U.S. patent application number 10/849635 was filed with the patent office on 2004-12-16 for androgen regulated prostate specific polypeptide-5 (arp-5).
This patent application is currently assigned to Institute for Systems Biology. Invention is credited to Lin, Biaoyang.
Application Number | 20040253622 10/849635 |
Document ID | / |
Family ID | 27805612 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040253622 |
Kind Code |
A1 |
Lin, Biaoyang |
December 16, 2004 |
Androgen regulated prostate specific polypeptide-5 (ARP-5)
Abstract
The present invention provides novel androgen regulated nucleic
acid molecules. Related polypeptides and diagnostic methods also
are provided.
Inventors: |
Lin, Biaoyang; (Bothell,
WA) |
Correspondence
Address: |
Cathryn Campbell
McDERMOTT, WILL & EMERY
Suite 700
4370 La Jolla Village Drive
San Diego
CA
92122
US
|
Assignee: |
Institute for Systems
Biology
|
Family ID: |
27805612 |
Appl. No.: |
10/849635 |
Filed: |
May 19, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10849635 |
May 19, 2004 |
|
|
|
09821812 |
Mar 28, 2001 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
536/23.2 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2600/136 20130101; Y10S 530/821 20130101 |
Class at
Publication: |
435/006 ;
536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
1. to 61. Canceled.
62. A substantially pure ARP5 polypeptide, comprising substantially
an amino acid sequence having at least 40% amino acid identity with
SEQ ID No:9.
63. The substantially pure ARP5 polypeptide of claim 62, comprising
the amino acid sequence shown as SEQ ID NO:9.
64. A substantially pure ARP5 polypeptide fragment, comprising at
least eight contiguous amino acids of SEQ ID NO:9.
65 to 71. Canceled
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates generally to cancer and, more
specifically, to prostate-specific genes that can be used to
diagnose and treat prostate cancer.
[0003] 2. Background Information
[0004] Cancer is currently the second leading cause of mortality in
the United States. However, it is estimated that by the year 2000
cancer will surpass heart disease and become the leading cause of
death in the United States. Prostate cancer is the most common
non-cutaneous cancer in the United States and the second leading
cause of male cancer mortality.
[0005] Cancerous tumors result when a cell escapes from its normal
growth regulatory mechanisms and proliferates in an uncontrolled
fashion. As a result of such uncontrolled proliferation, cancerous
tumors usually invade neighboring tissues and spread by lymph or
blood stream to create secondary or metastatic growths in other
tissues. If untreated, cancerous tumors follow a fatal course.
Prostate cancer, due to its slow growth profile, is an excellent
candidate for early detection and therapeutic intervention.
[0006] During the last decade, most advances in prostate cancer
research have focused on prostate specific antigen (PSA), a member
of the serine protease family that exhibits a prostate-specific
expression profile. Serum PSA remains the most widely used tumor
marker for monitoring prostate cancer, but its specificity is
limited by a high frequency of falsely elevated values in men with
benign prostatic hyperplasia (BPH). Other biomarkers of prostate
cancer progression have proven to be of limited clinical use in
recent surveys because they are not uniformly elevated in men with
advanced prostate cancer. Due to the limitations of currently
available biomarkers, the identification and characterization of
prostate specific genes is essential to the development of more
accurate diagnostic methods and therapeutic targets. In many cases,
the clinical potential of novel tumor markers can be optimized by
utilizing them in combination with other tumor markers in the
development of diagnostic and treatment modalities.
[0007] Normal prostate tissue consists of three distinct
non-stromal cell populations, luminal secretory cells, basal cells,
and endocrine paracrine cells. Phenotypic similarities between
normal luminal cells and prostate cancer cells, including the
expression of PSA, have suggested that prostate adenocarcinomas
derive from luminal cells. However, a number of recent studies
suggest that at least some prostate cancers can arise from the
transformation of basal cells and report the expression of various
genes in normal prostate basal cells as well as in prostate
carcinoma cells. These genes include prostate stem cell antigen
(PSCA), c-met and Bcl-2. Because none of these genes is universally
expressed in all basal cells and prostate carcinomas, the utility
of these genes as diagnostic markers is limited. Likewise, because
PSA is expressed in luminal secretory cells in normal prostate
tissue, this antigen has limited utility as a marker for basal cell
derived carcinomas.
[0008] Thus, there exists a need for the identification of
additional prostate specific genes that can be used as diagnostic
markers and therapeutic targets for prostate cancer. The present
invention satisfies this need and provides related advantages as
well.
SUMMARY OF THE INVENTION
[0009] The present invention provides androgen responsive prostate
specific (ARP) nucleic acid and polypeptide molecules.
[0010] The present invention provides a substantially pure ARP1
nucleic acid molecule containing substantially the nucleotide
sequence shown as SEQ ID NO:1. The invention also provides a
substantially pure ARP1 nucleic acid molecule containing at least
10 contiguous nucleotides of nucleotides 722 to 1026 of SEQ ID
NO:1. In one embodiment, the substantially pure ARP1 nucleic acid
molecule includes at least 15 contiguous nucleotides of nucleotides
722 to 1026 of SEQ ID NO:1.
[0011] The present invention also provides a method of diagnosing
or predicting susceptibility to a prostate neoplastic condition in
an individual by contacting a sample from the individual with an
ARP1 nucleic acid molecule that includes at least 10 contiguous
nucleotides of SEQ ID NO:1, determining a test expression level of
ARP1 RNA in the sample, and comparing the test expression level to
a non-neoplastic control expression level of ARP1 RNA, where an
altered test expression level as compared to the control expression
level indicates the presence of a prostate neoplastic condition in
the individual. In one embodiment, the sample is prostate tissue.
In another embodiment, the sample is blood, urine or semen. In yet
another embodiment, the ARP1 nucleic acid molecule has a length of
15 to 18 nucleotides.
[0012] The present invention further provides a method for treating
or reducing the severity of a prostate neoplastic condition in an
individual by administering to the individual an ARP1 regulatory
agent.
[0013] Further provided by the invention is a substantially pure
ARP2 nucleic acid molecule containing substantially the nucleotide
sequence shown as SEQ ID NO:2. The invention also provides a
substantially pure ARP2 nucleic acid molecule that includes at
least 10 contiguous nucleotides of nucleotides 1128 to 4509 of SEQ
ID NO:2. In one embodiment, such a substantially pure ARP2 nucleic
acid molecule includes at least 15 contiguous nucleotides of
nucleotides 1128 to 4509 of SEQ ID NO:2.
[0014] The present invention additionally provides a method of
diagnosing or predicting susceptibility to a prostate neoplastic
condition in an individual by contacting a sample from the
individual with an ARP2 nucleic acid molecule that contains at
least 10 contiguous nucleotides of nucleotides 1128 to 4509 of SEQ
ID NO:2, determining a test expression level of ARP2 RNA in the
sample, and comparing the test expression level to a non-neoplastic
control expression level of ARP2 RNA, where an altered test
expression level as compared to the control expression level
indicates the presence of a prostate neoplastic condition in the
individual. A method of the invention can be practiced, for
example, with a sample of prostate tissue, or a sample or blood,
urine or semen. An ARP2 nucleic acid molecule useful in a
diagnostic method of the invention can be, for example, 15 to 18
nucleotides in length.
[0015] The present invention also provides a method for treating or
reducing the severity of a prostate neoplastic condition in an
individual by administering to the individual an ARP2 regulatory
agent.
[0016] The present invention additionally provides a substantially
pure ARP3 nucleic acid molecule containing a nucleic acid sequence
that encodes an ARP3 polypeptide having at least 45% amino acid
identity with SEQ ID NO:5. In one embodiment, the substantially
pure ARP3 nucleic acid molecule contains a nucleic acid sequence
encoding the amino acid sequence shown as SEQ ID NO:5. In another
embodiment, the substantially pure ARP3 nucleic acid molecule
contains the nucleotide sequence shown as SEQ ID NO:4.
[0017] The present invention further provides a method of
diagnosing or predicting susceptibility to a prostate neoplastic
condition in an individual by contacting a sample from the
individual with an ARP3 nucleic acid molecule containing at least
10 contiguous nucleotides of SEQ ID NO:4, determining a test
expression level of ARP3 RNA in the sample, and comparing the test
expression level to a non-neoplastic control expression level of
ARP3 RNA, where an altered test expression level as compared to the
control expression level indicates the presence of a prostate
neoplastic condition in the individual. In one embodiment, the
sample from the individual is prostate tissue. In another
embodiment, the sample from the individual is blood, urine or
semen. In yet a further embodiment, the ARP3 nucleic acid molecule
is 15 to 18 nucleotides in length.
[0018] In addition, the present invention provides a substantially
pure ARP3 polypeptide which contains an amino acid sequence having
at least 45% amino acid identity with SEQ ID NO:5. In one
embodiment, the substantially pure ARP3 polypeptide includes the
amino acid sequence shown as SEQ ID NO:5. The present invention
also provides a substantially pure ARP3 polypeptide fragment that
includes at least eight contiguous amino acids of SEQ ID NO:5.
Further provided by the invention is a binding agent that
selectively binds an ARP3 polypeptide having at least 45% amino
acid identity with SEQ ID NO: 5. In one embodiment, the binding
agent that selectively binds an ARP3 polypeptide is an
antibody.
[0019] The present invention also provides a method of diagnosing
or predicting susceptibility to a prostate neoplastic condition in
an individual by contacting a specimen from the individual with a
binding agent that selectively binds an ARP3 polypeptide having at
least 45% amino acid identity with SEQ ID NO: 5, determining a test
expression level of ARP3 polypeptide in the specimen, and comparing
the test expression level to a non-neoplastic control expression
level of ARP3 polypeptide, where an altered test expression level
as compared to the control expression level indicates the presence
of a prostate neoplastic condition in the individual. A specimen
useful in a diagnostic method of the invention can be, for example,
prostate tissue, or can be, for example, blood, serum, urine or
serum. A binding agent useful for determining a test expression
level of ARP3 polypeptide in a method of the invention can be, for
example, an antibody.
[0020] The present invention further provides a method for treating
or reducing the severity of a prostate neoplastic condition in an
individual by administering to said individual an ARP3 regulatory
agent.
[0021] Also provided by the invention is a substantially pure ARP4
nucleic acid molecule that contains a nucleic acid sequence
encoding an ARP4 polypeptide having at least 50% amino acid
identity with SEQ ID NO:7. In one embodiment, the substantially
pure ARP4 nucleic acid molecule contains a nucleic acid sequence
encoding the amino acid sequence shown as SEQ ID NO:7. In another
embodiment, the substantially pure ARP4 nucleic acid molecule
includes the nucleotide sequence shown as SEQ ID NO:6.
[0022] The present invention also provides a substantially pure
ARP4 nucleic acid molecule containing at least 10 contiguous
nucleotides of nucleotides 821 to 1940 of SEQ ID NO:6. In one
embodiment, the substantially pure ARP4 nucleic acid molecule
includes at least 15 contiguous nucleotides of nucleotides 821 to
1940 of SEQ ID NO:6.
[0023] The present invention further provides a method of
diagnosing or predicting susceptibility to a prostate neoplastic
condition in an individual by contacting a sample from the
individual with an ARP4 nucleic acid molecule containing at least
10 contiguous nucleotides of SEQ ID NO:6, determining a test
expression level of ARP4 RNA in the sample, and comparing the test
expression level to a non-neoplastic control expression level of
ARP4 RNA, where an altered test expression level as compared to the
control expression level indicates the presence of a prostate
neoplastic condition in the individual. A sample useful in the
invention can be, for example, prostate tissue, or can be, for
example, blood, urine or semen. An ARP4 nucleic acid molecule
useful in a diagnostic method of the invention can have, for
example, a length of 15 to 18 nucleotides.
[0024] The present invention additionally provides a substantially
pure ARP4 polypeptide containing an amino acid sequence having at
least 50% amino acid identity with SEQ ID NO:7. Such a
substantially pure ARP4 polypeptide can contain, for example, the
amino acid sequence shown as SEQ ID NO:7. The present invention
also provides a substantially pure ARP4 polypeptide fragment
including at least eight contiguous amino acids of SEQ ID NO:7. The
invention additionally provides a binding agent that selectively
binds an ARP4 polypeptide having at least 50% amino acid identity
with SEQ ID NO:7. In one embodiment, the binding agent that
selectively binds an ARP4 polypeptide is an antibody.
[0025] The present invention also provides a method of diagnosing
or predicting susceptibility to a prostate neoplastic condition in
an individual by contacting a specimen from the individual with a
binding agent that selectively binds an ARP4 polypeptide having at
least 50% amino acid identity with SEQ ID NO: 7, determining a test
expression level of ARP4 polypeptide in the specimen, and comparing
the test expression level to a non-neoplastic control expression
level of ARP4 polypeptide, where an altered test expression level
as compared to the control expression level indicates the presence
of a prostate neoplastic condition in the individual. In one
embodiment, the specimen from the individual is prostate tissue,
and, in another embodiment, the specimen from the individual is
blood, serum, urine or semen. A particularly useful binding agent
that selectively binds an ARP4 polypeptide is an antibody.
[0026] The present invention further provides a method for treating
or reducing the severity of a prostate neoplastic condition in an
individual by administering to the individual an ARP4 regulatory
agent.
[0027] Further provided by the invention is a substantially pure
ARP5 nucleic acid molecule which contains a nucleic acid sequence
encoding an ARP5 polypeptide having at least 40% amino acid
identity with SEQ ID NO:9. In one embodiment, the substantially
pure ARP5 nucleic acid molecule contains a nucleic acid sequence
encoding the amino acid sequence shown as SEQ ID NO:9. In another
embodiment, the substantially pure ARP5 nucleic acid molecule
contains the nucleotide sequence shown as SEQ ID NO:8.
[0028] The present invention provides a substantially pure ARP5
nucleic acid molecule containing at least 10 contiguous nucleotides
of nucleotides 565 to 1276 of SEQ ID NO:8. In one embodiment, the
substantially pure ARP5 nucleic acid molecule includes at least 15
contiguous nucleotides of nucleotides 565 to 1276 of SEQ ID
NO:8.
[0029] In addition, the present invention provides a method of
diagnosing or predicting susceptibility to a prostate neoplastic
condition in an individual by contacting a sample from the
individual with an ARP5 nucleic acid molecule which includes at
least 10 contiguous nucleotides of SEQ ID NO:8, determining a test
expression level of ARP5 RNA in the sample, and comparing the test
expression level to a non-neoplastic control expression level of
ARP5 RNA, where an altered test expression level as compared to the
control expression level indicates the presence of a prostate
neoplastic condition in the individual. In one embodiment, a sample
used in a method of the invention is prostate tissue. In another
embodiment, a sample used in a method of the invention is blood,
urine or semen. In a further embodiment, the ARP5 nucleic acid
molecule has a length of 15 to 18 nucleotides.
[0030] The present invention also provides a substantially pure
ARP5 polypeptide that contains an amino acid sequence having at
least 40% amino acid identity with SEQ ID NO:9. In one embodiment,
the substantially pure ARP5 polypeptide contains the amino acid
sequence shown as SEQ ID NO:9. The present invention also provides
a substantially pure ARP5 polypeptide fragment including at least
eight contiguous amino acids of SEQ ID NO:9. Further provided by
the invention is a binding agent that selectively binds an ARP5
polypeptide having at least 40% amino acid identity with SEQ ID
NO:9, which can be, for example, an antibody.
[0031] The present invention also provides a method of diagnosing
or predicting susceptibility to a prostate neoplastic condition in
an individual by contacting a specimen from the individual with a
binding agent that selectively binds an ARP5 polypeptide having at
least 40% amino acid identity with SEQ ID NO: 9, determining a test
expression level of ARP5 polypeptide in the specimen, and comparing
the test expression level to a non-neoplastic control expression
level of ARP5 polypeptide, where an altered test expression level
as compared to the control expression level indicates the presence
of a prostate neoplastic condition in the individual. A specimen
useful in the invention can be, for example, prostate tissue, or
can be, for example, blood, serum, urine or semen. A binding agent
useful in the invention can be, for example, an antibody.
[0032] Also provided by the invention is a method for treating or
reducing the severity of a prostate neoplastic condition in an
individual by administering to the individual an ARP5 regulatory
agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 shows the nucleotide (SEQ ID NO:1) sequence of
ARP1.
[0034] FIG. 2 shows the nucleotide (SEQ ID NO: 2) and amino acid
sequence (SEQ ID NO: 3) of ARP2.
[0035] FIG. 3 shows the nucleotide (SEQ ID NO:4) and amino acid
sequence (SEQ ID NO:5) of ARP3.
[0036] FIG. 4 shows the nucleotide (SEQ ID NO:6) and amino acid
sequence (SEQ ID NO:7) of ARP4.
[0037] FIG. 5 shows the nucleotide (SEQ ID NO:8) and amino acid
(SEQ ID NO:9) sequence of ARP5.
[0038] FIG. 6 shows multiple tissue Northern blot analysis of ARP4
expression.
DETAILED DESCRIPTION OF THE INVENTION
[0039] This invention is directed to the discovery of androgen
regulated prostate (ARP) expressed nucleic acid molecules. The
androgen regulated prostate expressed nucleic acid molecules and
encoded gene products are useful as diagnostic markers for
neoplastic conditions of the prostate, and, further, are targets
for therapy.
[0040] As disclosed herein in Example I, the ARP1 cDNA is an
androgen-regulated sequence. The ARP1 nucleic acid sequence is
disclosed herein in FIG. 1. As further disclosed herein, the ARP2
cDNA is another androgen-regulated cDNA, which contains 4509
nucleotides and is predicted to encode a protein of 252 amino acids
(see FIG. 2). The androgen-regulated ARP3 cDNA contains 2213
nucleotides and is predicted to encode a protein of 538 amino acids
(see Example I and FIG. 3). As further disclosed herein, the
androgen-regulated ARP4 and ARP5 cDNAs are alternatively spliced
mRNA from the same gene. A single nucleotide insertion results in a
frameshift change in the coding region. As shown in FIG. 4, the
ARP4 nucleic acid sequence contains 4433 nucleotides and is
predicted to encode a protein of 141 amino acids. The alternatively
spliced ARP5 nucleic acid sequence contains 1276 nucleotides and is
predicted to encode a protein of 425 amino acids (see FIG. 5).
