U.S. patent application number 09/738884 was filed with the patent office on 2002-03-21 for isolated human phospholipase proteins, nucleic acid molecules encoding human phospholipase proteins, and uses thereof.
Invention is credited to Beasley, Ellen M., Di Francesco, Valentina, Guegler, Karl, Ketchum, Karen A..
Application Number | 20020034806 09/738884 |
Document ID | / |
Family ID | 26926182 |
Filed Date | 2002-03-21 |
United States Patent
Application |
20020034806 |
Kind Code |
A1 |
Guegler, Karl ; et
al. |
March 21, 2002 |
ISOLATED HUMAN PHOSPHOLIPASE PROTEINS, NUCLEIC ACID MOLECULES
ENCODING HUMAN PHOSPHOLIPASE PROTEINS, AND USES THEREOF
Abstract
The present invention provides amino acid sequences of peptides
that are encoded by genes within the human genome, the
phospholipase peptides of the present invention. The present
invention specifically provides isolated peptide and nucleic acid
molecules, methods of identifying orthologs and paralogs of the
phospholipase peptides, and methods of identifying modulators of
the phospholipase peptides.
Inventors: |
Guegler, Karl; (Menlo Park,
CA) ; Beasley, Ellen M.; (Darnestown, MD) ;
Ketchum, Karen A.; (Germantown, MD) ; Di Francesco,
Valentina; (Rockville, MD) |
Correspondence
Address: |
CELERA GENOMICS CORP.
ATTN: ROBERT A. MILLMAN, PATENT DIRECTOR
45 WEST GUDE DRIVE
C2-4#20
ROCKVILLE
MD
20850
US
|
Family ID: |
26926182 |
Appl. No.: |
09/738884 |
Filed: |
December 18, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60232632 |
Sep 14, 2000 |
|
|
|
Current U.S.
Class: |
435/196 ;
435/325; 435/6.14; 435/69.1; 435/7.1; 530/388.1; 536/23.2;
800/8 |
Current CPC
Class: |
A01K 2217/05 20130101;
C12N 9/20 20130101; A61P 35/00 20180101; A61K 38/00 20130101 |
Class at
Publication: |
435/196 ;
530/388.1; 536/23.2; 435/6; 435/7.1; 435/69.1; 435/325; 800/8 |
International
Class: |
C12N 009/16; C12P
021/02; C12N 005/06; A01K 067/00; C12Q 001/68; G01N 033/53; C07H
021/04 |
Claims
That which is claimed is:
1. An isolated peptide consisting of an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic
variant of an amino acid sequence shown in SEQ ID NO:2, wherein
said allelic variant is encoded by a nucleic acid molecule that
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid
sequence of an ortholog of an amino acid sequence shown in SEQ ID
NO:2, wherein said ortholog is encoded by a nucleic acid molecule
that hybridizes under stringent conditions to the opposite strand
of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a
fragment of an amino acid sequence shown in SEQ ID NO:2, wherein
said fragment comprises at least 10 contiguous amino acids.
2. An isolated peptide comprising an amino acid sequence selected
from the group consisting of: (a) an amino acid sequence shown in
SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said allelic
variant is encoded by a nucleic acid molecule that hybridizes under
stringent conditions to the opposite strand of a nucleic acid
molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of
an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein
said ortholog is encoded by a nucleic acid molecule that hybridizes
under stringent conditions to the opposite strand of a nucleic acid
molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino
acid sequence shown in SEQ ID NO:2, wherein said fragment comprises
at least 10 contiguous amino acids.
3. An isolated antibody that selectively binds to a peptide of
claim 2.
4. An isolated nucleic acid molecule consisting of a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide
sequence hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a
nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO:2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous
amino acids; and (e) a nucleotide sequence that is the complement
of a nucleotide sequence of (a)-(d).
5. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide
sequence hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a
nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO:2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous
amino acids; and (e) a nucleotide sequence that is the complement
of a nucleotide sequence of (a)-(d).
6. A gene chip comprising a nucleic acid molecule of claim 5.
7. A transgenic non-human animal comprising a nucleic acid molecule
of claim 5.
8. A nucleic acid vector comprising a nucleic acid molecule of
claim 5.
9. A host cell containing the vector of claim 8.
10. A method for producing any of the peptides of claim 1
comprising introducing a nucleotide sequence encoding any of the
amino acid sequences in (a)-(d) into a host cell, and culturing the
host cell under conditions in which the peptides are expressed from
the nucleotide sequence.
11. A method for producing any of the peptides of claim 2
comprising introducing a nucleotide sequence encoding any of the
amino acid sequences in (a)-(d) into a host cell, and culturing the
host cell under conditions in which the peptides are expressed from
the nucleotide sequence.
12. A method for detecting the presence of any of the peptides of
claim 2 in a sample, said method comprising contacting said sample
with a detection agent that specifically allows detection of the
presence of the peptide in the sample and then detecting the
presence of the peptide.
13. A method for detecting the presence of a nucleic acid molecule
of claim 5 in a sample, said method comprising contacting the
sample with an oligonucleotide that hybridizes to said nucleic acid
molecule under stringent conditions and determining whether the
oligonucleotide binds to said nucleic acid molecule in the
sample.
14. A method for identifying a modulator of a peptide of claim 2,
said method comprising contacting said peptide with an agent and
determining if said agent has modulated the function or activity of
said peptide.
15. The method of claim 14, wherein said agent is administered to a
host cell comprising an expression vector that expresses said
peptide.
16. A method for identifying an agent that binds to any of the
peptides of claim 2, said method comprising contacting the peptide
with an agent and assaying the contacted mixture to determine
whether a complex is formed with the agent bound to the
peptide.
17. A pharmaceutical composition comprising an agent identified by
the method of claim 16 and a pharmaceutically acceptable carrier
therefor.
18. A method for treating a disease or condition mediated by a
human phospholipase protein, said method comprising administering
to a patient a pharmaceutically effective amount of an agent
identified by the method of claim 16.
19. A method for identifying a modulator of the expression of a
peptide of claim 2, said method comprising contacting a cell
expressing said peptide with an agent, and determining if said
agent has modulated the expression of said peptide.
20. An isolated human phospholipase peptide having an amino acid
sequence that shares at least 70% homology with an amino acid
sequence shown in SEQ ID NO:2.
21. A peptide according to claim 20 that shares at least 90 percent
homology with an amino acid sequence shown in SEQ ID NO:2.
22. An isolated nucleic acid molecule encoding a human
phospholipase peptide, said nucleic acid molecule sharing at least
80 percent homology with a nucleic acid molecule shown in SEQ ID
NOS:1 or 3.
23. A nucleic acid molecule according to claim 22 that shares at
least 90 percent homology with a nucleic acid molecule shown in SEQ
ID NOS:1 or 3.
Description
RELATED APPLICATIONS
[0001] The present application claims priority to Provisional
Application U.S. Serial No. 60/232,632, filed Sep. 14, 2000 (Atty.
Docket CL000849-PROV).
FIELD OF THE INVENTION
[0002] The present invention is in the field of phospholipase
proteins that are related to the phospholipase C subfamily,
recombinant DNA molecules, and protein production. The present
invention specifically provides novel peptides and proteins that
effect protein phosphorylation and nucleic acid molecules encoding
such peptide and protein molecules, all of which are useful in the
development of human therapeutics and diagnostic compositions and
methods.
BACKGROUND OF THE INVENTION
[0003] Phospholipases
[0004] There are three major families of known human phospholipase
enzymes: Phospholipase A2, Phospholipase C, and Phospholipase
D.
[0005] Enzymes in the Phospholipase A2 family ("P1A2") hydrolyze
the sn-2 fatty acid acyl ester bond of phosphoglycerides, releasing
free fatty acids and lysophospholipids. The P1A2s constitute a
diverse family of enzymes with respect to sequence, function,
localization and divalent cation requirements. They play an
important role in a variety of cellular processes, including the
digestion and metabolism of phospholipids as well as the production
of precursors for inflammatory reactions. The P1A2s have been
classified into at least 5 groups (although different
classification schemes exist and up to 10 groups have been
identified by some authorities) based on their size, structure and
need for divalent cations. Groups I, II and III all contain
secreted forms of P1A, which are extracellular enzymes that have a
low molecular mass and require calcium ions for catalysis. Groups
IV and V contain cytosolic forms of P1A2s that have a high
molecular mass and do not necessarily require calcium ions.
[0006] Amongst the best characterized of the P1A2 phospholipases
are digestive enzymes secreted as zymogens by the pancreas. These
enzymes, which are involved in the hydrolysis of dietary
phospholipids, have strong homology to the venom phospholipases of
snakes. Other P1A2s play important roles in the control of
signaling cascades such as the cytosolic P1A2, Group IVA enzyme
("PLA2G4A") which catalyzes the release of arachidonic acid from
membrane phospholipids. Arachidonic acid serves as a precursor for
a wide spectrum of biological effectors, collectively known as
eicosanoids (and including the prostaglandin group of molecules)
that are involved in hemodynamic regulation, inflammatory responses
and other cellular processes.
[0007] Another biologically active phospholipid,
platelet-activating factor ("PAF") is hydrolyzed to
metabolically-inactive degradation products by the group VII P1A2
known as PAF acetylhydrolase. Deficiency of PAF acetylhydrolase has
been reported in patients with systemic lupus erythematosis and
increased levels of PAF have been reported in children with acute
asthmatic attacks. Elevated levels of the group II P1A2 known as
PLA2G2A have been reported in plasma and synovial fluid in patients
with inflammatory arthritis. Studies of a mouse colon cancer model
showed that alleles of the murine ortholog of this gene were able
to modify the number of tumors that developed in animals with
multiple intestinal neoplasia (a mouse model of the human disorder
known as familial adenomtous polyposis). Subsequent studies in
humans showed mutations in PLA2G2A were associated with the risk of
developing colorectal cancer. PLA2G2A is presumed to act through
altering cellular microenvironments within the intestinal crypts of
the colonic mucosa, although the precise mechanism by which this
effect is exerted is not clear.
[0008] Enzymes in the Phospholipase C ("PLC") family catalyze the
hydrolysis of the plasma membrane phospholipids, phosphatidyl
inositol phosphate ("PIP") or phosphatidylinositol 4,5-biphosphate
("PIP2"), generating as products the second messengers,
1,4,5-inositol triphosphate ("IP3") and 1,2-diacylglycerol ("DAG").
Molecules belonging to the PLC gene family are divided into
subfamilies, PLC-beta, PLC-gamma and PLC-delta. PLC-delta is
distinguished from PLC-gamma by lack of the SH2 and SH3 domains
that are essential for activation of PLC-gamma by tyrosine protein
kinases. PLC-delta is distinguished from PLC-beta by lack of the
C-terminal region of PLC-beta that is responsible for binding and
activation of G proteins. Various PLC enzymes play important roles
in signal transduction cascades throughout the body. Activating
signals include hormones, growth factors and neurotransmitters. One
of the functions of IP2 is to modulate intracellular calcium levels
while DAG is involved in the activation of certain protein kinases
and can promote membrane fusion in processes involving vesicular
trafficking.
[0009] The novel human protein provided by the present invention is
related to the phospholipase C family, and shows a particularly
high degree of similarity to enzymes of the phospholipase C-delta
subclass. PLC-delta proteins may be associated with abnormal
calcium homeostasis and increased intracellular calcium ion
concentrations, conditions commonly associated with hypertension
(Yagisawa et al., J Hypertens 1991 Nov;9(11):997-1004).
Furthermore, the gene encoding PLC-delta1 is located just distal to
a region of chromosome 3 that is deleted in a lung cancer cell line
(Ishikawa et al., Cytogenet Cell Genet 1997;78(1):58-60),
suggesting an involvement in cancers such as lung cancer.
Additionally, a mutation in the pleckstrin homology domain of
PLC-delta1 has been found in a patient with early-onset sporadic
Alzheimer's diseases (Shimohama et al., Biochem. Biophys. Res.
Commun. 245: 722-728, 1998), and mutations in the pleckstrin
homology domain are known to occur in Bruton agammaglobulinemia
(Shimohama et al., Biochem. Biophys. Res. Commun. 245: 722-728,
1998). For a further review of PLC proteins, particularly proteins
of the PLC-delta subclass, see: Leonis et al., Biochem Biophys Res
Commun Jul. 16, 1996;224(2):382-90; Cheng et al., J Biol Chem Mar.
10, 1995;270(10):5495-505; Suh et al., Cell Jul. 15,
1988;54(2):161-9; Milting et al., J Muscle Res Cell Motil 1996
Feb;17(1):79-84; and Lyu et al., Mammalian Genome 7: 501-504,
1996.
[0010] Enzymes in the Phospholipase D ("PLD") family catalyze the
hydrolysis of phosphatidylcholine ("PC") and other phospholipids to
produce phosphatidic acid. A range of agonists acting through G
protein-coupled receptors and receptor tyrosine kinases stimulate
this hydrolysis. Phosphatidic acid appears to be important as a
second messenger capable of activating a diverse range of signaling
pathways. PC-specific PLD activity has been implicated in numerous
cellular pathways, including signal transduction, membrane
trafficking, the regulation of mitosis, regulated secretion,
cytoskeletal reorganization, transcriptional regulation and
cell-cycle control. Many proteins are attached to the plasma
membrane via a glysylphosphatidylinositol ("GPI") anchor.
Phosphatidylinositol-glycan ("PIG")-specific PLDs selectively
hydrolyze the inositol phosphate linkage, allowing release of the
protein.
[0011] Phospholipase proteins, particularly members of the
phospholipase C subfamily, are a major target for drug action and
development. Accordingly, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown members of this subfamily of phospholipase proteins. The
present invention advances the state of the art by providing
previously unidentified human phospholipase proteins that have
homology to members of the phospholipase C subfamily.
SUMMARY OF THE INVENTION
[0012] The present invention is based in part on the identification
of amino acid sequences of human phospholipase peptides and
proteins that are related to the phospholipase C subfamily, as well
as allelic variants and other mammalian orthologs thereof. These
unique peptide sequences, and nucleic acid sequences that encode
these peptides, can be used as models for the development of human
therapeutic targets, aid in the identification of therapeutic
proteins, and serve as targets for the development of human
therapeutic agents that modulate phospholipase activity in cells
and tissues that express the phospholipase. Experimental data as
provided in FIG. 1 indicates expression in humans in pancreas
adenocarcinomas, lung, brain anaplastic oligodendrogliomas,
placenta choriocarcinomas, pancreas adenocarcinomas, uterus
endometrium, colon adenocarcinomas, eye retinoblastomas, and fetal
brain.
DESCRIPTION OF THE FIGURE SHEETS
[0013] FIG. 1 provides the nucleotide sequence of a cDNA molecule
that encodes the phospholipase protein of the present invention.
(SEQ ID NO:1). In addition, structure and functional information is
provided, such as ATG start, stop and tissue distribution, where
available, that allows one to readily determine specific uses of
inventions based on this molecular sequence. Experimental data as
provided in FIG. 1 indicates expression in humans in pancreas
adenocarcinomas, lung, brain anaplastic oligodendrogliomas,
placenta choriocarcinomas, pancreas adenocarcinomas, uterus
endometrium, colon adenocarcinomas, eye retinoblastomas, and fetal
brain.
[0014] FIG. 2 provides the predicted amino acid sequence of the
phospholipase of the present invention. (SEQ ID NO:2) In addition
structure and functional information such as protein family,
function, and modification sites is provided where available,
allowing one to readily determine specific uses of inventions based
on this molecular sequence.
[0015] FIG. 3 provides genomic sequences that span the gene
encoding the phospholipase protein of the present invention. (SEQ
ID NO:3) In addition structure and functional information, such as
intron/exon structure, promoter location, etc., is provided where
available, allowing one to readily determine specific uses of
inventions based on this molecular sequence. As illustrated in FIG.
3, SNPs were identified at 16 different nucleotide positions.
DETAILED DESCRIPTION OF THE INVENTION
[0016] General Description
[0017] The present invention is based on the sequencing of the
human genome. During the sequencing and assembly of the human
genome, analysis of the sequence information revealed previously
unidentified fragments of the human genome that encode peptides
that share structural and/or sequence homology to
protein/peptide/domains identified and characterized within the art
as being a phospholipase protein or part of a phospholipase protein
and are related to the phospholipase C subfamily. Utilizing these
sequences, additional genomic sequences were assembled and
transcript and/or cDNA sequences were isolated and characterized.
Based on this analysis, the present invention provides amino acid
sequences of human phospholipase peptides and proteins that are
related to the phospholipase C subfamily, nucleic acid sequences in
the form of transcript sequences, cDNA sequences and/or genomic
sequences that encode these phospholipase peptides and proteins,
nucleic acid variation (allelic information), tissue distribution
of expression, and information about the closest art known
protein/peptide/domain that has structural or sequence homology to
the phospholipase of the present invention.
[0018] In addition to being previously unknown, the peptides that
are provided in the present invention are selected based on their
ability to be used for the development of commercially important
products and services. Specifically, the present peptides are
selected based on homology and/or structural relatedness to known
phospholipase proteins of the phospholipase C subfamily and the
expression pattern observed. Experimental data as provided in FIG.
1 indicates expression in humans in pancreas adenocarcinomas, lung,
brain anaplastic oligodendrogliomas, placenta choriocarcinomas,
pancreas adenocarcinomas, uterus endometrium, colon
adenocarcinomas, eye retinoblastomas, and fetal brain. The art has
clearly established the commercial importance of members of this
family of proteins and proteins that have expression patterns
similar to that of the present gene. Some of the more specific
features of the peptides of the present invention, and the uses
thereof, are described herein, particularly in the Background of
the Invention and in the annotation provided in the Figures, and/or
are known within the art for each of the known phospholipase C
family or subfamily of phospholipase proteins.
[0019] Specific Embodiments
[0020] Peptide Molecules
[0021] The present invention provides nucleic acid sequences that
encode protein molecules that have been identified as being members
of the phospholipase family of proteins and are related to the
phospholipase C subfamily (protein sequences are provided in FIG.
2, transcript/cDNA sequences are provided in FIG. 1 and genomic
sequences are provided in FIG. 3). The peptide sequences provided
in FIG. 2, as well as the obvious variants described herein,
particularly allelic variants as identified herein and using the
information in FIG. 3, will be referred herein as the phospholipase
peptides of the present invention, phospholipase peptides, or
peptides/proteins of the present invention.
[0022] The present invention provides isolated peptide and protein
molecules that consist of, consist essentially of, or comprise the
amino acid sequences of the phospholipase peptides disclosed in the
FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1,
transcript/cDNA or FIG. 3, genomic sequence), as well as all
obvious variants of these peptides that are within the art to make
and use. Some of these variants are described in detail below.
[0023] As used herein, a peptide is said to be "isolated" or
"purified" when it is substantially free of cellular material or
free of chemical precursors or other chemicals. The peptides of the
present invention can be purified to homogeneity or other degrees
of purity. The level of purification will be based on the intended
use. The critical feature is that the preparation allows for the
desired function of the peptide, even if in the presence of
considerable amounts of other components (the features of an
isolated nucleic acid molecule is discussed below).
