U.S. patent application number 11/708858 was filed with the patent office on 2010-10-07 for transgenic animals comprising gene fusions.
This patent application is currently assigned to Regents of the University of Michigan. Invention is credited to Chinnaiyan Arul, Bharathi Laxman, Rohit Mera, Scott Tomlins.
Application Number | 20100257617 11/708858 |
Document ID | / |
Family ID | 37865521 |
Filed Date | 2010-10-07 |
United States Patent
Application |
20100257617 |
Kind Code |
A1 |
Arul; Chinnaiyan ; et
al. |
October 7, 2010 |
Transgenic animals comprising gene fusions
Abstract
The present invention relates to compositions and methods for
cancer diagnosis, research and therapy, including but not limited
to, cancer markers. In particular, the present invention relates to
recurrent gene fusion nucleic acids and transgenic animals
comprising recurrent gene fusion nucleic acids.
Inventors: |
Arul; Chinnaiyan; (Plymouth,
MI) ; Laxman; Bharathi; (Ann Arbor, MI) ;
Tomlins; Scott; (Ann Arbor, MI) ; Mera; Rohit;
(Ann Arbor, MI) |
Correspondence
Address: |
Casimir Jones, S.C.
2275 DEMING WAY, SUITE 310
MIDDLETON
WI
53562
US
|
Assignee: |
Regents of the University of
Michigan
Ann Arbor
MI
|
Family ID: |
37865521 |
Appl. No.: |
11/708858 |
Filed: |
February 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11519397 |
Sep 12, 2006 |
7718369 |
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11708858 |
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60716436 |
Sep 12, 2005 |
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60779041 |
Mar 3, 2006 |
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60730358 |
Oct 27, 2005 |
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60795590 |
Apr 28, 2006 |
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Current U.S.
Class: |
800/10 ; 800/13;
800/18 |
Current CPC
Class: |
C12Q 2600/136 20130101;
C12Q 2600/156 20130101; C12Q 2600/106 20130101; A01K 2227/105
20130101; A01K 2217/072 20130101; C07K 2319/00 20130101; C07K 14/82
20130101; C12N 9/6445 20130101; C12N 9/6424 20130101; A01K 67/0275
20130101; C12N 15/113 20130101; C12N 2310/11 20130101; C12N 2310/14
20130101; C12Q 2600/112 20130101; G01N 33/57434 20130101; A01K
2267/0331 20130101; A01K 2217/206 20130101; C12Q 1/6886 20130101;
C12Q 2600/158 20130101; Y10S 435/81 20130101; C12Q 2600/118
20130101 |
Class at
Publication: |
800/10 ; 800/13;
800/18 |
International
Class: |
A01K 67/027 20060101
A01K067/027 |
Goverment Interests
[0002] This invention was made with government support under grant
number prostate SPORE P50CA69568 awarded by the National Institutes
of Health, grant number RO1 CA97063 awarded by the National
Institutes of Health, grant number U01 CA111275 awarded by the
National Institutes of Health, grant number AG021404 awarded by the
National Institutes of Health, grant number CA046592 awarded by the
National Institutes of Health, and grant number ARMY
W81XWH-05-1-0173 awarded by the Department of Defense. The
government has certain rights in the invention.
Claims
1. A transgenic animal comprising a genome that comprises a
heterologous gene, wherein said heterologous gene comprises a gene
fusion having a 5' portion from a transcriptional regulatory region
of an androgen regulated gene and a 3' portion from an ETS family
member gene.
2. The transgenic animal of claim 1, wherein the androgen regulated
gene is selected from the group consisting of TMPRSS2 and PSA.
3. The transgenic animal of claim 2, wherein the transcriptional
regulatory region of the androgen regulated gene comprises a
promoter region of the androgen regulated gene.
4. The transgenic animal of claim 3, wherein the promoter region of
the androgen regulated gene comprises an androgen response element
(ARE) of the androgen regulated gene.
5. The transgenic animal of claim 1, wherein the ETS family member
gene is selected from the group consisting of ERG, ETV1 (ER81),
FLI1, ETS1, ETS2, ELK1, ETV6 (TEL1), ETV7 (TEL2), GABP.alpha.,
ELF1, ETV4 (E1AF; PEA3), ETV5 (ERM), ERF, PEA3/E1AF, PU.1,
ESE1/ESX, SAP1 (ELK4), ETV3 (METS), EWS/FLI1, ESE1, ESE2 (ELF5),
ESE3, PDEF, NET (ELK3; SAP2), NERF (ELF2), and FEV.
6. The transgenic animal of claim 1, wherein said gene fusions are
selected from the group consisting of SEQ ID NOs: 269-306.
7. The transgenic animal of claim 1, wherein said animal is a
mouse.
8. The transgenic animal of claim 1, wherein said animal displays
symptoms of prostatic intraepithelial neoplasia.
9. The transgenic animal of claim 1, wherein said animal displays
symptoms of prostate cancer.
Description
[0001] This application is a continuation in part of application
Ser. No. 11/519,397, filed Sep. 12, 2006, which claims priority to
provisional patent application Ser. Nos. 60/716,436, filed Sep. 12,
2005, 60/779,041, filed Mar. 3, 2006, 60/730,358, filed Oct. 27,
2005, and 60/795,590, filed Apr. 28, 2006, each of which is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to compositions and methods
for cancer diagnosis, research and therapy, including but not
limited to, cancer markers. In particular, the present invention
relates to recurrent gene fusion nucleic acids and transgenic
animals comprising recurrent gene fusion nucleic acids.
BACKGROUND OF THE INVENTION
[0004] A central aim in cancer research is to identify altered
genes that are causally implicated in oncogenesis. Several types of
somatic mutations have been identified including base
substitutions, insertions, deletions, translocations, and
chromosomal gains and losses, all of which result in altered
activity of an oncogene or tumor suppressor gene. First
hypothesized in the early 1900's, there is now compelling evidence
for a causal role for chromosomal rearrangements in cancer (Rowley,
Nat Rev Cancer 1: 245 (2001)). Reccurent chromosomal aberrations
were thought to be primarily characteristic of leukemias,
lymphomas, and sarcomas. Epithelial tumors (carcinomas), which are
much more common and contribute to a relatively large fraction of
the morbidity and mortality associated with human cancer, comprise
less than 1% of the known, disease-specific chromosomal
rearrangements (Mitelman, Mutat Res 462: 247 (2000)). While
hematological malignancies are often characterized by balanced,
disease-specific chromosomal rearrangements, most solid tumors have
a plethora of non-specific chromosomal aberrations. It is thought
that the karyotypic complexity of solid tumors is due to secondary
alterations acquired through cancer evolution or progression.
[0005] Two primary mechanisms of chromosomal rearrangements have
been described. In one mechanism, promoter/enhancer elements of one
gene are rearranged adjacent to a proto-oncogene, thus causing
altered expression of an oncogenic protein. This type of
translocation is exemplified by the apposition of immunoglobulin
(IG) and T-cell receptor (TCR) genes to MYC leading to activation
of this oncogene in B- and T-cell malignancies, respectively
(Rabbitts, Nature 372: 143 (1994)). In the second mechanism,
rearrangement results in the fusion of two genes, which produces a
fusion protein that may have a new function or altered activity.
The prototypic example of this translocation is the BCR-ABL gene
fusion in chronic myelogenous leukemia (CML) (Rowley, Nature 243:
290 (1973); de Klein et al., Nature 300: 765 (1982)). Importantly,
this finding led to the rational development of imatinib mesylate
(Gleevec), which successfully targets the BCR-ABL kinase (Deininger
et al., Blood 105: 2640 (2005)). Thus, identifying recurrent gene
rearrangements in common epithelial tumors may have profound
implications for cancer drug discovery efforts as well as patient
treatment.
SUMMARY OF THE INVENTION
[0006] The present invention relates to compositions and methods
for cancer diagnosis, research and therapy, including but not
limited to, cancer markers. In particular, the present invention
relates to recurrent gene fusion nucleic acids and transgenic
animals comprising recurrent gene fusion nucleic acids.
[0007] Accordingly, in some embodiments, the present invention
provides a composition comprising an isolated gene fusion nucleic
acid having a 5' portion from a transcriptional regulatory region
of an androgen regulated gene and a 3' portion from an ETS family
member gene. In some embodiments, the androgen regulated gene is
TMPRSS2 or PSA. In certain embodiments, the transcriptional
regulatory region of the androgen regulated gene comprises a
promoter region of the androgen regulated gene (e.g., an androgen
response element (ARE) of the androgen regulated gene). In some
embodiments, the ETS family member gene is ERG, ETV1 (ER81), FLI1,
ETS1, ETS2, ELK1, ETV6 (TEL1), ETV7 (TEL2), GABP.alpha., ELF1, ETV4
(E1AF; PEA3), ETV5 (ERM), ERF, PEA3/E1AF, PU.1, ESE1/ESX, SAP1
(ELK4), ETV3 (METS), EWS/FLI1, ESE1, ESE2 (ELF5), ESE3, PDEF, NET
(ELK3; SAP2), NERF (ELF2), or FEV. In some embodiments, the gene
fusions are selected from SEQ ID NOs: 269-306.
[0008] In other embodiments, the present invention provide a vector
comprising the gene fusion nucleic acid compositions described
herein.
[0009] The present invention further comprises a transgenic animal
comprising a genome that comprises a heterologous nucleic acid,
wherein said heterologous nucleic acid comprises a gene fusion
having a 5' portion from a transcriptional regulatory region of an
androgen regulated gene and a 3' portion from an ETS family member
gene. In some embodiments, the animal is a mouse. In certain
embodiments, the transgenic mouse develops prostate cancer or
pre-cancerous lesions (e.g., prostatic intraepithelial
neoplasia).
[0010] Additional embodiments of the present invention are provided
in the description and examples below.
DESCRIPTION OF THE FIGURES
[0011] FIG. 1 shows the Cancer Outlier Profile Analysis (COPA) of
microarray data. (A) ETV1 (left panels) and ERG (middle panels)
expression (normalized expression units) are shown from all
profiled samples in two large scale gene expression studies. (B) As
in (A), except data from laser capture microdissected samples were
used. (C) As in (A), except oncogenes (FGFR3 and CCND1) with known
translocations to the immunoglobulin heavy chain promoter (IgH) in
multiple myeloma were examined.
[0012] FIG. 2 shows the identification and characterization of
TMPRSS2:ETV1 and TMPRSS2:ERG gene fusions in prostate cancer (PCA).
(A) Prostate cancer cell lines (DuCaP, LnCaP and VCaP) and hormone
refractory metastatic (MET) prostate cancer tissues were analyzed
for ERG (.box-solid.) and ETV1 (.quadrature.) mRNA expression by
quantitative PCR (QPCR). (B) Loss of over-expression of ETV1 exons
2 and 3 in MET26 compared to LNCaP cells. (C) Schematic of 5' RNA
ligase-mediated rapid amplification of cDNA ends (RLM-RACE) results
for ETV1 in MET26-LN and ERG in MET28-LN revealing gene fusions
with TMPRSS2. (D) Validation of TMPRSS2:ETV1 expression using
translocation-specific QPCR in MET26-LN and MET26-RP. (E)
Validation of TMPRSS2:ERG expression using translocation-specific
QPCR in cell lines and PCA specimens.
[0013] FIG. 3 shows interphase fluorescence in situ hybridization
(FISH) on formalin-fixed paraffin embedded tissue sections that
confirms TMPRSS2:ETV1 gene fusion and ERG gene rearrangement. (A
and B) show two-color, fusion-signal approach to detect the fusion
of TMPRSS2 (green signal) and ETV1 (red signal). (C and D)
Detection of ERG gene rearrangements using a two-color split-signal
approach with two probes spanning the 5' (green signal) and 3' (red
signal) regions of ERG. (E) Matrix representation of FISH results
using the same probes as (A-D) on an independent tissue microarray
containing cores from 13 cases of clinically localized prostate
cancer (PCA) and 16 cases of metastatic prostate cancer (MET).
[0014] FIG. 4 shows androgen regulation of ERG in prostate cancer
cells carrying the TMPRSS2:ERG translocation.
[0015] FIG. 5 shows Cancer Outlier Profile Analysis (COPA). FIG. 5A
shows a schematic of COPA analysis. FIG. 5B shows that RUNX1T1
(ETO) had the highest scoring outlier profile at the 90th
percentile in the Valk et al. acute myeloid leukemia dataset
(n=293).
[0016] FIG. 6 shows a schematic of RNA ligase-mediated rapid
amplification of cDNA ends (RLM-RACE) results for ETV1 in MET26-LN
and ERG in PCA4 revealing gene fusions with TMPRSS2 (TMPRSS2:ERGb
fusion).
[0017] FIG. 7 shows over-expression of ETS family members in
prostate cancer. Expression of all monitored ETS family members in
profiled benign prostate, prostatic intraepithelial neoplasia
(PIN), clinically localized prostate cancer and metastatic prostate
cancer from grossly dissected tissue (A) or tissue isolated by
laser capture microdissection (B) was visualized using
Oncomine.
[0018] FIG. 8 shows over expression of TMPRSS2 and ETV4 loci in a
prostate cancer case that over-expresses ETV4. A. Expression of the
indicated exons or region of ETV4 in pooled benign prostate tissue
(CPP), prostate cancers that did not over-express ETV4 and were
either TMPRSS2:ERG positive (PCA1-2) or negative (PCA3-4), and the
prostate cancer case from our LCM cohort with ETV4
over-overexpression (PCA5). B. RLM-RACE reveals fusion of sequences
upstream of TMPRSS2 with ETV4 in PCA5. C. Expression of
TMPRSS2:ETV4a and TMPRSS2:ETV4b in PCA5 by QPCR. D. Interphase
fluorescence in situ hybridization on formalin-fixed
paraffin-embedded tissue confirms fusion of TMPRSS2 and ETV4 loci
in PCA5.
[0019] FIG. 9 shows mRNA sequences of exemplary ETS family
genes.
[0020] FIG. 10 shows the mRNA sequence of TMPRSS2.
[0021] FIG. 11 shows TMPRSS2:ERG gene fusion analysis by FISH.
Panel A: Ideogram, depicting a break apart assay for the indirect
detection of TMPRSS2:ERG fusion. Panel B: Interphase nuclei of a
stromal cell (left) and a prostate cancer gland (right). Panel C:
Interphase nuclei of prostate cancer glands showing break apart and
simultaneous deletion as indicated by loss of the telomeric probe
(100.times. oil immersion objective magnification). Panel D.
Magnified view of boxed area in C demonstrating two nuclei with
break apart and loss of the telomeric probe. (60.times. oil
immersion objective magnification).
[0022] FIG. 12 shows Genomic deletions on chromosome 21 between ERG
and TMPRSS2. Panel A: Samples, including 6 cell lines, 13
xenografts and 11 metastatic PCA samples, were characterized for
TMPRSS2:ERG and TMPRSS2:ETV1 status (gray bars for negative and
blue bar for positive status), by qPCR and/or by FISH. Panel B:
Magnification of the green framed box in A. Panel C: Magnification
of the black framed box in A.
[0023] FIG. 13 shows TMPRSS2:ERG rearrangement in clinically
localized prostate cancer and association with pathological
parameters. Panel A. The TMPRSS2:ERG rearrangement was identified
in 49.2% of the primary PCA samples and 41.2% in the hormone naive
metastatic LN samples. Panel B. TMPRSS2:ERG rearranged tumors with
deletions tended to be observed in a higher percentage of PCA cases
with advanced tumor stage (p=0.03).
[0024] FIG. 14 shows known genes located on 21q22-23 between ERG
(centromeric) and TMPRSS2 (telomeric). Genes above the black line
are oriented 5'-centromeric to 3'-telomeric and genes below the
black line are oriented 5'-telomeric to 3'-centromeric. In the
lower half of the image, a magnification of the ERG locus is
depicted with FISH probes.
[0025] FIG. 15 shows `heterogenous` prostate cancer case
predominantly showing TMPRSS2:ERG rearrangement with the deletion
(nucleus on the right) and only small areas showing the TMPRSS2:ERG
rearrangement without the deletion (nucleus on the left).
[0026] FIG. 16 shows meta-analysis of genes located between TMPRSS2
and ERG across 8 published expression array datasets.
[0027] FIG. 17 shows that the FISH assay detects the characteristic
deletion associated with TMPRSS2:ERG gene fusion, which is
associated with disease progression. Panels A and B: For analyzing
the ERG rearrangement on chromosome 21q22.2, a break apart probe
system was applied, consisting of the Biotin-14-dCTP labeled BAC
clone RP11-24A11 (eventually conjugated to produce a red signal)
and the Digoxigenin-dUTP labeled BAC clone RP11-137J13 (eventually
conjugated to produce a green signal), spanning the neighboring
centromeric and telomeric region of the ERG locus, respectively.
Using this break apart probe system, a nucleus without ERG
rearrangement exhibits two pairs of juxtaposed red and green
signals. Juxtaposed red-green signals form a yellow fusion signal
(Panel B, arrow). Panel C: In a cumulative incidence regression
model, TMPRSS2:ERG was evaluated as a determinant for the
cumulative incidence or metastases or prostate cancer-specific
death.
[0028] FIG. 18 shows FLI1 overexpression without fusion
transcript.
[0029] FIG. 19 shows induction of ERG protein expression by
androgen in TMPRSS2-ERG+ cells.
[0030] FIG. 20 shows a schematic of the endogenous and fusion ERG
polypeptides.
[0031] FIG. 21 shows Nuclear interactors for ERG2.
[0032] FIG. 22 shows sequences for peptide antibody and aqua probe
generation against ERG1.
[0033] FIG. 23 shows sequences for peptide antibody and aqua probe
generation against ETV1.
[0034] FIG. 24 shows sequences for peptide antibody and aqua probe
generation against FLI1.
[0035] FIG. 25 shows sequences for peptide antibody and aqua probe
generation against ETV4.
[0036] FIG. 26 shows the over-expression and androgen regulation of
ETV1 in the LNCaP prostate cancer cell line. FIG. 26A shows
expression signature of androgen-regulated genes in VCaP and LNCaP
prostate cancer cell lines. FIG. 26B shows confirmation of PSA
induction by androgen in both VCaP and LNCaP cells by quantitative
PCR (QPCR). FIG. 26C shows ETV1 induction by androgen in LNCaP
cells. FIG. 26D shows that ETV1 is markedly over-expressed in LNCaP
cells.
[0037] FIG. 27 shows rearrangement of ETV1 in LNCaP cells. FIG. 27A
shows a schematic of BACs used as probes for fluorescence in situ
hybridization (FISH). FIG. 27B shows that RP11-124L22 and
RP11-1149J13 co-localize to chromosome 7 in normal peripheral
lymphocytes (NPLs). FIG. 27C shows localization of BAC #1 and BAC
#4 on metaphase spreads (top panel) and interphase cells (bottom
panel) was determined in the near tetraploid LNCaP cell line. FIG.
27D shows signal from RP11-124L22 localizes to chromosome 14 in
LNCaP cells.
[0038] FIG. 28 shoes that the entire ETV1 locus is inserted into
chromosome 14 in LNCaP cells. FIG. 28A shows a schematic of BACs
used in this experiment. FIG. 28B shows localization of RP11-124L22
(BAC #1) and RP11-313C20 (BAC #2) on metaphase spreads (top panel)
and interphase cells (bottom panel) was determined by FISH in LNCaP
cells.
[0039] FIG. 29 shows siRNA knockdown of ETV1 in LnCaP.
[0040] FIG. 30 shows siRNA knockdown of ERG in VCAP.
[0041] FIG. 31 shows viral overexpression systems.
[0042] FIG. 32 shows a schematic of transgenic mice.
[0043] FIG. 33 shows detection of ERG and ETV1 transcripts in
urine. FIG. 33A shows detection of ERG and ETV1 in LNCaP (high ETV1
expression) or VCaP (high ERG and TMPRSS2:ERG expression) prostate
cancer cells. FIG. 33B shows detection of ERG and ETV1 in urine of
patients suspected of having prostate cancer.
[0044] FIG. 34 shows assays used to detect TMPRSS2:ETS gene fusions
in prostate cancer. FIG. 34A shows break apart assays for TMPRSS2
and ERG. An ERG rearrangement positive case (without deletion), as
indicated by one pair of split 5' and 3' signals, is shown in the
left panel. A TMPRSS2 rearrangement positive case (with deletion),
as indicated by a loss of one 3' signal, is shown in the right
panel. FIG. 34B shows a fusion assay for TMPRSS2:ETV1 gene fusions.
FIG. 34C shows a break apart assay for ETV4.
[0045] FIG. 35 shows TMPRSS2, ERG, ETV1 and ETV4 rearrangements as
detected by FISH. FIG. 35A shows a Table of results for
rearrangements in. TMPRSS2, ERG, ETV1 and ETV4 as detected by the
assays shown in FIG. 34. FIG. 34B shows a heat map representation
of the TMPRSS2, ERG, ETV1 and ETV4 status from the 38 cases where
all four assays were evaluable as described in A. FIG. 34C shows a
heat map representation of cases with discordant TMPRSS2 and ETS
rearrangement status.
[0046] FIG. 36 shows the sequences of gene fusions of the present
invention.
[0047] FIG. 37 shows primers and probes for FLI-1 expression
analysis.
[0048] FIG. 38 shows construction and expression verification of
transgenes recapitulating TMPRSS2:ETS gene fusions. A. Structures
of human TMPRSS2 (NM.sub.--005656.2, pink); ERG1
(NM.sub.--182918.2, blue) and ETV1 (NM.sub.--004956.3, green). B.
Quantitative real time PCR (QPCR).
[0049] FIG. 39 shows histological characterization of TMRPSS2:ETS
transgenic mice demonstrates mouse prostatic intraepithelial
neoplasia (mPIN) at 12 weeks of age. Prostates from 12 week old
transgenic mice expressing (A) ERG (Pb-ERG1) or (B) ETV1 (Pb-ETV1)
were characterized after H&E staining and the anterior prostate
is shown.
DEFINITIONS
[0050] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below:
[0051] As used herein, the term "animal" refers to any animal
(e.g., a mammal), including, but not limited to, humans, non-human
primates, rodents (e.g., mice, rats, etc.), flies, and the
like.
[0052] As used herein, the term "non-human animals" refers to all
non-human animals including, but are not limited to, vertebrates
such as rodents, non-human primates, ovines, bovines, ruminants,
lagomorphs, porcines, caprines, equines, canines, felines, ayes,
etc.
[0053] The term "transgene" as used herein refers to a foreign,
heterologous, or autologous gene that is placed into an organism by
introducing the gene into newly fertilized eggs or early embryos.
The term "foreign gene" refers to any nucleic acid (e.g., gene
sequence) that is introduced into the genome of an animal by
experimental manipulations and may include gene sequences found in
that animal so long as the introduced gene does not reside in the
same location as does the naturally-occurring gene.
[0054] As used herein, the term "transgenic animal" refers to any
animal containing a transgene.
[0055] As used herein, the term "gene fusion" refers to a chimeric
genomic DNA, a chimeric messenger RNA, a truncated protein or a
chimeric protein resulting from the fusion of at least a portion of
a first gene to at least a portion of a second gene. The gene
fusion need not include entire genes or exons of genes.
[0056] As used herein, the term "transcriptional regulatory region"
refers to the non-coding upstream regulatory sequence of a gene,
also called the 5' untranslated region (5'UTR).
[0057] As used herein, the term "androgen regulated gene" refers to
a gene or portion of a gene whose expression is initiated or
enhanced by an androgen (e.g., testosterone). The promoter region
of an androgen regulated gene may contain an "androgen response
element" that interacts with androgens or androgen signaling
molecules (e.g., downstream signaling molecules).
[0058] As used herein the term "symptoms of prostatic
intraepithelial neoplasia (PIN)" refers to any symptom of PIN
(e.g., in a transgenic animal). Examples include, but are not
limited to, pathological evidence of PIN (e.g., in a prostate
biopsy), increased levels of prostate specific antigen, enlarged
prostate, etc.
[0059] As used herein the term "prostate cancer" refers to any
symptom of PIN (e.g., in a transgenic animal). Examples include,
but are not limited to, pathological evidence of PIN (e.g., in a
prostate biopsy), increased levels of prostate specific antigen,
etc.
[0060] As used herein, the terms "detect", "detecting", or
"detection" may describe either the general act of discovering or
discerning or the specific observation of a detectably labeled
composition.
[0061] As used herein, the term "inhibits at least one biological
activity of a gene fusion" refers to any agent that decreases any
activity of a gene fusion of the present invention (e.g.,
including, but not limited to, the activities described herein),
via directly contacting gene fusion protein, contacting gene fusion
mRNA or genomic DNA, causing conformational changes of gene fusion
polypeptides, decreasing gene fusion protein levels, or interfering
with gene fusion interactions with signaling partners, and
affecting the expression of gene fusion target genes. Inhibitors
also include molecules that indirectly regulate gene fusion
biological activity by intercepting upstream signaling
molecules.
[0062] As used herein, the term "siRNAs" refers to small
interfering RNAs. In some embodiments, siRNAs comprise a duplex, or
double-stranded region, of about 18-25 nucleotides long; often
siRNAs contain from about two to four unpaired nucleotides at the
3' end of each strand. At least one strand of the duplex or
double-stranded region of a siRNA is substantially homologous to,
or substantially complementary to, a target RNA molecule. The
strand complementary to a target RNA molecule is the "antisense
strand;" the strand homologous to the target RNA molecule is the
"sense strand," and is also complementary to the siRNA antisense
strand. siRNAs may also contain additional sequences; non-limiting
examples of such sequences include linking sequences, or loops, as
well as stem and other folded structures. siRNAs appear to function
as key intermediaries in triggering RNA interference in
invertebrates and in vertebrates, and in triggering
sequence-specific RNA degradation during posttranscriptional gene
silencing in plants.
[0063] The term "RNA interference" or "RNAi" refers to the
silencing or decreasing of gene expression by siRNAs. It is the
process of sequence-specific, post-transcriptional gene silencing
in animals and plants, initiated by siRNA that is homologous in its
duplex region to the sequence of the silenced gene. The gene may be
endogenous or exogenous to the organism, present integrated into a
chromosome or present in a transfection vector that is not
integrated into the genome. The expression of the gene is either
completely or partially inhibited. RNAi may also be considered to
inhibit the function of a target RNA; the function of the target
RNA may be complete or partial.
[0064] As used herein, the term "stage of cancer" refers to a
qualitative or quantitative assessment of the level of advancement
of a cancer. Criteria used to determine the stage of a cancer
include, but are not limited to, the size of the tumor and the
extent of metastases (e.g., localized or distant).
[0065] As used herein, the term "gene transfer system" refers to
any means of delivering a composition comprising a nucleic acid
sequence to a cell or tissue. For example, gene transfer systems
include, but are not limited to, vectors (e.g., retroviral,
adenoviral, adeno-associated viral, and other nucleic acid-based
delivery systems), microinjection of naked nucleic acid,
polymer-based delivery systems (e.g., liposome-based and metallic
particle-based systems), biolistic injection, and the like. As used
herein, the term "viral gene transfer system" refers to gene
transfer systems comprising viral elements (e.g., intact viruses,
modified viruses and viral components such as nucleic acids or
proteins) to facilitate delivery of the sample to a desired cell or
tissue. As used herein, the term "adenovirus gene transfer system"
refers to gene transfer systems comprising intact or altered
viruses belonging to the family Adenoviridae.
[0066] As used herein, the term "site-specific recombination target
sequences" refers to nucleic acid sequences that provide
recognition sequences for recombination factors and the location
where recombination takes place.
[0067] As used herein, the term "nucleic acid molecule" refers to
any nucleic acid containing molecule, including but not limited to,
DNA or RNA. The term encompasses sequences that include any of the
known base analogs of DNA and RNA including, but not limited to,
4-acetylcytosine, 8-hydroxy-N-6-methyladenosine,
aziridinylcytosine, pseudoisocytosine,
5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0068] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide, precursor, or RNA (e.g., rRNA; tRNA).
The polypeptide can be encoded by a full length coding sequence or
by any portion of the coding sequence so long as the desired
activity or functional properties (e.g., enzymatic activity, ligand
binding, signal transduction, immunogenicity, etc.) of the
full-length or fragment are retained. The term also encompasses the
coding region of a structural gene and the sequences located
adjacent to the coding region on both the 5' and 3' ends for a
distance of about 1 kb or more on either end such that the gene
corresponds to the length of the full-length mRNA. Sequences
located 5' of the coding region and present on the mRNA are
referred to as 5' non-translated sequences. Sequences located 3' or
downstream of the coding region and present on the mRNA are
referred to as 3' non-translated sequences. The term "gene"
encompasses both cDNA and genomic forms of a gene. A genomic form
or clone of a gene contains the coding region interrupted with
non-coding sequences termed "introns" or "intervening regions" or
"intervening sequences." Introns are segments of a gene that are
transcribed into nuclear RNA (hnRNA); introns may contain
regulatory elements such as enhancers. Introns are removed or
"spliced out" from the nuclear or primary transcript; introns
therefore are absent in the messenger RNA (mRNA) transcript. The
mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide.
[0069] As used herein, the term "heterologous gene" refers to a
gene that is not in its natural environment. For example, a
heterologous gene includes a gene from one species introduced into
another species. A heterologous gene also includes a gene native to
an organism that has been altered in some way (e.g., mutated, added
in multiple copies, linked to non-native regulatory sequences,
etc). Heterologous genes are distinguished from endogenous genes in
that the heterologous gene sequences are typically joined to DNA
sequences that are not found naturally associated with the gene
sequences in the chromosome or are associated with portions of the
chromosome not found in nature (e.g., genes expressed in loci where
the gene is not normally expressed).
[0070] As used herein, the term "oligonucleotide," refers to a
short length of single-stranded polynucleotide chain.
Oligonucleotides are typically less than 200 residues long (e.g.,
between 15 and 100), however, as used herein, the term is also
intended to encompass longer polynucleotide chains.
Oligonucleotides are often referred to by their length. For example
a 24 residue oligonucleotide is referred to as a "24-mer".
Oligonucleotides can form secondary and tertiary structures by
self-hybridizing or by hybridizing to other polynucleotides. Such
structures can include, but are not limited to, duplexes, hairpins,
cruciforms, bends, and triplexes.
[0071] As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides) related by the base-pairing rules. For
example, the sequence "5'-A-G-T-3'," is complementary to the
sequence "3'-T-C-A-5'." Complementarity may be "partial," in which
only some of the nucleic acids' bases are matched according to the
base pairing rules. Or, there may be "complete" or "total"
complementarity between the nucleic acids. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of hybridization between
nucleic acid strands. This is of particular importance in
amplification reactions, as well as detection methods that depend
upon binding between nucleic acids.
[0072] The term "homology" refers to a degree of complementarity.
There may be partial homology or complete homology (i.e.,
identity). A partially complementary sequence is a nucleic acid
molecule that at least partially inhibits a completely
complementary nucleic acid molecule from hybridizing to a target
nucleic acid is "substantially homologous." The inhibition of
hybridization of the completely complementary sequence to the
target sequence may be examined using a hybridization assay
(Southern or Northern blot, solution hybridization and the like)
under conditions of low stringency. A substantially homologous
sequence or probe will compete for and inhibit the binding (i.e.,
the hybridization) of a completely homologous nucleic acid molecule
to a target under conditions of low stringency. This is not to say
that conditions of low stringency are such that non-specific
binding is permitted; low stringency conditions require that the
binding of two sequences to one another be a specific (i.e.,
selective) interaction. The absence of non-specific binding may be
tested by the use of a second target that is substantially
non-complementary (e.g., less than about 30% identity); in the
absence of non-specific binding the probe will not hybridize to the
second non-complementary target.
[0073] When used in reference to a double-stranded nucleic acid
sequence such as a cDNA or genomic clone, the term "substantially
homologous" refers to any probe that can hybridize to either or
both strands of the double-stranded nucleic acid sequence under
conditions of low stringency as described above.
[0074] A gene may produce multiple RNA species that are generated
by differential splicing of the primary RNA transcript. cDNAs that
are splice variants of the same gene will contain regions of
sequence identity or complete homology (representing the presence
of the same exon or portion of the same exon on both cDNAs) and
regions of complete non-identity (for example, representing the
presence of exon "A" on cDNA 1 wherein cDNA 2 contains exon "B"
instead). Because the two cDNAs contain regions of sequence
identity they will both hybridize to a probe derived from the
entire gene or portions of the gene containing sequences found on
both cDNAs; the two splice variants are therefore substantially
homologous to such a probe and to each other.
[0075] When used in reference to a single-stranded nucleic acid
sequence, the term "substantially homologous" refers to any probe
that can hybridize (i.e., it is the complement of) the
single-stranded nucleic acid sequence under conditions of low
stringency as described above.
[0076] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids.
Hybridization and the strength of hybridization (i.e., the strength
of the association between the nucleic acids) is impacted by such
factors as the degree of complementary between the nucleic acids,
stringency of the conditions involved, the T.sub.m of the formed
hybrid, and the G:C ratio within the nucleic acids. A single
molecule that contains pairing of complementary nucleic acids
within its structure is said to be "self-hybridized."
[0077] As used herein the term "stringency" is used in reference to
the conditions of temperature, ionic strength, and the presence of
other compounds such as organic solvents, under which nucleic acid
hybridizations are conducted. Under "low stringency conditions" a
nucleic acid sequence of interest will hybridize to its exact
complement, sequences with single base mismatches, closely related
sequences (e.g., sequences with 90% or greater homology), and
sequences having only partial homology (e.g., sequences with 50-90%
homology). Under "medium stringency conditions," a nucleic acid
sequence of interest will hybridize only to its exact complement,
sequences with single base mismatches, and closely relation
sequences (e.g., 90% or greater homology). Under "high stringency
conditions," a nucleic acid sequence of interest will hybridize
only to its exact complement, and (depending on conditions such a
temperature) sequences with single base mismatches. In other words,
under conditions of high stringency the temperature can be raised
so as to exclude hybridization to sequences with single base
mismatches.
[0078] "High stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/lNaH.sub.2PO.sub.4H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 0.1.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
[0079] "Medium stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 1.0.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
[0080] "Low stringency conditions" comprise conditions equivalent
to binding or hybridization at 42.degree. C. in a solution
consisting of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l
NaH.sub.2PO.sub.4H.sub.2O and 1.85 g/l EDTA, pH adjusted to 7.4
with NaOH), 0.1% SDS, 5.times.Denhardt's reagent
[50.times.Denhardt's contains per 500 ml: 5 g Ficoll (Type 400,
Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 .mu.g/ml denatured
salmon sperm DNA followed by washing in a solution comprising
5.times.SSPE, 0.1% SDS at 42.degree. C. when a probe of about 500
nucleotides in length is employed.
[0081] The art knows well that numerous equivalent conditions may
be employed to comprise low stringency conditions; factors such as
the length and nature (DNA, RNA, base composition) of the probe and
nature of the target (DNA, RNA, base composition, present in
solution or immobilized, etc.) and the concentration of the salts
and other components (e.g., the presence or absence of formamide,
dextran sulfate, polyethylene glycol) are considered and the
hybridization solution may be varied to generate conditions of low
stringency hybridization different from, but equivalent to, the
above listed conditions. In addition, the art knows conditions that
promote hybridization under conditions of high stringency (e.g.,
increasing the temperature of the hybridization and/or wash steps,
the use of formamide in the hybridization solution, etc.) (see
definition above for "stringency").
[0082] As used herein, the term "amplification oligonucleotide"
refers to an oligonucleotide that hybridizes to a target nucleic
acid, or its complement, and participates in a nucleic acid
amplification reaction. An example of an amplification
oligonucleotide is a "primer" that hybridizes to a template nucleic
acid and contains a 3' OH end that is extended by a polymerase in
an amplification process. Another example of an amplification
oligonucleotide is an oligonucleotide that is not extended by a
polymerase (e.g., because it has a 3' blocked end) but participates
in or facilitates amplification. Amplification oligonucleotides may
optionally include modified nucleotides or analogs, or additional
nucleotides that participate in an amplification reaction but are
not complementary to or contained in the target nucleic acid.
Amplification oligonucleotides may contain a sequence that is not
complementary to the target or template sequence. For example, the
5' region of a primer may include a promoter sequence that is
non-complementary to the target nucleic acid (referred to as a
"promoter-primer"). Those skilled in the art will understand that
an amplification oligonucleotide that functions as a primer may be
modified to include a 5' promoter sequence, and thus function as a
promoter-primer. Similarly, a promoter-primer may be modified by
removal of, or synthesis without, a promoter sequence and still
function as a primer. A 3' blocked amplification oligonucleotide
may provide a promoter sequence and serve as a template for
polymerization (referred to as a "promoter-provider").
[0083] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, that is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product that is
complementary to a nucleic acid strand is induced, (i.e., in the
presence of nucleotides and an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer is
preferably single stranded for maximum efficiency in amplification,
but may alternatively be double stranded. If double stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and the use
of the method.
[0084] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, that is
capable of hybridizing to at least a portion of another
oligonucleotide of interest. A probe may be single-stranded or
double-stranded. Probes are useful in the detection, identification
and isolation of particular gene sequences. It is contemplated that
any probe used in the present invention will be labeled with any
"reporter molecule," so that is detectable in any detection system,
including, but not limited to enzyme (e.g., ELISA, as well as
enzyme-based histochemical assays), fluorescent, radioactive, and
luminescent systems. It is not intended that the present invention
be limited to any particular detection system or label.
[0085] The term "isolated" when used in relation to a nucleic acid,
as in "an isolated oligonucleotide" or "isolated polynucleotide"
refers to a nucleic acid sequence that is identified and separated
from at least one component or contaminant with'which it is
ordinarily associated in its natural source. Isolated nucleic acid
is such present in a form or setting that is different from that in
which it is found in nature. In contrast, non-isolated nucleic
acids as nucleic acids such as DNA and RNA found in the state they
exist in nature. For example, a given DNA sequence (e.g., a gene)
is found on the host cell chromosome in proximity to neighboring
genes; RNA sequences, such as a specific mRNA sequence encoding a
specific protein, are found in the cell as a mixture with numerous
other mRNAs that encode a multitude of proteins. However, isolated
nucleic acid encoding a given protein includes, by way of example,
such nucleic acid in cells ordinarily expressing the given protein
where the nucleic acid is in a chromosomal location different from
that of natural cells, or is otherwise flanked by a different
nucleic acid sequence than that found in nature. The isolated
nucleic acid, oligonucleotide, or polynucleotide may be present in
single-stranded or double-stranded form. When an isolated nucleic
acid, oligonucleotide or polynucleotide is to be utilized to
express a protein, the oligonucleotide or polynucleotide will
contain at a minimum the sense or coding strand (i.e., the
oligonucleotide or polynucleotide may be single-stranded), but may
contain both the sense and anti-sense strands (i.e., the
oligonucleotide or polynucleotide may be double-stranded).
[0086] As used herein, the term "purified" or "to purify" refers to
the removal of components (e.g., contaminants) from a sample. For
example, antibodies are purified by removal of contaminating
non-immunoglobulin proteins; they are also purified by the removal
of immunoglobulin that does not bind to the target molecule. The
removal of non-immunoglobulin proteins and/or the removal of
immunoglobulins that do not bind to the target molecule results in
an increase in the percent of target-reactive immunoglobulins in
the sample. In another example, recombinant polypeptides are
expressed in bacterial host cells and the polypeptides are purified
by the removal of host cell proteins; the percent of recombinant
polypeptides is thereby increased in the sample.
DETAILED DESCRIPTION OF THE INVENTION
[0087] The present invention is based on the discovery of recurrent
gene fusions in prostate cancer. The present invention provides
diagnostic, research, and therapeutic methods that either directly
or indirectly detect or target the gene fusions. The present
invention also provides compositions for diagnostic, research, and
therapeutic purposes.
I. Gene Fusions
[0088] The present invention identifies recurrent gene fusions
indicative of prostate cancer. The gene fusions are the result of a
chromosomal rearrangement of an androgen regulated gene (ARG) and
an ETS family member gene. Despite their recurrence, the junction
where the ARG fuses to the ETS family member gene varies. The gene
fusions typically comprise a 5' portion from a transcriptional
regulatory region of an ARG and a 3' portion from an ETS family
member gene. In some embodiments, the present invention provides
gene fusion nucleic acids as well as vectors and transgenic animals
comprising the gene fusion nucleic acids. The recurrent gene
fusions have use as diagnostic markers, in research applications,
and as clinical targets for prostate cancer.
[0089] A. Androgen Regulated Genes Genes regulated by androgenic
hormones are of critical importance for the normal physiological
function of the human prostate gland. They also contribute to the
development and progression of prostate carcinoma. Recognized ARGs
include, but are not limited to: TMPRSS2; PSA; PSMA; KLK2; SNRK;
Seladin-1; and, FKBP51 (Paoloni-Giacobino et al., Genomics 44: 309
(1997); Velasco et al., Endocrinology 145(8): 3913 (2004)). TMPRSS2
(NM.sub.--005656), in particular, has been demonstrated to be
highly expressed in prostate epithelium relative to other normal
human tissues (Lin et al., Cancer Research 59: 4180 (1999)). The
TMPRSS2 gene is located on chromosome 21. This gene is located at
41,750,797-41,801,948 by from the pter (51,151 total bp; minus
strand orientation). The human TMPRSS2 protein sequence may be
found at GenBank accession no. AAC51784 (Swiss Protein accession
no. 015393)) and the corresponding cDNA at GenBank accession no.
U75329 (see also, Paoloni-Giacobino, et al., Genomics 44: 309
(1997)).
[0090] The transcriptional regulatory region of an ARG may contain
coding or non-coding regions of the ARG, including the promoter
region. The promoter region of the ARG may further contain an
androgen response element (ARE) of the ARG. The promoter region for
TMPRSS2, in particular, is provided by GenBank accession number
AJ276404.
[0091] B. ETS Family Member Genes
[0092] The ETS family of transcription factors regulate the
intra-cellular signaling pathways controlling gene expression. As
downstream effectors, they activate or repress specific target
genes. As upstream effectors, they are responsible for the spacial
and temporal expression of numerous growth factor receptors. Almost
30 members of this family have been identified and implicated in a
wide range of physiological and pathological processes. These
include, but are not limited to: ERG; ETV1 (ER81); FLI1; ETS1;
ETS2; ELK1; ETV6 (TEL1); ETV7 (TEL2); GABP.alpha.; ELF1; ETV4
(E1AF; PEA3); ETV5 (ERM); ERF; PEA3/E1AF; PU.1; ESE1/ESX; SAP1
(ELK4); ETV3 (METS); EWS/FLI1; ESE1; ESE2 (ELF5); ESE3; PDEF; NET
(ELK3; SAP2); NERF (ELF2); and FEV. Exemplary ETS family member
gene sequences are given in FIG. 9.
[0093] ERG (NM.sub.--004449), in particular, has been demonstrated
to be highly expressed in prostate epithelium relative to other
normal human tissues. The ERG gene is located on chromosome 21. The
gene is located at 38,675,671-38,955,488 base pairs from the pter.
The ERG gene is 279,817 total bp; minus strand orientation. The
corresponding ERG cDNA and protein sequences are given at GenBank
accesssion no. M17254 and GenBank accession no. NP04440 (Swiss
Protein acc. no. P11308), respectively.
[0094] The ETV1 gene is located on chromosome 7 (GenBank accession
nos. NC.sub.--000007.11; NC.sub.--086703.11; and
NT.sub.--007819.15). The gene is located at 13,708330-13,803,555
base pairs from the pter. The ETV1 gene is 95,225 by total, minus
strand orientation. The corresponding ETV1 cDNA and protein
sequences are given at GenBank accession no. NM.sub.--004956 and
GenBank accession no. NP.sub.--004947 (Swiss protein acc. no.
P50549), respectively.
[0095] The human ETV4 gene is located on chromosome 14 (GenBank
accession nos. NC.sub.--000017.9; NT.sub.--010783.14; and
NT.sub.--086880.1). The gene is at 38,960,740-38,979,228 base pairs
from the pter. The ETV4 gene is 18,488 by total, minus strand
orientation. The corresponding ETV4 cDNA and protein sequences are
given at GenBank accession no. NM.sub.--001986 and GenBank
accession no. NP.sub.--01977 (Swiss protein acc. no. P43268),
respectively.
[0096] C. ARG/ETS Gene Fusions
As described above, the present invention provides fusions of an
ARG to an ETS family member gene. Exemplary gene fusion sequences
are given in FIG. 36. For all involved genes (TMPRSS2, ERG, ETV1
and ETV4), the GenBank reference sequence ID's are provided and the
exons are aligned using the May 2004 assembly of the UCSC Human
Genome. For all identified fusions, FIG. 36 provides a complete
sequence from the beginning of the TMPRSS2 gene through the fusion
and the stop codon of the ETS family member gene. The deposited
GenBank sequence for each of the published variants is also
provided. Some TMPRSS2:ERG and TMPRSS2:ETV1 fusions are described
by the breakpoint exons of TMPRSS2 and the ETS family member gene.
For example, TMPRSS2:ERGa, which fuses exon 1 of TMPRSS2 to exons 4
through 11 of ERG, is identified as TMPRSS2:ERG(1,4).
[0097] The fusion of an ARG to an ETS family member gene is
detectable as DNA, RNA or protein. Initially, the gene fusion is
detectable as a chromosomal rearrangement of genomic DNA having a
5' portion from a transcriptional regulatory region of the ARG and
a 3' portion from the ETS family member gene. Once transcribed, the
gene fusion is detectable as a chimeric mRNA having a 5' portion
from the transcriptional regulatory region of the ARG and a 3'
portion from the ETS family member gene. Once translated, the gene
fusion is detectable as an amino-terminally truncated ETS family
member protein resulting from the fusion of the transcriptional
regulatory region of the ARG to the ETS family member gene; a
chimeric protein having an amino-terminal portion from the
transcriptional regulatory region of the ARG and a carboxy-terminal
portion from the ETS family member gene; or, an upregulated, but
otherwise indistinguishable, native ETS family member protein. The
truncated ETS family member protein and chimeric protein may differ
from their respective native proteins in amino acid sequence,
post-translational processing and/or secondary, tertiary or
quaternary structure. Such differences, if present, can be used to
identify the presence of the gene fusion. Specific methods of
detection are described in more detail below.
[0098] Certain gene fusions are more common than others in prostate
cancer. The present invention identifies 50-80% of prostate cancers
as having recurrent gene fusions of TMPRSS2 with ERG, ETV1, ETV4,
or FLI1. Of those, 50-70% are TMPRSS2-ERG, 50%-60% of which result
from the deletion of genetic information between the TMPRSS2 and
ERG locus on chromosome 21 (described in more detail below), 5-10%
are TMPRSS2-ETV1, 1-2% are TMPRSS2-ETV4, and 1-2% are
TMPRSS2--FLI1.
[0099] Experiments conducted during the course of development of
the present invention indicated that certain fusion genes express
fusion transcripts, while others do not express a functional
transcript (Tomlins et al., Science, 310: 644-648 (2005); Tomlins
et al., Cancer Research 66: 3396-3400 (2006)).
[0100] Further experiments conducted during the course of
development of the present invention identified significant genomic
deletions located between TMPRSS2 and ERG on chromosome 21q22.2-3.
Deletions were seen in TMPRSS2:ERG fusion positive PCA samples. The
deletions appear in a consensus area but show variability within
this area. In previously published work by Paris et al. (Hum. Mol.
Genet. 13:1303-13 (2004)), CGH analysis detected deletions in the
CTD-210307 BAC that is 6 kb centromeric from TMPRSS2. These
deletions were observed in 12.5% (9/72) of clinically localized PCA
samples and 33% (5/15) of the metastatic PCA samples. These results
support the SNP array data from the current study and suggests that
either PCA deletions become more common with progression or that
deletions are identified more often in PCA that tend to progress
more rapidly. Given the striking intra-tumoral homogeneity of the
TMPRSS2:ERG rearrangements, it is more likely that these molecular
sub-types are associated with different disease progression
characteristics.
[0101] One hundred eighteen clinically localized PCA cases with
49.2% harboring rearrangement of ERG were evaluated. Intronic
deletions were observed in 60.3% of these TMPRSS2:ERG fusion
positive cases. Almost all PCA samples with marked over expression
of ERG have a rearrangement, and the over expression occurs in
about the same number of cases as the rearrangement. Using
Oncomine, a publicly available compendium of gene expression data,
4 significantly down regulated genes located in the area of the
common deletion site were identified (FIG. 16). The present
invention is not limited to a particular mechanism. Indeed, an
understanding of the mechanism is not necessary to practice the
present invention. Nonetheless, the results suggest that nearly
half of all PCAs can be defined by the TMPRSS2:ERG rearrangement.
The majority of these tumors demonstrate an intronic deletion,
which according to the oligonucleotide SNP array genomic analysis
is variable in size. However, approximately 30-40% did not
demonstrate a deletion and thus might harbor a balanced
translocation of TMRPSS2 and ERG. This variability in the extent of
the deletion may be associated with disease progression as has been
observed with CML. The current study identified significant
clinical associations with tumor stage and lymph node status.
TMPRSS2:ERG rearranged tumors with deletion also showed a trend
towards higher rates of PSA biochemical failure. Additional
experiments conducted during the course of development of the
present invention explored the risk of developing metastases or
prostate cancer specific death based on the presence of the
TMPRSS2:ERG gene fusion in a watchful waiting cohort of early
prostate cancers with long term follow-up. The frequency of the
TMPRSS2:ERG gene fusion was assessed using 92 cases. The frequency
of TMPRSS2:ERG gene fusion in this population-based cohort was
15.2% (14/92), lower than the 50% frequency observed in two
hospital-based cohorts. The present invention is not limited to a
particular mechanism. Indeed, an understanding of the mechanism is
not necessary to practice the present invention. Nonetheless, this
difference in TMPRSS2:ERG gene fusion prostate cancers may be due
to ethnic and racial genetic differences. These differences may
also be explained by the lower percentage of high grade cases in
this watchful waiting cohort as compared to the other
non-population based studies.
[0102] A significant association between TMPRSS2:ERG gene fusion
and development of distant metastases and prostate cancer specific
death was observed with a cumulative incidence ratio of 3.6
(P=0.004, 95% confidence interval=1.5 to 8.9). These data suggest
that TMPRSS2:ERG gene fusion prostate cancers have a more
aggressive phenotype. Further experiments indicated that genomic
deletions in the TMPRSS2:ERG gene fusion were correlated with
advanced and/or metastatic prostate cancer (See e.g., Example
5).
[0103] The present invention has also demonstrated that androgen
can induce the overexpression of ERG, presumably through AREs, in a
TMPRSS2-ERG-positive cell line. The present invention is not
limited to a particular mechanism. Indeed, an understanding of the
mechanism is not necessary to practice the present invention.
Nonetheless, collectively, the results suggest that dysregulation
of ETS family activity through AREs upstream of TMPRSS2 may drive
prostate cancer development.
[0104] It is contemplated that the presence, molecular sub-type or
amount of gene fusion expression is correlated with the stage,
aggressiveness or progression of the disease, or the presence or
risk of metastasis. It is further contemplated that similar
recurrent gene fusions involving ETS family member genes occur in
other epithelial cancers.
II. Antibodies
[0105] The gene fusion proteins of the present invention, including
fragments, derivatives and analogs thereof, may be used as
immunogens to produce antibodies having use in the diagnostic,
research, and therapeutic methods described below. The antibodies
may be polyclonal or monoclonal, chimeric, humanized, single chain
or Fab fragments. Various procedures known to those of ordinary
skill in the art may be used for the production and labeling of
such antibodies and fragments. See, e.g., Burns, ed.,
Immunochemical Protocols, 3.sup.rd ed., Humana Press (2005); Harlow
and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory (1988); Kozbor et al., Immunology Today 4: 72 (1983);
Kohler and Milstein, Nature 256: 495 (1975). Antibodies or
fragments exploiting the differences between the truncated ETS
family member protein or chimeric protein and their respective
native proteins are particularly preferred.
III. Diagnostic Applications
[0106] The present invention provides DNA, RNA and protein based
diagnostic methods that either directly or indirectly detect the
gene fusions. The present invention also provides compositions and
kits for diagnostic purposes.
[0107] The diagnostic methods of the present invention may be
qualitative or quantitative. Quantitative diagnostic methods may be
used, for example, to discriminate between indolent and aggressive
cancers via a cutoff or threshold level. Where applicable,
qualitative or quantitative diagnostic methods may also include
amplification of target, signal or intermediary (e.g., a universal
primer).
[0108] An initial assay may confirm the presence of a gene fusion
but not identify the specific fusion. A secondary assay is then
performed to determine the identity of the particular fusion, if
desired. The second assay may use a different detection technology
than the initial assay.
[0109] The gene fusions of the present invention may be detected
along with other markers in a multiplex or panel format. Markers
are selected for their predictive value alone or in combination
with the gene fusions. Exemplary prostate cancer markers include,
but are not limited to: AMACR/P504S (U.S. Pat. No. 6,262,245); PCA3
(U.S. Pat. No. 7,008,765); PCGEM1 (U.S. Pat. No. 6,828,429);
prostein/P501S, P503S, P504S, P509S, P510S, prostase/P703P, P710P
(U.S. Publication No. 20030185830); and, those disclosed in U.S.
Pat. Nos. 5,854,206 and 6,034,218, and U.S. Publication No.
20030175736, each of which is herein incorporated by reference in
its entirety. Markers for other cancers, diseases, infections, and
metabolic conditions are also contemplated for inclusion in a
multiplex of panel format.
[0110] The diagnostic methods of the present invention may also be
modified with reference to data correlating particular gene fusions
with the stage, aggressiveness or progression of the disease or the
presence or risk of metastasis. Ultimately, the information
provided by the methods of the present invention will assist a
physician in choosing the best course of treatment for a particular
patient.
[0111] A. Sample
[0112] Any patient sample suspected of containing the gene fusions
may be tested according to the methods of the present invention. By
way of non-limiting examples, the sample may be tissue (e.g., a
prostate biopsy sample or a tissue sample obtained by
prostatectomy), blood, urine, semen, prostatic secretions or a
fraction thereof (e.g., plasma, serum, urine supernatant, urine
cell pellet or prostate cells). A urine sample is preferably
collected immediately following an attentive digital rectal
examination (DRE), which causes prostate cells from the prostate
gland to shed into the urinary tract.
[0113] The patient sample typically requires preliminary processing
designed to isolate or enrich the sample for the gene fusions or
cells that contain the gene fusions. A variety of techniques known
to those of ordinary skill in the art may be used for this purpose,
including but not limited: centrifugation; immunocapture; cell
lysis; and, nucleic acid target capture (See, e.g., EP Pat. No. 1
409 727, herein incorporated by reference in its entirety).
[0114] B. DNA and RNA Detection
[0115] The gene fusions of the present invention may be detected as
chromosomal rearrangements of genomic DNA or chimeric mRNA using a
variety of nucleic acid techniques known to those of ordinary skill
in the art, including but not limited to: nucleic acid sequencing;
nucleic acid hybridization; and, nucleic acid amplification.
[0116] 1. Sequencing
[0117] Illustrative non-limiting examples of nucleic acid
sequencing techniques include, but are not limited to, chain
terminator (Sanger) sequencing and dye terminator sequencing. Those
of ordinary skill in the art will recognize that because RNA is
less stable in the cell and more prone to nuclease attack
experimentally RNA is usually reverse transcribed to DNA before
sequencing.
[0118] Chain terminator sequencing uses sequence-specific
termination of a DNA synthesis reaction using modified nucleotide
substrates. Extension is initiated at a specific site on the
template DNA by using a short radioactive, or other labeled,
oligonucleotide primer complementary to the template at that
region. The oligonucleotide primer is extended using a DNA
polymerase, standard four deoxynucleotide bases, and a low
concentration of one chain terminating nucleotide, most commonly a
di-deoxynucleotide. This reaction is repeated in four separate
tubes with each of the bases taking turns as the
di-deoxynucleotide. Limited incorporation of the chain terminating
nucleotide by the DNA polymerase results in a series of related DNA
fragments that are terminated only at positions where that
particular di-deoxynucleotide is used. For each reaction tube, the
fragments are size-separated by electrophoresis in a slab
polyacrylamide gel or a capillary tube filled with a viscous
polymer. The sequence is determined by reading which lane produces
a visualized mark from the labeled primer as you scan from the top
of the gel to the bottom.
[0119] Dye terminator sequencing alternatively labels the
terminators. Complete sequencing can be performed in a single
reaction by labeling each of the di-deoxynucleotide
chain-terminators with a separate fluorescent dye, which fluoresces
at a different wavelength.
[0120] 2. Hybridization
[0121] Illustrative non-limiting examples of nucleic acid
hybridization techniques include, but are not limited to, in situ
hybridization (ISH), microarray, and Southern or Northern blot.
[0122] In situ hybridization (ISH) is a type of hybridization that
uses a labeled complementary DNA or RNA strand as a probe to
localize a specific DNA or RNA sequence in a portion or section of
tissue (in situ), or, if the tissue is small enough, the entire
tissue (whole mount ISH). DNA ISH can be used to determine the
structure of chromosomes. RNA ISH is used to measure and localize
mRNAs and other transcripts within tissue sections or whole mounts.
Sample cells and tissues are usually treated to fix the target
transcripts in place and to increase access of the probe. The probe
hybridizes to the target sequence at elevated temperature, and then
the excess probe is washed away. The probe that was labeled with
either radio-, fluorescent- or antigen-labeled bases is localized
and quantitated in the tissue using either autoradiography,
fluorescence microscopy or immunohistochemistry, respectively. ISH
can also use two or more probes, labeled with radioactivity or the
other non-radioactive labels, to simultaneously detect two or more
transcripts.
[0123] 2.1 FISH
[0124] In some embodiments, fusion sequences are detected using
fluorescence in situ hybridization (FISH). The preferred FISH
assays for the present invention utilize bacterial artificial
chromosomes (BACs). These have been used extensively in the human
genome sequencing project (see Nature 409: 953-958 (2001)) and
clones containing specific BACs are available through distributors
that can be located through many sources, e.g., NCBI. Each BAC
clone from the human genome has been given a reference name that
unambiguously identifies it. These names can be used to find a
corresponding GenBank sequence and to order copies of the clone
from a distributor.
[0125] In some embodiments, the detection assay is a FISH assay
utilizing a probe for ETV1 (e.g., bac RP11-692L4), a set of probes
for c-ERG:t-ERG break apart (e.g., bac RP11-24A11 and as a probe
for t-ERG RP11-372017 or RP11-137J13). In other embodiments, the
FISH assay is performed by testing for ETV1 deletion or
amplification with a set of probes, wherein one probe spans the
ETV1 locus (e.g., bac RP11-692L4) and the other probe hybridizes to
chromosome 7 (e.g., a probe on the centromere of the chromosome).
In still further embodiments, the method is performed by testing
for ERG deletion or amplification with a set of probes, one
spanning the ERG locus (e.g., bac RP11-476D17) and one reference
probe on chromosome 21 (e.g., PR11-32L6; RP11-752M23; RP11-1107H21;
RP11-639A7 or RP11-1077M21). In yet other embodiments, the method
is performed by testing for TMPRSS2 deletion/amplification with a
set of probes, one spanning the TMPRSS2 (e.g., RP11-121A5;
RP11-120C17; PR11-814F13; or RR11-535H11) locus and one reference
probe on chromosome 21 (e.g., PR11-32L6; RP11-752M23; RP11-1107H21;
RP11-639A7 or RP11-1077M21). In some embodiments, the method
further comprises a hybridization using a probe selected from the
group including, but not limited to RF'11-121A5; RP11-120C17;
PR11-814F13; and RR11-535H11.
[0126] The present invention further provides a method of
performing a FISH assay on human prostate cells, human prostate
tissue or on the fluid surrounding said human prostate cells or
human prostate tissue. In some embodiments, the assay comprises a
hybridization step utilizing a probe selected from the group
including, but not limited to, RP11-372017; RP11-137J13;
RP11-692L4; RP11-476D17; PR11-32L6; RP11-752M23; RP11-1107H21;
RP11-639A7; RP11-1077M21; RP11-121A5; RP11-120C17; PR11-814F13; and
RR11-535H11.
[0127] Specific BAC clones that can be used in FISH protocols to
detect rearrangements relevant to the present invention are as
follows: [0128] For testing for an ETV1-TMPRSS2 fusion, one probe
spanning the ETV1 and one spanning the TMPRSS2 locus may be used:
[0129] BAC for ETV1: RP11-692L4 [0130] BAC for TMPRSS2: RP11-121A5,
(RP11-120C17, PR11-814F13, RR11-535H11) [0131] Testing ERG
translocation with set of probes for c-ERG:t-ERG break apart:
[0132] BAC for c-ERG: RP11-24A11 [0133] BACs for t-ERG:
RP11-372017, RP11-137J13 [0134] Testing ETV1 deletion/amplification
with set of probes, one spanning the ETV1 locus and one reference
probe on chromosome 7: [0135] BAC for ETV1: RP11-692L4 [0136]
Testing ERG deletion/amplification with set of probes, one spanning
the ERG locus and one reference probe on chromosome 21: [0137] BAC
for ERG: RP11-476D17 [0138] BACs for reference probe on chromosome
21: * [0139] Testing TMPRSS2 deletion/amplification with set of
probes, one spanning the TMPRSS2 locus and one reference probe on
chromosome 21: [0140] BACs for TMPRSS2: RP11-121A5, (RP11-120C17,
PR11-814F13, RR11-535H11) [0141] BACs for reference probe on
chromosome 21: PR11-32L6, RP11-752M23, RP11-1107H21, RP11-639A 7,
(RP11-1077M21).
[0142] The most preferred probes for detecting a deletion mutation
resulting in a fusion between TMPRSS2 and ERG are RP11-24A11 and
RP11-137J13. These probes, or those described above, are labeled
with appropriate fluorescent or other markers and then used in
hybridizations. The Examples section provided herein sets forth one
particular protocol that is effective for measuring deletions but
one of skill in the art will recognize that many variations of this
assay can be used equally well. Specific protocols are well known
in the art and can be readily adapted for the present invention.
Guidance regarding methodology may be obtained from many references
including: In situ Hybridization: Medical Applications (eds. G. R.
Coulton and J. de Belleroche), Kluwer Academic Publishers, Boston
(1992); In situ Hybridization: In Neurobiology; Advances in
Methodology (eds. J. H. Eberwine, K. L. Valentino, and J. D.
Barchas), Oxford University Press Inc., England (1994); In situ
Hybridization: A Practical Approach (ed. D. G. Wilkinson), Oxford
University Press Inc., England (1992)); Kuo, et al., Am. J. Hum.
Genet. 49:112-119 (1991); Klinger, et al., Am. J. Hum. Genet.
51:55-65 (1992); and Ward, et al., Am. J. Hum. Genet. 52:854-865
(1993)). There are also kits that are commercially available and
that provide protocols for performing FISH assays (available from
e.g., Oncor, Inc., Gaithersburg, Md.). Patents providing guidance
on methodology include U.S. Pat. Nos. 5,225,326; 5,545,524;
6,121,489 and 6,573,043. All of these references are hereby
incorporated by reference in their entirety and may be used along
with similar references in the art and with the information
provided in the Examples section herein to establish procedural
steps convenient for a particular laboratory.
[0143] Table 13 below shows additional BAC clones that find use as
FISH probes.
TABLE-US-00001 TABLE 13 Gene Chromosome RefSeq 5' BAC 3' BAC Paired
EHF 11p13 NM_012153 RP5-1135K18 RP5-1002E13 2 ELF1 13q14 NM_172373
RP11-88n4 RP11-53f19 ELF2 4q28 NM_201999.1 RP11-22o8 RP11-375P1
ELF3 1q32 NM_004433 RP11-25B7 RP11-246J15 ELF4 Xq25 NM_001421
RP5-875H3 RP4-753P9 ELF5 11p13 NM_001422.2 RP5-1002E13 RP5-1135K18
2 ELK1 Xp11 NM_005229 RP1-54B20 RP1-306D1 ELK3 12q22 NM_005230
RP11-69E3 RP11-510I5 ELK4 1q32 NM_001973.2 RP11-131E5 RP11-249h15
ERF 19q13 NM_006494.1 RP11-208I3 RP11-317E13 ERG 21q22 NM_004449.3
RP11-137J13 RP11-24A11 1 ETS1 11q24 NM_005238.2 RP11-254C5
RP11-112m22 ETS2 21q22 NM_005239.4 RP11-24A11 RP11-137J13 1 ETV1
7p21 NM_004956.3 RP11-1149J13 RP11-34C22 ETV2 19q13 NM_014209.1
RP11-32h17 RP11-92j4 ETV3 1q23 NM_005240.1 RP11-91G5 RP11-1038N13 3
ETV4 17q21 NM_001986.1 RP11-436J4 RP11-100E5 ETV5 3q27 NM_004454.1
RP11-379C23 RP11-1144N13 ETV6 12p13 NM_001987.3 RP11-90N7 RP11-59h1
ETV7 6p21 NM_016135.2 RP3-431A14 RP1-179N16 FEV 2q35 NM_017521.2
RP11-316O14 RP11-129D2 FLI1 11q24 NM_002017.2 RP11-112M22
RP11-75P14 FLJ16478 1q23 NM_001004341 RP11-91G5 RP11-1038N13 3
SPDEF 6p21 NM_012391.1 RP11-79j23 RP11-119c22 SPI1 11p11
NM_016135.2 RP11-56e13 RP11-29o22 SPIB 19q13 NM_003121.2
RP11-510I16 RP11-26P14 SPIC 12q23 NM_152323.1 RP11-426H24
RP11-938C1 TMPRSS2 21q22 NM_005656.2 RP11-35C4 RP11-120C17
[0144] 2.2 Microarrays
[0145] Different kinds of biological assays are called microarrays
including, but not limited to: DNA microarrays (e.g., cDNA
microarrays and oligonucleotide microarrays); protein microarrays;
tissue microarrays; transfection or cell microarrays; chemical
compound microarrays; and, antibody microarrays. A DNA microarray,
commonly known as gene chip, DNA chip, or biochip, is a collection
of microscopic DNA spots attached to a solid surface (e.g., glass,
plastic or silicon chip) forming an array for the purpose of
expression profiling or monitoring expression levels for thousands
of genes simultaneously. The affixed DNA segments are known as
probes, thousands of which can be used in a single DNA microarray.
Microarrays can be used to identify disease genes by comparing gene
expression in disease and normal cells. Microarrays can be
fabricated using a variety of technologies, including but not
limiting: printing with fine-pointed pins onto glass slides;
photolithography using pre-made masks; photolithography using
dynamic micromirror devices; ink-jet printing; or, electrochemistry
on microelectrode arrays.
[0146] Southern and Northern blotting is used to detect specific
DNA or RNA sequences, respectively. DNA or RNA extracted from a
sample is fragmented, electrophoretically separated on a matrix
gel, and transferred to a membrane filter. The filter bound DNA or
RNA is subject to hybridization with a labeled probe complementary
to the sequence of interest. Hybridized probe bound to the filter
is detected. A variant of the procedure is the reverse Northern
blot, in which the substrate nucleic acid that is affixed to the
membrane is a collection of isolated DNA fragments and the probe is
RNA extracted from a tissue and labeled.
[0147] 3. Amplification
[0148] Chromosomal rearrangements of genomic DNA and chimeric mRNA
may be amplified prior to or simultaneous with detection.
Illustrative non-limiting examples of nucleic acid amplification
techniques include, but are not limited to, polymerase chain
reaction (PCR), reverse transcription polymerase chain reaction
(RT-PCR), transcription-mediated amplification (TMA), ligase chain
reaction (LCR), strand displacement amplification (SDA), and
nucleic acid sequence based amplification (NASBA). Those of
ordinary skill in the art will recognize that certain amplification
techniques (e.g., PCR) require that RNA be reversed transcribed to
DNA prior to amplification (e.g., RT-PCR), whereas other
amplification techniques directly amplify RNA (e.g., TMA and
NASBA).
[0149] The polymerase chain reaction (U.S. Pat. Nos. 4,683,195,
4,683,202, 4,800,159 and 4,965,188, each of which is herein
incorporated by reference in its entirety), commonly referred to as
PCR, uses multiple cycles of denaturation, annealing of primer
pairs to opposite strands, and primer extension to exponentially
increase copy numbers of a target nucleic acid sequence. In a
variation called RT-PCR, reverse transcriptase (RT) is used to make
a complementary DNA (cDNA) from mRNA, and the cDNA is then
amplified by PCR to produce multiple copies of DNA. For other
various permutations of PCR see, e.g., U.S. Pat. Nos. 4,683,195,
4,683,202 and 4,800,159; Mullis et al., Meth. Enzymol. 155: 335
(1987); and, Murakawa et al., DNA 7: 287 (1988), each of which is
herein incorporated by reference in its entirety.
[0150] Transcription mediated amplification (U.S. Pat. Nos.
5,480,784 and 5,399,491, each of which is herein incorporated by
reference in its entirety), commonly referred to as TMA,
synthesizes multiple copies of a target nucleic acid sequence
autocatalytically under conditions of substantially constant
temperature, ionic strength, and pH in which multiple RNA copies of
the target sequence autocatalytically generate additional copies.
See, e.g., U.S. Pat. Nos. 5,399,491 and 5,824,518, each of which is
herein incorporated by reference in its entirety. In a variation
described in U.S. Publ. No. 20060046265 (herein incorporated by
reference in its entirety), TMA optionally incorporates the use of
blocking moieties, terminating moieties, and other modifying
moieties to improve TMA process sensitivity and accuracy.
[0151] The ligase chain reaction (Weiss, R., Science 254: 1292
(1991), herein incorporated by reference in its entirety), commonly
referred to as LCR, uses two sets of complementary DNA
oligonucleotides that hybridize to adjacent regions of the target
nucleic acid. The DNA oligonucleotides are covalently linked by a
DNA ligase in repeated cycles of thermal denaturation,
hybridization and ligation to produce a detectable double-stranded
ligated oligonucleotide product.
[0152] Strand displacement amplification (Walker, G. et al., Proc.
Natl. Acad. Sci. USA 89: 392-396 (1992); U.S. Pat. Nos. 5,270,184
and 5,455,166, each of which is herein incorporated by reference in
its entirety), commonly referred to as SDA, uses cycles of
annealing pairs of primer sequences to opposite strands of a target
sequence, primer extension in the presence of a dNTPaS to produce a
duplex hemiphosphorothioated primer extension product,
endonuclease-mediated nicking of a hemimodified restriction
endonuclease recognition site, and polymerase-mediated primer
extension from the 3' end of the nick to displace an existing
strand and produce a strand for the next round of primer annealing,
nicking and strand displacement, resulting in geometric
amplification of product. Thermophilic SDA (tSDA) uses thermophilic
endonucleases and polymerases at higher temperatures in essentially
the same method (EP Pat. No. 0 684 315).
[0153] Other amplification methods include, for example: nucleic
acid sequence based amplification (U.S. Pat. No. 5,130,238, herein
incorporated by reference in its entirety), commonly referred to as
NASBA; one that uses an RNA replicase to amplify the probe molecule
itself (Lizardi et al., BioTechnol. 6: 1197 (1988), herein
incorporated by reference in its entirety), commonly referred to as
Q.beta. replicase; a transcription based amplification method (Kwoh
et al., Proc. Natl. Acad. Sci. USA 86:1173 (1989)); and,
self-sustained sequence replication (Guatelli et al., Proc. Natl.
Acad. Sci. USA 87: 1874 (1990), each of which is herein
incorporated by reference in its entirety). For further discussion
of known amplification methods see Persing, David H., "In Vitro
Nucleic Acid Amplification Techniques" in Diagnostic Medical
Microbiology: Principles and Applications (Persing et al., Eds.),
pp. 51-87 (American Society for Microbiology, Washington, D.C.
(1993)).
[0154] 4. Detection Methods
[0155] Non-amplified or amplified gene fusion nucleic acids can be
detected by any conventional means. For example, the gene fusions
can be detected by hybridization with a detectably labeled probe
and measurement of the resulting hybrids. Illustrative non-limiting
examples of detection methods are described below.
[0156] One illustrative detection method, the Hybridization
Protection Assay (HPA) involves hybridizing a chemiluminescent
oligonucleotide probe (e.g., an acridinium ester-labeled (AE)
probe) to the target sequence, selectively hydrolyzing the
chemiluminescent label present on unhybridized probe, and measuring
the chemiluminescence produced from the remaining probe in a
luminometer. See, e.g., U.S. Pat. No. 5,283,174 and Norman C.
Nelson et al., Nonisotopic Probing, Blotting, and Sequencing, ch.
17 (Larry J. Kricka ed., 2d ed. 1995, each of which is herein
incorporated by reference in its entirety).
[0157] Another illustrative detection method provides for
quantitative evaluation of the amplification process in real-time.
Evaluation of an amplification process in "real-time" involves
determining the amount of amplicon in the reaction mixture either
continuously or periodically during the amplification reaction, and
using the determined values to calculate the amount of target
sequence initially present in the sample. A variety of methods for
determining the amount of initial target sequence present in a
sample based on real-time amplification are well known in the art.
These include methods disclosed in U.S. Pat. Nos. 6,303,305 and
6,541,205, each of which is herein incorporated by reference in its
entirety. Another method for determining the quantity of target
sequence initially present in a sample, but which is not based on a
real-time amplification, is disclosed in U.S. Pat. No. 5,710,029,
herein incorporated by reference in its entirety.
[0158] Amplification products may be detected in real-time through
the use of various self-hybridizing probes, most of which have a
stem-loop structure. Such self-hybridizing probes are labeled so
that they emit differently detectable signals, depending on whether
the probes are in a self-hybridized state or an altered state
through hybridization to a target sequence. By way of non-limiting
example, "molecular torches" are a type of self-hybridizing probe
that includes distinct regions of self-complementarity (referred to
as "the target binding domain" and "the target closing domain")
which are connected by a joining region (e.g., non-nucleotide
linker) and which hybridize to each other under predetermined
hybridization assay conditions. In a preferred embodiment,
molecular torches contain single-stranded base regions in the
target binding domain that are from 1 to about 20 bases in length
and are accessible for hybridization to a target sequence present
in an amplification reaction under strand displacement conditions.
Under strand displacement conditions, hybridization of the two
complementary regions, which may be fully or partially
complementary, of the molecular torch is favored, except in the
presence of the target sequence, which will bind to the
single-stranded region present in the target binding domain and
displace all or a portion of the target closing domain. The target
binding domain and the target closing domain of a molecular torch
include a detectable label or a pair of interacting labels (e.g.,
luminescent/quencher) positioned so that a different signal is
produced when the molecular torch is self-hybridized than when the
molecular torch is hybridized to the target sequence, thereby
permitting detection of probe:target duplexes in a test sample in
the presence of unhybridized molecular torches. Molecular torches
and a variety of types of interacting label pairs are disclosed in
U.S. Pat. No. 6,534,274, herein incorporated by reference in its
entirety.
[0159] Another example of a detection probe having
self-complementarity is a "molecular beacon." Molecular beacons
include nucleic acid molecules having a target complementary
sequence, an affinity pair (or nucleic acid arms) holding the probe
in a closed conformation in the absence of a target sequence
present in an amplification reaction, and a label pair that
interacts when the probe is in a closed conformation. Hybridization
of the target sequence and the target complementary sequence
separates the members of the affinity pair, thereby shifting the
probe to an open conformation. The shift to the open conformation
is detectable due to reduced interaction of the label pair, which
may be, for example, a fluorophore and a quencher (e.g., DABCYL and
EDANS). Molecular beacons are disclosed in U.S. Pat. Nos. 5,925,517
and 6,150,097, herein incorporated by reference in its
entirety.
[0160] Other self-hybridizing probes are well known to those of
ordinary skill in the art. By way of non-limiting example, probe
binding pairs having interacting labels, such as those disclosed in
U.S. Pat. No. 5,928,862 (herein incorporated by reference in its
entirety) might be adapted for use in the present invention. Probe
systems used to detect single nucleotide polymorphisms (SNPs) might
also be utilized in the present invention. Additional detection
systems include "molecular switches," as disclosed in U.S. Publ.
No. 20050042638, herein incorporated by reference in its entirety.
Other probes, such as those comprising intercalating dyes and/or
fluorochromes, are also useful for detection of amplification
products in the present invention. See, e.g., U.S. Pat. No.
5,814,447 (herein incorporated by reference in its entirety).
[0161] C. Protein Detection
[0162] The gene fusions of the present invention may be detected as
truncated ETS family member proteins or chimeric proteins using a
variety of protein techniques known to those of ordinary skill in
the art, including but not limited to: protein sequencing; and,
immunoassays.
[0163] 1. Sequencing
[0164] Illustrative non-limiting examples of protein sequencing
techniques include, but are not limited to, mass spectrometry and
Edman degradation.
[0165] Mass spectrometry can, in principle, sequence any size
protein but becomes computationally more difficult as size
increases. A protein is digested by an endoprotease, and the
resulting solution is passed through a high pressure liquid
chromatography column. At the end of this column, the solution is
sprayed out of a narrow nozzle charged to a high positive potential
into the mass spectrometer. The charge on the droplets causes them
to fragment until only single ions remain. The peptides are then
fragmented and the mass-charge ratios of the fragments measured.
The mass spectrum is analyzed by computer and often compared
against a database of previously sequenced proteins in order to
determine the sequences of the fragments. The process is then
repeated with a different digestion enzyme, and the overlaps in
sequences are used to construct a sequence for the protein.
[0166] In the Edman degradation reaction, the peptide to be
sequenced is adsorbed onto a solid surface (e.g., a glass fiber
coated with polybrene). The Edman reagent, phenylisothiocyanate
(PTC), is added to the adsorbed peptide, together with a mildly
basic buffer solution of 12% trimethylamine, and reacts with the
amine group of the N-terminal amino acid. The terminal amino acid
derivative can then be selectively detached by the addition of
anhydrous acid. The derivative isomerizes to give a substituted
phenylthiohydantoin, which can be washed off and identified by
chromatography, and the cycle can be repeated. The efficiency of
each step is about 98%, which allows about 50 amino acids to be
reliably determined.
[0167] 2. Immunoassays
[0168] Illustrative non-limiting examples of immunoassays include,
but are not limited to: immunoprecipitation; Western blot; ELISA;
immunohistochemistry; immunocytochemistry; flow cytometry; and,
immuno-PCR. Polyclonal or monoclonal antibodies detectably labeled
using various techniques known to those of ordinary skill in the
art (e.g., colorimetric, fluorescent, chemiluminescent or
radioactive) are suitable for use in the immunoassays.
[0169] Immunoprecipitation is the technique of precipitating an
antigen out of solution using an antibody specific to that antigen.
The process can be used to identify protein complexes present in
cell extracts by targeting a protein believed to be in the complex.
The complexes are brought out of solution by insoluble
antibody-binding proteins isolated initially from bacteria, such as
Protein A and Protein G. The antibodies can also be coupled to
sepharose beads that can easily be isolated out of solution. After
washing, the precipitate can be analyzed using mass spectrometry,
Western blotting, or any number of other methods for identifying
constituents in the complex.
[0170] A Western blot, or immunoblot, is a method to detect protein
in a given sample of tissue homogenate or extract. It uses gel
electrophoresis to separate denatured proteins by mass. The
proteins are then transferred out of the gel and onto a membrane,
typically polyvinyldiflroride or nitrocellulose, where they are
probed using antibodies specific to the protein of interest. As a
result, researchers can examine the amount of protein in a given
sample and compare levels between several groups.
[0171] An ELISA, short for Enzyme-Linked ImmunoSorbent Assay, is a
biochemical technique to detect the presence of an antibody or an
antigen in a sample. It utilizes a minimum of two antibodies, one
of which is specific to the antigen and the other of which is
coupled to an enzyme. The second antibody will cause a chromogenic
or fluorogenic substrate to produce a signal. Variations of ELISA
include sandwich ELISA, competitive ELISA, and ELISPOT. Because the
ELISA can be performed to evaluate either the presence of antigen
or the presence of antibody in a sample, it is a useful tool both
for determining serum antibody concentrations and also for
detecting the presence of antigen.
[0172] Immunohistochemistry and immunocytochemistry refer to the
process of localizing proteins in a tissue section or cell,
respectively, via the principle of antigens in tissue or cells
binding to their respective antibodies. Visualization is enabled by
tagging the antibody with color producing or fluorescent tags.
Typical examples of color tags include, but are not limited to,
horseradish peroxidase and alkaline phosphatase. Typical examples
of fluorophore tags include, but are not limited to, fluorescein
isothiocyanate (FITC) or phycoerythrin (PE).
[0173] Flow cytometry is a technique for counting, examining and
sorting microscopic particles suspended in a stream of fluid. It
allows simultaneous multiparametric analysis of the physical and/or
chemical characteristics of single cells flowing through an
optical/electronic detection apparatus. A beam of light (e.g., a
laser) of a single frequency or color is directed onto a
hydrodynamically focused stream of fluid. A number of detectors are
aimed at the point where the stream passes through the light beam;
one in line with the light beam (Forward Scatter or FSC) and
several perpendicular to it (Side Scatter (SSC) and one or more
fluorescent detectors). Each suspended particle passing through the
beam scatters the light in some way, and fluorescent chemicals in
the particle may be excited into emitting light at a lower
frequency than the light source. The combination of scattered and
fluorescent light is picked up by the detectors, and by analyzing
fluctuations in brightness at each detector, one for each
fluorescent emission peak, it is possible to deduce various facts
about the physical and chemical structure of each individual
particle. FSC correlates with the cell volume and SSC correlates
with the density or inner complexity of the particle (e.g., shape
of the nucleus, the amount and type of cytoplasmic granules or the
membrane roughness).
[0174] Immuno-polymerase chain reaction (IPCR) utilizes nucleic
acid amplification techniques to increase signal generation in
antibody-based immunoassays. Because no protein equivalence of PCR
exists, that is, proteins cannot be replicated in the same manner
that nucleic acid is replicated during PCR, the only way to
increase detection sensitivity is by signal amplification. The
target proteins are bound to antibodies which are directly or
indirectly conjugated to oligonucleotides. Unbound antibodies are
washed away and the remaining bound antibodies have their
oligonucleotides amplified. Protein detection occurs via detection
of amplified oligonucleotides using standard nucleic acid detection
methods, including real-time methods.
[0175] D. Data Analysis
[0176] In some embodiments, a computer-based analysis program is
used to translate the raw data generated by the detection assay
(e.g., the presence, absence, or amount of a given marker or
markers) into data of predictive value for a clinician. The
clinician can access the predictive data using any suitable means.
Thus, in some preferred embodiments, the present invention provides
the further benefit that the clinician, who is not likely to be
trained in genetics or molecular biology, need not understand the
raw data. The data is presented directly to the clinician in its
most useful form. The clinician is then able to immediately utilize
the information in order to optimize the care of the subject.
[0177] The present invention contemplates any method capable of
receiving, processing, and transmitting the information to and from
laboratories conducting the assays, information provides, medical
personal, and subjects. For example, in some embodiments of the
present invention, a sample (e.g., a biopsy or a serum or urine
sample) is obtained from a subject and submitted to a profiling
service (e.g., clinical lab at a medical facility, genomic
profiling business, etc.), located in any part of the world (e.g.,
in a country different than the country where the subject resides
or where the information is ultimately used) to generate raw data.
Where the sample comprises a tissue or other biological sample, the
subject may visit a medical center to have the sample obtained and
sent to the profiling center, or subjects may collect the sample
themselves (e.g., a urine sample) and directly send it to a
profiling center. Where the sample comprises previously determined
biological information, the information may be directly sent to the
profiling service by the subject (e.g., an information card
containing the information may be scanned by a computer and the
data transmitted to a computer of the profiling center using an
electronic communication systems). Once received by the profiling
service, the sample is processed and a profile is produced (i.e.,
expression data), specific for the diagnostic or prognostic
information desired for the subject.
[0178] The profile data is then prepared in a format suitable for
interpretation by a treating clinician. For example, rather than
providing raw expression data, the prepared format may represent a
diagnosis or risk assessment (e.g., likelihood of cancer being
present) for the subject, along with recommendations for particular
treatment options. The data may be displayed to the clinician by
any suitable method. For example, in some embodiments, the
profiling service generates a report that can be printed for the
clinician (e.g., at the point of care) or displayed to the
clinician on a computer monitor.