[0041] Based on these novel prostate-expressed sequences, the
invention provides methods for diagnosing prostate neoplastic
conditions. An ARP nucleic acid molecule or polypeptide of the
invention can be used alone or in combination with other molecules
as a specific marker for prostate cells or prostate neoplastic
conditions.
[0042] The present invention provides a substantially pure ARP1
nucleic acid molecule containing substantially the nucleotide
sequence shown as SEQ ID NO:1. The invention also provides a
substantially pure ARP1 nucleic acid molecule containing at least
10 contiguous nucleotides of nucleotides 722 to 1026 of SEQ ID
NO:1. In one embodiment, the substantially pure ARP1 nucleic acid
molecule includes at least 15 contiguous nucleotides of nucleotides
722 to 1026 of SEQ ID NO:1.
[0043] The present invention further provides a substantially pure
ARP2 nucleic acid molecule containing substantially the nucleotide
sequence shown as SEQ ID NO:2. The invention also provides a
substantially pure ARP2 nucleic acid molecule that includes at
least 10 contiguous nucleotides of nucleotides 1128 to 4509 of SEQ
ID NO:2. In one embodiment, such a substantially pure ARP2 nucleic
acid molecule includes at least 15 contiguous nucleotides of
nucleotides 1128 to 4509 of SEQ ID NO:2.
[0044] The present invention additionally provides a substantially
pure ARP3 nucleic acid molecule containing a nucleic acid sequence
that encodes an ARP3 polypeptide having at least 45% amino acid
identity with SEQ ID NO:5. In one embodiment, the substantially
pure ARP3 nucleic acid molecule contains a nucleic acid sequence
encoding the amino acid sequence shown as SEQ ID NO:5. In another
embodiment, the substantially pure ARP3 nucleic acid molecule
contains the nucleotide sequence shown as SEQ ID NO:4.
[0045] Also provided by the invention is a substantially pure ARP4
nucleic acid molecule that contains a nucleic acid sequence
encoding an ARP4 polypeptide having at least 50% amino acid
identity with SEQ ID NO:7. In one embodiment, the substantially
pure ARP4 nucleic acid molecule contains a nucleic acid sequence
encoding the amino acid sequence shown as SEQ ID NO:7. In another
embodiment, the substantially pure ARP4 nucleic acid molecule
includes the nucleotide sequence shown as SEQ ID NO:6. The present
invention further provides a substantially pure ARP4 nucleic acid
molecule containing at least 10 contiguous nucleotides of
nucleotides 821 to 1940 of SEQ ID NO:6. Such an ARP4 nucleic acid
molecule can include, for example, at least 15 contiguous
nucleotides of nucleotides 821 to 1940 of SEQ ID NO:6.
[0046] Further provided by the invention is a substantially pure
ARP5 nucleic acid molecule which contains a nucleic acid sequence
encoding an ARP5 polypeptide having at least 40% amino acid
identity with SEQ ID NO:9. In one embodiment, the substantially
pure ARP5 nucleic acid molecule contains a nucleic acid sequence
encoding the amino acid sequence shown as SEQ ID NO:9. In another
embodiment, the substantially pure ARP5 nucleic acid molecule
contains the nucleotide sequence shown as SEQ ID NO:8. The
invention also provides a substantially pure ARP5 nucleic acid
molecule containing at least 10 contiguous nucleotides of
nucleotides 565 to 1276 of SEQ ID NO:8. In one embodiment, the
substantially pure ARP5 nucleic acid molecule includes at least 15
contiguous nucleotides of nucleotides 565 to 1276 of SEQ ID
NO:8.
[0047] The nucleic acid molecules of the invention corresponding to
unique sequences are useful in a variety of diagnostic procedures
which employ probe hybridization methods. One advantage of
employing nucleic acid hybridization in diagnostic procedures is
that very small amounts of sample can be used because the analyte
nucleic acid molecule can be amplified to many copies by, for
example, polymerase chain reaction (PCR) or other well known
methods for nucleic acid molecule amplification and synthesis.
[0048] As used herein, the term "nucleic acid molecule" means a
single- or double-stranded DNA or RNA molecule including, for
example, genomic DNA, cDNA and mRNA. The term is intended to
include nucleic acid molecules of both synthetic and natural
origin. A nucleic acid molecule of natural origin can be derived
from any animal, such as a human, non-human primate, mouse, rat,
rabbit, bovine, porcine, ovine, canine, feline, or amphibian, or
from a lower eukaryote. A nucleic acid molecule of the invention
can be of linear, circular or branched configuration, and can
represent either the sense or antisense strand, or both, of a
native nucleic acid molecule. A nucleic acid molecule of the
invention can further incorporate a detectable moiety such as a
radiolabel, a fluorochrome, a ferromagnetic substance, a
luminescent tag or a detectable moiety such as biotin.
[0049] As used herein, the term "substantially pure nucleic acid
molecule" means a nucleic acid molecule that is substantially free
from cellular components or other contaminants that are not the
desired molecule. A substantially pure nucleic acid molecule can
also be sufficiently homogeneous so as to resolve as a band by gel
electrophoresis, and generate a nucleotide sequence profile
consistent with a predominant species.
[0050] In particular embodiments, the present invention provides a
substantially pure ARP1 nucleic acid molecule containing at least
10 contiguous nucleotides of nucleotides 722 to 1026 of SEQ ID
NO:1; a substantially pure ARP2 nucleic acid molecule that includes
at least 10 contiguous nucleotides of nucleotides 1128 to 4509 of
SEQ ID NO:2; a substantially pure ARP4 nucleic acid molecule
containing at least 10 contiguous nucleotides of nucleotides 821 to
1940 of SEQ ID NO:6; and a substantially pure ARP5 nucleic acid
molecule containing at least 10 contiguous nucleotides of
nucleotides 565 to 1276 of SEQ ID NO:8. Such a nucleic acid
molecule is a portion of a full-length nucleic acid molecule having
the ability to selectively hybridize with the parent nucleic acid
molecule. As used herein, the term selectively hybridize means an
ability to bind the parent nucleic acid molecule without
substantial cross-reactivity with a molecule that is not the parent
nucleic acid molecule.
[0051] Therefore, the term selectively hybridize includes specific
hybridization where there is little or no detectable
cross-reactivity with other nucleic acid molecules. The term also
includes minor cross-reactivity with other molecules provided
hybridization to the parent nucleic acid molecule is
distinguishable from hybridization to the cross-reactive species.
Thus, a nucleic acid molecule of the invention can be used, for
example, as a PCR primer to selectively amplify a parent nucleic
acid molecule; as a selective primer for 5' or 3' RACE to determine
additional 5' or 3' sequence of a parent nucleic acid molecule; as
a selective probe to identify or isolate a parent nucleic acid
molecule on a RNA or DNA blot, or within a genomic or cDNA library;
or as a selective inhibitor of transcription or translation of an
ARP in a tissue, cell or cell extract.
[0052] Several specific nucleic acid sequences are excluded as
nucleic acid molecules of the invention. An ARP1 nucleic acid
molecule of the invention containing at least 10 contiguous
nucleotides of nucleotides 722 to 1026 of SEQ ID NO: 1 excludes one
or both of AA404252, AI133138, or any subportion thereof.
Similarly, an ARP4 nucleic acid molecule of the invention
containing at least 10 contiguous nucleotides of nucleotides 821 to
1940 of SEQ ID NO:6 specifically excludes one or any combination of
AW861164, AW856874, AI299663, H59488, W60959, AA659693, AW961788,
AA249370, AL133779, or any subportion thereof. In addition, an ARP5
nucleic acid molecule of the invention containing at least 10
contiguous nucleotides of nucleotides 565 to 1276 specifically
excludes one or any combination of AW861164, AW856874, or BF130410,
or a subportion thereof.
[0053] In one embodiment, an ARP3 nucleic acid molecule of the
invention specifically excludes the nucleotide sequence AK002597.
In another embodiment, an ARP4 nucleic acid molecule of the
invention specifically excludes the nucleotide sequence
AK012931.
[0054] A nucleic acid molecule of the invention includes at least
10 contiguous nucleotides corresponding to the reference nucleic
acid molecule, and can include at least 11, 12, 13, 14, 15, 16, 17,
18, 19, 20 or at least 25 nucleotides and, if desired, can include
at least 30, 40, 50, 100, 300 or 500 nucleotides or up to the full
length of the reference nucleic acid molecule. Nucleic acid
molecules of such lengths are able to selectively hybridize with
the subject nucleic acid molecule in a variety of detection formats
described herein.
[0055] As used herein, the term "substantially the nucleotide
sequence" in reference to a nucleic acid molecule or nucleic acid
probe of the invention includes sequences having one or more
additions, deletions or substitutions with respect to the reference
sequence, so long as the nucleic acid molecule retains its ability
to selectively hybridize with the subject nucleic acid
molecule.
[0056] Nucleic acid molecules of the invention are useful, in part,
as hybridization probes in diagnostic procedures. The nucleic acid
molecules can be as long as the full length transcript or as short
as about 10-15 nucleotides, for example, 15-18 nucleotides in
length. A nucleic acid molecule of the invention that is not a
full-length sequence can correspond to coding region or
untranslated region sequence. The particular application and degree
of desired specificity will be one consideration well known to
those skilled in the art in selecting a nucleic acid molecule for a
particular application. For example, if it is desired to detect an
ARP and other related species, the probe can correspond to a coding
sequence and be used in low stringency hybridization conditions.
Alternatively, using high stringency conditions with a probe of the
invention will select a specific ARP1, ARP2, ARP3, ARP4, or ARP5
nucleic acid molecule. Untranslated region sequences corresponding
to an ARP transcript also can be used to construct probes since
there is little evolutionary pressure to conserve non-coding
domains. Nucleic acid molecules as small as 15 nucleotides are
statistically unique sequences within the human genome. Therefore,
fragments of 15 nucleotides or more of the ARP sequences disclosed
herein as SEQ ID NOS: 1, 2, 4, 6, and 8 can be constructed from
essentially any region of an ARP cDNA, mRNA or promoter/regulatory
region and be capable of uniquely hybridizing to ARP DNA or
RNA.
[0057] A nucleic acid molecule of the invention can be produced
recombinantly or chemically synthesized using methods well known in
the art. Additionally, an ARP nucleic acid molecule can be labeled
with a variety of detectable labels including, for example,
radioisotopes, fluorescent tags, reporter enzymes, biotin and other
ligands for use as a probe in a hybridization method. Such
detectable labels can additionally be coupled with, for example,
calorimetric or photometric indicator substrate for
spectrophotometric detection. Methods for labeling and detecting
nucleic acid molecules are well known in the art and can be found
described in, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview,
N.Y. (1989), and Ausubel et al., Current Protocols in Molecular
Biology (Supplement 47), John Wiley & Sons, New York
(1999).
[0058] The nucleic acid molecules of the invention can be
hybridized under various stringency conditions readily determined
by one skilled in the art. Depending on the particular assay, one
skilled in the art can readily vary the stringency conditions to
optimize detection of an ARP nucleic acid molecule.
[0059] In general, the stability of a hybrid is a function of the
ion concentration and temperature. Typically, a hybridization
reaction is performed under conditions of lower stringency,
followed by washes of varying, but higher, stringency. Moderately
stringent hybridization refers to conditions that permit a nucleic
acid molecule such as a probe to bind a complementary nucleic acid
molecule. The hybridized nucleic acid molecules generally have at
least 60% identity, at least 75% identity, at least 85% identity;
or at least 90% identity with the parent or target nucleic acid
sequence. Moderately stringent conditions are conditions equivalent
to hybridization in 50% formamide, 5.times. Denhardt's solution,
5.times.SSPE, 0.2% SDS at 42.degree. C., followed by washing in
0.2.times.SSPE, 0.2% SDS, at 42.degree. C. High stringency
conditions can be provided, for example, by hybridization in 50%
formamide, 5.times. Denhart's solution, 5.times.SSPE, 0.2% SDS at
42.degree. C., followed by washing in 0.1.times.SSPE, and 0.1% SDS
at 65.degree. C.
[0060] The term low stringency hybridization means conditions
equivalent to hybridization in 10% formamide, 5.times. Denhart's
solution, 6.times.SSPE, 0.2% SDS at 22.degree. C., followed by
washing in 1.times.SSPE, 0.2% SDS, at 37.degree. C. Denhart's
solution contains 1% Ficoll, 1% polyvinylpyrolidine, and 1% bovine
serum albumin (BSA). 20.times.SSPE (sodium chloride, sodium
phosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M
sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other
suitable moderate stringency and high stringency hybridization
buffers and conditions are well known to those of skill in the art
and are described, for example, in Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press,
Plainview, N.Y. (1989); and Ausubel et al., supra, 1999). Nucleic
acid molecules encoding polypeptides hybridize under moderately
stringent or high stringency conditions to substantially the entire
sequence, or substantial portions, for example, typically at least
15-30 nucleotides of an ARP nucleic acid sequence.
[0061] The invention also provides a modification of an ARP
nucleotide sequence that hybridizes to an ARP nucleic acid
molecule, for example, an ARP nucleic acid molecule referenced
herein as SEQ ID NO:1, 2, 4, 6 or 8, under moderately stringent
conditions. Modifications of ARP nucleotide sequences, where the
modification has at least 60% identity to an ARP nucleotide
sequence, are also provided. The invention also provides
modification of an ARP nucleotide sequence having at least 65%
identity, at least 70% identity, at least 75% identity, at least
80% identity, at least 85% identity, at least 90% identity, or at
least 95% identity to SEQ ID NO: 1, 2, 4, 6 or 8.
[0062] Identity of any two nucleic acid sequences can be determined
by those skilled in the art based, for example, on a BLAST 2.0
computer alignment, using default parameters. BLAST 2.0 searching
is available at http://www.ncbi.nlm.nih.gov/gorf/bl2.html., as
described by Tatiana et al., FEMS Microbiol Lett. 174:247-250
(1999); Altschul et al., Nucleic Acids Res., 25:3389-3402
(1997).
[0063] The present invention further provides substantially pure
ARP polypeptides encoded by the prostate-expressed nucleic acid
molecules of the invention. Thus, the invention provides a
substantially pure ARP3 polypeptide which contains an amino acid
sequence having at least 45% amino acid identity with SEQ ID NO:5.
A substantially pure ARP3 polypeptide of the invention can have,
for example, the amino acid sequence shown as SEQ ID NO:5. The
present invention also provides a substantially pure ARP3
polypeptide fragment, which includes at least eight contiguous
amino acids of SEQ ID NO:5.
[0064] The present invention also provides a substantially pure
ARP4 polypeptide containing an amino acid sequence having at least
50% amino acid identity with SEQ ID NO:7. In one embodiment, the
substantially pure ARP4 polypeptide contains the amino acid
sequence shown as SEQ ID NO:7. The present invention also provides
a substantially pure ARP4 polypeptide fragment including at least
eight contiguous amino acids of SEQ ID NO:7.
[0065] The present invention additionally provides a substantially
pure ARP5 polypeptide, which contains an amino acid sequence having
at least 40% amino acid identity with SEQ ID NO:9. Such a
substantially pure ARP5 polypeptide of the invention can contain,
for example, the amino acid sequence shown as SEQ ID NO:9. The
present invention also provides a substantially pure ARP5
polypeptide fragment including at least eight contiguous amino
acids of SEQ ID NO:9.
[0066] Exemplary polypeptide fragments include those fragments
having amino acids 1 to 8, 2 to 9, 3 to 10, of SEQ ID NO: 5, 7 or
9. The invention also encompasses other polypeptide fragments which
are potential antigenic fragments capable of eliciting an immune
response, and thereby generating antibodies selective for an ARP3,
ARP4 or ARP5 polypeptide of the invention. It is understood that,
while eight residues is the minimum length of a polypeptide
fragment of the invention, a fragment can be longer and can include
9, 10, 11, 12, 13, 14, 15, 18, 20, 25, 30, 35, 40, 45 or more
contiguous amino acids of the amino acid sequence shown as SEQ ID
NO: 5 in FIG. 3, the amino acid sequence shown as SEQ ID NO: 7 in
FIG. 4, or the amino acid sequence shown a SEQ ID NO: 9 in FIG.
5.
[0067] The term "ARP3 polypeptide" as used herein, means a
polypeptide that is structurally similar to a human ARP3 (SEQ ID
NO: 5) and that has at least one biological activity of human ARP3.
Such an ARP3 polypeptide has 45% or more amino acid sequence
identity to SEQ ID NO:5 and can have, for example, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to
human ARP3 (SEQ ID NO:5). Percent amino acid identity can be
determined using Clustal W version 1.7 (Thompson et al., Nucleic
Acids Res. 22:4673-4680 (1994)).
[0068] Thus, it is clear to the skilled person that the term "ARP3
polypeptide" encompasses polypeptides with one or more naturally
occurring or non-naturally occurring amino acid substitutions,
deletions or insertions as compared to SEQ ID NO:5, provided that
the peptide has at least 45% amino acid identity with SEQ ID NO: 5
and retains at least one biological activity of human ARP3. An ARP3
polypeptide can be, for example, a naturally occurring variant of
human ARP3 (SEQ ID NO:5); a species homolog including mammalian and
non-mammalian homologs and murine, bovine, and primate homologs; an
ARP3 polypeptide mutated by recombinant techniques, and the like.