[0024] In some uses, "substantially free of cellular material"
includes preparations of the peptide having less than about 30% (by
dry weight) other proteins (i.e., contaminating protein), less than
about 20% other proteins, less than about 10% other proteins, or
less than about 5% other proteins. When the peptide is
recombinantly produced, it can also be substantially free of
culture medium, i.e., culture medium represents less than about 20%
of the volume of the protein preparation.
[0025] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the peptide in which it
is separated from chemical precursors or other chemicals that are
involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the phospholipase peptide having less than
about 30% (by dry weight) chemical precursors or other chemicals,
less than about 20% chemical precursors or other chemicals, less
than about 10% chemical precursors or other chemicals, or less than
about 5% chemical precursors or other chemicals.
[0026] The isolated phospholipase peptide can be purified from
cells that naturally express it, purified from cells that have been
altered to express it (recombinant), or synthesized using known
protein synthesis methods. Experimental data as provided in FIG. 1
indicates expression in humans in pancreas adenocarcinomas, lung,
brain anaplastic oligodendrogliomas, placenta choriocarcinomas,
pancreas adenocarcinomas, uterus endometrium, colon
adenocarcinomas, eye retinoblastomas, and fetal brain. For example,
a nucleic acid molecule encoding the phospholipase peptide is
cloned into an expression vector, the expression vector introduced
into a host cell and the protein expressed in the host cell. The
protein can then be isolated from the cells by an appropriate
purification scheme using standard protein purification techniques.
Many of these techniques are described in detail below.
[0027] Accordingly, the present invention provides proteins that
consist of the amino acid sequences provided in FIG. 2 (SEQ ID
NO:2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence
of such a protein is provided in FIG. 2. A protein consists of an
amino acid sequence when the amino acid sequence is the final amino
acid sequence of the protein.
[0028] The present invention further provides proteins that consist
essentially of the amino acid sequences provided in FIG. 2 (SEQ ID
NO:2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists
essentially of an amino acid sequence when such an amino acid
sequence is present with only a few additional amino acid residues,
for example from about 1 to about 100 or so additional residues
typically from 1 to about 20 additional residues in the final
protein.
[0029] The present invention further provides proteins that
comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2),
for example, proteins encoded by the transcript/cDNA nucleic acid
sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences
provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid
sequence when the amino acid sequence is at least part of the final
amino acid sequence of the protein. In such a fashion, the protein
can be only the peptide or have additional amino acid molecules,
such as amino acid residues (contiguous encoded sequence) that are
naturally associated with it or heterologous amino acid
residues/peptide sequences. Such a protein can have a few
additional amino acid residues or can comprise several hundred or
more additional amino acids. The preferred classes of proteins that
are comprised of the phospholipase peptides of the present
invention are the naturally occurring mature proteins. A brief
description of how various types of these proteins can be
made/isolated is provided below.
[0030] The phospholipase peptides of the present invention can be
attached to heterologous sequences to form chimeric or fusion
proteins. Such chimeric and fusion proteins comprise a
phospholipase peptide operatively linked to a heterologous protein
having an amino acid sequence not substantially homologous to the
phospholipase peptide. "Operatively linked" indicates that the
phospholipase peptide and the heterologous protein are fused
in-frame. The heterologous protein can be fused to the N-terminus
or C-terminus of the phospholipase peptide.
[0031] In some uses, the fusion protein does not affect the
activity of the phospholipase peptide per se. For example, the
fusion protein can include, but is not limited to, enzymatic fusion
proteins, for example beta-galactosidase fusions, yeast two-hybrid
GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig
fusions. Such fusion proteins, particularly poly-His fusions, can
facilitate the purification of recombinant phospholipase peptide.
In certain host cells (e.g., mammalian host cells), expression
and/or secretion of a protein can be increased by using a
heterologous signal sequence.
[0032] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. In another embodiment, the
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (see Ausubel et al., Current
Protocols in Molecular Biology, 1992). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A phospholipase peptide-encoding
nucleic acid can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the phospholipase
peptide.
[0033] As mentioned above, the present invention also provides and
enables obvious variants of the amino acid sequence of the proteins
of the present invention, such as naturally occurring mature forms
of the peptide, allelic/sequence variants of the peptides,
non-naturally occurring recombinantly derived variants of the
peptides, and orthologs and paralogs of the peptides. Such variants
can readily be generated using art-known techniques in the fields
of recombinant nucleic acid technology and protein biochemistry. It
is understood, however, that variants exclude any amino acid
sequences disclosed prior to the invention.
[0034] Such variants can readily be identified/made using molecular
techniques and the sequence information disclosed herein. Further,
such variants can readily be distinguished from other peptides
based on sequence and/or structural homology to the phospholipase
peptides of the present invention. The degree of homology/identity
present will be based primarily on whether the peptide is a
functional variant or non-functional variant, the amount of
divergence present in the paralog family and the evolutionary
distance between the orthologs.
[0035] To determine the percent identity of two amino acid
sequences or 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, at
least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of
a reference sequence is aligned for comparison purposes. 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.
[0036] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a
preferred embodiment, the percent identity between two amino acid
sequences is determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossom 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 (Devereux, J., et
al., Nucleic Acids Res. 12(1):387 (1984)) (available at
http:/www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or
6. In another embodiment, the percent identity between two amino
acid or nucleotide sequences is determined using the algorithm of
E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4.
[0037] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against sequence 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. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches
can be performed with the NBLAST program, score=100, wordlength=12
to obtain nucleotide sequences homologous to the 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 the proteins of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used.
[0038] Full-length pre-processed forms, as well as mature processed
forms, of proteins that comprise one of the peptides of the present
invention can readily be identified as having complete sequence
identity to one of the phospholipase peptides of the present
invention as well as being encoded by the same genetic locus as the
phospholipase peptide provided herein. The gene encoding the novel
phospholipase protein of the present invention is located on a
genome component that has been mapped to human chromosome 17 (as
indicated in FIG. 3), which is supported by multiple lines of
evidence, such as STS and BAC map data.
[0039] Allelic variants of a phospholipase peptide can readily be
identified as being a human protein having a high degree
(significant) of sequence homology/identity to at least a portion
of the phospholipase peptide as well as being encoded by the same
genetic locus as the phospholipase peptide provided herein. Genetic
locus can readily be determined based on the genomic information
provided in FIG. 3, such as the genomic sequence mapped to the
reference human. The gene encoding the novel phospholipase protein
of the present invention is located on a genome component that has
been mapped to human chromosome 17 (as indicated in FIG. 3), which
is supported by multiple lines of evidence, such as STS and BAC map
data. As used herein, two proteins (or a region of the proteins)
have significant homology when the amino acid sequences are
typically at least about 70-80%, 80-90%, and more typically at
least about 90-95% or more homologous. A significantly homologous
amino acid sequence, according to the present invention, will be
encoded by a nucleic acid sequence that will hybridize to a
phospholipase peptide encoding nucleic acid molecule under
stringent conditions as more fully described below.
[0040] FIG. 3 provides information on SNPs that have been found in
the gene encoding the phospholipase proteins of the present
invention. SNPs were identified at 16 different nucleotide
positions. SNPs outside the ORF and in introns may affect
control/regulatory elements.
[0041] Paralogs of a phospholipase peptide can readily be
identified as having some degree of significant sequence
homology/identity to at least a portion of the phospholipase
peptide, as being encoded by a gene from humans, and as having
similar activity or function. Two proteins will typically be
considered paralogs when the amino acid sequences are typically at
least about 60% or greater, and more typically at least about 70%
or greater homology through a given region or domain. Such paralogs
will be encoded by a nucleic acid sequence that will hybridize to a
phospholipase peptide encoding nucleic acid molecule under moderate
to stringent conditions as more fully described below.
[0042] Orthologs of a phospholipase peptide can readily be
identified as having some degree of significant sequence
homology/identity to at least a portion of the phospholipase
peptide as well as being encoded by a gene from another organism.
Preferred orthologs will be isolated from mammals, preferably
primates, for the development of human therapeutic targets and
agents. Such orthologs will be encoded by a nucleic acid sequence
that will hybridize to a phospholipase peptide encoding nucleic
acid molecule under moderate to stringent conditions, as more fully
described below, depending on the degree of relatedness of the two
organisms yielding the proteins.
[0043] Non-naturally occurring variants of the phospholipase
peptides of the present invention can readily be generated using
recombinant techniques. Such variants include, but are not limited
to deletions, additions and substitutions in the amino acid
sequence of the phospholipase peptide. For example, one class of
substitutions are conserved amino acid substitution. Such
substitutions are those that substitute a given amino acid in a
phospholipase peptide by another amino acid of like
characteristics. Typically seen as conservative substitutions are
the replacements, one for another, among the aliphatic amino acids
Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser
and Thr; exchange of the acidic residues Asp and Glu; substitution
between the amide residues Asn and Gln; exchange of the basic
residues Lys and Arg; and replacements among the aromatic residues
Phe and Tyr. Guidance concerning which amino acid changes are
likely to be phenotypically silent are found in Bowie et al.,
Science 247:1306-1310 (1990).
[0044] Variant phospholipase peptides can be fully functional or
can lack function in one or more activities, e.g. ability to bind
substrate, ability to phosphorylate substrate, ability to mediate
signaling, etc. Fully functional variants typically contain only
conservative variation or variation in non-critical residues or in
non-critical regions. FIG. 2 provides the result of protein
analysis and can be used to identify critical domains/regions.
Functional variants can also contain substitution of similar amino
acids that result in no change or an insignificant change in
function. Alternatively, such substitutions may positively or
negatively affect function to some degree.
[0045] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0046] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science 244:1081-1085 (1989)), particularly using the results
provided in FIG. 2. The latter procedure introduces single alanine
mutations at every residue in the molecule. The resulting mutant
molecules are then tested for biological activity such as
phospholipase activity or in assays such as an in vitro
proliferative activity. Sites that are critical for binding
partner/substrate binding can also be determined by structural
analysis such as crystallization, nuclear magnetic resonance or
photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904
(1992); de Vos et al. Science 255:306-312 (1992)).
[0047] The present invention further provides fragments of the
phospholipase peptides, in addition to proteins and peptides that
comprise and consist of such fragments, particularly those
comprising the residues identified in FIG. 2. The fragments to
which the invention pertains, however, are not to be construed as
encompassing fragments that may be disclosed publicly prior to the
present invention.
[0048] As used herein, a fragment comprises at least 8, 10, 12, 14,
16, or more contiguous amino acid residues from a phospholipase
peptide. Such fragments can be chosen based on the ability to
retain one or more of the biological activities of the
phospholipase peptide or could be chosen for the ability to perform
a function, e.g. bind a substrate or act as an immunogen.
Particularly important fragments are biologically active fragments,
peptides that are, for example, about 8 or more amino acids in
length. Such fragments will typically comprise a domain or motif of
the phospholipase peptide, e.g., active site, a transmembrane
domain or a substrate-binding domain. Further, possible fragments
include, but are not limited to, domain or motif containing
fragments, soluble peptide fragments, and fragments containing
immunogenic structures. Predicted domains and functional sites are
readily identifiable by computer programs well known and readily
available to those of skill in the art (e.g., PROSITE analysis).
The results of one such analysis are provided in FIG. 2.
[0049] Polypeptides often contain amino acids other than the 20
amino acids commonly referred to as the 20 naturally occurring
amino acids. Further, many amino acids, including the terminal
amino acids, may be modified by natural processes, such as
processing and other post-translational modifications, or by
chemical modification techniques well known in the art. Common
modifications that occur naturally in phospholipase peptides are
described in basic texts, detailed monographs, and the research
literature, and they are well known to those of skill in the art
(some of these features are identified in FIG. 2).
[0050] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0051] Such modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993). Many
detailed reviews are available on this subject, such as by Wold,
F., Posttranslational Covalent Modification of Proteins, B. C.
Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.
(Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y.
Acad. Sci. 663:48-62 (1992)).
[0052] Accordingly, the phospholipase peptides of the present
invention also encompass derivatives or analogs in which a
substituted amino acid residue is not one encoded by the genetic
code, in which a substituent group is included, in which the mature
phospholipase peptide is fused with another compound, such as a
compound to increase the half-life of the phospholipase peptide
(for example, polyethylene glycol), or in which the additional
amino acids are fused to the mature phospholipase peptide, such as
a leader or secretory sequence or a sequence for purification of
the mature phospholipase peptide or a pro-protein sequence.
[0053] Protein/Peptide Uses
[0054] The proteins of the present invention can be used in
substantial and specific assays related to the functional
information provided in the Figures; to raise antibodies or to
elicit another immune response; as a reagent (including the labeled
reagent) in assays designed to quantitatively determine levels of
the protein (or its binding partner or ligand) in biological
fluids; and as markers for tissues in which the corresponding
protein is preferentially expressed (either constitutively or at a
particular stage of tissue differentiation or development or in a
disease state). Where the protein binds or potentially binds to
another protein or ligand (such as, for example, in a
phospholipase-effector protein interaction or phospholipase-ligand
interaction), the protein can be used to identify the binding
partner/ligand so as to develop a system to identify inhibitors of
the binding interaction. Any or all of these uses are capable of
being developed into reagent grade or kit format for
commercialization as commercial products.
[0055] Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include "Molecular Cloning: A Laboratory Manual", 2d ed., Cold
Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.
Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular
Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel
eds., 1987.
[0056] The potential uses of the peptides of the present invention
are based primarily on the source of the protein as well as the
class/action of the protein. For example, phospholipases isolated
from humans and their human/mammalian orthologs serve as targets
for identifying agents for use in mammalian therapeutic
applications, e.g. a human drug, particularly in modulating a
biological or pathological response in a cell or tissue that
expresses the phospholipase. Experimental data as provided in FIG.
1 indicates that phospholipase proteins of the present invention
are expressed in humans in pancreas adenocarcinomas, lung, brain
anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. Specifically, a virtual northern
blot shows expression in pancreas adenocarcinomas, lung, brain
anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, and eye
retinoblastomas. In addition, PCR-based tissue screening panels
indicate expression in fetal brain. A large percentage of
pharmaceutical agents are being developed that modulate the
activity of phospholipase proteins, particularly members of the
phospholipase C subfamily (see Background of the Invention). The
structural and functional information provided in the Background
and Figures provide specific and substantial uses for the molecules
of the present invention, particularly in combination with the
expression information provided in FIG. 1. Experimental data as
provided in FIG. 1 indicates expression in humans in pancreas
adenocarcinomas, lung, brain anaplastic oligodendrogliomas,
placenta choriocarcinomas, pancreas adenocarcinomas, uterus
endometrium, colon adenocarcinomas, eye retinoblastomas, and fetal
brain. Such uses can readily be determined using the information
provided herein, that which is known in the art, and routine
experimentation.
[0057] The proteins of the present invention (including variants
and fragments that may have been disclosed prior to the present
invention) are useful for biological assays related to
phospholipases that are related to members of the phospholipase C
subfamily. Such assays involve any of the known phospholipase
functions or activities or properties useful for diagnosis and
treatment of phospholipase-related conditions that are specific for
the subfamily of phospholipases that the one of the present
invention belongs to, particularly in cells and tissues that
express the phospholipase. Experimental data as provided in FIG. 1
indicates that phospholipase proteins of the present invention are
expressed in humans in pancreas adenocarcinomas, lung, brain
anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. Specifically, a virtual northern
blot shows expression in pancreas adenocarcinomas, lung, brain
anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, and eye
retinoblastomas. In addition, PCR-based tissue screening panels
indicate expression in fetal brain.
[0058] The proteins of the present invention are also useful in
drug screening assays, in cell-based or cell-free systems.
Cell-based systems can be native, i.e., cells that normally express
the phospholipase, as a biopsy or expanded in cell culture.
Experimental data as provided in FIG. 1 indicates expression in
humans in pancreas adenocarcinomas, lung, brain anaplastic
oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. In an alternate embodiment,
cell-based assays involve recombinant host cells expressing the
phospholipase protein.
[0059] The polypeptides can be used to identify compounds that
modulate phospholipase activity of the protein in its natural state
or an altered form that causes a specific disease or pathology
associated with the phospholipase. Both the phospholipases of the
present invention and appropriate variants and fragments can be
used in high-throughput screens to assay candidate compounds for
the ability to bind to the phospholipase. These compounds can be
further screened against a functional phospholipase to determine
the effect of the compound on the phospholipase activity. Further,
these compounds can be tested in animal or invertebrate systems to
determine activity/effectiveness. Compounds can be identified that
activate (agonist) or inactivate (antagonist) the phospholipase to
a desired degree.
[0060] Further, the proteins of the present invention can be used
to screen a compound for the ability to stimulate or inhibit
interaction between the phospholipase protein and a molecule that
normally interacts with the phospholipase protein, e.g. a substrate
or a component of the signal pathway that the phospholipase protein
normally interacts (for example, another phospholipase). Such
assays typically include the steps of combining the phospholipase
protein with a candidate compound under conditions that allow the
phospholipase protein, or fragment, to interact with the target
molecule, and to detect the formation of a complex between the
protein and the target or to detect the biochemical consequence of
the interaction with the phospholipase protein and the target, such
as any of the associated effects of signal transduction such as
protein phosphorylation, cAMP turnover, and adenylate cyclase
activation, etc.
[0061] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al., Nature
354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0062] One candidate compound is a soluble fragment of the receptor
that competes for substrate binding. Other candidate compounds
include mutant phospholipases or appropriate fragments containing
mutations that affect phospholipase function and thus compete for
substrate. Accordingly, a fragment that competes for substrate, for
example with a higher affinity, or a fragment that binds substrate
but does not allow release, is encompassed by the invention.
[0063] The invention further includes other end point assays to
identify compounds that modulate (stimulate or inhibit)
phospholipase activity. The assays typically involve an assay of
events in the signal transduction pathway that indicate
phospholipase activity. Thus, the phosphorylation of a substrate,
activation of a protein, a change in the expression of genes that
are up- or down-regulated in response to the phospholipase protein
dependent signal cascade can be assayed.
[0064] Any of the biological or biochemical functions mediated by
the phospholipase can be used as an endpoint assay. These include
all of the biochemical or biochemical/biological events described
herein, in the references cited herein, incorporated by reference
for these endpoint assay targets, and other functions known to
those of ordinary skill in the art or that can be readily
identified using the information provided in the Figures,
particularly FIG. 2. Specifically, a biological function of a cell
or tissues that expresses the phospholipase can be assayed.
Experimental data as provided in FIG. 1 indicates that
phospholipase proteins of the present invention are expressed in
humans in pancreas adenocarcinomas, lung, brain anaplastic
oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. Specifically, a virtual northern
blot shows expression in pancreas adenocarcinomas, lung, brain
anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, and eye
retinoblastomas. In addition, PCR-based tissue screening panels
indicate expression in fetal brain.
[0065] Binding and/or activating compounds can also be screened by
using chimeric phospholipase proteins in which the amino terminal
extracellular domain, or parts thereof, the entire transmembrane
domain or subregions, such as any of the seven transmembrane
segments or any of the intracellular or extracellular loops and the
carboxy terminal intracellular domain, or parts thereof, can be
replaced by heterologous domains or subregions. For example, a
substrate-binding region can be used that interacts with a
different substrate then that which is recognized by the native
phospholipase. Accordingly, a different set of signal transduction
components is available as an end-point assay for activation. This
allows for assays to be performed in other than the specific host
cell from which the phospholipase is derived.
