U.S. patent application number 10/784089 was filed with the patent office on 2004-12-23 for 32544, novel human phospholipase c and uses thereof.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Meyers, Rachel, Silos-Santiago, Inmaculada.
Application Number | 20040259199 10/784089 |
Document ID | / |
Family ID | 22932296 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040259199 |
Kind Code |
A1 |
Meyers, Rachel ; et
al. |
December 23, 2004 |
32544, Novel human phospholipase C and uses thereof
Abstract
The invention provides isolated nucleic acids molecules,
designated 32544 nucleic acid molecules, which encode novel
phospholipase family members. The invention also provides antisense
nucleic acid molecules, recombinant expression vectors containing
32544 nucleic acid molecules, host cells into which the expression
vectors have been introduced, and nonhuman transgenic animals in
which a 32544 gene has been introduced or disrupted. The invention
still further provides isolated 32544 proteins, fusion proteins,
antigenic peptides and anti-32544 antibodies. Diagnostic methods
utilizing compositions of the invention are also provided.
Inventors: |
Meyers, Rachel; (Newton,
MA) ; Silos-Santiago, Inmaculada; (Cambridge,
MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
40 Landsdowne Street
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
22932296 |
Appl. No.: |
10/784089 |
Filed: |
February 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10784089 |
Feb 20, 2004 |
|
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09927112 |
Aug 10, 2001 |
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60246808 |
Nov 8, 2000 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.2 |
Current CPC
Class: |
C12Q 2600/106 20130101;
C12Q 2600/136 20130101; C12Q 1/6886 20130101; A01K 2217/05
20130101; A01K 2217/075 20130101; C07K 2319/00 20130101; A61K
2039/505 20130101; A61K 48/00 20130101; C12Q 1/6883 20130101; C12Y
301/04003 20130101; C12N 9/16 20130101 |
Class at
Publication: |
435/069.1 ;
536/023.2; 530/350; 435/320.1; 435/325 |
International
Class: |
C07H 021/04; C07K
014/705 |
Claims
What is claimed is:
1. An isolated 32544 nucleic acid molecule selected from the group
consisting of: a) a nucleic acid molecule comprising a nucleotide
sequence which is at least 60% identical to the nucleotide sequence
of SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
______; b) a nucleic acid molecule comprising a fragment of at
least 15 nucleotides of the nucleotide sequence of SEQ ID NO:1, SEQ
ID NO:3, or the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC as Accession Number ______; c) a
nucleic acid molecule which encodes a polypeptide comprising the
amino acid sequence of SEQ ID NO:2, or the amino acid sequence
encoded by the cDNA insert of the plasmid deposited with the ATCC
as Accession Number ______; d) a nucleic acid molecule which
encodes a fragment of a polypeptide comprising the amino acid
sequence of SEQ ID NO:2, or the amino acid sequence encoded by the
cDNA insert of the plasmid ATCC as Accession Number ______, wherein
the fragment comprises at least 15 contiguous amino acids of SEQ ID
NO:2, or the amino acid sequence encoded by the cDNA insert of the
plasmid deposited with the ATCC as Accession Number ______; e) a
nucleic acid molecule which encodes a naturally occurring allelic
variant of a polypeptide comprising the amino acid sequence of SEQ
ID NO:2, or the amino acid sequence encoded by the cDNA insert of
the plasmid deposited with the ATCC as Accession Number ______,
wherein the nucleic acid molecule hybridizes to a nucleic acid
molecule comprising SEQ ID NO:1, SEQ ID NO:3, or a complement
thereof, under stringent conditions; f) a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or
the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC as Accession Number ______; and g) a nucleic acid
molecule which encodes a polypeptide comprising the amino acid
sequence of SEQ ID NO:2, or the amino acid sequence encoded by the
cDNA insert of the plasmid deposited with the ATCC as Accession
Number ______.
2. The isolated nucleic acid molecule of claim 1, which is the
nucleotide sequence SEQ ID NO:1.
3. A host cell which contains the nucleic acid molecule of claim
1.
4. An isolated 32544 polypeptide selected from the group consisting
of: a) a polypeptide which is encoded by a nucleic acid molecule
comprising a nucleotide sequence which is at least 60% identical to
a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1,
SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC as Accession Number ______, or a
complement thereof; b) a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:2, or
the amino acid sequence encoded by the cDNA insert of the plasmid
deposited with the ATCC as Accession Number ______, wherein the
polypeptide is encoded by a nucleic acid molecule which hybridizes
to a nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:3, or
a complement thereof under stringent conditions; c) a fragment of a
polypeptide comprising the amino acid sequence of SEQ ID NO:2, or
the amino acid sequence encoded by the cDNA insert of the plasmid
deposited with the ATCC as Accession Number ______, wherein the
fragment comprises at least 15 contiguous amino acids of SEQ ID
NO:2; and d) the amino acid sequence of SEQ ID NO:2.
5. An antibody which selectively binds to a polypeptide of claim
4.
6. A method for producing a polypeptide selected from the group
consisting of: a) a polypeptide comprising the amino acid sequence
of SEQ ID NO:2, or the amino acid sequence encoded by the cDNA
insert of the plasmid deposited with the ATCC as Accession Number
______; b) a polypeptide comprising a fragment of the amino acid
sequence of SEQ ID NO:2, or the amino acid sequence encoded by the
cDNA insert of the plasmid deposited with the ATCC as Accession
Number ______, wherein the fragment comprises at least 15
contiguous amino acids of SEQ ID NO:2, or the amino acid sequence
encoded by the cDNA insert of the plasmid deposited with the ATCC
as Accession Number ______; c) a naturally occurring allelic
variant of a polypeptide comprising the amino acid sequence of SEQ
ID NO:2, or the amino acid sequence encoded by the cDNA insert of
the plasmid deposited with the ATCC as Accession Number ______,
wherein the polypeptide is encoded by a nucleic acid molecule which
hybridizes to a nucleic acid molecule comprising SEQ ID NO:1 or SEQ
ID NO:3; and d) the amino acid sequence of SEQ ID NO:2; comprising
culturing the host cell of claim 3 under conditions in which the
nucleic acid molecule is expressed.
7. A method for detecting the presence of a nucleic acid molecule
of claim 1 or a polypeptide encoded by the nucleic acid molecule in
a sample, comprising: a) contacting the sample with a compound
which selectively hybridizes to the nucleic acid molecule of claim
1 or binds to the polypeptide encoded by the nucleic acid molecule;
and b) determining whether the compound hybridizes to the nucleic
acid or binds to the polypeptide in the sample.
8. A kit comprising a compound which selectively hybridizes to a
nucleic acid molecule of claim 1 or binds to a polypeptide encoded
by the nucleic acid molecule and instructions for use.
9. A method for identifying a compound which binds to a polypeptide
or modulates the activity of the polypeptide of claim 4 comprising
the steps of: a) contacting a polypeptide, or a cell expressing a
polypeptide of claim 4 with a test compound; and b) determining
whether the polypeptide binds to the test compound or determining
the effect of the test compound on the activity of the
polypeptide.
10. A method for modulating the activity of a polypeptide of claim
4 comprising contacting the polypeptide or a cell expressing the
polypeptide with a compound which binds to the polypeptide in a
sufficient concentration to modulate the activity of the
polypeptide.
11. A method of identifying a nucleic acid molecule associated with
a disorder comprising: a) contacting a sample from a subject with
or at risk of developing a disorder comprising nucleic acid
molecules with a hybridization probe comprising at least 25
contiguous nucleotides of SEQ ID NO:1 defined in claim 2; and b)
detecting the presence of a nucleic acid molecule in the sample
that hybridizes to the probe, thereby identifying a nucleic acid
molecule associated with a disorder.
12. A method of identifying a nucleic acid associated with a
disorder comprising: a) contacting a sample from a subject having a
disorder or at risk of developing a disorder comprising nucleic
acid molecules with a first and a second amplification primer, the
first primer comprising at least 25 contiguous nucleotides of SEQ
ID NO:1 defined in claim 2 and the second primer comprising at
least 25 contiguous nucleotides from the complement of SEQ ID NO:1;
b) incubating the sample under conditions that allow nucleic acid
amplification; and c) detecting the presence of a nucleic acid
molecule in the sample that is amplified, thereby identifying the
nucleic acid molecule associated with a disorder.
13. A method of identifying a polypeptide associated with a
disorder comprising: a) contacting a sample comprising polypeptides
with a 32544 binding partner of the 32544 polypeptide defined in
claim 4; and b) detecting the presence of a polypeptide in the
sample that binds to the 32544 binding partner, thereby identifying
the polypeptide associated with a disorder.
14. A method of identifying a subject having a disorder or at risk
for developing a disorder comprising: a) contacting a sample
obtained from the subject comprising nucleic acid molecules with a
hybridization probe comprising at least 25 contiguous nucleotides
of SEQ ID NO:1 defined in claim 2; and b) detecting the presence of
a nucleic acid molecule in the sample that hybridizes to the probe,
thereby identifying a subject having a disorder or at risk for
developing a disorder.
15. A method of identifying a subject having a disorder or at risk
for developing a disorder comprising: a) contacting a sample
obtained from the subject comprising nucleic acid molecules with a
first and a second amplification primer, the first primer
comprising at least 25 contiguous nucleotides of SEQ ID NO:1
defined in claim 2 and the second primer comprising at least 25
contiguous nucleotides from the complement of SEQ ID NO:1; b)
incubating the sample under conditions that allow nucleic acid
amplification; and c) detecting the presence of a nucleic acid
molecule in the sample that is amplified, thereby identifying a
subject having a disorder or at risk for developing a disorder.
16. A method of identifying a subject having a disorder or at risk
for developing a disorder comprising: a) contacting a sample
obtained from the subject comprising polypeptides with a 32544
binding partner of the 32544 polypeptide defined in claim 4; and b)
detecting the presence of a polypeptide in the sample that binds to
the 32544 binding partner, thereby identifying a subject having a
disorder or at risk for developing a disorder.
17. A method for identifying a compound capable of treating a
disorder characterized by aberrant 32544 nucleic acid expression or
32544 polypeptide activity comprising assaying the ability of the
compound to modulate 32544 nucleic acid expression or 32544
polypeptide activity, thereby identifying a compound capable of
treating a disorder characterized by aberrant 32544 nucleic acid
expression or 32544 polypeptide activity.
18. A method for treating a subject having a disorder or at risk of
developing a disorder comprising administering to the subject a
32544 modulator of the nucleic acid molecule defined in claim 1 or
the polypeptide encoded by the nucleic acid molecule or contacting
a cell with a 32544 modulator.
19. The method of claim 18, wherein the 32544 modulator is a) a
small molecule; b) peptide; c) phosphopeptide; d) anti-32544
antibody; e) a 32544 polypeptide comprising the amino acid sequence
of SEQ ID NO:2, or a fragment thereof; f) a 32544 polypeptide
comprising an amino acid sequence which is at least 90 percent
identical to the amino acid sequence of SEQ ID NO:2, wherein the
percent identity is calculated using the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4; or g) an isolated
naturally occurring allelic variant of a polypeptide consisting of
the amino acid sequence of SEQ ID NO:2, wherein the polypeptide is
encoded by a nucleic acid molecule which hybridizes to a complement
of a nucleic acid molecule consisting of SEQ ID NO:1 at 6.times.SSC
at 45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 65.degree. C.
20. The method of claim 18, wherein the 32544 modulator is a) an
antisense 32544 nucleic acid molecule; b) is a ribozyme; c) the
nucleotide sequence of SEQ ID NO:1, or a fragment thereof; d) a
nucleic acid molecule encoding a polypeptide comprising an amino
acid sequence which is at least 90 percent identical to the amino
acid sequence of SEQ ID NO:2, wherein the percent identity is
calculated using the ALIGN program for comparing amino acid
sequences, a PAM120 weight residue table, a gap length penalty of
12, and a gap penalty of 4; e) a nucleic acid molecule encoding a
naturally occurring allelic variant of a polypeptide comprising the
amino acid sequence of SEQ ID NO:2, wherein the nucleic acid
molecule which hybridizes to a complement of a nucleic acid
molecule consisting of SEQ ID NO:1 at 6.times.SSC at 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
65.degree. C.; or f) a gene therapy vector.
21. A method for evaluating the efficacy of a treatment of a
disorder, in a subject, comprising: treating a subject with a
protocol under evaluation; assessing the expression level of a
32544 nucleic acid molecule defined in claim 1 or 32544 polypeptide
encoded by the 32544 nucleic acid molecule, wherein a change in the
expression level of 32544 nucleic acid or 32544 polypeptide after
the treatment, relative to the level before the treatment, is
indicative of the efficacy of the treatment of a disorder.
22. A method of diagnosing a disorder in a subject, comprising:
evaluating the expression or activity of a 32544 nucleic acid
molecule defined in claim 1 or a 32544 polypeptide encoded by the
32544 nucleic acid molecule, such that a difference in the level of
32544 nucleic acid or 32544 polypeptide relative to a normal
subject or a cohort of normal subjects is indicative of a
disorder.
23. The method defined in claim 18, wherein the disorder is a pain
disorder.
Description
[0001] This application claims priority on U.S. Application Ser.
No. 60/246,808 filed 8 Nov. 2000, which is relied on and
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Phospholipase C (PLC) belongs to a family of enzymes, also
known as disulfide isomerases, which play an important role in
mediating signal transduction pathways. Many extracellular
signaling molecules including hormones, growth factors,
neurotransmitters, and immunoglobulins bind to their respective
cell surface receptors and activate PLCs. Activated PLCs then
catalyze the hydrolysis of phosphatidyl-inositol-4,5-bisphosphate
(PIP2), a component of the plasma membrane, to produce
diacylglycerol and inositol 1,4,5-trisphosphate (IP3).
[0003] In their respective biochemical pathways, IP3 and
diacylglycerol serve as second messengers and trigger a series of
intracellular responses. IP3 induces the release of calcium from
internal cellular storage, and diacylglycerol activates protein
kinase C (PKC). Both pathways are part of transmembrane signal
transduction mechanisms, which regulate numerous cellular
processes, including secretion, neural activity, metabolism, and
proliferation.
[0004] PLC molecules have been found in a broad spectrum of
organisms including bacteria, simple eukaryotes, plants and animals
(Munnik et al., Biochim. Biophys. Acta. 1389:222-272, (1998)).
Several distinct isoforms of PLC have been identified in animals
and are categorized as PLC-beta, PLC-gamma, and PLC-delta. Subtypes
are designated by adding Arabic numbers after the Greek letters,
eg., PLC-beta-1. PLCs have a molecular mass of 62-68 kDa, and their
amino acid sequences show two regions of significant
similarity.
SUMMARY OF THE INVENTION
[0005] The present invention is based, in part, on the discovery of
a novel human phospholipase, referred to herein as "32544". The
nucleotide sequence of a cDNA encoding 32544 is shown in SEQ ID
NO:1, and the amino acid sequence of a 32544 polypeptide is shown
in SEQ ID NO:2. In addition, the nucleotide sequence of the coding
region is depicted in SEQ ID NO:3.
[0006] Accordingly, in one aspect, the invention features a nucleic
acid molecule which encodes a 32544 protein or polypeptide, e.g., a
biologically active portion of the 32544 protein. In a preferred
embodiment, the isolated nucleic acid molecule encodes a
polypeptide having the amino acid sequence of SEQ ID NO:2. In other
embodiments, the invention provides an isolated 32544 nucleic acid
molecule having the nucleotide sequence shown in SEQ ID NO:1, SEQ
ID NO:3, or the sequence of the DNA insert of the plasmid deposited
with ATCC Accession Number ______. In still other embodiments, the
invention provides nucleic acid molecules that are substantially
identical (e.g., naturally occurring allelic variants) to the
nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the
sequence of the DNA insert of the plasmid deposited with ATCC
Accession Number ______. In other embodiments, the invention
provides a nucleic acid molecule which hybridizes under stringent
hybridization conditions to a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the sequence of
the DNA insert of the plasmid deposited with ATCC Accession Number
______, wherein the nucleic acid encodes a full length 32544
protein or an active fragment thereof.
[0007] In a related aspect, the invention further provides nucleic
acid constructs which include a 32544 nucleic acid molecule
described herein. In certain embodiments, the nucleic acid
molecules of the invention are operatively linked to native or
heterologous regulatory sequences. Also included, are vectors and
host cells containing the 32544 nucleic acid molecules of the
invention e.g., vectors and host cells suitable for producing 32544
nucleic acid molecules and polypeptides.
[0008] In another related aspect, the invention provides nucleic
acid fragments suitable as primers or hybridization probes for the
detection of 32544-encoding nucleic acids.
[0009] In still another related aspect, isolated nucleic acid
molecules that are antisense to a 32544 encoding nucleic acid
molecule are provided.
[0010] In another aspect, the invention features, 32544
polypeptides, and biologically active or antigenic fragments
thereof that are useful, e.g., as reagents or targets in assays
applicable to treatment and diagnosis of 32544-mediated or related
disorders. In another embodiment, the invention provides 32544
polypeptides having a 32544 activity. Preferred polypeptides are
32544 proteins including at least one domain, e.g., a PLC-X domain
(from about amino acids 323-468 of SEQ ID NO:2), a PLC-Y domain
(from about amino acids 621-736 of SEQ ID NO:2), a calcium binding
(C2) domain (from about amino acids 756-848 of SEQ ID NO:2), or a
pleckstrin homology (PH) domain (from about amino acids 0.44-151 of
SEQ ID NO:2), and, preferably, having a 32544 activity, e.g., an
activity as described herein, e.g., the ability to catalyze the
hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (PIP2)
producing diacylglycerol and inositol 1,4,5-trisphosphate
(IP3).
[0011] In other embodiments, the invention provides 32544
polypeptides, e.g., a 32544 polypeptide having the amino acid
sequence shown in SEQ ID NO:2; the amino acid sequence encoded by
the cDNA insert of the plasmid deposited with ATCC Accession Number
______; an amino acid sequence that is substantially identical to
the amino acid sequence shown in SEQ ID NO:2; or an amino acid
sequence encoded by a nucleic acid molecule having a nucleotide
sequence which hybridizes under stringent hybridization conditions
to a nucleic acid molecule comprising the nucleotide sequence of
SEQ ID NO:1, SEQ ID NO:3, or the sequence of the DNA insert of the
plasmid deposited with ATCC.
[0012] Accession Number ______, wherein the nucleic acid encodes a
full length 32544 protein or an active fragment thereof.
[0013] In a related aspect, the invention further provides nucleic
acid constructs which include a 32544 nucleic acid molecule
described herein.
[0014] In a related aspect, the invention provides 32544
polypeptides or fragments operatively linked to non-32544
polypeptides to form fusion proteins.
[0015] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably specifically bind 32544 polypeptides.
[0016] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the 32544 polypeptides or nucleic acids.
[0017] In still another aspect, the invention provides a process
for modulating 32544 polypeptide or nucleic acid expression or
activity, e.g. using the screened compounds. In certain
embodiments, the methods involve treatment of conditions related to
aberrant activity or expression of the 32544 polypeptides or
nucleic acids, such as conditions involving aberrant or deficient
cellular proliferation or differentiation.
[0018] The invention also provides assays for determining the
activity of or the presence or absence of 32544 polypeptides or
nucleic acid molecules in a biological sample, including for
disease diagnosis.
[0019] In further aspect the invention provides assays for
determining the presence or absence of a genetic alteration in a
32544 polypeptide or nucleic acid molecule, including for disease
diagnosis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1A-C depicts a cDNA sequence (SEQ ID NO:1) and
predicted amino acid sequence (SEQ ID NO:2) of human 32544. The
methionine-initiated open reading frame of human 32544 (without the
5' and 3' untranslated regions) extends from nucleotide position 1
to position 3621 of SEQ ID NO:3, not including the terminal
codon.
[0021] FIG. 2 depicts a hydropathy plot of human 32544. Relatively
hydrophobic residues are shown above the dashed horizontal line,
and relatively hydrophilic residues are below the dashed horizontal
line. The location of the transmembrane domains, and the
extracellular and intracellular portions is also indicated. The
cysteine residues (cys) and N-glycosylation sites (Ngly) are
indicated by short vertical lines just below the hydropathy trace.
The numbers corresponding to the amino acid sequence of human 32544
are indicated. Polypeptides of the invention include fragments
which include: all or part of a hydrophobic sequence, e.g., a
sequence above the dashed line, e.g., the sequence from about amino
acid 25 to 40, from about 180 to 190, and from about 815 to 830 SEQ
ID NO:2; all or part of a hydrophilic sequence, e.g., a sequence
below the dashed line, e.g., the sequence from about amino acid 60
to 80, from about 160 to 180, and from about 590 to 610 of SEQ ID
NO:2; a sequence which includes a Cys, or a glycosylation site.
[0022] FIG. 3 depicts an alignment of the PH domain of human 32544
with a consensus amino acid sequence derived from a hidden Markov
model (HMM) from PFAM. The upper sequences are the consensus amino
acid sequence (SEQ ID NO:4), while the lower amino acid sequences
correspond to amino acids 44 to 151 of SEQ ID NO:2.
[0023] FIGS. 4a-b depict an alignment of the EF hand domain of
human 32544 with a consensus amino acid sequence derived from a
hidden Markov model (HMM) from PFAM. The upper sequences are the
consensus amino acid sequence (SEQ ID NOs:5-6), while the lower
amino acid sequences correspond to amino acids 169 to 197 and 205
to 234 of SEQ ID NO:2.
[0024] FIG. 5 depicts an alignment of the
phosphatidylinositol-specific phospholipase X domain of human 32544
with a consensus amino acid sequence derived from a hidden Markov
model (HMM) from PFAM. The upper sequences are the consensus amino
acid sequence (SEQ ID NO:7), while the lower amino acid sequences
correspond to amino acids 323 to 468 of SEQ ID NO:2.
[0025] FIG. 6 depicts an alignment of the
phosphatidylinositol-specific phospholipase Y domain of human 32544
with a consensus amino acid sequence derived from a hidden Markov
model (HMM) from PFAM. The upper sequences are the consensus amino
acid sequence (SEQ ID NO:8), while the lower amino acid sequences
correspond to amino acids 621 to 736 of SEQ ID NO:2.
[0026] FIG. 7 depicts an alignment of the C2 domain of human 32544
with a consensus amino acid sequence derived from a hidden Markov
model (HMM) from PFAM. The upper sequences are the consensus amino
acid sequence (SEQ ID NO:9), while the lower amino acid sequences
correspond to amino acids 756 to 848 of SEQ ID NO:2.
[0027] FIG. 8 depicts a BLAST alignment of human 32544 with a
consensus amino acid sequence derived from a ProDomain "KIAA0450"
(PD183899) (Release 2001.1;
http://www.toulouse.inra.fr/prodom.html). The lower sequence is
amino acid residues 101 to 425 of the 425 amino acid consensus
sequence (SEQ ID NO:10), while the upper amino acid sequence
corresponds to the "KIAA0450" domain of human 32544, amino acid
residues 883 to 1207 of SEQ ID NO:2.
[0028] FIGS. 9a-c depict a BLAST alignment of human 32544 with a
consensus amino acid sequence derived from a ProDomain
"phospholipase phosphodiesterase hydrolase
phosphoinositide-specific 1-phosphatidylinositol-45-bisphosphate
degradation tranducer lipid beta" (PD001214) (Release 2001.1;
http://www.toulouse.inra.fr/prodom.html). The lower sequence is
amino acid residues 2 to 159, 162 to 202, and 151 to 168 of the 219
amino acid consensus sequence (SEQ ID NOs:11-13), while the upper
amino acid sequence corresponds to the "phospholipase
phosphodiesterase hydrolase phosphoinositide-specific
1-phosphatidylinositol-45-bisphosphate degradation tranducer lipid
beta" domain of human 32544, amino acid residues 307 to 456, 514 to
562, and 742 to 759 of SEQ ID NO:2. FIG. 9a depicts the first local
alignment, FIG. 9b the second, and FIG. 9c the third.
[0029] FIG. 10 depicts a BLAST alignment of human 32544 with a
consensus amino acid sequence derived from a ProDomain
"phospholipase C delta calcium-binding PLC-III hydrolase
phosphodiesterase lipid PLC-delta-1
1-phosphatidylinositol-45-bisphosphate" (PD186804) (Release 2001.1;
http://www.toulouse.inra.fr/prodom.html). The lower sequence is
amino acid residues 14 to 194 of the 203 amino acid consensus
sequence (SEQ ID NO:14), while the upper amino acid sequence
corresponds to the "phospholipase C delta calcium-binding PLC-III
hydrolase phosphodiesterase lipid PLC-delta-1
1-phosphatidylinositol-45-bisphosphat- e" domain of human 32544,
amino acid residues 41 to 214 of SEQ ID NO:2.
[0030] FIG. 11 depicts a BLAST alignment of human 32544 with a
consensus amino acid sequence derived from a ProDomain
"phospholipase binding C KDA-INS145P3 K10F12.3" (PD023751) (Release
2001.1; http://www.toulouse.inra.fr/prodom.html). The lower
sequence is amino acid residues 2 to 135 of the 136 amino acid
consensus sequence (SEQ ID NO:15), while the upper amino acid
sequence corresponds to the "phospholipase binding C KDA-INS145P3
K10F12.3" domain of human 32544, amino acid residues 174 to 304 of
SEQ ID NO:2.
[0031] FIG. 12 depicts a BLAST alignment of human 32544 with a
consensus amino acid sequence derived from a ProDomain "FLJ12548
similar FIS cDNA phosphatidylinositol-45-bisphosphate NT2RM4000657
delta phosphodiesterase weakly 1-" (PD308221) (Release 2001.1;
http://www.toulouse.inra.fr/prodom- .html). The lower sequence is
amino acid residues 2 to 93 of the 187 amino acid consensus
sequence (SEQ ID NO:16), while the upper amino acid sequence
corresponds to the "FLJ12548 similar FIS cDNA
phosphatidylinositol-45-bisphosphate NT2RM4000657 delta
phosphodiesterase weakly 1-" domain of human 32544, amino acid
residues 851 to 944 of SEQ ID NO:2.
[0032] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DETAILED DESCRIPTION
[0033] Human 32544
[0034] The human 32544 sequence (FIG. 1; SEQ ID NO:1), which is
approximately 4635 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
3621 nucleotides (nucleotides 435-4055 of SEQ ID NO:1; SEQ ID
NO:3), not including the terminal codon. The coding sequence
encodes a 1207 amino acid protein (SEQ ID NO:2).
[0035] This mature protein form is approximately 1207 amino acid
residues in length (from about amino acid 1 to amino acid 1207 of
SEQ ID NO:2). Human 32544 may contain the following regions or
other structural features:
[0036] a tyrosine kinase phosphorylation site (PS00007) from about
amino acid residues 620-628 of SEQ ID NO:2;
[0037] three amidation sites (PS00009) from about amino acid
residues 465-468, 546-549, and 935-938 of SEQ ID NO:2;
[0038] an EF-hand calcium-binding domain (PS00018) from about amino
acid residues 178-190;
[0039] four N-glycosylation sites (PS00001) located at about amino
acids 290-293, 303-306, 472-475, and 534-537 of SEQ ID NO:2;
[0040] one cAMP- and cGMP-dependent protein kinase phosphorylation
site (PS00004) at about amino acids 581-584 of SEQ ID NO:2;
[0041] seventeen predicted protein kinase C sites (PS00005) at
about amino acids 79-81, 124-126, 235-237, 387-389, 504-506,
512-514, 543-545, 565-567, 577-579, 615-617, 681-683, 881-883,
918-920, 932-934, 980-982, 1111-1113, and 1180-1182 of SEQ ID NO:2;
and
[0042] eighteen predicted casein kinase II phosphorylation sites
(PS00006) located at about amino acids 91-94, 124-127, 134-137,
154-157, 173-176, 186-189, 236-239, 276-279, 350-353, 445-448,
483-486, 487-490, 550-553, 558-561, 591-594, 649-652, 812-815, and
1100-1103 of SEQ ID NO:2.
[0043] For general information regarding PFAM identifiers, PS
prefix and PF prefix domain identification numbers, refer to
Sonnhammer et al. (1997) Protein 28:405-420 and
http//www.psc.edu/general/software/packages- /pfam/pfam.html.
[0044] A plasmid containing the nucleotide sequence encoding human
32544 was deposited with American Type Culture Collection (ATCC),
10801 University Boulevard, Manassas, Va. 20110-2209, on and
assigned Accession Number ______. This deposit will be maintained
under the terms of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purposes of
Patent Procedure. This deposit was made merely as a convenience for
those of skill in the art and is not an admission that a deposit is
required under 35 U.S.C. .sctn.112.
[0045] To identify the presence of a "phospholipase family" domain
in a 32544-protein sequence (Pfam accession number 00387 and
00388), and make the determination that a polypeptide or protein of
interest has a particular profile, the amino acid sequence of the
protein can be searched against a database of HMMs (e.g., the Pfam
database, release 2.1) using the default parameters
(http://www.sanger.ac.uk/Software/Pfam/- HMM_search). For example,
the hmmsf program, which is available as part of the HMMER package
of search programs, is a family specific default program for
MILPAT0063 and a score of 15 is the default threshold score for
determining a hit. Alternatively, the threshold score for
determining a hit can be lowered (e.g., to 8 bits). A description
of the Pfam database can be found in Sonhammer et al., (1997)
Proteins 28(3):405-420 and a detailed description of HMMs can be
found, for example, in Gribskov et al., (1990) Meth. Enzymol.
183:146-159; Gribskov et al., (1987) Proc. Natl. Acad. Sci. USA
84:4355-4358; Krogh et al., (1994) J. Mol. Biol. 235:1501-1531; and
Stultz et al., (1993) Protein Sci. 2:305-314, the contents of which
are incorporated herein by reference.
[0046] The 32544 protein contains a significant number of
structural characteristics in common with members of the
phospholipase family. The term "family" when referring to the
protein and nucleic acid molecules of the invention means two or
more proteins or nucleic acid molecules having a common structural
domain or motif and having sufficient amino acid or nucleotide
sequence homology as defined herein. Such family members can be
naturally or non-naturally occurring and can be from either the
same or different species. For example, a family can contain a
first protein of human origin as well as other distinct proteins of
human origin, or alternatively, can contain homologues of non-human
origin, e.g., rat or mouse proteins. Members of a family can also
have common functional domains such as a pleckstrin homology
domain, a phosphatidylinositol-spec- ific phospholipase domain X
(PLC-X) domain, a phosphatidylinositol-specifi- c phospholipase
domain Y (PLC-Y) domain, an EF hand domain, or a C2 domain. Members
of this family can also have common functional characteristics,
e.g., the ability to hydrolyze phosphatidylinositols.
[0047] A 32544 polypeptide can include an "EF hand domain" or
regions homologous with an "EF hand domain". As used herein, the
term "EF hand domain" refers to a protein domain having an amino
acid sequence of about 10 to 50, preferably about 10 to 40, more
preferably about 29-30 amino acid residues. By an "EF hand domain"
is meant a domain that consists of a twelve-residue loop flanked-on
both side by a twelve residue alpha-helical domain. In an EF-hand
loop the calcium ion is coordinated in a pentagonal bipyramidal
configuration. The six residues involved in the binding are in
positions 1, 3, 5, 7, 9 and 12; these residues are denoted by X, Y,
Z, -Y, -X and -Z. The invariant Glu or Asp at position 12 provides
two oxygens for liganding Ca (bidentate ligand). As used herein,
the term "EF hand" includes an amino acid sequence of about 28
amino acid residues in length and having a bit score for the
alignment of the sequence to the EF hand domain (HMM) of at least
10. Preferably, an EF hand domain includes at least about 10-50
amino acids, more preferably about 10-35 amino acid residues, or
about 20-30 amino acids and has a bit score for the alignment of
the sequence to the EF hand domain (HMM) of at least 15, 20, or
greater. An alignment of the EF hand domain (amino acids 169 to 197
and 205 to 234 of SEQ ID NO:2) of human EF hand with a consensus
amino acid sequence derived from a hidden Markov model is depicted
in FIG. 4.
[0048] Preferably, the EF hand domain includes the following amino
acid consensus sequence having Prosite signature as PS00018, or
sequences homologous thereto:
D-x-[DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]-{GP}-[LIVMC]-[D-
ENQSTAGC]-x(2)-[DE]-[LIVMFYW] (SEQ ID NO:17). In the above
conserved motif, and other motifs described herein, the standard
IUPAC one-letter code for the amino acids is used. Each element in
the pattern is separated by a dash (-); square brackets ([ ])
indicate the particular residues that are accepted at that
position; x indicates that any residue is accepted at that
position; and numbers in parentheses (( )) indicate the number of
residues represented by the accompanying amino acid. The EF hand
domain of 32544 is found in the N-terminal cytoplasmic domain.