In view of the above definition, it is clear to the skilled person
that the C. elegans polypeptide encoded by T25F10.5 (T29520), which
shares 39.7% amino acid identity with human ARP3 (SEQ ID NO:5), is
not encompassed by the invention. In a particular embodiment, the
mouse cDNA amino acid sequence encoded by AK002597 is specifically
excluded from the definition of an ARP3 polypeptide.
[0069] The term "ARP4 polypeptide" as used herein, means a
polypeptide that is structurally similar to a human ARP4 (SEQ ID
NO: 7) and that has at least one biological activity of human ARP4.
Such an ARP4 polypeptide has 50% or more amino acid sequence
identity to SEQ ID NO:5 and can have, for example 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95% or more amino acid sequence identity
to human ARP4 (SEQ ID NO:7). Percent amino acid identity can be
determined using Clustal W version 1.7 as described above.
[0070] Thus, the term "ARP4 polypeptide" encompasses polypeptides
with one or more naturally occurring or non-naturally occurring
amino acid substitutions, deletions or insertions as compared to
SEQ ID NO:7, provided that the peptide has at least 50% amino acid
identity with SEQ ID NO: 7 and retains at least one biological
activity of human ARP4. An ARP4 polypeptide can be, for example, a
naturally occurring variant of human ARP4 (SEQ ID NO:7); a species
homolog including mammalian and non-mammalian homologs and murine,
bovine, and primate homologs; an ARP4 polypeptide mutated by
recombinant techniques; and the like. In view of the above
definition, it is clear to the skilled person that the Drosophila
polypeptide encoded by AE003831 (AAF58858), which shares 45% amino
acid identity with human ARP4 (SEQ ID NO:7), is not encompassed by
the invention. In a particular embodiment, the mouse cDNA amino
acid sequence encoded by AK012931 is specifically excluded from the
definition of an ARP4 polypeptide.
[0071] The term "ARP5 polypeptide" as used herein, means a
polypeptide that is structurally similar to a human ARP5 (SEQ ID
NO: 9) and that has at least one biological activity of human ARP5.
Such an ARP5 polypeptide has 40% or more amino acid sequence
identity to SEQ ID NO:9 and can have, for example 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to
human ARP5 (SEQ ID NO:9). Percent amino acid identity can be
determined using Clustal W version 1.7 (Thompson et al., supra,
1994).
[0072] The term "ARP5 polypeptide" encompasses polypeptides with
one or more naturally occurring or non-naturally occurring amino
acid substitutions, deletions or insertions as compared to SEQ ID
NO:9, provided that the peptide has at least 40% amino acid
identity with SEQ ID NO: 9 and retains at least one biological
activity of human ARP5. An ARP5 polypeptide can be, for example, a
naturally occurring variant of human ARP5 (SEQ ID NO:9); a species
homolog including mammalian and non-mammalian homologs and murine,
bovine, and primate homologs; an ARP4 polypeptide mutated by
recombinant techniques, and the like. In view of the above
definition, it is clear to the skilled person that the Drosophila
polypeptide encoded by AE003831 (AAF58858), which shares 35% amino
acid identity with human ARP5 (SEQ ID NO:9), is not encompassed by
the invention.
[0073] Modifications to ARP3, ARP4 and ARP5 polypeptides of SEQ ID
NOS:5, 7, or 9 that are encompassed within the invention include,
for example, an addition, deletion, or substitution of one or more
conservative or non-conservative amino acid residues; substitution
of a compound that mimics amino acid structure or function; or
addition of chemical moieties such as amino or acetyl groups.
[0074] The present invention provides a binding agent that
selectively binds an ARP3 polypeptide having at least 45% amino
acid identity with SEQ ID NO:5. The present invention also provides
a binding agent that selectively binds an ARP4 polypeptide having
at least 50% amino acid identity with SEQ ID NO:7. Further provided
by the invention is a binding agent that selectively binds an ARP5
polypeptide having at least 40% amino acid identity with SEQ ID
NO:9. Particularly useful binding agents of the invention are
polyclonal and monoclonal antibodies and binding portions
thereof.
[0075] As used herein, the term "binding agent" when used in
reference to a specified ARP polypeptide, means a compound,
including a simple or complex organic molecule, a metal containing
compound, carbohydrate, peptide, protein, peptidomimetic,
glycoprotein, lipoprotein, lipid, nucleic acid molecule, antibody,
or the like that selectively binds the specified ARP3, ARp4 or ARP5
polypeptide, or fragment thereof. For example, a binding agent can
be a polypeptide that selectively binds with high affinity or
avidity to the specified ARP polypeptide, without substantial
cross-reactivity to other unrelated polypeptides. The affinity of a
binding agent that selectively binds an ARP polypeptide generally
is greater than about 10.sup.5 M.sup.-1 and can be greater than
about 10.sup.6 M.sup.-1. A binding agent also can bind with high
affinity; such an agent generally binds with an affinity greater
than 10.sup.8 M.sup.-1 to 10.sup.9 M.sup.-1. Specific examples of
such selective binding agents include a polyclonal or monoclonal
antibody selective for an ARP3, ARP4 or ARP5 polypeptide, or a
fragment thereof containing at least eight contiguous amino acids
of SEQ ID NO: 5, 7 or 9; or a nucleic acid molecule, nucleic acid
analog, or small organic molecule, identified, for example, by
affinity screening of the appropriate library. For certain
applications, a binding agent can be utilized that preferentially
recognizes a particular conformational or post-translationally
modified state of the specified ARP polypeptide. The binding agent
can be labeled with a detectable moiety, if desired, or rendered
detectable by specific binding to a detectable secondary binding
agent.
[0076] As used herein, the term "antibody" is used in its broadest
sense to mean polyclonal and monoclonal antibodies, including
antigen binding fragments of such antibodies. As used herein, the
term antigen means a native or synthesized fragment of a
polypeptide of the invention. Such an antibody of the invention, or
antigen binding fragment of such an antibody, is characterized by
having specific binding activity for the specified ARP3, ARP4 or
ARP5 polypeptide, or fragment thereof, of at least about
1.times.10.sup.5 M.sup.-1. Thus, Fab, F(ab').sub.2, Fd and Fv
fragments of an anti-ARP antibody, which retain specific binding
activity for an ARP polypeptide of the invention, or fragment
thereof, are included within the definition of an antibody.
Specific binding activity can be readily determined by one skilled
in the art, for example, by comparing the binding activity of the
antibody to the specified ARP polypeptide, or fragment thereof,
versus a control polypeptide that does not include a polypeptide of
the invention. Methods of preparing polyclonal or monoclonal
antibodies are well known to those skilled in the art (see, for
example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press (1988)).
[0077] The term antibody also includes naturally occurring
antibodies as well as ncn-naturally occurring antibodies,
including, for example, single chain antibodies, chimeric,
bifunctional and humanized antibodies, as well as antigen-binding
fragments thereof. Such non-naturally occurring antibodies can be
constructed using solid phase peptide synthesis, produced
recombinantly or obtained, for example, by screening combinatorial
libraries consisting of variable heavy chains and variable light
chains as described by Huse et al. (Science 246:1275-1281 (1989)).
These and other methods of making, for example, chimeric,
humanized, CDR-grafted, single chain, and bifunctional antibodies
are well known to those skilled in the art (Winter and Harris,
Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546
(1989); Harlow and Lane, supra, 1988); Hilyard et al., Protein
Engineering: A practical approach (IRL Press 1992); Borrabeck,
Antibody Engineering, 2d ed. (Oxford University Press 1995)).
[0078] An antibody of the invention can be prepared using as an
immunogen an ARP3, ARP4 or ARP5 polypeptide of the invention, which
can be prepared from natural sources or produced recombinantly, or
a polypeptide fragment of the invention, which contains at least 8
contiguous amino acids of SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO:
9. Such polypeptide fragments are functional antigenic fragments if
the antigenic peptides can be used to generate an antibody
selective for an ARP polypeptide of the invention. As is well known
in the art, a non-immunogenic or weakly immunogenic ARP polypeptide
of the invention, or polypeptide fragment thereof, can be made
immunogenic by coupling the hapten to a carrier molecule such as
bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
Various other carrier molecules and methods for coupling a hapten
to a carrier molecule are well known in the art (see, for example,
Harlow and Lane, supra, 1988). An immunogenic ARP polypeptide
fragment of the invention can also be generated by expressing the
peptide portion as a fusion protein, for example, to glutathione S
transferase (GST), polyHis or the like. Methods for expressing
peptide fusions are well known to those skilled in the art (Ausubel
et al., Current Protocols in Molecular Biology (Supplement 47),
John Wiley & Sons, New York (1999)).
[0079] Methods of diagnosing or predicting susceptibility to a
prostate neoplastic condition in an individual further are provided
by the invention. In particular, the present invention provides a
method of diagnosing or predicting susceptibility to a prostate
neoplastic condition in an individual by contacting a sample from
the individual with an ARP1 nucleic acid molecule that includes at
least 10 contiguous nucleotides of SEQ ID NO:1, determining a test
expression level of ARP1 RNA in the sample, and comparing the test
expression level to a non-neoplastic control expression level of
ARP1 RNA, where an altered test expression level as compared to the
control expression level indicates the presence of a prostate
neoplastic condition in the individual. In one embodiment, the ARP1
nucleic acid molecule has a length of 15 to 18 nucleotides.
[0080] The present invention additionally provides a method of
diagnosing or predicting susceptibility to a prostate neoplastic
condition in an individual by contacting a sample from the
individual with an ARP2 nucleic acid molecule that contains at
least 10 contiguous nucleotides of nucleotides 1128 to 4509 of SEQ
ID NO:2, determining a test expression level of ARP2 RNA in the
sample, and comparing the test expression level to a non-neoplastic
control expression level of ARP2 RNA, where an altered test
expression level as compared to the control expression level
indicates the presence of a prostate neoplastic condition in the
individual. An ARP2 nucleic acid molecule useful in the invention
can contain, for example 15 to 18 nucleotides.
[0081] The present invention further provides a method of
diagnosing or predicting susceptibility to a prostate neoplastic
condition in an individual by contacting a sample from the
individual with an ARP3 nucleic acid molecule containing at least
10 contiguous nucleotides of SEQ ID NO:4, determining a test
expression level of ARP3 RNA in the sample, and comparing the test
expression level to a non-neoplastic control expression level of
ARP3 RNA, where an altered test expression level as compared to the
control expression level indicates the presence of a prostate
neoplastic condition in the individual. An ARP3 nucleic acid
molecule useful in the invention can contain, for example 15 to 18
nucleotides.
[0082] The present invention further provides a method of
diagnosing or predicting susceptibility to a prostate neoplastic
condition in an individual by contacting a sample from said
individual with an ARP4 nucleic acid molecule containing at least
10 contiguous nucleotides of SEQ ID NO:6, determining a test
expression level of ARP4 RNA in the sample, and comparing the test
expression level to a non-neoplastic control expression level of
ARP4 RNA, where an altered test expression level as compared to the
control expression level indicates the presence of a prostate
neoplastic condition in the individual. In one embodiment, the ARP4
nucleic acid molecule is 15 to 18 nucleotides in length.
[0083] Further provided by the invention is a method of diagnosing
or predicting susceptibility to a prostate neoplastic condition in
an individual by contacting a sample from said individual with an
ARP5 nucleic acid molecule includes at least 10 contiguous
nucleotides of SEQ ID NO:8, determining a test expression level of
ARP5 RNA in the sample, and comparing the test expression level to
a non-neoplastic control expression level of ARP5 RNA, where an
altered test expression level as compared to the control expression
level indicates the presence of a prostate neoplastic condition in
the individual. In one embodiment the ARP5 nucleic acid molecule is
15 to 18 nucleotides in length.
[0084] In the diagnostic methods of the invention, the sample can
be, for example, a prostate tissue, or can be, for example, a fluid
such as blood, urine or semen. The non-neoplastic control
expression level can be determined, for example, using a normal
prostate cell or an androgen-dependent cell line.
[0085] As described herein, the term "prostate neoplastic
condition" means a benign or malignant or metastatic prostate
lesion of proliferating cells. For example, primary prostate tumors
are classified into stages TX, T0, T1, T2, T3, and T4. Metastatic
prostate cancer is classified into stages D1, D2, and D3. The term
further includes prostate neoplasm. Each of the above conditions is
encompassed within the term "prostate neoplastic condition."
[0086] As used herein, the term "sample" means any biological
fluid, cell, tissue, organ or portion thereof, that includes or
potentially includes an ARP nucleic acid molecule. The term sample
includes materials present in an individual as well as materials
obtained or derived from the individual. For example, a sample can
be a histologic section of a specimen obtained by biopsy, or cells
that are placed in or adapted to tissue culture. A sample further
can be a subcellular fraction or extract, or a crude or
substantially pure nucleic acid molecule. A sample can be prepared
by methods known in the art suitable for the particular format of
the detection method.
[0087] As used herein, the term "test expression level" is used in
reference to ARP RNA expression or to ARP polypeptide expression as
discussed below and means the extent, amount or rate of synthesis
of the specified ARP RNA or polypeptide. The amount or rate of
synthesis can be determined by measuring the accumulation or
synthesis of the specified ARP RNA or polypeptide, or by measuring
an activity associated with a polypeptide of the invention.
[0088] As used herein, an "altered test expression level" means a
test expression level that is either elevated or reduced as
compared to a control expression level. One skilled in the art
understands that such an elevation or reduction is not within the
inherent variability of the assay and generally is an expression
level that is at least two-fold elevated or reduced. An altered
test expression level can be, for example, two-fold, five-fold,
ten-fold, 100-fold, 200-fold, or 1000-fold increased in the extent,
amount or rate of synthesis of the specified RNA or polypeptide as
compared to a control expression level of the specified ARP RNA or
polypeptide. An altered test expression level also can be, for
example, two-fold, five-fold, ten-fold, 100-fold, 200-fold, or
1000-fold decreased in the extent, amount or rate of synthesis of
the specified ARP RNA or polypeptide compared to a control
expression level of the same ARP RNA or polypeptide.
[0089] As used herein, the term "non-neoplastic control expression
level" means an ARP RNA expression level or to an ARP polypeptide
expression level as discussed below used as a baseline for
comparison to a test expression level. For example, a suitable
control expression level can be the expression level of ARP nucleic
acid or polypeptide from a non-neoplastic prostate cell or a fluid
sample obtained from a normal individual. Another suitable
non-neoplastic control is a prostate cell line that is
androgen-dependent. It is understood that ARP nucleic acid or
polypeptide expression levels determined in cell lines generally
are determined under androgen-depleted growth conditions which can
correlate to non-neoplastic control expression levels. The response
of an androgen-depleted androgen-dependent prostate cell line to
androgen stimulation will be indicative of ARP nucleic acid or
polypeptide expression levels in neoplastic cells. The control
expression level can be determined simultaneously with one or more
test samples or, alternatively, expression levels can be
established for a particular type of sample and standardized to
internal or external parameters such as protein or nucleic acid
content, cell number or mass of tissue. Such standardized control
samples can then be directly compared with results obtained from
the test sample. As indicated above, an increase of two-fold or
more, for example, of a test expression level of the specified ARP
nucleic acid or polypeptide indicates the presence of a prostate
neoplastic condition or pathology in the tested individual.
[0090] A detectable label can be useful in a method of the
invention and refers to a molecule that renders a nucleic acid
molecule of the invention detectable by an analytical method. An
appropriate detectable label depends on the particular assay
format; such labels are well known by those skilled in the art. For
example, a detectable label selective for a nucleic acid molecule
can be a complementary nucleic acid molecule, such as a
hybridization probe, that selectively hybridizes to the nucleic
acid molecule. A hybridization probe can be labeled with a
measurable moiety, such as a radioisotope, fluorochrome,
chemiluminescent marker, biotin, or other moiety known in the art
that is measurable by analytical methods. A detectable label also
can be a nucleic acid molecule without a measurable moiety. For
example, PCR or RT-PCR primers can be used without conjugation to
selectively amplify all or a desired portion of the nucleic acid
molecule. The amplified nucleic acid molecules can then be detected
by methods known in the art.
[0091] The present invention also provide diagnostic methods that
rely on a binding agent that selectively binds the specified ARP. A
method of the invention for diagnosing or predicting susceptibility
to a prostate neoplastic condition in an individual is practiced by
contacting a specimen from an individual with a binding agent that
selectively binds an ARP3 polypeptide having at least 45% amino
acid identity with SEQ ID NO:5; determining a test expression level
of ARP3 polypeptide in the specimen; and comparing the test
expression level to a non-neoplastic control expression level of
ARP3 polypeptide, where an altered test expression level as
compared to the control expression level indicates the presence of
a prostate neoplastic condition in the individual.
[0092] The present invention also provides a method for diagnosing
or predicting susceptibility to a prostate neoplastic condition in
an individual by contacting a specimen from the individual with a
binding agent that selectively binds an ARP4 polypeptide having at
least 50% amino acid identity with SEQ ID NO:7; determining a test
expression level of ARP4 polypeptide in the specimen; and comparing
the test expression level to a non-neoplastic control expression
level of ARP4 polypeptide, where an altered test expression level
as compared to the control expression level indicates the presence
of a prostate neoplastic condition in the individual.
[0093] Further provided by the invention is a method for diagnosing
or predicting susceptibility to a prostate neoplastic condition in
an individual by contacting a specimen from an individual with a
binding agent that selectively binds an ARP5 polypeptide having at
least 40% amino acid identity with SEQ ID NO:9; determining a test
expression level of ARP5 polypeptide in the specimen; and comparing
the test expression level to a non-neoplastic control expression
level of ARP5 polypeptide, where an altered test expression level
as compared to the control expression level indicates the presence
of a prostate neoplastic condition in the individual.
