U.S. patent application number 10/273334 was filed with the patent office on 2003-07-10 for gene family with transformation modulating activity.
Invention is credited to Brody, Jonathan R., Kadkol, Shrihari S., Kocheavar, Gerald J., Pasternack, Gary R..
Application Number | 20030129631 10/273334 |
Document ID | / |
Family ID | 26750308 |
Filed Date | 2003-07-10 |
United States Patent
Application |
20030129631 |
Kind Code |
A1 |
Pasternack, Gary R. ; et
al. |
July 10, 2003 |
Gene family with transformation modulating activity
Abstract
pp32 is a member of a highly conserved family of
differentiation-regulated nuclear proteins that is highly expressed
in nearly all human prostatic adenocarcinomas of Gleason Grade
.gtoreq.5. This contrasts with the low percentage of prostate
tumors that express molecular alterations in proto-oncogens or
demonstrate tumor suppressor mutation or loss of heterozygosity. By
analysis of specimens of human prostatic adenocarcinoma and paired
adjacent normal prostate from three individual patients, the
inventors have shown that normal prostate continues to express
normal pp32, whereas three of three sets of RT-PCR-amplified
transcripts from prostatic adenocarcinomas display multiple
cancer-associated coding sequence changes. The cancer-associated
sequence changes appear to be functionally significant. Normal pp32
exerts antineoplastic effects through suppression of
transformation. In contrast, cancer-associated pp32 variants
augment, rather than inhibit, transformation.
Inventors: |
Pasternack, Gary R.;
(Baltimore, MD) ; Kocheavar, Gerald J.; (College
Station, TX) ; Brody, Jonathan R.; (Potomac, MD)
; Kadkol, Shrihari S.; (Ellicott City, MD) |
Correspondence
Address: |
BROBECK, PHLEGER & HARRISON, LLP
ATTN: INTELLECTUAL PROPERTY DEPARTMENT
1333 H STREET, N.W. SUITE 800
WASHINGTON
DC
20005
US
|
Family ID: |
26750308 |
Appl. No.: |
10/273334 |
Filed: |
October 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10273334 |
Oct 18, 2002 |
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09591500 |
Jun 12, 2000 |
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09591500 |
Jun 12, 2000 |
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PCT/US98/26433 |
Dec 11, 1998 |
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60069677 |
Dec 12, 1997 |
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Current U.S.
Class: |
435/6.11 ;
435/199; 435/320.1; 435/325; 435/6.14; 435/69.3; 435/7.23;
536/23.2 |
Current CPC
Class: |
C07K 14/47 20130101;
C12Q 2600/112 20130101; A61K 38/00 20130101; C12Q 2600/136
20130101; C12Q 1/6886 20130101 |
Class at
Publication: |
435/6 ; 435/7.23;
435/69.3; 435/199; 435/320.1; 435/325; 536/23.2 |
International
Class: |
C12Q 001/68; G01N
033/574; C07H 021/04; C12N 009/22; C12P 021/02; C12N 005/06 |
Goverment Interests
[0001] The work leading to this invention was supported in part by
Grant No. RO1 CA 54404 from the National Institutes of Health. The
U.S. Government retains certain rights in this invention.
Claims
1. An isolated DNA molecule comprising at least a sequence of 18
contiguous nucleotides selected from the sequence consisting of
base pairs 4894-4942 of the sequence in FIG. 2 or the corresponding
sequence from FIG. 5, or a sequence complementary thereto, said DNA
molecule also containing non-mammalian DNA sequence and being
substantially free of human DNA molecules.
2. An isolated DNA molecule comprising at least a sequence of 18
contiguous nucleotides selected from a sequence which encodes the
amino acids from residue 146-163 of the amino acid sequence of
pp32r1 or the corresponding sequence of pp32r2.
3. An isolated nucleic acid probe of at least 15 nucleotides which
specifically hybridizes on Northern blot with nucleic acid encoding
the amino acids from residue 146-163 of the amino acid sequence of
pp32r1 or the corresponding sequence of pp32r2.
4. An isolated nucleic acid probe comprising a sequence of at least
8 contiguous nucleotides unique to pp32r1 or pp32r2.
5. A nucleic acid molecule produced by recombinant methods, wherein
said nucleic acid molecule encodes at least the amino acids from
residue 146-163 of sequence of the amino acid sequence of pp32r1 or
the corresponding sequence of pp32r2.
6. The nucleic acid molecule according to claim 5, wherein said
nucleic acid molecule is an expression vector which expresses said
amino acid sequence.
7. A recombinant cell containing the nucleic acid molecule of claim
6.
8. A nucleic acid molecule produced by recombinant methods, said
nucleic acid molecule containing a sequence encoding at least the
amino acids from residue 146-163 of sequence of the amino acid
sequence of pp32r1 or the corresponding sequence of pp32r2, said
sequence being operatively linked to a promoter in antisense
orientation.
9. A pair of nucleic acid primers each of which comprises at least
10 contiguous nucleotides, at least one of said primers being
selected from or complementary to the sequence of pp32r1, wherein
nucleic acid amplification of human chromosome 4 or a transcript
thereof using said pair of nucleic acid primers will produce an
amplified nucleic acid encoding residues 146-163 of the sequence of
pp32r1.
10. A diagnostic method for predicting malignant potential of
neuroendocrine, neural, mesenchymal, lymphoid, epithelial or germ
cell derived tumors, comprising: providing a sample of human
neuroendocrine, neural, mesenchymal, lymphoid, epithelial or germ
cell derived tissue; and determining, in the sample, levels or
intracellular sites of expression of a gene product expressed from
a gene sequence which encodes residues 146-163 of the sequence of
pp32r1 or the corresponding sequence of pp32r2.
11. A diagnostic method for predicting malignant potential of
neuroendocrine, neural, mesenchymal, lymphoid, epithelial or germ
cell derived tumors, comprising: providing a sample of human
neuroendocrine, neural, mesenchymal, lymphoid, epithelial or germ
cell derived tumor tissue; and determining, in the sample, levels
or intracellular sites of expression of a gene product expressed
from a gene sequence which encodes residues 146-163 of the sequence
of pp32r1 or the corresponding sequence of pp32r2.
12. The method of claim 11, wherein the gene product is mRNA.
13. The method of claim 12, wherein the mRNA is extracted from the
sample and quantitated.
14. The method of claim 12, wherein the level of mRNA is determined
by in situ hybridization to a section of the tissue sample.
15. The method of claim 12, wherein the mRNA is quantitated by
polymerase chain reaction.
16. The method according to claim 11, wherein the gene product is
protein.
17. The method according to claim 16, wherein the method further
comprises reacting the sample with an antibody that specifically
binds to a polypeptide consisting of the sequence of pp32r1, but
does not specifically bind to a polypeptide consisting of the
sequence of pp32 or pp32r2, or an antibody that specifically binds
to a polypeptide consisting of the sequence of pp32r2, but does not
specifically bind to a polypeptide consisting of the sequence of
pp32 or pp32r1
18. The method according to claim 11, wherein the tissue is a
carcinoma.
19. The method according to claim 11, wherein the tissue is a
carcinoma or sarcoma of a tissue selected from the group consisting
of epithelial, lymphoid, hematopoietic, mesenchymal, central
nervous system and peripheral nervous system tissues.
20. The method according to claim 19, wherein the tissue is
selected from the group consisting of colon carcinoma, prostate
carcinoma and non-Hodgkin's lymphoma.
21. An antibody that specifically binds to a polypeptide consisting
of the sequence of pp32r1 or pp32r2, but does not specifically bind
to a polypeptide consisting of the sequence of pp32.
22. The antibody of claim 21, wherein the antibody is a monoclonal
antibody.
23. An isolated DNA molecule comprising an androgen-activated
transcriptional promoter.
24. The isolated DNA molecule of claim 23, wherein the promoter
comprises a transcription initiation site and a binding site for a
steroid hormone receptor protein positioned within 10,000
nucleotide base pairs (bp) of the transcription initiation site,
preferably 5,000 bp, more preferably 3000 bp.
25. The isolated DNA molecule of claim 24, further comprising at
least one binding site for steroid hormone receptor proteins
positioned within 2000 nucleotide base pairs (bp) of the
transcription initiation site, preferably a plurality of binding
sites for steroid hormone receptor proteins are positioned within
2000 bp of the transcription initiation site, more preferably, at
least 5 binding sites for steroid hormone receptor proteins are so
positioned.
26. The isolated DNA molecule of claim 25, wherein the binding
sites for steroid hormone receptor proteins are selected from the
group of steroid receptor protein binding sites listed on Table
1.
27. The isolated DNA molecule of claim 24, further comprising an
open reading frame comprising at least one exon of a protein coding
sequence, wherein said open reading frame is operatively linked to
said androgen-activated transcriptional promoter.
28. The isolated DNA molecule of claim 27, wherein transcriptional
activity of the promoter is regulated by steroids.
29. A method of screening a compound for pharmacological activity
comprising: culturing a cell transfected with the DNA molecule of
claim 27; and determining expression of the protein coding sequence
in the presence and absence of the compound.
30. The method of claim 29, wherein the expression determined is
RNA expression or protein expression.
31. The DNA molecule of claim 23, wherein the DNA molecule is a DNA
vector.
Description
BACKGROUND
[0002] 1. File of the Invention
[0003] This invention is directed to various members of a gene
family with transformation modulating activity, and to diagnostic
and gene therapy techniques based on the variants.
[0004] 2. Review of Related Art
[0005] Prostatic adenocarcinoma is the most frequent malignancy in
adult men with approximately 317,000 new cases diagnosed each year
(Parker, et al., CA, 46:8-27, 1996). In spite of the capabilities
for early diagnosis and treatment (Potosky, et al., JAMA,
273:548-552, 1995), it represents the second leading cause of
cancer death in men following lung cancer.
[0006] To date, the study of alterations in specific genes has not
been particularly rewarding in primary prostate cancer. Most
alterations in the widely studied oncogenes and tumor suppressor
genes occur in only 20-30% of primary prostate carcinomas, except
for the myc gene, where overexpression has been observed in as many
as 50-60% of such cases (Fleming, et al., Cancer Res.,
46:1535-1538, 1986). Up to 40% of primary prostate cancers studied
by comparative genomic hybridization display chromosomal
aberrations (Visakorpi, et al., Cancer Res., 55:342-347, 1995),
although such alterations occur more frequently as tumors recur and
become refractory to hormonal therapy. Characterization of
candidate proto-oncogenes or tumor suppressor genes at such altered
loci may eventually shed light on tumor progression in the
prostate.
[0007] pp32 (GenBank HSU73477) is a highly conserved nuclear
phosphoprotein. Increased expression of pp32 or closely related
species is a frequent feature of clinical cancers. For example, in
human prostate cancer, high-level expression of RNA hybridizing
with pp32 probes occurs in nearly 90% of clinically significant
prostate cancers, in contrast to the substantially lower
frequencies of alterations of other oncogenes and tumor suppressors
(See U.S. Pat. No. 5,726,018, incorporated herein by reference).
Molecular Features and Activities of pp32.
[0008] pp32 is a nuclear phosphoprotein that is
differentiation-regulated during differentiation of adult prostatic
epithelium (Walensky, et al., Cancer Res. 53:4720-4726, 1993). The
human pp32 cDNA sequence (Gen-Bank U73477) is 1052 bp in length and
encodes a protein of 249 amino acids. The protein is composed of
two domains: an amino terminal amphipathic .alpha.-helical region
containing a leucine zipper, and a highly acidic carboxyl terminal
region. The murine and human forms of pp32 are highly conserved
with over 90% nucleic acid homology and over 95% protein-level
homology.
[0009] Human pp32 has been isolated independently by a number of
groups. Vaesen et al. ("Purification and characterization of two
putative HLA class II associated proteins: PHAPI and PHAPII." Biol.
Chem. Hoppe-Seyler., 375:113-126, 1994) cloned an essentially
equivalent molecule, termed PHAPI, from an EBV-transformed human
B-lymphoblastoid cell line; PHAPII, cloned by the same strategy, is
unrelated to pp32. This study identified PHAPI through its
association in solution with human HLA class II protein, noting
membrane and cytoplasmic localization as well as nuclear; the gene
has putatively been localized to chromosome 15q22.3-q23 by
fluorescent in situ hybridization (Fink, et al., "Localization of
the gene encoding the putative human HLA class II-associated
protein (PHAPI) to chromosome 15q22.3-q23 by fluorescence in situ
hybridization." Genomics, 29:309-310, 1995). More recently, a group
studying inhibitors of protein phosphatases identified pp32 as
IIPP2a, an inhibitor of protein phosphatase 2a (Li, et al.,
"Molecular Identification of II PP2A, a novel potent-heat-stable
inhibitor protein of protein phosphatase 2A." Biochemistry
35:6998-7002, 1996); another phosphatase inhibitor, I2PP2a, is
unrelated to pp32. Interestingly, another recent report (Ulitzur,
et al., "Biochemical characterization of mapmodulin, a protein that
binds microtubule-associated proteins." Journal of Biological
Chemistry 272:30577-30582, 1997) identified pp32 as a
cytoskeletally-associated cytosolic protein in CHO cells. It is not
clear whether this finding stems from a difference in system, or
whether pp32 can localize to the cytoplasm under certain
circumstances. pp32 has also been identified as LANP, a leucine
rich nuclear protein in the central nervous system (Matsuoka, et
al., "A nuclear factor containing the leucine-rich repeats
expressed in murine cerebellar neurons. Proc Natl Acad Sci USA
91:9670-9674, 1994).
[0010] There are also a number of reports of gene products bearing
lesser degrees of homology to pp32. The Vaesen group has identified
a series of unpublished sequences, termed PHAPI2a (EMBL Locus
HSPHAPI2A) and PHAPI2b (EMBL Locus HSPHAPI2B), also cloned from an
EBV-transformed human B-lymphoblastoid cell line. These variant
pp32 sequences are distinct from the sequences reported herein,
representing the April protein instead. April, cloned from human
pancreas, is shorter than PHAPI2a by two N-terminal amino acids
(Mencinger, et al., "Expression analysis and chromosomal mapping of
a novel human gene, APRIL, encoding an acidic protein rich in
leucines." Biochimica et Biophysica Acta, 1395:176-180, 1998, see
EMBL Locus HSAPRIL); PHAPI2b is identical to a subset of APRIL.
Silver-stainable protein SSP29 (unpublished GenBank Locus HSU70439)
was cloned from HeLa cells and is identical to PHAPI2a.
[0011] The nuclear phosphoprotein pp32 has been linked to
proliferation. Malek and associates reported that various
neoplastic cell lines showed markedly elevated expression levels
and that bacterial polysaccharide induced expression of pp32
epitopes by B lymphocytes upon polyclonal expansion (Malek, et al.,
J. Biol. Chem., 265:13400-13409, 1990). Walensky and associates
reported that levels of pp32 expression, measured by in situ
hybridization, increased in direct relation to increasing Gleason
grade of human prostatic cancers.
[0012] pp32 cDNA probes hybridize strongly with prostatic
adenocarcinoma, whereas the hybridization signal in normal prostate
is confined to basal cells. Polyclonal anti-pp32 antibodies react
strongly with sections of human prostatic adenocarcinoma. The
antibodies and riboprobes used by the investigators in previous
studies are consistent with cross-reactivities of the reagents with
all reported members of the pp32 nuclear phosphoprotein family,
therefore, while previous descriptions focused upon pp32, it cannot
be excluded that homologous proteins were detected.
SUMMARY OF THE INVENTION
[0013] In one aspect, this invention provides a DNA molecule
containing at least a portion of the sequence consisting of base
pairs 4894-4942 of the sequence shown in FIG. 2 or its complement.
Alternatively, the DNA molecule may contain at least a portion
consisting of base pairs 4879-4927, or base pairs 4858-4927.
Alternatively, this invention provides a DNA molecule that contains
at least a portion of a nucleotide sequence encoding amino acid
residues 146-163 of tumor-derived pp32r1 sequence; preferably the
DNA encodes all of that segment. In one mode, the DNA molecule is
an expression vector which expresses said amino acid sequence, and
the invention also includes a recombinant cell containing the
expression vector. In another mode, the DNA molecule has the
particular sequence operatively linked to a promoter in antisense
orientation. In another alternative, this invention provides a DNA
probe which specifically hybridizes on Northern blot with nucleic
acid encoding the amino acids from residue 146-163 of the
tumor-derived pp32r1 sequence, a preferred probe would have a
sequence of at least 8 contiguous nucleotides "unique" to the
nucleotide sequence of the pp32r1 variant as described herein. In
yet another alternative, the invention provides a pair of nucleic
acid primers each of which comprises at least 10 contiguous
nucleotides, at least one of the primers binding specifically to
the pp32r1 sequence, where if the primers are used in nucleic acid
amplification of a suitable source of human nucleic acid, the
amplification will produce an amplified nucleic acid encoding at
least residues 146-163 of the pp32r1 sequence.
[0014] In still another aspect, this invention provides antibodies
that specifically bind the tumor derived pp32, but do not bind to
normal pp32. Preferably, these antibodies are monoclonal
antibodies. The invention also provides polypeptides containing
epitopes that bind these antibodies.
[0015] In yet another aspect, this invention provides diagnostic
methods for predicting malignant potential of neuroendocrine,
neural, mesenchymal, lymphoid, epithelial or germ cell derived
tumors by determining, in a sample of human neuroendocrine, neural,
mesenchymal, lymphoid, epithelial or germ cell derived tissue, the
level of, or the intracellular sites of expression of, a gene
product expressed from a gene sequence which encodes, inter alia,
residues 146-163 of tumor derived pp32r1. Where the gene product is
mRNA, the mRNA is extracted from the sample and quantitated,
optionally by PCR, or the level of mRNA may be determined by in
situ hybridization to a section of the tissue sample. Where the
gene product is protein, the determination may include reacting the
sample with an antibody that specifically binds to tumor derived
pp32, but not to normal pp32. Preferably, the tissue sample is
carcinoma tissue. e.g., carcinoma or sarcoma of a tissue selected
from the group consisting of epithelial, lymphoid, hematopoietic,
mesenchymal, central nervous system and peripheral nervous system
tissues, including colon carcinoma, prostate carcinoma and
non-Hodgkin's lymphoma.
[0016] In still another aspect, this invention provides an
androgen-activated transcriptional promoter which may be inserted
into recombinant DNA molecules. The minimal promoter is made up of
a transcription initiation site and at least one binding site for a
steroid hormone receptor protein. Typically the consensus sequence
for the steroid hormone receptor protein binding site is positioned
within 5000 nucleotide base pairs (bp), more preferably within 3000
bp, or even fewer bp of the transcription initiation site: In a
preferred mode, a number of binding sites for steroid hormone
receptor proteins are positioned within that distance of the
transcription initiation site, the promoter may contain five, ten
or even 25 steroid hormone receptor protein binding sites.
Preferably, the binding site(s) for steroid hormone receptor
protein binding are selected from the consensus sequences listed on
Table 1. In a preferred mode of the invention, the
androgen-activated transcriptional promoter is operatively linked
to an open reading frame comprising at least one exon of a protein
coding sequence, operative linking of the open reading frame
thereby providing an expression vector in which expression of the
open reading frame is regulated by steroids.
[0017] In another aspect, this invention provides a method for
screening candidate compounds for pharmacological activity by (1)
culturing a cell transfected with the DNA molecule containing the
androgen-activated transcriptional promoter which is operatively
linked to an open reading frame comprising at least one exon of a
protein coding sequence, and (2) determining expression of the open
reading frame in the presence and absence of the compound. In a
preferred mode the androgen-activated promoter may be all or an
operative portion of the sequence in FIG. 2 which is up-stream of
the translation initiation site, or alternatively the
androgen-activated promoter may be the 2700 bp of the sequence in
FIG. 2 which is upstream from the translation initiation site.
[0018] pp32 is a member of a highly conserved family of
differentiation-regulated nuclear proteins that is highly expressed
in nearly all human prostatic adenocarcinomas of Gleason Grade
.gtoreq.5. This contrasts with the low percentage of prostate
tumors that express molecular alterations in proto-oncogenes or
demonstrate tumor suppressor mutation or loss of heterozygosity. By
analysis of specimens of human prostatic adenocarcinoma and paired
adjacent normal prostate from three individual patients, the
inventors have shown that normal prostate continues to express
normal pp32, whereas three of three sets of RT-PCR-amplified
transcripts from prostatic adenocarcinomas display multiple
cancer-associated coding sequence changes. The cancer-associated
sequence changes appear to be functionally significant. Normal pp32
exerts antineoplastic effects through suppression of
transformation. In contrast, cancer-associated pp32 variants
augment, rather than inhibit, transformation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1A shows detection of pp32-related mRNA in benign
prostate and prostate cancer tissue sections by in situ
hybridization.
[0020] FIG. 1B shows immunohistochemical stain of prostate cancer
sections with anti-pp32 antibodies.
[0021] FIG. 2 shows the genomic sequence of variant pp32r1 isolated
from human placenta.
[0022] FIG. 3 provides a base-by-base comparison of the sequence of
pp32r1 (top) with normal human pp32 (bottom). The numbering system
for pp32r1 corresponds to FIG. 1, and the numbering system for
normal pp32 is taken from Chen, et al. Nucleotide base differences
are underlined in the pp32r1 sequence. Sequences within the normal
pp32 sequence missing in pp32r1 are represented by dashes. The open
reading frame for pp32r1 is indicated by overlining.
[0023] FIG. 4 shows the alignment of the pp32r1 amino acid sequence
(top) with normal human pp32 (bottom). Residue changes are
underlined in the pp32r1 sequence. Amino acids missing in the
pp32r1 sequence compared to normal pp32 are represented by
dashes.
[0024] FIG. 5 shows the genomic sequence of variant pp32r2.
[0025] FIG. 6A shows RT-PCR amplification of pp32 and pp32 variants
from human prostate cancer and prostate cancer cell line.
[0026] FIG. 6B shows cleavase fragment length polymorphism analysis
of pp32 detects variant pp32 transcripts in human prostate
cancer.
[0027] FIG. 7 shows the alignment of nucleic acid (A) and amino
acid (B) sequences from human prostatic adenocarcinoma and
prostatic adenocarcinoma cell lines with pp32.
[0028] FIG. 8 is a bar graph showing ras+myc induced transformed
focus formation. Co-transfection with a pp32 expression vector
reduces transformation, while co-transfection with a pp32r1
expression vector stimulates transformation.
[0029] FIG. 9 is a bar graph showing pp32r1 stimulation of ras+myc
induced transformed focus formation. Co-transfection with a pp32
expression vector reduces transformation, while co-transfection
with expression vectors for pp32r1 sequences from prostate cancer
cell lines stimulate transformation.
[0030] FIG. 10 is a graph of transformation assay results for cells
transfected with variant pp32 species, which are shown to stimulate
transformation with variable potency.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The inventors have discovered that phenotypic changes in
pp32 are a common feature of human prostate cancer. Previous data
show that 87% of prostate cancers of Gleason Score 5 and above
express pp32 or closely-related transcripts (U.S. Pat. No.
5,734,022, incorporated herein by reference). This is striking in
comparison to the frequency of molecular alterations in other
widely studied oncogenes and tumor suppressor genes in primary
prostatic adenocarcinoma, which occur in a substantially smaller
proportion of cases. For example, myc overexpression (Fleming, et
al.) occurs in around 60% of cases, and p53 is abnormal in only
around 25% of primary tumors (Isaacs, et al., in "Genetic
Alterations in Prostate Cancer." Cold Spring Harbor Symposia on
Quantitative Biology, 59:653-659, 1994).
[0032] Several lines of evidence suggest that pp32 may act as a
tumor suppressor. Functionally, pp32 inhibits transformation in
vitro by oncogene pairs such as ras with myc, mutant p53, Ela, or
jun, or human papilloma virus E6 and E7 (Chen, et al., "Structure
of pp32, an acidic nuclear protein which inhibits oncogene-induced
formation of transformed foci." Molecular Biology of the Cell,
7:2045-2056, 1996). pp32 also inhibits growth of transformed cells
in soft agar (Chen, et al.). In another system, ras-transfected
NIH3T3 cells previously transfected to overexpress normal human
pp32 do not form foci in vitro or, preliminarily, do not form
tumors in nude mice, unlike control cells. In contrast, knockout of
endogenous pp32 in the same system by an antisense pp32 expression
construct markedly augments tumorigenesis (Example 12 below).
[0033] In clinical prostate cancer, the situation at first appears
counterintuitive. Most human prostate cancers seem to express high
levels of pp32 by in situ hybridization (see Example 1 below) and
stain intensely with anti-pp32 antibodies. Because pp32 inhibits
oncogene-mediated transformation (Chen, et al.), its paradoxical
expression in cancer was investigated at the sequence level. The
paradoxical question of why prostate cancers seem to express
high-levels of an anti-oncogenic protein was addressed by comparing
the sequence and function of pp32 species from paired normal
prostate and adjacent prostatic carcinoma from three patients as
well as from four prostate cancer cell lines. It is demonstrated
herein that pp32 is a member of a closely-related gene family, and
that alternate expression of these closely-related genes located on
different chromosomes modulates oncogenic potential in human
prostate cancer. The variant pp32 species expressed in prostate
cancer are closely related to pp32.
[0034] The present data indicate that prostate cancers express
variant pp32 transcripts, whereas adjacent normal prostate
expresses normal pp32. Two instances clearly show that expression
of alternate genes on different chromosomes can lead to the
phenotypic switch, rather than mutation or alternate splicing. This
switch in molecular phenotype is accompanied by a switch in
functional pp32 phenotype. Normal pp32 is anti-oncogenic in
character, in contrast to the pro-oncogenic variant transcripts
that foster oncogene-mediated transformation. The high frequency of
this abnormality suggests that expression of variant pp32 species
may play an etiologic role in the development of human prostate
cancer. In addition, these findings have significant diagnostic and
prognostic implications.