[0049] A 32544 polypeptide can include a "pleckstrin homology (PH)
domain" or regions homologous with a "PH domain". As used herein,
the term "PH domain" refers to a protein domain having an amino
acid sequence of about 10 to 200, preferably about 50 to 150, more
preferably about 108 amino acid residues. By "PH domain" is meant a
domain that can function as a recognition site for a
phosphatidylinositol, e.g., a 3,4,5-trisphosphate (PIP3) or another
kinase ligand product, and can function as a means to localize PLC
to the cytoplasmic face of the plasma membrane. As used herein, the
term "PH domain" includes an amino acid sequence of about 108 amino
acid residues in length and having a bit score for the alignment of
the sequence to the PH domain (HMM) of at least 10. Preferably, a
PH domain includes at least about 10-200 amino acids, more
preferably about 15-150 amino acid residues, or about 50-110 amino
acids and has a bit score for the alignment of the sequence to the
PH domain (HMM) of at least 15, 20, or greater. An alignment of the
PH domain (amino acids 44 to 151 of SEQ ID NO:2) of human PH with a
consensus amino acid sequence derived from a hidden Markov model is
depicted in FIG. 3.
[0050] In a preferred embodiment 32544 polypeptide or protein has a
"PH domain" or a region which includes at least about 10-200 amino
acids, more preferably about 15-150 amino acid residues, or about
50-110 amino acid residues and has at least about 60%, 70% 80% 90%
95%, 99%, or 100% homology with a "PH domain," e.g., the PH domain
of human 32544 (e.g., residues 44-151 of SEQ ID NO:2).
[0051] A 32544 polypeptide can also include a
"phosphatidylinositol-specif- ic phospholipase C domain X (referred
to herein as "PLC-X domain")" or regions homologous with a "PLC-X
domain". As used herein, the term "PLC-X domain" refers to a
protein domain having an amino acid sequence of about 8 to 200,
preferably about 15 to 170, more preferably about 145 amino acid
residues. By "PLC-X domain" is meant a subdomain that composes the
catalytic site of the phospholipase, e.g., PLC-X subdomain can fold
together with another subdomain, e.g.,
phosphatidylinositol-specific phospholipase C domain Y such that a
functioning catalytic site that hydrolyzes a phosphatidylinositol
is formed, e.g., phosphatidylinositol 4,5-bisphosphate, is
formed.
[0052] The "PLC-X domain" includes an amino acid sequence of about
145 amino acid residues in length and can have a bit score for the
alignment of the sequence to the phosphatidylinositol-specific
phospholipase-C domain X (HMM) of at least 50. Preferably, a PLC-X
domain includes at least about 15-170 amino acids, or at least
about 20-150, or about 145 amino acids and has a bit score for the
alignment of the sequence to the phosphatidylinositol-specific
phospholipase-C domain X (HMM) of at least 60, 70, 80, 90, 100,
150, 200, 250, or greater. An alignment of the PLC-X domain (amino
acids 323 to 468 of SEQ ID NO:2) of human 32544 with a consensus
amino acid sequence derived from a hidden Markov model is depicted
in FIG. 5.
[0053] In a preferred embodiment, the 32544 polypeptide or protein
has a "PLC-X" or a region which includes at least about 8-200, more
preferably about 15-170 or 20-150 amino acid residues and has at
least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a
"PLC-X domain," e.g., the PLC-X domain of human 32544 (e.g.,
residues 323-468 of SEQ ID NO:2).
[0054] A 32544 polypeptide can include a
"phosphatidylinositol-specific phospholipase C domain Y (referred
to herein as PLC-Y domain)" or regions homologous with a "PLC-Y
domain". As used herein, the term "PLC-Y domain" refers to a
protein domain having an amino acid sequence of about 8 to 200,
preferably about 15 to 170, more preferably about 117 amino acid
residues. By "PLC-Y domain" is meant a subdomain that composes the
catalytic site of the phospholipase, e.g., the subdomain can fold
together with another subdomain, e.g., PLC-X domain such that a
functioning catalytic site that hydrolyzes a phosphatidylinositol,
e.g., phosphatidylinositol 4,5-bisphosphate, is formed.
[0055] The "PLC-Y domain" includes an amino acid sequence of about
117 amino acid residues in length and can have a bit score for the
alignment of the sequence to the PLC-Y domain (HMM) of at least 50.
Preferably, a PLC-Y domain includes at least about 15-170 amino
acids, or at least about 20-150, or about 117 amino acids and has a
bit score for the alignment of the sequence to the PLC-Y domain
(HMM) of at least 60, 70, 80, 90, 100, 110, 120, 140, 160, 180, or
greater. An alignment of the PLC-Y domain (amino acids 621 to 736
of SEQ ID NO:2) of human 32544 with a consensus amino acid sequence
derived from a hidden Markov model is depicted in FIG. 6.
[0056] In a preferred embodiment 32544 polypeptide or protein has a
"PLC-Y domain" or a region which includes at least about 8-200,
more preferably about 15-170 or 20-150 amino acid residues and has
at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a
"PLC-Y domain" e.g., PLC-Y domain of human 32544 (e.g., residues
621-736 of SEQ ID NO:2).
[0057] A 32544 polypeptide can include a "calcium (Ca2+) binding
domain (referred to as "C2 domain") or regions-homologous with a
"C2 domain". As used herein, the term "C2 domain" refers to a
protein domain having an amino acid sequence of about 8 to 200,
preferably about 15 to 170, more preferably about 20 to 100, or
still more preferably about 90 amino acid residues. By "C2 domain"
is meant a domain that can mediate interaction with calcium or
phospholipids.
[0058] The "C2 domain" includes an amino acid sequence of about 90
amino acid residues in length and can have a bit score for the
alignment of the sequence to the phosphatidylinositol-specific
phospholipase-C domain Y (HMM) of at least 50. Preferably, a C2
domain includes at least about 8-200, or at least about 15-170, or
at least 20-100, or about 90 amino acids and has a bit score for
the alignment of the sequence to the C2 domain (HMM) of at least
60, 70, 80, 85, or greater. An alignment of the C2 domain (amino
acids 756 to 848 of SEQ ID NO:2) of human 32544 with a consensus
amino acid sequence derived from a hidden Markov model is depicted
in FIG. 7.
[0059] In a preferred embodiment, a 32544 polypeptide or protein
has a "C2 domain" or a region which includes at least about 10-200,
more preferably about 15-170 or 20-100 amino acid residues and has
at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a
"C2 domain," e.g., the C2 domain of human 32544 (e.g., residues
756-848 of SEQ ID NO:2).
[0060] To identify the presence of a "PH" domain, "PLC-X domain,"
"PLC-Y domain," or a "C2" domain in a 32544 protein sequence, and
make the determination that a polypeptide or protein of interest
has a particular profile, the amino acid sequence of the protein
can be searched against a database of HMMs (e.g., the Pfam
database, release 2.1) using the default parameters
(http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example,
the hmmsf program, which is available as part of the HMMER package
of search programs, is a family specific default program for
MILPAT0063 and a score of 15 is the default threshold score for
determining a hit. Alternatively, the threshold score for
determining a hit can be lowered (e.g., to 8 bits). A description
of the Pfam database can be found in Sonhammer et al. (1997)
Proteins 28(3):405-420 and a detailed description of HMMs can be
found, for example, in Gribskov et al.(1990) Meth. Enzymol.
183:146-159; Gribskov et al.(1987) Proc. Natl. Acad. Sci. USA
84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; and
Stultz et al.(1993) Protein Sci. 2:305-314, the contents of which
are incorporated herein by reference. A search was performed
against the HMM database resulting in the identification of a "PH
domain" domain in the amino acid sequence of human 32544 at about
residues 44-151 of SEQ ID NO:2 (see FIG. 3); a "C2 domain" in the
amino acid sequence of human 32544 at about residues 756-848 of SEQ
ID NO:2 (see FIG. 7); an EF hand domain in the amino acid sequence
of human 32544 at about residues 169 to 197 of SEQ. ID. NO: 2 (see
FIG. 4); a "PLC-Y domain" in the amino acid sequence of human 32544
at about residues 621-736 of SEQ ID NO:2 (see FIG. 6); and a "PLC-X
domain" in the amino acid sequence of human 32544 at about residues
323-468 of SEQ ID NO:2 (see FIG. 5).
[0061] To identify the presence of a "phospholipase" domain such as
a PI-PLC-X domain in a 32544 protein sequence, and make the
determination that a polypeptide or protein of interest has a
particular profile, the amino acid sequence of the protein can be
searched against a database of domains, e.g., the ProDom database
(Corpet et al. (1999), Nucl. Acids Res. 27:263-267). The ProDom
protein domain database consists of an automatic compilation of
homologous domains. Current versions of ProDom are built using
recursive PSI-BLAST searches (Altschul S F et al. (1997) Nucleic
Acids Res. 25:3389-3402; Gouzy et al. (1999) Computers and
Chemistry 23:333-340) of the SWISS-PROT 38 and TREMBL protein
databases. The database automatically generates a consensus
sequence for each domain. A BLAST search was performed against the
HMM database resulting in the identification of a phospholipase
domain in the amino acid sequence of human 32544.
[0062] The phospholipase domain is homologous to ProDom family PD
183899 ("KIAA0450" SEQ ID NO:10, ProDomain Release 2001.1;
http://www.toulouse.inra.fr/prodom.html). An alignment of the
phospholipase domain (amino acids 883 to 1207 of SEQ ID NO:2) of
human 32544 with a consensus amino acid sequence (SEQ ID NO:10)
derived from a hidden Markov model is depicted in FIG. 8. The
consensus sequence for SEQ ID NO:8 is 77% identical over amino
acids 883 to 1207 of SEQ ID NO:2 as shown in FIG. 8.
[0063] The phospholipase domain is homologous to ProDom family
PD001214 ("phospholipase phosphodiesterase hydrolase
phosphoinositide-specific 1-phosphatidylinositol-45-bisphosphate
degradation tranducer lipid beta" SEQ ID NOs:11-13, ProDomain
Release 2001.1; http://www.toulouse.inra.fr/p- rodom.html). An
alignment of the phospholipase domain (amino acids 307 to 456, 514
to 562, and 742 to 759 of SEQ ID NO:2) of human 32544 with a
consensus amino acid sequence (SEQ ID NO:10) derived from a hidden
Markov model is depicted in FIG. 9. The consensus sequence for SEQ
ID NO:11 is 55% identical over amino acids 307 to 456 of SEQ ID
NO:2; the consensus sequence for SEQ ID NO:12 is 30% identical over
amino acids 514 to 562 of SEQ ID NO:2; and the consensus sequence
for SEQ ID NO:13 is 33% identical over amino acids 742 to 759 of
SEQ ID NO:2 as shown in FIG. 9.
[0064] The phospholipase domain is homologous to ProDom family
PD186804 ("phospholipase C delta calcium-binding PLC-III hydrolase
phosphodiesterase lipid PLC-delta-1
1-phosphatidylinositol-45-bisphosphat- e" SEQ ID NO:14, ProDomain
Release 2001.1; http://www.toulouse.inra.fr/pro- dom.html). An
alignment of the phospholipase domain (amino acids 41 to 214 of SEQ
ID NO:2) of human 32544 with a consensus amino acid sequence (SEQ
ID NO:14) derived from a hidden Markov model is depicted in FIG.
10. The consensus sequence for SEQ ID NO:14 is 37% identical over
amino acids 41 to 214 of SEQ ID NO:2 as shown in FIG. 10.
[0065] The phospholipase domain is homologous to ProDom family
PD023751 ("phospholipase binding C KDA-INS145P3 K10F12.3" SEQ ID
NO:15, ProDomain Release 2001.1;
http://www.toulouse.inra.fr/prodom.html). An alignment of the
phospholipase domain (amino acids 174 to 304 of SEQ ID NO:2) of
human 32544 with a consensus amino acid sequence (SEQ ID NO:15)
derived from a hidden Markov model is depicted in FIG. 11. The
consensus sequence for SEQ ID NO:14 is 39% identical over amino
acids 174 to 304 of SEQ ID NO:2 as shown in FIG. 11.
[0066] The phospholipase domain is homologous to ProDom family
PD308221 ("FLJ12548 similar FIS cDNA
phosphatidylinositol-45-bisphosphate NT2RM4000657 delta
phosphodiesterase weakly 1-" SEQ ID NO:16, ProDomain Release
2001.1; http://www.toulouse.inra.fr/prodom.html). An alignment of
the phospholipase domain (amino acids 851 to 944 of SEQ ID NO:2) of
human 32544 with a consensus amino acid sequence (SEQ ID NO:15)
derived from a hidden Markov model is depicted in FIG. 12. The
consensus sequence for SEQ ID NO:16 is 39% identical over amino
acids 851 to 944 of SEQ ID NO:2 as shown in FIG. 12.
[0067] An additional method to identify the presence of a
"phospholipase" domain in a 32544 protein sequence, and make the
determination that a polypeptide or protein of interest has a
particular profile, the amino acid sequence of the protein can be
searched against a SMART database (Simple Modular Architecture
Research Tool, http://smart.embl-heidelberg.- de/) of HMMs as
described in Schultz et al. (1998), Proc. Natl. Acad. Sci. USA
95:5857 and Schultz et al. (2000) Nucl. Acids Res 28:231. The
database contains domains identified by profiling with the hidden
Markov models of the HMMer2 search program (R. Durbin et al. (1998)
Biological sequence analysis: probabilistic models of proteins and
nucleic acids. Cambridge University Press.;
http://hmmer.wustl.edu/). The database also is extensively
annotated and monitored by experts to enhance accuracy. A search
was performed against the HMM database resulting in the
identification of a "PH_update" domain in the amino acid sequence
of human 32544 at about residues 44 to 153 of SEQ ID NO:2 (see FIG.
1); a "efh.sub.--1" domain in the amino acid sequence of human
32544 at about residues 169 to 197 and 205 to 234 of SEQ ID NO:2; a
"plcx.sub.--3" domain in the amino acid sequence of human 32544 at
about residues 322 to 467 of SEQ ID NO:2; a "plcy.sub.--3" domain
in the amino acid sequence of human 32544 at about residues 622 to
736 of SEQ ID NO:2; and a "C2.sub.--3c" domain in the amino acid
sequence of human 32544 at about residues 755 to 863 of SEQ ID
NO:2.
[0068] A 32544 polypeptide can include at least one, two, and
preferably three "transmembrane domains" or regions homologous with
a "transmembrane domain". As used herein, the term "transmembrane
domain" includes an amino acid sequence of about 10 to 40 amino
acid residues in length and spans the plasma membrane.
Transmembrane domains are rich in hydrophobic residues, e.g., at
least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a
transmembrane domain are hydrophobic, e.g., leucines, isoleucines,
tyrosines, or tryptophans. Transmembrane domains typically have
alpha-helical structures and are described in, for example,
Zagotta, W. N. et al., (1996) Annual Rev. Neurosci. 19:235-263, the
contents of which are incorporated herein by reference.
[0069] In a preferred embodiment, a 32544 polypeptide or protein
has at least one, two, and preferably three "transmembrane domains"
or a region which includes at least about 12 to 35 more preferably
about 15 to 30 or 15 to 25 amino acid residues and has at least
about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a
"transmembrane domain," e.g., the transmembrane domains of human
32544 (e.g., residues 817 to 834, 1153 to 1176, and 1183 to 1200 of
SEQ ID NO:2). The transmembrane domains of human 32544 is
visualized in the hydropathy plot (FIG. 2) as regions of about 15
to 25 amino acids where the hydropathy trace is mostly above the
horizontal line.
[0070] To identify the presence of a "transmembrane" domain in a
32544 protein sequence, and make the determination that a
polypeptide or protein of interest has a particular profile, the
amino acid sequence of the protein can be analyzed by a
transmembrane prediction method that predicts the secondary
structure and topology of integral membrane proteins based on the
recognition of topological models (MEMSAT, Jones et al., (1994)
Biochemistry 33:3038-3049).
[0071] A 32544 polypeptide can include at least one, two, three,
and preferably four "non-transmembrane regions." As used herein,
the term "non-transmembrane region" includes an amino acid sequence
not identified as a transmembrane domain. The non-transmembrane
regions in 32544 are located at about amino acids 1 to 816, 835 to
1152, 1177 to 1182, and 1201 to 1207 of SEQ ID NO:2.
[0072] The non-transmembrane regions of 32544 include at least one,
and preferably two cytoplasmic regions. When located at the
N-terminus, the cytoplasmic region is referred to herein as the
"N-terminal cytoplasmic domain." As used herein, an "N-terminal
cytoplasmic domain" includes an amino acid sequence having about 1
to 1000, preferably about 1 to 900, more preferably about 1 to 850,
or even more preferably about 1 to 816 amino acid residues in
length and is located inside of a cell or within the cytoplasm of a
cell. The C-terminal amino acid residue of an "N-terminal
cytoplasmic domain" is adjacent to an N-terminal amino acid residue
of a transmembrane domain in a 32544 protein. For example, an
N-terminal cytoplasmic domain is located at about amino acid
residues 1 to 816 of SEQ ID NO:2.
[0073] In a preferred embodiment, a polypeptide or protein has an
N-terminal cytoplasmic domain or a region which includes at least
about 5, preferably about 1 to 1000, and more preferably about 1 to
850 amino acid residues and has at least about 60%, 70% 80% 90%
95%, 99%, or 100% homology with an "N-terminal cytoplasmic domain,"
e.g., the N-terminal cytoplasmic domain of human 32544 (e.g.,
residues 1 to 816 of SEQ ID NO:2).
[0074] In another embodiment, a 32544 protein includes at least one
cytoplasmic loop. As used herein, the term "loop" includes an amino
acid sequence that resides outside of a phospholipid membrane,
having a length of at least about 4, preferably about 5 to 10, more
preferably about 6 to 8 amino acid residues, and has an amino acid
sequence that connects two transmembrane domains within a protein
or polypeptide. Accordingly, the N-terminal amino acid of a loop is
adjacent to a C-terminal amino acid of a transmembrane domain in a
32544 molecule, and the C-terminal amino acid of a loop is adjacent
to an N-terminal amino acid of a transmembrane domain in a 32544
molecule. As used herein, a "cytoplasmic loop" includes a loop
located inside of a cell or within the cytoplasm of a cell. For
example, a "cytoplasmic loop" can be found at about amino acid
residues 1177 to 1182 of SEQ ID NO:2.
[0075] In a preferred embodiment, a 32544 polypeptide or protein
has a cytoplasmic loop or a region which includes at least about 4,
preferably about 5 to 10, and more preferably about 6 to 8 amino
acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or
100% homology with a cytoplasmic loop," e.g., a cytoplasmic loop of
human 32544 (e.g., residues 1177 to 1182 of SEQ ID NO:2).
[0076] The non-transmembrane regions of 32544 also include at least
one, and preferably two, non-cytoplasmic regions. When located at
the C-terminus, the non-cytoplasmic region is referred to herein as
the "C-terminal non-cytoplasmic domain." As used herein, a
"C-terminal non-cytoplasmic domain" includes an amino acid sequence
having about 1 to 10, preferably about 1 to 9, more preferably
about 1 to 8, or even more preferably about 1 to 7 amino acid
residues in length and is located outside of a cell. The N-terminal
amino acid residue of an "C-terminal non-cytoplasmic domain" is
adjacent to a C-terminal amino acid residue of a transmembrane
domain in a 32544 protein. For example, a C-terminal
non-cytoplasmic domain is located at about amino acid residues 1201
to 1207 of SEQ ID NO:2.
[0077] In a preferred embodiment, a polypeptide or protein has a
C-terminal non-cytoplasmic domain or a region which includes at
least about 5, preferably about 1 to 10, and more preferably about
1 to 7 amino acid residues and has at least about 60%, 70% 80% 90%
95%, 99%, or 100% homology with a "C-terminal non-cytoplasmic
domain," e.g., the C-terminal cytoplasmic domain of human 32544
(e.g., residues 1201 to 1207 of SEQ ID NO:2).
[0078] In another embodiment, a 32544 protein includes at least one
non-cytoplasmic loop. As used herein, a "non-cytoplasmic loop"
includes an amino acid sequence located outside of a cell or within
an intracellular organelle. Non-cytoplasmic loops include
extracellular domains (i.e., outside of the cell) and intracellular
domains (i.e., within the cell). When referring to membrane-bound
proteins found in intracellular organelles (e.g., mitochondria,
endoplasmic reticulum, peroxisomes microsomes, vesicles, endosomes,
and lysosomes), non-cytoplasmic loops include those domains of the
protein that reside in the lumen of the organelle or the matrix or
the intermembrane space. For example, a "non-cytoplasmic loop" can
be found at about amino acid residues 835 to 1152 of SEQ ID
NO:2.
[0079] In a preferred embodiment, a 32544 polypeptide or protein
has at least one non-cytoplasmic loop or a region which includes at
least about 4, preferably about 5 to 350, more preferably about 6
to 319 amino acid residues and has at least about 60%, 70% 80% 90%
95%, 99%, or 100% homology with a "non-cytoplasmic loop," e.g., at
least one non-cytoplasmic loop of human 32544 (e.g., residues 835
to 1152 of SEQ ID NO:2).
[0080] A 32544 family member can include at least one PI-PLC-X
domain, at least one PI-PLC-Y domain, at least one C2 domain, at
least one, preferably two EF hand domains, at least one PH domain
domain; and at least one two, three, four, five, six and preferably
seven transmembrane or non-transmembrane domains. Furthermore, a
32544 family member can include at least one tyrosine kinase
phosphorylation site (PS00007); at least one, two, preferably three
amidation sites (PS00009); at least one EF-hand calcium-binding
domain (PS00018); at least one, two, three, preferably four
N-glycosylation sites (PS00001); at least one cAMP-and
cGMP-dependent protein kinase phosphorylation site (PS00004); at
least one, two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, fifteen, sixteen and preferably
seventeen protein kinase C sites (PS00005); and at least one, two,
three, four, five, six, seven, eight, nine, ten, eleven, twelve,
thirteen, fourteen, fifteen, sixteen, seventeen and preferably
eighteen casein kinase II phosphorylation sites (PS00006).
[0081] As the 32544 polypeptides of the invention may modulate
32544-mediated activities, they may be useful as of for developing
novel diagnostic and therapeutic agents for 32544-mediated or
related disorders, as described below. As used herein, a "32544
activity", "biological activity of 32544" or "functional activity
of 32544", refers to an activity exerted by a 32544 protein,
polypeptide or nucleic acid molecule on e.g., a 32544-responsive
cell or on a 32544 substrate, e.g., a protein substrate, as
determined in vivo or in vitro. In one embodiment, a 32544 activity
is a direct activity, such as an association with a 32544 target
molecule. A "target molecule" or "binding partner" is a molecule
with which a 32544 protein binds or interacts with in nature. In an
exemplary embodiment, it is a receptor, e.g., a tyrosine kinase
receptor. In another embodiment, 32544 can associate with a second
messenger molecule such as a specialized adaptor molecule; with
inositol phosphates and inositol lipids; membrane proteins; or with
a guanine nucleotide binding-regulatory protein (G-protein). A
32544 activity can also be an indirect activity, e.g., a cellular
signaling activity mediated by interaction of the 32544 protein
with a receptor or another signaling molecule. For example, the
32544 proteins of the present invention can have one or more of the
following activities: (1) transduction of transmembrane signals;
(2) lipid-metabolizing activity, e.g., 32544 can catalyze the
hydrolysis of phosphatidyl-inositol-4,5-bisp- hophate (PIP2)
producing diacylglycerol and inositol 1,4,5-trisphophate; (3) the
regulation of transmission of signals from cellular receptors such
as hormones such as serotonin, growth factors such as
platelet-derived growth factor (PDGF), fibroblast growth factor
(FGF), and nerve growth factor (NGF), neurotransmitters and
immunoglobulins; (4) modulation of cell proliferation; (5)
modulation of cell differenciation; (6) modulation of cell
migration; (7) modulation of fertilization; (8) modulation of
hypertension; (9) treatment of pain; and (10) the ability to
antagonize or inhibit, competitively or non-competitively, any of
1-9.
[0082] Based on the above-described sequence similarities, the
32544 molecules of the present invention are predicted to have
similar biological activities as members of the PLC family. Members
of the PLC family play a very important role in transmembrane
signal transduction. Extracellular signaling molecules including
hormones, growth factors, neurotransmitters, and immunoglobulins
bind to their respective cell surface receptors and activate
phospholipase-C. PLC molecules have many functions including:
glycogenolysis in liver cells, histamine secretion by mast cells,
serotonin release by blood platelets, aggregation by blood
platelets, insulin release by pancreatic islet cells, epinephrine
secretion by adrenal chromaffin cells, and smooth muscle
contraction. In general, biological systems that are activated by
receptor tyrosine kinase cause the activation of phospholipase-C.
The role of an activated PLC is to catalyze the hydrolysis of
phosphatidyl-inositol-4,5-bisphospha- te (PIP2), a minor component
of the plasma membrane, to produce diacylglycerol and inositol
1,4,5-trisphosphate (IP3). Inositol trisphosphate releases calcium
from intracellular stores and increases the influx of calcium from
the extracellular fluid. The calcium ions directly regulate target
enzymes and indirectly affect other enzymes by functioning as a
second messenger and interacting with calcium-binding proteins,
such as troponin C and calmodulin. For example, calcium ions
regulate muscle contraction, glycogen breakdown and exocytosis.
Diacylglycerol, a product of the hydrolysis by PLCs, acts as a
second messenger by activating protein kinase C. Activated protein
kinase C phosphorylates a great number of intracellular proteins at
the serine and threonine residues and modulates different signaling
pathways. For example, the phosphorylation of glycogen synthase by
protein kinase C stops the synthesis of glycogen. Moreover, protein
kinase C controls cell division and proliferation. Both pathways
are part of transmembrane signal transduction mechanisms, which
regulate cellular processes, which include secretion, neural
activity, metabolism, differenciation and proliferation.
[0083] Thus, the 32544 molecules can act as novel diagnostic
targets and therapeutic agents for controlling disorders caused by
abnormal or aberrant PLC activity. Evidence indicates that a high
percentage of primary human mammary carcinomas concomitantly show
abnormally high levels of PLC-gamma-1 (Kassis et al., Clin Cancer
Res., Aug;5(8):2251-60, 1999). Likewise, studies on spontaneous
hypertensive rats have suggested that one of the main causes for
the hypertension is an abnormal activation of PLC-delta-1 resulting
from point mutations in the X and Y regions of the PLC amino acid
sequence (Sanada et al., Hypertension 33(4):1036-42, 1999).
Therefore, the 32544 molecules can act as novel diagnostic targets
and therapeutic agents for controlling one or more of cellular
proliferative and/or differentiative disorders, disorders
associated with bone metabolism, immune disorders, hematopoietic
disorders, cardiovascular disorders, liver disorders, viral
diseases, pain or metabolic disorders.
[0084] Further, it has been shown that the activation of PLC-delta
isozymes, a member of phosphoinositide-specific phospholipase C
family, may occur as an event secondary to the receptor-mediated
activation of other PLC isozymes or calcium channels. More
recently, it has been also shown that phospholipase C-delta 1 is
activated upon bradykinin stimulation. In this situation it appears
that PLC-delta 1 is activated by capacitative calcium entry
occurring subsequent to IP3 production and calcium release after
PLC-beta activation. This regulation of PLC-delta 1 has an
important meaning since it may represent a positive feedback
mechanism in that the signaling mediated by PLC-beta linked
receptors can be potentiated and prolonged. In addition, the
activation of PLC-delat 1 after bradykinin stimulation is
associated with ah increase in intracellular calcium leading to the
potentiation of norepinephrine (the main neurotransmitter in
sympathetic neurons) secretion. Furthermore, expression of
PLC-delta 1 increases in neurons after glutamate treatment. This
increase can be prevented by removal of extracellular calcium, NMDA
antagonist or NO synthase inhibitor.
[0085] Phospholipase C-delta 1 appears to be an important mediator
in the signaling pathways of hyperlagesic agents, like bradykinin,
and glutamate, one of the main neurotransmiters in the spinal cord
involved in nociceptive transmission. Furthermore, phospholipase
C-delta 1 induces release of a sympathetic neurotransmitter that
has been involved in maintenance of some types of pain states.
Thus, antagonizing 32544 may be beneficial for the treatment of
pain.
[0086] As used herein, a "phospholipase family-associated disorder"
includes a disorder, disease or condition which is caused or
characterized by a misregulation (e.g., downregulation or
upregulation) of a phospholipase family-mediated activity.
Phospholipase family-associated disorders can detrimentally affect
cellular functions such as cellular proliferation, growth,
differentiation, or migration, cellular regulation of homeostasis,
inter- or intra-cellular communication; tissue function, such as
cardiac function or musculoskeletal function; systemic responses in
an organism, such as nervous system responses, hormonal responses
(e.g., insulin response), or immune responses; and protection of
cells from toxic compounds (e.g., carcinogens, toxins, mutagens,
and toxic byproducts of metabolic activity (e.g., reactive oxygen
species)). Accordingly, 32544 protein may mediate various
disorders, including cellular proliferative and/or differentiative
disorders, hormonal disorders, immune disorders, brain disorders,
heart disorders, blood vessel disorders, platelet disorders or pain
or metabolic disorders. As the 32544 polypeptides of the invention
may modulate 32544 mediated activities, they may be useful for
developing novel diagnostic and therapeutic agents for
32544-mediated or related disorders, as described below.
[0087] Phospholipase-family proteins are key molecules involved in
signal transduction through the hydrolysis of phosphatidylinositol
into inositol 1,4,5-trisphosphate and diacylglycerol. Abnormalities
in neurotransmitter signalling coupled to
phosphatidylinositol-specific phospholipase have been reported in
brain tissue from patients with schizophrenia. The regulation of
phospholipase in different regions of the brain result in increased
enzyme levels in some regions and decreased enzyme levels in other
regions. Due to the activity of phospholipase in response to
G-protein-coupled receptors by neuroendocrine growth factors,
phosphatidylinositol specific phospholipase has also been suggested
to be involved in small cell lung carcinoma and non-small cell lung
carcinoma proliferation. The 32544 polypeptides share a common
domain with known phospholipase-family members and is expected to
have similar effects in cellular metabolism. Accordingly, 32544 may
play a role in signal transduction and alteration of
neurotransmitter signalling and thus the 32544 compositions of the
invention (e.g., nucleic acids, polypeptides, proteins, antibodies)
can be used to modulate cellular immune response, and furthermore
can be used in screening assays to identify agents for modulating
cellular signal transduction, as well as in detection or diagnostic
assays to identify conditions such as schizophrenia, small cell
lung carcinoma and non-small cell lung carcinoma.
[0088] Examples of cellular proliferative and/or differentiative
disorders include cancer, e.g., carcinoma, sarcoma, metastatic
disorders or hematopoietic neoplastic disorders, e.g., leukemias. A
metastatic tumor can arise from a multitude of primary tumor types,
including but not limited to those of prostate, colon, lung, breast
and liver origin.
[0089] As used herein, the terms "cancer", "hyperproliferative" and
"neoplastic" refer to cells having the capacity for autonomous
growth, i.e., an abnormal state or condition characterized by
rapidly proliferating cell growth. Hyperproliferative and
neoplastic disease states may be categorized as pathologic, i.e.,
characterizing or constituting a disease state, or may be
categorized as non-pathologic, i.e., a deviation from normal but
not associated with a disease state. The term is meant to include
all types of cancerous growths or oncogenic processes, metastatic
tissues or malignantly transformed cells, tissues, or organs,
irrespective of histopathologic type or stage of invasiveness.
"Pathologic hyperproliferative" cells occur in disease states
characterized by malignant tumor growth. Examples of non-pathologic
hyperproliferative cells include proliferation of cells associated
with wound repair.
[0090] The terms "cancer" or "neoplasms" include malignancies of
the various organ systems, such as affecting lung, breast, thyroid,
lymphoid, gastrointestinal, and genito-urinary tract, as well as
adenocarcinomas which include malignancies such as most colon
cancers, renal-cell carcinoma, prostate cancer and/or testicular
tumors, non-small cell carcinoma of the lung, cancer of the small
intestine and cancer of the esophagus.