[0179] In some embodiments, the information is first analyzed at
the point of care or at a regional facility. The raw data is then
sent to a central processing facility for further analysis and/or
to convert the raw data to information useful for a clinician or
patient. The central processing facility provides the advantage of
privacy (all data is stored in a central facility with uniform
security protocols), speed, and uniformity of data analysis. The
central processing facility can then control the fate of the data
following treatment of the subject. For example, using an
electronic communication system, the central facility can provide
data to the clinician, the subject, or researchers.
[0180] In some embodiments, the subject is able to directly access
the data using the electronic communication system. The subject may
chose further intervention or counseling based on the results. In
some embodiments, the data is used for research use. For example,
the data may be used to further optimize the inclusion or
elimination of markers as useful indicators of a particular
condition or stage of disease.
[0181] E. In Vivo Imaging
[0182] The gene fusions of the present invention may also be
detected using in vivo imaging techniques, including but not
limited to: radionuclide imaging; positron emission tomography
(PET); computerized axial tomography, X-ray or magnetic resonance
imaging method, fluorescence detection, and chemiluminescent
detection. In some embodiments, in vivo imaging techniques are used
to visualize the presence of or expression of cancer markers in an
animal (e.g., a human or non-human mammal). For example, in some
embodiments, cancer marker mRNA or protein is labeled using a
labeled antibody specific for the cancer marker. A specifically
bound and labeled antibody can be detected in an individual using
an in vivo imaging method, including, but not limited to,
radionuclide imaging, positron emission tomography, computerized
axial tomography, X-ray or magnetic resonance imaging method,
fluorescence detection, and chemiluminescent detection. Methods for
generating antibodies to the cancer markers of the present
invention are described below.
[0183] The in vivo imaging methods of the present invention are
useful in the diagnosis of cancers that express the cancer markers
of the present invention (e.g., prostate cancer). In vivo imaging
is used to visualize the presence of a marker indicative of the
cancer. Such techniques allow for diagnosis without the use of an
unpleasant biopsy. The in vivo imaging methods of the present
invention are also useful for providing prognoses to cancer
patients. For example, the presence of a marker indicative of
cancers likely to metastasize can be detected. The in vivo imaging
methods of the present invention can further be used to detect
metastatic cancers in other parts of the body.
[0184] In some embodiments, reagents (e.g., antibodies) specific
for the cancer markers of the present invention are fluorescently
labeled. The labeled antibodies are introduced into a subject
(e.g., orally or parenterally). Fluorescently labeled antibodies
are detected using any suitable method (e.g., using the apparatus
described in U.S. Pat. No. 6,198,107, herein incorporated by
reference).
[0185] In other embodiments, antibodies are radioactively labeled.
The use of antibodies for in vivo diagnosis is well known in the
art. Sumerdon et al., (Nucl. Med. Biol 17:247-254 [1990] have
described an optimized antibody-chelator for the
radioimmunoscintographic imaging of tumors using Indium-111 as the
label. Griffin et al., (J Clin One 9:631-640 [1991]) have described
the use of this agent in detecting tumors in patients suspected of
having recurrent colorectal cancer. The use of similar agents with
paramagnetic ions as labels for magnetic resonance imaging is known
in the art (Lauffer, Magnetic Resonance in Medicine 22:339-342
[1991]). The label used will depend on the imaging modality chosen.
Radioactive labels such as Indium-111, Technetium-99m, or
Iodine-131 can be used for planar scans or single photon emission
computed tomography (SPECT). Positron emitting labels such as
Fluorine-19 can also be used for positron emission tomography
(PET). For MRI, paramagnetic ions such as Gadolinium (III) or
Manganese (II) can be used.
[0186] Radioactive metals with half-lives ranging from 1 hour to
3.5 days are available for conjugation to antibodies, such as
scandium-47 (3.5 days) gallium-67 (2.8 days), gallium-68 (68
minutes), technetium-99m (6 hours), and indium-111 (3.2 days), of
which gallium-67, technetium-99m, and indium-111 are preferable for
gamma camera imaging, gallium-68 is preferable for positron
emission tomography.
[0187] A useful method of labeling antibodies with such radiometals
is by means of a bifunctional chelating agent, such as
diethylenetriaminepentaacetic acid (DTPA), as described, for
example, by Khaw et al. (Science 209:295 [1980]) for In-111 and
Tc-99m, and by Scheinberg et al. (Science 215:1511 [1982]). Other
chelating agents may also be used, but the
1-(p-carboxymethoxybenzyl)EDTA and the carboxycarbonic anhydride of
DTPA are advantageous because their use permits conjugation without
affecting the antibody's immunoreactivity substantially.
[0188] Another method for coupling DPTA to proteins is b.sub.y use
of the cyclic anhydride of DTPA, as described by Hnatowich et al.
(Int. J. Appl. Radiat. Isot. 33:327 [1982]) for labeling of albumin
with In-111, but which can be adapted for labeling of antibodies. A
suitable method of labeling antibodies with Tc-99m which does not
use chelation with DPTA is the pretinning method of Crockford et
al., (U.S. Pat. No. 4,323,546, herein incorporated by
reference).
[0189] A preferred method of labeling immunoglobulins with Tc-99m
is that described by Wong et al. (Int. J. Appl. Radiat. Isot.,
29:251 [1978]) for plasma protein, and recently applied
successfully by Wong et al. (J. Nucl. Med., 23:229 [1981]) for
labeling antibodies.
[0190] In the case of the radiometals conjugated to the specific
antibody, it is likewise desirable to introduce as high a
proportion of the radiolabel as possible into the antibody molecule
without destroying its immunospecificity. A further improvement may
be achieved by effecting radiolabeling in the presence of the
specific cancer marker of the present invention, to insure that the
antigen binding site on the antibody will be protected. The antigen
is separated after labeling.
[0191] In still further embodiments, in vivo biphotonic imaging
(Xenogen, Almeda, Calif.) is utilized for in vivo imaging. This
real-time in vivo imaging utilizes luciferase. The luciferase gene
is incorporated into cells, microorganisms, and animals (e.g., as a
fusion protein with a cancer marker of the present invention). When
active, it leads to a reaction that emits light. A CCD camera and
software is used to capture the image and analyze it.
[0192] F. Compositions & Kits
[0193] Compositions for use in the diagnostic methods of the
present invention include, but are not limited to, probes,
amplification oligonucleotides, and antibodies. Particularly
preferred compositions detect a product only when an ARG fuses to
ETS family member gene. These compositions include: a single
labeled probe comprising a sequence that hybridizes to the junction
at which a 5' portion from a transcriptional regulatory region of
an ARG fuses to a 3' portion from an ETS family member gene (i.e.,
spans the gene fusion junction); a pair of amplification
oligonucleotides wherein the first amplification oligonucleotide
comprises a sequence that hybridizes to a transcriptional
regulatory region of an ARG and the second amplification
oligonucleotide comprises a sequence that hybridizes to an ETS
family member gene; an antibody to an amino-terminally truncated
ETS family member protein resulting from a fusion of a
transcriptional regulatory region of an ARG to an ETS family member
gene; or, an antibody to a chimeric protein having an
amino-terminal portion from a transcriptional regulatory region of
an ARG and a carboxy-terminal portion from an ETS family member
gene. Other useful compositions, however, include: a pair of
labeled probes wherein the first labeled probe comprises a sequence
that hybridizes to a transcriptional regulatory region of an ARG
and the second labeled probe comprises a sequence that hybridizes
to an ETS family member gene.
[0194] Any of these compositions, alone or in combination with
other compositions of the present invention, may be provided in the
form of a kit. For example, the single labeled probe and pair of
amplification oligonucleotides may be provided in a kit for the
amplification and detection of gene fusions of the present
invention. Kits may further comprise appropriate controls and/or
detection reagents.
[0195] The probe and antibody compositions of the present invention
may also be provided in the form of an array.
IV. Prognostic Applications
[0196] Experiments conducted during the course of development of
the present invention demonstrated a close correlation between gene
fusions of the present invention and the prognosis of patients with
prostate cancer (See e.g., Example 5 below). Especially in cases
where a fusion results from a deletion of the genomic DNA lying
between TMPRSS2 and ERG, it has been found that cancer cells assume
a more aggressive phenotype. Thus, in some embodiments, assays that
are capable of detecting gene fusions between TMPRSS2 and ERG in
which there has been a deletion of intervening DNA are used to
provide prognoses and help physicians decide on an appropriate
therapeutic strategy. For example, in some embodiments, patients
with tumors having this particular rearrangement are treated more
intensively since their prognosis is significantly worse than
patients that lack the rearrangement.
[0197] Any assay may be used to determine whether cells are present
having a rearrangement of the type discussed above (e.g., those
described above).
[0198] Although the present invention will most preferably be used
in connection with obtaining a prognosis for prostate cancer
patients, other epithelial cell tumors may also be examined and the
assays and probes described herein may be used in determining
whether cancerous cells from these tumors have rearrangements that
are likely to make them particularly aggressive, i.e., likely to be
invasive and metastatic. Examples of tumors that may be
characterized using this procedure include tumors of the breast,
lung, colon, ovary, uterus, esophagus, stomach, liver, kidney,
brain, skin and muscle. The assays will also be of value to
researchers studying these cancers in cell lines and animal
models.
[0199] Further experiments conducted during the course of
development of the present invention demonstrated that chromosomal
deletions can be detected by assaying samples to determine whether
there is a loss of expression of one or more genes located in the
deleted region. For example, approximately 2.8 megabases of genomic
DNA is typically deleted in forming a fusion between TMPRSS2 and
ERG and at least four genes lying in this area are lost when this
occurs. These are the ETS2 gene, the WRB gene, the PCP4 gene and
the MX1 gene. A decrease in one or more of these in cancerous
prostate cells suggests a poor prognosis.
[0200] Accordingly, in some embodiments, the present invention
provides a method of assaying epithelial cells for the deletion of
chromosomal DNA indicative of a cancer-associated rearrangement,
comprising performing a FISH assay using at least a first and a
second probe, wherein the first probe is at least 15 nucleotides in
length (e.g., at least 15, 20, 35, etc.); is bound to a first
fluorescent label; and hybridizes under stringent conditions to a
first sequence in the human genome wherein the first sequence
includes at least a portion of either an androgen responsive gene
(e.g., the TMPRSS2 gene) or a ETS family gene (e.g., the ERG gene,
the ETV1 gene, or the ETV4 gene); and the second probe: is at least
15 nucleotides in length; is bound to a second fluorescent label
that is different from the first fluorescent label; and hybridizes
under stringent conditions to a second sequence in the human genome
that is different from the first sequence and which includes at
least a portion of an androgen responsive gene (e.g., the TMPRSS2
gene) or a ETS family gene (e.g., the ERG gene, the ETV1 gene, or
the ETV4 gene).
[0201] In further embodiments, the present invention provides a
method for assaying epithelial cells (e.g., prostate cells) for a
deletion of genomic DNA indicative of a cancer-associated
rearrangement, comprising: obtaining a test sample of epithelial
cells; assaying the sample of epithelial cells to determine the
level of expression of one or more genes selected from the group
including, but not limited to, ETS2; WRB; PCP4; and MX1; comparing
the expression level determined in step b) with the level in a
control sample; and concluding that a deletion has occurred if the
level of expression determined for the gene in the test sample is
lower than that for a control sample.
V. Drug Screening Applications
[0202] In some embodiments, the present invention provides drug
screening assays (e.g., to screen for anticancer drugs). The
screening methods of the present invention utilize cancer markers
identified using the methods of the present invention (e.g.,
including but not limited to, the gene fusions described herein).
For example, in some embodiments, the present invention provides
methods of screening for compounds that alter (e.g., decrease) the
expression of cancer marker genes. The compounds or agents may
interfere with transcription, by interacting, for example, with the
promoter region. The compounds or agents may interfere with mRNA
produced from the fusion (e.g., by RNA interference, antisense
technologies, etc.). The compounds or agents may interfere with
pathways that are upstream or downstream of the biological activity
of the fusion. In some embodiments, candidate compounds are
antisense or interfering RNA agents (e.g., oligonucleotides)
directed against cancer markers. In other embodiments, candidate
compounds are antibodies or small molecules that specifically bind
to a cancer marker regulator or expression products of the present
invention and inhibit its biological function.
[0203] In one screening method, candidate compounds are evaluated
for their ability to alter cancer marker expression by contacting a
compound with a cell expressing a cancer marker and then assaying
for the effect of the candidate compounds on expression. In some
embodiments, the effect of candidate compounds on expression of a
cancer marker gene is assayed for by detecting the level of cancer
marker mRNA expressed by the cell. mRNA expression can be detected
by any suitable method. In other embodiments, the effect of
candidate compounds on expression of cancer marker genes is assayed
by measuring the level of polypeptide encoded by the cancer
markers. The level of polypeptide expressed can be measured using
any suitable method, including but not limited to, those disclosed
herein.
[0204] Specifically, the present invention provides screening
methods for identifying modulators, i.e., candidate or test
compounds or agents (e.g., proteins, peptides, peptidomimetics,
peptoids, small molecules or other drugs) which bind to cancer
markers of the present invention, have an inhibitory (or
stimulatory) effect on, for example, cancer marker expression or
cancer marker activity, or have a stimulatory or inhibitory effect
on, for example, the expression or activity of a cancer marker
substrate. Compounds thus identified can be used to modulate the
activity of target gene products (e.g., cancer marker genes) either
directly or indirectly in a therapeutic protocol, to elaborate the
biological function of the target gene product, or to identify
compounds that disrupt normal target gene interactions. Compounds
that inhibit the activity or expression of cancer markers are
useful in the treatment of proliferative disorders, e.g., cancer,
particularly prostate cancer.
[0205] In one embodiment, the invention provides assays for
screening candidate or test compounds that are substrates of a
cancer marker protein or polypeptide or a biologically active
portion thereof. In another embodiment, the invention provides
assays for screening candidate or test compounds that bind to or
modulate the activity of a cancer marker protein or polypeptide or
a biologically active portion thereof.
[0206] In certain embodiments, drug screening methods of the
present invention utilize transgenic animals comprising gene fusion
nucleic acids. In some embodiments, the transgenic animals have
tumors indicative of cancer (e.g., prostate cancer). For example,
in some embodiments, test compounds are administered to transgenic
animals of the present invention and the effect of the test
compound on transgene expression and/or function is assayed. In
other embodiments, test compounds are administered to transgenic
animals of the present invention and the effect of the test
compound on cancer in the animal is assessed. For example, in some
embodiments, the effect of the test compounds on the rate of
formation of precancerous lesions (e.g., prostatic intraepithelial
neoplasia), tumors (e.g., prostate tumors) and metastatic cancer
(e.g., prostate cancer) is assessed. In other embodiments, the
effect of the test compounds on progression of cancer (e.g.,
development of cancerous lesions from precancerous lesions or
development of metastatic cancer is assayed). In yet other
embodiments, the effect of test compounds on the rate of growth or
shrinkage of tumors is assayed. In still further embodiments, the
rate or recurrence following removal of cancers from transgenic
animals is assayed in the presence of test compounds.
[0207] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including biological libraries; peptoid
libraries (libraries of molecules having the functionalities of
peptides, but with a novel, non-peptide backbone, which are
resistant to enzymatic degradation but which nevertheless remain
bioactive; see, e.g., Zuckennann et al., J. Med. Chem. 37: 2678-85
[1994]); spatially addressable parallel solid phase or solution
phase libraries; synthetic library methods requiring deconvolution;
the `one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library and peptoid library approaches are preferred for use with
peptide libraries, while the other four approaches are applicable
to peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam (1997) Anticancer Drug Des. 12:145).
[0208] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al., Proc. Natl.
Acad. Sci. U.S.A. 90:6909 [1993]; Erb et al., Proc. Nad. Acad. Sci.
USA 91:11422 [1994]; Zuckermann et al., J. Med. Chem. 37:2678
[1994]; Cho et al., Science 261:1303 [1993]; Carrell et al., Angew.
Chem. Int. Ed. Engl. 33.2059 [1994]; Carell et al., Angew. Chem.
Int. Ed. Engl. 33:2061 [1994]; and Gallop et al., J. Med. Chem.
37:1233 [1994].
[0209] Libraries of compounds may be presented in solution (e.g.,
Houghten, Biotechniques 13:412-421 [1992]), or on beads (Lam,
Nature 354:82-84 [1991]), chips (Fodor, Nature 364:555-556 [1993]),
bacteria or spores (U.S. Pat. No. 5,223,409; herein incorporated by
reference), plasmids (Cull et al., Proc. Nad. Acad. Sci. USA
89:18651869 [1992]) or on phage (Scott and Smith, Science
249:386-390 [1990]; Devlin Science 249:404-406 [1990]; Cwirla et
al., Proc. Natl. Acad. Sci. 87:6378-6382 [1990]; Felici, J. Mol.
Biol. 222:301 [1991]).
[0210] In one embodiment, an assay is a cell-based assay in which a
cell that expresses a cancer marker mRNA or protein or biologically
active portion thereof is contacted with a test compound, and the
ability of the test compound to the modulate cancer marker's
activity is determined. Determining the ability of the test
compound to modulate cancer marker activity can be accomplished by
monitoring, for example, changes in enzymatic activity, destruction
or mRNA, or the like.
[0211] The ability of the test compound to modulate cancer marker
binding to a compound, e.g., a cancer marker substrate or
modulator, can also be evaluated. This can be accomplished, for
example, by coupling the compound, e.g., the substrate, with a
radioisotope or enzymatic label such that binding of the compound,
e.g., the substrate, to a cancer marker can be determined by
detecting the labeled compound, e.g., substrate, in a complex.
[0212] Alternatively, the cancer marker is coupled with a
radioisotope or enzymatic label to monitor the ability of a test
compound to modulate cancer marker binding to a cancer marker
substrate in a complex. For example, compounds (e.g., substrates)
can be labeled with .sup.125I, .sup.35S .sup.14C or .sup.3H, either
directly or indirectly, and the radioisotope detected by direct
counting of radioemmission or by scintillation counting.
Alternatively, compounds can be enzymatically labeled with, for
example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
[0213] The ability of a compound (e.g., a cancer marker substrate)
to interact with a cancer marker with or without the labeling of
any of the interactants can be evaluated. For example, a
microphysiorneter can be used to detect the interaction of a
compound with a cancer marker without the labeling of either the
compound or the cancer marker (McConnell et al. Science
257:1906-1912 [1992]). As used herein, a "microphysiometer" (e.g.,
Cytosensor) is an analytical instrument that measures the rate at
which a cell acidifies its environment using a light-addressable
potentiometric sensor (LAPS). Changes in this acidification rate
can be used as an indicator of the interaction between a compound
and cancer markers.
[0214] In yet another embodiment, a cell-free assay is provided in
which a cancer marker protein or biologically active portion
thereof is contacted with a test compound and the ability of the
test compound to bind to the cancer marker protein, mRNA, or
biologically active portion thereof is evaluated. Preferred
biologically active portions of the cancer marker proteins or mRNA
to be used in assays of the present invention include fragments
that participate in interactions with substrates or other proteins,
e.g., fragments with high surface probability scores.
[0215] Cell-free assays involve preparing a reaction mixture of the
target gene protein and the test compound under conditions and for
a time sufficient to allow the two components to interact and bind,
thus forming a complex that can be removed and/or detected.
[0216] The interaction between two molecules can also be detected,
e.g., using fluorescence energy transfer (FRET) (see, for example,
Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos et al.,
U.S. Pat. No. 4,968,103; each of which is herein incorporated by
reference). A fluorophore label is selected such that a first donor
molecule's emitted fluorescent energy will be absorbed by a
fluorescent label on a second, `acceptor` molecule, which in turn
is able to fluoresce due to the absorbed energy.
[0217] Alternately, the `donor` protein molecule may simply utilize
the natural fluorescent energy of tryptophan residues. Labels are
chosen that emit different wavelengths of light, such that the
`acceptor` molecule label may be differentiated from that of the
`donor`. Since the efficiency of energy transfer between the labels
is related to the distance separating the molecules, the spatial
relationship between the molecules can be assessed. In a situation
in which binding occurs between the molecules, the fluorescent
emission of the `acceptor` molecule label should be maximal. A FRET
binding event can be conveniently measured through standard
fluorometric detection means well known in the art (e.g., using a
fluorimeter).
[0218] In another embodiment, determining the ability of the cancer
marker protein or mRNA to bind to a target molecule can be
accomplished using real-time Biomolecular Interaction Analysis
(BIA) (see, e.g., Sjolander and Urbaniczky, Anal. Chem.
63:2338-2345 [1991] and Szabo et al. Curr. Opin. Struct. Biol.
5:699-705 [1995]). "Surface plasmon resonance" or "BIA" detects
biospecific interactions in real time, without labeling any of the
interactants (e.g., BIAcore). Changes in the mass at the binding
surface (indicative of a binding event) result in alterations of
the refractive index of light near the surface (the optical
phenomenon of surface plasmon resonance (SPR)), resulting in a
detectable signal that can be used as an indication of real-time
reactions between biological molecules.
[0219] In one embodiment, the target gene product or the test
substance is anchored onto a solid phase. The target gene
product/test compound complexes anchored on the solid phase can be
detected at the end of the reaction. Preferably, the target gene
product can be anchored onto a solid surface, and the test
compound, (which is not anchored), can be labeled, either directly
or indirectly, with detectable labels discussed herein.
[0220] It may be desirable to immobilize cancer markers, an
anti-cancer marker antibody or its target molecule to facilitate
separation of complexed from non-complexed forms of one or both of
the proteins, as well as to accommodate automation of the assay.
Binding of a test compound to a cancer marker protein, or
interaction of a cancer marker protein with a target molecule in
the presence and absence of a candidate compound, can be
accomplished in any vessel suitable for containing the reactants.
Examples of such vessels include microtiter plates, test tubes, and
micro-centrifuge tubes. In one embodiment, a fusion protein can be
provided which adds a domain that allows one or both of the
proteins to be bound to a matrix. For example,
glutathione-S-transferase-cancer marker fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione Sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione-derivatized microtiter plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or cancer marker protein, and the
mixture incubated under conditions conducive for complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the beads or microtiter plate wells are washed to
remove any unbound components, the matrix immobilized in the case
of beads, complex determined either directly or indirectly, for
example, as described above.
[0221] Alternatively, the complexes can be dissociated from the
matrix, and the level of cancer markers binding or activity
determined using standard techniques. Other techniques for
immobilizing either cancer markers protein or a target molecule on
matrices include using conjugation of biotin and streptavidin.
Biotinylated cancer marker protein or target molecules can be
prepared from biotin-NHS (N-hydroxy-succinimide) using techniques
known in the art (e.g., biotinylation kit, Pierce Chemicals,
Rockford, EL), and immobilized in the wells of streptavidin-coated
96 well plates (Pierce Chemical).
[0222] In order to conduct the assay, the non-immobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously non-immobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
non-immobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the immobilized component (the
antibody, in turn, can be directly labeled or indirectly labeled
with, e.g., a labeled anti-IgG antibody).
[0223] This assay is performed utilizing antibodies reactive with
cancer marker protein or target molecules but which do not
interfere with binding of the cancer markers protein to its target
molecule. Such antibodies can be derivatized to the wells of the
plate, and unbound target or cancer markers protein trapped in the
wells by antibody conjugation. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the cancer marker protein or target
molecule, as well as enzyme-linked assays which rely on detecting
an enzymatic activity associated with the cancer marker protein or
target molecule.
[0224] Alternatively, cell free assays can be conducted in a liquid
phase. In such an assay, the reaction products are separated from
unreacted components, by any of a number of standard techniques,
including, but not limited to: differential centrifugation (see,
for example, Rivas and Minton, Trends Biochem Sci 18:284-7 [1993]);
chromatography (gel filtration chromatography, ion-exchange
chromatography); electrophoresis (see, e.g., Ausubel et al., eds.
Current Protocols in Molecular Biology 1999, J. Wiley: New York.);
and immunoprecipitation (see, for example, Ausubel et al., eds.
Current Protocols in Molecular Biology 1999, J. Wiley: New York).
Such resins and chromatographic techniques are known to one skilled
in the art (See e.g., Heegaard J. Mol. Recognit 11:141-8 [1998];
Hageand Tweed J. Chromatogr. Biomed. Sci. App 1 699:499-525
[1997]). Further, fluorescence energy transfer may also be
conveniently utilized, as described herein, to detect binding
without further purification of the complex from solution.
[0225] The assay can include contacting the cancer markers protein,
mRNA, or biologically active portion thereof with a known compound
that binds the cancer marker to form an assay mixture, contacting
the assay mixture with a test compound, and determining the ability
of the test compound to interact with a cancer marker protein or
mRNA, wherein determining the ability of the test compound to
interact with a cancer marker protein or mRNA includes determining
the ability of the test compound to preferentially bind to cancer
markers or biologically active portion thereof, or to modulate the
activity of a target molecule, as compared to the known
compound.
[0226] To the extent that cancer markers can, in vivo, interact
with one or more cellular or extracellular macromolecules, such as
proteins, inhibitors of such an interaction are useful. A
homogeneous assay can be used can be used to identify
inhibitors.
[0227] For example, a preformed complex of the target gene product
and the interactive cellular or extracellular binding partner
product is prepared such that either the target gene products or
their binding partners are labeled, but the signal generated by the
label is quenched due to complex formation (see, e.g., U.S. Pat.
No. 4,109,496, herein incorporated by reference, that utilizes this
approach for immunoassays). The addition of a test substance that
competes with and displaces one of the species from the preformed
complex will result in the generation of a signal above background.
In this way, test substances that disrupt target gene
product-binding partner interaction can be identified.
Alternatively, cancer markers protein can be used as a "bait
protein" in a two-hybrid assay or three-hybrid assay (see, e.g.,
U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223-232 [1993];
Madura et al., J. Biol. Chem. 268.12046-12054 [1993]; Bartel et
al., Biotechniques 14:920-924 [1993]; Iwabuchi et al., Oncogene
8:1693-1696 [1993]; and Brent WO 94/10300; each of which is herein
incorporated by reference), to identify other proteins, that bind
to or interact with cancer markers ("cancer marker-binding
proteins" or "cancer marker-bp") and are involved in cancer marker
activity. Such cancer marker-bps can be activators or inhibitors of
signals by the cancer marker proteins or targets as, for example,
downstream elements of a cancer markers-mediated signaling
pathway.
[0228] Modulators of cancer markers expression can also be
identified. For example, a cell or cell free mixture is contacted
with a candidate compound and the expression of cancer marker mRNA
or protein evaluated relative to the level of expression of cancer
marker mRNA or protein in the absence of the candidate compound.
When expression of cancer marker mRNA or protein is greater in the
presence of the candidate compound than in its absence, the
candidate compound is identified as a stimulator of cancer marker
mRNA or protein expression. Alternatively, when expression of
cancer marker mRNA or protein is less (i.e., statistically
significantly less) in the presence of the candidate compound than
in its absence, the candidate compound is identified as an
inhibitor of cancer marker mRNA or protein expression. The level of
cancer markers mRNA or protein expression can be determined by
methods described herein for detecting cancer markers mRNA or
protein.
[0229] A modulating agent can be identified using a cell-based or a
cell free assay, and the ability of the agent to modulate the
activity of a cancer markers protein can be confirmed in vivo,
e.g., in an animal such as an animal model for a disease (e.g., an
animal with prostate cancer or metastatic prostate cancer; or an
animal harboring a xenograft of a prostate cancer from an animal
(e.g., human) or cells from a cancer resulting from metastasis of a
prostate cancer (e.g., to a lymph node, bone, or liver), or cells
from a prostate cancer cell line.
[0230] This invention further pertains to novel agents identified
by the above-described screening assays (See e.g., below
description of cancer therapies). Accordingly, it is within the
scope of this invention to further use an agent identified as
described herein (e.g., a cancer marker modulating agent, an
antisense cancer marker nucleic acid molecule, a siRNA molecule, a
cancer marker specific antibody, or a cancer marker-binding
partner) in an appropriate animal model (such as those described
herein) to determine the efficacy, toxicity, side effects, or
mechanism of action, of treatment with such an agent. Furthermore,
novel agents identified by the above-described screening assays can
be, e.g., used for treatments as described herein.
VI. Therapeutic Applications
[0231] In some embodiments, the present invention provides
therapies for cancer (e.g., prostate cancer). In some embodiments,
therapies directly or indirectly target cancer markers (e.g.,
including but not limited to, ERG, ETV1, and ETV4 gene fusions with
TMPRSS2).
[0232] A. RNA Interference and Antisense Therapies
[0233] In some embodiments, the present invention targets the
expression of cancer markers. For example, in some embodiments, the
present invention employs compositions comprising oligomeric
antisense or RNAi compounds, particularly oligonucleotides (e.g.,
those identified in the drug screening methods described above),
for use in modulating the function of nucleic acid molecules
encoding cancer markers of the present invention, ultimately
modulating the amount of cancer marker expressed.
[0234] 1. RNA Interference (RNAi)
[0235] In some embodiments, RNAi is utilized to inhibit fusion
protein function. RNAi represents an evolutionary conserved
cellular defense for controlling the expression of foreign genes in
most eukaryotes, including humans. RNAi is typically triggered by
double-stranded RNA (dsRNA) and causes sequence-specific mRNA
degradation of single-stranded target RNAs homologous in response
to dsRNA. The mediators of mRNA degradation are small interfering
RNA duplexes (siRNAs), which are normally produced from long dsRNA
by enzymatic cleavage in the cell. siRNAs are generally
approximately twenty-one nucleotides in length (e.g. 21-23
nucleotides in length), and have a base-paired structure
characterized by two nucleotide 3'-overhangs. Following the
introduction of a small RNA, or RNAi, into the cell, it is believed
the sequence is delivered to an enzyme complex called
RISC(RNA-induced silencing complex). RISC recognizes the target and
cleaves it with an endonuclease. It is noted that if larger RNA
sequences are delivered to a cell, RNase III enzyme (Dicer)
converts longer dsRNA into 21-23 nt ds siRNA fragments. In some
embodiments, RNAi oligonucleotides are designed to target the
junction region of fusion proteins.
[0236] Chemically synthesized siRNAs have become powerful reagents
for genome-wide analysis of mammalian gene function in cultured
somatic cells. Beyond their value for validation of gene function,
siRNAs also hold great potential as gene-specific therapeutic
agents (Tuschl and Borkhardt, Molecular Intervent. 2002;
2(3):158-67, herein incorporated by reference).
[0237] The transfection of siRNAs into animal cells results in the
potent, long-lasting post-transcriptional silencing of specific
genes (Caplen et al, Proc Natl Acad Sci U.S.A. 2001; 98: 9742-7;
Elbashir et al., Nature. 2001; 411:494-8; Elbashir et al., Genes
Dev. 2001; 15: 188-200; and Elbashir et al., EMBO J. 2001; 20:
6877-88, all of which are herein incorporated by reference).
Methods and compositions for performing RNAi with siRNAs are
described, for example, in U.S. Pat. No. 6,506,559, herein
incorporated by reference.
[0238] siRNAs are extraordinarily effective at lowering the amounts
of targeted RNA, and by extension proteins, frequently to
undetectable levels. The silencing effect can last several months,
and is extraordinarily specific, because one nucleotide mismatch
between the target RNA and the central region of the siRNA is
frequently sufficient to prevent silencing (Brummelkamp et al,
Science 2002; 296:550-3; and Holen et al, Nucleic Acids Res. 2002;
30:1757-66, both of which are herein incorporated by reference). An
important factor in the design of siRNAs is the presence of
accessible sites for siRNA binding. Bahoia et al., (J. Biol. Chem.,
2003; 278: 15991-15997; herein incorporated by reference) describe
the use of a type of DNA array called a scanning array to find
accessible sites in mRNAs for designing effective siRNAs. These
arrays comprise oligonucleotides ranging in size from monomers to a
certain maximum, usually Comers, synthesized using a physical
barrier (mask) by stepwise addition of each base in the sequence.
Thus the arrays represent a full oligonucleotide complement of a
region of the target gene. Hybridization of the target mRNA to
these arrays provides an exhaustive accessibility profile of this
region of the target mRNA. Such data are useful in the design of
antisense oligonucleotides (ranging from 7mers to 25mers), where it
is important to achieve a compromise between oligonucleotide length
and binding affinity, to retain efficacy and target specificity
(Sohail et al, Nucleic Acids Res., 2001; 29(10): 2041-2045).
Additional methods and concerns for selecting siRNAs are described
for example, in WO 05054270, WO05038054A1, WO03070966A2, J Mol
Biol. 2005 May 13; 348(4):883-93, J Mol Biol. 2005 May 13;
348(4):871-81, and Nucleic Acids Res. 2003 Aug. 1; 31(15):4417-24,
each of which is herein incorporated by reference in its entirety.
In addition, software (e.g., the MWG online siMAX siRNA design
tool) is commercially or publicly available for use in the
selection of siRNAs.
[0239] 2. Antisense
[0240] In other embodiments, fusion protein expression is modulated
using antisense compounds that specifically hybridize with one or
more nucleic acids encoding cancer markers of the present
invention. The specific hybridization of an oligomeric compound
with its target nucleic acid interferes with the normal function of
the nucleic acid. This modulation of function of a target nucleic
acid by compounds that specifically hybridize to it is generally
referred to as "antisense." The functions of DNA to be interfered
with include replication and transcription. The functions of RNA to
be interfered with include all vital functions such as, for
example, translocation of the RNA to the site of protein
translation, translation of protein from the RNA, splicing of the
RNA to yield one or more mRNA species, and catalytic activity that
may be engaged in or facilitated by the RNA. The overall effect of
such interference with target nucleic acid function is modulation
of the expression of cancer markers of the present invention. In
the context of the present invention, "modulation" means either an
increase (stimulation) or a decrease (inhibition) in the expression
of a gene. For example, expression may be inhibited to potentially
prevent tumor proliferation.
[0241] It is preferred to target specific nucleic acids for
antisense. "Targeting" an antisense compound to a particular
nucleic acid, in the context of the present invention, is a
multistep process. The process usually begins with the
identification of a nucleic acid sequence whose function is to be
modulated. This may be,' for example, a cellular gene (or mRNA
transcribed from the gene) whose expression is associated with a
particular disorder or disease state, or a nucleic acid molecule
from an infectious agent. In the present invention, the target is a
nucleic acid molecule encoding a cancer marker of the present
invention. The targeting process also includes determination of a
site or sites within this gene for the antisense interaction to
occur such that the desired effect, e.g., detection or modulation
of expression of the protein, will result. Within the context of
the present invention, a preferred intragenic site is the region
encompassing the translation initiation or termination codon of the
open reading frame (ORF) of the gene. Since the translation
initiation codon is typically 5'-AUG (in transcribed mRNA
molecules; 5'-ATG in the corresponding DNA molecule), the
translation initiation codon is also referred to as the "AUG
codon," the "start codon" or the "AUG start codon". A minority of
genes have a translation initiation codon having the RNA sequence
5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been
shown to function in vivo. Thus, the terms "translation initiation
codon" and "start codon" can encompass many codon sequences, even
though the initiator amino acid in each instance is typically
methionine (in eukaryotes) or formylmethionine (in prokaryotes).
Eukaryotic and prokaryotic genes may have two or more alternative
start codons, any one of which may be preferentially utilized for
translation initiation in a particular cell type or tissue, or
under a particular set of conditions. In the context of the present
invention, "start codon" and "translation initiation codon" refer
to the codon or codons that are used in vivo to initiate
translation of an mRNA molecule transcribed from a gene encoding a
tumor antigen of the present invention, regardless of the
sequence(s) of such codons.
[0242] Translation termination codon (or "stop codon") of a gene
may have one of three sequences (i.e., 5'-UAA, 5'-UAG and 5'-UGA;
the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA,
respectively). The terms "start codon region" and "translation
initiation codon region" refer to a portion of such an mRNA or gene
that encompasses from about 25 to about 50 contiguous nucleotides
in either direction (i.e., 5' or 3') from a translation initiation
codon. Similarly, the terms "stop codon region" and "translation
termination codon region" refer to a portion of such an mRNA or
gene that encompasses from about 25 to about 50 contiguous
nucleotides in either direction (i.e., 5' or 3') from a translation
termination codon.
[0243] The open reading frame (ORF) or "coding region," which
refers to the region between the translation initiation codon and
the translation termination codon, is also a region that may be
targeted effectively. Other target regions include the 5'
untranslated region (5' UTR), referring to the portion of an mRNA
in the 5' direction from the translation initiation codon, and thus
including nucleotides between the 5' cap site and the translation
initiation codon of an mRNA or corresponding nucleotides on the
gene, and the 3' untranslated region (3' UTR), referring to the
portion of an mRNA in the 3' direction from the translation
termination codon, and thus including nucleotides between the
translation termination codon and 3' end of an mRNA or
corresponding nucleotides on the gene. The 5' cap of an mRNA
comprises an N7-methylated guanosine residue joined to the 5'-most
residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap
region of an mRNA is considered to include the 5' cap structure
itself as well as the first 50 nucleotides adjacent to the cap. The
cap region may also be a preferred target region.
[0244] Although some eukaryotic mRNA transcripts are directly
translated, many contain one or more regions, known as "introns,"
that are excised from a transcript before it is translated. The
remaining (and therefore translated) regions are known as "exons"
and are spliced together to form a continuous mRNA sequence. mRNA
splice sites (i.e., intron-exon junctions) may also be preferred
target regions, and are particularly useful in situations where
aberrant splicing is implicated in disease, or where an
overproduction of a particular mRNA splice product is implicated in
disease. Aberrant fusion junctions due to rearrangements or
deletions are also preferred targets. It has also been found that
introns can also be effective, and therefore preferred, target
regions for antisense compounds targeted, for example, to DNA or
pre-mRNA.
[0245] In some embodiments, target sites for antisense inhibition
are identified using commercially available software programs
(e.g., Biognostik, Gottingen, Germany; SysArris Software,
Bangalore, India; Antisense Research Group, University of
Liverpool, Liverpool, England; GeneTrove, Carlsbad, Calif.). In
other embodiments, target sites for antisense inhibition are
identified using the accessible site method described in PCT Publ.
No. WO0198537A2, herein incorporated by reference.
[0246] Once one or more target sites have been identified,
oligonucleotides are chosen that are sufficiently complementary to
the target (i.e., hybridize sufficiently well and with sufficient
specificity) to give the desired effect. For example, in preferred
embodiments of the present invention, antisense oligonucleotides
are targeted to or near the start codon.
[0247] In the context of this invention, "hybridization," with
respect to antisense compositions and methods, means hydrogen
bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen
hydrogen bonding, between complementary nucleoside or nucleotide
bases. For example, adenine and thymine are complementary
nucleobases that pair through the formation of hydrogen bonds. It
is understood that the sequence of an antisense compound need not
be 100% complementary to that of its target nucleic acid to be
specifically hybridizable. An antisense compound is specifically
hybridizable when binding of the compound to the target DNA or RNA
molecule interferes with the normal function of the target DNA or
RNA to cause a loss of utility, and there is a sufficient degree of
complementarity to avoid non-specific binding of the antisense
compound to non-target sequences under conditions in which specific
binding is desired (i.e., under physiological conditions in the
case of in vivo assays or therapeutic treatment, and in the case of
in vitro assays, under conditions in which the assays are
performed).
[0248] Antisense compounds are commonly used as research reagents
and diagnostics. For example, antisense oligonucleotides, which are
able to inhibit gene expression with specificity, can be used to
elucidate the function of particular genes. Antisense compounds are
also used, for example, to distinguish between functions of various
members of a biological pathway.
[0249] The specificity and sensitivity of antisense is also applied
for therapeutic uses. For example, antisense oligonucleotides have
been employed as therapeutic moieties in the treatment of disease
states in animals and man. Antisense oligonucleotides have been
safely and effectively administered to humans and numerous clinical
trials are presently underway. It is thus established that
oligonucleotides are useful therapeutic modalities that can be
configured to be useful in treatment regimes for treatment of
cells, tissues, and animals, especially humans.
[0250] While antisense oligonucleotides are a preferred form of
antisense compound, the present invention comprehends other
oligomeric antisense compounds, including but not limited to
oligonucleotide mimetics such as are described below. The antisense
compounds in accordance with this invention preferably comprise
from about 8 to about 30 nucleobases (i.e., from about 8 to about
30 linked bases), although both longer and shorter sequences may
find use with the present invention. Particularly preferred
antisense compounds are antisense oligonucleotides, even more
preferably those comprising from about 12 to about 25
nucleobases.
[0251] Specific examples of preferred antisense compounds useful
with the present invention include oligonucleotides containing
modified backbones or non-natural internucleoside linkages. As
defined in this specification, oligonucleotides having modified
backbones include those that retain a phosphorus atom in the
backbone and those that do not have a phosphorus atom in the
backbone. For the purposes of this specification, modified
oligonucleotides that do not have a phosphorus atom in their
internucleoside backbone can also be considered to be
oligonucleosides.
[0252] Preferred modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free acid forms are also
included.
[0253] Preferred modified oligonucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; amide backbones; and others having mixed N,
O, S and CH.sub.2 component parts.
[0254] In other preferred oligonucleotide mimetics, both the sugar
and the internucleoside linkage (i.e., the backbone) of the
nucleotide units are replaced with novel groups. The base units are
maintained for hybridization with an appropriate nucleic acid
target compound. One such oligomeric compound, an oligonucleotide
mimetic that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar-backbone of an oligonucleotide is replaced
with an amide containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to aza nitrogen atoms of the amide
portion of the backbone. Representative United States patents that
teach the preparation of PNA compounds include, but are not limited
to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of
which is herein incorporated by reference. Further teaching of PNA
compounds can be found in Nielsen et al., Science 254:1497
(1991).
[0255] Most preferred embodiments of the invention are
oligonucleotides with phosphorothioate backbones and
oligonucleosides with heteroatom backbones, and in particular
--CH.sub.2, --NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2--, and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of
the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
preferred are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0256] Modified oligonucleotides may also contain one or more
substituted sugar moieties. Preferred oligonucleotides comprise one
of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-,
S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein
the alkyl, alkenyl and alkynyl may be substituted or unsubstituted
C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and
alkynyl. Particularly preferred are
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or
O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3,
SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3,
NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A preferred
modification includes 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta
78:486 [1995]) i.e., an alkoxyalkoxy group. A further preferred
modification includes 2'-dimethylaminooxyethoxy (i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group), also known as 2'-DMAOE,
and 2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2.
[0257] Other preferred modifications include
2'-methoxy(2'-O--CH.sub.3),
2'-aminopropoxy(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro
(2'-F). Similar modifications may also be made at other positions
on the oligonucleotide, particularly the 3' position of the sugar
on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides
and the 5' position of 5' terminal nucleotide. Oligonucleotides may
also have sugar mimetics such as cyclobutyl moieties in place of
the pentofuranosyl sugar.
[0258] Oligonucleotides may also include nucleobase (often referred
to in the art simply as "base") modifications or substitutions. As
used herein, "unmodified" or "natural" nucleobases include the
purine bases adenine (A) and guanine (G), and the pyrimidine bases
thymine (T), cytosine (C) and uracil (U). Modified nucleobases
include other synthetic and natural nucleobases such as
5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and
cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,
8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other
5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further
nucleobases include those disclosed in U.S. Pat. No. 3,687,808.
Certain of these nucleobases are particularly useful for increasing
the binding affinity of the oligomeric compounds of the invention.
These include 5-substituted pyrimidines, 6-azapyrimidines and N-2,
N-6 and O-6 substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2. .degree. C. and are presently preferred base
substitutions, even more particularly when combined with
2'-O-methoxyethyl sugar modifications.
[0259] Another modification of the oligonucleotides of the present
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates that enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. Such
moieties include but are not limited to lipid moieties such as a
cholesterol moiety, cholic acid, a thioether, (e.g.,
hexyl-5-tritylthiol), a thiocholesterol, an aliphatic chain, (e.g.,
dodecandiol or undecyl residues), a phospholipid, (e.g.,
di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or a
polyethylene glycol chain or adamantane acetic acid, a palmityl
moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol
moiety.
[0260] One skilled in the relevant art knows well how to generate
oligonucleotides containing the above-described modifications. The
present invention is not limited to the antisense oligonucleotides
described above. Any suitable modification or substitution may be
utilized.
[0261] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an oligonucleotide.
The present invention also includes antisense compounds that are
chimeric compounds. "Chimeric" antisense compounds or "chimeras,"
in the context of the present invention, are antisense compounds,
particularly oligonucleotides, which contain two or more chemically
distinct regions, each made up of at least one monomer unit, i.e.,
a nucleotide in the case of an oligonucleotide compound. These
oligonucleotides typically contain at least one region wherein the
oligonucleotide is modified so as to confer upon the
oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target nucleic acid. An additional region of the
oligonucleotide may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a
cellular endonuclease that cleaves the RNA strand of an RNA:DNA
duplex. Activation of RNase H, therefore, results in cleavage of
the RNA target, thereby greatly enhancing the efficiency of
oligonucleotide inhibition of gene expression. Consequently,
comparable results can often be obtained with shorter
oligonucleotides when chimeric oligonucleotides are used, compared
to phosphorothioate deoxyoligonucleotides hybridizing to the same
target region. Cleavage of the RNA target can be routinely detected
by gel electrophoresis and, if necessary, associated nucleic acid
hybridization techniques known in the art.
[0262] Chimeric antisense compounds of the present invention may be
formed as composite structures of two or more oligonucleotides,
modified oligonucleotides, oligonucleosides and/or oligonucleotide
mimetics as described above.
[0263] The present invention also includes pharmaceutical
compositions and formulations that include the antisense compounds
of the present invention as described below.
[0264] B. Gene Therapy
[0265] The present invention contemplates the use of any genetic
manipulation for use in modulating the expression of cancer markers
of the present invention. Examples of genetic manipulation include,
but are not limited to, gene knockout (e.g., removing the fusion
gene from the chromosome using, for example, recombination),
expression of antisense constructs with or without inducible
promoters, and the like. Delivery of nucleic acid construct to
cells in vitro or in vivo may be conducted using any suitable
method. A suitable method is one that introduces the nucleic acid
construct into the cell such that the desired event occurs (e.g.,
expression of an antisense construct). Genetic therapy may also be
used to deliver siRNA or other interfering molecules that are
expressed in vivo (e.g., upon stimulation by an inducible promoter
(e.g., an androgen-responsive promoter)).
[0266] Introduction of molecules carrying genetic information into
cells is achieved by any of various methods including, but not
limited to, directed injection of naked DNA constructs, bombardment
with gold particles loaded with said constructs, and macromolecule
mediated gene transfer using, for example, liposomes, biopolymers,
and the like. Preferred methods use gene delivery vehicles derived
from viruses, including, but not limited to, adenoviruses,
retroviruses, vaccinia viruses, and adeno-associated viruses.
Because of the higher efficiency as compared to retroviruses,
vectors derived from adenoviruses are the preferred gene delivery
vehicles for transferring nucleic acid molecules into host cells in
vivo. Adenoviral vectors have been shown to provide very efficient
in vivo gene transfer into a variety of solid tumors in animal
models and into human solid tumor xenografts in immune-deficient
mice. Examples of adenoviral vectors and methods for gene transfer
are described in PCT publications WO 00/12738 and WO 00/09675 and
U.S. Pat. Nos. 6,033,908, 6,019,978, 6,001,557, 5,994,132,
5,994,128, 5,994,106, 5,981,225, 5,885,808, 5,872,154, 5,830,730,
and 5,824,544, each of which is herein incorporated by reference in
its entirety.
[0267] Vectors may be administered to subject in a variety of ways.
For example, in some embodiments of the present invention, vectors
are administered into tumors or tissue associated with tumors using
direct injection. In other embodiments, administration is via the
blood or lymphatic circulation (See e.g., PCT publication 99/02685
herein incorporated by reference in its entirety). Exemplary dose
levels of adenoviral vector are preferably 10.sup.8 to 10.sup.11
vector particles added to the perfusate.
[0268] C. Antibody Therapy
[0269] In some embodiments, the present invention provides
antibodies that target prostate tumors that express a cancer marker
of the present invention (e.g., a gene fusion of the present
invention). Any suitable antibody (e.g., monoclonal, polyclonal, or
synthetic) may be utilized in the therapeutic methods disclosed
herein. In preferred embodiments, the antibodies used for cancer
therapy are humanized antibodies. Methods for humanizing antibodies
are well known in the art (See e.g., U.S. Pat. Nos. 6,180,370,
5,585,089, 6,054,297, and 5,565,332; each of which is herein
incorporated by reference).
[0270] In some embodiments, the therapeutic antibodies comprise an
antibody generated against a cancer marker of the present invention
(e.g., a gene fusion of the present invention), wherein the
antibody is conjugated to a cytotoxic agent. In such embodiments, a
tumor specific therapeutic agent is generated that does not target
normal cells, thus reducing many of the detrimental side effects of
traditional chemotherapy. For certain applications, it is
envisioned that the therapeutic agents will be pharmacologic agents
that will serve as useful agents for attachment to antibodies,
particularly cytotoxic or otherwise anticellular agents having the
ability to kill or suppress the growth or cell division of
endothelial cells. The present invention contemplates the use of
any pharmacologic agent that can be conjugated to an antibody, and
delivered in active form. Exemplary anticellular agents include
chemotherapeutic agents, radioisotopes, and cytotoxins. The
therapeutic antibodies of the present invention may include a
variety of cytotoxic moieties, including but not limited to,
radioactive isotopes (e.g., iodine-131, iodine-123, technicium-99m,
indium-111, rhenium-188, rhenium-186, gallium-67, copper-67,
yttrium-90, iodine-125 or astatine-211), hormones such as a
steroid, antimetabolites such as cytosines (e.g., arabinoside,
fluorouracil, methotrexate or aminopterin; an anthracycline;
mitomycin C), vinca alkaloids (e.g., demecolcine; etoposide;
mithramycin), and antitumor alkylating agent such as chlorambucil
or melphalan. Other embodiments may include agents such as a
coagulant, a cytokine, growth factor, bacterial endotoxin or the
lipid A moiety of bacterial endotoxin. For example, in some
embodiments, therapeutic agents will include plant-, fungus- or
bacteria-derived toxin, such as an A chain toxins, a ribosome
inactivating protein, .alpha.-sarcin, aspergillin, restrictocin, a
ribonuclease, diphtheria toxin or pseudomonas exotoxin, to mention
just a few examples. In some preferred embodiments, deglycosylated
ricin A chain is utilized.
[0271] In any event, it is proposed that agents such as these may,
if desired, be successfully conjugated to an antibody, in a manner
that will allow their targeting, internalization, release or
presentation to blood components at the site of the targeted tumor
cells as required using known conjugation technology (See, e.g.,
Ghose et al., Methods Enzymol., 93:280 [1983]).
[0272] For example, in some embodiments the present invention
provides immunotoxins targeted a cancer marker of the present
invention (e.g., ERG or ETV1 fusions). Immunotoxins are conjugates
of a specific targeting agent typically a tumor-directed antibody
or fragment, with a cytotoxic agent, such as a toxin moiety. The
targeting agent directs the toxin to, and thereby selectively
kills, cells carrying the targeted antigen. In some embodiments,
therapeutic antibodies employ crosslinkers that provide high in
vivo stability (Thorpe et al., Cancer Res., 48:6396 [1988]).
[0273] In other embodiments, particularly those involving treatment
of solid tumors, antibodies are designed to have a cytotoxic or
otherwise anticellular effect against the tumor vasculature, by
suppressing the growth or cell division of the vascular endothelial
cells. This attack is intended to lead to a tumor-localized
vascular collapse, depriving the tumor cells, particularly those
tumor cells distal of the vasculature, of oxygen and nutrients,
ultimately leading to cell death and tumor necrosis.
[0274] In preferred embodiments, antibody based therapeutics are
formulated as pharmaceutical compositions as described below. In
preferred embodiments, administration of an antibody composition of
the present invention results in a measurable decrease in cancer
(e.g., decrease or elimination of tumor).
[0275] D. Pharmaceutical Compositions
[0276] The present invention further provides pharmaceutical
compositions (e.g., comprising pharmaceutical agents that modulate
the expression or activity of gene fusions of the present
invention). The pharmaceutical compositions of the present
invention may be administered in a number of ways depending upon
whether local or systemic treatment is desired and upon the area to
be treated. Administration may be topical (including ophthalmic and
to mucous membranes including vaginal and rectal delivery),
pulmonary (e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal), oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; or
intracranial, e.g., intrathecal or intraventricular,
administration.
[0277] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like may be necessary or desirable.
[0278] Compositions and formulations for oral administration
include powders or granules, suspensions or solutions in water or
non-aqueous media, capsules, sachets or tablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable.
[0279] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions that may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
[0280] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compositions may be
generated from a variety of components that include, but are not
limited to, preformed liquids, self-emulsifying solids and
self-emulsifying semisolids.
[0281] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0282] The compositions of the present invention may be formulated
into any of many possible dosage forms such as, but not limited to,
tablets, capsules, liquid syrups, soft gels, suppositories, and
enemas. The compositions of the present invention may also be
formulated as suspensions in aqueous, non-aqueous or mixed media.
Aqueous suspensions may further contain substances that increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0283] In one embodiment of the present invention the
pharmaceutical compositions may be formulated and used as foams.
Pharmaceutical foams include formulations such as, but not limited
to, emulsions, microemulsions, creams, jellies and liposomes. While
basically similar in nature these formulations vary in the
components and the consistency of the final product.
[0284] Agents that enhance uptake of oligonucleotides at the
cellular level may also be added to the pharmaceutical and other
compositions of the present invention. For example, cationic
lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationic
glycerol derivatives, and polycationic molecules, such as
polylysine (WO 97/30731), also enhance the cellular uptake of
oligonucleotides.
[0285] The compositions of the present invention may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions. Thus, for example, the compositions
may contain additional, compatible, pharmaceutically-active
materials such as, for example, antipruritics, astringents, local
anesthetics or anti-inflammatory agents, or may contain additional
materials useful in physically formulating various dosage forms of
the compositions of the present invention, such as dyes, flavoring
agents, preservatives, antioxidants, opacifiers, thickening agents
and stabilizers. However, such materials, when added, should not
unduly interfere with the biological activities of the components
of the compositions of the present invention. The formulations can
be sterilized and, if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously interact with the nucleic acid(s) of the
formulation.
[0286] Certain embodiments of the invention provide pharmaceutical
compositions containing (a) one or more antisense compounds and (b)
one or more other chemotherapeutic agents that function by a
non-antisense mechanism. Examples of such chemotherapeutic agents
include, but are not limited to, anticancer drugs such as
daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin,
nitrogen mustard, chlorambucil, melphalan, cyclophosphamide,
6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil
(5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine,
vincristine, vinblastine, etoposide, teniposide, cisplatin and
diethylstilbestrol (DES). Anti-inflammatory drugs, including but
not limited to nonsteroidal anti-inflammatory drugs and
corticosteroids, and antiviral drugs, including but not limited to
ribivirin, vidarabine, acyclovir and ganciclovir, may also be
combined in compositions of the invention. Other non-antisense
chemotherapeutic agents are also within the scope of this
invention. Two or more combined compounds may be used together or
sequentially.
[0287] Dosing is dependent on severity and responsiveness of the
disease state to be treated, with the course of treatment lasting
from several days to several months, or until a cure is effected or
a diminution of the disease state is achieved. Optimal dosing
schedules can be calculated from measurements of drug accumulation
in the body of the patient. The administering physician can easily
determine optimum dosages, dosing methodologies and repetition
rates. Optimum dosages may vary depending on the relative potency
of individual oligonucleotides, and can generally be estimated
based on EC.sub.50s found to be effective in in vitro and in vivo
animal models or based on the examples described herein. In
general, dosage is from 0.01 .mu.g to 100 g per kg of body weight,
and may be given once or more daily, weekly, monthly or yearly. The
treating physician can estimate repetition rates for dosing based
on measured residence times and concentrations of the drug in
bodily fluids or tissues. Following successful treatment, it may be
desirable to have the subject undergo maintenance therapy to
prevent the recurrence of the disease state, wherein the
oligonucleotide is administered in maintenance doses, ranging from
0.01 .mu.g to 100 g per kg of body weight, once or more daily, to
once every 20 years.
VII. Transgenic Animals
[0288] The present invention contemplates the generation of
transgenic animals comprising an exogenous cancer marker nucleic
acid (e.g., gene fusion) of the present invention or mutants and
variants thereof (e.g., truncations or single nucleotide
polymorphisms). In preferred embodiments, the transgenic animal
displays an altered expression profile (e.g., increased or
decreased presence of markers) as compared to wild-type animals. In
other embodiments, transgenic animals display an altered phenotype
(e.g., presence of cancer or pre-cancerous lesions) as compared to
wild-type animals. Methods for analyzing the presence or absence of
such phenotypes include but are not limited to, those disclosed
herein. In some preferred embodiments, the transgenic animals
further display an increased or decreased growth of tumors or
evidence of cancer. Exemplary transgenic animals of the present
invention are described in Example 19 below.
[0289] The transgenic animals of the present invention find use in
drug (e.g., cancer therapy) screens. In some embodiments, test
compounds (e.g., a drug that is suspected of being useful to treat
cancer) and control compounds (e.g., a placebo) are administered to
the transgenic animals and the control animals and the effects
evaluated.
[0290] The transgenic animals can be generated via a variety of
methods. In some embodiments, embryonal cells at various
developmental stages are used to introduce transgenes for the
production of transgenic animals. Different methods are used
depending on the stage of development of the embryonal cell. The
zygote is the best target for micro-injection. In the mouse, the
male pronucleus reaches the size of approximately 20 micrometers in
diameter that allows reproducible injection of 1-2 picoliters (pl)
of DNA solution. The use of zygotes as a target for gene transfer
has a major advantage in that in most cases the injected DNA will
be incorporated into the host genome before the first cleavage
(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 [1985]).
As a consequence, all cells of the transgenic non-human animal will
carry the incorporated transgene. This will in general also be
reflected in the efficient transmission of the transgene to
offspring of the founder since 50% of the germ cells will harbor
the transgene. U.S. Pat. No. 4,873,191 describes a method for the
micro-injection of zygotes; the disclosure of this patent is
incorporated herein in its entirety.
[0291] In other embodiments, retroviral infection is used to
introduce transgenes into a non-human animal. In some embodiments,
the retroviral vector is utilized to transfect oocytes by injecting
the retroviral vector into the perivitelline space of the oocyte
(U.S. Pat. No. 6,080,912, incorporated herein by reference). In
other embodiments, the developing non-human embryo can be cultured
in vitro to the blastocyst stage. During this time, the blastomeres
can be targets for retroviral infection (Janenich, Proc. Natl.
Acad. Sci. USA 73:1260 [1976]). Efficient infection of the
blastomeres is obtained by enzymatic treatment to remove the zona
pellucida (Hogan et al., in Manipulating the Mouse Embryo, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1986]).
The viral vector system used to introduce the transgene is
typically a replication-defective retrovirus carrying the transgene
(Jahner et al., Proc. Natl. Acad. Sci. USA 82:6927, [1985]).
Transfection is easily and efficiently obtained by culturing the
blastomeres on a monolayer of virus-producing cells (Stewart, et
al., EMBO J., 6:383 [1987]). Alternatively, infection can be
performed at a later stage. Virus or virus-producing cells can be
injected into the blastocoele (Jahner et al., Nature 298:623
[1982]). Most of the founders will be mosaic for the transgene
since incorporation occurs only in a subset of cells that form the
transgenic animal. Further, the founder may contain various
retroviral insertions of the transgene at different positions in
the genome that generally will segregate in the offspring. In
addition, it is also possible to introduce transgenes into the
germline, albeit with low efficiency, by intrauterine retroviral
infection of the midgestation embryo (Jahner et al., supra [1982]).
Additional means of using retroviruses or retroviral vectors to
create transgenic animals known to the art involve the
micro-injection of retroviral particles or mitomycin C-treated
cells producing retrovirus into the perivitelline space of
fertilized eggs or early embryos (PCT International Application WO
90/08832 [1990], and Haskell and Bowen, Mol. Reprod. Dev., 40:386
[1995]).
[0292] In other embodiments, the transgene is introduced into
embryonic stem cells and the transfected stem cells are utilized to
form an embryo. ES cells are obtained by culturing pre-implantation
embryos in vitro under appropriate conditions (Evans et al., Nature
292:154 [1981]; Bradley et al., Nature 309:255 [1984]; Gossler et
al., Proc. Acad. Sci. USA 83:9065 [1986]; and Robertson et al.,
Nature 322:445 [1986]). Transgenes can be efficiently introduced
into the ES cells by DNA transfection by a variety of methods known
to the art including calcium phosphate co-precipitation, protoplast
or spheroplast fusion, lipofection and DEAE-dextran-mediated
transfection. Transgenes may also be introduced into ES cells by
retrovirus-mediated transduction or by micro-injection. Such
transfected ES cells can thereafter colonize an embryo following
their introduction into the blastocoel of a blastocyst-stage embryo
and contribute to the germ line of the resulting chimeric animal
(for review, See, Jaenisch, Science 240:1468 [1988]). Prior to the
introduction of transfected ES cells into the blastocoel, the
transfected ES cells may be subjected to various selection
protocols to enrich for ES cells which have integrated the
transgene assuming that the transgene provides a means for such
selection. Alternatively, the polymerase chain reaction may be used
to screen for ES cells that have integrated the transgene. This
technique obviates the need for growth of the transfected ES cells
under appropriate selective conditions prior to transfer into the
blastocoel.
[0293] In still other embodiments, homologous recombination is
utilized to knock-out gene function or create deletion mutants
(e.g., truncation mutants). Methods for homologous recombination
are described in U.S. Pat. No. 5,614,396, incorporated herein by
reference.
EXPERIMENTAL
[0294] The following examples are provided in order to demonstrate
and further illustrate certain preferred embodiments and aspects of
the present invention and are not to be construed as limiting the
scope thereof.
Example 1
ERG and ETV1 Gene Fusions
A. Materials and Methods
Cancer Outlier Profile Analysis (COPA)
[0295] COPA analysis was performed on 132 gene expression data sets
in Oncomine 3.0 comprising 10,486 microarray experiments. In
addition, data from 99 amplified laser-capture microdissected
prostate tissue samples were included in the COPA analysis. COPA
has three steps. First, gene expression values are median centered,
setting each gene's median expression value to zero. Second, the
median absolute deviation (MAD) is calculated and scaled to 1 by
dividing each gene expression value by its MAD. Median and MAD were
used for transformation as opposed to mean and standard deviation
so that outlier expression values do not unduly influence the
distribution estimates, and are thus preserved post-normalization.
Third, the 75th, 90th, and 95th percentiles of the transformed
expression values are tabulated for each gene and then genes are
rank-ordered by their percentile scores, providing a prioritized
list of outlier profiles.
Samples
[0296] Tissues utilized were from the radical prostatectomy series
at the University of Michigan and from the Rapid Autopsy Program
(Shah et al., Cancer Res 64, 9209 (Dec. 15, 2004)), which are both
part of University of Michigan Prostate Cancer Specialized Program
of Research Excellence (S.P.O.R.E.) Tissue Core.
[0297] Tissues were also obtained from a radical prostatectomy
series at the University Hospital Ulm (Ulm, Germany). All samples
were collected from consented patients with prior institutional
review board approval at each respective institution. Total RNA
from all samples was isolated with Trizol (Invitrogen) according to
the manufacturer's instructions. Total RNA was also isolated from
RWPE, PC3, PC3+AR (Dai et al., Steroids 61, 531 (1996)), LNCaP,
VCaP and DuCaP cell lines. RNA integrity was verified by denaturing
formaldehyde gel electrophoresis or the Agilent Bioanalyzer 2100. A
commercially available pool of benign prostate tissue total RNA
(CPP, Clontech) was also used.
Quantitative PCR (QPCR)
[0298] Quantitative PCR (QPCR) was performed using SYBR Green dye
on an Applied Biosystems 7300 Real Time PCR system essentially as
described (Chinnaiyan et al., Cancer Res 65, 3328 (2005); Rubin et
al., Cancer Res 64, 3814 (2004)). Briefly, 1-5 .mu.g of total RNA
was reverse transcribed into cDNA using SuperScript III
(Invitrogen) in the presence of random primers or random primers
and oligo dT primers. All reactions were performed with SYBR Green
Master Mix (Applied Biosystems) and 25 ng of both the forward and
reverse primer using the manufacturer's recommended thermocycling
conditions. All reactions were subjected to melt curve analysis and
products from selected experiments were resolved by electrophoreses
on 1.5% agarose gels. For each experiment, threshold levels were
set during the exponential phase of the QPCR reaction using
Sequence Detection Software version 1.2.2 (Applied Biosystems). The
amount of each target gene relative to the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample
was determined using the comparative threshold cycle (Ct) method
(Applied Biosystems User Bulletin #2), with the cDNA sample serving
as the calibrator for each experiment described in the figure
legend. All oligonucleotide primers were synthesized by Integrated
DNA Technologies. GAPDH primers were as described (Vandesompele et
al., Genome Biol 3, RESEARCH0034 (2002)) and all other primers are
listed (Table 4). Approximately equal efficiencies of the primers
were confirmed using serial dilutions of prostate cancer cDNA or
plasmid templates in order to use the comparative Ct method.
RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE)
[0299] RNA ligase mediated rapid amplification of cDNA ends was
performed using the GeneRacer RLM-RACE kit (Invitrogen), according
to the manufacturer's instructions. Initially, samples were
selected based on expression of ERG or ETV1 by QPCR. Five
micrograms of total RNA was treated with calf intestinal
phosphatase to remove 5' phosphates from truncated mRNA and
non-mRNA and decapped with tobacco acid pyrophosphatase. The
GeneRace RNA Oligo was ligated to full length transcripts and
reverse transcribed using SuperScript III. To obtain 5' ends,
first-strand cDNA was amplified with Platinum Taq High Fidelity
(Invitrogen) using the GeneRacer 5' Primer and ETV1 exon 4-5_r for
ETV1 or the GeneRacer 5' Primer and ERG exon 4a_r or ERG exon 4b_r
for ERG. Primer sequences are given (Table S2). Products were
resolved by electrophoresis on 1.5% agarose gels and bands were
excised, purified and TOPO TA cloned into pCR 4-TOPO. Purified
plasmid DNA from at least 4 colonies was sequenced bi-directionally
using M13 Reverse and M13 Forward (-20) primers or T3 and T7
primers on an ABI Model 3730 automated sequencer by the University
of Michigan DNA Sequencing Core. RLM-RACEd cDNA was not used for
the other assays.
Reverse-Transcription PCR for TMPRSS2:ERG Fusion
[0300] After identifying TMPRSS2:ERG positive cases using QPCR as
described above, the same cDNA samples were PCR amplified with
Platinum Taq High Fidelity and TPRSS2:ERG primers. Products were
resolved by electrophoresis, cloned into pCR 4-TOPO and sequenced
as described above.
In Vitro Androgen Responsiveness
[0301] RWPE, LNCaP, VCap DuCaP, PC3 and PC3 cells stably
transfected with the human androgen receptor (PC3+AR) (3) were
treated for 24 h with 1% ethanol control or 1 nM of the synthetic
androgen R1881. Total RNA was isolated and subjected to reverse
transcription and QPCR as described above with ERG exon 5-6_f and
_r primers. The relative amount of ERG/GAPDH for each sample was
calibrated to the RWPE control sample.
Fluorescence In Situ Hybridization (FISH)
[0302] Formalin-fixed paraffin-embedded (FFPE) tissue sections from
normal peripheral lymphocytes and the metastatic prostate cancer
samples MET-26 and MET-28 were used for interphase fluorescence in
situ hybridization (FISH) analysis. In addition, interphase FISH
was performed on a tissue microarray containing cores from FFPE
sections of 13 clinically localized prostate cancer and 16
metastatic prostate cancer samples. A two-color, two-signal
approach was employed to evaluate the fusion of TMPRSS2 and ETV1,
with probes spanning most of the respective gene loci. The
biotin-14-dCTP BAC clone RP11-124L22 was used for the ETV1 locus
and the digoxin-dUTP labeled BAC clone RPP11-35CD was used for the
TMPRSS2 locus. For analyzing gene rearrangements involving ERG, a
split-signal probe strategy was used, with two probes spanning the
ERG locus (biotin-14-dCTP labeled BAC clone RP11-476D17 and
digoxin-dUTP labeled BAC clone RP11-95I21). All BAC clones were
obtained from the Children's Hospital of Oakland Research Institute
(CHORI). Prior to tissue analysis, the integrity and purity of all
probes were verified by hybridization to metaphase spreads of
normal peripheral lymphocytes. Tissue hybridization, washing and
color detection were performed as described (Rubin et al., Cancer
Res 64, 3814 (2004); Garraway et al., Nature 436, 117 (2005)).
B. Results
Cancer Outlier Profile Analysis
[0303] In recent years, gene expression profiling with DNA
microarrays has become a common method to study the cancer
transcriptome. Microarray studies have provided great insight into
the molecular heterogeneity of cancer, often identifying novel
molecular subtypes of disease that correspond to tumor histology,
patient outcome, and treatment response (Valk et al., N Engl J Med
350, 1617 (2004)). However, in general, transcriptome analysis has
not led to the discovery of novel causal cancer genes. It was
hypothesized that rearrangements and highlevel copy number changes
that result in marked over-expression of an oncogene should be
evident in transcriptome data, but not necessarily by traditional
analytical approaches.
[0304] In the majority of cancer types, heterogeneous patterns of
oncogene activation have been observed, thus traditional analytical
methods that search for common activation of genes across a class
of cancer samples (e.g., t-test or signal-to-noise ratio) will fail
to find such oncogene expression profiles. Instead, a method that
searches for marked over-expression in a subset of cases is needed.
Experiments conducted during the course of development of the
present invention resulted in the development of Cancer Outlier
Profile Analysis (COPA). COPA seeks to accentuate and identify
outlier profiles by applying a simple numerical transformation
based on the median and median absolute deviation of a gene
expression profile (Ross et al., Blood 102, 2951 (2003)). This
approach is illustrated in FIG. 5A. COPA was applied to the
Oncomine database (Bittner et al., Nature 406, 536 (2000)), which
comprised a compendium of 132 gene expression datasets representing
10,486 microarray experiments. COPA correctly identified several
outlier profiles for genes in specific cancer types in which a
recurrent rearrangement or high-level amplification is known to
occur. The analysis was focused on outlier profiles of known causal
cancer genes, as defined by the Cancer Gene Census (Vasselli et
al., Proc Natl Acad Sci USA 100, 6958 (2003)), that ranked in the
top 10 outlier profiles in an Oncomine dataset (Table 1 and Table
3). For example, in the Valk et al. acute myeloid leukemia (AML)
dataset, RUNX1T1 (ETO) had the strongest outlier profile at the
95th percentile, consistent with this gene's known translocation
and oncogenic activity in a subset of AML (Davis et al., Proc Natl
Acad Sci USA 100, 6051 (2003)) (Table 1). The outlier profile
precisely associated with cases that had a documented t(8; 21)
translocation which fuses RUNX1 (AML1) and RUNX1T1 (ETO) (FIG. 5B).
Similarly, in the Ross et al. acute lymphoblastic leukemia (ALL)
dataset, PBX1 showed the strongest outlier profile at the 90th
percentile, consistent with the E2A-PBX1 translocation known to
occur in a subset of ALL (Segal et al., J Clin Oncol 21, 1775
(2003)) (Table 1). Again, the outlier expression profile perfectly
correlated with the characterized t(1; 19) E2A-PBX1 translocation
in this panel of ALLs (FIG. S1C).
Identification of Outlier Profiles for ETS Family Members ERG and
ETV1 in Prostate Cancer
[0305] Novel COPA predictions were next examined. In several
independent datasets, COPA identified strong outlier profiles in
prostate cancer for ERG and ETV1, two ETS family transcription
factors that are known to be involved in oncogenic translocations
in Ewing's sarcoma and myeloid leukemias (Lapointe et al., Proc
Natl Acad Sci USA 101, 811 (2004); Tian et al., N Engl J Med 349,
2483 (2003)). In the Dhanasekaran et al. (Keats et al., Blood 105,
4060 (2005)), Welsh et al. (Dhanasekaran et al., Faseb J 19, 243
(2005)) and Lapointe et al. (Wang et al., Lancet 365, 671 (2005))
prostate cancer gene expression datasets, ERG had the highest
scoring outlier profile at the 75th percentile (Table 1), while in
the Lapointe et al. and Tomlins et al. (Welsh et al., Cancer Res
61, 5974 (2001)) datasets, ETV1 had the highest scoring outlier
profile at the 90th percentile (Table 1). In total, COPA ranked ERG
or ETV1 within the top ten outlier genes nine times in seven
independent prostate cancer profiling studies. Both ERG and ETV1
are involved in oncogenic translocations in Ewing's sarcoma. Fusion
of the 5' activation domain of the EWS gene to the highly conserved
3' DNA binding domain of an ETS family member, such as ERG (t(21;
22)(q22; q12)) or ETV1 (t(7; 22)(p21; q12)), is characteristic of
Ewing's sarcoma (Lapoint et al., supra; Zhan et al., Blood 99, 1745
(2002); Fonseca et al., Cancer Res 64, 1546 (2004)). Because
translocations involving ETS family members are functionally
redundant in oncogenic transformation, only one type of
translocation is typically observed in each case of Ewing's
sarcoma.
[0306] It was contemplated that if ERG and ETV1 are similarly
involved in the development of prostate cancer, their outlier
profiles should be mutually exclusive, that is, each case should
over-express only one of the two genes. Mutations in functionally
redundant genes, or genes in the same oncogenic pathway, are
unlikely to be co-selected for in neoplastic progression. The joint
expression profiles of ERG and ETV1 was examined across several
prostate cancer datasets and it was found that they showed mutually
exclusive outlier profiles. ERG and ETV1 expression profiles from
two large-scale transcriptome studies (Wang et al., supra; Cheok et
al., Nat Genet 34, 85 (2003)), which profiled grossly dissected
prostate tissues using different microarray platforms, were
identified (FIG. 1A, left and middle panels). The study by Lapointe
et al. profiled benign prostate tissue, clinically localized
prostate cancer, and metastatic prostate cancer, with ERG and ETV1
outlier expression restricted to prostate cancer and metastatic
prostate cancer, while the study by Glinsky et al. profiled
clinically localized prostate cancer samples only. In both studies,
prostate cancers exclusively expressed ERG or ETV1 (FIG. 1A, right
panel). Similar results were found in a profiling study of 99
prostate tissue samples obtained by laser capture microdissection
(LCM) (Welsh et al., supra). In addition to exclusive outlier
expression of either ERG or ETV1 (FIG. 1B, right panel), results
from the LCM study demonstrated that ETV1 and ERG are only
over-expressed in epithelial cells from prostate cancer or
metastatic prostate cancer, but not in the putative precursor
lesion prostatic intraepithelial neoplasia (PIN) or adjacent benign
epithelia. To directly determine whether the observed exclusive
outlier pattern is consistent with other translocations where an
activating gene can fuse with multiple partners, the Zhan et al.
multiple myeloma dataset (Dhanasekaran et al., Nature 412, 822
(2001)) was examined. Recurrent fusions of the immunoglobulin heavy
chain promoter to CCND1 or FGFR3, t(11,14) or t(4,14) respectively,
characterize specific subsets of multiple myeloma (Wigle et al.,
Cancer Res 62, 3005 (2002)). These translocations were reflected in
the outlier profile analysis (FIG. 1C), as CCND1 was the highest
scoring outlier at the 75th percentile and FGFR3 was the third
highest scoring outlier at the 95th percentile (Table 1). Except
for two cases, myeloma samples showed exclusive over-expression of
CCND1 or FGFR3 (FIG. 1C, right panel). Taken together, the outlier
profiles of ERG and ETV1 across multiple prostate cancer data sets
are consistent with other causal mutations in various human
malignancies. The exclusive over-expression of ERG or ETV1 in
individual prostate cancer samples is consistent with other
neoplasms in which an activating gene can fuse with biologically
redundant partner genes, such as in multiple myeloma.
Discovery of a Recurrent Gene Fusion of TMPRSS2 to ERG or ETV1 in
Prostate Cancer.
[0307] The mechanism of ERG and ETV1 over-expression in individual
prostate cancer samples was next determined. Prostate cancer cell
lines and clinical specimens that over-expressed ERG or ETV1 were
identified by performing quantitative PCR (QPCR) (FIG. 2A). The
LNCaP prostate cancer cell line and two specimens obtained from a
patient who died of hormone refractory metastatic prostate cancer
(MET-26RP, residual primary carcinoma in the prostate and MET-26LN,
a lymph node metastasis) markedly over-expressed ETV1 by QPCR (FIG.
2A). Five independent metastatic foci from different anatomical
locations as well as the residual carcinoma in the prostate from
this patient also over-expressed ETV1 by DNA microarray analysis
(Welsh et al., supra), suggesting that ETV1 activation occurred in
the primary tumor before widespread metastasis. A lymph node
metastasis was also identified from a second patient who died of
hormone refractory metastatic prostate cancer (MET-28LN) and two
prostate cancer cell lines, VCaP and DuCaP, that over-expressed ERG
(FIG. 2A). These cell lines were independently isolated from a
vertebral metastasis (VCaP) and a dural metastasis (DuCaP) from a
third patient with hormone-refractory prostate cancer (Golub et
al., Science 286, 531 (1999); Rosenwald et al., Cancer Cell 3, 185
(2003)). The common over-expression of ERG in these two cell lines
again suggests that ERG activation occurred before widespread
metastasis. Taken together, these results suggest that specific
genetic events may activate ERG or ETV1 in individual samples
during prostate tumorigenesis.
[0308] In an effort to characterize these genetic events, samples
with high ERG or ETV1 expression were tested for chromosomal
amplifications at their respective loci (7p21.2 and 21q22.3). By
QPCR on genomic DNA, amplification of ERG or ETV1 in samples with
respective transcript over-expression (Sotiriou et al., Proc Natl
Acad Sci USA 100, 10393 (2003)) was not found. Next, the occurrence
of DNA rearrangements was assayed. Because the primers used for the
QPCR described above were located 5' to the known breakpoints for
ERG and ETV1 in Ewing's sarcoma, it was unlikely that the same
translocations occur in prostate cancer. Accordingly, the
expression level of ETV1 exons was measured by exonwalking QPCR in
the samples identified above that displayed ETV1 over-expression.
Five primer pairs spanning ETV1 exons 2 through 7 were used and
LNCaP cells showed essentially uniform over-expression of all
measured ETV1 exons, and both MET26 specimens showed >90%
reduction in the expression of ETV1 exons 2 and 3 compared to exons
4-7 (FIG. 2B). Potential explanations for this result include
alternative splicing, a novel cancer-specific isoform or an
unreported rearrangement.
[0309] In order to characterize the full length ETV1 transcript, 5'
RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) was
performed on LNCaP cells and MET26-LN. In addition, RLM-RACE was
performed to obtain the full length transcript of ERG in MET28-LN.
For PCR amplification of ETV1 from the RLM-RACE cDNA, a forward
primer complementary to the RNA-oligonucleotide ligated to the 5'
end of complete transcripts and a reverse primer in exon 4, the
5'-most exon that was over-expressed in both LNCaP cells and
MET26-LN was used. Utilizing a similar strategy as described above,
it was determined that exon 4 of ERG was over-expressed in
MET28-LN. A reverse primer in this exon was utilized for PCR
amplification of RLM-RACE cDNA. Sequencing of the cloned products
revealed fusions of the prostate specific gene TMPRSS2 (28)
(21q22.2) with ETV1 in MET26-LN and with ERG in MET28-LN (FIG. 2C).
In MET26-LN, two RLM-RACE PCR products were identified. The first
product, TMPRSS2:ETV1a, resulted in a fusion of the complete exon 1
of TMPRSS2 with the beginning of exon 4 of ETV1 (FIG. 2C). The
second product, TMPRSS2:ETV1b, resulted in a fusion of exons 1 and
2 of TMPRSS2 with the beginning of exon 4 of ETV1 (FIG. 6). Both
products are consistent with the exon-walking QPCR described above,
where MET26-LN showed loss of over-expression in exons 2 and 3. In
MET28-LN, a single RLM-RACE PCR product was identified and
sequencing revealed a fusion of the complete exon 1 of TMPRSS2 with
the beginning of exon 4 of ERG (TMPRSS2:ERGa) (FIG. 2C).
Validation of TMPRSS2:ERG and TMPRSS2:ETV1 Gene Fusions in Prostate
Cancer
[0310] Based on these results, QPCR primer pairs were designed with
forward primers in TMPRSS2 and reverse primers in exon 4 of ERG and
ETV1. SYBR Green QPCR was performed using both primer pairs across
a panel of samples from 42 cases of clinically localized prostate
cancer and metastatic prostate cancer, with representative results
depicted (FIGS. 2, D and E). These results demonstrate that only
samples with high levels of ETV1 or ERG express the respective
fusion product with TMPRSS2. Although QPCR resulted in measurable
product after 35 cycles in some negative samples, melt curve
analysis revealed distinct products in positive and negative
samples, and gel electrophoresis of products after the 40 cycle
QPCR analysis revealed only primer dimmers in negative fusion
samples (FIGS. 2, D and E). The formation of primer dimers may in
part be explained by the difficulty in designing primers entirely
in exon 1 of TMPRSS2 due to the high GC content (80.3%). However,
the specific expression of TMPRSS2:ERGa, TMPRSS2:ETVla and
TMPRSS2:ETV1b fusions was confirmed using Taqman QPCR, with the
forward primer spanning the respective fusion, and in each case,
products were only detected in the same cases as the SYBR Green
QPCR (Sotiriou et al., supra). To further confirm the specificity
of the primers used for SYBR Green QPCR and the amplicons, standard
reverse-transcription PCR was performed with the same primers as
the SYBR Green QPCR on a panel of samples that expressed
TMPRSS2:ERGa. Similar sized products were obtained and sequencing
of cloned products confirmed the presence of TMPRSS2:ERGa. Two
cases, PCA16 and PCA17, which expressed high levels of ETV1 or ERG
respectively, but showed no evidence of the translocation by QPCR
(FIGS. 2, D and E) were identified. RLM-RACE supported these
results, as sequencing of the product produced with ETV1 primers in
PCA16 revealed no evidence of a fusion transcript and no product
could be obtained with ERG primers in PCA17. Similar results were
obtained for LNCaP cells, with no evidence of a fusion by RLMRACE
or QPCR, consistent with the exon walking QPCR described above.
Summary of Evidence for TMPRSS2 Fusion Transcripts with ETS Family
Members in Prostate Cancer Samples
[0311] Results from three different assays for the TMPRSS2:ERG and
TMPRSS2:ETV1 fusion transcripts including sequencing of RLM-RACE
products, QPCR and sequencing of RT-PCR products are summarized in
Table 2. In addition to QPCR for TMPRSS2 fusions being performed in
all samples, the existence of these fusions was confirmed using
several techniques on selected samples. For example, in PCA1
(prostate cancer sample 1), TMPRSS2:ERGa was identified using
sequencing of RLMRACE products, QPCR and sequencing of RT-PCR
products. By QPCR melt curve analysis and gel electrophoresis of
QPCR products, PCA4 produced a larger amplicon than expected.
Subsequent RLM-RACE analysis confirmed a fusion of the complete
exon 1 of TMPRSS2 with the beginning of exon 2 of ERG
(TMPRSS2:ERGb) (FIG. 6). Taqman QPCR with the forward primer
spanning the TMPRSS2:ERGb junction confirmed the presence of
TMPRSS2:ERGb only in PCA4 and Taqman QPCR with the forward primer
spanning the TMPRSS2:ERGa junction did not produce a product in
this specimen (27). Evidence for the TMPRSS2:ERG and TMPRSS2:ETV1
fusions were only found in cases that over-expressed ERG or ETV1
respectively, by QPCR or DNA microarray. These results are in
agreement with the exclusive expression observed in the outlier
analysis.