[0094] In a method of the invention, the specimen can contain, for
example, a prostate cell or prostate tissue and, in one embodiment,
is a fluid such as blood, serum, urine or semen. The control
expression level can be determined, for example, using a normal
prostate cell or an androgen-dependent cell line. In addition, a
binding agent selective for a polypeptide of the invention can be,
for example, an antibody, and, if desired, can further include a
detectable label.
[0095] As used herein, the term "specimen" means any biological
material including fluid, cell, tissue, organ or portion thereof,
that contains or potentially contains an ARP polypeptide of the
invention. The term specimen includes materials present in an
individual as well as materials obtained or derived from the
individual. For example, a specimen can be a histologic section
obtained by biopsy, or cells that are placed in or adapted to
tissue culture. A specimen further can be a subcellular fraction or
extract, or a crude or substantially pure protein preparation. A
specimen can be prepared by methods known in the art suitable for
the particular format of the detection method.
[0096] In methods of the invention, the specimen can be, for
example, a prostate cell or prostate tissue such as a tissue
biopsy. A specimen can also be a fluid sample, for example, blood,
serum, urine or semen. A normal specimen can be, for example, a
normal prostate cell or an androgen-dependent cell line.
[0097] These diagnostic methods of the invention rely on a binding
agent. As described above, the term "binding agent" when used in
reference to an ARP polypeptide, is intended to mean a compound,
including a simple or complex organic molecule, a metal containing
compound, carbohydrate, peptide, protein, peptidomimetic,
glycoprotein, lipoprotein, lipid, nucleic acid molecule, antibody,
or the like that selectively binds the specified ARP polypeptide,
or fragment thereof. The binding agent can be labeled with a
detectable moiety, if desired, or rendered detectable by specific
binding to a detectable secondary binding agent. Exemplary binding
agents are discussed hereinabove.
[0098] A prostate neoplastic condition is a benign or malignant
prostate lesion of proliferating cells. Prostate neoplastic
conditions include, for example, prostate interepithelial neoplasia
(PIN) and prostate cancer. Prostate cancer is an uncontrolled
proliferation of prostate cells which can invade and destroy
adjacent tissues as well as metastasize. Primary prostate tumors
can be classified into stages TX, T0, T1, T2, T3, and T4 and
metastatic tumors can be classified into stages D1, D2 and D3.
Similarly, there are classifications known by those skilled in the
art for the progressive stages of precancerous lesions or PIN. The
methods herein are applicable for the diagnosis or treatment of any
or all stages of prostate neoplastic conditions.
[0099] The methods of the invention are also applicable to prostate
pathologies other than neoplastic conditions. Such other
pathologies include, for example, benign prostatic hyperplasia
(BPH) and prostatitis. BPH is one of the most common diseases in
adult males. Histological evidence of BPH has been found in more
than 40% of men in their fifties and almost 90% of men in their
eighties. The disease results from the accumulation of
non-malignant nodules arising in a small region around the proximal
segment of the prostatic urethra which leads to an increase in
prostate volume. If left untreated, BPH can result in acute and
chronic, retention of urine, renal failure secondary to obstructive
uropathy, serious urinary tract infection and irreversible bladder
decompensation. Prostatitis is an infection of the prostate. Other
prostate pathologies known to those skilled in the art exist as
well and are similarly applicable for diagnosis or treatment using
the methods of the invention. Various neoplastic conditions of the
prostate as well as prostate pathologies can be found described in,
for example, Campbell's Urology, Seventh Edition, W. B. Saunders
Company, Philadelphia (1998). Therefore, the methods of the
invention are applicable to both prostate neoplastic conditions and
prostate pathologies.
[0100] Therefore, the invention provides a method for both
diagnosing and prognosing a prostate neoplastic condition including
prostate cancer and prostate interepithelial neoplasia as well as
other prostate pathologies such as BPH and prostatitis.
[0101] The invention provides a method of diagnosing or predicting
prostate neoplastic conditions based on a finding of a positive
correlation between a test expression level of an ARP polypeptide
or nucleic acid in neoplastic cells of the prostate and the degree
or extent of the neoplastic condition or pathology. The diagnostic
methods of the invention are applicable to numerous prostate
neoplastic conditions and pathologies as described above. One
consequence of progression into these neoplastic and pathological
conditions can be altered expression of ARP polypeptide or nucleic
acid in prostate tissue. The alteration in ARP polypeptide or
nucleic acid expression in individuals suffering from a prostate
neoplastic condition can be measured by comparing the amount of ARP
polypeptide or nucleic acid to that found, for example, in normal
prostate tissue samples or in normal blood or serum samples. A
two-fold or more increase or decrease in a test expression level in
a prostate cell sample relative to a non-neoplastic control
expression sample obtained, for example, from normal prostate cells
or from an androgen-dependent cell line is indicative of a prostate
neoplastic condition or pathology. Similarly, an alteration in ARP
polypeptide or nucleic acid expression leading to an increased or
decreased secretion into the blood or other circulatory fluids of
the individual compared to a non-neoplastic control blood or fluid
samples also can be indicative of a prostate neoplastic condition
or pathology. For example, an alteration in ARP polypeptide or
nucleic acid expression can lead to a two-fold, five-fold,
ten-fold, 100-fold, 200-fold or 1000-fold increased secretion into
the blood or other circulatory fluids of the individual compared to
a non-neoplastic control blood or fluid samples. As another
example, an alteration in ARP polypeptide or nucleic acid
expression can lead to a two-fold, five-fold, ten-fold, 100-fold,
200-fold or 1000-fold decreased secretion into the blood or other
circulatory fluids of the individual compared to a non-neoplastic
control blood or fluid samples.
[0102] As a diagnostic indicator, an ARP polypeptide or nucleic
acid molecule can be used qualitatively to positively identify a
prostate neoplastic condition or pathology as described above.
Alternatively, ARP polypeptide or nucleic acid molecule also can be
used quantitatively to determine the degree or susceptibility of a
prostate neoplastic condition or pathology. For example, successive
increases or decreases in the expression levels of ARP polypeptide
or nucleic acid can be used as a predictive indicator of the degree
or severity of a prostate neoplastic condition or pathology. For
example, increased expression can lead to a rise in accumulated
levels and can be positively correlated with increased severity of
a neoplastic condition of the prostate. A higher level of ARP
polypeptide or nucleic acid expression can be correlated with a
later stage of a prostate neoplastic condition or pathology. For
example, increases in expression levels of two-fold or more
compared to a normal sample can be indicative of at least prostate
neoplasia. ARP polypeptide or nucleic acid molecule also can be
used quantitatively to distinguish between pathologies and
neoplastic conditions as well as to distinguish between the
different types of neoplastic conditions.
[0103] Correlative alterations can be determined by comparison of
ARP polypeptide or nucleic acid expression from the individual
having, or suspected of having, a neoplastic condition of the
prostate to expression levels of ARP polypeptide or nucleic acid
from known specimens or samples determined to exhibit a prostate
neoplastic condition. Alternatively, correlative alterations also
can be determined by comparison of a test expression level of ARP
polypeptide or nucleic acid expression to expression levels of
other known markers of prostate cancer such as prostate specific
antigen (PSA), glandular kallikrein 2 (hK2) and prostase/PRSS18.
These other known markers can be used, for example, as an internal
or external standard for correlation of stage-specific expression
with altered ARP polypeptide or nucleic acid expression and
severity of the neoplastic or pathological condition. Conversely, a
regression in the severity of a prostate neoplastic condition or
pathology can be followed by a corresponding reversal in ARP
polypeptide or nucleic acid expression levels and can similarly be
assessed using the methods described herein.
[0104] Given the teachings and guidance provided herein, those
skilled in the art will know or can determine the stage or severity
of a prostate neoplastic condition or pathology based on a
determination of ARP polypeptide or nucleic acid expression and
correlation with a prostate neoplastic condition or pathology. A
correlation can be determined using known procedures and marker
comparisons as described herein. For a review of recognized values
for such other marker in normal versus pathological tissues, see,
for example, Campbell's Urology, Seventh Edition, W. B. Saunders
Company, Philadelphia (1998).
[0105] The use of ARP polypeptide or nucleic acid expression levels
in prostate cells, the circulatory system and urine as a diagnostic
indicator of a prostate pathology allows for early diagnosis as a
predictive indicator when no physiological or pathological symptoms
are apparent. The methods are particularly applicable to any males
over age 50, African-American males and males with familial history
of prostate neoplastic conditions or pathologies. The diagnostic
methods of the invention also are particularly applicable to
individuals predicted to be at risk for prostate neoplastic
conditions or pathologies by reliable prognostic indicators prior
to onset of overt clinical symptoms. All that is necessary is to
determine the ARP polypeptide or nucleic acid prostate tissue or
circulatory or bodily fluid expression levels to determine whether
there is altered ARP polypeptide or nucleic acid levels in the
individual suspected of having a prostate pathology compared to a
control expression level such as the level observed in normal
individuals. Those skilled in the art will know by using routine
examinations and practices in the field of medicine those
individuals who are applicable candidates for diagnosis by the
methods of the invention.
[0106] For example, individuals suspected of having a prostate
neoplastic condition or pathology can be identified by exhibiting
presenting signs of prostate cancer which include, for example, a
palpable nodule (>50% of the cases), dysuria, cystitis and
prostatitis, frequency, urinary retention, or decreased urine
stream. Signs of advanced disease include pain, uremia, weight loss
and systemic bleeding. Prognostic methods of this invention are
applicable to individuals after diagnosis of a prostate neoplastic
condition, for example, to monitor improvements or identify
residual neoplastic prostate cells using, for example, imaging
methods known in the art and which target ARP polypeptide or
nucleic acid. Therefore, the invention also provides a method of
predicting the onset of a prostate neoplastic condition or
pathology by determining an altered test expression level of one of
the ARP nucleic acid molecules or polypeptides of the
invention.
[0107] The diagnostic methods of the invention are applicable for
use with a variety of different types of samples or specimens
isolated or obtained from an individual having, or suspected of
having a prostate neoplastic condition or prostate pathology. For
example, samples applicable for use in one or more diagnostic
formats of the invention include tissue and cell samples. A tissue
or cell sample or specimen can be obtained, for example, by biopsy
or surgery. As described below, and depending on the format of the
method, the tissue can be used whole or subjected to various
methods known in the art to disassociate the sample or specimen
into smaller pieces, cell aggregates or individual cells.
Additionally, when combined with amplification methods such as
polymerase chain reaction (PCR), a single prostate cell can be a
sample sufficient for use in diagnostic assays of the invention
which employ hybridization detection methods. Similarly, when
measuring ARP polypeptide or activity levels, amplification of the
signal with enzymatic coupling or photometric enhancement can be
employed using only a few or a small number of cells.
[0108] Whole tissue obtained from a prostate biopsy or surgery is
one example of a prostate cell sample or specimen. Whole tissue
prostate cell samples or specimens can be assayed employing any of
the formats described below. For example, the prostate tissue
sample can be mounted and hybridized in situ with ARP nucleic acid
probes. Similar histological formats employing protein detection
methods and in situ activity assays also can be used to detect an
ARP polypeptide in whole tissue prostate cell specimens. Protein
detection methods include, for example, staining with an ARP
specific antibody and activity assays. Such histological methods as
well as others well known to those skilled in the art are
applicable for use in the diagnostic methods of the invention using
whole tissue as the source of a prostate cell specimen. Methods for
preparing and mounting the samples and specimens are similarly well
known in the art.
[0109] Individual prostate cells and cell aggregates from an
individual having, or suspected of having a prostate neoplastic
condition or pathology also are prostate cell samples which can be
analyzed for an altered test expression level in a method of the
invention. The cells can be grown in culture and analyzed in situ
using procedures such as those described above. Whole cell samples
expressing cell surface markers associated with ARP polypeptide or
nucleic acid expression can be rapidly tested using fluorescent or
magnetic activated cell sorting (FACS or MACS) with labeled binding
agents selective for the surface marker or using binding agents
selective for epithelial or prostate cell populations, for example,
and then determining a test expression level of a specified ARP
polypeptide or nucleic acid within this population. The test
expression level can be determined using, for example, binding
agents selective for polypeptides of the invention or by
hybridization to a specific nucleic acid molecule of the invention.
Other methods for measuring the expression level of ARP polypeptide
or nucleic acid in whole cell samples are known in the art and are
similarly applicable in any of the diagnostic formats described
below.
[0110] The tissue or whole cell prostate cell sample or specimen
obtained from an individual also can be analyzed for increased ARP
polypeptide or nucleic acid expression by lysing the cell and
measuring a test expression levels of ARP polypeptide or nucleic
acid in the lysate, a fractionated portion thereof or a purified
component thereof using any of diagnostic formats described herein.
For example, if a hybridization format is used, ARP RNA can be
amplified directly from the lysate using PCR, or other
amplification procedures well known in the art such as RT-PCR, 5'
or 3' RACE to directly measure the expression levels of ARP nucleic
acid molecules. RNA also can be isolated and probed directly such
as by solution hybridization or indirectly by hybridization to
immobilized RNA. Similarly, when determining a test expression
level of ARP using polypeptide detection formats, lysates can be
assayed directly, or they can be further fractionated to enrich for
ARP polypeptide and its corresponding activity. Numerous other
methods applicable for use with whole prostate cell samples are
well known to those skilled in the art and can accordingly be used
in the methods of the invention.
[0111] The prostate tissue or cell sample or specimen can be
obtained directly from the individual or, alternatively, it can be
obtained from other sources for testing. Similarly, a cell sample
can be tested when it is freshly isolated or it can be tested
following short or prolonged periods of cryopreservation without
substantial loss in accuracy or sensitivity. If the sample is to be
tested following an indeterminate period of time, it can be
obtained and then cryopreserved, or stored at 4.degree. C. for
short periods of time, for example. An advantage of the diagnostic
methods of the invention is that they do not require histological
analysis of the sample. As such, the sample can be initially
disaggregated, lysed, fractionated or purified and the active
component stored for later diagnosis.
[0112] The diagnostic methods of the invention are applicable for
use with a variety of different types of samples and specimens
other than prostate cell samples. For example, an ARP polypeptide
or fragment thereof that is released into the extracellular space,
including circulatory fluids as well as other bodily fluids, can be
detected in a method of the invention. In such a case, the
diagnostic methods of the invention are practiced with fluid
samples collected from an individual having, or suspected of having
a neoplastic condition of the prostate or a prostate pathology.
[0113] Fluid samples and specimens, which can be measured for ARP
polypeptide or nucleic acid expression levels, include, for
example, blood, serum, lymph, urine and semen. Other bodily fluids
are known to those skilled in the art and are similarly applicable
for use as a sample or specimen in the diagnostic methods of the
invention. One advantage of analyzing fluid samples or specimens is
that they are readily obtainable, in sufficient quantity, without
invasive procedures as required by biopsy and surgery. Analysis of
fluid samples or specimens such as blood, serum and urine will
generally be in the diagnostic formats described herein which
measure ARP polypeptide levels or activity. As the ARP related
polypeptide is circulating in a soluble form, the methods will be
similar to those which measure expression levels from cell lysates,
fractionated portions thereof or purified components.
[0114] Prostate neoplastic conditions and prostate pathologies can
be diagnosed, predicted or prognosed by measuring a test expression
level of ARP polypeptide or nucleic acid in a prostate cell sample,
circulating fluid or other bodily fluid obtained from the
individual. As described herein, a test or control expression level
can be measured by a variety of methods known in the art. For
example, a test expression level of a specified ARP can be
determined by measuring the amount of ARP RNA or polypeptide in a
sample or specimen from the individual. Alternatively, a test
expression level of ARP can be determined by measuring the amount
of an ARP activity in a specimen, the amount of activity being
indicative of the specified ARP polypeptide expression level.
[0115] One skilled in the art can readily determine an appropriate
assay system given the teachings and guidance provided herein and
choose a method based on measuring ARP RNA, polypeptide or
activity. Considerations such as the sample or specimen type,
availability and amount will also influence selection of a
particular diagnostic format. For example, if the sample or
specimen is a prostate cell sample and there is only a small amount
available, then diagnostic formats which measure the amount of ARP
RNA by, for example, PCR amplification, or which measure
ARP-related cell surface polypeptide by, for example, FACS analysis
can be appropriate choices for determining a test expression level.
Alternatively, if the specimen is a blood sample and the user is
analysing numerous different samples simultaneous, such as in a
clinical setting, then a multisample format, such as an Enzyme
Linked Immunoabsorbant Assay (ELISA), which measures the amount of
an ARP polypeptide can be an appropriate choice for determining a
test expression level of a specified ARP. Additionally, ARP nucleic
acid molecules released into bodily fluids from the neoplastic or
pathological prostate cells can also be analyzed by, for example,
PCR or RT-PCR. Those skilled in the art will know, or can determine
which format is amenable for a particular application and which
methods or modifications known within the art are compatible with a
particular type of format.
[0116] Hybridization methods are applicable for measuring the
amount of ARP RNA as an indicator of ARP expression levels. There
are numerous methods well known in the art for detecting nucleic
acid molecules by specific or selective hybridization with a
complementary nucleic acid molecule. Such methods include both
solution hybridization procedures and solid-phase hybridization
procedures where the probe or sample is immobilized to a solid
support. Descriptions for such methods can be found in, for
example, Sambrook et al., supra, and in Ausubel et al., supra.
Specific examples of such methods include PCR and other
amplification methods such as RT-PCR, 5' or 3' RACE, RNase
protection, RNA blot, dot blot or other membrane-based
technologies, dip stick, pin, ELISA or two-dimensional arrays
immobilized onto chips as a solid support. These methods can be
performed using either qualitative or quantitative measurements,
all of which are well known to those skilled in the art.
[0117] PCR or RT-PCR can be used with isolated RNA or crude cell
lysate preparations. As described previously, PCR is advantageous
when there is limiting amounts of starting material. A further
description of PCR methods can be found in, for example,
Dieffenbach, C. W., and Dveksler, G. S., PCR Primer: A Laboratory
Manual, Cold Spring Harbor Press, Plainview, N.Y. (1995).