[0035] Definitions
[0036] In describing the present invention, the following
terminology is used in accordance with the definitions set out
below.
[0037] Nucleic Acids
[0038] In discussing the structure of particular double-stranded
DNA molecules, sequences may be described herein according to the
normal convention of giving only the sequence in the 5' to 3'
direction along the nontranscribed stand of DNA (i.e., the-strand
having a sequence homologous to the mRNA).
[0039] A DNA sequence "corresponds" to an amino acid sequence if
translation of the DNA sequence in accordance with the genetic code
yields the amino acid sequence (i.e., the DNA sequence "encodes"
the amino acid sequence): one DNA sequence "corresponds" to another
DNA sequence if the two sequences encode the same amino acid
sequence.
[0040] Two DNA sequences are "substantially similar" when at least
about 90% (preferably at least about 94%, and most preferably at
least about 96%) of the nucleotides match over the defined length
of the DNA sequences. Sequences that are substantially similar can
be identified by the assay procedures described below or by
isolating and sequencing the DNA molecules. See e.g., Maniatis et
al., infra, DNA Cloning, vols. 1 and II infra: Nucleic Acid
Hybridization, infra.
[0041] A "heterologous" region or domain of a DNA construct is an
identifiable segment of DNA within a larger DNA molecule that is
not found in association with the larger molecule in nature. Thus,
when the heterologous region encodes a mammalian gene, the gene
will usually be flanked by DNA that does not flank the mammalian
genomic DNA in the genome of the source organism. Another example
of a heterologous region is a construct where the coding sequence
itself is not found in nature (e.g., a cDNA where the genomic
coding sequence contains introns, or synthetic sequences having
codons different than the native gene). Allelic variations or
naturally-occurring mutational events do not give rise to a
heterologous region of DNA as defined herein.
[0042] A "coding sequence" or "open reading frame" is an in-frame
sequence of codons that (in view of the genetic code) correspond to
or encode a protein or peptide sequence. Two coding sequences
correspond to each other if the sequences or their complementary
sequences encode the same amino acid sequences. A coding sequence
in association with appropriate regulatory sequences may be
transcribed and translated into a polypeptide in vivo. A
polydenylation signal and transcription termination sequence will
usually be located 3' to the coding sequence. A "promoter sequence"
is a DNA regulatory region capable of binding RNA polymerase in a
cell and initiating transcription of a downstream (3' direction)
coding sequence. Promoter sequences typically contain additional
sites for binding of regulatory molecules (e.g., transcription
factors) which affect the transcription of the coding sequence. A
coding sequence is "under the control" of the promoter sequence or
"operatively linked" to the promoter when RNA polymerase binds the
promoter sequence in a cell and transcribes the coding sequence
into mRNA, which is then in turn translated into the protein
encoded by the coding sequence.
[0043] Vectors are used to introduce a foreign substance, such as
DNA, RNA or protein, into an organism. Typical vectors include
recombinant viruses (for DNA) and liposomes (for protein). A "DNA
vector" is a replicon, such as plasmid, phase or cosmid, to which
another DNA segment may be attached so as to bring about the
replication of the attached segment. An "expression vector" is a
DNA vector which contains regulatory sequences which will direct
protein synthesis by an appropriate host cell. This usually means a
promoter to bind RNA polymerase and initiate transcription of mRNA,
as well as ribosome binding sites and initiation signals to direct
translation of the mRNA into a polypeptide. Incorporation of a DNA
sequence into an expression vector at the proper site and in
correct reading frame, followed by transformation of an appropriate
host cell by the vector, enables the production of a protein
encoded by said DNA sequence.
[0044] An expression vector may alternatively contain an antisense
sequence, where a small DNA fragment, corresponding to all or part
of an mRNA sequence, is inserted in opposite orientation into the
vector after a promoter. As a result, the inserted DNA will be
transcribed to produce an RNA which is complementary to and capable
of binding or hybridizing with the mRNA. Upon binding to the mRNA,
translation of the mRNA is prevented, and consequently the protein
coded for by the mRNA is not produced. Production and use of
antisense expression vectors is described in more detail in U.S.
Pat. No. 5,107,065 (describing and exemplifying antisense
regulation of genes in plants) and U.S. Pat. No. 5,190,931
(describing antisense regulation of genes in both prokaryotes and
eukarvotes and exemplifying prokaryotes), both of which are
incorporated herein by reference.
[0045] "Amplification" of nucleic acid sequences is the in vitro
production of multiple copies of a particular nucleic acid
sequence. The amplified sequence is usually in the form of DNA. A
variety of techniques for carrying out such amplification are
described in a review article by Van Brunt (1990, Bio/Technol.,
8(4):291-294). Polymerase chain reaction or PCR is a prototype of
nucleic acid amplification and use of PCR herein should be
considered exemplary of other suitable amplification
techniques.
[0046] Polypeptides
[0047] For the purposes of defining the present invention, two
proteins are homologous if 80% of the amino acids in their
respective amino acid sequences are the same; for proteins of
differing length, the sequences will be at least 80% identical over
the sequence which is in common (i.e., the length of the shorter
protein).
[0048] Two amino acid sequences are "substantially similar" when at
least about 87% of the amino acids match over the defined length of
the amino acid sequences, preferably a match of at least about 89%,
more preferably a match of at least about 95%. Typically, two amino
acid sequences which are similar will differ by only conservative
substitutions.
[0049] "Conservative amino acid substitutions" are the substitution
of one amino acid residue in a sequence by another residue of
similar properties, such that the secondary and tertiary structure
of the resultant peptides are substantially the same. Conservative
amino acid substitutions occur when an amino acid has substantially
the same charge or hydrophobicity as the amino acid for which it is
substituted and the substitution has no significant effect on the
local conformation of the protein. Amino acid pairs which may be
conservatively substituted for one another are well-known to those
of ordinary skill in the art.
[0050] The polypeptides of this invention encompass pp32r1 and
pp32r1 analogs, pp32r2 and pp32r2 analogs, along with other
variants of pp32 and their analogs. pp32r1 and pp32r2 are naturally
occurring, mature proteins, and further encompass all precursors
and allelic variations of pp32r1 and pp32r2, as well as including
forms of heterogeneous molecular weight that may result from
inconsistent processing in vivo. An example of the pp32r1 sequence
is shown in FIG. 3, top line. "pp32r1 analogs" are a class of
peptides which includes:
[0051] 1) "Allelic variations of pp32r1," which are polypeptides
which are substantially similar to pp32r1. Preferably the amino
acid sequence of the allelic variation is encoded by a nucleic acid
sequence that differs from the sequence of pp32r1 by one nucleotide
in 300;
[0052] 2) "Truncated pp32r1 peptides," which include fragments of
either pp32 or allelic variations of pp32r1 that preferably retain
either (i) an amino acid sequence unique to pp32r1, (ii) an epitope
unique to pp32r1 or (iii) pp32r1 activity;
[0053] 3) "pp32r1 fusion proteins," which include heterologous
polypeptides which are made up of one of the above polypeptides
(pp32r1, allelic variations of pp32r1 or truncated pp32r1 peptides)
fused to any heterologous amino acid sequence.
[0054] "Unique" sequences of the pp32r1 variant according to this
invention, either amino acid sequences or nucleic acid sequences
which encode them, are sequences which are identical to a sequence
of a pp32r1 polypeptide, but which differ in at least one amino
acid or nucleotide residue from the sequences of human pp32
(Genbank Locus HSU73477), murine pp32 (Genbank Locus MMU73478),
human cerebellar leucine rich acidic nuclear protein (LANP)
(Genbank Locus AF025684), murine LANP (Genbank Locus AF022957).
IIPP2a or human potent heat-stable protein phospatase 2a inhibitor
(Genbank Locus HSU60823), SSP29 (Genbank Locus HSU70439), HLA-DR
associated protein 1 (Genbank Locus HSPPHAPI, Accession No.
X75090), PHAPI2a (EMBL Locus HSPHAPI2A, Genbank Accession No.
Y07569), PHAPI2b (EMBL Locus HSPHAPI2B, Genbank Accession No.
Y07570), and April (EMBL Locus HSAPRIL), and preferably, are not
found elsewhere in the human genome. (A list of these sequences is
provided in Table 3A.) Similarly, an epitope is "unique" to pp32r1
polypeptides if it is found on pp32r1 polypeptides but not found on
any members of the set of proteins listed above. Analogs of pp32r2
and unique pp32r2 sequences are defined similarly. Of course,
unique sequences of pp32r1 are not found in pp32r2 and vice
versa.
[0055] "Variants of pp32" are homologous proteins which differ from
pp32 by at least 2 amino acids. In particular, sequence comparison
between pp32 and a variant will demonstrate at least one segment of
10 amino acids in which the sequence differs by at least two (2)
amino acids. More typically a variant will exhibit at least two
such 10 amino acid segments. Preferably, variants of pp32 in
accordance with this invention will exhibit differences in
functional activity from pp32. In particular, pp32r1 and pp32r2 are
variants of pp32 whose activity includes stimulation of
transformation in the rat fibroblast transformation assay described
herein.
[0056] A composition comprising a selected component A is
"substantially free" of another component B when component A makes
up at least about 75% by weight of the combined weight of
components A and B. Preferably, selected component A comprises at
least about 90% by weight of the combined weight, most preferably
at least about 99% by weight of the combined weight. In the case of
a composition comprising a selected biologically active protein,
which is substantially free of contaminating proteins, it is
sometimes preferred that the composition having the activity of the
protein of interest contain species with only a single molecular
weight (i.e.. a "homogeneous" composition).
[0057] As used herein, a "biological sample" refers to a sample of
tissue or fluid isolated from a individual, including but not
limited to, for example, plasma, serum, spinal fluid, lymph fluid,
the external sections of the skin, respiratory, intestinal, and
genitourinary tracts, tears, saliva, milk, blood cells, tumors,
organs, and also samples of in vivo cell culture constituents
(including but not limited to conditioned medium resulting from the
growth of cells in cell culture medium, putatively virally infected
cells, recombinant cells, and cell components).
[0058] "Human tissue" is an aggregate of human cells which may
constitute a solid mass. This term also encompasses a suspension of
human cells, such as blood cells, or a human cell line.
[0059] The term "immunoglobulin molecule" encompasses whole
antibodies made up of four immunoglobulin peptide chains, two heavy
chains and two light chains, as well as immunoglobulin fragments.
"Immunoglobulin fragments" are protein molecules related to
antibodies, which are known to retain the epitopic binding
specificity of the original antibody such as Fab, F(ab)'.sub.2, Fv,
etc. Two polypeptides are "immunologically cross-reactive" when
both polypeptides react with the same polyclonal antiserum.
[0060] General Methods
[0061] The practice of the present invention employs, unless
otherwise indicated, conventional molecular biology, microbiology,
and recombinant DNA techniques within the skill of the art. Such
techniques are well known to the skilled worker and are explained
fully in the literature. See, e.g., Maniatis, Fritsch &
Sambrook, "Molecular Cloning: A Laboratory Manual" (1982); "DNA
Cloning: A Practical Approach," Volumes I and II (D. N. Glover,
ed., 1985); "Oligonucleotide Synthesis" (M. J. Gait. ed., 1984);
"Nucleic Acid Hybridization" (B. D. Hames & S. J. Higgins,
eds., 1985): "Transcription and Translation" (B. D. Hames & S.
J. Higgins, eds., 1984): "Animal Cell Culture" (R. I. Freshney,
ed., 1986); "Immobilized Cells and Enzymes" (IRL Press, 1986); B.
Perbal, "A Practical Guide to Molecular Cloning" (1984), and
Sambrook, et al., "Molecular Cloning: a Laboratory Manual"
(1989).
[0062] pp32 Related Genomic DNA
[0063] Screening a human genomic library in bacteriophages with
probes generated from human pp32 cDNA yielded a new sequence that
contained an open reading frame encoding a protein homologous with
pp32 (see Example 2; pp32 sequence, reported in Chen, et al., Mol.
Biol. Cell, 7:2045-2056, 1996). While the pp32r1 and pp32r2
sequences (see FIGS. 2 and 5) are substantially homologous to pp32,
multiple single nucleotide base changes and short deletions suggest
that they are encoded by gene distinct from pp32 gene. The pp32
family also includes substantially homologous polypeptides reported
by others: HLA-DR associated protein 1 (Vaesen, 1994), leucine-rich
acidic nuclear protein (Matsuoka, 1994), and protein phosphatase 2A
inhibitor (Li, 1996).
[0064] DNA segments or oligonucleotides having specific sequences
can be synthesized chemically or isolated by one of several
approaches. The basic strategies for identifying, amplifying and
isolating desired DNA sequences as well as assembling them into
larger DNA molecules containing the desired sequence domains in the
desired order, are well known to those of ordinary skill in the
art. See. e.g., Sambrook, et al., (1989); B. Perbal, (1984).
Preferably, DNA segments corresponding to all or a part of the cDNA
or genomic sequence of pp32r1 may be isolated individually using
the polymerase chain reaction (M. A. Innis, et al., "PCR Protocols:
A Guide To Methods and Applications." Academic Press, 1990). A
complete sequence may be assembled from overlapping
oligonucleotides prepared by standard methods and assembled into a
complete coding sequence. See, e.g., Edge (1981) Nature 292:756:
Nambair, et al. (1984) Science 223:1299: Jay, et al. (1984) J.
Biol. Chem., 259:6311.
[0065] The assembled sequence can be cloned into any suitable
vector or replicon and maintained there in a composition which is
substantially free of vectors that do not contain the assembled
sequence. This provides a reservoir of the assembled sequence, and
segments or the entire sequence can be extracted from the reservoir
by excising from DNA in the reservoir material with restriction
enzymes or by PCR amplification. Numerous cloning vectors are known
to those of skill in the art, and the selection of an appropriate
cloning vector is a matter of choice (see, e.g., Sambrook, et al.,
incorporated herein by reference). The construction of vectors
containing desired DNA segments linked by appropriate DNA sequences
is accomplished by techniques similar to those used to construct
the segments. These vectors may be constructed to contain
additional DNA segments, such as bacterial origins of replication
to make shuttle vectors (for shuttling between prokaryotic hosts
and mammalian hosts), etc.
[0066] Procedures for construction and expression of proteins of
defined sequence are well known in the art. A DNA sequence encoding
pp32r1, pp32r2, or an analog of either pp31R1 or pp32r2, can be
synthesized chemically or prepared from the wild-type sequence by
one of several approaches, including primer extension, linker
insertion and PCR (see, e.g., Sambrook, et al.). Mutants can be
prepared by these techniques having additions, deletions and
substitutions in the wild-type sequence. It is preferable to test
the mutants to confirm that they are the desired sequence by
sequence analysis and/or the assays described below. Mutant protein
for testing may be prepared by placing the coding sequence for the
polypeptide in a vector under the control of a promoter, so that
the DNA sequence is transcribed into RNA and translated into
protein in a host cell transformed by this (expression) vector. The
mutant protein may be produced by growing host cells transfected by
an expression vector containing the coding sequence for the mutant
under conditions whereby the polypeptide is expressed. The
selection of the appropriate growth conditions is within the skill
of the art.
[0067] The assembled sequence can be cloned into any suitable
vector or replicon and maintained there in a composition which is
substantially free of vectors that do not contain the assembled
sequence. This provides a reservoir of the assembled sequence, and
segments or the entire sequence can be extracted from the reservoir
by excising from DNA in the reservoir material with restriction
enzymes or by PCR amplification. Numerous cloning vectors are known
to those of skill in the art, and the selection of an appropriate
cloning vector is a matter of choice (see, e.g., Sambrook, et al.,
incorporated herein by reference). The construction of vectors
containing desired DNA segments linked by appropriate DNA sequences
is accomplished by techniques similar to those used to construct
the segments. These vectors may be constructed to contain
additional DNA segments, such as bacterial origins of replication
to make shuttle vectors (for shuttling between prokaryotic hosts
and mammalian hosts), etc.
[0068] Producing the Recombinant Peptide
[0069] Preferably, DNA from the selected clones should be subcloned
into an expression vector, and the protein expressed by cells
transformed with the vector should be tested for immunoreactivity
with antibodies against the recombinant protein of this invention
prepared as described below. Such subcloning is easily within the
skill of the ordinary worker in the art in view of the present
disclosure. The amino acid coding region of the DNA sequence of
this invention may be longer or shorter than the coding region of
the disclosed sequence, so long as the recombinant peptide
expressed by the DNA sequence retains at least one epitope
cross-reactive with antibodies which are specifically
immunoreactive with pp32r1, pp32r2; or other pp32 variant as
desired. The preparation of selected clones which contain DNA
sequences corresponding to all or part of the sequence of pp32r1 or
pp32r2 may be accomplished by those of ordinary skill in the art
using conventional molecular biology techniques along with the
information provided in this specification.
[0070] It is possible to purify a pp32 variant protein such as
pp32r1, which is cross-reactive with antibodies specific for pp32,
from an appropriate tissue/fluid source; however, a cross-reactive
pp32 variant, or analog thereof, may also be produced by
recombinant methods from a DNA sequence encoding such a protein or
polypeptide. Polypeptides corresponding to the recombinant protein
of this invention may be obtained by transforming cells with an
expression vector containing DNA from a clone selected from an
mammalian (preferably human) library as described herein. Suitable
expression vector and host cell systems are well known to those of
ordinary skill in the art, and are taught, for instance, in
Sambrook, et al., 1989. The peptide may be obtained by growing the
transformed cells in culture under conditions wherein the cloned
DNA is expressed. Of course, the peptide expressed by the clone may
be longer or shorter than pp32r1 or pp32r2, so long as the peptides
are immunologically cross-reactive. Depending on the expression
vector chosen, the peptide may be expressed as a fusion protein or
a mature protein which is secreted or retained intracellularly, or
as an inclusion protein. The desired polypeptides can be recovered
from the culture by well-known procedures, such as centrifugation,
filtration, extraction, and the like, with or without cell rupture,
depending on how the peptide was expressed. The crude aqueous
solution or suspension may be enriched for the desired peptide by
protein purification techniques well known to those skilled in the
art. Preparation of the polypeptides may include biosynthesis of a
protein including extraneous sequence which may be removed by
post-culture processing.
[0071] Using the nucleotide sequences disclosed herein and the
polypeptides expressed from them, antibodies can be obtained which
have high binding affinity for pp32r1 or pp32r2, but much lower
affinity for pp32 and/or other variants of pp32. Such antibodies,
whether monoclonal or purified polyclonal antibodies can be used to
specifically detect pp32r1 or pp32r2. Techniques for preparing
polypeptides, antibodies and nucleic acid probes for use in
diagnostic assays, as well as diagnostic procedures suitable for
detection of pp32 are described in U.S. Pat. Nos. 5,726,018 and
5,734,022, incorporated herein by reference, and these techniques
may be applied to pp32r1 or pp32r2 by substitution of the nucleic
acid sequences disclosed herein. Similar substitution may be
applied to other variants of pp32.
[0072] pp32r1 Promoter Sequence
[0073] Multiple consensus sequences for binding active steroid
receptors found in genomic sequences upstream from the pp32r1
coding region are consistent with hormone regulation of gene
expression. The consensus sequences were associated with the both
induction and repression of expression by steroid hormones. The
combination of both positively and negatively acting elements
suggests complex regulation of pp32r1 expression.
[0074] Possible steroid hormone regulation of pp32r1 expression is
important in regard to prostate cancer. While about one-half of
treated patients initially respond to androgen ablation, subsequent
hormone refraction and continued aggressive tumor growth is common
(Garnick, M. B., "Prostate Cancer," in Scientific American
Medicine, Dale, D. C. and Federman, D. D. Eds., Scientific American
Inc., New York. 1995). Many different steroid hormones regulate the
growth of prostate cancer cells (Huggins, et al., "Studies on
prostate cancer: I. The effect of castration, of estrogen, and of
androgen injection on serum phosphatases in metastatic carcinoma of
the prostate," Cancer Res., 1:293, 1941). These findings
established a basis for androgen ablation therapy for the treatment
of metastatic prostate cancer.
[0075] The present invention provides androgen-activated promoters
based on the upstream portion of the genomic sequence in FIG. 2.
The promoter sequence provided by this invention is bounded at its
3' terminus by the translation start codon of a coding sequence and
extends upstream (5' direction) to include at least the number of
bases or elements necessary to initiate transcription at levels
above background. Within the promoter sequence will be found a
transcription initiation site (conveniently defined by mapping with
nuclease S1), a protein binding domain (consensus sequence) within
about 100 bases upstream of the transcription initiation site
generally designated the TATA box (a binding site for TATA box
binding proteins and RNA polymerase), and various other protein
binding domains (consensus sequences) upstream of the TATA box that
modulate the basic transcriptional activity of the transcription
initiation site and the TATA box. The various other protein binding
domains preferably contain recognition sequences shown in Table 1
for binding (1) androgen receptors, estrogen receptors,
glucocorticoid receptors, and progesterone receptors; (2)
transcription factors containing the leucine zipper motif
including, but not limited to Fos, Jun, JunB, and Myc; and, (3)
certain tissue specific transcription factors including, but not
limited to GATA-1 and GATA-2. The various other protein binding
domains upstream of the TATA box may contribute to specificity
(tissue specific expression), accuracy (proper initiation), and
strength (transcription frequency) of the promoter. The promoter
elements may serve overlapping functions so that the promoter may
function in the absence of subsets of these elements.
[0076] Therapy
[0077] Inhibition of function of protransforming variants of pp32
by any means would be expected to be an avenue of therapy.
[0078] U.S. Pat. No. 5,726,018, incorporated herein by reference,
describes various therapeutic avenues which may be applied by the
skilled worker based on the nucleotides and protein sequences
disclosed herein. In a particular embodiment, all or a portion of
the sequence of pp32r1 or pp32r2 may be supplied in the antisense
orientation to block expression of the variants found in carcinomas
particularly prostate cancer. Suitable methods for preparation of
antisense expression vectors and administration of antisense
therapy may be found in U.S. Pat. No. 5,756,676, incorporated
herein by reference. Prescreening of the patient population using
the diagnostic methods described herein to identify patients having
tumors expressing the particular pp32 variant is preferred.
[0079] Screening for compounds having therapeutic effects in
prostate cancer may also be facilitated by the present invention.
Studies which may be used to screen candidate compounds are
described in U.S. Pat. No. 5,756,676, incorporated herein by
reference, modified by the use of cell lines which express
particular variants of pp32 (see, e.g., Examples below). Compounds
which affect steroid dependent protein expression may also be
detected according to this invention by similar screening studies
using an androgen-activated promoter as provided herein operatively
coupled to a DNA sequence whose expression may be detected. (Marker
sequences are well known in the art, see, e.g., Sambrook, et al.,
and selection of an appropriate detectable expression marker is a
routine matter for the skilled worker.) Screening by testing the
effect of candidate compounds on recombinant cells containing an
expression vector having an androgen-activated promoter operatively
coupled to an expression marker, with appropriate controls, is
within the skill of the art, in view of the promoter sequences
provided herein. In one aspect, this invention provides a method
for screening candidate compounds for pharmacological activity by
(1) culturing a cell transfected with the DNA molecule containing
an androgen-activated transcriptional promoter which is operatively
linked to an open reading frame comprising at least one exon of a
protein coding sequence, and (2) determining expression of the open
reading frame in the presence and absence of the compound. In a
preferred mode the androgen activated promoter may be the portion
of the sequence in FIG. 2 which is up-stream of the translation
initiation site, or alternatively the androgen activated promoter
may be the 2700 bp upstream from the translation initiation
site.
[0080] Diagnostic Methods Based on the pp32 Gene Family
[0081] In one aspect, this invention provides methods for detecting
and distinguishing among members of the pp32 gene family. As
explained herein, the presence of one or more members of the gene
family may be detected using assays based on common structures
among the members resulting from common or similar sequences. For
example, polyclonal antibodies elicited by pp32 will cross-react
with pp32r1 and pp32r2, including various alleles of these pp32
variants. Similarly, the full coding region of the pp32 cDNA will
hybridize under suitable conditions with nucleic acid encoding any
of the variants, as shown by the in situ detection of the variants
in tumor sections which were subsequently shown to contain either
pp32r1 or pp32r2 allelic forms (Example 1). Selection of conditions
that promote the immune cross-reactivity or cross-hybridization
necessary for such detection is within the skill of the art, in
view of the examples provided herein. For example, by using large
nucleotide probes in hybridization experiments, the effects of one
or a few differences in sequence may be overcome, i.e., larger
probes will bind to more dissimilar target sequences, in contrast
to shorter probes for which each nucleotide makes a larger
percentage contribution to the affinity, and a single nucleotide
alteration will cause a greater relative reduction in hybridization
efficiency. Typically probes of 50 or more nucleotides are used to
find homologues to a given sequence, and the studies reported in
Example 1 used the entire sequence of pp32 as a probe to find cells
expressing homologous members of the gene family other than pp32.
Likewise, polyclonal antisera elicited to an antigen having
multiple epitopes is more likely to cross-react with a second
antigen that has a few of the same epitopes along with many
different epitopes, while a monoclonal antibody or even a purified
polyclonal antiserum might not bind to the second antigen.