Fluorescence In Situ Hybridization (FISH) Confirms TMPRSS2:ETV1
Translocation and ERG Rearrangement
[0312] After confirming the existence of the TMPRSS2:ETV1 and
TMPRSS2:ERG fusion transcripts, evidence of these rearrangements at
the chromosomal level was obtained using interphase fluorescence in
situ hybridization (FISH) on formalin fixed paraffin embedded
(FFPE) specimens. Two different probe strategies were employed: a
twocolor, fusion-signal approach to detect TMPRSS2:ETV1
translocations and a two-color, split-signal approach to detect
rearrangements of the ERG locus. These probe strategies were
validated on the two cases initially used for RLM-RACE, MET26 and
MET28 (FIG. 3). Using probes for TMPRSS2 and ETV1, normal
peripheral lymphocytes (NPLs) demonstrated a pair of red and a pair
of green signals (FIG. 3A). MET26 showed fusion of one pair of
signals, indicative of probe overlap (FIG. 3B, yellow arrowhead),
consistent with the expression of the TMPRSS2:ETV1 transcript in
this sample. In addition, consistent low-level amplification of the
ETV1 locus was identified, as indicated by the two remaining
signals for ETV1 (FIG. 3B, red arrowheads). Similarly, using probes
spanning the 5' and 3' region of the ERG locus, a pair of yellow
signals in NPLs was observed (FIG. 3C). In MET28, one pair of
probes split into separate green and red signals, indicative of a
rearrangement at the ERG locus (FIG. 3D, green and red arrows).
This result is consistent with the expression of the TMPRSS2:ERG
transcript in this case. Based on these results, the individual
FISH analyses described above were performed on serial tissue
microarrays containing cores from 13 cases of localized prostate
cancer and 16 cases of metastatic prostate cancer (FIG. 3E). As
indicated by the matrix, 23 of 29 cases (79.3%) showed evidence of
TMPRSS2:ETV1 fusion (7 cases) or ERG rearrangement (16 cases). In
addition, 12 of 29 cases (41.4%) showed evidence of low level
amplification at the ETV1 locus. Previous reports have identified
the genomic location of ETV1, 7p, as one of the most commonly
amplified regions in localized and metastatic prostate cancer
(Slamon et al., Science 235, 177 (1987)). However it does not
appear that 7p amplification drives ETV1 expression, as ETV1
amplification occurred in 6 cases with ERG rearrangements and our
transcript data demonstrates that 0 of 19 samples with high ERG
expression and the TMPRSS2:ERG fusion also have high ETV1
expression. Furthermore, when both ETV1 amplification and the
TMPRSS2:ETV1 fusion were present by FISH, only the individual ETV1
signal was amplified and not the fused signal. Nevertheless,
results from this FISH analysis demonstrate the presence of
TMPRSS2:ETV1 and ERG rearrangements at the genomic level consistent
with the transcript data described above.
[0313] TMPRSS2 is an androgen-regulated gene and fusion with ERG
results in androgen regulation of ERG. TMPRSS2 was initially
identified as a prostate-specific gene whose expression was
increased by androgen in LNCaP cells and also contains androgen
responsive elements (AREs) in its promoter (Huang et al., Lancet
361, 1590 (2003); Schwartz et al., Cancer Res 62, 4722 (2002)).
Subsequent studies have confirmed high expression in normal and
neoplastic prostate tissue and demonstrated that TMPRSS2 is
androgen-regulated in androgen-sensitive prostate cell lines
(Schwartz et al., Cancer Res 62, 4722 (2002); Ferrando et al.,
Cancer Cell 1, 75 (2002); Chen et al., Mol Biol Cell 14, 3208
(2003); LaTulippe et al., Cancer Res 62, 4499 (2002)). In addition,
while androgen does not increase the expression of TMPRSS2 in the
androgen insensitive prostate cancer cell line PC3, stable
expression of the androgen receptor in PC3 cells resulted in
TMPRSS2 becoming androgen responsive (Schwartz et al., supra;
Ferrando et al., supra; Chen et al., supra; LaTulippe et al.,
supra). In contrast, microarray studies of LNCaP prostate cell
lines treated with androgen have not identified ERG or ETV1 as
being androgen-responsive (Jain et al., Cancer Res 64, 3907 (2004))
and examination of their promoter sequences did not reveal
consensus AREs (Sotiriou et al., supra). It was contemplated that
the TMPRSS2:ERGa fusion in DuCaP and VCaP cell lines, which was
confirmed by three independent assays in each cell line (Table 2),
would result in the androgen regulation of ERG. Using QPCR to assay
for ERG expression, it was confirmed that even though ERG was
highly expressed in both VCaP and DuCaP cells, treatment with the
synthetic androgen R1881 increased the expression of ERG 2.57 fold
in DuCaP cells and 5.02 fold in VCaP cells compared to untreated
controls (FIG. 4). Expression of ERG was minimal and essentially
unchanged after R1881 treatment in RWPE (1.37 fold), LnCaP (0.86
fold), PC3 (1.28 fold) and PC3 cells expressing the androgen
receptor (0.73 fold) compared to untreated controls.
[0314] Microarray analysis of the same samples confirmed that ERG
was only up-regulated in response to androgen in DuCaP and VCaP
cells (Sotiriou et al., supra). The present invention is not
limited to a particular mechanism. Indeed, an understanding of the
mechanism is not necessary to practice the present invention.
Nonetheless, it is contemplated that these results suggest a
possible mechanism for the aberrant expression of ERG or ETV1 in
prostate cancer when respective fusions with TMPRSS2 are
present.
Table 1. Cancer Outlier Profile Analysis (COPA). Genes known to
undergo causal mutations in cancer that had strong outlier
profiles. "X", signifies literature evidence for acquired
pathogenomic translocation. "XX" signifies literature evidence for
the specific translocation as well as the samples in the specific
study that were characterized for that translocation. "Y" signifies
consistent with known amplification. "**" signifies ERG and ETV1
outlier profiles in prostate cancer.
TABLE-US-00002 TABLE 1 Rank % Score Study Cancer Gene Evidence 1 95
20.056 Valk et al., N Engl J Leukemia RUNX1T1 XX Med 350, 1617
(2004) 1 95 15.4462 Vasselli et al., Renal PRO1073 X PNAS USA 100,
6958 (2003) 1 90 12.9581 Ross et al., Blood Leukemia PBX1 XX 102,
2951 (2003). 1 95 10.03795 Lapointe et al., Prostate ETV1 ** PNAS
USA 101, 811 (Jan. 20, 2004) 1 90 9.1163 Prostate ETV1 ** 1 90
7.4557 Tian et al., N Engl J Myeloma WHSC1 X Med 349, 2483 (2003) 1
75 5.4071 Dhanasekaran et al., Prostate ERG ** Nature 412, 822
(2001) 1 75 4.3628 Welsh et al., Cancer Prostate ERG ** Res 61,
5974 (2001) 1 75 4.3425 Zhan et al., Blood Myeloma CCND1 X 99, 1745
(2002) 1 75 3.4414 Lapointe et al., Prostate ERG ** supra 1 75
3.3875 Dhanasekaran et al., Prostate ERG ** Faseb J 19, 243 (2005)
2 90 6.7029 Prostate ERG ** 3 95 13.3478 Zhan et al., supra Myeloma
FGFR3 X 4 75 2.5728 Huang et al., Lancet Breast ERBB2 Y 361, 1590
(2003) 6 90 6.6079 Sotiriou et al., Breast ERBB2 Y PNAS USA 100,
10393 (2003) 9 95 17.1698 Glinsky et al., J Prostate ETV1 ** Clin
Invest 113, 913 (2004) 9 90 6.60865 Nielsen et al., Sarcoma SSX1 X
Lancet 359, 1301 (2002) 9 75 2.2218 Yu et al., J Clin Prostate ERG
** Oncol 22, 2790 (2004)
Table 2 shows a summary of TMPRSS2 fusion to ETS family member
status in prostate cancer samples and cell lines. For all assays,
positive results are indicated by "+" and negative results are
indicated by "-". Blank cells indicate that the specific assay was
not performed for that sample. Over-expression of ERG or ETV1 by
quantitative PCR (QPCR) is indicated and samples marked with an
asterisk indicate the sample was also assessed by cDNA microarray
and over-expression was confirmed. In order to detect TMPRSS2:ERG
or TMPRSS2:ETV1 gene fusions, selected samples were subjected to
RLM-RACE for the over-expressed ETS family member and samples with
the TMPRSS2 fusion after sequencing are indicated. All samples were
assayed for TMPRSS2:ETV1 and TMPRSS2:ERG expression by QPCR.
Selected cases were also amplified by standard
reverse-transcription PCR (RT-PCR) using the same TMPRSS2 fusion
primers as for QPCR and amplicons were sequenced. Samples with
evidence for TMPRSS2:ETV1 or TMPRSS2:ERG fusion are indicated in
the final column.
TABLE-US-00003 TABLE 2 TMPRSS2:ETS family member gene fusion assays
TMPRSS2:ETS RLM- family QPCR RACE QPCR QPCR RT-PCR member Case
Sample Expression sequencing TMPRSS2:ETV1 TMPRSS2:ERG sequencing
fusion 1 MET26- ETV1* + + - + LN 1 MET26- ETV1* + - + RP 2 MET28-B
ERG - + + 2 MET28- ERG - + + PTLN 2 MET28- ERG - + + 41 2 MET28-
ERG + - + + LN 3 MET16- ERG - + + 44 3 MET16- ERG - + + 47 4 MET3
ERG* - + + 5 MET18- ERG* - + + + 23 6 PCA1 ERG* + - + + + 7 PCA2
ERG* - + + + 8 PCA3 ERG* - + + + 9 PCA4 ERG* + - + + 10 PCA5 ERG* +
- + + 11 PCA6 ERG* - + + 12 PCA7 ERG* + - + + 13 PCA8 ERG* - + + 14
PCA9 ERG* - + + 15 PCA10 ERG* - + + 16 PCA11 ERG* - + + 17 PCA12
ERG* - + + 18 PCA13 ERG* - + + 19 PCA14 ERG* - + + 20 PCA15 ERG* -
+ + 21 PCA16 ETV1* - - - - 22 PCA17 ERG* - - - - 23 MET30- -- - - -
LN 24 MET17- -- - - - 12 25 MET20- -- - - - 76 26 MET22- -- - - -
61 27 MET5-7 -- - - - 28 PCA18 -- - - - 29 PCA19 -- - - - 30 PCA20
-- - - - 31 PCA21 -- - - - 32 PCA22 -- - - - 33 PCA23 -- - - - 34
PCA24 -- - - - 35 PCA25 -- - - - 36 PCA26 -- - - - 37 PCA27 -- - -
- 38 PCA28 -- - - - 39 PCA29 -- - - - 40 PCA30 -- - - - 41 PCA31 --
- - - 42 PCA32 -- - - - Cell VCap ERG + - + + line Cell DUCaP ERG -
+ + + line Cell LnCaP ETV1 - - - - line Cell DU145 -- - - - line
Cell PC3 -- - - - line Cell RWPE -- - - - line
Table 3. Cancer Outlier Profile Analysis (COPA). Genes that are
known to undergo causal mutations in cancer that had an outlier
profile in the top 10 of a study in Oncomine are shown. "X",
signifies literature evidence for acquired pathognomonic
translocation. "XX" signifies literature evidence for the specific
translocation as well as that the samples in the specific study
were characterized for that translocation. "Y" signifies consistent
with known amplification. "**" signifies ERG and ETV1 outlier
profiles in prostate cancer.
TABLE-US-00004 TABLE 3 Rank % Score Study Cancer Reference Gene
Evidence 1 90 21.9346 Bittner et al. Melanoma Nature CDH1 406, 536
(2000) 1 95 20.056 Valk et al. Leukemia Nature RUNX1T1 XX 406, 536
(2000) 1 95 15.4462 Vasselli et al. Renal PNAS PRO1073 X (12) USA
100, 6958 (2003) 1 95 14.2008 Segal et al. Sarcoma J Clin MYH11
Oncol 21, 1775 (2003) 1 90 12.9581 Ross et al. Leukemia Blood PBX1
XX 102, 2951 (2003) 1 95 10.03795 Lapointe et Prostate PNAS ETV1 **
al. USA 101, 811 (2004) 1 90 9.1163 Prostate ETV1 ** 1 90 7.4557
Tian et al. Myeloma N Engl J WHSC1 X (16) Med 349, 2483 (2003) 1 75
5.4071 Dhanasekaran Prostate Faseb J ERG ** et al. 19, 243 (2005) 1
75 5.2067 Wang et al. Breast Lancet FOXO3A 365, 671 (2005) 1 75
4.3628 Welsh et al. Prostate Cancer ERG ** Res 61, 5974 (2001) 1 75
4.3425 Zhan et al. Myeloma Blood 99, CCND1 X (21) 1745 (2002) 1 75
3.724 Cheok et al. Leukemia Nat Genet PCSK7 34, 85 (May, 2003) 1 75
3.4414 Lapointe et Prostate PNAS ERG ** al. USA 101, 811 (2004) 1
75 3.3875 Dhanasekaran Prostate Nature ERG ** et al. 412, 822
(2001) 1 75 2.5913 Wigle et al. Lung Cancer IGH@ Res 62, 3005
(2002) 2 90 12.7953 Ross et al. Leukemia Blood HOXA9 102, 2951
(2003) 2 95 9.2916 Golub et al. Leukemia Science TRA@ 286, 531
(1999) 2 95 9.2916 Golub et al. Leukemia Science TRD@ 286, 531
(1999) 2 90 8.2292 Cheok et al. Leukemia Nat Genet SSX2 34, 85
(May, 2003) 2 90 6.7029 Prostate ERG ** 3 95 13.3478 Zhan et al.
Myeloma Blood 99, FGFR3 X (21) 1745 (2002) 3 95 10.2267 Cheok et
al. Leukemia Nat Genet ARHGAP26 34, 85 (May, 2003) 3 90 5.9174
Prostate REL 3 75 2.6162 Rosenwald et Lymphoma Cancer TCL1A al.
Cell 3, 185 (2003) 3 75 2.036 Sotiriou et al. Breast PNAS RAD51L1
USA 100, 10393 (2003) 4 75 8.4985 Bittner et al. Melanoma Nature
TP53 406, 536 (2000) 4 90 5.4881 Golub et al. Leukemia Science LCK
286, 531 (1999) 4 75 2.5728 Huang et al. Breast Lancet ERBB2 Y(29)
361, 1590 (2003) 4 75 2.0229 Schwartz et Ovarian Cancer IGL@ al.
Res 62, 4722 (2002) 6 90 17.3733 Ferrando et Leukemia Cancer ZBTB16
al. Cell 1, 75 (2002) 6 95 9.1267 Chen et al. Gastric Mol Biol
FGFR2 Cell 14, 3208 (2003) 6 90 6.6079 Sotiriou et al. Breast PNAS
ERBB2 Y(29) USA 100, 10393 (2003) 6 75 5.7213 LaTulippe et al.
Prostate Cancer NF1 Res 62, 4499 (2002) 6 75 5.2752 Jain et al.
Endocrine Cancer PHOX2B Res 64, 3907 (2004) 6 90 4.8383 Lapointe et
al. Prostate PNAS LAF4 USA 101, 811 (2004) 6 90 4.1779 Alizadeh et
al. Lymphoma Nature IRTA1 403, 503 (2000) 6 90 3.6325 Rosenwald et
al. Lymphoma N Engl J IRTA1 Med 346, 1937 (2002) 6 75 1.85865 Chen
et al. Liver Mol Biol HMGA1 Cell 13, 1929 (2002) 7 95 4.7561 Alon
et al. Colon Proc Natl NONO Acad Sci USA 96, 6745 (1999) 7 75
1.8133 Chen et al. Liver Mol Biol GPC3 Cell 13, 1929 (2002) 8 90
4.7068 Lacayo et al. Leukemia Blood EVI1 104, 2646 (2004) 8 90
4.7068 Lacayo et al. Leukemia Blood MDS1 104, 2646 (2004) 9 95
17.1698 Glinsky et al. Prostate J Clin ETV1 ** Invest 113, 913
(2004) 9 90 15.3889 Ferrando et al. Leukemia Ferrando MN1 et al.,
Cancer Cell 1, 75 (2002) 9 90 6.60865 Nielsen et al. Sarcoma Lancet
SSX1 X (42) 359, 1301 (2002) 9 90 4.4875 Lapointe et al. Prostate
PNAS CHEK2 USA 101, 811 (2004) 9 75 2.2218 Yu et al. Prostate J
Clin ERG ** Oncol 22, 2790 (2004) 10 95 10.6036 Segal et al.
Sarcoma Segal et KIT al., J Clin Oncol 21, 1775 (2003)
Table 4. Oligonucleotide primers used in this study. For all
primers, the gene, bases and exons (according to alignment of the
reference sequences described in the text with the May 2004
assembly of the human genome using the UCSC Genome Browser) are
listed. Forward primers are indicated with "f" and reverse primers
with "r".
TABLE-US-00005 TABLE 4 ##STR00001## ##STR00002##
Example 2
ETV4 Gene Fusions
A. Materials and Methods
ETS Family Expression in Profiling Studies
[0315] To investigate the expression of ETS family members in
prostate cancer, two prostate cancer profiling studies were
utilized (Lapointe et al., Proc Natl Acad Sci USA 2004; 101:811-6
and Tomlins et al., Science 2005; 310:644-8) present in the
Oncomine database (Rhodes et al., Neoplasia 2004; 6:1-6). Genes
with an ETS domain were identified by the Interpro filter `Ets`
(Interpro ID: IPRO00418). Heatmap representations were generated in
Oncomine using the `median-center per gene` option, and the color
contrast was set to accentuate ERG and ETV1 differential
expression.
Samples
[0316] Prostate cancer tissues (PCA1-5) were from the radical
prostatectomy series at the University of Michigan, which is part
of the University of Michigan Prostate Cancer Specialized Program
of Research Excellence (S.P.O.R.E.) Tissue Core. All samples were
collected with informed consent of the patients and prior
institutional review board approval. Total RNA was isolated with
Trizol (Invitrogen, Carlsbad, Calif.) according to the
manufacturer's instructions. A commercially available pool of
benign prostate tissue total RNA (CPP, Clontech, Mountain View,
Calif.) was also used.
Quantitative PCR (QPCR)
[0317] QPCR was performed using SYBR Green dye on an Applied
Biosystems 7300 Real Time PCR system (Applied Biosystems, Foster
City, Calif.) as described (Tomlins et al., supra). The amount of
each target gene relative to the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample
was reported. The relative amount of the target gene was calibrated
to the relative amount from the pool of benign prostate tissue
(CPP). All oligonucleotide primers were synthesized by Integrated
DNA Technologies (Coralville, Iowa). GAPDH primers were as
described (Vandesompele et al., Genome Biol 2002; 3:RESEARCH0034).
Primers for exons of ETV4 were as follows (listed 5' to 3'):
ETV4_exon2-f: CCGGATGGAGCGGAGGATGA (SEQ ID NO:21), ETV4_exon2-r:
CGGGCGATTTGCTGCTGAAG (SEQ ID NO:22), ETV4_exon3-f:
GCCGCCCCTCGACTCTGAA (SEQ ID NO:23), ETV4_exon4-r:
GAGCCACGTCTCCTGGAAGTGACT (SEQ ID NO:24), ETV4_exon11-f:
CTGGCCGGTTCTTCTGGATGC (SEQ ID NO:25), ETV4_exon12-r:
CGGGCCGGGGAATGGAGT (SEQ ID NO:26), ETV4.sub.--3'UTR-f:
CCTGGAGGGTACCGGTTTGTCA (SEQ ID NO:27), ETV4.sub.--3'UTR-r:
CCGCCTGCCTCTGGGAACAC (SEQ ID NO:28). Exons were numbered by
alignment of the RefSeq for ETV4 (NM.sub.--001986.1) with the May
2004 freeze of the human genome using the UCSC Genome Browser. For
QPCR confirmation of TMPRSS2:ETV4 fusion transcripts,
TMPRSS2:ETV4a-f (AAATAAGTTTGTAAGAGGAGCCTCAGCATC (SEQ ID NO:29)) and
TMPRSS2:ETV4b-f (ATCGTAAAGAGCTTTTCTCCCCGC (SEQ ID NO:30)), which
detects both TMPRSS2:ETV4a and TMPRSS2;ETV4b transcripts, were used
with ETV4_exon4-r.
RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE)
[0318] RLM-RACE was performed using the GeneRacer RLM-RACE kit
(Invitrogen), according to the manufacturer's instructions as
described (Tomlins et al., supra). To obtain the 5' end of ETV4,
first-strand cDNA from PCA5 was amplified using the GeneRacer 5'
Primer and ETV4_exon4-r or ETV4_exon7-r (GAAAGGGCTGTAGGGGCGACTGT
(SEQ ID NO:31)). Products were cloned and sequenced as described
(Tomlins et al., supra). Equivalent 5' ends of the TMPRSS2:ETV4
transcripts were obtained from both primer pairs.
Fluorescence In Situ Hybridization (FISH)
[0319] Formalin-fixed paraffin-embedded (FFPE) tissue sections were
used for interphase FISH. Deparaffinized tissue was treated with
0.2 M HCl for 10 min, 2.times.SSC for 10 min at 80.degree. C. and
digested with Proteinase K (Invitrogen) for 10 min. The tissues and
BAC probes were co-denatured for 5 min at 94.degree. C. and
hybridized overnight at 37.degree. C. Post-hybridization washing
was with 2.times.SSC with 0.1% Tween-20 for 5 min and fluorescent
detection was performed using anti-digoxigenin conjugated to
fluorescein (Roche Applied Science, Indianapolis, Ind.) and
streptavidin conjugated to Alexa Fluor 594 (Invitrogen). Slides
were counterstained and mounted in ProLong Gold Antifade Reagent
with DAPI (Invitrogen). Slides were examined using a Leica DMRA
fluorescence microscope (Leica, Deerfield, Ill.) and imaged with a
CCD camera using the CytoVision software system (Applied Imaging,
Santa Clara, Calif.).
[0320] All BACs were obtained from the BACPAC Resource Center
(Oakland, Calif.) and probe locations were verified by
hybridization to metaphase spreads of normal peripheral
lymphocytes. For detection of TMPRSS2:ETV4 fusion, RP11-35C4 (5' to
TMPRSS2) was used with multiple BACs located 3' to ETV4 (distal to
ETV4 to proximal: RP11-266124, RP11-242D8, and RP11-100E5). For
detection of ETV4 rearrangements, RP11-436J4 (5' to ETV4) was used
with the multiple BACs 3' to ETV4. For each hybridization, areas of
cancerous cells were identified by a pathologist and 100 cells were
counted per sample. The reported cell count for TMPRSS2:ETV4
fusions used RP11-242D8 and similar results were obtained with all
3' ETV4 BACs. To exclude additional rearrangements in PCA5, FISH
was performed with two probes 3' to ETV4 (RP11-266124 and
RP11-242D8), ERG split signal probes (RP11-95I21 and RP11-476D17)
and TMPRSS2:ETV1 fusion probes (RP11-35C4 and RP11-124L22). BAC DNA
was isolated using a QIAFilter Maxi Prep kit (Qiagen, Valencia,
Calif.) and probes were synthesized using digoxigenin- or
biotin-nick translation mixes (Roche Applied Science).
B. Results
[0321] The initial COPA screen led to the characterization of
TMPRSS2 fusions with ERG or ETV1 (Example 1). It was further
contemplated that prostate cancers negative for these gene fusions
harbor rearrangements involving other ETS family members. By
interrogating the expression of all ETS family members monitored in
prostate cancer profiling studies from the Oncomine database
(Rhodes et al., supra), marked over-expression of the ETS family
member ETV4 was identified in a single prostate cancer case from
each of two studies--one profiling grossly dissected tissues
(Lapointe et al., supra) (FIG. 7A) and the other profiling laser
capture microdissected (LCM) tissuesl (FIG. 7B). As these cases did
not over-express ERG or ETV1, and no benign prostate tissues showed
over-expression, it was contemplated that fusion with TMPRSS2 was
responsible for the over-expression of ETV4 in these cases.
Although ELF3 was also over-expressed in a fraction of prostate
cancer cases, in both studies normal prostate tissue samples also
showed marked ELF3 over-expression, indicating that a gene fusion
driving expression in both benign and cancerous tissue is unlikely.
Thus, the ETV4 over-expressing case (designated here as PCA5) was
further analyzed.
[0322] Total RNA was isolated from PCA5 and exon-walking
quantitative PCR was used (QPCR) to confirm the over-expression of
ETV4. QPCR demonstrated that exons 3' to exon 2 of ETV4 were
markedly over-expressed in this case compared to pooled benign
prostate tissue (CPP) (.about.900 fold) and prostate cancers that
did not over-express ETV4 and were either TMPRSS2:ERG positive
(PCA1-2) or negative (PCA3-4) (FIG. 8A). However, a dramatic
decrease (>99%) in the expression of exon 2 of ETV4 relative to
distal regions in PCA5 was observed, indicating a possible fusion
with TMPRSS2, as observed previously in TMPRSS2:ERG and
TMPRSS2:ETV1 positive cases (Tomlins et al., supra).
[0323] To identify the 5' end of the ETV4 transcript in PCA5,
RNA-ligase mediated rapid amplification of cDNA ends (RLM-RACE) was
performed using a reverse primer in exon 7. RLM-RACE revealed two
transcripts, each containing 5' ends consisting of sequence located
approximately 8 kb upstream of TMPRSS2 fused to sequence from ETV4
(FIG. 8B). Specifically, the 5' end of TMPRSS2:ETV4a has 47 base
pairs from this region upstream of TMPRSS2, while the 5' end of
TMPRSS2:ETV4b has the same terminal 13 base pairs. These 5' ends of
both transcripts were fused to the same contiguous stretch
consisting of the 9 base pairs of the intron immediately 5' to exon
3 of ETV4 and the reported reference sequence of exons 3 through
the reverse primer in exon 7 of ETV4.
[0324] The existence of both transcripts in PCA5 and their absence
in CPP and PCA1-4 was confirmed using QPCR. To further exclude the
presence of fusion transcripts involving known exons from TMPRSS2,
QPCR was performed using a forward primer in exon 1 of TMPRSS2 and
the ETV4 exon 4 reverse primer, and as expected, no product was
detected in CPP or PCA1-5.
[0325] Whether other prostate cancers with ETV4 dysregulation might
contain TMPRSS2:ETV4 fusion transcripts structurally more similar
to TMPRSS2:ERG and TMPRSS2:ETV1 transcripts (which involve known
exons from TMPRSS2) is unknown. The TMPRSS2:ETV4 fusions reported
here do not contain the well characterized AREs immediately
upstream of TMPRSS2. However, evidence exists for androgen
responsive enhancers located upstream of the TMPRSS2 sequences
present in the TMPRSS2:ETV4 transcripts described here (Rabbitts,
Nature 1994; 372:143-9). Nevertheless, the marked over-expression
of only ETV4 exons involved in the fusion transcript strongly
suggests that the gene fusion is responsible for the dysregulation
of ETV4. Together, the structure of the TMPRSS2:ETV4 fusion
transcripts supports the conclusion that the regulatory elements
upstream of TMPRSS2, rather than transcribed TMPRSS2 sequences,
drive the dysregulation of ETS family members.
[0326] To confirm the fusion of the genomic loci surrounding
TMPRSS2 (21q22) and ETV4 (17q21) as demonstrated by RLM-RACE and
QPCR, interphase fluorescence in situ hybridization (FISH) was
used. Using probes 5' to TMPRSS2 and 3' to ETV4, fusion of TMPRSS2
and ETV4 loci was observed in 65% of cancerous cells from PCA5
(FIG. 8D). As further confirmation of the rearrangement of ETV4,
using probes 5' and 3' to ETV4, 64% of cancerous cells from PCA5
showed split signals. FISH was also performed on PCA5 using two
probes 3' to ETV4, ERG split signal probes and TMPRSS2:ETV1 fusion
probes to exclude additional rearrangements, with negative results
obtained for each hybridization.
[0327] Taken together, the results highlight the use of carefully
examining outlier profiles in tumor gene expression data, as most
analytical methods discount profiles that do not show consistent
deregulation (Eisen et al., Proc Natl Acad Sci USA 1998;
95:14863-8; Golub et al., Science 1999; 286:531-7; Tusher et al.,
Proc Natl Acad Sci USA 2001; 98:5116-21) and would thus fail to
identify ETV4 in prostate cancer, which appears rare (2 of 98
cases). Combined with the identification of TMPRSS2:ERG and
TMPRSS2:ETV1 fusions, the results presented here show that
dysregulation of ETS family members mediated by subversion of AREs
or enhancers upstream of TMPRSS2 is a hallmark of prostate
tumorigenesis.
Example 3
Detection of Gene Fusion RNA
[0328] This example describes target capture, amplification and
qualitative detection of RNA (NT) containing the sequences of the
four gene fusions in four separate qualitative assays:
TMPRSS2:ETV1a, TMPRSS2:ETV1b, TMPRSS2:ERGa and TMPRSS2:ERGb with
APTIMA formulation reagents and HPA detection each spiked with the
appropriate target specific oligonucleotides, primers and probes.
Table 5 shows sequences of oligonucleotides used in the assay.
TABLE-US-00006 TABLE 5 SEQ ID Gene Fusion Sequence (5' to NO
TMPRSS2 exon1/Target Capture
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTTCUCGAUUCGUCCUCCG 59 TMPRSS2
exon1/Target Capture
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTTAUCCGCGCUCGAUUCGUC 60 TMPRSS2
exon1/Non-T7 GAGGGCGAGGGGCGGGGAGCGCC 61 TMPRSS2 exon2/Non-T7
CCTATCATTACTCGATGCTGTTGATAACAGC 62 ETV1a/b exon4/T7
AATTTAATACGACTCACTATAGGGAGAAACTTTCAGCCTGATA 63 ERGb exon2/T7
AATTTAATACGACTCACTATAGGGAGACTCTGTGAGTCATTTGTCTTGCTT 64 ERGa
exon4/T7 AATTTAATACGACTCACTATAGGGAGAGCACACTCAAACAACGACTG 65
TMPRSS2exon1: ETV1a Junction/AE GCGCGGCAG-CUCAGGUACCUGAC 66
TMPRSS2exon2: ETV1b Junction/AE GCUUUGAACUCA-CUCAGGUACCUGAC 67
TMPRSS2exon1: ERGa Junction/AE GAGCGCGGCAG-GAAGCCUUAUCAGUUG 68
TMPRSS2exon1: ERGb Junction/AE GAGCGCGGCAG-GUUAUUCCAGGAUCUUU 69
A. Materials and Methods
RNA Target Capture
[0329] Lysis buffer contained 15 mM sodium phosphate monobasic
monohydrate, 15 mM sodium phosphate dibasic anhydrous, 1.0 mM EDTA
disodium dihydrate, 1.0 mM EGTA free acid, and 110 mM lithium
lauryl sulfate, pH 6.7.
[0330] Target capture reagent contained 250 mM HEPES, 310 mM
lithium hydroxide, 1.88 M lithium chloride, 100 mM EDTA free acid,
at pH 6.4, and 250 .mu.g/ml 1 micron magnetic particles SERA-MAG
MG-CM Carboxylate Modified (Seradyn, Inc., Indianapolis, Ind.)
having dT).sub.14 oligomers covalently bound thereto.
[0331] Wash solution contained 10 mM HEPES, 6.5 mM sodium
hydroxide, 1 mM EDTA, 0.3% (v/v) ethanol, 0.02% (w/v) methyl
paraben, 0.01% (w/v) propyl paraben, 150 mM sodium chloride, 0.1%
(w/v) lauryl sulfate, sodium (SDS), at pH 7.5.
RNA Amplification & Detection
[0332] Amplification reagent was a lyophilized form of a 3.6 mL
solution containing 26.7 mM rATP, 5.0 mM rCTP, 33.3 mM rGTP and 5.0
mM rUTP, 125 mM HEPES, 8% (w/v) trehalose dihydrate, 1.33 mM dATP,
1.33 mM dCTP, 1.33 mM dGTP and 1.33 mM dTTP, at pH 7.5. The
Amplification reagent was reconstituted in 9.7 mL of the
amplification reagent reconstitution solution (see below). Before
use, 15 pmol each of primer oligomers was added.
[0333] Amplification reagent reconstitution solution contained 0.4%
(v/v) ethanol, 0.10% (w/v) methyl paraben, 0.02% (w/v) propyl
paraben, 33 mM KCl, 30.6 mM MgCl.sub.2, 0.003% phenol red.
[0334] Enzyme reagent was a lyophilized form of a 1.45 mL solution
containing 20 mM HEPES, 125 mM N-acetyl-L-cysteine, 0.1 mM EDTA
disodium dihydrate, 0.2% (v/v) TRITON7 X-100 detergent, 0.2 M
trehalose dihydrate, 0.90 RTU/mL Moloney murine leukemia virus
(MMLV) reverse transcriptase, and 0.20 U/mL T7 RNA polymerase, at
pH 7.0. One unit (RTU) of activity is defined as the synthesis and
release of 5.75 fmol cDNA in 15 minutes at 37.degree. C. for MMLV
reverse transcriptase, and for T7 RNA polymerase, one unit (U) of
activity is defined as the production of 5.0 fmol RNA transcript in
20 minutes at 37.degree. C. Enzyme reagent was reconstituted in 3.6
mL of the enzyme reagent reconstitution solution (see below).
[0335] Enzyme reagent reconstitution solution contained 50 mM
HEPES, 1 mM EDTA, 10% (v/v) TRITON7 X-100, 120 mM potassium
chloride, 20% (v/v) glycerol anhydrous, at pH 7.0.
[0336] Hybridization reagent contained 100 mM succinic acid free
acid, 2% (w/v) lithium lauryl sulfate, 100 mM lithium hydroxide, 15
mM aldrithiol-2, 1.2 M lithium chloride, 20 mM EDTA free acid, 3.0%
(v/v) ethanol, at pH 4.7.
[0337] Selection reagent contained 600 mM boric acid, 182.5 mM
sodium hydroxide, 1% (v/v) TRITON7 X-100, at pH 8.5.
[0338] The detection reagents comprised detect reagent I, which
contained 1 mM nitric acid and 32 mM hydrogen peroxide, and detect
reagent II, which contained 1.5 M sodium hydroxide.
B. Assay Protocol
Target Capture
[0339] 1. Prepare samples by making dilutions of IVT stock solution
into STM at indicated copy levels for 400 .mu.L sample per reaction
tube. [0340] 2. Using the repeat pipettor, add 100 .mu.L of the TCR
with the TCO to the appropriate reaction tube. [0341] 3. Using the
micropipettor, add 400 .mu.L of each sample to the properly
labeled. [0342] 4. Cover the tubes with the sealing card(s) and
shake the rack gently by hand. Do not vortex. Incubate the rack at
62.degree..+-.1.degree. C. in a water bath for 30.+-.5 minutes.
[0343] 5. Remove the rack from the water bath and blot bottoms of
tubes dry on absorbent material. [0344] 6. Ensure the sealing cards
are firmly seated. If necessary, replace with new sealing cards and
seal tightly. [0345] 7. Without removing sealing cards, incubate
the rack at room temperature for 30.+-.5 minutes. [0346] 8. Place
the rack on the TCS magnetic base for 5 to 10 minutes. [0347] 9.
Prime the dispense station pump lines by pumping APTIMA Wash
Solution through the dispense manifold. Pump enough liquid through
the system so that there are no air bubbles in the line and all 10
nozzles are delivering a steady stream of liquid. [0348] 10. Turn
on the vacuum pump and disconnect the aspiration manifold at the
first connector between the aspiration manifold and the trap
bottle. Ensure that the vacuum gauge reads greater than 25 inHg. It
may take 15 seconds to achieve this reading. Reconnect the
manifold, and ensure the vacuum gauge is between 7 and 12 inHg.
Leave the vacuum pump on until all target capture steps are
completed. [0349] 11. Firmly attach the aspiration manifold to the
first set of tips. Aspirate all liquid by lowering the tips into
the first TTU until the tips come into brief contact with the
bottoms of the tubes. Do not hold the tips in contact with the
bottoms of the tubes. [0350] 12. After the aspiration is complete,
eject the tips into their original tip cassette. Repeat the
aspiration steps for the remaining TTUs, using a dedicated tip for
each specimen. [0351] 13. Place the dispense manifold over each TTU
and, using the dispense station pump, deliver 1.0 mL of APTIMA Wash
Solution into each tube of the TTU. [0352] 14. Cover tubes with a
sealing card and remove the rack from the TCS. Vortex once on the
multi-tube vortex mixer. [0353] 15. Place rack on the TCS magnetic
base for 5 to 10 minutes. [0354] 16. Aspirate all liquid as in
steps 13 and 14. [0355] 17. After the final aspiration, remove the
rack from the TCS base and visually inspect the tubes to ensure
that all liquid has been aspirated. If any liquid is visible, place
the rack back onto the TCS base for 2 minutes, and repeat the
aspiration for that TTU using the same tips used previously for
each specimen.
Primer Annealing and Amplification
[0355] [0356] 1. Using the repeat pipettor, add 75 .mu.L of the
reconstituted Amplification Reagent containing the analyte specific
primers to each reaction tube. All reaction mixtures in the rack
should now be red in color. [0357] 2. Using the repeat pipettor,
add 200 .mu.L of Oil Reagent. [0358] 3. Cover the tubes with a
sealing card and vortex on the multi-tube vortex mixer. [0359] 4.
Incubate the rack in a water bath at 62.degree..+-.1.degree. C. for
10.+-.5 minutes. [0360] 5. Transfer the rack into a water bath at
42.degree..+-.1.degree. C. for 5.+-.2 minutes. [0361] 6. With the
rack in the water bath, carefully remove the sealing card and,
using the repeat pipettor, add 25 .mu.L of the reconstituted Enzyme
Reagent to each of the reaction mixtures. All reactions should now
be orange in color. [0362] 7. Immediately cover the tubes with a
fresh sealing card, remove from the water bath, and mix the
reactions by gently shaking the rack by hand. [0363] 8. Incubate
the rack at 42.degree..+-.1.degree. C. for 60.+-.15 minutes.
Hybridization
[0363] [0364] 1. Remove the rack from the pre-amplification water
bath and transfer to the post-amplification area. Add 100 .mu.L of
the reconstituted Probe Reagent with analyte specific probe, using
the repeat pipettor. All reaction mixtures should now be yellow in
color. [0365] 2. Cover tubes with a sealing card and vortex for 10
seconds on the multi-tube vortex mixer. [0366] 2. Incubate the rack
in a 62.degree..+-.1.degree. C. water bath for 20.+-.5 minutes.
[0367] 3. Remove the rack from the water bath and incubate at room
temperature for 5.+-.1 minutes
Selection
[0367] [0368] 1. Using the repeat pipettor, add 250 .mu.L of
Selection Reagent to each tube. All reactions should now be red in
color. [0369] 2. Cover tubes with a sealing card, vortex for 10
seconds or until the color is uniform, and incubate the rack in a
water bath at 62.degree..+-.1.degree. C. for 10.+-.1 minutes:
[0370] 3. Remove the rack from the water bath. Incubate the rack at
room temperature for 15.+-.3 minutes.
Reading the TTUs
[0370] [0371] 1. Ensure there are sufficient volumes of Auto
Detection Regents I and II to complete the tests. [0372] 2. Prepare
the LEADER Luminometer by placing one empty TTU in cassette
position number 1 and perform the WASH protocol. [0373] 3. Load the
TTUs into the luminometer and run the HC+Rev B protocol.
C. Results
[0374] The results are shown in Tables 6-9 for 4 assays with each
of the TMPRSS2:ERG and TMPRSS2:ETV1 gene fusion IVTs spiked into
TCR.
TABLE-US-00007 TABLE 6 TMPRSS2:ETV1a (copies IVT/reaction) RLU 0
4,945 0 4,599 10 2,185,959 10 2,268,090 10 2,284,908 100 2,270,369
100 2,302,023 100 2,272,735 1,000 2,279,627 1,000 2,285,742
TABLE-US-00008 TABLE 7 TMPRSS2:ETV1b (copies IVT/reaction) RLU 0
7,743 0 6,622 0 7,370 0 6,181 0 7,409 10 7,712 10 7,178 10 7,302 10
8,430 10 8,331 100 774,792 100 285,712 100 3,361,878 100 1,349,368
100 2,757,334 1,000 3,647,502 1,000 3,790,087 1,000 3,813,812 1,000
3,753,743 1,000 3,667,242
TABLE-US-00009 TABLE 8 TMPRSS2:ERGa (copies IVT/reaction) RLU 0
7,938 0 7,505 10 2,043,379 10 387,408 10 978,457 100 2,332,764 100
2,445,544 100 2,530,239
TABLE-US-00010 TABLE 9 TMPRSS2:ERGb (copies IVT/reaction) RLU 0
5,978 0 6,284 10 2,700,069 10 2,768,541 100 2,883,091 100 2,779,233
1,000 2,857,247 1,000 2,957,914
Example 4
FISH Assay for Gene Fusions
[0375] This Example describes the use of fluorescence in situ
hybridization (FISH), to demonstrate that 23 of 29 prostate cancer
samples harbor rearrangements in ERG or ETV1. Cell line experiments
suggest that the androgen-responsive promoter elements of TMPRSS2
mediate the overexpression of ETS family members in prostate
cancer. These results have implications in the development of
carcinomas and the molecular diagnosis and treatment of prostate
cancer.
[0376] Below is a list of the specific BAC probes used in FISH
assays.
[0377] Clinical FISH Assay for Testing Aberrations in ETS Family
Members by FISH [0378] Testing ETV1-TMPRSS2 fusion with one probe
spanning the ETV1 and one spanning the TMPRSS2 locus [0379] BAC for
ETV1: RP11-692L4 [0380] BAC for TMPRSS2: RP11-121A5, (RP11-120C17,
PR11-814F13, RR11-535H11) [0381] Testing ERG translocation with set
of probes for c-ERG:t-ERG break apart: [0382] BAC for c-ERG:
RP11-24A11 [0383] BACs for t-ERG: RP11-372017, RP11-137J13 [0384]
Testing ETV1 deletion/amplification with set of probes, one
spanning the ETV1 locus and one reference probe on chromosome 7:
[0385] BAC for ETV1: RP11-692L4 [0386] BAC for reference robe on
chromosome 7: A commercial probe on centromere of chr. [0387]
Testing ERG deletion/amplification with set of probes, one spanning
the ERG locus and one reference probe on chromosome 21: [0388] BAC
for ERG: RP11-476D17 [0389] BACs for reference probe on chromosome
21: * [0390] Testing TMPRSS2 deletion/amplification with set of
probes, one spanning the TMPRSS2 locus and one reference probe on
chromosome 21: [0391] BACs for TMPRSS2: RP11-121A5, (RP11-120C17,
PR11-814F13, RR11-535H11) [0392] BACs for reference probe on
chromosome 21: * *BACs for reference probe on chromosome 21:
PR11-32L6, RP11-752M23, RP11-1107H21, RP11-639A7,
(RP11-1077M21)
Example 5
TMPRSS2:ERG Fusion Associated Deletions
[0393] This example describes the presence of common deletions
located between ERG and TMPRSS2 on chromosome 21q22.2-3 associated
with the TMPRSS2:ERG fusion. Associations between disease
progression and clinical outcome were examined using a wide range
of human PCA samples, 6 cell lines, and 13 xenografts.
A. Materials and Methods
Clinical Samples
[0394] Prostate samples used for this study were collected under an
IRB approved protocol. All clinically localized PCA samples were
characterized by one pathologist and assigned a Gleason score to
eliminate inter-observer differences in pathology reporting.
Clinically localized PCA samples were collected as part of an
on-going research protocol at the University of Ulm. The hormone
refractory samples were taken from the Rapid Autopsy Program of the
University of Michigan.
[0395] The FISH experiments were conducted on two PCA outcome
arrays, which were composed of 897 tissue cores (histospots) from
214 patients. A summary of the patient demographics is presented in
Table 10. All patients had undergone radical prostatectomy with
pelvic lymphadenectomy at the University of Ulm (Ulm, Germany)
between 1989 and 2001. Pre-operative PSA ranged between 1 and 314
ng/ml (mean 36 ng/ml). Mean and maximum follow-up was 3.4 and 8.4
yrs, respectively.
Cell Lines and Xenografts
[0396] Androgen independent (PC-3, DU-145, HPV10, and 22Rv1) and
androgen sensitive (LNCaP) PCA cell lines were purchased from the
American Type Culture Collection (Manassas, Va.) and maintained in
their defined medium. HPV10 was derived from cells from a
high-grade PCA (Gleason score 4+4=8), which were transformed by
transfection with HPV18 DNA(18). 22Rv1 is a human PCA epithelial
cell line derived from a xenograft that was serially propagated in
mice after castration-induced regression and relapse of the
parental, androgen-dependent CWR22 xenograft. The VCAP cell line
was from a vertebral metastatic lesion as part of the Rapid Autopsy
program at the University of Michigan.
[0397] LuCaP 23.1, 35, 73, 77, 81, 86.2, 92.1, and 105 were derived
from patients with androgen independent hormone-refractory disease
PCA. LuCaP 49 and 115 are from patients with androgen dependent
PCA. LuCaP 58 is derived from an untreated patient with clinically
advanced metastatic disease and LuCaP 96 was established from a
prostate derived tumor growing in a patient with hormone refractory
PCA. LuCaP 49 (established from an omental mass) and LuCaP 93 are
hormone-insensitive (androgen receptor [AR]-negative) small cell
PCAs. These two xenografts demonstrate a neuroendocrine phenotype.
LuCaP 23.1 is maintained in SCID mice, and other xenografts are
maintained by implanting tumors in male BALB/c nu/nu mice.
Determining TMPRSS2:ERG Fusion Status Using Interphase FISH
[0398] The FISH analysis for the translocation of TMPRSS2:ERG is
described above and previously (Tomlins, et al., Science 310:644-8
(2005)). This break apart assay is presented in FIGS. 11 and 14.
For analyzing the ERG rearrangement on chromosome 21q22.2, a break
apart probe system was applied, consisting of the Biotin-14-dCTP
labeled BAC clone RP11-24A11 (eventually conjugated to produce a
red signal) and the Digoxigenin-dUTP labeled BAC clone RP11-137J13
(eventually conjugated to produce a green signal), spanning the
neighboring centromeric and telomeric region of the ERG locus,
respectively. All BAC clones were obtained from the BACPAC Resource
Center, Children's Hospital Oakland Research Institute (CHORD,
Oakland, Calif.
[0399] Using this break apart probe system, a nucleus without ERG
rearrangement exhibits two pairs of juxtaposed red and green
signals. Juxtaposed red-green signals form a yellow fusion signal.
A nucleus with an ERG rearrangement shows break apart of one
juxtaposed red-green signal pair resulting in a single red and
green signal for the translocated allele and a combined yellow
signal for the non-translocated allele in each cell. Prior to
tissue analysis, the integrity and purity of all probes were
verified by hybridization to normal peripheral lymphocyte metaphase
spreads. Tissue hybridization, washing, and fluorescence detection
were performed as described previously (Garraway, et al., Nature
436:117-22 (2005); Rubin, et al., Cancer Res. 64:3814-22 (2004)).
At least one TMA core could be evaluated in 59% PCA cases from two
TMAs. The technical difficulties with this assay included the
absence of diagnostic material to evaluate, weak probe signals, and
overlapping cells preventing an accurate diagnosis. The remainder
of the analysis focused on the 118 cases of clinically localized
PCA that could be evaluated. 15 cases had corresponding hormone
naive metastatic lymph node samples that could also be
evaluated.
[0400] The samples were analyzed under a 100.times. oil immersion
objective using an Olympus BX-51 fluorescence microscope equipped
with appropriate filters, a CCD (charge-coupled device) camera and
the CytoVision FISH imaging and capturing software (Applied
Imaging, San Jose, Calif.). Evaluation of the tests was
independently performed by two pathologists both with experience in
analyzing interphase FISH experiments. For each case, it was
attempted to score at least 100 nuclei per case. If significant
differences between the results of both pathologists were found,
the case was refereed by a third pathologist.
Oligonucleotide SNP Array Analysis
[0401] Although SNP arrays were intended for genotyping alleles,
the SNP array data can provide information on
Loss-of-Heterozygosity (Lieberfarb, et al., Cancer Res 63:4781-5
(2003); Lin, et al., Bioinformatics 20:1233-40 (2004)) and
detection of copy number alterations (Zhao, et al., Cancer Cell
3:483-95 (2003)). Using SNP array analysis, it was possible to
identify and validate amplified genes in various cancers including
melanoma (MITF) (Garraway, et al., Nature 436:117-22 (2005)) and
PCA (TPD52) (Rubin, et al., Cancer Res. 64:3814-22 (2004)).
[0402] SNP detection on the 100K array began with a reduction in
genome representation. Two aliquots of 250 ng of genomic DNA were
digested separately with XbaI HindIII. The digested fragments were
independently ligated to an oligonucleotide linker. The resulting
products were amplified using a single PCR primer under conditions
in which 200-2000 by PCR fragments were amplified. These fragments
represent a sub-fraction of the genome. The SNPs tiled on the
arrays have been pre-selected as they lie within these XbaI and
HindIII fragments and have been validated as robustly detected on
the arrays. The derived amplified pools of DNA were then labeled,
fragmented further and hybridized to separate HindIII and XbaI
oligonucleotide SNP arrays.
[0403] Arrays were scanned with a GeneChip Scanner 3000. Genotyping
calls and signal quantification were obtained with GeneChip
Operating System 1.1.1 and Affymetrix Genotyping Tools 2.0
software. Only arrays with genotyping call rates exceeding 90% were
analyzed further. Raw data files were pre-processed and visualized
in dChipSNP Lin, et al., Bioinformatics 20:1233-40 (2004)). In
particular, preprocessing included array data normalization to a
baseline array using a set of invariant probes and subsequent
processing to obtain single intensity values for each SNP on each
sample using a model based (PM/MM)method (Li, et al., Proc. Nat'l
Acad. Sci. USA 98:31-6 (2001)).
Quantitative PCR for TMPRSS2:ERG and TMPRSS2:ETV1 Fusion
Transcripts
[0404] QPCR was performed using SYBR Green dye (Qiagen) on a DNA
engine Opticon 2 machine from MJ Research. Total RNA was reverse
transcribed into cDNA using TAQMAN reverse transcription reagents
(Applied Biosystems) in the presence of random Hexamers. All QPCR
reactions were performed with SYBR Green Master Mix (Qiagen). All
Oligonucleotide primers were designed at Integrated DNA
Technologies. Primers that were described by Tomlin et al. (Science
310:644-8 (2005)) and are specific for the fusion were
utilized:
TABLE-US-00011 (SEQ ID NO: 55) TMPRSS2: ERG_f:
TAGGCGCGAGCTAAGCAGGAG, (SEQ ID NO: 56) TMPRSS2: ERG_r:
GTAGGCACACTCAAACAACGACTGG, (SEQ ID NO: 57) TMPRSS2: ETV1_f
CGCGAGCTAAGCAGGAGGC, (SEQ ID NO: 58) TMPRSS2: ETV-1_r:
CAGGCCATGAAAAGCCAAACTT.
[0405] GAPDH primers were previously described (Vandesompele, et
al., Genome Biol 3: RESEARCH 0034 (2002)). 10 .mu.Mol of forward
and reverse primer were used and procedures were performed
according to the manufacturer's recommended thermocycling
conditions. Threshold levels were set during the exponential phase
of the QPCR reaction using Opticon Monitor analysis software
version 2.02. The amount of each target gene relative to the
housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
for each sample was determined using the comparative threshold
cycle (Ct) method (Applied Biosystems User Bulletin #2). All
reactions were subjected to melt curve analysis and products from
selected experiments were resolved by electrophoreses on 2% agarose
gel.
Statistics
[0406] The clinical and pathology parameters were explored for
associations with rearrangement status and with the presence of the
deletion. Chi-squared test and Fisher exact test were used
appropriately. Kaplan-Meier analysis was used to generate
prostate-specific antigen recurrence free survival curves of the
pathology and the genomic alteration parameters. Log-rank test was
used to evaluate statistical significance of associations. Patients
with prior neo-adjuvant hormone ablation therapy were excluded. All
statistics were performed using SPSS 13.0 for Windows (SPSS Inc.,
Chicago, Ill.) with a significance level of 0.05.
B. Results
[0407] Detection of Deletions on Chromosome 21 Associated with the
TMPRSS2:ERG Gene Rearrangement
[0408] In order to characterize the frequency of the TMPRSS2:ERG
rearrangement in PCA, a modified FISH assay from the assay
described by Tomlins, et al. (Science 310:644-8 (2005)) was
utilized. The original FISH assay used two probes located on ERG at
the centromeric 3' and telomeric 5' ends. The new assay moved the
5' probe in a telomeric direction (FIG. 14). Using a PCA screening
tissue microarray (TMA), it was observed that approximately 70% of
PCA demonstrating TMPRSS2:ERG rearrangement (FIGS. 11A and 11B)
also showed a loss of the green signal corresponding to the
telomeric 5' ERG probe (FIGS. 11C and 11D), suggesting that this
chromosomal region was deleted. 100K oligonucleotide SNP arrays
were used to characterize the extent of these deletions. By
interrogating 30 PCA samples, including cell lines, xenografts and
hormone naive and hormone refractory metastatic PCA samples,
genomic loss between ERG and TMPRSS2 on chromosome 21q23 was
identified (FIG. 12A-C).
[0409] The rearrangement status for TMPRSS2:ERG and TMPRSS2:ETV1
was determined for these 30 PCA by FISH and/or qPCR (FIG. 12A, gray
and light blue bar). Discrete genomic loss was observed in
TMPRSS2:ERG rearrangement positive samples involving an area
between TMPRSS2 and the ERG loci for LuCaP 49, LuCaP 93, ULM LN 13,
MET6-9,MET18-2, MET24-28, and MET28-27. The extent of these
discrete deletions was heterogeneous. More extensive genomic loss
on chromosome 21 including the area between TMPRSS2 and the ERG
loci was observed in LuCaP 35, LuCaP 86.2, LuCaP 92:1, and MET3-81.
The VCaP cell line and the xenograft LuCap 23.1 did not demonstrate
loss in this region. For a subset of samples 45% (5 out of 11) the
deletion occurs in proximity of ERG intron 3. For a majority of
samples 64% (7 out of 11) the deletion ends in proximity of the SNP
located on TMPRSS2 (the next SNP in the telomeric direction is
about 100K by distant). The VCaP cell line shows copy number gain
along the entire chromosome 21.
[0410] For TMPRSS2:ERG rearrangement positive tumors, 71% (5 of 7)
hormone refractory PCA demonstrate a deletion between TMPRSS2 and
the ERG loci whereas deletion was only identified in 25% (1 of 4)
hormone naive metastatic PCA samples (ULM LN 13). There is
significant homogeneity for the deletion borders with two distinct
sub-classes, distinguished by the start point of the
deletion--either at 38.765 Mb or 38.911 Mb. None of the standard
PCA cell lines (PC-3, LNCaP, DU-145, or CWR22 (22Rv1)) demonstrated
the TMPRSS2:ERG or TMPRSS2:ETV1 fusion. Several of the LuCap
xenografts demonstrate TMPRSS2:ERG fusion with deletion including
LuCaP 49 (established from an omental mass) and LuCaP 93, both
hormone-insensitive (androgen receptor [AR]-negative) small-cell
PCAs.
[0411] Copy number gain of ERG was observed in a small subset of
cases both with and without the TMPRSS2:ERG rearrangement. The VCaP
cell line derived from a hormone refractory PCA demonstrated
significant copy number gain on chromosome 21 (FIG. 12A-C), which
was confirmed by FISH.
TMPRSS2:ERG rearrangement in Primary Prostate Cancer Samples and
Hormone Naive Lymph Node Metastases
[0412] To characterize the frequency and potential clinical
significance of these observations, 118 clinically localized PCA
cases were examined by FISH. The clinical and pathology
demographics are presented in Table 10. This cohort of patients is
at high risk of disease recurrence as demonstrated by high tumor
grades (Gleason grade), pathology stage, and pre-treatment PSA
levels. Using standard tissue sections from this cohort, where the
large areas of the PCA could be studied microscopically, the
TMPRSS2:ERG rearrangement was observed to be homogeneous for a
given tumor. The TMA experiments confirmed these observations. In
PCA cases where 3-6 cores were taken from different areas of the
tumor, 100% concordance was observed for TMPRSS2:ERG rearrangement
status (i.e. present or absent). It was also observed that in cases
with, the TMPRSS2:ERG rearrangement with deletion, the deletion was
observed in all of the TMA cores from the same patient in 97.9%
(94/96) of the cases.
TABLE-US-00012 TABLE 10 Clinical and Pathological Demographics of
118 Men with Clinically Localized Prostate Cancer Treated by Radial
Protatectomy* Count Column N % Age <=median 55 50.0% >median
55 50.0% Preoperative PSA <=4 6 8.2% (ng/ml) >4 and <10 13
17.8% >=10 54 74.0% Gleason Score Sum <7 7 6.0% =7 51 43.6%
>7 59 50.4% Nuclear Grade 1 -- -- 2 38 35.5% 3 69 64.5%
Pathology Stage (pT) PT2 26 22.2% PT3a 34 29.1% PT3b 57 48.7%
Surgical Margins status Negative 30 27.8% Positive 78 72.2% Lymph
Node Status N.sub.0 52 44.1% (pN) N.sub.1 56 47.5% N.sub.2 10 8.5%
PSA Recurrence no 34 48.6% yes 36 51.4% *Not all data points were
available for all 118 cases
[0413] The TMPRSS2:ERG rearrangement was identified in 49.2% of the
primary PCA samples and 41.2% in the hormone naive metastatic LN
samples (FIG. 13A). Deletion of the telomeric probe (green signal)
(FIG. 1C-D) was observed in 60.3% (35/58) of the primary PCA
samples and 42.9% (3/7) of the hormone naive lymph node tumors with
TMPRSS2:ERG rearrangement.
[0414] In the 15 cases where there was matched primary and hormone
naive lymph node tumors, there was 100% concordance for TMPRSS2:ERG
rearrangement status with 47% (7 of 15) of the pairs demonstrating
the rearrangement. Deletion of the telomeric (green signal) probe
was concordantly seen in 42.9% (3 of 7) of the pairs.
TMPRSS2:ERG Rearrangement Status and Prostate Cancer
Progression
[0415] The associations between rearrangement status and clinical
and pathological parameters were observed (FIG. 13). TMPRSS2:ERG
rearrangement with deletion was observed in a higher percentage of
PCA cases with advanced tumor stage (pT) (p=0.03) (FIG. 13B), and
the presence of metastatic disease to regional pelvic lymph nodes
(pN.sub.0 versus pN.sub.1-2) (p=0.02). Associations between
TMPRSS2:ERG rearrangement with and without deletion and clinical
outcome as determined by prostate specific antigen (PSA)
biochemical failure for 70 patients where follow up data was
available were also assesed. Gleason grade, tumor stage, nuclear
grade and lymph node status were good predictors of PSA biochemical
failure (all p-values <0.0005). A trend was observed at the
univariate level suggesting a PSA recurrence free survival
advantage in TMPRSS2:ERG rearranged PCA cases without deletion as
determined by the FISH assay.
Example 6
TMPRSS2:ERG Gene Fusion Associated with Lethal Prostate Cancer
[0416] In previous studies, the gene fusions of the 5'-untranslated
region of TMPRSS2 (21q22.3) with the ETS transcription factor
family members, either ERG (21q22.2), ETV1 (7p21.2) (Tomlins, et
al., Science 310:644-8 (2005)), or ETV4 (Tomlins, et al., Cancer
Res. 66(7):3396-400 (2006)) provide a mechanism for the over
expression of the ETS genes in the majority of prostate cancers.
Furthermore, the fusion of an androgen regulated gene, TMPRSS2, and
an oncogene suggests that disease progression may vary based on
these molecular subtypes. The most common mechanism for gene fusion
is loss of about 2.8 megabases of genomic DNA between TMPRSS2 and
ERG (FIGS. 17A and B). This example describes the risk of
metastases or prostate cancer specific death based on the presence
of the common TMPRSS2:ERG gene fusion.
A. Methods
[0417] The study population comprises men with early prostate
cancer (T1a-b, Nx, M0) diagnosed at the Orebro University Hospital,
Sweden, between 1977 and 1991 by transurethral resection of the
prostate (TURP) or transvesical adenoma enucleation for symptomatic
benign prostatic hyperplasia as described by Andren et al. (J.
Urol. 175(4): 1337-40 (2006)). Baseline evaluation at diagnosis
included physical examination, chest radiography, bone scan and
skeletal radiography (if needed). Nodal staging was not carried
out. Because this evaluation provided no evidence for distant
metastases, patients were followed expectantly and received
clinical exams, laboratory tests and bone scans every 6 months
during the first 2 years after diagnosis and subsequently at
12-month intervals. Patients, who developed metastases, as
determined by bone scan, were treated with androgen deprivation
therapy if they exhibited symptoms.
[0418] The cause of death in the cohort was determined by review of
medical records by the study investigators. A validation study
regarding cause of death compared to the Swedish Death Register
showed greater than 90% concordance, with no systematic under- or
over-reporting of any cause of death (Johansson, et al., Lancet
1(8642):799-803 (1989)). Follow-up of the cohort with respect to
mortality was 100% and no patients were lost to follow-up through
October 2005. The study endpoint was defined as development of
distant metastases or prostate cancer specific death (median
follow-up time 9.1 years, maximum 27 years).
[0419] All TURP samples were reviewed by one pathologist to confirm
a diagnosis of prostate cancer, determine the Gleason score and
nuclear grade, and estimate the tumor burden as previously
described (J. Urol. 175(4):1337-40 (2006)). A tissue microarray was
assembled using a manual arrayer (Rubin, et al., Cancer Epidemiol.
Biomarkers Prev. 14(6):1424-32 (2005)). The frequency of the
TMPRSS2:ERG rearrangement in prostate cancer was assessed using a
modified florescence in situ hybridization (FISH) assay from the
assay originally described by Tomlins et al (Science 310:644-8
(2005)). The new assay moved the 5' probe approximately 600 kb in a
telomeric direction. At least one TMA core could be evaluated in 92
of the prostate cancer cases.
B. Results
[0420] In this population-based cohort of men diagnosed with
localized cancer, the frequency of TMPRSS2:ERG fusion was 15.2%
(14/92) (FIGS. 17A and B). TMPRSS2:ERG fusion positive tumors were
more likely to have a higher Gleason score (two-sided P=0.014)
(Table 11). To assess the relation of fusion status and lethal
prostate cancer, cumulative incidence regression was used. A
significant association between the presence of the TMPRSS2:ERG
gene fusion and metastases or disease specific death (FIG. 17C)
with a cumulative incidence ratio (CIR) of 3.6 (P=0.004, 95%
confidence interval [CI]=1.5 to 8.9) was observed. When adjusting
for Gleason Score, the OR was 2.4 (P=0.07 and 95% CI=0.9 to 6.1).
The present invention is not limited to a particular mechanism.
Indeed, an understanding of the mechanism is not necessary to
practive the present invention. Nonetheless, it is contemplated
that, based on the homogeneity of the TMPRSS2:ERG gene fusion in
cells in a given tumor and its presence only in invasive prostate
cancers (compared to Prostatic Intraepithelial Neoplasia), it is
contemplated that this is an early event, which might, in part,
contribute to the biology behind the phenotype of the Gleason
patterns.
TABLE-US-00013 TABLE 11 Prognostic Factors for a Cohort of Men
Expectantly Managed for Localized Prostate Cancer Stratified by the
TMPRSS2:ERG Gene Fusion Status TMPRSS2:ERG Fusion Status Negative
Positive Variable value* P No. of patients 78 14 Age at diagnosis,
y 73 (60 to 103) 73 (58 to 90) .683 Gleason Score** Gleason Score
< 7 48 (61.5%) 3 (21.4%) .014 Gleason Score = 7 20 (25.6%) 6
(42.9%) Gleason Score > 7 10 (12.8%) 5 (35.7%) Pathologic Stage
pT1a 28 (35.9%) 2 (14.3%) .112 pT1b 50 (64.1%) 12 (85.7%) Nuclear
grade*** 1 53 (67.9%) 7 (53.8%) .585 2 18 (23.1%) 4 (30.8%) 3 7
(9.0%) 2 (15.4%) Status**** Survived 12 years 20 (25.6%) 1 (7.1%)
.016 without metastases or cancer death Death due to other 45
(57.7%) 6 (42.9%) causes within 12 years Distant metastases or 13
(16.7%) 7 (50.0%) death due to prostate Cancer *Clinical parameters
of subjects having the TMPRSS2:ERG fusion and of subjects not
having the TMPRSS2:ERG fusion were compared by use of t tests or
chi-square tests for continuous variable and categorical variables,
respectively. **Gleason Score is obtained by summing the major and
minor Gleason patterns. ***For one case nuclear grade was not
assessed. ****Individuals who lived at least 12 years and have not
developed metastases or died of prostate cancer as of October 2005
are classified as long-term survivors. Individuals who lived less
than 12 years and did not develop metastases are classified as
short-term survivors.
Example 7
Detection of TMPRSS2:ETS Fusions in the Urine of Patients with
Prostate Cancer
A. Materials and Methods
Urine Collection, RNA Isolation and Amplification
[0421] Urine samples were obtained from patients following a
digital rectal exam before either needle biopsy or radical
prostatectomy. Urine was voided into urine collection cups
containing DNA/RNA preservative (Sierra Diagnostics). For isolation
of RNA, a minimum of 30 ml of urine were centrifuged at 400 rpm for
15 min at 4.degree. C. RNAlater (Ambion) was added to the urine
sediments and stored at -20.degree. C. until RNA isolation. Total
RNA was isolated using a Qiagen RNeasy Micro kit according to the
manufacturer's instructions. Total RNA was amplified using an
OmniPlex Whole Transcriptome Amplification (WTA) kit (Rubicon
Genomics) according to the manufacturer's instructions (Tomlins et
al., Neoplasia 8:153 [2006]). Twenty five nanograms of total RNA
were used for WTA library synthesis and the cDNA library was
subjected to one round of WTA PCR amplification. Amplified product
was purified using a QIAquick PCR Purification kit (Qiagen). For
cell line proof of concept experiments, the indicated number of
VCaP or LNCaP cells was spiked into 1 ml of sterile urine and the
samples were processed as for voided urine.
Quantitative PCR
[0422] Quantitative PCR (QPCR) was used to detect ERG, ETV1 and
TMPRSS2:ERG transcripts from WTA amplified cDNA essentially as
described (Tomlins et al., Neoplasia 8:153 [2006], Tomlins et al.,
Science 310:644 [2005], Example 1 above). For each QPCR reaction,
10 ng of WTA amplified cDNA was used as template. Reactions for
ERG, ETV1, PSA and GAPDH used 2.times. Power SYBR Green Master Mix
(Applied Biosystems) and 25 ng of both the forward and reverse
primers. Reactions for TMPRSS2:ERGa used 2.times. Taqman Universal
PCR Master Mix and a final concentration of 900 nM forward and
reverse primers, and 250 nM probe. For the Taqman assay, samples
with Ct values greater than 38 cycles were considered to show no
amplification. For all samples, the amount of ERG and ETV1 were
normalized to the amount of GAPDH. Samples with inadequate
amplification of PSA, indicating poor recovery of prostate cells in
the urine, were excluded from further analysis. ERG (exon5.sub.--6
forward) and ETV1 (exon6.sub.--7).sup.2, GAPDH.sup.3, and PSA.sup.4
primers were as described. The Taqman primers and probe (MGB
labeled) specific for TMPRSS2:ERGa are as follows:
TABLE-US-00014 TM-ERGa2_MGB-f; CGCGGCAGGAAGCCTTA (SEQ ID NO: 70)
TM-ERGa2_MGB-r; TCCGTAGGCACACTCAAACAAC, (SEQ ID NO: 71)
TM-ERGa2_MGB-probe; 5'-MGB-CAGTTGTGAGTGAGGACC-NFQ-3' (SEQ ID NO:
72)
Fluorescene In Situ Hybridization (FISH)
[0423] Four .mu.m thick formalin-fixed paraffin-embedded (FFPE)
sections from matched needle biopsies were used for interphase
fluorescence in situ hybridization (FISH), processed and hybridized
as described previously (Example 2 and Tomlins et al., Cancer Res
66:3396 [2006]). BAC probes to detect ERG rearrangements,
RP11-95I21 (5' to ERG) and RP11-476D17 (3' to ERG) were prepared as
described previously (Tomlins et al., Cancer Res 66:3396 [2006];
Tomlins et al., Science 310:644 [2005]; Examples 1 and 2
above).
B. Results
[0424] This example describes a non-invasive method to detect
prostate cancer by the presence of TMPRSS2:ETS fusion transcripts
in prostate cancer cells shed into the urine after a digital rectal
exam. Results are shown in FIG. 33. As a proof of concept, sterile
urine spiked with prostate cancer cell lines expressing high levels
of ERG and TMPRSS2:ERG (VCaP) or high levels of ETV1 (LNCaP) was
used. As shown in FIG. 33A, it was possible to detect ERG
over-expression exclusively in VCaP at 1,600 cells and ETV1
over-expression exclusively in LNCaP at 16,000 cells by
quantitative PCR (QPCR).
[0425] By correlating the number of spiked VCaP and LNCaP cells to
GAPDH C.sub.t (threshold cycle) values, it was observed that, in
some cases, urine obtained from patients after a digital rectal
exam contained insufficient cell numbers to reliably detect ERG or
ETV1 over-expression. Thus, total RNA collected from the urine of
patients suspected of having prostate cancer was amplified using
OmniPlex Whole Transcriptome Amplification before QPCR analysis.
Using this strategy, a cohort of 16 patients where urine was
obtained after a digital rectal exam before a needle biopsy to
detect prostate cancer was assessed. Subsequent assessment of
needle biopsies demonstrated that this cohort contained 4 patients
with benign prostates, 1 with high grade prostatic intraepithelial
neoplasia (HGPIN) and 11 with prostate cancer. In addition, a
cohort of 3 patients with prostate cancer where urine was collected
after a digital rectal exam before radical prostatectomy was
assessed.
[0426] Cohort characteristics are presented in Table 12. Each urine
specimen was from a unique patient and was assigned an ID. The
source of the sample (pre biopsy or radical prostatectomy (RP) is
indicated. The diagnosis following needle biopsy (including benign,
high grade prostatic intraepithelial neoplasia (HGPIN), and
prostate cancer (PCa)) is indicated. For patients diagnosed as
having prostate cancer following needle biopsy, major Gleason,
minor Gleason, and Gleason sum score are indicated. For all
patients, pre biopsy PSA (ng/ml) and age are reported, if
available.
TABLE-US-00015 TABLE 12 Biopsy Biopsy Biopsy Sample Gleason Gleason
Gleason Pre-Biopsy source Diagnosis Major Minor Score PSA (ng/ml)
Pre-Biopsy Benign 4.7 Pre-Biopsy Benign 8.3 Pre-Biopsy Benign 6.7
Pre-Biopsy Benign 4 Pre-Biopsy HGPIN 9.7 Pre-Biopsy Pca 3 4 7 3.3
Pre-Biopsy Pca 3 3 6 5.99 Pre-Biopsy Pca 3 3 6 2.8 Pre-Biopsy Pca 3
3 6 5.9 Pre-Biopsy Pca 4 4 8 10.6 Pre-Biopsy Pca Pre-Biopsy Pca 4 5
9 11.8 Pre-Biopsy Pca 3 4 7 5.5 Pre-Biopsy Pca 3 3 6 3.8 Pre-Biopsy
Pca 4 5 9 19.3 Pre-Biopsy Pca-treated 3 3 6 Pre-RP Pca Pre-RP Pca
Pre-RP Pca
[0427] From the needle biopsy cohort, 5 patients were identified
with marked over-expression of ERG, 1 of which was diagnosed by
needle biopsy as having HGPIN, while the other 4 were diagnosed as
having prostate cancer. From the radical prostatectomy cohort, 1 of
3 patients with prostate cancer were identified as having high ERG
expression (FIG. 33B). ETV1 over-expression was not detected in any
patients from either cohort. To confirm the expression of
TMPRSS2:ERG in the samples which over-expressed ERG, a TaqMan
primer/probe assay designed to specifically amplify TMPRSS2:ERGa
was utilized. This assay robustly amplified product from VCaP
cells, which express TMPRSS2:ERGa (Tomlins et al., Science 310:644
[2005]). In addition, 5 of the 6 urine samples from patients with
prostate cancer that over-expressed ERG also expressed TMPRSS2:ERGa
(Ct values 29.8-38.9), while 0 of the 10 samples from patients
without ERG over-expression expressed TMPRSS2:ERGa. As one sample
over-expressed ERG without expression of TMPRSS2:ERGa, it is likely
that this sample expresses other isoforms of the fusion transcript,
such as TMPRSS2:ERGb or more recently identified fusion transcripts
(Soller et al., Genes Chromosomes Cancer 45:717 [2006]; Yoshimoto
et al., Neoplasia 8:465:2006). To confirm that the presence of
TMPRSS2:ERG fusion transcripts indicates the presence of
TMPRSS2:ERG positive cancerous tissue, fluorescence in situ
hybridization (FISH) was performed using probes designed to detect
ERG rearrangements on matched tissue sections from representative
cases. Matched tissue was obtained from three patients with
detectable TMPRSS2:ERG transcripts in the urine and a diagnosis of
cancer, one patient with detectable TMPRSS2:ERG transcripts in the
urine and a diagnosis of high grade PIN, and two patients without
detectable TMPRSS2:ERG transcripts and a diagnosis of cancer. As
shown in FIG. 33B, both patients diagnosed with cancer but without
detectable TMPRSS2:ERG transcripts in their urine did not harbor
ERG rearrangements in cancerous tissue by FISH. All three patients
diagnosed with cancer and with detectable TMPRSS2:ERG transcripts
in their urine also showed ERG rearrangements in cancerous tissue
by FISH. Finally, the patient with a diagnosis of high grade PIN
with detectable TMPRSS2:ERG in their urine did not show ERG
rearrangements in high grade PIN tissue. This indicates that this
patient may have undiagnosed cancer elsewhere in the prostate,
resulting in the presence of detectable TMPRSS2:ERG transcripts in
their urine.
Example 8
TMPRSS2 and ETS Family Genes Fusions in Prostate Cancer
[0428] This study describes a comprehensive analysis of the
frequency for the TMPRSS2 and ETS family genes rearrangements in a
screening-based cohort of 111 American men surgically treated for
clinically localized prostate cancer.
A. Materials and Methods
[0429] Study Population, Clinical data and Prostate Sample
Collection:
[0430] As a source of clinically localized prostate cancers, a
tissue microarray (TMA) containing--cores representing cancer and
benign tissue was constructed from 111 men who underwent radical
prostatectomy at the University of Michigan as the primary
monotherapy (i.e., no adjuvant or neoadjuvant hormonal or radiation
therapy). The radical prostatectomy series is part of the
University of Michigan Prostate Cancer Specialized Program of
Research Excellence (SPORE) Tissue Core. Three cores (0.6 mm in
diameter) were taken from each representative tissue block to
construct the TMA. The TMA construction protocol has been described
(Kononen et al., Nat. Med. 4:844 [1998]; Rubin et al., Am J surg
Pathol 26:312 [2002]). Detailed clinical, pathological, and TMA
data re maintained on a secure relational database as previously
described (Manley et al., Am J. Pathol. 159:837 [2001]).
Assessment of TMPRSS2-ETS Gene Fusion Using an Interphase
Fluorescence In Situ Hybridization Assay
[0431] Four .mu.m thick tissue micro array sections were used for
interphase fluorescence in situ hybridization (FISH), processed and
hybridized as described previously (Tomlins et al., Science 310:644
[2005]; Tomlins et al., Cancer Res 66:3396 [2006]). Slides were
examined using an Axioplan ImagingZ1 microscope (Carl Zeiss) and
imaged with a CCD camera using the ISIS software system in Metafer
image analysis system (Meta Systems, Altlussheim, Germany). FISH
signals were scored manually (100.times. oil immersion) by
pathologists in morphologically intact and non-overlapping nuclei
and a minimum of 30 cells or the maximum numbers of cancer cells
available in three cores from a case were recorded. Cases without
30 evaluable cells were reported as insufficient hybridization. All
BACs were obtained from the BACPAC Resource Center (Oakland,
Calif.), and probe locations were verified by hybridization to
metaphase spreads of normal peripheral lymphocytes. For detection
of TMPRSS2, ERG and ETV4 rearrangements we used the following
probes: RP11-35C4 (5' to TMPRSS2) and RP11-120C17 (3' to TMPRSS2),
RP11-95I21 (5' to ERG) and RP11-476D17 (3' to ERG), and RP11-100E5
(5' to ETV4) and RP11-436J4 (3' to ETV4). For detection of
TMPSS2-ETV1 fusion, RP11-35C4 (5' to TMPRSS2) was used with
RP11-124L22 (3' to ETV1). BAC DNA was isolated using a QIAFilter
Maxi Prep kit (Qiagen, Valencia, Calif.), and probes were
synthesized using digoxigenin- or biotin-nick translation mixes
(Roche Applied Science, Indianapolis, Ind.).
[0432] The digoxigenin and biotin labeled probes were detected
using fluorescein conjugated anti-digoxigenin antibodies (Roche
Applied Science) and Alexa 594 conjugated streptavidin (Invitrogen,
Carlsbad, Calif.), respectively.
[0433] A break apart (TMPRSS2, ERG, ETV4) or fusion (TMPRSS2-ETV1)
probe strategy was employed to detect rearrangements at the
chromosomal level. Normal signal patterns for TMPRSS2, ERG and ETV4
in DAPI stained nuclei were indicated by two pairs of colocalized
green and red signals. For these probes, a rearrangement was
confirmed by break apart of one of the two colocalized signals. For
TMPRSS2-ETV1 fusion, two pairs of separate red and green were
recorded as normal, while one pair of separate and one pair of
colocalized signals was recorded as a rearrangement.
B. Results and Discussion
[0434] This example describes a comprehensive analysis outlining
the signature of TMPRSS2 and ETS transcription factor genes
rearrangement in a large screening-based cohort of American men
surgically treated for clinically localized prostate cancer. A
TMPRSS2 split probe FISH assay approach was used to detect the
overall frequency of gene rearrangement in prostate cancer with
known ETS family partners ERG, ETV1, ETV4 and other unknown
partners, as shown in FIG. 34. It was hypothesized that prostate
cancers negative for three known ETS partners (ERG, ETV1 and ETV4)
may harbor rearrangements involving other ETS family members. The
results demonstrate complex molecular signature of TMPRSS and ETS
family genes rearrangement in clinically localized prostate cancer
(FIGS. 35A and B). Overall TMPRSS2 was rearranged in 65% of
evaluable cases, while ERG, ETV1 and ETV4 were rearranged in 55%,
2% and 2% of evaluable cases (FIG. 35A). In 40.5% of cases with
TMPRSS2 rearrangement, loss of the 3' probe was observed,
consistent with a chromosomal deletion between TMPRSS2 and ERG as a
mechanism of gene fusion. These results confirm the high frequency
of TMPRSS2:ETS fusions in prostate cancer and confirm previous
studies showing that TMPRSS2:ERG are by far the most common type
(Tomlins et al., Science 310:644; Perner et al., Cancer Res 66:3396
[2006]; Yoshimoto et al., Neoplasia 8:4665 [2006]; Soller et al.,
Genes Chromosomes Cancer 45:717 [2006]; Wang et al., Cancer Res
66:8347 [2006] and above examples).
[0435] Similar results were observed when the cohort was limited to
just those cases where all four probes were evaluable (FIGS. 35A
and B). This analysis confirmed that TMPRSS2:ETS rearrangements are
mutually exclusive, as no cases showed rearrangments of multiple
ETS family members. This analysis also demonstrates that a single
TMPRSS2 assay can effectively detect almost all ETS rearrangements,
as 23 of the 24 cases with ERG, ETV1 or ETV4 rearrangement were
detected by the TMPRSS2 assay. In all 9 cases where the 5' ERG
probe was deleted, deletion of the 3' TMPRSS2 probe was
identified.
[0436] Furthermore, two cases were identified with break apart of
the TMPRSS2 probes, indicating a rearrangement, without
rearrangement of ERG, ETV1 or ETV4 (cases 32 and 36) and cases with
TMPRSS2 rearrangement without ERG rearrangement where ETV1 and/or
ETV4 could not be evaluated. These cases suggest that TMPRSS2 may
be partnering with novel ETS family members or unrelated oncogenes
in prostate cancer.
[0437] Together, these results suggest that a single TMPRSS2 assay
can provide diagnostic and prognostic information in prostate
cancer.
Example 9
PSA Gene Fusions
[0438] FISH experiments were used to identify cases that show a
split signal by FISH for probes located 5' and 3' to PSA. The 5'
and 3' BACs used to detect the PSA split are RP11-510116 and
RP11-26P14, respectively. A partner for the PSA gene fusion has not
yet been identified. These same probes also pick up a split in the
ETS family member SPIB, as it is located very close to PSA.
Example 10
FLI1 Overexpression
[0439] FLI1 expression was assayed in different cell samples not
harboring a FLI1 gene fusion. The expression of 5' and 3' exons of
FLI1 was measured from a case with high FLI1 expression. Results
are shown in FIG. 18. No difference in the 5' and 3' transcript
abundance was detected. RACE also did not indicate a fusion
transcript. FLI1 was overexpressed in prostate cancer relative to
control samples. Primers for Fli1 amplification, as well as TaqMan
probes, are shown in FIG. 37.
[0440] FISH was also used to identify samples that have split
signals for FLI1, indicating a rearrangement, but these cases do
not have TMPRSS2:FLI1 fusion by FISH. BAC probes are shown in Table
13. These cases also have high FLI1 expression.
Example 11
Tissue Microarrays
[0441] Tissue microarrays were used to assay for the presence of
gene fusions. TMAs used included prostate cancer progression array,
prostate cancer outcome array, warm autopsy array, prostate cancer
screening array, Erg negative prostate cancer array, and individual
prostate cancer cases. The following gene probes were used on
tissue microarrays: TMPRSS2-ETV1 fusion probes, Erg split probes,
TMPRSS2 split probes, ETV1 split probes, ETV4 split probes, and FL1
split probes.
[0442] In addition, Erg split probes were used on an outcome array.
The results are as follows: negative cases: 30, positive case: 29,
marginal cases: 1. There was a weak association of Erg positive
cases with higher Gleason score (.gtoreq.7).
[0443] Protein arrays and mass spec were used to identify nuclear
interactors for ERG2. The results are shown in FIG. 21.
Example 12
Androgen Regulation of ERG Expression
[0444] This Example describes the androgen regulation of Erg
expression. LNCap (TMPRSS2-ERG-) and VCaP (TMPRSS2-ERG+) cell lines
were used. The cells were contacted with varying amounts of R1881
for 48 hrs. Expression of Erg, PSA (+control) and beta-tubulin
(-control) were assayed. The results are shown in FIG. 19. ERG
expression was found to be androgen dependent in the VCaP, but not
the LNCap cells.
Example 13
Peptide Antibody and Aqua Probe Generation
[0445] FIGS. 22-25 shows sequences (underlined) of ERG1, ETV1,
FLI-1, and ETV4 for use in peptide antibody generation and for
making aqua probes. Primers are designed by Applied Biosystems for
all ETS family members. Expression is monitored in prostate cancer
cases, with high expression being an indicator of a possible gene
fusion and an indicator for FISH.
Example 14
ETV1 in LnCaP Cells
[0446] This Example describes an analysis of the transcriptional
response to androgen in VCaP and LNCaP. In addition to detecting a
number of transcripts differentially expressed in both cell lines
were identified, such as PSA, a number of transcripts uniquely
dysregulated in VCaP or LNCaP cells were also identified. This
analysis identified ETV1 as being exclusively responsive to
androgen in LNCaP cells. Combined with the over-expression of ETV1
in LNCaP cells, FISH was used to interrogate the ETV1 loci in LNCaP
cells.