[0091] The term "carcinoma" is art recognized and refers to
malignancies of epithelial or endocrine tissues including
respiratory system carcinomas, gastrointestinal system carcinomas,
genitourinary system carcinomas, testicular carcinomas, breast
carcinomas, prostatic carcinomas, endocrine system carcinomas, and
melanomas. Exemplary carcinomas include those forming from tissue
of the cervix, lung, prostate, breast, head and neck, colon and
ovary. The term also includes carcinosarcomas, e.g., which include
malignant tumors composed of carcinomatous and sarcomatous tissues.
An "adenocarcinoma" refers to a carcinoma derived from glandular
tissue or in which the tumor cells form recognizable glandular
structures.
[0092] The term "sarcoma" is art recognized and refers to malignant
tumors of mesenchymal derivation.
[0093] The 32544 nucleic acid and protein of the invention can be
used to monitor, treat and/or diagnose a variety of proliferative
disorders. E.g., such disorders include hematopoietic neoplastic
disorders. As used herein, the term "hematopoietic neoplastic
disorders" includes diseases involving hyperplastic/neoplastic
cells of hematopoietic origin, e.g., arising from myeloid, lymphoid
or erythroid lineages, or precursor cells thereof. Preferably, the
diseases arise from poorly differentiated acute leukemias, e.g.,
erythroblastic leukemia and acute megakaryoblastic leukemia.
Additional exemplary myeloid disorders include, but are not limited
to, acute promyeloid leukemia (APML), acute myelogenous leukemia
(AML) and chronic myelogenous-leukemia (CML) (reviewed in Vaickus,
L., (1991) Crit. Rev. in Oncol./Hemotol. 11:267-97); lymphoid
malignancies include, but are not limited to acute lymphoblastic
leukemia (ALL) which includes B-lineage ALL and T-lineage ALL,
chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),
hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
Additional-forms of malignant lymphomas include, but are not
limited to non-Hodgkin lymphoma and variants thereof, peripheral T
cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous
T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF),
Hodgkin's disease and Reed-Sternberg disease.
[0094] Phospholipase family-associated or related disorders also
include hormonal disorders, such as conditions or diseases in which
the production and/or regulation of hormones in an organism is
aberrant. Examples of such disorders and diseases include type I
and type II diabetes mellitus, pituitary disorders (e.g., growth
disorders), thyroid disorders (e.g., hypothyroidism or
hyperthyroidism), and reproductive or fertility disorders (e.g.,
disorders which affect the organs of the reproductive system, e.g.,
the prostate gland, the uterus, or the vagina; disorders which
involve an imbalance in the levels of a reproductive hormone in a
subject; disorders affecting the ability of a subject to reproduce;
and disorders affecting secondary sex characteristic development,
e.g., adrenal hyperplasia).
[0095] Phospholipase family-associated or related disorders also
include immune disorders, such as autoimmune disorders or immune
deficiency disorders, e.g., congenital X-linked infantile
hypogammaglobulinemia, transient hypogammaglobulinemia, common
variable immunodeficiency, selective IgA deficiency, chronic
mucocutaneous candidiasis, or severe combined immunodeficiency.
[0096] Disorders involving the brain include, but are not limited
to, disorders involving neurons, and disorders involving glia, such
as astrocytes, oligodendrocytes, ependymal cells, and microglia;
cerebral edema, raised intracranial pressure and herniation, and
hydrocephalus; malformations and developmental diseases, such as
neural tube defects, forebrain anomalies, posterior fossa
anomalies, and syringomyelia and hydromyelia; perinatal brain
injury; cerebrovascular diseases, such as those related to hypoxia,
ischemia, and infarction, including hypotension, hypoperfusion, and
low-flow states--global cerebral ischemia and focal cerebral
ischemia--infarction from obstruction of local blood supply,
intracranial hemorrhage, including intracerebral (intraparenchymal)
hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms,
and vascular malformations, hypertensive cerebrovascular disease,
including lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-borne
(Arbo) viral encephalitis, Herpes simplex virus Type I, Herpes
simplex virus Type 2, Varicella-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including HIV-1 meningoencephalitis (subacute
encephalitis), vacuolar myelopathy, AIDS-associated myopathy,
peripheral neuropathy, and ADS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyelinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyelitis and acute
necrotizing hemorrhagic encephalomyelitis, and other diseases with
demyelination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer disease and Pick
disease, degenerative diseases of basal ganglia and brain stem,
including Parkinsonism, idiopathic Parkinson disease (paralysis
agitans), progressive supranuclear palsy, corticobasal degenration,
multiple system atrophy, including striatonigral degenration,
Shy-Drager syndrome, and olivopontocerebellar atrophy, and
Huntington disease; spinocerebellar degenerations, including
spinocerebellar ataxias, including Friedreich ataxia, and
ataxia-telanglectasia, degenerative diseases affecting motor
neurons, including amyotrophic lateral sclerosis (motor neuron
disease), bulbospinal atrophy (Kennedy syndrome), and spinal
muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin B.sub.1) deficiency and vitamin B.sub.12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycemia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastoma multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain
stem glioma, oligodendroglioma, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), and neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type 1
neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease.
[0097] Disorders involving the heart, include but are not limited
to, heart failure, including but not limited to, cardiac
hypertrophy, left-sided heart failure, and right-sided heart
failure; ischemic heart disease, including but not limited to
angina pectoris, myocardial infarction, chronic ischemic heart
disease, and sudden cardiac death; hypertensive heart disease,
including but not limited to, systemic (left-sided) hypertensive
heart disease and pulmonary (right-sided) hypertensive heart
disease; valvular heart disease, including but not limited to,
valvular degeneration caused by calcification, such as calcific
aortic stenosis, calcification of a congenitally bicuspid aortic
valve, and mitral annular calcification, and myxomatous
degeneration of the mitral valve (mitral valve prolapse), rheumatic
fever and rheumatic heart disease, infective endocarditis, and
noninfected vegetations, such as nonbacterial thrombotic
endocarditis and endocarditis of systemic lupus erythematosus
(Libman-Sacks disease), carcinoid heart disease, and complications
of artificial valves; myocardial disease, including but not limited
to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, and myocarditis; pericardial disease, including but
not limited to, pericardial effusion and hemopericardium and
pericarditis, including acute pericarditis and healed pericarditis,
and rheumatoid heart disease; neoplastic heart disease, including
but not limited to, primary cardiac tumors, such as myxoma, lipoma,
papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac
effects of noncardiac neoplasms; congenital heart disease,
including but not limited to, left to right shunts--late cyanosis,
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosus, and atrioventricular septal defect,
right-to-left shunts--early cyanosis, such as tetralogy of fallot,
transposition of great arteries, truncus arteriosus, tricuspid
atresia, and total anomalous pulmonary venous connection,
obstructive congenital anomalies, such as coarctation of aorta,
pulmonary-stenosis and atresia, and aortic stenosis and atresia,
and disorders involving cardiac transplantation.
[0098] Disorders involving blood vessels include, but are not
limited to, responses of vascular cell walls to injury, such as
endothelial dysfunction and endothelial activation and intimal
thickening; vascular diseases including, but not limited to,
congenital anomalies, such as arteriovenous fistula,
atherosclerosis, and hypertensive vascular disease, such as
hypertension; inflammatory disease--the vasculitides, such as giant
cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa
(classic), Kawasaki syndrome (mucocutaneous lymph node syndrome),
microscopic polyanglitis (microscopic polyarteritis,
hypersensitivity or leukocytoclastic anglitis), Wegener
granulomatosis, thromboanglitis obliterans (Buerger disease),
vasculitis associated with other disorders, and infectious
arteritis; Raynaud disease; aneurysms and dissection, such as
abdominal aortic aneurysms, syphilitic (luetic) aneurysms, and
aortic dissection (dissecting hematoma); disorders of veins and
lymphatics, such as varicose veins, thrombophlebitis and
phlebothrombosis, obstruction of superior vena cava (superior vena
cava syndrome), obstruction of inferior vena cava (inferior vena
cava syndrome), and lymphangitis and lymphedema; tumors, including
benign tumors and tumor-like conditions, such as hemangioma,
lymphangioma, glomus tumor (glomangioma), vascular ectasias, and
bacillary angiomatosis, and intermediate-grade (borderline
low-grade malignant) tumors, such as Kaposi sarcoma and
hemangloendothelioma, and malignant tumors, such as angiosarcoma
and hemangiopericytoma; and pathology of therapeutic interventions
in vascular disease, such as balloon angioplasty and related
techniques and vascular replacement, such as coronary artery bypass
graft surgery.
[0099] Additionally, 32544 may play an important role in the
regulation of metabolism or pain disorders. Diseases of metabolic
imbalance include, but are not limited to, obesity, anorexia
nervosa, cachexia, lipid disorders, and diabetes. Examples of pain
disorders include, but are not limited to, pain response elicited
during various forms of tissue injury, e.g., inflammation,
infection, and ischemia, usually referred to as hyperalgesia
(described in, for example, Fields, H. L., (1987) Pain, New
York:McGraw-Hill); pain associated with muscoloskeletal disorders,
e.g., joint pain; tooth pain; headaches; pain associated with
surgery; pain related to irritable bowel syndrome; or chest
pain.
[0100] The 32544 protein, fragments thereof, and derivatives and
other variants of the sequence in SEQ ID NO:2 are collectively
referred to as "polypeptides or proteins of the invention" or
"32544 polypeptides or proteins". Nucleic acid molecules encoding
such polypeptides or proteins are collectively referred to as
"nucleic acids of the invention" or "32544 nucleic acids." 32544
molecules refer to 32544 nucleic acids, polypeptides, and
antibodies.
[0101] As used herein, the term "nucleic acid molecule" includes
DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules
(e.g., an mRNA) and analogs of the DNA or RNA generated, e.g., by
the use of nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0102] The term "isolated or purified nucleic acid molecule"
includes nucleic acid molecules which are separated from other
nucleic acid molecules which are present in the natural source of
the nucleic acid. For example, with regards to genomic DNA, the
term "isolated" includes nucleic acid molecules which are separated
from the chromosome with which the genomic DNA is naturally
associated. Preferably, an "isolated" nucleic acid is free of
sequences which naturally flank the nucleic acid (i.e., sequences
located at the 5' and/or 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
For example, in various embodiments, the isolated nucleic acid
molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,
0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences which
naturally flank the nucleic acid molecule in genomic DNA of the
cell from which the nucleic acid is derived. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically
synthesized.
[0103] As used herein, the term "hybridizes under stringent
conditions" describes conditions for hybridization and washing.
Stringent conditions are known to those skilled in the art and can
be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous
methods are described in that reference and either can be used. A
preferred, example of stringent hybridization conditions are
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 50.degree. C. Another example of
stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
55.degree. C. A further example of stringent hybridization
conditions are hybridization in 6.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by one or more
washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C. Preferably,
stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
65.degree. C. Particularly preferred stringency conditions (and the
conditions that should be used if the practitioner is uncertain
about what conditions should be applied to determine if a molecule
is within a hybridization limitation of the invention) are 0.5M
Sodium Phosphate, 7% SDS at 65.degree. C., followed by one or more
washes at 0.2.times.SSC, 1% SDS at 65.degree. C. Preferably, an
isolated nucleic acid molecule of the invention that hybridizes
under stringent conditions to the sequence of SEQ ID NO:1, or SEQ
ID NO:3, corresponds to a naturally-occurring nucleic acid
molecule.
[0104] As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural
protein).
[0105] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules which include an open reading frame
encoding a 32544 protein, preferably a mammalian 32544 protein, and
can further include non-coding regulatory sequences, and
introns.
[0106] An "isolated" or "purified" polypeptide or protein is
substantially free of cellular material or other contaminating
proteins from the cell or tissue source from which the protein is
derived, or substantially free from chemical precursors or other
chemicals when chemically synthesized. In one embodiment, the
language "substantially free" means preparation of 32544 protein
having less than about 30%, 20%, 10% and more preferably 5% (by dry
weight), of non-32544 protein (also referred to herein as a
"contaminating protein"), or of chemical precursors or non-32544
chemicals. When the 32544 protein or biologically active portion
thereof is recombinantly produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, more preferably less than about
10%, and most preferably less than about 5% of the volume of the
protein preparation. The invention includes isolated or purified
preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry
weight.
[0107] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of 32544 (e.g., the sequence
of SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
______) without abolishing or more preferably, without
substantially altering a biological activity, whereas an
"essential" amino acid residue results in such a change. For
example, amino acid residues that are conserved among the
polypeptides of the present invention, e.g., those present in the
phospholipase family domain, are predicted to be particularly
unamenable to alteration.
[0108] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a 32544 protein is
preferably replaced with another amino acid residue from the same
side chain family. Alternatively, in another embodiment, mutations
can be introduced randomly along all or part of a 32544 coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for 32544 biological activity to identify
mutants that retain activity. Following mutagenesis of SEQ ID NO:1,
SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC as Accession Number ______, the encoded
protein can be expressed recombinantly and the activity of the
protein can be As used herein, a "biologically active portion" of a
32544 protein includes a fragment of a 32544 protein which
participates in an interaction between a 32544 molecule and a
non-32544 molecule. Biologically active portions of a 32544 protein
include peptides comprising amino acid sequences sufficiently
homologous to or derived from the amino acid sequence of the 32544
protein, e.g., the amino acid sequence shown in SEQ ID NO:2, which
include less amino acids than the full length 32544 proteins, and
exhibit at least one activity of a 32544 protein. Typically,
biologically active portions comprise a domain or motif with at
least one activity of the 32544 protein, e.g., (1) transmembrane
signal transduction; (2) lipid-metabolizing activity, e.g., 32544
can catalyze the hydrolysis of
phosphatidyl-inositol-4,5-bisphophate (PIP2) producing
diacylglycerol and inositol 1, 4, 5-trisphophate; (3) the
regulation of transmission of signals from cellular receptors,
e.g., hormones, growth factors, neurotransmitters and
immunoglobulins; (4) modulation of cell proliferation; (5)
modulation of cell differenciation; (6) modulation of cell
migration; (7) modulation of fertilization; and (8) modulation of
hypertension. A biologically active portion of a 32544 protein can
be a polypeptide which is, for example, 10, 25, 50, 100, 200 or
more amino acids in length. Biologically active portions of a 32544
protein can be used as targets for developing agents which modulate
a 32544 mediated activity, e.g., phospholipase family activity.
[0109] Calculations of homology or sequence identity between
sequences (the terms are used interchangeably herein) are performed
as follows.
[0110] To determine the percent identity of two amino acid
sequences, or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, 90%, 100% of the length
of the reference sequence (e.g., when aligning a second sequence to
the 32544 amino acid sequence of SEQ ID NO:2 having 1207 amino acid
residues, at least 362, preferably at least 483, more preferably at
least 604, even more preferably at least 724, and even more
preferably at least 845, 966 or 1086 amino acid residues are
aligned). The amino acid residues or nucleotides at corresponding
amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino
acid residue or nucleotide as the corresponding position in the
second sequence, then the molecules are identical at that position
(as used herein amino acid or nucleic acid "identity" is equivalent
to amino acid or nucleic acid "homology"). The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which need to be introduced
for optimal alignment of the two sequences.
[0111] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch (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
Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In
yet another preferred embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (available at http://www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred
set of parameters (and the one that should be used if the
practitioner is uncertain about what parameters should be applied
to determine if a molecule is within a sequence identity or
homology limitation of the invention) is using a Blossum 62 scoring
matrix with a gap open penalty of 12, a gap extend penalty of 4,
and a frameshift gap penalty of 5.
[0112] The percent identity between two amino acid or nucleotide
sequences can be determined using the algorithm of E. Meyers 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.
[0113] The nucleic acid and protein sequences described herein can
be used as a "query sequence" to perform a search against public
databases to, for example, identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al., (1990) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to 32544 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 32544 protein molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
[0114] "Misexpression or aberrant expression", as used herein,
refers to a non-wild type pattern of gene expression, at the RNA or
protein level. It includes: expression at non-wild type levels,
i.e., over or under expression; a pattern of expression that
differs from wild type in terms of the time or stage at which the
gene is expressed, e.g., increased or decreased expression (as
compared with wild type) at a predetermined developmental period or
stage; a pattern of expression that differs from wild type in terms
of decreased expression (as compared with wild type) in a
predetermined cell type or tissue type; a pattern of expression
that differs from wild type in terms of the splicing size, amino
acid sequence, post-transitional modification, or biological
activity of the expressed polypeptide; a pattern of expression that
differs from wild type in terms of the effect of an environmental
stimulus or extracellular stimulus on expression of the gene, e.g.,
a pattern of increased or decreased expression (as compared with
wild type) in the presence of an increase or decrease in the
strength of the stimulus.
[0115] "Subject", as used herein, can refer to a mammal, e.g., a
human, or to an experimental or animal or disease model. The
subject can also be a non-human animal, e.g., a horse, cow, goat,
or other domestic animal.
[0116] A "purified preparation of cells", as used herein, refers
to, in the case of plant or animal cells, an in vitro preparation
of cells and not an entire intact plant or animal. In the case of
cultured cells or microbial cells, it consists of a preparation of
at least 10% and more preferably 50% of the subject cells.
[0117] Various aspects of the invention are described in further
detail below.
[0118] Isolated Nucleic Acid Molecules
[0119] In one aspect, the invention provides, an isolated or
purified, nucleic acid molecule that encodes a 32544 polypeptide
described herein, e.g., a full length 32544 protein or a fragment
thereof, e.g., a biologically active portion of 32544 protein. Also
included is a nucleic acid fragment suitable for use as a
hybridization probe, which can be used, e.g., to a identify nucleic
acid molecule encoding a polypeptide of the invention, 32544 mRNA,
and fragments suitable for use as primers, e.g., PCR primers for
the amplification or mutation of nucleic acid molecules.
[0120] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO:1, or
the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC as Accession Number ______, or a portion of any of these
nucleotide sequences. In one embodiment, the nucleic acid molecule
includes sequences encoding the human 32544 protein (i.e., "the
coding region", from nucleotides 435-4055 of SEQ ID NO:1, not
including the terminal codon), as well as 5' untranslated sequences
(nucleotides 1-434 of SEQ ID NO:1). Alternatively, the nucleic acid
molecule can include only the coding region of SEQ ID NO:1 (e.g.,
nucleotides 435-4055 of SEQ ID NO:1, corresponding to SEQ ID NO:3)
and, e.g., no flanking sequences which normally accompany the
subject sequence. In another embodiment, the nucleic acid molecule
encodes a sequence corresponding to the mature protein of SEQ ID
NO:2.
[0121] In another embodiment, an isolated nucleic acid molecule of
the invention includes a nucleic acid molecule which is a
complement of the nucleotide sequence shown in SEQ ID NO:1; SEQ ID
NO:3, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, or a portion of any
of these nucleotide sequences. In other embodiments, the nucleic
acid molecule of the invention is sufficiently complementary to the
nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as Accession Number ______ such that it can hybridize to the
nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as Accession Number ______, thereby forming a stable
duplex.
[0122] In one embodiment, an isolated nucleic acid molecule of the
present invention includes a nucleotide sequence which is at least
about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more homologous to the nucleotide sequence
shown in SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of
the DNA insert of the plasmid deposited with ATCC as Accession
Number ______. In the case of an isolated nucleic acid molecule
which is longer than or equivalent in length to the reference
sequence, e.g., SEQ ID NO:1, or SEQ ID NO:3, the comparison is made
with the full length of the reference sequence. Where the isolated
nucleic acid molecule is shorter than the reference sequence, e.g.,
shorter than SEQ ID NO:1, or SEQ ID NO:3, the comparison is made to
a segment of the reference sequence of the same length (excluding
any loop required by the homology calculation).
[0123] 32544 Nucleic Acid Fragments
[0124] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3,
or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______. For example, such a
nucleic acid molecule can include a fragment which can be used as a
probe or primer or a fragment encoding a portion of a 32544
protein, e.g., an immunogenic or biologically active portion of a
32544 protein. A fragment can comprise e.g., nucleotides 275 to 598
of SEQ ID NO:1, which encodes a PH domain of human 32544;
nucleotides 1112 to 1549 of SEQ ID NO:1, which encodes the PLC-X
domain of human 32544; or nucleotides 2006 to 2353 of SEQ ID NO:1,
which encodes the PLC-Y domain of human 32544. The nucleotide
sequence determined from the cloning of the 32544 gene allows for
the generation of probes and primers designed for use in
identifying and/or cloning other 32544 family members, or fragments
thereof, as well as 32544 homologues, or fragments thereof, from
other species.
[0125] In another embodiment, a nucleic acid includes a nucleotide
sequence that includes part, or all, of the coding region and
extends into either (or both) the 5' or 3' noncoding region. Other
embodiments include a fragment which includes a nucleotide sequence
encoding an amino acid fragment described herein. Nucleic acid
fragments can encode a specific domain or site described herein or
fragments thereof, particularly fragments thereof which are at
least 150 amino acids in length. Fragments also include nucleic
acid sequences corresponding to specific amino acid sequences
described above or fragments thereof. Nucleic acid fragments should
not to be construed as encompassing those fragments that may have
been disclosed prior to the invention.
[0126] A nucleic acid fragment can include a sequence corresponding
to a domain, region, or functional site described herein. A nucleic
acid fragment can also include one or more domain, region, or
functional site described herein. Thus, for example, the nucleic
acid fragment can include a PH domain and a C2 domain. In a
preferred embodiment the fragment is at least, 50, 100, 200, 300,
400, 500, 600, 700, or 900 base pairs in length.
[0127] 32544 probes and primers are provided. Typically a
probe/primer is an isolated or purified oligonucleotide. The
oligonucleotide typically includes a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 7, 12
or 15, preferably about 20 or 25, more preferably about 30, 35, 40,
45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or
antisense sequence of SEQ ID NO:1, SEQ ID NO:3, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number ______, or of a naturally occurring allelic
variant or mutant of SEQ ID NO:1, SEQ ID NO:3, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number ______.
[0128] In a preferred embodiment the nucleic acid is a probe which
is at least 5 or 10, and less than 200, more preferably less than
100, or less than 50, base pairs in length. It should be identical,
or differ by 1, or less than in 5 or 10 bases, from a sequence
disclosed herein. If alignment is needed for this comparison the
sequences should be aligned for maximum homology. "Looped" out
sequences from deletions or insertions, or mismatches, are
considered differences.
[0129] A probe or primer can be derived from the sense or
anti-sense strand of a nucleic acid which encodes: PH domain (from
about amino acids 44-151 of SEQ ID NO:2); a PLC-X domain (from
about amino acids 323-468 of SEQ ID NO:2); a PLC-Y domain (from
about amino acids 621-736 of SEQ ID NO:2); or a C2 domain (from
about amino acids 756-848 of SEQ ID NO:2). In another embodiment a
set of primers is provided, e.g., primers suitable for use in a
PCR, which can be used to amplify a selected region of a 32544
sequence. The primers should be at least 5, 10, or 50 base pairs in
length and less than 100, or less than 200, base pairs in length.
The primers should be identical, or differs by one base from a
sequence disclosed herein or from a naturally occurring variant.
E.g., primers suitable for amplifying a domain or region described
herein are provided, e.g., all or a portion of any of the following
regions: a PLC-X domain (from about amino acids 323-468 of SEQ ID
NO:2), a PLC-Y (from about amino acids 621-736 of SEQ ID NO:2), a
C2 domain (from about amino acids 756-848 of SEQ ID NO:2), or a PH
domain (from about amino acids 44-151 of SEQ ID NO:2).
[0130] In another embodiment a set of primers is provided, e.g.,
primers suitable for use in a PCR, which can be used to amplify a
selected region of a 32544 sequence, e.g., a region described
herein. The primers should be at least 5, 10, or 50 base pairs in
length and less than 100, or less than 200, base pairs in length.
The primers should be identical, or differs by one base from a
sequence disclosed herein or from a naturally occurring variant.
E.g., primers suitable for amplifying all or a portion of any of
the following regions are provided: a phospholipase family domain
(e.g., about nucleotides 1401-1838 of SEQ ID NO:1).
[0131] A nucleic acid fragment can encode an epitope bearing region
of a polypeptide described herein.
[0132] A nucleic acid fragment encoding a "biologically active
portion of a 32544 polypeptide" can be prepared by isolating a
portion of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or
the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC as Accession Number ______, which encodes a polypeptide
having a 32544 biological activity (e.g., the biological activities
of the 32544 proteins as described herein), expressing the encoded
portion of the 32544 protein (e.g., by recombinant expression in
vitro) and assessing the activity of the encoded portion of the
32544 protein. For example, a nucleic acid fragment encoding a
biologically active portion of 32544 includes a phospholipase
family domain (e.g., about nucleotides 1401-1838 of SEQ ID NO:1). A
nucleic acid fragment encoding a biologically active portion of a
32544 polypeptide, may comprise a nucleotide sequence which is
greater than 300-1200 or more nucleotides in length.
[0133] In preferred embodiments, nucleic acids include a nucleotide
sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000,
1100, 1200 nucleotides in length and hybridizes under stringent
hybridization conditions to a nucleic acid molecule of SEQ ID NO:1,
or SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC as Accession Number ______.
[0134] 32544 Nucleic Acid Variants
[0135] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO:1, SEQ
ID NO:3, or the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC as Accession Number ______. Such
differences can be due to degeneracy of the genetic code (and
result in a nucleic acid which encodes the same 32544 proteins as
those encoded by the nucleotide sequence disclosed herein. In
another embodiment, an isolated nucleic acid molecule of the
invention has a nucleotide sequence encoding a protein having an
amino acid sequence which differs, by at least 1, but less than 5,
10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO:2.
If alignment is needed for this comparison the sequences should be
aligned for maximum homology. "Looped" out sequences from deletions
or insertions, or mismatches, are considered differences.
[0136] Nucleic acids of the inventor can be chosen for having
codons, which are preferred, or non preferred, for a particular
expression system. E.g., the nucleic acid can be one in which at
least one colon, at preferably at least 10%, or 20% of the codons
has been altered such that the sequence is optimized for expression
in E. coli, yeast, human, insect, or CHO cells.
[0137] Nucleic acid variants can be naturally occurring, such as
allelic variants (same locus), homologs (different locus), and
orthologs (different organism) or can be non-naturally occurring.
Non-naturally occurring variants can be made by mutagenesis
techniques, including those applied to polynucleotides, cells, or
organisms. The variants can contain nucleotide substitutions,
deletions, inversions and insertions. Variation can occur in either
or both the coding and non-coding regions. The variations can
produce both conservative and non-conservative amino acid
substitutions (as compared in the encoded product).
[0138] In a preferred embodiment, the nucleic acid differs from
that of SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
______, e.g., as follows: by at least one but less than 10, 20, 30,
or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of
the in the subject nucleic acid. If necessary for this analysis the
sequences should be aligned for maximum homology. "Looped" out
sequences from deletions or insertions, or mismatches, are
considered differences.
[0139] Orthologs, homologs, and allelic variants can be identified
using methods known in the art. These variants comprise a
nucleotide sequence encoding a polypeptide that is 50%, at least
about 55%, typically at least about 70-75%, more typically at least
about 80-85%, and most typically at least about 90-95% or more
identical to the amino acid sequence shown in SEQ ID NO:2 or a
fragment of this sequence. Such nucleic acid molecules can readily
be obtained as being able to hybridize under stringent conditions,
to the nucleotide sequence shown in SEQ ID NO:3 or a fragment of
this sequence. Nucleic acid molecules corresponding to orthologs,
homologs, and allelic variants of the 32544 cDNAs of the invention
can further be isolated by mapping to the same chromosome or locus
as the 32544 gene. Preferred variants include those that are
correlated with phospholipase family activity.
[0140] Allelic variants of 32544, e.g., human 32544, include both
functional and non-functional proteins. Functional allelic variants
are naturally occurring amino acid sequence variants of the 32544
protein within a population that maintain the ability to function
similar to the 32544 protein, e.g., to hydrolyze
phosphatidylinositol 4,5-bisphosphate to produce inositol
1,4,5-trisphosphate and diacylglycerol.
[0141] Functional allelic variants will typically contain only
conservative substitution of one or more amino acids of SEQ ID
NO:2, or substitution, deletion or insertion of non-critical
residues in non-critical regions of the protein. Non-functional
allelic variants are naturally-occurring amino acid sequence
variants of the 32544, e.g., human 32544, protein within a
population that do not have the ability to hydrolyze
phosphatidylinositol. Non-functional allelic variants Will
typically contain a non-conservative substitution, a deletion, or
insertion, or premature truncation of the amino acid sequence of
SEQ ID NO:2, or a substitution, insertion, or deletion in critical
residues or critical regions of the protein.
[0142] Moreover, nucleic acid molecules encoding other 32544 family
members and, thus, which have a nucleotide sequence which differs
from the 32544 sequences of SEQ ID NO:1, SEQ ID NO:3, or the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as Accession Number ______ are intended to be within the scope
of the invention.
[0143] Antisense Nucleic Acid Molecules, Ribozymes and Modified
32544 Nucleic Acid Molecules
[0144] In another aspect, the invention features, an isolated
nucleic acid molecule which is antisense to 32544. An "antisense"
nucleic acid can include a nucleotide sequence which is
complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. The antisense
nucleic acid can be complementary to an entire 32544 coding strand,
or to only a portion thereof (e.g., the coding region of human
32544 corresponding to SEQ ID NO:3). In another embodiment, the
antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding
32544 (e.g., the 5' and 3' untranslated regions).
[0145] An antisense nucleic acid can be designed such that it is
complementary to the entire coding region of 32544 mRNA, but more
preferably is an oligonucleotide which is antisense to only a
portion of the coding or noncoding region of 32544 mRNA. For
example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of 32544 mRNA, e.g.,
between the -10 and +10 regions of the target gene nucleotide
sequence of interest. An antisense oligonucleotide can be, for
example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, or more nucleotides in length.
[0146] An antisense nucleic acid of the invention can be
constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used. The antisense nucleic acid also can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0147] The antisense nucleic acid molecules of the invention are
typically administered to a subject (e.g., by direct injection at a
tissue site), or generated in situ such that they hybridize with or
bind to cellular mRNA and/or genomic DNA encoding a 32544 protein
to thereby inhibit expression of the protein, e.g., by inhibiting
transcription and/or translation. Alternatively, antisense nucleic
acid molecules can be modified to target selected cells and then
administered systemically. For systemic administration, antisense
molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell surface, e.g.,
by linking the antisense nucleic acid molecules to peptides or
antibodies which bind to cell surface receptors or antigens. The
antisense nucleic acid molecules can also be delivered to cells
using the vectors described herein. To achieve sufficient
intracellular concentrations of the antisense molecules, vector
constructs in which the antisense nucleic acid molecule is placed
under the control of a strong pol II or pol III promoter are
preferred.
[0148] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al., (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al., (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., (1987)
FEBS Lett. 215:327-330).
[0149] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. A ribozyme having specificity for a
32544-encoding nucleic acid can include one or more sequences
complementary to the nucleotide sequence of a 32544 cDNA disclosed
herein (i.e., SEQ ID NO:1, or SEQ ID NO:3), and a sequence having
known catalytic sequence responsible for mRNA cleavage (see U.S.
Pat. No. 5,093,246 or Haselhoff and Gerlach, (1988) Nature
334:585-591). For example, a derivative of a Tetrahymena L-19 IVS
RNA can be constructed in which the nucleotide sequence of the
active site is complementary to the nucleotide sequence to be
cleaved in a 32544-encoding mRNA. See, e.g., Cech et al. U.S. Pat.
No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.
Alternatively, 32544 mRNA can be used to select a catalytic RNA
having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science
261:1411-1418.
[0150] 32544 gene expression can be inhibited by targeting
nucleotide sequences complementary to the regulatory region of the
32544 (e.g., the 32544 promoter and/or enhancers) to form triple
helical structures that prevent transcription of the 32544 gene in
target cells. See generally, Helene, C., (1991) Anticancer Drug
Des. 6(6):569-84; Helene, C. et al., (1992) Ann. N.Y. Acad. Sci.
660:27-36; and Maher, L. J., (1992) Bioassays 14(12):807-15. The
potential sequences that can be targeted for triple helix formation
can be increased by creating a so-called "switchback" nucleic acid
molecule. Switchback molecules are synthesized in an alternating
5'-3',3'-5' manner, such that they base pair with first one strand
of a duplex and then the other, eliminating the necessity for a
sizeable stretch of either purines or pyrimidines to be present on
one strand of a duplex.