Multisample formats such as an ELISA or two-dimensional array offer
the advantage of analyzing numerous, different samples in a single
assay. Solid-phase dip stick-based methods offer the advantage of
being able to rapidly analyze a patient's fluid sample and obtain
an immediate result.
[0118] Nucleic acid molecules useful for measuring a test
expression level of a specified ARP RNA are disclosed herein above.
Briefly, for detection by hybridization, an ARP nucleic acid
molecule having a detectable label is added to a prostate cell
sample or a fluid sample obtained from the individual having, or
suspected of having a prostate neoplastic condition or pathology
under conditions which allow annealing of the molecule to an ARP
RNA. Methods for detecting ARP RNA in a sample can include the use
of, for example, RT-PCR. Conditions are well known in the art for
both solution and solid phase hybridization procedures. Moreover,
optimization of hybridization conditions can be performed, if
desired, by hybridization of an aliquot of the sample at different
temperatures, durations and in different buffer conditions. Such
procedures are routine and well known to those skilled in the art.
Following annealing, the sample is washed and the signal is
measured and compared with a suitable control or standard value.
The magnitude of the hybridization signal is directly proportional
to the expression levels of ARP RNA.
[0119] The diagnostic procedures described herein can additionally
be used in conjunction with other prostate markers, such as
prostate specific antigen, human glandular kallikrein 2 (hk2) and
prostase/PRSS18 for simultaneous or independent corroboration of a
sample. Additionally, ARP polypeptide or nucleic acid expression
can be used, for example, in combination with other markers to
further distinguish normal basal cells, secretory cells and
neoplastic cells of the prostate. Moreover, ARP polypeptide or
nucleic acid expression can be used in conjunction with smooth
muscle cell markers to distinguish between pathological conditions
such as benign prostate hypertrophy (BPH) and neoplasia. Those
skilled in the art will know which markers are applicable for use
in conjunction with ARP polypeptide or nucleic acid to delineate
more specific diagnostic information such as that described
above.
[0120] The invention also provides diagnostic methods based on
determining whether there is an altered test expression level of an
ARP3, ARP4 or ARP5 polypeptide using a binding agent that
selectively binds the recited polypeptide. Essentially all modes of
affinity binding assays are applicable for use in determining a
test expression level of an ARP polypeptide in a method of the
invention. Such methods are rapid, efficient and sensitive.
Moreover, affinity binding methods are simple and can be modified
to be performed under a variety of clinical settings and conditions
to suit a variety of particular needs. Affinity binding assays
which are known and can be used in the methods of the invention
include both soluble and solid phase formats. A specific example of
a soluble phase affinity binding assay is immunoprecipitation using
an ARP selective antibody or other binding agent. Solid phase
formats are advantageous in that they are rapid and can be
performed easily and simultaneously on multiple different samples
without losing sensitivity or accuracy. Moreover, solid phase
affinity binding assays are further amenable to high throughput and
ultra high throughput screening and automation.
[0121] Specific examples of solid phase affinity binding assays
include immunoaffinity binding assays such as an ELISA and
radioimmune assay (RIA). Other solid phase affinity binding assays
are known to those skilled in the art and are applicable to the
methods of the invention. Although affinity binding assays are
generally formatted for use with an antibody binding molecule that
is selective for the analyte or ligand of interest, essentially any
binding agent can be alternatively substituted for the selectively
binding antibody. Such binding agents include, for example,
macromolecules such as polypeptides, peptides, nucleic acid
molecules, lipids and sugars as well as small molecule compounds.
Methods are known in the art for identifying such molecules which
bind selectively to a particular analyte or ligand and include, for
example, surface display libraries and combinatorial libraries.
Thus, for a molecule other than an antibody to be used in an
affinity binding assay, all that is necessary is for the binding
agent to exhibit selective binding activity for a polypeptide of
the invention.
[0122] Various modes of affinity binding formats are similarly
known which can be used in the diagnostic methods of the invention.
For the purpose of illustration, particular embodiments of such
affinity binding assays will be described further in reference to
immunoaffinity binding assays. The various modes of affinity
binding assays, such as immunoaffinity binding assays, include, for
example, solid phase ELISA and RIA as well as modifications
thereof. Such modifications thereof include, for example, capture
assays and sandwich assays as well as the use of either mode in
combination with a competition assay format. The choice of which
mode or format of immunoaffinity binding assay to use will depend
on the intent of the user. Such methods can be found described in
common laboratory manuals such as Harlow and Lane, Using
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, New York (1999).
[0123] As with the hybridization methods described previously, the
diagnostic formats employing affinity binding can be used in
conjunction with a variety of detection labels and systems known in
the art to quantitate amounts of a polypeptide of the invention in
the analyzed sample. Detection systems include the detection of
bound polypeptide on the invention by both direct and indirect
means. Direct detection methods include labeling of the
ARP-selective antibody or binding agent. Indirect detection systems
include, for example, the use of labeled secondary antibodies and
binding agents.
[0124] Secondary antibodies, labels and detection systems are well
known in the art and can be obtained commercially or by techniques
well known in the art. The detectable labels and systems employed
with the ARP-selective binding agent should not impair binding of
the agent to the corresponding ARP polypeptide. Moreover, multiple
antibody and label systems can be employed for detecting the bound
ARP-selective antibody to enhance the sensitivity of the binding
assay if desired.
[0125] As with the hybridization formats described previously,
detectable labels can be essentially any label that can be
quantitated or measured by analytical methods. Such labels include,
for example, enzymes, radioisotopes, fluorochromes as well as
chemi- and bioluminescent compounds. Specific examples of enzyme
labels include horseradish peroxidase (HRP), alkaline phosphatase
(AP), .beta.-galactosidase, urease and luciferase.
[0126] A horseradish-peroxidase detection system can be used, for
example, with the chromogenic substrate tetramethylbenzidine (TMB),
which yields a soluble product in the presence of hydrogen peroxide
that is detectable by measuring absorbance at 450 nm. An alkaline
phosphatase detection system can be used with the chromogenic
substrate p-nitrophenyl phosphate, for example, which yields a
soluble product readily detectable by measuring absorbance at 405
nm. Similarly, a .beta.-galactosidase detection system can be used
with the chromogenic substrate
o-nitrophenyl-.beta.-D-galactopyranoside (ONPG), which yields a
soluble product detectable by measuring absorbance at 410 nm, or a
urease detection system can be used with a substrate such as
urea-bromocresol purple (Sigma Immunochemicals, St. Louis, Mo.).
Luciferin is the substrate compound for luciferase which emits
light following ATP-dependent oxidation.
[0127] Fluorochrome detection labels are rendered detectable
through the emission of light of ultraviolet or visible wavelength
after excitation by light or another energy source. DAPI,
fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin,
R-phycoerythrin, rhodamine, Texas red and lissamine are specific
examples of fluorochrome detection labels that can be utilized in
the affinity binding formats of the invention. A particularly
useful fluorochrome is fluorescein or rhodamine.
[0128] Chemiluminescent as well as bioluminescent detection labels
are convenient for sensitive, non-radioactive detection of an ARP
polypeptide and can be obtained commercially from various sources
such as Amersham Lifesciences, Inc. (Arlington Heights, Ill.).
[0129] Alternatively, radioisotopes can be used as detectable
labels in the methods of the invention. Iodine-125 is a specific
example of a radioisotope useful as a detectable label.
[0130] Signals from detectable labels can be analyzed, for example,
using a spectrophotometer to detect color from a chromogenic
substrate; a fluorometer to detect fluorescence in the presence of
light of a certain wavelength; or a radiation counter to detect
radiation, such as a gamma counter for detection of iodine-125. For
detection of an enzyme-linked secondary antibody, for example, a
quantitative analysis of the amount of bound agent can be made
using a spectrophotometer such as an EMAX Microplate Reader
(Molecular Devices, Menlo Park, Calif.) in accordance with the
manufacturer's instructions. If desired, the assays of the
invention can be automated or performed robotically, and the signal
from multiple samples can be detected simultaneously.
[0131] The diagnostic formats of the present invention can be
forward, reverse or simultaneous as described in U.S. Pat. No.
4,376,110 and No. 4,778,751. Separation steps for the various assay
formats described herein, including the removal of unbound
secondary antibody, can be performed by methods known in the art
(Harlow and Lane, supra). For example, washing with a suitable
buffer can be followed by filtration, aspiration, vacuum or
magnetic separation as well as by centrifugation.
[0132] A binding agent selective for an ARP polypeptide also can be
utilized in imaging methods that are targeted at ARP expressing
prostate cells. These imaging techniques have utility in
identification of residual neoplastic cells at the primary site
following standard treatments including, for example, radical
prostatectomy, radiation or hormone therapy. In addition, imaging
techniques that detect neoplastic prostate cells have utility in
detecting secondary sites of metastasis. A binding agent that
selectively binds ARP3, ARP4 or ARP5 can be radiolabeled with, for
example, .sup.111indium and infused intravenously as described by
Kahn et al., Journal of Urology 152:1952-1955 (1994). The binding
agent selective for an ARP polypeptide can be, for example, a
monoclonal antibody selective for an ARP polypeptide. Imaging can
be accomplished by, for example, radioimmunoscintigraphy as
described by Kahn et al., supra.
[0133] In one embodiment, the invention provides a method of
diagnosing or predicting the susceptibility of a prostate
neoplastic condition in an individual suspected of having a
neoplastic condition of the prostate, where a test expression level
of an ARP polypeptide is determined by measuring the amount of
ARP3, ARP4, or ARP5 polypeptide activity. The method is practiced
by contacting a specimen from the individual with an agent that
functions to measure an activity associated with an ARP3, ARP4, or
ARP5 polypeptide of the invention.
[0134] As with the hybridization and affinity binding formats
described above, activity assays similarly can be performed using
essentially identical methods and modes of analysis. Therefore,
solution and solid phase modes, including multisample ELISA, RIA
and two-dimensional array procedures are applicable for use in
measuring an activity associated with an ARP polypeptide. The
activity can be measured by, for example, incubating an agent that
functions to measure an activity associated with an ARP polypeptide
with the sample and determining the amount of product formed that
corresponds to ARP3, ARP4 or ARP5 polypeptide activity. The amount
of product formed will directly correlate with the ARP3, ARP4 or
ARP5 polypeptide activity in the specimen and therefore, with the
expression levels of the corresponding polypeptide of the invention
in the specimen.
[0135] The invention further provides a method of identifying a
compound that inhibits ARP3, ARP4 or ARP5 polypeptide activity. The
method consists of contacting a specimen containing an ARP
polypeptide and an agent that functions to measure an activity
associated with an ARP polypeptide with a test compound under
conditions that allow formation of a product that corresponds to an
ARP polypeptide activity and measuring the amount of product
formed, where a decrease in the amount of product formed in the
presence of the test compound compared to the absence of the test
compound indicates that the compound has ARP polypeptide inhibitory
activity. Similarly, compounds that increase the activity of an ARP
polypeptide also can be identified. A test compound added to a
specimen containing an ARP polypeptide and an agent that functions
to measure an activity associated with an ARP polypeptide which
increases the amount of product formed compared to the absence of
the test compound indicates that the compound increases the
corresponding ARP polypeptide activity. Therefore, the invention
provides a method of identifying compounds that modulate the
activity of an ARP polypeptide. The ARP polypeptide containing
specimen used for such a method can be serum, prostate tissue, a
prostate cell population or a recombinant cell population
expressing an ARP polypeptide.
[0136] Those compounds having inhibitory activity are considered as
potential ARP polypeptide antagonists and further as potential
therapeutic agents for treatment of neoplastic conditions of the
prostate. Similarly, those compounds which increase an ARP
polypeptide activity are considered as potential ARP polypeptide
agonists and further as potential therapeutic agents for the
treatment of neoplastic conditions of the prostate. Each of these
classes of compounds is encompassed by the term ARP regulatory
agent as defined herein.
[0137] Within the biological arts, the term "about" when used in
reference to a particular activity or measurement is intended to
refer to the referenced activity or measurement as being within a
range of values encompassing the referenced value and within
accepted standards of a credible assay within the art, or within
accepted statistical variance of a credible assay within the
art.
[0138] A reaction system for identifying a compound that inhibits
or enhances an ARP polypeptide activity can be performed using
essentially any source of ARP polypeptide activity. Such sources
include, for example, a prostate cell sample, lysate or
fractionated portion thereof; a bodily fluid such as blood, serum
or urine from an individual with a prostate neoplastic condition; a
recombinant cell or soluble recombinant source, and an in vitro
translated source. The ARP polypeptide source is combined with an
agent that functions to measure an activity associated with an ARP
polypeptide as described above and incubated in the presence or
absence of a test inhibitory compound. The amount of product that
corresponds to an ARP polypeptide activity that is formed in the
presence of the test compound is compared with that in the absence
of the test compound. Those test compounds which inhibit product
formation are considered to be ARP polypeptide inhibitors. For
example, a test compound can inhibit product formation by at least
50%, 80%, 90%, 95%, 99%, 99.5% or 99.9%. Similarly, those compounds
which increase product formation are considered to be ARP
polypeptide enhancers or activators. For example, a test compound
can increase product formation by at least two-fold, five-fold,
ten-fold, 100-fold, 200-fold or 1000-fold. ARP polypeptide
inhibitors and activators can then be subjected to further in vitro
or in vivo testing to confirm that they inhibit an ARP polypeptide
activity in cellular and animal models.
[0139] Suitable test compounds for the inhibition or enhancement
assays can be any substance, molecule, compound, mixture of
molecules or compounds, or any other composition which is suspected
of being capable of inhibiting an ARP polypeptide activity in vivo
or in vitro. The test compounds can be macromolecules, such as
biological polymers, including proteins, polysaccharides and
nucleic acid molecules. Sources of test compounds which can be
screened for ARP polypeptide inhibitory activity include, for
example, libraries of peptides, polypeptides, DNA, RNA and small
organic compounds. The test compounds can be selected randomly and
tested by the screening methods of the present invention. Test
compounds are administered to the reaction system at a
concentration in the range from about 1 pM to 1 mM.
[0140] Methods for producing pluralities of compounds to use in
screening for compounds that modulate the activity of an ARP
polypeptide, including chemical or biological molecules that are
inhibitors or enhancers of an ARP activity such as simple or
complex organic molecules, metal-containing compounds,
carbohydrates, peptides, proteins, peptidomimetics, glycoproteins,
lipoproteins, nucleic acid molecules, antibodies, and the like, are
well known in the art and are described, for example, in Huse, U.S.
Pat. No. 5,264,563; Francis et al., Curr. Opin. Chem. Biol.
2:422-428 (1998); Tietze et al., Curr. Biol., 2:363-371 (1998);
Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al., Med. Res. Rev.
15:481-496 (1995); and the like. Libraries containing large numbers
of natural and synthetic compounds also can be obtained from
commercial sources. Combinatorial libraries of molecules can be
prepared using well known combinatorial chemistry methods (Gordon
et al., J. Med. Chem. 37: 1233-1251 (1994); Gordon et al., J. Med.
Chem. 37: 1385-1401 (1994); Gordon et al., Acc. Chem. Res.
29:144-154 (1996); Wilson and Czarnik, eds., Combinatorial
Chemistry: Synthesis and Application, John Wiley &. Sons, New
York (1997)).
[0141] Therefore, the invention provides a method of identifying a
compound that inhibits or enhances an ARP polypeptide activity
where the sample further consists of a prostate cell lysate, a
recombinant cell lysate expressing an ARP polypeptide, an in vitro
translation lysate containing an ARP mRNA, a fraction of a prostate
cell lysate, a fraction of a recombinant cell lysate expressing an
ARP polypeptide, a fractionated sample of an in vitro translation
lysate containing an ARP mRNA or an isolated ARP polypeptide. The
method can be performed in single or multiple sample format.
[0142] In another embodiment, polypeptides of the invention can be
used as vaccines to prophylactically treat individuals for the
occurrence of a prostate neoplastic condition or pathology. Such
vaccines can be used to induce B or T cell immune responses or both
aspects of the individuals endogenous immune mechanisms. The mode
of administration and formulations to induce either or both of
these immune responses are well known to those skilled in the art.
For example, polypeptides can be administered in many possible
formulations, including pharmaceutically acceptable mediums. They
can be administered alone or, for example, in the case of a
peptide, the peptide can be conjugated to a carrier, such as KLH,
in order to increase its immunogenicity. The vaccine can include or
be administered in conjunction with an adjuvant, various of which
are known to those skilled in the art. After initial immunization
with the vaccine, further boosters can be provided if desired.
Therefore, the vaccines are administered by conventional methods in
dosages which are sufficient to elicit an immunological response,
which can be easily determined by those skilled in the art.
Alternatively, the vaccines can contain anti-idiotypic antibodies
which are internal images of polypeptides of the invention. Methods
of making, selecting and administering such anti-idiotype vaccines
are well known in the art. See, for example, Eichmann, et al., CRC
Critical Reviews in Immunology 7:193-227 (1987). In addition, the
vaccines can contain an ARP nucleic acid molecule. Methods for
using nucleic acid molecules such as DNA as vaccines are well known
to those skilled in the art (see, for example, Donnelly et al.
(Ann. Rev. Immunol. 15:617-648 (1997)); Felgner et al. (U.S. Pat.
No. 5,580,859, issued Dec. 3, 1996); Felgner (U.S. Pat. No.
5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S. Pat. No.
5,679,647, issued Oct. 21, 1997)).