A. Materials And Methods
Cell Lines
[0447] The prostate cancer cell lines LNCaP (originally derived
from a lymph node prostate cancer metastasis) and VCaP (Korenchuk,
S. et al., In vivo 15, 163-8 (2001)) (originally derived from a
vertebral prostate cancer metastasis) were used for this study. For
microarray studies, VCaP and LNCaP cells were grown in
charcoal-stripped serum containing media for 24 hours before
treatment for 48 hours with 0.1% ethanol or 1 nM of the synthetic
androgen methyltrienolone (R1881, NEN Life Science Products,
Boston, Mass.) dissolved in ethanol. For quantitative PCR (QPCR)
studies, cells were grown in charcoal-stripped serum containing
media for 24 hours, preincubated with 0.1% ethanol, Casodex
dissolved in acetone (10 uM, bicalutamide, AstraZeneca
Pharmaceuticals, Wilmington, Del.) or flutamide dissolved in
ethanol (10 uM, Sigma, St. Louis, Mo.). After 2 hours, 0.1% ethanol
or 0.5 nM of R1881 was added and the cells were harvested after 48
hours. Total RNA was isolated from all samples with Trizol
(Invitrogen, Carlsbad, Calif.) according to the manufacturer's
instructions. RNA integrity was verified by denaturing formaldehyde
gel electrophoresis or the Agilent Bioanalyzer 2100 (Agilent
Technologies, Palo Alto, Calif.).
Microarray Analysis
[0448] The cDNA microarrays used for this study were constructed
essentially as described, except the array contains 32,448
features. Protocols for printing and postprocessing of arrays are
available on the Internet. cDNA microarray analysis was done
essentially as described. Briefly, total RNA from control and R1881
treated VCaP and LNCaP cell lines were reverse transcribed and
labeled with cy5 fluorescent dye. Pooled total RNA from control
VCaP or LNCaP samples were reverse transcribed and labeled with cy3
fluorescent dye for all hybridizations from the respective cell
lines. The labeled products were then mixed and hybridized to the
cDNA arrays. Images were flagged and normalized using the Genepix
software package (Axon Instruments Inc., Union City, Calif.). Data
were median-centered by arrays and only genes that had expression
values in at least 80% of the samples were used in the
analysis.
Quantitative PCR (QPCR)
[0449] QPCR was performed using SYBR Green dye on an Applied
Biosystems 7300 Real Time PCR system (Applied Biosystems, Foster
City, Calif.) as described (Tomlins et al., Cancer Res 66, 3396-400
(2006); Tomlins et al., Science 310, 644-8 (2005)). The amount of
each target gene relative to the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample
was reported. The relative amount of the target gene in each cell
line and/or experiment was calibrated to controls. All
oligonucleotide primers were synthesized by Integrated DNA
Technologies (Coralville, Iowa). GAPDH (Vandesompele et al., Genome
Biol 3, RESEARCH0034 (2002)), PSA (Specht et al., Am J Pathol 158,
419-29 (2001)), ERG (Exon 5-6_f and Exon 5-6_r) and ETV1 (Exon
6-7_f and Exon 6-7_r) primers (Tomlins et al., Science 310, 644-8
(2005)) were as described.
Fluorescence In Situ Hybridization (FISH)
[0450] Metaphase spreads were prepared from normal peripheral
lymphocytes (NPLs) and LNCaP cells using standard techniques.
Slides were treated with 2.times.SSC for 2 min, 70% ethanol for 2
min and 100% ethanol for 2 min before addition of the probe. Slides
were coverslipped and incubated at 75.degree. for 2 min and
hybridized overnight at 37.degree. C. Post-hybridization washing
was with 2.times.SSC at 42.degree. C. for 5 min, followed by 3
washes in PBST. Fluorescent detection was performed using
anti-digoxigenin conjugated to fluorescein (Roche Applied Science,
Indianapolis, Ind.) and streptavidin conjugated to Alexa Fluor 594
(Invitrogen, Carlsbad, Calif.). Slides were counterstained and
mounted in ProLong Gold Antifade Reagent with DAPI (Invitrogen).
Slides were examined using a Zeiss Axio Imager Z1 fluorescence
microscope (Zeiss, Thornwood, N.Y.) and imaged with a CCD camera
using ISIS software (Metasystems, Altlussheim, Germany).
[0451] All BACs were obtained from the BACPAC Resource Center
(Oakland, Calif.) and probe locations were verified by
hybridization to metaphase spreads of normal peripheral
lymphocytes. For hybridization to the ETV1 region on chromosome 7p,
four BACs were used (telomeric to centromeric): RP11-124L22,
RP11-313C20, RP11-703A4 and RP11-1149J13. For localization to
chromosome 14q, the FISH mapped BAC RP11-483K13, which we also
confirmed as hybridizing to 14q using NPLs. BAC DNA was isolated
using a QIAFilter Maxi Prep kit (Qiagen, Valencia, Calif.) and
probes were synthesized using digoxigenin- or biotin-nick
translation mixes (Roche Applied Science).
B. Results
[0452] Results are shown in FIGS. 26-28. FIG. 26 shows the
over-expression and androgen regulation of ETV1 in the LNCaP
prostate cancer cell line. FIG. 26A shows expression signature of
androgen-regulated genes in VCaP and LNCaP prostate cancer cell
lines. Heatmap of genes showing induction or repression in either
cell line (3,499 features, p<0.05 and fold change ratio>=1.5)
by 1 nM synthetic androgen R1881 (green) compared to vehicle
treatment (gray). Each row represents a gene; each column
represents a sample. Yellow and blue cells indicate over- or
under-expression, respectively, according to the color scale. Gray
cells indicate missing data. Values for each cell line are centered
on the corresponding control samples. The locations of PSA, ERG and
ETV1 in the heatmap are indicated and their expression is shown in
the inset. FIG. 26B shows confirmation of PSA induction by androgen
in both VCaP and LNCaP cells by quantitative PCR (QPCR). The
relative expression of PSA (normalized to GAPDH) in LNCaP (red) and
VCaP (blue) cell lines was determined by QPCR. Cells were treated
with vehicle or 1 nM R1881 for 48 hours in the presence or absence
of the anti-androgens Casodex or Flutamide as indicated. The
relative amount of PSA in each sample was calibrated to the amount
in the control sample for each cell line. FIG. 26C shows ETV1
induction by androgen in LNCaP cells. Using the same samples as B,
the relative amount of ETV1 was determined by QPCR. FIG. 26D shows
that ETV1 is markedly over-expressed in LNCaP cells. The relative
expression of PSA, ETV1 and ERG were determined in the 48 hour
control samples from each cell line by QPCR. The relative amount of
target gene in each sample was calibrated to the average amount of
PSA from both cell lines. The fold difference in ERG and ETV1
expression between LNCaP and VCaP is indicated.
[0453] FIG. 27 shows rearrangement of ETV1 in LNCaP cells. FIG. 27A
shows a schematic of BACs used as probes for fluorescence in situ
hybridization (FISH). The location and coordinates at 7p21
(including the ETV1 locus and surrounding BACs) and 14q32 was
determined on the May 2004 freeze of the human genome using the
UCSC Genome Browser. BACs used in this study are indicated as
numbered rectangles. The location of ETV1 and DGKB are shown with
the arrowhead indicating the direction of transcription. FIG. 27B
shows that RP11-124L22 and RP11-1149J13 co-localize to chromosome 7
in normal peripheral lymphocytes (NPLs). Localization of
RP11-124L22 (BAC #1) and RP11-1149J13 (BAC #4) on metaphase spreads
(top panel) or interphase cells (bottom panel) was determined by
FISH in NPLs. For all metaphase pictures, signals on chromosome 7
are indicated by arrows, while signals on chromosome 14 are
indicated by arrowheads of the corresponding probe color. Higher
magnification of informative regions of metaphase spreads are shown
in boxes. FIG. 27C shows localization of BAC #1 and BAC #4 on
metaphase spreads (top panel) and interphase cells (bottom panel)
was determined in the near tetraploid LNCaP cell line. Two
co-localized signals on chromosome 7, two red signals on chromosome
7 and two green signals on a different chromosome were observed.
FIG. 27D shows signal from RP11-124L22 localizes to chromosome 14
in LNCaP cells. As in C, except RP11-124L22 (BAC #1) was
co-hybridized with RP11-483K13 (BAC #5, FISH mapped to chromosome
14q) on LNCaP metaphase spreads. Four red signals from RP11-483K13
localize to chromosome 14q; two green signals localize to
chromosome 7p and two green signals localize to chromosome 14q.
[0454] FIG. 28 shoes that the entire ETV1 locus is inserted into
chromosome 14 in LNCaP cells. FIG. 28A shows a schematic of BACs
used in this experiment. FIG. 28B shows localization of RP11-124L22
(BAC #1) and RP11-313C20 (BAC #2) on metaphase spreads (top panel)
and interphase cells (bottom panel) was determined by FISH in LNCaP
cells. In metaphase spreads, two pairs of co-localized signals were
observed on chromosome 7 (yellow arrows) and chromosome 14 (yellow
arrowheads).
[0455] These results demonstrate that the entire ETV1 locus is
translocated from chromosome 7 to chromosome 14. Although the
genomic sequence upstream of the insertion on chromosome 14 is
unknown, it is likely that this region contains AREs, which drive
the high level of ETV1 observed only in LNCaP cells and the
androgen responsiveness. These results suggest that LNCaP cells
find use as an in vitro model of ETS gene fusions seen in human
prostate cancers.
Example 15
Knockdown of ETS Family Members in PCA
[0456] This Example describes the knockdown of ETS family members
in prostate cancer. siRNAs were used to knockdown expression of
ETV1 and ERG in LnCaP and VCAP. Quantitative PCR was used to
confirm the knockdown. Results are shown in FIGS. 29 and 30. The
knockdown did not affect proliferation. Lentivirus expressing shRNA
are generated for stable knockdowns.
[0457] Microarrays were performed on Agilent 44K Whole Genome
arrays to determine which genes were differentially expressed when
ERG expression was knocked down in VCaP cells (which have the
TMPRSS2:ERG fusion). For this experiment, three conditions were
used: knockdown using Dharmacon siRNA for ERG (ERGsi), knockdown of
luciferase (control), and untransfected (untrans) VCaP cells. Three
hybridizations of ERG/untrans and two of control/untrans were
performed. The genes were called as present in all five
experiments, had standard deviations less than 0.5 (of the average
for both conditions), and showed a fold difference between the ERG
and control of <0.75 or >1.5. The ERGdif field indicates the
fold difference between the ERG and control knockdown experiments,
so value less than one means the gene is underexpressed in the ERG
knockdown (ERG itself ranks 81st in this analysis).
Example 16
Transgenic Mice
[0458] Transgenic mice that over express gene fusions of the
present invention, as well as ETS and androgen responsive genes are
generated. FIG. 31 shows viral overexpression systems for use in
generating mice. FIG. 32 shows a schematic of genomic insertions in
transgenic mice. Such mice find use in research (e.g., mechanistic
studies) and drug screening applications.
Example 17
Identification of TMPRSS2:ERGa
[0459] As described above (Example 1), fusions of TMPRSS2 to ERG
were observed. To determine the expressed protein from the
TMPRSS2:ERGa gene fusion, PCR was used to amplify the portion of
ERG (NM.sub.--004449) from the fusion breakpoint at the beginning
of exon 4 to the presumed stop codon in exon 11, inserting a
3.times. Flag tag immediately upstream of the stop codon, from the
VCaP prostate cancer cell line. The product was TA cloned into
pCR8/GW/TOPO TA (Invitrogen) and bi directionally sequenced.
Sequencing revealed the presence of two distinct isoforms, herein
designated as ERG1 (includes exon 6 from ERG isoform 1
(NM.sub.--182918,
GGGGTGCAGCTTTTATTTTCCCAAATACTTCAGTATATCCTGAAGCTACGCAA
AGAATTACAACTAGGCCAG; SEQ ID NO:73) and ERG2 (does not include this
exon). The product was Gateway cloned into the pLenti6/V5-DEST
destination vector. This plasmid was transfected directly into
PHINX cells for ERG protein production.
A. Methods
[0460] Transfection Assay: Phinx cells were transfected with either
ERG2 or the empty vector using Fugene transfection reagent (Roche)
as per manufacturer's instructions. A total of ten 150 mm diameter
plates were used for each construct. The cells were harvested 48 h
post-transfection and used for immunoprecipitation assay as
described below.
[0461] Protein Lysis and Immunoprecipitation: Cells were washed in
ice cold PBS containing protease inhibitors and lysed by
homogenization in TBS containing 1% NP40. The supernatant
containing proteins were estimated for their protein content using
Bradfords Protein Assay (Biorad Laboratories, Hercules, Calif.) as
per manufacturer's instructions. Equal amounts of protein
(approximately 30 mg in 15 ml buffer) from all samples were used
for immunoprecipitation studies. About 200 microlitres of a 50%
slurry of EZVIEW Red ANTI-FLAG M2 Affinity Gel (Sigma, St Louis,
Mo.) was added to each sample and incubated overnight at 4 C. The
immunoprecipitate was washed thrice each with TBS containing 0.1%
NP40 and TBS alone. Bound proteins were eluted using FLAG peptide
(Sigma, St Louis, Mo.) as per manufacturer's instruction. The
elution was performed three times. Proteins in the eluate were
precipitated using 50% TCA (Sigma, St Louis, Mo.). The precipitate
was washed thrice with ice cold acetone, resuspended in Laemmeli
buffer and electrophoresed on 4-20% BIS-TRIS gel (Invitrogen
Corporation, Carlsbad, Calif.). The gels were stained with mass
spectrometry compatible silver stain (Silver Quest, Invitrogen
Corporation, Carlsbad, Calif.). Bands corresponding to ERG2 and the
corresponding region in the vector lane were excised into 6 pieces
of 1 cm each. Each of the gel pieces were labeled bands 1-6
starting from higher molecular weight region on the gel moving
down. Thus Band 1 corresponds to the region containing high
molecular weight proteins while band 6 corresponds to region of low
molecular weight. Based on its native molecular mass of ERG2
(approximately 55 KDa) would migrate in Bands 4 and 5. ERG2
sequence identification was repeated three times and the data was
consolidated from all the experiments.
Protein Identification
[0462] The gel bands were collected, destained using the destaining
solution provided in the Silver Stain Kit as per manufacturers
instruction (Invitrogen Corporation, Carlsbad, Calif.). In gel
digestion was performed using Porcine Trypsin (1:50, Promega
Corporation, Madison, Wis.) in 1M Ammonium Bicarbonate, pH 9. The
digestion was performed for 16 h at 37.degree. C. At the end of 24
h the trypsin activity was stopped using 3% formic acid. The
peptides were extracted using 50% Acetonitrile. The peptides were
dried and resuspended in 2% Acetonitrile containing 0.1% formic
acid and separated by reversed-phase chromatography using a 0.075
mm.times.150 mm C18 column attached to a Paradigm HPLC pump
(Michrome Bio Resources Inc.). Peptides were eluted using a 45-min
gradient from 5 to 95% B (0.1% formic acid/95% acetonitrile), where
solvent A was 0.1% formic acid/2% acetonitrile. A Finnigan LTQ mass
spectrometer (Thermo Electron Corp.) was used to acquire spectra,
the instrument operating in data-dependent mode with dynamic
exclusion enabled. The MS/MS spectra on three most abundant peptide
ions in full MS scan were obtained. The spectra are searched using
the MASCOT search tool against the composite, non-identical NCBI
human reference sequence database. These database search results
are validated for peptide assignment accuracy using the
PeptideProphet program. This is a mixture model; an expectation
maximization evaluation assigning a probability of correct peptide
identification based on search result scores and various peptide
features including the number of typtic termini. A second program,
ProteinProphet, is used to group peptides by protein and combine
their probabilities to assign a probability of a correct protein
assignment. Discriminatory power increases with the subsequent
re-estimation of individual peptide probabilities by way of their
NSP value, or number of sibling peptides, which amounts to peptide
grouping information and the status of a possible multi-hit
protein.
Results:
TABLE-US-00016 [0463] TABLE 14 COVERAGE MAP (ERG2) NOTE: E*BAND*-*
represent ERG2 peptides in ERG1 experiments MIQTVPDPAA HI . . .
(SEQ ID NO: 74) SEQ ID BAND05-20060217 NCBI N-terminal, NO
MASTIKEALS VVSEDQSLFE CAYGTPHLAK TEMTASSSSD 40 74 SSSSD
BAND03-20060206 75 YGQTSKMSPR VPQQDWLSQP PARVTIKMEC NPSQVNGSRN 80
76 VPQQDWLSQP PAR BAND01-20060217 77 VPQQDWLSQP PAR BAND02-20060206
78 VPQQDWLSQP PAR BAND02-20060209 79 VPQQDWLSQP PAR BAND02-20060217
80 YGQTSKMS VPQQDWLSQP PAR BAND03-20060206 81 VPQQDWLSQP PAR
BAND03-20060209 82 VPQQDWLSQP PAR BAND03-20060217 83 VPQQDWLSQP PAR
BAND04-20060206 84 VPQQDWLSQP PAR MEC NPSQVNGSR BAND04-20060209 85
VPQQDWLSQP PAR BAND04-20060217 86 VPQQDWLSQP PAR BAND05-20060217 87
SPDECSVAKG GKMVGSPDTV GMNYGSYMEE KHMPPPNMTT 120 88 HMPPPNMTT
BAND01-20060206 89 HMPPPNMTT BAND02-20060206 90 HMPPPNMTT
BAND02-20060209 91 NYGSYMEE KHMP BAND02-20060217 92 MVGSPDTV
GMNYGSYMEE KHMPPPNMTT BAND03-20060206 93 HMPPPNMTT BAND03-20060209
94 HMPPPNMTT BAND04-20060206 95 MVGSPDTV GMNYGSYMEE KHMPPPNMTT
BAND04-20060209 96 MVGSPDTV GMNYGSYMEE KHMPPPNMTT BAND04-20060217
97 NERRVIVPAD PTLWSTDHVR QWLEWAVKEY GLPDVNILLF 160 98 NER VIVPAD
PTLWSTDHVR QWLEWAVKEY GLPDVNILLF BAND01-20060206 99 NER EY
GLPDVNILLF BAND02-20060206 100 NER BAND02-20060209 101 NER VIVPAD
PTLWSTDHVR QWLEWAVK BAND03-20060206 102 NERRVIVPAD PTLWSTDHVR EY
GLPDVNILLF BAND03-20060209 103 NER VIVPAD PTLWSTDHVR QWLEWAVKEY
GLPDVNILLF BAND04-20060206 104 NERRVIVPAD PTLWSTDHVR QWLEWAVKEY
GLPDVNILLF BAND04-20060209 105 NERRVIVPAD PTLWSTDHVR
BAND04-20060217 106 EY GLPDVNILLF BAND05-20060206 107 QNIDGKELCK
MTKDDFQRLT PSYNADILLS HLHYLRETPL 200 108 QNIDGK LT PSYNADILLS
HLHYLRETPL BAND01-20060206 109 ETPL BAND01-20060217 110 QNIDGK ETPL
BAND02-20060206 111 ETPL BAND02-20060217 112 ETPL BAND03-20060206
113 QNIDGK LT PSYNADILLS HLHYLRETPL BAND03-20060209 114 ETPL
BAND03-20060217 115 QNIDGK LT PSYNADILLS HLHYLRETPL BAND04-20060206
116 QNIDGK LT PSYNADILLS HLHYLRETPL BAND04-20060209 117 LT
PSYNADILLS HLHYLRETPL BAND04-20060217 118 QNIDGK BAND05-20060206
119 PSYNADILLS HLHYLRETPL BAND05-20060217 120 PHLTSDDVDK ALQNSPRLMH
ARNTGGAAFI FPNTSVYPEA 240 121 PRLMH ARNT BAND01-20060206 122
PHLTSDDVDK BAND01-20060206 123 PHLTSDDVDK ALQNSPR BAND01-20060217
124 PHLTSDDVDK ALQNSPR BAND02-20060206 125 PHLTSDDVDK ALQNSPR
BAND02-20060217 126 PHLTSDDVDK ALQNSPR BAND03-20060206 127
PHLTSDDVDK BAND03-20060209 128 PHLTSDDVDK ALQNSPR BAND03-20060217
129 PHLTSDDVDK ALQNSPR BAND04-20060206 130 PHLTSDDVDK ALQNSPR
BAND04-20060209 131 RNT BAND04-20060209 132 PHLTSDDVDK ALQNSPR
BAND04-20060217 133 PHLTSDDVDK ALQNSPRL BAND05-20060217 134
TQRITTRPDL PYEPPRRSAW TGHGHPTPQS KAAQPSPSTV 280 135 DL PYEPPR
BAND01-20060206 136 SAW TGHGHPTPQS KAAQPSPSTV BAND01-20060206 137
PYEPPRR BAND01-20060217 138 SAW TGHGHPTPQS KAAQPSPSTV
BAND02-20060206 139 SAW TGHGHPTPQS KAAQPSPSTV BAND02-20060209 140
PYEPPRRSAW TGHGHPTPQS KAAQPSPSTV BAND02-20060217 141 SAW TGHGHPTPQS
KAAQPSPSTV BAND03-20060206 142 SAW TGHGHPTPQS KAAQPSPSTV
BAND03-20060209 143 DL PYEPPRR BAND03-20060217 144 PYEPPRRSAW
TGHGHPTPQS KAAQPSPSTV BAND04-20060206 145 DL PYEPPRRSAW TGHGHPTPQS
KAAQPSPSTV BAND04-20060209 146 DL PYEPPRR BAND04-20060209 147 DL
PYEPPRRSAW TGHGHPTPQS KAAQPSPSTV BAND04-20060217 148 AAQPSPSTV
BAND05-20060206 149 SAW TGHGHPTPQS KAAQPSPSTV BAND05-20060209 150
DL PYEPPRRSAW TGHGHPTPQS KAAQPSPSTV BAND05-20060217 151 SAW
TGHGHPTPQS KAAQPSPSTV BAND06-20060209 PKTEDQRPQL DPYQILGPTS
SRLANPGSGQ IQLWQFLLEL 320 152 PK BAND01-20060206 153 TEDQRPQL
DPYQILGPTS SR BAND01-20060217 154 PKTEDQRPQL DPYQILGPTS SR
BAND02-20060206 155 PKTEDQRPQL DPYQILGPTS SR BAND02-20060209 156
PKTEDQRPQL DPYQILGPTS SR BAND02-20060217 157 PKTEDQRPQL DPYQILGPTS
SR BAND03-20060206 158 PKTEDQRPQL DPYQILGPTS SR BAND03-20060209 159
TEDQRPQL DPYQILGPTS SR BAND03-20060217 160 PKTEDQRPQL DPYQILGPTS SR
BAND04-20060206 161 PKTEDQRPQL DPYQILGPTS SR BAND04-20060209 162
PKTEDQRPQL DPYQILGPTS SR BAND04-20060217 163 PK BAND05-20060206 164
PKTEDQRPQL DPYQILGPTS SR BAND05-20060209 165 PKTEDQRPQL DPYQILGPTS
SR BAND05-20060217 166 PK BAND06-20060209 167 LSDSSNSSCI TWEGTNGEFK
MTDPDEVARR WGERKSKPNM 360 168 MTDPDEVAR BAND01-20060206 169
MTDPDEVAR BAND02-20060206 170 MTDPDEVAR BAND03-20060206 171
MTDPDEVAR BAND03-20060209 172 MTDPDEVAR BAND04-20060206 173
MTDPDEVARR BAND04-20060209 174 TDPDEVARR KSKPNM BAND04-20060217 175
MTDPDEVAR BAND05-20060209 176 KSKPNM BAND05-20060217 177 NYDKLSRALR
YYYDKNIMTK VHGKRYAYKF DFHGIAQALQ 400 178 F DFHGIAQALQ
BAND02-20060206 179 F DFHGIAQALQ BAND02-20060209 180 F DFHGIAQALQ
BAND03-20060206 181 F DFHGIAQALQ BAND03-20060209 182 YYYDKNIMTK
YAYKF DFHGIAQALQ BAND04-20060209 183 NYDKLSR BAND04-20060217 184
NYDKLSR YYYDKNIMTK BAND05-20060217 185 PHPPESSLYK YPSDLPYMGS
YHAHPQKMNF VAPHPPALPV 440 186 PHPPESSLYK BAND02-20060206 187
PHPPESSLYK YPSDLPYMGS YHAH BAND02-20060209 188 PHPPESSLYK
YPSDLPYMGS YHAHPQK BAND03-20060206 189 PHPPESSLYK YPSDLPYMGS
YHAHPQK BAND03-20060209 190 YPSDLPYMGS YHAHPQK BAND04-20060206 191
PHPPESSLYK YPSDLPYMGS YHAHPQK BAND04-20060209 192 TSSSFFAAPN
PYWNSPTGGI YPNTRLPTSH MPSHLGTYY 479 193 NSPTG BAND02-20060217
194
SPTGGI YPNTR BAND04-20060209 195
The table shows the coverage map for ERG2 obtained over 3 different
experiments. The underlined amino acid sequence corresponds to the
in silico translated sequence of ERG1 that was cloned from VCAP
cells. The aminoacid sequence GGAAFI FPNTSVYPEATQRITTRP (SEQ ID
NO:196) corresponds to the exon that is specific to ERG1 and is
missing in ERG2. The remaining amino acid sequence correspond to
ERG2 sequence identified in each of the three experiments. ERG2 was
identified in Bands 1-5 in all the experiments. The peptide
sequences for ERG2 obtained in each of these bands is illustrated.
A very high coverage of the ERG2 protein was observed over the
three experiments. The coverage map showed that the coverage of
peptides in the N-terminal region of the cloned protein,
corresponding to the first 50 amino acid residues were rarely
observed in the mass spectrometry coverage map. However, the
peptide VPQQDWLSQP (SEQ ID NO:197) that starts with amino acid
valine was found to be highly abundant and thus identified in all
the experiments. Closer evaluation suggested that amino acid in the
47.sup.th position was an in frame Methionine. The lack of any
peptide upstream (Nterminus) of the 47 th methionine in multiple
experiments confirms that it is the N-terminal amino acid of ERG2.
Further, the presence of a Arginine residue at the 50.sup.th
position makes it a potential tryptic cleavage site. Digestion by
trypsin at this site would result in a shorter N-terminal peptide
MSPR, which is too small for identification by ion trap mass
spectrometer and a longer C-terminal peptide VPQQDWLSQP (SEQ ID
NO:198), which was identified in all the experiments. Also the
peptide sequence MIQTVPDPAA HI (SEQ ID NO:199) was identified in a
single experiment at a very low probability score. This maps to the
N-terminus of ERG as reported in NCBI. This sequence was not a part
of the ectopically overexpressed construct that was cloned from the
VCAP cells. This could have been obtained from the in vivo ERG that
is expressed in PHINX cells and thus may represent part of the ERG
associated with benign cells. Thus, in summary, the results
indicate that the third Methionine is the translational Start site
for the TMPRSS2-ERG fusion product. MASTIKEALS VVSEDQSLFE
CAYGTPHLAK TEMTA YGQTSKMSPR VPQQDWLSQP (SEQ ID NO:200) The First
Methionine is the translational START Site for endogenous ERG.
TABLE-US-00017 MIQTVPDPAA HI (SEQ ID NO: 201)
FIG. 20 shows a schematic of the endogenous and fusion
polypeptides.
Example 18
FISH Analysis on Urine Samples
[0464] To isolate and prepare prostatic cells from urine, .about.30
ml of urine is collected following an attentive digital rectal
exam. Immediately, 15 ml of PreservCyt is added, and the sample is
centrifuged at 4000 rpm in a 50 ml tube for 10 min at room
temperature. The supernatant is discarded, the pellet is
resuspended in 15 ml of 0.75 M KCl for 15 min at room temperature,
and centrifuged at 4000 rpm in a 50 ml tube for 10 min at room
temperature. The supernatant is discarded, and the pellet is
resuspended in 10 ml of a 3:1 ratio of methanol:glacial acetic
acid. This is then centrifuged at 4000 rpm for 8 min. The
supernatant is discarded, except for 200 .mu.l, and the pellet is
resuspended. The resuspended pellet is then dropped onto glass
slides and allowed to air dry. Hybridization and probe preparation
are as in Example 2 above, with the ERG 5'/3' and TMPRSS 5'/3'
probe pairs.
Example 19
Transgenic Mice
[0465] Transgenic mouse models of TMPRSS2:ETS fusions were
generated by prostate specific over-expression of the fused
portions of ERG and ETV1. By 12 weeks, 5 of 5 ERG and ETV1
transgenic mice developed mouse prostatic intraepithelial neoplasia
(mPIN), the precursor lesion of prostate cancer. These are the
first in vivo models of chromosomal rearrangements in a common
epithelial cancer and represent authentic oncogene driven models of
prostate cancer.
[0466] The results are shown in FIGS. 38-39. FIG. 38 shows
construction and expression verification of transgenes
recapitulating TMPRSS2:ETS gene fusions. FIG. 38A shows structures
of human TMPRSS2 (NM.sub.--005656.2), ERG1 (NM.sub.--182918.2) and
ETV1 (NM.sub.--004956.3) are shown, with coding regions in darker
colors. Numbered boxes represent exons, and the last base of
indicated exons is given according to alignment with the March 2006
freeze of the Human Genome using the UCSC Genome Browser. The
position of stop codons for ERG1 and ETV1 are indicated. The
structure of TMPRSS2:ERGa and TMPRSS2:ETV1 from human prostate
cancer samples are indicated. To recapitulate these fusions, cDNA
encoding the regions of ERG and ETV1 with a c-terminal 3.times.
FLAG tag (3.times.) involved in TMPRSS2 fusions were cloned along
with the bovine growth hormone polyA site (PA-BGH, yellow)
downstream of the modified small composite probasin promoter
(ARR2Pb, pink). FIG. 38B shows quantitative real time PCR (QPCR) to
confirm transgene expression. Expression of ERG and ETV1 was
determined in the anterior prostate (AP), dorsolateral prostate
(DLP), ventral prostate (VP) and liver from representative
transgenic mice expressing ERG (Pb-ERG1) or ETV1 (Pb-ETV1).
[0467] FIG. 39 shows that histological characterization of
TMRPSS2:ETS transgenic mice demonstrates mouse prostatic
intraepithelial neoplasia (mPIN) at 12 weeks of age. Prostates from
12 week old transgenic mice expressing (A) ERG (Pb-ERG1) or (B)
ETV1 (Pb-ETV1) were characterized after H&E staining and the
anterior prostate is shown. Both mice show mPIN, with transgenic
ETV1 mice showing cribiform architecture.
[0468] All publications, patents, patent applications and accession
numbers mentioned in the above specification are herein
incorporated by reference in their entirety. Although the invention
has been described in connection with specific embodiments, it
should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications and variations of the described compositions and
methods of the invention will be apparent to those of ordinary
skill in the art and are intended to be within the scope of the
following claims.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 307 <210> SEQ ID NO 1 <211> LENGTH: 24 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
1 aacagagatc tggctcatga ttca 24 <210> SEQ ID NO 2 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 cttctgcaag ccatgtttcc tgta 24 <210>
SEQ ID NO 3 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 3
aggaaacatg gcttgcagaa gctc 24 <210> SEQ ID NO 4 <211>
LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 4 tctggtacaa actgctcatc attgtc 26 <210>
SEQ ID NO 5 <211> LENGTH: 26 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 5
ctcaggtacc tgacaatgat gagcag 26 <210> SEQ ID NO 6 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 6 catggactgt ggggttcttt cttg 24 <210>
SEQ ID NO 7 <211> LENGTH: 26 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 7
aacagccctt taaattcagc tatgga 26 <210> SEQ ID NO 8 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 8 ggagggcctc attcccactt g 21 <210> SEQ
ID NO 9 <211> LENGTH: 22 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 9 ctaccccatg
gaccacagat tt 22 <210> SEQ ID NO 10 <211> LENGTH: 22
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 10 cttaaagcct tgtggtggga ag 22 <210>
SEQ ID NO 11 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 11
cgcagagtta tcgtgccagc agat 24 <210> SEQ ID NO 12 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 12 ccatattctt tcaccgccca ctcc 24 <210>
SEQ ID NO 13 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 13
cgactggagc acgaggacac tga 23 <210> SEQ ID NO 14 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 14 catggactgt ggggttcttt cttg 24 <210>
SEQ ID NO 15 <211> LENGTH: 22 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 15
ggcgttccgt aggcacactc aa 22 <210> SEQ ID NO 16 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 16 cctggctggg ggttgagaca 20 <210> SEQ
ID NO 17 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 17 taggcgcgag
ctaagcagga g 21 <210> SEQ ID NO 18 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 18 gtaggcacac tcaaacaacg actgg 25 <210>
SEQ ID NO 19 <211> LENGTH: 19 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 19
cgcgagctaa gcaggaggc 19 <210> SEQ ID NO 20 <211>
LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 20 caggccatga aaagccaaac tt 22 <210>
SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 21
ccggatggag cggaggatga 20 <210> SEQ ID NO 22 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 22 cgggcgattt gctgctgaag 20 <210> SEQ
ID NO 23 <211> LENGTH: 19 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 23 gccgcccctc
gactctgaa 19 <210> SEQ ID NO 24 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 24 gagccacgtc tcctggaagt gact 24 <210>
SEQ ID NO 25 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 25
ctggccggtt cttctggatg c 21 <210> SEQ ID NO 26 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 26 cgggccgggg aatggagt 18 <210> SEQ ID
NO 27 <211> LENGTH: 22 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 27 cctggagggt
accggtttgt ca 22 <210> SEQ ID NO 28 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 28 ccgcctgcct ctgggaacac 20 <210> SEQ
ID NO 29 <211> LENGTH: 30 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 29 aaataagttt
gtaagaggag cctcagcatc 30 <210> SEQ ID NO 30 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 30 atcgtaaaga gcttttctcc ccgc 24 <210>
SEQ ID NO 31 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 31
gaaagggctg taggggcgac tgt 23 <210> SEQ ID NO 32 <211>
LENGTH: 327 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 32 ccgtcggcgc cgagggagtt agtgcgaccc
ggctcggcgc gcacggccaa ggcacgcgcg 60 ctggcacacg cgggcgcgga
cacgcgcgga cacacacgtg cgggacacgc cctcccccga 120 cggcggcgct
aacctctcgg ttattccagg atctttggag acccgaggaa agccgtgttg 180
accaaaagca agacaaatga ctcacagaga aaaaagatgg cagaaccaag ggcaactaaa
240 gccgtcaggt tctgaacagc tggtagatgg gctggcttac tgaaggacat
gattcagact 300 gtcccggacc cagcagctca tatcaag 327 <210> SEQ ID
NO 33 <211> LENGTH: 6158 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 33 gttgatagaa
gtccagatcc tgaggaaatc tccagctaaa tgctcaaaat ataaaatact 60
gagctgagat ttgcgaagag cagcagcatg gatggatttt atgaccagca agtgccttac
120 atggtcacca atagtcagcg tgggagaaat tgtaacgaga aaccaacaaa
tgtcaggaaa 180 agaaaattca ttaacagaga tctggctcat gattcagaag
aactctttca agatctaagt 240 caattacagg aaacatggct tgcagaagct
caggtacctg acaatgatga gcagtttgta 300 ccagactatc aggctgaaag
tttggctttt catggcctgc cactgaaaat caagaaagaa 360 ccccacagtc
catgttcaga aatcagctct gcctgcagtc aagaacagcc ctttaaattc 420
agctatggag aaaagtgcct gtacaatgtc agtgcctatg atcagaagcc acaagtggga
480 atgaggccct ccaacccccc cacaccatcc agcacgccag tgtccccact
gcatcatgca 540 tctccaaact caactcatac accgaaacct gaccgggcct
tcccagctca cctccctcca 600 tcgcagtcca taccagatag cagctacccc
atggaccaca gatttcgccg ccagctttct 660 gaaccctgta actcctttcc
tcctttgccg acgatgccaa gggaaggacg tcctatgtac 720 caacgccaga
tgtctgagcc aaacatcccc ttcccaccac aaggctttaa gcaggagtac 780
cacgacccag tgtatgaaca caacaccatg gttggcagtg cggccagcca aagctttccc
840 cctcctctga tgattaaaca ggaacccaga gattttgcat atgactcaga
agtgcctagc 900 tgccactcca tttatatgag gcaagaaggc ttcctggctc
atcccagcag aacagaaggc 960 tgtatgtttg aaaagggccc caggcagttt
tatgatgaca cctgtgttgt cccagaaaaa 1020 ttcgatggag acatcaaaca
agagccagga atgtatcggg aaggacccac ataccaacgg 1080 cgaggatcac
ttcagctctg gcagtttttg gtagctcttc tggatgaccc ttcaaattct 1140
cattttattg cctggactgg tcgaggcatg gaatttaaac tgattgagcc tgaagaggtg
1200 gcccgacgtt ggggcattca gaaaaacagg ccagctatga actatgataa
acttagccgt 1260 tcactccgct attactatga gaaaggaatt atgcaaaagg
tggctggaga gagatatgtc 1320 tacaagtttg tgtgtgatcc agaagccctt
ttctccatgg cctttccaga taatcagcgt 1380 ccactgctga agacagacat
ggaacgtcac atcaacgagg aggacacagt gcctctttct 1440 cactttgatg
agagcatggc ctacatgccg gaagggggct gctgcaaccc ccacccctac 1500
aacgaaggct acgtgtatta acacaagtga cagtcaagca gggcgttttt gcgcttttcc
1560 ttttttctgc aagatacaga gaattgctga atctttgttt tatttctgtt
gtttgtattt 1620 tatttttaaa taataataca caaaaagggg cttttcctgt
tgcattattc tatggtctgc 1680 catggactgt gcactttatt tgagggtggg
tgggagtaat ctaaacattt attctgtgta 1740 acaggaagct aatgggtgaa
tgggcagagg gatttgggga ttacttttta cttaggcttg 1800 ggatggggtc
ctacaagttt tgagtatgat gaaactatat catgtctgtt tgatttcata 1860
acaacataag ataatgttta ttttatcggg gtatctatgg tacagttaat ttcacgttgt
1920 gtaaatatcc acttggagac tatttgcctt gggcattttc ccctgtcatt
tatgagtctc 1980 tgcaggtgta caaaaaaacc ccaatctact gtaaatggca
gtttaattgt tagaaatgac 2040 tgtttttgca ccacttgtaa aaaggtattt
agcgattgca tttgctgttt gttgttttat 2100 tttgctttat atatgacttg
cagaggataa ccataaaatg ggtaattctc tctgaagttg 2160 aataatcacc
atgactgtaa atgaggggca caattttgga ctctggcgcc aaactgagtc 2220
ataggccagt agcattacgt gtatctggtg ccaccttgct gtttagatac aaatcatacc
2280 gtcttttaaa tattttgaag cccatttcag ttaaataatg acatgtcatg
gtcctttgga 2340 atcttcattt aaatgttaaa tctggaatca aaatgaagca
aaaaatatct gtctcctttt 2400 cactttcttc agtacataaa tacattattt
aatcaataag aattaactgt actaaatcat 2460 gtattatgct gttctagtta
cagcaaacac tctttaagaa aaatatccaa tacactaaat 2520 aggtactata
gtaattttta gacatggtac ccattgatat gcatttaaac cttttactgc 2580
tgtgttatgt tgataacata tataaatatt agataatgct aatgcttctg ctgctgtctt
2640 ttctgtaata ttctctttca tgctgaattt actatgacca tttataagca
gtgcagttaa 2700 ctacagatag catttcagga caaaatagat gactcaaacc
atttattgct taaaaaatag 2760 cttacgccat gctatgctat aagcagcttt
tatgcacatt gacaaatgaa gagtaagctt 2820 cagcttgcta aaggaaactg
tggaaccttt tgtaactttt ggtgatatgg aaaattattt 2880 acaaaccgtc
aaagaatatg aggaagttgc tgtatgacat agtgctggca ctgatattat 2940
ccatcatctc tttttggaca cttctgtaaa tgtgattgga ttgtttgaaa gaagatttaa
3000 agtttcaaag ttttttgttc tgtttttgct ttgcatttgg agaaaatatt
gaaagcaggg 3060 tatgttgttt cattcacctt gaaaaaacca tgagtaaatg
gggatataga atctctgaat 3120 agctcgctaa aagattcaag caagggacat
gaattttgtt ccatctatca ataatatcca 3180 gaagaacaac ttttttaaag
agtctatagc aaaaagcaaa aaaaaaaaaa aattctaaac 3240 acaaagtcaa
aataaaccta ttgtaaaagc atttcgtgat gagcatgaaa aagattgttt 3300
aaagatgatc cccccagcta cccattttcc aaaactacac agatcacagc tcatttctct
3360 aagtggagca gttatcaaga aacccaaaca ccaaaattgc tactcttcac
atttaatcct 3420 acaaaaagta ctccaatttc aaaatatgta tgtaacctgc
gatttcaatg attgttgttc 3480 atatacatca tgtattattt tggcccattt
tgggcctaaa aaagaaaact atgccttaaa 3540 aatcagaacc ttttctcccc
actatgctta tgtggccatc tacagcactt agaataaaaa 3600 cagatgttaa
aatattcagt gaaagtttta ttggaaaaag gaattgagat atataattga 3660
gatttggtga aattgaagga gaaaatttaa gtgagtcttt aaaatatatt ctgaatgaaa
3720 actgtattga ggattcattt ttgttccttt tttttctttt tctcttttct
cctttttctt 3780 ctttttaata gtctagtttt agtcagtcag tgaggaagaa
ttgggccatg ctaacgttat 3840 cacaagagaa caatggcaga aatggtatta
gttatataat atttaaggac aaactatatg 3900 ttttgctgtt ttaacgtagt
gactcactga actaaataca taattgacca acattaagtg 3960 tatttccaat
acagaagggt tgaaaatatt acattataaa ctcttttgaa aaatgtatct 4020
aaaatttttt aagttctgtt ttgattccac tttttggttg agtttttatg tttttgtttt
4080 caggtagatt aataaatctg gcagctgatt tctgcaagat tcttgtgttt
tgaatttctc 4140 attgaattgg ctactcaaac atagaaatca tttgttaatg
atgtaatgtc ttctctcagc 4200 ttttatcttc actgctgttt gctgtctctt
gatgatgaca tgttaatacc caatagatta 4260 attgcaacaa acacttatac
tcaaataact aagtaaaaat aatttttctt gttatgtcca 4320 tgaaaagtgc
ttcagaataa aaatccacaa gactgacagt gcagaacatt tttctcaaat 4380
catgggcgga tcttggaggt ctagtttccc gtagatgctg taaccaatta ccacaacttc
4440 agtaatttac acaaatttat cttatagttc tggaggcaga agttcaaaag
aagccttaag 4500 agactaaaac caagatgtcc ttaggtctgg ttccttctgg
aggctccagg ggagattctt 4560 ccagctttca cttctagagt ctgctgacat
tccttggctc ctggctacat cacttcaatc 4620 tctgcttcca tggtcacata
ctcttctact atagtcaaat ttccttcctg cctcttataa 4680 ggatgcttgt
gattacattt aggggatgct cagataatcc aggacaatct ctccatctca 4740
agatccttaa cttaatgacg tgtgccaagt ccctttggct agataattat tcataggtcc
4800 cagggattag gacatggatg taaggggtga gggcagggct gttattcaga
acaccgcacg 4860 gaggaggaag actgtgtagc aaagactcta attgatttac
tcaggaacag tggagttctg 4920 ctgagggatc taggatttga aagtactaga
gtttgctttt atttaccact gagatatttt 4980 ccccttattc tgcataaata
attttgaaaa ctttctatat taaatttcaa ctattccact 5040 aaaatgtctg
gtaatcacat caagccttta gattattcaa atccttcccc agcccccagg 5100
aaaacactaa gtcatgaaac agaaaaacag aaggtatgat aataatagta ataacagtta
5160 aatcagtggt ctaatccaga ttttattttt taatacattt cttttggtgt
taatatgggt 5220 tactatgtga tcttatcatt tgctagtgat tattacttat
taggtaagaa caatgtgtaa 5280 aatatgtcta ttactcaaaa gaacaattgc
aaaatgagtc aacttatctt tatataacca 5340 ggaaagaaat atattgccag
aagctacaga attttgccag atgataggga tttctaaaat 5400 gagccacttt
gtctatcatg cagccttttc agagcttgta atgagaaaac attacagagg 5460
agaaggtcat ttggatgttt gttacttgga atcctagaaa acaaaaacta aaatttaaaa
5520 ataagaagtg agtaagctat tttccatttg cgatttggta tggagaagag
aggaaataga 5580 attattaaaa aaatacaaat tgggtaaaag tgatggtgga
aaaaatataa agaaggcaaa 5640 tgtacatatt aagcaattct actaagaatt
ggaaaaatca agtttcaaaa agatggtaat 5700 agttgggcat gatactagaa
aatttcaccc agtttattca gagctcaact agtactttta 5760 ggacttcttt
ttttatatac atgagactca ctttgacata cttaaaaaaa aaacagttta 5820
tggaaagtac agtttaagag gagaatttga ttagactaag tggatatctt tatagaaata
5880 ttaatgattt cagaattttc agttacaagt gtatataccg tggctattgt
ttatggattc 5940 atatgtaagg tagggtcttt tttgcatata gactccagta
ttagttactt tcattctaaa 6000 attatattta tgcttctatg gggaagaaaa
tttttaattc acttggttgt attaaaatta 6060 tacttacggt ttgagaaaac
atgctatgaa aatcatgatt atagcaaatt aaatatgctc 6120 aaaatttaaa
tctaaaataa aagcccagaa actgaaaa 6158 <210> SEQ ID NO 34
<211> LENGTH: 5228 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 34 cgggcgaggg
ccgggcagga ggagcgggcg cggcgcgggc gaggctggga cccgagcgcg 60
ctcacttcgc cgcaaagtgc caacttcccc tggagtgccg ggcgcgcacc gtccgggcgc
120 gggggaaaga aaggcagcgg gaatttgaga tttttgggaa gaaagtcgga
tttcccccgt 180 ccccttcccc ctgttactaa tcctcattaa aaagaaaaac
aacagtaact gcaaacttgc 240 taccatcccg tacgtccccc actcctggca
ccatgaaggc ggccgtcgat ctcaagccga 300 ctctcaccat catcaagacg
gaaaaagtcg atctggagct tttcccctcc ccggatatgg 360 aatgtgcaga
tgtcccacta ttaactccaa gcagcaaaga aatgatgtct caagcattaa 420
aagctacttt cagtggtttc actaaagaac agcaacgact ggggatccca aaagaccccc
480 ggcagtggac agaaacccat gttcgggact gggtgatgtg ggctgtgaat
gaattcagcc 540 tgaaaggtgt agacttccag aagttctgta tgaatggagc
agccctctgc gccctgggta 600 aagactgctt tctcgagctg gccccagact
ttgttgggga catcttatgg gaacatctag 660 agatcctgca gaaagaggat
gtgaaaccat atcaagttaa tggagtcaac ccagcctatc 720 cagaatcccg
ctatacctcg gattacttca ttagctatgg tattgagcat gcccagtgtg 780
ttccaccatc ggagttctca gagcccagct tcatcacaga gtcctatcag acgctccatc
840 ccatcagctc ggaagagctc ctctccctca agtatgagaa tgactacccc
tcggtcattc 900 tccgagaccc tctccagaca gacaccttgc agaatgacta
ctttgctatc aaacaagaag 960 tcgtcacccc agacaacatg tgcatgggga
ggaccagtcg tggtaaactc gggggccagg 1020 actcttttga aagcatagag
agctacgata gttgtgatcg cctcacccag tcctggagca 1080 gccagtcatc
tttcaacagc ctgcagcgtg ttccctccta tgacagcttc gactcagagg 1140
actatccggc tgccctgccc aaccacaagc ccaagggcac cttcaaggac tatgtgcggg
1200 accgtgctga cctcaataag gacaagcctg tcattcctgc tgctgcccta
gctggctaca 1260 caggcagtgg accaatccag ctatggcagt ttcttctgga
attactcact gataaatcct 1320 gtcagtcttt tatcagctgg acaggagatg
gctgggaatt caaactttct gacccagatg 1380 aggtggccag gagatgggga
aagaggaaaa acaaacctaa gatgaattat gagaaactga 1440 gccgtggcct
acgctactat tacgacaaaa acatcatcca caagacagcg gggaaacgct 1500
acgtgtaccg ctttgtgtgt gacctgcaga gcctgctggg gtacacccct gaggagctgc
1560 acgccatgct ggacgtcaag ccagatgccg acgagtgatg gcactgaagg
ggctggggaa 1620 accctgctga gaccttccaa ggacagccgt gttggttgga
ctctgaattt tgaattgtta 1680 ttctattttt tattttccag aactcatttt
ttaccttcag gggtgggagc taagtcagtt 1740 gcagctgtaa tcaattgtgc
gcagttggga aaggaaagcc aggacttgtg gggtgggtgg 1800 gaccagaaat
tcttgagcaa attttcagga gagggagaag ggccttctca gaagcttgaa 1860
ggctctggct taacagagaa agagactaat gtgtccaatc atttttaaaa atcatccatg
1920 aaaaagtgtc ttgagttgtg gacccattag caagtgacat tgtcacatca
gaactcatga 1980 aactgatgta aggcaattaa tttgcttctg tttttaggtc
tgggagggca aaaaagaggt 2040 gggtgggatg aaacatgttt tgggggggga
tgcactgaaa atctgagaac tatttaccta 2100 tcactctagt tttgaagcaa
agatggactt cagtggggag gggccaaaac cgttgttgtg 2160 ttaaaattta
ttttattaaa ttttgtgcca gtattttttt tcttaaaaat cgtcttaagc 2220
tctaaggtgg tctcagtatt gcaatatcat gtaagtttgt ttttatttgc cggctgagga
2280 ttctgtcaca atgaaagaaa actgtttata tagaccccat tggaaaagca
aaacgctctc 2340 actgagatca gggatcccaa attcatggga cttatataag
aaggacaatt aatgctgatt 2400 tgggtacagg ggaattatgt gtgtgaatgt
catctacaat taaaaaaaat tagcacatcc 2460 ctttacttac ttgttatcag
tggattctcg gggtttggac ttaatgttga gctaagaagc 2520 attaagtctt
tgaactgaat gtattttgca tccctggttt tggacgacag taaacgtagg 2580
agcactgttg aagtcctgga agggagatcg aaggaggaag attgacttgg ttctttctta
2640 gtcctatatc tgtagcatag atgacttgga ataaaagctg tatgcatggg
cattacccct 2700 caggtcctaa gaaataagtc ctgaatgcat gtcgttccaa
actaacactc tgtaattttt 2760 cttttatgtc ttattttcca agagtcctcc
attttttgca ccccctcacc gccaactctg 2820 ttattcagta gagagaagtg
tacggctttc tgattggtga gtgaaaaagt aacttgagac 2880 acgacctaag
ttgaagagtt tagacttgct gagttttaga agtgatggaa attaagagag 2940
catttcaata aaatgtgact tggctgtctt tggaagagaa gtgcaaggct ttcctttgaa
3000 gaatttaaat tagtccggta ggatgtcagg tgagactgtg tatgcaaaat
gaatggcaca 3060 ggtgatgcca gggcctcttg cttgggtctg atgtcttggc
acagggtaag tgaaggttaa 3120 ttccagaaga gaggaatgac ttgaaggcaa
aggaaactaa ggaaggaggt tcagtgagga 3180 aaataaggtt gtccatgaga
tttgaataga tttttagttc ccccaaggtt taaatacaaa 3240 catagtcaag
caaggtagtc atctttctgc tggttgtgag ggggaatctg aaaatggagt 3300
tttagaggaa aagtcaacat ctaactagtg aggaaaagtg cctaatacaa ttagaatctc
3360 cctcactcta tagttgccca gttgaaagga taaggaggag gggtggcttt
tatggacttc 3420 catgagagaa ggaaagaaat atttcaggta agcttctcag
ggctggccct ttttgggatt 3480 tggatgagaa attggaagta ctaactactt
tctagcatat ctttaagaaa attgattgtt 3540 atttactccc agatcctctt
gcagacccag aattatcagg aacatagctc tgtgattcat 3600 gagtgtcccc
atactgatga attggagcat ccatatggaa agcaaaggca gaattatccc 3660
agctgtatta ttttgatctt ttggatgcag gtgccttaat gaagctctca aaatatttta
3720 ggagctgctc agggagtgtt gggtggaact gtttggacta cattgttttc
tcttagatta 3780 tgtgattttt gttgggcact ggcaaaaggt gtgtgtgtga
atgtgtgcat gtgtgtgaat 3840 gttgtgtgtg tgtgtgtgtg tgtgtgtgtg
tgtgtgtgtg tttgcagaca tgcaaaactg 3900 cagctgaaat aataccttag
atttctaggt aagtctttcc acatttcaat aatgggtaag 3960 agtagaacca
gggccgggta tcaattattg cttgctgttt gcaaccaggc ataaaatcac 4020
tttctcaaat catccaccgt tcctattaaa tttatgccgg aaactctcct tctgtgagta
4080 taactcctgc agttcctata gcagataaga tataagaaag tgcctcctag
tgctcctccg 4140 cccgcttgtt tgctaaaatt ccctttctct ctaagtccac
cattttcaag atttgtagat 4200 agtgtattag ttaagacagc tttgtcgatc
tggccagatg ttttttctcc tttgtccaaa 4260 ggccagagac catcccagga
agagtggtgg gtggtttata cactggaaat gttgcgttta 4320 tgctttttaa
aaacacacgt taacttcaga ggaaggatgg gcaaatctgg tctagctggg 4380
tgaaaccctt attttcccag agatgcctta acctttgttg gttttggctt tagggttcag
4440 agtcactttt gttcccttct ccattctgga gagggacttc ccctacatag
agccctgatt 4500 tttgtggctg tggggattgg aggtagcatt caaagatcag
atgtgctttt cctcactttg 4560 gagatgaaca ctctgggttt tacagcatta
acctgcctaa ccttcatggt gagaaataca 4620 ccatctctct tctagtcatg
ctgtgcatgc cgcttactct gttggggtct atataaattt 4680 gttgaactct
tacctacatt ccaaagaagt ttcaaggaac cataaatata tgtatacata 4740
tacatatata aaatatatat attaaaataa aattatcagg aatactgcct cagttattga
4800 actttttttt ttaagaatac ttttttttta agctgagaag tatagggatg
aaaaagatgt 4860 tatattgtgt ttgactattt tccaacttgt attttcatat
aatttatatt ttttaaaagc 4920 tgaaaattta gaagcaagat gaaaaaaagg
aaaagcaggt gctttttaaa aatcagaact 4980 gaggtagctt agagatgtag
cgatgtaagt gtcgatgttt ttttaaaaaa aaatgcaaaa 5040 aaattcttat
ggcggagttt tttgtttgtt tattttagta gctgatgctg gcacatcatt 5100
ttgctggaga gttttttata tactgtagcc tgatttcata ttgtatttta aactgtgtga
5160 aattaaaaac aaagaatttc attcataaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 5220 aaaaaaaa 5228 <210> SEQ ID NO 35 <211>
LENGTH: 3672 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 35 gcccggttac ttcctccaga gactgacgag
tgcggtgtcg ctccagctca gagctcccgg 60 agccgcccgg ccagcgtccg
gcctccctga tcgtctctgg ccggcgccct cgccctcgcc 120 cggcgcgcac
cgagcagccg cgggcgccga gcagccaccg tcccgaccaa gcgccggccc 180
tgcccgcagc ggcaggatga atgatttcgg aatcaagaat atggaccagg tagcccctgt
240 ggctaacagt tacagaggga cactcaagcg ccagccagcc tttgacacct
ttgatgggtc 300 cctgtttgct gtttttcctt ctctaaatga agagcaaaca
ctgcaagaag tgccaacagg 360 cttggattcc atttctcatg actccgccaa
ctgtgaattg cctttgttaa ccccgtgcag 420 caaggctgtg atgagtcaag
ccttaaaagc taccttcagt ggcttcaaaa aggaacagcg 480 gcgcctgggc
attccaaaga acccctggct gtggagtgag caacaggtat gccagtggct 540
tctctgggcc accaatgagt tcagtctggt gaacgtgaat ctgcagaggt tcggcatgaa
600 tggccagatg ctgtgtaacc ttggcaagga acgctttctg gagctggcac
ctgactttgt 660 gggtgacatt ctctgggaac atctggagca aatgatcaaa
gaaaaccaag aaaagacaga 720 agatcaatat gaagaaaatt cacacctcac
ctccgttcct cattggatta acagcaatac 780 attaggtttt ggcacagagc
aggcgcccta tggaatgcag acacagaatt accccaaagg 840 cggcctcctg
gacagcatgt gtccggcctc cacacccagc gtactcagct ctgagcagga 900
gtttcagatg ttccccaagt ctcggctcag ctccgtcagc gtcacctact gctctgtcag
960 tcaggacttc ccaggcagca acttgaattt gctcaccaac aattctggga
ctcccaaaga 1020 ccacgactcc cctgagaacg gtgcggacag cttcgagagc
tcagactccc tcctccagtc 1080 ctggaacagc cagtcgtcct tgctggatgt
gcaacgggtt ccttccttcg agagcttcga 1140 agatgactgc agccagtctc
tctgcctcaa taagccaacc atgtctttca aggattacat 1200 ccaagagagg
agtgacccag tggagcaagg caaaccagtt atacctgcag ctgtgctggc 1260
cggcttcaca ggaagtggac ctattcagct gtggcagttt ctcctggagc tgctatcaga
1320 caaatcctgc cagtcattca tcagctggac tggagacgga tgggagttta
agctcgccga 1380 ccccgatgag gtggcccgcc ggtggggaaa gaggaaaaat
aagcccaaga tgaactacga 1440 gaagctgagc cggggcttac gctactatta
cgacaagaac atcatccaca agacgtcggg 1500 gaagcgctac gtgtaccgct
tcgtgtgcga cctccagaac ttgctggggt tcacgcccga 1560 ggaactgcac
gccatcctgg gcgtccagcc cgacacggag gactgaggtc gccgggacca 1620
ccctgagccg gccccaggct cgtggactga gtgggaagcc catcctgacc agctgctccg
1680 aggacccagg aaaggcagga ttgaaaatgt ccaggaaagt ggccaagaag
cagtggcctt 1740 attgcatccc aaaccacgcc tcttgaccag gctgcctccc
ttgtggcagc aacggcacag 1800 ctaattctac tcacagtgct tttaagtgaa
aatggtcgag aaagaggcac caggaagccg 1860 tcctggcgcc tggcagtccg
tgggacggga tggttctggc tgtttgagat tctcaaagga 1920 gcgagcatgt
cgtggacaca cacagactat ttttagattt tcttttgcct tttgcaacca 1980
ggaacagcaa atgcaaaaac tctttgagag ggtaggaggg tgggaaggaa acaaccatgt
2040 catttcagaa gttagtttgt atatattatt ataatcttat aattgttctc
agaatccctt 2100 aacagttgta tttaacagaa attgtatatt gtaatttaaa
ataattatat aactgtattt 2160 gaaataagaa ttcagacatc tgaggtttta
tttcattttt caatagcaca tatggaattt 2220 tgcaaagatt taatctgcca
agggccgact aagagaagtt gtaaagtatg tattatttac 2280 atttaataga
cttacaggga taaggcctgt ggggggtaat ccctgctttt tgtgtttttt 2340
tgtttgtttg tttgtttgtt tttggggggt tttcttgcct tggttgtctg gcaaggactt
2400 tgtacatttg ggagttttta tgagaaactt aaatgttatt atctgggctt
atatctggcc 2460 tctgctttct cctttaattg taaagtaaaa gctataaagc
agtatttttc ttgacaaatg 2520 gcatatgttt tccacttctt tgcatgcgtt
taagtcagtt tatacacaaa atggatttta 2580 ttttttagtt taactgtgtt
tctccgacag ctcacctctc tctgaccacc cagccatttc 2640 cttcctgtgc
tccacgttct tctgtgtgat taaaataaga atattatttt tggaaatatg 2700
caactccttt tcagagatca ggagggattt atgtagcagc tatttttact gcaaaagtaa
2760 ttcactggaa aaaaaatgta atttgtaaga aagctttatt tttatctcag
ctctatgtaa 2820 agttaaagtt actgtacaga gctgaaggac ggggggcggt
aggggtcttg atgaaacctc 2880 ttgaacgaag cacagtttgt cccatctttg
ttcactcgtg tgtctcaacc atcttaatag 2940 catgctgctc ctttttgctc
agtgtccaca gcaagatgac gtgattctta ttttcttgga 3000 cacagactat
tctgaggcac agagcgggga cttaagatgg gaaagagaaa gcatcggagc 3060
cattcattcg gagaaaacgt tttgatcaaa atggagactt ttgtagtcgt ttcaaaagag
3120 cacctgagtc atgtgtattc ccggccttta taaatgaccc ggtcaagttg
gtttcaaagt 3180 ccgacaggct tgtctgttta ctagctgcgt ggccttggac
gggtggctga catctgtaaa 3240 gaatcctcct gtgatgaaac tgaggaatcg
ggtggccggg caagctggga agagcaaagc 3300 cagagctgcg ctgcctcaat
acccacaaaa gaccattccc agtatacata agcacaggat 3360 gtttttctca
agagggatgt atttatcact tggacatctg tttataatat aaacagacat 3420
gtgactggga acatcttgct gccaaaagaa tcctaggcag tggctcattg tatgtgaggt
3480 tgaaccacgt gaaattgcca atattaggct ggcttttatc tacaaagaag
gagtttcatg 3540 gggttcagcc taacagttat ggaaactaca gtccttataa
accattggca tggtaataaa 3600 cagatcttaa gtataaaaat tttgtaattg
ggcctttact ctctcaataa taaagtattt 3660 tgtttatata aa 3672
<210> SEQ ID NO 36 <211> LENGTH: 2668 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 36
cgctacacac aggtacccct gggatggcgt gagcactccc ccagcgatgg acccatctgt
60 gacgctgtgg cagtttctgc tgcagctgct gagagagcaa ggcaatggcc
acatcatctc 120 ctggacttca cgggatggtg gtgaattcaa gctggtggat
gcagaggagg tggcccggct 180 gtgggggcta cgcaagaaca agaccaacat
gaattacgac aagctcagcc gggccttgcg 240 gtactactat gacaagaaca
tcatccgcaa ggtgagcggc cagaagttcg tctacaagtt 300 tgtgtcctac
cctgaggtcg cagggtgctc cactgaggac tgcccgcccc agccagaggt 360
gtctgttacc tccaccatgc caaatgtggc ccctgctgct atacatgccg ccccagggga
420 cactgtctct ggaaagccag gcacacccaa gggtgcagga atggcaggcc
caggcggttt 480 ggcacgcagc agccggaacg agtacatgcg ctcgggcctc
tattccacct tcaccatcca 540 gtctctgcag ccgcagccac cccctcatcc
tcggcctgct gtggtgctcc ccagtgcagc 600 tcctgcaggg gcagcagcgc
ccccctcggg gagcaggagc accagtccaa gccccttgga 660 ggcctgtctg
gaggctgaag aggccggctt gcctctgcag gtcatcctga ccccgcccga 720
ggccccaaac ctgaaatcgg aagagcttaa tgtggagccg ggtttgggcc gggctttgcc
780 cccagaagtg aaagtagaag ggcccaagga agagttggaa gttgcggggg
agagagggtt 840 tgtgccagaa accaccaagg ccgagccaga agtccctcca
caggagggcg tgccagcccg 900 gctgcccgcg gttgttatgg acaccgcagg
gcaggcgggc ggccatgcgg cttccagccc 960 tgagatctcc cagccgcaga
agggccggaa gccccgggac ctagagcttc cactcagccc 1020 gagcctgcta
ggtgggccgg gacccgaacg gaccccagga tcgggaagtg gctccggcct 1080
ccaggctccg gggccggcgc tgaccccatc cctgcttcct acgcatacat tgaccccggt
1140 gctgctgaca cccagctcgc tgcctcctag cattcacttc tggagcaccc
tgagtcccat 1200 tgcgccccgt agcccggcca agctctcctt ccagtttcca
tccagtggca gcgcccaggt 1260 gcacatccct tctatcagcg tggatggcct
ctcgaccccc gtggtgctct ccccagggcc 1320 ccagaagcca tgactactac
caccaccacc accacccctt ctggggtcac tccatccatg 1380 ctctctccag
ccagccatct caaggagaaa catagttcaa ctgaaagact catgctctga 1440
ttgtggtggg gtggggatcc ttgggaagaa ttactcccaa gagtaactct cattatctcc
1500 tccacagaaa acacacagct tccacaactt ctctgttttc tgtcagtccc
ccagtggccg 1560 cccttacacg tctcctactt caatggtagg ggcggtttat
ttatttattt tttgaaggcc 1620 actgggagga gcctgaccta accttttagg
gtggttagga catctccccc acctccccac 1680 ttttttcccc aagacaagac
aatcgaggtc tggcttgaga acgacctttc tttctttatt 1740 tctcagcctg
cccttgggga gatgagggag ccctgtctgc gtttttggat gtgagtagaa 1800
gagttagttt gttttgtttt attattcctg gccatactca ggggtccagg aagaatttgt
1860 accatttaat gggttgggag tcttggccaa ggaagaatca cacccttgga
atagaaattt 1920 ccacctcccc aacctttctc tcagacagct tatccttttc
aaccaacttt ttggccaggg 1980 aggaatgtcc cttttgttct tccccctgag
aagccattcc tttgtctgcc aacctccctg 2040 gggtcctgcc tgtttcctcc
caatggaggg tttttttggg gggtggtccc cgtctggggg 2100 gcccctccag
ccagtactcc aggtctccct gtctctcccc cgctgccatt ttgatagtat 2160
aatctatttt taaatggggc ttttcaatag gggagaggga gtcatctctt cctatatttg
2220 gtggggtggg tgggaaggaa gggatttggg ggggaatctt cctgccgcct
cccccactcc 2280 aagtgtttat ttttgatacc aaacatgaat tttcagttcc
ctccctccca gccccccaat 2340 ttcctgcggg cgggtacaaa ggaccctttc
aatgtccctg gagttgggag ggaggaatgg 2400 gggacataaa gcctgtcctg
tctctattct aggcaagaga gagtgggttc aaaagactcc 2460 tgggctcacc
tgttagcgct ggcccagccc aggccttggg acctgggggt tggtgatttg 2520
ggggacagtg ctacactcgt ctccactgtt tgttttactt ccccaaaatg gacctttttt
2580 ttttctaaag agtcccagag aatggggaat tgttcctgta aatatatatt
tttcaaagtg 2640 aaaaaaaaaa aaaaaaaaaa aaaaaaaa 2668 <210> SEQ
ID NO 37 <211> LENGTH: 5992 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 37 gcgtcccggg
tccccgcgcc gcgccgcgac ctgcagaccc cgccgccgcg ctcgggcccg 60
tctcccacgc ccccgccgcc ccgcgcgccc aactccgccg gccgccccgc cccgccccgc
120 gcgctccaga cccccggggc ggctgccggg agagatgctg gaagaaactt
cttaaatgac 180 cgcgtctggc tggccgtgga gcctttctgg gttggggaga
ggaaaggaaa gtggaaaaaa 240 cctgagaact tcctgatctc tctcgctgtg
agacatgtct gagactcctg ctcagtgtag 300 cattaagcag gaacgaattt
catatacacc tccagagagc ccagtgccga gttacgcttc 360 ctcgacgcca
cttcatgttc cagtgcctcg agcgctcagg atggaggaag actcgatccg 420
cctgcctgcg cacctgcgct tgcagccaat ttactggagc agggatgacg tagcccagtg
480 gctcaagtgg gctgaaaatg agttttcttt aaggccaatt gacagcaaca
cgtttgaaat 540 gaatggcaaa gctctcctgc tgctgaccaa agaggacttt
cgctatcgat ctcctcattc 600 aggtgatgtg ctctatgaac tccttcagca
tattctgaag cagaggaaac ctcggattct 660 tttttcacca ttcttccacc
ctggaaactc tatacacaca cagccggagg tcatactgca 720 tcagaaccat
gaagaagata actgtgtcca gaggaccccc aggccatccg tggataatgt 780
gcaccataac cctcccacca ttgaactgtt gcaccgctcc aggtcaccta tcacgacaaa
840 tcaccggcct tctcctgacc ccgagcagcg gcccctccgg tcccccctgg
acaacatgat 900 ccgccgcctc tccccggctg agagagctca gggacccagg
ccgcaccagg agaacaacca 960 ccaggagtcc taccctctgt cagtgtctcc
catggagaat aatcactgcc cagcgtcctc 1020 cgagtcccac ccgaagccat
ccagcccccg gcaggagagc acacgcgtga tccagctgat 1080 gcccagcccc
atcatgcacc ctctgatcct gaacccccgg cactccgtgg atttcaaaca 1140
gtccaggctc tccgaggacg ggctgcatag ggaagggaag cccatcaacc tctctcatcg
1200 ggaagacctg gcttacatga accacatcat ggtctctgtc tccccgcctg
aagagcacgc 1260 catgcccatt gggagaatag cagactgtag actgctttgg
gattacgtct atcagttgct 1320 ttctgacagc cggtacgaaa acttcatccg
atgggaggac aaagaatcca aaatattccg 1380 gatagtggat cccaacggac
tggctcgact gtggggaaac cataagaaca gaacaaacat 1440 gacctatgag
aaaatgtcca gagccctgcg ccactactac aaactaaaca ttatcaggaa 1500
ggagccagga caaaggcttt tgttcaggtt tatgaaaacc ccagatgaaa tcatgagtgg
1560 ccgaacagac cgtctggagc acctagagtc ccaggagctg gatgaacaaa
tataccaaga 1620 agatgaatgc tgaaggaacc aacagtccac ctcagcgggc
cagcagccca gggaacccct 1680 gcccaccagg attgctggaa gtgtgacgga
gcaggcgggc tgaggagagt ggaaaaggaa 1740 gcgacccaga aatggcaggg
acacttctct tgcagaccaa gagggaccct ggagcacctt 1800 agacaaacta
cccagcacag gcggggctgg aattctggcg gagggcatga gcctgggact 1860
ccatgtcacg tttccttctg atttggaatc tctccatctg taattcctca ccctcaccct
1920 tccaccgttg ttagtatcat ggtgtttttg tttttgtttt tgttttaaga
acctgcagtt 1980 tgactcttca tcgttcatct aggggaagac atctgatgtt
gttttcctat ggaaatatat 2040 atctattata tatatatttt ttgcaaatct
cacaaagtgc ggcaagccca gctggtcagg 2100 aaagagaata cttgcagagg
ggttcaggtt cctctttttc ctgccacgtg gatcaggtct 2160 gttcctgtta
ctgttgggtc ttggctgaaa aaaaaaaatg cttttaaaaa agataaaatg 2220
aaaaggagag ctctcttttt ctctctcttg ctctgttctt cccttggtcc cctctgtcct
2280 cccgccctgc ctgcagttga gattcagatg ccttctgaca gagttcagcc
tcttggagag 2340 tcttggggat tgttggcacc taaacagaat cagtgacccg
ggtgctttgt ggccagcagc 2400 acagaatcaa acccgcatcc cagcattggg
ccacccatct gagggaggcc aaaatcatca 2460 cagatgctgc tgtgctgcag
acagatacat gctagtccag agagccgccc ctgagatggc 2520 tgtgagaacc
atgtgtctaa ggcgtaagat aaggatggaa ggctgtccaa gttatttgga 2580
aggcctcggc agcttgggat tagcttggga gcgcagcgct gcaaagtgga aaatatgaaa
2640 agaccacaca ggcccagcag tccagaaact gggcaaaaat attctgcagt
ggggatttat 2700 ttttccaaag caggtaacag aggctagtga gaaagaaaag
ctcctctctg ctccattcca 2760 aaggccatct tgtggtcagt ttcatgccct
cacctgattt tttttttttt tttttttttt 2820 caattcctaa ccttttttaa
agtttcctgg tctccactgg acacagagct ttggagacgg 2880 aggatcccag
agggcagtct cagttgcaat cagtgtgtgc ccagcctggg cagacaggaa 2940
attcctcgga tacattattt tttctttctt tcatagctgt gtctcagaaa ggacccattt
3000 gtggctcttt ttcacctcaa aataagatcg atggtatctt gtaaaatgag
ggtagtgcca 3060 cttcttagta tttttgaaag ctgttttaga tttttttttt
ttttcctttt ctagccatct 3120 aaattgactc ttccaatata ggtctcagaa
atccaatatt tggagtacaa tttcttttaa 3180 tccagattac acctgcctta
caaagcaccc cctccttgtt cccctctgtt tcctctactc 3240 agttggggga
gaaactcaca gctcctccgg gatacatatg tgccctcagc agcagctccc 3300
aggtgaagtt accagacccc tgggcttctc cccagctttt tctgagttga gtcagacatg
3360 tagagtttgg gtcacacagg caagaggaat tttccctcgg ccttactgac
aaggacacca 3420 acctagggtg caaacagatg gactatggtt caaggacact
ggaattgagg agctgatcaa 3480 ggctctcttc agccttgctc tgtccctgcc
tcttatcaga gcacaggtag acacacgggc 3540 atagccagcc cactcctact
gtcacaggcg ccccaccatt caaccttccg ggaggtcagg 3600 gaccttctat
atgaggcgag tgggtctcag tctgcttgaa tggtgatgag attctgctgg 3660
atctcagcac gctgcaggtg tcttttgaga gcattcagta ggacatggtg atccctattt
3720 cagcctctaa gatgactggt attctatctg aaatgcagag attaagccaa
atacctgatg 3780 tattgtgaaa gccactgatt ttaagaatgg agagaaaggg
attttttact gcatccctct 3840 gtatgaatat gaaatcagag accagggcat
gatgttgcta ggattagagc ctctcagtct 3900 ggcctcttca cccaagtgca
agaactcagt ctcttactgt tcaaagaatc ttaacagttg 3960 aattatggag
ggaaattccc ttttgcccca agcatttcta tatttaaagc aatatcccag 4020
gagaatatgt tagacttagg atgatacctt cagccacttg aagaagaaat agaaggcgct
4080 cattccaata tagtctttat ttcccattca gatacaggtt gagcatccct
aatctgaaca 4140 gttaaaaccc ccaaatgccc caaaatccaa accttcctga
acgctatgac accatgagtg 4200 gaaaattcca cacctaacaa acacatttgc
tttcttatgg ttcaatgtac acaaactgtt 4260 ttatatagaa aatgatttca
aatatcataa aattaccttc aggctatgtg tataaagtat 4320 atatgagcca
taaatgaatt ttgtgtttag actttgtgtc catccccaag atctctcatt 4380
ttatatatat atatatatat atatatatat atatatatat atatatatac acacacacac
4440 acatacacaa atattccagg atacaaaaaa aaacatttaa aaatccgaga
cccagaacac 4500 ttctggtccc aagcatttca gataagggat atcaatctgt
actaccaata aggatttcgt 4560 aattccccta actgcaaatg tcctcttcat
ttgttcttta tgagaaaacc cgggtagtgc 4620 cagcacctgg atacagtatt
tacaccctgc agaccctaaa gatttcagat tcagttagca 4680 aaccttgatg
aagcacctgc tggacactga gggacccaaa gctcaatcag ccataatccc 4740
tgctttcaga gtttatattg tacctgccta atccacccgg cgtgactcat ttcaacacta
4800 agtactaggg gtgttgtcag gagacaaatc tgaagtcagg agaggaaaat
gcaaaggagc 4860 cctgccgtgt gatggatgtg cattctcact tgggtcttga
agttctcatt cctacatctc 4920 aagctagcca ggcagtctcc tctctatcag
aagaaagcac tggtaattgg ctagactggc 4980 tatgttgaag gtaacatgaa
ctctaagatc ttgacccagg gcgacttggt tttgcttaag 5040 gtggcatcac
caatgttcca aatcctttag ggagatgagg gtatccccac agaaaaagag 5100
gaataataga ccaatggatt ttctcctttc accagtatgt ttggaaccct ctgatccaat
5160 gtcctttgat actgatctct tgtccaaatg agaatgtcgc tttagctgaa
attcaaatgg 5220 ctgtgacaat ttaccgaaat gatgaagtaa ccaccattcc
cacctttcac tgcctaggct 5280 ccaagtctga atacattttt gaaataggaa
ctcccttttg caaaaaagaa acctgggtgt 5340 cagggaggtg aagtgacttg
ccctaggagc agacagcatg ccaagaatgg aattaggctc 5400 aggatccagc
ctgggctcac cctgtgtggc tcattcccac ccaggaaact gaagataaaa 5460
gatttgggaa aacacaccaa gaaaaagggg cagttttctt tgcccaagca tttggtgcta
5520 gttagaggct gttcactctc tcctgctcct cttcggagta gaaataaagg
ctgtgacaca 5580 aggaagccag tggggtggga gggaggcacc ataatccctc
cctaaaaccc acagaagact 5640 aacctgatac tcttttgacc caactgcatc
aacactaaac agctgcagac cccctgaatc 5700 tttcacacat gccaagtgaa
cattcttgat gatttctctt tgtgaccgca accacctgca 5760 aaccagaacg
actctagaat ttccttcccc gccccccttt ttgtttagtt tctaatctct 5820
tgtttatgag gtgtggggtt tataagggac tgaatcaaat gaatgtaaca aaaaagaaaa
5880 aaaaaacaaa aaaaaatgcc ttttctcagg gccagtgagt tgcaaataat
ttttaaagaa 5940 aagcctataa ttacatcatc tcaataaatt ttttataaaa
aaaaaaaaaa aa 5992 <210> SEQ ID NO 38 <211> LENGTH:
1670 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 38 gatttcctcc cacgcgacct tccagttctc
ggagccaggt taggggtttg gcggaggagg 60 actgcggggc gcgggcctag
ggccccagca gccacggcca ggggagcgct caagacagaa 120 agccggtggc
ttcctcacct ccacctgtaa tgcaggaggg agaattggct atttctccta 180
taagccctgt ggcagccatg cctcccctag gcacccacgt gcaagccaga tgtgaagctc
240 aaattaacct gctgggtgaa ggggggatct gcaagctgcc aggaagactc
cgcatccagc 300 ccgcactgtg gagcagggag gacgtgctgc actggctgcg
ctgggcagag caggagtact 360 ctctgccatg caccgcggag cacgggttcg
agatgaacgg acgcgccctc tgcatcctca 420 ccaaggacga cttccggcac
cgtgcgccca gctcaggtga cgtcctgtat gagctgctcc 480 agtacatcaa
gacccagcgg cgagccctgg tgtgtggacc cttttttgga gggatcttca 540
ggctgaagac gcccacccag cactctccag tccccccgga agaggtgact ggcccctctc
600 agatggacac ccgaaggggc cacctgctgc agccaccaga cccagggctt
accagcaact 660 tcggccacct ggatgaccct ggcctggcaa ggtggacccc
tggcaaggag gagtccctca 720 acttatgtca ctgtgcagag ctcggctgca
ggacccaggg ggtctgttcc ttccccgcga 780 tgccgcaggc ccccattgac
ggcaggatcg ctgactgccg cctgctgtgg gattacgtgt 840 atcagctgct
ccttgatacc cgatatgagc cctacatcaa gtgggaagac aaggacgcca 900
agatcttccg agttgtggat ccaaatgggc tcgccagact ctggggaaat cacaagaacc
960 gggtgaacat gacctacgag aagatgtctc gtgccctgcg ccactattat
aagcttaata 1020 tcattaagaa ggaaccgggg cagaaactcc tgttcagatt
tctaaagact ccgggaaaga 1080 tggtccagga caagcacagc cacctggagc
cgctggagag ccaggagcag gacagaatag 1140 agttcaagga caagaggcca
gaaatctctc cgtgaggggc aggtggactc caggcacccg 1200 gtaccgatgg
ggcagggacc gagtctccca tgaaggcaga ctcctcctcc cagcagagca 1260