[0151] The invention also provides detectably labeled
oligonucleotide primer and probe molecules. Typically, such labels
are chemiluminescent, fluorescent, radioactive, or
calorimetric.
[0152] A 32544 nucleic acid molecule can be modified at the base
moiety, sugar moiety or phosphate backbone to improve, e.g., the
stability, hybridization, or solubility of the molecule. For
example, the deoxyribose phosphate backbone of the nucleic acid
molecules can be modified to generate peptide nucleic acids (see
Hyrup B. et al., (1996) Bioorganic & Medicinal Chemistry 4 (1):
5-23). As used herein, the terms "peptide nucleic acid" or "PNA"
refers to a nucleic acid mimic, e.g., a DNA mimic, in which the
deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of a PNA can allow for specific hybridization to
DNA and RNA under conditions of low ionic strength. The synthesis
of PNA oligomers can be performed using standard solid phase
peptide synthesis protocols as described in Hyrup B. et al., (1996)
supra; Perry-O'Keefe et al., Proc. Natl. Acad. Sci. 93:
14670-675.
[0153] PNAs of 32544 nucleic acid molecules can be used in
therapeutic and diagnostic applications. For example, PNAs can be
used as antisense or antigene agents for sequence-specific
modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs
of 32544 nucleic acid molecules can also be used in the analysis of
single base pair mutations in a gene, (e.g., by PNA-directed PCR
clamping); as `artificial restriction enzymes` when used in
combination with other enzymes, (e.g., S1 nucleases (Hyrup B.,
(1996) supra)); or as probes or primers for DNA sequencing or
hybridization (Hyrup B. et al., (1996) supra; Perry-O'Keefe
supra).
[0154] In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al., (1989) Proc. Natl.
Acad. Sci. USA 86:6553-6556; Lemaitre et al., (1987) Proc. Natl.
Acad. Sci. USA 84:648-652; PCT Publication No. WO88/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In
addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (See, e.g., Krol et al.,
(1988) Bio-Techniques 6:958-976) or intercalating agents. (See,
e.g., Zon, (1988) Pharm. Res. 5:539-549). To this end, the
oligonucleotide may be conjugated to another molecule, (e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, or hybridization-triggered cleavage agent).
[0155] The invention also includes molecular beacon oligonucleotide
primer and probe molecules having at least one region which is
complementary to a 32544 nucleic acid of the invention, two
complementary regions one having a fluorophore and one a quencher
such that the molecular beacon is useful for quantitating the
presence of the 32544 nucleic acid of the invention in a sample.
Molecular beacon nucleic acids are described, for example, in
Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al., U.S.
Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.
[0156] Isolated 32544 Polypeptides
[0157] In another aspect, the invention features, an isolated 32544
protein, or fragment, e.g., a biologically active portion, for use
as immunogens or antigens to raise or test (or more generally to
bind) anti-32544 antibodies. 32544 protein can be isolated from
cells or tissue sources using standard protein purification
techniques. 32544 protein or fragments thereof can be produced by
recombinant DNA techniques or synthesized chemically.
[0158] Polypeptides of the invention include those which arise as a
result of the existence of multiple genes, alternative
transcription events, alternative RNA splicing events, and
alternative translational and postranslational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same postranslational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of postranslational modifications, e.g., gylcosylation or
cleavage, present when expressed in a native cell.
[0159] In a preferred embodiment, a 32544 polypeptide has one or
more of the following characteristics:
[0160] (i) it has the ability to promote the hydrolysis of a
phosphatidylinositol;
[0161] (ii) it has the ability to promote the transduction of
transmembrane signals;
[0162] (iii) it has lipid-metabolizing activity, e.g., 32544 can
catalyze the hydrolysis of phosphatidyl-inositol-4,5-bisphophate
(PIP2) producing diacylglycerol and inositol
1,4,5-trisphophate;
[0163] (iv) it has the ability to regulate the transmission of
signals from cellular receptors such as hormones such as serotonin,
growth factors such as platelet-derived growth factor (PDGF),
fibroblast growth factor (FGF), and nerve growth factor (NGF),
neurotransmitters and immunoglobulins;
[0164] (v) it has the ability to modulate cell proliferation;
[0165] (vi) it has the ability to modulate cell
differenciation;
[0166] (vii) it has the ability to modulate cell migration;
[0167] (viii) it has the ability to modulate fertilization;
[0168] (ix) it has the ability to modulate hypertension;
[0169] (x) it has the ability to treat pain;
[0170] (xi) it has a molecular weight (e.g., deduced molecular
weight), amino acid composition or other physical characteristic of
32544 of SEQ ID NO:2;
[0171] (xii) it has an overall sequence similarity of at least 60%,
more preferably at least 70, 80, 90, or 95%, with a polypeptide of
SEQ ID NO:2;
[0172] (xiii) it has a PH domain which is preferably about 70%,
80%, 90% or 95% identical to amino acid residues 44-151 of SEQ ID
NO:2;
[0173] (xiv) it has a PLC-X domain which is preferably about 70%,
80%, 90% or 95% identical to amino acid residues 323-468 of SEQ ID
NO:2;
[0174] (xv) it has a PLC-Y domain which is preferably about 70%,
80%, 90% or 95% identical to amino acid residues 621-736 of SEQ ID
NO:2;
[0175] (xvi) it has a C2 domain which is preferably about 70%, 80%,
90% or 95% identical to amino acid residues 756-848 of SEQ ID NO:2;
or
[0176] (xvii) it has at least 70%, preferably 80%, and most
preferably 90% identity to the cysteines found amino acid sequence
of the native protein.
[0177] In a preferred embodiment the 32544 protein, or fragment
thereof, differs from the corresponding sequence in SEQ ID NO: 2.
In one embodiment, it differs by at least one but by less than 15,
10 or 5 amino acid residues. In another it differs from the
corresponding sequence in SEQ ID NO: 2 by at least one residue but
less than 20%, 15%, 10% or 5% of the residues in it differ from the
corresponding sequence in S-EQ ID NO: 2. The differences are,
preferably, differences or changes at a non essential residue or a
conservative substitution. In a preferred embodiment, the
differences are not in one or more of the following domains: the PH
domain, PLC-X domain, PLC-Y domain, the EF hand domain or the C2
domain. In another preferred embodiment one or more differences are
in one or more of the following domains: the PH domain, PLC-X
domain, PLC-Y domain, EF hand domain or the C2 domain.
[0178] Other embodiments include a protein that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such 32544 proteins
differ in amino acid sequence from SEQ ID NO:2, yet retain
biological activity.
[0179] In one embodiment, a biologically active portion of a 32544
protein includes at least one or more of the following domains: the
PH domain, PLC-X domain, PLC-Y domain, or the C2 domain. Moreover,
other biologically active portions, in which other regions of the
protein are deleted, can be prepared by recombinant techniques and
evaluated for one or more of the functional activities of a native
32544 protein.
[0180] In a preferred embodiment, the 32544 protein has an amino
acid sequence shown in SEQ ID NO:2. In other embodiments, the 32544
protein is substantially identical to SEQ ID NO:2. In yet another
embodiment, the 32544 protein is substantially identical to SEQ ID
NO:2 and retains the functional activity of the protein of SEQ ID
NO:2, as described in detail above. Accordingly, in another
embodiment, the 32544 protein is a protein which includes an amino
acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 98% or more identical to SEQ ID NO:2.
[0181] 32544 Chimeric or Fusion Proteins
[0182] In another aspect, the invention provides 32544 chimeric or
fusion proteins. As used herein, a 32544 "chimeric protein" or
"fusion protein" includes a 32544 polypeptide linked to a non-32544
polypeptide. A "non-32544 polypeptide" refers to a polypeptide
having an amino acid sequence corresponding to a protein which is
not substantially homologous to the 32544 protein, e.g., a protein
which is different from the 32544 protein and which is derived from
the same or a different organism. The 32544 polypeptide of the
fusion protein can correspond to all-or a portion e.g., a fragment
described herein of a 32544 amino acid sequence. In a preferred
embodiment, a 32544 fusion protein includes at least one (or two)
biologically active portion of a 32544 protein. The non-32544
polypeptide can be fused to the N-terminus or C-terminus of the
32544 polypeptide.
[0183] The fusion protein can include a moiety which has a high
affinity for a ligand. For example, the fusion protein can be a
GST-32544 fusion protein in which the 32544 sequences are fused to
the C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant 32544. Alternatively,
the fusion protein can be a 32544 protein containing a heterologous
signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian host cells), expression and/or secretion of 32544 can be
increased through use of a heterologous signal sequence.
[0184] Fusion proteins can include all or a part of a serum
protein, e.g., an IgG constant region, or human serum albumin.
[0185] The 32544 fusion proteins of the invention can be
incorporated into pharmaceutical compositions and administered to a
subject in vivo. The 32544 fusion proteins can be used to affect
the bioavailability of a 32544 substrate. 32544 fusion proteins may
be useful therapeutically for the treatment of disorders caused by,
for example, (i) aberrant modification or mutation of a gene
encoding a 32544 protein; (ii) mis-regulation of the 32544 gene;
and (iii) aberrant post-translational modification of a 32544
protein.
[0186] Moreover, the 32544-fusion proteins of the invention can be
used as immunogens to produce anti-32544 antibodies in a subject,
to purify 32544 ligands and in screening assays to identify
molecules which inhibit the interaction of 32544 with a 32544
substrate.
[0187] Expression vectors are commercially available that already
encode a fusion moiety (e.g., a GST polypeptide). A 32544-encoding
nucleic acid can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the 32544 protein.
[0188] Variants of 32544 Proteins
[0189] In another aspect, the invention also features a variant of
a 32544 polypeptide, e.g., which functions as an agonist (mimetics)
or as an antagonist. Variants of the 32544 proteins can be
generated by mutagenesis, e.g., discrete point mutation, the
insertion or deletion of sequences or the truncation of a 32544
protein. An agonist of the 32544 proteins can retain substantially
the same, or a subset, of the biological activities of the
naturally occurring form of a 32544 protein. An antagonist of a
32544 protein can inhibit one or more of the activities of the
naturally occurring form of the 32544 protein by, for example,
competitively modulating a 32544-mediated activity of a 32544
protein. Thus, specific biological effects can be elicited by
treatment with a variant of limited function. Preferably, treatment
of a subject with a variant having a subset of the biological
activities of the naturally occurring form of the protein has fewer
side effects in a subject relative to treatment with the naturally
occurring form of the 32544 protein.
[0190] Variants of a 32544 protein can be identified by screening
combinatorial libraries of mutants, e.g., truncation mutants, of a
32544 protein for agonist or antagonist activity.
[0191] Libraries of fragments e.g., N terminal, C terminal, or
internal fragments, of a 32544 protein coding sequence can be used
to generate a variegated population of fragments for screening and
subsequent selection of variants of a 32544 protein.
[0192] Variants in which a cysteine residues is added or deleted or
in which a residue which is glycosylated is added or deleted are
particularly preferred.
[0193] Methods for screening gene products of combinatorial
libraries made by point mutations or truncation, and for screening
cDNA libraries for gene products having a selected property.
Recursive ensemble mutagenesis (REM), a new technique which
enhances the frequency of functional mutants in the libraries, can
be used in combination with the screening assays to identify 32544
variants (Arkin and Yourvan, (1992) Proc. Natl. Acad. Sci. USA
89:7811-7815; Delgrave et al., (1993) Protein Engineering
6(3):327-331).
[0194] Cell based assays can be exploited to analyze a variegated
32544 library. For example, a library of expression vectors can be
transfected into a cell line, e.g., a cell line, which ordinarily
responds to 32544 in a substrate-dependent manner. The transfected
cells are then contacted with 32544 and the effect of the
expression of the mutant on signaling by the 32544 substrate can be
detected, e.g., by measuring inositol 1,4,5-trisphosphate or
diacylglycerol. Plasmid DNA can then be recovered from the cells
which score for inhibition, or alternatively, potentiation of
signaling by the 32544 substrate, and the individual clones further
characterized.
[0195] In another aspect, the invention features a method of making
a 32544 polypeptide, e.g., a peptide having a non-wild type
activity, e.g., an antagonist, agonist, or super agonist of a
naturally occurring 32544 polypeptide, e.g., a naturally occurring
32544 polypeptide. The method includes: altering the sequence of a
32544 polypeptide, e.g., altering the sequence, e.g., by
substitution or deletion of one or more residues of a non-conserved
region, a domain or residue disclosed herein, and testing the
altered polypeptide for the desired activity.
[0196] In another aspect, the invention features a method of making
a fragment or analog of a 32544 polypeptide a biological activity
of a naturally occurring 32544 polypeptide. The method includes:
altering the sequence, e.g., by substitution or deletion of one or
more residues, of a 32544 polypeptide, e.g., altering the sequence
of a non-conserved region, or a domain or residue described herein,
and testing the altered polypeptide for the desired activity.
[0197] Anti-32544 Antibodies
[0198] In another aspect, the invention provides an anti-32544
antibody. The term "antibody" as used herein refers to an
immunoglobulin molecule or immunologically active portion thereof,
i.e., an antigen-binding portion. Examples of immunologically
active portions of immunoglobulin molecules include F(ab) and
F(ab').sub.2 fragments which can be generated by treating the
antibody with an enzyme such as pepsin.
[0199] The antibody can be a polyclonal, monoclonal, recombinant,
e.g., a chimeric or humanized, fully human, non-human, e.g.,
murine, or single chain antibody. In a preferred embodiment it has
effector function and can fix complement. The antibody can be
coupled to a toxin or imaging agent.
[0200] A full-length 32544 protein or, antigenic peptide fragment
of 32544 can be used as an immunogen or can be used to identify
anti-32544 antibodies made with other immunogens, e.g., cells,
membrane preparations, and the like. The antigenic peptide of 32544
should include at least 8 amino acid residues of the amino acid
sequence shown in SEQ ID NO:2 and encompasses an epitope of 32544.
Preferably, the antigenic peptide includes at least 10 amino acid
residues, more preferably at least 15 amino acid residues, even
more preferably at least 20 amino acid residues, and most
preferably at least 30 amino acid residues.
[0201] Fragments of 32544 which include, e.g., residues 960-980 of
SEQ ID NO:2 can be, e.g., used as immunogens, or used to be
hydrophilic regions of the 32544 protein. Similarly, a fragment of
32544 which includes, e.g., residues 1010-1025 of SEQ ID NO:2 can
be used to make an antibody against what is believed to be a
hydrophobic region of the 32544 protein; a fragment of 32544 which
includes, e.g., residues 323-468 of SEQ ID NO:2 can be used to make
an antibody against what is believed to be the phospholipase family
region of the 32544 protein.
[0202] Antibodies reactive with, or specific for, any of these
regions, or other regions or domains described herein are
provided.
[0203] In a preferred embodiment the antibody fails to bind an Fc
receptor, e.g. it is a type which does not support Fc receptor
binding or has been modified, e.g., by deletion or other mutation,
such that is does not have a functional Fc receptor binding
region.
[0204] Preferred epitopes encompassed by the antigenic peptide are
regions of 32544 are located on the surface of the protein, e.g.,
hydrophilic regions, as well as regions with high antigenicity. For
example, an Emini surface probability analysis of the human 32544
protein sequence can be used to indicate the regions that have a
particularly high probability of being localized to the surface of
the 32544 protein and are thus likely to constitute surface
residues useful for targeting antibody production.
[0205] In a preferred embodiment the antibody binds an epitope on
any domain or region on 32544 proteins described herein.
[0206] Chimeric, humanized, but most preferably, completely human
antibodies are desirable for applications which include repeated
administration, e.g., therapeutic treatment (and some diagnostic
applications) of human patients.
[0207] The anti-32544 antibody can be a single chain antibody. A
single-chain antibody (scFV) may be engineered (see, for example,
Colcher, D. et al., Ann. NY Acad. Sci. 1999 Jun. 30;880:263-80; and
Reiter, Y., Clin. Cancer Res. 1996 February;2(2):245-52). The
single chain antibody can be dimerized or multimerized to generate
multivalent antibodies having specificities for different epitopes
of the same target 32544 protein.
[0208] An anti-32544 antibody (e.g., monoclonal antibody) can be
used to isolate 32544 by standard techniques, such as affinity
chromatography or immunoprecipitation. Moreover, an anti-32544
antibody can be used to detect 32544 protein (e.g., in a cellular
lysate or cell supernatant) in order to evaluate the abundance and
pattern of expression of the protein. Anti-32544 antibodies can be
used diagnostically to monitor protein levels in tissue as part of
a clinical testing procedure, e.g., to, for example, determine the
efficacy of a given treatment regimen. Detection can be facilitated
by coupling (i.e., physically linking) the antibody to a detectable
substance (i.e., antibody labeling). Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0209] Recombinant Expression Vectors, Host Cells and Genetically
Engineered Cells
[0210] In another aspect, the invention includes, vectors,
preferably expression vectors, containing a nucleic acid encoding a
polypeptide described herein. As used herein, the term "vector"
refers to a nucleic acid molecule capable of transporting another
nucleic acid to which it has been linked and can include a plasmid,
cosmid or viral vector. The vector can be capable of autonomous
replication or it can integrate into a host DNA. Viral vectors
include, e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses.
[0211] A vector can include a 32544 nucleic acid in a form suitable
for expression of the nucleic acid in a host cell. Preferably the
recombinant expression vector includes one or more regulatory
sequences operatively linked to the nucleic acid sequence to be
expressed. The term "regulatory sequence" includes promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence, as well as
tissue-specific regulatory and/or inducible sequences. The design
of the expression vector can depend on such factors as the choice
of the host cell to be transformed, the level of expression of
protein desire, and the like. The expression vectors of the
invention can be introduced into host cells to thereby produce
proteins or polypeptides, including fusion proteins or
polypeptides, encoded by nucleic acids as described herein (e.g.,
32544 proteins, mutant forms of 32544 proteins, fusion proteins,
and the like).
[0212] The recombinant expression vectors of the invention can be
designed for expression of 32544 proteins in prokaryotic or
eukaryotic cells. For example, polypeptides of the invention can be
expressed in E. coli, insect cells (e.g., using baculovirus
expression vectors), yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0213] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
protein to enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include
Factor Xa, thrombin and enterokinase. Typical fusion expression
vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and
Johnson, K. S., (1988) Gene 67:31-40), pMAL (New England Biolabs,
Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant protein.
[0214] Purified fusion proteins can be used in 32544 activity
assays, (e.g., direct assays or competitive assays described in
detail below), or to generate antibodies specific for 32544
proteins. In a preferred embodiment, a fusion protein expressed in
a retroviral expression vector of the present invention can be used
to infect bone marrow cells which are subsequently transplanted
into irradiated recipients. The pathology of the subject recipient
is then examined after sufficient time has passed (e.g., six (6)
weeks).
[0215] To maximize recombinant protein expression in E. coli is to
express the protein in host bacteria with an impaired capacity to
proteolytically cleave the recombinant protein (Gottesman, S., Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990) 119-128). Another strategy is to alter the
nucleic acid sequence of the nucleic acid to be inserted into an
expression vector so that the individual codons for each amino acid
are those preferentially utilized in E. coli (Wada et al., (1992)
Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid
sequences of the invention can be carried out by standard DNA
synthesis techniques.
[0216] The 32544 expression vector can be a yeast expression
vector, a vector for expression in insect cells, e.g., a
baculovirus expression vector or a vector suitable for expression
in mammalian cells.
[0217] When used in mammalian cells, the expression vector's
control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40.
[0218] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al., (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton,
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore, (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al., (1983) Cell 33:729-740; Queen and
Baltimore, (1983) Cell 33:741-748), neuron-specific promoters
(e.g., the neurofilament promoter; Byrne and Ruddle, (1989) Proc.
Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters
(Edlund et al., (1985) Science 230:912-916), and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example, the murine hox promoters (Kessel and Gruss, (1990) Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman, (1989) Genes Dev. 3:537-546).
[0219] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. Regulatory sequences
e.g., viral promoters and/or enhancers) operatively linked to a
nucleic acid cloned in the antisense orientation can be chosen
which direct the constitutive, tissue specific or cell type
specific expression of antisense RNA in a variety of cell types.
The antisense expression vector can be in the form of a recombinant
plasmid, phagemid or attenuated virus. For a discussion of the
regulation of gene expression using antisense genes see Weintraub,
H. et al., Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0220] Another aspect the invention provides a host cell which
includes a nucleic acid molecule described herein, e.g., a 32544
nucleic acid molecule within a recombinant expression vector or a
32544 nucleic acid molecule containing sequences which allow it to
homologously recombine into a specific site of the host cell's
genome. The terms "host cell" and "recombinant host cell" are used
interchangeably herein. Such terms refer not only to the particular
subject cell but rather also to the progeny or potential progeny of
such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term as used herein.
[0221] A host cell can be any prokaryotic or eukaryotic cell. For
example, a 32544 protein can be expressed in bacterial cells such
as E. coli, insect cells, yeast or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to those skilled in the art.
[0222] Vector DNA can be introduced into host cells via
conventional transformation or transfection techniques. As used
herein, the terms "transformation" and "transfection" are intended
to refer to a variety of art-recognized techniques for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or electroporation
A host cell of the invention can be used to produce (i.e., express)
a 32544 protein. Accordingly, the invention further provides
methods for producing a 32544 protein using the host cells of the
invention. In one embodiment, the method includes culturing the
host cell of the invention (into which a recombinant expression
vector encoding a 32544 protein has been introduced) in a suitable
medium such that a 32544 protein is produced. In another
embodiment, the method further includes isolating a 32544 protein
from the medium or the host cell.
[0223] In another aspect, the invention features, a cell or
purified preparation of cells which include a 32544 transgene, or
which otherwise misexpress 32544. The cell preparation can consist
of human or non-human cells, e.g., rodent cells, e.g., mouse or rat
cells, rabbit cells, or pig cells. In preferred embodiments, the
cell or cells include a 32544 transgene, e.g., a heterologous form
of a 32544, e.g., a gene derived from humans (in the case of a
non-human cell). The 32544 transgene can be misexpressed, e.g.,
overexpressed or underexpressed. In other preferred embodiments,
the cell or cells include a gene which misexpress an endogenous
32544, e.g., a gene the expression of which is disrupted, e.g., a
knockout. Such cells can serve as a model for studying disorders
which are related to mutated or mis-expressed 32544 alleles or for
use in drug screening.
[0224] In another aspect, the invention features, a human cell,
e.g., a hematopoietic stem cell, transformed with nucleic acid
which encodes a subject 32544 polypeptide.
[0225] Also provided are cells or a purified preparation thereof,
e.g., human cells, in which an endogenous 32544 is under the
control of a regulatory sequence that does not normally control the
expression of the endogenous 32544 gene. The expression
characteristics of an endogenous gene within a cell, e.g., a cell
line or microorganism, can be modified by inserting a heterologous
DNA regulatory element into the genome of the cell such that the
inserted regulatory element is operably linked to the endogenous
32544 gene. For example, an endogenous 32544 gene, e.g., a gene
which is "transcriptionally silent," e.g., not normally expressed,
or expressed only at very low levels, may be activated by inserting
a regulatory element which is capable of promoting the expression
of a normally expressed gene product in that cell. Techniques such
as targeted homologous recombinations, can be used to insert the
heterologous DNA as described in, e.g., Chappel, U.S. Pat. No.
5,272,071; WO 91/06667, published on May 16, 1991.
[0226] Transgenic Animals
[0227] The invention provides non-human transgenic animals. Such
animals are useful for studying the function and/or activity of a
32544 protein and for identifying and/or evaluating modulators of
32544 activity. As used herein, a "transgenic animal" is a
non-human animal, preferably a mammal, more preferably a rodent
such as a rat or mouse, in which one or more of the cells of the
animal includes a transgene. Other examples of transgenic animals
include non-human primates, sheep, dogs, cows, goats, chickens,
amphibians, and the like. A transgene is exogenous DNA or a
rearrangement, e.g., a deletion of endogenous chromosomal DNA,
which preferably is integrated into or occurs in the genome of the
cells of a transgenic animal. A transgene can direct the expression
of an encoded gene product in one or more cell types or tissues of
the transgenic animal, other transgenes, e.g., a knockout, reduce
expression. Thus, a transgenic animal can be one in which an
endogenous 32544 gene has been altered by, e.g., by homologous
recombination between the endogenous gene and an exogenous DNA
molecule introduced into a cell of the animal, e.g., an embryonic
cell of the animal, prior to development of the animal.
[0228] Intronic sequences and polyadenylation signals can also be
included in the transgene to increase the efficiency of expression
of the transgene. A tissue-specific regulatory sequence(s) can be
operably linked to a transgene of the invention to direct
expression of a 32544 protein to particular cells. A transgenic
founder animal can be identified based upon the presence of a 32544
transgene in its genome and/or expression of 32544 mRNA in tissues
or cells of the animals. A transgenic founder animal can then be
used to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene encoding a 32544 protein
can further be bred to other transgenic animals carrying other
transgenes.
[0229] 32544 proteins or polypeptides can be expressed in
transgenic animals or plants, e.g., a nucleic acid encoding the
protein or polypeptide can be introduced into the genome of an
animal. In preferred embodiments the nucleic acid is placed under
the control of a tissue specific promoter, e.g., a milk or egg
specific promoter, and recovered from the milk or eggs produced by
the animal. Suitable animals are mice, pigs, cows, goats, and
sheep.
[0230] The invention also includes a population of cells from a
transgenic animal, as discussed herein.
[0231] Uses
[0232] The nucleic acid molecules, proteins, protein homologues,
and antibodies described herein can be used in one or more of the
following methods: a) screening assays; b) predictive medicine
(e.g., diagnostic assays, prognostic assays, monitoring clinical
trials, and pharmacogenetics); and c) methods of treatment (e.g.,
therapeutic and prophylactic).
[0233] The isolated nucleic acid molecules of the invention can be
used, for example, to express a 32544 protein (e.g., via a
recombinant expression vector in a host cell in gene therapy
applications), to detect a 32544 mRNA (e.g., in a biological
sample) or a genetic alteration in a 32544 gene, and to modulate
32544 activity, as described further below. The 32544 proteins can
be used to treat disorders characterized by insufficient or
excessive production of a 32544 substrate or production of 32544
inhibitors. In addition, the 32544 proteins can be used to screen
for naturally occurring 32544 substrates, to screen for drugs or
compounds which modulate 32544 activity, as well as to treat
disorders characterized by insufficient or excessive production of
32544 protein or production of 32544 protein forms which have
decreased, aberrant or unwanted activity compared to 32544
wild-type protein. Such disorders include those characterized by
aberrant signaling or aberrant, e.g., hyperproliferative, cell
growth. Moreover, the anti-32544 antibodies of the invention can be
used to detect and isolate 32544 proteins, regulate the
bioavailability of 32544 proteins, and modulate 32544 activity.
[0234] A method of evaluating a compound for the ability to
interact with, e.g., bind, a subject 32544 polypeptide is provided.
The method includes: contacting the compound with the subject 32544
polypeptide; and evaluating ability of the compound to interact
with, e.g., to bind or form a complex with the subject 32544
polypeptide. This method can be performed in vitro, e.g., in a cell
free system, or in vivo, e.g., in a two-hybrid interaction trap
assay. This method can be used to identify naturally occurring
molecules which interact with subject 32544 polypeptide. It can
also be used to find natural or synthetic inhibitors of subject
32544 polypeptide. Screening methods are discussed in more detail
below.
[0235] Screening Assays:
[0236] The invention provides methods (also referred to herein as
"screening assays") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., proteins, peptides,
peptidomimetics, peptoids, small molecules or other drugs) which
bind to 32544 proteins, have a stimulatory or inhibitory effect on,
for example, 32544 expression or 32544 activity; or have a
stimulatory or inhibitory effect on, for example, the expression or
activity of a 32544 substrate. Compounds thus identified can be
used to modulate the activity of target gene products (e.g., 32544
genes) in a therapeutic protocol, to elaborate the biological
function of the target gene product, or to identify compounds
that-disrupt normal target gene interactions.
[0237] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a
32544 protein or polypeptide or a biologically active portion
thereof. In another embodiment, the invention provides assays for
screening candidate or test compounds which bind to or modulate the
activity of a 32544 protein or polypeptide or a biologically active
portion thereof.
[0238] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries; peptoid
libraries [libraries of molecules having the functionalities of
peptides, but with a novel, non-peptide backbone which are
resistant to enzymatic degradation but which nevertheless remain
bioactive] (see, e.g., Zuckermann, R. N. et al., J. Med. Chem.
1994, 37: 2678-85); spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution; the `one-bead one-compound` library method; and
synthetic library methods using affinity chromatography selection.
The biological library and peptoid library approaches are limited
to peptide libraries, while the other four approaches are
applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des.
12:145).
[0239] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al., (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al., (1994). J. Med. Chem.
37:2678; Cho et al., (1993) Science 261:1303; Carrell et al.,
(1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., (1994)
Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al., (1994)
J. Med. Chem. 37:1233.
[0240] Libraries of compounds may be presented in solution (e.g.,
Houghten, (1992) Biotechniques 13:412-421), or on beads (Lam,
(1991) Nature 354:82-84), chips (Fodor, (1993) Nature 364:555-556),
bacteria or spores (Ladner, U.S. Pat. No. 5,223,409), plasmids
(Cull et al., (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on
phage (Scott and Smith, (1990) Science 249:386-390); (Devlin,
(1990) Science 249:404-406); (Cwirla et al., (1990) Proc. Natl.
Acad. Sci. 87:6378-6382); (Felici, (1991) J. Mol. Biol.
222:301-310); (Ladner supra.).
[0241] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a 32544 protein or biologically active portion
thereof is contacted with a test compound, and the ability of the
test compound to modulate 32544 activity is determined. Determining
the ability of the test compound to modulate 32544 activity can be
accomplished by monitoring, for example, phospholipase family
activity. The cell, for example, can be of mammalian origin, e.g.,
human. Cell homogenates, or fractions, preferably membrane
containing fractions, can also be tested.
[0242] The ability of the test compound to modulate 32544 binding
to a compound, e.g., a 32544 substrate, or to bind to 32544 can
also be evaluated. This can be accomplished, for example, by
coupling the compound, e.g., the substrate, with a radioisotope or
enzymatic label such that binding of the compound, e.g., the
substrate, to 32544 can be determined by detecting the labeled
compound, e.g., substrate, in a complex. Alternatively, 32544 could
be coupled with a radioisotope or enzymatic label to monitor the
ability of a test compound to modulate 32544 binding to a 32544
substrate in a complex. For example, compounds (e.g., 32544
substrates) can be labeled with .sup.125I, .sup.35S, .sup.14C, or
.sup.3H, either directly or indirectly, and the radioisotope
detected by direct counting of radioemmission or by scintillation
counting. Alternatively, compounds can be enzymatically labeled
with, for example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
[0243] The ability of a compound (e.g., a 32544 substrate) to
interact with 32544 with or without the labeling of any of the
interactants can be evaluated. For example, a microphysiometer can
be used to detect the interaction of a compound with 32544 without
the labeling of either the compound or the 32544. McConnell, H. M.
et al., (1992) Science 257:1906-1912. As used herein, a
"microphysiometer" (e.g., Cytosensor) is an analytical instrument
that measures the rate at which a cell acidifies its environment
using a light-addressable potentiometric sensor (LAPS). Changes in
this acidification rate can be used as an indicator of the
interaction between a compound and 32544.
[0244] In yet another embodiment, a cell-free assay is provided in
which a 32544 protein or biologically active portion thereof is
contacted with a test compound and the ability of the test compound
to bind to the 32544 protein or biologically active portion thereof
is evaluated. Preferred biologically active portions of the 32544
proteins to be used in assays of the present invention include
fragments which participate in interactions with non-32544
molecules, e.g., fragments with high surface probability
scores.
[0245] Soluble and/or membrane-bound forms of isolated proteins
(e.g., 32544 proteins or biologically active portions thereof) can
be used in the cell-free assays of the invention. When
membrane-bound forms of the protein are used, it may be desirable
to utilize a solubilizing agent. Examples of such solubilizing
agents include non-ionic detergents such as n-octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton.RTM. X-100, Triton.RTM. X-114,
Thesit.RTM., Isotridecypoly(ethylene glycol ether).sub.n,
3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),
3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane
sulfonate (CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane
sulfonate.
[0246] Cell-free assays involve preparing a reaction mixture of the
target gene protein and the test compound under conditions and for
a time sufficient to allow the two components to interact and bind,
thus forming a complex that can be removed and/or detected.