[0082] In addition to determining the presence of one or more
members of the pp32 gene family, this invention also provides
methods for distinguishing among members. Determining which pp32
variant may be useful, for instance, to determine whether a
transfomation promoting or suppressing variant is present in a
tissue sample. Suitable methods for distinguishing include both
immunoassay and nucleic acid binding assays. Preferred are methods
which can detect a 10-fold difference in the affinity of the
detecting ligand (e.g., antibody or oligonucleotide) for the target
analyte. Such methods are well documented for other systems, and
may be adopted to distnguish between pp32 variants by routine
modification of such methods in view of the guidance provided
herein.
[0083] Protein level assays may rely on monoclonal or purified
polyclonal antibodies of relatively greater affinity for one
variant compared to another (see, e.g., Smith, et al. ("Kinetics in
interactions between antibodies and haptens," Biochemistry,
14(7):1496-1502, 1975, which shows that the major kinetic variable
governing antibody-hapten interactions is the rate of dissociation
of the complex, and that the strength of antibody-hapten
association is determined principally by the activation energy for
dissociation), and Pontarotti, et al.("Monoclonal antibodies to
antitumor Vinca alkaloids: thermodynamics and kinetics," Molecular
Immunology, 22(3):277-84, 1985, which describes a set of monoclonal
antibodies that bind various dimeric alkaloids and can distinguish
among the alkaloid haptens due to different relative affinities of
the various monoclonal antibodies for particular dimeric
alkaloids), each of which is incorporated herein by reference).
Suitable modifications of the conditions for immunoassays to
emphasize the relative affinity of monoclonal antibodies with
different affinity are also discussed in U.S. Pat. No. 5,759,791,
incorporated herein by reference.
[0084] A number of methods are available which are capable of
distinguishing between nucleic acid sequences which differ in
sequence by as little as one nucleotide. For example, the ligase
chain reaction has been used to detect point mutations in various
genes (see. e.g., Abravaya, et al., "Detection of point mutations
with a modified ligase chain reaction (Gap-LCR)." Nucleic Acids
Research, 23(4):675-82, 1995, or Pfeffer, et al., "A lipase chain
reaction targeting two adjacent nucleotides allows the
differentiation of cowpox virus from other Orthopoxvirus species,"
Journal of Virological Methods, 49(3):353-60, 1994, each of which
is incorporated herein by reference). Amplification of a sequence
by PCR also may be used to distinguish sequences by selection of
suitable primers, for example, short primers, preferably 10-15
matching nucleotides, where at least one of the primers has on the
3' end a unique base that matches one variant but not other
variants, and using annealing conditions under which the primer
having the unique base has at least a ten-fold difference in
dissociation rate between the fully matching variants and variants
which do not fully match. Similar differentiation may be achieved
in other methods dependent on hybridization by using short probes
(typically under 50 bp, preferably 25 bp or less more preferably
less than 20 bp or even 10-12 bp) by adjusting conditions in
hybridization reactions to achieve at least a ten-fold difference
in dissociation rate for the probes between the fully matching
variants and variants which do not fully match. Cleavase fragment
length polymorphism may also be used, and a specific example below
provides guidance from which the skilled worker will be able to
design similar studies by routine selection of other cleavase
enzymes in view of the sequences provided herein.
[0085] The diagnostic methods of this invention may be used for
prognostic purposes and patient differentiation as described
herein. In particular, the methods of this invention allow
differentiation between products expressed from the various
sequences disclosed in FIG. 7. Preferred methods are those that
detect and/or differentiate, between pp32, pp32r1, and/or pp32r2.
Situations in which differentiation between pp32 variants will be
of benefit will be readily apparent to the skilled clinician, in
view of the present disclosure. Selection among the diagnostic
methods provided by this invention of a suitable technique to
achieve the desired benefit is a routine matter for the skilled
clinician.
EXAMPLES
[0086] In order to facilitate a more complete understanding of the
invention, a number of Examples are provided below. However, the
scope of the invention is not limited to specific embodiments
disclosed in these Examples, which are for purposes of illustration
only.
Example 1
[0087] Cellular Location of pp32 Expression
[0088] pp32 mRNA can be detected by in situ hybridization with a
pp32 probe under stringent conditions.
[0089] In situ hybridization. Bases 1-298 of the pp32 cDNA sequence
(GenBank HSU73477) were subcloned into the Bluescript vector by
standard techniques. Digoxigenin labeled anti-sense and sense RNA
probes were generated using a commercially available kit
(Boehringer Mannheim). Vector DNA linearized with BamHI and Xhol
served as template for antisense and sense probe generation
respectively. In vitro transcription was performed for 2 hours at
37.degree. in a final volume of 20 .mu.l which contained 1 .mu.g of
template DNA, 2 U/.mu.l of either T3 or T7 RNA polymerase. 1
U/.mu.l ribonuclease inhibitor, 1 mM each of ATP, CTP, GTP, 0.65 mM
UTP, 0.35 mM digoxigenin-11-UTP, 40 mM Tris-HCl pH 8.0, 10 mM NaCl,
10 mM DTT, 6 mM MgCl.sub.2 and 2 mM spermidine. The reaction was
stopped by adding 2 .mu.l of 0.2M EDTA, pH 8. 0 and the synthesized
transcripts were precipitated for 30 min at -70.degree. with 2.2
.mu.l of 4 M LiCl and 75 .mu.l of pre-chilled ethanol. RNA was
pelleted by centrifugation, washed with 80% ethanol, mildly dried
and dissolved in 100 .mu.l of DEPC treated water. Yields of labeled
probe were determined by an enzyme linked irrimunoassay using a
commercially available kit (Boehringer Mannheim). Non-radioactive
in situ hybridization was performed with anti-sense and sense pp32
RNA probes generated by in vitro transcription (see U.S. Pat. No.
5,726,018, incorporated herein by reference). FIG. 1A shows that
normal prostatic basal cells are positive, whereas the clear,
differentiated glandular cells are negative. In contrast, prostatic
adenocarcinoma, shown at left, is strikingly positive. Note that
the signal is cytoplasmic since it is mRNA and not the protein that
is detected in this assay.
[0090] pp32 displays a distinctive pattern of expression in vivo
(Chen, et al.; Malek. et al., "Identification and preliminary
characterization of two related proliferation-associated nuclear
phosphoproteins." Journal of Biological Chemistry, 265:13400-13409,
1990; Walensky, et al., "A novel M(r) 32,000 nuclear phosphoprotein
is selectively expressed in cells competent for self-renewal."
Cancer Research 53:4720-4716, 1993). In normal peripheral tissues,
expression is restricted to stem-like cell populations such as
crypt epithelial cells in the gut and basal epithelium in the skin:
in the adult central nervous system, cerebral cortical neurons and
Purkinje cells also express pp32. In normal prostate, basal cells
express pp32, whereas pp32 mRNA is not detectable by in situ
hybridization in differentiated glandular cells (FIG. 1A). In
contrast, strong in situ hybridization to pp32 probes is found in
nearly all clinically significant human prostatic adenocarcinomas.
87% of human prostatic adenocarcinomas of Gleason Score 5 and above
express mRNA that hybridizes strongly with probes to pp32 in
contrast to only 11% of prostate cancers of Gleason Score 4 and
below in a study of 55 patients.
[0091] Immunohistochemistry. Formalin-fixed, paraffin-embedded
tissue was sectioned at 4 .mu.M, deparaffinized, hydrated,
processed for heat-induced antigen retrieval at 95.degree. in 0.01
M citrate buffer, pH 6.0, for 20 min (Cattoretti, et al., "Antigen
unmasking on formalin-fixed, paraffin-embedded tissue sections,"
Journal of Pathology 171:83-98, 1993), then incubated overnight at
room temperature with a {fraction (1/20)} dilution of anti-pp32
antibody. Following washing, the slide was sequentially developed
with biotinylated swine-anti-rabbit IgG at {fraction (1/100)}
(Dako), strepavidin peroxidase (Dako), and diaminobenzidine. FIG.
1B shows a representative high-grade human prostate cancer stained
with affinity-purified rabbit polyclonal anti-pp32 antibody (Gusev,
et al., "pp32 overexpression induces nuclear pleomorphism in rat
prostatic carcinoma cells." Cell Proliferation 29:643-653, 1996).
The left-hand panel shows a representative field at 250x: the
rectangle indicates the area shown in computer venerated detail in
the right-hand panel. Strongly hybridizing tumors show intense
immunopositivity with antibodies to pp32, indicating that they
express pp32 or immunologically related proteins (FIGS. 1A and
1B).
Example 2
[0092] ESTs Corresponding to pp32
[0093] Several potential variant pp32 species have been identified
in the prostate cancer expressed sequence tag libraries of the
NCI's Cancer Genome Anatomy Project. Clone 588488 encodes a protein
that is 96% identical to APRIL, although absent retrieval and
sequencing of the full clone, it is impossible to tell whether the
entire EST clone encodes a pp32 related sequence; neither is it
possible to assess the biologic function of this molecule at this
time. Nevertheless, it is apparent that the sequenced portion
encodes a protein bearing great similarity to pp32. This EST does
not appear in the database for normal prostate. As with the variant
pp32 species recovered from prostate cancer, generation of this
molecule by mutation would require a complex mechanism.
[0094] pp32-related genes are present in other organisms. The
existence of a pp32 gene family in rodent would be consistent with
the existence of a comparably sized family in human. A murine pp32
(GenBank U73478) has 89% amino acid identity to pp32, but less
identity to pp32r1 and APRIL. (The murine cerebellar leucine rich
acidic nuclear protein has a single amino acid substitution
relative to murine pp32.) We additionally identified a murine EST,
GenBank AA066733, with closest identity to APRIL protein at 85%
identity over 148 amino acids of a predicted open reading frame.
Several other murine EST's. AA212094 and W82526, are closely
related to the pp32 family but are not significantly more related
to either pp32, pp32r1, or APRIL. A human homologue of such a gene
would be expected to encode a fourth member of this gene family. We
identified EST's predicted to encode pp32-related proteins in C.
elegans, schistosomes, zebrafish, and Drosophila (data not shown).
However, these sequences may not represent the complete extent of
the pp32 gene family in these organisms, and thus are not
informative for the likely size of the mammalian pp32 gene
family.
Example 3
[0095] The Structure of a Gene Encoding a Relative of the pp32
Family
[0096] Screening a human genomic library in bacteriophages with
probes generated from human pp32 cDNA yielded a new sequence that
contained an open reading frame encoding a protein homologous with
pp32.
[0097] Screening a Human Genomic Library in Bacteriophages for pp32
cDNA.
[0098] A genomic library from human placenta in the Lambda Fix II
vector was expressed in E. coli strain XL-1 Blue MRA (Stratagene
#946206). Screening for bacteriophage clones containing DNA inserts
homologous with pp32 cDNA followed routine procedures (Sambrook, et
al.). Briefly, nitrocellulose filters that had overlain
bacteriophage plaques were hybridized with P-32 labeled probes for
pp32 cDNA. The probes were prepared by the random primer method
(Stratagene #300385) using pp32 cDNA as a template (Chen, et al.,
Molec. Biol. Cell, 7:2045-2056,1996.). Reactive bacteriophage
plaques were plugged and the bacteriophages were eluted,
reexpressed, and rescreened with pp32 cDNA probes until pure.
Bacteriophage DNA was prepared by the plate lysate method
(Sambrook, et al.).
[0099] Identifying Restriction Fragments within Bacteriophage DNA
Containing Sequences Homologous with pp32 cDNA.
[0100] DNA from a bacteriophage clone containing pp32 cDNA
sequences was digested with HindIII. Using routine methods, the
restriction fragments were separated by agarose gel
electrophoresis, transferred in alkaline buffer to positively
charged nylon filters, and hybridized with probes that were
selective for the 5' and 3' ends of the pp32 cDNA (Sambrook, et
al.). The 5' and 3' probes were prepared as described above except
that the products of polymerase chain reactions (PCR) were used as
templates for the labeling reactions (Sailki, et al., Science,
239:487-491, 1988.). One PCR product was a 249 base pair segment of
pp32 cDNA containing nucleotides 32 through 279. It was the result
of a reaction using a pp32cDNA template and the primers
[0101] 5'-TATGCTAGCGGGTTCGGGGTTTATTG-3' and
[0102] 5'-GATTCTAGATGGTAAGTTTGCGATTGAGG-3' (primer set A).
[0103] The other product was a 263 base pair segment of pp32 cDNA
including nucleotides 677 through 938. It was the result of a
reaction using a pp32 cDNA template and the primers
[0104] 5'-GAATCTAGAAGGAGGAGGAAGGTGAAGAG-3' and
[0105] 5'-CTATCTAGATTCAGGGGGCAGGATTAGAG-3' (primer set B).
[0106] The PCR reactions included 35 cycles of one minute
denaturations at 95.degree. C., one minute primer annealings at
50.degree. C., and one minute extensions at 72.degree. C. (cycling
program A). A 4.7 kb HindIII restriction fragment that hybridized
with the 5' probe, but not with the 3' probe and a 0.9 kb HindIII
fragment that hybridized with the 3' probe, but not with the 5'
probe were subcloned into pBluescript (Gibco) by routine methods
(Sambrook, et al.). The nucleotide sequences of both strands of
purified plasmid DNA containing the inserts were determined by
automated procedures (DNA Analysis Facility, Johns Hopkins
University School of Medicine).
[0107] Completion of Sequencing by Direct Sequencing of PCR
Products. Alignment of the sequences of the 4.7 and 0.9 kb HindIII
restriction fragments with pp32 cDNA showed about 90% homologies
between the 3' end of the 4.7 kb fragment and the 5' region of pp32
cDNA and the 5' end of the 0.9 kb fragment and the 3' region of the
pp32 cDNA. There was an unaligned 199 base pair gap of pp32 cDNA
sequence between the ends of the restriction fragments. Primers
were designed to specifically anneal to relative pp32 sequences on
both sides of the sequence gap. The primer sequences were
[0108] 5'-GAGGTTTATTGATTGAATTCGGCT-3' and
[0109] 5'-CCCCAGTACACTTTTCCCGTCTCA-3' (primer set C).
[0110] Polymerase chain reactions followed cycling program A with
primer set C and pure bacteriophage DNA as a template. The 943 base
pair products were shown by ethidium bromide staining agarose gels,
extracted from excised fragments of low melt agarose (NuSieve)
electrophoresis gels, and sequenced by automated procedures as
described above.
[0111] A sequence of 5,785 bases was obtained from the human
placental genomic library bacteriophage clone containing segments
homologous with pp32 cDNA (FIG. 2). This sequence was deposited in
Genbank under Accession No. U71084, Locus HSU71084. The sequence
has an open reading frame extending from nucleotides 4,453 to
5,154. Analysis of the nucleotide sequence upstream of the open
reading frame revealed consensus sequences for active steroid
hormone receptors at over twenty positions (Table 1).
[0112] Sequence analysis of the open reading frame showed 94%
sequence homology to pp32 (FIG. 3). Alignment of the-open reading
frame sequence to pp32 cDNA revealed 33 scattered, solitary base
differences and clustered differences of two and seven bases. There
were two internal deletions of three and nine bases. The open
reading frame encoded a polypeptide containing 234 amino acid
residues with 88% protein-level homology to pp32 (FIG. 4).
Alignment of the translated sequence to the pp32 amino acid
sequence revealed 18 scattered, solitary amino acid residue
differences, three differences in clusters of two residues, and one
difference in a clusters of four residues. There were two internal
deletions of one and three residues and a terminal deletion of
eleven residues. The translated sequence contained 69 acidic
residues, 26 fewer than pp32.
Example 4
[0113] Chromosome Mapping of pp32r1
[0114] The pp32r1 gene maps to chromosome 4 as determined by PCR of
the NIGMS monochromosomal panel 2 (Drwinga, et al., "NIGMS
human/rodent somatic cell hybrid mapping panels 1 and 2," Genomics
16:311314, 1993) followed by sequencing of the PCR product.
Interestingly, the full sequence of pp32r1 including 4364
nucleotides of sequence 5' to the start ATG contained over 400
matches in a blastn search of the non-redundant GenBank database.
These matches were to two short regions of about 278 and 252 base
pairs (nucleotides 674-952 and 2542-2794) that represent repeats in
opposite orientations. The repeats are significantly related to
elements on many chromosomes.
[0115] The human pp32 gene has been mapped to chromosome
15q22.3-q23 by fluorescence in situ hybridization (Fink, et al.). A
Unigene entry for pp32 (Hs. 76689; HLA-DR associated protein 1)
lists 93 EST sequences corresponding to this gene, 12 of which
contain a mapped sequence-tagged site (STS). These STS sites are
all reported to map to chromosome 15, as are many of the pp32 EST's
analyzed by electronic PCR (http://www.ncbi.nlm.nih.gov). APRIL
protein was also mapped to chromosome 15q25 (Mencinger, et al.;
GenBank Y07969).
Example 5
[0116] Sequence Analysis of pp32r2
[0117] A pp32-related sequence (designated pp32r2) has been
identified on chromosome 12 by methods analogous to those described
in Example 2 for isolation of the unique intronless pp32-related
gene pp32r1, found on chromosome 4. It was initially thought that
the chromosome 12 sequence, encoding a truncated protein, might
represent a pseudogene; however that interpretation has been
reassessed in view of the present findings. The sequence has been
designated pp32r2, and is recorded in Genbank as locus AF008216:
the sequence of pp32r2 is shown in FIG. 5. By BESTFIT analysis
(Genetics Computer Group. Inc., Wisconsin Package, version 9.1,
Madison, Wis., 1997), pp32r2 is 99.5% identical to FT1.11, FT2.4
and T1, showing four nucleotide differences over the 875 nucleotide
overlap of the sequences: this level of variation is consistent
with a polymorphism. Similarly, BESTFIT analysis shows that PP32R1
is 99.6 % identical to FT3.3 and 99.4% identical to FT2.2,
displaying four and five nucleotide differences, respectively (see
FIG. 7 below).
Example 6
[0118] Sequence Comparison of Multiple Clones
[0119] Screening of a human placental genomic library in Lambda Fix
II vector (Stratagene #946206) with P-32 labeled probes for pp32
cDNA yielded a clone of approximately 23 kb. 4.7 kb and 0.9 kb
HindIII restriction fragments of this clone hybridized with probes
for pp32 cDNA. The 4.7 kb clone aligned with the 5' portion of the
pp32 cDNA sequence, and the 0.9 kb fragment aligned with the 3'
end. A small discontinuity of 0.2 kb was sequenced from a bridging
PCR product. No introns were identified.
[0120] Cultured cells including the whole human embryonic line
FSH173WE and the prostatic cancer cell lines PC-3 and LNCaP
(American Type Culture Collection) were grown under recommended
tissue culture conditions. Poly A+RNA was prepared by oligo dT
adsorption (MicroFasTrack, Invitrogen) and used as a template for
the, generation of cDNA through reactions with reverse
transcriptase and random hexamers (GeneAmp RNA PCR Kit, Perkin
Elmer). The cDNA sequences encoding the open reading frame were
amplified by nested PCR using primers specifically selective for
the genomic sequence over pp32 sequences. The final 298 base pair
products were seen by ethidium bromide staining agarose
electrophoretic gels.
[0121] Using procedures similar to those described in Example 3,
except without the need for nested primers in most cases,
transcripts from DU-145 cells and from numerous patients were
sequenced for comparison to the transcripts from the above samples.
The results are shown in Table 2. A summary of the degree of
identity between various transcripts is provided in Table 3.
Example 7
[0122] Sequence Variation for Individual Isolates of Different Cell
Lines and Tumor Tissue
[0123] The explanation for the apparent discordant expression of
p32 in cancer is that prostate tumors do not generally express
pp32, but rather express variant pp32 species that promote
transformation, instead of inhibiting it.
[0124] RT-PCR and CFLP. Sequences were reverse-transcribed and
amplified using bases 32 to 52 of HSU73477 as a forward primer and
9 19 to 938 of the same sequence as a reverse primer in conjunction
with the Titan One-Tube RT-PCR kit (Boehringer). Reverse
transcription was carried out at 50.degree. for 45 min followed by
incubation at 94.degree. for 2 min; the subsequent PCR utilized 45
cycles of 92.degree. for 45, 55.degree. for 45 sec. and 68.degree.
for 1 min with a final extension at 68.degree. for 10 min in a PTC
100 thermocycler (MJ Research). Template RNA was isolated from cell
lines or frozen tumor samples using RNAzol B (Tel-Test) according
to the manufacturer's instructions, then digested with RNAse-free
DNAse 1 (Boehringer). pCMV32 was used as a positive control without
reverse transcription. The cleavage assay was performed according
to the manufacturer's specifications (Life Technologies) with
digestion at 55.degree. for 10 min at 0.2 mM MnCl.sub.2 and
electrophoresed on a 6% denaturing polyacrylamide sequencing
gel.
[0125] At the level of RTPCR, paired normal prostate and prostatic
adenocarcinoma from three patients yielded amplification products
(FIG. 6A) ranging from 889 to 909 bp. The reaction employed
consensus primers capable of ampliring the full-length coding
sequence from pp32 and the two closely-related intronless genomic
sequences pp32r1 and pp32r2. The sole difference noted was a
diminished amplicon yield from normal tissue as compared to
neoplastic. Four human prostatic adenocarcinoma cell lines, DU-145,
LNCaP, PC-3, and TSUPR-1, also yielded similar products.
[0126] FIG. 6A shows RT-PCR amplified DNA from human prostate and
prostate cancer cell lines. Lane a is an undigested control whose
band migrated substantially slower than the digestion produces;
samples in all other lanes were digested with cleavage as
described. The lanes show: 1 kb ladder (Lif Technologies), A;
pCMV32, B; DU-145, C; LNCaP, D; PC-3, E; TSUPr-1, F; a
representative sample, FT-1, without reverse transcription, G; FN-1
H; FT-1, I; FN-2, J; FT-2, K; FN-3, L; FT3, M; negative control
with template omitted. FN indicates frozen benign prostate and the
number indicates the patient: FT indicates frozen prostatic
adenocarcinoma and the number indicates the patient. Numbers on the
left-hand side of the figure indicate the size in kb of a reference
1 kb DNA ladder (Life Technologies).
[0127] Qualitative differences between normal and neoplastic tissue
began to emerge when the RT-PCR products were subcloned and
analyzed by cleavage fragment length polymorphism analysis (CFLP)
and sequence analysis. FIG. 6B shows a cleavase fragment length
polymorphism analysis of cloned cDNA amplified by RT-PCR from human
prostatic adenocarcinoma, adjacent normal prostate, and human
prostatic adenocarcinoma cell lines using primers derived from the
normal pp32 cDNA sequence. The lanes show individual RT-PCR-derived
clones from the DU-145, LNCaP, PC-3 and TSUPr1 cell lines, from
frozen prostate cancer (FT), and from frozen normal prostate (FN):
a, undigested normal pp32 cDNA, be normal pp32cDNA: c, DU-145-1; d,
DU-145-3; e, DU-145-5; f, LNCaP-3; g, PC3-3; h, PC3-8; i, TSUPr1,
-I; j, TSUPr1-3; k, TSUPr1-6; 1, FT1.11; m, FT1.7; n, FT2.2; o,
FT2.4; p, FT3.18; q, FT3.3; r, FN3.17; s, FN2.1. LNCaP expresses
normal pp32. The band shifts correspond to sequence differences.
All clones of RT-PCR product from normal prostate tissue displayed
a normal CFLP pattern that corresponded precisely to that obtained
from cloned pp32 cDNA template (GenBank HSU73477, FIG. 6B).
Prostatic adenocarcinomas yielded four distinct CFLP patterns upon
similar analysis, of which three were unique and one mimicked the
normal pp32 pattern. Examination of DU-145, PC-3, and TSUPR-1 cell
lines yielded substantially similar results whereas LnCaP yielded
only a normal pp32 CFLP pattern. Further analysis at the sequence
level confirmed that normal prostate and LnCaP contained solely
normal pp32 transcripts.
[0128] Transcripts obtained from prostatic adenocarcinomas and from
most cell lines represented closely-related variant species of
pp32, summarized in Table 1. These transcripts varied from 92.4% to
95.9% nucleotide identity to normal pp32 cDNA (Genetics Computer
Group, Inc., Wisconsin Package, version 9.1, Madison, Wis., 1997).
Of the sixteen variant transcripts obtained, fifteen had open
reading frames encoding proteins ranging from 89.3% to 99.6%
identity to normal pp32. The table summarizes data obtained for
variant pp32 transcripts obtained from human prostatic
adenocarcinoma and prostate cancer cell lines. Sequences falling
into closely related groups are indicated by the group letters
(A,B,C); U indicates unassigned sequences not clearly falling into
a group. The origin of each sequence is: FT, frozen tumor followed
by patient number, decimal point, and clone number; D, DU-145
followed by clone number (as are all cell line sequences); P, PC3;
and T, TSUPr1. Nucleotide identity, gaps in the nucleotide sequence
alignment, and protein identity were determined from BESTFIT
alignments with the normal pp32 cDNA and protein sequences. The
effect on transformation is described as: stimulates, more foci
obtained when transfected with ras+myc than with ras-myc+vector
control: inactive, equivalent foci obtained as with ras+myc+vector
control; and suppresses, fewer foci obtained as with ras+myc+vector
control.
[0129] The predicted protein sequences fell into three discrete
groups: [1] truncated sequences spanning the N-terminal 131 amino
acids of pp32, of which one such sequence substantially equivalent
to pp32r2 was obtained identically from two of three patients and
from the TSUPR-1 cell line; [2] sequences more closely homologous
to a distinct pp32-related gene, pp32r1 than to pp32, and [3]
heterogeneous pp32-related sequences. Tumors from two of the three
patients analyzed contained no detectable normal pp32 transcripts.