gcaggatccc cagccagact ctgtacccac aggattacag ccattgcttg ggaaggctgg
1320 gaggcctccc atccaggaca ctgggggcag gagtgtcatc ttttgggcag
ggcaatcctg 1380 gggctaaatg aggtacaggg gaatggactc tcccctactg
cacccctggg agaggaagcc 1440 aggcaccgat agagcaccca gccccacccc
tgtaaatgga atttaccaga tgaagggaat 1500 gaagtccctc actgagcctc
agatttcctc acctgtgaaa tgggctgagg caggaaatgg 1560 gaaaaagtgt
tagtgcttcc aggcggcact gacagcctca gtaacaataa aaacaatggt 1620
agctgaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1670
<210> SEQ ID NO 39 <211> LENGTH: 4071 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 39
gagtccagcc gctggtgcgc ggagcggttc accgtcttcg gagcggttcg gcccagcctt
60 tcgcccaggc gcccaggccc gctgcgcgcg tgcgtgagcg cgcctgcgcc
gccagggccg 120 ctgcaagggg aggagagcgg ccgcctcagg aggatccctt
ttcccccaga aattactcaa 180 tgctgaaacc tctcaaagtg gtattagaga
cgctgaaagc accatggacg ggttttatga 240 tcagcaagtc ccttttatgg
tcccagggaa atctcgatct gaggaatgca gagggcggcc 300 tgtgattgac
agaaagagga agtttttgga cacagatctg gctcacgatt ctgaagagct 360
atttcaggat ctcagtcaac ttcaagaggc ttggttagct gaagcacaag ttcctgatga
420 tgaacagttt gtcccagatt ttcagtctga taacctggtg cttcatgccc
cacctccaac 480 caagatcaaa cgggagctgc acagcccctc ctctgagctg
tcgtcttgta gccatgagca 540 ggctcttggt gctaactatg gagaaaagtg
cctctacaac tattgtgcct atgataggaa 600 gcctccctct gggttcaagc
cattaacccc tcctacaacc cccctctcac ccacccatca 660 gaatccccta
tttcccccac ctcaggcaac tctgcccacc tcagggcatg cccctgcagc 720
tggcccagtt caaggtgtgg gccccgcccc cgccccccat tcgcttccag agcctggacc
780 acagcagcaa acatttgcgg tcccccgacc accacatcag cccctgcaga
tgccaaagat 840 gatgcctgaa aaccagtatc catcagaaca gagatttcag
agacaactgt ctgaaccctg 900 ccaccccttc cctcctcagc caggagttcc
tggagataat cgccccagtt accatcggca 960 aatgtcagaa cctattgtcc
ctgcagctcc cccgccccct cagggattca aacaagaata 1020 ccatgaccca
ctctatgaac atggggtccc gggcatgcca gggcccccag cacacgggtt 1080
ccagtcacca atgggaatca agcaggagcc tcgggattac tgcgtcgatt cagaagtgcc
1140 taactgccag tcatcctaca tgagaggggg ttatttctcc agcagccatg
aaggtttttc 1200 atatgaaaaa gatccccgat tatactttga cgacacttgt
gttgtgcctg agagactgga 1260 aggcaaagtc aaacaggagc ctaccatgta
tcgagagggg cccccttacc agaggcgagg 1320 ttcccttcag ctgtggcagt
tcctggtcac ccttcttgat gacccagcca atgcccactt 1380 cattgcctgg
acaggtcgag gcatggagtt caagctgata gaaccggaag aggttgctcg 1440
gcgctggggc atccagaaga accggccagc catgaactat gacaagctga gccgctctct
1500 ccgctattac tatgaaaagg gcatcatgca gaaggtggct ggagagcgat
acgtctacaa 1560 atttgtctgt gacccagatg ccctcttctc catggctttc
ccggataacc agcgtccgtt 1620 cctgaaggca gagtccgagt gccacctcag
cgaggaggac accctgccgc tgacccactt 1680 tgaagacagc cccgcttacc
tcctggacat ggaccgctgc agcagcctcc cctatgccga 1740 aggctttgct
tactaagttt ctgagtggcg gagtggccaa accctagagc tagcagttcc 1800
cattcaggca aacaagggca gtggttttgt ttgtgttttt ggttgttcct aaagcttgcc
1860 ctttgagtat tatctggaga acccaagctg tctctggatt ggcaccctta
aagacagata 1920 cattggctgg ggagtgggaa cagggagggg cagaaaacca
ccaaaaggcc agtgcctcaa 1980 ctcttgattc tgatgaggtt tctgggaaga
gatcaaaatg gagtctcctt accatggaca 2040 atacatgcaa agcaatatct
tgttcaggtt agtacccgca aaacgggaca tgatgtgaca 2100 atctcgatcg
atcatggact actaaatggc ctttacatag aagggctctg atttgcacaa 2160
tttgttgaaa aatcacaaac ccatagaaaa gtgagtaggc taagttgggg aggctcaaac
2220 cattaagggt taaaaataca tcttaaacat tggaaagctc ttctagctga
atctgaaata 2280 ttaccccttg tctagaaaaa ggggggcagt cagaacagct
gttccccact ccgtgttctc 2340 aaaatcataa accatggcta ctcttgggaa
ccacccggcc atgtggtcgc caagtagagc 2400 aagccccctt tctcttccca
atcacgtggc tgagtgtgga tgacttttat tttaggagaa 2460 gggcgattaa
cacttttgac agtattttgt tttgccctga tttgggggat tgttttgttt 2520
tggtggttgt tttggaaaaa cagtttataa actgattttt gtagttttgg tatttaaagc
2580 aaaaaaacga aaaacaaaaa acaaaaacaa accttttggt aatgtgcact
gtgtctttag 2640 ccagggccgt gcaacttatg aagacactgc agcttgagag
gggctttgct gaggcttccc 2700 cttggccatg tgaaagcccg ccttgttgcc
tgctttgtgc tttctgcacc agacaacctg 2760 atggaacatt tgcacctgag
ttgtacattt ttgaagtgtg cagggcagcc tggacacaag 2820 cttagattct
ctatgtatag ttccccgtgt tcactaacat gccctctctg gaaagcatat 2880
gtatataaca tgtgtcatgt cctttggaaa cctggtcacc tggtgaaaac ccttgggatt
2940 cttccctggg catgactgat gacaatttcc atttcatcag tttgttttgt
tttccttttt 3000 ctttaaatct tggactttaa accctacctg tgtgattcag
tagggtttga gacttagctg 3060 tgatactgac aggtaagcaa cagtgctagc
attctagatt cctgcctttt tttaaaaaga 3120 aattattctc attgctgtat
tatattggaa aagttttaaa caaccaagct aaagctatgt 3180 gaaagttgag
ctcaaagtag aggaaaagtt actggtggta ccttgctgcc tgctctgctg 3240
gtagaattct gtgctccccg tgacacttag tacattaaga atgactacac tgttcctcgt
3300 atgtgaagga ggcagtgctg actccgtgag tgtgagacac gtgctttgaa
ctgcttttct 3360 attcatggag cactccatag tctcaaactg tcccccttat
gaccaacagc acatttgtga 3420 agaggttcgc agggataagg ggtgcacttt
atagctatgg aaacatgaga ttctcctcta 3480 ttggaagcta attagcccac
aaaggtggta aacctgtaga ttgggcctta attagcattg 3540 tactctaatc
aaaggactct ttctaaacca tatttatagc tttcttaacc tacacatagt 3600
ctatacatag atgcatattt tacccccagc tggctagaga tttatttgtt gtaaatgctg
3660 tatagatttg gttttccttt ctttacttac cctggtttgg attttttttt
tttttttttt 3720 tgaatggatt tatgctgtct tagcaatatg acaataatcc
tctgtagctt gagctacccc 3780 tcccctgctg taacttacgt gacctgtgct
gtcactgggc ataggacagc ggcatcacgg 3840 ttgcattccc attggactca
tgcacctccc ggatggtttt tgtttttttc gggggttctt 3900 tggggtttgt
ttgtttgctt cttttccaga gtgtggaaag tctacagtgc agaaaggctt 3960
gaacctgcca gctgatttga aatactttca ccctgcgcag ggccgtatgc atcctgccaa
4020 gctgcgttat attctgtact gtgtacaata aagaagtttg cttttcgttt a 4071
<210> SEQ ID NO 40 <211> LENGTH: 3499 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 40
gccaagaagc ttgagagaag aaaaatttca gaaaaattgt ctcaatttga ctagaatatc
60 aatgaaccag gaaaactgaa gcaccttccc taaagaaaac ttgggtatac
aattactcca 120 cagacagagc tgagggtttt ttacccaaat cagtcactgg
attttgctgc ctgatacgtg 180 aatcttcttg gaatttttct catgtggatc
taaggggaat gctttattat ggctgctgtt 240 gtccaacaga acgacctagt
atttgaattt gctagtaacg tcatggagga tgaacgacag 300 cttggtgatc
cagctatttt tcctgccgta attgtggaac atgttcctgg tgctgatatt 360
ctcaatagtt atgccggtct agcctgtgtg gaagagccca atgacatgat tactgagagt
420 tcactggatg ttgctgaaga agaaatcata gacgatgatg atgatgacat
cacccttaca 480 gttgaagctt cttgtcatga cggggatgaa acaattgaaa
ctattgaggc tgctgaggca 540 ctcctcaata tggattcccc tggccctatg
ctggatgaaa aacgaataaa taataatata 600 tttagttcac ctgaagatga
catggttgtt gccccagtca cccatgtgtc cgtcacatta 660 gatgggattc
ctgaagtgat ggaaacacag caggtgcaag aaaaatatgc agactcaccg 720
ggagcctcat caccagaaca gcctaagagg aaaaaaggaa gaaaaactaa accaccacga
780 ccagattccc cagccactac gccaaatata tctgtgaaga agaaaaacaa
agatggaaag 840 ggaaacacaa tttatctttg ggagttttta ctggcactgc
tccaggacaa ggctacttgt 900 cctaaataca tcaagtggac ccagcgagag
aaaggcattt ttaaattggt ggattctaaa 960 gcagtgtcca ggttgtgggg
gaagcacaaa aacaaacctg atatgaatta tgagaccatg 1020 ggaagagcac
tcaggtacta ttaccaaagg ggtattctgg caaaagtgga aggtcagcgc 1080
ttggtgtatc agtttaaaga aatgccaaaa gatcttatat atataaatga tgaggatcca
1140 agttccagca tagagtcttc agatccatca ctatcttcat cagccacttc
aaataggaat 1200 caaaccagcc ggtcgagagt atcttcaagt ccaggggtaa
aaggaggagc cactacagtt 1260 ctaaaaccag ggaattctaa agctgcaaaa
cccaaagatc ctgtggaagt tgcacaacca 1320 tcagaagttt tgaggacagt
gcagcccacg cagtctccat atcctaccca gctcttccgg 1380 actgttcatg
tagtacagcc agtacaggct gtcccagagg gagaagcagc tagaaccagt 1440
accatgcagg atgaaacatt aaattcttcc gttcagagta ttaggactat acaggctcca
1500 acccaagttc cagtggttgt gtctcctagg aatcagcagt tgcatacagt
aacactccaa 1560 acagtgccac tcacaacagt tatagccagc acagatccat
cagcaggtac tggatctcag 1620 aagtttattt tacaagccat tccatcatca
cagcccatga cagtactgaa agaaaatgtc 1680 atgctgcagt cacaaaaggc
gggctctcct ccttcaattg tcttgggccc tgcccaggtt 1740 cagcaggtcc
ttactagcaa tgttcagacc atttgcaatg gaaccgtcag tgtggcttcc 1800
tctccatcct tcagtgctac tgcacctgtg gtgacctttt ctcctcgcag ttcacagctg
1860 gttgctcacc cacctggcac tgtaatcact tcagttatca aaactcaaga
aacaaaaact 1920 cttacacagg aagtagagaa aaaggaatct gaagatcatt
tgaaagagaa cactgagaaa 1980 acggagcagc agccacagcc ttatgtgatg
gtagtgtcca gttccaatgg atttacttct 2040 caggtagcta tgaaacaaaa
cgaactgctg gaacccaact ctttttagtt aatataccaa 2100 agcttatgaa
taattgtttg ttaattgaac attttcaatt atatgcagac tgactgattc 2160
taagataaat tctaaggagg tttctaattt tgtaattgtt aaaaatagag ttaattttga
2220 ctttgttaga tgagggagga aaactcaact gtttctcttt gttatctaaa
tgtttcagaa 2280 ttcaatcgtg aaggaacagg cattttacac tatgaagaca
ttcttttgag atttttattt 2340 cagttgctat atcataagca tttttaaagt
ttcttttcta attttacatt gtattagatt 2400 ttctgattct tttgtaaata
cagaacttaa atagaaggca acaggaaatt tatataggaa 2460 ctattttcat
tccacttgtg taagttaagt cttgactctt tcaaatgcaa aaaacctatt 2520
ttatgctttg ttaaaattat ggtgtcactt agattgactt tagttgactg cactatataa
2580 tatagaacta tgaatatgta gaataacatg aaaaattgga ggtgctggtg
gtatggctga 2640 ccctgtttca gaagcaggat agtataaaag catcagccta
agaatggcac tcccactaac 2700 tagctatgta atcttgacct ctttgggctt
tagttcctct cataaaagga agagatgtat 2760 tggattagac tagattatca
ccactttctc ttctagttct aattttttta attctaatac 2820 ctatattttc
aagttatgtc aattaaatca ttatcaggtt atttcctaat gtaagaatag 2880
ctaaaatgtt gcagagaaat aagtgaccca acaaaattta ttcatctgtt atgggtaaga
2940 tctgccataa attcttccta aataatttgt ttactaactc tttaggccac
tgtgctttgc 3000 ggtccattag taaacttgtg ttgctaagtg ctaaacagaa
tactgctatt ttgagagagt 3060 caagactctt tcttaagggc caagaaagca
acttgagcct tgggctaatc tggctgagta 3120 gtcagttata aaagcataat
tgctttatat tttggatcat tttttactgg gggcggactt 3180 ggggggggtt
gcatacaaag ataacatata tatccaactt tctgaaatga aatgttttta 3240
gattactttt tcaactgtaa ataatgtaca tttaatgtca caagaaaaaa atgtcttctg
3300 caaattttct agtataacag aaatttttgt agatgaaaaa aatcattatg
tttagaggtc 3360 taatgctatg ttttcatatt acagagtgaa tttgtattta
aacaaaaatt taaattttgg 3420 aatcctctaa acatttttgt atctttaatt
ggtttattat taaataaatc atataaaaat 3480 tctcaaaaaa aaaaaaaaa 3499
<210> SEQ ID NO 41 <211> LENGTH: 2212 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 41
gcccggctcc tgggagcagg tctcggcccc cgcttggggc cccggccgtg cggccggagg
60 gagcggccgg atggagcgga ggatgaaagc cggatacttg gaccagcaag
tgccctacac 120 cttcagcagc aaatcgcccg gaaatgggag cttgcgcgaa
gcgctgatcg gcccgctggg 180 gaagctcatg gacccgggct ccctgccgcc
cctcgactct gaagatctct tccaggatct 240 aagtcacttc caggagacgt
ggctcgctga agctcaggta ccagacagtg atgagcagtt 300 tgttcctgat
ttccattcag aaaacctagc tttccacagc cccaccacca ggatcaagaa 360
ggagccccag agtccccgca cagacccggc cctgtcctgc agcaggaagc cgccactccc
420 ctaccaccat ggcgagcagt gcctttactc cagtgcctat gaccccccca
gacaaatcgc 480 catcaagtcc cctgcccctg gtgcccttgg acagtcgccc
ctacagccct ttccccgggc 540 agagcaacgg aatttcctga gatcctctgg
cacctcccag ccccaccctg gccatgggta 600 cctcggggaa catagctccg
tcttccagca gcccctggac atttgccact ccttcacatc 660 tcagggaggg
ggccgggaac ccctcccagc cccctaccaa caccagctgt cggagccctg 720
cccaccctat ccccagcaga gctttaagca agaataccat gatcccctgt atgaacaggc
780 gggccagcca gccgtggacc agggtggggt caatgggcac aggtacccag
gggcgggggt 840 ggtgatcaaa caggaacaga cggacttcgc ctacgactca
gatgtcaccg ggtgcgcatc 900 aatgtacctc cacacagagg gcttctctgg
gccctctcca ggtgacgggg ccatgggcta 960 tggctatgag aaacctctgc
gaccattccc agatgatgtc tgcgttgtcc ctgagaaatt 1020 tgaaggagac
atcaagcagg aaggggtcgg tgcatttcga gaggggccgc cctaccagcg 1080
ccggggtgcc ctgcagctgt ggcaatttct ggtggccttg ctggatgacc caacaaatgc
1140 ccatttcatt gcctggacgg gccggggaat ggagttcaag ctcattgagc
ctgaggaggt 1200 cgccaggctc tggggcatcc agaagaaccg gccagccatg
aattacgaca agctgagccg 1260 ctcgctccga tactattatg agaaaggcat
catgcagaag gtggctggtg agcgttacgt 1320 gtacaagttt gtgtgtgagc
ccgaggccct cttctctttg gccttcccgg acaatcagcg 1380 tccagctctc
aaggctgagt ttgaccggcc tgtcagtgag gaggacacag tccctttgtc 1440
ccacttggat gagagccccg cctacctccc agagctggct ggccccgccc agccatttgg
1500 ccccaagggt ggctactctt actagccccc agcggctgtt ccccctgccg
caggtgggtg 1560 ctgccctgtg tacatataaa tgaatctggt gttggggaaa
ccttcatctg aaacccacag 1620 atgtctctgg ggcagatccc cactgtccta
ccagttgccc tagcccagac tctgagctgc 1680 tcaccggagt cattgggaag
gaaaagtgga gaaatggcaa gtctagagtc tcagaaactc 1740 ccctgggggt
ttcacctggg ccctggagga attcagctca gcttcttcct aggtccaagc 1800
cccccacacc ttttccccaa ccacagagaa caagagtttg ttctgttctg ggggacagag
1860 aaggcgcttc ccaacttcat actggcagga gggtgaggag gttcactgag
ctccccagat 1920 ctcccactgc ggggagacag aagcctggac tctgccccac
gctgtggccc tggagggtac 1980 cggtttgtca gttcttggtg ctctgtgttc
ccagaggcag gcggaggttg aagaaaggaa 2040 cctgggatga ggggtgctgg
gtataagcag agagggatgg gttcctgctc caagggaccc 2100 tttgcctttc
ttctgccctt tcctaggccc aggcctgggt ttgtacttcc acctccacca 2160
catctgccag accttaataa aggcccccac ttctcccaaa aaaaaaaaaa aa 2212
<210> SEQ ID NO 42 <211> LENGTH: 2667 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 42
tctgagaggc gaggccgggt gaggcggcga gggcggcccg acgggcgcgg gacgggacgg
60 ggcagcgagg gcgccgggag ccgcggcccg gaatcggggc gcttcgcccc
gggcccccca 120 gcatgaagac cccggcggac acagggtttg ccttcccgga
ttgggcctac aagccagagt 180 cgtcccctgg ctcaaggcag atccagctgt
ggcactttat cctggagctg ctgcggaagg 240 aggagtacca gggcgtcatt
gcctggcagg gggactacgg ggaattcgtc atcaaagacc 300 ctgatgaggt
ggcccggctg tggggcgttc gcaagtgcaa gccccagatg aattacgaca 360
agctgagccg ggccctgcgc tattactata acaagcgcat tctgcacaag accaagggga
420 aacggttcac ctacaagttc aatttcaaca aactggtgct ggtcaattac
ccattcattg 480 atgtggggtt ggctgggggt gcagtgcccc agagtgcccc
gccagtgccg tcgggtggta 540 gccacttccg cttccctccc tcaacgccct
ccgaggtgct gtcccccacc gaggaccccc 600 gctcaccacc agcctgctct
tcatcttcat cttccctctt ctcggctgtg gtggcccgcc 660 gcctgggccg
aggctcagtc agtgactgta gtgatggcac gtcagagctg gaggaaccgc 720
tgggagagga tccccgcgcc cgaccacccg gccctccgga tctgggtgcc ttccgagggc
780 ccccgctggc ccgcctgccc catgaccctg gtgtcttccg agtctatccc
cggcctcggg 840 gtggccctga acccctcagc cccttccctg tgtcgcctct
ggccggtcct ggatccctgc 900 tgccccctca gctctccccg gctctgccca
tgacgcccac ccacctggcc tacactccct 960 cgcccacgct gagcccgatg
taccccagtg gtggcggggg gcccagcggc tcagggggag 1020 gctcccactt
ctccttcagc cctgaggaca tgaaacggta cctgcaggcc cacacccaaa 1080
gcgtctacaa ctaccacctc agcccccgcg ccttcctgca ctaccctggg ctggtggtgc
1140 cccagcccca gcgccctgac aagtgcccgc tgccgcccat ggcacccgag
accccaccgg 1200 tcccctcctc ggcctcgtca tcctcttctt cttcttcctc
cccattcaag tttaagctcc 1260 agcggccccc actcggacgc cggcagcggg
cagctgggga gaaggccgta gccgctgctg 1320 acaagagcgg tggcagtgca
ggcgggctgg ctgagggggc aggggcgcta gccccaccgc 1380 ccccgccacc
acagatcaag gtggagccca tctcggaagg cgagtcggag gaggtagagg 1440
tgactgacat cagtgatgag gatgaggaag acggggaggt gttcaagacg ccccgtgccc
1500 cacctgcacc ccctaagcct gagcccggcg aggcacccgg ggcatcccag
tgcatgcccc 1560 tcaagctacg ctttaagcgg cgctggagtg aagactgtcg
cctcgaaggg ggtgggggcc 1620 ccgctggggg ctttgaggat gagggtgagg
acaagaaggt gcgtggggag gggcctgggg 1680 aggctggggg gcccctcacc
ccaaggcggg tgagctctga cctccagcat gccacggccc 1740 agctctccct
ggagcaccga gactcctgag ggctgtgggc aggggacctg tgtgccccgc 1800
accccccatg cttcttttgc tgccttaagc cccctatgcc ctggaggtga gggcagctct
1860 cttgtctctt ccctgcctcc tcccttttcc ctccccacat tttgtataaa
actttaattt 1920 ctttttttta aaaatggtgg gggtgggtgg gtgcccaggg
ctaggggcta ttccctgtct 1980 ctgtgggttt ctaagctctg ggcaaattgg
tggtaggggg agggaggggg aagttaaggg 2040 ggtcacctcc attctgggga
atttatattt gaattgaggc tttggcctta acacccagga 2100 acttttctat
tacaatcgct taggaagtaa agccttgtct ccctccctgt tctctgcctc 2160
ttgtacccct ctgacccacc cgctctgccc cactcccagc cctcctcagc cccagccctg
2220 cctgccctgc ccctccaggg ggccatgagt gcctaggttt ctcatacccc
acaaggtcac 2280 agcaggggag ggagggacaa ttttataatg aaccaaaaat
tccatgtgtt ggggggtggg 2340 gggcggagga gggtgagggg tgccgcccat
gggccacaaa tctctacaag tgcctgctat 2400 ccctctccca ctccccaccc
cagcaccggt ccaacccctt catccccagc tgctcctagg 2460 actggcccat
gggcaggcgg gtggggggat gggaaggggg tgccctgaaa ccaaactgga 2520
agccccctct gcctcccagc tggggcctct ggggtggggt ggggggctgt ggtcaagcct
2580 tattctgtat tggggactga gggtgggggg agtagagggg ccgctggaga
atgtattcaa 2640 aacaataaac tttggacctt tggaaaa 2667 <210> SEQ
ID NO 43 <211> LENGTH: 1364 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 43 aaaatcagga
acttgtgctg gccctgcaat gtcaagggag ggggctcacc cagggctcct 60
gtagctcagg gggcaggcct gagccctgca cccgccccac gaccgtccag cccctgacgg
120 gcaccccatc ctgaggggct ctgcattggc ccccaccgag gcaggggatc
tgaccgactc 180 ggagcccggc tggatgttac aggcgtgcaa aatggaaggg
tttcccctcg tcccccctcc 240 atcagaagac ctggtgccct atgacacgga
tctataccaa cgccaaacgc acgagtatta 300 cccctatctc agcagtgatg
gggagagcca tagcgaccat tactgggact tccaccccca 360 ccacgtgcac
agcgagttcg agagcttcgc cgagaacaac ttcacggagc tccagagcgt 420
gcagcccccg cagctgcagc agctctaccg ccacatggag ctggagcaga tgcacgtcct
480 cgataccccc atggtgccac cccatcccag tcttggccac caggtctcct
acctgccccg 540 gatgtgcctc cagtacccat ccctgtcccc agcccagccc
agctcagatg aggaggaggg 600 cgagcggcag agccccccac tggaggtgtc
tgacggcgag gcggatggcc tggagcccgg 660 gcctgggctc ctgcctgggg
agacaggcag caagaagaag atccgcctgt accagttcct 720 gttggacctg
ctccgcagcg gcgacatgaa ggacagcatc tggtgggtgg acaaggacaa 780
gggcaccttc cagttctcgt ccaagcacaa ggaggcgctg gcgcaccgct ggggcatcca
840 gaagggcaac cgcaagaaga tgacctacca gaagatggcg cgcgcgctgc
gcaactacgg 900 caagacgggc gaggtcaaga aggtgaagaa gaagctcacc
taccagttca gcggcgaagt 960 gctgggccgc gggggcctgg ccgagcggcg
ccacccgccc cactgagccc gcagcccccg 1020 ccggccccgc caggcctccc
cgctggccat agcattaagc cctcgcccgg cccggacaca 1080 gggaggacgc
tcccggggcc cagaggcagg actgtggcgg gccgggctcc gtcacccgcc 1140
cctcccccca ctccaggccc cctccacatc ccgcttcgcc tccctccagg actccacccc
1200 ggctcccgac gccagctggg cgtcagaccc accggcaacc ttgcagagga
cgacccgggg 1260 tactgccttg ggagtctcaa gtccgtatgt aaatcagatc
tcccctctca cccctcccac 1320 ccattaacct cctcccaaaa aacaagtaaa
gttattctca atcc 1364 <210> SEQ ID NO 44 <211> LENGTH:
3034 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 44 tttcttgtta aacaaacacc taatttattt
cttggaggtt ttgttcagct gtcctaattt 60 atgactttac attccttctg
gtgctaaact gctcaagtag cctcttgtat caagtgtgac 120 ctgattcctt
aagaatttta cttaatgaga acctctaagc tagaaactct tgctaggtgt 180
ttcatgcacc ttattttctt taatcattac aacaactcta agattgggtt ctctccacct
240 tataaatgat gactgtttta gagaggttaa ggttgcttaa aattggtgag
ttagtgaggg 300 gtagagccac gaatggattt ctggtcgctg cctccatcgt
cagggcaagc ttttcccacg 360 actccagcgc ttccatttgt cagtccccag
gctagaaagc cacagtgcta atttagtatt 420 tatcaagcgt ttgtagtgtc
ctgggatctg gcacttcgat gagaaagctg tgacggcccc 480 aacttctaac
agcgagtggt aaggaggacg agggacacag gagggaggag actctcccca 540
aagcttagca ccaacagaag tggtcccccg caggttgctc tgcgagcgcc acctcttccc
600 tccaaccgag gagaaagtgg cgcgcctttg aggagtccga ggtcccggcc
caggcggcag 660 cttgggtcct ggcgggttcc ggacgggcgc ctcagggacc
tggaagcaac cgcaccgaac 720 gcgacggaga gcggcgagac gactccagga
ggcgcccgag ctacatcccc cggccacacc 780 aaacccgggt ttgctggcag
acgcggctca cgacacccct tagggtcgca gcccctcccc 840 cggaagtgac
gtgtagcgac tacggcgtct gggagggacc caggagcagt cggggggttt 900
gagagtggcg gcggccgcgg agggcctggc aggccccgcc gctgcaagga acgccccgaa
960 cgcgcgcgcc cggcgtgtag cggccccaag acccgcgccg ccgctgccgc
gtgcgggggc 1020 ggggagggcg gggcgccagg agccgcggcg gcgggagatg
cgggcggctg cgggcacccg 1080 gcgggctcgg cttggccgcc gccgccttct
acggctccgc cgcgggggtc gcagcggctg 1140 ccgcgccgtc ctcgagtttc
cagcgtgagg aggaggctga gggcggagag gcgcatcgtg 1200 ttcgaggcgg
agaccgaggg ggagccccgc gcgcggcgtc gctcattgct atggacagtg 1260
ctatcaccct gtggcagttc cttcttcagc tcctgcagaa gcctcagaac aagcacatga
1320 tctgttggac ctctaatgat gggcagttta agcttttgca ggcagaagag
gtggctcgtc 1380 tctgggggat tcgcaagaac aagcctaaca tgaattatga
caaactcagc cgagccctca 1440 gatactatta tgtaaagaat atcatcaaaa
aagtgaatgg tcagaagttt gtgtacaagt 1500 ttgtctctta tccagagatt
ttgaacatgg atccaatgac agtgggcagg attgagggtg 1560 actgtgaaag
tttaaacttc agtgaagtca gcagcagttc caaagatgtg gagaatggag 1620
ggaaagataa accacctcag cctggtgcca agacctctag ccgcaatgac tacatacact
1680 ctggcttata ttcttcattt actctcaact ctttgaactc ctccaatgta
aagcttttca 1740 aattgataaa gactgagaat ccagccgaga aactggcaga
gaaaaaatct cctcaggagc 1800 ccacaccatc tgtcatcaaa tttgtcacga
caccttccaa aaagccaccg gttgaacctg 1860 ttgctgccac catttcaatt
ggcccaagta tttctccatc ttcagaagaa actatccaag 1920 ctttggagac
attggtttcc ccaaaactgc cttccctgga agccccaacc tctgcctcta 1980
acgtaatgac tgcttttgcc accacaccac ccatttcgtc cataccccct ttgcaggaac
2040 ctcccagaac accttcacca ccactgagtt ctcacccaga catcgacaca
gacattgatt 2100 cagtggcttc tcagccaatg gaacttccag agaatttgtc
actggagcct aaagaccagg 2160 attcagtctt gctagaaaag gacaaagtaa
ataattcatc aagatccaag aaacccaaag 2220 ggttagaact ggcacccacc
cttgtgatca cgagcagtga tccaagccca ctgggaatac 2280 tgagcccatc
tctccctaca gcttctctta caccagcatt tttttcacag acacccatca 2340
tactgactcc aagccccttg ctctccagta tccacttctg gagtactctc agtcctgttg
2400 ctcccctaag tccagccaga ctgcaaggtg ctaacacact tttccagttt
ccttctgtac 2460 tgaacagtca tgggccattc actctgtctg ggctggatgg
accttccacc cctggcccat 2520 tttccccaga cctacagaag acataaccta
tgcacttgtg gaatgagaga accgaggaac 2580 gaagaaacag acattcaaca
tgattgcatt tgaagtgagc aattgatagt tctacaatgc 2640 tgataataga
ctattgtgat ttttgccatt ccccattgaa aacatctttt taggattctc 2700
tttgaatagg actcaagttg gactatatgt ataaaaatgc cttaattgga gtctaaactc
2760 cacctccctc tgtcttttcc ttttcttttt ctttccttcc ttccttttct
tttctccttt 2820 aaaaatattt tgagctttgt gctgaagaag tttttggtgg
gctttagtga ctgtgctttg 2880 caaaagcaat taagaacaaa gttactcctt
ctggctattg ggaccctttg gccaggaaaa 2940 attatgctta gaatctatta
tttaaagaaa tatttgtgaa atgaaaaaaa aaaaaaaaaa 3000 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaa 3034 <210> SEQ ID NO 45
<211> LENGTH: 3077 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 45 tttcttgtta
aacaaacacc taatttattt cttggaggtt ttgttcagct gtcctaattt 60
atgactttac attccttctg gtgctaaact gctcaagtag cctcttgtat caagtgtgac
120 ctgattcctt aagaatttta cttaatgaga acctctaagc tagaaactct
tgctaggtgt 180 ttcatgcacc ttattttctt taatcattac aacaactcta
agattgggtt ctctccacct 240 tataaatgat gactgtttta gagaggttaa
ggttgcttaa aattggtgag ttagtgaggg 300 gtagagccac gaatggattt
ctggtcgctg cctccatcgt cagggcaagc ttttcccacg 360 actccagcgc
ttccatttgt cagtccccag gctagaaagc cacagtgcta atttagtatt 420
tatcaagcgt ttgtagtgtc ctgggatctg gcacttcgat gagaaagctg tgacggcccc
480 aacttctaac agcgagtggt aaggaggacg agggacacag gagggaggag
actctcccca 540 aagcttagca ccaacagaag tggtcccccg caggttgctc
tgcgagcgcc acctcttccc 600 tccaaccgag gagaaagtgg cgcgcctttg
aggagtccga ggtcccggcc caggcggcag 660 cttgggtcct ggcgggttcc
ggacgggcgc ctcagggacc tggaagcaac cgcaccgaac 720 gcgacggaga
gcggcgagac gactccagga ggcgcccgag ctacatcccc cggccacacc 780
aaacccgggt ttgctggcag acgcggctca cgacacccct tagggtcgca gcccctcccc
840 cggaagtgac gtgtagcgac tacggcgtct gggagggacc caggagcagt
cggggggttt 900 gagagtggcg gcggccgcgg agggcctggc aggccccgcc
gctgcaagga acgccccgaa 960 cgcgcgcgcc cggcgtgtag cggccccaag
acccgcgccg ccgctgccgc gtgcgggggc 1020 ggggagggcg gggcgccagg
agccgcggcg gcgggagatg cgggcggctg cgggcacccg 1080 gcgggctcgg
cttggccgcc gccgccttct acggctccgc cgcgggggtc gcagcggctg 1140
ccgcgccgtc ctcgagtttc cagcgtgagg aggaggctga gggcggagag gcgcatcgtg
1200 ttcgaggcgg agaccgaggg ggagccccgc gcgcggcgtc gctcattgct
atggacagtg 1260 ctatcaccct gtggcagttc cttcttcagc tcctgcagaa
gcctcagaac aagcacatga 1320 tctgttggac ctctaatgat gggcagttta
agcttttgca ggcagaagag gtggctcgtc 1380 tctgggggat tcgcaagaac
aagcctaaca tgaattatga caaactcagc cgagccctca 1440 gatactatta
tgtaaagaat atcatcaaaa aagtgaatgg tcagaagttt gtgtacaagt 1500
ttgtctctta tccagagatt ttgaacatgg atccaatgac agtgggcagg attgagggtg
1560 actgtgaaag tttaaacttc agtgaagtca gcagcagttc caaagatgtg
gagaatggag 1620 ggaaagataa accacctcag cctggtgcca agacctctag
ccgcaatgac tacatacact 1680 ctggcttata ttcttcattt actctcaact
ctttgaactc ctccaatgta aagcttttca 1740 aattgataaa gactgagaat
ccagccgaga aactggcaga gaaaaaatct cctcaggagc 1800 ccacaccatc
tgtcatcaaa tttgtcacga caccttccaa aaagccaccg gttgaacctg 1860
ttgctgccac catttcaatt ggcccaagta tttctccatc ttcagaagaa actatccaag
1920 ctttggagac attggtttcc ccaaaactgc cttccctgga agccccaacc
tctgcctcta 1980 acgtaatgac tgcttttgcc accacaccac ccatttcgtc
cataccccct ttgcaggaac 2040 ctcccagaac accttcacca ccactgagtt
ctcacccaga catcgacaca gacattgatt 2100 cagtggcttc tcagccaatg
gaacttccag agaatttgtc actggagcct aaagaccagg 2160 attcagtctt
gctagaaaag gacaaagtaa ataattcatc aagatccaag aaacccaaag 2220
ggttagaact ggcacccacc cttgtgatca cgagcagtga tccaagccca ctgggaatac
2280 tgagcccatc tctccctaca gcttctctta caccagcatt tttttcacag
gtagcttgct 2340 cgctctttat ggtgtcacca ttgctttcat ttatttgccc
ttttaagcaa atccagaatt 2400 tatacactca agtttgcttt ctgttactta
ggtttgtctt agaaaggtta tgtgtgactg 2460 tcatgtgaaa gttaccccat
ttctcatctt aattaggatt gctaaaatag aaagtttgga 2520 gtattttctt
aaaaaattca ttgttctaca agtaaataaa tattttgatt tttctatttc 2580
ctcctaaaga aagtacacac actctctcgc tctctctcgg tcttataaaa ctcgttggtg
2640 tcttataaaa caaacagtga taatctcaag ttagaaaaca gtaggtcctg
agaaccataa 2700 gaaaaatgac tggtgtgatg ttgagtaaca agttggtaca
gttactttag ctatttatta 2760 acttgctcat ctcatagaac attttagtag
atttttcaca cacctcatta ttaaaaaaaa 2820 acaaacatgc tggtgtcttg
gttacccatt attcctctgt acctgaattc aggttggttt 2880 ttctatttgg
aaaagacttt ataaatgttg gcttaaaaag aggttgagca ccagaatctc 2940
agaatttacc accaaagaac tcatccatgt aaccaaaaac cacttgtacc cccaaaaact
3000 attgaaataa aaatttaaaa aattttaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 3060 aaaaaaaaaa aaaaaaa 3077 <210> SEQ ID NO 46
<211> LENGTH: 1443 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 46 ggggggggtg
gatgaggagg agccggagac gccgcggagg agaccggacc gaagacggac 60
cgtgccggga agagcaggcg ggtgaaaatg aaagccggct gtagcatcgt ggaaaagcca
120 gaaggaggtg gagggtatca gtttcctgac tgggcctaca aaacagagtc
atccccaggc 180 tcccggcaga tccagctgtg gcacttcatc ctggagctgc
tgcagaagga agagttccgc 240 catgtcatcg cctggcagca gggagagtac
ggggaatttg tcatcaagga tccagatgag 300 gtggcccgcc tctggggccg
caggaaatgc aaaccacaga tgaattatga caagctgagc 360 cgggccctca
gatactatta caacaagagg atccttcata aaacaaaagg gaaaagattt 420
acctataaat ttaacttcaa caagctggtg atgcccaact acccattcat caacattcgg
480 tcaagtggta agatacaaac tcttttggta gggaattaat tttgaattga
aaagaatttt 540 taaaaatcca aatctaagac atggcatgtt taggaagatt
ttagaaacac taaaataatg 600 tgatcctttg gattgcctca atgttcttac
tcaagtcatc tcacttataa ggagagttat 660 aggctattca gtatcaagat
agatttcttt ggtttatttg gttggttccc ttttctgcat 720 attgtttgta
atctcctaga tactattacg ctatcttgtt tgggaatgat gtttcatagg 780
tttgtgatga tctttacgtt caggactcag ttttaacacc cagcccagtg gttctttcat
840 agatgggaac ctgtttctac aaacacttcc gattttctgt gaaactacca
agctctccct 900 tatcaagtga atatcatcaa aaccacagca tccttgatca
gagaaggggg aggttcacat 960 gtttgcagtg aaaagcagtg tctttgatct
gcaacagcaa atcctcagag aaaaagattc 1020 tggggttact tgaccttctc
tcctgttaag tgcagtaggg cttcccctct tgactttcct 1080 ggttatagct
ttccatcaca gctccccaca ttctctcttg atgttgaaag cagtctctca 1140
aaagactttg ttgttgtgtg gttttttgtt tgtgattttt ttccttatgc aaatcatact
1200 cctgcccaag aaaatacagt agttcccctt atctgagcag tatatgttct
aagaccccta 1260 gtagattcgc aaaccacaga tagtaccaaa ctccattcat
atatatgatg ttttttcttc 1320 ccttaacccc actcatatgt atctgtgata
acgtttaatt tataaattag gcacagtaag 1380 agattaatga caataataaa
atagaaaaat tataaaaaaa aaaaaaaaaa aaaaaaaaaa 1440 aaa 1443
<210> SEQ ID NO 47 <211> LENGTH: 229 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 47
cccactagtt acccacccca aactggatcc tacagccaag ctccaagtca atatagccaa
60 cagagcagca gctacgggca gcagaatccg tatcagatcc tgggcccgac
cagcagtcgc 120 ctagccaacc ctggaagcgg gcagatccag ctgtggcaat
tcctcctgga gctgctctcc 180 gacagcgcca acgccagctg tatcacctgg
gaggggacca acggggagt 229 <210> SEQ ID NO 48 <211>
LENGTH: 2319 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 48 cacaaggcta caggtgtctt tatttccact
gcacgctggt gctgggagcg cctgccttct 60 cttgccttga aagcctcctc
tttggaccta gccaccgctg ccctcacggt aatgttggac 120 tcggtgacac
acagcacctt cctgcctaat gcatccttct gcgatcccct gatgtcgtgg 180
actgatctgt tcagcaatga agagtactac cctgcctttg agcatcagac agcctgtgac
240 tcatactgga catcagtcca ccctgaatac tggactaagc gccatgtgtg
ggagtggctc 300 cagttctgct gcgaccagta caagttggac accaattgca
tctccttctg caacttcaac 360 atcagtggcc tgcagctgtg cagcatgaca
caggaggagt tcgtcgaggc agctggcctc 420 tgcggcgagt acctgtactt
catcctccag aacatccgca cacaaggtta ctcctttttt 480 aatgacgctg
aagaaagcaa ggccaccatc aaagactatg ctgattccaa ctgcttgaaa 540
acaagtggca tcaaaagtca agactgtcac agtcatagta gaacaagcct ccaaagttct
600 catctatggg aatttgtacg agacctgctt ctatctcctg aagaaaactg
tggcattctg 660 gaatgggaag atagggaaca aggaattttt cgggtggtta
aatcggaagc cctggcaaag 720 atgtggggac aaaggaagaa aaatgacaga
atgacatatg aaaagttgag cagagccctg 780 agatactact ataaaacagg
aattttggag cgggttgacc gaaggttagt gtacaaattt 840 ggaaaaaatg
cacacgggtg gcaggaagac aagctatgat ctgctccagg catcaagctc 900
attttatgga tttctgtctt ttaaaacaat cagattgcaa tagacattcg aaaggcttca
960 ttttcttctc tttttttttt aacctgcaaa catgctgata aaatttctcc
acatctcagc 1020 ttacatttgg attcagagtt gttgtctacg gagggtgaga
gcagaaactc ttaagaaatc 1080 ctttcttctc cctaagggga tgaggggatg
atcttttgtg gtgtcttgat caaactttat 1140 tttcctagag ttgtggaatg
acaacagccc atgccattga tgctgatcag agaaaaacta 1200 ttcaattctg
ccattagaga cacatccaat gctcccatcc caaaggttca aaagttttca 1260
aataactgtg gcagctcacc aaaggtgggg gaaagcatga ttagtttgca ggttatggta
1320 ggagagggtg agatataaga catacatact ttagatttta aattattaaa
gtcaaaaatc 1380 catagaaaag tatccctttt tttttttttt gagacgggtt
ctcactatgt tgcccagggc 1440 tggtcttgaa ctcctatgct caagtgatcc
tcccacctcg gcctcccaaa gtactgtgat 1500 tacaagcgtg agccacggca
cctgggcaga aaagtatctt aattaatgaa agagctaagc 1560 catcaagctg
ggacttaatt ggatttaaca taggttcaca gaaagtttcc taaccagagc 1620
atctttttga ccactcagca aaacttccac agacatcctt ctggacttaa acacttaaca
1680 ttaaccacat tattaattgt tgctgagttt attccccctt ctaactgatg
gctggcatct 1740 gatatgcaga gttagtcaac agacactggc atcaattaca
aaatcactgc tgtttctgtg 1800 attcaagctg tcaacacaat aaaatcgaaa
ttcattgatt ccatctctgg tccagatgtt 1860 aaacgtttat aaaaccggaa
atgtcctaac aactctgtaa tggcaaatta aattgtgtgt 1920 cttttttgtt
ttgtctttct acctgatgtg tattcaagtg ctataacacg tatttccttg 1980
acaaaaatag tgacagtgaa ttcacactaa taaatgttca taggttaaag tctgcactga
2040 cattttctca tcaatcactg gtatgtaagt tatcagtgac tgacagctag
gtggactgcc 2100 cctaggactt ctgtttcacc agagcaggaa tcaagtggtg
aggcactgaa tcgctgtaca 2160 ggctgaagac ctccttatta gagttgaact
tcaaagtaac ttgttttaaa aaatgtgaat 2220 tactgtaaaa taatctattt
tggattcatg tgttttccag gtggatatag tttgtaaaca 2280 atgtgaataa
agtatttaac atgtaaaaaa aaaaaaaaa 2319 <210> SEQ ID NO 49
<211> LENGTH: 3617 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 49 acccgtggtg
ccccatccct ataggagctg gtgagattgc agcctgctgc ctcccctcca 60
tcagccacag ctattggatt tcccacccag aatctttagg taaatgagat catgattctg
120 gaaggaggtg gtgtaatgaa tctcaacccc ggcaacaacc tccttcacca
gccgccagcc 180 tggacagaca gctactccac gtgcaatgtt tccagtgggt
tttttggagg ccagtggcat 240 gaaattcatc ctcagtactg gaccaagtac
caggtgtggg agtggctcca gcacctcctg 300 gacaccaacc agctggatgc
caattgtatc cctttccaag agttcgacat caacggcgag 360 cacctctgca
gcatgagttt gcaggagttc acccgggcgg cagggacggc ggggcagctc 420
ctctacagca acttgcagca tctgaagtgg aacggccagt gcagtagtga cctgttccag
480 tccacacaca atgtcattgt caagactgaa caaactgagc cttccatcat
gaacacctgg 540 aaagacgaga actatttata tgacaccaac tatggtagca
cagtagattt gttggacagc 600 aaaactttct gccgggctca gatctccatg
acaaccacca gtcaccttcc tgttgcagag 660 tcacctgata tgaaaaagga
gcaagacccc cctgccaagt gccacaccaa aaagcacaac 720 ccgagaggga
ctcacttatg ggaattcatc cgcgacatcc tcttgaaccc agacaagaac 780
ccaggattaa taaaatggga agaccgatct gagggcgtct tcaggttctt gaaatcagag
840 gcagtggctc agctatgggg taaaaagaag aacaacagca gcatgaccta
tgaaaagctc 900 agccgagcta tgagatatta ctacaaaaga gaaattctgg
agcgtgtgga tggacgaaga 960 ctggtatata aatttgggaa gaatgcccga
ggatggagag aaaatgaaaa ctgaagctgc 1020 caatactttg gacacaaacc
aaaacacaca ccaaataatc agaaacaaag aactcctgga 1080 cgtaaatatt
tcaaagacta cttttctctg atatttatgt accatgaggg gaacaagaaa 1140
ctacttctaa cgggaagaag aaacactaca gtcgattaaa aaaattattt tgttacttcg
1200 aagtatgtcc tatatgggga aaaaacgtac acagttttct gtgaaatatg
atgctgtatg 1260 tggttgtgat tttttttcac ctctattgtg aattcttttt
cactgcaaga gtaacaggat 1320 ttgtagcctt gtgcttcttg ctaagagaaa
gaaaaacaaa atcagagggc attaaatgtt 1380 ttgtatgtga catgatttag
aaaaaggtga tgcatcctcc tcacataagc atccatatgg 1440 cttcgtcaag
ggaggtgaac attgttgctg agttaaattc cagggtctca gatggttagg 1500
acaaagtgga tggatgccgg gaagtttaac ctgagcctta ggatccaatg agtggagaat
1560 ggggacttcc aaaacccaag gttggctata atctctgcat aaccacatga
cttggaatgc 1620 ttaaatcagc aagaagaata atggtggggt ctttatactc
attcaggaat ggtttatctg 1680 atgccagggc tgtcttcctt tctccccttt
ggatggttgg tgaaatactt taattgccct 1740 gtctgctcac ttctagctat
ttaagagaga acccagcttg gttctttttt gctccaagtg 1800 cttaaaaata
agttggaaaa aggagacggt ggtgtggaaa tggctgaaga gtttgctctt 1860
gtatccctat agtccaaggt ttctcaatct gcacaattga catttttggc cggagtgttc
1920 tttgtggtga gggctttcct gtgcattgta agatgttcag cagtatccac
tcatggtctc 1980 taaccacttg acaccagaaa ccccccagct gtgataacgc
aaaatgtctc tagacatcac 2040 caaatgttcc ctgggggtgg caaatttgcc
cttgattgag aaccaccagt ttagctagtc 2100 aatatgagga tggtggttta
ttctcagaag aaaaagatat gtaaggtctt ttagctcctt 2160 agagtgaagc
aaaagcaaga cttcaacctc aacctatctt tatgttttaa atgttaggga 2220
caataagttg aaatagctag aggagcttct tttcagaacc ccagatgaga gccaatgtca
2280 gataaagtaa gcatagtaat gtagcaggaa ctacaataga agacattttc
actggaatta 2340 caaagcagaa ttaaaattat attgtagaag gaaacaccaa
gaaaagaatt tccagggaaa 2400 atcctctttg caggtattaa ttcttataat
tttttgtctt ttggattatc tgtttactgt 2460 ctcatctgaa ctgatcccag
gtgaacggtt tattgcctag atttgtactc agaggaattt 2520 tttttgtttt
gttttgtctt ttaagaaagg aaagaaagga tgaaaaaaat aaacagaaaa 2580
ctcagctcag gcacaattgt caccaaggag ttaaaagctt cttcttcaat agaggaattg
2640 ttctgggggt cctggagact taccattgag ccatgcaatc tgggaagcac
aggaataagt 2700 agacactttg aaaatggatt tgaatgttct catccctttt
gcagcttttc tttttggctc 2760 tctcatgtcc ttggcttgct cctctattct
acctctcttt ctccagcaat aatatgcaaa 2820 tgaagacatg tatccataag
aaggagtgct cttcatcaac taatagagca cctaccacag 2880 tgtcatacct
ggtagaggtg agcaattcat attcaaaggt tgcaaagtgt ttgtaatata 2940
ttcatgaggc tggaagtaag aagaattaaa aatttgtcct aattacaatg agaaccattc
3000 taggtagtga tcttggagca cacatgaata actttctgaa ggtgcaacca
aatccatttt 3060 tatttctgcc tggcttggtc acctctgtaa aggtttaact
tagtgttgtc aagtaacagt 3120 tactgaaaga gctgagaaaa agaacaatga
acagcaacga tcttgactgt gcaactcaga 3180 cattcctgca gaaaagacat
atgttgcttt acaagaaggc caaagaacta tggggccttc 3240 ccagcatttg
actgttcatt gcatagaatg aattaaatat ccagttactt gaatgggtat 3300
aacgcatgaa tatttgtgtg tctgtgtgtg tgtctgagtt gtgtgatttt attaggggca
3360 tctgccaatt ctctcactgt ggttccttct ctgactttgc ctgttcatca
tctaaggagg 3420 ctagatcctt cgctgacttc accattcctc aaacctgtaa
gtttctcact tcttccaaat 3480 tggctttggc tctttctgca acctttccat
tcaagagcaa tctttgctaa ggagtaagtg 3540 aatgtgaaga gtaccaacta
caacaattct acagataatt agtggattgt gttgtttgtt 3600 gagagtgaag gtttctt
3617 <210> SEQ ID NO 50 <211> LENGTH: 1894 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
50 gtctgacttc ctcccagcac attcctgcac tctgccgtgt ccacactgcc
ccacagaccc 60 agtcctccaa gcctgctgcc agctccctgc aagcccctca
ggttgggcct tgccacggtg 120 ccagcaggca gccctgggct gggggtaggg
gactccctac aggcacgcag ccctgagacc 180 tcagagggcc accccttgag
ggtggccagg cccccagtgg ccaacctgag tgctgcctct 240 gccaccagcc
ctgctggccc ctggttccgc tggcccccca gatgcctggc tgagacacgc 300
cagtggcctc agctgcccac acctcttccc ggcccctgaa gttggcactg cagcagacag
360 ctccctgggc accaggcagc taacagacac agccgccagc ccaaacagca
gcggcatggg 420 cagcgccagc ccgggtctga gcagcgtatc ccccagccac
ctcctgctgc cccccgacac 480 ggtgtcgcgg acaggcttgg agaaggcggc
agcgggggca gtgggtctcg agagacggga 540 ctggagtccc agtccacccg
ccacgcccga gcagggcctg tccgccttct acctctccta 600 ctttgacatg
ctgtaccctg aggacagcag ctgggcagcc aaggcccctg gggccagcag 660
tcgggaggag ccacctgagg agcctgagca gtgcccggtc attgacagcc aagccccagc
720 gggcagcctg gacttggtgc ccggcgggct gaccttggag gagcactcgc
tggagcaggt 780 gcagtccatg gtggtgggcg aagtgctcaa ggacatcgag
acggcctgca agctgctcaa 840 catcaccgca gatcccatgg actggagccc
cagcaatgtg cagaagtggc tcctgtggac 900 agagcaccaa taccggctgc
cccccatggg caaggccttc caggagctgg cgggcaagga 960 gctgtgcgcc
atgtcggagg agcagttccg ccagcgctcg cccctgggtg gggatgtgct 1020
gcacgcccac ctggacatct ggaagtcagc ggcctggatg aaagagcgga cttcacctgg
1080 ggcgattcac tactgtgcct cgaccagtga ggagagctgg accgacagcg
aggtggactc 1140 atcatgctcc gggcagccca tccacctgtg gcagttcctc
aaggagttgc tactcaagcc 1200 ccacagctat ggccgcttca ttaggtggct
caacaaggag aagggcatct tcaaaattga 1260 ggactcagcc caggtggccc
ggctgtgggg catccgcaag aaccgtcccg ccatgaacta 1320 cgacaagctg
agccgctcca tccgccagta ttacaagaag ggcatcatcc ggaagccaga 1380
catctcccag cgcctcgtct accagttcgt gcaccccatc tgagtgcctg gcccagggcc
1440 tgaaacccgc cctcaggggc ctctctcctg cctgccctgc ctcagccagg
ccctgagatg 1500 ggggaaaacg ggcagtctgc tctgctgctc tgaccttcca
gagcccaagg tcagggaggg 1560 gcaaccaact gccccagggg gatatgggtc
ctctggggcc ttcgggacca tggggcaggg 1620 gtgcttcctc ctcaggccca
gctgctcccc tggaggacag agggagacag ggctgctccc 1680 caacacctgc
ctctgacccc agcatttcca gagcagagcc tacagaaggg cagtgactcg 1740
acaaaggcca caggcagtcc aggcctctct ctgctccatc cccctgcctc ccattctgca
1800 ccacacctgg catggtgcag ggagacatct gcacccctga gttgggcagc
caggagtgcc 1860 cccgggaatg gataataaag atactagaga actg 1894
<210> SEQ ID NO 51 <211> LENGTH: 2180 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 51
gggcggaaaa gcctgtttac acagactgca caccgcctgg ggaataatgc agtaaaggaa
60 gtgagccggc tcggcctgac tgctccaact tcctgctctc acacacacca
gaggggaaaa 120 aaaaagagga gcgagagaaa gaaaaaaagg gggaaaaatc
aggatctcat tacaagagcc 180 acagaccgtc tgcagacgcc tgtcagcatg
gaaagtcggg ggctttcgcc cgggtcctcc 240 tagaaattcc ccccgaagaa
gactccccca catctgggta tggagagtgc aatcacgctg 300 tggcagttcc
tgttgcagtt gctgctggat cagaaacatg agcatttgat ctgctggacc 360
tcgaacgatg gtgaattcaa gctcctcaaa gcagaagaag tggccaagct gtggggactc
420 cgaaaaaaca aaacaaatat gaactatgat aagctgagca gagccctgcg
atactattat 480 gacaagaaca tcatcaagaa ggtgatcggg cagaagtttg
tgtacaagtt tgtctctttc 540 ccggagatcc tgaagatgga tcctcacgcg
gtggagatca gccgggagag ccttctgctg 600 caggacagcg actgcaaggc
gtctccggag ggccgcgagg cccacaaaca cggcctggcc 660 gccctcagaa
gcacgagccg caacgaatac atccactcag gcctgtactc gtccttcacc 720
attaattccc tgcagaaccc accagacgcc ttcaaggcca tcaagacgga gaagctggag
780 gagccgcccg aagacagccc ccccgtggaa gaagtcagga ctgtgatcag
gtttgtgacc 840 aataaaaccg acaagcacgt caccaggccg gtggtgtccc
tgccttccac gtcagaggct 900 gcggcggcgt ccgccttcct ggcctcgtcc
gtctcggcca agatctcctc tttaatgttg 960 ccaaacgctg ccagtatttc
atccgcctca cccttctcat ctcggtcccc gtccctgtcc 1020 cccaactcac
ccctcccttc tgaacacaga agcctcttcc tggaggccgc ctgccatgac 1080
tccgattccc tggagccctt gaacctgtca tcgggctcca agaccaagtc tccatctctt
1140 cccccaaagg ccaaaaaacc caaaggcttg gaaatctcag cgcccccgct
ggtgctctcc 1200 ggcaccgaca tcggctccat cgccctcaac agcccagccc
tcccctcggg atccctcacc 1260 ccagccttct tcaccgcaca gacaccaaat
ggattgcttc tgactccgag tccactgctc 1320 tccagcatac atttctggag
cagccttagt ccagttgctc cgctgagtcc tgccaggctg 1380 caagggccaa
gcacgctgtt ccagttcccc acactgctta atggccacat gccagtgcca 1440
atccccagtc tggacagagc tgcttctcca gtactgcttt cttcaaactc tcagaaatcc
1500 tgatgacgtc tggccacaat taaggactca ttaactgatg aaacaaattt
gtccccacgg 1560 gctagtttac ctgtgtcgtg agaaggacat tgtgaaactc
ttgttaattt ggtttgcact 1620 tttcataaca tggatagtct agatttatgt
tagcatttta aaaactgttt ttgatatatt 1680 caagtatata tgaaaatctg
tttggcatta agtgaatttt aatgtttttg tttttatatc 1740 cttttagctc
ttaagtgttg aacactgttg acagtgaaga acttttctta atggttttca 1800
gtataactaa taaggatgtg aagctttttt ctctttagtt ctgagtatgc taaactgtgt
1860 gcttatatag actataacca gttgtgcctt cctttgcatt taatgtaaat
gaatgattta 1920 tatatttttt agtattaaga ggaaatgttt gaaagatgaa
aattagtatc aaacagctct 1980 ctagtagaat ttcattattt ttcaccagtg
ggcaatatga aagcatatat cacgttttgt 2040 tttactttca attgtataag
aattgcctta gaacctcttt tgaactgaaa ttcagtaaat 2100 gtccaagtaa
tgtttttata ataaactaag ccatatttag acaataaaca tcgaaaaaaa 2160
aaaaaaaaaa aaaaaaaaaa 2180 <210> SEQ ID NO 52 <211>
LENGTH: 3171 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 52 gttgccagct gcggcggccg ccacagccac
agccgccgcc gccgccgccg ccgcccctgc 60 ccctgccgcc cctgcccctg
ccgttaggtg gtggggtttc tcagcccggc ggcgggaggc 120 gggccggcct
cggcttcctg tcggaggacg cgcaaggatc cgggcgtcgg agtgtgtgcg 180
agtgcgtgag tgtgtgtcgg tcgcacggcg tgtgtctccg gccgcgggtt ccgcctcctc
240 ccctgccgcc gctgctcacg gtgtaagtca atgtgaagca gcagctccag
ccccgggata 300 aacatggcga cgtctctgca tgagggaccc acgaaccagc
tggatctgct catccgggcc 360 gtggaagcat cagttcacag cagtaatgca
cactgtacag ataagacaat tgaagctgct 420 gaagccctgc ttcatatgga
atctcctacc tgcttgaggg attcaagaag tcctgtggaa 480 gtgtttgttc
ctccttgtgt atcaactcca gaattcatcc atgctgctat gaggccagat 540
gtcattacag aaactgtagt ggaggtgtca actgaagagt ctgaacccat ggatacctct
600 cctattccaa catcaccaga tagccatgaa ccaatgaaaa agaaaaaagt
tggccgtaaa 660 ccaaagaccc agcaatcacc aatttccaat gggtctcctg
agttaggtat aaagaagaaa 720 ccaagagaag gaaaaggaaa cacaacctat
ttgtgggagt ttcttttaga tctacttcaa 780 gataaaaata cttgtcccag
gtatattaaa tggactcaga gagaaaaagg catattcaag 840 ctggtggatt
caaaggctgt ctctaagctt tggggaaagc ataagaacaa accagacatg 900
aactatgaaa ccatgggacg agctttgaga tactactacc aaaggggaat tcttgcaaag
960 gttgaaggac agaggcttgt atatcagttc aaggatatgc cgaaaaacat
agtggtcata 1020 gatgatgaca aaagtgaaac ctgtaatgaa gatttagcag
gaactactga tgaaaaatca 1080 ttagaacgag tgtcactgtc tgcagaaagt
ctcctgaaag cagcatcctc tgttcgcagt 1140 ggaaaaaatt catcccctat
aaactgctcc agagcagaga agggtgtagc tagagttgtg 1200 aatatcactt
cccctgggca cgatgcttca tccaggtctc ctactaccac tgcatctgtg 1260
tcagcaacag cagctccaag gacagttcgt gtggcaatgc aggtacctgt tgtaatgaca
1320 tcattgggtc agaaaatttc aactgtggca gttcagtcag ttaatgcagg
tgcaccatta 1380 ataaccagca ctagtccaac aacagcgacc tctccaaagg
tagtcattca gacaatccct 1440 actgtgatgc cagcttctac tgaaaatgga
gacaaaatca ccatgcagcc tgccaaaatt 1500 attaccatcc cagctacaca
gcttgcacag tgtcaactgc agacaaagtc aaatctgact 1560 ggatcaggaa
gcattaacat tgttggaacc ccattggctg tgagagcact tacccctgtt 1620
tcaatagccc atggtacacc tgtaatgaga ctatcaatgc ctactcagca ggcatctggc
1680 cagactcctc ctcgagttat cagtgcagtc ataaaggggc cagaggttaa
atcggaagca 1740 gtggcaaaaa agcaagaaca tgatgtgaaa actttgcagc
tagtagaaga aaaaccagca 1800 gatggaaata agacagtgac ccacgtagtg
gttgtcagtg cgccttcagc tattgccctt 1860 cctgtaacta tgaaaacaga
aggactagtg acatgtgaga aataaaatag cagctccacc 1920 atggacttca
ggctgttagt ggcagtactg acataaacat ttgcaaggga agtcatcaag 1980
aaaagtcaaa gaagacttta aaacattttt aatgcatata caaaaacaat cagacttact
2040 ggaaataaat tacctatccc atgtttcagt gggaaatgaa ctacatattg
agatgctgac 2100 agaaaactgc ctcttacagt aggaaacaac tgaacccatc
aataagaaaa aggatcgaaa 2160 gggaccaagc agctcactac gatatcaagt
tacactaaga cttggaacac taacattctg 2220 taagaggtta tatagttttc
agtgggaggg gttgggatgg gtaatctcat tgttacatat 2280 agcaattttt
gatgcatttt atatgcatac cagcaattat tactgtgttc gcacagttct 2340
cacttaactg gtgctatgtg aagactctgc taatataggt attttagaat gtgaattgaa
2400 gaatggatcc caaaaacttc agaaagagga tagcaaaaaa agatctagtg
cgattttata 2460 tatatatata tatatatata catacatata tatatatcat
atagcttaag ctgatttaaa 2520 acaaaggcct tagactaatt ttcgattttc
tttcttgaaa taagctaatg gcttgtttgt 2580 gtaaagcttt tttattaaaa
gaaaaatttt aaaaatcttg tacctagcac agtattgtta 2640 tagaatatac
atgtaacatt ttatatggta gtttaagtct gtcagtttct taattgtgga 2700
caaattaaca gttggctctg gccttttgct gtaacatgcc tgtgtcactc acttagcctt
2760 ggcatttgtg cagacatacc attttcagtt ctgctgtcac ttggaagttc
aggctcagca 2820 tgaatttttg gcaggtagct ctaatacctg gagttttctt
tgtttttttt tctttttttt 2880 agttgaagtt tatgagggaa ataccagtgt
tcagttttga actataatag tttgtatatt 2940 caacatttga agtatattct
attttgttgt actcttgttt caaagtgtat tcaagtaggt 3000 tttctgaaat
atagaaatga aatttatctt ctgttttggt ctctggtgat attttaaaca 3060
atatttaaaa gtcagtatag aagtgtttta gttaggaagt gataaaacat ctctcttctc
3120 cttcccaact actgcatgaa gaaattctac ttccattata ttaatatttg g 3171
<210> SEQ ID NO 53 <211> LENGTH: 3218 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 53
aaaatagtga aggatgctta gactacttaa catacaaact gctttctggt taatcatctt
60 tagaagactg gatttctgga tatctactcc actccatctc tattgacttt
taaaacatga 120 taatgcaaac ctataacact ggcaaccatc agtgaacctt
taatttcatt gattaatagc 180 gtttgaagct tcctcaggga ataacaatga
catcagcagt ggttgacagt ggaggtacta 240 ttttggagct ttccagcaat
ggagtagaaa atcaagagga aagtgaaaag gtttctgaat 300 atccagcagt
gattgtggag ccagttccaa gtgccagatt agagcagggc tatgcagccc 360
aggttctggt ttatgatgat gagacttata tgatgcaaga tgtggcagaa gaacaagaag
420 ttgagaccga gaatgtggaa acagtggaag catcagttca cagcagtaat
gcacactgta 480 cagataagac aattgaagct gctgaagccc tgcttcatat
ggaatctcct acctgcttga 540 gggattcaag aagtcctgaa ttcatccatg
ctgctatgag gccagatgtc attacagaaa 600 ctgtagtgga ggtgtcaact
gaagagtctg aacccatgga tacctctcct attccaacat 660 caccagatag
ccatgaacca atgaaaaaga aaaaagttgg ccgtaaacca aagacccagc 720
aatcaccaat ttccaatggg tctcctgagt taggtataaa gaagaaacca agagaaggaa
780 aaggaaacac aacctatttg tgggagtttc ttttagatct acttcaagat
aaaaatactt 840 gtcccaggta tattaaatgg actcagagag aaaaaggcat
attcaagctg gtggattcaa 900 aggctgtctc taagctttgg ggaaagcata
agaacaaacc agacatgaac tatgaaacca 960 tgggacgagc tttgagatac
tactaccaaa ggggaattct tgcaaaggtt gaaggacaga 1020 ggcttgtata
tcagttcaag gatatgccga aaaacatagt ggtcatagat gatgacaaaa 1080
gtgaaacctg taatgaagat ttagcaggaa ctactgatga aaaatcatta gaacgagtgt
1140 cactgtctgc agaaagtctc ctgaaagcag catcctctgt tcgcagtgga
aaaaattcat 1200 cccctataaa ctgctccaga gcagagaagg gtgtagctag
agttgtgaat atcacttccc 1260 ctgggcacga tgcttcatcc aggtctccta
ctaccactgc atctgtgtca gcaacagcag 1320 ctccaaggac agttcgtgtg
gcaatgcagg tacctgttgt aatgacatca ttgggtcaga 1380 aaatttcaac
tgtggcagtt cagtcagtta atgcaggtgc accattaata accagcacta 1440
gtccaacaac agcgacctct ccaaaggtag tcattcagac aatccctact gtgatgccag
1500 cttctactga aaatggagac aaaatcacca tgcagcctgc caaaattatt
accatcccag 1560 ctacacagct tgcacagtgt caactgcaga caaagtcaaa
tctgactgga tcaggaagca 1620 ttaacattgt tggaacccca ttggctgtga
gagcacttac ccctgtttca atagcccatg 1680 gtacacctgt aatgagacta
tcaatgccta ctcagcaggc atctggccag actcctcctc 1740 gagttatcag
tgcagtcata aaggggccag aggttaaatc ggaagcagtg gcaaaaaagc 1800
aagaacatga tgtgaaaact ttgcagctag tagaagaaaa accagcagat ggaaataaga
1860 cagtgaccca cgtagtggtt gtcagtgcgc cttcagctat tgcccttcct
gtaactatga 1920 aaacagaagg actagtgaca tgtgagaaat aaaatagcag
ctccaccatg gacttcaggc 1980 tgttagtggc agtactgaca taaacatttg
caagggaagt catcaagaaa agtcaaagaa 2040 gactttaaaa catttttaat
gcatatacaa aaacaatcag acttactgga aataaattac 2100 ctatcccatg
tttcagtggg aaatgaacta catattgaga tgctgacaga aaactgcctc 2160
ttacagtagg aaacaactga acccatcaat aagaaaaagg atcgaaaggg accaagcagc
2220 tcactacgat atcaagttac actaagactt ggaacactaa cattctgtaa
gaggttatat 2280 agttttcagt gggaggggtt gggatgggta atctcattgt
tacatatagc aatttttgat 2340 gcattttata tgcataccag caattattac
tgtgttcgca cagttctcac ttaactggtg 2400 ctatgtgaag actctgctaa
tataggtatt ttagaatgtg aattgaagaa tggatcccaa 2460 aaacttcaga
aagaggatag caaaaaaaga tctagtgcga ttttatatat atatatatat 2520
atatatacat acatatatat atatcatata gcttaagctg atttaaaaca aaggccttag
2580 actaattttc gattttcttt cttgaaataa gctaatggct tgtttgtgta
aagctttttt 2640 attaaaagaa aaattttaaa aatcttgtac ctagcacagt
attgttatag aatatacatg 2700 taacatttta tatggtagtt taagtctgtc
agtttcttaa ttgtggacaa attaacagtt 2760 ggctctggcc ttttgctgta
acatgcctgt gtcactcact tagccttggc atttgtgcag 2820 acataccatt
ttcagttctg ctgtcacttg gaagttcagg ctcagcatga atttttggca 2880
ggtagctcta atacctggag ttttctttgt ttttttttct tttttttagt tgaagtttat
2940 gagggaaata ccagtgttca gttttgaact ataatagttt gtatattcaa
catttgaagt 3000 atattctatt ttgttgtact cttgtttcaa agtgtattca
agtaggtttt ctgaaatata 3060 gaaatgaaat ttatcttctg ttttggtctc
tggtgatatt ttaaacaata tttaaaagtc 3120 agtatagaag tgttttagtt
aggaagtgat aaaacatctc tcttctcctt cccaactact 3180 gcatgaagaa
attctacttc cattatatta atatttgg 3218 <210> SEQ ID NO 54
<211> LENGTH: 1901 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 54 gcggcgagtg
gagcgggagc cgactggaag aagggctcta gggagggggc tgtggctgct 60
ggggtccgag gtggggccgg gtacaccagc cccatcactg tttgcagaga gtcagggagg
120 cggaaaagac acgcgctcta ggctcccatc agggcacatg gcccgggccc
atcccccgcg 180 cgtctccccg gctgcggggc gcggggggct gccgggtgcg
cttggctgtg gcgcggcgcg 240 ttggagactt tattgcgatg ggacgataag
aggggcgggg gcggggtcct gggggccgag 300 gcggcagcgc tttaattaaa
acggaaattg cggccccggg ccgcgcgggg gccggagggt 360 tccaagcggc
cccttagctg gaagcgtttc tccaggaccc ccccgcaacc cccgccacgc 420
ccgggctgcc ccctcccgcc aggccctgcc ggacccggcg ccgtcttctc ctccttgtca
480 cccgcggtcg cttcgggcgg ggatcggtgc caccgagcgc aaagcctgcc
tcgcccccct 540 tccccgtccc ccccatctcc caccgcccag tccccggcgg
cgatgagaca gagcggcgcc 600 tcccagcccc tgctgatcaa catgtacctg
ccagatcccg tcggagacgg tctcttcaag 660 gacgggaaga acccgagctg
ggggccgctg agccccgcgg ttcagaaagg cagcggacag 720 atccagctgt
ggcagtttct gctggagctg ctggctgacc gcgcgaacgc cggctgcatc 780
gcgtgggagg gcggtcacgg cgagttcaag ctcacggacc cggacgaggt ggcgcggcgg
840 tggggcgagc gcaagagcaa gcccaacatg aactacgaca agctgagccg
cgccctgcgc 900 tactactacg acaagaacat catgagcaag gtgcatggca
agcgctacgc ctaccgcttc 960 gacttccagg gcctggcgca ggcctgccag
ccgccgcccg cgcacgctca tgccgccgcc 1020 gcagctgctg ccgccgccgc
ggccgcccag gacggcgcgc tctacaagct gcccgccggc 1080 ctcgccccgc
tgcccttccc cggcctctcc aaactcaacc tcatggccgc ctcggccggg 1140
gtcgcgcccg ccggcttctc ctactggccg ggcccgggcc ccgccgccac cgctgccgcc
1200 gccaccgccg cgctctaccc cagtcccagc ttgcagcccc cgcccgggcc
cttcggggcc 1260 gtggccgcag cctcgcactt ggggggccat taccactaga
cggggcggtc gggtgcctgc 1320 ggcctcgccc gcacgcctag agtctcgccc
gatcccatcg gcatcccggg gagggcccgg 1380 gagcctccgt caaccgtcct
ctaatccaga gtttactcca cctgccgcac ttagcagggg 1440 gacgggaccg
aagctccctc aatccttgtc tggtactaga tttgctcctg tcccaccccg 1500
cagtcccctg aggagggcga tgtgcgccct ctttcacttt ttttcttcta ggtctccagg
1560 tcccggaggg gatttgtgga cctctcttgt ctccccacca ctccagtgca
tttccgcctg 1620 gctcctagaa gccccattca atatcactac tctttaacga
gtgccaaatc ttttcccact 1680 tttgctcttc cccaaggaac tgctcccacc
tcagcacgtg gaggcctctc acggtcctcc 1740 ttcctgggac ctgagcaggt
ttggtgaaag ccaccgtcct ccgtgacaca cggccccctt 1800 cctcctgtcc
ccacactccc aggagaaact cccggtgtgt ttctgaccct ttcagcccca 1860
ttaaagctcc tgagctctca aaaaaaaaaa aaaaaaaaaa a 1901 <210> SEQ
ID NO 55 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 55 taggcgcgag
ctaagcagga g 21 <210> SEQ ID NO 56 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 56 gtaggcacac tcaaacaacg actgg 25 <210>
SEQ ID NO 57 <211> LENGTH: 19 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 57
cgcgagctaa gcaggaggc 19 <210> SEQ ID NO 58 <211>
LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 58 caggccatga aaagccaaac tt 22 <210>
SEQ ID NO 59 <211> LENGTH: 49 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 59
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa tttcucgauu cguccuccg 49
<210> SEQ ID NO 60 <211> LENGTH: 51 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 60
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa tttauccgcg cucgauucgu c 51
<210> SEQ ID NO 61 <211> LENGTH: 23 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 61
gagggcgagg ggcggggagc gcc 23 <210> SEQ ID NO 62 <211>
LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 62 cctatcatta ctcgatgctg ttgataacag c 31
<210> SEQ ID NO 63 <211> LENGTH: 43 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 63
aatttaatac gactcactat agggagaaac tttcagcctg ata 43 <210> SEQ
ID NO 64 <211> LENGTH: 51 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 64 aatttaatac
gactcactat agggagactc tgtgagtcat ttgtcttgct t 51 <210> SEQ ID
NO 65 <211> LENGTH: 47 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 65 aatttaatac
gactcactat agggagagca cactcaaaca acgactg 47 <210> SEQ ID NO
66 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 66 gcgcggcagc
ucagguaccu gac 23 <210> SEQ ID NO 67 <211> LENGTH: 26
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 67 gcuuugaacu cacucaggua ccugac 26
<210> SEQ ID NO 68 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 68
gagcgcggca ggaagccuua ucaguug 27 <210> SEQ ID NO 69
<211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 69 gagcgcggca gguuauucca
ggaucuuu 28 <210> SEQ ID NO 70 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 70 cgcggcagga agcctta 17 <210> SEQ ID
NO 71 <211> LENGTH: 22 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 71 tccgtaggca
cactcaaaca ac 22 <210> SEQ ID NO 72 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 72 cagttgtgag tgaggacc 18 <210> SEQ ID
NO 73 <211> LENGTH: 72 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 73 ggggtgcagc
ttttattttc ccaaatactt cagtatatcc tgaagctacg caaagaatta 60
caactaggcc ag 72 <210> SEQ ID NO 74 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 74 Met Ala Ser Thr Ile Lys Glu Ala Leu Ser
Val Val Ser Glu Asp Gln 1 5 10 15 Ser Leu Phe Glu Cys Ala Tyr Gly
Thr Pro His Leu Ala Lys Thr Glu 20 25 30 Met Thr Ala Ser Ser Ser
Ser Asp 35 40 <210> SEQ ID NO 75 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 75 Ser Ser Ser Ser Asp 1 5 <210> SEQ ID
NO 76 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 76 Tyr Gly Gln Thr Ser
Lys Met Ser Pro Arg Val Pro Gln Gln Asp Trp 1 5 10 15 Leu Ser Gln
Pro Pro Ala Arg Val Thr Ile Lys Met Glu Cys Asn Pro 20 25 30 Ser
Gln Val Asn Gly Ser Arg Asn 35 40 <210> SEQ ID NO 77
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 77 Val Pro Gln Gln Asp Trp Leu
Ser Gln Pro Pro Ala Arg 1 5 10 <210> SEQ ID NO 78 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 78 Val Pro Gln Gln Asp Trp Leu Ser Gln Pro
Pro Ala Arg 1 5 10 <210> SEQ ID NO 79 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 79 Val Pro Gln Gln Asp Trp Leu Ser Gln Pro
Pro Ala Arg 1 5 10 <210> SEQ ID NO 80 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 80 Val Pro Gln Gln Asp Trp Leu Ser Gln Pro
Pro Ala Arg 1 5 10 <210> SEQ ID NO 81 <211> LENGTH: 21
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 81 Tyr Gly Gln Thr Ser Lys Met Ser Val Pro
Gln Gln Asp Trp Leu Ser 1 5 10 15 Gln Pro Pro Ala Arg 20
<210> SEQ ID NO 82 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 82 Val
Pro Gln Gln Asp Trp Leu Ser Gln Pro Pro Ala Arg 1 5 10 <210>
SEQ ID NO 83 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 83 Val Pro
Gln Gln Asp Trp Leu Ser Gln Pro Pro Ala Arg 1 5 10 <210> SEQ
ID NO 84 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 84 Val Pro Gln Gln Asp
Trp Leu Ser Gln Pro Pro Ala Arg 1 5 10 <210> SEQ ID NO 85
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 85 Val Pro Gln Gln Asp Trp Leu
Ser Gln Pro Pro Ala Arg Met Glu Cys 1 5 10 15 Asn Pro Ser Gln Val
Asn Gly Ser Arg 20 25 <210> SEQ ID NO 86 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 86 Val Pro Gln Gln Asp Trp Leu Ser Gln Pro
Pro Ala Arg 1 5 10 <210> SEQ ID NO 87 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 87 Val Pro Gln Gln Asp Trp Leu Ser Gln Pro
Pro Ala Arg 1 5 10 <210> SEQ ID NO 88 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 88 Ser Pro Asp Glu Cys Ser Val Ala Lys Gly
Gly Lys Met Val Gly Ser 1 5 10 15 Pro Asp Thr Val Gly Met Asn Tyr
Gly Ser Tyr Met Glu Glu Lys His 20 25 30 Met Pro Pro Pro Asn Met
Thr Thr 35 40 <210> SEQ ID NO 89 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 89 His Met Pro Pro Pro Asn Met Thr Thr 1 5
<210> SEQ ID NO 90 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 90 His
Met Pro Pro Pro Asn Met Thr Thr 1 5 <210> SEQ ID NO 91
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 91 His Met Pro Pro Pro Asn Met
Thr Thr 1 5 <210> SEQ ID NO 92 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 92 Asn Tyr Gly Ser Tyr Met Glu Glu Lys His
Met Pro 1 5 10 <210> SEQ ID NO 93 <211> LENGTH: 28
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 93 Met Val Gly Ser Pro Asp Thr Val Gly Met
Asn Tyr Gly Ser Tyr Met 1 5 10 15 Glu Glu Lys His Met Pro Pro Pro
Asn Met Thr Thr 20 25 <210> SEQ ID NO 94 <211> LENGTH:
9 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 94 His Met Pro Pro Pro Asn Met Thr Thr 1 5
<210> SEQ ID NO 95 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 95 His
Met Pro Pro Pro Asn Met Thr Thr 1 5 <210> SEQ ID NO 96
<211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 96 Met Val Gly Ser Pro Asp Thr
Val Gly Met Asn Tyr Gly Ser Tyr Met 1 5 10 15 Glu Glu Lys His Met
Pro Pro Pro Asn Met Thr Thr 20 25 <210> SEQ ID NO 97
<211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 97 Met Val Gly Ser Pro Asp Thr
Val Gly Met Asn Tyr Gly Ser Tyr Met 1 5 10 15 Glu Glu Lys His Met
Pro Pro Pro Asn Met Thr Thr 20 25 <210> SEQ ID NO 98
<211> LENGTH: 40 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 98 Asn Glu Arg Arg Val Ile Val
Pro Ala Asp Pro Thr Leu Trp Ser Thr 1 5 10 15 Asp His Val Arg Gln
Trp Leu Glu Trp Ala Val Lys Glu Tyr Gly Leu 20 25 30 Pro Asp Val
Asn Ile Leu Leu Phe 35 40 <210> SEQ ID NO 99 <211>
LENGTH: 39 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 99 Asn Glu Arg Val Ile Val Pro Ala Asp Pro
Thr Leu Trp Ser Thr Asp 1 5 10 15 His Val Arg Gln Trp Leu Glu Trp
Ala Val Lys Glu Tyr Gly Leu Pro 20 25 30 Asp Val Asn Ile Leu Leu
Phe 35 <210> SEQ ID NO 100 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
100 Asn Glu Arg Glu Tyr Gly Leu Pro Asp Val Asn Ile Leu Leu Phe 1 5
10 15 <210> SEQ ID NO 101 <211> LENGTH: 3 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
101 Asn Glu Arg 1 <210> SEQ ID NO 102 <211> LENGTH: 27
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 102 Asn Glu Arg Val Ile Val Pro Ala Asp Pro
Thr Leu Trp Ser Thr Asp 1 5 10 15 His Val Arg Gln Trp Leu Glu Trp
Ala Val Lys 20 25 <210> SEQ ID NO 103 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 103 Asn Glu Arg Arg Val Ile Val Pro Ala Asp
Pro Thr Leu Trp Ser Thr 1 5 10 15 Asp His Val Arg Glu Tyr Gly Leu
Pro Asp Val Asn Ile Leu Leu Phe 20 25 30 <210> SEQ ID NO 104
<211> LENGTH: 39 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 104 Asn Glu Arg Val Ile Val Pro
Ala Asp Pro Thr Leu Trp Ser Thr Asp 1 5 10 15 His Val Arg Gln Trp
Leu Glu Trp Ala Val Lys Glu Tyr Gly Leu Pro 20 25 30 Asp Val Asn
Ile Leu Leu Phe 35 <210> SEQ ID NO 105 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 105 Asn Glu Arg Arg Val Ile Val Pro Ala Asp
Pro Thr Leu Trp Ser Thr 1 5 10 15 Asp His Val Arg Gln Trp Leu Glu
Trp Ala Val Lys Glu Tyr Gly Leu 20 25 30 Pro Asp Val Asn Ile Leu
Leu Phe 35 40 <210> SEQ ID NO 106 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 106 Asn Glu Arg Arg Val Ile Val Pro Ala Asp
Pro Thr Leu Trp Ser Thr 1 5 10 15 Asp His Val Arg 20 <210>
SEQ ID NO 107 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 107 Glu
Tyr Gly Leu Pro Asp Val Asn Ile Leu Leu Phe 1 5 10 <210> SEQ
ID NO 108 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 108 Gln Asn Ile Asp
Gly Lys Glu Leu Cys Lys Met Thr Lys Asp Asp Phe 1 5 10 15 Gln Arg
Leu Thr Pro Ser Tyr Asn Ala Asp Ile Leu Leu Ser His Leu 20 25 30
His Tyr Leu Arg Glu Thr Pro Leu 35 40 <210> SEQ ID NO 109
<211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 109 Gln Asn Ile Asp Gly Lys Leu
Thr Pro Ser Tyr Asn Ala Asp Ile Leu 1 5 10 15 Leu Ser His Leu His
Tyr Leu Arg Glu Thr Pro Leu 20 25 <210> SEQ ID NO 110
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 110 Glu Thr Pro Leu 1
<210> SEQ ID NO 111 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 111
Gln Asn Ile Asp Gly Lys Glu Thr Pro Leu 1 5 10 <210> SEQ ID
NO 112 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 112 Glu Thr Pro Leu 1
<210> SEQ ID NO 113 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 113
Glu Thr Pro Leu 1 <210> SEQ ID NO 114 <211> LENGTH: 28
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 114 Gln Asn Ile Asp Gly Lys Leu Thr Pro Ser
Tyr Asn Ala Asp Ile Leu 1 5 10 15 Leu Ser His Leu His Tyr Leu Arg
Glu Thr Pro Leu 20 25 <210> SEQ ID NO 115 <211> LENGTH:
4 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 115 Glu Thr Pro Leu 1 <210> SEQ ID NO
116 <211> LENGTH: 28 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 116 Gln Asn Ile Asp
Gly Lys Leu Thr Pro Ser Tyr Asn Ala Asp Ile Leu 1 5 10 15 Leu Ser
His Leu His Tyr Leu Arg Glu Thr Pro Leu 20 25 <210> SEQ ID NO
117 <211> LENGTH: 28 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 117 Gln Asn Ile Asp
Gly Lys Leu Thr Pro Ser Tyr Asn Ala Asp Ile Leu 1 5 10 15 Leu Ser
His Leu His Tyr Leu Arg Glu Thr Pro Leu 20 25 <210> SEQ ID NO
118 <211> LENGTH: 22 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 118 Leu Thr Pro Ser
Tyr Asn Ala Asp Ile Leu Leu Ser His Leu His Tyr 1 5 10 15 Leu Arg
Glu Thr Pro Leu 20 <210> SEQ ID NO 119 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 119 Gln Asn Ile Asp Gly Lys 1 5 <210>
SEQ ID NO 120 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 120 Pro
Ser Tyr Asn Ala Asp Ile Leu Leu Ser His Leu His Tyr Leu Arg 1 5 10
15 Glu Thr Pro Leu 20 <210> SEQ ID NO 121 <211> LENGTH:
40 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 121 Pro His Leu Thr Ser Asp Asp Val Asp Lys
Ala Leu Gln Asn Ser Pro 1 5 10 15 Arg Leu Met His Ala Arg Asn Thr
Gly Gly Ala Ala Phe Ile Phe Pro 20 25 30 Asn Thr Ser Val Tyr Pro
Glu Ala 35 40 <210> SEQ ID NO 122 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 122 Pro Arg Leu Met His Ala Arg Asn Thr 1 5
<210> SEQ ID NO 123 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 123
Pro His Leu Thr Ser Asp Asp Val Asp Lys 1 5 10 <210> SEQ ID
NO 124 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 124 Pro His Leu Thr
Ser Asp Asp Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5 10 15 Arg
<210> SEQ ID NO 125 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 125
Pro His Leu Thr Ser Asp Asp Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5
10 15 Arg <210> SEQ ID NO 126 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 126 Pro His Leu Thr Ser Asp Asp Val Asp Lys
Ala Leu Gln Asn Ser Pro 1 5 10 15 Arg <210> SEQ ID NO 127
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 127 Pro His Leu Thr Ser Asp Asp
Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5 10 15 Arg <210> SEQ
ID NO 128 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 128 Pro His Leu Thr
Ser Asp Asp Val Asp Lys 1 5 10 <210> SEQ ID NO 129
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 129 Pro His Leu Thr Ser Asp Asp
Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5 10 15 Arg <210> SEQ
ID NO 130 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 130 Pro His Leu Thr
Ser Asp Asp Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5 10 15 Arg
<210> SEQ ID NO 131 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 131
Pro His Leu Thr Ser Asp Asp Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5
10 15 Arg <210> SEQ ID NO 132 <211> LENGTH: 3