[0247] In one embodiment, assays are performed where the ability of
an agent to block phospholipase family activity within a cell is
evaluated.
[0248] The interaction between two molecules can also be detected,
e.g., using fluorescence energy transfer (FET) (see, for example,
Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al.,
U.S. Pat. No. 4,868,103). A fluorophore label on the first, `donor`
molecule is selected such that its emitted fluorescent energy will
be absorbed by a fluorescent label on a second, `acceptor`
molecule, which in turn is able to fluoresce due to the absorbed
energy. Alternately, the `donor` protein molecule may simply
utilize the natural fluorescent energy of tryptophan residues.
Labels are chosen that emit different wavelengths of light, such
that the `acceptor` molecule label may be differentiated from that
of the `donor`. Since the efficiency of energy transfer between the
labels is related to the distance separating the molecules, the
spatial relationship between the molecules can be assessed. In a
situation in which binding occurs between the molecules; the
fluorescent emission of the `acceptor` molecule label in the assay
should be maximal. An FET binding event can be conveniently
measured through standard fluorometric detection means well known
in the art (e.g., using a fluorimeter).
[0249] In another embodiment, determining the ability of the 32544
protein to bind to a target molecule can be accomplished using
real-time Biomolecular Interaction Analysis (BIA) (see, e.g.,
Sjolander, S. and Urbaniczky, C., (1991) Anal. Chem. 63:2338-2345
and Szabo et al., (1995) Curr. Opin. Struct. Biol. 5:699-705).
"Surface plasmon resonance" or "BIA" detects biospecific
interactions in real time, without labeling any of the interactants
(e.g., BIAcore). Changes in the mass at the binding surface
(indicative of a binding event) result in alterations of the
refractive index of light near the surface (the optical phenomenon
of surface plasmon resonance (SPR)), resulting in a detectable
signal which can be used as an indication of real-time reactions
between biological molecules.
[0250] In one embodiment, the target gene product or the test
substance is anchored onto a solid phase. The target gene
product/test compound complexes anchored on the solid phase can be
detected at the end of the reaction. Preferably, the target gene
product can be anchored onto a solid surface, and the test
compound, (which is not anchored), can be labeled, either directly
or indirectly, with detectable labels discussed herein.
[0251] It may be desirable to immobilize either 32544, an
anti-32544 antibody or its target molecule to facilitate separation
of complexed from uncomplexed forms of one or both of the proteins,
as well as to accommodate automation of the assay. Binding of a
test compound to a 32544 protein, or interaction of a 32544 protein
with a target molecule in the presence and absence of a candidate
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtiter plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/32544 fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtiter plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or 32544 protein, and the mixture
incubated under conditions conducive to complex-formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtiter plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described above. Alternatively, the complexes can be dissociated
from the matrix, and the level of 32544 binding or activity
determined using standard techniques.
[0252] Other techniques for immobilizing either a 32544 protein or
a target molecule on matrices include using conjugation of biotin
and streptavidin. Biotinylated 32544 protein or target molecules
can be prepared from biotin-NHS(N-hydroxy-succinimide) using
techniques known in the art (e.g., biotinylation kit, Pierce
Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
[0253] In order to conduct the assay, the non-immobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously non-immobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
non-immobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the immobilized component (the
antibody, in turn, can be directly labeled or indirectly labeled
with, e.g., a labeled anti-Ig antibody).
[0254] In one embodiment, this assay is performed utilizing
antibodies reactive with 32544 protein or target molecules but
which do not interfere with binding of the 32544 protein to its
target molecule. Such antibodies can be derivatized to the wells of
the plate, and unbound target or 32544 protein trapped in the wells
by antibody conjugation. Methods for detecting such complexes, in
addition to those described above for the GST-immobilized
complexes, include immunodetection of complexes using antibodies
reactive with the 32544 protein or target molecule, as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the 32544 protein or target molecule.
[0255] Alternatively, cell free assays can be conducted in a liquid
phase. In such an assay, the reaction products are separated from
unreacted components, by any of a number of standard techniques,
including but not limited to: differential centrifugation (see, for
example, Rivas, G., and Minton, A. P., Trends Biochem Sci 1993
August;18(8):284-7); chromatography (gel filtration chromatography,
ion-exchange chromatography); electrophoresis (see, e.g., Ausubel,
F. et al., eds. Current Protocols in Molecular Biology 1999, J.
Wiley: New York.); and immunoprecipitation (see, for example,
Ausubel, F. et al., eds. Current Protocols in Molecular Biology
1999, J. Wiley: New York). Such resins and chromatographic
techniques are known to one skilled in the art (see, e.g.,
Heegaard, N. H., J. Mol. Recognit. 1998 Winter;11(1-6):141-8; Hage,
D. S., and Tweed, S. A., J. Chromatogr. B Biomed. Sci. Appl. 1997
Oct. 10;699 (1-2):499-525). Further, fluorescence energy transfer
may also be conveniently utilized, as described herein, to detect
binding without further purification of the complex from
solution.
[0256] In a preferred embodiment, the assay includes contacting the
32544 protein or biologically active portion thereof with a known
compound which binds 32544 to form an assay mixture, contacting the
assay mixture with a test compound, and determining the ability of
the test compound to interact with a 32544 protein, wherein
determining the ability of the test compound to interact with a
32544 protein includes determining the ability of the test compound
to preferentially bind to 32544 or biologically active portion
thereof, or to modulate the activity of a target molecule, as
compared to the known compound.
[0257] The target gene products of the invention can, in vivo,
interact with one or more cellular or extracellular macromolecules,
such as proteins. For the purposes of this discussion, such
cellular and extracellular macromolecules are referred to herein as
"binding partners." Compounds that disrupt such interactions can be
useful in regulating the activity of the target gene product. Such
compounds can include, but are not limited to molecules such as
antibodies, peptides, and small molecules. The preferred target
genes/products for use in this embodiment are the 32544 genes
herein identified. In an alternative embodiment, the invention
provides methods for determining the ability of the test compound
to modulate the activity of a 32544 protein through modulation of
the activity of a downstream effector of a 32544 target molecule.
For example, the activity of the effector molecule on an
appropriate target can be determined, or the binding of the
effector to an appropriate target can be determined, as previously
described.
[0258] To identify compounds that interfere with the interaction
between the target gene product and its cellular or extracellular
binding partner(s), e.g., a substrate, a reaction mixture
containing the target gene product and the binding partner is
prepared, under conditions and for a time sufficient, to allow the
two products to form complex. In order to test an inhibitory agent,
the reaction mixture is provided in the presence and absence of the
test compound. The test compound can be initially included in the
reaction mixture, or can be added at a time subsequent to the
addition of the target gene and its cellular or extracellular
binding partner. Control reaction mixtures are incubated without
the test compound or with a placebo. The formation of any complexes
between the target gene product and the cellular or extracellular
binding partner is then detected. The formation of a complex in the
control reaction, but not in the reaction mixture containing the
test compound, indicates that the compound interferes with the
interaction of the target gene product and the interactive binding
partner. Additionally, complex formation within reaction mixtures
containing the test compound and normal target gene product can
also be compared to complex formation within reaction mixtures
containing the test compound and mutant target gene product. This
comparison can be important in those cases wherein it is desirable
to identify compounds that disrupt interactions of mutant but not
normal target gene products.
[0259] These assays can be conducted in a heterogeneous or
homogeneous format. Heterogeneous assays involve anchoring either
the target gene product or the binding partner onto a solid phase,
and detecting complexes anchored on the solid phase at the end of
the reaction. In homogeneous assays, the entire reaction is carried
out in a liquid phase. In either approach, the order of addition of
reactants can be varied to obtain different information about the
compounds being tested. For example, test compounds that interfere
with the interaction between the target gene products and the
binding partners, e.g., by competition, can be identified by
conducting the reaction in the presence of the test substance.
Alternatively, test compounds that disrupt preformed complexes,
e.g., compounds with higher binding constants that displace one of
the components from the complex, can be tested by adding the test
compound to the reaction mixture after complexes have been formed.
The various formats are briefly described below.
[0260] In a heterogeneous assay system, either the target gene
product or the interactive cellular or extracellular binding
partner, is anchored onto a solid surface (e.g., a microtiter
plate), while the non-anchored species is labeled, either directly
or indirectly. The anchored species can be immobilized by
non-covalent or covalent attachments. Alternatively, an immobilized
antibody specific for the species to be anchored can be used to
anchor the species to the solid surface.
[0261] In order to conduct the assay, the partner of the
immobilized species is exposed to the coated surface with or
without the test compound. After the reaction is complete,
unreacted components are removed (e.g., by washing) and any
complexes formed will remain immobilized on the solid surface.
Where the non-immobilized species is pre-labeled, the detection of
label immobilized on the surface indicates that complexes were
formed. Where the non-immobilized species is not pre-labeled, an
indirect label can be used to detect complexes anchored on the
surface; e.g., using a labeled antibody specific for the initially
non-immobilized species (the antibody, in turn, can be directly
labeled or indirectly labeled with, e.g., a labeled anti-Ig
antibody). Depending upon the order of addition of reaction
components, test compounds that inhibit complex formation or that
disrupt preformed complexes can be detected.
[0262] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected; e.g., using an immobilized antibody specific for one of
the binding components to anchor any complexes formed in solution,
and a labeled antibody specific for the other partner to detect
anchored complexes. Again, depending upon the order of addition of
reactants to the liquid phase, test compounds that inhibit complex
or that disrupt preformed complexes can be identified.
[0263] In an alternate embodiment of the invention, a homogeneous
assay can be used. For example, a preformed complex of the target
gene product and the interactive cellular or extracellular binding
partner product is prepared in that either the target gene products
or their binding partners are labeled, but the signal generated by
the label is quenched due to complex formation (see, e.g., U.S.
Pat. No. 4,109,496 that utilizes this approach for immunoassays).
The addition of a test substance that competes with and displaces
one of the species from the preformed complex will result in the
generation of a signal above background. In this way, test
substances that disrupt target gene product-binding partner
interaction can be identified.
[0264] In yet another aspect, the 32544 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 32544
("32544-binding proteins" or "32544-bp") and are involved in 32544
activity. Such 32544-bps can be activators or inhibitors of signals
by the 32544 proteins or 32544 targets as, for example, downstream
elements of a 32544-mediated signaling pathway.
[0265] 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 32544
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
(Alternatively the: 32544 protein can be the fused to the activator
domain.) If the "bait" and the "prey" proteins are able to
interact, in vivo, forming a 32544-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 32544 protein.
[0266] In another embodiment, modulators of 32544 expression are
identified. For example, a cell or cell free mixture is contacted
with a candidate compound and the expression of 32544 mRNA or
protein evaluated relative to the level of expression of 32544 mRNA
or protein in the absence of the candidate compound. When
expression of 32544 mRNA or protein is greater in the presence of
the candidate compound than in its absence, the candidate compound
is identified as a stimulator of 32544 mRNA or protein expression.
Alternatively, when expression of 32544 mRNA or protein is less
(statistically significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of 32544 mRNA or protein expression. The level of
32544 mRNA or protein expression can be determined by methods
described herein for detecting 32544 mRNA or protein.
[0267] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a cell
free assay, and the ability of the agent to modulate the activity
of a 32544 protein can be confirmed in vivo, e.g., in an
animal.
[0268] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein (e.g., a 32544 modulating agent, an antisense
32544 nucleic acid molecule, a 32544-specific antibody, or a
32544-binding partner) in an appropriate animal model to determine
the efficacy, toxicity, side effects, or mechanism of action, of
treatment with such an agent. Furthermore, novel agents identified
by the above-described screening assays can be used for treatments
as described herein.
[0269] Detection Assays
[0270] Portions or fragments of the nucleic acid sequences
identified herein can be used as polynucleotide reagents. For
example, these sequences can be used to: (i) map their respective
genes on a chromosome e.g., to locate gene regions associated with
genetic disease or to associate 32544 with a disease; (ii) identify
an individual from a minute biological sample (tissue typing); and
(iii) aid in forensic identification of a biological sample. These
applications are described in the subsections below.
[0271] Chromosome Mapping
[0272] The 32544 nucleotide sequences or portions thereof can be
used to map the location of the 32544 genes on a chromosome. This
process is called chromosome mapping. Chromosome mapping is useful
in correlating the 32544 sequences with genes associated with
disease.
[0273] Briefly, 32544 genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp in length) from the
32544 nucleotide sequences. These primers can then be used for PCR
screening of somatic cell hybrids containing individual human
chromosomes. Only those hybrids containing the human gene
corresponding to the 32544 sequences will yield an amplified
fragment.
[0274] A panel of somatic cell hybrids in which each cell line
contains either a single human chromosome or a small number of
human chromosomes, and a full set of mouse chromosomes, can allow
easy mapping of individual genes to specific human chromosomes.
(D'Eustachio P. et al., (1983) Science 220:919-924).
[0275] Other mapping strategies e.g., in situ hybridization
(described in Fan, Y. et al., (1990) Proc. Natl. Acad. Sci. USA,
87:6223-27), pre-screening with labeled flow-sorted chromosomes,
and pre-selection by hybridization to chromosome specific cDNA
libraries can be used to map 32544 to a chromosomal location.
[0276] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. The FISH technique can be
used with a DNA sequence as short as 500 or 600 bases. However,
clones larger than 1,000 bases have a higher likelihood of binding
to a unique chromosomal location with sufficient signal intensity
for simple detection. Preferably 1,000 bases, and more preferably
2,000 bases will suffice to get good results at a reasonable amount
of time. For a review of this technique, see Verma et al., Human
Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York
1988).
[0277] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0278] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland, J. et al., (1987) Nature, 325:783-787.
[0279] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the 32544 gene, can be determined. If a mutation is observed in
some or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
[0280] Tissue Typing
[0281] 32544 sequences can be used to identify individuals from
biological samples using, e.g., restriction fragment length
polymorphism (RFLP). In this technique, an individual's genomic DNA
is digested with one or more restriction enzymes, the fragments
separated, e.g., in a Southern blot, and probed to yield bands for
identification. The sequences of the present invention are useful
as additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[0282] Furthermore, the sequences of the present invention can also
be used to determine the actual base-by-base DNA sequence of
selected portions of an individual's genome. Thus, the 32544
nucleotide sequences described herein can be used to prepare two
PCR primers from the 5' and 3' ends of the sequences. These primers
can then be used to amplify an individual's DNA and subsequently
sequence it. Panels of corresponding DNA sequences from
individuals, prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences.
[0283] Allelic variation occurs to some degree in the coding
regions of these sequences, and to a greater degree in the
noncoding regions. Each of the sequences described herein can, to
some degree, be used as a standard against which DNA from an
individual can be compared for identification purposes. Because
greater numbers of polymorphisms occur in the noncoding regions,
fewer sequences are necessary to differentiate individuals. The
noncoding sequences of SEQ ID NO:1 can provide positive individual
identification with a panel of perhaps 10 to 1,000 primers which
each Yield a noncoding amplified sequence of 100 bases. If
predicted coding sequences, such as those in SEQ ID NO:3 are used,
a more appropriate number of primers for positive individual
identification would be 500-2,000.
[0284] If a panel of reagents from 32544 nucleotide sequences
described herein is used to generate a unique identification
database for an individual, those same reagents can later be used
to identify tissue from that individual. Using the unique
identification database, positive identification of the individual,
living or dead, can be made from extremely small tissue
samples.
[0285] Use of Partial 32544 Sequences in Forensic Biology DNA-based
identification techniques can also be used in forensic biology. To
make such an identification, PCR technology can be used to amplify
DNA sequences taken from very small biological samples such as
tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva,
or semen found at a crime scene. The amplified sequence can then be
compared to a standard, thereby allowing identification of the
origin of the biological sample.
[0286] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As mentioned
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to
noncoding regions of SEQ ID NO:1 (e.g., fragments derived from the
noncoding regions of SEQ ID NO:1 having a length of at least 20
bases, preferably at least 30 bases) are particularly appropriate
for this use.
[0287] The 32544 nucleotide sequences described herein can further
be used to provide polynucleotide reagents, e.g., labeled or
labelable probes which can be used in, for example, an in situ
hybridization technique, to identify a specific tissue, e.g., a
tissue containing phospholipase family activity. This can be very
useful in cases where a forensic pathologist is presented with a
tissue of unknown origin. Panels of such 32544 probes can be used
to identify tissue by species and/or by organ type.
[0288] In a similar fashion, these reagents, e.g., 32544 primers or
probes can be used to screen tissue culture for contamination (i.e.
screen for the presence of a mixture of different types of cells in
a culture).
[0289] Predictive Medicine
[0290] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual.
[0291] Generally, the invention provides, a method of determining
if a subject is at risk for a disorder related to a lesion in or
the misexpression of a gene which encodes 32544.
[0292] Such disorders include, e.g., a disorder associated with the
misexpression of 32544, or lipid metabolism related disorder.
[0293] The method includes one or more of the following:
[0294] detecting, in a tissue of the subject, the presence or
absence of a mutation which affects the expression of the 32544
gene, or detecting the presence or absence of a mutation in a
region which controls the expression of the gene, e.g., a mutation
in the 5' control region;
[0295] detecting, in a tissue of the subject, the presence or
absence of a mutation which alters the structure of the 32544
gene;
[0296] detecting, in a tissue of the subject, the misexpression of
the 32544 gene, at the mRNA level, e.g., detecting a non-wild type
level of a mRNA;
[0297] detecting, in a tissue of the subject, the misexpression of
the gene, at the protein level, e.g., detecting a non-wild type
level of a 32544 polypeptide.
[0298] In preferred embodiments the method includes: ascertaining
the existence of at least one of: a deletion of one or more
nucleotides from the 32544 gene; an insertion of one or more
nucleotides into the gene, a point mutation, e.g., a substitution
of one or more nucleotides of the gene, a gross chromosomal
rearrangement of the gene, e.g., a translocation, inversion, or
deletion.
[0299] For example, detecting the genetic lesion can include: (i)
providing a probe/primer including an oligonucleotide containing a
region of nucleotide sequence which hybridizes to a sense or
antisense sequence from SEQ ID NO:1 naturally occurring mutants
thereof or 5' or 3' flanking sequences naturally associated with
the 32544 gene; (ii) exposing the probe/primer to nucleic acid of
the tissue; and detecting, by hybridization, e.g., in situ
hybridization, of the probe/primer to the nucleic acid, the
presence or absence of the genetic lesion.
[0300] In preferred embodiments detecting the misexpression
includes ascertaining the existence of at least one of: an
alteration in the level of a messenger RNA transcript of the 32544
gene; the presence of a non-wild type splicing pattern of a
messenger RNA transcript of the gene; or a non-wild type level of
32544.
[0301] Methods of the invention can be used prenatally or to
determine if a subject's offspring will be at risk for a
disorder.
[0302] In preferred embodiments the method includes determining the
structure of a 32544 gene, an abnormal structure being indicative
of risk for the disorder.
[0303] In preferred embodiments the method includes contacting a
sample form the subject with an antibody to the 32544 protein or a
nucleic acid, which hybridizes specifically with the gene. These
and other embodiments are discussed below.
[0304] Diagnostic and Prognostic Assays
[0305] The presence, level, or absence of 32544 protein or nucleic
acid in a biological sample can be evaluated by obtaining a
biological sample from a test subject and contacting the biological
sample with a compound or an agent capable of detecting 32544
protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes
32544 protein such that the presence of 32544 protein or nucleic
acid is detected in the biological sample. The term "biological
sample" includes tissues, cells and biological fluids isolated from
a subject, as well as tissues, cells and fluids present within a
subject. A preferred biological sample is serum. The level of
expression of the 32544 gene can be measured in a number of ways,
including, but not limited to: measuring the "mRNA encoded by the
32544 genes; measuring the amount of protein encoded by the 32544
genes; or measuring the activity of the protein encoded by the
32544 genes.
[0306] The level of mRNA corresponding to the 32544 gene in a cell
can be determined both by in situ and by in vitro formats.
[0307] The isolated mRNA can be used in hybridization or
amplification assays that include, but are not limited to, Southern
or Northern analyses, polymerase chain reaction analyses and probe
arrays. One preferred diagnostic method for the detection of mRNA
levels involves contacting the isolated mRNA with a nucleic acid
molecule (probe) that can hybridize to the mRNA encoded by the gene
being detected. The nucleic acid probe can be, for example, a
full-length 32544 nucleic acid, such as the nucleic acid of SEQ ID
NO:1, or the DNA insert of the plasmid deposited with ATCC as
Accession Number ______, or a portion thereof, such as an
oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to 32544 mRNA or genomic DNA. Other
suitable probes for use in the diagnostic assays are described
herein.
[0308] In one format, mRNA (or cDNA) is immobilized on a surface
and contacted with the probes, for example by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probes are immobilized on a surface and the mRNA (or cDNA) is
contacted with the probes, for example, in a two-dimensional gene
chip array. A skilled artisan can adapt known mRNA detection
methods for use in detecting the level of mRNA encoded by the 32544
genes.
[0309] The level of mRNA in a sample that is encoded by one of
32544 can be evaluated with nucleic acid amplification, e.g., by
rtPCR (Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain
reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA 88:189-193),
self sustained sequence replication (Guatelli et al., 1990, Proc.
Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification
system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988,
Bio/Technology 6:1197), rolling circle replication (Lizardi et al.,
U.S. Pat. No. 5,854,033) or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques known in the art. As used herein, amplification primers
are defined as being a pair of nucleic acid molecules that can
anneal to 5' or 3' regions of a gene (plus and minus strands,
respectively, or vice-versa) and contain a short region in between.
In general, amplification primers are from about 10 to 30
nucleotides in length and flank a region from about 50 to 200
nucleotides in length. Under appropriate conditions and with
appropriate reagents, such primers permit the amplification of a
nucleic acid molecule comprising the nucleotide sequence flanked by
the primers.
[0310] For in situ methods, a cell or tissue sample can be
prepared/processed and immobilized on a support, typically a glass
slide, and then contacted with a probe that can hybridize to mRNA
that encodes the 32544 gene being analyzed.
[0311] In another embodiment, the methods further contacting a
control sample with a compound or agent capable of detecting 32544
mRNA, or genomic DNA, and comparing the presence of 32544 mRNA or
genomic DNA in the control sample with the presence of 32544 mRNA
or genomic DNA in the test sample.
[0312] A variety of methods can be used to determine the level of
protein encoded by 32544. In general, these methods include
contacting an agent that selectively binds to the protein, such as
an antibody with a sample, to evaluate the level of protein in the
sample. In a preferred embodiment, the antibody bears a detectable
label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab').sub.2) can be used. The term "labeled", with regard to the
probe or antibody, is intended to encompass direct labeling of the
probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with a detectable
substance. Examples of detectable substances are provided
herein.
[0313] The detection methods can be used to detect 32544 protein in
a biological sample in vitro as well as in vivo. In vitro
techniques for detection of 32544 protein include enzyme linked
immuno sorb ent assays (ELISAs), immunoprecipitations, immuno
fluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and
Western blot analysis. In vivo techniques for detection of 32544
protein include introducing into a subject a labeled anti-32544
antibody. For example, the antibody can be labeled with a
radioactive marker whose presence and location in a subject can be
detected by standard imaging techniques.
[0314] In another embodiment, the methods further include
contacting the control sample with a compound or agent capable of
detecting 32544 protein, and comparing the presence of 32544
protein in the control sample with the presence of 32544 protein in
the test sample.
[0315] The invention also includes kits for detecting the presence
of 32544 in biologic sample. For example, the kit can include a
compound or agent capable of detecting 32544 protein or mRNA in a
biological sample; and a standard. The compound or agent can be
packaged in a suitable container. The kit can further comprise
instructions for using the kit to detect 32544 protein or nucleic
acid. For antibody-based kits, the kit can include: (1) a first
antibody (e.g., attached to a solid support) which binds to a
polypeptide corresponding to a marker of the invention; and,
optionally, (2) a second, different antibody which binds to either
the polypeptide or the first antibody and is conjugated to a
detectable agent.
[0316] For oligonucleotide-based kits, the kit can include: (1) an
oligonucleotide, e.g., a detectably labeled oligonucleotide, which
hybridizes to a nucleic acid sequence encoding a polypeptide
corresponding to a marker of the invention or (2) a pair of primers
useful for amplifying a nucleic acid molecule corresponding to a
marker of the invention. The kit can also includes a buffering
agent, a preservative, or a protein-stabilizing agent. The kit can
also includes components necessary for detecting the detectable
agent (e.g., an enzyme or a substrate). The kit can also contain a
control sample or a series of control samples which can be assayed
and compared to the test sample contained. Each component of the
kit can be enclosed within an individual container and all of the
various containers can be within a single package, along with
instructions for interpreting the results of the assays performed
using the kit.
[0317] The diagnostic methods described herein can identify
subjects having, or at risk of developing, a disease or disorder
associated with misexpressed or aberrant or unwanted 32544
expression or activity. As used herein, the term "unwanted"
includes an unwanted phenomenon involved in a biological response
such as pain or deregulated cell proliferation.
[0318] In one embodiment, a disease or disorder associated with
aberrant or unwanted 32544 expression or activity is identified. A
test sample is obtained from a subject and 32544 protein or nucleic
acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level,
e.g., the presence or absence, of 32544 protein or nucleic acid is
diagnostic for a subject having or at risk of developing a disease
or disorder associated with aberrant or unwanted 32544 expression
or activity. As used herein, a "test sample" refers to a biological
sample obtained from a subject of interest, including a biological
fluid (e.g., serum), cell sample, or tissue.
[0319] The prognostic assays described herein can be used to
determine whether a subject can be administered an agent (e.g., an
agonist, antagonist, peptidomimetic, protein, peptide, nucleic
acid, small molecule, or other drug candidate) to treat a disease
or disorder associated with aberrant or unwanted 32544 expression
or activity. For example, such methods can be used to determine
whether a subject can be effectively treated with an agent for a
cellular growth related disorder.
[0320] The methods of the invention can also be used to detect
genetic alterations in a 32544 gene, thereby determining if a
subject with the altered gene is at risk for a disorder
characterized by misregulation in 32544 protein activity or nucleic
acid expression, such as a cellular growth related disorder. In
preferred embodiments, the methods include detecting, in a sample
from the subject, the presence or absence of a genetic alteration
characterized by at least one of an alteration affecting the
integrity of a gene encoding a 32544-protein, or the mis-expression
of the 32544 gene. For example, such genetic alterations can be
detected by ascertaining the existence of at least one of 1) a
deletion of one or more nucleotides from a 32544 gene; 2) an
addition of one or more nucleotides to a 32544 gene; 3) a
substitution of one or more nucleotides of a 32544 gene, 4) a
chromosomal rearrangement of a 32544 gene; 5) an alteration in the
level of a messenger RNA transcript of a 32544 gene, 6) aberrant
modification of a 32544 gene, such as of the methylation pattern of
the genomic DNA, 7) the presence of a non-wild type splicing
pattern of a messenger RNA transcript of a 32544 gene, 8) a
non-wild type level of a 32544-protein, 9) allelic loss of a 32544
gene, and 10) inappropriate post-translational modification of a
32544-protein.
[0321] An alteration can be detected without a probe/primer in a
polymerase chain reaction, such as anchor PCR or RACE PCR, or,
alternatively, in a ligation chain reaction (LCR), the latter of
which can be particularly useful for detecting point mutations in
the 32544-gene.
[0322] This method can include the steps of collecting a sample of
cells from a subject, isolating nucleic acid (e.g., genomic, mRNA
or both) from the sample, contacting the nucleic acid sample with
one or more primers which specifically hybridize to a 32544 gene
under conditions such that hybridization and amplification of the
32544-gene (if present) occurs, and detecting the presence or
absence of an amplification product, or detecting the size of the
amplification product and comparing the length to a control sample.
It is anticipated that PCR and/or LCR may be desirable to use as a
preliminary amplification step in conjunction with any of the
techniques used for detecting mutations described herein.
[0323] Alternative amplification methods include: self sustained
sequence replication (Guatelli, J. C. et al., (1990) Proc. Natl.
Acad. Sci. USA 87:1874-1878), transcriptional amplification system
(Kwoh, D. Y. et al., (1989) Proc. Natl. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al., (1988)
Bio-Technology 6:1197), or other nucleic acid amplification
methods, followed by the detection of the amplified molecules using
techniques known to those of skill in the art.
[0324] In another embodiment, mutations in a 32544 gene from a
sample cell can be identified by detecting alterations in
restriction enzyme cleavage patterns. For example, sample and
control DNA is isolated, amplified (optionally), digested with one
or more restriction endonucleases, and fragment length sizes are
determined, e.g., by gel electrophoresis and compared. Differences
in fragment length sizes between sample and control DNA indicates
mutations in the sample DNA. Moreover, the use of sequence specific
ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used
to score for the presence of specific mutations by development or
loss of a ribozyme cleavage site.
[0325] In other embodiments, genetic mutations in 32544 can be
identified by hybridizing a sample and control nucleic acids, e.g.,
DNA or RNA, two-dimensional arrays, e.g., chip based arrays. Such
arrays include a plurality of addresses, each of which is
positionally distinguishable from the other. A different probe is
located at each address of the plurality. The arrays can have a
high density of addresses, e.g., can contain hundreds or thousands
of oligonucleotides probes (Cronin, M. T. et al., (1996) Human
Mutation 7: 244-255; Kozal, M. J. et al., (1996) Nature Medicine
2:753-759). For example, genetic mutations in 32544 can be
identified in two dimensional arrays containing light-generated DNA
probes as described in Cronin, M. T. et al., supra. Briefly, a
first hybridization array of probes can be used to scan through
long stretches of DNA in a sample and control to identify base
changes between the sequences by making linear arrays of sequential
overlapping probes. This step allows the identification of point
mutations. This step is followed by a second hybridization array
that allows the characterization of specific mutations by using
smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel
probe sets, one complementary to the wild-type gene and the other
complementary to the mutant gene.
[0326] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
32544 gene and detect mutations by comparing the sequence of the
sample 32544 with the corresponding wild-type (control) sequence.
Automated sequencing procedures can be utilized when performing the
diagnostic assays ((1995) Biotechniques 19:448), including
sequencing by mass spectrometry.
[0327] Other methods for detecting mutations in the 32544 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al., (1985) Science 230:1242; Cotton et al., (1988) Proc. Natl.
Acad. Sci. USA 85:4397; Saleeba et al., (1992) Methods Enzymol.
217:286-295).
[0328] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in 32544
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et
al., (1994) Carcinogenesis 15:1657-1662; U.S. Pat. No.
5,459,039).
[0329] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in 32544 genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al., (1989) Proc. Natl. Acad.
Sci. USA: 86:2766, see also Cotton, (1993) Mutat. Res. 285:125-144;
and Hayashi, (1992) Genet. Anal. Tech. Appl. 9:73-79).
Single-stranded DNA fragments of sample and control 32544 nucleic
acids will be denatured and allowed to renature. The secondary
structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The DNA
fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In a preferred embodiment, the subject method
utilizes heteroduplex analysis to separate double stranded
heteroduplex molecules on the basis of changes in electrophoretic
mobility (Keen et al., (1991) Trends Genet. 7:5).
[0330] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al., (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner, (1987) Biophys.
Chem. 265:12753).
[0331] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension (Saiki et al., (1986) Nature 324:163); Saiki et al.,
(1989) Proc. Natl. Acad. Sci. USA 86:6230).
[0332] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al., (1989)
Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one
primer where, under appropriate conditions, mismatch can prevent,
or reduce polymerase extension (Prossner, (1993) Tibtech 11:238).
In addition it may be desirable to introduce a novel restriction
site in the region of the mutation to create cleavage-based
detection (Gasparini et al., (1992) Mol. Cell Probes 6:1). It is
anticipated that in certain embodiments amplification may also be
performed using Taq ligase for amplification (Barany, (1991) Proc.
Natl. Acad. Sci USA 88:189). In such cases, ligation will occur
only if there is a perfect match at the 3' end of the 5' sequence
making it possible to detect the presence of a known mutation at a
specific site by looking for the presence or absence of
amplification.
[0333] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving a 32544 gene.
[0334] Use of 32544 Molecules as Surrogate Markers
[0335] The 32544 molecules of the invention are also useful as
markers of disorders or disease states, as markers for precursors
of disease states, as markers for predisposition of disease states,
as markers of drug activity, or as markers of the pharmacogenomic
profile of a subject. Using the methods described herein, the
presence, absence and/or quantity of the 32544 molecules of the
invention may be detected, and may be correlated with one or more
biological states in vivo. For example, the 32544 molecules of the
invention may serve as surrogate markers for one or more disorders
or disease states or for conditions leading up to disease states.