Two of twelve cloned transcripts from the third patient tumor were
normal by CFLP pattern, with sequence confirmation of normality on
one clone. Two clones from cell lines were normal by CFLP
screening, but were later shown to represent variant-sequences.
[0130] FIGS. 7A and 7B show a multiple pairwise alignment of
nucleotide and predicted protein sequences for all transcripts
(Smith, et al., "Identification of common molecular subsequences,"
J. Mol. Biol., 147:195-197 1981). The figures were compiled with
the GCG Pileup and Pretty programs (Smith, et al.). Differences
from the consensus sequences are shown as indicated, agreement with
the consensus sequence is shown as a blank. Normal human pp32 is
designated hpp32. Sequences from the TSUPr1, PC3, and DU-145 cell
lines are as indicated. The designation FT indicates sequence
derived from a frozen human prostatic adenocarcinoma. Only the
normal pp32 sequence. hpp32, was obtained from normal prostate
adjacent to tumor tissue. FIG. 8A shows alignment of the amplicon
nucleotide sequences with pp32 and the predicted amplicon from
pp32r1: FIG. 8B shows alignment of the predicted protein sequences.
One sequence (FT 1.11), independently obtained three times from two
separate patients and the TSUPR-1 cell line, is shown only once in
the diagram. The pileup and pairwise alignments illustrate several
important points: [1] there is a high degree of sequence
conservation at both the nucleotide and predicted amino acid
levels; [2] the sequence differences are distributed throughout the
length of the sequence without obvious hotspots; [3] there is no
obvious clustering or segmentation of sequence differences: and [4]
the variant sequences fall into the previously described groups.
These points are detailed in FIGS. 8A and 8B.
Example 8
[0131] Diagnostic Method to Distinguish Among Family Members
[0132] The three members of the pp32 family which are expressed in
human prostate cancer are pp32, pp32r1 and pp32r2. Whereas pp32
suppresses in vitro transformation and in vivo tumorigenesis in
model systems, pp32r1 and pp32r2 are pro-transforming and are
tumorigenic in the same systems. It is possible to determine which
of the three members is expressed in a tissue sample by using a
protocol similar to that described in Example 7.
[0133] Analysis from freshly frozen human tissue and cell lines.
Total RNA is extracted from freshly frozen human tissues or human
cancer cell lines and subjected to reverse transcription and
polymerase chain reaction amplification with single set of primers
capable of amplifying the entire coding region of the cDNA of all
the three genes. A suitable set of primers is:
[0134] Upper: 5'GGGTTCGGGGTTTATTG3'--This corresponds to bp32 to
bp48 of the pp32 cDNA sequence (Genbank U73477)
[0135] Lower: 5'CTCTAATCCTGCCCCCTGAA3'--This corresponds to bp919
to bp938 of the pp32 cDNA sequence (Genbank U73477)
[0136] The observed amplicon sizes with this primer set are
pp32-907 bp, pp32r1-889 bp and pp32r2-900 bp. The three cDNAs are
distinguished from each other by restriction enzyme digestion with
the following enzymes--EcoR I, Hind III and Xho I. The resultant
digest is run on a 2.5% agarose gel to positively identify the
three different cDNAs. The table below lists the sizes of the bands
observed The bolded numbers indicate the band sizes useful for
identification of the three cDNAs.
1TABLE 4A Expected band sizes upon restriction digestion of the
RT-PCR product from fresh tissue and cell lines EcoR I/Hind III
EcoR I/Xho I Undigested EcoR I Double digest Double digest hpp32
907 21,177,709 21,177,69,640 21,177,709 pp32r1 889 21,177,691
21,19,66,198,427 21,177,691 pp32r2 900 21,879 21,244,635
21,385,494
[0137] Analysis from formalin fixed and paraffin embedded tissue. A
similar approach is followed for identification of pp32, pp32r1 and
pp32r2 transcripts from formalin fixed and paraffin embedded
tissues. Total RNA is extracted and subjected to reverse
transcription and PCR amplification with a single set of primers
capable of amplifying a stretch of 200 bp from all the three cDNAs.
A suitable set of primers is:
[0138] Upper primer--from bp394 to bp414 of the pp32 cDNA sequence
(Genbank U73477)
[0139] Lower primer--from bp609 to bp629 of the pp32 cDNA sequence
(Genbank U73477)
[0140] The three cDNAs are distinguished from each other by
restriction enzyme digestion with the following enzymes--Hind III,
Xho I and BseR 1. The resultant digest is run on a 3% agarose gel
to positively identify the three different cDNAs. The table below
lists the sizes of the bands observed. The bolded numbers indicate
the band sizes useful for identification of the three cDNAs.
2TABLE 5A Expected band sizes upon restriction digestion of the
RT-PCR product from formalin fixed and paraffin embedded tissues
Undigested Hind III Xho I BseR I hpp32 200 200 200 80,120 pp32r1
200 100,100 200 200 pp32r2 200 200 44,156 80,120
Example 9
[0141] pp32r1 Augments Oncogene-Mediated Transformation of Rat
Embryo Fibroblasts.
[0142] pp32r1 was subcloned into a eukaryotic expression vector
under the CMV promoter and analyzed for its effect on
ras+myc-mediated formation of transformed foci in rat embryo
fibroblasts. Genomic sequences including the entire coding region
for pp32r1 were amplified by PCR and subcloned into the eukaryotic
TA cloning and expression vector pCR3.1 vector (Invitrogen) which
contains a CMV promoter. The assay was performed as described (Chen
et al. Mol Biol Cell, 7:2045-56, 1996) with each T75 flask
receiving 5 micrograms of pEJ-ras, and/or 10 micrograms of
pMLV-c-myc, pCMV32, pp32r1 in PCR3.1, or PCR 3.1 alone. After 14
days, transformed colonies were enumerated. FIG. 8 shows the
results. The data represent the average of seven replicates from
two separate experiments in duplicate and one in triplicate. The
error bars indicate standard error of the mean. In contrast to
pp32, which consistently suppresses focus formation induced by
ras+myc and other oncogene pairs, pp32r1 caused a statistically
significant stimulation of focus formation with p=0.004 by an
unpaired t-test.
Example 10
[0143] Effect of Transcripts from Various Cell Lines on Rat
Fibroblast Transformation Assays
[0144] Expression constructs prepared as described above from PC-3
and DU-145 cells were tested in the rat embryo fibroblast
transformation assay described by Chen, et al., Mol Biol Cell.,
7:2045-56, 1996, incorporated herein by reference. The results are
shown in FIG. 9. Transcripts from the two cell lines stimulated
ras+myc induction of transformed rat embryo fibroblast foci, in
contrast to normal pp32, which suppressed transformation. The
figure shows the mean.+-.the standard deviation, except for DU-145,
which represents a single determination.
Example 11
[0145] Transformation Activity of Various Isolates from Patient
Tumors
[0146] The variant transcripts isolated from prostate cancer
patients differ significantly from pp32 in sequence. The isolated
transcripts were found to stimulate transformation. Transformation
assay. Rat embryo fibroblasts were transfected with the indicated
constructs as described (Chen, et al.) and transformed foci
enumerated. For each experiment, approximately 1.times.10.sup.6
cells were plated per T75 flask and incubated for 2 to 3 d prior to
transfection to achieve approximately 40% confluency. For each
flask of primary rat embryo fibroblasts, the plasmids indicated in
each experiment were added in the following amounts: pEJ-ras, 5
.mu.g; and pMLV-c-myc, pCMV32, pCMVneo, or variant pp32 constructs
in pCR3.1 (Invitrogen), 10 .mu.g. Plasmids were prepared in two
volumes Lipofectin (2 .mu.l lipofectin per .mu.g DNA) then gently
mixed by inversion in 1.5 ml OPTIMEM in sterile 15 ml polystyrene
tubes and allowed to incubate at room temperature for >15 min.
For experiments with more than one flask, mixtures of all reagents
were increased in proportion to the numbers of flasks required for
each transfection. Cells were washed once with OPTIMEM (Gibco-BRL),
and then fed with 6 ml of OPTIMEM and 1.5 ml of the DNA/Lipofectin
mix. After overnight incubation, the cells were grown in standard
media and refed with fresh media twice weekly. Foci were counted
fourteen days post-transfection. FIG. 10 summarizes four separate
experiments. Each data point represents the results from an
individual flask expressed as the percent foci obtained in the
contemporaneous control of ras+myc+vector.
[0147] FIG. 10 shows that expressed variant transcripts from
prostate cancer cell lines and from human prostatic adenocarcinoma
generally produce increased numbers of transformed foci when
co-transfected with ras and myc as compared to the number of foci
obtained when ras and myc are transfected with blank vector.
Variant pp32 transcripts from DU-145 (D3), and from three prostate
cancers (FT 1.7, FT 2.2 and FT3.18) yield increased numbers of
transformed foci over those produced by ras and myc alone with
blank vector. This stands in marked contrast to normal pp32, which
consistently suppresses transformation. These activities are also
summarized in Table 1.
Example 12
[0148] Effect of pp32 Variants on Tumorigenesis in Vivo
[0149] Experiments testing the effect of transfection of NIH3T3
cells on tumorigenesis in vivo are consistent with in vitro results
in rat embryo fibroblasts. NIH3T3 cells were stably transfected by
lipofection with the pp32 species indicated in Table 6A carried in
the pCR3.1-Uni CMV-driven mammalian expression vector (Invitrogen).
The G418-resistant clones employed in these experiments were all
shown by genomic PCR to carry the indicated pp32 species. For
analysis of tumorigenesis, 5.times.10.sup.6 cells in 100
microliters of unsupplemented Dulbecco's modified Eagle's medium
without phenol red were injected into the flanks of female athymic
nude mice on an outbred background of greater than six weeks in age
(Harlan). For logistical reasons, inoculations of the various
groups were staggered over a seven day period. Each group of mice
was euthanized precisely seven weeks after inoculation. Where a
mouse had a tumor, the tumor was dissected, measured, and weighed,
and Table 6A reports the average weight of tumors in mice injected
with cells carrying various vectors. One tumor from each group was
examined histologically. All tumors were fibrosarcomas without
noteworthy inflammation present. Data obtained with NIH3T3 cells
indicate that NIH3T3 cells stably transfected with the variant pp32
species P3, P8, FT1.7, FT2.2, and FT2.4 form tumors when inoculated
into nude mice. In contrast, NIH3T3 cells stably transfected to
express human pp32 fail to form tumors in vivo even when further
transfected with ras. Lines of NIH3T3 cells were also established
that were stably transfected with expression constructs encoding
pp32 or pp32-antisense. Basal expression of pp32 is essential for
maintenance of contact inhibition and serum-dependent cell growth:
antisense ablation of endogenous pp32 synthesis permitted cells to
grow normally following serum withdrawal. Constitutive
over-expression of pp32 potently suppressed ras-mediated
transformation of NIH3T3 cells in vitro and tumorigenesis in vivo.
In contrast, antisense ablation of endogenous pp32 dramatically
increased the number and size of ras-transformed foci; in vivo,
tumors obtained from ras-transformed antisense pp32 cells were
approximately 50-fold greater in mass than tumors obtained from
ras-transformed control cells.
[0150] For purposes of clarity of understanding, the foregoing
invention has been described in some detail by way of illustration
and example in conjunction with specific embodiments, although
other aspects, advantages and modifications will be apparent to
those skilled in the art to which the invention pertains. The
foregoing description and examples are intended to illustrate, but
not limit the scope of the invention. Modifications of the
above-described modes for carrying out the invention that are
apparent to persons of skill in medicine, immunology, hybridoma
technology, pharmacology, pathology, and/or related fields are
intended to be within the scope of the invention, which is limited
only by the appended claims.
[0151] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporate reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
3TABLE 1 Consensus Position Strand Sequence Factor 4 C TTTCCT PEA3
21 N CAAGGTCA ELP 23 N AGGTCA PPAR 32 C CCCTAA TBF1 41 N CTTGGC
NF-1 (-like proteins) 81 N TAAACAC Pit-1 82 N AAACACA HiNF-A 113 C
CTTCCC c-Ets-2 118 N CTATCA GATA-1 122 N CAGTTG c-Myc 212 C
AATAAATA TFIID 213 N ATAAATA ETF 247 N TATCTA NIT2 261 C AAGGAA
c-Ets-2 262 N AGGAAA PEA3 283 C TTTTTCTTTTTC Hb 320 C TTATAT GAL4
333 N TAAAAAA TBP 349 N TTATACATT TBP 363 C AAGGAA c-Ets-2 394 C
TTTCTATA TBP 398 N TATAAA TBP 398 N TATAAA TFIID 411 C CTGAATT
Pit-1 420 N TGTCCC GR 423 C CCCTAA TBF1 434 N TTCCTT c-Ets-2 447 C
CTTCCC c-Ets-2 514 N TTATCTCT GATA-1 514 C TTATCT GATA-2 515 N
TATCTC NIT2 537 N TATGCA EFII 553 N AAGTCA GCN4 608 N TGACTA GCN4
628 N CCTCCCAAC LyF-1 640 N TGTCCT GR 648 N TTAAAATTCA 1-Oct 648 N
TTAAAATTCA 4-Oct 649 N TAAAAT F2F 649 N TAAAAT Pit-1 661 N TAAAAAA
TBP 673 N CTTGGC NF-1 (-like proteins) 725 N AGGCGG Sp1 729 N
GGGCGG ETF 729 N GGGCGG Sp1 729 C GGGCGG Sp1 741 N AGGTCA PPAR 793
N TATAAATA B factor 793 N TATAAA TBP 793 N TATAAATA TFIID 793 N
TATAAAT TMF 794 N ATAAATA ETF 809 N TTATCT GATA-1 809 C TTATCT
GATA-2 815 N GGGTGTGG TEF-2 826 C CACATG muEBP-C2 826 C CACATG
TFE3-S 826 N CACATG USF 978 N ATGTAAAACA 1-Oct 978 N ATGTAAAACA
2-Oct 978 N ATGTAAAACA NF-IL-2A 1000 N ATGTCAGA CSBP-1 1006 N
GATTTC H4TF-1 1034 C TTTTCAT Pit-1 1047 N AAGATAAAACC RVF 1048 C
AGATAA GATA-1 1048 N AGATAA GATA-2 1049 N GATAAA TFIID 1083 C
GCCAAG NF-1 (-like proteins) 1124 N CGCCAT UCRF-L 1163 C GACCTG
TGT3 1307 N CAGTCA GCN4 1347 C TGCATA EFII 1373 C AGAACA AR 1373 N
AGAACAT GR 1373 N AGAACA GR 1373 C AGAACA GR 1373 N AGAACA PR 1373
C AGAACA PR 1373 N AGAACA PR A 1373 C AGAACA PR A 1393 C TCACTT
IRF-1 1393 C TCACTT IRF-2 1395 C ACTTCCT E1A-F 1423 N TTATCT GATA-1
1423 C TTATCT GATA-2 1424 N TATCTA NIT2 1452 N TTACTC GCN4 1471 N
TGGGTCA c-Fos 1471 N TGGGTCA c-Jun 1471 N TGGGTCA ER 1496 N TCTCTTA
c-Myc 1511 N TATAAA TBP 1511 N TATAAA TFIID 1549 C TTTGAA TFIID
1568 C AATGTATAA TBP 1581 C TTTGAA TFIID 1590 C AGATAA GATA-1 1590
N AGATAA GATA-2 1591 C GATAATTG Dfd 1657 C AGGACA GR 1670 C ATTTTA
F2F 1670 C ATTTTA Pit-1 1671 C TTTTATA B factor 1671 C TTTTATA Dr1
1671 C TTTTATA En 1671 C TTTTATA TBP 1671 C TTTTATA TBP-1 1671 C
TTTTATA TFIIA 1671 C TTTTATA TFIIB 1671 C TTTTATA TFIID 1671 C
TTTTATA TFIIE 1671 C TTTTATA TFIIF 1671 C TTTTATA TRF 1672 C TTTATA
TBP 1694 C AATAAATA TFIID 1695 N ATAAATA ETF 1733 N AGGAAA PEA3
1749 C TTATAT GAL4 1783 N TAACTCA AP-1 1829 C TAGATA NIT2 1857 N
CGCCAT UCRF-L 1875 N TTCTGGGAA IL-6 RE-BP 1895 N TGACTA GCN4 1899 N
TATTTAA TBP 1942 N ATATAA GAL4 1985 C TTTATA TBP 1985 C TTTATA
TFIID 2010 C AATAAATA TFIID 2011 N ATAAATA ETF 2058 C TGCATA EFII
2095 N CAGTCA GCN4 2146 C AAGGAA c-Ets-2 2147 N AGGAAA PEA3 2190 N
AGGAAA PEA3 2220 C GGCACA GR 2252 N CCAATAG gammaCAAT 2286 N TGTGCC
GR 2292 N ATGGGA PTF1-beta 2314 N TATGCA EFII 2328 C GGCACA GR 2350
C ATGATAAG GATA-1 2351 N TGATAAG GATA-1 2363 N GGGAAG c-Ets-2 2367
N AGCCACT CP2 2369 C CCACTGGGGA AP-2 2404 N TAAAAT F2F 2404 N
TAAAAT F2F 2404 N TAAAAT Pit-1 2409 N TTGTCATA 77 + 82K protein
2409 N TTGTCATA VETF 2415 N TATCTA NIT2 2451 C TTTATC TFIID 2452 N
TTATCT GATA-1 2452 C TTATCT GATA-2 2486 N CTCTCTCTCTCTC GAGA factor
2644 N AGGCGG Sp1 2658 N ACAGCTG GT-IIBalpha 2658 N ACAGCTG
GT-IIBbeta 2709 C GGCCAGGC AP-2 2723 N TGAACT GR 2731 C TGACCT PPAR
2731 C TGACCTCA URTF 2753 N CTTGGC NF-1 (-like proteins) 2818 C
TGATGTCA AP-1 2818 C TGATGTCA c-Fos 2818 C TGATGTCA c-Jun 2818 C
TGATGTCA CREB 2845 N GGGAAG c-Ets-2 2858 N AGATAG GATA-1 2858 C
AGATAG GATA-1 2864 C AGTTCA GR 2899 N ATATAA GAL4 2900 N TATAAAA B
factor 2900 N TATAAAA Dr1 2900 N TATAAAA En 2900 N TATAAAA TBP 2900
N TATAAA TBP 2900 N TATAAAA TBP-1 2900 N TATAAAA TFIIA 2900 N
TATAAAA TFIIB 2900 N TATAAAA TFIID 2900 N TATAAAA TFIIE 2900 N
TATAAAA TFIIF 2900 N TATAAAA TRF 2921 C TTTGAA TFIID 2924 C GAAATC
H4TF-1 2930 C CATTAG Is1-1 2948 C TGTACA GR 2948 C TGTACA PR 2948 C
TGTACA PR A 2964 C ATTTGAGAA VITF 3030 N AGTGTTCT GR 3032 N TGTTCT
AR 3032 N TGTTCT GR 3032 C TGTTCT GR 3032 N TGTTCT PR 3032 C TGTTCT
PR 3032 N TGTTCT PR A 3032 C TGTTCT PR A 3104 C GGATTATT T11 3106 C
ATTATTAA AFP1 3111 N TAAAAT F2F 3111 N TAAAAT Pit-1 3125 C ATTTTA
F2F 3125 C ATTTTA Pit-1 3142 N TGTGAT GR 3169 N GTTTTATT HOXD10
3169 N GTTTTATT HOXD8 3169 N GTTTTATT HOXD9 3175 C TTTGAA TFIID
3185 N TTGCTCA Zta 3206 N GATTTC H4TF-1 3212 N AGGAAA PEA3 3238 C
ATTTTA F2F 3238 C ATTTTA Pit-1 3256 C TTTGAA TFIID 3266 N TTGCTCA
Zta 3320 C ATTTTA F2F 3320 C ATTTTA Pit-1 3358 N ATGGGA PTF1-beta
3360 C GGGACA GR 3440 C CACTCA GCN4 3460 C TTTCCT PEA3 3483 N
GACACA GR 3491 C TTTCCT PEA3 3495 N CTAATG Is1-l 3523 C AGAACA AR
3523 N AGAACA GR 3523 C AGAACACT GR 3523 C AGAACA GR 3523 N AGAACA
PR 3523 C AGAACA PR 3523 N AGAACA PR A 3523 C AGAACA PR A 3538 C
TTTATC TFIID 3539 N TTATCT GATA-1 3539 C TTATCT GATA-2 3551 N
TGAGTG GCN4 3569 C TCCCAT PTF1-beta 3594 N TTAGGG TBF1 3653 C
CCTGCTGAA LyF-1 3668 N CTCATGA 1-Oct 3668 N CTCATGA 2-Oct 3668 N
CTCATGA Oct-2B 3668 N CTCATGA Oct-2B 3668 N CTCATGA Oct-2C 3679 C
TGTGTAA Zta 3685 C AGAACT GR 3712 C TTTCCT PEA3 3713 N TTCCTT