[0066] The proteins of the present invention are also useful in
competition binding assays in methods designed to discover
compounds that interact with the phospholipase (e.g. binding
partners and/or ligands). Thus, a compound is exposed to a
phospholipase polypeptide under conditions that allow the compound
to bind or to otherwise interact with the polypeptide. Soluble
phospholipase polypeptide is also added to the mixture. If the test
compound interacts with the soluble phospholipase polypeptide, it
decreases the amount of complex formed or activity from the
phospholipase target. This type of assay is particularly useful in
cases in which compounds are sought that interact with specific
regions of the phospholipase. Thus, the soluble polypeptide that
competes with the target phospholipase region is designed to
contain peptide sequences corresponding to the region of
interest.
[0067] To perform cell free drug screening assays, it is sometimes
desirable to immobilize either the phospholipase protein, or
fragment, or its target molecule to facilitate separation of
complexes from uncomplexed forms of one or both of the proteins, as
well as to accommodate automation of the assay.
[0068] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase fusion
proteins can be adsorbed onto glutathione sepharose beads (Sigma
Chemical, St. Louis, Mo.) or glutathione derivatized microtitre
plates, which are then combined with the cell lysates (e.g.,
.sup.35S-labeled) and the candidate compound, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads are washed to remove any unbound label, and the matrix
immobilized and radiolabel determined directly, or in the
supernatant after the complexes are dissociated. Alternatively, the
complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of phospholipase-binding protein found in
the bead fraction quantitated from the gel using standard
electrophoretic techniques. For example, either the polypeptide or
its target molecule can be immobilized utilizing conjugation of
biotin and streptavidin using techniques well known in the art.
Alternatively, antibodies reactive with the protein but which do
not interfere with binding of the protein to its target molecule
can be derivatized to the wells of the plate, and the protein
trapped in the wells by antibody conjugation. Preparations of a
phospholipase-binding protein and a candidate compound are
incubated in the phospholipase protein-presenting wells and the
amount of complex trapped in the well can be quantitated. 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 phospholipase protein
target molecule, or which are reactive with phospholipase protein
and compete with the target molecule, as well as enzyme-linked
assays which rely on detecting an enzymatic activity associated
with the target molecule.
[0069] Agents that modulate one of the phospholipases of the
present invention can be identified using one or more of the above
assays, alone or in combination. It is generally preferable to use
a cell-based or cell free system first and then confirm activity in
an animal or other model system. Such model systems are well known
in the art and can readily be employed in this context.
[0070] Modulators of phospholipase protein activity identified
according to these drug screening assays can be used to treat a
subject with a disorder mediated by the phospholipase pathway, by
treating cells or tissues that express the phospholipase.
Experimental data as provided in FIG. 1 indicates expression in
humans in pancreas adenocarcinomas, lung, brain anaplastic
oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. These methods of treatment
include the steps of administering a modulator of phospholipase
activity in a pharmaceutical composition to a subject in need of
such treatment, the modulator being identified as described
herein.
[0071] In yet another aspect of the invention, the phospholipase
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 the
phospholipase and are involved in phospholipase activity. Such
phospholipase-binding proteins are also likely to be involved in
the propagation of signals by the phospholipase proteins or
phospholipase targets as, for example, downstream elements of a
phospholipase-mediated signaling pathway. Alternatively, such
phospholipase-binding proteins are likely to be phospholipase
inhibitors.
[0072] 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
phospholipase 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. If the "bait" and the "prey" proteins are
able to interact, in vivo forming a phospholipase-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
phospholipase protein.
[0073] 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 in an appropriate animal model. For example, an
agent identified as described herein (e.g., a
phospholipase-modulating agent, an antisense phospholipase nucleic
acid molecule, a phospholipase-specific antibody, or a
phospholipase-binding partner) can be used in an animal or other
model to determine the efficacy, toxicity, or side effects of
treatment with such an agent. Alternatively, an agent identified as
described herein can be used in an animal or other model to
determine the mechanism of action of such an agent. Furthermore,
this invention pertains to uses of novel agents identified by the
above-described screening assays for treatments as described
herein.
[0074] The phospholipase proteins of the present invention are also
useful to provide a target for diagnosing a disease or
predisposition to disease mediated by the peptide. Accordingly, the
invention provides methods for detecting the presence, or levels
of, the protein (or encoding mRNA) in a cell, tissue, or organism.
Experimental data as provided in FIG. 1 indicates expression in
humans in pancreas adenocarcinomas, lung, brain anaplastic
oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. The method involves contacting a
biological sample with a compound capable of interacting with the
phospholipase protein such that the interaction can be detected.
Such an assay can be provided in a single detection format or a
multi-detection format such as an antibody chip array.
[0075] One agent for detecting a protein in a sample is an antibody
capable of selectively binding to protein. A biological sample
includes tissues, cells and biological fluids isolated from a
subject, as well as tissues, cells and fluids present within a
subject.
[0076] The peptides of the present invention also provide targets
for diagnosing active protein activity, disease, or predisposition
to disease, in a patient having a variant peptide, particularly
activities and conditions that are known for other members of the
family of proteins to which the present one belongs. Thus, the
peptide can be isolated from a biological sample and assayed for
the presence of a genetic mutation that results in aberrant
peptide. This includes amino acid substitution, deletion,
insertion, rearrangement, (as the result of aberrant splicing
events), and inappropriate post-translational modification.
Analytic methods include altered electrophoretic mobility, altered
tryptic peptide digest, altered phospholipase activity in
cell-based or cell-free assay, alteration in substrate or
antibody-binding pattern, altered isoelectric point, direct amino
acid sequencing, and any other of the known assay techniques useful
for detecting mutations in a protein. Such an assay can be provided
in a single detection format or a multi-detection format such as an
antibody chip array.
[0077] In vitro techniques for detection of peptide include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence using a detection
reagent, such as an antibody or protein binding agent.
Alternatively, the peptide can be detected in vivo in a subject by
introducing into the subject a labeled anti-peptide antibody or
other types of detection agent. 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.
Particularly useful are methods that detect the allelic variant of
a peptide expressed in a subject and methods which detect fragments
of a peptide in a sample.
[0078] The peptides are also useful in pharmacogenomic analysis.
Pharmacogenomics deal with clinically significant hereditary
variations in the response to drugs due to altered drug disposition
and abnormal action in affected persons. See, e.g., Eichelbaum, M.
(Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and
Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical
outcomes of these variations result in severe toxicity of
therapeutic drugs in certain individuals or therapeutic failure of
drugs in certain individuals as a result of individual variation in
metabolism. Thus, the genotype of the individual can determine the
way a therapeutic compound acts on the body or the way the body
metabolizes the compound. Further, the activity of drug
metabolizing enzymes effects both the intensity and duration of
drug action. Thus, the pharmacogenomics of the individual permit
the selection of effective compounds and effective dosages of such
compounds for prophylactic or therapeutic treatment based on the
individual's genotype. The discovery of genetic polymorphisms in
some drug metabolizing enzymes has explained why some patients do
not obtain the expected drug effects, show an exaggerated drug
effect, or experience serious toxicity from standard drug dosages.
Polymorphisms can be expressed in the phenotype of the extensive
metabolizer and the phenotype of the poor metabolizer. Accordingly,
genetic polymorphism may lead to allelic protein variants of the
phospholipase protein in which one or more of the phospholipase
functions in one population is different from those in another
population. The peptides thus allow a target to ascertain a genetic
predisposition that can affect treatment modality. Thus, in a
ligand-based treatment, polymorphism may give rise to amino
terminal extracellular domains and/or other substrate-binding
regions that are more or less active in substrate binding, and
phospholipase activation. Accordingly, substrate dosage would
necessarily be modified to maximize the therapeutic effect within a
given population containing a polymorphism. As an alternative to
genotyping, specific polymorphic peptides could be identified.
[0079] The peptides are also useful for treating a disorder
characterized by an absence of, inappropriate, or unwanted
expression of the protein. Experimental data as provided in FIG. 1
indicates expression in humans in pancreas adenocarcinomas, lung,
brain anaplastic oligodendrogliomas, placenta choriocarcinomas,
pancreas adenocarcinomas, uterus endometrium, colon
adenocarcinomas, eye retinoblastomas, and fetal brain. Accordingly,
methods for treatment include the use of the phospholipase protein
or fragments.
[0080] Antibodies
[0081] The invention also provides antibodies that selectively bind
to one of the peptides of the present invention, a protein
comprising such a peptide, as well as variants and fragments
thereof. As used herein, an antibody selectively binds a target
peptide when it binds the target peptide and does not significantly
bind to unrelated proteins. An antibody is still considered to
selectively bind a peptide even if it also binds to other proteins
that are not substantially homologous with the target peptide so
long as such proteins share homology with a fragment or domain of
the peptide target of the antibody. In this case, it would be
understood that antibody binding to the peptide is still selective
despite some degree of cross-reactivity.
[0082] As used herein, an antibody is defined in terms consistent
with that recognized within the art: they are multi-subunit
proteins produced by a mammalian organism in response to an antigen
challenge. The antibodies of the present invention include
polyclonal antibodies and monoclonal antibodies, as well as
fragments of such antibodies, including, but not limited to, Fab or
F(ab').sub.2, and Fv fragments.
[0083] Many methods are known for generating and/or identifying
antibodies to a given target peptide. Several such methods are
described by Harlow, Antibodies, Cold Spring Harbor Press,
(1989).
[0084] In general, to generate antibodies, an isolated peptide is
used as an immunogen and is administered to a mammalian organism,
such as a rat, rabbit or mouse. The full-length protein, an
antigenic peptide fragment or a fusion protein can be used.
Particularly important fragments are those covering functional
domains, such as the domains identified in FIG. 2, and domain of
sequence homology or divergence amongst the family, such as those
that can readily be identified using protein alignment methods and
as presented in the Figures.
[0085] Antibodies are preferably prepared from regions or discrete
fragments of the phospholipase proteins. Antibodies can be prepared
from any region of the peptide as described herein. However,
preferred regions will include those involved in function/activity
and/or phospholipase/binding partner interaction. FIG. 2 can be
used to identify particularly important regions while sequence
alignment can be used to identify conserved and unique sequence
fragments.
[0086] An antigenic fragment will typically comprise at least 8
contiguous amino acid residues. The antigenic peptide can comprise,
however, at least 10, 12, 14, 16 or more amino acid residues. Such
fragments can be selected on a physical property, such as fragments
correspond to regions that are located on the surface of the
protein, e.g., hydrophilic regions or can be selected based on
sequence uniqueness (see FIG. 2).
[0087] Detection on an antibody of the present invention can be
facilitated by coupling (i.e., physically linking) the antibody to
a detectable substance. 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.
[0088] Antibody Uses
[0089] The antibodies can be used to isolate one of the proteins of
the present invention by standard techniques, such as affinity
chromatography or immunoprecipitation. The antibodies can
facilitate the purification of the natural protein from cells and
recombinantly produced protein expressed in host cells. In
addition, such antibodies are useful to detect the presence of one
of the proteins of the present invention in cells or tissues to
determine the pattern of expression of the protein among various
tissues in an organism and over the course of normal development.
Experimental data as provided in FIG. 1 indicates that
phospholipase proteins of the present invention are expressed in
humans in pancreas adenocarcinomas, lung, brain anaplastic
oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. Specifically, a virtual northern
blot shows expression in pancreas adenocarcinomas, lung, brain
anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, and eye
retinoblastomas. In addition, PCR-based tissue screening panels
indicate expression in fetal brain. Further, such antibodies can be
used to detect protein in situ, in vitro, or in a cell lysate or
supernatant in order to evaluate the abundance and pattern of
expression. Also, such antibodies can be used to assess abnormal
tissue distribution or abnormal expression during development or
progression of a biological condition. Antibody detection of
circulating fragments of the full length protein can be used to
identify turnover.
[0090] Further, the antibodies can be used to assess expression in
disease states such as in active stages of the disease or in an
individual with a predisposition toward disease related to the
protein's function. When a disorder is caused by an inappropriate
tissue distribution, developmental expression, level of expression
of the protein, or expressed/processed form, the antibody can be
prepared against the normal protein. Experimental data as provided
in FIG. 1 indicates expression in humans in pancreas
adenocarcinomas, lung, brain anaplastic oligodendrogliomas,
placenta choriocarcinomas, pancreas adenocarcinomas, uterus
endometrium, colon adenocarcinomas, eye retinoblastomas, and fetal
brain. If a disorder is characterized by a specific mutation in the
protein, antibodies specific for this mutant protein can be used to
assay for the presence of the specific mutant protein.
[0091] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Experimental data as provided in FIG. 1 indicates
expression in humans in pancreas adenocarcinomas, lung, brain
anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. The diagnostic uses can be
applied, not only in genetic testing, but also in monitoring a
treatment modality. Accordingly, where treatment is ultimately
aimed at correcting expression level or the presence of aberrant
sequence and aberrant tissue distribution or developmental
expression, antibodies directed against the protein or relevant
fragments can be used to monitor therapeutic efficacy.
[0092] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic proteins
can be used to identify individuals that require modified treatment
modalities. The antibodies are also useful as diagnostic tools as
an immunological marker for aberrant protein analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[0093] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression in
humans in pancreas adenocarcinomas, lung, brain anaplastic
oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. Thus, where a specific protein
has been correlated with expression in a specific tissue,
antibodies that are specific for this protein can be used to
identify a tissue type.
[0094] The antibodies are also useful for inhibiting protein
function, for example, blocking the binding of the phospholipase
peptide to a binding partner such as a substrate. These uses can
also be applied in a therapeutic context in which treatment
involves inhibiting the protein's function. An antibody can be
used, for example, to block binding, thus modulating (agonizing or
antagonizing) the peptides activity. Antibodies can be prepared
against specific fragments containing sites required for function
or against intact protein that is associated with a cell or cell
membrane. See FIG. 2 for structural information relating to the
proteins of the present invention.
[0095] The invention also encompasses kits for using antibodies to
detect the presence of a protein in a biological sample. The kit
can comprise antibodies such as a labeled or labelable antibody and
a compound or agent for detecting protein in a biological sample;
means for determining the amount of protein in the sample; means
for comparing the amount of protein in the sample with a standard;
and instructions for use. Such a kit can be supplied to detect a
single protein or epitope or can be configured to detect one of a
multitude of epitopes, such as in an antibody detection array.
Arrays are described in detail below for nuleic acid arrays and
similar methods have been developed for antibody arrays.
[0096] Nucleic Acid Molecules
[0097] The present invention further provides isolated nucleic acid
molecules that encode a phospholipase peptide or protein of the
present invention (cDNA, transcript and genomic sequence). Such
nucleic acid molecules will consist of, consist essentially of, or
comprise a nucleotide sequence that encodes one of the
phospholipase peptides of the present invention, an allelic variant
thereof, or an ortholog or paralog thereof.
[0098] As used herein, an "isolated" nucleic acid molecule is one
that is separated from other nucleic acid present in the natural
source of the nucleic acid. Preferably, an "isolated" nucleic acid
is free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
However, there can be some flanking nucleotide sequences, for
example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less,
particularly contiguous peptide encoding sequences and peptide
encoding sequences within the same gene but separated by introns in
the genomic sequence. The important point is that the nucleic acid
is isolated from remote and unimportant flanking sequences such
that it can be subjected to the specific manipulations described
herein such as recombinant expression, preparation of probes and
primers, and other uses specific to the nucleic acid sequences.
[0099] Moreover, an "isolated" nucleic acid molecule, such as a
transcript/cDNA molecule, can be substantially free of other
cellular material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0100] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0101] Accordingly, the present invention provides nucleic acid
molecules that consist of the nucleotide sequence shown in FIG. 1
or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists
of a nucleotide sequence when the nucleotide sequence is the
complete nucleotide sequence of the nucleic acid molecule.
[0102] The present invention further provides nucleic acid
molecules that consist essentially of the nucleotide sequence shown
in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3,
genomic sequence), or any nucleic acid molecule that encodes the
protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule
consists essentially of a nucleotide sequence when such a
nucleotide sequence is present with only a few additional nucleic
acid residues in the final nucleic acid molecule.
[0103] The present invention further provides nucleic acid
molecules that comprise the nucleotide sequences shown in FIG. 1 or
3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises
a nucleotide sequence when the nucleotide sequence is at least part
of the final nucleotide sequence of the nucleic acid molecule. In
such a fashion, the nucleic acid molecule can be only the
nucleotide sequence or have additional nucleic acid residues, such
as nucleic acid residues that are naturally associated with it or
heterologous nucleotide sequences. Such a nucleic acid molecule can
have a few additional nucleotides or can comprises several hundred
or more additional nucleotides. A brief description of how various
types of these nucleic acid molecules can be readily made/isolated
is provided below.
[0104] In FIGS. 1 and 3, both coding and non-coding sequences are
provided. Because of the source of the present invention, humans
genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1),
the nucleic acid molecules in the Figures will contain genomic
intronic sequences, 5' and 3' non-coding sequences, gene regulatory
regions and non-coding intergenic sequences. In general such
sequence features are either noted in FIGS. 1 and 3 or can readily
be identified using computational tools known in the art. As
discussed below, some of the non-coding regions, particularly gene
regulatory elements such as promoters, are useful for a variety of
purposes, e.g. control of heterologous gene expression, target for
identifying gene activity modulating compounds, and are
particularly claimed as fragments of the genomic sequence provided
herein.
[0105] The isolated nucleic acid molecules can encode the mature
protein plus additional amino or carboxyl-terminal amino acids, or
amino acids interior to the mature peptide (when the mature form
has more than one peptide chain, for instance). Such sequences may
play a role in processing of a protein from precursor to a mature
form, facilitate protein trafficking, prolong or shorten protein
half-life or facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in situ,
the additional amino acids may be processed away from the mature
protein by cellular enzymes.
[0106] As mentioned above, the isolated nucleic acid molecules
include, but are not limited to, the sequence encoding the
phospholipase peptide alone, the sequence encoding the mature
peptide and additional coding sequences, such as a leader or
secretory sequence (e.g., a pre-pro or pro-protein sequence), the
sequence encoding the mature peptide, with or without the
additional coding sequences, plus additional non-coding sequences,
for example introns and non-coding 5' and 3' sequences such as
transcribed but non-translated sequences that play a role in
transcription, mRNA processing (including splicing and
polyadenylation signals), ribosome binding and stability of mRNA.
In addition, the nucleic acid molecule may be fused to a marker
sequence encoding, for example, a peptide that facilitates
purification.
[0107] Isolated nucleic acid molecules can be in the form of RNA,
such as mRNA, or in the form DNA, including cDNA and genomic DNA
obtained by cloning or produced by chemical synthetic techniques or
by a combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[0108] The invention further provides nucleic acid molecules that
encode fragments of the peptides of the present invention as well
as nucleic acid molecules that encode obvious variants of the
phospholipase proteins of the present invention that are described
above. Such nucleic acid molecules may be naturally occurring, such
as allelic variants (same locus), paralogs (different locus), and
orthologs (different organism), or may be constructed by
recombinant DNA methods or by chemical synthesis. Such
non-naturally occurring variants may be made by mutagenesis
techniques, including those applied to nucleic acid molecules,
cells, or organisms. Accordingly, as discussed above, 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.