[0143] The invention additionally provides a method of treating or
reducing the severity of a prostate neoplastic condition. The
method is practiced by administering to an individual having a
prostate neoplastic condition or other prostatic pathology an ARP1,
ARP2, ARP3, ARP4 or ARP5 regulatory agent. A "regulatory agent"
means an agent that inhibits or enhances a biological activity of
the specified ARP polypeptide. Such an ARP regulatory agent can
effect the amount of ARP polypeptide produced or can inhibit or
enhance activity without effecting the amount of polypeptide. Such
an ARP regulatory agent can be, for example, a dominant negative
form of ARP1, ARP2, ARp3, Arp4 or Arp5; a ARP3, ARP4 or ARP
selective binding agent, or an antisense molecule. One skilled in
the art understands that such an ARP1, ARP2, ARP3, ARP4 or ARP5
regulatory agent can be an agent that selectively regulates a
biological activity of the specified ARP polypeptide or,
alternatively, can be a non-selective agent that, in addition to
regulating a biological activity of the specified polypeptide, also
regulates the activity of one or more polypeptides.
[0144] A ARP regulatory agent can cause a two-fold, five-fold,
ten-fold, 20-fold, 100-fold or more reduction in the amount or
activity of an ARP polypeptide. As another example, a regulatory
agent can cause a two-fold, five-fold, ten-fold, 20-fold, 100-fold
or more increase in the amount or activity of an ARP polypeptide or
nucleic acid. ARP regulatory agents include ARP nucleic acid
molecules, including antisense nucleic acid molecules, and other
non-ARP nucleic acid molecules; binding agents including
antibodies, and compounds identified by the methods described
herein. Such regulatory agents can be useful as therapeutics for
treating or reducing the severity of an individual with a prostate
neoplastic condition or pathology.
[0145] One type of ARP regulatory agent is an inhibitor, means an
agent effecting a decrease in the extent, amount or rate of ARP
polypeptide expression or activity. An example of an ARP inhibitor
is an ARP antisense nucleic acid molecule or a transcriptional
inhibitor that binds to an ARP 5' promoter/regulatory region.
[0146] The term inhibitory amount means the amount of an inhibitor
necessary to effect a reduction in the extent, amount or rate of
ARP polypeptide. For example, an inhibitory amount of inhibitor can
cause a two-fold, five-fold, ten-fold, 20-fold, 100-fold or more
reduction in the amount or activity of an ARP polypeptide of the
invention.
[0147] Such inhibitors can be produced using methods which are
generally known in the art, and include the use of a purified ARP
polypeptide to produce antibodies or to screen libraries of
compounds, as described previously, for those which specifically
bind a corresponding ARP polypeptide. For example, in one aspect,
antibodies which are selective for an ARP polypeptide of the
invention can be used directly as an antagonist, or indirectly as a
targeting or delivery mechanism for bringing a cytotoxic or
cytostatic agent to neoplastic prostate cells. Such agents can be,
for example, radioisotopes. The antibodies can be generated using
methods that are well known in the art and include, for example,
polyclonal, monoclonal, chimeric, humanized single chain, Fab
fragments, and fragments produced by a Fab expression library.
[0148] In another embodiment of the invention, ARP polynucleotides,
or any fragment thereof, or antisense molecules, can be used as an
ARP regulatory agent in a method of the invention. In one aspect,
antisense molecules to an ARP encoding nucleic acid molecules can
be used to block the transcription or translation of the
corresponding mRNA. Specifically, cells can be transformed with
sequences complementary to a nucleic acid molecule of the
invention. Such methods are well known in the art, and sense or
antisense oligonucleotides or larger fragments, can be designed
from various locations along the coding or control regions of
sequences encoding ARP polypeptides or nucleic acids. Thus,
antisense molecules may be used to modulate an ARP activity, or to
achieve regulation of an ARP gene function.
[0149] Expression vectors derived from retroviruses, adenovirus,
adeno-associated virus (AAV), herpes or vaccinia viruses, or from
various bacterial plasmids can be used for delivery of antisense
nucleotide sequences to the prostate cell population. The viral
vector selected should be able to infect the tumor cells and be
safe to the host and cause minimal cell transformation. Retroviral
vectors and adenoviruses offer an efficient, useful, and presently
the best-characterized means of introducing and expressing foreign
genes efficiently in mammalian cells. These vectors are well known
in the art and have very broad host and cell type ranges, express
genes stably and efficiently. Methods which are well known to those
skilled in the art can be used to construct such recombinant
vectors and are described in Sambrook et al., supra. Even in the
absence of integration into the DNA, such vectors can continue to
transcribe RNA molecules until they are disabled by endogenous
nucleases. Transient expression can last for a month or more with a
non-replicating vector and even longer if appropriate replication
elements are part of the vector system.
[0150] Ribozymes, which are enzymatic RNA molecules, can also be
used to catalyze the specific cleavage of an ARP mRNA. The
mechanism of ribozyme action involves sequence-specific
hybridization of the ribozyme molecule to complementary target ARP
RNA, followed by endonucleolytic cleavage. Specific ribozyme
cleavage sites within any potential RNA target are identified by
scanning an ARP RNA for ribozyme cleavage sites which include the
following sequences: GUA, GUU, and GUC. Once identified, short RNA
sequences of between 15 and 20 ribonucleotides corresponding to the
region of the target gene containing the cleavage site can be
evaluated for secondary structural features which can render the
oligonucleotide inoperable. The suitability of candidate targets
can also be evaluated by testing accessibility to hybridization
with complementary oligonucleotides using ribonuclease protection
assays. Antisense molecules and ribozymes of the invention can be
prepared by any method known in the art for the synthesis of
nucleic acid molecules.
[0151] In another embodiment, an ARP promoter and regulatory region
can be used for constructing vectors for prostate cancer gene
therapy. The promoter and regulatory region can be fused to a
therapeutic gene for prostate specific expression. This method can
include the addition of one or more enhancer elements which amplify
expression of the heterologous therapeutic gene without
compromising tissue specificity. Methods for identifying a gene
promoter and regulatory region are well known to those skilled in
the art, for example, by selecting an appropriate primer from the
5' end of the coding sequence and isolating the promoter and
regulatory region from genomic DNA.
[0152] Examples of therapeutic genes that are candidates for
prostate gene therapy utilizing an ARP promoter include suicide
genes. The expression of suicide genes produces a protein or agent
that directly or indirectly inhibits neoplastic prostate cell
growth or promotes neoplastic prostate cell death. Suicide genes
include genes encoding enzymes, oncogenes, tumor suppressor genes,
genes encoding toxins, genes encoding cytokines, or a gene encoding
oncostatin. The therapeutic gene can be expressed using the vectors
described previously for antisense expression.
[0153] In accordance with another embodiment of the present
invention, there are provided diagnostic systems, for example, in
kit form. Such a diagnostic system contains at least one nucleic
acid molecule or antibody of the invention in a suitable packaging
material. The diagnostic kits containing nucleic acid molecules are
derived from ARP nucleic acid molecules described herein. A
diagnostic system of the invention can be useful for assaying for
the presence or absence of an ARP nucleic acid molecule in either
genomic DNA or mRNA.
[0154] A suitable diagnostic system includes at least one ARP
nucleic acid molecule or antibody, as a separately packaged
chemical reagent(s) in an amount sufficient for at least one assay.
For a diagnostic kit containing a nucleic acid molecule of the
invention, the kit will generally contain two or more nucleic acid
molecules. When the diagnostic kit is to be used in PCR, the kit
can further contain at least two oligonucleotides that can serve as
primers for PCR. Those of skill in the art can readily incorporate
nucleic acid molecules antibodies of the invention into kit form in
combination with appropriate buffers and solutions for the practice
of the invention methods as described herein. A kit containing an
ARP polypeptide-specific antibody can contain a reaction cocktail
that provides the proper conditions for performing an assay, for
example, an ELISA or other immunoassay, for determining the level
of expression of a corresponding ARP polypeptide in a specimen, and
can contain control samples that contain known amounts of a
corresponding ARP polypeptide and, if desired, a second antibody
selective for the corresponding anti-ARP antibody.
[0155] The contents of the kit of the invention, for example, ARP
nucleic acid molecules or antibodies, are contained in packaging
material, which can provide a sterile, contaminant-free
environment. In addition, the packaging material contains
instructions indicating how the materials within the kit can be
employed both to detect the presence or absence of a particular
nucleic acid sequence or polypeptide of the invention or to
diagnose the presence of, or a predisposition for a condition
associated with the presence or absence of a nucleic acid sequence
or polypeptide of the invention such as prostate cancer. The
instructions for use typically include a tangible expression
describing the reagent concentration or at least one assay method
parameter, such as the relative amounts of reagent and sample to be
admixed, maintenance time periods for reagent/sample admixtures,
temperature, buffer conditions, and the like.
[0156] All journal article, reference, and patent citations
provided above, in parentheses or otherwise, whether previously
stated or not, are incorporated herein by reference.
[0157] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also included within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention.
EXAMPLE I
Isolation of ARP cDNA
[0158] This example describes the isolation of ARP1, ARP2, ARP3,
ARP4 AND ARP5 cDNAs.
[0159] To identify genes transcriptionally regulated by androgens,
microarrays containing prostate derived cDNAs were screened using
RNA from a prostate cell line. Those RNAs showing increased
expression levels in response to androgen stimulation were
identified and characterized further. Specifically, the microarrays
were constructed from a non-redundant set of 1500 prostate-derived
cDNA clones identified from the Prostate Expression Database, a
public sequence repository of expressed sequence tag (EST) data
derived from human prostate cDNA libraries (Hawkins et al., Nucleic
Acids Res. 27: 204-208 (1999)). The 1500 prostate cDNA clones were
selected from the Prostate Expression Database by randomly
selecting the first 1500 non-redundant clones that were in the
database. The 1500 prostate cDNA clones were randomly selected from
the database using the criteria of taking the first 1500
non-redundant clones in the database and available based on their
prior characterization as prostate-derived. Individual clones from
the library were obtained and inserts were amplified using primers
corresponding to priming sites flanking the insert cloning site of
the pSport1 plasmid (Life Technologies, Germantown, Md.) as
previously described by Nelson et al. (Genomics 47:12-25 (1998)).
PCR products were purified through Sephacryl S500 (Pharmacia,
Kalamazoo, Mich.), mixed 1:1 with denaturing Reagent D (Amersham,
Piscataway, N.J.), and spotted in duplicate onto coated Type IV
glass microscope slides (Amersham, Piscataway, N.J.) using a
Molecular Dynamics (Sunnyvale, Calif.) Gen II robotic spotting
tool. After spotting, the slides were air-dried and UV-crosslinked
with 500 mJ of energy.
[0160] To identify genes transcriptionally regulated by androgens,
the microarrays of prostate derived cDNAs were profiled using total
RNA isolated from LNCaP cells cultured for 72 hours either in the
presence or absence of the synthetic androgen R1881 (NEN Life
Sciences Products, Boston, Mass.). Total RNA was prepared using
TRIzol (Life Technologies, Germantown, Md.) according to the
manufacturer's directions. The integrity of the RNA preparation was
checked on a standard formaldehyde agarose gel. Fifty .mu.g of the
total RNA was digested with 1 .mu.l of RNase-free DNase (Promega,
Madison, Wis.) (1.mu./.mu.l) in 1.times. first strand cDNA
synthesis buffer (Gibco-BRL, Germantown, Md.) at 37.degree. C. for
30 minutes. The reaction mix was then extracted with
Phenol/chloroform (1:1) and RNA was precipitated with ethanol. The
mRNA was isolated from the DNA-free total RNA using a Dynabeads
mRNA purification kit (Dynal, Lake Success, N.Y.).
[0161] Fluorescence-labeled probes were constructed from the
above-isolated mRNA as follows. Briefly, 1 .mu.g polyA+ RNA or 30
.mu.g total RNA in a reaction volume of 20 .mu.l containing 1 .mu.l
anchored oligo-dT primer (Amersham, Piscataway, N.J.), 0.05 mM
Cy3-dCTP (Amersham, Piscatawy, N.J.), 0.05 mM dCTP, 0.1 mM each
dGTP, dATP, dTTP, and 200 U Superscript II reverse transcriptase
(Life Technologies, Germantown, Md.) were incubated at 42.degree.
C. for 90 minutes followed by heating to 94.degree. C. for 3
minutes. Unlabeled RNA was hydrolyzed by the addition of 1 .mu.l of
5M NaOH and heating to 37.degree. C. for 10 minutes. One .mu.l of
5M HCl and 5 .mu.l of 1 M Tris-HCl (pH 7.5) were added to
neutralize the base. Unincorporated nucleotides and salts were
removed by chromatography (Qiagen, Valencia, Calif.), and the cDNA
was eluted in 30 .mu.l distilled water.
[0162] Microarray hybridization was performed as follows. One .mu.g
of dA/dT 12-18 (Pharmacia, Kalamazoo, Mich.) and 1 .mu.g of Cot1
DNA (Life Technologies, Germantown, Md.) were added to the probe,
heat denatured at 94.degree. C. for minutes, combined with an equal
volume of 2.times. microarray hybridization solution (Amersham,
Piscataway, N.J.) and placed onto the microarray slide with a
coverslip. Hybridization was carried out in a humid chamber at
52.degree. C. for 16 hours. The slides were washed once with
1.times.SSC, 0.2% SDS at room temperature for 5 minutes, then twice
washed with 0.1.times.SSC, 0.2% SDS at room temperature for 10
minutes. After washing, the slide was rinsed in distilled water to
remove trace salts and dried.
[0163] Analysis of the microarray slides to identify
androgen-regulated prostate genes was performed as follows.
Fluorescence intensities of the immobilized targets were measured
using a laser confocal microscope (Molecular Dynamics, Sunnyvale,
Calif.). Intensity data were integrated at a pixel resolution of 10
micrometers using approximately 20 pixels per spot, and recorded at
16 bits. Local background hybridization signals were subtracted
prior to comparing spot intensities and determining expression
ratios. For each experiment, each cDNA was represented twice on
each slide, and the experiments were performed in duplicate
producing four data points per cDNA clone per hybridization probe.
Intensity ratios for each cDNA clone hybridized with probes derived
from androgen-stimulated LNCaP and androgen-starved LNCaP were
calculated (stimulated intensity/starved intensity). A gene
expression level change was treated as significantly different
between the two conditions if all four replicate spots for a given
cDNA demonstrated a ratio greater than 2 or less than 1/2 and the
signal intensity was greater than 2 standard deviations above the
image background. It had been determined previously that expression
ratios less than 2-fold are not reproducible in this system.
[0164] Of a total of 1500 distinct cDNAs represented on the
microarray, several were identified as giving a differential signal
with the androgen-stimulated probe as compared to the
non-stimulated probe.
[0165] Additional cDNA clones were obtained from screening human
prostate 5' stretch cDNA (ClonTech, Inc.) With the original cDNA
clones and sequences. RACE was performed using Marathon Ready human
prostate cDNAs from ClonTech and using cDNA prepared from
androgen-stimulated LNCaP cells with the Marathon cDNA
amplification kit (ClonTech Inc.) according to manufacturer's
protocol.
[0166] As shown in FIG. 1, the ARP1 cDNA has a nucleotide sequence
of 1026 nucleotides. As shown in FIG. 2, ARP2 cDNA contains 4509
nucleotides predicted to encode a polypeptide 252 amino acids long.
As shown in FIG. 3, ARP3 cDNA contains 2213 nucleotides predicted
to encode a polypeptide 538 amino acids long.
[0167] The ARP4 cDNA was isolated as described above. RACE was
performed using the following primers: RC55
(5'-TGAGGTATCCCAGAGCAAACACAAAGCAG-3'; SEQ ID NO: 10) and RC202
(5'-TCAGTTCTTCATCCTTCCGAAACATCCC-3'; SEQ ID NO: 11). RACE reactions
were performed according to the standard ClonTech protocol, and the
resulting nucleic acids sequenced by standard methods. Two related
cDNAs were obtained derived from alternately spliced mRNAs. As
shown in FIG. 4, ARP4 cDNA contains 4433 nucleotides predicted to
encode a protein of 141 amino acids. ARP5 cDNA, shown in FIG. 5,
contains 1276 nucleotides predicted to encode a protein of 425
amino acids. The alternate splice junction is at nucleotide 401
resulting in a relative frameshift in the mRNAs.
[0168] Expression analysis of ARP4 was performed using a multiple
tissue Northern blot and a 5D6 probe containing nucleotides 1814 to
4433 of SEQ ID NO: 6. As shown in FIG. 6, ARP4 is most abundantly
expressed in prostate, testis and ovary tissues.