c-Ets-2 3717 N TTGCTCA Zta 3727 C AAAACATAAAT ssARS-T 3749 N
TAAAAAA TBP 3784 C CACTCA GCN4 3791 C ATTTTA F2F 3791 C ATTTTA
Pit-1 3815 N TATCTA NIT2 3829 C TAGATA NIT2 3859 C AGAACA AR 3859 N
AGAACAG GR 3859 N AGAACA GR 3859 C AGAACA GR 3859 N AGAACA PR 3859
C AGAACA PR 3859 N AGAACA PR A 3859 C AGAACA PR A 3860 N GAACAG LVa
3877 C ATCACA GR 3886 N TGAGTCA AP-1 3886 C TGAGTCA AP-1 3886 C
TGAGTCA c-Fos 3886 C TGAGTCA c-Jun 3886 C TGAGTCA FraI 3886 C
TGAGTCA NF-E2 3887 C GAGTCA GCN4 3931 N AGATAG GATA-1 3931 C AGATAG
GATA-1 3960 N TTGGCA NF-1/L 3965 C ATTTTA F2F 3965 C ATTTTA Pit-1
4026 N TATTTAA TBP 4037 N TGTGAT GR 4040 N GATGCAT Pit-1 4Q42 C
TGCATA EFII 4079 N TTCAAAG SRY 4079 N TTCAAAG TCF-1A 4079 N TTCAAA
TFIID 4097 N CAGGTC TGT3 4140 N TGATTCA AP-1 4140 C TGATTCA AP-1
4140 N TGATTC GCN4 4164 N GGGAGTG p300 4205 C AGATAA GATA-1 4205 N
AGATAA GATA-2 4219 C TTAGTCAC AP-1 4219 C TTAGTCA AP-1 4219 C
TTAGTCAC c-Fos 4219 C TTAGTCAC c-Jun 4219 C TTAGTCA c-Jun 4219 C
TTAGTCA Jun-D 4220 C TAGTCA GCN4 4271 N TGTTCT AR 4271 N TGTTCT GR
4271 C TGTTCT GR 4271 N TGTTCT PR 4271 C TGTTCT PR 4271 N TGTTCT PR
A 4271 C TGTTCT PR A 4280 C TGACCCA c-Fos 4280 C TGACCCA c-Jun 4280
C TGACCCA ER 4292 C CTTATCAG GATA-1 4292 C CTTATCA GATA-1 4361 N
TTCAAAG SRY 4361 N TTCAAAG TCF-1A 4361 N TTCAAA TFIID
[0152]
4TABLE 2 COMPARISON OF ALL PROTEIN SEQUENCES 1 15 16 30 31 45 46 60
61 75 TSU6 MEMGRRIHLELRNGT PSDVKELVLDNSRSN EGKLEGLTDEFEELE
FLSTINVGLTSIANL PKLNKLKKLELSSNR D3 MEMGRRIHLELRNRT PSDVKELVLDNSRSN
EGKLEGLTDEFEELE FLSTINVGLTSIANL PKLNKLKKLELSDNR PG MEMGKWIHLELRNRT
PSDVKELFLDNSQSN EGKLEGLADEFEELE LLNTINIGLSSIANL AKLNKLKKLELSSNR
FT1.11 MEMGKWIHLELRNRT PSDVKELFLDNSQSN EGKLEGLTDEFEELE
LLNTINIGLTSIANL PKLNKLKKLELSSNR TSU1 MEMGKWIHLELRNRT
PSDVKELFLDNSQSN EGKLEGLTDEFEELE LLNTINIGLTSIANL PKLNKLKKLELSSNR
FT3.18 MEMGKWIHLELRNRT PSDVKELFLDNSQSN EGKLEGLTDEFEELE
LLNTINIGLTSIANL PKLNKLKKLELSSNR FT2.4 MEMGKWIHLELRNRT
PSDVKELFLDNSQSN EGKLEGLTDEFEELE LLNTINIGLTSIANL PKLNKLKKLELSSNR
FT2.2 MEMGRRIHSELRNRA PSDVKELVLDNSRSN EGKLEALTDEFEELE
FLSKINGGLTSISDL PKL-KLRKLEL---K KG MEMGRRIHSELRNRA PSDVKELALDNSRSN
EGKLEALTDEFEELE FLSKINGGLTSISDL PKL-KLRKLEL---R FT1.7
MEMGRRIHLELRNRT PSDVKELVLDNSRSN EGKLEGLTDEFEELE FLSTINVGLTSIANL
PKL-KLRKLEL---R P3 MEMGKWIHLELRNRT PSDVKELFLDNSQSN EGKLEGLTDEFEELE
LLNTINIGLTSIANL PKLNKLKKLELSSNR L3 MEMGRRIHLELRNRT PSDVKELVLDNSRSN
EGKLEGLTDEFEELE FLSTINVGLTSIANL PKLNKLKKLELSDNR pp32
MEMGRRIHLELRNRT PSDVKELVLDNSRSN EGKLEGLTDEFEELE FLSTINVGLTSIANL
PKLNKLKKLELSDNR P8 MEMGRRIHLELRNRT PSDVKELVLDNSRSN EGKLEGLTDEFEELE
FLSTINVGLTSIANL PKLNKLKKLELSSNR 76 90 91 105 106 120 121 135 136
150 TSU6 ASVGLEVLAEKCPNL 90 IHLNLSGNKIKDLST IEPLKKLENLESLDL
FTCEVTNLNNY---- --------------- D3 VSGGLEVLAEKCPNL 90
IHLNLSGNKIKDLST IEPLKKLENLESLDL FTCEVTNLNNY---- --------------- PG
ASVGLEVLAEKCPNL 90 IHLNLSGNKIKDLST IEPLKKLENLESLDL FTCEVTNLNNY----
--------------- FT1.11 ASVGLEVLAEKCPNL 90 IHLNLSGNKIKDLST
IEPLKKLENLESLDL FTCEVTNLNNY---- --------------- TSU1
ASVGLEVLAEKCPNL 90 IHLNLSGNKIKDLST IEPLKKLENLESLDL FTCEVTNLNNY----
--------------- FT3.18 ASVGLEVLAEKCPNL 90 IHLNLSGNKIKDLST
IEPLKKLENLESLDL FTCEVTNLNNY---- --------------- FT2.4
ASVGLEVLAEKCPNL 90 IHLNLSGNKIKDLST IEPLKKLENLESLDL FTCEVTNLNNY----
--------------- FT2.2 VSGGLEVLAEKCPNL 86 THLYLSGNKIKDLST
IEPLKQLENLKSLDL FNCEVTNLNDYGENV FKLLLQLTYLDSCYW KG VSGGLEVLAEKCPNL
86 THLYLSGNKIKDLST IEPLKQLENLKSLDL FNCEVTNLNDYGENV FKLLLQLTYLDSCYW
FT1.7 VSGGLEVLAEKCPNL 86 THLYLSGNKIKDLST IEPLKQLENLKSLDL
FNCEVTNLNDYGENV FKLLLQLTYLDSCYW P3 ASVGLEVLAEKCPNL 90
IHLNLSGNKIKDLST IEPLKKLENLESLDL FTCEVTNLNNYRENV FKLLPQLTYLDGYDR L3
VSGGLEVLAEKCPNL 90 THLNLSGNKIKDLST IEPLKKLENLESLDL FNCEVTNLNDYRENV
FKLLPQLTYLDGYDR pp32 VSGGLEVLAEKCPNL 90 THLNLSGNKIKDLST
IEPLKKLENLESLDL FNCEVTNLNDYRENV FKLLPQLTYLDGYDR P8 ASVGLEVLAEKCPNL
90 IHLNLSGNKIKDLST IEPLKKLENLESLDL SNCEVTNLNDYRENV FKLLPQLTYLDGYDR
151 165 166 180 181 195 196 210 TSU6 ---------------
--------------- 131 --------------- --------------- D3
--------------- --------------- 131 --------------- ---------------
PG --------------- --------------- 131 ---------------
--------------- FT1.11 --------------- --------------- 131
--------------- --------------- TSU1 ---------------
--------------- 131 --------------- --------------- FT3.18
--------------- --------------- 131 --------------- ---------------
FT2.4 --------------- --------------- 131 ---------------
--------------- FT2.2 DHKEAPYSDIEDHVE GLDDEEEGEHEEEYD 176
EDAQVVEDEEGEEEE EEGEEEDVSGGDEED KG DHKEAPYSDIEDHVE GLDDEEEGEHEEEYD
176 EDAQVVEDEEGEEEE EEGEEEDVSGGDEED FT1.7 DHKEAPYSDIEDHVE
GLDDEEEGEHEEEYD 176 EDAQVVEDEEGEEGE EEGEEEDVSGGDEED P3
DDKEAPDSDAEGYVE GLDDEEEDEDEEEYD 180 EDAQVVEDEEDEDEE EEGEEEDVSGEEEED
L3 DDKEAPDSDAEGYVE GLDDEEEDEDEEEYD 180 EDAQVVEDEEDEDEE
EEGEEEDVSGEEEED pp32 DDKEAPDSDAEGYVE GLDDEEEDEDEEEYD 180
EDAQVVEDEEDEDEE EEGEEEDVSGEEEED P8 DDKEAPDSDAEGYVE GLDDEEEDEDEEEYD
180 EDAQVVEDEEDEDEE EEGEEEDVSGEEEED 211 225 226 240 241 TSU6
--------------- --------------- --------- 131 D3 ---------------
--------------- --------- 131 PG --------------- ---------------
--------- 131 FT1.11 --------------- --------------- --------- 131
TSU1 --------------- --------------- --------- 131 FT3.18
--------------- --------------- --------- 131 FT2.4 ---------------
--------------- --------- 131 FT2.2 EEGYNDGEVDGEEDE EELGEEERGQKRK--
--------- 234 KG EEGYNDGEVDGEEDE EELGEEERGQKRK-- --------- 234
FT1.7 EEGYNDGEVDDEEDE EELGEEERGQKRKRE PEDEGEDDD 245 P3
EEGYNDGEVDDEEDE EELGEEERGQKRKRE PEDEGEDDD 249 L3 EEGYNDGEVDDEEDE
EELGEEERGQKRKRE PEDEGEDDD 249 pp32 EEGYNDGEVDDEEDE EELGEEERGQKRKRE
PEDEGEDDD 249 P8 EEGYNDGEVDDEEDE EELGEEERGQKRKRE PEDEGEDDD 249 TSU6
and TSU1 from TSU cell line; D3 from DU-145 cell line; P3 and P8
from PC-3 cell line; FT1, FT2 and FT3 from patient carcinoma; LE
from LNCAP; KQ from placenta
[0153]
5 TABLE 3 Comparison to pp32 Sequences % Identity % Similarity
CLONE cDNA Protein Protein D3, DU-145 cells 95 90 95 P3, PC-3 86 94
96 P8, PC-3 98 97 97 FT1.11 97 86 92 FT1.7 95 95 95 FT2.2 94 85 88
FT2.4 99 86 92 FT3.18 99 90 94
[0154]
6TABLE 1A Effect on Oncogene- Sequence Nucleotide Protein Identity
Mediated Sequence Group Identity with pp32 Gaps with pp32
Transformation Comment FT1.3 A 99.8 100 Not Tested Identical to
pp32 D1 A 99.9 100 Not tested identical to pp32 with 2 silent nt
changes L3 A 99.9 100 Not Tested D3 U 95.8 0 96.9 Generally Encodes
truncated variant Stimulatory pp32 D5 U 99.6 0 99.6 Not Tested
FT1.2 U 92.9 1 Not tested No ORF P3 U 96.5 1 94.4 Slightly
Stimulatory P8 U 98.7 0 98.0 Variable FT1.11 B 92.4 2 89.3 Not
Tested All sequences identical, appears to be product of pp32r2
FT2.4 B 92.4 2 89.3 Variable T1 B 92.4 2 89.3 T6 U 94.2 1 93.9 Not
Tested Encodes truncated variant pp32 FT3.18 U 94.7 2 89.3
Stimulatory Encodes truncated variant pp32 FT2.2 C 94.4 3 87.6
Stimulatory Sequences differ by 1 nt appears to be product of
pp32r1 FT3.3 C 94.4 3 87.6 not tested FT1.7 U 95.9 2 91.4
Stimulatory
[0155]
7TABLE 2A Genbank Protein Accession Length Human pp32 Human pp32r1
Human pp32r2 Human April Murine pp32 Human pp32 HSU73477 249 100%
88% Identity 84% Identity 0 71% Identity 89% Identity 2 gaps; Z =
77 gaps; Z = 73 3 gaps; Z = 58 1 gap; Z = 87 Human pp32r1 AF008216
234 100% Identity 785 Identity 2 61% Identity 90% identity 3 gaps;
Z = 65 5 gaps; Z = 15 gaps; Z = 64 Human pp32r2 HSU71084 131 100%
Identity 61% Identity 77% Identity 3 gaps; Z = 52 1 gap; Z = 80
Human April Y07969 249 100% 71% Identity 4 gaps; Z = 68 Murine pp32
U734778 247 100% Identity Percent amino acid identity of pp32 and
related proteins. Sequences were aligned using the GAP program (7).
The number of gaps in the alignment is indicated as well as the Z
score, a statistical measure of protein relatedness derived from 50
comparisons of randomized protein sequences
[0156]
8TABLE 3A pp32 Homologs human pp32 (Genbank Locus HSU73477) murine
pp32 (Genbank Locus MMU73478) human cerebellar leucine rich acidic
nuclear protein (LANP) (Genbank Locus AF025684) murine LANP
(Genbank Locus AF022957) murine RFC1 (Genbank Locus MUSMRFC,
Accession NO. L23755) HPP2a or human potent heat-stable protein
phospatase 2a inhibitor (Genbank Locus HSU60823) SSP29 (Genbank
Locus HSU70439) HLA-DR associated protein I (Genbank Locus
HSPPHAPI, Accession No. X75090) PHAPI2a (EMBL Locus HSPHAPI2A,
Genbank Accession No. Y07569) PHAPI2b (EMBL Locus HSPHAPI2B,
Genbank Accession No. Y07570) April (EMBL Locus HSAPRIL)
[0157]
9TABLE 6A Tumorigenicity in Nude Mice of NIH3T3 Cells Transfected
with pp32 and pp32 Variants pp32 Species Clone Tumors/ Average
Tumor Weight FT1.7 1 3/3 14.9 .+-. 2.1 2.sup.1 3/3 13.3 .+-. 3.7
FT2.2 1 3/3 10.5 .+-. 2.8 2 3/3 3.8 .+-. 2.1 FT2.4 1 3/3.sup.6 1.3
.+-. 0.9 2 3/3 13.8 .+-. 3.3 D3 5.sup.2 0/3 6.sup.2 0/3 P3 11 3/3
5.7 .+-. 0.5 14.sup.3 3/3 2.1 .+-. 1.2 P8 1.sup.4 3/3 6.4 .+-. 5.3
2 3/3 11.3 .+-. 3.9 4.sup.5 3/3 10.1 .+-. 4.8 L3 (pp32) 5.sup.5 0/3
6.sup.4 0/3 Vector Control 2.sup.3 0/3 3.sup.1 0/3 .sup.1FT1.7,
clone 2 and Vector Control, clone 3 were tested on contralateral
sides of a single group of animals. .sup.2D3 clone 5 was tested on
the contralateral sides of a group of animals simultaneously
injected with NIH3T3 cells transfected with a clone of pp32r1 (data
not shown). D3 clone 6 was tested on the contralateral sides of a
group of animals simultaneously injected with a second clone of
NIH3T3 cells transfected with pp32r1 (data not show). .sup.3P3,
clone 14 and Vector Control, clone 2 were tested on contralateral
sides of a single group of animals. .sup.4P8, clone 1 and pp32,
clone 6 were tested on contralateral sides of a single group of
animals. .sup.5P8, clone 4 and pp32, clone 5 were tested on
contralateral sides of a single group of animals. .sup.6One tumor
in this group, weighing 0.5 gm, was detected only upon post mortem
dissection.
[0158]
Sequence CWU 1
1
51 1 5785 DNA Homo sapiens CDS (4453)..(5154) 1 aagctttcct
gatctctaaa tcaaggtcag ctccctaagc tcttggctcc cgtactgaaa 60
ctttttctta tgtaactctc ataaacacat agcataatgt tttgcatgtt tttcttccct
120 atcagttgca agttccagca gagctgatat attttcattt cattcgctac
tatagcccta 180 gagcctgaca tagtttctgg ctgtgaatgc tcaataaata
tttgtttaat tgagtagaaa 240 cataaagtat ctatttcatt gaaggaaaga
ataattagct acatttttct ttttcttgcc 300 ttaatatttg aggaatttgc
ttatatgtca taataaaaaa gttaaagcct tatacattat 360 actaaggaat
ttggacatta aattcaagct agcctttcta taaacaaaat actgaatttc 420
tgtccctaaa tttgttcctt ccctattctt ccccattgag atgacaccaa atccctctag
480 ctgctcaaac caagtacccg tatgttattc ttaattatct ctttaccttg
cttctcatat 540 gcaatttgtt aacaagtcat cttcagtctg tatccattat
tctccctttc cagaccacca 600 acatgtcttg actatactgc tacaatagcc
tcccaactct tgtcctactt aaaattcatt 660 gtaaaaaatc agtcttggcc
gggcacggtg gctcacacct ataatcccag cactttggga 720 gtcccaggcg
ggcgggtcac gaggtcaaga gatggagacc atcatggcca acatggtgaa 780
accctgtctc tactataaat acaaaaaaat tatctgggtg tggtggcaca tgcctgtaat
840 cccaactact agggaggctg aggcaggaga atcgcttgaa cctgggaggc
ggaggttgca 900 gtgagccgag atcgcaccat tgcactccag cctggcaaca
gagcgagact ccatcccaaa 960 acaaaacaaa acaaaaccat gtaaaacatg
tctgtaaaac atgtcagatt tcgtgttcag 1020 aagtcttaca tgtcttttca
ttatgctaag ataaaaccca aatgcatttt cttggtttct 1080 aaagccaaga
aaataagagt tgctttcagc aaccttgttt cttccgccat gcttttccct 1140
agctcactct ttttaggcaa gtcgacctga ttttctttct gttagtctgt ttctgcctcg
1200 tggtctggct ttctttctgt tagtctgttt ccacctcgtg gtcttggtcc
tggctcttca 1260 ttctgcctgg aatgctctcc actccagatc cttactagat
cttagctcag tcatcaccct 1320 cgcaggaaga tcttccaacc attcacctgc
atacacctat ggctgctccc tagagaacat 1380 cattctgttt tcttcacttc
ctagcactta ctgctttctg aaattatcta ctttgattgt 1440 ttatttcttt
ctttactctt actaggatac ctgggtcatt aaaggaggga tatttctctc 1500
ttatttactg ttataaactt aatgcttagg ctgtagaagt tatacaatat ttgaagaata
1560 aatcgttaaa tgtataacat ttttgaagaa agataattgt gggatccatt
tagtttgcaa 1620 acatttgatc tgtgtgttag acagaaggcc atggtaaagg
acaaagacat attttatagg 1680 actgtaccct gaaaaataaa taaacttgaa
ccagttatac aagacttatg tgcaggaaac 1740 aggtaccagt tatatttaga
aatggtaaat caccttctaa gcataactca gagcacaata 1800 tattagaggg
tagagagaga agtgcgtctt agatattggt aatcatatta ggactgacgc 1860
catccttgat ttttcttctg ggaaacagct caaaatgact atttaatgtt tacaatgata
1920 tcttgcatct tgccagtaaa taatataata gacactagga atccaaattg
taagatgaac 1980 aagtctttat agagggagag ccaaatacac aataaataac
acaaggtggt aaatgcagta 2040 atacaaacat acataccatg cataggagtg
cagagaaggt gtgcttctcc gaatgcagtc 2100 acccagaaag tccttctgta
gaaagggata tcttaaatgg tgcttaaagg aaaagtaacc 2160 aaaggcaact
aaagattgca aggaggtccc aggaaaaagc aaaagaacca aaggtacata 2220
ggcacaaaag tagcctgcct tcctgggaac ttccaatagt ttgctggagc acacagttag
2280 aagtactgtg ccatgggagc aaagactgaa gacatatgca ggttcaaggg
cacagagccc 2340 catatatgtc atgataagat attgggaagc cactggggag
ctactgaaac tttaagcagg 2400 gaaataaaat tgtcatatct acaccttaga
aatttgattt ttttctcttc ttttatcttc 2460 tcttctcctc tcttctctct
ctctctctct ctctctctct gtgtgtgtgt gtgtgtgtgt 2520 gtgtgtgtgt
gacagagtcc tgctctgtca cccaggctgg agtgtagtgg agtgatctcc 2580
gcttactgca gtctctgcct ctcaagcgat tccctgcctc agcctcccga gtagctggga
2640 ttacaggcgg gctctacaac agctggctaa cttttgtatt ttttggtaac
aaccaggttt 2700 taccatgttg gccaggctgg tcttgaactc ctgacctcag
gtgatctgcc tgccttggct 2760 ttccaaagtg ctgggattac aggcgtgagc
caccctgcct ggtgtagaag tttgattttg 2820 atgtcagtgt ggtagatgaa
tttgtgggaa gcaaaacaag atagagttca atgacagtga 2880 aaagtttatt
gtataagcta tataaaagaa aatgttgaag gtttgaaatc cattagtggc 2940
agtaagggtg tacagaacga aactatttga gaagtacaca aggcaagtct tactttcaag
3000 gcagtttatg taagctcatt caattgtctc agtgttcttg ctatgtgtgg
gttataggat 3060 ttggaacata tgatcaatct gagcacacat cagtaaactg
aataggatta ttaaaatcca 3120 caagcatttt actagtggaa tctgtgatat
tttctagcta ctcttgcttg ttttatttga 3180 atcttttgct catatcctat
agtaaagatt tcaggaaata tatttttatt tgcctagaat 3240 tttagccttt
tagttttttg aatctattgc tcatattctt atagtaagag tttcagggaa 3300
tgtatttcta tttgtctgga attttagcct ttcaggtttt tgagcccctc ttttgcttat
3360 gggacatagt atgagacaag atgaaatgat acttctattc ccaattcact
gatggggaaa 3420 atgaagcaaa aaatgttatt cactcaaggc ttctgccatg
tttcctggtg gaattacggc 3480 tcagacacaa atttcctaat gcctgtgctg
ctaacttctc aatagaacac tatattaatt 3540 tatcttcttc ctgagtgttt
ttccacaaat cccatagcct gtgaaaagat tgttttaggg 3600 aaatattatt
tttaatatag catattttgt caatgtggga cataggacta gtacctgctg 3660
aaaaccatct catgatcctt gtgtaagaac taattcacac tagaaatact attttccttg
3720 ctcattaaaa acataaatgt ctcagaaagt aaaaaattat tcctctctaa
ataaacatac 3780 atgccactca aattttattc ctctaccact tgccgtatct
aaacctagtt agatactttg 3840 gttttaggta taatctgaca gaacagatac
aaccaagatc acattgtgag tcagaagtgg 3900 aaaattcata attcatgatg
ataccaataa aagatagatt tagcttttta caggatgttt 3960 ttggcatttt
attctttcat ttgaggggag atctcaccaa aatatgtctt tcatggttca 4020
ttgtgttatt taatttctgt gatgcatatt ctcaggttac tttaaaccta gtctatagat
4080 tcaaagatat cccgtgtcag gtctctaaaa gtaaaaagaa aaatgggtac
ttgtgaaggc 4140 tgattcacag taagtagtgt agaggggagt gccttgtgta
ttcacaaatt atcaacgtga 4200 gcatcagata agattttctt tagtcacaca
cacctacctt cttactagga agatccatat 4260 acttgaataa ttgttctgct
tgacccaggt tacttatcag tccctttatt ataatatttg 4320 taaatattgg
ggctcgagaa ccgagcggag ctggttgagt cttcaaagtc ctaaaacgtg 4380
cggccgtggg ttcgaggttt attgattgaa ttcggctggc acgagagcct ctgcagacag
4440 agagcgcgag ag atg gag atg ggc aga cgg att cat tca gag ctg cgg
aac 4491 Met Glu Met Gly Arg Arg Ile His Ser Glu Leu Arg Asn 1 5 10
agg gcg ccc tct gat gtg aaa gaa ctt gcc ctg gac aac agt cgg tcg
4539 Arg Ala Pro Ser Asp Val Lys Glu Leu Ala Leu Asp Asn Ser Arg
Ser 15 20 25 aat gaa ggc aaa ctc gaa gcc ctc aca gat gaa ttt gaa
gaa ctg gaa 4587 Asn Glu Gly Lys Leu Glu Ala Leu Thr Asp Glu Phe
Glu Glu Leu Glu 30 35 40 45 ttc tta agt aaa atc aac gga ggc ctc acc
tca atc tca gac tta cca 4635 Phe Leu Ser Lys Ile Asn Gly Gly Leu
Thr Ser Ile Ser Asp Leu Pro 50 55 60 aag tta aag ttg aga aag ctt
gaa cta aga gtc tca ggg ggc ctg gaa 4683 Lys Leu Lys Leu Arg Lys
Leu Glu Leu Arg Val Ser Gly Gly Leu Glu 65 70 75 gta ttg gca gaa
aag tgt cca aac ctc acg cat cta tat tta agt ggc 4731 Val Leu Ala
Glu Lys Cys Pro Asn Leu Thr His Leu Tyr Leu Ser Gly 80 85 90 aac
aaa att aaa gac ctc agc aca ata gag cca ctg aaa cag tta gaa 4779
Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Gln Leu Glu 95
100 105 aac ctc aag agc tta gac ctt ttc aat tgc gag gta acc aac ctg
aac 4827 Asn Leu Lys Ser Leu Asp Leu Phe Asn Cys Glu Val Thr Asn
Leu Asn 110 115 120 125 gac tac gga gaa aac gtg ttc aag ctt ctc ctg
caa ctc aca tat ctc 4875 Asp Tyr Gly Glu Asn Val Phe Lys Leu Leu
Leu Gln Leu Thr Tyr Leu 130 135 140 gac agc tgt tac tgg gac cac aag
gag gcc cct tac tca gat att gag 4923 Asp Ser Cys Tyr Trp Asp His
Lys Glu Ala Pro Tyr Ser Asp Ile Glu 145 150 155 gac cac gtg gag ggc
ctg gat gac gag gag gag ggt gag cat gag gag 4971 Asp His Val Glu
Gly Leu Asp Asp Glu Glu Glu Gly Glu His Glu Glu 160 165 170 gag tat
gat gaa gat gct cag gta gtg gaa gat gag gag ggc gag gag 5019 Glu
Tyr Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu Gly Glu Glu 175 180
185 gag gag gag gaa ggt gaa gag gag gac gtg agt gga ggg gac gag gag
5067 Glu Glu Glu Glu Gly Glu Glu Glu Asp Val Ser Gly Gly Asp Glu
Glu 190 195 200 205 gat gaa gaa ggt tat aac gat gga gag gta gat ggc
gag gaa gat gaa 5115 Asp Glu Glu Gly Tyr Asn Asp Gly Glu Val Asp
Gly Glu Glu Asp Glu 210 215 220 gaa gag ctt ggt gaa gaa gaa agg ggt
cag aag cga aaa tgagaacctg 5164 Glu Glu Leu Gly Glu Glu Glu Arg Gly
Gln Lys Arg Lys 225 230 aagatgaggg agaagatgat gactaagtag aataacctat
tttgaaaaat tcctattgtg 5224 atttgactgt ttttacccat atcccctccc
ccctccaatc ctgccccctg aaacttactt 5284 ttttctgatt gtaacattgc