[0109] The present invention further provides non-coding fragments
of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred
non-coding fragments include, but are not limited to, promoter
sequences, enhancer sequences, gene modulating sequences and gene
termination sequences. Such fragments are useful in controlling
heterologous gene expression and in developing screens to identify
gene-modulating agents. A promoter can readily be identified as
being 5' to the ATG start site in the genomic sequence provided in
FIG. 3.
[0110] A fragment comprises a contiguous nucleotide sequence
greater than 12 or more nucleotides. Further, a fragment could at
least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length
of the fragment will be based on its intended use. For example, the
fragment can encode epitope bearing regions of the peptide, or can
be useful as DNA probes and primers. Such fragments can be isolated
using the known nucleotide sequence to synthesize an
oligonucleotide probe. A labeled probe can then be used to screen a
cDNA library, genomic DNA library, or mRNA to isolate nucleic acid
corresponding to the coding region. Further, primers can be used in
PCR reactions to clone specific regions of gene.
[0111] A probe/primer typically comprises substantially a purified
oligonucleotide or oligonucleotide pair. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, 20, 25, 40, 50 or
more consecutive nucleotides.
[0112] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. As described in the Peptide
Section, these variants comprise a nucleotide sequence encoding a
peptide that is typically 60-70%, 70-80%, 80-90%, and more
typically at least about 90-95% or more homologous to the
nucleotide sequence shown in the Figure sheets or a fragment of
this sequence. Such nucleic acid molecules can readily be
identified as being able to hybridize under moderate to stringent
conditions, to the nucleotide sequence shown in the Figure sheets
or a fragment of the sequence. Allelic variants can readily be
determined by genetic locus of the encoding gene. The gene encoding
the novel phospholipase protein of the present invention is located
on a genome component that has been mapped to human chromosome 17
(as indicated in FIG. 3), which is supported by multiple lines of
evidence, such as STS and BAC map data.
[0113] FIG. 3 provides information on SNPs that have been found in
the gene encoding the phospholipase proteins of the present
invention. SNPs were identified at 16 different nucleotide
positions. SNPs outside the ORF and in introns may affect
control/regulatory elements.
[0114] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a peptide at
least 60-70% homologous to each other typically remain hybridized
to each other. The conditions can be such that sequences at least
about 60%, at least about 70%, or at least about 80% or more
homologous to each other typically remain hybridized to each other.
Such 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. One example of stringent
hybridization conditions are hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45 C., followed by one or
more washes in 0.2.times.SSC, 0.1% SDS at 50-65 C. Examples of
moderate to low stringency hybridization conditions are well known
in the art.
[0115] Nucleic Acid Molecule Uses
[0116] The nucleic acid molecules of the present invention are
useful for probes, primers, chemical intermediates, and in
biological assays. The nucleic acid molecules are useful as a
hybridization probe for messenger RNA, transcript/cDNA and genomic
DNA to isolate full-length cDNA and genomic clones encoding the
peptide described in FIG. 2 and to isolate cDNA and genomic clones
that correspond to variants (alleles, orthologs, etc.) producing
the same or related peptides shown in FIG. 2. As illustrated in
FIG. 3, SNPs were identified at 16 different nucleotide
positions.
[0117] The probe can correspond to any sequence along the entire
length of the nucleic acid molecules provided in the Figures.
Accordingly, it could be derived from 5' noncoding regions, the
coding region, and 3' noncoding regions. However, as discussed,
fragments are not to be construed as encompassing fragments
disclosed prior to the present invention.
[0118] The nucleic acid molecules are also useful as primers for
PCR to amplify any given region of a nucleic acid molecule and are
useful to synthesize antisense molecules of desired length and
sequence.
[0119] The nucleic acid molecules are also useful for constructing
recombinant vectors. Such vectors include expression vectors that
express a portion of, or all of, the peptide sequences. Vectors
also include insertion vectors, used to integrate into another
nucleic acid molecule sequence, such as into the cellular genome,
to alter in situ expression of a gene and/or gene product. For
example, an endogenous coding sequence can be replaced via
homologous recombination with all or part of the coding region
containing one or more specifically introduced mutations.
[0120] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0121] The nucleic acid molecules are also useful as probes for
determining the chromosomal positions of the nucleic acid molecules
by means of in situ hybridization methods. The gene encoding the
novel phospholipase protein of the present invention is located on
a genome component that has been mapped to human chromosome 17 (as
indicated in FIG. 3), which is supported by multiple lines of
evidence, such as STS and BAC map data.
[0122] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0123] The nucleic acid molecules are also useful for designing
ribozymes corresponding to all, or a part, of the mRNA produced
from the nucleic acid molecules described herein.
[0124] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0125] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0126] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0127] The nucleic acid molecules are also useful as hybridization
probes for determining the presence, level, form and distribution
of nucleic acid expression. Experimental data as provided in FIG. 1
indicates that phospholipase proteins of the present invention are
expressed in humans in pancreas adenocarcinomas, lung, brain
anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. Specifically, a virtual northern
blot shows expression in pancreas adenocarcinomas, lung, brain
anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, and eye
retinoblastomas. In addition, PCR-based tissue screening panels
indicate expression in fetal brain. Accordingly, the probes can be
used to detect the presence of, or to determine levels of, a
specific nucleic acid molecule in cells, tissues, and in organisms.
The nucleic acid whose level is determined can be DNA or RNA.
Accordingly, probes corresponding to the peptides described herein
can be used to assess expression and/or gene copy number in a given
cell, tissue, or organism. These uses are relevant for diagnosis of
disorders involving an increase or decrease in phospholipase
protein expression relative to normal results.
[0128] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[0129] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a phospholipase protein,
such as by measuring a level of a phospholipase-encoding nucleic
acid in a sample of cells from a subject e.g., mRNA or genomic DNA,
or determining if a phospholipase gene has been mutated.
Experimental data as provided in FIG. 1 indicates that
phospholipase proteins of the present invention are expressed in
humans in pancreas adenocarcinomas, lung, brain anaplastic
oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. Specifically, a virtual northern
blot shows expression in pancreas adenocarcinomas, lung, brain
anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, and eye
retinoblastomas. In addition, PCR-based tissue screening panels
indicate expression in fetal brain.
[0130] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate phospholipase nucleic acid
expression.
[0131] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the phospholipase gene, particularly
biological and pathological processes that are mediated by the
phospholipase in cells and tissues that express it. Experimental
data as provided in FIG. 1 indicates expression in humans in
pancreas adenocarcinomas, lung, brain anaplastic
oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas., uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. The method typically includes
assaying the ability of the compound to modulate the expression of
the phospholipase nucleic acid and thus identifying a compound that
can be used to treat a disorder characterized by undesired
phospholipase nucleic acid expression. The assays can be performed
in cell-based and cell-free systems. Cell-based assays include
cells naturally expressing the phospholipase nucleic acid or
recombinant cells genetically engineered to express specific
nucleic acid sequences.
[0132] The assay for phospholipase nucleic acid expression can
involve direct assay of nucleic acid levels, such as mRNA levels,
or on collateral compounds involved in the signal pathway. Further,
the expression of genes that are up- or down-regulated in response
to the phospholipase protein signal pathway can also be assayed. In
this embodiment the regulatory regions of these genes can be
operably linked to a reporter gene such as luciferase.
[0133] Thus, modulators of phospholipase gene expression can be
identified in a method wherein a cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of phospholipase mRNA in the presence of the candidate
compound is compared to the level of expression of phospholipase
mRNA in the absence of the candidate compound. The candidate
compound can then be identified as a modulator of nucleic acid
expression based on this comparison and be used, for example to
treat a disorder characterized by aberrant nucleic acid expression.
When expression of mRNA is statistically significantly greater in
the presence of the candidate compound than in its absence, the
candidate compound is identified as a stimulator of nucleic acid
expression. When nucleic acid expression is statistically
significantly less in the presence of the candidate compound than
in its absence, the candidate compound is identified as an
inhibitor of nucleic acid expression.
[0134] The invention further provides methods of treatment, with
the nucleic acid as a target, using a compound identified through
drug screening as a gene modulator to modulate phospholipase
nucleic acid expression in cells and tissues that express the
phospholipase. Experimental data as provided in FIG. 1 indicates
that phospholipase proteins of the present invention are expressed
in humans in pancreas adenocarcinomas, lung, brain anaplastic
oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. Specifically, a virtual northern
blot shows expression in pancreas adenocarcinomas, lung, brain
anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, and eye
retinoblastomas. In addition, PCR-based tissue screening panels
indicate expression in fetal brain. Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or nucleic acid expression.
[0135] Alternatively, a modulator for phospholipase nucleic acid
expression can be a small molecule or drug identified using the
screening assays described herein as long as the drug or small
molecule inhibits the phospholipase nucleic acid expression in the
cells and tissues that express the protein. Experimental data as
provided in FIG. 1 indicates expression in humans in pancreas
adenocarcinomas, lung, brain anaplastic oligodendrogliomas,
placenta choriocarcinomas, pancreas adenocarcinomas, uterus
endometrium, colon adenocarcinomas, eye retinoblastomas, and fetal
brain.
[0136] The nucleic acid molecules are also useful for monitoring
the effectiveness of modulating compounds on the expression or
activity of the phospholipase gene in clinical trials or in a
treatment regimen. Thus, the gene expression pattern can serve as a
barometer for the continuing effectiveness of treatment with the
compound, particularly with compounds to which a patient can
develop resistance. The gene expression pattern can also serve as a
marker indicative of a physiological response of the affected cells
to the compound. Accordingly, such monitoring would allow either
increased administration of the compound or the administration of
alternative compounds to which the patient has not become
resistant. Similarly, if the level of nucleic acid expression falls
below a desirable level, administration of the compound could be
commensurately decreased.
[0137] The nucleic acid molecules are also useful in diagnostic
assays for qualitative changes in phospholipase nucleic acid
expression, and particularly in qualitative changes that lead to
pathology. The nucleic acid molecules can be used to detect
mutations in phospholipase genes and gene expression products such
as mRNA. The nucleic acid molecules can be used as hybridization
probes to detect naturally occurring genetic mutations in the
phospholipase gene and thereby to determine whether a subject with
the mutation is at risk for a disorder caused by the mutation.
Mutations include deletion, addition, or substitution of one or
more nucleotides in the gene, chromosomal rearrangement, such as
inversion or transposition, modification of genomic DNA, such as
aberrant methylation patterns or changes in gene copy number, such
as amplification. Detection of a mutated form of the phospholipase
gene associated with a dysfunction provides a diagnostic tool for
an active disease or susceptibility to disease when the disease
results from overexpression, underexpression, or altered expression
of a phospholipase protein.
[0138] Individuals carrying mutations in the phospholipase gene can
be detected at the nucleic acid level by a variety of techniques.
FIG. 3 provides information on SNPs that have been found in the
gene encoding the phospholipase proteins of the present invention.
SNPs were identified at 16 different nucleotide positions. SNPs
outside the ORF and in introns may affect control/regulatory
elements. The gene encoding the novel phospholipase protein of the
present invention is located on a genome component that has been
mapped to human chromosome 17 (as indicated in FIG. 3), which is
supported by multiple lines of evidence, such as STS and BAC map
data. Genomic DNA can be analyzed directly or can be amplified by
using PCR prior to analysis. RNA or cDNA can be used in the same
way. In some uses, detection of the mutation involves the use of a
probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S.
Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR,
or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,
Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et
al., PNAS 91:360-364 (1994)), the latter of which can be
particularly useful for detecting point mutations in the gene (see
Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method
can include the steps of collecting a sample of cells from a
patient, isolating nucleic acid (e.g., genomic, mRNA or both) from
the cells of the sample, contacting the nucleic acid sample with
one or more primers which specifically hybridize to a gene under
conditions such that hybridization and amplification of the 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. Deletions and
insertions can be detected by a change in size of the amplified
product compared to the normal genotype. Point mutations can be
identified by hybridizing amplified DNA to normal RNA or antisense
DNA sequences.
[0139] Alternatively, mutations in a phospholipase gene can be
directly identified, for example, by alterations in restriction
enzyme digestion patterns determined by gel electrophoresis.
[0140] Further, sequence-specific ribozymes (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.
Perfectly matched sequences can be distinguished from mismatched
sequences by nuclease cleavage digestion assays or by differences
in melting temperature.
[0141] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method. Furthermore, sequence differences
between a mutant phospholipase gene and a wild-type gene can be
determined by direct DNA sequencing. A variety of automated
sequencing procedures can be utilized when performing the
diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448),
including sequencing by mass spectrometry (see, e.g., PCT
International Publication No. WO 94/16101; Cohen et al., Adv.
Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.
Biotechnol. 38:147-159 (1993)).
[0142] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.,
Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988);
Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144
(1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79
(1992)), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al., Nature
313:495 (1985)). Examples of other techniques for detecting point
mutations include selective oligonucleotide hybridization,
selective amplification, and selective primer extension.
[0143] The nucleic acid molecules are also useful for testing an
individual for a genotype that while not necessarily causing the
disease, nevertheless affects the treatment modality. Thus, the
nucleic acid molecules can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship).
Accordingly, the nucleic acid molecules described herein can be
used to assess the mutation content of the phospholipase gene in an
individual in order to select an appropriate compound or dosage
regimen for treatment. FIG. 3 provides information on SNPs that
have been found in the gene encoding the phospholipase proteins of
the present invention. SNPs were identified at 16 different
nucleotide positions. SNPs outside the ORF and in introns may
affect control/regulatory elements.
[0144] Thus nucleic acid molecules displaying genetic variations
that affect treatment provide a diagnostic target that can be used
to tailor treatment in an individual. Accordingly, the production
of recombinant cells and animals containing these polymorphisms
allow effective clinical design of treatment compounds and dosage
regimens.
[0145] The nucleic acid molecules are thus useful as antisense
constructs to control phospholipase gene expression in cells,
tissues, and organisms. A DNA antisense nucleic acid molecule is
designed to be complementary to a region of the gene involved in
transcription, preventing transcription and hence production of
phospholipase protein. An antisense RNA or DNA nucleic acid
molecule would hybridize to the mRNA and thus block translation of
mRNA into phospholipase protein.
[0146] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of phospholipase
nucleic acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired phospholipase nucleic acid
expression. This technique involves cleavage by means of ribozymes
containing nucleotide sequences complementary to one or more
regions in the mRNA that attenuate the ability of the mRNA to be
translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the phospholipase protein, such as
substrate binding.
[0147] The nucleic acid molecules also provide vectors for gene
therapy in patients containing cells that are aberrant in
phospholipase gene expression. Thus, recombinant cells, which
include the patient's cells that have been engineered ex vivo and
returned to the patient, are introduced into an individual where
the cells produce the desired phospholipase protein to treat the
individual.
[0148] The invention also encompasses kits for detecting the
presence of a phospholipase nucleic acid in a biological sample.
Experimental data as provided in FIG. 1 indicates that
phospholipase proteins of the present invention are expressed in
humans in pancreas adenocarcinomas, lung, brain anaplastic
oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, eye
retinoblastomas, and fetal brain. Specifically, a virtual northern
blot shows expression in pancreas adenocarcinomas, lung, brain
anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreas
adenocarcinomas, uterus endometrium, colon adenocarcinomas, and eye
retinoblastomas. In addition, PCR-based tissue screening panels
indicate expression in fetal brain. For example, the kit can
comprise reagents such as a labeled or labelable nucleic acid or
agent capable of detecting phospholipase nucleic acid in a
biological sample; means for determining the amount of
phospholipase nucleic acid in the sample; and means for comparing
the amount of phospholipase nucleic acid in the sample with 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 phospholipase protein mRNA or DNA.
[0149] Nucleic Acid Arrays
[0150] The present invention further provides nucleic acid
detection kits, such as arrays or microarrays of nucleic acid
molecules that are based on the sequence information provided in
FIGS. 1 and 3 (SEQ ID NOS:1 and 3).
[0151] As used herein "Arrays" or "Microarrays" refers to an array
of distinct polynucleotides or oligonucleotides synthesized on a
substrate, such as paper, nylon or other type of membrane, filter,
chip, glass slide, or any other suitable solid support. In one
embodiment, the microarray is prepared and used according to the
methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT
application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996;
Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc.
Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated
herein in their entirety by reference. In other embodiments, such
arrays are produced by the methods described by Brown et al., U.S.
Pat. No. 5,807,522.
[0152] The microarray or detection kit is preferably composed of a
large number of unique, single-stranded nucleic acid sequences,
usually either synthetic antisense oligonucleotides or fragments of
cDNAs, fixed to a solid support. The oligonucleotides are
preferably about 6-60 nucleotides in length, more preferably 15-30
nucleotides in length, and most preferably about 20-25 nucleotides
in length. For a certain type of microarray or detection kit, it
may be preferable to use oligonucleotides that are only 7-20
nucleotides in length. The microarray or detection kit may contain
oligonucleotides that cover the known 5', or 3', sequence,
sequential oligonucleotides which cover the full length sequence;
or unique oligonucleotides selected from particular areas along the
length of the sequence. Polynucleotides used in the microarray or
detection kit may be oligonucleotides that are specific to a gene
or genes of interest.
[0153] In order to produce oligonucleotides to a known sequence for
a microarray or detection kit, the gene(s) of interest (or an ORF
identified from the contigs of the present invention) is typically
examined using a computer algorithm which starts at the 5' or at
the 3' end of the nucleotide sequence. Typical algorithms will then
identify oligomers of defined length that are unique to the gene,
have a GC content within a range suitable for hybridization, and
lack predicted secondary structure that may interfere with
hybridization. In certain situations it may be appropriate to use
pairs of oligonucleotides on a microarray or detection kit. The
"pairs" will be identical, except for one nucleotide that
preferably is located in the center of the sequence. The second
oligonucleotide in the pair (mismatched by one) serves as a
control. The number of oligonucleotide pairs may range from two to
one million. The oligomers are synthesized at designated areas on a
substrate using a light-directed chemical process. The substrate
may be paper, nylon or other type of membrane, filter, chip, glass
slide or any other suitable solid support.
[0154] In another aspect, an oligonucleotide may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, as described in PCT
application WO95/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference. In another
aspect, a "gridded" array analogous to a dot (or slot) blot may be
used to arrange and link cDNA fragments or oligonucleotides to the
surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedures. An array, such as those
described above, may be produced by hand or by using available
devices (slot blot or dot blot apparatus), materials (any suitable
solid support), and machines (including robotic instruments), and
may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or
any other number between two and one million which lends itself to
the efficient use of commercially available instrumentation.