As used herein, a "surrogate marker" is an objective biochemical
marker which correlates with the absence or presence of a disease
or disorder, or with the progression of a disease or disorder
(e.g., with the presence or absence of a tumor). The presence or
quantity of such markers is independent of the disease. Therefore,
these markers may serve to indicate whether a particular course of
treatment is effective in lessening a disease state or disorder.
Surrogate markers are of particular use when the presence or extent
of a disease state or disorder is difficult to assess through
standard methodologies (e.g., early stage tumors), or when an
assessment of disease progression is desired before a potentially
dangerous clinical endpoint is reached (e.g., an assessment of
cardiovascular disease may be made using cholesterol levels as a
surrogate marker, and an analysis of HIV infection may be made
using HIV RNA levels as a surrogate marker, well in advance of the
undesirable clinical outcomes of myocardial infarction or
fully-developed AIDS). Examples of the use of surrogate markers in
the art include: Koomen et al. (2000) J. Mass. Spectrom. 35:
258-264; and James (1994) AIDS Treatment News Archive 209.
[0336] The 32544 molecules of the invention are also useful as
pharmacodynamic markers. As used herein, a "pharmacodynamic marker"
is an objective biochemical marker which correlates specifically
with drug effects. The presence or quantity of a pharmacodynamic
marker is not related to the disease state or disorder for which
the drug is being administered; therefore, the presence or quantity
of the marker is indicative of the presence or activity of the drug
in a subject. For example, a pharmacodynamic marker may be
indicative of the concentration of the drug in a biological tissue,
in that the marker is either expressed or transcribed or not
expressed or transcribed in that tissue in relationship to the
level of the drug. In this fashion, the distribution or uptake of
the drug may be monitored by the pharmacodynamic marker. Similarly,
the presence or quantity of the pharmacodynamic marker may be
related to the presence or quantity of the metabolic product of a
drug, such that the presence or quantity of the marker is
indicative of the relative breakdown rate of the drug in vivo.
Pharmacodynamic markers are of particular use in increasing the
sensitivity of detection of drug effects, particularly when the
drug is administered in low doses. Since even a small amount of a
drug may be sufficient to activate multiple rounds of marker (e.g.,
a 32544 marker) transcription or expression, the amplified marker
may be in a quantity which is more readily detectable than the drug
itself. Also, the marker may be more easily detected due to the
nature of the marker itself, for example, using the methods
described herein, anti-32544 antibodies may be employed in an
immune-based detection system for a 32544 protein marker, or
32544-specific radiolabeled probes may be used to detect a 32544
mRNA marker. Furthermore, the use of a pharmacodynamic marker may
offer mechanism-based prediction of risk due to drug treatment
beyond the range of possible direct observations. Examples of the
use of pharmacodynamic markers in the art include: Matsuda et al.
U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect.
90: 229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl.
3: S21-S24; and Nicolau (1999) Am, J Health-Syst. Pharm. 56 Suppl.
3: S16-S20.
[0337] The 32544 molecules of the invention are also useful as
pharmacogenomic markers. As used herein, a "pharmacogenomic marker"
is an objective biochemical marker which correlates with a specific
clinical drug response or susceptibility in a subject (see, e.g.,
McLeod et al. (1999) Eur. J. Cancer 35(12): 1650-1652). The
presence or quantity of the pharmacogenomic marker is related to
the predicted response of the subject to a specific drug or class
of drugs prior to administration of th e drug. By assessing the
presence or quantity of one or more pharmacogenomic markers in a
subject, a drug therapy which is most appropriate for the subject,
or which is predicted to have a greater degree of success, may be
selected. For example, based on the presence or quantity of RNA, or
protein (e.g., 32544 protein or RNA) for specific tumor markers in
a subject, a drug or course of treatment may be selected that is
optimized for the treatment of the specific tumor likely to be
present in the subject. Similarly, the presence or absence of a
specific sequence mutation in 32544 DNA may correlate 32544 drug
response. The use of pharmacogenomic markers therefore permits the
application of the most appropriate treatment for each subject
without having to administer the therapy.
[0338] Pharmaceutical Compositions
[0339] The nucleic acid and polypeptides, fragments thereof, as
well as anti-32544 antibodies (also referred to herein as "active
compounds") of the invention can be incorporated into
pharmaceutical compositions. Such compositions typically include
the nucleic acid molecule, protein, or antibody and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" includes solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. Supplementary active compounds can
also be incorporated into the compositions.
[0340] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e.g., inhalation), transdermal (topical),
transmucosal, and rectal administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0341] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It should be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifingal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0342] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0343] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0344] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0345] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0346] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0347] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0348] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
[0349] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit high therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0350] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0351] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
protein or polypeptide can be administered one time per week for
between about 1 to 10 weeks, preferably between 2 to 8 weeks, more
preferably between about 3 to 7 weeks, and even more preferably for
about 4, 5, or 6 weeks. The skilled artisan will appreciate that
certain factors may influence the dosage and timing required to
effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of a protein, polypeptide, or antibody can include
a single treatment or, preferably, can include a series of
treatments.
[0352] For antibodies, the preferred dosage is 0.1 mg/kg of body
weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act
in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually
appropriate. Generally, partially human antibodies and fully human
antibodies have a longer half-life within the human body than other
antibodies. Accordingly, lower dosages and less frequent
administration is often possible. Modifications such as lipidation
can be used to stabilize antibodies and to enhance uptake and
tissue penetration (e.g., into the brain). A method for lipidation
of antibodies is described by Cruikshank et al., ((1997) J.
Acquired Immune Deficiency Syndromes and Human Retrovirology
14:193).
[0353] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics (e.g., peptoids), amino
acids, amino acid analogs, polynucleotides, polynucleotide analogs,
nucleotides, nucleotide analogs, organic or inorganic compounds
(i.e., including heteroorganic and organometallic compounds) having
a molecular weight less than about 10,000 grams per mole, organic
or inorganic compounds having a molecular weight less than about
5,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 1,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 500
grams per mole, and salts, esters, and other pharmaceutically
acceptable forms of such compounds.
[0354] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram. It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. When one or more of these small molecules is to be
administered to an animal (e.g., a human) in order to modulate
expression or activity of a polypeptide or nucleic acid of the
invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0355] An antibody (or fragment thereof) may be conjugated to a
therapeutic moiety such as a cytotoxin, a therapeutic agent or a
radioactive metal ion. A cytotoxin or cytotoxic agent includes any
agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0356] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
.alpha.-interferon, .beta.-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophase colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0357] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980. The nucleic acid molecules of the
invention can be inserted into vectors and used as gene therapy
vectors. Gene therapy vectors can be delivered to a subject by, for
example, intravenous injection, local administration (see U.S. Pat.
No. 5,328,470) or by stereotactic injection (see e.g., Chen et al.,
(1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells which
produce the gene delivery system.
[0358] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0359] Methods of Treatment:
[0360] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant or unwanted 32544 expression or activity. "Treatment", as
used herein, is defined as the application or administration of a
therapeutic agent to a patient, or application or administration of
a therapeutic agent to an isolated tissue or cell line from a
patient, who has a disease, a symptom of disease or a
predisposition toward a disease, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve or affect
the disease, the symptoms of disease or the predisposition toward
disease. A therapeutic agent includes, but is not limited to, small
molecules, peptides, antibodies, ribozymes and antisense
oligonucleotides. With regards to both prophylactic and therapeutic
methods of treatment, such treatments may be specifically tailored
or modified, based on knowledge obtained from the field of
pharmacogenomics. "Pharmacogenomics", as used herein, refers to the
application of genomics technologies such as gene sequencing,
statistical genetics, and gene expression analysis to drugs in
clinical development and on the market. More specifically, the term
refers the study of how a patient's genes determine his or her
response to a drug (e.g., a patient's "drug response phenotype", or
"drug response genotype".) Thus, another aspect of the invention
provides methods for tailoring an individual's prophylactic or
therapeutic treatment with either the 32544 molecules of the
present invention or 32544 modulators according to that
individual's drug response genotype. Pharmacogenomics allows a
clinician or physician to target prophylactic or therapeutic
treatments to patients who will most benefit from the treatment and
to avoid treatment of patients who will experience toxic
drug-related side effects.
[0361] In one aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant or unwanted 32544 expression or activity, by administering
to the subject a 32544 or an agent which modulates 32544 expression
or at least one 32544 activity. Subjects at risk for a disease
which is caused or contributed to by aberrant or unwanted 32544
expression or activity can be identified by, for example, any or a
combination of diagnostic or prognostic assays as described herein.
Administration of a prophylactic agent can occur prior to the
manifestation of symptoms characteristic of the 32544 aberrance,
such that a disease or disorder is prevented or, alternatively,
delayed in its progression. Depending on the type of 32544
aberrance, for example, a 32544, 32544 agonist or 32544 antagonist
agent can be used for treating the subject. The appropriate agent
can be determined based on screening assays described herein.
[0362] It is possible that some 32544 disorders can be caused, at
least in part, by an abnormal level of gene product, or by the
presence of a gene product exhibiting abnormal activity. As such,
the reduction in the level and/or activity of such gene products
would bring about the amelioration of disorder-symptoms.
[0363] As discussed, successful treatment of 32544 disorders can be
brought about by techniques that serve to inhibit the expression or
activity of target gene products. For example, compounds, e.g., an
agent identified using an assays described above, that proves to
exhibit negative modulatory activity, can be used in accordance
with the invention to prevent and/or ameliorate symptoms of 32544
disorders. Such molecules can include, but are not limited to
peptides, phosphopeptides, small organic or inorganic molecules, or
antibodies (including, for example, polyclonal, monoclonal,
humanized, anti-idiotypic, chimeric or single chain antibodies, and
FAb, F(ab').sub.2 and FAb expression library fragments, scFV
molecules, and epitope-binding fragments thereof).
[0364] Further, antisense and ribozyme molecules that inhibit
expression of the target gene can also be used in accordance with
the invention to reduce the level of target gene expression, thus
effectively reducing the level of target gene activity. Still
further, triple helix molecules can be utilized in reducing the
level of target gene activity. Antisense, ribozyme and triple helix
molecules are discussed above.
[0365] It is possible that the use of antisense, ribozyme, and/or
triple helix molecules to reduce or inhibit mutant gene expression
can also reduce or inhibit the transcription (triple helix) and/or
translation (antisense, ribozyme) of mRNA produced by normal target
gene alleles, such that the concentration of normal target gene
product present can be lower than is necessary for a normal
phenotype. In such cases, nucleic acid molecules that encode and
express target gene polypeptides exhibiting normal target gene
activity can be introduced into cells via gene therapy method.
Alternatively, in instances in that the target gene encodes an
extracellular protein, it can be preferable to co-administer normal
target gene protein into the cell or tissue in order to maintain
the requisite level of cellular or tissue target gene activity.
[0366] Another method by which nucleic acid molecules may be
utilized in treating or preventing a disease characterized by 32544
expression is through the use of aptamer molecules specific for
32544 protein. Aptamers are nucleic acid molecules having a
tertiary structure which permits them to specifically bind to
protein ligands (see, e.g., Osborne, et al., Curr. Opin. Chem. Biol
1997, 1(1): 5-9; and Patel, D. J., Curr. Opin. Chem. Biol. 1997
June;1(1):32-46). Since nucleic acid molecules may in many cases be
more conveniently introduced into target cells than therapeutic
protein molecules may be, aptamers offer a method by which 32544
protein activity may be specifically decreased without the
introduction of drugs or other molecules which may have pluripotent
effects.
[0367] Antibodies can be generated that are both specific for
target gene product and that reduce target gene product activity.
Such antibodies may, therefore, by administered in instances
whereby negative modulatory techniques are appropriate for the
treatment of 32544 disorders. For a description of antibodies, see
the Antibody section above.
[0368] In circumstances wherein injection of an animal or a human
subject with a 32544 protein or epitope for stimulating antibody
production is harmful to the subject, it is possible to generate an
immune response against 32544 through the use of anti-idiotypic
antibodies (see, for example, Herlyn, D., Ann. Med.
1999;31(1):66-78; and Bhattacharya-Chatterjee, M., and Foon, K. A.,
Cancer Treat. Res. 1998;94:51-68). If an anti-idiotypic antibody is
introduced into a mammal or human subject, it should stimulate the
production of anti-anti-idiotypic antibodies, which should be
specific to the 32544 protein. Vaccines directed to a disease
characterized by 32544 expression may also be generated in this
fashion.
[0369] In instances where the target antigen is intracellular and
whole antibodies are used, internalizing antibodies may be
preferred. Lipofectin or liposomes can be used to deliver the
antibody or a fragment of the Fab region that binds to the target
antigen into cells. Where fragments of the antibody are used, the
smallest inhibitory fragment that binds to the target antigen is
preferred. For example, peptides having an amino acid sequence
corresponding to the Fv region of the antibody can be used.
Alternatively, single chain neutralizing antibodies that bind to
intracellular target antigens can also be administered. Such single
chain antibodies can be administered, for example, by expressing
nucleotide sequences encoding single-chain antibodies within the
target cell population (see e.g., Marasco et al., (1993, Proc.
Natl. Acad. Sci. USA 90:7889-7893).
[0370] The identified compounds that inhibit target gene
expression, synthesis and/or activity can be administered to a
patient at therapeutically effective doses to prevent, treat or
ameliorate 32544 disorders. A therapeutically effective dose refers
to that amount of the compound sufficient to result in amelioration
of symptoms of the disorders.
[0371] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
that exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects can be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0372] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound that achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma can
be measured, for example, by high performance liquid
chromatography.
[0373] Another example of determination of effective dose for an
individual is the ability to directly assay levels of "free" and
"bound" compound in the serum of the test subject. Such assays may
utilize antibody mimics and/or "biosensors" that have been created
through molecular imprinting techniques. The compound which is able
to modulate 32544 activity is used as a template, or "imprinting
molecule", to spatially organize polymerizable monomers prior to
their polymerization with catalytic reagents. The subsequent
removal of the imprinted molecule leaves a polymer matrix which
contains a repeated "negative image" of the compound and is able to
selectively rebind the molecule under biological assay conditions.
A detailed review of this technique can be seen in Ansell, R. J. et
al., (1996) Current Opinion in Biotechnology 7:89-94 and in Shea,
K. J., (1994) Trends in Polymer Science 2:166-173. Such "imprinted"
affinity matrixes are amenable to ligand-binding assays, whereby
the immobilized monoclonal antibody component is replaced by an
appropriately imprinted matrix. An example of the use of such
matrixes in this way can be seen in Vlatakis, G. et al., (1993)
Nature 361:645-647. Through the use of isotope-labeling, the "free"
concentration of compound which modulates the expression or
activity of 32544 can be readily monitored and used in calculations
of IC.sub.50.
[0374] Such "imprinted" affinity matrixes can also be designed to
include fluorescent groups whose photon-emitting properties
measurably change upon local and selective binding of target
compound. These changes can be readily assayed in real time using
appropriate fiberoptic devices, in turn allowing the dose in a test
subject to be quickly optimized based on its individual IC.sub.50.
A rudimentary example of such a "biosensor" is discussed in Kriz,
D. et al., (1995) Analytical Chemistry 67:2142-2144.
[0375] Another aspect of the invention pertains to methods of
modulating 32544 expression or activity for therapeutic purposes.
Accordingly, in an exemplary embodiment, the modulatory method of
the invention involves contacting a cell with a 32544 or agent that
modulates one or more of the activities of 32544 protein activity
associated with the cell. An agent that modulates 32544 protein
activity can be an agent as described herein, such as a nucleic
acid or a protein, a naturally-occurring target molecule of a 32544
protein (e.g., a 32544 substrate or receptor), a 32544 antibody, a
32544 agonist or antagonist, a peptidomimetic of a 32544 agonist or
antagonist, or other small molecule.
[0376] In one embodiment, the agent stimulates one or 32544
activities. Examples of such stimulatory agents include active
32544 protein and a nucleic acid molecule encoding 32544. In
another embodiment, the agent inhibits one or more 32544
activities. Examples of such inhibitory agents include antisense
32544 nucleic acid molecules, anti-32544 antibodies, and 32544
inhibitors. These modulatory methods can be performed in vitro
(e.g., by culturing the cell with the agent) or, alternatively, in
vivo (e.g., by administering the agent to a subject). As such, the
present invention provides methods of treating an individual
afflicted with a disease or disorder characterized by aberrant or
unwanted expression or activity of a 32544 protein or nucleic acid
molecule. In one embodiment, the method involves administering an
agent (e.g., an agent identified by a screening assay described
herein), or combination of agents that modulates (e.g., upregulates
or downregulates) 32544 expression or activity. In another
embodiment, the method involves administering a 32544 protein or
nucleic acid molecule as therapy to compensate for reduced,
aberrant, or unwanted 32544 expression or activity.
[0377] Stimulation of 32544 activity is desirable in situations in
which 32544 is abnormally downregulated and/or in which increased
32544 activity is likely to have a beneficial effect. For example,
stimulation of 32544 activity is desirable in situations in which a
32544 is downregulated and/or in which increased 32544 activity is
likely to have a beneficial effect. Likewise, inhibition of 32544
activity is desirable in situations in which 32544 is abnormally
upregulated and/or in which decreased 32544 activity is likely to
have a beneficial effect.
[0378] The 32544 molecules can act as novel diagnostic targets and
therapeutic agents for controlling one or more of cellular
proliferative and/or differentiative disorders, cardiovascular
disorders, immune disorders, brain disorders, and pain disorders or
metabolic disorders as described above, as well as disorders
associated with bone metabolism, hematopoietic disorders, liver
disorders, or viral diseases.
[0379] Aberrant expression and/or activity of 32544 molecules may
mediate disorders associated with bone metabolism. "Bone
metabolism" refers to direct or indirect effects in the formation
or degeneration of bone structures e.g., bone formation, bone
resorption, etc., which may ultimately affect the concentrations in
serum of calcium and phosphate. This term also includes activities
mediated by 32544 molecules effects in bone cells, e.g. osteoclasts
and osteoblasts, that may in turn result in bone formation and
degeneration. For example 32544 molecules may support different
activities of bone resorbing osteoclasts such as the stimulation of
differentiation of monocytes and mononuclear phagocytes into
osteoclasts. Accordingly, 32544 molecules that modulate the
production of bone cells can influence bone formation and
degeneration, and thus may be used to treat bone disorders.
Examples of such disorders include, but are not limited to,
osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis
fibrosa cystica, renal osteodystrophy, osteosclerosis,
anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta
ossium, secondary hyperparathyrodism, hypoparathyroidism,
hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced
metabolism, medullary carcinoma, chronic renal disease, rickets,
sarcoidosis, glucocorticoid antagonism, malabsorption syndrome,
steatorrhea, tropical sprue, idiopathic hypercalcemia and milk
fever.
[0380] Examples of hematopoietic disorders include, but are not
limited to, autoimmune diseases (including, for example, diabetes
mellitus, arthritis (including rheumatoid arthritis, juvenile
rheumatoid arthritis, osteoarthritis, psoriatic arthritis),
multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic
lupus erythematosis, autoimmune thyroiditis, dermatitis (including
atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's
Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Graves' disease, sarcoidosis,
primary biliary cirrhosis, uveitis posterior, and interstitial lung
fibrosis), graft-versus-host disease, cases of transplantation, and
allergy such as, atopic allergy.
[0381] Disorders which may be treated or diagnosed by methods
described herein include, but are not limited to disorders
associated with an accumulation in the liver of fibrous tissue,
such as that resulting from an imbalance between production and
degradation of the extracellular matrix accompanied by the collapse
and condensation of preexisting fibers. The methods described
herein can be used to diagnose or treat hepatocellular necrosis or
injury induced by a wide variety of agents including processes
which disturb homeostasis, such as an inflammatory process, tissue
damage resulting from toxic injury or altered hepatic blood flow,
and infections (e.g., bacterial, viral and parasitic). For example,
the methods can be used for the early detection of hepatic injury,
such as portal hypertension or hepatic fibrosis. In addition, the
methods can be employed to detect liver fibrosis attributed to
inborn errors of metabolsim, for example, fibrosis resulting from a
storage disorder such as Gaucher's disease (lipid abnormalities) or
a glycogen storage disease, A1-antitrypsin deficiency; a disorder
mediating the accumulation (e.g., storage) of an exogenous
substance, for example, hemochromatosis (iron-overload syndrome)
and copper storage diseases (Wilson's disease), disorders resulting
in the accumulation of a toxic metabolite (e.g., tyrosinemia,
fructosemia and galactosemia) and peroxisomal disorders (e.g.,
Zellweger syndrome). Additionally, the methods described herein may
be useful for the early detection and treatment of liver injury
associated with the administration of various chemicals or drugs,
such as for example, methotrexate, isonizaid, oxyphenisatin,
methyldopa, chlorpromazine, tolbutamide or alcohol, or which
represents a hepatic manifestation of a vascular disorder such as
obstruction of either the intrahepatic or extrahepatic bile flow or
an alteration in hepatic circulation resulting, for example, from
chronic heart failure, veno-occlusive disease, portal vein
thrombosis or Budd-Chiari syndrome.
[0382] Additionally, 32544 molecules may play an important role in
the etiology of certain viral diseases, including but not limited
to, Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV).
Modulators of 32544 activity could be used to control viral
diseases. The modulators can be used in the treatment and/or
diagnosis of viral infected tissue or virus-associated tissue
fibrosis, especially liver and liver fibrosis. Also, 32544
modulators can be used in the treatment and/or diagnosis of
virus-associated carcinoma, especially hepatocellular cancer.
[0383] Pharmacogenomics
[0384] The 32544 molecules of the present invention, as well as
agents, or modulators which have a stimulatory or inhibitory effect
on 32544 activity (e.g., 32544 gene expression) as identified by a
screening assay described herein can be administered to individuals
to treat (prophylactically or therapeutically) 32544 associated
disorders (e.g., cellular growth related disorders) associated with
aberrant or unwanted 32544 activity. In conjunction with such
treatment, pharmacogenomics (i.e., the study of the relationship
between an individual's genotype and that individual's response to
a foreign compound or drug) may be considered. Differences in
metabolism of therapeutics can lead to severe toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the pharmacologically active drug. Thus, a
physician or clinician may consider applying knowledge obtained in
relevant pharmacogenomics studies in determining whether to
administer a 32544 molecule or 32544 modulator as well as tailoring
the dosage and/or therapeutic regimen of treatment with a 32544
molecule or 32544 modulator.
[0385] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol.
Physiol. 23(10-11):983-985 and Linder, M. W. et al. (1997) Clin.
Chem. 43(2):254-266. In general, two types of pharmacogenetic
conditions can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body (altered
drug action) or genetic conditions transmitted as single factors
altering the way the body acts on drugs (altered drug metabolism).
These pharmacogenetic conditions can occur either as rare genetic
defects or as naturally-occurring polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0386] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase-II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high-resolution map can be generated from a
combination of some ten million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[0387] Alternatively, a method termed the "candidate gene
approach", can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drug's
target is known (e.g., a 32544 protein of the present invention),
all common variants of that gene can be fairly easily identified in
the population and it can be determined if having one version of
the gene versus another is associated with a particular drug
response.
[0388] Alternatively, a method termed the "gene expression
profiling", can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a 32544 molecule or 32544 modulator of the present
invention) can give an indication whether gene pathways related to
toxicity have been turned on.
[0389] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment of an individual. This knowledge, when applied to dosing
or drug selection, can avoid adverse reactions or therapeutic
failure and thus enhance therapeutic or prophylactic efficiency
when treating a subject with a 32544 molecule or 32544 modulator,
such as a modulator identified by one of the exemplary screening
assays described herein.
[0390] The present invention further provides methods for
identifying new agents, or combinations, that are based on
identifying agents that modulate the activity of one or more of the
gene products encoded by one or more of the 32544 genes of the
present invention, wherein these products may be associated with
resistance of the cells to a therapeutic agent. Specifically, the
activity of the proteins encoded by the 32544 genes of the present
invention can be used as a basis for identifying agents for
overcoming agent resistance. By blocking the activity of one or
more of the resistance proteins, target cells, e.g., cancer cells,
will become sensitive to treatment with an agent that the
unmodified target cells were resistant to.
[0391] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a 32544 protein can be applied in
clinical trials. For example, the effectiveness of an agent
determined by a screening assay as described herein to increase
32544 gene expression, protein levels, or upregulate 32544
activity, can be monitored in clinical trials of subjects
exhibiting decreased 32544 gene expression, protein levels, or
downregulated 32544 activity. Alternatively, the effectiveness of
an agent determined by a screening assay to decrease 32544 gene
expression, protein levels, or downregulate 32544 activity, can be
monitored in clinical trials of subjects exhibiting increased 32544
gene expression, protein levels, or upregulated 32544 activity. In
such clinical trials, the expression or activity of a 32544 gene,
and preferably, other genes that have been implicated in, for
example, a 32544-associated disorder can be used as a "read out" or
markers of the phenotype of a particular cell.
[0392] Other Embodiments
[0393] In another aspect, the invention features, a method of
analyzing a plurality of capture probes. The method can be used,
e.g., to analyze gene expression. The method includes: providing a
two dimensional array having a plurality of addresses, each address
of the plurality being positionally distinguishable from each other
address of the plurality, and each address of the plurality having
a unique capture probe, e.g., a nucleic acid or peptide sequence;
contacting the array with a 32544, preferably purified, nucleic
acid, preferably purified, polypeptide, preferably purified, or
antibody, and thereby evaluating the plurality of capture probes.
Binding, e.g., in the case of a nucleic acid, hybridization with a
capture probe at an address of the plurality, is detected, e.g., by
signal generated from a label attached to the 32544 nucleic acid,
polypeptide, or antibody.
[0394] The capture probes can be a set of nucleic acids from a
selected sample, e.g., a sample of nucleic acids derived from a
control or non-stimulated tissue or cell.
[0395] The method can include contacting the 32544 nucleic acid,
polypeptide, or antibody with a first array having a plurality of
capture probes and a second array having a different plurality of
capture probes. The results of each hybridization can be compared,
e.g., to analyze differences in expression between a first and
second sample. The first plurality of capture probes can be from a
control sample, e.g., a wild type, normal, or non-diseased,
non-stimulated, sample, e.g., a biological fluid, tissue, or cell
sample. The second plurality of capture probes can be from an
experimental sample, e.g., a mutant type, at risk, disease-state or
disorder-state, or stimulated, sample, e.g., a biological fluid,
tissue, or cell sample.
[0396] The plurality of capture probes can be a plurality of
nucleic acid probes each of which specifically hybridizes, with an
allele of 32544. Such methods can be used to diagnose a subject,
e.g., to evaluate risk for a disease or disorder, to evaluate
suitability of a selected treatment for a subject, to evaluate
whether a subject has a disease or disorder. 32544 is associated
with phospholipase family activity, thus it is useful for disorders
associated with abnormal lipid metabolism.
[0397] The method can be used to detect SNPs, as described
above.
[0398] In another aspect, the invention features, a method of
analyzing a plurality of probes. The method is useful, e.g., for
analyzing gene expression. The method includes: providing a two
dimensional array having a plurality of addresses, each address of
the plurality being positionally distinguishable from each other
address of the plurality having a unique capture probe, e.g.,
wherein the capture probes are from a cell or subject which express
or mis express 32544 or from a cell or subject in which a 32544
mediated response has been elicited, e.g., by contact of the cell
with 32544 nucleic acid or protein, or administration to the cell
or subject 32544 nucleic acid or protein; contacting the array with
one or more inquiry probe, wherein an inquiry probe can be a
nucleic acid, polypeptide, or antibody (which is preferably other
than 32544 nucleic acid, polypeptide, or antibody); providing a two
dimensional array having a plurality of addresses, each address of
the plurality being positionally distinguishable from each other
address of the plurality, and each address of the plurality having
a unique capture probe, e.g., wherein the capture probes are from a
cell or subject which does not express 32544 (or does not express
as highly as in the case of the 32544 positive plurality of capture
probes) or from a cell or subject which in which a 32544 mediated
response has not been elicited (or has been elicited to a lesser
extent than in the first sample); contacting the array with one or
more inquiry probes (which is preferably other than a 32544 nucleic
acid, polypeptide or antibody), and thereby evaluating the
plurality of capture probes. Binding, e.g., in the case of a
nucleic acid, hybridization with a capture probe at an address of
the plurality, is detected, e.g., by signal generated from a label
attached to the nucleic acid, polypeptide, or antibody.
[0399] In another aspect, the invention features, a method of
analyzing 32544, e.g., analyzing structure, function, or
relatedness to other nucleic acid or amino acid sequences. The
method includes: providing a 32544 nucleic acid or amino acid
sequence; comparing the 32544 sequence with one or more preferably
a plurality of sequences from a collection of sequences, e.g., a
nucleic acid or protein sequence database; to thereby analyze
32544.
[0400] Preferred databases include GenBank.TM.. The method can
include evaluating the sequence identity between a 32544 sequence
and a database sequence. The method can be performed by accessing
the database at a second site, e.g., over the internet.
[0401] In another aspect, the invention features, a set of
oligonucleotides, useful, e.g., for identifying SNP's, or
identifying specific alleles of 32544. The set includes a plurality
of oligonucleotides, each of which has a different nucleotide at an
interrogation position, e.g., an SNP or the site of a mutation. In
a preferred embodiment, the oligonucleotides of the plurality
identical in sequence with one another (except for differences in
length). The oligonucleotides can be provided with different
labels, such that an oligonucleotides which hybridizes to one
allele provides a signal that is distinguishable from an
oligonucleotides which hybridizes to a second allele.
[0402] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are incorporated herein by
reference.
EXAMPLES
Example 1
Identification and Characterization of Human 32544 cDNA
[0403] The human 32544 sequence (FIG. 1; SEQ ID NO:1), which is
approximately 4635 nucleotides long including untranslated regions,
contains a predicted methionine-initiated coding sequence of about
3621 nucleotides (nucleotides 435 to 4055 of SEQ ID NO:1; SEQ ID
NO:3). The coding sequence encodes a 1207 amino acid protein (SEQ
ID NO:2).
Example 2
Tissue Distribution of 32544 mRNA
[0404] Northern blot hybridizations with various RNA samples can be
performed under standard conditions and washed under stringent
conditions, i.e., 0.2.times.SSC at 65.degree. C. A DNA probe
corresponding to all or a portion of the 32544 cDNA (SEQ ID NO:1)
can be used. The DNA was radioactively labeled with .sup.32P-dCTP
using the Prime-It Kit (Stratagene, La Jolla, Calif.) according to
the instructions of the supplier. Filters containing mRNA from
mouse hematopoietic and endocrine tissues, and cancer cell lines
(Clontech, Palo Alto, Calif.) can be probed in ExpressHyb
hybridization solution (Clontech) and washed at high stringency
according to manufacturer's recommendations.
Example 3
Gene Expression Analysis
[0405] Total RNA was prepared from various human tissues by a
single step extraction method using RNA STAT-60 according to the
manufacturer's instructions (TelTest, Inc). Each RNA preparation
was treated with DNase I (Ambion) at 37.degree. C. for 1 hour.
DNAse I treatment was determined to be complete if the sample
required at least 38 PCR amplification cycles to reach a threshold
level of fluorescence using .beta.-2 microglobulin as an internal
amplicon reference. The integrity of the RNA samples following
DNase I treatment was confirmed by agarose gel electrophoresis and
ethidium bromide staining. After phenol extraction cDNA was
prepared from the sample using the SUPERSCRIP.TM. Choice System
following the manufacturer's instructions (GibcoBRL). A negative
control of RNA without reverse transcriptase was mock reverse
transcribed for each RNA sample.
[0406] Human 32544 expression was measured by TaqMan.RTM.
quantitative PCR (Perkin Elmer Applied Biosystems) in cDNA prepared
from a variety of normal and diseased (e.g., cancerous) human
tissues or cell lines.