Sequence CWU 1
1
11 1 1026 DNA Homo sapiens 1 tagtttgtat ttttcattac cagcaagggt
aaacagttat ccatgaccca tttctatgtt 60 ctcgtggcat gcttccatgt
actgcctctg catgcagcag gccacctcgg gcagagccta 120 aagcatgtga
taaatgaaat gctatcacaa tacaggttgt gtctgaaaaa caaatggcaa 180
cttattatcc aagatcaatg aaggaaaaag caaatttact aaaatatttc tttatttgaa
240 taaggtcaat gccatttctt gaattccagc tagcatcaaa taatcaggaa
aaaaaaaact 300 tgacaaaatg ttatccaatt gaaattgaca gtggatagaa
aaccctttta aactttaagt 360 aatgtcataa aagaaatata ttaaacaagc
aacagacaga tctaaaaagt tccaagtgtg 420 gatttcacat tagatcttat
aaattaaaaa aatcctcaat ataatcattt gttcactatc 480 ttctttcaat
aagcacatgg acagggaaag ataatcacac cttaatattc acaactgcta 540
tttgtgttct ttacaaaaat tgtatctctg caatgcagtg aggcaggcaa tcccttgttc
600 aagtcatttc tgttttccct aagttatcaa aaagtacaac tgtctgatat
aaattgttac 660 cataatcaca atcaggaagg caaagaagct ttagcaggca
ggcttgaaga tgggagtttt 720 catggcttga ccatgaatga tctcaagatg
atttcataag attaaaagcc atcacgaaaa 780 tactgaaagc aacaggtaat
aatctggatt cagtctgtag ttgctcatga accacgcgtt 840 ttaataaaag
gaacattaag taaattgtag gtataaaaga atcagtgcat atctgttaat 900
gtcattgaca ataaaaatat attatcttct cagctcagct ctaaattaac aaaacaccta
960 tttttttttt cccactcctc attttagtgg ttctcaaaca ttggtgtgct
cagaatctcc 1020 tgaggt 1026 2 4527 DNA Homo sapiens CDS
(96)...(851) 2 tccttgggtt cgggtgaaag cgcctggggg ttcgtggcca
tgatccccga gctgctggag 60 aactgaaggc ggacagtctc ctgcgaaacc aggca atg
gcg gag ctg gag ttt 113 Met Ala Glu Leu Glu Phe 1 5 gtt cag atc atc
atc atc gtg gtg gtg atg atg gtg atg gtg gtg gtg 161 Val Gln Ile Ile
Ile Ile Val Val Val Met Met Val Met Val Val Val 10 15 20 atc acg
tgc ctg ctg agc cac tac aag ctg tct gca cgg tcc ttc atc 209 Ile Thr
Cys Leu Leu Ser His Tyr Lys Leu Ser Ala Arg Ser Phe Ile 25 30 35
agc cgg cac agc cag ggg cgg agg aga gaa gat gcc ctg tcc tca gaa 257
Ser Arg His Ser Gln Gly Arg Arg Arg Glu Asp Ala Leu Ser Ser Glu 40
45 50 gga tgc ctg tgg ccc tcg gag agc aca gtg tca ggc aac gga atc
cca 305 Gly Cys Leu Trp Pro Ser Glu Ser Thr Val Ser Gly Asn Gly Ile
Pro 55 60 65 70 gag ccg cag gtc tac gcc ccg cct cgg ccc acc gac cgc
ctg gcc gtg 353 Glu Pro Gln Val Tyr Ala Pro Pro Arg Pro Thr Asp Arg
Leu Ala Val 75 80 85 ccg ccc ttc gcc cag cgg gag cgc ttc cac cgc
ttc cag ccc acc tat 401 Pro Pro Phe Ala Gln Arg Glu Arg Phe His Arg
Phe Gln Pro Thr Tyr 90 95 100 ccg tac ctg cag cac gag atc gac ctg
cca ccc acc atc tcg ctg tca 449 Pro Tyr Leu Gln His Glu Ile Asp Leu
Pro Pro Thr Ile Ser Leu Ser 105 110 115 gac ggg gag gag ccc cca ccc
tac cag ggc ccc tgc acc ctc cag ctt 497 Asp Gly Glu Glu Pro Pro Pro
Tyr Gln Gly Pro Cys Thr Leu Gln Leu 120 125 130 cgg gac ccc gag cag
cag ctg gaa ctg aac cgg gag tcg gtg cgc gca 545 Arg Asp Pro Glu Gln
Gln Leu Glu Leu Asn Arg Glu Ser Val Arg Ala 135 140 145 150 ccc cca
aac aga acc atc ttc gac agt gac ctg atg gat agt gcc agg 593 Pro Pro
Asn Arg Thr Ile Phe Asp Ser Asp Leu Met Asp Ser Ala Arg 155 160 165
ctg ggc ggc ccc tgc ccc ccc agc agt aac tcg ggc atc agc gcc acg 641
Leu Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly Ile Ser Ala Thr 170
175 180 tgc tac ggc agc ggc ggg cgc atg gag ggg ccg ccg ccc acc tac
agc 689 Cys Tyr Gly Ser Gly Gly Arg Met Glu Gly Pro Pro Pro Thr Tyr
Ser 185 190 195 gag gtc atc ggc cac tac ccg ggg tcc tcc ttc cag cac
cag cag agc 737 Glu Val Ile Gly His Tyr Pro Gly Ser Ser Phe Gln His
Gln Gln Ser 200 205 210 agt ggg ccg ccc tcc ttg ctg gag ggg acc cgg
ctc cac cac aca cac 785 Ser Gly Pro Pro Ser Leu Leu Glu Gly Thr Arg
Leu His His Thr His 215 220 225 230 atc gcg ccc cta gag agc gca gcc
atc tgg agc aaa gag aag gat aaa 833 Ile Ala Pro Leu Glu Ser Ala Ala
Ile Trp Ser Lys Glu Lys Asp Lys 235 240 245 cag aaa gga cac cct ctc
tagggtcccc aggggggccg ggctggggct 881 Gln Lys Gly His Pro Leu 250
gcgtaggtga aaaggcagaa cactccgcgc ttcttagaag aggagtgaga ggaaggcggg
941 gggcgcagca acgcatcgtg tggccctccc ctcccacctc cctgtgtata
aatatttaca 1001 tgtgatgtct ggtctgaatg cacaagctaa gagagcttgc
aaaaaaaaaa agaaaaaaga 1061 aaaaaaaaaa ccacgtttct ttgttgagct
gtgtcttgaa ggcaaaagaa aaaaaatttc 1121 tacagtagtc tttcttgttt
ctagttgagc tgcgtgcgtg aatgcttatt ttcttttgtt 1181 tatgataatt
tcacttaact ttaaagacat atttgcacaa aacctttgtt taaagatctg 1241
caatattata tatataaata tatataagat aagagaaact gtatgtgcga gggcaggagt
1301 atttttgtat tagaagaggc ctattaaaaa aaaaagttgt tttctgaact
agaagaggaa 1361 aaaaatggca atttttgagt gccaagtcag aaagtgtgta
ttaccttgta aagaaaaaaa 1421 ttacaaagca ggggtttaga gttatttata
taaatgttga gattttgcac tattttttaa 1481 tataaatatg tcagtgcttg
cttgatggaa acttctcttg tgtctgttga gactttaagg 1541 gagaaatgtc
ggaatttcag agtcgcctga cggcagaggg tgagcccccg tggagtctgc 1601
agagaggcct tggccaggag cggcgggctt tcccgagggg ccactgtccc tgcagagtgg
1661 atgcttctgc ctagtgacag gttatcacca cgttatatat tccctaccga
aggagacacc 1721 ttttcccccc tgacccagaa cagcctttaa atcacaagca
aaataggaaa gttaaccacg 1781 gaggcaccga gttccaggta gtggttttgc
ctttcccaaa aatgaaaata aactgttacc 1841 gaaggaatta gtttttcctc
ttcttttttc caactgtgaa ggtccccgtg gggtggagca 1901 tggtgcccct
cacaagccgc agcggctggt gcccgggcta ccagggacat gccagagggc 1961
tcgatgactt gtctctgcag ggcgctttgg tggttgttca gctggctaaa ggttcaccgg
2021 tgaaggcagg tgcggtaact gccgcactgg accctaggaa gccccaggta
ttcgcaatct 2081 gacctcctcc tgtctgtttc ccttcacgga tcaattctca
cttaagaggc caataaacaa 2141 cccaacatga aaaggtgaca agcctgggtt
tctcccagga taggtgaaag ggttaaaatg 2201 agtaaagcag ttgagcaaac
accaacccga gcttcgggcg cagaattctt caccttctct 2261 tcccctttcc
atctcctttc cccgcggaaa caacgcttcc cttctggtgt gtctgttgat 2321
ctgtgttttc atttacatct ctcttagact ccgctcttgt tctccaggtt ttcaccagat
2381 agatttgggg ttggcgggac ctgctggtga cgtgcaggtg aaggacagga
aggggcatgt 2441 gagcgtaaat agaggtgacc agaggagagc atgaggggtg
gggctttggg acccaccggg 2501 gccagtggct ggagcttgac gtctttcctc
cccatggggg tgggagggcc cccagctgga 2561 agagcagact cccagctgct
accccctccc ttcccatggg agtggctttc cattttgggc 2621 agaatgctga
ctagtagact aacataaaag atataaaagg caataactat tgtttgtgag 2681
caactttttt ataacttcca aaacaaaaac ctgagcacag ttttgaagtt ctagccactc
2741 gagctcatgc atgtgaaacg tgtgctttac gaaggtggca gctgacagac
gtgggctctg 2801 catgccgcca gcctagtaga aagttctcgt tcattggcaa
cagcagaacc tgcctctccg 2861 tgaagtcgtc agcctaaaat ttgtttctct
cttgaagagg attctttgaa aaggtcctgc 2921 agagaaatca gtacaggtta
tcccgaaagg tacaaggacg cacttgtaaa gatgattaaa 2981 acgtatcttt
cctttatgtg acgcgtctct agtgccttac tgaagaagca gtgacactcc 3041
cgtcgctcgg tgaggacgtt cccggacagt gcctcactca cctgggactg gtatcccctc
3101 ccagggtcca ccaagggctc ctgcttttca gacaccccat catcctcgcg
cgtcctcacc 3161 ctgtctctac cagggaggtg cctagcttgg tgaggttact
cctgctcctc caaccttttt 3221 ttgccaaggt ttgtacacga ctcccatcta
ggctgaaaac ctagaagtgg accttgtgtg 3281 tgtgcatggt gtcagcccaa
agccaggctg agacagtcct catatcctct tgagccaaac 3341 tgtttgggtc
tcgttgcttc atggtatggt ctggatttgt gggaatggct ttgcgtgaga 3401
aaggggagga gagtggttgc tgccctcagc cggcttgagg acagagcctg tccctctcat
3461 gacaactcag tgttgaagcc cagtgtcctc agcttcatgt ccagtggatg
gcagaagttc 3521 atggggtagt ggcctctcaa aggctgggcg catcccaaga
cagccagcag gttgtctctg 3581 gaaacgacca gagttaagct ctcggcttct
ctgctgaggg tgcacccttt cctctagatg 3641 gtagttgtca cgttatcttt
gaaaactctt ggactgctcc tgaggaggcc ctcttttcca 3701 gtaggaagtt
agatgggggt tctcagaagt ggctgattgg aaggggacaa gcttcgtttc 3761
aggggtctgc cgttccatcc tggttcagag aaggccgagc gtggctttct ctagccttgt
3821 cactgtctcc ctgcctgtca atcaccacct ttccyccaga ggaggaaaat
tatctcccct 3881 gcaaagcccg gttctacaca gatttcacaa attgtgctaa
gaaccgtccg tgttctcaga 3941 aagcccagtg tttttgcaaa gaatgaaaag
ggaccccata tgtagcaaaa atcagggctg 4001 ggggagagcc gggttcattc
cctgtcctca ttggtcgtcc ctatgaattg tacgtttcag 4061 agaaattttt
tttcctatgt gcaacacgaa gcttccagaa ccataaaata tcccgtcgat 4121
aaggaaagaa aatgtcgttg ttgttgtttt tctggaaact gcttgaaatc ttgctgtact
4181 atagagctca gaaggacaca gcccgtcctc ccctgcctgc ctgattccat
ggctgttgtg 4241 ctgattccaa tgctttcacg ttggttcctg gcgtgggaac
tgctctcctt tgcagcccca 4301 tttcccaagc tctgttcaag ttaaacttat
gtaagctttc cgtggcatgc ggggcgcgca 4361 cccacgtccc cgctgcgtaa
gactctgtat ttggatgcca atccacaggc ctgaagaaac 4421 tgcttgttgt
gtatcagtaa tcattagtgg caatgatgac attctgaaaa gctgcaatac 4481
ttatacaata aattttacaa ttctttggaa aaaaaaaaaa aaaaaa 4527 3 252 PRT
Homo sapiens 3 Met Ala Glu Leu Glu Phe Val Gln Ile Ile Ile Ile Val
Val Val Met 1 5 10 15 Met Val Met Val Val Val Ile Thr Cys Leu Leu
Ser His Tyr Lys Leu 20 25 30 Ser Ala Arg Ser Phe Ile Ser Arg His
Ser Gln Gly Arg Arg Arg Glu 35 40 45 Asp Ala Leu Ser Ser Glu Gly
Cys Leu Trp Pro Ser Glu Ser Thr Val 50 55 60 Ser Gly Asn Gly Ile
Pro Glu Pro Gln Val Tyr Ala Pro Pro Arg Pro 65 70 75 80 Thr Asp Arg
Leu Ala Val Pro Pro Phe Ala Gln Arg Glu Arg Phe His 85 90 95 Arg
Phe Gln Pro Thr Tyr Pro Tyr Leu Gln His Glu Ile Asp Leu Pro 100 105
110 Pro Thr Ile Ser Leu Ser Asp Gly Glu Glu Pro Pro Pro Tyr Gln Gly
115 120 125 Pro Cys Thr Leu Gln Leu Arg Asp Pro Glu Gln Gln Leu Glu
Leu Asn 130 135 140 Arg Glu Ser Val Arg Ala Pro Pro Asn Arg Thr Ile
Phe Asp Ser Asp 145 150 155 160 Leu Met Asp Ser Ala Arg Leu Gly Gly
Pro Cys Pro Pro Ser Ser Asn 165 170 175 Ser Gly Ile Ser Ala Thr Cys
Tyr Gly Ser Gly Gly Arg Met Glu Gly 180 185 190 Pro Pro Pro Thr Tyr
Ser Glu Val Ile Gly His Tyr Pro Gly Ser Ser 195 200 205 Phe Gln His
Gln Gln Ser Ser Gly Pro Pro Ser Leu Leu Glu Gly Thr 210 215 220 Arg
Leu His His Thr His Ile Ala Pro Leu Glu Ser Ala Ala Ile Trp 225 230
235 240 Ser Lys Glu Lys Asp Lys Gln Lys Gly His Pro Leu 245 250 4
2213 DNA Homo sapiens CDS (1)...(1611) misc_feature (1)...(2213)
Xaa=any amino acid 4 ggg ggg ctg aca aca act gtg ata ggt acg agg
ctg ggt gtg gat cgg 48 Gly Gly Leu Thr Thr Thr Val Ile Gly Thr Arg
Leu Gly Val Asp Arg 1 5 10 15 ccg agg ctc tcc tgg agc gct ggg cct
tcg ctg gcc gca ccg gca gcc 96 Pro Arg Leu Ser Trp Ser Ala Gly Pro
Ser Leu Ala Ala Pro Ala Ala 20 25 30 atg agc tcg gag atg gag ccg
ctg ctc ctg gcc tgg agc tat ttt agg 144 Met Ser Ser Glu Met Glu Pro
Leu Leu Leu Ala Trp Ser Tyr Phe Arg 35 40 45 cgc agg aag ttc cag
ctc tgc gcc gat cta tgc acg cag atg ctg gag 192 Arg Arg Lys Phe Gln
Leu Cys Ala Asp Leu Cys Thr Gln Met Leu Glu 50 55 60 aag tcc cct
tat gac cag gca gct tgg atc tta aaa gca aga gcg cta 240 Lys Ser Pro
Tyr Asp Gln Ala Ala Trp Ile Leu Lys Ala Arg Ala Leu 65 70 75 80 aca
gaa atg gta tac ata gat gaa att gat gta gat cag gaa gga att 288 Thr
Glu Met Val Tyr Ile Asp Glu Ile Asp Val Asp Gln Glu Gly Ile 85 90
95 gca gaa atg atg ctg gat gaa aat gct ata gct caa gtt cca cgc cct
336 Ala Glu Met Met Leu Asp Glu Asn Ala Ile Ala Gln Val Pro Arg Pro
100 105 110 gga acg tct ttg aaa ctc cct gga act aat cag aca gga ggg
cct agc 384 Gly Thr Ser Leu Lys Leu Pro Gly Thr Asn Gln Thr Gly Gly
Pro Ser 115 120 125 cag gcc gtt agg cca atc aca caa gct gga aga ccc
att aca ggt ttc 432 Gln Ala Val Arg Pro Ile Thr Gln Ala Gly Arg Pro
Ile Thr Gly Phe 130 135 140 ctc agg ccc agc acg cag agt gga agg cca
ggc act atg gaa cag gct 480 Leu Arg Pro Ser Thr Gln Ser Gly Arg Pro
Gly Thr Met Glu Gln Ala 145 150 155 160 atc aga aca ccc aga acc gcc
tac aca gcc cgc cct atc acc agc tcc 528 Ile Arg Thr Pro Arg Thr Ala
Tyr Thr Ala Arg Pro Ile Thr Ser Ser 165 170 175 tcc gga aga ttt gtc
agg ctg gga acg gct tcc atg ctt aca agt cct 576 Ser Gly Arg Phe Val
Arg Leu Gly Thr Ala Ser Met Leu Thr Ser Pro 180 185 190 gat gga cca
ttt ata aat tta tct agg ctg aat tta aca aag tat tcc 624 Asp Gly Pro
Phe Ile Asn Leu Ser Arg Leu Asn Leu Thr Lys Tyr Ser 195 200 205 cag
aaa cct aag ttg gca aag gct tgt ttg agt ata tct ttc atc atg 672 Gln
Lys Pro Lys Leu Ala Lys Ala Cys Leu Ser Ile Ser Phe Ile Met 210 215
220 aaa atg atg tta aga ctg ctt tgg atc tgg ctg gcc ctc tcc aca gaa