[0155] In order to conduct sample analysis using a microarray or
detection kit, the RNA or DNA from a biological sample is made into
hybridization probes. The mRNA is isolated, and cDNA is produced
and used as a template to make antisense RNA (aRNA). The aRNA is
amplified in the presence of fluorescent nucleotides, and labeled
probes are incubated with the microarray or detection kit so that
the probe sequences hybridize to complementary oligonucleotides of
the microarray or detection kit. Incubation conditions are adjusted
so that hybridization occurs with precise complementary matches or
with various degrees of less complementarity. After removal of
nonhybridized probes, a scanner is used to determine the levels and
patterns of fluorescence. The scanned images are examined to
determine degree of complementarity and the relative abundance of
each oligonucleotide sequence on the microarray or detection kit.
The biological samples may be obtained from any bodily fluids (such
as blood, urine, saliva, phlegm, gastric juices, etc.), cultured
cells, biopsies, or other tissue preparations. A detection system
may be used to measure the absence, presence, and amount of
hybridization for all of the distinct sequences simultaneously.
This data may be used for large-scale correlation studies on the
sequences, expression patterns, mutations, variants, or
polymorphisms among samples.
[0156] Using such arrays, the present invention provides methods to
identify the expression of the phospholipase proteins/peptides of
the present invention. In detail, such methods comprise incubating
a test sample with one or more nucleic acid molecules and assaying
for binding of the nucleic acid molecule with components within the
test sample. Such assays will typically involve arrays comprising
many genes, at least one of which is a gene of the present
invention and or alleles of the phospholipase gene of the present
invention. FIG. 3 provides information on SNPs that have been found
in the gene encoding the phospholipase proteins of the present
invention. SNPs were identified at 16 different nucleotide
positions. SNPs outside the ORF and in introns may affect
control/regulatory elements.
[0157] Conditions for incubating a nucleic acid molecule with a
test sample vary. Incubation conditions depend on the format
employed in the assay, the detection methods employed, and the type
and nature of the nucleic acid molecule used in the assay. One
skilled in the art will recognize that any one of the commonly
available hybridization, amplification or array assay formats can
readily be adapted to employ the novel fragments of the Human
genome disclosed herein. Examples of such assays can be found in
Chard, T, An Introduction to Radioimmunoassay and Related
Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands
(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,
Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3
(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
[0158] The test samples of the present invention include cells,
protein or membrane extracts of cells. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts used as
the sample to be assayed. Methods for preparing nucleic acid
extracts or of cells are well known in the art and can be readily
be adapted in order to obtain a sample that is compatible with the
system utilized.
[0159] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0160] Specifically, the invention provides a compartmentalized kit
to receive, in close confinement, one or more containers which
comprises: (a) a first container comprising one of the nucleic acid
molecules that can bind to a fragment of the Human genome disclosed
herein; and (b) one or more other containers comprising one or more
of the following: wash reagents, reagents capable of detecting
presence of a bound nucleic acid.
[0161] In detail, a compartmentalized kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers, strips of
plastic, glass or paper, or arraying material such as silica. Such
containers allows one to efficiently transfer reagents from one
compartment to another compartment such that the samples and
reagents are not cross-contaminated, and the agents or solutions of
each container can be added in a quantitative fashion from one
compartment to another. Such containers will include a container
which will accept the test sample, a container which contains the
nucleic acid probe, containers which contain wash reagents (such as
phosphate buffered saline, Tris-buffers, etc.), and containers
which contain the reagents used to detect the bound probe. One
skilled in the art will readily recognize that the previously
unidentified phospholipase gene of the present invention can be
routinely identified using the sequence information disclosed
herein can be readily incorporated into one of the established kit
formats which are well known in the art, particularly expression
arrays.
[0162] Vectors/host cells
[0163] The invention also provides vectors containing the nucleic
acid molecules described herein. The term "vector" refers to a
vehicle, preferably a nucleic acid molecule, which can transport
the nucleic acid molecules. When the vector is a nucleic acid
molecule, the nucleic acid molecules are covalently linked to the
vector nucleic acid. With this aspect of the invention, the vector
includes a plasmid, single or double stranded phage, a single or
double stranded RNA or DNA viral vector, or artificial chromosome,
such as a BAC, PAC, YAC, OR MAC.
[0164] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the nucleic acid molecules. Alternatively, the
vector may integrate into the host cell genome and produce
additional copies of the nucleic acid molecules when the host cell
replicates.
[0165] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
nucleic acid molecules. The vectors can function in prokaryotic or
eukaryotic cells or in both (shuttle vectors).
[0166] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the nucleic acid
molecules such that transcription of the nucleic acid molecules is
allowed in a host cell. The nucleic acid molecules can be
introduced into the host cell with a separate nucleic acid molecule
capable of affecting transcription. Thus, the second nucleic acid
molecule may provide a trans-acting factor interacting with the
cis-regulatory control region to allow transcription of the nucleic
acid molecules from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself. It is understood,
however, that in some embodiments, transcription and/or translation
of the nucleic acid molecules can occur in a cell-free system.
[0167] The regulatory sequence to which the nucleic acid molecules
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lambda., the lac, TRP,
and TAC promoters from E. coli, the early and late promoters from
SV40, the CMV immediate early promoter, the adenovirus early and
late promoters, and retrovirus long-terminal repeats.
[0168] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0169] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al., Molecular
Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1989).
[0170] A variety of expression vectors can be used to express a
nucleic acid molecule. Such vectors include chromosomal, episomal,
and virus-derived vectors, for example vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, including yeast artificial chromosomes,
from viruses such as baculoviruses, papovaviruses such as SV40,
Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses,
and retroviruses. Vectors may also be derived from combinations of
these sources such as those derived from plasmid and bacteriophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning
and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1989).
[0171] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e. tissue specific) or may provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0172] The nucleic acid molecules can be inserted into the vector
nucleic acid by well-known methodology. Generally, the DNA sequence
that will ultimately be expressed is joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known to those of ordinary skill in the art.
[0173] The vector containing the appropriate nucleic acid molecule
can be introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[0174] As described herein, it may be desirable to express the
peptide as a fusion protein. Accordingly, the invention provides
fusion vectors that allow for the production of the peptides.
Fusion vectors can increase the expression of a recombinant
protein, increase the solubility of the recombinant protein, and
aid in the purification of the protein by acting for example as a
ligand for affinity purification. A proteolytic cleavage site may
be introduced at the junction of the fusion moiety so that the
desired peptide can ultimately be separated from the fusion moiety.
Proteolytic enzymes include, but are not limited to, factor Xa,
thrombin, and enterophospholipase. Typical fusion expression
vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), 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. Examples of suitable inducible
non-fusion E. coli expression vectors include pTrc (Amann et al.,
Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene
Expression Technology: Methods in Enzymology 185:60-89 (1990)).
[0175] Recombinant protein expression can be maximized in host
bacteria by providing a genetic background wherein the host cell
has 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).
Alternatively, the sequence of the nucleic acid molecule of
interest can be altered to provide preferential codon usage for a
specific host cell, for example E. coli. (Wada et al., Nucleic
Acids Res. 20:2111-2118 (1992)).
[0176] The nucleic acid molecules can also be expressed by
expression vectors that are operative in yeast. Examples of vectors
for expression in yeast e.g., S. cerevisiae include pYepSec1
(Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al.,
Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0177] The nucleic acid molecules can also be expressed in insect
cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL
series (Lucklow et al., Virology 170:31-39 (1989)).
[0178] In certain embodiments of the invention, the nucleic acid
molecules described herein are expressed in mammalian cells using
mammalian expression vectors. Examples of mammalian expression
vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC
(Kaufman et al., EMBO J. 6:187-195 (1987)).
[0179] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
nucleic acid molecules. The person of ordinary skill in the art
would be aware of other vectors suitable for maintenance
propagation or expression of the nucleic acid molecules described
herein. These are found for example 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.
[0180] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the nucleic
acid molecule sequences described herein, including both coding and
non-coding regions. Expression of this antisense RNA is subject to
each of the parameters described above in relation to expression of
the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0181] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0182] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook,
et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989).
[0183] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the nucleic acid molecules can be introduced
either alone or with other nucleic acid molecules that are not
related to the nucleic acid molecules such as those providing
trans-acting factors for expression vectors. When more than one
vector is introduced into a cell, the vectors can be introduced
independently, co-introduced or joined to the nucleic acid molecule
vector.
[0184] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0185] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the nucleic acid molecules described
herein or may be on a separate vector. Markers include tetracycline
or ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0186] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0187] Where secretion of the peptide is desired, which is
difficult to achieve with multi-transmembrane domain containing
proteins such as phospholipases, appropriate secretion signals are
incorporated into the vector. The signal sequence can be endogenous
to the peptides or heterologous to these peptides.
[0188] Where the peptide is not secreted into the medium, which is
typically the case with phospholipases, the protein can be isolated
from the host cell by standard disruption procedures, including
freeze thaw, sonication, mechanical disruption, use of lysing
agents and the like. The peptide can then be recovered and purified
by well-known purification methods including ammonium sulfate
precipitation, acid extraction, anion or cationic exchange
chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0189] It is also understood that depending upon the host cell in
recombinant production of the peptides described herein, the
peptides can have various glycosylation patterns, depending upon
the cell, or maybe non-glycosylated as when produced in bacteria.
In addition, the peptides may include an initial modified
methionine in some cases as a result of a host-mediated
process.
[0190] Uses of vectors and host cells
[0191] The recombinant host cells expressing the peptides described
herein have a variety of uses. First, the cells are useful for
producing a phospholipase protein or peptide that can be further
purified to produce desired amounts of phospholipase protein or
fragments. Thus, host cells containing expression vectors are
useful for peptide production.
[0192] Host cells are also useful for conducting cell-based assays
involving the phospholipase protein or phospholipase protein
fragments, such as those described above as well as other formats
known in the art. Thus, a recombinant host cell expressing a native
phospholipase protein is useful for assaying compounds that
stimulate or inhibit phospholipase protein function.