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 132 Arg Asn Thr 1 <210> SEQ ID NO 133
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 133 Pro His Leu Thr Ser Asp Asp
Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5 10 15 Arg <210> SEQ
ID NO 134 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 134 Pro His Leu Thr
Ser Asp Asp Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5 10 15 Arg Leu
<210> SEQ ID NO 135 <211> LENGTH: 40 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 135
Thr Gln Arg Ile Thr Thr Arg Pro Asp Leu Pro Tyr Glu Pro Pro Arg 1 5
10 15 Arg Ser Ala Trp Thr Gly His Gly His Pro Thr Pro Gln Ser Lys
Ala 20 25 30 Ala Gln Pro Ser Pro Ser Thr Val 35 40 <210> SEQ
ID NO 136 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 136 Asp Leu Pro Tyr
Glu Pro Pro Arg 1 5 <210> SEQ ID NO 137 <211> LENGTH:
23 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 137 Ser Ala Trp Thr Gly His Gly His Pro Thr
Pro Gln Ser Lys Ala Ala 1 5 10 15 Gln Pro Ser Pro Ser Thr Val 20
<210> SEQ ID NO 138 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 138
Pro Tyr Glu Pro Pro Arg Arg 1 5 <210> SEQ ID NO 139
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 139 Ser Ala Trp Thr Gly His Gly
His Pro Thr Pro Gln Ser Lys Ala Ala 1 5 10 15 Gln Pro Ser Pro Ser
Thr Val 20 <210> SEQ ID NO 140 <211> LENGTH: 23
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 140 Ser Ala Trp Thr Gly His Gly His Pro Thr
Pro Gln Ser Lys Ala Ala 1 5 10 15 Gln Pro Ser Pro Ser Thr Val 20
<210> SEQ ID NO 141 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 141
Pro Tyr Glu Pro Pro Arg Arg Ser Ala Trp Thr Gly His Gly His Pro 1 5
10 15 Thr Pro Gln Ser Lys Ala Ala Gln Pro Ser Pro Ser Thr Val 20 25
30 <210> SEQ ID NO 142 <211> LENGTH: 23 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
142 Ser Ala Trp Thr Gly His Gly His Pro Thr Pro Gln Ser Lys Ala Ala
1 5 10 15 Gln Pro Ser Pro Ser Thr Val 20 <210> SEQ ID NO 143
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 143 Ser Ala Trp Thr Gly His Gly
His Pro Thr Pro Gln Ser Lys Ala Ala 1 5 10 15 Gln Pro Ser Pro Ser
Thr Val 20 <210> SEQ ID NO 144 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 144 Asp Leu Pro Tyr Glu Pro Pro Arg Arg 1 5
<210> SEQ ID NO 145 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 145
Pro Tyr Glu Pro Pro Arg Arg Ser Ala Trp Thr Gly His Gly His Pro 1 5
10 15 Thr Pro Gln Ser Lys Ala Ala Gln Pro Ser Pro Ser Thr Val 20 25
30 <210> SEQ ID NO 146 <211> LENGTH: 32 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
146 Asp Leu Pro Tyr Glu Pro Pro Arg Arg Ser Ala Trp Thr Gly His Gly
1 5 10 15 His Pro Thr Pro Gln Ser Lys Ala Ala Gln Pro Ser Pro Ser
Thr Val 20 25 30 <210> SEQ ID NO 147 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 147 Asp Leu Pro Tyr Glu Pro Pro Arg Arg 1 5
<210> SEQ ID NO 148 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 148
Asp Leu Pro Tyr Glu Pro Pro Arg Arg Ser Ala Trp Thr Gly His Gly 1 5
10 15 His Pro Thr Pro Gln Ser Lys Ala Ala Gln Pro Ser Pro Ser Thr
Val 20 25 30 <210> SEQ ID NO 149 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 149 Ala Ala Gln Pro Ser Pro Ser Thr Val 1 5
<210> SEQ ID NO 150 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 150
Ser Ala Trp Thr Gly His Gly His Pro Thr Pro Gln Ser Lys Ala Ala 1 5
10 15 Gln Pro Ser Pro Ser Thr Val 20 <210> SEQ ID NO 151
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 151 Asp Leu Pro Tyr Glu Pro Pro
Arg Arg Ser Ala Trp Thr Gly His Gly 1 5 10 15 His Pro Thr Pro Gln
Ser Lys Ala Ala Gln Pro Ser Pro Ser Thr Val 20 25 30 <210>
SEQ ID NO 152 <211> LENGTH: 40 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 152 Pro
Lys Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5 10
15 Gly Pro Thr Ser Ser Arg Leu Ala Asn Pro Gly Ser Gly Gln Ile Gln
20 25 30 Leu Trp Gln Phe Leu Leu Glu Leu 35 40 <210> SEQ ID
NO 153 <211> LENGTH: 2 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 153 Pro Lys 1
<210> SEQ ID NO 154 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 154
Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu Gly Pro 1 5
10 15 Thr Ser Ser Arg 20 <210> SEQ ID NO 155 <211>
LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 155 Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu
Asp Pro Tyr Gln Ile Leu 1 5 10 15 Gly Pro Thr Ser Ser Arg 20
<210> SEQ ID NO 156 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 156
Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5
10 15 Gly Pro Thr Ser Ser Arg 20 <210> SEQ ID NO 157
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 157 Pro Lys Thr Glu Asp Gln Arg
Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5 10 15 Gly Pro Thr Ser Ser
Arg 20 <210> SEQ ID NO 158 <211> LENGTH: 22 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
158 Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu
1 5 10 15 Gly Pro Thr Ser Ser Arg 20 <210> SEQ ID NO 159
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 159 Pro Lys Thr Glu Asp Gln Arg
Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5 10 15 Gly Pro Thr Ser Ser
Arg 20 <210> SEQ ID NO 160 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
160 Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu Gly Pro
1 5 10 15 Thr Ser Ser Arg 20 <210> SEQ ID NO 161 <211>
LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 161 Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu
Asp Pro Tyr Gln Ile Leu 1 5 10 15 Gly Pro Thr Ser Ser Arg 20
<210> SEQ ID NO 162 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 162
Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5
10 15 Gly Pro Thr Ser Ser Arg 20 <210> SEQ ID NO 163
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 163 Pro Lys Thr Glu Asp Gln Arg
Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5 10 15 Gly Pro Thr Ser Ser
Arg 20 <210> SEQ ID NO 164 <211> LENGTH: 2 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
164 Pro Lys 1 <210> SEQ ID NO 165 <211> LENGTH: 22
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 165 Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu
Asp Pro Tyr Gln Ile Leu 1 5 10 15 Gly Pro Thr Ser Ser Arg 20
<210> SEQ ID NO 166 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 166
Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5
10 15 Gly Pro Thr Ser Ser Arg 20 <210> SEQ ID NO 167
<211> LENGTH: 2 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 167 Pro Lys 1 <210> SEQ ID
NO 168 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 168 Leu Ser Asp Ser
Ser Asn Ser Ser Cys Ile Thr Trp Glu Gly Thr Asn 1 5 10 15 Gly Glu
Phe Lys Met Thr Asp Pro Asp Glu Val Ala Arg Arg Trp Gly 20 25 30
Glu Arg Lys Ser Lys Pro Asn Met 35 40 <210> SEQ ID NO 169
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 169 Met Thr Asp Pro Asp Glu Val
Ala Arg 1 5 <210> SEQ ID NO 170 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 170 Met Thr Asp Pro Asp Glu Val Ala Arg 1 5
<210> SEQ ID NO 171 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 171
Met Thr Asp Pro Asp Glu Val Ala Arg 1 5 <210> SEQ ID NO 172
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 172 Met Thr Asp Pro Asp Glu Val
Ala Arg 1 5 <210> SEQ ID NO 173 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 173 Met Thr Asp Pro Asp Glu Val Ala Arg 1 5
<210> SEQ ID NO 174 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 174
Met Thr Asp Pro Asp Glu Val Ala Arg Arg 1 5 10 <210> SEQ ID
NO 175 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 175 Thr Asp Pro Asp
Glu Val Ala Arg Arg Lys Ser Lys Pro Asn Met 1 5 10 15 <210>
SEQ ID NO 176 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 176 Met
Thr Asp Pro Asp Glu Val Ala Arg 1 5 <210> SEQ ID NO 177
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 177 Lys Ser Lys Pro Asn Met 1 5
<210> SEQ ID NO 178 <211> LENGTH: 40 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 178
Asn Tyr Asp Lys Leu Ser Arg Ala Leu Arg Tyr Tyr Tyr Asp Lys Asn 1 5
10 15 Ile Met Thr Lys Val His Gly Lys Arg Tyr Ala Tyr Lys Phe Asp
Phe 20 25 30 His Gly Ile Ala Gln Ala Leu Gln 35 40 <210> SEQ
ID NO 179 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 179 Phe Asp Phe His
Gly Ile Ala Gln Ala Leu Gln 1 5 10 <210> SEQ ID NO 180
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 180 Phe Asp Phe His Gly Ile Ala
Gln Ala Leu Gln 1 5 10 <210> SEQ ID NO 181 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 181 Phe Asp Phe His Gly Ile Ala Gln Ala Leu
Gln 1 5 10 <210> SEQ ID NO 182 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 182 Phe Asp Phe His Gly Ile Ala Gln Ala Leu
Gln 1 5 10 <210> SEQ ID NO 183 <211> LENGTH: 25
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 183 Tyr Tyr Tyr Asp Lys Asn Ile Met Thr Lys
Tyr Ala Tyr Lys Phe Asp 1 5 10 15 Phe His Gly Ile Ala Gln Ala Leu
Gln 20 25 <210> SEQ ID NO 184 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 184 Asn Tyr Asp Lys Leu Ser Arg 1 5
<210> SEQ ID NO 185 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 185
Asn Tyr Asp Lys Leu Ser Arg Tyr Tyr Tyr Asp Lys Asn Ile Met Thr 1 5
10 15 Lys <210> SEQ ID NO 186 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 186 Pro His Pro Pro Glu Ser Ser Leu Tyr Lys
Tyr Pro Ser Asp Leu Pro 1 5 10 15 Tyr Met Gly Ser Tyr His Ala His
Pro Gln Lys Met Asn Phe Val Ala 20 25 30 Pro His Pro Pro Ala Leu
Pro Val 35 40 <210> SEQ ID NO 187 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 187 Pro His Pro Pro Glu Ser Ser Leu Tyr Lys 1
5 10 <210> SEQ ID NO 188 <211> LENGTH: 24 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
188 Pro His Pro Pro Glu Ser Ser Leu Tyr Lys Tyr Pro Ser Asp Leu Pro
1 5 10 15 Tyr Met Gly Ser Tyr His Ala His 20 <210> SEQ ID NO
189 <211> LENGTH: 27 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 189 Pro His Pro Pro
Glu Ser Ser Leu Tyr Lys Tyr Pro Ser Asp Leu Pro 1 5 10 15 Tyr Met
Gly Ser Tyr His Ala His Pro Gln Lys 20 25 <210> SEQ ID NO 190
<211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 190 Pro His Pro Pro Glu Ser Ser
Leu Tyr Lys Tyr Pro Ser Asp Leu Pro 1 5 10 15 Tyr Met Gly Ser Tyr
His Ala His Pro Gln Lys 20 25 <210> SEQ ID NO 191 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 191 Tyr Pro Ser Asp Leu Pro Tyr Met Gly Ser
Tyr His Ala His Pro Gln 1 5 10 15 Lys <210> SEQ ID NO 192
<211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 192 Pro His Pro Pro Glu Ser Ser
Leu Tyr Lys Tyr Pro Ser Asp Leu Pro 1 5 10 15 Tyr Met Gly Ser Tyr
His Ala His Pro Gln Lys 20 25 <210> SEQ ID NO 193 <211>
LENGTH: 39 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 193 Thr Ser Ser Ser Phe Phe Ala Ala Pro Asn
Pro Tyr Trp Asn Ser Pro 1 5 10 15 Thr Gly Gly Ile Tyr Pro Asn Thr
Arg Leu Pro Thr Ser His Met Pro 20 25 30 Ser His Leu Gly Thr Tyr
Tyr 35 <210> SEQ ID NO 194 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
194 Asn Ser Pro Thr Gly 1 5 <210> SEQ ID NO 195 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 195 Ser Pro Thr Gly Gly Ile Tyr Pro Asn Thr
Arg 1 5 10 <210> SEQ ID NO 196 <211> LENGTH: 24
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 196 Gly Gly Ala Ala Phe Ile Phe Pro Asn Thr
Ser Val Tyr Pro Glu Ala 1 5 10 15 Thr Gln Arg Ile Thr Thr Arg Pro
20 <210> SEQ ID NO 197 <211> LENGTH: 10 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
197 Val Pro Gln Gln Asp Trp Leu Ser Gln Pro 1 5 10 <210> SEQ
ID NO 198 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 198 Val Pro Gln Gln
Asp Trp Leu Ser Gln Pro 1 5 10 <210> SEQ ID NO 199
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 199 Met Ile Gln Thr Val Pro Asp
Pro Ala Ala His Ile 1 5 10 <210> SEQ ID NO 200 <211>
LENGTH: 55 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 200 Met Ala Ser Thr Ile Lys Glu Ala Leu Ser
Val Val Ser Glu Asp Gln 1 5 10 15 Ser Leu Phe Glu Cys Ala Tyr Gly
Thr Pro His Leu Ala Lys Thr Glu 20 25 30 Met Thr Ala Tyr Gly Gln
Thr Ser Lys Met Ser Pro Arg Val Pro Gln 35 40 45 Gln Asp Trp Leu
Ser Gln Pro 50 55 <210> SEQ ID NO 201 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 201 Met Ile Gln Thr Val Pro Asp Pro Ala Ala
His Ile 1 5 10 <210> SEQ ID NO 202 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 202 gctgcagact tggccaaatg gac 23 <210>
SEQ ID NO 203 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 203
tcaccaccga cagagcctcc tta 23 <210> SEQ ID NO 204 <211>
LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 204 accacatgaa tggatccagg gagtct 26
<210> SEQ ID NO 205 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 205
accagcttgc tgcatttgct aacg 24 <210> SEQ ID NO 206 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 206 ctccgcgcca ccaccctcta 20 <210> SEQ
ID NO 207 <211> LENGTH: 24 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 207 ggccagcagt
gaactttccc tgag 24 <210> SEQ ID NO 208 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 208 ctcctcccaa catgaccacc aac 23 <210>
SEQ ID NO 209 <211> LENGTH: 22 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 209
gtctgcgggg acgatgactc tc 22 <210> SEQ ID NO 210 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 210 catgtgaggc aatggctgga gtg 23 <210>
SEQ ID NO 211 <211> LENGTH: 26 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 211
ccatgttctg gaaaaaggat gtgtcg 26 <210> SEQ ID NO 212
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 212 ccttggaggg gcacaaacga t 21
<210> SEQ ID NO 213 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 213
ggtcgggccc aggatctgat ac 22 <210> SEQ ID NO 214 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 214 cgccaacgcc agctgtatca c 21 <210>
SEQ ID NO 215 <211> LENGTH: 19 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 215
agcgcctggc cacctcatc 19 <210> SEQ ID NO 216 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 216 atgtttttat gaccaaagca gtttcttgtc 30
<210> SEQ ID NO 217 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 217
atgacgggtt aagtccatga ttctgtg 27 <210> SEQ ID NO 218
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 218 cgaaagctgc tcaaccatct c 21
<210> SEQ ID NO 219 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 219
taactgagga cgctggtctt ca 22 <210> SEQ ID NO 220 <211>
LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 220 ccacagtgcc caaaa 15 <210> SEQ ID NO
221 <400> SEQUENCE: 221 000 <210> SEQ ID NO 222
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 222 cggcaaaaga tatgcttaca aattt
25 <210> SEQ ID NO 223 <211> LENGTH: 19 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
223 gacgactcgg tcggatgtg 19 <210> SEQ ID NO 224 <211>
LENGTH: 14 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 224 cacggcattg ccca 14 <210> SEQ ID NO
225 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 225 cgcggcagga agcctta
17 <210> SEQ ID NO 226 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
226 tccgtaggca cactcaaaca ac 22 <210> SEQ ID NO 227
<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 227 cagttgtgag tgaggacc 18
<210> SEQ ID NO 228 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 228
gctcgctccg atactattat gagaa 25 <210> SEQ ID NO 229
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 229 cacacacaaa cttgtacacg taacg
25 <210> SEQ ID NO 230 <211> LENGTH: 16 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
230 accagccacc ttctgc 16 <210> SEQ ID NO 231 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 231 ggtcgaggca tggaatttaa actga 25
<210> SEQ ID NO 232 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 232
gctggcctgt ttttctgaat gc 22 <210> SEQ ID NO 233 <211>
LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 233 tcgggccacc tcttc 15 <210> SEQ ID NO
234 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 234 Met Ile Gln Thr
Val Pro Asp Pro Ala Ala His Ile 1 5 10 <210> SEQ ID NO 235
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 235 Ser Ala Trp Thr Gly His Gly
His Pro Thr Pro Gln Ser Lys Ala Ala 1 5 10 15 Gln Pro Ser Pro Ser
Thr Val 20 <210> SEQ ID NO 236 <211> LENGTH: 42
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: NON_CONS <222>
LOCATION: (18)..(19) <400> SEQUENCE: 236 Met Ile Gln Thr Val
Pro Asp Pro Ala Ala Ser His Ile Lys Glu Ala 1 5 10 15 Leu Ser Gly
Gly Ala Ala Phe Ile Phe Pro Asn Thr Ser Val Tyr Pro 20 25 30 Glu
Ala Thr Gln Arg Ile Thr Thr Arg Pro 35 40 <210> SEQ ID NO 237
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 237 Met Ile Gln Thr Val Pro Asp
Pro Ala Ala Ser His Ile Lys Glu Ala 1 5 10 15 Leu Ser <210>
SEQ ID NO 238 <211> LENGTH: 42 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: NON_CONS <222> LOCATION: (10)..(11) <220>
FEATURE: <221> NAME/KEY: NON_CONS <222> LOCATION:
(14)..(15) <220> FEATURE: <221> NAME/KEY: NON_CONS
<222> LOCATION: (18)..(19) <400> SEQUENCE: 238 Met Ala
Ser Thr Ile Lys Glu Ala Leu Ser Met Thr Ala Ser Met Ser 1 5 10 15
Pro Arg Gly Gly Ala Ala Phe Ile Phe Pro Asn Thr Ser Val Tyr Pro 20
25 30 Glu Ala Thr Gln Arg Ile Thr Thr Arg Pro 35 40 <210> SEQ
ID NO 239 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
NON_CONS <222> LOCATION: (10)..(11) <220> FEATURE:
<221> NAME/KEY: NON_CONS <222> LOCATION: (14)..(15)
<400> SEQUENCE: 239 Met Ala Ser Thr Ile Lys Glu Ala Leu Ser
Met Thr Ala Ser Met Ser 1 5 10 15 Pro Arg <210> SEQ ID NO 240
<211> LENGTH: 479 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 240 Met Ala Ser Thr Ile Lys Glu
Ala Leu Ser Val Val Ser Glu Asp Gln 1 5 10 15 Ser Leu Phe Glu Cys
Ala Tyr Gly Thr Pro His Leu Ala Lys Thr Glu 20 25 30 Met Thr Ala
Ser Ser Ser Ser Asp Tyr Gly Gln Thr Ser Lys Met Ser 35 40 45 Pro
Arg Val Pro Gln Gln Asp Trp Leu Ser Gln Pro Pro Ala Arg Val 50 55
60 Thr Ile Lys Met Glu Cys Asn Pro Ser Gln Val Asn Gly Ser Arg Asn
65 70 75 80 Ser Pro Asp Glu Cys Ser Val Ala Lys Gly Gly Lys Met Val
Gly Ser 85 90 95 Pro Asp Thr Val Gly Met Asn Tyr Gly Ser Tyr Met
Glu Glu Lys His 100 105 110 Met Pro Pro Pro Asn Met Thr Thr Asn Glu
Arg Arg Val Ile Val Pro 115 120 125 Ala Asp Pro Thr Leu Trp Ser Thr
Asp His Val Arg Gln Trp Leu Glu 130 135 140 Trp Ala Val Lys Glu Tyr
Gly Leu Pro Asp Val Asn Ile Leu Leu Phe 145 150 155 160 Gln Asn Ile
Asp Gly Lys Glu Leu Cys Lys Met Thr Lys Asp Asp Phe 165 170 175 Gln
Arg Leu Thr Pro Ser Tyr Asn Ala Asp Ile Leu Leu Ser His Leu 180 185
190 His Tyr Leu Arg Glu Thr Pro Leu Pro His Leu Thr Ser Asp Asp Val
195 200 205 Asp Lys Ala Leu Gln Asn Ser Pro Arg Leu Met His Ala Arg
Asn Thr 210 215 220 Gly Gly Ala Ala Phe Ile Phe Pro Asn Thr Ser Val
Tyr Pro Glu Ala 225 230 235 240 Thr Gln Arg Ile Thr Thr Arg Pro Asp
Leu Pro Tyr Glu Pro Pro Arg 245 250 255 Arg Ser Ala Trp Thr Gly His
Gly His Pro Thr Pro Gln Ser Lys Ala 260 265 270 Ala Gln Pro Ser Pro
Ser Thr Val Pro Lys Thr Glu Asp Gln Arg Pro 275 280 285 Gln Leu Asp
Pro Tyr Gln Ile Leu Gly Pro Thr Ser Ser Arg Leu Ala 290 295 300 Asn
Pro Gly Ser Gly Gln Ile Gln Leu Trp Gln Phe Leu Leu Glu Leu 305 310
315 320 Leu Ser Asp Ser Ser Asn Ser Ser Cys Ile Thr Trp Glu Gly Thr
Asn 325 330 335 Gly Glu Phe Lys Met Thr Asp Pro Asp Glu Val Ala Arg
Arg Trp Gly 340 345 350 Glu Arg Lys Ser Lys Pro Asn Met Asn Tyr Asp
Lys Leu Ser Arg Ala 355 360 365 Leu Arg Tyr Tyr Tyr Asp Lys Asn Ile
Met Thr Lys Val His Gly Lys 370 375 380 Arg Tyr Ala Tyr Lys Phe Asp
Phe His Gly Ile Ala Gln Ala Leu Gln 385 390 395 400 Pro His Pro Pro
Glu Ser Ser Leu Tyr Lys Tyr Pro Ser Asp Leu Pro 405 410 415 Tyr Met
Gly Ser Tyr His Ala His Pro Gln Lys Met Asn Phe Val Ala 420 425 430
Pro His Pro Pro Ala Leu Pro Val Thr Ser Ser Ser Phe Phe Ala Ala 435
440 445 Pro Asn Pro Tyr Trp Asn Ser Pro Thr Gly Gly Ile Tyr Pro Asn
Thr 450 455 460 Arg Leu Pro Thr Ser His Met Pro Ser His Leu Gly Thr
Tyr Tyr 465 470 475 <210> SEQ ID NO 241 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 241 Gly Ala Ala Phe Ile Phe Pro Asn Thr Ser
Val Tyr Pro 1 5 10 <210> SEQ ID NO 242 <211> LENGTH:
477 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 242 Met Asp Gly Phe Tyr Asp Gln Gln Val Pro
Tyr Met Val Thr Asn Ser 1 5 10 15 Gln Arg Gly Arg Asn Cys Asn Glu
Lys Pro Thr Asn Val Arg Lys Arg 20 25 30 Lys Phe Ile Asn Arg Asp
Leu Ala His Asp Ser Glu Glu Leu Phe Gln 35 40 45 Asp Leu Ser Gln
Leu Gln Glu Thr Trp Leu Ala Glu Ala Gln Val Pro 50 55 60 Asp Asn
Asp Glu Gln Phe Val Pro Asp Tyr Gln Ala Glu Ser Leu Ala 65 70 75 80
Phe His Gly Leu Pro Leu Lys Ile Lys Lys Glu Pro His Ser Pro Cys 85
90 95 Ser Glu Ile Ser Ser Ala Cys Ser Gln Glu Gln Pro Phe Lys Phe
Ser 100 105 110 Tyr Gly Glu Lys Cys Leu Tyr Asn Val Ser Ala Tyr Asp
Gln Lys Pro 115 120 125 Gln Val Gly Met Arg Pro Ser Asn Pro Pro Thr
Pro Ser Ser Thr Pro 130 135 140 Val Ser Pro Leu His His Ala Ser Pro
Asn Ser Thr His Thr Pro Lys 145 150 155 160 Pro Asp Arg Ala Phe Pro
Ala His Leu Pro Pro Ser Gln Ser Ile Pro 165 170 175 Asp Ser Ser Tyr
Pro Met Asp His Arg Phe Arg Arg Gln Leu Ser Glu 180 185 190 Pro Cys
Asn Ser Phe Pro Pro Leu Pro Thr Met Pro Arg Glu Gly Arg 195 200 205
Pro Met Tyr Gln Arg Gln Met Ser Glu Pro Asn Ile Pro Phe Pro Pro 210
215 220 Gln Gly Phe Lys Gln Glu Tyr His Asp Pro Val Tyr Glu His Asn
Thr 225 230 235 240 Met Val Gly Ser Ala Ala Ser Gln Ser Phe Pro Pro
Pro Leu Met Ile 245 250 255 Lys Gln Glu Pro Arg Asp Phe Ala Tyr Asp
Ser Glu Val Pro Ser Cys 260 265 270 His Ser Ile Tyr Met Arg Gln Glu
Gly Phe Leu Ala His Pro Ser Arg 275 280 285 Thr Glu Gly Cys Met Phe
Glu Lys Gly Pro Arg Gln Phe Tyr Asp Asp 290 295 300 Thr Cys Val Val
Pro Glu Lys Phe Asp Gly Asp Ile Lys Gln Glu Pro 305 310 315 320 Gly
Met Tyr Arg Glu Gly Pro Thr Tyr Gln Arg Arg Gly Ser Leu Gln 325 330
335 Leu Trp Gln Phe Leu Val Ala Leu Leu Asp Asp Pro Ser Asn Ser His
340 345 350 Phe Ile Ala Trp Thr Gly Arg Gly Met Glu Phe Lys Leu Ile
Glu Pro 355 360 365 Glu Glu Val Ala Arg Arg Trp Gly Ile Gln Lys Asn
Arg Pro Ala Met 370 375 380 Asn Tyr Asp Lys Leu Ser Arg Ser Leu Arg
Tyr Tyr Tyr Glu Lys Gly 385 390 395 400 Ile Met Gln Lys Val Ala Gly
Glu Arg Tyr Val Tyr Lys Phe Val Cys 405 410 415 Asp Pro Glu Ala Leu
Phe Ser Met Ala Phe Pro Asp Asn Gln Arg Pro 420 425 430 Leu Leu Lys
Thr Asp Met Glu Arg His Ile Asn Glu Glu Asp Thr Val 435 440 445 Pro
Leu Ser His Phe Asp Glu Ser Met Ala Tyr Met Pro Glu Gly Gly 450 455
460 Cys Cys Asn Pro His Pro Tyr Asn Glu Gly Tyr Val Tyr 465 470 475
<210> SEQ ID NO 243 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 243
Pro His Ser Pro Cys Ser Glu Ile Ser Ser Ala Cys Ser Gln 1 5 10
<210> SEQ ID NO 244 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 244
Thr Pro Ser Ser Thr Pro Val Ser Pro Leu His His Ala 1 5 10
<210> SEQ ID NO 245 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 245
Arg Ala Phe Pro Ala His Leu Pro Pro Ser Gln Ser Ile Pro Asp Ser 1 5
10 15 <210> SEQ ID NO 246 <211> LENGTH: 452 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
246 Met Asp Gly Thr Ile Lys Glu Ala Leu Ser Val Val Ser Asp Asp Gln
1 5 10 15 Ser Leu Phe Asp Ser Ala Tyr Gly Ala Ala Ala His Leu Pro
Lys Ala 20 25 30 Asp Met Thr Ala Ser Gly Ser Pro Asp Tyr Gly Gln
Pro His Lys Ile 35 40 45 Asn Pro Leu Pro Pro Gln Gln Glu Trp Ile
Asn Gln Pro Val Arg Val 50 55 60 Asn Val Lys Arg Glu Tyr Asp His
Met Asn Gly Ser Arg Glu Ser Pro 65 70 75 80 Val Asp Cys Ser Val Ser
Lys Cys Ser Lys Leu Val Gly Gly Gly Glu 85 90 95 Ser Asn Pro Met
Asn Tyr Asn Ser Tyr Met Asp Glu Lys Asn Gly Pro 100 105 110 Pro Pro
Pro Asn Met Thr Thr Asn Glu Arg Arg Val Ile Val Pro Ala 115 120 125
Asp Pro Thr Leu Trp Thr Gln Glu His Val Arg Gln Trp Leu Glu Trp 130
135 140 Ala Ile Lys Glu Tyr Ser Leu Met Glu Ile Asp Thr Ser Phe Phe
Gln 145 150 155 160 Asn Met Asp Gly Lys Glu Leu Cys Lys Met Asn Lys
Glu Asp Phe Leu 165 170 175 Arg Ala Thr Thr Leu Tyr Asn Thr Glu Val
Leu Leu Ser His Leu Ser 180 185 190 Tyr Leu Arg Glu Ser Ser Leu Leu
Ala Tyr Asn Thr Thr Ser His Thr 195 200 205 Asp Gln Ser Ser Arg Leu
Ser Val Lys Glu Asp Pro Ser Tyr Asp Ser 210 215 220 Val Arg Arg Gly
Ala Trp Gly Asn Asn Met Asn Ser Gly Leu Asn Lys 225 230 235 240 Ser
Pro Pro Leu Gly Gly Ala Gln Thr Ile Ser Lys Asn Thr Glu Gln 245 250
255 Arg Pro Gln Pro Asp Pro Tyr Gln Ile Leu Gly Pro Thr Ser Ser Arg
260 265 270 Leu Ala Asn Pro Gly Ser Gly Gln Ile Gln Leu Trp Gln Phe
Leu Leu 275 280 285 Glu Leu Leu Ser Asp Ser Ala Asn Ala Ser Cys Ile
Thr Trp Glu Gly 290 295 300 Thr Asn Gly Glu Phe Lys Met Thr Asp Pro
Asp Glu Val Ala Arg Arg 305 310 315 320 Trp Gly Glu Arg Lys Ser Lys
Pro Asn Met Asn Tyr Asp Lys Leu Ser 325 330 335 Arg Ala Leu Arg Tyr
Tyr Tyr Asp Lys Asn Ile Met Thr Lys Val His 340 345 350 Gly Lys Arg
Tyr Ala Tyr Lys Phe Asp Phe His Gly Ile Ala Gln Ala 355 360 365 Leu
Gln Pro His Pro Thr Glu Ser Ser Met Tyr Lys Tyr Pro Ser Asp 370 375
380 Ile Ser Tyr Met Pro Ser Tyr His Ala His Gln Gln Lys Val Asn Phe
385 390 395 400 Val Pro Pro His Pro Ser Ser Met Pro Val Thr Ser Ser
Ser Phe Phe 405 410 415 Gly Ala Ala Ser Gln Tyr Trp Thr Ser Pro Thr
Gly Gly Ile Tyr Pro 420 425 430 Asn Pro Asn Val Pro Arg His Pro Asn
Thr His Val Pro Ser His Leu 435 440 445 Gly Ser Tyr Tyr 450
<210> SEQ ID NO 247 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 247
Ser Leu Phe Asp Ser Ala Tyr Gly Ala Ala Ala His Leu Pro 1 5 10
<210> SEQ ID NO 248 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 248
His Lys Ile Asn Pro Leu Pro Pro Gln Ile Asn Gln Pro Val Arg Val 1 5
10 15 Asn Val <210> SEQ ID NO 249 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 249 Arg Glu Ser Pro Val Asp Cys Ser Val Ser
Lys Cys Ser Lys Leu Val 1 5 10 15 Gly <210> SEQ ID NO 250
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 250 Thr Gln Glu His Val Arg Gln
1 5 <210> SEQ ID NO 251 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
251 Ser Ser Arg Leu Ser Val Lys Glu 1 5 <210> SEQ ID NO 252
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 252 Pro Asn Thr His Val Pro Ser
His Leu 1 5 <210> SEQ ID NO 253 <211> LENGTH: 484
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 253 Met Glu Arg Arg Met Lys Ala Gly Tyr Leu
Asp Gln Gln Val Pro Tyr 1 5 10 15 Thr Phe Ser Ser Lys Ser Pro Gly
Asn Gly Ser Leu Arg Glu Ala Leu 20 25 30 Ile Gly Pro Leu Gly Lys
Leu Met Asp Pro Gly Ser Leu Pro Pro Leu 35 40 45 Asp Ser Glu Asp
Leu Phe Gln Asp Leu Ser His Phe Gln Glu Thr Trp 50 55 60 Leu Ala
Glu Ala Gln Val Pro Asp Ser Asp Glu Gln Phe Val Pro Asp 65 70 75 80
Phe His Ser Glu Asn Leu Ala Phe His Ser Pro Thr Thr Arg Ile Lys 85
90 95 Lys Glu Pro Gln Ser Pro Arg Thr Asp Pro Ala Leu Ser Cys Ser
Arg 100 105 110 Lys Pro Pro Leu Pro Tyr His His Gly Glu Gln Cys Leu
Tyr Ser Ser 115 120 125 Ala Tyr Asp Pro Pro Arg Gln Ile Ala Ile Lys
Ser Pro Ala Pro Gly 130 135 140 Ala Leu Gly Gln Ser Pro Leu Gln Pro
Phe Pro Arg Ala Glu Gln Arg 145 150 155 160 Asn Phe Leu Arg Ser Ser
Gly Thr Ser Gln Pro His Pro Gly His Gly 165 170 175 Tyr Leu Gly Glu
His Ser Ser Val Phe Gln Gln Pro Leu Asp Ile Cys 180 185 190 His Ser
Phe Thr Ser Gln Gly Gly Gly Arg Glu Pro Leu Pro Ala Pro 195 200 205
Tyr Gln His Gln Leu Ser Glu Pro Cys Pro Pro Tyr Pro Gln Gln Ser 210
215 220 Phe Lys Gln Glu Tyr His Asp Pro Leu Tyr Glu Gln Ala Gly Gln
Pro 225 230 235 240 Ala Val Asp Gln Gly Gly Val Asn Gly His Arg Tyr
Pro Gly Ala Gly 245 250 255 Val Val Ile Lys Gln Glu Gln Thr Asp Phe
Ala Tyr Asp Ser Asp Val 260 265 270 Thr Gly Cys Ala Ser Met Tyr Leu
His Thr Glu Gly Phe Ser Gly Pro 275 280 285 Ser Pro Gly Asp Gly Ala
Met Gly Tyr Gly Tyr Glu Lys Pro Leu Arg 290 295 300 Pro Phe Pro Asp
Asp Val Cys Val Val Pro Glu Lys Phe Glu Gly Asp 305 310 315 320 Ile
Lys Gln Glu Gly Val Gly Ala Phe Arg Glu Gly Pro Pro Tyr Gln 325 330
335 Arg Arg Gly Ala Leu Gln Leu Trp Gln Phe Leu Val Ala Leu Leu Asp
340 345 350 Asp Pro Thr Asn Ala His Phe Ile Ala Trp Thr Gly Arg Gly
Met Glu 355 360 365 Phe Lys Leu Ile Glu Pro Glu Glu Val Ala Arg Leu
Trp Gly Ile Gln 370 375 380 Lys Asn Arg Pro Ala Met Asn Tyr Asp Lys
Leu Ser Arg Ser Leu Arg 385 390 395 400 Tyr Tyr Tyr Glu Lys Gly Ile
Met Gln Lys Val Ala Gly Glu Arg Tyr 405 410 415 Val Tyr Lys Phe Val
Cys Glu Pro Glu Ala Leu Phe Ser Leu Ala Phe 420 425 430 Pro Asp Asn
Gln Arg Pro Ala Leu Lys Ala Glu Phe Asp Arg Pro Val 435 440 445 Ser
Glu Glu Asp Thr Val Pro Leu Ser His Leu Asp Glu Ser Pro Ala 450 455
460 Tyr Leu Pro Glu Leu Ala Gly Pro Ala Gln Pro Phe Gly Pro Lys Gly
465 470 475 480 Gly Tyr Ser Tyr <210> SEQ ID NO 254
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 254 Gly Tyr Leu Asp Gln Gln Val
Pro Tyr Thr Phe Ser 1 5 10 <210> SEQ ID NO 255 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 255 Leu Arg Glu Ala Leu Ile Gly Pro Leu Gly
Lys 1 5 10 <210> SEQ ID NO 256 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 256 Leu Phe Gln Asp Leu Ser His 1 5
<210> SEQ ID NO 257 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 257
Ser Glu Asn Leu Ala Phe His 1 5 <210> SEQ ID NO 258
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 258 Thr Asp Pro Ala Leu Ser Cys
1 5 <210> SEQ ID NO 259 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
259 Ser Arg Lys Pro Pro Leu Pro Tyr His His Gly Glu Gln Cys Leu Tyr
1 5 10 15 Ser Ser Ala Tyr 20 <210> SEQ ID NO 260 <211>
LENGTH: 25 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 260 Asp Pro Pro Arg Gln Ile Ala Ile Lys Ser
Pro Ala Pro Gly Ala Leu 1 5 10 15 Gly Gln Ser Pro Leu Gln Pro Phe
Pro 20 25 <210> SEQ ID NO 261 <211> LENGTH: 21
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 261 Ser Gln Pro His Pro Gly His Tyr Leu Gly
Glu His Ser Ser Val Phe 1 5 10 15 Gln Gln Pro Leu Asp 20
<210> SEQ ID NO 262 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 262
Ile Cys His Ser Phe 1 5 <210> SEQ ID NO 263 <211>
LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 263 Glu Pro Leu Pro Ala Pro Tyr Gln His Gln
Leu Ser Glu Pro Cys Pro 1 5 10 15 Pro Tyr Pro Gln Gln 20
<210> SEQ ID NO 264 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 264
Tyr His Asp Pro Leu Tyr Glu Gln Gly Gln Pro Ala Val Asp Gln 1 5 10
15 <210> SEQ ID NO 265 <211> LENGTH: 10 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
265 Arg Tyr Pro Gly Ala Gly Val Val Ile Lys 1 5 10 <210> SEQ
ID NO 266 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 266 Tyr Asp Ser Asp
Val Thr Gly Cys Ala Ser Met Tyr 1 5 10 <210> SEQ ID NO 267
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 267 Tyr Glu Lys Pro Leu Arg Pro
Phe Pro Asp Asp Val 1 5 10 <210> SEQ ID NO 268 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 268 Cys Val Val Pro Glu 1 5 <210> SEQ
ID NO 269 <211> LENGTH: 71 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 269 cgcgagctaa
gcaggaggcg gaggcggagg cggagggcga ggggcgggga gcgccgcctg 60
gagcgcggca g 71 <210> SEQ ID NO 270 <211> LENGTH: 142
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 270 cgcgagctaa gcaggaggcg gaggcggagg
cggagggcga ggggcgggga gcgccgcctg 60 gagcgcggca ggtcatattg
aacattccag atacctatca ttactcgatg ctgttgataa 120 cagcaagatg
gctttgaact ca 142 <210> SEQ ID NO 271 <211> LENGTH: 365
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 271 cgcgagctaa gcaggaggcg gaggcggagg
cggagggcga ggggcgggga gcgccgcctg 60 gagcgcggca ggtcatattg
aacattccag atacctatca ttactcgatg ctgttgataa 120 cagcaagatg
gctttgaact cagggtcacc accagctatt ggaccttact atgaaaacca 180
tggataccaa ccggaaaacc cctatcccgc acagcccact gtggtcccca ctgtctacga
240 ggtgcatccg gctcagtact acccgtcccc cgtgccccag tacgccccga
gggtcctgac 300 gcaggcttcc aaccccgtcg tctgcacgca gcccaaatcc
ccatccggga cagtgtgcac 360 ctcaa 365 <210> SEQ ID NO 272
<211> LENGTH: 452 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 272 cgcgagctaa gcaggaggcg
gaggcggagg cggagggcga ggggcgggga gcgccgcctg 60 gagcgcggca
ggtcatattg aacattccag atacctatca ttactcgatg ctgttgataa 120
cagcaagatg gctttgaact cagggtcacc accagctatt ggaccttact atgaaaacca
180 tggataccaa ccggaaaacc cctatcccgc acagcccact gtggtcccca
ctgtctacga 240 ggtgcatccg gctcagtact acccgtcccc cgtgccccag
tacgccccga gggtcctgac 300 gcaggcttcc aaccccgtcg tctgcacgca
gcccaaatcc ccatccggga cagtgtgcac 360 ctcaaagact aagaaagcac
tgtgcatcac cttgaccctg gggaccttcc tcgtgggagc 420 tgcgctggcc
gctggcctac tctggaagtt ca 452 <210> SEQ ID NO 273 <211>
LENGTH: 572 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 273 cgcgagctaa gcaggaggcg gaggcggagg
cggagggcga ggggcgggga gcgccgcctg 60 gagcgcggca ggtcatattg
aacattccag atacctatca ttactcgatg ctgttgataa 120 cagcaagatg
gctttgaact cagggtcacc accagctatt ggaccttact atgaaaacca 180
tggataccaa ccggaaaacc cctatcccgc acagcccact gtggtcccca ctgtctacga
240 ggtgcatccg gctcagtact acccgtcccc cgtgccccag tacgccccga
gggtcctgac 300 gcaggcttcc aaccccgtcg tctgcacgca gcccaaatcc
ccatccggga cagtgtgcac 360 ctcaaagact aagaaagcac tgtgcatcac
cttgaccctg gggaccttcc tcgtgggagc 420 tgcgctggcc gctggcctac
tctggaagtt catgggcagc aagtgctcca actctgggat 480 agagtgcgac
tcctcaggta cctgcatcaa cccctctaac tggtgtgatg gcgtgtcaca 540
ctgccccggc ggggaggacg agaatcggtg tg 572 <210> SEQ ID NO 274
<211> LENGTH: 67 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 274 ctttgataaa taagtttgta
agaggagcct cagcatcgta aagagctttt ctccccgctt 60 ctcgcag 67
<210> SEQ ID NO 275 <211> LENGTH: 33 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 275
atcgtaaaga gcttttctcc ccgcttctcg cag 33 <210> SEQ ID NO 276
<211> LENGTH: 102 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 276 gttattccag gatctttgga
gacccgagga aagccgtgtt gaccaaaagc aagacaaatg 60 actcacagag
aaaaaagatg gcagaaccaa gggcaactaa ag 102 <210> SEQ ID NO 277
<211> LENGTH: 86 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 277 ccgtcaggtt ctgaacagct
ggtagatggg ctggcttact gaaggacatg attcagactg 60 tcccggaccc
agcagctcat atcaag 86 <210> SEQ ID NO 278 <211> LENGTH:
218 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 278 gaagccttat cagttgtgag tgaggaccag
tcgttgtttg agtgtgccta cggaacgcca 60 cacctggcta agacagagat
gaccgcgtcc tcctccagcg actatggaca gacttccaag 120 atgagcccac
gcgtccctca gcaggattgg ctgtctcaac ccccagccag ggtcaccatc 180
aaaatggaat gtaaccctag ccaggtgaat ggctcaag 218 <210> SEQ ID NO
279 <211> LENGTH: 152 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 279 gaactctcct
gatgaatgca gtgtggccaa aggcgggaag atggtgggca gcccagacac 60
cgttgggatg aactacggca gctacatgga ggagaagcac atgccacccc caaacatgac
120 cacgaacgag cgcagagtta tcgtgccagc ag 152 <210> SEQ ID NO
280 <211> LENGTH: 204 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 280 atcctacgct
atggagtaca gaccatgtgc ggcagtggct ggagtgggcg gtgaaagaat 60
atggccttcc agacgtcaac atcttgttat tccagaacat cgatgggaag gaactgtgca
120 agatgaccaa ggacgacttc cagaggctca cccccagcta caacgccgac
atccttctct 180 cacatctcca ctacctcaga gaga 204 <210> SEQ ID NO
281 <211> LENGTH: 81 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 281 ctcctcttcc
acatttgact tcagatgatg ttgataaagc cttacaaaac tctccacggt 60
taatgcatgc tagaaacaca g 81 <210> SEQ ID NO 282 <211>
LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 282 ggggtgcagc ttttattttc ccaaatactt
cagtatatcc tgaagctacg caaagaatta 60 caactaggcc ag 72 <210>
SEQ ID NO 283 <211> LENGTH: 69 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 283
atttaccata tgagcccccc aggagatcag cctggaccgg tcacggccac cccacgcccc
60 agtcgaaag 69 <210> SEQ ID NO 284 <211> LENGTH: 57
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 284 ctgctcaacc atctccttcc acagtgccca
aaactgaaga ccagcgtcct cagttag 57 <210> SEQ ID NO 285
<211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 285 atccttatca gattcttgga
ccaacaagta gccgccttgc aaatccag 48 <210> SEQ ID NO 286
<211> LENGTH: 521 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 286 gcagtggcca gatccagctt
tggcagttcc tcctggagct cctgtcggac agctccaact 60 ccagctgcat
cacctgggaa ggcaccaacg gggagttcaa gatgacggat cccgacgagg 120
tggcccggcg ctggggagag cggaagagca aacccaacat gaactacgat aagctcagcc
180 gcgccctccg ttactactat gacaagaaca tcatgaccaa ggtccatggg
aagcgctacg 240 cctacaagtt cgacttccac gggatcgccc aggccctcca
gccccacccc ccggagtcat 300 ctctgtacaa gtacccctca gacctcccgt
acatgggctc ctatcacgcc cacccacaga 360 agatgaactt tgtggcgccc
caccctccag ccctccccgt gacatcttcc agtttttttg 420 ctgccccaaa
cccatactgg aattcaccaa ctgggggtat ataccccaac actaggctcc 480
ccaccagcca tatgccttct catctgggca cttactacta a 521 <210> SEQ
ID NO 287 <211> LENGTH: 54 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 287 ctcaggtacc
tgacaatgat gagcagtttg taccagacta tcaggctgaa agtt 54 <210> SEQ
ID NO 288 <211> LENGTH: 130 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 288 tggcttttca
tggcctgcca ctgaaaatca agaaagaacc ccacagtcca tgttcagaaa 60
tcagctctgc ctgcagtcaa gaacagccct ttaaattcag ctatggagaa aagtgcctgt
120 acaatgtcag 130 <210> SEQ ID NO 289 <211> LENGTH:
189 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 289 tgcctatgat cagaagccac aagtgggaat
gaggccctcc aaccccccca caccatccag 60 cacgccagtg tccccactgc
atcatgcatc tccaaactca actcatacac cgaaacctga 120 ccgggccttc
ccagctcacc tccctccatc gcagtccata ccagatagca gctaccccat 180
ggaccacag 189 <210> SEQ ID NO 290 <211> LENGTH: 248
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 290 atttcgccgc cagctttctg aaccctgtaa
ctcctttcct cctttgccga cgatgccaag 60 ggaaggacgt cctatgtacc
aacgccagat gtctgagcca aacatcccct tcccaccaca 120 aggctttaag
caggagtacc acgacccagt gtatgaacac aacaccatgg ttggcagtgc 180
ggccagccaa agctttcccc ctcctctgat gattaaacag gaacccagag attttgcata
240 tgactcag 248 <210> SEQ ID NO 291 <211> LENGTH: 69
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 291 aagtgcctag ctgccactcc atttatatga
ggcaagaagg cttcctggct catcccagca 60 gaacagaag 69 <210> SEQ ID
NO 292 <211> LENGTH: 69 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 292 gctgtatgtt
tgaaaagggc cccaggcagt tttatgatga cacctgtgtt gtcccagaaa 60 aattcgatg
69 <210> SEQ ID NO 293 <211> LENGTH: 170 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
293 gagacatcaa acaagagcca ggaatgtatc gggaaggacc cacataccaa
cggcgaggat 60 cacttcagct ctggcagttt ttggtagctc ttctggatga
cccttcaaat tctcatttta 120 ttgcctggac tggtcgaggc atggaattta
aactgattga gcctgaagag 170 <210> SEQ ID NO 294 <211>
LENGTH: 102 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 294 gtggcccgac gttggggcat tcagaaaaac
aggccagcta tgaactatga taaacttagc 60 cgttcactcc gctattacta
tgagaaagga attatgcaaa ag 102 <210> SEQ ID NO 295 <211>
LENGTH: 222 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 295 gtggctggag agagatatgt ctacaagttt
gtgtgtgatc cagaagccct tttctccatg 60 gcctttccag ataatcagcg
tccactgctg aagacagaca tggaacgtca catcaacgag 120 gaggacacag
tgcctctttc tcactttgat gagagcatgg cctacatgcc ggaagggggc 180
tgctgcaacc cccaccccta caacgaaggc tacgtgtatt aa 222 <210> SEQ
ID NO 296 <211> LENGTH: 93 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 296 aaatcgcccg
gaaatgggag cttgcgcgaa gcgctgatcg gcccgctggg gaagctcatg 60
gacccgggct ccctgccgcc ctcgactctg aag 93 <210> SEQ ID NO 297
<211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 297 atctcttcca ggatctaagt
cacttccagg agacgtggct cgctgaag 48 <210> SEQ ID NO 298
<211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 298 ctcaggtacc agacagtgat
gagcagtttg ttcctgattt ccattcagaa aacc 54 <210> SEQ ID NO 299
<211> LENGTH: 127 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 299 tagctttcca cagccccacc
accaggatca agaaggagcc ccagagtccc cgcacagacc 60 cggccctgtc
ctgcagcagg aagccgccac tcccctacca ccatggcgag cagtgccttt 120 actccag
127 <210> SEQ ID NO 300 <211> LENGTH: 162 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
300 tgcctatgac ccccccagac aaatcgccat caagtcccct gcccctggtg
cccttggaca 60 gtcgccccta cagccctttc cccgggcaga gcaacggaat
ttcctgagat cctctggcac 120 ctcccagccc caccctggcc atgggtacct
cggggaacat ag 162 <210> SEQ ID NO 301 <211> LENGTH: 266
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 301 ctccgtcttc cagcagcccc tggacatttg
ccactccttc acatctcagg gagggggccg 60 ggaacccctc ccagccccct
accaacacca gctgtcggag ccctgcccac cctatcccca 120 gcagagcttt
aagcaagaat accatgatcc cctgtatgaa caggcgggcc agccagccgt 180
ggaccagggt ggggtcaatg ggcacaggta cccaggggcg ggggtggtga tcaaacagga
240 acagacggac ttcgcctacg actcag 266 <210> SEQ ID NO 302
<211> LENGTH: 75 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 302 gtgtcaccgg gtgcgcatca
atgtacctcc acacagaggg cttctctggg ccctctccag 60 gtgacggggc catgg 75
<210> SEQ ID NO 303 <211> LENGTH: 69 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 303
gctatggcta tgagaaacct ctgcgaccat tcccagatga tgtctgcgtt gtccctgaga
60 aatttgaag 69 <210> SEQ ID NO 304 <211> LENGTH: 173
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 304 gagacatcaa gcaggaaggg gtcggtgcat
ttcgagaggg gccgccctac cagcgccggg 60 gtgccctgca gctgtggcaa
tttctggtgg ccttgctgga tgacccaaca aatgcccatt 120 tcattgcctg
gacgggccgg ggaatggagt tcaagctcat tgagcctgag gag 173 <210> SEQ
ID NO 305 <211> LENGTH: 102 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 305 gtcgccaggc
tctggggcat ccagaagaac cggccagcca tgaattacga caagctgagc 60
cgctcgctcc gatactatta tgagaaaggc atcatgcaga ag 102 <210> SEQ
ID NO 306 <211> LENGTH: 225 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 306 gtggctggtg
agcgttacgt gtacaagttt gtgtgtgagc ccgaggccct cttctctttg 60
gccttcccgg acaatcagcg tccagctctc aaggctgagt ttgaccggcc tgtcagtgag
120 gaggacacag tccctttgtc ccacttggat gagagccccg cctacctccc
agagctggct 180 ggccccgccc agccatttgg ccccaagggt ggctactctt actag
225 <210> SEQ ID NO 307 <211> LENGTH: 3226 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
307 cgcgagctaa gcaggaggcg gaggcggagg cggagggcga ggggcgggga
gcgccgcctg 60 gagcgcggca ggtcatattg aacattccag atacctatca
ttactcgatg ctgttgataa 120 cagcaagatg gctttgaact cagggtcacc
accagctatt ggaccttact atgaaaacca 180 tggataccaa ccggaaaacc
cctatcccgc acagcccact gtggtcccca ctgtctacga 240 ggtgcatccg
gctcagtact acccgtcccc cgtgccccag tacgccccga gggtcctgac 300
gcaggcttcc aaccccgtcg tctgcacgca gcccaaatcc ccatccggga cagtgtgcac
360 ctcaaagact aagaaagcac tgtgcatcac cttgaccctg gggaccttcc
tcgtgggagc 420 tgcgctggcc gctggcctac tctggaagtt catgggcagc
aagtgctcca actctgggat 480 agagtgcgac tcctcaggta cctgcatcaa
cccctctaac tggtgtgatg gcgtgtcaca 540 ctgccccggc ggggaggacg
agaatcggtg tgttcgcctc tacggaccaa acttcatcct 600 tcagatgtac
tcatctcaga ggaagtcctg gcaccctgtg tgccaagacg actggaacga 660
gaactacggg cgggcggcct gcagggacat gggctataag aataattttt actctagcca
720 aggaatagtg gatgacagcg gatccaccag ctttatgaaa ctgaacacaa
gtgccggcaa 780 tgtcgatatc tataaaaaac tgtaccacag tgatgcctgt
tcttcaaaag cagtggtttc 840 tttacgctgt atagcctgcg gggtcaactt
gaactcaagc cgccagagca ggatcgtggg 900 cggtgagagc gcgctcccgg
gggcctggcc ctggcaggtc agcctgcacg tccagaacgt 960 ccacgtgtgc
ggaggctcca tcatcacccc cgagtggatc gtgacagccg cccactgcgt 1020
ggaaaaacct cttaacaatc catggcattg gacggcattt gcggggattt tgagacaatc
1080 tttcatgttc tatggagccg gataccaagt agaaaaagtg atttctcatc
caaattatga 1140 ctccaagacc aagaacaatg acattgcgct gatgaagctg
cagaagcctc tgactttcaa 1200 cgacctagtg aaaccagtgt gtctgcccaa
cccaggcatg atgctgcagc cagaacagct 1260 ctgctggatt tccgggtggg
gggccaccga ggagaaaggg aagacctcag aagtgctgaa 1320 cgctgccaag
gtgcttctca ttgagacaca gagatgcaac agcagatatg tctatgacaa 1380
cctgatcaca ccagccatga tctgtgccgg cttcctgcag gggaacgtcg attcttgcca
1440 gggtgacagt ggagggcctc tggtcacttc gaagaacaat atctggtggc
tgatagggga 1500 tacaagctgg ggttctggct gtgccaaagc ttacagacca
ggagtgtacg ggaatgtgat 1560 ggtattcacg gactggattt atcgacaaat
gagggcagac ggctaatcca catggtcttc 1620 gtccttgacg tcgttttaca
agaaaacaat ggggctggtt ttgcttcccc gtgcatgatt 1680 tactcttaga
gatgattcag aggtcacttc atttttatta aacagtgaac ttgtctggct 1740
ttggcactct ctgccattct gtgcaggctg cagtggctcc cctgcccagc ctgctctccc
1800 taaccccttg tccgcaaggg gtgatggccg gctggttgtg ggcactggcg
gtcaagtgtg 1860 gaggagaggg gtggaggctg ccccattgag atcttcctgc
tgagtccttt ccaggggcca 1920 attttggatg agcatggagc tgtcacctct
cagctgctgg atgacttgag atgaaaaagg 1980 agagacatgg aaagggagac
agccaggtgg cacctgcagc ggctgccctc tggggccact 2040 tggtagtgtc
cccagcctac ctctccacaa ggggattttg ctgatgggtt cttagagcct 2100
tagcagccct ggatggtggc cagaaataaa gggaccagcc cttcatgggt ggtgacgtgg
2160 tagtcacttg taaggggaac agaaacattt ttgttcttat ggggtgagaa
tatagacagt 2220 gcccttggtg cgagggaagc aattgaaaag gaacttgccc
tgagcactcc tggtgcaggt 2280 ctccacctgc acattgggtg gggctcctgg
gagggagact cagccttcct cctcatcctc 2340 cctgaccctg ctcctagcac
cctggagagt gcacatgccc cttggtcctg gcagggcgcc 2400 aagtctggca
ccatgttggc ctcttcaggc ctgctagtca ctggaaattg aggtccatgg 2460
gggaaatcaa ggatgctcag tttaaggtac actgtttcca tgttatgttt ctacacattg
2520 ctacctcagt gctcctggaa acttagcttt tgatgtctcc aagtagtcca
ccttcattta 2580 actctttgaa actgtatcac ctttgccaag taagagtggt
ggcctatttc agctgctttg 2640 acaaaatgac tggctcctga cttaacgttc
tataaatgaa tgtgctgaag caaagtgccc 2700 atggtggcgg cgaagaagag
aaagatgtgt tttgttttgg actctctgtg gtcccttcca 2760 atgctgtggg
tttccaacca ggggaagggt cccttttgca ttgccaagtg ccataaccat 2820
gagcactact ctaccatggt tctgcctcct ggccaagcag gctggtttgc aagaatgaaa
2880 tgaatgattc tacagctagg acttaacctt gaaatggaaa gtcttgcaat
cccatttgca 2940 ggatccgtct gtgcacatgc ctctgtagag agcagcattc
ccagggacct tggaaacagt 3000 tggcactgta aggtgcttgc tccccaagac
acatcctaaa aggtgttgta atggtgaaaa 3060 cgtcttcctt ctttattgcc
ccttcttatt tatgtgaaca actgtttgtc tttttttgta 3120 tcttttttaa
actgtaaagt tcaattgtga aaatgaatat catgcaaata aattatgcga 3180
tttttttttc aaagcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 3226
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 307
<210> SEQ ID NO 1 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1
aacagagatc tggctcatga ttca 24 <210> SEQ ID NO 2 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 cttctgcaag ccatgtttcc tgta 24 <210>
SEQ ID NO 3 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 3
aggaaacatg gcttgcagaa gctc 24 <210> SEQ ID NO 4 <211>
LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 4 tctggtacaa actgctcatc attgtc 26 <210>
SEQ ID NO 5 <211> LENGTH: 26 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 5
ctcaggtacc tgacaatgat gagcag 26 <210> SEQ ID NO 6 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 6 catggactgt ggggttcttt cttg 24 <210>
SEQ ID NO 7 <211> LENGTH: 26 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 7
aacagccctt taaattcagc tatgga 26 <210> SEQ ID NO 8 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 8 ggagggcctc attcccactt g 21 <210> SEQ
ID NO 9 <211> LENGTH: 22 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 9 ctaccccatg
gaccacagat tt 22 <210> SEQ ID NO 10 <211> LENGTH: 22
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 10 cttaaagcct tgtggtggga ag 22 <210>
SEQ ID NO 11 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 11
cgcagagtta tcgtgccagc agat 24 <210> SEQ ID NO 12 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 12 ccatattctt tcaccgccca ctcc 24 <210>
SEQ ID NO 13 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 13
cgactggagc acgaggacac tga 23 <210> SEQ ID NO 14 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 14 catggactgt ggggttcttt cttg 24 <210>
SEQ ID NO 15 <211> LENGTH: 22 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 15
ggcgttccgt aggcacactc aa 22 <210> SEQ ID NO 16 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 16 cctggctggg ggttgagaca 20 <210> SEQ
ID NO 17 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 17 taggcgcgag
ctaagcagga g 21 <210> SEQ ID NO 18 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 18 gtaggcacac tcaaacaacg actgg 25 <210>
SEQ ID NO 19 <211> LENGTH: 19 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 19
cgcgagctaa gcaggaggc 19 <210> SEQ ID NO 20 <211>
LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 20 caggccatga aaagccaaac tt 22 <210>
SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 21
ccggatggag cggaggatga 20 <210> SEQ ID NO 22 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 22 cgggcgattt gctgctgaag 20 <210> SEQ
ID NO 23 <211> LENGTH: 19 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 23 gccgcccctc
gactctgaa 19 <210> SEQ ID NO 24 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 24 gagccacgtc tcctggaagt gact 24 <210>
SEQ ID NO 25 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 25 ctggccggtt cttctggatg c 21 <210> SEQ
ID NO 26 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 26 cgggccgggg aatggagt
18 <210> SEQ ID NO 27 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
27 cctggagggt accggtttgt ca 22 <210> SEQ ID NO 28 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 28 ccgcctgcct ctgggaacac 20 <210> SEQ
ID NO 29 <211> LENGTH: 30 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 29 aaataagttt
gtaagaggag cctcagcatc 30 <210> SEQ ID NO 30 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 30 atcgtaaaga gcttttctcc ccgc 24 <210>
SEQ ID NO 31 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 31
gaaagggctg taggggcgac tgt 23 <210> SEQ ID NO 32 <211>
LENGTH: 327 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 32 ccgtcggcgc cgagggagtt agtgcgaccc
ggctcggcgc gcacggccaa ggcacgcgcg 60 ctggcacacg cgggcgcgga
cacgcgcgga cacacacgtg cgggacacgc cctcccccga 120 cggcggcgct
aacctctcgg ttattccagg atctttggag acccgaggaa agccgtgttg 180
accaaaagca agacaaatga ctcacagaga aaaaagatgg cagaaccaag ggcaactaaa
240 gccgtcaggt tctgaacagc tggtagatgg gctggcttac tgaaggacat
gattcagact 300 gtcccggacc cagcagctca tatcaag 327 <210> SEQ ID
NO 33 <211> LENGTH: 6158 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 33 gttgatagaa
gtccagatcc tgaggaaatc tccagctaaa tgctcaaaat ataaaatact 60
gagctgagat ttgcgaagag cagcagcatg gatggatttt atgaccagca agtgccttac
120 atggtcacca atagtcagcg tgggagaaat tgtaacgaga aaccaacaaa
tgtcaggaaa 180 agaaaattca ttaacagaga tctggctcat gattcagaag
aactctttca agatctaagt 240 caattacagg aaacatggct tgcagaagct
caggtacctg acaatgatga gcagtttgta 300 ccagactatc aggctgaaag
tttggctttt catggcctgc cactgaaaat caagaaagaa 360 ccccacagtc
catgttcaga aatcagctct gcctgcagtc aagaacagcc ctttaaattc 420
agctatggag aaaagtgcct gtacaatgtc agtgcctatg atcagaagcc acaagtggga
480 atgaggccct ccaacccccc cacaccatcc agcacgccag tgtccccact
gcatcatgca 540 tctccaaact caactcatac accgaaacct gaccgggcct
tcccagctca cctccctcca 600 tcgcagtcca taccagatag cagctacccc
atggaccaca gatttcgccg ccagctttct 660 gaaccctgta actcctttcc
tcctttgccg acgatgccaa gggaaggacg tcctatgtac 720 caacgccaga
tgtctgagcc aaacatcccc ttcccaccac aaggctttaa gcaggagtac 780
cacgacccag tgtatgaaca caacaccatg gttggcagtg cggccagcca aagctttccc
840 cctcctctga tgattaaaca ggaacccaga gattttgcat atgactcaga
agtgcctagc 900 tgccactcca tttatatgag gcaagaaggc ttcctggctc
atcccagcag aacagaaggc 960 tgtatgtttg aaaagggccc caggcagttt
tatgatgaca cctgtgttgt cccagaaaaa 1020 ttcgatggag acatcaaaca
agagccagga atgtatcggg aaggacccac ataccaacgg 1080 cgaggatcac
ttcagctctg gcagtttttg gtagctcttc tggatgaccc ttcaaattct 1140
cattttattg cctggactgg tcgaggcatg gaatttaaac tgattgagcc tgaagaggtg
1200 gcccgacgtt ggggcattca gaaaaacagg ccagctatga actatgataa
acttagccgt 1260 tcactccgct attactatga gaaaggaatt atgcaaaagg
tggctggaga gagatatgtc 1320 tacaagtttg tgtgtgatcc agaagccctt
ttctccatgg cctttccaga taatcagcgt 1380 ccactgctga agacagacat
ggaacgtcac atcaacgagg aggacacagt gcctctttct 1440 cactttgatg
agagcatggc ctacatgccg gaagggggct gctgcaaccc ccacccctac 1500
aacgaaggct acgtgtatta acacaagtga cagtcaagca gggcgttttt gcgcttttcc
1560 ttttttctgc aagatacaga gaattgctga atctttgttt tatttctgtt
gtttgtattt 1620 tatttttaaa taataataca caaaaagggg cttttcctgt
tgcattattc tatggtctgc 1680 catggactgt gcactttatt tgagggtggg
tgggagtaat ctaaacattt attctgtgta 1740 acaggaagct aatgggtgaa
tgggcagagg gatttgggga ttacttttta cttaggcttg 1800 ggatggggtc
ctacaagttt tgagtatgat gaaactatat catgtctgtt tgatttcata 1860
acaacataag ataatgttta ttttatcggg gtatctatgg tacagttaat ttcacgttgt
1920 gtaaatatcc acttggagac tatttgcctt gggcattttc ccctgtcatt
tatgagtctc 1980 tgcaggtgta caaaaaaacc ccaatctact gtaaatggca
gtttaattgt tagaaatgac 2040 tgtttttgca ccacttgtaa aaaggtattt
agcgattgca tttgctgttt gttgttttat 2100 tttgctttat atatgacttg
cagaggataa ccataaaatg ggtaattctc tctgaagttg 2160 aataatcacc
atgactgtaa atgaggggca caattttgga ctctggcgcc aaactgagtc 2220
ataggccagt agcattacgt gtatctggtg ccaccttgct gtttagatac aaatcatacc
2280 gtcttttaaa tattttgaag cccatttcag ttaaataatg acatgtcatg
gtcctttgga 2340 atcttcattt aaatgttaaa tctggaatca aaatgaagca
aaaaatatct gtctcctttt 2400 cactttcttc agtacataaa tacattattt
aatcaataag aattaactgt actaaatcat 2460 gtattatgct gttctagtta
cagcaaacac tctttaagaa aaatatccaa tacactaaat 2520 aggtactata
gtaattttta gacatggtac ccattgatat gcatttaaac cttttactgc 2580
tgtgttatgt tgataacata tataaatatt agataatgct aatgcttctg ctgctgtctt
2640 ttctgtaata ttctctttca tgctgaattt actatgacca tttataagca
gtgcagttaa 2700 ctacagatag catttcagga caaaatagat gactcaaacc
atttattgct taaaaaatag 2760 cttacgccat gctatgctat aagcagcttt
tatgcacatt gacaaatgaa gagtaagctt 2820 cagcttgcta aaggaaactg
tggaaccttt tgtaactttt ggtgatatgg aaaattattt 2880 acaaaccgtc
aaagaatatg aggaagttgc tgtatgacat agtgctggca ctgatattat 2940
ccatcatctc tttttggaca cttctgtaaa tgtgattgga ttgtttgaaa gaagatttaa
3000 agtttcaaag ttttttgttc tgtttttgct ttgcatttgg agaaaatatt
gaaagcaggg 3060 tatgttgttt cattcacctt gaaaaaacca tgagtaaatg
gggatataga atctctgaat 3120 agctcgctaa aagattcaag caagggacat
gaattttgtt ccatctatca ataatatcca 3180 gaagaacaac ttttttaaag
agtctatagc aaaaagcaaa aaaaaaaaaa aattctaaac 3240 acaaagtcaa
aataaaccta ttgtaaaagc atttcgtgat gagcatgaaa aagattgttt 3300
aaagatgatc cccccagcta cccattttcc aaaactacac agatcacagc tcatttctct
3360 aagtggagca gttatcaaga aacccaaaca ccaaaattgc tactcttcac
atttaatcct 3420 acaaaaagta ctccaatttc aaaatatgta tgtaacctgc
gatttcaatg attgttgttc 3480 atatacatca tgtattattt tggcccattt
tgggcctaaa aaagaaaact atgccttaaa 3540 aatcagaacc ttttctcccc
actatgctta tgtggccatc tacagcactt agaataaaaa 3600 cagatgttaa
aatattcagt gaaagtttta ttggaaaaag gaattgagat atataattga 3660
gatttggtga aattgaagga gaaaatttaa gtgagtcttt aaaatatatt ctgaatgaaa
3720 actgtattga ggattcattt ttgttccttt tttttctttt tctcttttct
cctttttctt 3780 ctttttaata gtctagtttt agtcagtcag tgaggaagaa
ttgggccatg ctaacgttat 3840 cacaagagaa caatggcaga aatggtatta
gttatataat atttaaggac aaactatatg 3900 ttttgctgtt ttaacgtagt
gactcactga actaaataca taattgacca acattaagtg 3960 tatttccaat
acagaagggt tgaaaatatt acattataaa ctcttttgaa aaatgtatct 4020
aaaatttttt aagttctgtt ttgattccac tttttggttg agtttttatg tttttgtttt
4080 caggtagatt aataaatctg gcagctgatt tctgcaagat tcttgtgttt
tgaatttctc 4140 attgaattgg ctactcaaac atagaaatca tttgttaatg
atgtaatgtc ttctctcagc 4200 ttttatcttc actgctgttt gctgtctctt
gatgatgaca tgttaatacc caatagatta 4260 attgcaacaa acacttatac
tcaaataact aagtaaaaat aatttttctt gttatgtcca 4320 tgaaaagtgc
ttcagaataa aaatccacaa gactgacagt gcagaacatt tttctcaaat 4380
catgggcgga tcttggaggt ctagtttccc gtagatgctg taaccaatta ccacaacttc
4440 agtaatttac acaaatttat cttatagttc tggaggcaga agttcaaaag
aagccttaag 4500 agactaaaac caagatgtcc ttaggtctgg ttccttctgg
aggctccagg ggagattctt 4560 ccagctttca cttctagagt ctgctgacat
tccttggctc ctggctacat cacttcaatc 4620 tctgcttcca tggtcacata
ctcttctact atagtcaaat ttccttcctg cctcttataa 4680 ggatgcttgt
gattacattt aggggatgct cagataatcc aggacaatct ctccatctca 4740
agatccttaa cttaatgacg tgtgccaagt ccctttggct agataattat tcataggtcc
4800 cagggattag gacatggatg taaggggtga gggcagggct gttattcaga
acaccgcacg 4860 gaggaggaag actgtgtagc aaagactcta attgatttac
tcaggaacag tggagttctg 4920 ctgagggatc taggatttga aagtactaga
gtttgctttt atttaccact gagatatttt 4980 ccccttattc tgcataaata
attttgaaaa ctttctatat taaatttcaa ctattccact 5040 aaaatgtctg
gtaatcacat caagccttta gattattcaa atccttcccc agcccccagg 5100
aaaacactaa gtcatgaaac agaaaaacag aaggtatgat aataatagta ataacagtta
5160 aatcagtggt ctaatccaga ttttattttt taatacattt cttttggtgt
taatatgggt 5220 tactatgtga tcttatcatt tgctagtgat tattacttat
taggtaagaa caatgtgtaa 5280 aatatgtcta ttactcaaaa gaacaattgc
aaaatgagtc aacttatctt tatataacca 5340 ggaaagaaat atattgccag
aagctacaga attttgccag atgataggga tttctaaaat 5400 gagccacttt
gtctatcatg cagccttttc agagcttgta atgagaaaac attacagagg 5460
agaaggtcat ttggatgttt gttacttgga atcctagaaa acaaaaacta aaatttaaaa
5520 ataagaagtg agtaagctat tttccatttg cgatttggta tggagaagag
aggaaataga 5580 attattaaaa aaatacaaat tgggtaaaag tgatggtgga
aaaaatataa agaaggcaaa 5640 tgtacatatt aagcaattct actaagaatt
ggaaaaatca agtttcaaaa agatggtaat 5700 agttgggcat gatactagaa
aatttcaccc agtttattca gagctcaact agtactttta 5760 ggacttcttt
ttttatatac atgagactca ctttgacata cttaaaaaaa aaacagttta 5820
tggaaagtac agtttaagag gagaatttga ttagactaag tggatatctt tatagaaata
5880 ttaatgattt cagaattttc agttacaagt gtatataccg tggctattgt
ttatggattc 5940 atatgtaagg tagggtcttt tttgcatata gactccagta
ttagttactt tcattctaaa 6000 attatattta tgcttctatg gggaagaaaa
tttttaattc acttggttgt attaaaatta 6060 tacttacggt ttgagaaaac
atgctatgaa aatcatgatt atagcaaatt aaatatgctc 6120 aaaatttaaa
tctaaaataa aagcccagaa actgaaaa 6158 <210> SEQ ID NO 34
<211> LENGTH: 5228 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 34 cgggcgaggg
ccgggcagga ggagcgggcg cggcgcgggc gaggctggga cccgagcgcg 60
ctcacttcgc cgcaaagtgc caacttcccc tggagtgccg ggcgcgcacc gtccgggcgc
120 gggggaaaga aaggcagcgg gaatttgaga tttttgggaa gaaagtcgga
tttcccccgt 180 ccccttcccc ctgttactaa tcctcattaa aaagaaaaac
aacagtaact gcaaacttgc 240 taccatcccg tacgtccccc actcctggca
ccatgaaggc ggccgtcgat ctcaagccga 300 ctctcaccat catcaagacg
gaaaaagtcg atctggagct tttcccctcc ccggatatgg 360 aatgtgcaga
tgtcccacta ttaactccaa gcagcaaaga aatgatgtct caagcattaa 420
aagctacttt cagtggtttc actaaagaac agcaacgact ggggatccca aaagaccccc
480 ggcagtggac agaaacccat gttcgggact gggtgatgtg ggctgtgaat
gaattcagcc 540 tgaaaggtgt agacttccag aagttctgta tgaatggagc
agccctctgc gccctgggta 600 aagactgctt tctcgagctg gccccagact
ttgttgggga catcttatgg gaacatctag 660 agatcctgca gaaagaggat
gtgaaaccat atcaagttaa tggagtcaac ccagcctatc 720 cagaatcccg
ctatacctcg gattacttca ttagctatgg tattgagcat gcccagtgtg 780
ttccaccatc ggagttctca gagcccagct tcatcacaga gtcctatcag acgctccatc
840 ccatcagctc ggaagagctc ctctccctca agtatgagaa tgactacccc
tcggtcattc 900 tccgagaccc tctccagaca gacaccttgc agaatgacta
ctttgctatc aaacaagaag 960 tcgtcacccc agacaacatg tgcatgggga
ggaccagtcg tggtaaactc gggggccagg 1020 actcttttga aagcatagag
agctacgata gttgtgatcg cctcacccag tcctggagca 1080 gccagtcatc
tttcaacagc ctgcagcgtg ttccctccta tgacagcttc gactcagagg 1140
actatccggc tgccctgccc aaccacaagc ccaagggcac cttcaaggac tatgtgcggg
1200 accgtgctga cctcaataag gacaagcctg tcattcctgc tgctgcccta
gctggctaca 1260 caggcagtgg accaatccag ctatggcagt ttcttctgga
attactcact gataaatcct 1320 gtcagtcttt tatcagctgg acaggagatg
gctgggaatt caaactttct gacccagatg 1380 aggtggccag gagatgggga
aagaggaaaa acaaacctaa gatgaattat gagaaactga 1440 gccgtggcct
acgctactat tacgacaaaa acatcatcca caagacagcg gggaaacgct 1500
acgtgtaccg ctttgtgtgt gacctgcaga gcctgctggg gtacacccct gaggagctgc
1560 acgccatgct ggacgtcaag ccagatgccg acgagtgatg gcactgaagg
ggctggggaa 1620 accctgctga gaccttccaa ggacagccgt gttggttgga
ctctgaattt tgaattgtta 1680 ttctattttt tattttccag aactcatttt
ttaccttcag gggtgggagc taagtcagtt 1740 gcagctgtaa tcaattgtgc
gcagttggga aaggaaagcc aggacttgtg gggtgggtgg 1800 gaccagaaat
tcttgagcaa attttcagga gagggagaag ggccttctca gaagcttgaa 1860
ggctctggct taacagagaa agagactaat gtgtccaatc atttttaaaa atcatccatg
1920 aaaaagtgtc ttgagttgtg gacccattag caagtgacat tgtcacatca
gaactcatga 1980 aactgatgta aggcaattaa tttgcttctg tttttaggtc
tgggagggca aaaaagaggt 2040 gggtgggatg aaacatgttt tgggggggga
tgcactgaaa atctgagaac tatttaccta 2100 tcactctagt tttgaagcaa
agatggactt cagtggggag gggccaaaac cgttgttgtg 2160 ttaaaattta
ttttattaaa ttttgtgcca gtattttttt tcttaaaaat cgtcttaagc 2220
tctaaggtgg tctcagtatt gcaatatcat gtaagtttgt ttttatttgc cggctgagga
2280 ttctgtcaca atgaaagaaa actgtttata tagaccccat tggaaaagca
aaacgctctc 2340 actgagatca gggatcccaa attcatggga cttatataag
aaggacaatt aatgctgatt 2400 tgggtacagg ggaattatgt gtgtgaatgt
catctacaat taaaaaaaat tagcacatcc 2460 ctttacttac ttgttatcag
tggattctcg gggtttggac ttaatgttga gctaagaagc 2520 attaagtctt
tgaactgaat gtattttgca tccctggttt tggacgacag taaacgtagg 2580
agcactgttg aagtcctgga agggagatcg aaggaggaag attgacttgg ttctttctta
2640 gtcctatatc tgtagcatag atgacttgga ataaaagctg tatgcatggg
cattacccct 2700 caggtcctaa gaaataagtc ctgaatgcat gtcgttccaa
actaacactc tgtaattttt 2760 cttttatgtc ttattttcca agagtcctcc
attttttgca ccccctcacc gccaactctg 2820 ttattcagta gagagaagtg
tacggctttc tgattggtga gtgaaaaagt aacttgagac 2880 acgacctaag
ttgaagagtt tagacttgct gagttttaga agtgatggaa attaagagag 2940
catttcaata aaatgtgact tggctgtctt tggaagagaa gtgcaaggct ttcctttgaa
3000 gaatttaaat tagtccggta ggatgtcagg tgagactgtg tatgcaaaat
gaatggcaca 3060 ggtgatgcca gggcctcttg cttgggtctg atgtcttggc
acagggtaag tgaaggttaa 3120 ttccagaaga gaggaatgac ttgaaggcaa
aggaaactaa ggaaggaggt tcagtgagga 3180 aaataaggtt gtccatgaga
tttgaataga tttttagttc ccccaaggtt taaatacaaa 3240 catagtcaag
caaggtagtc atctttctgc tggttgtgag ggggaatctg aaaatggagt 3300
tttagaggaa aagtcaacat ctaactagtg aggaaaagtg cctaatacaa ttagaatctc
3360 cctcactcta tagttgccca gttgaaagga taaggaggag gggtggcttt
tatggacttc 3420 catgagagaa ggaaagaaat atttcaggta agcttctcag
ggctggccct ttttgggatt 3480 tggatgagaa attggaagta ctaactactt
tctagcatat ctttaagaaa attgattgtt 3540 atttactccc agatcctctt
gcagacccag aattatcagg aacatagctc tgtgattcat 3600 gagtgtcccc
atactgatga attggagcat ccatatggaa agcaaaggca gaattatccc 3660
agctgtatta ttttgatctt ttggatgcag gtgccttaat gaagctctca aaatatttta
3720 ggagctgctc agggagtgtt gggtggaact gtttggacta cattgttttc
tcttagatta 3780 tgtgattttt gttgggcact ggcaaaaggt gtgtgtgtga
atgtgtgcat gtgtgtgaat 3840 gttgtgtgtg tgtgtgtgtg tgtgtgtgtg
tgtgtgtgtg tttgcagaca tgcaaaactg 3900 cagctgaaat aataccttag
atttctaggt aagtctttcc acatttcaat aatgggtaag 3960 agtagaacca
gggccgggta tcaattattg cttgctgttt gcaaccaggc ataaaatcac 4020
tttctcaaat catccaccgt tcctattaaa tttatgccgg aaactctcct tctgtgagta
4080 taactcctgc agttcctata gcagataaga tataagaaag tgcctcctag
tgctcctccg 4140 cccgcttgtt tgctaaaatt ccctttctct ctaagtccac
cattttcaag atttgtagat 4200 agtgtattag ttaagacagc tttgtcgatc
tggccagatg ttttttctcc tttgtccaaa 4260 ggccagagac catcccagga
agagtggtgg gtggtttata cactggaaat gttgcgttta 4320 tgctttttaa
aaacacacgt taacttcaga ggaaggatgg gcaaatctgg tctagctggg 4380
tgaaaccctt attttcccag agatgcctta acctttgttg gttttggctt tagggttcag
4440 agtcactttt gttcccttct ccattctgga gagggacttc ccctacatag
agccctgatt 4500 tttgtggctg tggggattgg aggtagcatt caaagatcag
atgtgctttt cctcactttg 4560 gagatgaaca ctctgggttt tacagcatta
acctgcctaa ccttcatggt gagaaataca 4620 ccatctctct tctagtcatg
ctgtgcatgc cgcttactct gttggggtct atataaattt 4680 gttgaactct
tacctacatt ccaaagaagt ttcaaggaac cataaatata tgtatacata 4740
tacatatata aaatatatat attaaaataa aattatcagg aatactgcct cagttattga