tgtgggaatg agacgggaaa agtgtactgg gggttgtgga 5344 gggagggagg
gcaggaggcg gtggactaaa atactatttt tactgccaaa taaaataata 5404
tttgtaaata ttaactggga tactagcttt gtagaatgat tactattaat tattctctct
5464 ctctttttat ttttttacac attctattct tttaagtata gtccttttag
tccaaggaaa 5524 aggcactaca atccacttat taatgcttgc tactgtgttc
aagtaaaata agctccagga 5584 tttaacaaaa agaggaaaga aaatatttac
aatgaaaatg ttgctaaaaa tttaaaacaa 5644 attacagtaa atgtattgtt
aaagcaaatt ctatttttaa aatttattaa taaggaaata 5704 atttgctaaa
gcaaattttt ggaaaaataa taatgcactt tatacttgat tttatttatt 5764
aaaacaatga tttataagct t 5785 2 234 PRT Homo sapiens 2 Met Glu Met
Gly Arg Arg Ile His Ser Glu Leu Arg Asn Arg Ala Pro 1 5 10 15 Ser
Asp Val Lys Glu Leu Ala Leu Asp Asn Ser Arg Ser Asn Glu Gly 20 25
30 Lys Leu Glu Ala Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu Ser
35 40 45 Lys Ile Asn Gly Gly Leu Thr Ser Ile Ser Asp Leu Pro Lys
Leu Lys 50 55 60 Leu Arg Lys Leu Glu Leu Arg Val Ser Gly Gly Leu
Glu Val Leu Ala 65 70 75 80 Glu Lys Cys Pro Asn Leu Thr His Leu Tyr
Leu Ser Gly Asn Lys Ile 85 90 95 Lys Asp Leu Ser Thr Ile Glu Pro
Leu Lys Gln Leu Glu Asn Leu Lys 100 105 110 Ser Leu Asp Leu Phe Asn
Cys Glu Val Thr Asn Leu Asn Asp Tyr Gly 115 120 125 Glu Asn Val Phe
Lys Leu Leu Leu Gln Leu Thr Tyr Leu Asp Ser Cys 130 135 140 Tyr Trp
Asp His Lys Glu Ala Pro Tyr Ser Asp Ile Glu Asp His Val 145 150 155
160 Glu Gly Leu Asp Asp Glu Glu Glu Gly Glu His Glu Glu Glu Tyr Asp
165 170 175 Glu Asp Ala Gln Val Val Glu Asp Glu Glu Gly Glu Glu Glu
Glu Glu 180 185 190 Glu Gly Glu Glu Glu Asp Val Ser Gly Gly Asp Glu
Glu Asp Glu Glu 195 200 205 Gly Tyr Asn Asp Gly Glu Val Asp Gly Glu
Glu Asp Glu Glu Glu Leu 210 215 220 Gly Glu Glu Glu Arg Gly Gln Lys
Arg Lys 225 230 3 889 DNA Homo sapiens 3 gggttcgagg tttattgatt
gaattcggct ggcacgagag cctctgcaga cagagagcgc 60 gagagatgga
gatgggcaga cggattcatt cagagctgcg gaacagggcg ccctctgatg 120
tgaaagaact tgccctggac aacagtcggt cgaatgaagg caaactcgaa gccctcacag
180 atgaatttga agaactggaa ttcttaagta aaatcaacgg aggcctcacc
tcaatctcag 240 acttaccaaa gttaaagttg agaaagcttg aactaagagt
ctcagggggc ctggaagtat 300 tggcagaaaa gtgtccaaac ctcacgcatc
tatatttaag tggcaacaaa attaaagacc 360 tcagcacaat agagccactg
aaacagttag aaaacctcaa gagcttagac cttttcaatt 420 gcgaggtaac
caacctgaac gactacggag aaaacgtgtt caagcttctc ctgcaactca 480
catatctcga cagctgttac tgggaccaca aggaggcccc ttactcagat attgaggacc
540 acgtggaggg cctggatgac gaggaggagg gtgagcatga ggaggagtat
gatgaagatg 600 ctcaggtagt ggaagatgag gagggcgagg aggaggagga
ggaaggtgaa gaggaggacg 660 tgagtggagg ggacgaggag gatgaagaag
gttataacga tggagaggta gatggcgagg 720 aagatgaaga agagcttggt
gaagaagaaa ggggtcagaa gcgaaaatga gaacctgaag 780 atgagggaga
agatgatgac taagtagaat aacctatttt gaaaaattcc tattgtgatt 840
tgactgtttt tacccatatc ccctcccccc tccaatcctg ccccctgaa 889 4 907 DNA
Homo sapiens 4 gggttcgggg tttattgatt gaattcggct ggcgcgggag
cctctgcaga gagagagcgc 60 gagagatgga gatgggcaga cggattcatt
tagagctgcg gaacgggacg ccctctgatg 120 tgaaagaact tgtcctggac
aacagtcggt cgaatgaagg caaactcgaa ggcctcacag 180 atgaatttga
agaactggaa ttcttaagta caatcaacgt aggcctcacc tcaatcgcaa 240
acttaccaaa gttaaacaaa cttaagaagc ttgaactaag cagtaacaga gcctcagtgg
300 gcctagaagt attggcagaa aagtgtccaa acctcataca tctaaattta
agtggcaaca 360 aaattaaaga cctcagcaca atagagcccc tgaaaaagtt
agaaaacctc gagagcttag 420 accttttcac ttgcgaggta accaacctga
acaactactg agagaagatg ttcaagctcc 480 tcctgcaact cacatatctc
aacggctgtg acccggatga caaggaggcc cctaactcgg 540 atggtgaggg
ctttgtggag tgcctggatg acaaggagga ggatgaggat gaggaggagt 600
atgatgaaga tgctcaggta atggaagatg aggaggacga ggatgaggag gaggaacgtg
660 aagaggagga cgtgagtgga gacgaggagg agaaggatga aggttataac
aatggagagg 720 tagatgatga ggaagatgaa gaagagcttg gtgaagaaga
aaggggtcag aagcgaaaat 780 aagaaactga agatgaggga gaagacgatg
cctaagtgga ataatctatt ttgaaaaatt 840 ccttttgtga ttttactgtt
tttagccgta ccccctctcc ccccccactc taatcctgcc 900 ccctgaa 907 5 130
PRT Homo sapiens 5 Met Glu Met Gly Arg Arg Ile His Leu Glu Leu Arg
Asn Gly Thr Pro 1 5 10 15 Ser Asp Val Lys Glu Leu Val Leu Asp Asn
Ser Arg Ser Asn Glu Gly 20 25 30 Lys Leu Glu Gly Leu Thr Asp Glu
Phe Glu Glu Leu Glu Phe Leu Ser 35 40 45 Thr Ile Asn Val Gly Leu
Thr Ser Ile Ala Asn Leu Pro Lys Leu Asn 50 55 60 Lys Leu Lys Lys
Leu Glu Leu Ser Ser Asn Arg Ala Ser Val Gly Leu 65 70 75 80 Glu Val
Leu Ala Glu Lys Cys Pro Asn Leu Ile His Leu Asn Leu Ser 85 90 95
Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100
105 110 Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr Cys Glu Val Thr Asn
Leu 115 120 125 Asn Asn 130 6 907 DNA Homo sapiens 6 gggttcgggg
tttattgatt gaattcggct ggcacgagag cctctgcaga cagagagcgc 60
gagagacgga gatgggcaga cggattcatc tagagctgcg gaacagggcg ccctctgatg
120 tgaaagaact tgccctggac aacagtcggt cgaatgaagg caaactcgaa
gccctcacag 180 atgaatttga agaactggaa ttcttaagta aaatcaacgg
aggcctcacc tcaatctcag 240 acttaccaaa gttaaacaag ttgagaaagc
ttgaactaag cagtaacaga gtctcagggg 300 gcctggaagt attggcagaa
aagtgtccaa acctcacgca tctatattta agtggcaaca 360 aaattaaaga
cctcagcaca atagagccac tgaaacagtt agaaaacctc aagagcttag 420
accttttcaa ttgcgaggta accaacctga acgactacgg agaaaacgtg ttcaagcttc
480 tcctgcaact cacatatctc gacagctgtt actgggacca caaggaggcc
ccttactcag 540 atattgaggc ccacgtggag ggcctggatg acgaggagga
gggtgagcat gaggaggagt 600 atgatgaaga tgctcaggta gtggaagatg
aggagggcga ggaggaggag gaggaaggtg 660 aagaggagga cgtgagtgga
ggggacgagg aggatgaaga aggttataac gatggagagg 720 tagatggcga
ggaagatgaa gaagagcttg gtgaagaaga aaggggtcag aagcgaaaat 780
gagaacctga agatgaggga gaagatgatg actaagtaga ataacctatt ttgaaaaatt
840 cctattgtga tttgactgtt tttacccata tcccctctcc cccccccctc
taatcctgcc 900 ccctgaa 907 7 905 DNA Homo sapiens CDS (64)..(453) 7
gggttcgggg tttattggtt gaattccgct ggctcaggag cctctgcaga gaaagcgtga
60 gag atg gag atg ggc aaa tgg att cat tta gag ctg cgg aac agg acg
108 Met Glu Met Gly Lys Trp Ile His Leu Glu Leu Arg Asn Arg Thr 1 5
10 15 ccc tcc gat gtg aaa gaa ctt ttc ctg gac aac agt cag tca aat
gaa 156 Pro Ser Asp Val Lys Glu Leu Phe Leu Asp Asn Ser Gln Ser Asn
Glu 20 25 30 ggc aaa ttg gaa ggc ctc aca gat gaa ttt gaa gaa ctg
gaa tta tta 204 Gly Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu
Glu Leu Leu 35 40 45 aat aca atc aac ata ggc ctc acc tca att gca
aac ttg cca aag tta 252 Asn Thr Ile Asn Ile Gly Leu Thr Ser Ile Ala
Asn Leu Pro Lys Leu 50 55 60 aac aaa ctt aag aag ctt gaa cta agc
agt aac aga gcc tca gtg ggc 300 Asn Lys Leu Lys Lys Leu Glu Leu Ser
Ser Asn Arg Ala Ser Val Gly 65 70 75 cta gaa gta ttg gca gaa aag
tgt cca aac ctc ata cat cta aat tta 348 Leu Glu Val Leu Ala Glu Lys
Cys Pro Asn Leu Ile His Leu Asn Leu 80 85 90 95 agt ggc aac aaa att
aaa gac ctc agc aca ata gag ccc ctg aaa aag 396 Ser Gly Asn Lys Ile
Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys 100 105 110 tta gaa aac
ctc gag agc tta gac ctt ttc act tgc gag gta acc aac 444 Leu Glu Asn
Leu Glu Ser Leu Asp Leu Phe Thr Cys Glu Val Thr Asn 115 120 125 ctg
aac aac tactgagaaa agatgttcaa gctcctcctg caactcacat 493 Leu Asn Asn
130 atctcaacgg ctgtgacccg gatgacaagg aggcccctaa ctcggatggt
gagggctatg 553 tggagtgcct ggatgacaag gaggaggatg aggatgagga
ggagtatgat gaagatgctc 613 aggtaatgga agatgaggag gacgaggatg
aggaggagga acgtgaagag gaggacgtga 673 gtggagacga ggaggagaag
gatgaaggtt ataacaatgg agaggtagat gatgaggaag 733 atgaagaaga
gcttggtgaa gaagaaaggg gtcagaagcg aaaataagaa actgaagatg 793
agggagaaga cgatgcctaa gtggaataat ctattttgaa aaattccttt tgtgatttta
853 ctgtttttag ccgtatcccc tctccccccc cactctaatc ctgccccctg aa 905 8
130 PRT Homo sapiens 8 Met Glu Met Gly Lys Trp Ile His Leu Glu Leu
Arg Asn Arg Thr Pro 1 5 10 15 Ser Asp Val Lys Glu Leu Phe Leu Asp
Asn Ser Gln Ser Asn Glu Gly 20 25 30 Lys Leu Glu Gly Leu Thr Asp
Glu Phe Glu Glu Leu Glu Leu Leu Asn 35 40 45 Thr Ile Asn Ile Gly
Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu Asn 50 55 60 Lys Leu Lys
Lys Leu Glu Leu Ser Ser Asn Arg Ala Ser Val Gly Leu 65 70 75 80 Glu
Val
Leu Ala Glu Lys Cys Pro Asn Leu Ile His Leu Asn Leu Ser 85 90 95
Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100
105 110 Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr Cys Glu Val Thr Asn
Leu 115 120 125 Asn Asn 130 9 907 DNA Homo sapiens CDS (66)..(812)
9 gggttcgggg tttattgatt gaattccgcc ggcgcgggag cctctgcaga gagagagcgc
60 gagag atg gag atg ggc aga cgg att cat tta gag ctg cgg aac agg
acg 110 Met Glu Met Gly Arg Arg Ile His Leu Glu Leu Arg Asn Arg Thr
1 5 10 15 ccc tct gat gtg aaa gaa ctt gtc ctg gac aac agt cgg tcg
aat gaa 158 Pro Ser Asp Val Lys Glu Leu Val Leu Asp Asn Ser Arg Ser
Asn Glu 20 25 30 ggc aaa ctc gaa ggc ctc aca gat gaa ttt gaa gaa
ctg gaa ttc tta 206 Gly Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu
Leu Glu Phe Leu 35 40 45 agt aca atc aac gta ggc ctc acc tca atc
gca aac ttg cca aag tta 254 Ser Thr Ile Asn Val Gly Leu Thr Ser Ile
Ala Asn Leu Pro Lys Leu 50 55 60 aac aaa ctt aag aag ctt gaa cta
agc agt aac aga gcc tca gtg ggc 302 Asn Lys Leu Lys Lys Leu Glu Leu
Ser Ser Asn Arg Ala Ser Val Gly 65 70 75 cta gaa gta ttg gca gaa
aag tgt cca aac ctc ata cat cta aat tta 350 Leu Glu Val Leu Ala Glu
Lys Cys Pro Asn Leu Ile His Leu Asn Leu 80 85 90 95 agt ggc aac aaa
att aaa gac ctc agc aca ata gag cca ctg aaa aag 398 Ser Gly Asn Lys
Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys 100 105 110 tta gaa
aac ctc aag agc tta gac ctt tcc aat tgc gag gta acc aac 446 Leu Glu
Asn Leu Lys Ser Leu Asp Leu Ser Asn Cys Glu Val Thr Asn 115 120 125
ctg aac gac tac cga gaa aat gtg ttc aag ctc ctc ccg caa ctc aca 494
Leu Asn Asp Tyr Arg Glu Asn Val Phe Lys Leu Leu Pro Gln Leu Thr 130
135 140 tat ctc gac ggc tat gac cgg gac gac aag gag gcc cct gac tcg
gat 542 Tyr Leu Asp Gly Tyr Asp Arg Asp Asp Lys Glu Ala Pro Asp Ser
Asp 145 150 155 gct gag ggc tac gtg gag ggc ctg gat gat gag gag gag
gat gag gat 590 Ala Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu
Asp Glu Asp 160 165 170 175 gag gag gag tat gat gaa gat gct cag gta
gta gaa gat gag gag gac 638 Glu Glu Glu Tyr Asp Glu Asp Ala Gln Val
Val Glu Asp Glu Glu Asp 180 185 190 gag gat gag gag gag gaa ggt gaa
gag gag gac gtg agt gga gag gag 686 Glu Asp Glu Glu Glu Glu Gly Glu
Glu Glu Asp Val Ser Gly Glu Glu 195 200 205 gag gag gat gaa gaa ggt
tat aac gat gga gag gta gat gac gag gaa 734 Glu Glu Asp Glu Glu Gly
Tyr Asn Asp Gly Glu Val Asp Asp Glu Glu 210 215 220 gat gaa gaa gag
ctt ggt gaa gaa gaa agg ggt cag aag cga aaa cga 782 Asp Glu Glu Glu
Leu Gly Glu Glu Glu Arg Gly Gln Lys Arg Lys Arg 225 230 235 gaa cct
gaa gat gag gga gaa gat gat gac taagtggaat aacctatttt 832 Glu Pro
Glu Asp Glu Gly Glu Asp Asp Asp 240 245 gaaaaattcc tattgtgatt
tgactgtttt tacccatatc ccctctcccc cccccctcta 892 atcctgcccc ctgaa
907 10 249 PRT Homo sapiens 10 Met Glu Met Gly Arg Arg Ile His Leu
Glu Leu Arg Asn Arg Thr Pro 1 5 10 15 Ser Asp Val Lys Glu Leu Val
Leu Asp Asn Ser Arg Ser Asn Glu Gly 20 25 30 Lys Leu Glu Gly Leu
Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu Ser 35 40 45 Thr Ile Asn
Val Gly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu Asn 50 55 60 Lys
Leu Lys Lys Leu Glu Leu Ser Ser Asn Arg Ala Ser Val Gly Leu 65 70
75 80 Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Ile His Leu Asn Leu
Ser 85 90 95 Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu
Lys Lys Leu 100 105 110 Glu Asn Leu Lys Ser Leu Asp Leu Ser Asn Cys
Glu Val Thr Asn Leu 115 120 125 Asn Asp Tyr Arg Glu Asn Val Phe Lys
Leu Leu Pro Gln Leu Thr Tyr 130 135 140 Leu Asp Gly Tyr Asp Arg Asp
Asp Lys Glu Ala Pro Asp Ser Asp Ala 145 150 155 160 Glu Gly Tyr Val
Glu Gly Leu Asp Asp Glu Glu Glu Asp Glu Asp Glu 165 170 175 Glu Glu
Tyr Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu Asp Glu 180 185 190
Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp Val Ser Gly Glu Glu Glu 195
200 205 Glu Asp Glu Glu Gly Tyr Asn Asp Gly Glu Val Asp Asp Glu Glu
Asp 210 215 220 Glu Glu Glu Leu Gly Glu Glu Glu Arg Gly Gln Lys Arg
Lys Arg Glu 225 230 235 240 Pro Glu Asp Glu Gly Glu Asp Asp Asp 245
11 905 DNA Homo sapiens CDS (64)..(810) 11 gggttcgggg tttattggtt
gaattccgct ggctcaggag cctctgcaga gaaagcgtga 60 gag atg gag atg ggc
aaa tgg att cat tta gag ctg cgg aac agg acg 108 Met Glu Met Gly Lys
Trp Ile His Leu Glu Leu Arg Asn Arg Thr 1 5 10 15 ccc tcc gat gtg
aaa gaa ctt ttc ctg gac aac agt cag tca aat gaa 156 Pro Ser Asp Val
Lys Glu Leu Phe Leu Asp Asn Ser Gln Ser Asn Glu 20 25 30 ggc aaa
ttg gaa ggc ctc aca gat gaa ttt gaa gaa ctg gaa tta tta 204 Gly Lys
Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu Leu Leu 35 40 45
aat aca atc aac ata ggc ctc acc tca att gca aac ttg cca aag tta 252
Asn Thr Ile Asn Ile Gly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu 50
55 60 aac aaa ctt aag aag ctt gaa cta agc agt aac aga gcc tca gtg
ggc 300 Asn Lys Leu Lys Lys Leu Glu Leu Ser Ser Asn Arg Ala Ser Val
Gly 65 70 75 cta gaa gta ttg gca gaa aag tgt cca aac ctc ata cat
cta aat tta 348 Leu Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Ile His
Leu Asn Leu 80 85 90 95 agt ggc aac aaa att aaa gac ctc agc aca ata
gag ccc ctg aaa aag 396 Ser Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile
Glu Pro Leu Lys Lys 100 105 110 tta gaa aac ctc gag agc tta gac ctt
ttc act tgc gag gta acc aac 444 Leu Glu Asn Leu Glu Ser Leu Asp Leu
Phe Thr Cys Glu Val Thr Asn 115 120 125 ctg aac aac tac cga gaa aat
gtg ttc aag ctc ctc ccg caa ctc aca 492 Leu Asn Asn Tyr Arg Glu Asn
Val Phe Lys Leu Leu Pro Gln Leu Thr 130 135 140 tat ctc gac ggc tat
gac cgg gac gac aag gag gcc cct gac tcg gat 540 Tyr Leu Asp Gly Tyr
Asp Arg Asp Asp Lys Glu Ala Pro Asp Ser Asp 145 150 155 gct gag ggc
tac gtg gag ggc ctg gat gat gag gag gag gat gag gat 588 Ala Glu Gly
Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu Asp Glu Asp 160 165 170 175
gag gag gag tat gat gaa gat gct cag gta gtg gaa gac gag gag gac 636
Glu Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu Asp 180
185 190 gag gat gag gag gag gaa ggt gaa gag gag gac gtg agt gga gag
gag 684 Glu Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp Val Ser Gly Glu
Glu 195 200 205 gag gag gat gaa gaa ggt tat aac gat gga gag gta gat
gac gag gaa 732 Glu Glu Asp Glu Glu Gly Tyr Asn Asp Gly Glu Val Asp
Asp Glu Glu 210 215 220 gat gaa gaa gag ctt ggt gaa gaa gaa agg ggt
cag aag cga aaa cga 780 Asp Glu Glu Glu Leu Gly Glu Glu Glu Arg Gly
Gln Lys Arg Lys Arg 225 230 235 gaa cct gaa gat gag gga gaa gat gat
gac taagtggaat aacctatttt 830 Glu Pro Glu Asp Glu Gly Glu Asp Asp
Asp 240 245 gaaaaattcc tattgtgatt tgactgtttt tacccatatc ccctctcccc
cccccctcta 890 atcctgcccc ctgaa 905 12 249 PRT Homo sapiens 12 Met
Glu Met Gly Lys Trp Ile His Leu Glu Leu Arg Asn Arg Thr Pro 1 5 10
15 Ser Asp Val Lys Glu Leu Phe Leu Asp Asn Ser Gln Ser Asn Glu Gly
20 25 30 Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu Leu
Leu Asn 35 40 45 Thr Ile Asn Ile Gly Leu Thr Ser Ile Ala Asn Leu
Pro Lys Leu Asn 50 55 60 Lys Leu Lys Lys Leu Glu Leu Ser Ser Asn
Arg Ala Ser Val Gly Leu 65 70 75 80 Glu Val Leu Ala Glu Lys Cys Pro
Asn Leu Ile His Leu Asn Leu Ser 85 90 95 Gly Asn Lys Ile Lys Asp
Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100 105 110 Glu Asn Leu Glu
Ser Leu Asp Leu Phe Thr Cys Glu Val Thr Asn Leu 115 120 125 Asn Asn
Tyr Arg Glu Asn Val Phe Lys Leu Leu Pro Gln Leu Thr Tyr 130 135 140
Leu Asp Gly Tyr Asp Arg Asp Asp Lys Glu Ala Pro Asp Ser Asp Ala 145
150 155 160 Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu Asp Glu
Asp Glu 165 170 175 Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu Asp
Glu Glu Asp Glu 180 185 190 Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp
Val Ser Gly Glu Glu Glu 195 200 205 Glu Asp Glu Glu Gly Tyr Asn Asp
Gly Glu Val Asp Asp Glu Glu Asp 210 215 220 Glu Glu Glu Leu Gly Glu
Glu Glu Arg Gly Gln Lys Arg Lys Arg Glu 225 230 235 240 Pro Glu Asp
Glu Gly Glu Asp Asp Asp 245 13 907 DNA Homo sapiens CDS (66)..(812)
13 gggttcgggg tttattgatt gaattccgcc ggcgcgggag cctctgcaga
gagagagcgc 60 gagag atg gag atg ggc aga cgg att cat tta gag ctg cgg
aac agg acg 110 Met Glu Met Gly Arg Arg Ile His Leu Glu Leu Arg Asn
Arg Thr 1 5 10 15 ccc tct gat gtg aaa gaa ctt gtc ctg gac aac agt
cgg tcg aat gaa 158 Pro Ser Asp Val Lys Glu Leu Val Leu Asp Asn Ser
Arg Ser Asn Glu 20 25 30 ggc aaa ctc gaa ggc ctc aca gat gaa ttt
gaa gaa ctg gaa ttc tta 206 Gly Lys Leu Glu Gly Leu Thr Asp Glu Phe
Glu Glu Leu Glu Phe Leu 35 40 45 agt aca atc aac gta ggc ctc acc
tca atc gca aac tta cca aag tta 254 Ser Thr Ile Asn Val Gly Leu Thr
Ser Ile Ala Asn Leu Pro Lys Leu 50 55 60 aac aaa ctt aag aag ctt
gaa cta agc gat aac aga gtc tca ggg ggc 302 Asn Lys Leu Lys Lys Leu
Glu Leu Ser Asp Asn Arg Val Ser Gly Gly 65 70 75 ctg gaa gta ttg
gca gaa aag tgt ccg aac ctc acg cat cta aat tta 350 Leu Glu Val Leu
Ala Glu Lys Cys Pro Asn Leu Thr His Leu Asn Leu 80 85 90 95 agt ggc
aac aaa att aaa gac ctc agc aca ata gag cca ctg aaa aag 398 Ser Gly
Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys 100 105 110
tta gaa aac ctc aag agc tta gac ctt ttc aat tgc gag gta acc aac 446
Leu Glu Asn Leu Lys Ser Leu Asp Leu Phe Asn Cys Glu Val Thr Asn 115
120 125 ctg aac gac tac cga gaa aat gtg ttc aag ctc ctc ccg caa ctc
aca 494 Leu Asn Asp Tyr Arg Glu Asn Val Phe Lys Leu Leu Pro Gln Leu
Thr 130 135 140 tat ctc gac ggc tat gac cgg gac gac aag gag gcc cct
gac tcg gat 542 Tyr Leu Asp Gly Tyr Asp Arg Asp Asp Lys Glu Ala Pro
Asp Ser Asp 145 150 155 gct gag ggc tac gtg gag ggc ctg gat gat gag
gag gag gat gag gat 590 Ala Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu
Glu Glu Asp Glu Asp 160 165 170 175 gag gag gag tat gat gaa gat gct
cag gta gtg gaa gac gag gag gac 638 Glu Glu Glu Tyr Asp Glu Asp Ala
