arg Leu Gly Val Tyr 195 200 205 Pro
Thr Lys Lys His Leu Ile Phe Gly Asp Pro Lys Lys Leu Ile Thr 210 215
220 Glu Asp Phe Val Arg Gln Arg Tyr Leu Glu Tyr Arg Arg Ile Pro His
225 230 235 240 Thr Asp Pro Val Asp Tyr Glu Phe Gln Trp Gly Pro Arg
Thr Asn Leu 245 250 255 Glu Thr Ser Lys Met Lys Val Leu Lys Phe Val
Ala Lys Val His Asn 260 265 270 Gln Asp Pro Lys Asp Trp Pro Ala Gln
Tyr Cys Glu Ala Leu Ala Asp 275 280 285 Glu Glu Asn Arg Ala Arg Pro
Gln Pro Ser Gly Pro Ala Pro Ser Ser 290 295 300 42 915 DNA Homo
sapiens 42 atgttgcaaa aaccgaggaa ccggggccgc tctggcggcc aggccgagag
ggacagagac 60 tggagccata gcggaaaccc cggggcttcg cgggccgggg
aagacgcccg ggttctcaga 120 gacggctttg ccgaggaggc cccgagcacg
tcccgcgggc cgggcggctc gcaggggtcg 180 cagggcccct cgcctcaggg
cgcccgccgg gcccaggccg cccccgccgt ggggcccagg 240 agccagaagc
agctggagct gaaagtgtcc gagctggtgc agttcttgct gattaaagac 300
cagaagaaga ttccgatcaa gcgggccgac atactgaagc acgtcatcgg ggactacaag
360 gacatcttcc ccgacctctt caaacgggcc gccgagcgcc tccagtacgt
cttcgggtat 420 aagctggtgg aacttgaacc caagagcaac acttacatcc
tcatcaacac cctggagcct 480 gtggaggagg atgccgagat gaggggtgac
caaggcacgc ccactacggg cctcctgatg 540 atcgtcttag ggctcatctt
tatgaagggc aacaccatca aggaaactga agcctgggac 600 tttctgcggc
gcttaggggt ctaccccacc aagaagcatt taattttcgg agatccaaag 660
aaactcatta ctgaggactt tgtgcgacag cgttacctgg aataccggcg gataccccac
720 accgaccccg tcgactacga attccagtgg ggcccgcgaa ccaacctgga
aaccagcaag 780 atgaaagttc ttaagtttgt ggccaaggtc cataatcaag
accccaagga ctggccagcg 840 cagtactgtg aggctttggc agatgaggag
aacagggcca gacctcagcc tagtggccca 900 gctccatcct cttga 915 43 260
PRT Artificial Sequence MAGE family PFAM consensus domain 43 Arg
Gly Gln Lys Ser Gln Leu Cys Lys Arg Glu Glu Arg Leu Gln Ala 1 5 10
15 Arg Gly Glu Thr Gln Gly Leu Val Gly Ala Gln Ala Pro Ala Ala Glu
20 25 30 Glu Gln Gln Glu Glu Ala Ser Ser Ser Ser Pro Leu Gln Ser
Pro Val 35 40 45 Ser Leu Gly Val Ser Glu Asp Glu Ala Leu Val Leu
Gly Thr Leu Glu 50 55 60 Glu Val Pro Ala Ala Gly Gly Ser Pro Ser
Pro Pro Gln Ser Pro Gln 65 70 75 80 Gly Ser Pro Pro Glu Ser Pro Leu
Ala Ser Ser Thr Ile Ser Ala Val 85 90 95 Ala Ala Lys Thr Ser Trp
Thr Gln Ser Asp Glu Gly Ser Ser Ser Gln 100 105 110 Val Val Leu Gln
Glu Glu Glu Gly Pro Ser Thr Ser Gln Ala Leu Thr 115 120 125 Ser Thr
Glu Ser Leu Phe Arg Asp Ala Leu Asp Glu Lys Val Ala Glu 130 135 140
Leu Val Gln Phe Leu Leu Leu Lys Tyr Gln Met Lys Glu Pro Val Thr 145
150 155 160 Lys Ala Glu Met Leu Lys Ser Val Ile Lys Asn Tyr Lys Asp
His Phe 165 170 175 Pro Glu Ile Phe Arg Lys Ala Ser Glu Phe Leu Glu
Leu Val Phe Gly 180 185 190 Ile Asp Leu Lys Glu Val Asp Pro Thr Gly
His Ser Tyr Val Leu Val 195 200 205 Thr Lys Leu Gly Leu Ser Tyr Asp
Gly Leu Leu Ser Asp Asn Gln Gly 210 215 220 Met Pro Lys Thr Gly Leu
Leu Ile Ile Val Leu Gly Val Ile Phe Met 225 230 235 240 Lys Gly Asn
Cys Ala Pro Glu Glu Glu Ile Trp Glu Val Leu Ser Val 245 250 255 Leu
Gly Val Tyr 260
* * * * *