[0193] Host cells are also useful for identifying phospholipase
protein mutants in which these functions are affected. If the
mutants naturally occur and give rise to a pathology, host cells
containing the mutations are useful to assay compounds that have a
desired effect on the mutant phospholipase protein (for example,
stimulating or inhibiting function) which may not be indicated by
their effect on the native phospholipase protein.
[0194] Genetically engineered host cells can be further used to
produce non-human transgenic animals. A transgenic animal is
preferably a mammal, for example a rodent, such as a rat or mouse,
in which one or more of the cells of the animal include a
transgene. A transgene is exogenous DNA which is integrated into
the genome of a cell from which a transgenic animal develops and
which remains in the genome of the mature animal in one or more
cell types or tissues of the transgenic animal. These animals are
useful for studying the function of a phospholipase protein and
identifying and evaluating modulators of phospholipase protein
activity. Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, and amphibians.
[0195] A transgenic animal can be produced by introducing nucleic
acid into the male pronuclei of a fertilized oocyte, e.g., by
microinjection, retroviral infection, and allowing the oocyte to
develop in a pseudopregnant female foster animal. Any of the
phospholipase protein nucleotide sequences can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[0196] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the
phospholipase protein to particular cells.
[0197] Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described, for example,
in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of the transgene
in its genome and/or expression of transgenic 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 can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0198] In another embodiment, transgenic non-human animals can be
produced which contain selected systems that allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS
89:6232-6236 (1992). Another example of a recombinase system is the
FLP recombinase system of S. cerevisiae (O'Gorman et al. Science
251:1351-1355 (1991). If a cre/loxP recombinase system is used to
regulate expression of the transgene, animals containing transgenes
encoding both the Cre recombinase and a selected protein is
required. Such animals can be provided through the construction of
"double" transgenic animals, e.g., by mating two transgenic
animals, one containing a transgene encoding a selected protein and
the other containing a transgene encoding a recombinase.
[0199] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. Nature 385:810-813 (1997) and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter G.sub.0 phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyst and then transferred to pseudopregnant female
foster animal. The offspring born of this female foster animal will
be a clone of the animal from which the cell, e.g., the somatic
cell, is isolated.
[0200] Transgenic animals containing recombinant cells that express
the peptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
effect substrate binding, phospholipase protein activation, and
signal transduction, may not be evident from in vitro cell-free or
cell-based assays. Accordingly, it is useful to provide non-human
transgenic animals to assay in vivo phospholipase protein function,
including substrate interaction, the effect of specific mutant
phospholipase proteins on phospholipase protein function and
substrate interaction, and the effect of chimeric phospholipase
proteins. It is also possible to assess the effect of null
mutations, that is, mutations that substantially or completely
eliminate one or more phospholipase protein functions.
[0201] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the above-described modes for carrying out
the invention which are obvious to those skilled in the field of
molecular biology or related fields are intended to be within the
scope of the following claims.
Sequence CWU 1
1
5 1 2211 PRT Human 1 Ala Thr Gly Gly Gly Cys Cys Thr Gly Ala Cys
Gly Gly Ala Gly Gly 1 5 10 15 Ala Cys Gly Ala Gly Gly Ala Cys Gly
Thr Gly Cys Gly Cys Gly Cys 20 25 30 Cys Ala Thr Gly Cys Thr Gly
Cys Gly Gly Gly Gly Cys Thr Cys Cys 35 40 45 Cys Gly Gly Cys Thr
Cys Cys Gly Cys Ala Ala Gly Ala Thr Cys Cys 50 55 60 Gly Cys Thr
Cys Gly Cys Gly Cys Ala Cys Gly Thr Gly Gly Cys Ala 65 70 75 80 Cys
Ala Ala Gly Gly Ala Gly Cys Gly Gly Cys Thr Gly Thr Ala Cys 85 90
95 Cys Gly Gly Cys Thr Gly Cys Ala Gly Gly Ala Gly Gly Ala Cys Gly
100 105 110 Gly Cys Cys Thr Gly Ala Gly Cys Gly Thr Gly Thr Gly Gly
Thr Thr 115 120 125 Cys Cys Ala Gly Cys Gly Gly Cys Gly Cys Ala Thr
Cys Cys Cys Gly 130 135 140 Cys Gly Thr Gly Cys Gly Cys Cys Ala Thr
Cys Gly Cys Ala Gly Cys 145 150 155 160 Ala Cys Ala Thr Cys Thr Thr
Cys Thr Thr Cys Gly Thr Gly Cys Ala 165 170 175 Gly Cys Ala Cys Ala
Thr Cys Gly Ala Gly Gly Cys Gly Gly Thr Cys 180 185 190 Cys Gly Cys
Gly Ala Gly Gly Gly Cys Cys Ala Cys Cys Ala Gly Thr 195 200 205 Cys
Cys Gly Ala Gly Gly Gly Cys Cys Thr Gly Cys Gly Gly Cys Gly 210 215
220 Cys Thr Thr Cys Gly Gly Gly Gly Gly Thr Gly Cys Cys Thr Thr Cys
225 230 235 240 Gly Cys Gly Cys Cys Ala Gly Cys Gly Cys Gly Cys Thr
Gly Cys Cys 245 250 255 Thr Cys Ala Cys Cys Ala Thr Cys Gly Cys Cys
Thr Thr Cys Ala Ala 260 265 270 Gly Gly Gly Cys Cys Gly Cys Cys Gly
Cys Ala Ala Gly Ala Ala Cys 275 280 285 Cys Thr Gly Gly Ala Cys Cys
Thr Gly Gly Cys Gly Gly Cys Gly Cys 290 295 300 Cys Cys Ala Cys Gly
Gly Cys Thr Gly Ala Gly Gly Ala Ala Gly Cys 305 310 315 320 Gly Cys
Ala Gly Cys Gly Cys Thr Gly Gly Gly Thr Gly Cys Gly Cys 325 330 335
Gly Gly Thr Cys Thr Gly Ala Cys Cys Ala Ala Gly Cys Thr Cys Cys 340
345 350 Gly Cys Gly Cys Gly Cys Gly Cys Cys Thr Gly Gly Ala Cys Gly
Cys 355 360 365 Cys Ala Thr Gly Ala Gly Cys Cys Ala Gly Cys Gly Cys
Gly Ala Gly 370 375 380 Cys Gly Gly Cys Thr Ala Gly Ala Cys Cys Ala
Cys Thr Gly Gly Ala 385 390 395 400 Thr Cys Cys Ala Cys Thr Cys Cys
Thr Ala Thr Cys Thr Gly Cys Ala 405 410 415 Cys Cys Gly Gly Gly Cys
Thr Gly Ala Cys Thr Cys Cys Ala Ala Cys 420 425 430 Cys Ala Gly Gly
Ala Cys Ala Gly Cys Ala Ala Gly Ala Thr Gly Ala 435 440 445 Gly Cys
Thr Thr Cys Ala Ala Gly Gly Ala Gly Ala Thr Cys Ala Ala 450 455 460
Gly Ala Gly Cys Cys Thr Gly Cys Thr Gly Ala Gly Ala Ala Thr Gly 465
470 475 480 Gly Thr Cys Ala Ala Cys Gly Thr Gly Gly Ala Cys Ala Thr
Gly Ala 485 490 495 Ala Cys Gly Ala Cys Ala Thr Gly Thr Ala Cys Gly
Cys Cys Thr Ala 500 505 510 Cys Cys Thr Cys Cys Thr Cys Thr Thr Cys
Ala Ala Gly Gly Ala Gly 515 520 525 Thr Gly Thr Gly Ala Cys Cys Ala
Cys Thr Cys Cys Ala Ala Cys Ala 530 535 540 Ala Cys Gly Ala Cys Cys
Gly Thr Cys Thr Ala Gly Ala Gly Gly Gly 545 550 555 560 Gly Gly Cys
Thr Gly Ala Gly Ala Thr Cys Gly Ala Gly Gly Ala Gly 565 570 575 Thr
Thr Cys Cys Thr Gly Cys Gly Gly Cys Gly Gly Cys Thr Gly Cys 580 585
590 Thr Gly Ala Ala Gly Cys Gly Gly Cys Cys Gly Gly Ala Gly Cys Thr
595 600 605 Gly Gly Ala Gly Gly Ala Gly Ala Thr Cys Thr Thr Cys Cys
Ala Thr 610 615 620 Cys Ala Gly Thr Ala Cys Thr Cys Gly Gly Gly Cys
Gly Ala Gly Gly 625 630 635 640 Ala Cys Cys Gly Cys Gly Thr Gly Cys
Thr Gly Ala Gly Thr Gly Cys 645 650 655 Cys Cys Cys Thr Gly Ala Gly
Cys Thr Gly Cys Thr Gly Gly Ala Gly 660 665 670 Thr Thr Cys Cys Thr
Gly Gly Ala Gly Gly Ala Cys Cys Ala Gly Gly 675 680 685 Gly Cys Gly
Ala Gly Gly Ala Gly Gly Gly Cys Gly Cys Cys Ala Cys 690 695 700 Ala
Cys Thr Gly Gly Cys Cys Cys Gly Cys Gly Cys Cys Cys Ala Gly 705 710
715 720 Cys Ala Gly Cys Thr Cys Ala Thr Thr Cys Ala Gly Ala Cys Cys
Thr 725 730 735 Ala Thr Gly Ala Gly Cys Thr Cys Ala Ala Cys Gly Ala
Gly Ala Cys 740 745 750 Ala Gly Cys Cys Ala Ala Gly Cys Ala Gly Cys
Ala Thr Gly Ala Gly 755 760 765 Cys Thr Gly Ala Thr Gly Ala Cys Ala
Cys Thr Gly Gly Ala Thr Gly 770 775 780 Gly Cys Thr Thr Cys Ala Thr
Gly Ala Thr Gly Thr Ala Cys Cys Thr 785 790 795 800 Gly Thr Thr Gly
Thr Cys Gly Cys Cys Gly Gly Ala Gly Gly Gly Gly 805 810 815 Ala Cys
Thr Gly Cys Cys Thr Thr Gly Gly Ala Cys Ala Ala Cys Ala 820 825 830
Cys Cys Cys Ala Cys Ala Cys Gly Thr Gly Thr Gly Thr Gly Thr Thr 835
840 845 Cys Cys Ala Gly Gly Ala Cys Ala Thr Gly Ala Ala Cys Cys Ala
Gly 850 855 860 Cys Cys Cys Cys Thr Thr Gly Cys Cys Cys Ala Cys Thr
Ala Cys Thr 865 870 875 880 Thr Cys Ala Thr Cys Thr Cys Thr Thr Cys
Cys Thr Cys Cys Cys Ala 885 890 895 Cys Ala Ala Cys Ala Cys Cys Thr
Ala Thr Cys Thr Gly Ala Cys Thr 900 905 910 Gly Ala Cys Thr Cys Cys
Cys Ala Gly Ala Thr Cys Gly Gly Gly Gly 915 920 925 Gly Gly Cys Cys
Cys Ala Gly Cys Ala Gly Cys Ala Cys Cys Gly Ala 930 935 940 Gly Gly
Cys Cys Thr Ala Thr Gly Thr Thr Ala Gly Gly Gly Cys Cys 945 950 955
960 Thr Thr Thr Gly Cys Cys Cys Ala Gly Gly Gly Ala Thr Gly Cys Cys
965 970 975 Gly Cys Thr Gly Cys Gly Thr Gly Gly Ala Gly Cys Thr Gly
Gly Ala 980 985 990 Cys Thr Gly Cys Thr Gly Gly Gly Ala Gly Gly Gly
Gly Cys Cys Ala 995 1000 1005 Gly Gly Ala Gly Gly Gly Gly Ala Gly
Cys Cys Cys Gly Thr Cys Ala 1010 1015 1020 Thr Cys Thr Ala Thr Cys
Ala Thr Gly Gly Cys Cys Ala Thr Ala Cys 1025 1030 1035 1040 Cys Cys
Thr Cys Ala Cys Cys Thr Cys Cys Ala Ala Gly Ala Thr Thr 1045 1050
1055 Cys Thr Cys Thr Thr Cys Cys Gly Gly Gly Ala Cys Gly Thr Gly
Gly 1060 1065 1070 Cys Cys Cys Ala Ala Gly Cys Cys Gly Thr Gly Cys
Gly Cys Gly Ala 1075 1080 1085 Cys Cys Ala Thr Gly Cys Cys Thr Thr
Cys Ala Cys Gly Cys Thr Gly 1090 1095 1100 Thr Cys Cys Cys Cys Thr
Thr Ala Cys Cys Cys Thr Gly Thr Cys Ala 1105 1110 1115 1120 Thr Cys
Cys Thr Ala Thr Cys Cys Cys Thr Gly Gly Ala Gly Ala Ala 1125 1130
1135 Cys Cys Ala Cys Thr Gly Cys Gly Gly Gly Cys Thr Gly Gly Ala
Gly 1140 1145 1150 Cys Ala Gly Cys Ala Gly Gly Cys Thr Gly Cys Cys
Ala Thr Gly Gly 1155 1160 1165 Cys Cys Cys Gly Cys Cys Ala Cys Cys
Thr Cys Thr Gly Cys Ala Cys 1170 1175 1180 Cys Ala Thr Cys Cys Thr
Gly Gly Gly Gly Gly Ala Cys Ala Thr Gly 1185 1190 1195 1200 Cys Thr
Gly Gly Thr Gly Ala Cys Ala Cys Ala Gly Gly Cys Gly Cys 1205 1210
1215 Thr Gly Gly Ala Cys Thr Cys Cys Cys Cys Ala Ala Ala Thr Cys
Cys 1220 1225 1230 Cys Gly Ala Gly Gly Ala Gly Cys Thr Gly Cys Cys
Ala Thr Cys Cys 1235 1240 1245 Cys Cys Ala Gly Ala Gly Cys Ala Gly
Cys Thr Gly Ala Ala Gly Gly 1250 1255 1260 Gly Cys Cys Gly Gly Gly
Thr Cys Cys Thr Gly Gly Thr Gly Ala Ala 1265 1270 1275 1280 Gly Gly
Gly Ala Ala Ala Gly Ala Ala Gly Cys Thr Gly Cys Cys Cys 1285 1290
1295 Gly Cys Thr Gly Cys Thr Cys Gly Gly Ala Gly Cys Gly Ala Gly
Gly 1300 1305 1310 Ala Thr Gly Gly Cys Cys Gly Gly Gly Cys Thr Cys
Thr Gly Thr Cys 1315 1320 1325 Gly Gly Ala Thr Cys Gly Gly Gly Ala
Gly Gly Ala Gly Gly Gly Gly 1330 1335 1340 Gly Ala Gly Gly Ala Gly
Gly Ala Thr Gly Ala Cys Gly Ala Gly Gly 1345 1350 1355 1360 Ala Gly
Gly Ala Ala Gly Ala Ala Gly Ala Gly Gly Ala Gly Gly Thr 1365 1370
1375 Gly Gly Ala Gly Gly Cys Thr Gly Cys Ala Gly Cys Gly Cys Ala
Gly 1380 1385 1390 Ala Gly Gly Cys Gly Gly Cys Thr Gly Gly Cys Cys
Ala Ala Gly Cys 1395 1400 1405 Ala Gly Ala Thr Cys Thr Cys Cys Cys
Cys Gly Gly Ala Gly Cys Thr 1410 1415 1420 Gly Thr Cys Gly Gly Cys
Cys Cys Thr Gly Gly Cys Thr Gly Thr Gly 1425 1430 1435 1440 Thr Ala
Cys Thr Gly Cys Cys Ala Cys Gly Cys Cys Ala Cys Cys Cys 1445 1450
1455 Gly Cys Cys Thr Gly Cys Gly Gly Ala Cys Cys Cys Thr Gly Cys
Ala 1460 1465 1470 Cys Cys Cys Thr Gly Cys Cys Cys Cys Cys Ala Ala
Cys Gly Cys Cys 1475 1480 1485 Cys Cys Ala Cys Ala Ala Cys Cys Cys
Thr Gly Cys Cys Ala Gly Gly 1490 1495 1500 Thr Cys Ala Gly Cys Thr
Cys Cys Cys Thr Cys Ala Gly Cys Gly Ala 1505 1510 1515 1520 Gly Cys
Gly Cys Ala Ala Ala Gly Cys Cys Ala Ala Gly Ala Ala Ala 1525 1530
1535 Cys Thr Cys Ala Thr Thr Cys Gly Gly Gly Ala Gly Gly Cys Ala
Gly 1540 1545 1550 Gly Gly Ala Ala Cys Ala Gly Cys Thr Thr Thr Gly
Thr Cys Ala Gly 1555 1560 1565 Gly Cys Ala Cys Ala Ala Thr Gly Cys
Cys Cys Gly Cys Cys Ala Gly 1570 1575 1580 Cys Thr Gly Ala Cys Cys
Cys Gly Cys Gly Thr Gly Thr Ala Cys Cys 1585 1590 1595 1600 Cys Gly
Cys Thr Gly Gly Gly Gly Cys Thr Gly Cys Gly Gly Ala Thr 1605 1610
1615 Gly Ala Ala Cys Thr Cys Ala Gly Cys Cys Ala Ala Cys Thr Ala
Cys 1620 1625 1630 Ala Gly Thr Cys Cys Cys Cys Ala Gly Gly Ala Gly
Ala Thr Gly Thr 1635 1640 1645 Gly Gly Ala Ala Cys Thr Cys Gly Gly
Gly Cys Thr Gly Thr Cys Ala 1650 1655 1660 Gly Cys Thr Gly Gly Thr
Gly Gly Cys Cys Thr Thr Gly Ala Ala Cys 1665 1670 1675 1680 Thr Thr
Cys Cys Ala Gly Ala Cys Gly Cys Cys Ala Gly Gly Cys Thr 1685 1690
1695 Ala Cys Gly Ala Gly Ala Thr Gly Gly Ala Cys Cys Thr Cys Ala
Ala 1700 1705 1710 Thr Gly Cys Cys Gly Gly Gly Cys Gly Cys Thr Thr
Cys Cys Thr Ala 1715 1720 1725 Gly Thr Cys Ala Ala Thr Gly Gly Gly
Cys Ala Gly Thr Gly Thr Gly 1730 1735 1740 Gly Cys Thr Ala Cys Gly
Thr Cys Cys Thr Ala Ala Ala Ala Cys Cys 1745 1750 1755 1760 Thr Gly
Cys Cys Thr Gly Cys Cys Thr Gly Cys Gly Gly Cys Ala Ala 1765 1770
1775 Cys Cys Thr Gly Ala Cys Thr Cys Gly Ala Cys Cys Thr Thr Thr
Gly 1780 1785 1790 Ala Cys Cys Cys Cys Gly Ala Gly Thr Ala Cys Cys
Cys Ala Gly Gly 1795 1800 1805 Ala Cys Cys Thr Cys Cys Cys Ala Gly
Ala Ala Cys Cys Ala Cys Thr 1810 1815 1820 Cys Thr Cys Ala Gly Cys
Ala Thr Cys Cys Ala Gly Gly Thr Gly Cys 1825 1830 1835 1840 Thr Gly
Ala Cys Thr Gly Cys Ala Cys Ala Gly Cys Ala Gly Cys Thr 1845 1850
1855 Gly Cys Cys Cys Ala Ala Gly Cys Thr Gly Ala Ala Thr Gly Cys
Cys 1860 1865 1870 Gly Ala Gly Ala Ala Gly Cys Cys Ala Cys Ala Cys
Thr Cys Cys Ala 1875 1880 1885 Thr Thr Gly Thr Gly Gly Ala Cys Cys
Cys Cys Cys Thr Gly Gly Thr 1890 1895 1900 Gly Cys Gly Cys Ala Thr
Thr Gly Ala Gly Ala Thr Cys Cys Ala Thr 1905 1910 1915 1920 Gly Gly
Gly Gly Thr Gly Cys Cys Cys Gly Cys Ala Gly Ala Cys Thr 1925 1930
1935 Gly Thr Gly Cys Cys Cys Gly Gly Cys Ala Gly Gly Ala Gly Ala
Cys 1940 1945 1950 Thr Gly Ala Cys Thr Ala Cys Gly Thr Gly Cys Thr
Cys Ala Ala Cys 1955 1960 1965 Ala Ala Thr Gly Gly Cys Thr Thr Cys
Ala Ala Cys Cys Cys Cys Cys 1970 1975 1980 Gly Cys Thr Gly Gly Gly
Gly Gly Cys Ala Gly Ala Cys Cys Cys Thr 1985 1990 1995 2000 Gly Cys
Ala Gly Thr Thr Cys Cys Ala Gly Cys Thr Gly Cys Gly Gly 2005 2010
2015 Gly Cys Thr Cys Cys Gly Gly Ala Gly Cys Thr Gly Gly Cys Ala