[0407] Probes were designed by PrimerExpress software (PE
Biosystems) based on the sequence of the human 32544 gene. Each
human 32544 gene probe was labeled using FAM
(6-carboxyfluorescein), and the .beta.2-microglobulin reference
probe was labeled with a different fluorescent dye, VIC. The
differential labeling of the target gene and internal reference
gene thus enabled measurement in same well. Forward and reverse
primers and the probes for both .beta.2-microglobulin and target
gene were added to the TaqMan.RTM. Universal PCR Master Mix (PE
Applied Biosystems). Although the final concentration of primer and
probe could vary, each was internally consistent within a given
experiment. A typical experiment contained 200 nM of forward and
reverse primers plus 100 nM probe for .beta.-2 microglobulin and
600 nM forward and reverse primers plus 200 nM probe for the target
gene. TaqMan matrix experiments were carried out on an ABI PRISM
7700 Sequence Detection System (PE Applied Biosystems). The thermal
cycler conditions were as follows: hold for 2 min at 50.degree. C.
and 10 min at 95.degree. C., followed by two-step PCR for 40 cycles
of 95.degree. C. for 15 sec followed by 60.degree. C. for 1
min.
[0408] The following method was used to quantitatively calculate
human 32544 gene expression in the various tissues relative to
.beta.-2 microglobulin expression in the same tissue. The threshold
cycle (Ct) value is defined as the cycle at which a statistically
significant increase in fluorescence is detected. A lower Ct value
is indicative of a higher mRNA concentration. The Ct value of the
human 32544 gene is normalized by subtracting the Ct value of the
.beta.-2 microglobulin gene to obtain a .DELTA.Ct value using the
following formula: .DELTA.Ct=Ct.sub.human 59914 and
59921-Ct.sub..beta.-2 microglobulin. Expression is then calibrated
against a cDNA sample showing a comparatively low level of
expression of the human 32544 gene. The .DELTA.Ct value for the
calibrator sample is then subtracted from .DELTA.Ct for each tissue
sample according to the following formula:
.DELTA..DELTA.Ct=.DELTA.Ct-.sub.sample-.DELTA.Ct-.sub.calibrator.
Relative expression is then calculated using the arithmetic formula
given by 2-.DELTA.Ct. Expression of the target human 0.32544 gene
in each of the tissues tested is then graphically represented as
discussed in more detail below.
[0409] TaqMan real-time quantitative RT-PCR was used to detect the
presence of RNA transcript corresponding to human 32544 relative to
a no template control in a panel of human tissues or cells.
[0410] Taqman in Pain human panel phase I shows the highest levels
of expression in brain followed by spinal cord and skin as shown in
Table 1.
1TABLE 1 Phase 1.3.3 Expression of 32544 w/.beta.2 Tissue Type Mean
.beta. 2 Mean .differential..differential. Ct Expression Artery
normal 40 21.64 18.36 0 Vein normal 40 20.5 19.5 0 Aortic SMC EARLY
38.01 22.2 15.81 0 Coronary SMC 35.63 22.34 13.29 0 Static HUVEC
32.74 20.54 12.21 0.2111 Shear HUVEC 30.81 20.8 10.01 0.9698 Heart
normal 31.85 18.59 13.26 0.1023 Heart CHF 32.4 18.99 13.4 0.0922
Kidney 29.62 20.52 9.11 1.816 Skeletal Muscle 37.45 21.58 15.88 0
Adipose normal 37.2 19.82 17.38 0 Pancreas 32.05 21.75 10.3 0.7932
primary osteoblasts 32.79 18.95 13.85 0.068 Osteoclasts (diff)
34.15 17.5 16.64 0.0098 Skin normal 28.11 21.22 6.89 8.4315 Spinal
cord normal 26.61 20.09 6.51 10.9343 Brain Cortex normal 25.2 21.34
3.86 69.1082 Brain Hypothalamus normal 26.95 21.68 5.27 25.9162
Nerve 35.86 23.53 12.33 0 DRG (Dorsal Root Ganglion) 31.93 21.82
10.11 0.908 Glial Cells (Astrocytes) 33 22.48 10.52 0.6834
Glioblastoma 27.9 18.22 9.68 1.2191 Breast normal 30.43 21.04 9.39
1.4853 Breast tumor 27.82 18.72 9.1 1.8223 Ovary normal 32.47 20.2
12.27 0.2032 Ovary Tumor 33.69 21.48 12.21 0.2118 Prostate Normal
27.92 20.09 7.83 4.41 Prostate Tumor 26.64 18.15 8.49 2.7813
Epithelial Cells (Prostate) 25.16 21.37 3.79 72.544 Colon normal
29.86 18.44 11.41 0.3662 Colon Tumor 29.11 18.75 10.36 0.7635 Lung
normal 31.66 18.36 13.3 0.0992 Lung tumor 25.68 18.57 7.12 7.189
Lung COPD 30.35 19.09 11.26 0.4092 Colon IBD 31.69 18.66 13.04
0.1191 Liver normal 31.84 20.32 11.53 0.3382 Liver fibrosis 32.08
21.57 10.51 0.6858 Dermal Cells-fibroblasts 33.99 20.45 13.54 0.084
Spleen normal 28.81 19.6 9.21 1.6885 Tonsil normal 25.63 17.43 8.19
3.4242 Lymph node 27.29 18.74 8.54 2.6773 Small Intestine 30.25
19.73 10.52 0.681 Skin-Decubitus 29.14 20.52 8.62 2.5417 Synovium
31.92 20.16 11.77 0.2873 BM-MNC (Bone marrow 26.13 16.98 9.15
1.7603 mononuclear cells) Activated PBMC 29.23 16.22 13.02
0.1208
[0411]
2TABLE 2 Human Panel Phase I (Pain) Tissue Type 32544 .beta.2.803
.differential.Ct Expression Adrenal Gland 29.91 18.78 11.14 0.44
Brain 24.65 20.35 4.30 50.77 Heart 37.29 18.67 18.62 0.00 Kidney
28.78 18.68 10.10 0.91 Liver 30.86 19.12 11.74 0.29 Lung 30.90
16.68 14.22 0.05 Mammary Gland 28.45 18.34 10.11 0.90 Pancreas
31.06 21.66 9.40 1.49 Placenta 35.88 18.68 17.20 0.01 Prostate
26.57 18.13 8.44 2.88 Salivary Gland 27.37 18.68 8.70 2.41 Muscle
32.42 21.00 11.42 0.36 Sm. Intestine 29.76 19.11 10.66 0.62 Spleen
28.46 16.57 11.90 0.26 Stomach 30.91 18.63 12.28 0.20 Teste 27.58
20.20 7.38 6.02 Thymus 26.63 18.11 8.52 2.72 Trachea 27.11 18.68
8.43 2.90 Uterus 30.85 18.70 12.15 0.22 Spinal Cord 25.65 19.21
6.44 11.52 DRG 33.43 19.26 14.17 0.05 Skin 25.85 18.90 6.96
8.06
[0412] Taqman experiments with the rat probe showed similar pattern
of expression in the Phase I panel than the human probe. This rat
Phase I panel also showed high levels of expression of 32544 in the
sympathetic neurons of the superior cervical ganglia.
3TABLE 3 Rat Panel Phase I Tissue r32544 18S .differential.Ct
Expression Brain 25.07 12.12 12.96 0.13 Spinal Cord 29.68 12.74
16.95 0.01 DRG 32.10 12.37 19.73 0.00 SCG 26.18 13.21 12.97 0.12
Optic Nerve 28.44 11.66 16.78 0.01 Hairy Skin 28.39 13.40 14.99
0.03 Gastro Muscle 29.87 12.07 17.80 0.00 Heart 35.78 12.92 22.86
0.00 Kidney 35.65 13.59 22.06 0.00 Liver 39.15 12.83 26.32 0.00
Lung 32.41 13.35 19.06 0.00 Spleen 34.22 12.68 21.54 0.00 Aorta
32.66 12.69 19.98 0.00 Adrenal Gland 32.73 12.21 20.52 0.00
Salivary Gland 32.09 12.28 19.81 0.00 Thyroid 27.45 12.14 15.31
0.02 Prostate 32.75 12.23 20.52 0.00 Thymus 31.06 12.30 18.77 0.00
Trachea 28.79 13.18 15.61 0.02 Esophagus 27.26 12.02 15.24 0.03
Duodenum 29.89 13.49 16.40 0.01 Diaphragm 36.36 13.31 23.06 0.00
Colon 28.43 13.87 14.56 0.04
[0413] Rat Panel Phase II (Table 4) showed no regulation of 32544
in the DRG after CFA treatment. However, 32544 is up-regulated
about six fold in DRG after CCI and about 10 folds in axotomized
DRG.
4TABLE 4 Rat Panel Phase II Tissue r32544 18S .differential.Ct
Expression Nave DRG 32.18 11.44 20.74 0.001 I DRG CCI 3 29.00 11.50
17.50 0.005 I DRG CCI 7 28.80 11.18 17.62 0.005 I DRG CCI 10 29.16
11.09 18.07 0.004 I DRG CCI 14 31.93 13.19 18.75 0.002 I DRG CCI 28
29.87 11.26 18.61 0.002 Nave DRG 32.03 11.50 20.53 0.001 I DRG CFA
1 32.53 11.24 21.30 0.000 I DRG CFA 3 32.60 11.37 21.24 0.000 I DRG
CFA 7 32.50 11.37 21.13 0.000 I DRG CFA 10 32.45 11.31 21.14 0.000
I DRG CFA 14 32.21 11.33 20.88 0.001 I DRG CFA 28 32.41 11.34 21.08
0.000 Nave DRG 32.35 11.42 20.93 0.001 I DRG AXT 1 30.26 11.01
19.25 0.002 I DRG AXT 3 28.42 11.61 16.82 0.009 I DRG AXT 7 27.96
11.44 16.52 0.011 I DRG AXT 14 27.53 11.36 16.18 0.014
[0414]
5TABLE 5 Rat Panel Phase III Tissue r32544 18S .differential.Ct
Expression Nave SC 25.13 11.76 13.37 0.09 I SC CCI 3 23.63 11.13
12.50 0.17 I SC CCI 7 24.35 12.08 12.27 0.20 I SC CCI 10 24.28
12.47 11.81 0.28 I SC CCI 14 24.25 11.53 12.72 0.15 I SC CCI 28
24.83 12.13 12.70 0.15 Nave SC 25.27 11.83 13.44 0.09 I SC CFA 3
24.73 11.63 13.10 0.11 I SC CFA 7 24.66 11.63 13.03 0.12 I SC CFA
10 24.61 11.37 13.24 0.10 I SC CFA 14 24.84 12.28 12.56 0.17 I SC
CFA 28 24.08 12.14 11.95 0.25 Nave SC 25.33 11.81 13.52 0.09 I SC
AXT 1 24.64 11.99 12.65 0.16 I SC AXT 3 24.86 11.71 13.15 0.11 I SC
AXT 7 24.88 11.59 13.29 0.10 I SC AXT 14 23.75 12.63 11.12 0.45
Example 4
In Situ Hybridization of 33544
[0415] Human ISH hybridization probe e+f shows expression of the
33544 gene in human brain, DRG and spinal cord. Human ISH probes do
not cross-react with rat tissues. Probe e+f cross-react with monkey
tissues, however, it shows higher background compared to the human
tissues. In the spinal cord 33544 is expressed in the dorsal horn
and in the DRG only in a small subpopulation of neurons.
Example 5
Recombinant Expression of 32544 in Bacterial Cells
[0416] In this example, 32544 is expressed as a recombinant
glutathione-S-transferase (GST) fusion polypeptide in E. coli and
the fusion polypeptide is isolated and characterized. Specifically,
32544 is fused to GST and this fusion polypeptide is expressed in
E. coli, e.g., strain PEB199. Expression of the GST-25934 fusion
protein in PEB199 is induced with IPTG. The recombinant fusion
polypeptide is purified from crude bacterial lysates of the induced
PEB199 strain by affinity chromatography on glutathione beads.
Using polyacrylamide gel electrophoretic analysis of the
polypeptide purified from the bacterial lysates, the molecular
weight of the resultant fusion polypeptide is determined.
Example 6
Expression of Recombinant 32544 Protein in COS Cells
[0417] To express the 32544 gene in COS cells, the pcDNA/Amp vector
by Invitrogen Corporation (San Diego, Calif.) is used. This vector
contains an SV40 origin of replication, an ampicillin resistance
gene, an E. coli replication origin, a CMV promoter followed by a
polylinker region, and an SV40 intron and polyadenylation site. A
DNA fragment encoding the entire 32544 protein and an HA tag
(Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to
its 3' end of the fragment is cloned into the polylinker region of
the vector, thereby placing the expression of the recombinant
protein under the control of the CMV promoter.
[0418] To construct the plasmid, the 32544 DNA sequence is
amplified by PCR using two primers. The 5' primer contains the
restriction site of interest followed by approximately twenty
nucleotides of the 32544 coding sequence starting from the
initiation codon; the 3' end sequence contains complementary
sequences to the other restriction site of interest, a translation
stop codon, the HA tag or FLAG tag and the last 20 nucleotides of
the 32544 coding sequence. The PCR amplified fragment and the
pcDNA/Amp vector are digested with the appropriate restriction
enzymes and the vector is dephosphorylated using the CIAP enzyme
(New England Biolabs, Beverly, Mass.). Preferably the two
restriction sites chosen are different so that the 32544 gene is
inserted in the correct orientation. The ligation mixture is
transformed into E. coli cells (strains HB101, DH5.alpha., SURE,
available from Stratagene Cloning Systems, La Jolla, Calif., can be
used), the transformed culture is plated on ampicillin media
plates, and resistant colonies are selected. Plasmid DNA is
isolated from transformants and examined by restriction analysis
for the presence of the correct fragment.
[0419] COS cells are subsequently transfected with the
32544-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium
chloride co-precipitation methods, DEAE-dextran-mediated
transfection, lipofection, or electroporation. Other suitable
methods for transfecting host cells can be found in Sambrook, J.,
Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory
Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989. The expression of
the 32544 polypeptide is detected by radiolabelling
(.sup.35S-methionine or .sup.35S-cysteine available from NEN,
Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and
Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1988) using an HA
specific monoclonal antibody. Briefly, the cells are labeled for 8
hours with .sup.35S-methionine (or .sup.35S-cysteine). The culture
media are then collected and the cells are lysed using detergents
(RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM
Tris, pH 7.5). Both the cell lysate and the culture media are
precipitated with an HA specific monoclonal antibody. Precipitated
polypeptides are then analyzed by SDS-PAGE.
[0420] Alternatively, DNA containing the 32544 coding sequence is
cloned directly into the polylinker of the pcDNA/Amp vector using
the appropriate restriction sites. The resulting plasmid is
transfected into COS cells in the manner described above, and the
expression of the 32544 polypeptide is detected by radiolabelling
and immunoprecipitation using a 32544 specific monoclonal
antibody.
Equivalents
[0421] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
17 1 4635 DNA Homo sapiens CDS (435)...(4058) 1 tcgcgatcta
gaactagtgg cacggctcct gcactcccac tgccgcagga actgctcagg 60
aacctgccgg tctccggctg ggacggtggc tggatcagct caagcctcca gggccctgag
120 gctgaggggc tgagtgctca ttccagccgc ctcggggaac ccgggctggg
agaccccatg 180 cctgggggtg agcctggagc cagggcagtg cggtgagagg
ctccggagag agggctgggc 240 accaccaggc ttgggtgtgt gatgcgctgc
tggcccaggc tacaccccga caagggacac 300 cgggggccct gggagcagag
agacctcaga gcagcctcct cctgcctcct gtggacggcc 360 ggccccagct
ggtgatccca gccagtccca gctttcagtt gctgccccca ccgacagtcc 420
tcagtccctc catg atg gct ccc ccg aca gcc ggc ccc ctt cct ggc cca 470
Met Ala Pro Pro Thr Ala Gly Pro Leu Pro Gly Pro 1 5 10 gct ctt ccg
cct gag gac cca ggg ccg gat ccg gag agc agg tgg ctt 518 Ala Leu Pro
Pro Glu Asp Pro Gly Pro Asp Pro Glu Ser Arg Trp Leu 15 20 25 ttc
ttg agc gcc aac att ctg ccc gtg gtg gag cgg tgc atg ggt gcc 566 Phe
Leu Ser Ala Asn Ile Leu Pro Val Val Glu Arg Cys Met Gly Ala 30 35
40 atg caa gag ggg atg cag atg gtg aag ctg cgt ggc ggc tcc aag ggc
614 Met Gln Glu Gly Met Gln Met Val Lys Leu Arg Gly Gly Ser Lys Gly
45 50 55 60 ctg gtc cgc ttc tac tac ctg gac gag cac cgc tcc tgc atc
cgc tgg 662 Leu Val Arg Phe Tyr Tyr Leu Asp Glu His Arg Ser Cys Ile
Arg Trp 65 70 75 agg ccc tca cgc aag aac gag aag gcc aag atc tcc
atc gac tcc atc 710 Arg Pro Ser Arg Lys Asn Glu Lys Ala Lys Ile Ser
Ile Asp Ser Ile 80 85 90 cag gag gtg agt gag ggg cgg cag tcg gag
gtc ttc cag cgc tac cct 758 Gln Glu Val Ser Glu Gly Arg Gln Ser Glu
Val Phe Gln Arg Tyr Pro 95 100 105 gac ggc agc ttc gac ccc aac tgc
tgc ttc agc atc tac cac ggc agc 806 Asp Gly Ser Phe Asp Pro Asn Cys
Cys Phe Ser Ile Tyr His Gly Ser 110 115 120 cac cgc gag tcg ctg gac
ctg gtc tcc acc agc agc gag gtg gcg cgc 854 His Arg Glu Ser Leu Asp
Leu Val Ser Thr Ser Ser Glu Val Ala Arg 125 130 135 140 acc tgg gtc
act ggc ctg cgc tac ctc atg gcc ggc atc agc gac gag 902 Thr Trp Val
Thr Gly Leu Arg Tyr Leu Met Ala Gly Ile Ser Asp Glu 145 150 155 gac
agc ctg gct cgc cgc cag cgc acc agg gac cag tgg ctg aag cag 950 Asp
Ser Leu Ala Arg Arg Gln Arg Thr Arg Asp Gln Trp Leu Lys Gln 160 165
170 acg ttt gac gag gcc gac aag aac ggg gat ggc agc ctg agc att ggc
998 Thr Phe Asp Glu Ala Asp Lys Asn Gly Asp Gly Ser Leu Ser Ile Gly
175 180 185 gag gtc ctg cag ctg ctg cac aag ctc aac gtg aac ctg ccc
cgg cag 1046 Glu Val Leu Gln Leu Leu His Lys Leu Asn Val Asn Leu
Pro Arg Gln 190 195 200 agg gtg aag cag atg ttc agg gaa gcg gac acg
gat gac cac caa ggg 1094 Arg Val Lys Gln Met Phe Arg Glu Ala Asp
Thr Asp Asp His Gln Gly 205 210 215 220 acg ctg ggt ttt gaa gag ttc
tgt gcc ttc tac aag atg atg tcc acc 1142 Thr Leu Gly Phe Glu Glu
Phe Cys Ala Phe Tyr Lys Met Met Ser Thr 225 230 235 cgc cgg gac ctc
tac ctg ctc atg ctg acc tac agc aac cac aag gac 1190 Arg Arg Asp
Leu Tyr Leu Leu Met Leu Thr Tyr Ser Asn His Lys Asp 240 245 250 cac
ctg gat gcc gcc agc ctg cag cgc ttc ctg cag gtg gag cag aag 1238
His Leu Asp Ala Ala Ser Leu Gln Arg Phe Leu Gln Val Glu Gln Lys 255
260 265 atg gcg ggt gtg acc ctc gag agc tgc cag gac atc atc gag cag
ttt 1286 Met Ala Gly Val Thr Leu Glu Ser Cys Gln Asp Ile Ile Glu
Gln Phe 270 275 280 gag cca tgc cca gaa aac aag agt aag ggg ctg ctg
ggc att gat ggc 1334 Glu Pro Cys Pro Glu Asn Lys Ser Lys Gly Leu
Leu Gly Ile Asp Gly 285 290 295 300 ttc acc aac tac acc agg agc cct
gct ggt gac atc ttc aac cct gag 1382 Phe Thr Asn Tyr Thr Arg Ser
Pro Ala Gly Asp Ile Phe Asn Pro Glu 305 310 315 cac cac cat gtg cac
cag gac atg acg cag ccg ctg agc cac tac ttc 1430 His His His Val
His Gln Asp Met Thr Gln Pro Leu Ser His Tyr Phe 320 325 330 atc acc
tcg tcc cac aac acc tac ctc gtg ggt gac cag ctc atg tcc 1478 Ile
Thr Ser Ser His Asn Thr Tyr Leu Val Gly Asp Gln Leu Met Ser 335 340
345 cag tca cgg gtg gac atg tat gct tgg gtc ctg cag gct ggc tgc cgc
1526 Gln Ser Arg Val Asp Met Tyr Ala Trp Val Leu Gln Ala Gly Cys
Arg 350 355 360 tgc gtg gag gtg gac tgc tgg gat ggg ccc gac ggg gag
ccc att gtg 1574 Cys Val Glu Val Asp Cys Trp Asp Gly Pro Asp Gly
Glu Pro Ile Val 365 370 375 380 cac cat ggc tac act ctg act tcc aag
atc ctc ttc aaa gac gtc att 1622 His His Gly Tyr Thr Leu Thr Ser
Lys Ile Leu Phe Lys Asp Val Ile 385 390 395 gaa acc atc aac aaa tat
gcc ttc atc aag aat gag tac cca gtg atc 1670 Glu Thr Ile Asn Lys
Tyr Ala Phe Ile Lys Asn Glu Tyr Pro Val Ile 400 405 410 ctg tcc atc
gaa aac cac tgc agt gtc atc cag cag aag aaa atg gcc 1718 Leu Ser
Ile Glu Asn His Cys Ser Val Ile Gln Gln Lys Lys Met Ala 415 420 425
cag tat ctg act gac atc ctt ggg gac aag ctg gac ctg tca tca gtg
1766 Gln Tyr Leu Thr Asp Ile Leu Gly Asp Lys Leu Asp Leu Ser Ser
Val 430 435 440 agc agt gaa gat gcc acc aca ctc ccc tct cca cag atg
ctc aag ggc 1814 Ser Ser Glu Asp Ala Thr Thr Leu Pro Ser Pro Gln
Met Leu Lys Gly 445 450 455 460 aag atc ctc gtg aag ggg aag aag ctc
cca gcc aac atc agc gag gat 1862 Lys Ile Leu Val Lys Gly Lys Lys
Leu Pro Ala Asn Ile Ser Glu Asp 465 470 475 gcg gag gaa ggc gag gtg
tct gat gag gac agt gct gat gag att gac 1910 Ala Glu Glu Gly Glu
Val Ser Asp Glu Asp Ser Ala Asp Glu Ile Asp 480 485 490 gat gac tgc
aag ctc ctc aat ggg gat gca tcc acc aat cga aag cgt 1958 Asp Asp
Cys Lys Leu Leu Asn Gly Asp Ala Ser Thr Asn Arg Lys Arg 495 500 505
gta gaa aac act gct aag agg aaa ctg gat tcc ctc atc aaa gag tcg
2006 Val Glu Asn Thr Ala Lys Arg Lys Leu Asp Ser Leu Ile Lys Glu
Ser 510 515 520 aag att cgg gac tgt gag gac ccc aac aac ttc tcc gtc
tcc aca ctg 2054 Lys Ile Arg Asp Cys Glu Asp Pro Asn Asn Phe Ser
Val Ser Thr Leu 525 530 535 540 tcc cca tct gga aag ctc gga cgc aag
agc aag gct gaa gag gac gtg 2102 Ser Pro Ser Gly Lys Leu Gly Arg
Lys Ser Lys Ala Glu Glu Asp Val 545 550 555 gag tct ggg gag gat gcc
ggg gcc agc aga cgc aat ggc cgc ctc gtc 2150 Glu Ser Gly Glu Asp
Ala Gly Ala Ser Arg Arg Asn Gly Arg Leu Val 560 565 570 gtg gga agc
ttc tcc agg cgc aag aag aag ggc agc aag ctg aag aag 2198 Val Gly
Ser Phe Ser Arg Arg Lys Lys Lys Gly Ser Lys Leu Lys Lys 575 580 585
gcg gcc agc gtg gag gag gga gat gag ggt cag gac tcc ccg gga ggc
2246 Ala Ala Ser Val Glu Glu Gly Asp Glu Gly Gln Asp Ser Pro Gly
Gly 590 595 600 cag agc cga ggg gcg acc cgg cag aag aag acc atg aag
ctg tcc cgg 2294 Gln Ser Arg Gly Ala Thr Arg Gln Lys Lys Thr Met
Lys Leu Ser Arg 605 610 615 620 gcc ctc tct gac ctg gtg aag tac acc
aag tcc gtg gcc acc cac gac 2342 Ala Leu Ser Asp Leu Val Lys Tyr
Thr Lys Ser Val Ala Thr His Asp 625 630 635 ata gag atg gag gcg gcg
tcc agc tgg cag gtg tcg tcc ttc agc gag 2390 Ile Glu Met Glu Ala
Ala Ser Ser Trp Gln Val Ser Ser Phe Ser Glu 640 645 650 acc aag gcc
cac cag att ctg cag cag aag ccg gcg cag tac cta cgc 2438 Thr Lys
Ala His Gln Ile Leu Gln Gln Lys Pro Ala Gln Tyr Leu Arg 655 660 665
ttc aac cag cag cag ctc tcc cgc atc tac ccc tcc tcc tac cgt gtg
2486 Phe Asn Gln Gln Gln Leu Ser Arg Ile Tyr Pro Ser Ser Tyr Arg
Val 670 675 680 gac tcc agc aac tac aac ccg cag ccc ttc tgg aac gcc
ggc tgc caa 2534 Asp Ser Ser Asn Tyr Asn Pro Gln Pro Phe Trp Asn
Ala Gly Cys Gln 685 690 695 700 atg gtt gcc ctg aac tac cag tca gag
ggg cgg atg ctg cag ctg aac 2582 Met Val Ala Leu Asn Tyr Gln Ser
Glu Gly Arg Met Leu Gln Leu Asn 705 710 715 cga gcc aag ttc agc gcc
aac ggt ggc tgc ggc tac gta ctc aag cct 2630 Arg Ala Lys Phe Ser
Ala Asn Gly Gly Cys Gly Tyr Val Leu Lys Pro 720 725 730 ggg tgc atg
tgc cag ggc gtg ttc aac ccc aac tcg gag gac ccc ctg 2678 Gly Cys
Met Cys Gln Gly Val Phe Asn Pro Asn Ser Glu Asp Pro Leu 735 740 745
ccc ggg cag ctc aag aag cag ctg gtg ctc cgg atc atc agt ggc cag
2726 Pro Gly Gln Leu Lys Lys Gln Leu Val Leu Arg Ile Ile Ser Gly
Gln 750 755 760 cag ctt ccc aag ccg cgc gac tcc atg ctg ggg gac cgt
ggg gag atc 2774 Gln Leu Pro Lys Pro Arg Asp Ser Met Leu Gly Asp
Arg Gly Glu Ile 765 770 775 780 atc gac ccc ttt gtg gag gtg gag atc
att ggg ctc cct gtg gac tgc 2822 Ile Asp Pro Phe Val Glu Val Glu
Ile Ile Gly Leu Pro Val Asp Cys 785 790 795 agc agg gag cag acc cgc
gtg gtg gac gac aac ggg ttc aac ccc acc 2870 Ser Arg Glu Gln Thr
Arg Val Val Asp Asp Asn Gly Phe Asn Pro Thr 800 805 810 tgg gag gag
acc ctg gtt ttc atg gtg cac atg ccg gag atc gcg ctg 2918 Trp Glu
Glu Thr Leu Val Phe Met Val His Met Pro Glu Ile Ala Leu 815 820 825
gtc cgc ttc ctc gtc tgg gac cac gat ccc atc ggg cgt gac ttc att
2966 Val Arg Phe Leu Val Trp Asp His Asp Pro Ile Gly Arg Asp Phe
Ile 830 835 840 ggc cag agg acg ctg gcc ttc agc agc atg atg cca ggc
tac aga cac 3014 Gly Gln Arg Thr Leu Ala Phe Ser Ser Met Met Pro
Gly Tyr Arg His 845 850 855 860 gtg tac cta gaa ggg atg gaa gag gcc
tcc atc ttc gtg cat gtg gct 3062 Val Tyr Leu Glu Gly Met Glu Glu
Ala Ser Ile Phe Val His Val Ala 865 870 875 gtc agt gac atc agc ggt
aag gtc aag cag gct ctg ggc cta aaa ggc 3110 Val Ser Asp Ile Ser
Gly Lys Val Lys Gln Ala Leu Gly Leu Lys Gly 880 885 890 ctc ttc ctc
cga ggc cca aag ccc ggc tcg ctg gac agt cat gct gct 3158 Leu Phe
Leu Arg Gly Pro Lys Pro Gly Ser Leu Asp Ser His Ala Ala 895 900 905