Gln Val Val Glu Asp Glu Glu Asp 180 185 190 gag gat gag gag gag gaa
ggt gaa gag gag gac gtg agt gga gag gag 686 Glu Asp Glu Glu Glu Glu
Gly Glu Glu Glu Asp Val Ser Gly Glu Glu 195 200 205 gag gag gat gaa
gaa ggt tat aac gat gga gag gta gat gac gag gaa 734 Glu Glu Asp Glu
Glu Gly Tyr Asn Asp Gly Glu Val Asp Asp Glu Glu 210 215 220 gat gaa
gaa gag ctt ggt gaa gaa gaa agg ggt cag aag cga aaa cga 782 Asp Glu
Glu Glu Leu Gly Glu Glu Glu Arg Gly Gln Lys Arg Lys Arg 225 230 235
gaa cct gaa gat gag gga gaa gat gat gac taagtggaat aacctatttt 832
Glu Pro Glu Asp Glu Gly Glu Asp Asp Asp 240 245 gaaaaattcc
tattgtgatt tgactgtttt tacccatatc ccctctcccc cccccctcta 892
atcctgcccc ctgaa 907 14 249 PRT Homo sapiens 14 Met Glu Met Gly Arg
Arg Ile His Leu Glu Leu Arg Asn Arg Thr Pro 1 5 10 15 Ser Asp Val
Lys Glu Leu Val Leu Asp Asn Ser Arg Ser Asn Glu Gly 20 25 30 Lys
Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu Ser 35 40
45 Thr Ile Asn Val Gly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu Asn
50 55 60 Lys Leu Lys Lys Leu Glu Leu Ser Asp Asn Arg Val Ser Gly
Gly Leu 65 70 75 80 Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Thr His
Leu Asn Leu Ser 85 90 95 Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile
Glu Pro Leu Lys Lys Leu 100 105 110 Glu Asn Leu Lys Ser Leu Asp Leu
Phe Asn Cys Glu Val Thr Asn Leu 115 120 125 Asn Asp Tyr Arg Glu Asn
Val Phe Lys Leu Leu Pro Gln Leu Thr Tyr 130 135 140 Leu Asp Gly Tyr
Asp Arg Asp Asp Lys Glu Ala Pro Asp Ser Asp Ala 145 150 155 160 Glu
Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu Asp Glu Asp Glu 165 170
175 Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu Asp Glu
180 185 190 Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp Val Ser Gly Glu
Glu Glu 195 200 205 Glu Asp Glu Glu Gly Tyr Asn Asp Gly Glu Val Asp
Asp Glu Glu Asp 210 215 220 Glu Glu Glu Leu Gly Glu Glu Glu Arg Gly
Gln Lys Arg Lys Arg Glu 225 230 235 240 Pro Glu Asp Glu Gly Glu Asp
Asp Asp 245 15 895 DNA Homo sapiens CDS (66)..(767) 15 gggttcgggg
tttattgatt gaattcggct ggcacgagag cctctgcaga cagagagcgc 60 gagag atg
gag atg ggc aga cgg att cat tca gag ctg cgg aac agg gcg 110 Met Glu
Met Gly Arg Arg Ile His Ser Glu Leu Arg Asn Arg Ala 1 5 10 15 ccc
tct gat gtg aaa gaa ctt gcc ctg gac aac agt cgg tcg aat gaa 158 Pro
Ser Asp Val Lys Glu Leu Ala Leu Asp Asn Ser Arg Ser Asn Glu 20 25
30 ggc aaa ctc gaa gcc ctc aca gat gaa ttt gaa gaa ctg gaa ttc tta
206 Gly Lys Leu Glu Ala Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu
35 40 45 agt aaa atc aac gga ggc ctc acc tca atc tca gac tta cca
aag tta 254 Ser Lys Ile Asn Gly Gly Leu Thr Ser Ile Ser Asp Leu Pro
Lys Leu 50 55 60 aag ttg aga aag ctt gaa cta aga gtc tca ggg ggc
ctg gaa gta ttg 302 Lys Leu Arg Lys Leu Glu Leu Arg Val Ser Gly Gly
Leu Glu Val Leu 65 70 75 gca gaa aag tgt cca aac ctc acg cat cta
tat tta agt ggc aac aaa 350 Ala Glu Lys Cys Pro Asn Leu Thr His Leu
Tyr Leu Ser Gly Asn Lys 80 85 90 95 att aaa gac ctc agc aca ata gag
cca ctg aaa cag tta gaa aac ctc 398 Ile Lys Asp Leu Ser Thr Ile Glu
Pro Leu Lys Gln Leu Glu Asn Leu 100 105 110 aag agc tta gac ctt ttc
aat tgc gag gta acc aac ctg aac gac tac 446 Lys Ser Leu Asp Leu Phe
Asn Cys Glu Val Thr Asn Leu Asn Asp Tyr 115 120 125 gga gaa aac gtg
ttc aag ctt ctc ctg caa ctc aca tat ctc gac agc 494 Gly Glu Asn Val
Phe Lys Leu Leu Leu Gln Leu Thr Tyr Leu Asp Ser 130 135 140 tgt tac
tgg gac cac aag gag gcc cct tac tca gat att gag gac cac 542 Cys Tyr
Trp Asp His Lys Glu Ala Pro Tyr Ser Asp Ile Glu Asp His 145 150 155
gtg gag ggc ctg gat gac gag gag gag ggt gag cat gag gag gag tat 590
Val Glu Gly Leu Asp Asp Glu Glu Glu Gly Glu His Glu Glu Glu Tyr 160
165 170 175 gat gaa gat gct cag gta gtg gaa gat gag gag ggc gag gag
gag gag 638 Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu Gly Glu Glu
Glu Glu
180 185 190 gag gaa ggt gaa gag gag gac gtg agt gga ggg gac ggg gag
gat gaa 686 Glu Glu Gly Glu Glu Glu Asp Val Ser Gly Gly Asp Gly Glu
Asp Glu 195 200 205 gaa ggt tat aac gat gga gag gta gat ggc gag gaa
gat gaa gaa gag 734 Glu Gly Tyr Asn Asp Gly Glu Val Asp Gly Glu Glu
Asp Glu Glu Glu 210 215 220 ctt ggt gaa gaa gaa agg ggt cag aag cga
aaa tgagaacctg aagatgaggg 787 Leu Gly Glu Glu Glu Arg Gly Gln Lys
Arg Lys 225 230 agaagatgat gactaagtag aataacctat tttgaaaaat
tcctattgtg atttgactgt 847 ttttacccat atcccatctc ccccccccct
ctaatcctgc cccctgaa 895 16 234 PRT Homo sapiens 16 Met Glu Met Gly
Arg Arg Ile His Ser Glu Leu Arg Asn Arg Ala Pro 1 5 10 15 Ser Asp
Val Lys Glu Leu Ala Leu Asp Asn Ser Arg Ser Asn Glu Gly 20 25 30
Lys Leu Glu Ala Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu Ser 35
40 45 Lys Ile Asn Gly Gly Leu Thr Ser Ile Ser Asp Leu Pro Lys Leu
Lys 50 55 60 Leu Arg Lys Leu Glu Leu Arg Val Ser Gly Gly Leu Glu
Val Leu Ala 65 70 75 80 Glu Lys Cys Pro Asn Leu Thr His Leu Tyr Leu
Ser Gly Asn Lys Ile 85 90 95 Lys Asp Leu Ser Thr Ile Glu Pro Leu
Lys Gln Leu Glu Asn Leu Lys 100 105 110 Ser Leu Asp Leu Phe Asn Cys
Glu Val Thr Asn Leu Asn Asp Tyr Gly 115 120 125 Glu Asn Val Phe Lys
Leu Leu Leu Gln Leu Thr Tyr Leu Asp Ser Cys 130 135 140 Tyr Trp Asp
His Lys Glu Ala Pro Tyr Ser Asp Ile Glu Asp His Val 145 150 155 160
Glu Gly Leu Asp Asp Glu Glu Glu Gly Glu His Glu Glu Glu Tyr Asp 165
170 175 Glu Asp Ala Gln Val Val Glu Asp Glu Glu Gly Glu Glu Glu Glu
Glu 180 185 190 Glu Gly Glu Glu Glu Asp Val Ser Gly Gly Asp Gly Glu
Asp Glu Glu 195 200 205 Gly Tyr Asn Asp Gly Glu Val Asp Gly Glu Glu
Asp Glu Glu Glu Leu 210 215 220 Gly Glu Glu Glu Arg Gly Gln Lys Arg
Lys 225 230 17 905 DNA Homo sapiens CDS (64)..(453) 17 gggttcgggg
tttattggtt gaattccgct ggctcgagag cctctggaga gaaagcgtga 60 gag atg
gag atg ggc aaa tgg att cat tta gag ctg cgg aac agg acg 108 Met Glu
Met Gly Lys Trp Ile His Leu Glu Leu Arg Asn Arg Thr 1 5 10 15 ccc
tcc gat gtg aaa gaa ctt ttc ctg gac aac agt cag tca aat gaa 156 Pro
Ser Asp Val Lys Glu Leu Phe Leu Asp Asn Ser Gln Ser Asn Glu 20 25
30 ggc aaa ttg gaa ggc ctc aca gat gaa ttt gag gaa ctg gaa tta tta
204 Gly Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu Leu Leu
35 40 45 aat aca atc aac ata ggc ctc acc tca att gca aac ttg cca
aag tta 252 Asn Thr Ile Asn Ile Gly Leu Thr Ser Ile Ala Asn Leu Pro
Lys Leu 50 55 60 aac aaa ctt aag aag ctt gaa cta agc agt aac aga
gcc tca gtg ggc 300 Asn Lys Leu Lys Lys Leu Glu Leu Ser Ser Asn Arg
Ala Ser Val Gly 65 70 75 cta gaa gta ttg gca gaa aag tgt cca aac
ctc ata cat cta aat tta 348 Leu Glu Val Leu Ala Glu Lys Cys Pro Asn
Leu Ile His Leu Asn Leu 80 85 90 95 agt ggc aac aaa att aaa gac ctc
agc aca ata gag ccc ctg aaa aag 396 Ser Gly Asn Lys Ile Lys Asp Leu
Ser Thr Ile Glu Pro Leu Lys Lys 100 105 110 tta gaa aac ctt gag agc
tta gac ctt ttc act tgc gag gta acc aac 444 Leu Glu Asn Leu Glu Ser
Leu Asp Leu Phe Thr Cys Glu Val Thr Asn 115 120 125 ctg aac aac
tactgagaaa agatgttcaa gctcctcctg caactcacat 493 Leu Asn Asn 130
atctcaacgg ctgtgacccg gatgacaagg aggcccctaa ctcggatggt gagggctacg
553 tggagggcct ggacgatgag gaggaggatg aggatgagga ggagtatgat
gaagatgctc 613 aggtagtgga agacgaggag gacgaggatg aggaggagga
aggtgaagag gaggacgtga 673 gtggagagga ggaggaggat gaagaaggtt
ataacgatgg agaggtagat gacgaggaag 733 atgaagaaga gcttggtgaa
gaagaaaggg gtcagaagcg aaaacgagaa cctgaagatg 793 agggagaaga
tgatgactaa gtggaataac ctattttgaa aaattcctat tgtgatttga 853
ctgtttttag ccgtatcccc tctccccccc cactctaatc ctgccccctg aa 905 18
130 PRT Homo sapiens 18 Met Glu Met Gly Lys Trp Ile His Leu Glu Leu
Arg Asn Arg Thr Pro 1 5 10 15 Ser Asp Val Lys Glu Leu Phe Leu Asp
Asn Ser Gln Ser Asn Glu Gly 20 25 30 Lys Leu Glu Gly Leu Thr Asp
Glu Phe Glu Glu Leu Glu Leu Leu Asn 35 40 45 Thr Ile Asn Ile Gly
Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu Asn 50 55 60 Lys Leu Lys
Lys Leu Glu Leu Ser Ser Asn Arg Ala Ser Val Gly Leu 65 70 75 80 Glu
Val Leu Ala Glu Lys Cys Pro Asn Leu Ile His Leu Asn Leu Ser 85 90
95 Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu
100 105 110 Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr Cys Glu Val Thr
Asn Leu 115 120 125 Asn Asn 130 19 905 DNA Homo sapiens CDS
(64)..(453) 19 gggttcgggg tttattggtt gaattccgct ggctcaggag
cctctgcaga gaaagcgtga 60 gag atg gag atg ggc aaa tgg att cat tta
gag ctg cgg aac agg acg 108 Met Glu Met Gly Lys Trp Ile His Leu Glu
Leu Arg Asn Arg Thr 1 5 10 15 ccc tcc gat gtg aaa gaa ctt ttc ctg
gac aac agt cag tca aat gaa 156 Pro Ser Asp Val Lys Glu Leu Phe Leu
Asp Asn Ser Gln Ser Asn Glu 20 25 30 ggc aaa ttg gaa ggc ctc aca
gat gaa ttt gaa gaa ctg gaa tta tta 204 Gly Lys Leu Glu Gly Leu Thr
Asp Glu Phe Glu Glu Leu Glu Leu Leu 35 40 45 aat aca atc aac ata
ggc ctc acc tca att gca aac ttg cca aag tta 252 Asn Thr Ile Asn Ile
Gly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu 50 55 60 aac aaa ctt
aag aag ctt gaa cta agc agt aac aga gcc tca gtg ggc 300 Asn Lys Leu
Lys Lys Leu Glu Leu Ser Ser Asn Arg Ala Ser Val Gly 65 70 75 cta
gaa gta ttg gca gaa aag tgt cca aac ctc ata cat cta aat tta 348 Leu
Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Ile His Leu Asn Leu 80 85
90 95 agt ggc aac aaa att aaa gac ctc agc aca ata gag ccc ctg aaa
aag 396 Ser Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys
Lys 100 105 110 tta gaa aac ctc gag agc tta gac ctt ttc act tgc gag
gta acc aac 444 Leu Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr Cys Glu
Val Thr Asn 115 120 125 ctg aac aac tactgagaaa agatgttcaa
gctcctcctg caactcacat 493 Leu Asn Asn 130 atctcaacgg ctgtgacccg
gatgacaagg aggcccctaa ctcggatggt gagggctttg 553 tggagtgcct
ggatgacaag gaggaggatg aggatgagga ggagtatgat gaagatgctc 613
aggtaatgga agatgaggag gacgaggatg aggaggagga acgtgaagag gaggacgtga
673 gtggagacga ggaggagaag gatgaaggtt ataacaatgg agaggtagat
gatgaggaag 733 atgaagaaga gcttggtgaa gaagaaaggg gtcagaagcg
aaaataagaa actgaagatg 793 agggagaaga cgatgcctaa gtggaataat
ctattttgaa aaattccttt tgtgatttta 853 ctgtttttag ccgtatcccc
tctccccccc cactctaatc ctgccccctg aa 905 20 130 PRT Homo sapiens 20
Met Glu Met Gly Lys Trp Ile His Leu Glu Leu Arg Asn Arg Thr Pro 1 5
10 15 Ser Asp Val Lys Glu Leu Phe Leu Asp Asn Ser Gln Ser Asn Glu
Gly 20 25 30 Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu
Leu Leu Asn 35 40 45 Thr Ile Asn Ile Gly Leu Thr Ser Ile Ala Asn
Leu Pro Lys Leu Asn 50 55 60 Lys Leu Lys Lys Leu Glu Leu Ser Ser
Asn Arg Ala Ser Val Gly Leu 65 70 75 80 Glu Val Leu Ala Glu Lys Cys
Pro Asn Leu Ile His Leu Asn Leu Ser 85 90 95 Gly Asn Lys Ile Lys
Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100 105 110 Glu Asn Leu
Glu Ser Leu Asp Leu Phe Thr Cys Glu Val Thr Asn Leu 115 120 125 Asn
Asn 130 21 895 DNA Homo sapiens CDS (66)..(767) 21 gggttcgggg
tttattgatt gaattcggct ggcacgagag cctctgcaga cagagagcgc 60 gagag atg
gag atg ggc aga cgg att cat tca gag ctg cgg aac agg gcg 110 Met Glu
Met Gly Arg Arg Ile His Ser Glu Leu Arg Asn Arg Ala 1 5 10 15 ccc
tct gat gtg aaa gaa ctt gtc ctg gac aac agt cgg tcg aat gaa 158 Pro
Ser Asp Val Lys Glu Leu Val Leu Asp Asn Ser Arg Ser Asn Glu 20 25
30 ggc aaa ctc gaa gcc ctc aca gat gaa ttt gaa gaa ctg gaa ttc tta
206 Gly Lys Leu Glu Ala Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu
35 40 45 agt aaa atc aac gga ggc ctc acc tca atc tca gac tta cca
aag tta 254 Ser Lys Ile Asn Gly Gly Leu Thr Ser Ile Ser Asp Leu Pro
Lys Leu 50 55 60 aag ttg aga aag ctt gaa cta aaa gtc tca ggg ggc
ctg gaa gta ttg 302 Lys Leu Arg Lys Leu Glu Leu Lys Val Ser Gly Gly
Leu Glu Val Leu 65 70 75 gca gaa aag tgt cca aac ctc acg cat cta
tat tta agt ggc aac aaa 350 Ala Glu Lys Cys Pro Asn Leu Thr His Leu
Tyr Leu Ser Gly Asn Lys 80 85 90 95 att aaa gac ctc agc aca ata gag
cca ctg aaa cag tta gaa aac ctc 398 Ile Lys Asp Leu Ser Thr Ile Glu
Pro Leu Lys Gln Leu Glu Asn Leu 100 105 110 aag agc tta gac ctt ttc
aat tgc gag gta acc aac ctg aac gac tac 446 Lys Ser Leu Asp Leu Phe
Asn Cys Glu Val Thr Asn Leu Asn Asp Tyr 115 120 125 gga gaa aac gtg
ttc aag ctt ctc ctg caa ctc aca tat ctc gac agc 494 Gly Glu Asn Val
Phe Lys Leu Leu Leu Gln Leu Thr Tyr Leu Asp Ser 130 135 140 tgt tac
tgg gac cac aag gag gcc cct tac tca gat att gag gac cac 542 Cys Tyr
Trp Asp His Lys Glu Ala Pro Tyr Ser Asp Ile Glu Asp His 145 150 155
gtg gag ggc ctg gat gac gag gag gag ggt gag cat gag gag gag tat 590
Val Glu Gly Leu Asp Asp Glu Glu Glu Gly Glu His Glu Glu Glu Tyr 160
165 170 175 gat gaa gat gct cag gta gtg gaa gat gag gag ggc gag gag
gag gag 638 Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu Gly Glu Glu
Glu Glu 180 185 190 gag gaa ggt gaa gag gag gac gtg agt gga ggg gac
gag gag gat gaa 686 Glu Glu Gly Glu Glu Glu Asp Val Ser Gly Gly Asp
Glu Glu Asp Glu 195 200 205 gaa ggt tat aac gat gga gag gta gat ggc
gag gaa gat gaa gaa gag 734 Glu Gly Tyr Asn Asp Gly Glu Val Asp Gly
Glu Glu Asp Glu Glu Glu 210 215 220 ctt ggt gaa gaa gaa agg ggt cag
aag cga aaa tgagaacctg aagatgaggg 787 Leu Gly Glu Glu Glu Arg Gly
Gln Lys Arg Lys 225 230 agaagatgat gactaagtag aataacctat tttgaaaaat
tcctattgtg atttgactgt 847 ttttacccat atcccccctc ccccccccct
ctaatcctgc cccctgaa 895 22 234 PRT Homo sapiens 22 Met Glu Met Gly
Arg Arg Ile His Ser Glu Leu Arg Asn Arg Ala Pro 1 5 10 15 Ser Asp
Val Lys Glu Leu Val Leu Asp Asn Ser Arg Ser Asn Glu Gly 20 25 30
Lys Leu Glu Ala Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu Ser 35
40 45 Lys Ile Asn Gly Gly Leu Thr Ser Ile Ser Asp Leu Pro Lys Leu
Lys 50 55 60 Leu Arg Lys Leu Glu Leu Lys Val Ser Gly Gly Leu Glu
Val Leu Ala 65 70 75 80 Glu Lys Cys Pro Asn Leu Thr His Leu Tyr Leu
Ser Gly Asn Lys Ile 85 90 95 Lys Asp Leu Ser Thr Ile Glu Pro Leu
Lys Gln Leu Glu Asn Leu Lys 100 105 110 Ser Leu Asp Leu Phe Asn Cys
Glu Val Thr Asn Leu Asn Asp Tyr Gly 115 120 125 Glu Asn Val Phe Lys
Leu Leu Leu Gln Leu Thr Tyr Leu Asp Ser Cys 130 135 140 Tyr Trp Asp
His Lys Glu Ala Pro Tyr Ser Asp Ile Glu Asp His Val 145 150 155 160
Glu Gly Leu Asp Asp Glu Glu Glu Gly Glu His Glu Glu Glu Tyr Asp 165
170 175 Glu Asp Ala Gln Val Val Glu Asp Glu Glu Gly Glu Glu Glu Glu
Glu 180 185 190 Glu Gly Glu Glu Glu Asp Val Ser Gly Gly Asp Glu Glu
Asp Glu Glu 195 200 205 Gly Tyr Asn Asp Gly Glu Val Asp Gly Glu Glu
Asp Glu Glu Glu Leu 210 215 220 Gly Glu Glu Glu Arg Gly Gln Lys Arg
Lys 225 230 23 895 DNA Homo sapiens CDS (66)..(767) 23 gggttcgggg
tttattgatt gaattccgcc ggcgcgggag cctctgcaga gagggagcgc 60 gagag atg
gag atg ggc aga cgg att cat tta gag ctg cgg aac agg acg 110 Met Glu
Met Gly Arg Arg Ile His Leu Glu Leu Arg Asn Arg Thr 1 5 10 15 ccc
tct gat gtg aaa gaa ctt gtc ctg gac aac agt cgg tcg aat gaa 158 Pro
Ser Asp Val Lys Glu Leu Val Leu Asp Asn Ser Arg Ser Asn Glu 20 25
30 ggc aaa ctc gaa ggc ctc aca gat gaa ttt gaa gaa ctg gaa ttc tta
206 Gly Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu
35 40 45 agt aca atc aac gta ggc ctc acc tca atc gca aac tta cca
aag tta 254 Ser Thr Ile Asn Val Gly Leu Thr Ser Ile Ala Asn Leu Pro
Lys Leu 50 55 60 aag ttg aga aag ctt gaa cta aga gtc tca ggg ggc
ctg gaa gta ttg 302 Lys Leu Arg Lys Leu Glu Leu Arg Val Ser Gly Gly
Leu Glu Val Leu 65 70 75 gca gaa aag tgt cca aac ctc acg cac cta
tat tta agt ggc aac aaa 350 Ala Glu Lys Cys Pro Asn Leu Thr His Leu
Tyr Leu Ser Gly Asn Lys 80 85 90 95 att aaa gac ctc agc aca ata gag
cca ctg aaa cag tta gaa aac ctc 398 Ile Lys Asp Leu Ser Thr Ile Glu
Pro Leu Lys Gln Leu Glu Asn Leu 100 105 110 aag agc tta gac ctt ttc
aat tgc gag gta acc aac ctg aac gac tac 446 Lys Ser Leu Asp Leu Phe
Asn Cys Glu Val Thr Asn Leu Asn Asp Tyr 115 120 125 gga gaa aac gtg
ttc aag ctt ctc ctg caa ctc aca tat ctc gac agc 494 Gly Glu Asn Val
Phe Lys Leu Leu Leu Gln Leu Thr Tyr Leu Asp Ser 130 135 140 tgt tac
tgg gac cac aag gag gcc cct tac tca gat att gag gac cac 542 Cys Tyr
Trp Asp His Lys Glu Ala Pro Tyr Ser Asp Ile Glu Asp His 145 150 155
gtg gag ggc ctg gat gac gag gag gag ggt gag cat gag gag gag tat 590
Val Glu Gly Leu Asp Asp Glu Glu Glu Gly Glu His Glu Glu Glu Tyr 160
165 170 175 gat gaa gat gct cag gta gtg gaa gat gag gag ggc gag gag
ggg gag 638 Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu Gly Glu Glu
Gly Glu 180 185 190 gag gaa ggt gaa gag gag gac gtg agt gga ggg gac
gag gag gat gaa 686 Glu Glu Gly Glu Glu Glu Asp Val Ser Gly Gly Asp
Glu Glu Asp Glu 195 200 205 gaa ggt tat aac gat gga gag gta gat gac
gag gaa gat gaa gaa gag 734 Glu Gly Tyr Asn Asp Gly Glu Val Asp Asp
Glu Glu Asp Glu Glu Glu 210 215 220 ctt ggt gaa gaa gaa agg ggt cag
aag cga aaa cgagaacctg aagatgaggg 787 Leu Gly Glu Glu Glu Arg Gly
Gln Lys Arg Lys 225 230 agaagatgat gactaagtgg aataacctat tttgaaaaat
tcctattgtg atttgactgt 847 ttttacccat atcccctctc ccccccccct
ctaatcctgc cccctgaa 895 24 234 PRT Homo sapiens 24 Met Glu Met Gly
Arg Arg Ile His Leu Glu Leu Arg Asn Arg Thr Pro 1 5 10 15 Ser Asp
Val Lys Glu Leu Val Leu Asp Asn Ser Arg Ser Asn Glu Gly 20 25 30
Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu Ser 35
40 45 Thr Ile Asn Val Gly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu
Lys 50 55 60 Leu Arg Lys Leu Glu Leu Arg Val Ser Gly Gly Leu Glu
Val Leu Ala 65 70 75 80 Glu Lys Cys Pro Asn Leu Thr His Leu Tyr Leu
Ser Gly Asn Lys Ile 85 90 95 Lys Asp Leu Ser Thr Ile Glu Pro Leu
Lys Gln Leu Glu Asn Leu Lys 100 105 110 Ser Leu Asp Leu Phe Asn Cys
Glu Val Thr Asn Leu Asn Asp Tyr Gly 115 120 125 Glu Asn Val Phe Lys
Leu Leu Leu Gln Leu Thr Tyr Leu Asp Ser Cys 130 135 140 Tyr Trp Asp
His Lys Glu Ala Pro Tyr Ser Asp Ile Glu Asp His Val 145 150 155 160
Glu Gly Leu Asp Asp Glu Glu
Glu Gly Glu His Glu Glu Glu Tyr Asp 165 170 175 Glu Asp Ala Gln Val
Val Glu Asp Glu Glu Gly Glu Glu Gly Glu Glu 180 185 190 Glu Gly Glu
Glu Glu Asp Val Ser Gly Gly Asp Glu Glu Asp Glu Glu 195 200 205 Gly
Tyr Asn Asp Gly Glu Val Asp Asp Glu Glu Asp Glu Glu Glu Leu 210 215