Cys 2020 2025 2030 Thr Gly Gly Thr Cys Cys Gly Gly Thr Thr Thr Gly
Thr Gly Gly Thr 2035 2040 2045 Gly Gly Ala Ala Gly Ala Thr Thr Ala
Thr Gly Ala Cys Gly Cys Cys 2050 2055 2060 Ala Cys Cys Thr Cys Cys
Cys Cys Cys Ala Ala Thr Gly Ala Cys Thr 2065 2070 2075 2080 Thr Thr
Gly Thr Gly Gly Gly Cys Cys Ala Gly Thr Thr Thr Ala Cys 2085 2090
2095 Ala Cys Thr Gly Cys Cys Thr Cys Thr Thr Ala Gly Cys Ala Gly
Cys 2100 2105 2110 Cys Thr Ala Ala Ala Gly Cys Ala Ala Gly Gly Gly
Thr Ala Cys Cys 2115 2120 2125 Gly Cys Cys Ala Cys Ala Thr Ala Cys
Ala Cys Cys Thr Gly Cys Thr 2130 2135 2140 Thr Thr Cys Cys Ala Ala
Gly Gly Ala Cys Gly Gly Gly Gly Cys Cys 2145 2150 2155 2160 Thr Cys
Ala Cys Thr Gly Thr Cys Ala Cys Cys Ala Gly Cys Cys Ala 2165 2170
2175 Cys Gly Cys Thr Cys Thr Thr Cys Ala Thr Cys Cys Ala Ala Ala
Thr 2180 2185 2190 Cys Cys Gly Cys Ala Thr Cys Cys Ala Gly Cys Gly
Cys Thr Cys Cys 2195 2200 2205 Thr Gly Ala 2210 2 736 PRT Human 2
Met Gly Leu Thr Glu Asp Glu Asp Val Arg Ala Met Leu Arg Gly Ser 1 5
10 15 Arg Leu Arg Lys Ile Arg Ser Arg Thr Trp His Lys Glu Arg Leu
Tyr 20 25 30 Arg Leu Gln Glu Asp Gly Leu Ser Val Trp Phe Gln Arg
Arg Ile Pro 35 40 45 Arg Ala Pro Ser Gln His Ile Phe Phe Val Gln
His Ile Glu Ala Val 50 55 60 Arg Glu Gly His Gln Ser Glu Gly Leu
Arg Arg Phe Gly Gly Ala Phe 65 70 75 80 Ala Pro Ala Arg Cys Leu Thr
Ile Ala Phe Lys Gly Arg Arg Lys Asn 85 90 95 Leu Asp Leu Ala Ala
Pro Thr Ala Glu Glu Ala Gln Arg Trp Val Arg 100 105 110 Gly Leu Thr
Lys Leu Arg Ala Arg Leu Asp Ala Met Ser Gln Arg Glu 115 120 125 Arg
Leu Asp His Trp Ile His Ser Tyr Leu His Arg Ala Asp Ser Asn 130 135
140 Gln Asp Ser Lys Met Ser Phe Lys Glu Ile Lys Ser Leu Leu Arg Met
145 150 155 160 Val Asn Val Asp Met Asn Asp Met Tyr Ala Tyr Leu Leu
Phe Lys Glu 165 170 175 Cys Asp His Ser Asn Asn Asp Arg Leu Glu Gly
Ala Glu Ile Glu Glu 180 185 190 Phe Leu Arg Arg Leu Leu Lys Arg Pro
Glu Leu Glu Glu Ile Phe His 195 200 205 Gln Tyr Ser Gly Glu Asp Arg
Val Leu Ser Ala
Pro Glu Leu Leu Glu 210 215 220 Phe Leu Glu Asp Gln Gly Glu Glu Gly
Ala Thr Leu Ala Arg Ala Gln 225 230 235 240 Gln Leu Ile Gln Thr Tyr
Glu Leu Asn Glu Thr Ala Lys Gln His Glu 245 250 255 Leu Met Thr Leu
Asp Gly Phe Met Met Tyr Leu Leu Ser Pro Glu Gly 260 265 270 Thr Ala
Leu Asp Asn Thr His Thr Cys Val Phe Gln Asp Met Asn Gln 275 280 285
Pro Leu Ala His Tyr Phe Ile Ser Ser Ser His Asn Thr Tyr Leu Thr 290
295 300 Asp Ser Gln Ile Gly Gly Pro Ser Ser Thr Glu Ala Tyr Val Arg
Ala 305 310 315 320 Phe Ala Gln Gly Cys Arg Cys Val Glu Leu Asp Cys
Trp Glu Gly Pro 325 330 335 Gly Gly Glu Pro Val Ile Tyr His Gly His
Thr Leu Thr Ser Lys Ile 340 345 350 Leu Phe Arg Asp Val Ala Gln Ala
Val Arg Asp His Ala Phe Thr Leu 355 360 365 Ser Pro Tyr Pro Val Ile
Leu Ser Leu Glu Asn His Cys Gly Leu Glu 370 375 380 Gln Gln Ala Ala
Met Ala Arg His Leu Cys Thr Ile Leu Gly Asp Met 385 390 395 400 Leu
Val Thr Gln Ala Leu Asp Ser Pro Asn Pro Glu Glu Leu Pro Ser 405 410
415 Pro Glu Gln Leu Lys Gly Arg Val Leu Val Lys Gly Lys Lys Leu Pro
420 425 430 Ala Ala Arg Ser Glu Asp Gly Arg Ala Leu Ser Asp Arg Glu
Glu Gly 435 440 445 Glu Glu Asp Asp Glu Glu Glu Glu Glu Glu Val Glu
Ala Ala Ala Gln 450 455 460 Arg Arg Leu Ala Lys Gln Ile Ser Pro Glu
Leu Ser Ala Leu Ala Val 465 470 475 480 Tyr Cys His Ala Thr Arg Leu
Arg Thr Leu His Pro Ala Pro Asn Ala 485 490 495 Pro Gln Pro Cys Gln
Val Ser Ser Leu Ser Glu Arg Lys Ala Lys Lys 500 505 510 Leu Ile Arg
Glu Ala Gly Asn Ser Phe Val Arg His Asn Ala Arg Gln 515 520 525 Leu
Thr Arg Val Tyr Pro Leu Gly Leu Arg Met Asn Ser Ala Asn Tyr 530 535
540 Ser Pro Gln Glu Met Trp Asn Ser Gly Cys Gln Leu Val Ala Leu Asn
545 550 555 560 Phe Gln Thr Pro Gly Tyr Glu Met Asp Leu Asn Ala Gly
Arg Phe Leu 565 570 575 Val Asn Gly Gln Cys Gly Tyr Val Leu Lys Pro
Ala Cys Leu Arg Gln 580 585 590 Pro Asp Ser Thr Phe Asp Pro Glu Tyr
Pro Gly Pro Pro Arg Thr Thr 595 600 605 Leu Ser Ile Gln Val Leu Thr
Ala Gln Gln Leu Pro Lys Leu Asn Ala 610 615 620 Glu Lys Pro His Ser
Ile Val Asp Pro Leu Val Arg Ile Glu Ile His 625 630 635 640 Gly Val
Pro Ala Asp Cys Ala Arg Gln Glu Thr Asp Tyr Val Leu Asn 645 650 655
Asn Gly Phe Asn Pro Arg Trp Gly Gln Thr Leu Gln Phe Gln Leu Arg 660
665 670 Ala Pro Glu Leu Ala Leu Val Arg Phe Val Val Glu Asp Tyr Asp
Ala 675 680 685 Thr Ser Pro Asn Asp Phe Val Gly Gln Phe Thr Leu Pro
Leu Ser Ser 690 695 700 Leu Lys Gln Gly Tyr Arg His Ile His Leu Leu
Ser Lys Asp Gly Ala 705 710 715 720 Ser Leu Ser Pro Ala Thr Leu Phe
Ile Gln Ile Arg Ile Gln Arg Ser 725 730 735 3 13953 DNA Human 3
ttcccccaac aggttcctgg ggagctgagg gtaaggggcc agaagtaggg gccccctcca
60 ttgggagcct gcccaagtgg gggcccagtc aggcgttcag gctgcctgtg
ttgatggcct 120 gtttgcagaa ggctctggac agagcaggcc ccggtgactc
acccccattt ccggccttag 180 tcactcatct cccccacacc catgggcttg
gcatcagagg gctggcctag gtaggggagg 240 cagccccagc tgcagagcct
ggttgtcatg gcaatgagca gtcccccttc ccatggccaa 300 gggggtgtag
gaggatgtgc ctaagtgtag tggcgctgca ggctcccctc tgccctcacc 360
gtgtcattgc tcaggatggg accatttaga ggctacctgg acctggtggg gggggggggt
420 ctggtgagcc cctcggcctg gcacgtgccg tgtacaccca gctctttgtc
tggaaagagg 480 agggttccac tttccccttc ctgaccaggg ggtaccgagt
gggcattcac tcagagggcc 540 acagtctaga ccctgaggga ccctctggag
ctgttagggc cacaggctgg ccccaagggg 600 tcatggcctc ttaaggaggt
ggatttctcc tcttgcccct gagaccctga ttttcagtca 660 tggtcaaagt
gggttttaaa aatacagatg cccaggcccc atcccacacc agctgggtga 720
gatgccctgg gctgtggtgt ctgcagcctc ccctgggtaa ttccgatgca cagctaaggt
780 tgagaaaccc tgacctgcga ggcctctgcc ctgcgaggcc tctgccctga
actctggaca 840 gatggggcag agctggggat agaattcact tagtcccccc
atctgtccgt ctacctgtct 900 gccccttcag tcaacactag ctggtatcct
ggctctggga tgagcactgt ggtgaggaga 960 taagacctca ttccgccctc
agatttatta aagtatatag gtgggttttg tgttttattt 1020 taattaaagc
aaaaagtgct gttagtggtc agagagggag aatgtccact tgaggacaaa 1080
tcaaggaagg gttcctggag gaggtgatat ctgaactggt ccttcaagga ctcagatagg
1140 aggaagagaa gggagaagga tcctacaaaa gggtccaaga gagggtgaga
ggagatggtc 1200 cagagatata gactcaattc cttaggtcag aatttgcaat
tgggcaagcc agggccttgg 1260 ggtcacaaag ccaaacccag tattaggggc
ttgtcgaaat aaagggagga agagaggccc 1320 tctgtcttac ttcttttggc
ccacacaggg gttattgatt tttatttttt ttcaatttga 1380 tttcgttgtc
aacattagaa tatctggagg cttcacacac agatctatat tcggttctct 1440
ggaaactccg ggccctcatt cactccgggc tgtgccccac ctccaaaggc catggactct
1500 ccgtttaccc agttcccacc tcctctattg tcctctgact caaaaatctt
tggttgtgac 1560 tctcgttgag gcattgtaga gcagggtgtg gcactgtttg
ggctgggcat cacgtgttcc 1620 agggggccag agccaggaga gggccgaaac
tctccggggc acagtgtcgg tcacgctgta 1680 gaaacctctg aattttggat
tttgcagcag gtatgattgt ggtctccatt ttatggatgg 1740 agaacatgag
gcttagtgac aggttagtgc cactggggcc atgtattaaa taactgggaa 1800
gtgacagtgt tgagacttac tccccagact tcggaattca gaagcactag aagagccgtg
1860 cagtgcaggg ccaaaggtca gaggcgctgg accccgacct ccagagccca
aagtggcttc 1920 taccagtatg gaattctgac aacttactta agcttccctg
agcctcagtt tccttgtctg 1980 ttagtacatg agcagggcct gcacacagga
gatgctttta tttatttatt ttttagagac 2040 agagtctcac tctgttgccc
aagctggagt gcagtggcgc aatcttggct cactgcaacc 2100 tccgcctccc
ggattcaagt gattctcatg cctcagcctc ccatgcctgg agatcagctg 2160
ggcagggctg atcctggagt ccttggcctg tgaccctgga gccagggtgg cctctgctgt
2220 cctccttgtc tttgggaaag cagaggcaag gtgcttgggt tacagcaaga
gagatctagg 2280 ttagatggtg agaccttcct gatggctacg gtttcacatc
aagggaaaaa ggctgttggt 2340 ggcaaattct ggacttgaag gctggaagcc
atgtggtggg ggatagtggc agaaaaggca 2400 attttttttt ttttttgtta
agacgcagtc tcactctgtc gcttaggctg cagtgcagtg 2460 gcacaatctt
ggctcatggc aacctcagcc tcctaagtag ctgggattac aggcatgtgc 2520
caccacaccc agctaatttt gtatttttag tagaggtggg gttgcaccat gttggccagg
2580 ctggtctcga actcccgacc tcaagtgatc cacccacctc ggcctcccaa
gtgctgagat 2640 tacaggcgtg cagccgcgcc ctgccagaaa gggaaccttt
agtgaggcct gagtcccagg 2700 cattgtccga gggcagttcc acactgaggg
actgcagagg ccgctgcttg gaaggaagga 2760 gggagatggg gtacgggttg
gggggcgctt agggctccaa agcctaaagc caaacccagt 2820 gcaggaggct
tcttacaatc ggggaggagg ggagtcctgt gagggggagg gaagcttact 2880
gtgggatgaa ggcctgggtt tggagaggct ggaggatggg gcagcaggtc ctatcggggg
2940 agggcagggg agcctctggg caggtacggc ctgacgcccc gggtcctccc
gccccgccag 3000 gcctgacgga ggacgaggac gtgcgcgcca tgctgcgggg
ctcccggctc cgcaagatcc 3060 gctcgcgcac gtggcacaag gagcggctgt
accggctgca ggaggacggc ctgagcgtgt 3120 ggttccagcg gcgcatcccg
cgtgcgccat cgcagcacat ctgtgagagg ccggggagcg 3180 ccgggcggac
agggaggtgg ggacaggccc ggtcctccgc cctgacccgg ccctcctctc 3240
agtcttcgtg cagcacatcg aggcggtccg cgagggccac cagtccgagg gcctgcggcg
3300 cttcgggggt gccttcgcgc cagcgcgctg cctcaccatc gccttcaagg
gccgccgcaa 3360 gaacctggac ctggcggcgc ccacggctga ggaagcgcag
cgctgggtgc gcggtctgac 3420 caagctccgc gcgcgcctgg acgccatgag
ccagcgcgag cggctagacc aatatcctgc 3480 cggggctggg agggagggcc
ccatccttgg agagccctaa ccttggagag ccccatcctt 3540 ggagagcccc
cacgcccagg gaaggcaggg cccctgtcct gagggcgcac ggtctgggtc 3600
ggagcccttg gtcttcgaaa ctgagggtgg ggacacgcgc cctggctagg cccaagctcc
3660 caccaggagg ggtggcgcgg cgcctccttc agggaggcct gaggccttct
gagggtgaag 3720 acagggcctc ctgtctttgg taagccccag tcctgcggag
gagacactgc taccgtgctc 3780 ggggagtgca caggccaagg ggaaagtaca
gcccctaaac tggggatccc ccagacccag 3840 gtgggggtga cttaccagcc
agttacctga gggctggggg cagcctggcg tgttgctgtt 3900 atttcctgag
cacccacacc tggatccact cctatctgca ccgggctgac tccaaccagg 3960
acagcaagat gagcttcaag gagatcaaga gcctgctgag aatggtcaac gtggacatga
4020 acgacatgta cgcctacctc ctcttcaagg tgggcttccg ccctgaaccc
cagcccctgg 4080 ctctgccatc actctgacct ctgcctgccc ccagccatct
gaatcatcag ctcccctgcc 4140 ctctccatct gcagcttttt tggagcgagc
agcctttccc tgcacaaagg caacatctgg 4200 ggttagttag ccttttgcct
cccctgaggc cactgggcca ggaagggagt ggggagccgg 4260 ggcccagaac
tttcctcatc ctcagttcct acccctccaa agtgagcagc aaagctgttt 4320
tcatttccct gagtctgcaa ggcacctttt gcacgtgggg gcacaggcag gagccagcct
4380 acaggtgtgc atgtgcacgg atacacgtgt gcacccttct ccacgcagac
cctccactgt 4440 ggcacgtgtt ggcacctcat gcatgggagc acaggggcac
acaggagtgg gctctgcagc 4500 tatggcccag ctggagccca tggcagacct
ctgtgtgcat gtggatggtg ggacccacct 4560 gtgaccccga aagacaggag
agcccatggc agctggaccc ctgggccaca gcttcacagg 4620 gatgctccat
gaattctcat tgacttgttc aatgacgctg ggtgtgaata tgtgcatgtc 4680
aggatagtaa cgtgtccacc taggggagtg tgagggccca agggccctgt cttggcccag
4740 ggtcaccatt tttccttcca catcttgatt cctggggtag gtcatggtgc
agaggggcag 4800 gcactgaacc agagagacct tttaagtcca gccctgccgg
ccaggctcag tggctcaccc 4860 ctgtcatccc agcactttga gaggcctagg
cgggaggatt gcttgaagcc aggagtttga 4920 gatcagcttg ggcaacacag
caagacccca tctctacaaa aaatacaaaa attagccagg 4980 tgcagtggtg
cacaccagtg gtcccagctg tccagctatt tggaaggctg aggtgggagg 5040
atcacttgag atcacgcgtt ggaagctgca gtgagctatg atcatgccac tgcgctccag
5100 cctgggtaac agagcaagac caaacaaaca aacaaaccta aaaaacaaac
caaaaagtcc 5160 tgccctgtaa tttaccagct gtgtgagcta gagtcacaag
tcttactctg tttttccacc 5220 tgtgaaatgg ggaccattgc cttctcagat
gtggtagatg acagatgggt caaaggggcc 5280 aggtttcctg gagtgtctgc
ctcctctgag cttctcctgc tgctccccag gagtgtgacc 5340 actccaacaa
cgaccgtcta gagggggctg agatcgagga gttcctgcgg cggctgctga 5400
agcggccgga gctggaggag atcttccatc agtactcggg cgaggaccgc gtgctgagtg
5460 cccctgagct gctggagttc ctggaggacc agggcgagga gggcgccaca
ctggcccgcg 5520 cccagcagct cattcagacc tatgagctca acgagacagg
tggggggggt ggcacggcca 5580 ggtcgtggca cctgaaaggg ctgtgttccc
agctaagcgg gttctgctgc gggctggcgc 5640 gggcatcatc tcgggcaggt
tacctcacct cctccagact cagcttccac atgtgtaaaa 5700 tggcacaaca
gtttgtcttc ctcccagggt ctggtcgaag gttaagtggc atgacttacg 5760
tggagtctag tcagtagatt gtctagtcat tattcctaag ctgagtgggc cttgggggca
5820 ttggaagctg gacatggggg atccctatgt ggcctgatgc cctctcctgc
cacccctgca 5880 gccaagcagc atgagctgat gacactggat ggcttcatga
tgtacctgtt gtcgccggag 5940 ggggctgcct tggacaacac ccacacgtgt
gtgttccagg acatgaacca gccccttgcc 6000 cactacttca tctcttcctc
ccacaacacc tatctgactg actcccagat cggggggccc 6060 agcagcaccg
aggcctatgt taggtactgt agcagggggt gaatgtttcc gggagcccca 6120
cctggggaga gacatgggct ggcttcctgg ggccttcctg gcccagcctg cctgctgccc
6180 tgctctgtgg cactctggca acaccttgcc cgtccaccca tctgtccaca
cacagggcct 6240 ttgcccaggg atgccgctgc gtggagctgg actgctggga
ggggccagga ggggagcccg 6300 tcatctatca tggccatacc ctcacctcca
agattctctt ccgggacgtg gtccaagccg 6360 tgcgcgacca tgccttcacg
gtgagcccct gggatgccca gccccagccc cacagccttc 6420 ccaatgacct
ccgtccgcac tcacatgcca gctgccctcc gtccttccag aggaaaccct 6480
tgcccagaga atcattcatt caccaagtgc tgctgggata agtggccaat ggggtggcat
6540 aaagctgaat gagatgccac cctcatattt gggaaatatg ttccctctct
catatttggg 6600 aaatgtccta aaaatcacag agtttccaga agaaaacctt
tgggggaggg aaggacattt 6660 taatcatgac ttccaaagaa taaataagct
gtaagagaag ctcaccagtg gcatctgcat 6720 aaacactgca actttggtca
cagtgtgcaa atcttcccat cagcaaatga tgggaggaga 6780 gttttcacat
cactgacata gtcaatgtca ttcacatgta aaaagcattt atgggaccag 6840
gtgcggtggc tcacacccgt attcccagca ctctgggagg ctgacgcgag gatcacttga
6900 ggccaggaat tccagaccag cctgagcaaa tagctacatc ctgtctcaac
aaaaataaaa 6960 attagctgag tgtgggggca tgtgcctgta ggctgagtta
ctcaggaggc tgaggtgggg 7020 ggatcacttg agcccaggac tggagaacac
agggagactg actgtgccac tgcacttcag 7080 cctgggtgat ggagagagac
caaagcatag acaacaacaa caaaaaatcg gccaggcgtg 7140 gtggctctca
cctgtaatcc cagcactttg ggaggctgag gtgggcagat cacctgaggt 7200
caggagtttg agaccagcct ggccaacatg gtgaaacctc atctctatta aaaatacaaa
7260 aattagccgg gtgtggtggc gggcgcctgt aatcccagct acttaggagg
ctgaggcagg 7320 gagaatcgct tgaacccggg agatggaatt gtagtgagct
gagattgcac cactatactc 7380 cagcatgggc gacagagcaa cactccatct
caaagaaaaa aaaaataatt cctgcagccg 7440 ccctcagcct cctggacagt
ctgctgcttc cctgggttct ccagagccct gcctttgcct 7500 gcctgtcctg
aagggtctgg gagccagatg ttgggagata ctggcagagg gaggggagtg 7560
gctctgagct gctgcccttc cccccacagc tgtcccctta ccctgtcatc ctatccctgg
7620 agaaccactg cgggctggag cagcaggctg ccatggcccg ccacctctgc
accatcctgg 7680 gggacatgct ggtgacacag gcgctggact ccccaaatcc
cgaggagctg ccatccccag 7740 aggtgacgcc ccccagcccc tagtctgggt
ggagggaggt ctggtgggag tccgcctccc 7800 cgcactgctc gtgccacctc
tgtggctcag gacccctcac ctcttgctgc cttaactttt 7860 ctctgttcct
ctgttcatct gaggtctgcc atcccctggt cttgtccttc tgctgccctc 7920
tggctctcca ttcttgacta gaccccccca cacacacacc ctcttctgta acctggcttt
7980 gccctgctgg ccctgtgtcc ctgcccgtag agtcctgcag gctctgccat
tcccacctgc 8040 aaggccccgc ttgacacttt ctccacctgg aagtcccttc
tcggccatcc acacttgcca 8100 agcgtcacct gcttagaatt cagctcaact
tcagccgcct cttcctgtgg ccttatccac 8160 ttccccaact ctcgggcctg