ggg cgg ccc ccg gcc cgg ccc tcc gtt agc cag cgg atc ctg cgg cgc
3206 Gly Arg Pro Pro Ala Arg Pro Ser Val Ser Gln Arg Ile Leu Arg
Arg 910 915 920 acg gcc agc gcc ccg acc aag agc cag aag ccg ggc cgc
agg ggc ttc 3254 Thr Ala Ser Ala Pro Thr Lys Ser Gln Lys Pro Gly
Arg Arg Gly Phe 925 930 935 940 ccg gag ctg gtc ctg ggt aca cgg gac
aca ggc tcc aag ggg gtg gca 3302 Pro Glu Leu Val Leu Gly Thr Arg
Asp Thr Gly Ser Lys Gly Val Ala 945 950 955 gac gat gtg gtg ccc ccc
ggg ccc gga cct gct ccg gaa gcc cca gcc 3350 Asp Asp Val Val Pro
Pro Gly Pro Gly Pro Ala Pro Glu Ala Pro Ala 960 965 970 cag gag ggg
ccc ggc agc ggc agc ccc cga ggt aag gcg cca gct gcg 3398 Gln Glu
Gly Pro Gly Ser Gly Ser Pro Arg Gly Lys Ala Pro Ala Ala 975 980 985
gtg gca gag aag agc cct gtg cga gtg cgg ccc ccg cgt gtc ctg gac
3446 Val Ala Glu Lys Ser Pro Val Arg Val Arg Pro Pro Arg Val Leu
Asp 990 995 1000 ggc ccc ggg cct gct ggg atg gcc gcc aca tgc atg
aag tgt gtg gtg 3494 Gly Pro Gly Pro Ala Gly Met Ala Ala Thr Cys
Met Lys Cys Val Val 1005 1010 1015 1020 gga tcc tgc gcc ggc gtg aac
acc ggg ggc ctg cag agg gag cgg cca 3542 Gly Ser Cys Ala Gly Val
Asn Thr Gly Gly Leu Gln Arg Glu Arg Pro 1025 1030 1035 ccc agc ccg
ggg cct gca agc agg cag gca gcc att cgc cag cag ccc 3590 Pro Ser
Pro Gly Pro Ala Ser Arg Gln Ala Ala Ile Arg Gln Gln Pro 1040 1045
1050 cgg gcc cgg gct gac tca ctg ggg gcc ccc tgc tgt ggc ctg gac
cct 3638 Arg Ala Arg Ala Asp Ser Leu Gly Ala Pro Cys Cys Gly Leu
Asp Pro 1055 1060 1065 cac gct atc ccg ggg aga agc aga gag gcc ccc
aag ggt cct ggg gcc 3686 His Ala Ile Pro Gly Arg Ser Arg Glu Ala
Pro Lys Gly Pro Gly Ala 1070 1075 1080 tgg agg cag ggt cca ggc ggt
agc ggc tcc atg tcc tcg gac tcc agc 3734 Trp Arg Gln Gly Pro Gly
Gly Ser Gly Ser Met Ser Ser Asp Ser Ser 1085 1090 1095 1100 agc cca
gac agc ccg ggc atc ccc gaa agg tcc ccc cgc tgg cct gag 3782 Ser
Pro Asp Ser Pro Gly Ile Pro Glu Arg Ser Pro Arg Trp Pro Glu 1105
1110 1115 ggt gcc tgc agg caa ccg ggg gcc ctg cag gga gag atg agt
gcc ttg 3830 Gly Ala Cys Arg Gln Pro Gly Ala Leu Gln Gly Glu Met
Ser Ala Leu 1120 1125 1130 ttt gct caa aag ctg gag gag atc agg agt
aaa tcc ccc atg ttc tcc 3878 Phe Ala Gln Lys Leu Glu Glu Ile Arg
Ser Lys Ser Pro Met Phe Ser 1135 1140 1145 gcc ggt aag ccc ctc ttg
ccc tgc gtg gtc ctc ccg cac gcc cct ggc 3926 Ala Gly Lys Pro Leu
Leu Pro Cys Val Val Leu Pro His Ala Pro Gly 1150 1155 1160 atg gct
ggg cct ggg tca cct gct gct gct tct gcg tgg acg gtg tcg 3974 Met
Ala Gly Pro Gly Ser Pro Ala Ala Ala Ser Ala Trp Thr Val Ser 1165
1170 1175 1180 cct cgt gtg ctc gtg ctc gtg gct ctg tat ccg tgg cac
tgt ctc cgt 4022 Pro Arg Val Leu Val Leu Val Ala Leu Tyr Pro Trp
His Cys Leu Arg 1185 1190 1195 ggc act ctg ctc cct tgg ctt gcc tgt
ggc cca tag ccccagccct 4068 Gly Thr Leu Leu Pro Trp Leu Ala Cys Gly
Pro * 1200 1205 cctgtctgag cttgaggccc tgggacttgg gtggagctgg
tttgaggccc gacaggctgg 4128 gaagaaccag ctgctcttgc tgagggtctg
gggccgggac tgtggcctga catgctgggc 4188 ccctccggct gggcgcttcc
ccaaactcac ctcctgggcg gctggcgacc tgcatggccc 4248 ctgatgcctt
tcctgggact gggggccatg taccatccca ttcccacctc cctctagggc 4308
aggctccagg ggtccctact gggaagtctg atgtgggcag gtagtgcagc tgctgggcgt
4368 ctcctgcgcc cctgggacgc ctggagcctg ctgagtgctg cgtggagtag
attccctggg 4428 ccccagggct tcgctgcttt gggctgaagc accccactag
aagggtgtct ccttagcctg 4488 gagggaggga catacacgga gcccgcccca
caccaccctg cccctccaga cccccctgac 4548 caagctttcc tttctgcccc
cacccacgct tgcctccgta gttaggaact gagagcggcg 4608 agtgacaggt
aacggggccc agccccg 4635 2 1207 PRT Homo sapiens 2 Met Ala Pro Pro
Thr Ala Gly Pro Leu Pro Gly Pro Ala Leu Pro Pro 1 5 10 15 Glu Asp
Pro Gly Pro Asp Pro Glu Ser Arg Trp Leu Phe Leu Ser Ala 20 25 30
Asn Ile Leu Pro Val Val Glu Arg Cys Met Gly Ala Met Gln Glu Gly 35
40 45 Met Gln Met Val Lys Leu Arg Gly Gly Ser Lys Gly Leu Val Arg
Phe 50 55 60 Tyr Tyr Leu Asp Glu His Arg Ser Cys Ile Arg Trp Arg
Pro Ser Arg 65 70 75 80 Lys Asn Glu Lys Ala Lys Ile Ser Ile Asp Ser
Ile Gln Glu Val Ser 85 90 95 Glu Gly Arg Gln Ser Glu Val Phe Gln
Arg Tyr Pro Asp Gly Ser Phe 100 105 110 Asp Pro Asn Cys Cys Phe Ser
Ile Tyr His Gly Ser His Arg Glu Ser 115 120 125 Leu Asp Leu Val Ser
Thr Ser Ser Glu Val Ala Arg Thr Trp Val Thr 130 135 140 Gly Leu Arg
Tyr Leu Met Ala Gly Ile Ser Asp Glu Asp Ser Leu Ala 145 150 155 160
Arg Arg Gln Arg Thr Arg Asp Gln Trp Leu Lys Gln Thr Phe Asp Glu 165
170 175 Ala Asp Lys Asn Gly Asp Gly Ser Leu Ser Ile Gly Glu Val Leu
Gln 180 185 190 Leu Leu His Lys Leu Asn Val Asn Leu Pro Arg Gln Arg
Val Lys Gln 195 200 205 Met Phe Arg Glu Ala Asp Thr Asp Asp His Gln
Gly Thr Leu Gly Phe 210 215 220 Glu Glu Phe Cys Ala Phe Tyr Lys Met
Met Ser Thr Arg Arg Asp Leu 225 230 235 240 Tyr Leu Leu Met Leu Thr
Tyr Ser Asn His Lys Asp His Leu Asp Ala 245 250 255 Ala Ser Leu Gln
Arg Phe Leu Gln Val Glu Gln Lys Met Ala Gly Val 260 265 270 Thr Leu
Glu Ser Cys Gln Asp Ile Ile Glu Gln Phe Glu Pro Cys Pro 275 280 285
Glu Asn Lys Ser Lys Gly Leu Leu Gly Ile Asp Gly Phe Thr Asn Tyr 290
295 300 Thr Arg Ser Pro Ala Gly Asp Ile Phe Asn Pro Glu His His His
Val 305 310 315
320 His Gln Asp Met Thr Gln Pro Leu Ser His Tyr Phe Ile Thr Ser Ser
325 330 335 His Asn Thr Tyr Leu Val Gly Asp Gln Leu Met Ser Gln Ser
Arg Val 340 345 350 Asp Met Tyr Ala Trp Val Leu Gln Ala Gly Cys Arg
Cys Val Glu Val 355 360 365 Asp Cys Trp Asp Gly Pro Asp Gly Glu Pro
Ile Val His His Gly Tyr 370 375 380 Thr Leu Thr Ser Lys Ile Leu Phe
Lys Asp Val Ile Glu Thr Ile Asn 385 390 395 400 Lys Tyr Ala Phe Ile
Lys Asn Glu Tyr Pro Val Ile Leu Ser Ile Glu 405 410 415 Asn His Cys
Ser Val Ile Gln Gln Lys Lys Met Ala Gln Tyr Leu Thr 420 425 430 Asp
Ile Leu Gly Asp Lys Leu Asp Leu Ser Ser Val Ser Ser Glu Asp 435 440
445 Ala Thr Thr Leu Pro Ser Pro Gln Met Leu Lys Gly Lys Ile Leu Val
450 455 460 Lys Gly Lys Lys Leu Pro Ala Asn Ile Ser Glu Asp Ala Glu
Glu Gly 465 470 475 480 Glu Val Ser Asp Glu Asp Ser Ala Asp Glu Ile
Asp Asp Asp Cys Lys 485 490 495 Leu Leu Asn Gly Asp Ala Ser Thr Asn
Arg Lys Arg Val Glu Asn Thr 500 505 510 Ala Lys Arg Lys Leu Asp Ser
Leu Ile Lys Glu Ser Lys Ile Arg Asp 515 520 525 Cys Glu Asp Pro Asn
Asn Phe Ser Val Ser Thr Leu Ser Pro Ser Gly 530 535 540 Lys Leu Gly
Arg Lys Ser Lys Ala Glu Glu Asp Val Glu Ser Gly Glu 545 550 555 560
Asp Ala Gly Ala Ser Arg Arg Asn Gly Arg Leu Val Val Gly Ser Phe 565
570 575 Ser Arg Arg Lys Lys Lys Gly Ser Lys Leu Lys Lys Ala Ala Ser
Val 580 585 590 Glu Glu Gly Asp Glu Gly Gln Asp Ser Pro Gly Gly Gln
Ser Arg Gly 595 600 605 Ala Thr Arg Gln Lys Lys Thr Met Lys Leu Ser
Arg Ala Leu Ser Asp 610 615 620 Leu Val Lys Tyr Thr Lys Ser Val Ala
Thr His Asp Ile Glu Met Glu 625 630 635 640 Ala Ala Ser Ser Trp Gln
Val Ser Ser Phe Ser Glu Thr Lys Ala His 645 650 655 Gln Ile Leu Gln
Gln Lys Pro Ala Gln Tyr Leu Arg Phe Asn Gln Gln 660 665 670 Gln Leu
Ser Arg Ile Tyr Pro Ser Ser Tyr Arg Val Asp Ser Ser Asn 675 680 685
Tyr Asn Pro Gln Pro Phe Trp Asn Ala Gly Cys Gln Met Val Ala Leu 690
695 700 Asn Tyr Gln Ser Glu Gly Arg Met Leu Gln Leu Asn Arg Ala Lys
Phe 705 710 715 720 Ser Ala Asn Gly Gly Cys Gly Tyr Val Leu Lys Pro
Gly Cys Met Cys 725 730 735 Gln Gly Val Phe Asn Pro Asn Ser Glu Asp
Pro Leu Pro Gly Gln Leu 740 745 750 Lys Lys Gln Leu Val Leu Arg Ile
Ile Ser Gly Gln Gln Leu Pro Lys 755 760 765 Pro Arg Asp Ser Met Leu
Gly Asp Arg Gly Glu Ile Ile Asp Pro Phe 770 775 780 Val Glu Val Glu
Ile Ile Gly Leu Pro Val Asp Cys Ser Arg Glu Gln 785 790 795 800 Thr
Arg Val Val Asp Asp Asn Gly Phe Asn Pro Thr Trp Glu Glu Thr 805 810
815 Leu Val Phe Met Val His Met Pro Glu Ile Ala Leu Val Arg Phe Leu
820 825 830 Val Trp Asp His Asp Pro Ile Gly Arg Asp Phe Ile Gly Gln
Arg Thr 835 840 845 Leu Ala Phe Ser Ser Met Met Pro Gly Tyr Arg His
Val Tyr Leu Glu 850 855 860 Gly Met Glu Glu Ala Ser Ile Phe Val His
Val Ala Val Ser Asp Ile 865 870 875 880 Ser Gly Lys Val Lys Gln Ala
Leu Gly Leu Lys Gly Leu Phe Leu Arg 885 890 895 Gly Pro Lys Pro Gly
Ser Leu Asp Ser His Ala Ala Gly Arg Pro Pro 900 905 910 Ala Arg Pro
Ser Val Ser Gln Arg Ile Leu Arg Arg Thr Ala Ser Ala 915 920 925 Pro
Thr Lys Ser Gln Lys Pro Gly Arg Arg Gly Phe Pro Glu Leu Val 930 935
940 Leu Gly Thr Arg Asp Thr Gly Ser Lys Gly Val Ala Asp Asp Val Val
945 950 955 960 Pro Pro Gly Pro Gly Pro Ala Pro Glu Ala Pro Ala Gln
Glu Gly Pro 965 970 975 Gly Ser Gly Ser Pro Arg Gly Lys Ala Pro Ala
Ala Val Ala Glu Lys 980 985 990 Ser Pro Val Arg Val Arg Pro Pro Arg
Val Leu Asp Gly Pro Gly Pro 995 1000 1005 Ala Gly Met Ala Ala Thr
Cys Met Lys Cys Val Val Gly Ser Cys Ala 1010 1015 1020 Gly Val Asn
Thr Gly Gly Leu Gln Arg Glu Arg Pro Pro Ser Pro Gly 1025 1030 1035
1040 Pro Ala Ser Arg Gln Ala Ala Ile Arg Gln Gln Pro Arg Ala Arg
Ala 1045 1050 1055 Asp Ser Leu Gly Ala Pro Cys Cys Gly Leu Asp Pro
His Ala Ile Pro 1060 1065 1070 Gly Arg Ser Arg Glu Ala Pro Lys Gly
Pro Gly Ala Trp Arg Gln Gly 1075 1080 1085 Pro Gly Gly Ser Gly Ser
Met Ser Ser Asp Ser Ser Ser Pro Asp Ser 1090 1095 1100 Pro Gly Ile
Pro Glu Arg Ser Pro Arg Trp Pro Glu Gly Ala Cys Arg 1105 1110 1115
1120 Gln Pro Gly Ala Leu Gln Gly Glu Met Ser Ala Leu Phe Ala Gln
Lys 1125 1130 1135 Leu Glu Glu Ile Arg Ser Lys Ser Pro Met Phe Ser
Ala Gly Lys Pro 1140 1145 1150 Leu Leu Pro Cys Val Val Leu Pro His
Ala Pro Gly Met Ala Gly Pro 1155 1160 1165 Gly Ser Pro Ala Ala Ala
Ser Ala Trp Thr Val Ser Pro Arg Val Leu 1170 1175 1180 Val Leu Val
Ala Leu Tyr Pro Trp His Cys Leu Arg Gly Thr Leu Leu 1185 1190 1195
1200 Pro Trp Leu Ala Cys Gly Pro 1205 3 3624 DNA Homo sapiens 3
atggctcccc cgacagccgg cccccttcct ggcccagctc ttccgcctga ggacccaggg
60 ccggatccgg agagcaggtg gcttttcttg agcgccaaca ttctgcccgt
ggtggagcgg 120 tgcatgggtg ccatgcaaga ggggatgcag atggtgaagc
tgcgtggcgg ctccaagggc 180 ctggtccgct tctactacct ggacgagcac
cgctcctgca tccgctggag gccctcacgc 240 aagaacgaga aggccaagat
ctccatcgac tccatccagg aggtgagtga ggggcggcag 300 tcggaggtct
tccagcgcta ccctgacggc agcttcgacc ccaactgctg cttcagcatc 360
taccacggca gccaccgcga gtcgctggac ctggtctcca ccagcagcga ggtggcgcgc
420 acctgggtca ctggcctgcg ctacctcatg gccggcatca gcgacgagga
cagcctggct 480 cgccgccagc gcaccaggga ccagtggctg aagcagacgt
ttgacgaggc cgacaagaac 540 ggggatggca gcctgagcat tggcgaggtc
ctgcagctgc tgcacaagct caacgtgaac 600 ctgccccggc agagggtgaa
gcagatgttc agggaagcgg acacggatga ccaccaaggg 660 acgctgggtt
ttgaagagtt ctgtgccttc tacaagatga tgtccacccg ccgggacctc 720
tacctgctca tgctgaccta cagcaaccac aaggaccacc tggatgccgc cagcctgcag
780 cgcttcctgc aggtggagca gaagatggcg ggtgtgaccc tcgagagctg
ccaggacatc 840 atcgagcagt ttgagccatg cccagaaaac aagagtaagg
ggctgctggg cattgatggc 900 ttcaccaact acaccaggag ccctgctggt
gacatcttca accctgagca ccaccatgtg 960 caccaggaca tgacgcagcc
gctgagccac tacttcatca cctcgtccca caacacctac 1020 ctcgtgggtg
accagctcat gtcccagtca cgggtggaca tgtatgcttg ggtcctgcag 1080
gctggctgcc gctgcgtgga ggtggactgc tgggatgggc ccgacgggga gcccattgtg
1140 caccatggct acactctgac ttccaagatc ctcttcaaag acgtcattga
aaccatcaac 1200 aaatatgcct tcatcaagaa tgagtaccca gtgatcctgt
ccatcgaaaa ccactgcagt 1260 gtcatccagc agaagaaaat ggcccagtat
ctgactgaca tccttgggga caagctggac 1320 ctgtcatcag tgagcagtga
agatgccacc acactcccct ctccacagat gctcaagggc 1380 aagatcctcg
tgaaggggaa gaagctccca gccaacatca gcgaggatgc ggaggaaggc 1440
gaggtgtctg atgaggacag tgctgatgag attgacgatg actgcaagct cctcaatggg
1500 gatgcatcca ccaatcgaaa gcgtgtagaa aacactgcta agaggaaact
ggattccctc 1560 atcaaagagt cgaagattcg ggactgtgag gaccccaaca
acttctccgt ctccacactg 1620 tccccatctg gaaagctcgg acgcaagagc
aaggctgaag aggacgtgga gtctggggag 1680 gatgccgggg ccagcagacg
caatggccgc ctcgtcgtgg gaagcttctc caggcgcaag 1740 aagaagggca
gcaagctgaa gaaggcggcc agcgtggagg agggagatga gggtcaggac 1800
tccccgggag gccagagccg aggggcgacc cggcagaaga agaccatgaa gctgtcccgg
1860 gccctctctg acctggtgaa gtacaccaag tccgtggcca cccacgacat
agagatggag 1920 gcggcgtcca gctggcaggt gtcgtccttc agcgagacca
aggcccacca gattctgcag 1980 cagaagccgg cgcagtacct acgcttcaac
cagcagcagc tctcccgcat ctacccctcc 2040 tcctaccgtg tggactccag
caactacaac ccgcagccct tctggaacgc cggctgccaa 2100 atggttgccc
tgaactacca gtcagagggg cggatgctgc agctgaaccg agccaagttc 2160
agcgccaacg gtggctgcgg ctacgtactc aagcctgggt gcatgtgcca gggcgtgttc
2220 aaccccaact cggaggaccc cctgcccggg cagctcaaga agcagctggt
gctccggatc 2280 atcagtggcc agcagcttcc caagccgcgc gactccatgc
tgggggaccg tggggagatc 2340 atcgacccct ttgtggaggt ggagatcatt
gggctccctg tggactgcag cagggagcag 2400 acccgcgtgg tggacgacaa
cgggttcaac cccacctggg aggagaccct ggttttcatg 2460 gtgcacatgc
cggagatcgc gctggtccgc ttcctcgtct gggaccacga tcccatcggg 2520
cgtgacttca ttggccagag gacgctggcc ttcagcagca tgatgccagg ctacagacac
2580 gtgtacctag aagggatgga agaggcctcc atcttcgtgc atgtggctgt
cagtgacatc 2640 agcggtaagg tcaagcaggc tctgggccta aaaggcctct
tcctccgagg cccaaagccc 2700 ggctcgctgg acagtcatgc tgctgggcgg
cccccggccc ggccctccgt tagccagcgg 2760 atcctgcggc gcacggccag
cgccccgacc aagagccaga agccgggccg caggggcttc 2820 ccggagctgg
tcctgggtac acgggacaca ggctccaagg gggtggcaga cgatgtggtg 2880
ccccccgggc ccggacctgc tccggaagcc ccagcccagg aggggcccgg cagcggcagc
2940 ccccgaggta aggcgccagc tgcggtggca gagaagagcc ctgtgcgagt
gcggcccccg 3000 cgtgtcctgg acggccccgg gcctgctggg atggccgcca
catgcatgaa gtgtgtggtg 3060 ggatcctgcg ccggcgtgaa caccgggggc
ctgcagaggg agcggccacc cagcccgggg 3120 cctgcaagca ggcaggcagc
cattcgccag cagccccggg cccgggctga ctcactgggg 3180 gccccctgct
gtggcctgga ccctcacgct atcccgggga gaagcagaga ggcccccaag 3240
ggtcctgggg cctggaggca gggtccaggc ggtagcggct ccatgtcctc ggactccagc
3300 agcccagaca gcccgggcat ccccgaaagg tccccccgct ggcctgaggg
tgcctgcagg 3360 caaccggggg ccctgcaggg agagatgagt gccttgtttg
ctcaaaagct ggaggagatc 3420 aggagtaaat cccccatgtt ctccgccggt
aagcccctct tgccctgcgt ggtcctcccg 3480 cacgcccctg gcatggctgg
gcctgggtca cctgctgctg cttctgcgtg gacggtgtcg 3540 cctcgtgtgc
tcgtgctcgt ggctctgtat ccgtggcact gtctccgtgg cactctgctc 3600
ccttggcttg cctgtggccc atag 3624 4 85 PRT Artificial Sequence
Consensus amino acid 4 Val Ile Lys Glu Gly Trp Leu Leu Lys Lys Ser
Lys Ser Trp Lys Lys 1 5 10 15 Arg Tyr Phe Val Leu Phe Asn Asn Val
Leu Leu Tyr Tyr Lys Asp Ser 20 25 30 Lys Lys Lys Pro Lys Gly Ser
Ile Pro Leu Ser Gly Cys Gln Val Glu 35 40 45 Lys Pro Asp Lys Asn
Cys Phe Glu Ile Arg Thr Asp Arg Thr Leu Leu 50 55 60 Leu Gln Ala
Glu Ser Glu Glu Glu Arg Lys Glu Trp Val Lys Ala Ile 65 70 75 80 Gln
Ser Ala Ile Arg 85 5 29 PRT Artificial Sequence Consensus amino
acid 5 Glu Leu Lys Glu Ala Phe Lys Glu Phe Asp Lys Asp Gly Asp Gly
Lys 1 5 10 15 Ile Ser Phe Glu Glu Phe Lys Ala Ala Leu Lys Lys Leu
20 25 6 29 PRT Artificial Sequence Consensus amino acid 6 Glu Leu
Lys Glu Ala Phe Lys Glu Phe Asp Lys Asp Gly Asp Gly Lys 1 5 10 15
Ile Ser Phe Glu Glu Phe Lys Ala Ala Leu Lys Lys Leu 20 25 7 153 PRT
Artificial Sequence Consensus amino acid 7 Asp Met Ser Ile Pro Leu
Ser His Tyr Phe Ile Ser Ser Ser His Asn 1 5 10 15 Thr Tyr Leu Thr
Gly Lys Gln Leu Trp Gly Lys Ser Ser Val Glu Ser 20 25 30 Tyr Arg
Gln Gln Leu Asp Ala Gly Cys Arg Cys Val Glu Leu Asp Cys 35 40 45
Trp Asp Gly Lys Pro Asp Asp Glu Pro Ile Ile Tyr His Gly His Thr 50
55 60 Leu Thr Leu Glu Ile Lys Leu Lys Asp Val Leu Glu Ala Ile Lys
Asp 65 70 75 80 Phe Ala Phe Lys Pro Thr Ser Pro Tyr Pro Val Ile Leu
Ser Leu Glu 85 90 95 Asn His Cys Asn Ser Asp Asp Gln Gln Arg Lys
Met Ala Lys Tyr Phe 100 105 110 Lys Glu Ile Phe Gly Asp Met Leu Leu
Thr Lys Pro Thr Leu Asp Ser 115 120 125 Leu Thr Thr Glu Pro Gly Leu
Pro Leu Pro Ser Leu Lys Asp Leu Arg 130 135 140 Gly Lys Ile Leu Leu
Lys Asn Lys Lys 145 150 8 128 PRT Artificial Sequence Consensus
amino acid 8 Glu Leu Ser Asn Leu Val Asn Tyr Ile Gln Ser Ile Lys
Phe Arg Ser 1 5 10 15 Phe Glu Leu Ser Gly Glu Glu Lys Asn Thr Ser
Tyr Glu Ile Ser Ser 20 25 30 Phe Ser Glu Arg Lys Val Lys Ala Lys
Lys Leu Leu Lys Glu Ser Pro 35 40 45 Val Glu Phe Val Lys Tyr Asn
Lys Arg Gln Leu Ser Arg Val Tyr Pro 50 55 60 Lys Gly Thr Arg Val
Asp Ser Ser Asn Phe Met Pro Gln Val Phe Trp 65 70 75 80 Asn Ala Gly
Cys Gln Met Val Ala Leu Asn Phe Gln Thr Ser Asp Leu 85 90 95 Pro
Met Gln Ile Asn Asp Gly Met Phe Glu Tyr Asn Gly Gly Gln Pro 100 105
110 Asp Gly Ser Phe Lys Ser Gly Tyr Leu Leu Lys Pro Glu Phe Leu Arg
115 120 125 9 95 PRT Artificial Sequence Consensus amino acid 9 Leu
Thr Val Thr Val Ile Glu Ala Arg Asn Leu Pro Lys Met Asp Lys 1 5 10
15 Val Asn Gly Arg Leu Ser Asp Pro Tyr Val Lys Val Ser Leu Leu Gly
20 25 30 Asp Lys Lys Asp Leu Lys Lys Phe Lys Thr Lys Val Val Lys
Lys Thr 35 40 45 Asn Gly Leu Asn Pro Val Trp Asn Glu Glu Thr Phe
Val Phe Glu Lys 50 55 60 Val Pro Leu Pro Glu Leu Ala Ser Lys Thr
Leu Arg Phe Ala Val Tyr 65 70 75 80 Asp Glu Asp Arg Phe Ser Arg Asp
Asp Phe Ile Gly Gln Val Thr 85 90 95 10 325 PRT Artificial Sequence
Consensus amino acid 10 Gln Val Lys Gln Ala Leu Gly Leu Lys Gly Leu
Phe Leu Arg Gly Pro 1 5 10 15 Lys Pro Gly Ser Leu Asp Ser His Ala
Ala Gly Arg Pro Pro Ala Arg 20 25 30 Pro Ser Val Ser Gln Arg Ile
Leu Arg Arg Thr Ala Ser Ala Pro Thr 35 40 45 Lys Ser Gln Lys Pro
Gly Arg Arg Gly Phe Pro Glu Leu Val Leu Gly 50 55 60 Thr Arg Asp
Thr Gly Ser Lys Gly Val Ala Asp Asp Val Val Pro Pro 65 70 75 80 Gly
Pro Gly Pro Ala Pro Glu Ala Pro Ala Gln Glu Gly Pro Gly Ser 85 90
95 Gly Ser Pro Arg Gly Lys Ala Pro Ala Ala Val Ala Glu Lys Ser Pro
100 105 110 Val Arg Val Arg Pro Pro Arg Val Leu Asp Gly Pro Gly Pro
Ala Gly 115 120 125 Met Ala Ala Thr Cys Met Lys Cys Val Val Gly Ser
Cys Ala Gly Val 130 135 140 Asn Thr Gly Gly Leu Gln Arg Glu Arg Pro
Pro Ser Pro Gly Pro Ala 145 150 155 160 Ser Arg Gln Ala Ala Ile Arg
Gln Gln Pro Arg Ala Arg Ala Asp Ser 165 170 175 Leu Gly Ala Pro Cys
Cys Gly Leu Asp Pro His Ala Ile Pro Gly Arg 180 185 190 Ser Arg Glu
Ala Pro Lys Gly Pro Gly Ala Trp Arg Gln Gly Pro Gly 195 200 205 Gly
Ser Gly Ser Met Ser Ser Asp Ser Ser Ser Pro Asp Ser Pro Gly 210 215
220 Ile Pro Glu Arg Ser Pro Arg Trp Pro Glu Gly Ala Cys Arg Gln Pro
225 230 235 240 Gly Ala Leu Gln Gly Glu Met Ser Ala Leu Phe Ala Gln
Lys Leu Glu 245 250 255 Glu Ile Arg Ser Lys Ser Pro Met Phe Ser Ala
Gly Lys Pro Leu Leu 260 265 270 Pro Cys Val Val Leu Pro His Ala Pro
Gly Met Ala Gly Pro Gly Ser 275 280 285 Pro Ala Ala Ala Ser Ala Trp
Thr Val Ser Pro Arg Val Leu Val Leu 290 295 300 Val Ala Leu Tyr Pro
Trp His Cys Leu Arg Gly Thr Leu Leu Pro Trp 305 310 315 320 Leu Ala
Cys Gly Pro 325 11 158 PRT Artificial Sequence Consensus amino acid
11 Ser Pro Asp Cys Asn Val Phe Asp Pro Glu His Lys Gln Val His Gln
1 5 10 15 Asp Met Asn Gln Pro Leu Ser His Tyr Phe Ile Asn Ser Ser
His Asn 20 25 30 Thr Tyr Leu Thr Gly Asn Gln Leu Ser Ser Gly Glu
Ser Ser Val Glu 35 40 45 Met Tyr Arg Gln Ala Leu Leu Lys Gly Cys
Arg Cys
Ile Glu Leu Asp 50 55 60 Cys Trp Asp Gly Lys Asp Gly Asp Pro Glu
Pro Ile Ile Thr His Gly 65 70 75 80 His Thr Met Thr Thr Glu Ile Ser
Phe Lys Asp Cys Leu Glu Ala Ile 85 90 95 Lys Glu His Ala Phe Val
Thr Ser Glu Tyr Pro Val Ile Leu Ser Leu 100 105 110 Glu Asn His Cys
Asp Ser Thr Pro Gln Gln Gln Ala Lys Met Ala Glu 115 120 125 Tyr Cys
Lys Glu Val Phe Gly Asp Met Leu Phe Thr Glu Pro Leu Glu 130 135 140
Glu Ser Pro Leu Glu Pro Gly Lys Glu Leu Pro Ser Pro Glu 145 150 155
12 41 PRT Artificial Sequence Consensus amino acid 12 Lys Arg Lys
Ile Leu Ile Lys Asn Lys Lys Leu Lys Glu His Ser Glu 1 5 10 15 Glu
Lys Glu Ser Glu Glu Lys Lys Thr Asp Glu Glu Thr Glu Ser Glu 20 25
30 Glu Glu Asp Glu Met Gly Ser Asp Ala 35 40 13 18 PRT Artificial
Sequence Consensus amino acid 13 Pro Gly Lys Glu Leu Pro Ser Pro
Glu Glu Leu Lys Arg Lys Ile Leu 1 5 10 15 Ile Lys 14 181 PRT
Artificial Sequence Consensus amino acid 14 Cys Leu Gln Phe Met Gln
Lys Gly Ser Glu Leu Lys Lys Val Arg Ser 1 5 10 15 Asn Ser Trp Lys
Tyr Asn Arg Tyr Phe Thr Leu Asp Asp Asp Met Gln 20 25 30 Thr Leu
Trp Trp Glu Pro His Trp Phe Ser Lys Lys Asp Ser Glu Lys 35 40 45
Pro Lys Phe Asp Ile Ser Asp Ile Lys Glu Ile Arg Met Gly Lys Asn 50
55 60 Thr Glu Thr Phe Arg Asn Asn Gly Lys Glu Phe Gln Ile Gln Glu
Pro 65 70 75 80 Glu Asp Cys Cys Phe Ser Ile Ile Phe Gly Glu Asn Tyr
Phe His Glu 85 90 95 Ser Leu Asp Leu Val Ala Asn Ser Ala Asp Val
Ala Asn Ile Trp Val 100 105 110 Ser Gly Leu Arg Tyr Leu Val Asp Tyr
Ala Lys His Met Leu Asp Asn 115 120 125 Tyr Gln Glu Gln Leu Asp Gln
Trp Leu Arg Glu Trp Phe Gln Gln Ala 130 135 140 Asp Arg Asn Lys Asp
Ser Arg Met Ser Phe Arg Glu Ala Gln Asn Leu 145 150 155 160 Leu Lys
Leu Met Asn Val Gln Met Asp Glu Glu Tyr Ala Phe Ser Ile 165 170 175
Phe Arg Glu Cys Asp 180 15 134 PRT Artificial Sequence Consensus
amino acid 15 Phe Asp Glu Phe Asp Thr Asp Gly Asn Gly His Leu Asp
Glu Gln Thr 1 5 10 15 Ala Phe Lys Cys Ile Lys His Leu Asn Pro Arg
Leu Lys His His Lys 20 25 30 Ile Thr Asn Lys Phe Lys Glu Ile Thr
Ile Lys Ser Lys Glu Lys Glu 35 40 45 Arg Thr Lys Ile Thr Lys Glu
His Phe Val Asp Leu Tyr Lys Glu Leu 50 55 60 Gly Thr Arg Pro Glu
Val Tyr Phe Leu Met Val Gln Tyr Ser Lys Asn 65 70 75 80 Lys Asp Tyr
Leu Asp Cys Gln Asp Leu Met Leu Phe Leu Glu Thr Glu 85 90 95 Gln
Gly Met Val His Val Thr Glu Asp Asn Cys Leu Asp Ile Ile Glu 100 105
110 Gln Tyr Glu Pro Cys Ser Glu Gly Arg Glu Asn Gly Trp Met Thr Ile
115 120 125 Asp Gly Phe Thr Ser Tyr 130 16 92 PRT Artificial
Sequence Consensus amino acid 16 Phe Ser Ser Leu Val Pro Gly Tyr
Arg His Val Tyr Leu Glu Gly Leu 1 5 10 15 Thr Glu Ala Ser Ile Phe
Val His Ile Thr Ile Asn Glu Ile Tyr Gly 20 25 30 Lys Asn Arg Gln
Leu Gln Gly Leu Lys Gly Leu Phe Asn Lys Asn Pro 35 40 45 Arg His
Ser Ser Ser Glu Asn Asn Ser His Tyr Val Arg Lys Arg Ser 50 55 60
Ile Gly Asp Arg Ile Leu Arg Arg Thr Ala Ser Ala Pro Ala Lys Gly 65
70 75 80 Arg Lys Lys Ser Lys Met Gly Phe Gln Glu Met Val 85 90 17
51 PRT Artificial Sequence Consensus amino acid 17 Asp Xaa Asp Asn
Ser Ile Leu Val Phe Tyr Trp Asp Glu Asn Ser Thr 1 5 10 15 Gly Asp
Asn Gln Gly His Arg Lys Gly Pro Leu Ile Val Met Cys Asp 20 25 30
Glu Asn Gln Ser Thr Ala Gly Cys Xaa Xaa Asp Glu Leu Ile Val Met 35
40 45 Phe Tyr Trp 50
* * * * *
References