220 Gly Glu Glu Glu Arg Gly Gln Lys Arg Lys 225 230 25 907 DNA Homo
sapiens 25 gggttcgggg tttattgatt gaattccgcc ggcgcgggag cctctgcaga
gagagagcgc 60 gagagatgga gatgggcaga cggattcatt tagagctgcg
gaacaggacg ccctctgatg 120 tgaaagaact tgtcctggac aacagtcggt
cgaatgaagg caaactcgag ggcctcacag 180 atgaatttga agaactggaa
ttcttaagta caatcaacgt aggcctcacc tcaatcgcaa 240 acttaccaaa
gttaaacaaa cttaagaagc ttgaactaag cgataacaga gtctcagggg 300
gcctggaagt attggcagaa aagtgtccga acctcacgca tctaaattta agtggcaaca
360 aaattaaaga cctcagcaca atagagccac tgaaaaagtt agaaaacctc
aagagcttag 420 accttttcaa ttgcgaggta accaacctga acgactaccg
agaaaatgtg ttcaagctcc 480 tcccgcaact cacatatctc gacggctatg
accgggacga caaggaggcc cctgactcgg 540 atgctgaggg ctacgtggag
ggcctggatg atgaggagga ggatgaggat gaggaggagt 600 atgatgaaga
tgctcaggta gtggaagacg aggaggacga ggatgaggag gaggaaggtg 660
aagaggagga cgtgagtgga gaggaggagg aggatgaaga aggttataac gatggagagg
720 tagatgacga ggaagatgaa gaagagcttg gtgaagaaga aaggggtcag
aagcgaaaac 780 gagaacctga agatgaggga gaagatgatg actaagtgga
ataacctatt ttgaaaaatt 840 cctattgtga tttgactgtt tttacccata
tcccctctcc cccccccctc taatcctgcc 900 ccctgaa 907 26 905 DNA Homo
sapiens CDS (64)..(453) 26 gggttcgggg tttattggtt gaattccgct
ggctcaggag cctctgcaga gaaagcgtga 60 gag atg gag atg ggc aaa tgg att
cat tta gag ctg cgg aac agg acg 108 Met Glu Met Gly Lys Trp Ile His
Leu Glu Leu Arg Asn Arg Thr 1 5 10 15 ccc tcc gat gtg aaa gaa ctt
ttc ctg gac aac agt cag tca aat gaa 156 Pro Ser Asp Val Lys Glu Leu
Phe Leu Asp Asn Ser Gln Ser Asn Glu 20 25 30 ggc aaa ttg gaa ggc
ctc aca gat gaa ttt gaa gaa ctg gaa tta tta 204 Gly Lys Leu Glu Gly
Leu Thr Asp Glu Phe Glu Glu Leu Glu Leu Leu 35 40 45 aat aca atc
aac ata ggc ctc acc tca att gca aac ttg cca aag tta 252 Asn Thr Ile
Asn Ile Gly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu 50 55 60 aac
aaa ctt aag aag ctt gaa cta agc agt aac aga gcc tca gtg ggc 300 Asn
Lys Leu Lys Lys Leu Glu Leu Ser Ser Asn Arg Ala Ser Val Gly 65 70
75 cta gaa gta ttg gca gaa aag tgt cca aac ctc ata cat cta aat tta
348 Leu Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Ile His Leu Asn Leu
80 85 90 95 agt ggc aac aaa att aaa gac ctc agc aca ata gag ccc ctg
aaa aag 396 Ser Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu
Lys Lys 100 105 110 tta gaa aac ctc gag agc tta gac ctt ttc act tgc
gag gta acc aac 444 Leu Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr Cys
Glu Val Thr Asn 115 120 125 ctg aac aac tactgagaaa agatgttcaa
gctcctcctg caactcacat 493 Leu Asn Asn 130 atctcaacgg ctgtgacccg
gatgacaagg aggcccctaa ctcggatggt gagggctttg 553 tggagtgcct
ggatgacaag gaggaggatg aggatgagga ggagtatgat gaagatgctc 613
aggtaatgga agatgaggag gacgaggatg aggaggagga acgtgaagag gaggacgtga
673 gtggagacga ggaggagaag gatgaaggtt ataacaatgg agaggtagat
gatgaggaag 733 atgaagaaga gcttggtgaa gaagaaaggg gtcagaagcg
aaaataagaa actgaagatg 793 agggagaaga cgatgcctaa gtggaataat
ctattttgaa aaattccttt tgtgatttta 853 ctgtttttag ccgtatcccc
tctccccccc cactctaatc ctgccccctg aa 905 27 130 PRT Homo sapiens 27
Met Glu Met Gly Lys Trp Ile His Leu Glu Leu Arg Asn Arg Thr Pro 1 5
10 15 Ser Asp Val Lys Glu Leu Phe Leu Asp Asn Ser Gln Ser Asn Glu
Gly 20 25 30 Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu
Leu Leu Asn 35 40 45 Thr Ile Asn Ile Gly Leu Thr Ser Ile Ala Asn
Leu Pro Lys Leu Asn 50 55 60 Lys Leu Lys Lys Leu Glu Leu Ser Ser
Asn Arg Ala Ser Val Gly Leu 65 70 75 80 Glu Val Leu Ala Glu Lys Cys
Pro Asn Leu Ile His Leu Asn Leu Ser 85 90 95 Gly Asn Lys Ile Lys
Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100 105 110 Glu Asn Leu
Glu Ser Leu Asp Leu Phe Thr Cys Glu Val Thr Asn Leu 115 120 125 Asn
Asn 130 28 907 DNA Homo sapiens CDS (66)..(812) 28 gggttcgggg
tttattgatt gaattccgcc ggcgcgggag cctctgcaga gagagagcgc 60 gagag atg
gag atg ggc aga cgg att cat cta gag ctg cgg aac agg acg 110 Met Glu
Met Gly Arg Arg Ile His Leu Glu Leu Arg Asn Arg Thr 1 5 10 15 ccc
tct gat gtg aaa gaa ctt gtc ctg gtc aac agt cgg tcg aat gaa 158 Pro
Ser Asp Val Lys Glu Leu Val Leu Val Asn Ser Arg Ser Asn Glu 20 25
30 ggc aaa ctc gaa ggc ctc aca gat gaa ttt gaa gaa ctg gaa ttc tta
206 Gly Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu
35 40 45 agt aca atc aac gta ggc ctc acc tca atc gca aac tta cca
aag tta 254 Ser Thr Ile Asn Val Gly Leu Thr Ser Ile Ala Asn Leu Pro
Lys Leu 50 55 60 aac aaa ctt aag aag ctt gaa cta agc gat aac aga
gtc tca ggg ggc 302 Asn Lys Leu Lys Lys Leu Glu Leu Ser Asp Asn Arg
Val Ser Gly Gly 65 70 75 cta gaa gta ttg gca gaa aag tgt ccg aac
ctc acg cat cta aat tta 350 Leu Glu Val Leu Ala Glu Lys Cys Pro Asn
Leu Thr His Leu Asn Leu 80 85 90 95 agt ggc aac aaa att aaa gac ctc
agc aca ata gag cca ctg aaa aag 398 Ser Gly Asn Lys Ile Lys Asp Leu
Ser Thr Ile Glu Pro Leu Lys Lys 100 105 110 tta gaa aac ctc aag agc
tta gac ctt ttc aat tgc gag gta acc aac 446 Leu Glu Asn Leu Lys Ser
Leu Asp Leu Phe Asn Cys Glu Val Thr Asn 115 120 125 ctg aac gac tac
cga gaa aat gtg ttc aag ctc ctc ccg caa ctc aca 494 Leu Asn Asp Tyr
Arg Glu Asn Val Phe Lys Leu Leu Pro Gln Leu Thr 130 135 140 tat ctc
gac ggc tat gac cgg gac gac aag gag gcc cct gac tcg gat 542 Tyr Leu
Asp Gly Tyr Asp Arg Asp Asp Lys Glu Ala Pro Asp Ser Asp 145 150 155
gct gag ggc tac gtg gag ggc ctg gat gat gag gag gag gat gag gat 590
Ala Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu Asp Glu Asp 160
165 170 175 gag gag gag tat gat gaa gat gct cag gta gtg gaa gac gag
gag gac 638 Glu Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu Asp Glu
Glu Asp 180 185 190 gag gat gag gag gag gaa ggt gaa gag gag gac gtg
agt gga gag gag 686 Glu Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp Val
Ser Gly Glu Glu 195 200 205 gag gag gat gaa gaa ggt tat aac gat gga
gag gta gat gac gag gaa 734 Glu Glu Asp Glu Glu Gly Tyr Asn Asp Gly
Glu Val Asp Asp Glu Glu 210 215 220 gat gaa gaa gag ctt ggt gaa gaa
gaa agg ggt cag aag cga aaa cga 782 Asp Glu Glu Glu Leu Gly Glu Glu
Glu Arg Gly Gln Lys Arg Lys Arg 225 230 235 gaa cct gaa gat gag gga
gaa gat gat gac taagtggaat aacctatttt 832 Glu Pro Glu Asp Glu Gly
Glu Asp Asp Asp 240 245 gaaaaattcc tattgtgatt tgactgtttt tacccatatc
ccctctcccc cccccctcta 892 atcctgcccc ctgaa 907 29 249 PRT Homo
sapiens 29 Met Glu Met Gly Arg Arg Ile His Leu Glu Leu Arg Asn Arg
Thr Pro 1 5 10 15 Ser Asp Val Lys Glu Leu Val Leu Val Asn Ser Arg
Ser Asn Glu Gly 20 25 30 Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu
Glu Leu Glu Phe Leu Ser 35 40 45 Thr Ile Asn Val Gly Leu Thr Ser
Ile Ala Asn Leu Pro Lys Leu Asn 50 55 60 Lys Leu Lys Lys Leu Glu
Leu Ser Asp Asn Arg Val Ser Gly Gly Leu 65 70 75 80 Glu Val Leu Ala
Glu Lys Cys Pro Asn Leu Thr His Leu Asn Leu Ser 85 90 95 Gly Asn
Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100 105 110
Glu Asn Leu Lys Ser Leu Asp Leu Phe Asn Cys Glu Val Thr Asn Leu 115
120 125 Asn Asp Tyr Arg Glu Asn Val Phe Lys Leu Leu Pro Gln Leu Thr
Tyr 130 135 140 Leu Asp Gly Tyr Asp Arg Asp Asp Lys Glu Ala Pro Asp
Ser Asp Ala 145 150 155 160 Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu
Glu Glu Asp Glu Asp Glu 165 170 175 Glu Glu Tyr Asp Glu Asp Ala Gln
Val Val Glu Asp Glu Glu Asp Glu 180 185 190 Asp Glu Glu Glu Glu Gly
Glu Glu Glu Asp Val Ser Gly Glu Glu Glu 195 200 205 Glu Asp Glu Glu
Gly Tyr Asn Asp Gly Glu Val Asp Asp Glu Glu Asp 210 215 220 Glu Glu
Glu Leu Gly Glu Glu Glu Arg Gly Gln Lys Arg Lys Arg Glu 225 230 235
240 Pro Glu Asp Glu Gly Glu Asp Asp Asp 245 30 907 DNA Homo sapiens
CDS (66)..(455) 30 gggttcgggg tttattgatt gaattccgcc ggcgcgggag
cctctgcaga gagagagcgc 60 gagag atg gag atg ggc aga cgg att cat tta
gag ctg cgg aac agg acg 110 Met Glu Met Gly Arg Arg Ile His Leu Glu
Leu Arg Asn Arg Thr 1 5 10 15 ccc tct gat gtg aaa gaa ctt gtc ctg
gac aac agt cgg tcg aat gaa 158 Pro Ser Asp Val Lys Glu Leu Val Leu
Asp Asn Ser Arg Ser Asn Glu 20 25 30 ggc aaa ctc gaa ggc ctc aca
gat gaa ttt gaa gaa ctg gaa ttc tta 206 Gly Lys Leu Glu Gly Leu Thr
Asp Glu Phe Glu Glu Leu Glu Phe Leu 35 40 45 agt aca atc aac gta
ggc ctc acc tca atc gca aac tta cca aag tta 254 Ser Thr Ile Asn Val
Gly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu 50 55 60 aac aaa ctt
aag aag ctt gaa cta agc gat aac aga gtc tca ggg ggc 302 Asn Lys Leu
Lys Lys Leu Glu Leu Ser Asp Asn Arg Val Ser Gly Gly 65 70 75 cta
gaa gta ttg gca gaa aag tgt cca aac ctc ata cat cta aat tta 350 Leu
Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Ile His Leu Asn Leu 80 85
90 95 agt ggc aac aaa att aaa gac ctc agc aca ata gag ccc ctg aaa
aag 398 Ser Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys
Lys 100 105 110 tta gaa aac ctc gag agc tta gac ctt ttc act tgc gag
gta acc aac 446 Leu Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr Cys Glu
Val Thr Asn 115 120 125 ctg aac aac tactgagaaa agatgttcaa
gctcctcctg caactcacat 495 Leu Asn Asn 130 atctcaacgg ctgtgacccg
gatgacaagg aggcccctaa ctcggatggt gagggctttg 555 tggagtgcct
ggatgacaag gaggaggatg aggatgagga ggagtatgat gaagatgctc 615
aggtaatgga agatgaggag gacgaggatg aggaggagga acgtgaagag gaggacgtga
675 gtggagacga ggaggagaag gatgaaggtt ataacaatgg agaggtagat
gatgaggaag 735 atgaagaaga gcttggtgaa gaagaaaggg gtcagaagcg
aaaataagaa actgaagatg 795 agggagaaga cgatgcctaa gtggaataat
ctattttgaa aaattcctat tgtgatttga 855 ctgtttttac ccatatcccc
tctccccccc ccctctaatc ctgccccctg aa 907 31 130 PRT Homo sapiens 31
Met Glu Met Gly Arg Arg Ile His Leu Glu Leu Arg Asn Arg Thr Pro 1 5
10 15 Ser Asp Val Lys Glu Leu Val Leu Asp Asn Ser Arg Ser Asn Glu
Gly 20 25 30 Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu
Phe Leu Ser 35 40 45 Thr Ile Asn Val Gly Leu Thr Ser Ile Ala Asn
Leu Pro Lys Leu Asn 50 55 60 Lys Leu Lys Lys Leu Glu Leu Ser Asp
Asn Arg Val Ser Gly Gly Leu 65 70 75 80 Glu Val Leu Ala Glu Lys Cys
Pro Asn Leu Ile His Leu Asn Leu Ser 85 90 95 Gly Asn Lys Ile Lys
Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100 105 110 Glu Asn Leu
Glu Ser Leu Asp Leu Phe Thr Cys Glu Val Thr Asn Leu 115 120 125 Asn
Asn 130 32 908 DNA Homo sapiens 32 gggttcgggg tttattgatt gaattccgcc
ggcgcgggag cctctgcaga gagagagcgc 60 ggagagatgg agatgggcag
acggattcat ttagagctgc ggaacaggac gccctctgat 120 gtgaaagaac
ttgtcctgga caacagtcgg tcgaatgaag gcaaactcga aggcctcaca 180
gatgaatttg aagaactgga attcttaagt acaatcaacg taggcctcac ctcaatcgca
240 aacttaccaa agttaaacaa acttaagaag cttgaactaa gcgataacag
agtctcaggg 300 ggcctggaag tattggcaga aaagtgtccg aacctcacgc
atctaaattt aagtggcaac 360 aaaattaaag acctcagcac aatagagcca
ctgaaaaagt tagaaaacct caagagctta 420 gaccttttca attgcgaggt
aaccaacctg aacgactacc gagaaaatgt gttcaagctc 480 ctcccgcaac
tcacatatct cgacggctat gaccgggacg acaaggaggc ccctgactcg 540
gatgctgagg gctacgtgga gggcctggat gatgaggagg aggatgagga tgaggaggag
600 tatgatgaag atgctcaggt agtggaagac gaggaggacg aggatgagga
ggaggaaggt 660 gaagaggagg acgtgagtgg agaggaggag gaggatgaag
aaggttataa cgatggagag 720 gtagatgacg aggaagatga agaagagctt
ggtgaagaag aaaggggtca gaagcgaaaa 780 cgagaacctg aagatgaggg
agaagatgat gactaagtgg aataacctat tttgaaaaat 840 tcctattgtg
atttgactgt ttttacccat atcccctctc ccccccccct ctaatcctgc 900 cccctgaa
908 33 906 DNA Homo sapiens CDS (66)..(812) 33 gggttcgggg
tttattgatt gaattccgct ggcgcgggag cctctgcaga gagagagcgc 60 gagag atg
gag atg ggc aga cgg att cat tta gag ctg cgg aac agg acg 110 Met Glu
Met Gly Arg Arg Ile His Leu Glu Leu Arg Asn Arg Thr 1 5 10 15 ccc
tct gat gtg aaa gaa ctt gtc ctg gac aac agt cgg tcg aat gaa 158 Pro
Ser Asp Val Lys Glu Leu Val Leu Asp Asn Ser Arg Ser Asn Glu 20 25
30 ggc aaa ctc gaa ggc ctc aca gat gaa ttt gaa gaa ctg gaa ttc tta
206 Gly Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe Leu
35 40 45 agt aca atc aac gta ggc ctc acc tca atc gca aac tta cca
aag tta 254 Ser Thr Ile Asn Val Gly Leu Thr Ser Ile Ala Asn Leu Pro
Lys Leu 50 55 60 aac aaa ctt aag aag ctt gaa cta agc agt aac aga
gtc tca ggg ggc 302 Asn Lys Leu Lys Lys Leu Glu Leu Ser Ser Asn Arg
Val Ser Gly Gly 65 70 75 cta gaa gta ttg gca gaa aag tgt cca aac
ctc acg cat cta aat tta 350 Leu Glu Val Leu Ala Glu Lys Cys Pro Asn
Leu Thr His Leu Asn Leu 80 85 90 95 agt ggc aac aaa att aaa gac ctc
agc aca ata gag cca ctg aaa aag 398 Ser Gly Asn Lys Ile Lys Asp Leu
Ser Thr Ile Glu Pro Leu Lys Lys 100 105 110 tta gaa aac ctc aag agc
tta gac ctt ttc aat tgc gag gta acc aac 446 Leu Glu Asn Leu Lys Ser
Leu Asp Leu Phe Asn Cys Glu Val Thr Asn 115 120 125 ctg aac gac tac
cga gaa aat gtg ttc aag ctc ctc ctg caa ctc aca 494 Leu Asn Asp Tyr
Arg Glu Asn Val Phe Lys Leu Leu Leu Gln Leu Thr 130 135 140 tat ctc
gac ggc tgt gac cgg gac gac aag gag gcc cct gac tcg gat 542 Tyr Leu
Asp Gly Cys Asp Arg Asp Asp Lys Glu Ala Pro Asp Ser Asp 145 150 155
gct gag ggc tac gtg gag ggc ctg gat gac gag gag gag gat gag gat 590
Ala Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu Asp Glu Asp 160
165 170 175 gag gag gag tat gat gaa gat gct cag gta gtg gaa gat gag
gag gac 638 Glu Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu Asp Glu
Glu Asp 180 185 190 gag gat gag gag gag gaa ggt gaa gag gag gac gtg
agt gga gag gag 686 Glu Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp Val
Ser Gly Glu Glu 195 200 205 gag gag gat gaa gaa ggt tat aac gat gga
gag gta gat gac gag gaa 734 Glu Glu Asp Glu Glu Gly Tyr Asn Asp Gly
Glu Val Asp Asp Glu Glu 210 215 220 gat gaa gaa gag ctt ggt gaa gaa
gaa agg ggt cag aag cga aaa gag 782 Asp Glu Glu Glu Leu Gly Glu Glu
Glu Arg Gly Gln Lys Arg Lys Glu 225 230 235 aac ctg aag atg agg gag
aag atg atg act aagtggaata acctattttg 832 Asn Leu Lys Met Arg Glu
Lys Met Met Thr 240 245 aaaaattcct attgtgattt gactgttttt acccatatcc
cctctccccc ccccctctaa 892 tcctgccccc tgaa 906 34 249 PRT Homo
sapiens 34 Met Glu Met Gly Arg Arg Ile His Leu Glu Leu Arg Asn Arg
Thr Pro 1 5 10 15 Ser Asp Val Lys Glu Leu Val Leu Asp Asn Ser Arg
Ser Asn Glu Gly 20 25 30 Lys Leu Glu Gly Leu Thr Asp Glu Phe Glu
Glu Leu Glu Phe Leu Ser 35 40 45 Thr
Ile Asn Val Gly Leu Thr Ser Ile Ala Asn Leu Pro Lys Leu Asn 50 55
60 Lys Leu Lys Lys Leu Glu Leu Ser Ser Asn Arg Val Ser Gly Gly Leu
65 70 75 80 Glu Val Leu Ala Glu Lys Cys Pro Asn Leu Thr His Leu Asn
Leu Ser 85 90 95 Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro
Leu Lys Lys Leu 100 105 110 Glu Asn Leu Lys Ser Leu Asp Leu Phe Asn
Cys Glu Val Thr Asn Leu 115 120 125 Asn Asp Tyr Arg Glu Asn Val Phe
Lys Leu Leu Leu Gln Leu Thr Tyr 130 135 140 Leu Asp Gly Cys Asp Arg
Asp Asp Lys Glu Ala Pro Asp Ser Asp Ala 145 150 155 160 Glu Gly Tyr
Val Glu Gly Leu Asp Asp Glu Glu Glu Asp Glu Asp Glu 165 170 175 Glu
Glu Tyr Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu Asp Glu 180 185
190 Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp Val Ser Gly Glu Glu Glu
195 200 205 Glu Asp Glu Glu Gly Tyr Asn Asp Gly Glu Val Asp Asp Glu
Glu Asp 210 215 220 Glu Glu Glu Leu Gly Glu Glu Glu Arg Gly Gln Lys
Arg Lys Glu Asn 225 230 235 240 Leu Lys Met Arg Glu Lys Met Met Thr
245 35 26 DNA Homo sapiens 35 tatgctagcg ggttcggggt ttattg 26 36 29
DNA Homo sapiens 36 gattctagat ggtaagtttg cgattgagg 29 37 29 DNA
Homo sapiens 37 gaatctagaa ggaggaggaa ggtgaagag 29 38 29 DNA Homo
sapiens 38 ctatctagat tcagggggca ggattagag 29 39 24 DNA Homo
sapiens 39 gaggtttatt gattgaattc ggct 24 40 24 DNA Homo sapiens 40
ccccagtaca cttttcccgt ctca 24 41 12 DNA Artificial Sequence
recognition sequence 41 tttttctttt tc 12 42 10 DNA Artificial
Sequence recognition sequence 42 ttaaaattca 10 43 10 DNA Artificial
Sequence recognition sequence 43 atgtaaaaca 10 44 11 DNA Artificial
Sequence recognition sequence 44 aagataaaac c 11 45 10 DNA
Artificial Sequence recognition sequence 45 ccactgggga 10 46 13 DNA
Artificial Sequence recognition sequence 46 ctctctctct ctc 13 47 11
DNA Artificial Sequence recognition sequence 47 aaaacataaa t 11 48
131 PRT Homo sapiens 48 Met Glu Met Gly Lys Trp Ile His Leu Glu Leu
Arg Asn Arg Thr Pro 1 5 10 15 Ser Asp Val Lys Glu Leu Phe Leu Asp
Asn Ser Gln Ser Asn Glu Gly 20 25 30 Lys Leu Glu Gly Leu Ala Asp
Glu Phe Glu Glu Leu Glu Leu Leu Asn 35 40 45 Thr Ile Asn Ile Gly
Leu Ser Ser Ile Ala Asn Leu Ala Lys Leu Asn 50 55 60 Lys Leu Lys
Lys Leu Glu Leu Ser Ser Asn Arg Ala Ser Val Gly Leu 65 70 75 80 Glu
Val Leu Ala Glu Lys Cys Pro Asn Leu Ile His Leu Asn Leu Ser 85 90
95 Gly Asn Lys Ile Lys Asp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu
100 105 110 Glu Asn Leu Glu Ser Leu Asp Leu Phe Thr Cys Glu Val Thr
Asn Leu 115 120 125 Asn Asn Tyr 130 49 234 PRT Homo sapiens 49 Met
Glu Met Gly Arg Arg Ile His Ser Glu Leu Arg Asn Arg Ala Pro 1 5 10
15 Ser Asp Val Lys Glu Leu Ala Leu Asp Asn Ser Arg Ser Asn Glu Gly
20 25 30 Lys Leu Glu Ala Leu Thr Asp Glu Phe Glu Glu Leu Glu Phe
Leu Ser 35 40 45 Lys Ile Asn Gly Gly Leu Thr Ser Ile Ser Asp Leu
Pro Lys Leu Lys 50 55 60 Leu Arg Lys Leu Glu Leu Arg Val Ser Gly
Gly Leu Glu Val Leu Ala 65 70 75 80 Glu Lys Cys Pro Asn Leu Thr His
Leu Tyr Leu Ser Gly Asn Lys Ile 85 90 95 Lys Asp Leu Ser Thr Ile
Glu Pro Leu Lys Gln Leu Glu Asn Leu Lys 100 105 110 Ser Leu Asp Leu
Phe Asn Cys Glu Val Thr Asn Leu Asn Asp Tyr Gly 115 120 125 Glu Asn
Val Phe Lys Leu Leu Leu Gln Leu Thr Tyr Leu Asp Ser Cys 130 135 140
Tyr Trp Asp His Lys Glu Ala Pro Tyr Ser Asp Ile Glu Asp His Val 145
150 155 160 Glu Gly Leu Asp Asp Glu Glu Glu Gly Glu His Glu Glu Glu
Tyr Asp 165 170 175 Glu Asp Ala Gln Val Val Glu Asp Glu Glu Gly Glu
Glu Glu Glu Glu 180 185 190 Glu Gly Glu Glu Glu Asp Val Ser Gly Gly
Asp Glu Glu Asp Glu Glu 195 200 205 Gly Tyr Asn Asp Gly Glu Val Asp
Gly Glu Glu Asp Glu Glu Glu Leu 210 215 220 Gly Glu Glu Glu Arg Gly
Gln Lys Arg Lys 225 230 50 17 DNA Homo sapiens 50 gggttcgggg
tttattg 17 51 20 DNA Homo sapiens 51 ctctaatcct gccccctgaa 20
* * * * *
References