aggcggcctc ctgctctccc ccagacacca gagcgcgtcc 8220 tcggctctct
ccagacattt atcctgtcgg gctccttgag aaaactctcc tcctacttcc 8280
aatccccaac atgcaggcag ggttgactca ccctcgtgtg tgtgtgagat gtgatttttg
8340 ttattactgt gaaaatcaca cccgtatggt aaaagaaaac tgagctgtac
taagggacgc 8400 gatgagcagt gctcccctcc ctccactgac ctgtcctctt
tcacaagaag taaccatctc 8460 taactttctt gcatgtcctt caagaatttc
cccacacaca ttacctgtgt acatatgtgt 8520 actgtcttct cttttaaatg
gtaccaaaaa aggacatgtg attccaggtt gtagcctcac 8580 ttcttccacg
taagatcttc tttgaagagg gtccctttcc tttcagtcct gagctgtcgc 8640
gtgcattgta gtggctgctg tgtatattct gatgttgagg gaccttatta agcatccctt
8700 cctgatggcc tcccgggcca ttctccactc tttcctatta gaaacatcgc
tcatctctgt 8760 gtctcctttc ctgcctccca cccctagcca gcccctcaca
aggcggtggt tgaataactg 8820 actgactggc tgccacaggg gcgagaaggc
ctaacccttc atcctccacg ttggccccca 8880 cagcagctga agggccgggt
cctggtgaag ggaaagaagc tgcccgctgc tcggagcgag 8940 gatggccggg
ctctgtcgga tcgggaggag gaggaggagg atgacgagga ggaagaagag 9000
gaggtggagg ctgcagcgca gaggcggctg gtgagagctg ggatgggtgg ggtggggaag
9060 ggggggaagg tgggaggacg gatgtgggaa gcggggccca gctgaacccc
gctgagcctg 9120 gtctcctagg ccaagcagat ctccccggag ctgtcggccc
tggctgtgta ctgccacgcc 9180 acccgcctgc ggaccctgca ccctgccccc
aacgccccac aaccctgcca ggtcagctcc 9240 ctcagcgagc gcaaagccaa
gaaactcatt cgggaggcag gtaggagctg gggcactggg 9300 tgtctggggg
cgagagggtg agagacctcc tgaagggagt ctggaaagga gttgaggggg 9360
ctttggggga cagtaaagag gtcaagagga ccccaaagag gggctgggct gagggaatga
9420 gggccccgca catgccaggg tggatggagg ggagtggcac gctgagggcg
gcctgctgtc 9480 tgccttcaga gccctgtggt ggggccggcc atagagggtg
atgggtcagg ctgggctggg 9540 ggctggggga gcccagcagc tgctgcaaca
ctggggagga attcctgggg caggtgatgc 9600 tgagaggaca catcacctgg
aggactagga agcagccagg tgaagagggg agagcgcttt 9660 ccagacagga
ggaacaggtt gttgaaggcc tggggccttg gcctggagaa gaattccagg 9720
aaccacagtg gagctggagg gccgtgggca ttcagtgtac ttggttggaa ctttgtcagg
9780 agctgggaat tgggggctgg ggatgcaggg ccaggctgtg tggctggagg
gggtccctgc 9840 agcctcctca gtgagctccc ctctcactct aggtcagaag
agagtgagga gcgggggcag 9900 ggtgacctgg ggacgggctt gggctctgtt
ccctggaggt tacaggccgg ggctttgggt 9960 gagggacccc cggagtctgt
cacggtctca ccccaactct gccccaggga acagctttgt 10020 caggcacaat
gcccgccagc tgacccgcgt gtacccgctg gggctgcgga tgaactcagc 10080
caactacagt ccccaggaga tgtggaactc gggctgtcag ctgggtaagt gggggccaca
10140 ggaatgagag caggcggggt gccacagtgt gggggaggcc tcagcagtga
ggggcccaca 10200 cagccttcca gacggtggaa acacgcattt tgttgattat
ttttaaagtt gccagcaatg 10260 taggtgggaa aatcaaatca ggctcgtttg
ttcttgacag tgtacaggtc tcgcattttt 10320 atcatggtag aaattaaaat
ctccactccc atgtcccccg gacacacaca acccccacac 10380 acctggcagt
cccactaagt caacactgac aagggtgggc tctgggggac gctgagcccc 10440
accaaaggag ggctgtatcg tctccttggg gtaaggatgg aaaccccagg cttcctcaga
10500 cccctggctc tgacccaagc ctggccctga gggtgggggc ttttttagta
cctcccaccc 10560 cagtcttgag ggatcccagg gcagaagtac aggtgacagt
gggggctggg tccatgaccc 10620 cctcccctaa agtcatggtg ggagcagagc
agcactaagc agacagtggg accccaggcc 10680 ctttgtagag aggaatgcag
tcccctgaaa ctgagtagtg agaacagggc cgctaagagc 10740 ccctgctctg
accctgctgt gtccacagtg gccttgaact tccagacgcc aggctacgag 10800
atggacctca atgccgggcg cttcctagtc aatgggcagt gtggctacgt cctaaaacct
10860 gcctgcctgc ggcaacctga ctcgaccttt gaccccgagt acccaggacc
tcccagaacc 10920 actctcagca tccaggtcag caggctggaa cgggccccag
tgtggggtca gactggttct 10980 agaaaggctc tttgtgaggt ggaggaaggc
aagggaggct ggggacattt ggaagtgagg 11040 gtggtgtgag cttgaggccc
atgaggggat ggccaaggtg ggcgtgggcc ccagccactc 11100 cctccctcac
ccacaggtgc tgactgcaca gcagctgccc aagctgaatg ccgagaagcc 11160
acactccatt gtggaccccc tggtgcgcat tgagatccat ggggtgcccg cagactgtgc
11220 ccggcaggag actgactacg tgctcaacaa tggtgggcag ccactggcgg
aagtggggtt 11280 gggggagact gcagagagcg aagggtggtg cagggtgggt
cctggggccc ctgggctctg 11340 actgccatcc ctgtgcccta ggcttcaacc
cccgctgggg gcagaccctg cagttccagc 11400 tgcgggctcc ggagctggca
ctggtccggt ttgtggtgga agattatgac gccacctccc 11460 ccaatgactt
tgtgggccag tttacactgc ctcttagcag cctaaagcaa ggttggtgga 11520
ggctggtggg gatggagagg gatgtgggtc caggtcctgc agaccctgtt cacctttgtg
11580 cctcgaattt cctgccctga agcaggcccg ggctgggcca gacccccagg
gtgcacagag 11640 gacctgaggc ctggctgtcc tccctacagg gtaccgccac
atacacctgc tttccaagga 11700 cggggcctca ctgtcaccag ccacgctctt
catccaaatc cgcatccagc gctcctgagg 11760 gcccacctca ctcgccttgg
ggttctgcga gtgccagtcc acatcccctg cagagccctc 11820
tcctcctctg gagtcaggtg gtgggagtac cagcccccca gcccacccac ttggcccact
11880 cagcccattc accaggcgct ggtctcacct gggtgctgag ggctgcctgg
gcccctcctg 11940 aagaacagaa aggtgttcat gtgacttcag tgagctccaa
ccctggggcc ctgagatggc 12000 cccagctcct cttgtcctca gcccacccct
cattgtgact tatgaggagc aagcctgttg 12060 ctgccaggag acttggggag
caggacactt gtgggccctc agttcccctc tgtcctcccg 12120 tgggccatcc
cagcctcctt cccccagagg agcgcagtca ctccacttgg ccccgacccc 12180
gagcttagcc cctaagccct cctttacccc aggccttcct ggactcctcc ctccagctcc
12240 ggaacctgag ctccccttcc cttctcaaag caagaaggga gcgctgaggc
atgaagccct 12300 ggggaaactg gcagtaggtt ttggttttta ttttttgaga
cagggtctcg ctccgtcgcc 12360 caggctggag tgcaatgttg caatcatggc
tcactgcagc tttgaactcc caggctcaag 12420 cgatcctccc atctcagcct
cctgagtagc tgggactaca ggcacaggcc accaaacctg 12480 gctaatgttt
aaattttatg tagagatggc ggggggggcg ggggtctccc tatgttgccc 12540
aggctggtct cgagctcctg acctcaagtg atcctcctgc cccagcattc caaagtgctg
12600 ggattacagg tgtgagccac tgcctggcct ggccgtaggt ttgtaactgt
ttcatagaag 12660 agccctggag aagacagtag aatgagccta tctagtttaa
aaaataaaga agcacgttga 12720 tttcaccacc gccactccct gagcacctct
atgctgccac ccccacaggc ccatgaggag 12780 ggacagggct ggatgagttt
cttgggcctc tcccctcaac agggcatgga cttggctaag 12840 acagccaggg
ggtggcagca ggagatgcca cagctctaaa tagcccgatc cctgctcctg 12900
gcttaaagct gctgcgagta tcacaagact gtctgaggat gccaattcag caggtcagcg
12960 cctccccaca acacacacac acacacacac acacacacac tcacacatac
acagaggggc 13020 cttgaatgag gaggagacag gagtctgagg gaccctcaga
ctgccactct ccaggcttgg 13080 ctgggctcaa ggcccctgtc tgctcagtag
ggcaaatccc aaggacacgg gcttcccttc 13140 ctccagccag ccagcctttt
acacaaaggc tggaaacgct gcttgggccg cctctataga 13200 aatgccagga
gagcgcactg gacactaggc gacagaatct ggagaaaatc ctgcacccgc 13260
ccctgccagc agggtgaaat gtggaggcct ggtgttttag gttctgccag ccaacagtgc
13320 cggcactgga caagggcaga acttgacatt agtccccaga gggtggcctg
gaactgggct 13380 gggataggcc ttgaactggc cccaagcccc acagaaacca
aaacacaggg ctggggcaga 13440 ctccaccttc tagcaactcc aagaactaat
gcagaaagtg gcagtgtccc ccgctccttc 13500 atgctgggag tggcgtttgg
ctctggttaa tttgtgtgtc tgaaagagga taatcagaat 13560 caaaagggcc
cagaaggaat gggccgagcc aggcgggccg ggccaggcag cccgggcatc 13620
tggccctcag ggctgtcaga gggtcaggct gccaaactgc agcctcagga tgggaggcac
13680 agtgaggcta agtccagttt tagcaaatga agagccatta gcattgccca
gcccctgcag 13740 agccaggtct ggccaggggc tggctggggg gtctgggtca
ggtgagcacc agacccaggg 13800 ctgagtgccc atcctgcttg gcttctctgg
tttcgtatct ggtaccaaga ggaaggggtg 13860 aaggctagga tgagggccca
atactgagaa ggctgccttg aaagggcaag agtctttttt 13920 tttttttctt
ttgagacaaa gtcttgctcg tcc 13953 4 744 PRT Cricetulus griseus 4 Gly
Leu Gln Asp Asp Gln Asp Leu Gln Ala Leu Leu Lys Gly Ser Gln 1 5 10
15 Leu Leu Lys Val Lys Ser Ser Ser Trp Arg Arg Glu Arg Phe Tyr Lys
20 25 30 Leu Gln Glu Asp Cys Lys Thr Ile Trp Gln Glu Ser Arg Lys
Val Met 35 40 45 Arg Ser Pro Glu Ser Gln Leu Phe Ser Ile Glu Asp
Ile Gln Glu Val 50 55 60 Arg Met Gly His Arg Thr Glu Gly Leu Glu
Lys Phe Ala Arg Asp Ile 65 70 75 80 Pro Glu Asp Arg Cys Phe Ser Ile
Val Phe Lys Asp Gln Arg Asn Thr 85 90 95 Leu Asp Leu Ile Ala Pro
Ser Ser Ala Asp Ala Gln His Trp Val Gln 100 105 110 Gly Leu Arg Lys
Ile Ile His His Ser Gly Ser Met Asp Gln Arg Gln 115 120 125 Lys Leu
Gln His Trp Ile His Ser Cys Leu Arg Lys Ala Asp Lys Asn 130 135 140
Lys Asp Asn Lys Met Asn Phe Lys Glu Leu Lys Asp Phe Leu Lys Glu 145
150 155 160 Leu Asn Ile Gln Val Asp Asp Ser Tyr Ala Arg Lys Ile Phe
Arg Glu 165 170 175 Cys Asp His Ser Gln Thr Asp Ser Leu Glu Asp Glu
Glu Ile Glu Thr 180 185 190 Phe Tyr Lys Met Leu Thr Gln Arg Ala Glu
Ile Asp Arg Val Phe Ala 195 200 205 Glu Ala Ala Gly Ser Ala Glu Thr
Leu Ser Val Glu Lys Leu Val Thr 210 215 220 Phe Leu Gln His Gln Gln
Arg Glu Glu Ala Ala Gly Pro Ala Leu Ala 225 230 235 240 Leu Ser Leu
Ile Glu Arg Tyr Glu Pro Ser Glu Thr Ala Lys Ala Gln 245 250 255 Arg
Gln Met Thr Lys Asp Gly Phe Leu Met Tyr Leu Leu Ser Ala Asp 260 265
270 Gly Ser Ala Phe Ser Leu Ala His Arg Arg Val Tyr Gln Asp Met Asp
275 280 285 Gln Pro Leu Ser His Tyr Leu Val Ser Ser Ser His Asn Thr
Tyr Leu 290 295 300 Leu Glu Asp Gln Leu Thr Gly Pro Ser Ser Thr Glu
Ala Tyr Ile Arg 305 310 315 320 Ala Leu Cys Lys Gly Cys Arg Cys Leu
Glu Leu Asp Cys Trp Asp Gly 325 330 335 Pro Asn Gln Glu Pro Ile Ile
Tyr His Gly Tyr Thr Phe Thr Ser Lys 340 345 350 Ile Leu Phe Tyr Asp
Val Leu Arg Ala Ile Arg Asp Tyr Ala Phe Lys 355 360 365 Ala Ser Pro
Tyr Pro Val Ile Leu Ser Leu Glu Asn His Cys Ser Leu 370 375 380 Glu
Gln Gln Gln Val Met Ala Arg His Leu Lys Ala Ile Leu Gly Pro 385 390
395 400 Met Leu Leu Asp Gln Pro Leu Asp Gly Val Thr Met Ser Leu Pro
Ser 405 410 415 Pro Glu Gln Leu Lys Gly Lys Ile Leu Leu Lys Gly Lys
Lys Phe Gly 420 425 430 Gly Leu Leu Pro Ala Gly Gly Glu Asn Gly Pro
Glu Thr Thr Asp Val 435 440 445 Ser Asp Glu Asp Glu Ala Ala Glu Met
Glu Asp Glu Ala Val Arg Ser 450 455 460 Gln Val Gln Gln Lys Ser Lys
Glu Asp Lys Leu Asn Val Ala Pro Glu 465 470 475 480 Leu Ser Asp Met
Val Ile Tyr Cys Lys Ser Val His Phe Gly Gly Phe 485 490 495 Ser Asn
Pro Ser Thr Ser Gly Gln Ala Phe Tyr Glu Met Ala Ser Phe 500 505 510
Ser Glu Asn Arg Ala Leu Arg Leu Leu Gln Glu Ser Gly Asn Asn Phe 515
520 525 Val Arg His Asn Val Ser His Leu Ser Arg Ile Tyr Pro Ala Gly
Arg 530 535 540 Arg Thr Asp Ser Ser Asn Tyr Ser Pro Val Glu Met Trp
Asn Gly Gly 545 550 555 560 Cys Gln Ile Val Ala Leu Asn Phe Gln Thr
Pro Gly Pro Glu Met Asp 565 570 575 Val Tyr Leu Gly Arg Phe Gln Asp
Asn Gly Ala Cys Gly Tyr Val Leu 580 585 590 Lys Pro Ala Phe Leu Arg
Asp Pro Asp Thr Ala Phe Asn Pro Arg Ala 595 600 605 Leu Thr Gln Gly
Pro Trp Trp Ala Gln Lys Arg Leu Arg Val Arg Val 610 615 620 Ile Ser
Gly Gln Gln Leu Pro Lys Val Asn Lys Ser Lys Asn Ser Ile 625 630 635
640 Val Asp Pro Lys Val Ile Val Glu Val His Gly Val Gly Gln Asp Val
645 650 655 Ala Ser Arg Gln Thr Ala Val Ile Thr Asn Asn Gly Phe Asn
Pro Trp 660 665 670 Trp Asp Thr Glu Phe Glu Phe Glu Val Ala Val Pro
Asp Leu Ala Leu 675 680 685 Val Arg Phe Val Val Glu Asp Tyr Asp Ala
Ser Ser Lys Asn Asp Phe 690 695 700 Ile Gly Gln Ser Thr Ile Pro Trp
Asn Ser Leu Lys Gln Gly Tyr Arg 705 710 715 720 His Val His Leu Leu
Ser Lys Asn Gly Asp Gln His Pro Ser Ala Thr 725 730 735 Leu Phe Val
Lys Ile Ser Leu Gln 740 5 744 PRT Mus musculus 5 Gly Leu Gln Asp
Asp Pro Asp Leu Gln Ala Leu Leu Lys Gly Ser Gln 1 5 10 15 Leu Leu
Lys Val Lys Ser Ser Ser Trp Arg Arg Glu Arg Phe Tyr Lys 20 25 30
Leu Gln Glu Asp Cys Lys Thr Ile Trp Gln Glu Ser Arg Lys Val Met 35
40 45 Arg Ser Pro Glu Ser Gln Leu Phe Ser Ile Glu Asp Ile Gln Glu
Val 50 55 60 Arg Met Gly His Arg Thr Glu Gly Leu Glu Lys Phe Ala
Arg Asp Ile 65 70 75 80 Pro Glu Asp Arg Cys Phe Ser Ile Val Phe Lys
Asp Gln Arg Asn Thr 85 90 95 Leu Asp Leu Ile Ala Pro Ser Pro Ala
Asp Val Gln His Trp Val Gln 100 105 110 Gly Leu Arg Lys Ile Ile Asp
Arg Ser Gly Ser Met Asp Gln Arg Gln 115 120 125 Lys Leu Gln His Trp
Ile His Ser Cys Leu Arg Lys Ala Asp Lys Asn 130 135 140 Lys Asp Asn
Lys Met Asn Phe Lys Glu Val Lys Asp Phe Leu Lys Glu 145 150 155 160
Leu Asn Val Gln Val Asp Asp Ser Tyr Ala Arg Lys Ile Phe Arg Glu 165
170 175 Cys Asp His Ser Gln Thr Asp Ser Leu Glu Asp Glu Glu Ile Glu
Thr 180 185 190 Phe Tyr Arg Met Leu Thr Gln Arg Ala Glu Ile Asp Arg
Ala Phe Ala 195 200 205 Glu Ala Ala Gly Ser Ala Glu Thr Leu Ser Val
Glu Lys Leu Val Thr 210 215 220 Phe Leu Gln His Gln Gln Arg Glu Glu
Glu Ala Gly Pro Ala Leu Ala 225 230 235 240 Leu Ser Leu Ile Glu Arg
Tyr Glu Pro Ser Glu Thr Ala Lys Ala Gln 245 250 255 Arg Gln Met Thr
Lys Asp Gly Phe Leu Met Tyr Leu Leu Ser Ala Asp 260 265 270 Gly Asn
Ala Phe Ser Leu Ala His Arg Arg Val Tyr Gln Asp Met Asn 275 280 285
Gln Pro Leu Ser His Tyr Leu Val Ser Ser Ser His Asn Thr Tyr Leu 290
295 300 Leu Glu Asp Gln Leu Thr Gly Pro Ser Ser Thr Glu Ala Tyr Ile
Arg 305 310 315 320 Ala Leu Cys Lys Gly Cys Arg Cys Leu Glu Leu Asp
Cys Trp Asp Gly 325 330 335 Pro Asn Gln Glu Pro Ile Ile Tyr His Gly
Tyr Thr Phe Thr Ser Lys 340 345 350 Ile Leu Phe Cys Asp Val Leu Arg
Ala Ile Arg Asp Tyr Ala Phe Lys 355 360 365 Ala Ser Pro Tyr Pro Val
Ile Leu Ser Leu Glu Asn His Cys Ser Leu 370 375 380 Glu Gln Gln Arg
Val Met Ala His His Leu Arg Ala Ile Leu Gly Pro 385 390 395 400 Met
Leu Leu Asp Gln Pro Leu Asp Gly Val Thr Thr Ser Leu Pro Ser 405 410
415 Pro Glu Gln Leu Lys Glu Lys Ile Leu Leu Lys Gly Lys Lys Leu Gly
420 425 430 Gly Leu Leu Pro Ala Gly Gly Glu Asn Gly Pro Glu Ala Thr
Asp Val 435 440 445 Ser Asp Glu Asp Glu Ala Ala Glu Met Glu Asp Glu
Ala Val Arg Ser 450 455 460 Gln Val Gln His Lys Pro Lys Glu Asp Lys
Leu Lys Leu Val Pro Glu 465 470 475 480 Leu Ser Asp Met Val Ile Tyr
Cys Lys Ser Val His Phe Gly Gly Phe 485 490 495 Ser Ser Pro Ser Thr
Ser Gly Gln Ala Phe Tyr Glu Met Ala Ser Phe 500 505 510 Ser Glu Ser
Arg Ala Leu Arg Leu Leu Gln Glu Ser Gly Asn Ser Phe 515 520 525 Val
Arg His Asn Val Gly His Leu Ser Arg Ile Tyr Pro Ala Gly Trp 530 535
540 Arg Thr Asp Ser Ser Asn Tyr Ser Pro Val Glu Met Trp Asn Gly Gly
545 550 555 560 Cys Gln Ile Val Ala Leu Asn Phe Gln Thr Pro Gly Pro
Glu Met Asp 565 570 575 Val Tyr Leu Gly Cys Phe Gln Asp Asn Gly Gly
Cys Gly Tyr Val Leu 580 585 590 Lys Pro Ala Phe Leu Arg Asp Pro Asp
Thr Thr Phe Asn Ser Arg Ala 595 600 605 Leu Thr Gln Gly Pro Trp Trp
Ala Pro Lys Lys Leu Arg Val Trp Ile 610 615 620 Ile Ser Gly Gln Gln
Leu Pro Lys Val Asn Lys Asn Lys Asn Ser Ile 625 630 635 640 Val Asp
Pro Lys Val Ile Val Glu Ile His Gly Val Gly Gln Asp Val 645 650 655
Ala Ser Arg Gln Thr Ala Val Ile Thr Asn Asn Gly Phe Asn Pro Arg 660
665 670 Trp Asp Thr Glu Phe Glu Phe Val Val Ala Val Pro Asp Leu Ala
Leu 675 680 685 Val Arg Phe Met Val Glu Asp Tyr Asp Ser Ser Ser Lys
Asn Asp Phe 690 695 700 Ile Gly Gln Ser Thr Ile Pro Trp Asn Ser Leu
Lys Gln Gly Tyr Arg 705 710 715 720 His Val His Leu Leu Ser Lys Asn
Gly Asp Leu His Pro Ser Ala Thr 725 730 735 Leu Phe Val Lys Ile Ser
Ile Gln 740
* * * * *
References