4800 actttttttt ttaagaatac ttttttttta agctgagaag tatagggatg
aaaaagatgt 4860 tatattgtgt ttgactattt tccaacttgt attttcatat
aatttatatt ttttaaaagc 4920 tgaaaattta gaagcaagat gaaaaaaagg
aaaagcaggt gctttttaaa aatcagaact 4980 gaggtagctt agagatgtag
cgatgtaagt gtcgatgttt ttttaaaaaa aaatgcaaaa 5040 aaattcttat
ggcggagttt tttgtttgtt tattttagta gctgatgctg gcacatcatt 5100
ttgctggaga gttttttata tactgtagcc tgatttcata ttgtatttta aactgtgtga
5160 aattaaaaac aaagaatttc attcataaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 5220 aaaaaaaa 5228 <210> SEQ ID NO 35 <211>
LENGTH: 3672 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 35 gcccggttac ttcctccaga gactgacgag
tgcggtgtcg ctccagctca gagctcccgg 60 agccgcccgg ccagcgtccg
gcctccctga tcgtctctgg ccggcgccct cgccctcgcc 120 cggcgcgcac
cgagcagccg cgggcgccga gcagccaccg tcccgaccaa gcgccggccc 180
tgcccgcagc ggcaggatga atgatttcgg aatcaagaat atggaccagg tagcccctgt
240 ggctaacagt tacagaggga cactcaagcg ccagccagcc tttgacacct
ttgatgggtc 300 cctgtttgct gtttttcctt ctctaaatga agagcaaaca
ctgcaagaag tgccaacagg 360
cttggattcc atttctcatg actccgccaa ctgtgaattg cctttgttaa ccccgtgcag
420 caaggctgtg atgagtcaag ccttaaaagc taccttcagt ggcttcaaaa
aggaacagcg 480 gcgcctgggc attccaaaga acccctggct gtggagtgag
caacaggtat gccagtggct 540 tctctgggcc accaatgagt tcagtctggt
gaacgtgaat ctgcagaggt tcggcatgaa 600 tggccagatg ctgtgtaacc
ttggcaagga acgctttctg gagctggcac ctgactttgt 660 gggtgacatt
ctctgggaac atctggagca aatgatcaaa gaaaaccaag aaaagacaga 720
agatcaatat gaagaaaatt cacacctcac ctccgttcct cattggatta acagcaatac
780 attaggtttt ggcacagagc aggcgcccta tggaatgcag acacagaatt
accccaaagg 840 cggcctcctg gacagcatgt gtccggcctc cacacccagc
gtactcagct ctgagcagga 900 gtttcagatg ttccccaagt ctcggctcag
ctccgtcagc gtcacctact gctctgtcag 960 tcaggacttc ccaggcagca
acttgaattt gctcaccaac aattctggga ctcccaaaga 1020 ccacgactcc
cctgagaacg gtgcggacag cttcgagagc tcagactccc tcctccagtc 1080
ctggaacagc cagtcgtcct tgctggatgt gcaacgggtt ccttccttcg agagcttcga
1140 agatgactgc agccagtctc tctgcctcaa taagccaacc atgtctttca
aggattacat 1200 ccaagagagg agtgacccag tggagcaagg caaaccagtt
atacctgcag ctgtgctggc 1260 cggcttcaca ggaagtggac ctattcagct
gtggcagttt ctcctggagc tgctatcaga 1320 caaatcctgc cagtcattca
tcagctggac tggagacgga tgggagttta agctcgccga 1380 ccccgatgag
gtggcccgcc ggtggggaaa gaggaaaaat aagcccaaga tgaactacga 1440
gaagctgagc cggggcttac gctactatta cgacaagaac atcatccaca agacgtcggg
1500 gaagcgctac gtgtaccgct tcgtgtgcga cctccagaac ttgctggggt
tcacgcccga 1560 ggaactgcac gccatcctgg gcgtccagcc cgacacggag
gactgaggtc gccgggacca 1620 ccctgagccg gccccaggct cgtggactga
gtgggaagcc catcctgacc agctgctccg 1680 aggacccagg aaaggcagga
ttgaaaatgt ccaggaaagt ggccaagaag cagtggcctt 1740 attgcatccc
aaaccacgcc tcttgaccag gctgcctccc ttgtggcagc aacggcacag 1800
ctaattctac tcacagtgct tttaagtgaa aatggtcgag aaagaggcac caggaagccg
1860 tcctggcgcc tggcagtccg tgggacggga tggttctggc tgtttgagat
tctcaaagga 1920 gcgagcatgt cgtggacaca cacagactat ttttagattt
tcttttgcct tttgcaacca 1980 ggaacagcaa atgcaaaaac tctttgagag
ggtaggaggg tgggaaggaa acaaccatgt 2040 catttcagaa gttagtttgt
atatattatt ataatcttat aattgttctc agaatccctt 2100 aacagttgta
tttaacagaa attgtatatt gtaatttaaa ataattatat aactgtattt 2160
gaaataagaa ttcagacatc tgaggtttta tttcattttt caatagcaca tatggaattt
2220 tgcaaagatt taatctgcca agggccgact aagagaagtt gtaaagtatg
tattatttac 2280 atttaataga cttacaggga taaggcctgt ggggggtaat
ccctgctttt tgtgtttttt 2340 tgtttgtttg tttgtttgtt tttggggggt
tttcttgcct tggttgtctg gcaaggactt 2400 tgtacatttg ggagttttta
tgagaaactt aaatgttatt atctgggctt atatctggcc 2460 tctgctttct
cctttaattg taaagtaaaa gctataaagc agtatttttc ttgacaaatg 2520
gcatatgttt tccacttctt tgcatgcgtt taagtcagtt tatacacaaa atggatttta
2580 ttttttagtt taactgtgtt tctccgacag ctcacctctc tctgaccacc
cagccatttc 2640 cttcctgtgc tccacgttct tctgtgtgat taaaataaga
atattatttt tggaaatatg 2700 caactccttt tcagagatca ggagggattt
atgtagcagc tatttttact gcaaaagtaa 2760 ttcactggaa aaaaaatgta
atttgtaaga aagctttatt tttatctcag ctctatgtaa 2820 agttaaagtt
actgtacaga gctgaaggac ggggggcggt aggggtcttg atgaaacctc 2880
ttgaacgaag cacagtttgt cccatctttg ttcactcgtg tgtctcaacc atcttaatag
2940 catgctgctc ctttttgctc agtgtccaca gcaagatgac gtgattctta
ttttcttgga 3000 cacagactat tctgaggcac agagcgggga cttaagatgg
gaaagagaaa gcatcggagc 3060 cattcattcg gagaaaacgt tttgatcaaa
atggagactt ttgtagtcgt ttcaaaagag 3120 cacctgagtc atgtgtattc
ccggccttta taaatgaccc ggtcaagttg gtttcaaagt 3180 ccgacaggct
tgtctgttta ctagctgcgt ggccttggac gggtggctga catctgtaaa 3240
gaatcctcct gtgatgaaac tgaggaatcg ggtggccggg caagctggga agagcaaagc
3300 cagagctgcg ctgcctcaat acccacaaaa gaccattccc agtatacata
agcacaggat 3360 gtttttctca agagggatgt atttatcact tggacatctg
tttataatat aaacagacat 3420 gtgactggga acatcttgct gccaaaagaa
tcctaggcag tggctcattg tatgtgaggt 3480 tgaaccacgt gaaattgcca
atattaggct ggcttttatc tacaaagaag gagtttcatg 3540 gggttcagcc
taacagttat ggaaactaca gtccttataa accattggca tggtaataaa 3600
cagatcttaa gtataaaaat tttgtaattg ggcctttact ctctcaataa taaagtattt
3660 tgtttatata aa 3672 <210> SEQ ID NO 36 <211>
LENGTH: 2668 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 36 cgctacacac aggtacccct gggatggcgt
gagcactccc ccagcgatgg acccatctgt 60 gacgctgtgg cagtttctgc
tgcagctgct gagagagcaa ggcaatggcc acatcatctc 120 ctggacttca
cgggatggtg gtgaattcaa gctggtggat gcagaggagg tggcccggct 180
gtgggggcta cgcaagaaca agaccaacat gaattacgac aagctcagcc gggccttgcg
240 gtactactat gacaagaaca tcatccgcaa ggtgagcggc cagaagttcg
tctacaagtt 300 tgtgtcctac cctgaggtcg cagggtgctc cactgaggac
tgcccgcccc agccagaggt 360 gtctgttacc tccaccatgc caaatgtggc
ccctgctgct atacatgccg ccccagggga 420 cactgtctct ggaaagccag
gcacacccaa gggtgcagga atggcaggcc caggcggttt 480 ggcacgcagc
agccggaacg agtacatgcg ctcgggcctc tattccacct tcaccatcca 540
gtctctgcag ccgcagccac cccctcatcc tcggcctgct gtggtgctcc ccagtgcagc
600 tcctgcaggg gcagcagcgc ccccctcggg gagcaggagc accagtccaa
gccccttgga 660 ggcctgtctg gaggctgaag aggccggctt gcctctgcag
gtcatcctga ccccgcccga 720 ggccccaaac ctgaaatcgg aagagcttaa
tgtggagccg ggtttgggcc gggctttgcc 780 cccagaagtg aaagtagaag
ggcccaagga agagttggaa gttgcggggg agagagggtt 840 tgtgccagaa
accaccaagg ccgagccaga agtccctcca caggagggcg tgccagcccg 900
gctgcccgcg gttgttatgg acaccgcagg gcaggcgggc ggccatgcgg cttccagccc
960 tgagatctcc cagccgcaga agggccggaa gccccgggac ctagagcttc
cactcagccc 1020 gagcctgcta ggtgggccgg gacccgaacg gaccccagga
tcgggaagtg gctccggcct 1080 ccaggctccg gggccggcgc tgaccccatc
cctgcttcct acgcatacat tgaccccggt 1140 gctgctgaca cccagctcgc
tgcctcctag cattcacttc tggagcaccc tgagtcccat 1200 tgcgccccgt
agcccggcca agctctcctt ccagtttcca tccagtggca gcgcccaggt 1260
gcacatccct tctatcagcg tggatggcct ctcgaccccc gtggtgctct ccccagggcc
1320 ccagaagcca tgactactac caccaccacc accacccctt ctggggtcac
tccatccatg 1380 ctctctccag ccagccatct caaggagaaa catagttcaa
ctgaaagact catgctctga 1440 ttgtggtggg gtggggatcc ttgggaagaa
ttactcccaa gagtaactct cattatctcc 1500 tccacagaaa acacacagct
tccacaactt ctctgttttc tgtcagtccc ccagtggccg 1560 cccttacacg
tctcctactt caatggtagg ggcggtttat ttatttattt tttgaaggcc 1620
actgggagga gcctgaccta accttttagg gtggttagga catctccccc acctccccac
1680 ttttttcccc aagacaagac aatcgaggtc tggcttgaga acgacctttc
tttctttatt 1740 tctcagcctg cccttgggga gatgagggag ccctgtctgc
gtttttggat gtgagtagaa 1800 gagttagttt gttttgtttt attattcctg
gccatactca ggggtccagg aagaatttgt 1860 accatttaat gggttgggag
tcttggccaa ggaagaatca cacccttgga atagaaattt 1920 ccacctcccc
aacctttctc tcagacagct tatccttttc aaccaacttt ttggccaggg 1980
aggaatgtcc cttttgttct tccccctgag aagccattcc tttgtctgcc aacctccctg
2040 gggtcctgcc tgtttcctcc caatggaggg tttttttggg gggtggtccc
cgtctggggg 2100 gcccctccag ccagtactcc aggtctccct gtctctcccc
cgctgccatt ttgatagtat 2160 aatctatttt taaatggggc ttttcaatag
gggagaggga gtcatctctt cctatatttg 2220 gtggggtggg tgggaaggaa
gggatttggg ggggaatctt cctgccgcct cccccactcc 2280 aagtgtttat
ttttgatacc aaacatgaat tttcagttcc ctccctccca gccccccaat 2340
ttcctgcggg cgggtacaaa ggaccctttc aatgtccctg gagttgggag ggaggaatgg
2400 gggacataaa gcctgtcctg tctctattct aggcaagaga gagtgggttc
aaaagactcc 2460 tgggctcacc tgttagcgct ggcccagccc aggccttggg
acctgggggt tggtgatttg 2520 ggggacagtg ctacactcgt ctccactgtt
tgttttactt ccccaaaatg gacctttttt 2580 ttttctaaag agtcccagag
aatggggaat tgttcctgta aatatatatt tttcaaagtg 2640 aaaaaaaaaa
aaaaaaaaaa aaaaaaaa 2668 <210> SEQ ID NO 37 <211>
LENGTH: 5992 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 37 gcgtcccggg tccccgcgcc gcgccgcgac
ctgcagaccc cgccgccgcg ctcgggcccg 60 tctcccacgc ccccgccgcc
ccgcgcgccc aactccgccg gccgccccgc cccgccccgc 120 gcgctccaga
cccccggggc ggctgccggg agagatgctg gaagaaactt cttaaatgac 180
cgcgtctggc tggccgtgga gcctttctgg gttggggaga ggaaaggaaa gtggaaaaaa
240 cctgagaact tcctgatctc tctcgctgtg agacatgtct gagactcctg
ctcagtgtag 300 cattaagcag gaacgaattt catatacacc tccagagagc
ccagtgccga gttacgcttc 360 ctcgacgcca cttcatgttc cagtgcctcg
agcgctcagg atggaggaag actcgatccg 420 cctgcctgcg cacctgcgct
tgcagccaat ttactggagc agggatgacg tagcccagtg 480 gctcaagtgg
gctgaaaatg agttttcttt aaggccaatt gacagcaaca cgtttgaaat 540
gaatggcaaa gctctcctgc tgctgaccaa agaggacttt cgctatcgat ctcctcattc
600 aggtgatgtg ctctatgaac tccttcagca tattctgaag cagaggaaac
ctcggattct 660 tttttcacca ttcttccacc ctggaaactc tatacacaca
cagccggagg tcatactgca 720 tcagaaccat gaagaagata actgtgtcca
gaggaccccc aggccatccg tggataatgt 780 gcaccataac cctcccacca
ttgaactgtt gcaccgctcc aggtcaccta tcacgacaaa 840 tcaccggcct
tctcctgacc ccgagcagcg gcccctccgg tcccccctgg acaacatgat 900
ccgccgcctc tccccggctg agagagctca gggacccagg ccgcaccagg agaacaacca
960
ccaggagtcc taccctctgt cagtgtctcc catggagaat aatcactgcc cagcgtcctc
1020 cgagtcccac ccgaagccat ccagcccccg gcaggagagc acacgcgtga
tccagctgat 1080 gcccagcccc atcatgcacc ctctgatcct gaacccccgg
cactccgtgg atttcaaaca 1140 gtccaggctc tccgaggacg ggctgcatag
ggaagggaag cccatcaacc tctctcatcg 1200 ggaagacctg gcttacatga
accacatcat ggtctctgtc tccccgcctg aagagcacgc 1260 catgcccatt
gggagaatag cagactgtag actgctttgg gattacgtct atcagttgct 1320
ttctgacagc cggtacgaaa acttcatccg atgggaggac aaagaatcca aaatattccg
1380 gatagtggat cccaacggac tggctcgact gtggggaaac cataagaaca
gaacaaacat 1440 gacctatgag aaaatgtcca gagccctgcg ccactactac
aaactaaaca ttatcaggaa 1500 ggagccagga caaaggcttt tgttcaggtt
tatgaaaacc ccagatgaaa tcatgagtgg 1560 ccgaacagac cgtctggagc
acctagagtc ccaggagctg gatgaacaaa tataccaaga 1620 agatgaatgc
tgaaggaacc aacagtccac ctcagcgggc cagcagccca gggaacccct 1680
gcccaccagg attgctggaa gtgtgacgga gcaggcgggc tgaggagagt ggaaaaggaa
1740 gcgacccaga aatggcaggg acacttctct tgcagaccaa gagggaccct
ggagcacctt 1800 agacaaacta cccagcacag gcggggctgg aattctggcg
gagggcatga gcctgggact 1860 ccatgtcacg tttccttctg atttggaatc
tctccatctg taattcctca ccctcaccct 1920 tccaccgttg ttagtatcat
ggtgtttttg tttttgtttt tgttttaaga acctgcagtt 1980 tgactcttca
tcgttcatct aggggaagac atctgatgtt gttttcctat ggaaatatat 2040
atctattata tatatatttt ttgcaaatct cacaaagtgc ggcaagccca gctggtcagg
2100 aaagagaata cttgcagagg ggttcaggtt cctctttttc ctgccacgtg
gatcaggtct 2160 gttcctgtta ctgttgggtc ttggctgaaa aaaaaaaatg
cttttaaaaa agataaaatg 2220 aaaaggagag ctctcttttt ctctctcttg
ctctgttctt cccttggtcc cctctgtcct 2280 cccgccctgc ctgcagttga
gattcagatg ccttctgaca gagttcagcc tcttggagag 2340 tcttggggat
tgttggcacc taaacagaat cagtgacccg ggtgctttgt ggccagcagc 2400
acagaatcaa acccgcatcc cagcattggg ccacccatct gagggaggcc aaaatcatca
2460 cagatgctgc tgtgctgcag acagatacat gctagtccag agagccgccc
ctgagatggc 2520 tgtgagaacc atgtgtctaa ggcgtaagat aaggatggaa
ggctgtccaa gttatttgga 2580 aggcctcggc agcttgggat tagcttggga
gcgcagcgct gcaaagtgga aaatatgaaa 2640 agaccacaca ggcccagcag
tccagaaact gggcaaaaat attctgcagt ggggatttat 2700 ttttccaaag
caggtaacag aggctagtga gaaagaaaag ctcctctctg ctccattcca 2760
aaggccatct tgtggtcagt ttcatgccct cacctgattt tttttttttt tttttttttt
2820 caattcctaa ccttttttaa agtttcctgg tctccactgg acacagagct
ttggagacgg 2880 aggatcccag agggcagtct cagttgcaat cagtgtgtgc
ccagcctggg cagacaggaa 2940 attcctcgga tacattattt tttctttctt
tcatagctgt gtctcagaaa ggacccattt 3000 gtggctcttt ttcacctcaa
aataagatcg atggtatctt gtaaaatgag ggtagtgcca 3060 cttcttagta
tttttgaaag ctgttttaga tttttttttt ttttcctttt ctagccatct 3120
aaattgactc ttccaatata ggtctcagaa atccaatatt tggagtacaa tttcttttaa
3180 tccagattac acctgcctta caaagcaccc cctccttgtt cccctctgtt
tcctctactc 3240 agttggggga gaaactcaca gctcctccgg gatacatatg
tgccctcagc agcagctccc 3300 aggtgaagtt accagacccc tgggcttctc
cccagctttt tctgagttga gtcagacatg 3360 tagagtttgg gtcacacagg
caagaggaat tttccctcgg ccttactgac aaggacacca 3420 acctagggtg
caaacagatg gactatggtt caaggacact ggaattgagg agctgatcaa 3480
ggctctcttc agccttgctc tgtccctgcc tcttatcaga gcacaggtag acacacgggc
3540 atagccagcc cactcctact gtcacaggcg ccccaccatt caaccttccg
ggaggtcagg 3600 gaccttctat atgaggcgag tgggtctcag tctgcttgaa
tggtgatgag attctgctgg 3660 atctcagcac gctgcaggtg tcttttgaga
gcattcagta ggacatggtg atccctattt 3720 cagcctctaa gatgactggt
attctatctg aaatgcagag attaagccaa atacctgatg 3780 tattgtgaaa
gccactgatt ttaagaatgg agagaaaggg attttttact gcatccctct 3840
gtatgaatat gaaatcagag accagggcat gatgttgcta ggattagagc ctctcagtct
3900 ggcctcttca cccaagtgca agaactcagt ctcttactgt tcaaagaatc
ttaacagttg 3960 aattatggag ggaaattccc ttttgcccca agcatttcta
tatttaaagc aatatcccag 4020 gagaatatgt tagacttagg atgatacctt
cagccacttg aagaagaaat agaaggcgct 4080 cattccaata tagtctttat
ttcccattca gatacaggtt gagcatccct aatctgaaca 4140 gttaaaaccc
ccaaatgccc caaaatccaa accttcctga acgctatgac accatgagtg 4200
gaaaattcca cacctaacaa acacatttgc tttcttatgg ttcaatgtac acaaactgtt
4260 ttatatagaa aatgatttca aatatcataa aattaccttc aggctatgtg
tataaagtat 4320 atatgagcca taaatgaatt ttgtgtttag actttgtgtc
catccccaag atctctcatt 4380 ttatatatat atatatatat atatatatat
atatatatat atatatatac acacacacac 4440 acatacacaa atattccagg
atacaaaaaa aaacatttaa aaatccgaga cccagaacac 4500 ttctggtccc
aagcatttca gataagggat atcaatctgt actaccaata aggatttcgt 4560
aattccccta actgcaaatg tcctcttcat ttgttcttta tgagaaaacc cgggtagtgc
4620 cagcacctgg atacagtatt tacaccctgc agaccctaaa gatttcagat
tcagttagca 4680 aaccttgatg aagcacctgc tggacactga gggacccaaa
gctcaatcag ccataatccc 4740 tgctttcaga gtttatattg tacctgccta
atccacccgg cgtgactcat ttcaacacta 4800 agtactaggg gtgttgtcag
gagacaaatc tgaagtcagg agaggaaaat gcaaaggagc 4860 cctgccgtgt
gatggatgtg cattctcact tgggtcttga agttctcatt cctacatctc 4920
aagctagcca ggcagtctcc tctctatcag aagaaagcac tggtaattgg ctagactggc
4980 tatgttgaag gtaacatgaa ctctaagatc ttgacccagg gcgacttggt
tttgcttaag 5040 gtggcatcac caatgttcca aatcctttag ggagatgagg
gtatccccac agaaaaagag 5100 gaataataga ccaatggatt ttctcctttc
accagtatgt ttggaaccct ctgatccaat 5160 gtcctttgat actgatctct
tgtccaaatg agaatgtcgc tttagctgaa attcaaatgg 5220 ctgtgacaat
ttaccgaaat gatgaagtaa ccaccattcc cacctttcac tgcctaggct 5280
ccaagtctga atacattttt gaaataggaa ctcccttttg caaaaaagaa acctgggtgt
5340 cagggaggtg aagtgacttg ccctaggagc agacagcatg ccaagaatgg
aattaggctc 5400 aggatccagc ctgggctcac cctgtgtggc tcattcccac
ccaggaaact gaagataaaa 5460 gatttgggaa aacacaccaa gaaaaagggg
cagttttctt tgcccaagca tttggtgcta 5520 gttagaggct gttcactctc
tcctgctcct cttcggagta gaaataaagg ctgtgacaca 5580 aggaagccag
tggggtggga gggaggcacc ataatccctc cctaaaaccc acagaagact 5640
aacctgatac tcttttgacc caactgcatc aacactaaac agctgcagac cccctgaatc
5700 tttcacacat gccaagtgaa cattcttgat gatttctctt tgtgaccgca
accacctgca 5760 aaccagaacg actctagaat ttccttcccc gccccccttt
ttgtttagtt tctaatctct 5820 tgtttatgag gtgtggggtt tataagggac
tgaatcaaat gaatgtaaca aaaaagaaaa 5880 aaaaaacaaa aaaaaatgcc
ttttctcagg gccagtgagt tgcaaataat ttttaaagaa 5940 aagcctataa
ttacatcatc tcaataaatt ttttataaaa aaaaaaaaaa aa 5992 <210> SEQ
ID NO 38 <211> LENGTH: 1670 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 38 gatttcctcc
cacgcgacct tccagttctc ggagccaggt taggggtttg gcggaggagg 60
actgcggggc gcgggcctag ggccccagca gccacggcca ggggagcgct caagacagaa
120 agccggtggc ttcctcacct ccacctgtaa tgcaggaggg agaattggct
atttctccta 180 taagccctgt ggcagccatg cctcccctag gcacccacgt
gcaagccaga tgtgaagctc 240 aaattaacct gctgggtgaa ggggggatct
gcaagctgcc aggaagactc cgcatccagc 300 ccgcactgtg gagcagggag
gacgtgctgc actggctgcg ctgggcagag caggagtact 360 ctctgccatg
caccgcggag cacgggttcg agatgaacgg acgcgccctc tgcatcctca 420
ccaaggacga cttccggcac cgtgcgccca gctcaggtga cgtcctgtat gagctgctcc
480 agtacatcaa gacccagcgg cgagccctgg tgtgtggacc cttttttgga
gggatcttca 540 ggctgaagac gcccacccag cactctccag tccccccgga
agaggtgact ggcccctctc 600 agatggacac ccgaaggggc cacctgctgc
agccaccaga cccagggctt accagcaact 660 tcggccacct ggatgaccct
ggcctggcaa ggtggacccc tggcaaggag gagtccctca 720 acttatgtca
ctgtgcagag ctcggctgca ggacccaggg ggtctgttcc ttccccgcga 780
tgccgcaggc ccccattgac ggcaggatcg ctgactgccg cctgctgtgg gattacgtgt
840 atcagctgct ccttgatacc cgatatgagc cctacatcaa gtgggaagac
aaggacgcca 900 agatcttccg agttgtggat ccaaatgggc tcgccagact
ctggggaaat cacaagaacc 960 gggtgaacat gacctacgag aagatgtctc
gtgccctgcg ccactattat aagcttaata 1020 tcattaagaa ggaaccgggg
cagaaactcc tgttcagatt tctaaagact ccgggaaaga 1080 tggtccagga
caagcacagc cacctggagc cgctggagag ccaggagcag gacagaatag 1140
agttcaagga caagaggcca gaaatctctc cgtgaggggc aggtggactc caggcacccg
1200 gtaccgatgg ggcagggacc gagtctccca tgaaggcaga ctcctcctcc
cagcagagca 1260 gcaggatccc cagccagact ctgtacccac aggattacag
ccattgcttg ggaaggctgg 1320 gaggcctccc atccaggaca ctgggggcag
gagtgtcatc ttttgggcag ggcaatcctg 1380 gggctaaatg aggtacaggg
gaatggactc tcccctactg cacccctggg agaggaagcc 1440 aggcaccgat
agagcaccca gccccacccc tgtaaatgga atttaccaga tgaagggaat 1500
gaagtccctc actgagcctc agatttcctc acctgtgaaa tgggctgagg caggaaatgg
1560 gaaaaagtgt tagtgcttcc aggcggcact gacagcctca gtaacaataa
aaacaatggt 1620 agctgaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1670 <210> SEQ ID NO 39 <211> LENGTH: 4071
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 39 gagtccagcc gctggtgcgc ggagcggttc
accgtcttcg gagcggttcg gcccagcctt 60 tcgcccaggc gcccaggccc
gctgcgcgcg tgcgtgagcg cgcctgcgcc gccagggccg 120 ctgcaagggg
aggagagcgg ccgcctcagg aggatccctt ttcccccaga aattactcaa 180
tgctgaaacc tctcaaagtg gtattagaga cgctgaaagc accatggacg ggttttatga
240 tcagcaagtc ccttttatgg tcccagggaa atctcgatct gaggaatgca
gagggcggcc 300 tgtgattgac agaaagagga agtttttgga cacagatctg
gctcacgatt ctgaagagct 360
atttcaggat ctcagtcaac ttcaagaggc ttggttagct gaagcacaag ttcctgatga
420 tgaacagttt gtcccagatt ttcagtctga taacctggtg cttcatgccc
cacctccaac 480 caagatcaaa cgggagctgc acagcccctc ctctgagctg
tcgtcttgta gccatgagca 540 ggctcttggt gctaactatg gagaaaagtg
cctctacaac tattgtgcct atgataggaa 600 gcctccctct gggttcaagc
cattaacccc tcctacaacc cccctctcac ccacccatca 660 gaatccccta
tttcccccac ctcaggcaac tctgcccacc tcagggcatg cccctgcagc 720
tggcccagtt caaggtgtgg gccccgcccc cgccccccat tcgcttccag agcctggacc
780 acagcagcaa acatttgcgg tcccccgacc accacatcag cccctgcaga
tgccaaagat 840 gatgcctgaa aaccagtatc catcagaaca gagatttcag
agacaactgt ctgaaccctg 900 ccaccccttc cctcctcagc caggagttcc
tggagataat cgccccagtt accatcggca 960 aatgtcagaa cctattgtcc
ctgcagctcc cccgccccct cagggattca aacaagaata 1020 ccatgaccca
ctctatgaac atggggtccc gggcatgcca gggcccccag cacacgggtt 1080
ccagtcacca atgggaatca agcaggagcc tcgggattac tgcgtcgatt cagaagtgcc
1140 taactgccag tcatcctaca tgagaggggg ttatttctcc agcagccatg
aaggtttttc 1200 atatgaaaaa gatccccgat tatactttga cgacacttgt
gttgtgcctg agagactgga 1260 aggcaaagtc aaacaggagc ctaccatgta
tcgagagggg cccccttacc agaggcgagg 1320 ttcccttcag ctgtggcagt
tcctggtcac ccttcttgat gacccagcca atgcccactt 1380 cattgcctgg
acaggtcgag gcatggagtt caagctgata gaaccggaag aggttgctcg 1440
gcgctggggc atccagaaga accggccagc catgaactat gacaagctga gccgctctct
1500 ccgctattac tatgaaaagg gcatcatgca gaaggtggct ggagagcgat
acgtctacaa 1560 atttgtctgt gacccagatg ccctcttctc catggctttc
ccggataacc agcgtccgtt 1620 cctgaaggca gagtccgagt gccacctcag
cgaggaggac accctgccgc tgacccactt 1680 tgaagacagc cccgcttacc
tcctggacat ggaccgctgc agcagcctcc cctatgccga 1740 aggctttgct
tactaagttt ctgagtggcg gagtggccaa accctagagc tagcagttcc 1800
cattcaggca aacaagggca gtggttttgt ttgtgttttt ggttgttcct aaagcttgcc
1860 ctttgagtat tatctggaga acccaagctg tctctggatt ggcaccctta
aagacagata 1920 cattggctgg ggagtgggaa cagggagggg cagaaaacca
ccaaaaggcc agtgcctcaa 1980 ctcttgattc tgatgaggtt tctgggaaga
gatcaaaatg gagtctcctt accatggaca 2040 atacatgcaa agcaatatct
tgttcaggtt agtacccgca aaacgggaca tgatgtgaca 2100 atctcgatcg
atcatggact actaaatggc ctttacatag aagggctctg atttgcacaa 2160
tttgttgaaa aatcacaaac ccatagaaaa gtgagtaggc taagttgggg aggctcaaac
2220 cattaagggt taaaaataca tcttaaacat tggaaagctc ttctagctga
atctgaaata 2280 ttaccccttg tctagaaaaa ggggggcagt cagaacagct
gttccccact ccgtgttctc 2340 aaaatcataa accatggcta ctcttgggaa
ccacccggcc atgtggtcgc caagtagagc 2400 aagccccctt tctcttccca
atcacgtggc tgagtgtgga tgacttttat tttaggagaa 2460 gggcgattaa
cacttttgac agtattttgt tttgccctga tttgggggat tgttttgttt 2520
tggtggttgt tttggaaaaa cagtttataa actgattttt gtagttttgg tatttaaagc
2580 aaaaaaacga aaaacaaaaa acaaaaacaa accttttggt aatgtgcact
gtgtctttag 2640 ccagggccgt gcaacttatg aagacactgc agcttgagag
gggctttgct gaggcttccc 2700 cttggccatg tgaaagcccg ccttgttgcc
tgctttgtgc tttctgcacc agacaacctg 2760 atggaacatt tgcacctgag
ttgtacattt ttgaagtgtg cagggcagcc tggacacaag 2820 cttagattct
ctatgtatag ttccccgtgt tcactaacat gccctctctg gaaagcatat 2880
gtatataaca tgtgtcatgt cctttggaaa cctggtcacc tggtgaaaac ccttgggatt
2940 cttccctggg catgactgat gacaatttcc atttcatcag tttgttttgt
tttccttttt 3000 ctttaaatct tggactttaa accctacctg tgtgattcag
tagggtttga gacttagctg 3060 tgatactgac aggtaagcaa cagtgctagc
attctagatt cctgcctttt tttaaaaaga 3120 aattattctc attgctgtat
tatattggaa aagttttaaa caaccaagct aaagctatgt 3180 gaaagttgag
ctcaaagtag aggaaaagtt actggtggta ccttgctgcc tgctctgctg 3240
gtagaattct gtgctccccg tgacacttag tacattaaga atgactacac tgttcctcgt
3300 atgtgaagga ggcagtgctg actccgtgag tgtgagacac gtgctttgaa
ctgcttttct 3360 attcatggag cactccatag tctcaaactg tcccccttat
gaccaacagc acatttgtga 3420 agaggttcgc agggataagg ggtgcacttt
atagctatgg aaacatgaga ttctcctcta 3480 ttggaagcta attagcccac
aaaggtggta aacctgtaga ttgggcctta attagcattg 3540 tactctaatc
aaaggactct ttctaaacca tatttatagc tttcttaacc tacacatagt 3600
ctatacatag atgcatattt tacccccagc tggctagaga tttatttgtt gtaaatgctg
3660 tatagatttg gttttccttt ctttacttac cctggtttgg attttttttt
tttttttttt 3720 tgaatggatt tatgctgtct tagcaatatg acaataatcc
tctgtagctt gagctacccc 3780 tcccctgctg taacttacgt gacctgtgct
gtcactgggc ataggacagc ggcatcacgg 3840 ttgcattccc attggactca
tgcacctccc ggatggtttt tgtttttttc gggggttctt 3900 tggggtttgt
ttgtttgctt cttttccaga gtgtggaaag tctacagtgc agaaaggctt 3960
gaacctgcca gctgatttga aatactttca ccctgcgcag ggccgtatgc atcctgccaa
4020 gctgcgttat attctgtact gtgtacaata aagaagtttg cttttcgttt a 4071
<210> SEQ ID NO 40 <211> LENGTH: 3499 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 40
gccaagaagc ttgagagaag aaaaatttca gaaaaattgt ctcaatttga ctagaatatc
60 aatgaaccag gaaaactgaa gcaccttccc taaagaaaac ttgggtatac
aattactcca 120 cagacagagc tgagggtttt ttacccaaat cagtcactgg
attttgctgc ctgatacgtg 180 aatcttcttg gaatttttct catgtggatc
taaggggaat gctttattat ggctgctgtt 240 gtccaacaga acgacctagt
atttgaattt gctagtaacg tcatggagga tgaacgacag 300 cttggtgatc
cagctatttt tcctgccgta attgtggaac atgttcctgg tgctgatatt 360
ctcaatagtt atgccggtct agcctgtgtg gaagagccca atgacatgat tactgagagt
420 tcactggatg ttgctgaaga agaaatcata gacgatgatg atgatgacat
cacccttaca 480 gttgaagctt cttgtcatga cggggatgaa acaattgaaa
ctattgaggc tgctgaggca 540 ctcctcaata tggattcccc tggccctatg
ctggatgaaa aacgaataaa taataatata 600 tttagttcac ctgaagatga
catggttgtt gccccagtca cccatgtgtc cgtcacatta 660 gatgggattc
ctgaagtgat ggaaacacag caggtgcaag aaaaatatgc agactcaccg 720
ggagcctcat caccagaaca gcctaagagg aaaaaaggaa gaaaaactaa accaccacga
780 ccagattccc cagccactac gccaaatata tctgtgaaga agaaaaacaa
agatggaaag 840 ggaaacacaa tttatctttg ggagttttta ctggcactgc
tccaggacaa ggctacttgt 900 cctaaataca tcaagtggac ccagcgagag
aaaggcattt ttaaattggt ggattctaaa 960 gcagtgtcca ggttgtgggg
gaagcacaaa aacaaacctg atatgaatta tgagaccatg 1020 ggaagagcac
tcaggtacta ttaccaaagg ggtattctgg caaaagtgga aggtcagcgc 1080
ttggtgtatc agtttaaaga aatgccaaaa gatcttatat atataaatga tgaggatcca
1140 agttccagca tagagtcttc agatccatca ctatcttcat cagccacttc
aaataggaat 1200 caaaccagcc ggtcgagagt atcttcaagt ccaggggtaa
aaggaggagc cactacagtt 1260 ctaaaaccag ggaattctaa agctgcaaaa
cccaaagatc ctgtggaagt tgcacaacca 1320 tcagaagttt tgaggacagt
gcagcccacg cagtctccat atcctaccca gctcttccgg 1380 actgttcatg
tagtacagcc agtacaggct gtcccagagg gagaagcagc tagaaccagt 1440
accatgcagg atgaaacatt aaattcttcc gttcagagta ttaggactat acaggctcca
1500 acccaagttc cagtggttgt gtctcctagg aatcagcagt tgcatacagt
aacactccaa 1560 acagtgccac tcacaacagt tatagccagc acagatccat
cagcaggtac tggatctcag 1620 aagtttattt tacaagccat tccatcatca
cagcccatga cagtactgaa agaaaatgtc 1680 atgctgcagt cacaaaaggc
gggctctcct ccttcaattg tcttgggccc tgcccaggtt 1740 cagcaggtcc
ttactagcaa tgttcagacc atttgcaatg gaaccgtcag tgtggcttcc 1800
tctccatcct tcagtgctac tgcacctgtg gtgacctttt ctcctcgcag ttcacagctg
1860 gttgctcacc cacctggcac tgtaatcact tcagttatca aaactcaaga
aacaaaaact 1920 cttacacagg aagtagagaa aaaggaatct gaagatcatt
tgaaagagaa cactgagaaa 1980 acggagcagc agccacagcc ttatgtgatg
gtagtgtcca gttccaatgg atttacttct 2040 caggtagcta tgaaacaaaa
cgaactgctg gaacccaact ctttttagtt aatataccaa 2100 agcttatgaa
taattgtttg ttaattgaac attttcaatt atatgcagac tgactgattc 2160
taagataaat tctaaggagg tttctaattt tgtaattgtt aaaaatagag ttaattttga
2220 ctttgttaga tgagggagga aaactcaact gtttctcttt gttatctaaa
tgtttcagaa 2280 ttcaatcgtg aaggaacagg cattttacac tatgaagaca
ttcttttgag atttttattt 2340 cagttgctat atcataagca tttttaaagt
ttcttttcta attttacatt gtattagatt 2400 ttctgattct tttgtaaata
cagaacttaa atagaaggca acaggaaatt tatataggaa 2460 ctattttcat
tccacttgtg taagttaagt cttgactctt tcaaatgcaa aaaacctatt 2520
ttatgctttg ttaaaattat ggtgtcactt agattgactt tagttgactg cactatataa
2580 tatagaacta tgaatatgta gaataacatg aaaaattgga ggtgctggtg
gtatggctga 2640 ccctgtttca gaagcaggat agtataaaag catcagccta
agaatggcac tcccactaac 2700 tagctatgta atcttgacct ctttgggctt
tagttcctct cataaaagga agagatgtat 2760 tggattagac tagattatca
ccactttctc ttctagttct aattttttta attctaatac 2820 ctatattttc
aagttatgtc aattaaatca ttatcaggtt atttcctaat gtaagaatag 2880
ctaaaatgtt gcagagaaat aagtgaccca acaaaattta ttcatctgtt atgggtaaga
2940 tctgccataa attcttccta aataatttgt ttactaactc tttaggccac
tgtgctttgc 3000 ggtccattag taaacttgtg ttgctaagtg ctaaacagaa
tactgctatt ttgagagagt 3060 caagactctt tcttaagggc caagaaagca
acttgagcct tgggctaatc tggctgagta 3120 gtcagttata aaagcataat
tgctttatat tttggatcat tttttactgg gggcggactt 3180 ggggggggtt
gcatacaaag ataacatata tatccaactt tctgaaatga aatgttttta 3240
gattactttt tcaactgtaa ataatgtaca tttaatgtca caagaaaaaa atgtcttctg
3300 caaattttct agtataacag aaatttttgt agatgaaaaa aatcattatg
tttagaggtc 3360 taatgctatg ttttcatatt acagagtgaa tttgtattta
aacaaaaatt taaattttgg 3420 aatcctctaa acatttttgt atctttaatt
ggtttattat taaataaatc atataaaaat 3480 tctcaaaaaa aaaaaaaaa 3499
<210> SEQ ID NO 41 <211> LENGTH: 2212 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 41
gcccggctcc tgggagcagg tctcggcccc cgcttggggc cccggccgtg cggccggagg
60 gagcggccgg atggagcgga ggatgaaagc cggatacttg gaccagcaag
tgccctacac 120 cttcagcagc aaatcgcccg gaaatgggag cttgcgcgaa
gcgctgatcg gcccgctggg 180 gaagctcatg gacccgggct ccctgccgcc
cctcgactct gaagatctct tccaggatct 240 aagtcacttc caggagacgt
ggctcgctga agctcaggta ccagacagtg atgagcagtt 300 tgttcctgat
ttccattcag aaaacctagc tttccacagc cccaccacca ggatcaagaa 360
ggagccccag agtccccgca cagacccggc cctgtcctgc agcaggaagc cgccactccc
420 ctaccaccat ggcgagcagt gcctttactc cagtgcctat gaccccccca
gacaaatcgc 480 catcaagtcc cctgcccctg gtgcccttgg acagtcgccc
ctacagccct ttccccgggc 540 agagcaacgg aatttcctga gatcctctgg
cacctcccag ccccaccctg gccatgggta 600 cctcggggaa catagctccg
tcttccagca gcccctggac atttgccact ccttcacatc 660 tcagggaggg
ggccgggaac ccctcccagc cccctaccaa caccagctgt cggagccctg 720
cccaccctat ccccagcaga gctttaagca agaataccat gatcccctgt atgaacaggc
780 gggccagcca gccgtggacc agggtggggt caatgggcac aggtacccag
gggcgggggt 840 ggtgatcaaa caggaacaga cggacttcgc ctacgactca
gatgtcaccg ggtgcgcatc 900 aatgtacctc cacacagagg gcttctctgg
gccctctcca ggtgacgggg ccatgggcta 960 tggctatgag aaacctctgc
gaccattccc agatgatgtc tgcgttgtcc ctgagaaatt 1020 tgaaggagac
atcaagcagg aaggggtcgg tgcatttcga gaggggccgc cctaccagcg 1080
ccggggtgcc ctgcagctgt ggcaatttct ggtggccttg ctggatgacc caacaaatgc
1140 ccatttcatt gcctggacgg gccggggaat ggagttcaag ctcattgagc
ctgaggaggt 1200 cgccaggctc tggggcatcc agaagaaccg gccagccatg
aattacgaca agctgagccg 1260 ctcgctccga tactattatg agaaaggcat
catgcagaag gtggctggtg agcgttacgt 1320 gtacaagttt gtgtgtgagc
ccgaggccct cttctctttg gccttcccgg acaatcagcg 1380 tccagctctc
aaggctgagt ttgaccggcc tgtcagtgag gaggacacag tccctttgtc 1440
ccacttggat gagagccccg cctacctccc agagctggct ggccccgccc agccatttgg
1500 ccccaagggt ggctactctt actagccccc agcggctgtt ccccctgccg
caggtgggtg 1560 ctgccctgtg tacatataaa tgaatctggt gttggggaaa
ccttcatctg aaacccacag 1620 atgtctctgg ggcagatccc cactgtccta
ccagttgccc tagcccagac tctgagctgc 1680 tcaccggagt cattgggaag
gaaaagtgga gaaatggcaa gtctagagtc tcagaaactc 1740 ccctgggggt
ttcacctggg ccctggagga attcagctca gcttcttcct aggtccaagc 1800
cccccacacc ttttccccaa ccacagagaa caagagtttg ttctgttctg ggggacagag
1860 aaggcgcttc ccaacttcat actggcagga gggtgaggag gttcactgag
ctccccagat 1920 ctcccactgc ggggagacag aagcctggac tctgccccac
gctgtggccc tggagggtac 1980 cggtttgtca gttcttggtg ctctgtgttc
ccagaggcag gcggaggttg aagaaaggaa 2040 cctgggatga ggggtgctgg
gtataagcag agagggatgg gttcctgctc caagggaccc 2100 tttgcctttc
ttctgccctt tcctaggccc aggcctgggt ttgtacttcc acctccacca 2160
catctgccag accttaataa aggcccccac ttctcccaaa aaaaaaaaaa aa 2212
<210> SEQ ID NO 42 <211> LENGTH: 2667 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 42
tctgagaggc gaggccgggt gaggcggcga gggcggcccg acgggcgcgg gacgggacgg
60 ggcagcgagg gcgccgggag ccgcggcccg gaatcggggc gcttcgcccc
gggcccccca 120 gcatgaagac cccggcggac acagggtttg ccttcccgga
ttgggcctac aagccagagt 180 cgtcccctgg ctcaaggcag atccagctgt
ggcactttat cctggagctg ctgcggaagg 240 aggagtacca gggcgtcatt
gcctggcagg gggactacgg ggaattcgtc atcaaagacc 300 ctgatgaggt
ggcccggctg tggggcgttc gcaagtgcaa gccccagatg aattacgaca 360
agctgagccg ggccctgcgc tattactata acaagcgcat tctgcacaag accaagggga
420 aacggttcac ctacaagttc aatttcaaca aactggtgct ggtcaattac
ccattcattg 480 atgtggggtt ggctgggggt gcagtgcccc agagtgcccc
gccagtgccg tcgggtggta 540 gccacttccg cttccctccc tcaacgccct
ccgaggtgct gtcccccacc gaggaccccc 600 gctcaccacc agcctgctct
tcatcttcat cttccctctt ctcggctgtg gtggcccgcc 660 gcctgggccg
aggctcagtc agtgactgta gtgatggcac gtcagagctg gaggaaccgc 720
tgggagagga tccccgcgcc cgaccacccg gccctccgga tctgggtgcc ttccgagggc
780 ccccgctggc ccgcctgccc catgaccctg gtgtcttccg agtctatccc
cggcctcggg 840 gtggccctga acccctcagc cccttccctg tgtcgcctct
ggccggtcct ggatccctgc 900 tgccccctca gctctccccg gctctgccca
tgacgcccac ccacctggcc tacactccct 960 cgcccacgct gagcccgatg
taccccagtg gtggcggggg gcccagcggc tcagggggag 1020 gctcccactt
ctccttcagc cctgaggaca tgaaacggta cctgcaggcc cacacccaaa 1080
gcgtctacaa ctaccacctc agcccccgcg ccttcctgca ctaccctggg ctggtggtgc
1140 cccagcccca gcgccctgac aagtgcccgc tgccgcccat ggcacccgag
accccaccgg 1200 tcccctcctc ggcctcgtca tcctcttctt cttcttcctc
cccattcaag tttaagctcc 1260 agcggccccc actcggacgc cggcagcggg
cagctgggga gaaggccgta gccgctgctg 1320 acaagagcgg tggcagtgca
ggcgggctgg ctgagggggc aggggcgcta gccccaccgc 1380 ccccgccacc
acagatcaag gtggagccca tctcggaagg cgagtcggag gaggtagagg 1440
tgactgacat cagtgatgag gatgaggaag acggggaggt gttcaagacg ccccgtgccc
1500 cacctgcacc ccctaagcct gagcccggcg aggcacccgg ggcatcccag
tgcatgcccc 1560 tcaagctacg ctttaagcgg cgctggagtg aagactgtcg
cctcgaaggg ggtgggggcc 1620 ccgctggggg ctttgaggat gagggtgagg
acaagaaggt gcgtggggag gggcctgggg 1680 aggctggggg gcccctcacc
ccaaggcggg tgagctctga cctccagcat gccacggccc 1740 agctctccct
ggagcaccga gactcctgag ggctgtgggc aggggacctg tgtgccccgc 1800
accccccatg cttcttttgc tgccttaagc cccctatgcc ctggaggtga gggcagctct
1860 cttgtctctt ccctgcctcc tcccttttcc ctccccacat tttgtataaa
actttaattt 1920 ctttttttta aaaatggtgg gggtgggtgg gtgcccaggg
ctaggggcta ttccctgtct 1980 ctgtgggttt ctaagctctg ggcaaattgg
tggtaggggg agggaggggg aagttaaggg 2040 ggtcacctcc attctgggga
atttatattt gaattgaggc tttggcctta acacccagga 2100 acttttctat
tacaatcgct taggaagtaa agccttgtct ccctccctgt tctctgcctc 2160
ttgtacccct ctgacccacc cgctctgccc cactcccagc cctcctcagc cccagccctg
2220 cctgccctgc ccctccaggg ggccatgagt gcctaggttt ctcatacccc
acaaggtcac 2280 agcaggggag ggagggacaa ttttataatg aaccaaaaat
tccatgtgtt ggggggtggg 2340 gggcggagga gggtgagggg tgccgcccat
gggccacaaa tctctacaag tgcctgctat 2400 ccctctccca ctccccaccc
cagcaccggt ccaacccctt catccccagc tgctcctagg 2460 actggcccat
gggcaggcgg gtggggggat gggaaggggg tgccctgaaa ccaaactgga 2520
agccccctct gcctcccagc tggggcctct ggggtggggt ggggggctgt ggtcaagcct
2580 tattctgtat tggggactga gggtgggggg agtagagggg ccgctggaga
atgtattcaa 2640 aacaataaac tttggacctt tggaaaa 2667 <210> SEQ
ID NO 43 <211> LENGTH: 1364 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 43 aaaatcagga
acttgtgctg gccctgcaat gtcaagggag ggggctcacc cagggctcct 60
gtagctcagg gggcaggcct gagccctgca cccgccccac gaccgtccag cccctgacgg
120 gcaccccatc ctgaggggct ctgcattggc ccccaccgag gcaggggatc
tgaccgactc 180 ggagcccggc tggatgttac aggcgtgcaa aatggaaggg
tttcccctcg tcccccctcc 240 atcagaagac ctggtgccct atgacacgga
tctataccaa cgccaaacgc acgagtatta 300 cccctatctc agcagtgatg
gggagagcca tagcgaccat tactgggact tccaccccca 360 ccacgtgcac
agcgagttcg agagcttcgc cgagaacaac ttcacggagc tccagagcgt 420
gcagcccccg cagctgcagc agctctaccg ccacatggag ctggagcaga tgcacgtcct
480 cgataccccc atggtgccac cccatcccag tcttggccac caggtctcct
acctgccccg 540 gatgtgcctc cagtacccat ccctgtcccc agcccagccc
agctcagatg aggaggaggg 600 cgagcggcag agccccccac tggaggtgtc
tgacggcgag gcggatggcc tggagcccgg 660 gcctgggctc ctgcctgggg
agacaggcag caagaagaag atccgcctgt accagttcct 720 gttggacctg
ctccgcagcg gcgacatgaa ggacagcatc tggtgggtgg acaaggacaa 780
gggcaccttc cagttctcgt ccaagcacaa ggaggcgctg gcgcaccgct ggggcatcca
840 gaagggcaac cgcaagaaga tgacctacca gaagatggcg cgcgcgctgc
gcaactacgg 900 caagacgggc gaggtcaaga aggtgaagaa gaagctcacc
taccagttca gcggcgaagt 960 gctgggccgc gggggcctgg ccgagcggcg
ccacccgccc cactgagccc gcagcccccg 1020 ccggccccgc caggcctccc
cgctggccat agcattaagc cctcgcccgg cccggacaca 1080 gggaggacgc
tcccggggcc cagaggcagg actgtggcgg gccgggctcc gtcacccgcc 1140
cctcccccca ctccaggccc cctccacatc ccgcttcgcc tccctccagg actccacccc
1200 ggctcccgac gccagctggg cgtcagaccc accggcaacc ttgcagagga
cgacccgggg 1260 tactgccttg ggagtctcaa gtccgtatgt aaatcagatc
tcccctctca cccctcccac 1320 ccattaacct cctcccaaaa aacaagtaaa
gttattctca atcc 1364 <210> SEQ ID NO 44 <211> LENGTH:
3034 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 44 tttcttgtta aacaaacacc taatttattt
cttggaggtt ttgttcagct gtcctaattt 60 atgactttac attccttctg
gtgctaaact gctcaagtag cctcttgtat caagtgtgac 120 ctgattcctt
aagaatttta cttaatgaga acctctaagc tagaaactct tgctaggtgt 180
ttcatgcacc ttattttctt taatcattac aacaactcta agattgggtt ctctccacct
240 tataaatgat gactgtttta gagaggttaa ggttgcttaa aattggtgag
ttagtgaggg 300
gtagagccac gaatggattt ctggtcgctg cctccatcgt cagggcaagc ttttcccacg
360 actccagcgc ttccatttgt cagtccccag gctagaaagc cacagtgcta
atttagtatt 420 tatcaagcgt ttgtagtgtc ctgggatctg gcacttcgat
gagaaagctg tgacggcccc 480 aacttctaac agcgagtggt aaggaggacg
agggacacag gagggaggag actctcccca 540 aagcttagca ccaacagaag
tggtcccccg caggttgctc tgcgagcgcc acctcttccc 600 tccaaccgag
gagaaagtgg cgcgcctttg aggagtccga ggtcccggcc caggcggcag 660
cttgggtcct ggcgggttcc ggacgggcgc ctcagggacc tggaagcaac cgcaccgaac
720 gcgacggaga gcggcgagac gactccagga ggcgcccgag ctacatcccc
cggccacacc 780 aaacccgggt ttgctggcag acgcggctca cgacacccct
tagggtcgca gcccctcccc 840 cggaagtgac gtgtagcgac tacggcgtct
gggagggacc caggagcagt cggggggttt 900 gagagtggcg gcggccgcgg
agggcctggc aggccccgcc gctgcaagga acgccccgaa 960 cgcgcgcgcc
cggcgtgtag cggccccaag acccgcgccg ccgctgccgc gtgcgggggc 1020
ggggagggcg gggcgccagg agccgcggcg gcgggagatg cgggcggctg cgggcacccg
1080 gcgggctcgg cttggccgcc gccgccttct acggctccgc cgcgggggtc
gcagcggctg 1140 ccgcgccgtc ctcgagtttc cagcgtgagg aggaggctga
gggcggagag gcgcatcgtg 1200 ttcgaggcgg agaccgaggg ggagccccgc
gcgcggcgtc gctcattgct atggacagtg 1260 ctatcaccct gtggcagttc
cttcttcagc tcctgcagaa gcctcagaac aagcacatga 1320 tctgttggac
ctctaatgat gggcagttta agcttttgca ggcagaagag gtggctcgtc 1380
tctgggggat tcgcaagaac aagcctaaca tgaattatga caaactcagc cgagccctca
1440 gatactatta tgtaaagaat atcatcaaaa aagtgaatgg tcagaagttt
gtgtacaagt 1500 ttgtctctta tccagagatt ttgaacatgg atccaatgac
agtgggcagg attgagggtg 1560 actgtgaaag tttaaacttc agtgaagtca
gcagcagttc caaagatgtg gagaatggag 1620 ggaaagataa accacctcag
cctggtgcca agacctctag ccgcaatgac tacatacact 1680 ctggcttata
ttcttcattt actctcaact ctttgaactc ctccaatgta aagcttttca 1740
aattgataaa gactgagaat ccagccgaga aactggcaga gaaaaaatct cctcaggagc
1800 ccacaccatc tgtcatcaaa tttgtcacga caccttccaa aaagccaccg
gttgaacctg 1860 ttgctgccac catttcaatt ggcccaagta tttctccatc
ttcagaagaa actatccaag 1920 ctttggagac attggtttcc ccaaaactgc
cttccctgga agccccaacc tctgcctcta 1980 acgtaatgac tgcttttgcc
accacaccac ccatttcgtc cataccccct ttgcaggaac 2040 ctcccagaac
accttcacca ccactgagtt ctcacccaga catcgacaca gacattgatt 2100
cagtggcttc tcagccaatg gaacttccag agaatttgtc actggagcct aaagaccagg
2160 attcagtctt gctagaaaag gacaaagtaa ataattcatc aagatccaag
aaacccaaag 2220 ggttagaact ggcacccacc cttgtgatca cgagcagtga
tccaagccca ctgggaatac 2280 tgagcccatc tctccctaca gcttctctta
caccagcatt tttttcacag acacccatca 2340 tactgactcc aagccccttg
ctctccagta tccacttctg gagtactctc agtcctgttg 2400 ctcccctaag
tccagccaga ctgcaaggtg ctaacacact tttccagttt ccttctgtac 2460
tgaacagtca tgggccattc actctgtctg ggctggatgg accttccacc cctggcccat
2520 tttccccaga cctacagaag acataaccta tgcacttgtg gaatgagaga
accgaggaac 2580 gaagaaacag acattcaaca tgattgcatt tgaagtgagc
aattgatagt tctacaatgc 2640 tgataataga ctattgtgat ttttgccatt
ccccattgaa aacatctttt taggattctc 2700 tttgaatagg actcaagttg
gactatatgt ataaaaatgc cttaattgga gtctaaactc 2760 cacctccctc
tgtcttttcc ttttcttttt ctttccttcc ttccttttct tttctccttt 2820
aaaaatattt tgagctttgt gctgaagaag tttttggtgg gctttagtga ctgtgctttg
2880 caaaagcaat taagaacaaa gttactcctt ctggctattg ggaccctttg
gccaggaaaa 2940 attatgctta gaatctatta tttaaagaaa tatttgtgaa
atgaaaaaaa aaaaaaaaaa 3000 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa
3034 <210> SEQ ID NO 45 <211> LENGTH: 3077 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
45 tttcttgtta aacaaacacc taatttattt cttggaggtt ttgttcagct
gtcctaattt 60 atgactttac attccttctg gtgctaaact gctcaagtag
cctcttgtat caagtgtgac 120 ctgattcctt aagaatttta cttaatgaga
acctctaagc tagaaactct tgctaggtgt 180 ttcatgcacc ttattttctt
taatcattac aacaactcta agattgggtt ctctccacct 240 tataaatgat
gactgtttta gagaggttaa ggttgcttaa aattggtgag ttagtgaggg 300
gtagagccac gaatggattt ctggtcgctg cctccatcgt cagggcaagc ttttcccacg
360 actccagcgc ttccatttgt cagtccccag gctagaaagc cacagtgcta
atttagtatt 420 tatcaagcgt ttgtagtgtc ctgggatctg gcacttcgat
gagaaagctg tgacggcccc 480 aacttctaac agcgagtggt aaggaggacg
agggacacag gagggaggag actctcccca 540 aagcttagca ccaacagaag
tggtcccccg caggttgctc tgcgagcgcc acctcttccc 600 tccaaccgag
gagaaagtgg cgcgcctttg aggagtccga ggtcccggcc caggcggcag 660
cttgggtcct ggcgggttcc ggacgggcgc ctcagggacc tggaagcaac cgcaccgaac
720 gcgacggaga gcggcgagac gactccagga ggcgcccgag ctacatcccc
cggccacacc 780 aaacccgggt ttgctggcag acgcggctca cgacacccct
tagggtcgca gcccctcccc 840 cggaagtgac gtgtagcgac tacggcgtct
gggagggacc caggagcagt cggggggttt 900 gagagtggcg gcggccgcgg
agggcctggc aggccccgcc gctgcaagga acgccccgaa 960 cgcgcgcgcc
cggcgtgtag cggccccaag acccgcgccg ccgctgccgc gtgcgggggc 1020
ggggagggcg gggcgccagg agccgcggcg gcgggagatg cgggcggctg cgggcacccg
1080 gcgggctcgg cttggccgcc gccgccttct acggctccgc cgcgggggtc
gcagcggctg 1140 ccgcgccgtc ctcgagtttc cagcgtgagg aggaggctga
gggcggagag gcgcatcgtg 1200 ttcgaggcgg agaccgaggg ggagccccgc
gcgcggcgtc gctcattgct atggacagtg 1260 ctatcaccct gtggcagttc
cttcttcagc tcctgcagaa gcctcagaac aagcacatga 1320 tctgttggac
ctctaatgat gggcagttta agcttttgca ggcagaagag gtggctcgtc 1380
tctgggggat tcgcaagaac aagcctaaca tgaattatga caaactcagc cgagccctca
1440 gatactatta tgtaaagaat atcatcaaaa aagtgaatgg tcagaagttt
gtgtacaagt 1500 ttgtctctta tccagagatt ttgaacatgg atccaatgac
agtgggcagg attgagggtg 1560 actgtgaaag tttaaacttc agtgaagtca
gcagcagttc caaagatgtg gagaatggag 1620 ggaaagataa accacctcag
cctggtgcca agacctctag ccgcaatgac tacatacact 1680 ctggcttata
ttcttcattt actctcaact ctttgaactc ctccaatgta aagcttttca 1740
aattgataaa gactgagaat ccagccgaga aactggcaga gaaaaaatct cctcaggagc
1800 ccacaccatc tgtcatcaaa tttgtcacga caccttccaa aaagccaccg
gttgaacctg 1860 ttgctgccac catttcaatt ggcccaagta tttctccatc
ttcagaagaa actatccaag 1920 ctttggagac attggtttcc ccaaaactgc
cttccctgga agccccaacc tctgcctcta 1980 acgtaatgac tgcttttgcc
accacaccac ccatttcgtc cataccccct ttgcaggaac 2040 ctcccagaac
accttcacca ccactgagtt ctcacccaga catcgacaca gacattgatt 2100
cagtggcttc tcagccaatg gaacttccag agaatttgtc actggagcct aaagaccagg
2160 attcagtctt gctagaaaag gacaaagtaa ataattcatc aagatccaag
aaacccaaag 2220 ggttagaact ggcacccacc cttgtgatca cgagcagtga
tccaagccca ctgggaatac 2280 tgagcccatc tctccctaca gcttctctta
caccagcatt tttttcacag gtagcttgct 2340 cgctctttat ggtgtcacca
ttgctttcat ttatttgccc ttttaagcaa atccagaatt 2400 tatacactca
agtttgcttt ctgttactta ggtttgtctt agaaaggtta tgtgtgactg 2460
tcatgtgaaa gttaccccat ttctcatctt aattaggatt gctaaaatag aaagtttgga
2520 gtattttctt aaaaaattca ttgttctaca agtaaataaa tattttgatt
tttctatttc 2580 ctcctaaaga aagtacacac actctctcgc tctctctcgg
tcttataaaa ctcgttggtg 2640 tcttataaaa caaacagtga taatctcaag
ttagaaaaca gtaggtcctg agaaccataa 2700 gaaaaatgac tggtgtgatg
ttgagtaaca agttggtaca gttactttag ctatttatta 2760 acttgctcat
ctcatagaac attttagtag atttttcaca cacctcatta ttaaaaaaaa 2820
acaaacatgc tggtgtcttg gttacccatt attcctctgt acctgaattc aggttggttt
2880 ttctatttgg aaaagacttt ataaatgttg gcttaaaaag aggttgagca
ccagaatctc 2940 agaatttacc accaaagaac tcatccatgt aaccaaaaac
cacttgtacc cccaaaaact 3000 attgaaataa aaatttaaaa aattttaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3060 aaaaaaaaaa aaaaaaa 3077
<210> SEQ ID NO 46 <211> LENGTH: 1443 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 46
ggggggggtg gatgaggagg agccggagac gccgcggagg agaccggacc gaagacggac
60 cgtgccggga agagcaggcg ggtgaaaatg aaagccggct gtagcatcgt
ggaaaagcca 120 gaaggaggtg gagggtatca gtttcctgac tgggcctaca
aaacagagtc atccccaggc 180 tcccggcaga tccagctgtg gcacttcatc
ctggagctgc tgcagaagga agagttccgc 240 catgtcatcg cctggcagca
gggagagtac ggggaatttg tcatcaagga tccagatgag 300 gtggcccgcc
tctggggccg caggaaatgc aaaccacaga tgaattatga caagctgagc 360
cgggccctca gatactatta caacaagagg atccttcata aaacaaaagg gaaaagattt
420 acctataaat ttaacttcaa caagctggtg atgcccaact acccattcat
caacattcgg 480 tcaagtggta agatacaaac tcttttggta gggaattaat
tttgaattga aaagaatttt 540 taaaaatcca aatctaagac atggcatgtt
taggaagatt ttagaaacac taaaataatg 600 tgatcctttg gattgcctca
atgttcttac tcaagtcatc tcacttataa ggagagttat 660 aggctattca
gtatcaagat agatttcttt ggtttatttg gttggttccc ttttctgcat 720
attgtttgta atctcctaga tactattacg ctatcttgtt tgggaatgat gtttcatagg
780 tttgtgatga tctttacgtt caggactcag ttttaacacc cagcccagtg
gttctttcat 840 agatgggaac ctgtttctac aaacacttcc gattttctgt
gaaactacca agctctccct 900 tatcaagtga atatcatcaa aaccacagca
tccttgatca gagaaggggg aggttcacat 960 gtttgcagtg aaaagcagtg
tctttgatct gcaacagcaa atcctcagag aaaaagattc 1020 tggggttact
tgaccttctc tcctgttaag tgcagtaggg cttcccctct tgactttcct 1080
ggttatagct ttccatcaca gctccccaca ttctctcttg atgttgaaag cagtctctca
1140
aaagactttg ttgttgtgtg gttttttgtt tgtgattttt ttccttatgc aaatcatact
1200 cctgcccaag aaaatacagt agttcccctt atctgagcag tatatgttct
aagaccccta 1260 gtagattcgc aaaccacaga tagtaccaaa ctccattcat
atatatgatg ttttttcttc 1320 ccttaacccc actcatatgt atctgtgata
acgtttaatt tataaattag gcacagtaag 1380 agattaatga caataataaa
atagaaaaat tataaaaaaa aaaaaaaaaa aaaaaaaaaa 1440 aaa 1443
<210> SEQ ID NO 47 <211> LENGTH: 229 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 47
cccactagtt acccacccca aactggatcc tacagccaag ctccaagtca atatagccaa
60 cagagcagca gctacgggca gcagaatccg tatcagatcc tgggcccgac
cagcagtcgc 120 ctagccaacc ctggaagcgg gcagatccag ctgtggcaat
tcctcctgga gctgctctcc 180 gacagcgcca acgccagctg tatcacctgg
gaggggacca acggggagt 229 <210> SEQ ID NO 48 <211>
LENGTH: 2319 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 48 cacaaggcta caggtgtctt tatttccact
gcacgctggt gctgggagcg cctgccttct 60 cttgccttga aagcctcctc
tttggaccta gccaccgctg ccctcacggt aatgttggac 120 tcggtgacac
acagcacctt cctgcctaat gcatccttct gcgatcccct gatgtcgtgg 180
actgatctgt tcagcaatga agagtactac cctgcctttg agcatcagac agcctgtgac
240 tcatactgga catcagtcca ccctgaatac tggactaagc gccatgtgtg
ggagtggctc 300 cagttctgct gcgaccagta caagttggac accaattgca
tctccttctg caacttcaac 360 atcagtggcc tgcagctgtg cagcatgaca
caggaggagt tcgtcgaggc agctggcctc 420 tgcggcgagt acctgtactt
catcctccag aacatccgca cacaaggtta ctcctttttt 480 aatgacgctg
aagaaagcaa ggccaccatc aaagactatg ctgattccaa ctgcttgaaa 540
acaagtggca tcaaaagtca agactgtcac agtcatagta gaacaagcct ccaaagttct
600 catctatggg aatttgtacg agacctgctt ctatctcctg aagaaaactg
tggcattctg 660 gaatgggaag atagggaaca aggaattttt cgggtggtta
aatcggaagc cctggcaaag 720 atgtggggac aaaggaagaa aaatgacaga
atgacatatg aaaagttgag cagagccctg 780 agatactact ataaaacagg
aattttggag cgggttgacc gaaggttagt gtacaaattt 840 ggaaaaaatg
cacacgggtg gcaggaagac aagctatgat ctgctccagg catcaagctc 900
attttatgga tttctgtctt ttaaaacaat cagattgcaa tagacattcg aaaggcttca
960 ttttcttctc tttttttttt aacctgcaaa catgctgata aaatttctcc
acatctcagc 1020 ttacatttgg attcagagtt gttgtctacg gagggtgaga
gcagaaactc ttaagaaatc 1080 ctttcttctc cctaagggga tgaggggatg
atcttttgtg gtgtcttgat caaactttat 1140 tttcctagag ttgtggaatg
acaacagccc atgccattga tgctgatcag agaaaaacta 1200 ttcaattctg
ccattagaga cacatccaat gctcccatcc caaaggttca aaagttttca 1260
aataactgtg gcagctcacc aaaggtgggg gaaagcatga ttagtttgca ggttatggta
1320 ggagagggtg agatataaga catacatact ttagatttta aattattaaa
gtcaaaaatc 1380 catagaaaag tatccctttt tttttttttt gagacgggtt
ctcactatgt tgcccagggc 1440 tggtcttgaa ctcctatgct caagtgatcc
tcccacctcg gcctcccaaa gtactgtgat 1500 tacaagcgtg agccacggca
cctgggcaga aaagtatctt aattaatgaa agagctaagc 1560 catcaagctg
ggacttaatt ggatttaaca taggttcaca gaaagtttcc taaccagagc 1620
atctttttga ccactcagca aaacttccac agacatcctt ctggacttaa acacttaaca
1680 ttaaccacat tattaattgt tgctgagttt attccccctt ctaactgatg
gctggcatct 1740 gatatgcaga gttagtcaac agacactggc atcaattaca
aaatcactgc tgtttctgtg 1800 attcaagctg tcaacacaat aaaatcgaaa
ttcattgatt ccatctctgg tccagatgtt 1860 aaacgtttat aaaaccggaa
atgtcctaac aactctgtaa tggcaaatta aattgtgtgt 1920 cttttttgtt
ttgtctttct acctgatgtg tattcaagtg ctataacacg tatttccttg 1980
acaaaaatag tgacagtgaa ttcacactaa taaatgttca taggttaaag tctgcactga
2040 cattttctca tcaatcactg gtatgtaagt tatcagtgac tgacagctag
gtggactgcc 2100 cctaggactt ctgtttcacc agagcaggaa tcaagtggtg
aggcactgaa tcgctgtaca 2160 ggctgaagac ctccttatta gagttgaact
tcaaagtaac ttgttttaaa aaatgtgaat 2220 tactgtaaaa taatctattt
tggattcatg tgttttccag gtggatatag tttgtaaaca 2280 atgtgaataa
agtatttaac atgtaaaaaa aaaaaaaaa 2319 <210> SEQ ID NO 49
<211> LENGTH: 3617 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 49 acccgtggtg
ccccatccct ataggagctg gtgagattgc agcctgctgc ctcccctcca 60
tcagccacag ctattggatt tcccacccag aatctttagg taaatgagat catgattctg
120 gaaggaggtg gtgtaatgaa tctcaacccc ggcaacaacc tccttcacca
gccgccagcc 180 tggacagaca gctactccac gtgcaatgtt tccagtgggt
tttttggagg ccagtggcat 240 gaaattcatc ctcagtactg gaccaagtac
caggtgtggg agtggctcca gcacctcctg 300 gacaccaacc agctggatgc
caattgtatc cctttccaag agttcgacat caacggcgag 360 cacctctgca
gcatgagttt gcaggagttc acccgggcgg cagggacggc ggggcagctc 420
ctctacagca acttgcagca tctgaagtgg aacggccagt gcagtagtga cctgttccag
480 tccacacaca atgtcattgt caagactgaa caaactgagc cttccatcat
gaacacctgg 540 aaagacgaga actatttata tgacaccaac tatggtagca
cagtagattt gttggacagc 600 aaaactttct gccgggctca gatctccatg
acaaccacca gtcaccttcc tgttgcagag 660 tcacctgata tgaaaaagga
gcaagacccc cctgccaagt gccacaccaa aaagcacaac 720 ccgagaggga
ctcacttatg ggaattcatc cgcgacatcc tcttgaaccc agacaagaac 780
ccaggattaa taaaatggga agaccgatct gagggcgtct tcaggttctt gaaatcagag
840 gcagtggctc agctatgggg taaaaagaag aacaacagca gcatgaccta
tgaaaagctc 900 agccgagcta tgagatatta ctacaaaaga gaaattctgg
agcgtgtgga tggacgaaga 960 ctggtatata aatttgggaa gaatgcccga
ggatggagag aaaatgaaaa ctgaagctgc 1020 caatactttg gacacaaacc
aaaacacaca ccaaataatc agaaacaaag aactcctgga 1080 cgtaaatatt
tcaaagacta cttttctctg atatttatgt accatgaggg gaacaagaaa 1140
ctacttctaa cgggaagaag aaacactaca gtcgattaaa aaaattattt tgttacttcg
1200 aagtatgtcc tatatgggga aaaaacgtac acagttttct gtgaaatatg
atgctgtatg 1260 tggttgtgat tttttttcac ctctattgtg aattcttttt
cactgcaaga gtaacaggat 1320 ttgtagcctt gtgcttcttg ctaagagaaa
gaaaaacaaa atcagagggc attaaatgtt 1380 ttgtatgtga catgatttag
aaaaaggtga tgcatcctcc tcacataagc atccatatgg 1440 cttcgtcaag
ggaggtgaac attgttgctg agttaaattc cagggtctca gatggttagg 1500
acaaagtgga tggatgccgg gaagtttaac ctgagcctta ggatccaatg agtggagaat
1560 ggggacttcc aaaacccaag gttggctata atctctgcat aaccacatga
cttggaatgc 1620 ttaaatcagc aagaagaata atggtggggt ctttatactc
attcaggaat ggtttatctg 1680 atgccagggc tgtcttcctt tctccccttt
ggatggttgg tgaaatactt taattgccct 1740 gtctgctcac ttctagctat
ttaagagaga acccagcttg gttctttttt gctccaagtg 1800 cttaaaaata
agttggaaaa aggagacggt ggtgtggaaa tggctgaaga gtttgctctt 1860
gtatccctat agtccaaggt ttctcaatct gcacaattga catttttggc cggagtgttc
1920 tttgtggtga gggctttcct gtgcattgta agatgttcag cagtatccac
tcatggtctc 1980 taaccacttg acaccagaaa ccccccagct gtgataacgc
aaaatgtctc tagacatcac 2040 caaatgttcc ctgggggtgg caaatttgcc
cttgattgag aaccaccagt ttagctagtc 2100 aatatgagga tggtggttta
ttctcagaag aaaaagatat gtaaggtctt ttagctcctt 2160 agagtgaagc
aaaagcaaga cttcaacctc aacctatctt tatgttttaa atgttaggga 2220
caataagttg aaatagctag aggagcttct tttcagaacc ccagatgaga gccaatgtca
2280 gataaagtaa gcatagtaat gtagcaggaa ctacaataga agacattttc
actggaatta 2340 caaagcagaa ttaaaattat attgtagaag gaaacaccaa
gaaaagaatt tccagggaaa 2400 atcctctttg caggtattaa ttcttataat
tttttgtctt ttggattatc tgtttactgt 2460 ctcatctgaa ctgatcccag
gtgaacggtt tattgcctag atttgtactc agaggaattt 2520 tttttgtttt
gttttgtctt ttaagaaagg aaagaaagga tgaaaaaaat aaacagaaaa 2580
ctcagctcag gcacaattgt caccaaggag ttaaaagctt cttcttcaat agaggaattg
2640 ttctgggggt cctggagact taccattgag ccatgcaatc tgggaagcac
aggaataagt 2700 agacactttg aaaatggatt tgaatgttct catccctttt
gcagcttttc tttttggctc 2760 tctcatgtcc ttggcttgct cctctattct
acctctcttt ctccagcaat aatatgcaaa 2820 tgaagacatg tatccataag
aaggagtgct cttcatcaac taatagagca cctaccacag 2880 tgtcatacct
ggtagaggtg agcaattcat attcaaaggt tgcaaagtgt ttgtaatata 2940
ttcatgaggc tggaagtaag aagaattaaa aatttgtcct aattacaatg agaaccattc
3000 taggtagtga tcttggagca cacatgaata actttctgaa ggtgcaacca
aatccatttt 3060 tatttctgcc tggcttggtc acctctgtaa aggtttaact
tagtgttgtc aagtaacagt 3120 tactgaaaga gctgagaaaa agaacaatga
acagcaacga tcttgactgt gcaactcaga 3180 cattcctgca gaaaagacat
atgttgcttt acaagaaggc caaagaacta tggggccttc 3240 ccagcatttg
actgttcatt gcatagaatg aattaaatat ccagttactt gaatgggtat 3300
aacgcatgaa tatttgtgtg tctgtgtgtg tgtctgagtt gtgtgatttt attaggggca
3360 tctgccaatt ctctcactgt ggttccttct ctgactttgc ctgttcatca
tctaaggagg 3420 ctagatcctt cgctgacttc accattcctc aaacctgtaa
gtttctcact tcttccaaat 3480 tggctttggc tctttctgca acctttccat
tcaagagcaa tctttgctaa ggagtaagtg 3540 aatgtgaaga gtaccaacta
caacaattct acagataatt agtggattgt gttgtttgtt 3600 gagagtgaag gtttctt
3617 <210> SEQ ID NO 50 <211> LENGTH: 1894 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
50
gtctgacttc ctcccagcac attcctgcac tctgccgtgt ccacactgcc ccacagaccc
60 agtcctccaa gcctgctgcc agctccctgc aagcccctca ggttgggcct
tgccacggtg 120 ccagcaggca gccctgggct gggggtaggg gactccctac
aggcacgcag ccctgagacc 180 tcagagggcc accccttgag ggtggccagg
cccccagtgg ccaacctgag tgctgcctct 240 gccaccagcc ctgctggccc
ctggttccgc tggcccccca gatgcctggc tgagacacgc 300 cagtggcctc
agctgcccac acctcttccc ggcccctgaa gttggcactg cagcagacag 360
ctccctgggc accaggcagc taacagacac agccgccagc ccaaacagca gcggcatggg
420 cagcgccagc ccgggtctga gcagcgtatc ccccagccac ctcctgctgc
cccccgacac 480 ggtgtcgcgg acaggcttgg agaaggcggc agcgggggca
gtgggtctcg agagacggga 540 ctggagtccc agtccacccg ccacgcccga
gcagggcctg tccgccttct acctctccta 600 ctttgacatg ctgtaccctg
aggacagcag ctgggcagcc aaggcccctg gggccagcag 660 tcgggaggag
ccacctgagg agcctgagca gtgcccggtc attgacagcc aagccccagc 720
gggcagcctg gacttggtgc ccggcgggct gaccttggag gagcactcgc tggagcaggt
780 gcagtccatg gtggtgggcg aagtgctcaa ggacatcgag acggcctgca
agctgctcaa 840 catcaccgca gatcccatgg actggagccc cagcaatgtg
cagaagtggc tcctgtggac 900 agagcaccaa taccggctgc cccccatggg
caaggccttc caggagctgg cgggcaagga 960 gctgtgcgcc atgtcggagg
agcagttccg ccagcgctcg cccctgggtg gggatgtgct 1020 gcacgcccac
ctggacatct ggaagtcagc ggcctggatg aaagagcgga cttcacctgg 1080
ggcgattcac tactgtgcct cgaccagtga ggagagctgg accgacagcg aggtggactc
1140 atcatgctcc gggcagccca tccacctgtg gcagttcctc aaggagttgc
tactcaagcc 1200 ccacagctat ggccgcttca ttaggtggct caacaaggag
aagggcatct tcaaaattga 1260 ggactcagcc caggtggccc ggctgtgggg
catccgcaag aaccgtcccg ccatgaacta 1320 cgacaagctg agccgctcca
tccgccagta ttacaagaag ggcatcatcc ggaagccaga 1380 catctcccag
cgcctcgtct accagttcgt gcaccccatc tgagtgcctg gcccagggcc 1440
tgaaacccgc cctcaggggc ctctctcctg cctgccctgc ctcagccagg ccctgagatg
1500 ggggaaaacg ggcagtctgc tctgctgctc tgaccttcca gagcccaagg
tcagggaggg 1560 gcaaccaact gccccagggg gatatgggtc ctctggggcc
ttcgggacca tggggcaggg 1620 gtgcttcctc ctcaggccca gctgctcccc
tggaggacag agggagacag ggctgctccc 1680 caacacctgc ctctgacccc
agcatttcca gagcagagcc tacagaaggg cagtgactcg 1740 acaaaggcca
caggcagtcc aggcctctct ctgctccatc cccctgcctc ccattctgca 1800
ccacacctgg catggtgcag ggagacatct gcacccctga gttgggcagc caggagtgcc
1860 cccgggaatg gataataaag atactagaga actg 1894 <210> SEQ ID
NO 51 <211> LENGTH: 2180 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 51 gggcggaaaa
gcctgtttac acagactgca caccgcctgg ggaataatgc agtaaaggaa 60
gtgagccggc tcggcctgac tgctccaact tcctgctctc acacacacca gaggggaaaa
120 aaaaagagga gcgagagaaa gaaaaaaagg gggaaaaatc aggatctcat
tacaagagcc 180 acagaccgtc tgcagacgcc tgtcagcatg gaaagtcggg
ggctttcgcc cgggtcctcc 240 tagaaattcc ccccgaagaa gactccccca
catctgggta tggagagtgc aatcacgctg 300 tggcagttcc tgttgcagtt
gctgctggat cagaaacatg agcatttgat ctgctggacc 360 tcgaacgatg
gtgaattcaa gctcctcaaa gcagaagaag tggccaagct gtggggactc 420
cgaaaaaaca aaacaaatat gaactatgat aagctgagca gagccctgcg atactattat
480 gacaagaaca tcatcaagaa ggtgatcggg cagaagtttg tgtacaagtt
tgtctctttc 540 ccggagatcc tgaagatgga tcctcacgcg gtggagatca
gccgggagag ccttctgctg 600 caggacagcg actgcaaggc gtctccggag
ggccgcgagg cccacaaaca cggcctggcc 660 gccctcagaa gcacgagccg
caacgaatac atccactcag gcctgtactc gtccttcacc 720 attaattccc
tgcagaaccc accagacgcc ttcaaggcca tcaagacgga gaagctggag 780
gagccgcccg aagacagccc ccccgtggaa gaagtcagga ctgtgatcag gtttgtgacc
840 aataaaaccg acaagcacgt caccaggccg gtggtgtccc tgccttccac
gtcagaggct 900 gcggcggcgt ccgccttcct ggcctcgtcc gtctcggcca
agatctcctc tttaatgttg 960 ccaaacgctg ccagtatttc atccgcctca
cccttctcat ctcggtcccc gtccctgtcc 1020 cccaactcac ccctcccttc
tgaacacaga agcctcttcc tggaggccgc ctgccatgac 1080 tccgattccc
tggagccctt gaacctgtca tcgggctcca agaccaagtc tccatctctt 1140
cccccaaagg ccaaaaaacc caaaggcttg gaaatctcag cgcccccgct ggtgctctcc
1200 ggcaccgaca tcggctccat cgccctcaac agcccagccc tcccctcggg
atccctcacc 1260 ccagccttct tcaccgcaca gacaccaaat ggattgcttc
tgactccgag tccactgctc 1320 tccagcatac atttctggag cagccttagt
ccagttgctc cgctgagtcc tgccaggctg 1380 caagggccaa gcacgctgtt
ccagttcccc acactgctta atggccacat gccagtgcca 1440 atccccagtc
tggacagagc tgcttctcca gtactgcttt cttcaaactc tcagaaatcc 1500
tgatgacgtc tggccacaat taaggactca ttaactgatg aaacaaattt gtccccacgg
1560 gctagtttac ctgtgtcgtg agaaggacat tgtgaaactc ttgttaattt
ggtttgcact 1620 tttcataaca tggatagtct agatttatgt tagcatttta
aaaactgttt ttgatatatt 1680 caagtatata tgaaaatctg tttggcatta
agtgaatttt aatgtttttg tttttatatc 1740 cttttagctc ttaagtgttg
aacactgttg acagtgaaga acttttctta atggttttca 1800 gtataactaa
taaggatgtg aagctttttt ctctttagtt ctgagtatgc taaactgtgt 1860
gcttatatag actataacca gttgtgcctt cctttgcatt taatgtaaat gaatgattta
1920 tatatttttt agtattaaga ggaaatgttt gaaagatgaa aattagtatc
aaacagctct 1980 ctagtagaat ttcattattt ttcaccagtg ggcaatatga
aagcatatat cacgttttgt 2040 tttactttca attgtataag aattgcctta
gaacctcttt tgaactgaaa ttcagtaaat 2100 gtccaagtaa tgtttttata
ataaactaag ccatatttag acaataaaca tcgaaaaaaa 2160 aaaaaaaaaa
aaaaaaaaaa 2180 <210> SEQ ID NO 52 <211> LENGTH: 3171
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 52 gttgccagct gcggcggccg ccacagccac
agccgccgcc gccgccgccg ccgcccctgc 60 ccctgccgcc cctgcccctg
ccgttaggtg gtggggtttc tcagcccggc ggcgggaggc 120 gggccggcct
cggcttcctg tcggaggacg cgcaaggatc cgggcgtcgg agtgtgtgcg 180
agtgcgtgag tgtgtgtcgg tcgcacggcg tgtgtctccg gccgcgggtt ccgcctcctc
240 ccctgccgcc gctgctcacg gtgtaagtca atgtgaagca gcagctccag
ccccgggata 300 aacatggcga cgtctctgca tgagggaccc acgaaccagc
tggatctgct catccgggcc 360 gtggaagcat cagttcacag cagtaatgca
cactgtacag ataagacaat tgaagctgct 420 gaagccctgc ttcatatgga
atctcctacc tgcttgaggg attcaagaag tcctgtggaa 480 gtgtttgttc
ctccttgtgt atcaactcca gaattcatcc atgctgctat gaggccagat 540
gtcattacag aaactgtagt ggaggtgtca actgaagagt ctgaacccat ggatacctct
600 cctattccaa catcaccaga tagccatgaa ccaatgaaaa agaaaaaagt
tggccgtaaa 660 ccaaagaccc agcaatcacc aatttccaat gggtctcctg
agttaggtat aaagaagaaa 720 ccaagagaag gaaaaggaaa cacaacctat
ttgtgggagt ttcttttaga tctacttcaa 780 gataaaaata cttgtcccag
gtatattaaa tggactcaga gagaaaaagg catattcaag 840 ctggtggatt
caaaggctgt ctctaagctt tggggaaagc ataagaacaa accagacatg 900
aactatgaaa ccatgggacg agctttgaga tactactacc aaaggggaat tcttgcaaag
960 gttgaaggac agaggcttgt atatcagttc aaggatatgc cgaaaaacat
agtggtcata 1020 gatgatgaca aaagtgaaac ctgtaatgaa gatttagcag
gaactactga tgaaaaatca 1080 ttagaacgag tgtcactgtc tgcagaaagt
ctcctgaaag cagcatcctc tgttcgcagt 1140 ggaaaaaatt catcccctat
aaactgctcc agagcagaga agggtgtagc tagagttgtg 1200 aatatcactt
cccctgggca cgatgcttca tccaggtctc ctactaccac tgcatctgtg 1260
tcagcaacag cagctccaag gacagttcgt gtggcaatgc aggtacctgt tgtaatgaca
1320 tcattgggtc agaaaatttc aactgtggca gttcagtcag ttaatgcagg
tgcaccatta 1380 ataaccagca ctagtccaac aacagcgacc tctccaaagg
tagtcattca gacaatccct 1440 actgtgatgc cagcttctac tgaaaatgga
gacaaaatca ccatgcagcc tgccaaaatt 1500 attaccatcc cagctacaca
gcttgcacag tgtcaactgc agacaaagtc aaatctgact 1560 ggatcaggaa
gcattaacat tgttggaacc ccattggctg tgagagcact tacccctgtt 1620
tcaatagccc atggtacacc tgtaatgaga ctatcaatgc ctactcagca ggcatctggc
1680 cagactcctc ctcgagttat cagtgcagtc ataaaggggc cagaggttaa
atcggaagca 1740 gtggcaaaaa agcaagaaca tgatgtgaaa actttgcagc
tagtagaaga aaaaccagca 1800 gatggaaata agacagtgac ccacgtagtg
gttgtcagtg cgccttcagc tattgccctt 1860 cctgtaacta tgaaaacaga
aggactagtg acatgtgaga aataaaatag cagctccacc 1920 atggacttca
ggctgttagt ggcagtactg acataaacat ttgcaaggga agtcatcaag 1980
aaaagtcaaa gaagacttta aaacattttt aatgcatata caaaaacaat cagacttact
2040 ggaaataaat tacctatccc atgtttcagt gggaaatgaa ctacatattg
agatgctgac 2100 agaaaactgc ctcttacagt aggaaacaac tgaacccatc
aataagaaaa aggatcgaaa 2160 gggaccaagc agctcactac gatatcaagt
tacactaaga cttggaacac taacattctg 2220 taagaggtta tatagttttc
agtgggaggg gttgggatgg gtaatctcat tgttacatat 2280 agcaattttt
gatgcatttt atatgcatac cagcaattat tactgtgttc gcacagttct 2340
cacttaactg gtgctatgtg aagactctgc taatataggt attttagaat gtgaattgaa
2400 gaatggatcc caaaaacttc agaaagagga tagcaaaaaa agatctagtg
cgattttata 2460 tatatatata tatatatata catacatata tatatatcat
atagcttaag ctgatttaaa 2520 acaaaggcct tagactaatt ttcgattttc
tttcttgaaa taagctaatg gcttgtttgt 2580 gtaaagcttt tttattaaaa
gaaaaatttt aaaaatcttg tacctagcac agtattgtta 2640 tagaatatac
atgtaacatt ttatatggta gtttaagtct gtcagtttct taattgtgga 2700
caaattaaca gttggctctg gccttttgct gtaacatgcc tgtgtcactc acttagcctt
2760 ggcatttgtg cagacatacc attttcagtt ctgctgtcac ttggaagttc
aggctcagca 2820 tgaatttttg gcaggtagct ctaatacctg gagttttctt
tgtttttttt tctttttttt 2880
agttgaagtt tatgagggaa ataccagtgt tcagttttga actataatag tttgtatatt
2940 caacatttga agtatattct attttgttgt actcttgttt caaagtgtat
tcaagtaggt 3000 tttctgaaat atagaaatga aatttatctt ctgttttggt
ctctggtgat attttaaaca 3060 atatttaaaa gtcagtatag aagtgtttta
gttaggaagt gataaaacat ctctcttctc 3120 cttcccaact actgcatgaa
gaaattctac ttccattata ttaatatttg g 3171 <210> SEQ ID NO 53
<211> LENGTH: 3218 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 53 aaaatagtga
aggatgctta gactacttaa catacaaact gctttctggt taatcatctt 60
tagaagactg gatttctgga tatctactcc actccatctc tattgacttt taaaacatga
120 taatgcaaac ctataacact ggcaaccatc agtgaacctt taatttcatt
gattaatagc 180 gtttgaagct tcctcaggga ataacaatga catcagcagt
ggttgacagt ggaggtacta 240 ttttggagct ttccagcaat ggagtagaaa
atcaagagga aagtgaaaag gtttctgaat 300 atccagcagt gattgtggag
ccagttccaa gtgccagatt agagcagggc tatgcagccc 360 aggttctggt
ttatgatgat gagacttata tgatgcaaga tgtggcagaa gaacaagaag 420
ttgagaccga gaatgtggaa acagtggaag catcagttca cagcagtaat gcacactgta
480 cagataagac aattgaagct gctgaagccc tgcttcatat ggaatctcct
acctgcttga 540 gggattcaag aagtcctgaa ttcatccatg ctgctatgag
gccagatgtc attacagaaa 600 ctgtagtgga ggtgtcaact gaagagtctg
aacccatgga tacctctcct attccaacat 660 caccagatag ccatgaacca
atgaaaaaga aaaaagttgg ccgtaaacca aagacccagc 720 aatcaccaat
ttccaatggg tctcctgagt taggtataaa gaagaaacca agagaaggaa 780
aaggaaacac aacctatttg tgggagtttc ttttagatct acttcaagat aaaaatactt
840 gtcccaggta tattaaatgg actcagagag aaaaaggcat attcaagctg
gtggattcaa 900 aggctgtctc taagctttgg ggaaagcata agaacaaacc
agacatgaac tatgaaacca 960 tgggacgagc tttgagatac tactaccaaa
ggggaattct tgcaaaggtt gaaggacaga 1020 ggcttgtata tcagttcaag
gatatgccga aaaacatagt ggtcatagat gatgacaaaa 1080 gtgaaacctg
taatgaagat ttagcaggaa ctactgatga aaaatcatta gaacgagtgt 1140
cactgtctgc agaaagtctc ctgaaagcag catcctctgt tcgcagtgga aaaaattcat
1200 cccctataaa ctgctccaga gcagagaagg gtgtagctag agttgtgaat
atcacttccc 1260 ctgggcacga tgcttcatcc aggtctccta ctaccactgc
atctgtgtca gcaacagcag 1320 ctccaaggac agttcgtgtg gcaatgcagg
tacctgttgt aatgacatca ttgggtcaga 1380 aaatttcaac tgtggcagtt
cagtcagtta atgcaggtgc accattaata accagcacta 1440 gtccaacaac
agcgacctct ccaaaggtag tcattcagac aatccctact gtgatgccag 1500
cttctactga aaatggagac aaaatcacca tgcagcctgc caaaattatt accatcccag
1560 ctacacagct tgcacagtgt caactgcaga caaagtcaaa tctgactgga
tcaggaagca 1620 ttaacattgt tggaacccca ttggctgtga gagcacttac
ccctgtttca atagcccatg 1680 gtacacctgt aatgagacta tcaatgccta
ctcagcaggc atctggccag actcctcctc 1740 gagttatcag tgcagtcata
aaggggccag aggttaaatc ggaagcagtg gcaaaaaagc 1800 aagaacatga
tgtgaaaact ttgcagctag tagaagaaaa accagcagat ggaaataaga 1860
cagtgaccca cgtagtggtt gtcagtgcgc cttcagctat tgcccttcct gtaactatga
1920 aaacagaagg actagtgaca tgtgagaaat aaaatagcag ctccaccatg
gacttcaggc 1980 tgttagtggc agtactgaca taaacatttg caagggaagt
catcaagaaa agtcaaagaa 2040 gactttaaaa catttttaat gcatatacaa
aaacaatcag acttactgga aataaattac 2100 ctatcccatg tttcagtggg
aaatgaacta catattgaga tgctgacaga aaactgcctc 2160 ttacagtagg
aaacaactga acccatcaat aagaaaaagg atcgaaaggg accaagcagc 2220
tcactacgat atcaagttac actaagactt ggaacactaa cattctgtaa gaggttatat
2280 agttttcagt gggaggggtt gggatgggta atctcattgt tacatatagc
aatttttgat 2340 gcattttata tgcataccag caattattac tgtgttcgca
cagttctcac ttaactggtg 2400 ctatgtgaag actctgctaa tataggtatt
ttagaatgtg aattgaagaa tggatcccaa 2460 aaacttcaga aagaggatag
caaaaaaaga tctagtgcga ttttatatat atatatatat 2520 atatatacat
acatatatat atatcatata gcttaagctg atttaaaaca aaggccttag 2580
actaattttc gattttcttt cttgaaataa gctaatggct tgtttgtgta aagctttttt
2640 attaaaagaa aaattttaaa aatcttgtac ctagcacagt attgttatag
aatatacatg 2700 taacatttta tatggtagtt taagtctgtc agtttcttaa
ttgtggacaa attaacagtt 2760 ggctctggcc ttttgctgta acatgcctgt
gtcactcact tagccttggc atttgtgcag 2820 acataccatt ttcagttctg
ctgtcacttg gaagttcagg ctcagcatga atttttggca 2880 ggtagctcta
atacctggag ttttctttgt ttttttttct tttttttagt tgaagtttat 2940
gagggaaata ccagtgttca gttttgaact ataatagttt gtatattcaa catttgaagt
3000 atattctatt ttgttgtact cttgtttcaa agtgtattca agtaggtttt
ctgaaatata 3060 gaaatgaaat ttatcttctg ttttggtctc tggtgatatt
ttaaacaata tttaaaagtc 3120 agtatagaag tgttttagtt aggaagtgat
aaaacatctc tcttctcctt cccaactact 3180 gcatgaagaa attctacttc
cattatatta atatttgg 3218 <210> SEQ ID NO 54 <211>
LENGTH: 1901 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 54 gcggcgagtg gagcgggagc cgactggaag
aagggctcta gggagggggc tgtggctgct 60 ggggtccgag gtggggccgg
gtacaccagc cccatcactg tttgcagaga gtcagggagg 120 cggaaaagac
acgcgctcta ggctcccatc agggcacatg gcccgggccc atcccccgcg 180
cgtctccccg gctgcggggc gcggggggct gccgggtgcg cttggctgtg gcgcggcgcg
240 ttggagactt tattgcgatg ggacgataag aggggcgggg gcggggtcct
gggggccgag 300 gcggcagcgc tttaattaaa acggaaattg cggccccggg
ccgcgcgggg gccggagggt 360 tccaagcggc cccttagctg gaagcgtttc
tccaggaccc ccccgcaacc cccgccacgc 420 ccgggctgcc ccctcccgcc
aggccctgcc ggacccggcg ccgtcttctc ctccttgtca 480 cccgcggtcg
cttcgggcgg ggatcggtgc caccgagcgc aaagcctgcc tcgcccccct 540
tccccgtccc ccccatctcc caccgcccag tccccggcgg cgatgagaca gagcggcgcc
600 tcccagcccc tgctgatcaa catgtacctg ccagatcccg tcggagacgg
tctcttcaag 660 gacgggaaga acccgagctg ggggccgctg agccccgcgg
ttcagaaagg cagcggacag 720 atccagctgt ggcagtttct gctggagctg
ctggctgacc gcgcgaacgc cggctgcatc 780 gcgtgggagg gcggtcacgg
cgagttcaag ctcacggacc cggacgaggt ggcgcggcgg 840 tggggcgagc
gcaagagcaa gcccaacatg aactacgaca agctgagccg cgccctgcgc 900
tactactacg acaagaacat catgagcaag gtgcatggca agcgctacgc ctaccgcttc
960 gacttccagg gcctggcgca ggcctgccag ccgccgcccg cgcacgctca
tgccgccgcc 1020 gcagctgctg ccgccgccgc ggccgcccag gacggcgcgc
tctacaagct gcccgccggc 1080 ctcgccccgc tgcccttccc cggcctctcc
aaactcaacc tcatggccgc ctcggccggg 1140 gtcgcgcccg ccggcttctc
ctactggccg ggcccgggcc ccgccgccac cgctgccgcc 1200 gccaccgccg
cgctctaccc cagtcccagc ttgcagcccc cgcccgggcc cttcggggcc 1260
gtggccgcag cctcgcactt ggggggccat taccactaga cggggcggtc gggtgcctgc
1320 ggcctcgccc gcacgcctag agtctcgccc gatcccatcg gcatcccggg
gagggcccgg 1380 gagcctccgt caaccgtcct ctaatccaga gtttactcca
cctgccgcac ttagcagggg 1440 gacgggaccg aagctccctc aatccttgtc
tggtactaga tttgctcctg tcccaccccg 1500 cagtcccctg aggagggcga
tgtgcgccct ctttcacttt ttttcttcta ggtctccagg 1560 tcccggaggg
gatttgtgga cctctcttgt ctccccacca ctccagtgca tttccgcctg 1620
gctcctagaa gccccattca atatcactac tctttaacga gtgccaaatc ttttcccact
1680 tttgctcttc cccaaggaac tgctcccacc tcagcacgtg gaggcctctc
acggtcctcc 1740 ttcctgggac ctgagcaggt ttggtgaaag ccaccgtcct
ccgtgacaca cggccccctt 1800 cctcctgtcc ccacactccc aggagaaact
cccggtgtgt ttctgaccct ttcagcccca 1860 ttaaagctcc tgagctctca
aaaaaaaaaa aaaaaaaaaa a 1901 <210> SEQ ID NO 55 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 55 taggcgcgag ctaagcagga g 21 <210> SEQ
ID NO 56 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 56 gtaggcacac
tcaaacaacg actgg 25 <210> SEQ ID NO 57 <211> LENGTH: 19
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 57 cgcgagctaa gcaggaggc 19 <210> SEQ ID
NO 58 <211> LENGTH: 22 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 58 caggccatga
aaagccaaac tt 22 <210> SEQ ID NO 59 <211> LENGTH: 49
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 59 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
tttcucgauu cguccuccg 49 <210> SEQ ID NO 60 <211>
LENGTH: 51
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 60 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
tttauccgcg cucgauucgu c 51 <210> SEQ ID NO 61 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 61 gagggcgagg ggcggggagc gcc 23 <210>
SEQ ID NO 62 <211> LENGTH: 31 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 62
cctatcatta ctcgatgctg ttgataacag c 31 <210> SEQ ID NO 63
<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 63 aatttaatac gactcactat
agggagaaac tttcagcctg ata 43 <210> SEQ ID NO 64 <211>
LENGTH: 51 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 64 aatttaatac gactcactat agggagactc
tgtgagtcat ttgtcttgct t 51 <210> SEQ ID NO 65 <211>
LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 65 aatttaatac gactcactat agggagagca
cactcaaaca acgactg 47 <210> SEQ ID NO 66 <211> LENGTH:
23 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 66 gcgcggcagc ucagguaccu gac 23 <210>
SEQ ID NO 67 <211> LENGTH: 26 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 67
gcuuugaacu cacucaggua ccugac 26 <210> SEQ ID NO 68
<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 68 gagcgcggca ggaagccuua ucaguug
27 <210> SEQ ID NO 69 <211> LENGTH: 28 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
69 gagcgcggca gguuauucca ggaucuuu 28 <210> SEQ ID NO 70
<211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 70 cgcggcagga agcctta 17
<210> SEQ ID NO 71 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 71
tccgtaggca cactcaaaca ac 22 <210> SEQ ID NO 72 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 72 cagttgtgag tgaggacc 18 <210> SEQ ID
NO 73 <211> LENGTH: 72 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 73 ggggtgcagc
ttttattttc ccaaatactt cagtatatcc tgaagctacg caaagaatta 60
caactaggcc ag 72 <210> SEQ ID NO 74 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 74 Met Ala Ser Thr Ile Lys Glu Ala Leu Ser
Val Val Ser Glu Asp Gln 1 5 10 15 Ser Leu Phe Glu Cys Ala Tyr Gly
Thr Pro His Leu Ala Lys Thr Glu 20 25 30 Met Thr Ala Ser Ser Ser
Ser Asp 35 40 <210> SEQ ID NO 75 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 75 Ser Ser Ser Ser Asp 1 5 <210> SEQ ID
NO 76 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 76 Tyr Gly Gln Thr Ser
Lys Met Ser Pro Arg Val Pro Gln Gln Asp Trp 1 5 10 15 Leu Ser Gln
Pro Pro Ala Arg Val Thr Ile Lys Met Glu Cys Asn Pro 20 25 30 Ser
Gln Val Asn Gly Ser Arg Asn 35 40 <210> SEQ ID NO 77
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 77 Val Pro Gln Gln Asp Trp Leu
Ser Gln Pro Pro Ala Arg 1 5 10 <210> SEQ ID NO 78 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 78 Val Pro Gln Gln Asp Trp Leu Ser Gln Pro
Pro Ala Arg 1 5 10 <210> SEQ ID NO 79 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 79 Val Pro Gln Gln Asp Trp Leu Ser Gln Pro
Pro Ala Arg 1 5 10 <210> SEQ ID NO 80 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 80 Val Pro Gln Gln Asp Trp Leu Ser Gln Pro
Pro Ala Arg 1 5 10 <210> SEQ ID NO 81 <211> LENGTH: 21
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 81 Tyr Gly Gln Thr Ser Lys Met Ser Val Pro
Gln Gln Asp Trp Leu Ser 1 5 10 15 Gln Pro Pro Ala Arg 20
<210> SEQ ID NO 82 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 82 Val
Pro Gln Gln Asp Trp Leu Ser Gln Pro Pro Ala Arg
1 5 10 <210> SEQ ID NO 83 <211> LENGTH: 13 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
83 Val Pro Gln Gln Asp Trp Leu Ser Gln Pro Pro Ala Arg 1 5 10
<210> SEQ ID NO 84 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 84 Val
Pro Gln Gln Asp Trp Leu Ser Gln Pro Pro Ala Arg 1 5 10 <210>
SEQ ID NO 85 <211> LENGTH: 25 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 85 Val Pro
Gln Gln Asp Trp Leu Ser Gln Pro Pro Ala Arg Met Glu Cys 1 5 10 15
Asn Pro Ser Gln Val Asn Gly Ser Arg 20 25 <210> SEQ ID NO 86
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 86 Val Pro Gln Gln Asp Trp Leu
Ser Gln Pro Pro Ala Arg 1 5 10 <210> SEQ ID NO 87 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 87 Val Pro Gln Gln Asp Trp Leu Ser Gln Pro
Pro Ala Arg 1 5 10 <210> SEQ ID NO 88 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 88 Ser Pro Asp Glu Cys Ser Val Ala Lys Gly
Gly Lys Met Val Gly Ser 1 5 10 15 Pro Asp Thr Val Gly Met Asn Tyr
Gly Ser Tyr Met Glu Glu Lys His 20 25 30 Met Pro Pro Pro Asn Met
Thr Thr 35 40 <210> SEQ ID NO 89 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 89 His Met Pro Pro Pro Asn Met Thr Thr 1 5
<210> SEQ ID NO 90 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 90 His
Met Pro Pro Pro Asn Met Thr Thr 1 5 <210> SEQ ID NO 91
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 91 His Met Pro Pro Pro Asn Met
Thr Thr 1 5 <210> SEQ ID NO 92 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 92 Asn Tyr Gly Ser Tyr Met Glu Glu Lys His
Met Pro 1 5 10 <210> SEQ ID NO 93 <211> LENGTH: 28
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 93 Met Val Gly Ser Pro Asp Thr Val Gly Met
Asn Tyr Gly Ser Tyr Met 1 5 10 15 Glu Glu Lys His Met Pro Pro Pro
Asn Met Thr Thr 20 25 <210> SEQ ID NO 94 <211> LENGTH:
9 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 94 His Met Pro Pro Pro Asn Met Thr Thr 1 5
<210> SEQ ID NO 95 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 95 His
Met Pro Pro Pro Asn Met Thr Thr 1 5 <210> SEQ ID NO 96
<211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 96 Met Val Gly Ser Pro Asp Thr
Val Gly Met Asn Tyr Gly Ser Tyr Met 1 5 10 15 Glu Glu Lys His Met
Pro Pro Pro Asn Met Thr Thr 20 25 <210> SEQ ID NO 97
<211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 97 Met Val Gly Ser Pro Asp Thr
Val Gly Met Asn Tyr Gly Ser Tyr Met 1 5 10 15 Glu Glu Lys His Met
Pro Pro Pro Asn Met Thr Thr 20 25 <210> SEQ ID NO 98
<211> LENGTH: 40 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 98 Asn Glu Arg Arg Val Ile Val
Pro Ala Asp Pro Thr Leu Trp Ser Thr 1 5 10 15 Asp His Val Arg Gln
Trp Leu Glu Trp Ala Val Lys Glu Tyr Gly Leu 20 25 30 Pro Asp Val
Asn Ile Leu Leu Phe 35 40 <210> SEQ ID NO 99 <211>
LENGTH: 39 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 99 Asn Glu Arg Val Ile Val Pro Ala Asp Pro
Thr Leu Trp Ser Thr Asp 1 5 10 15 His Val Arg Gln Trp Leu Glu Trp
Ala Val Lys Glu Tyr Gly Leu Pro 20 25 30 Asp Val Asn Ile Leu Leu
Phe 35 <210> SEQ ID NO 100 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
100 Asn Glu Arg Glu Tyr Gly Leu Pro Asp Val Asn Ile Leu Leu Phe 1 5
10 15 <210> SEQ ID NO 101 <211> LENGTH: 3 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
101 Asn Glu Arg 1 <210> SEQ ID NO 102 <211> LENGTH: 27
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 102 Asn Glu Arg Val Ile Val Pro Ala Asp Pro
Thr Leu Trp Ser Thr Asp 1 5 10 15
His Val Arg Gln Trp Leu Glu Trp Ala Val Lys 20 25 <210> SEQ
ID NO 103 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 103 Asn Glu Arg Arg
Val Ile Val Pro Ala Asp Pro Thr Leu Trp Ser Thr 1 5 10 15 Asp His
Val Arg Glu Tyr Gly Leu Pro Asp Val Asn Ile Leu Leu Phe 20 25 30
<210> SEQ ID NO 104 <211> LENGTH: 39 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 104
Asn Glu Arg Val Ile Val Pro Ala Asp Pro Thr Leu Trp Ser Thr Asp 1 5
10 15 His Val Arg Gln Trp Leu Glu Trp Ala Val Lys Glu Tyr Gly Leu
Pro 20 25 30 Asp Val Asn Ile Leu Leu Phe 35 <210> SEQ ID NO
105 <211> LENGTH: 40 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 105 Asn Glu Arg Arg
Val Ile Val Pro Ala Asp Pro Thr Leu Trp Ser Thr 1 5 10 15 Asp His
Val Arg Gln Trp Leu Glu Trp Ala Val Lys Glu Tyr Gly Leu 20 25 30
Pro Asp Val Asn Ile Leu Leu Phe 35 40 <210> SEQ ID NO 106
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 106 Asn Glu Arg Arg Val Ile Val
Pro Ala Asp Pro Thr Leu Trp Ser Thr 1 5 10 15 Asp His Val Arg 20
<210> SEQ ID NO 107 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 107
Glu Tyr Gly Leu Pro Asp Val Asn Ile Leu Leu Phe 1 5 10 <210>
SEQ ID NO 108 <211> LENGTH: 40 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 108 Gln
Asn Ile Asp Gly Lys Glu Leu Cys Lys Met Thr Lys Asp Asp Phe 1 5 10
15 Gln Arg Leu Thr Pro Ser Tyr Asn Ala Asp Ile Leu Leu Ser His Leu
20 25 30 His Tyr Leu Arg Glu Thr Pro Leu 35 40 <210> SEQ ID
NO 109 <211> LENGTH: 28 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 109 Gln Asn Ile Asp
Gly Lys Leu Thr Pro Ser Tyr Asn Ala Asp Ile Leu 1 5 10 15 Leu Ser
His Leu His Tyr Leu Arg Glu Thr Pro Leu 20 25 <210> SEQ ID NO
110 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 110 Glu Thr Pro Leu 1
<210> SEQ ID NO 111 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 111
Gln Asn Ile Asp Gly Lys Glu Thr Pro Leu 1 5 10 <210> SEQ ID
NO 112 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 112 Glu Thr Pro Leu 1
<210> SEQ ID NO 113 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 113
Glu Thr Pro Leu 1 <210> SEQ ID NO 114 <211> LENGTH: 28
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 114 Gln Asn Ile Asp Gly Lys Leu Thr Pro Ser
Tyr Asn Ala Asp Ile Leu 1 5 10 15 Leu Ser His Leu His Tyr Leu Arg
Glu Thr Pro Leu 20 25 <210> SEQ ID NO 115 <211> LENGTH:
4 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 115 Glu Thr Pro Leu 1 <210> SEQ ID NO
116 <211> LENGTH: 28 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 116 Gln Asn Ile Asp
Gly Lys Leu Thr Pro Ser Tyr Asn Ala Asp Ile Leu 1 5 10 15 Leu Ser
His Leu His Tyr Leu Arg Glu Thr Pro Leu 20 25 <210> SEQ ID NO
117 <211> LENGTH: 28 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 117 Gln Asn Ile Asp
Gly Lys Leu Thr Pro Ser Tyr Asn Ala Asp Ile Leu 1 5 10 15 Leu Ser
His Leu His Tyr Leu Arg Glu Thr Pro Leu 20 25 <210> SEQ ID NO
118 <211> LENGTH: 22 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 118 Leu Thr Pro Ser
Tyr Asn Ala Asp Ile Leu Leu Ser His Leu His Tyr 1 5 10 15 Leu Arg
Glu Thr Pro Leu 20 <210> SEQ ID NO 119 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 119 Gln Asn Ile Asp Gly Lys 1 5 <210>
SEQ ID NO 120 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 120 Pro
Ser Tyr Asn Ala Asp Ile Leu Leu Ser His Leu His Tyr Leu Arg 1 5 10
15 Glu Thr Pro Leu 20 <210> SEQ ID NO 121 <211> LENGTH:
40 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 121
Pro His Leu Thr Ser Asp Asp Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5
10 15 Arg Leu Met His Ala Arg Asn Thr Gly Gly Ala Ala Phe Ile Phe
Pro 20 25 30 Asn Thr Ser Val Tyr Pro Glu Ala 35 40 <210> SEQ
ID NO 122 <211> LENGTH: 9 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 122 Pro Arg Leu Met
His Ala Arg Asn Thr 1 5 <210> SEQ ID NO 123 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 123 Pro His Leu Thr Ser Asp Asp Val Asp Lys 1
5 10 <210> SEQ ID NO 124 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
124 Pro His Leu Thr Ser Asp Asp Val Asp Lys Ala Leu Gln Asn Ser Pro
1 5 10 15 Arg <210> SEQ ID NO 125 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 125 Pro His Leu Thr Ser Asp Asp Val Asp Lys
Ala Leu Gln Asn Ser Pro 1 5 10 15 Arg <210> SEQ ID NO 126
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 126 Pro His Leu Thr Ser Asp Asp
Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5 10 15 Arg <210> SEQ
ID NO 127 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 127 Pro His Leu Thr
Ser Asp Asp Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5 10 15 Arg
<210> SEQ ID NO 128 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 128
Pro His Leu Thr Ser Asp Asp Val Asp Lys 1 5 10 <210> SEQ ID
NO 129 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 129 Pro His Leu Thr
Ser Asp Asp Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5 10 15 Arg
<210> SEQ ID NO 130 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 130
Pro His Leu Thr Ser Asp Asp Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5
10 15 Arg <210> SEQ ID NO 131 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 131 Pro His Leu Thr Ser Asp Asp Val Asp Lys
Ala Leu Gln Asn Ser Pro 1 5 10 15 Arg <210> SEQ ID NO 132
<211> LENGTH: 3 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 132 Arg Asn Thr 1 <210>
SEQ ID NO 133 <211> LENGTH: 17 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 133 Pro
His Leu Thr Ser Asp Asp Val Asp Lys Ala Leu Gln Asn Ser Pro 1 5 10
15 Arg <210> SEQ ID NO 134 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
134 Pro His Leu Thr Ser Asp Asp Val Asp Lys Ala Leu Gln Asn Ser Pro
1 5 10 15 Arg Leu <210> SEQ ID NO 135 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 135 Thr Gln Arg Ile Thr Thr Arg Pro Asp Leu
Pro Tyr Glu Pro Pro Arg 1 5 10 15 Arg Ser Ala Trp Thr Gly His Gly
His Pro Thr Pro Gln Ser Lys Ala 20 25 30 Ala Gln Pro Ser Pro Ser
Thr Val 35 40 <210> SEQ ID NO 136 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 136 Asp Leu Pro Tyr Glu Pro Pro Arg 1 5
<210> SEQ ID NO 137 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 137
Ser Ala Trp Thr Gly His Gly His Pro Thr Pro Gln Ser Lys Ala Ala 1 5
10 15 Gln Pro Ser Pro Ser Thr Val 20 <210> SEQ ID NO 138
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 138 Pro Tyr Glu Pro Pro Arg Arg
1 5 <210> SEQ ID NO 139 <211> LENGTH: 23 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
139 Ser Ala Trp Thr Gly His Gly His Pro Thr Pro Gln Ser Lys Ala Ala
1 5 10 15 Gln Pro Ser Pro Ser Thr Val 20 <210> SEQ ID NO 140
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 140 Ser Ala Trp Thr Gly His Gly
His Pro Thr Pro Gln Ser Lys Ala Ala 1 5 10 15 Gln Pro Ser Pro Ser
Thr Val 20
<210> SEQ ID NO 141 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 141
Pro Tyr Glu Pro Pro Arg Arg Ser Ala Trp Thr Gly His Gly His Pro 1 5
10 15 Thr Pro Gln Ser Lys Ala Ala Gln Pro Ser Pro Ser Thr Val 20 25
30 <210> SEQ ID NO 142 <211> LENGTH: 23 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
142 Ser Ala Trp Thr Gly His Gly His Pro Thr Pro Gln Ser Lys Ala Ala
1 5 10 15 Gln Pro Ser Pro Ser Thr Val 20 <210> SEQ ID NO 143
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 143 Ser Ala Trp Thr Gly His Gly
His Pro Thr Pro Gln Ser Lys Ala Ala 1 5 10 15 Gln Pro Ser Pro Ser
Thr Val 20 <210> SEQ ID NO 144 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 144 Asp Leu Pro Tyr Glu Pro Pro Arg Arg 1 5
<210> SEQ ID NO 145 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 145
Pro Tyr Glu Pro Pro Arg Arg Ser Ala Trp Thr Gly His Gly His Pro 1 5
10 15 Thr Pro Gln Ser Lys Ala Ala Gln Pro Ser Pro Ser Thr Val 20 25
30 <210> SEQ ID NO 146 <211> LENGTH: 32 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
146 Asp Leu Pro Tyr Glu Pro Pro Arg Arg Ser Ala Trp Thr Gly His Gly
1 5 10 15 His Pro Thr Pro Gln Ser Lys Ala Ala Gln Pro Ser Pro Ser
Thr Val 20 25 30 <210> SEQ ID NO 147 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 147 Asp Leu Pro Tyr Glu Pro Pro Arg Arg 1 5
<210> SEQ ID NO 148 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 148
Asp Leu Pro Tyr Glu Pro Pro Arg Arg Ser Ala Trp Thr Gly His Gly 1 5
10 15 His Pro Thr Pro Gln Ser Lys Ala Ala Gln Pro Ser Pro Ser Thr
Val 20 25 30 <210> SEQ ID NO 149 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 149 Ala Ala Gln Pro Ser Pro Ser Thr Val 1 5
<210> SEQ ID NO 150 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 150
Ser Ala Trp Thr Gly His Gly His Pro Thr Pro Gln Ser Lys Ala Ala 1 5
10 15 Gln Pro Ser Pro Ser Thr Val 20 <210> SEQ ID NO 151
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 151 Asp Leu Pro Tyr Glu Pro Pro
Arg Arg Ser Ala Trp Thr Gly His Gly 1 5 10 15 His Pro Thr Pro Gln
Ser Lys Ala Ala Gln Pro Ser Pro Ser Thr Val 20 25 30 <210>
SEQ ID NO 152 <211> LENGTH: 40 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 152 Pro
Lys Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5 10
15 Gly Pro Thr Ser Ser Arg Leu Ala Asn Pro Gly Ser Gly Gln Ile Gln
20 25 30 Leu Trp Gln Phe Leu Leu Glu Leu 35 40 <210> SEQ ID
NO 153 <211> LENGTH: 2 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 153 Pro Lys 1
<210> SEQ ID NO 154 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 154
Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu Gly Pro 1 5
10 15 Thr Ser Ser Arg 20 <210> SEQ ID NO 155 <211>
LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 155 Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu
Asp Pro Tyr Gln Ile Leu 1 5 10 15 Gly Pro Thr Ser Ser Arg 20
<210> SEQ ID NO 156 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 156
Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5
10 15 Gly Pro Thr Ser Ser Arg 20 <210> SEQ ID NO 157
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 157 Pro Lys Thr Glu Asp Gln Arg
Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5 10 15 Gly Pro Thr Ser Ser
Arg 20 <210> SEQ ID NO 158 <211> LENGTH: 22 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
158 Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu
1 5 10 15 Gly Pro Thr Ser Ser Arg 20 <210> SEQ ID NO 159
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 159
Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5
10 15 Gly Pro Thr Ser Ser Arg 20 <210> SEQ ID NO 160
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 160 Thr Glu Asp Gln Arg Pro Gln
Leu Asp Pro Tyr Gln Ile Leu Gly Pro 1 5 10 15 Thr Ser Ser Arg 20
<210> SEQ ID NO 161 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 161
Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5
10 15 Gly Pro Thr Ser Ser Arg 20 <210> SEQ ID NO 162
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 162 Pro Lys Thr Glu Asp Gln Arg
Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5 10 15 Gly Pro Thr Ser Ser
Arg 20 <210> SEQ ID NO 163 <211> LENGTH: 22 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
163 Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu
1 5 10 15 Gly Pro Thr Ser Ser Arg 20 <210> SEQ ID NO 164
<211> LENGTH: 2 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 164 Pro Lys 1 <210> SEQ ID
NO 165 <211> LENGTH: 22 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 165 Pro Lys Thr Glu
Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu 1 5 10 15 Gly Pro
Thr Ser Ser Arg 20 <210> SEQ ID NO 166 <211> LENGTH: 22
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 166 Pro Lys Thr Glu Asp Gln Arg Pro Gln Leu
Asp Pro Tyr Gln Ile Leu 1 5 10 15 Gly Pro Thr Ser Ser Arg 20
<210> SEQ ID NO 167 <211> LENGTH: 2 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 167
Pro Lys 1 <210> SEQ ID NO 168 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 168 Leu Ser Asp Ser Ser Asn Ser Ser Cys Ile
Thr Trp Glu Gly Thr Asn 1 5 10 15 Gly Glu Phe Lys Met Thr Asp Pro
Asp Glu Val Ala Arg Arg Trp Gly 20 25 30 Glu Arg Lys Ser Lys Pro
Asn Met 35 40 <210> SEQ ID NO 169 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 169 Met Thr Asp Pro Asp Glu Val Ala Arg 1 5
<210> SEQ ID NO 170 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 170
Met Thr Asp Pro Asp Glu Val Ala Arg 1 5 <210> SEQ ID NO 171
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 171 Met Thr Asp Pro Asp Glu Val
Ala Arg 1 5 <210> SEQ ID NO 172 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 172 Met Thr Asp Pro Asp Glu Val Ala Arg 1 5
<210> SEQ ID NO 173 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 173
Met Thr Asp Pro Asp Glu Val Ala Arg 1 5 <210> SEQ ID NO 174
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 174 Met Thr Asp Pro Asp Glu Val
Ala Arg Arg 1 5 10 <210> SEQ ID NO 175 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 175 Thr Asp Pro Asp Glu Val Ala Arg Arg Lys
Ser Lys Pro Asn Met 1 5 10 15 <210> SEQ ID NO 176 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 176 Met Thr Asp Pro Asp Glu Val Ala Arg 1 5
<210> SEQ ID NO 177 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 177
Lys Ser Lys Pro Asn Met 1 5 <210> SEQ ID NO 178 <211>
LENGTH: 40 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 178 Asn Tyr Asp Lys Leu Ser Arg Ala Leu Arg
Tyr Tyr Tyr Asp Lys Asn 1 5 10 15 Ile Met Thr Lys Val His Gly Lys
Arg Tyr Ala Tyr Lys Phe Asp Phe 20 25 30 His Gly Ile Ala Gln Ala
Leu Gln 35 40 <210> SEQ ID NO 179 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 179 Phe Asp Phe His Gly Ile Ala Gln Ala Leu
Gln 1 5 10 <210> SEQ ID NO 180 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 180 Phe Asp Phe His Gly Ile Ala Gln Ala Leu
Gln 1 5 10 <210> SEQ ID NO 181 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 181 Phe Asp Phe His Gly Ile Ala Gln Ala Leu
Gln 1 5 10 <210> SEQ ID NO 182 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 182 Phe Asp Phe His Gly Ile Ala Gln Ala Leu
Gln 1 5 10 <210> SEQ ID NO 183 <211> LENGTH: 25
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 183 Tyr Tyr Tyr Asp Lys Asn Ile Met Thr Lys
Tyr Ala Tyr Lys Phe Asp 1 5 10 15 Phe His Gly Ile Ala Gln Ala Leu
Gln 20 25 <210> SEQ ID NO 184 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 184 Asn Tyr Asp Lys Leu Ser Arg 1 5
<210> SEQ ID NO 185 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 185
Asn Tyr Asp Lys Leu Ser Arg Tyr Tyr Tyr Asp Lys Asn Ile Met Thr 1 5
10 15 Lys <210> SEQ ID NO 186 <211> LENGTH: 40
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 186 Pro His Pro Pro Glu Ser Ser Leu Tyr Lys
Tyr Pro Ser Asp Leu Pro 1 5 10 15 Tyr Met Gly Ser Tyr His Ala His
Pro Gln Lys Met Asn Phe Val Ala 20 25 30 Pro His Pro Pro Ala Leu
Pro Val 35 40 <210> SEQ ID NO 187 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 187 Pro His Pro Pro Glu Ser Ser Leu Tyr Lys 1
5 10 <210> SEQ ID NO 188 <211> LENGTH: 24 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
188 Pro His Pro Pro Glu Ser Ser Leu Tyr Lys Tyr Pro Ser Asp Leu Pro
1 5 10 15 Tyr Met Gly Ser Tyr His Ala His 20 <210> SEQ ID NO
189 <211> LENGTH: 27 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 189 Pro His Pro Pro
Glu Ser Ser Leu Tyr Lys Tyr Pro Ser Asp Leu Pro 1 5 10 15 Tyr Met
Gly Ser Tyr His Ala His Pro Gln Lys 20 25 <210> SEQ ID NO 190
<211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 190 Pro His Pro Pro Glu Ser Ser
Leu Tyr Lys Tyr Pro Ser Asp Leu Pro 1 5 10 15 Tyr Met Gly Ser Tyr
His Ala His Pro Gln Lys 20 25 <210> SEQ ID NO 191 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 191 Tyr Pro Ser Asp Leu Pro Tyr Met Gly Ser
Tyr His Ala His Pro Gln 1 5 10 15 Lys <210> SEQ ID NO 192
<211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 192 Pro His Pro Pro Glu Ser Ser
Leu Tyr Lys Tyr Pro Ser Asp Leu Pro 1 5 10 15 Tyr Met Gly Ser Tyr
His Ala His Pro Gln Lys 20 25 <210> SEQ ID NO 193 <211>
LENGTH: 39 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 193 Thr Ser Ser Ser Phe Phe Ala Ala Pro Asn
Pro Tyr Trp Asn Ser Pro 1 5 10 15 Thr Gly Gly Ile Tyr Pro Asn Thr
Arg Leu Pro Thr Ser His Met Pro 20 25 30 Ser His Leu Gly Thr Tyr
Tyr 35 <210> SEQ ID NO 194 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
194 Asn Ser Pro Thr Gly 1 5 <210> SEQ ID NO 195 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 195 Ser Pro Thr Gly Gly Ile Tyr Pro Asn Thr
Arg 1 5 10 <210> SEQ ID NO 196 <211> LENGTH: 24
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 196 Gly Gly Ala Ala Phe Ile Phe Pro Asn Thr
Ser Val Tyr Pro Glu Ala 1 5 10 15 Thr Gln Arg Ile Thr Thr Arg Pro
20 <210> SEQ ID NO 197 <211> LENGTH: 10 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
197 Val Pro Gln Gln Asp Trp Leu Ser Gln Pro 1 5 10 <210> SEQ
ID NO 198 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 198 Val Pro Gln Gln
Asp Trp Leu Ser Gln Pro 1 5 10 <210> SEQ ID NO 199
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 199 Met Ile Gln Thr Val Pro Asp
Pro Ala Ala His Ile 1 5 10 <210> SEQ ID NO 200 <211>
LENGTH: 55 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 200 Met Ala Ser Thr Ile Lys Glu Ala Leu Ser
Val Val Ser Glu Asp Gln 1 5 10 15 Ser Leu Phe Glu Cys Ala Tyr Gly
Thr Pro His Leu Ala Lys Thr Glu 20 25 30 Met Thr Ala Tyr Gly Gln
Thr Ser Lys Met Ser Pro Arg Val Pro Gln 35 40 45 Gln Asp Trp Leu
Ser Gln Pro 50 55 <210> SEQ ID NO 201 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 201 Met Ile Gln Thr Val Pro Asp Pro Ala Ala
His Ile 1 5 10 <210> SEQ ID NO 202 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 202 gctgcagact tggccaaatg gac 23 <210>
SEQ ID NO 203 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 203
tcaccaccga cagagcctcc tta 23 <210> SEQ ID NO 204 <211>
LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 204 accacatgaa tggatccagg gagtct 26
<210> SEQ ID NO 205 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 205
accagcttgc tgcatttgct aacg 24 <210> SEQ ID NO 206 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 206 ctccgcgcca ccaccctcta 20 <210> SEQ
ID NO 207 <211> LENGTH: 24 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 207 ggccagcagt
gaactttccc tgag 24 <210> SEQ ID NO 208 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 208 ctcctcccaa catgaccacc aac 23 <210>
SEQ ID NO 209 <211> LENGTH: 22 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 209
gtctgcgggg acgatgactc tc 22 <210> SEQ ID NO 210 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 210 catgtgaggc aatggctgga gtg 23 <210>
SEQ ID NO 211 <211> LENGTH: 26 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 211
ccatgttctg gaaaaaggat gtgtcg 26 <210> SEQ ID NO 212
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 212 ccttggaggg gcacaaacga t 21
<210> SEQ ID NO 213 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 213
ggtcgggccc aggatctgat ac 22 <210> SEQ ID NO 214 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 214 cgccaacgcc agctgtatca c 21 <210>
SEQ ID NO 215 <211> LENGTH: 19 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 215
agcgcctggc cacctcatc 19 <210> SEQ ID NO 216 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 216 atgtttttat gaccaaagca gtttcttgtc 30
<210> SEQ ID NO 217 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 217
atgacgggtt aagtccatga ttctgtg 27 <210> SEQ ID NO 218
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 218 cgaaagctgc tcaaccatct c 21
<210> SEQ ID NO 219 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 219
taactgagga cgctggtctt ca 22 <210> SEQ ID NO 220 <211>
LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 220 ccacagtgcc caaaa 15 <210> SEQ ID NO
221 <400> SEQUENCE: 221 000 <210> SEQ ID NO 222
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 222 cggcaaaaga tatgcttaca aattt
25 <210> SEQ ID NO 223 <211> LENGTH: 19 <212>
TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 223
gacgactcgg tcggatgtg 19 <210> SEQ ID NO 224 <211>
LENGTH: 14 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 224 cacggcattg ccca 14 <210> SEQ ID NO
225 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 225 cgcggcagga agcctta
17 <210> SEQ ID NO 226 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
226 tccgtaggca cactcaaaca ac 22 <210> SEQ ID NO 227
<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 227 cagttgtgag tgaggacc 18
<210> SEQ ID NO 228 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 228
gctcgctccg atactattat gagaa 25 <210> SEQ ID NO 229
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 229 cacacacaaa cttgtacacg taacg
25 <210> SEQ ID NO 230 <211> LENGTH: 16 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
230 accagccacc ttctgc 16 <210> SEQ ID NO 231 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 231 ggtcgaggca tggaatttaa actga 25
<210> SEQ ID NO 232 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 232
gctggcctgt ttttctgaat gc 22 <210> SEQ ID NO 233 <211>
LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 233 tcgggccacc tcttc 15 <210> SEQ ID NO
234 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 234 Met Ile Gln Thr
Val Pro Asp Pro Ala Ala His Ile 1 5 10 <210> SEQ ID NO 235
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 235 Ser Ala Trp Thr Gly His Gly
His Pro Thr Pro Gln Ser Lys Ala Ala 1 5 10 15 Gln Pro Ser Pro Ser
Thr Val 20 <210> SEQ ID NO 236 <211> LENGTH: 42
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: NON_CONS <222>
LOCATION: (18)..(19) <400> SEQUENCE: 236 Met Ile Gln Thr Val
Pro Asp Pro Ala Ala Ser His Ile Lys Glu Ala 1 5 10 15 Leu Ser Gly
Gly Ala Ala Phe Ile Phe Pro Asn Thr Ser Val Tyr Pro 20 25 30 Glu
Ala Thr Gln Arg Ile Thr Thr Arg Pro 35 40 <210> SEQ ID NO 237
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 237 Met Ile Gln Thr Val Pro Asp
Pro Ala Ala Ser His Ile Lys Glu Ala 1 5 10 15 Leu Ser <210>
SEQ ID NO 238 <211> LENGTH: 42 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: NON_CONS <222> LOCATION: (10)..(11) <220>
FEATURE: <221> NAME/KEY: NON_CONS <222> LOCATION:
(14)..(15) <220> FEATURE: <221> NAME/KEY: NON_CONS
<222> LOCATION: (18)..(19) <400> SEQUENCE: 238 Met Ala
Ser Thr Ile Lys Glu Ala Leu Ser Met Thr Ala Ser Met Ser 1 5 10 15
Pro Arg Gly Gly Ala Ala Phe Ile Phe Pro Asn Thr Ser Val Tyr Pro 20
25 30 Glu Ala Thr Gln Arg Ile Thr Thr Arg Pro 35 40 <210> SEQ
ID NO 239 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
NON_CONS <222> LOCATION: (10)..(11) <220> FEATURE:
<221> NAME/KEY: NON_CONS <222> LOCATION: (14)..(15)
<400> SEQUENCE: 239 Met Ala Ser Thr Ile Lys Glu Ala Leu Ser
Met Thr Ala Ser Met Ser 1 5 10 15 Pro Arg <210> SEQ ID NO 240
<211> LENGTH: 479 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 240 Met Ala Ser Thr Ile Lys Glu
Ala Leu Ser Val Val Ser Glu Asp Gln 1 5 10 15 Ser Leu Phe Glu Cys
Ala Tyr Gly Thr Pro His Leu Ala Lys Thr Glu 20 25 30 Met Thr Ala
Ser Ser Ser Ser Asp Tyr Gly Gln Thr Ser Lys Met Ser 35 40 45 Pro
Arg Val Pro Gln Gln Asp Trp Leu Ser Gln Pro Pro Ala Arg Val 50 55
60 Thr Ile Lys Met Glu Cys Asn Pro Ser Gln Val Asn Gly Ser Arg Asn
65 70 75 80 Ser Pro Asp Glu Cys Ser Val Ala Lys Gly Gly Lys Met Val
Gly Ser 85 90 95 Pro Asp Thr Val Gly Met Asn Tyr Gly Ser Tyr Met
Glu Glu Lys His 100 105 110 Met Pro Pro Pro Asn Met Thr Thr Asn Glu
Arg Arg Val Ile Val Pro 115 120 125 Ala Asp Pro Thr Leu Trp Ser Thr
Asp His Val Arg Gln Trp Leu Glu 130 135 140 Trp Ala Val Lys Glu Tyr
Gly Leu Pro Asp Val Asn Ile Leu Leu Phe 145 150 155 160 Gln Asn Ile
Asp Gly Lys Glu Leu Cys Lys Met Thr Lys Asp Asp Phe 165 170 175 Gln
Arg Leu Thr Pro Ser Tyr Asn Ala Asp Ile Leu Leu Ser His Leu
180 185 190 His Tyr Leu Arg Glu Thr Pro Leu Pro His Leu Thr Ser Asp
Asp Val 195 200 205 Asp Lys Ala Leu Gln Asn Ser Pro Arg Leu Met His
Ala Arg Asn Thr 210 215 220 Gly Gly Ala Ala Phe Ile Phe Pro Asn Thr
Ser Val Tyr Pro Glu Ala 225 230 235 240 Thr Gln Arg Ile Thr Thr Arg
Pro Asp Leu Pro Tyr Glu Pro Pro Arg 245 250 255 Arg Ser Ala Trp Thr
Gly His Gly His Pro Thr Pro Gln Ser Lys Ala 260 265 270 Ala Gln Pro
Ser Pro Ser Thr Val Pro Lys Thr Glu Asp Gln Arg Pro 275 280 285 Gln
Leu Asp Pro Tyr Gln Ile Leu Gly Pro Thr Ser Ser Arg Leu Ala 290 295
300 Asn Pro Gly Ser Gly Gln Ile Gln Leu Trp Gln Phe Leu Leu Glu Leu
305 310 315 320 Leu Ser Asp Ser Ser Asn Ser Ser Cys Ile Thr Trp Glu
Gly Thr Asn 325 330 335 Gly Glu Phe Lys Met Thr Asp Pro Asp Glu Val
Ala Arg Arg Trp Gly 340 345 350 Glu Arg Lys Ser Lys Pro Asn Met Asn
Tyr Asp Lys Leu Ser Arg Ala 355 360 365 Leu Arg Tyr Tyr Tyr Asp Lys
Asn Ile Met Thr Lys Val His Gly Lys 370 375 380 Arg Tyr Ala Tyr Lys
Phe Asp Phe His Gly Ile Ala Gln Ala Leu Gln 385 390 395 400 Pro His
Pro Pro Glu Ser Ser Leu Tyr Lys Tyr Pro Ser Asp Leu Pro 405 410 415
Tyr Met Gly Ser Tyr His Ala His Pro Gln Lys Met Asn Phe Val Ala 420
425 430 Pro His Pro Pro Ala Leu Pro Val Thr Ser Ser Ser Phe Phe Ala
Ala 435 440 445 Pro Asn Pro Tyr Trp Asn Ser Pro Thr Gly Gly Ile Tyr
Pro Asn Thr 450 455 460 Arg Leu Pro Thr Ser His Met Pro Ser His Leu
Gly Thr Tyr Tyr 465 470 475 <210> SEQ ID NO 241 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 241 Gly Ala Ala Phe Ile Phe Pro Asn Thr Ser
Val Tyr Pro 1 5 10 <210> SEQ ID NO 242 <211> LENGTH:
477 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 242 Met Asp Gly Phe Tyr Asp Gln Gln Val Pro
Tyr Met Val Thr Asn Ser 1 5 10 15 Gln Arg Gly Arg Asn Cys Asn Glu
Lys Pro Thr Asn Val Arg Lys Arg 20 25 30 Lys Phe Ile Asn Arg Asp
Leu Ala His Asp Ser Glu Glu Leu Phe Gln 35 40 45 Asp Leu Ser Gln
Leu Gln Glu Thr Trp Leu Ala Glu Ala Gln Val Pro 50 55 60 Asp Asn
Asp Glu Gln Phe Val Pro Asp Tyr Gln Ala Glu Ser Leu Ala 65 70 75 80
Phe His Gly Leu Pro Leu Lys Ile Lys Lys Glu Pro His Ser Pro Cys 85
90 95 Ser Glu Ile Ser Ser Ala Cys Ser Gln Glu Gln Pro Phe Lys Phe
Ser 100 105 110 Tyr Gly Glu Lys Cys Leu Tyr Asn Val Ser Ala Tyr Asp
Gln Lys Pro 115 120 125 Gln Val Gly Met Arg Pro Ser Asn Pro Pro Thr
Pro Ser Ser Thr Pro 130 135 140 Val Ser Pro Leu His His Ala Ser Pro
Asn Ser Thr His Thr Pro Lys 145 150 155 160 Pro Asp Arg Ala Phe Pro
Ala His Leu Pro Pro Ser Gln Ser Ile Pro 165 170 175 Asp Ser Ser Tyr
Pro Met Asp His Arg Phe Arg Arg Gln Leu Ser Glu 180 185 190 Pro Cys
Asn Ser Phe Pro Pro Leu Pro Thr Met Pro Arg Glu Gly Arg 195 200 205
Pro Met Tyr Gln Arg Gln Met Ser Glu Pro Asn Ile Pro Phe Pro Pro 210
215 220 Gln Gly Phe Lys Gln Glu Tyr His Asp Pro Val Tyr Glu His Asn
Thr 225 230 235 240 Met Val Gly Ser Ala Ala Ser Gln Ser Phe Pro Pro
Pro Leu Met Ile 245 250 255 Lys Gln Glu Pro Arg Asp Phe Ala Tyr Asp
Ser Glu Val Pro Ser Cys 260 265 270 His Ser Ile Tyr Met Arg Gln Glu
Gly Phe Leu Ala His Pro Ser Arg 275 280 285 Thr Glu Gly Cys Met Phe
Glu Lys Gly Pro Arg Gln Phe Tyr Asp Asp 290 295 300 Thr Cys Val Val
Pro Glu Lys Phe Asp Gly Asp Ile Lys Gln Glu Pro 305 310 315 320 Gly
Met Tyr Arg Glu Gly Pro Thr Tyr Gln Arg Arg Gly Ser Leu Gln 325 330
335 Leu Trp Gln Phe Leu Val Ala Leu Leu Asp Asp Pro Ser Asn Ser His
340 345 350 Phe Ile Ala Trp Thr Gly Arg Gly Met Glu Phe Lys Leu Ile
Glu Pro 355 360 365 Glu Glu Val Ala Arg Arg Trp Gly Ile Gln Lys Asn
Arg Pro Ala Met 370 375 380 Asn Tyr Asp Lys Leu Ser Arg Ser Leu Arg
Tyr Tyr Tyr Glu Lys Gly 385 390 395 400 Ile Met Gln Lys Val Ala Gly
Glu Arg Tyr Val Tyr Lys Phe Val Cys 405 410 415 Asp Pro Glu Ala Leu
Phe Ser Met Ala Phe Pro Asp Asn Gln Arg Pro 420 425 430 Leu Leu Lys
Thr Asp Met Glu Arg His Ile Asn Glu Glu Asp Thr Val 435 440 445 Pro
Leu Ser His Phe Asp Glu Ser Met Ala Tyr Met Pro Glu Gly Gly 450 455
460 Cys Cys Asn Pro His Pro Tyr Asn Glu Gly Tyr Val Tyr 465 470 475
<210> SEQ ID NO 243 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 243
Pro His Ser Pro Cys Ser Glu Ile Ser Ser Ala Cys Ser Gln 1 5 10
<210> SEQ ID NO 244 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 244
Thr Pro Ser Ser Thr Pro Val Ser Pro Leu His His Ala 1 5 10
<210> SEQ ID NO 245 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 245
Arg Ala Phe Pro Ala His Leu Pro Pro Ser Gln Ser Ile Pro Asp Ser 1 5
10 15 <210> SEQ ID NO 246 <211> LENGTH: 452 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
246 Met Asp Gly Thr Ile Lys Glu Ala Leu Ser Val Val Ser Asp Asp Gln
1 5 10 15 Ser Leu Phe Asp Ser Ala Tyr Gly Ala Ala Ala His Leu Pro
Lys Ala 20 25 30 Asp Met Thr Ala Ser Gly Ser Pro Asp Tyr Gly Gln
Pro His Lys Ile 35 40 45 Asn Pro Leu Pro Pro Gln Gln Glu Trp Ile
Asn Gln Pro Val Arg Val 50 55 60 Asn Val Lys Arg Glu Tyr Asp His
Met Asn Gly Ser Arg Glu Ser Pro 65 70 75 80 Val Asp Cys Ser Val Ser
Lys Cys Ser Lys Leu Val Gly Gly Gly Glu 85 90 95 Ser Asn Pro Met
Asn Tyr Asn Ser Tyr Met Asp Glu Lys Asn Gly Pro 100 105 110 Pro Pro
Pro Asn Met Thr Thr Asn Glu Arg Arg Val Ile Val Pro Ala 115 120 125
Asp Pro Thr Leu Trp Thr Gln Glu His Val Arg Gln Trp Leu Glu Trp 130
135 140 Ala Ile Lys Glu Tyr Ser Leu Met Glu Ile Asp Thr Ser Phe Phe
Gln 145 150 155 160 Asn Met Asp Gly Lys Glu Leu Cys Lys Met Asn Lys
Glu Asp Phe Leu 165 170 175 Arg Ala Thr Thr Leu Tyr Asn Thr Glu Val
Leu Leu Ser His Leu Ser 180 185 190 Tyr Leu Arg Glu Ser Ser Leu Leu
Ala Tyr Asn Thr Thr Ser His Thr 195 200 205 Asp Gln Ser Ser Arg Leu
Ser Val Lys Glu Asp Pro Ser Tyr Asp Ser 210 215 220 Val Arg Arg Gly
Ala Trp Gly Asn Asn Met Asn Ser Gly Leu Asn Lys 225 230 235 240
Ser Pro Pro Leu Gly Gly Ala Gln Thr Ile Ser Lys Asn Thr Glu Gln 245
250 255 Arg Pro Gln Pro Asp Pro Tyr Gln Ile Leu Gly Pro Thr Ser Ser
Arg 260 265 270 Leu Ala Asn Pro Gly Ser Gly Gln Ile Gln Leu Trp Gln
Phe Leu Leu 275 280 285 Glu Leu Leu Ser Asp Ser Ala Asn Ala Ser Cys
Ile Thr Trp Glu Gly 290 295 300 Thr Asn Gly Glu Phe Lys Met Thr Asp
Pro Asp Glu Val Ala Arg Arg 305 310 315 320 Trp Gly Glu Arg Lys Ser
Lys Pro Asn Met Asn Tyr Asp Lys Leu Ser 325 330 335 Arg Ala Leu Arg
Tyr Tyr Tyr Asp Lys Asn Ile Met Thr Lys Val His 340 345 350 Gly Lys
Arg Tyr Ala Tyr Lys Phe Asp Phe His Gly Ile Ala Gln Ala 355 360 365
Leu Gln Pro His Pro Thr Glu Ser Ser Met Tyr Lys Tyr Pro Ser Asp 370
375 380 Ile Ser Tyr Met Pro Ser Tyr His Ala His Gln Gln Lys Val Asn
Phe 385 390 395 400 Val Pro Pro His Pro Ser Ser Met Pro Val Thr Ser
Ser Ser Phe Phe 405 410 415 Gly Ala Ala Ser Gln Tyr Trp Thr Ser Pro
Thr Gly Gly Ile Tyr Pro 420 425 430 Asn Pro Asn Val Pro Arg His Pro
Asn Thr His Val Pro Ser His Leu 435 440 445 Gly Ser Tyr Tyr 450
<210> SEQ ID NO 247 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 247
Ser Leu Phe Asp Ser Ala Tyr Gly Ala Ala Ala His Leu Pro 1 5 10
<210> SEQ ID NO 248 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 248
His Lys Ile Asn Pro Leu Pro Pro Gln Ile Asn Gln Pro Val Arg Val 1 5
10 15 Asn Val <210> SEQ ID NO 249 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 249 Arg Glu Ser Pro Val Asp Cys Ser Val Ser
Lys Cys Ser Lys Leu Val 1 5 10 15 Gly <210> SEQ ID NO 250
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 250 Thr Gln Glu His Val Arg Gln
1 5 <210> SEQ ID NO 251 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
251 Ser Ser Arg Leu Ser Val Lys Glu 1 5 <210> SEQ ID NO 252
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 252 Pro Asn Thr His Val Pro Ser
His Leu 1 5 <210> SEQ ID NO 253 <211> LENGTH: 484
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 253 Met Glu Arg Arg Met Lys Ala Gly Tyr Leu
Asp Gln Gln Val Pro Tyr 1 5 10 15 Thr Phe Ser Ser Lys Ser Pro Gly
Asn Gly Ser Leu Arg Glu Ala Leu 20 25 30 Ile Gly Pro Leu Gly Lys
Leu Met Asp Pro Gly Ser Leu Pro Pro Leu 35 40 45 Asp Ser Glu Asp
Leu Phe Gln Asp Leu Ser His Phe Gln Glu Thr Trp 50 55 60 Leu Ala
Glu Ala Gln Val Pro Asp Ser Asp Glu Gln Phe Val Pro Asp 65 70 75 80
Phe His Ser Glu Asn Leu Ala Phe His Ser Pro Thr Thr Arg Ile Lys 85
90 95 Lys Glu Pro Gln Ser Pro Arg Thr Asp Pro Ala Leu Ser Cys Ser
Arg 100 105 110 Lys Pro Pro Leu Pro Tyr His His Gly Glu Gln Cys Leu
Tyr Ser Ser 115 120 125 Ala Tyr Asp Pro Pro Arg Gln Ile Ala Ile Lys
Ser Pro Ala Pro Gly 130 135 140 Ala Leu Gly Gln Ser Pro Leu Gln Pro
Phe Pro Arg Ala Glu Gln Arg 145 150 155 160 Asn Phe Leu Arg Ser Ser
Gly Thr Ser Gln Pro His Pro Gly His Gly 165 170 175 Tyr Leu Gly Glu
His Ser Ser Val Phe Gln Gln Pro Leu Asp Ile Cys 180 185 190 His Ser
Phe Thr Ser Gln Gly Gly Gly Arg Glu Pro Leu Pro Ala Pro 195 200 205
Tyr Gln His Gln Leu Ser Glu Pro Cys Pro Pro Tyr Pro Gln Gln Ser 210
215 220 Phe Lys Gln Glu Tyr His Asp Pro Leu Tyr Glu Gln Ala Gly Gln
Pro 225 230 235 240 Ala Val Asp Gln Gly Gly Val Asn Gly His Arg Tyr
Pro Gly Ala Gly 245 250 255 Val Val Ile Lys Gln Glu Gln Thr Asp Phe
Ala Tyr Asp Ser Asp Val 260 265 270 Thr Gly Cys Ala Ser Met Tyr Leu
His Thr Glu Gly Phe Ser Gly Pro 275 280 285 Ser Pro Gly Asp Gly Ala
Met Gly Tyr Gly Tyr Glu Lys Pro Leu Arg 290 295 300 Pro Phe Pro Asp
Asp Val Cys Val Val Pro Glu Lys Phe Glu Gly Asp 305 310 315 320 Ile
Lys Gln Glu Gly Val Gly Ala Phe Arg Glu Gly Pro Pro Tyr Gln 325 330
335 Arg Arg Gly Ala Leu Gln Leu Trp Gln Phe Leu Val Ala Leu Leu Asp
340 345 350 Asp Pro Thr Asn Ala His Phe Ile Ala Trp Thr Gly Arg Gly
Met Glu 355 360 365 Phe Lys Leu Ile Glu Pro Glu Glu Val Ala Arg Leu
Trp Gly Ile Gln 370 375 380 Lys Asn Arg Pro Ala Met Asn Tyr Asp Lys
Leu Ser Arg Ser Leu Arg 385 390 395 400 Tyr Tyr Tyr Glu Lys Gly Ile
Met Gln Lys Val Ala Gly Glu Arg Tyr 405 410 415 Val Tyr Lys Phe Val
Cys Glu Pro Glu Ala Leu Phe Ser Leu Ala Phe 420 425 430 Pro Asp Asn
Gln Arg Pro Ala Leu Lys Ala Glu Phe Asp Arg Pro Val 435 440 445 Ser
Glu Glu Asp Thr Val Pro Leu Ser His Leu Asp Glu Ser Pro Ala 450 455
460 Tyr Leu Pro Glu Leu Ala Gly Pro Ala Gln Pro Phe Gly Pro Lys Gly
465 470 475 480 Gly Tyr Ser Tyr <210> SEQ ID NO 254
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 254 Gly Tyr Leu Asp Gln Gln Val
Pro Tyr Thr Phe Ser 1 5 10 <210> SEQ ID NO 255 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 255 Leu Arg Glu Ala Leu Ile Gly Pro Leu Gly
Lys 1 5 10 <210> SEQ ID NO 256 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 256 Leu Phe Gln Asp Leu Ser His 1 5
<210> SEQ ID NO 257 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 257 Ser Glu Asn Leu Ala Phe His 1 5
<210> SEQ ID NO 258 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 258
Thr Asp Pro Ala Leu Ser Cys 1 5 <210> SEQ ID NO 259
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 259 Ser Arg Lys Pro Pro Leu Pro
Tyr His His Gly Glu Gln Cys Leu Tyr 1 5 10 15 Ser Ser Ala Tyr 20
<210> SEQ ID NO 260 <211> LENGTH: 25 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 260
Asp Pro Pro Arg Gln Ile Ala Ile Lys Ser Pro Ala Pro Gly Ala Leu 1 5
10 15 Gly Gln Ser Pro Leu Gln Pro Phe Pro 20 25 <210> SEQ ID
NO 261 <211> LENGTH: 21 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 261 Ser Gln Pro His
Pro Gly His Tyr Leu Gly Glu His Ser Ser Val Phe 1 5 10 15 Gln Gln
Pro Leu Asp 20 <210> SEQ ID NO 262 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 262 Ile Cys His Ser Phe 1 5 <210> SEQ
ID NO 263 <211> LENGTH: 21 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 263 Glu Pro Leu Pro
Ala Pro Tyr Gln His Gln Leu Ser Glu Pro Cys Pro 1 5 10 15 Pro Tyr
Pro Gln Gln 20 <210> SEQ ID NO 264 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 264 Tyr His Asp Pro Leu Tyr Glu Gln Gly Gln
Pro Ala Val Asp Gln 1 5 10 15 <210> SEQ ID NO 265 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 265 Arg Tyr Pro Gly Ala Gly Val Val Ile Lys 1
5 10 <210> SEQ ID NO 266 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
266 Tyr Asp Ser Asp Val Thr Gly Cys Ala Ser Met Tyr 1 5 10
<210> SEQ ID NO 267 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 267
Tyr Glu Lys Pro Leu Arg Pro Phe Pro Asp Asp Val 1 5 10 <210>
SEQ ID NO 268 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 268 Cys
Val Val Pro Glu 1 5 <210> SEQ ID NO 269 <211> LENGTH:
71 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 269 cgcgagctaa gcaggaggcg gaggcggagg
cggagggcga ggggcgggga gcgccgcctg 60 gagcgcggca g 71 <210> SEQ
ID NO 270 <211> LENGTH: 142 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 270 cgcgagctaa
gcaggaggcg gaggcggagg cggagggcga ggggcgggga gcgccgcctg 60
gagcgcggca ggtcatattg aacattccag atacctatca ttactcgatg ctgttgataa
120 cagcaagatg gctttgaact ca 142 <210> SEQ ID NO 271
<211> LENGTH: 365 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 271 cgcgagctaa gcaggaggcg
gaggcggagg cggagggcga ggggcgggga gcgccgcctg 60 gagcgcggca
ggtcatattg aacattccag atacctatca ttactcgatg ctgttgataa 120
cagcaagatg gctttgaact cagggtcacc accagctatt ggaccttact atgaaaacca
180 tggataccaa ccggaaaacc cctatcccgc acagcccact gtggtcccca
ctgtctacga 240 ggtgcatccg gctcagtact acccgtcccc cgtgccccag
tacgccccga gggtcctgac 300 gcaggcttcc aaccccgtcg tctgcacgca
gcccaaatcc ccatccggga cagtgtgcac 360 ctcaa 365 <210> SEQ ID
NO 272 <211> LENGTH: 452 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 272 cgcgagctaa
gcaggaggcg gaggcggagg cggagggcga ggggcgggga gcgccgcctg 60
gagcgcggca ggtcatattg aacattccag atacctatca ttactcgatg ctgttgataa
120 cagcaagatg gctttgaact cagggtcacc accagctatt ggaccttact
atgaaaacca 180 tggataccaa ccggaaaacc cctatcccgc acagcccact
gtggtcccca ctgtctacga 240 ggtgcatccg gctcagtact acccgtcccc
cgtgccccag tacgccccga gggtcctgac 300 gcaggcttcc aaccccgtcg
tctgcacgca gcccaaatcc ccatccggga cagtgtgcac 360 ctcaaagact
aagaaagcac tgtgcatcac cttgaccctg gggaccttcc tcgtgggagc 420
tgcgctggcc gctggcctac tctggaagtt ca 452 <210> SEQ ID NO 273
<211> LENGTH: 572 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 273 cgcgagctaa gcaggaggcg
gaggcggagg cggagggcga ggggcgggga gcgccgcctg 60 gagcgcggca
ggtcatattg aacattccag atacctatca ttactcgatg ctgttgataa 120
cagcaagatg gctttgaact cagggtcacc accagctatt ggaccttact atgaaaacca
180 tggataccaa ccggaaaacc cctatcccgc acagcccact gtggtcccca
ctgtctacga 240 ggtgcatccg gctcagtact acccgtcccc cgtgccccag
tacgccccga gggtcctgac 300 gcaggcttcc aaccccgtcg tctgcacgca
gcccaaatcc ccatccggga cagtgtgcac 360 ctcaaagact aagaaagcac
tgtgcatcac cttgaccctg gggaccttcc tcgtgggagc 420 tgcgctggcc
gctggcctac tctggaagtt catgggcagc aagtgctcca actctgggat 480
agagtgcgac tcctcaggta cctgcatcaa cccctctaac tggtgtgatg gcgtgtcaca
540 ctgccccggc ggggaggacg agaatcggtg tg 572 <210> SEQ ID NO
274 <211> LENGTH: 67 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 274 ctttgataaa
taagtttgta agaggagcct cagcatcgta aagagctttt ctccccgctt 60 ctcgcag
67
<210> SEQ ID NO 275 <211> LENGTH: 33 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 275
atcgtaaaga gcttttctcc ccgcttctcg cag 33 <210> SEQ ID NO 276
<211> LENGTH: 102 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 276 gttattccag gatctttgga
gacccgagga aagccgtgtt gaccaaaagc aagacaaatg 60 actcacagag
aaaaaagatg gcagaaccaa gggcaactaa ag 102 <210> SEQ ID NO 277
<211> LENGTH: 86 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 277 ccgtcaggtt ctgaacagct
ggtagatggg ctggcttact gaaggacatg attcagactg 60 tcccggaccc
agcagctcat atcaag 86 <210> SEQ ID NO 278 <211> LENGTH:
218 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 278 gaagccttat cagttgtgag tgaggaccag
tcgttgtttg agtgtgccta cggaacgcca 60 cacctggcta agacagagat
gaccgcgtcc tcctccagcg actatggaca gacttccaag 120 atgagcccac
gcgtccctca gcaggattgg ctgtctcaac ccccagccag ggtcaccatc 180
aaaatggaat gtaaccctag ccaggtgaat ggctcaag 218 <210> SEQ ID NO
279 <211> LENGTH: 152 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 279 gaactctcct
gatgaatgca gtgtggccaa aggcgggaag atggtgggca gcccagacac 60
cgttgggatg aactacggca gctacatgga ggagaagcac atgccacccc caaacatgac
120 cacgaacgag cgcagagtta tcgtgccagc ag 152 <210> SEQ ID NO
280 <211> LENGTH: 204 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 280 atcctacgct
atggagtaca gaccatgtgc ggcagtggct ggagtgggcg gtgaaagaat 60
atggccttcc agacgtcaac atcttgttat tccagaacat cgatgggaag gaactgtgca
120 agatgaccaa ggacgacttc cagaggctca cccccagcta caacgccgac
atccttctct 180 cacatctcca ctacctcaga gaga 204 <210> SEQ ID NO
281 <211> LENGTH: 81 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 281 ctcctcttcc
acatttgact tcagatgatg ttgataaagc cttacaaaac tctccacggt 60
taatgcatgc tagaaacaca g 81 <210> SEQ ID NO 282 <211>
LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 282 ggggtgcagc ttttattttc ccaaatactt
cagtatatcc tgaagctacg caaagaatta 60 caactaggcc ag 72 <210>
SEQ ID NO 283 <211> LENGTH: 69 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 283
atttaccata tgagcccccc aggagatcag cctggaccgg tcacggccac cccacgcccc
60 agtcgaaag 69 <210> SEQ ID NO 284 <211> LENGTH: 57
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 284 ctgctcaacc atctccttcc acagtgccca
aaactgaaga ccagcgtcct cagttag 57 <210> SEQ ID NO 285
<211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 285 atccttatca gattcttgga
ccaacaagta gccgccttgc aaatccag 48 <210> SEQ ID NO 286
<211> LENGTH: 521 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 286 gcagtggcca gatccagctt
tggcagttcc tcctggagct cctgtcggac agctccaact 60 ccagctgcat
cacctgggaa ggcaccaacg gggagttcaa gatgacggat cccgacgagg 120
tggcccggcg ctggggagag cggaagagca aacccaacat gaactacgat aagctcagcc
180 gcgccctccg ttactactat gacaagaaca tcatgaccaa ggtccatggg
aagcgctacg 240 cctacaagtt cgacttccac gggatcgccc aggccctcca
gccccacccc ccggagtcat 300 ctctgtacaa gtacccctca gacctcccgt
acatgggctc ctatcacgcc cacccacaga 360 agatgaactt tgtggcgccc
caccctccag ccctccccgt gacatcttcc agtttttttg 420 ctgccccaaa
cccatactgg aattcaccaa ctgggggtat ataccccaac actaggctcc 480
ccaccagcca tatgccttct catctgggca cttactacta a 521 <210> SEQ
ID NO 287 <211> LENGTH: 54 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 287 ctcaggtacc
tgacaatgat gagcagtttg taccagacta tcaggctgaa agtt 54 <210> SEQ
ID NO 288 <211> LENGTH: 130 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 288 tggcttttca
tggcctgcca ctgaaaatca agaaagaacc ccacagtcca tgttcagaaa 60
tcagctctgc ctgcagtcaa gaacagccct ttaaattcag ctatggagaa aagtgcctgt
120 acaatgtcag 130 <210> SEQ ID NO 289 <211> LENGTH:
189 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 289 tgcctatgat cagaagccac aagtgggaat
gaggccctcc aaccccccca caccatccag 60 cacgccagtg tccccactgc
atcatgcatc tccaaactca actcatacac cgaaacctga 120 ccgggccttc
ccagctcacc tccctccatc gcagtccata ccagatagca gctaccccat 180
ggaccacag 189 <210> SEQ ID NO 290 <211> LENGTH: 248
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 290 atttcgccgc cagctttctg aaccctgtaa
ctcctttcct cctttgccga cgatgccaag 60 ggaaggacgt cctatgtacc
aacgccagat gtctgagcca aacatcccct tcccaccaca 120 aggctttaag
caggagtacc acgacccagt gtatgaacac aacaccatgg ttggcagtgc 180
ggccagccaa agctttcccc ctcctctgat gattaaacag gaacccagag attttgcata
240 tgactcag 248 <210> SEQ ID NO 291 <211> LENGTH: 69
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 291 aagtgcctag ctgccactcc atttatatga
ggcaagaagg cttcctggct catcccagca 60 gaacagaag 69 <210> SEQ ID
NO 292 <211> LENGTH: 69 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 292 gctgtatgtt
tgaaaagggc cccaggcagt tttatgatga cacctgtgtt gtcccagaaa 60 aattcgatg
69 <210> SEQ ID NO 293 <211> LENGTH: 170 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
293
gagacatcaa acaagagcca ggaatgtatc gggaaggacc cacataccaa cggcgaggat
60 cacttcagct ctggcagttt ttggtagctc ttctggatga cccttcaaat
tctcatttta 120 ttgcctggac tggtcgaggc atggaattta aactgattga
gcctgaagag 170 <210> SEQ ID NO 294 <211> LENGTH: 102
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 294 gtggcccgac gttggggcat tcagaaaaac
aggccagcta tgaactatga taaacttagc 60 cgttcactcc gctattacta
tgagaaagga attatgcaaa ag 102 <210> SEQ ID NO 295 <211>
LENGTH: 222 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 295 gtggctggag agagatatgt ctacaagttt
gtgtgtgatc cagaagccct tttctccatg 60 gcctttccag ataatcagcg
tccactgctg aagacagaca tggaacgtca catcaacgag 120 gaggacacag
tgcctctttc tcactttgat gagagcatgg cctacatgcc ggaagggggc 180
tgctgcaacc cccaccccta caacgaaggc tacgtgtatt aa 222 <210> SEQ
ID NO 296 <211> LENGTH: 93 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 296 aaatcgcccg
gaaatgggag cttgcgcgaa gcgctgatcg gcccgctggg gaagctcatg 60
gacccgggct ccctgccgcc ctcgactctg aag 93 <210> SEQ ID NO 297
<211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 297 atctcttcca ggatctaagt
cacttccagg agacgtggct cgctgaag 48 <210> SEQ ID NO 298
<211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 298 ctcaggtacc agacagtgat
gagcagtttg ttcctgattt ccattcagaa aacc 54 <210> SEQ ID NO 299
<211> LENGTH: 127 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 299 tagctttcca cagccccacc
accaggatca agaaggagcc ccagagtccc cgcacagacc 60 cggccctgtc
ctgcagcagg aagccgccac tcccctacca ccatggcgag cagtgccttt 120 actccag
127 <210> SEQ ID NO 300 <211> LENGTH: 162 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
300 tgcctatgac ccccccagac aaatcgccat caagtcccct gcccctggtg
cccttggaca 60 gtcgccccta cagccctttc cccgggcaga gcaacggaat
ttcctgagat cctctggcac 120 ctcccagccc caccctggcc atgggtacct
cggggaacat ag 162 <210> SEQ ID NO 301 <211> LENGTH: 266
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 301 ctccgtcttc cagcagcccc tggacatttg
ccactccttc acatctcagg gagggggccg 60 ggaacccctc ccagccccct
accaacacca gctgtcggag ccctgcccac cctatcccca 120 gcagagcttt
aagcaagaat accatgatcc cctgtatgaa caggcgggcc agccagccgt 180
ggaccagggt ggggtcaatg ggcacaggta cccaggggcg ggggtggtga tcaaacagga
240 acagacggac ttcgcctacg actcag 266 <210> SEQ ID NO 302
<211> LENGTH: 75 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 302 gtgtcaccgg gtgcgcatca
atgtacctcc acacagaggg cttctctggg ccctctccag 60 gtgacggggc catgg 75
<210> SEQ ID NO 303 <211> LENGTH: 69 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 303
gctatggcta tgagaaacct ctgcgaccat tcccagatga tgtctgcgtt gtccctgaga
60 aatttgaag 69 <210> SEQ ID NO 304 <211> LENGTH: 173
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 304 gagacatcaa gcaggaaggg gtcggtgcat
ttcgagaggg gccgccctac cagcgccggg 60 gtgccctgca gctgtggcaa
tttctggtgg ccttgctgga tgacccaaca aatgcccatt 120 tcattgcctg
gacgggccgg ggaatggagt tcaagctcat tgagcctgag gag 173 <210> SEQ
ID NO 305 <211> LENGTH: 102 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 305 gtcgccaggc
tctggggcat ccagaagaac cggccagcca tgaattacga caagctgagc 60
cgctcgctcc gatactatta tgagaaaggc atcatgcaga ag 102 <210> SEQ
ID NO 306 <211> LENGTH: 225 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 306 gtggctggtg
agcgttacgt gtacaagttt gtgtgtgagc ccgaggccct cttctctttg 60
gccttcccgg acaatcagcg tccagctctc aaggctgagt ttgaccggcc tgtcagtgag
120 gaggacacag tccctttgtc ccacttggat gagagccccg cctacctccc
agagctggct 180 ggccccgccc agccatttgg ccccaagggt ggctactctt actag
225 <210> SEQ ID NO 307 <211> LENGTH: 3226 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
307 cgcgagctaa gcaggaggcg gaggcggagg cggagggcga ggggcgggga
gcgccgcctg 60 gagcgcggca ggtcatattg aacattccag atacctatca
ttactcgatg ctgttgataa 120 cagcaagatg gctttgaact cagggtcacc
accagctatt ggaccttact atgaaaacca 180 tggataccaa ccggaaaacc
cctatcccgc acagcccact gtggtcccca ctgtctacga 240 ggtgcatccg
gctcagtact acccgtcccc cgtgccccag tacgccccga gggtcctgac 300
gcaggcttcc aaccccgtcg tctgcacgca gcccaaatcc ccatccggga cagtgtgcac
360 ctcaaagact aagaaagcac tgtgcatcac cttgaccctg gggaccttcc
tcgtgggagc 420 tgcgctggcc gctggcctac tctggaagtt catgggcagc
aagtgctcca actctgggat 480 agagtgcgac tcctcaggta cctgcatcaa
cccctctaac tggtgtgatg gcgtgtcaca 540 ctgccccggc ggggaggacg
agaatcggtg tgttcgcctc tacggaccaa acttcatcct 600 tcagatgtac
tcatctcaga ggaagtcctg gcaccctgtg tgccaagacg actggaacga 660
gaactacggg cgggcggcct gcagggacat gggctataag aataattttt actctagcca
720 aggaatagtg gatgacagcg gatccaccag ctttatgaaa ctgaacacaa
gtgccggcaa 780 tgtcgatatc tataaaaaac tgtaccacag tgatgcctgt
tcttcaaaag cagtggtttc 840 tttacgctgt atagcctgcg gggtcaactt
gaactcaagc cgccagagca ggatcgtggg 900 cggtgagagc gcgctcccgg
gggcctggcc ctggcaggtc agcctgcacg tccagaacgt 960 ccacgtgtgc
ggaggctcca tcatcacccc cgagtggatc gtgacagccg cccactgcgt 1020
ggaaaaacct cttaacaatc catggcattg gacggcattt gcggggattt tgagacaatc
1080 tttcatgttc tatggagccg gataccaagt agaaaaagtg atttctcatc
caaattatga 1140 ctccaagacc aagaacaatg acattgcgct gatgaagctg
cagaagcctc tgactttcaa 1200 cgacctagtg aaaccagtgt gtctgcccaa
cccaggcatg atgctgcagc cagaacagct 1260 ctgctggatt tccgggtggg
gggccaccga ggagaaaggg aagacctcag aagtgctgaa 1320 cgctgccaag
gtgcttctca ttgagacaca gagatgcaac agcagatatg tctatgacaa 1380
cctgatcaca ccagccatga tctgtgccgg cttcctgcag gggaacgtcg attcttgcca
1440 gggtgacagt ggagggcctc tggtcacttc gaagaacaat atctggtggc
tgatagggga 1500 tacaagctgg ggttctggct gtgccaaagc ttacagacca
ggagtgtacg ggaatgtgat 1560 ggtattcacg gactggattt atcgacaaat
gagggcagac ggctaatcca catggtcttc 1620 gtccttgacg tcgttttaca
agaaaacaat ggggctggtt ttgcttcccc gtgcatgatt 1680 tactcttaga
gatgattcag aggtcacttc atttttatta aacagtgaac ttgtctggct 1740
ttggcactct ctgccattct gtgcaggctg cagtggctcc cctgcccagc ctgctctccc
1800 taaccccttg tccgcaaggg gtgatggccg gctggttgtg ggcactggcg
gtcaagtgtg 1860 gaggagaggg gtggaggctg ccccattgag atcttcctgc
tgagtccttt ccaggggcca 1920 attttggatg agcatggagc tgtcacctct
cagctgctgg atgacttgag atgaaaaagg 1980
agagacatgg aaagggagac agccaggtgg cacctgcagc ggctgccctc tggggccact
2040 tggtagtgtc cccagcctac ctctccacaa ggggattttg ctgatgggtt
cttagagcct 2100 tagcagccct ggatggtggc cagaaataaa gggaccagcc
cttcatgggt ggtgacgtgg 2160 tagtcacttg taaggggaac agaaacattt
ttgttcttat ggggtgagaa tatagacagt 2220 gcccttggtg cgagggaagc
aattgaaaag gaacttgccc tgagcactcc tggtgcaggt 2280 ctccacctgc
acattgggtg gggctcctgg gagggagact cagccttcct cctcatcctc 2340
cctgaccctg ctcctagcac cctggagagt gcacatgccc cttggtcctg gcagggcgcc
2400 aagtctggca ccatgttggc ctcttcaggc ctgctagtca ctggaaattg
aggtccatgg 2460 gggaaatcaa ggatgctcag tttaaggtac actgtttcca
tgttatgttt ctacacattg 2520 ctacctcagt gctcctggaa acttagcttt
tgatgtctcc aagtagtcca ccttcattta 2580 actctttgaa actgtatcac
ctttgccaag taagagtggt ggcctatttc agctgctttg 2640 acaaaatgac
tggctcctga cttaacgttc tataaatgaa tgtgctgaag caaagtgccc 2700
atggtggcgg cgaagaagag aaagatgtgt tttgttttgg actctctgtg gtcccttcca
2760 atgctgtggg tttccaacca ggggaagggt cccttttgca ttgccaagtg
ccataaccat 2820 gagcactact ctaccatggt tctgcctcct ggccaagcag
gctggtttgc aagaatgaaa 2880 tgaatgattc tacagctagg acttaacctt
gaaatggaaa gtcttgcaat cccatttgca 2940 ggatccgtct gtgcacatgc
ctctgtagag agcagcattc ccagggacct tggaaacagt 3000 tggcactgta
aggtgcttgc tccccaagac acatcctaaa aggtgttgta atggtgaaaa 3060
cgtcttcctt ctttattgcc ccttcttatt tatgtgaaca actgtttgtc tttttttgta
3120 tcttttttaa actgtaaagt tcaattgtga aaatgaatat catgcaaata
aattatgcga 3180 tttttttttc aaagcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa
3226
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