720 Lys Met Met Leu Arg Leu Leu Trp Ile Trp Leu Ala Leu Ser Thr Glu
225 230 235 240 cat tct cag tac aag gac tgg tgg tgg aaa gta cag att
gga aaa tgt 768 His Ser Gln Tyr Lys Asp Trp Trp Trp Lys Val Gln Ile
Gly Lys Cys 245 250 255 tac tac agg ttg gga atg tat cgt gaa gca gaa
aaa cag ttt aaa tca 816 Tyr Tyr Arg Leu Gly Met Tyr Arg Glu Ala Glu
Lys Gln Phe Lys Ser 260 265 270 gcc ctg aag cag cag gaa atg gta gat
aca ttt ctg tac ttg gca aaa 864 Ala Leu Lys Gln Gln Glu Met Val Asp
Thr Phe Leu Tyr Leu Ala Lys 275 280 285 gtt tat gtc tca ttg gat caa
cct gtg act gct tta aat ctt ttc aaa 912 Val Tyr Val Ser Leu Asp Gln
Pro Val Thr Ala Leu Asn Leu Phe Lys 290 295 300 caa ggc tta gat aag
ttt cca gga gaa gta acc ctg ctc tgt gga att 960 Gln Gly Leu Asp Lys
Phe Pro Gly Glu Val Thr Leu Leu Cys Gly Ile 305 310 315 320 gca aga
atc tat gag gaa atg aac aat atg tca tca gca gca gaa tat 1008 Ala
Arg Ile Tyr Glu Glu Met Asn Asn Met Ser Ser Ala Ala Glu Tyr 325 330
335 tac aaa gaa gtt ttg aaa caa gac aat act cat gtg gra gcc atc gca
1056 Tyr Lys Glu Val Leu Lys Gln Asp Asn Thr His Val Xaa Ala Ile
Ala 340 345 350 tgc att gga agc aac cac ttc tat tct gat cag cca gaa
ata gct ctc 1104 Cys Ile Gly Ser Asn His Phe Tyr Ser Asp Gln Pro
Glu Ile Ala Leu 355 360 365 cgg ttt tac agg cgg ctg ctg cag atg ggc
att tat aac ggc cag ctt 1152 Arg Phe Tyr Arg Arg Leu Leu Gln Met
Gly Ile Tyr Asn Gly Gln Leu 370 375 380 ttt aac aat ctg ggg ctg tgt
tgc ttc tat gcc cag cag tat gat atg 1200 Phe Asn Asn Leu Gly Leu
Cys Cys Phe Tyr Ala Gln Gln Tyr Asp Met 385 390 395 400 act ctg acc
tca ttt gaa cgt gcc ctt tct ttg gct gaa aat gaa gaa 1248 Thr Leu
Thr Ser Phe Glu Arg Ala Leu Ser Leu Ala Glu Asn Glu Glu 405 410 415
gag gca gct gat gtc tgg tac aac ttg gga cat gta gct gtg gga ata
1296 Glu Ala Ala Asp Val Trp Tyr Asn Leu Gly His Val Ala Val Gly
Ile 420 425 430 gga gat aca aat ttg gcc cat cag tgc ttc agg ctg gct
ctg gtc aac 1344 Gly Asp Thr Asn Leu Ala His Gln Cys Phe Arg Leu
Ala Leu Val Asn 435 440 445 aac aac aac cac gcc gag gcc tac aac aac
ctg gct gtg ctg gag atg 1392 Asn Asn Asn His Ala Glu Ala Tyr Asn
Asn Leu Ala Val Leu Glu Met 450 455 460 cgg aag ggc cac gtt gaa cag
gca agg gca cta tta caa act gca tca 1440 Arg Lys Gly His Val Glu
Gln Ala Arg Ala Leu Leu Gln Thr Ala Ser 465 470 475 480 tca tta gca
ccc cat atg tat gaa ccg cat ttt aat ttt gca aca atc 1488 Ser Leu
Ala Pro His Met Tyr Glu Pro His Phe Asn Phe Ala Thr Ile 485 490 495
tct gat aag att gga gat ctg cag aga agc tat gtt gct gcg cag aag
1536 Ser Asp Lys Ile Gly Asp Leu Gln Arg Ser Tyr Val Ala Ala Gln
Lys 500 505 510 tct gaa gca gca ttt cca gac cat gtg gac aca caa cat
tta att aaa 1584 Ser Glu Ala Ala Phe Pro Asp His Val Asp Thr Gln
His Leu Ile Lys 515 520 525 caa tta agg cag cat ttt gct atg ctc
tgattgttcc ttagaccaca 1631 Gln Leu Arg Gln His Phe Ala Met Leu 530
535 tatgttctta tgaagcagca ttatgcaagg ggaaaaaagc actatgtctg
tgtatgtatg 1691 tatatagtgt aatacgtata ttttaacaaa cctgtccttg
atattagtta aggtgacaca 1751 taagggtgac acagaatgtg taatgcaaat
ttcatagtaa tagtaacttt ataaaataat 1811 attataaaat acaggattta
aacctttcta aatagatcct gaaactgtct ctcacattat 1871 atagtagatg
tttgtttata atgtttacaa aacattttgg tgaatttcct caatgtttta 1931
taaatgtaca ttttttaagt ccttaagctg actcttagcc atcatgtagc ttaaggagtc
1991 tgaaatctgc cattaaaact gcacctttaa gccaggtgtg gtagcatgtg
cctatagtcc 2051 cagctacttg ggaggtggag gtgggaggat tataaataga
gactttcctt aagactttaa 2111 aaatgtattt aaaactattt tttattaaat
actttgtgat ttcctattaa gctttaaaat 2171 aaatcattgt gtaaaacacc
atcaaagcga taagctctgt aa 2213
5 537 PRT Homo sapiens VARIANT (1)...(537) Xaa = Any Amino Acid 5
Gly Gly Leu Thr Thr Thr Val Ile Gly Thr Arg Leu Gly Val Asp Arg 1 5
10 15 Pro Arg Leu Ser Trp Ser Ala Gly Pro Ser Leu Ala Ala Pro Ala
Ala 20 25 30 Met Ser Ser Glu Met Glu Pro Leu Leu Leu Ala Trp Ser
Tyr Phe Arg 35 40 45 Arg Arg Lys Phe Gln Leu Cys Ala Asp Leu Cys
Thr Gln Met Leu Glu 50 55 60 Lys Ser Pro Tyr Asp Gln Ala Ala Trp
Ile Leu Lys Ala Arg Ala Leu 65 70 75 80 Thr Glu Met Val Tyr Ile Asp
Glu Ile Asp Val Asp Gln Glu Gly Ile 85 90 95 Ala Glu Met Met Leu
Asp Glu Asn Ala Ile Ala Gln Val Pro Arg Pro 100 105 110 Gly Thr Ser
Leu Lys Leu Pro Gly Thr Asn Gln Thr Gly Gly Pro Ser 115 120 125 Gln
Ala Val Arg Pro Ile Thr Gln Ala Gly Arg Pro Ile Thr Gly Phe 130 135
140 Leu Arg Pro Ser Thr Gln Ser Gly Arg Pro Gly Thr Met Glu Gln Ala
145 150 155 160 Ile Arg Thr Pro Arg Thr Ala Tyr Thr Ala Arg Pro Ile
Thr Ser Ser 165 170 175 Ser Gly Arg Phe Val Arg Leu Gly Thr Ala Ser
Met Leu Thr Ser Pro 180 185 190 Asp Gly Pro Phe Ile Asn Leu Ser Arg
Leu Asn Leu Thr Lys Tyr Ser 195 200 205 Gln Lys Pro Lys Leu Ala Lys
Ala Cys Leu Ser Ile Ser Phe Ile Met 210 215 220 Lys Met Met Leu Arg
Leu Leu Trp Ile Trp Leu Ala Leu Ser Thr Glu 225 230 235 240 His Ser
Gln Tyr Lys Asp Trp Trp Trp Lys Val Gln Ile Gly Lys Cys 245 250 255
Tyr Tyr Arg Leu Gly Met Tyr Arg Glu Ala Glu Lys Gln Phe Lys Ser 260
265 270 Ala Leu Lys Gln Gln Glu Met Val Asp Thr Phe Leu Tyr Leu Ala
Lys 275 280 285 Val Tyr Val Ser Leu Asp Gln Pro Val Thr Ala Leu Asn
Leu Phe Lys 290 295 300 Gln Gly Leu Asp Lys Phe Pro Gly Glu Val Thr
Leu Leu Cys Gly Ile 305 310 315 320 Ala Arg Ile Tyr Glu Glu Met Asn
Asn Met Ser Ser Ala Ala Glu Tyr 325 330 335 Tyr Lys Glu Val Leu Lys
Gln Asp Asn Thr His Val Xaa Ala Ile Ala 340 345 350 Cys Ile Gly Ser
Asn His Phe Tyr Ser Asp Gln Pro Glu Ile Ala Leu 355 360 365 Arg Phe
Tyr Arg Arg Leu Leu Gln Met Gly Ile Tyr Asn Gly Gln Leu 370 375 380
Phe Asn Asn Leu Gly Leu Cys Cys Phe Tyr Ala Gln Gln Tyr Asp Met 385
390 395 400 Thr Leu Thr Ser Phe Glu Arg Ala Leu Ser Leu Ala Glu Asn
Glu Glu 405 410 415 Glu Ala Ala Asp Val Trp Tyr Asn Leu Gly His Val
Ala Val Gly Ile 420 425 430 Gly Asp Thr Asn Leu Ala His Gln Cys Phe
Arg Leu Ala Leu Val Asn 435 440 445 Asn Asn Asn His Ala Glu Ala Tyr
Asn Asn Leu Ala Val Leu Glu Met 450 455 460 Arg Lys Gly His Val Glu
Gln Ala Arg Ala Leu Leu Gln Thr Ala Ser 465 470 475 480 Ser Leu Ala
Pro His Met Tyr Glu Pro His Phe Asn Phe Ala Thr Ile 485 490 495 Ser
Asp Lys Ile Gly Asp Leu Gln Arg Ser Tyr Val Ala Ala Gln Lys 500 505
510 Ser Glu Ala Ala Phe Pro Asp His Val Asp Thr Gln His Leu Ile Lys
515 520 525 Gln Leu Arg Gln His Phe Ala Met Leu 530 535 6 4433 DNA
Homo sapiens CDS (1)...(423) 6 ata gga gtg gag aac atg cac aat tac
tgc ttt gtg ttt gct ctg gga 48 Ile Gly Val Glu Asn Met His Asn Tyr
Cys Phe Val Phe Ala Leu Gly 1 5 10 15 tac ctc aca gtg tgc caa gtt
act cga gtc tat atc ttt gac tat gga 96 Tyr Leu Thr Val Cys Gln Val
Thr Arg Val Tyr Ile Phe Asp Tyr Gly 20 25 30 caa tat tct gct gat
ttt tca ggc cca atg atg atc att act cag aag 144 Gln Tyr Ser Ala Asp
Phe Ser Gly Pro Met Met Ile Ile Thr Gln Lys 35 40 45 atc act agt
ttg gct tgc gaa ata cat gat ggg atg ttt cgg aag gat 192 Ile Thr Ser
Leu Ala Cys Glu Ile His Asp Gly Met Phe Arg Lys Asp 50 55 60 gaa
gaa ctg act tcc tca cag agg gat tta gct gta agg cgc atg cca 240 Glu
Glu Leu Thr Ser Ser Gln Arg Asp Leu Ala Val Arg Arg Met Pro 65 70
75 80 agc tta ctg gag tat ttg agt tac aac tgt aac ttc atg ggg atc
ctg 288 Ser Leu Leu Glu Tyr Leu Ser Tyr Asn Cys Asn Phe Met Gly Ile
Leu 85 90 95 gca ggc cca ctt tgc tct tac aaa gac tac att act ttc
att gaa ggc 336 Ala Gly Pro Leu Cys Ser Tyr Lys Asp Tyr Ile Thr Phe
Ile Glu Gly 100 105 110 aga tca tac cat atc aca caa tct ggt gaa aat
gga aaa gaa gag aca 384 Arg Ser Tyr His Ile Thr Gln Ser Gly Glu Asn
Gly Lys Glu Glu Thr 115 120 125 cag tat gaa aga aca gag cca tct cca
aat gta agg tca tgagatttat 433 Gln Tyr Glu Arg Thr Glu Pro Ser Pro
Asn Val Arg Ser 130 135 140 ctggagcctt tacagcatgt attgactgcg
gktgttcaga agctcttagt ttgtgggctg 493 tccttgttat ttcacttgac
catctgtaca acattacctg tggagtacaa cattgatgag 553 cattttcaag
ctacagcttc gtggccaaca aagattatct atctgtatat ctctcttttg 613
gctgccagac ccaaatacta ttttgcatgg acgctagctg actgccatwa ataatgctgc
673 aggctttggt ttcagagggt atgacgaaaa tggagcagct cgctgggact
taatttccaa 733 tttgagaatt caacaaatag agatgtcaac aagtttcaag
atgtttcttg ataattggaa 793 tattcagaca gctctttggc tcaaaaggtg
cgttccttca aaaacgatct ttagatgtgc 853 tttggcgtct agttctcgag
gttgagcttc attgagttca ggttcttgat taaattaacg 913 gtgttgagtg
acattgtgac ctcagtgtca gccgggaaac actgttagcc tcctcctaag 973
caagtcagta tcgaatgaga actattttgg cttgagtcac gaatgcagct atcctgcagg
1033 tgcagctatc ctgccctctc aagcctcctt taaaggcctc tgccaatgtc
agaggtcacc 1093 agtatcctcc tttgcagctc ctgattgtgt tcagtagaga
tgtggtttaa attaacaagt 1153 gcctgcacaa gcacagtact tatgcctggg
tactccagaa cagtcctggt tttaaatatt 1213 tcaattcaac aaatcttkat
ttgttaggca agggaaacaa acatgagtaa gataaaaaga 1273 ctcagctcct
gaaagtgaaa gagttcacaa ttttattaaa gacacggtgg tgtaatcaga 1333
cacatgctgt tccctgtggt gaggatgagg agagagaaag caggaacagc gagggcacag
1393 agggatgcgg gaagaacttc ctacaagtgt gggtgcttga gctgaggttt
gtgtcaggag 1453 cgtgtctcgt gaacagggca aggtagaggc aagccaggct
gggtggagta acaggtgcga 1513 aggacagagc tggggaacag cacactctcc
caggggttct cttatcgtcc ctgtgagcac 1573 attgccctat cttgaattta
cttcataaaa aacggcccct ataacgatac ggtgataagc 1633 agcctttttt
tatagtgtcc ttttttaaat gacaaattaa acatctttat cccttgagat 1693
ggctagcata cgctgtcatc tcttcacagt gcctggcagt ctccccagtg gctgcagatc
1753 ctctgagcta atctgttgtg ttattttttg ttattgttat aatttaaatt
tgatacctta 1813 ggggaaactt tattttcagc tgagttctct atccctgtca
tagaagaatt gtagactaag 1873 cacagtctat ctgccggaag gagtagtgtt
attaggtcag ttgaaagtta ttgatttttt 1933 ttaaataaaa taatgtagga
taaaagcaac cttactcttt ttgtaaattg tatagactcc 1993 caaatactag
aaatgatcat ttaagttact atatatacca atatatatac tatatatacc 2053
aataagaaga tgagaattaa ctttatgttc ctaaatttga cacttaatag ctatagcctc
2113 cctgagatca tagagaagtg attgcctaag ataagttgta tttgtttttc
tagttaccct 2173 aaatcctgtc aggtaataaa agaatgatca ttgcaggctt
tgtaaactcg ggtcactcac 2233 tccacttggc tctccatgtt tttcatggtt
tctagggtgt gttatgaacg aacctccttc 2293 agtccaacta tccagacgtt
cattctctct gccatttggc acggggtata cccaggatat 2353 tatctaacgt
ttctaacagg ggtgttaatg acattagcag caagagctgt aagtatcaag 2413
aattttattt tacaattcaa tggtccactt gaactgttaa aaaggctgag tacatctctc
2473 ttacaaggta gaccctcttt ccttggtcgt ggtcagtatt gtcctttcca
ctagaagcga 2533 ggtgtgtact gcgtgcatgt ttgctgagcg ctcaccacgg
gctaggctcc atgcccagtt 2593 cctgtgagga gaaaacacgt ttctatgtgc
ccggcaggta ggaggcactc acaaaatgtt 2653 actttgtctt tacagaattt
tctgaaggag agataaaaac tgagttaaat aaagatgatc 2713 agaatggatg
agaaataact ttagacatta tttcattgaa ccttcccaac tgaaattatt 2773
ttatgatgtt ataacatgga tagtaactca agtagcaata agttacacag ttgtgccatt
2833 tgtgcttctt tctataaaac catcactcac gttttacagc tcctggtatt
attgcctgca 2893 cattcttggt atcttagtat tattgttgtt gccagtgaaa
aaaactcaaa gaagaaagaa 2953 tacacatgaa aacattcagc tctcacaatc
caaaaagttt gatgaaggag aaaattcttt 3013 gggacagaac agtttttcta
caacaaacaa tgtttgcaat cagaatcaag aaatagcctc 3073 gagacattca
tcactaaagc agtgatcggg aaggctctga gggctgtttt ttttttttga 3133
tgttaacaga aaccaatctt agcacctttt caaggggttt gagtttgttg gaaaagcagt
3193 taactggggg gaaatggaca gttatagata aggaatttcc tgtacaccag
attggaaatg 3253 gagtgaaaca agccctccca tgccatgtcc ccgtgggcca
cgccttatgt aagaatattt 3313 ccatatttca gtgggcactc ccaacctcag
cacttgtccg tagggtcaca cgcgtgccct 3373 gttgctgaat gtatgttgcg
tatcccaagg cactgaagag gtggaaaaat aatcgtgtca 3433 atctggatga
tagagagaaa ttaacttttc caaatgaatg tcttgcctta aaccctctat 3493
ttcctaaaat attgttccta aatggtattt tcaagtgtaa tattgtgaga acgctactgc
3553 agtagttgat gttgtgtgct gtaaaggatt ttaggaggaa tttgaaacag
gatatttaag 3613 agtgtggata tttttaaaat gcaataaaca tctcagtatt
tgaagggttt tcttaaagta 3673 tgtcaaatga ctacaatcca tagtgaaact
gtaaacagta atggacgcca aattataggt 3733 agctgatttt gctggagagt
ttaattacct tgtgcagtca aagagcgctt ccagaaggaa 3793 tctcttaaaa
cataatgaga ggtttggtaa tgtgatattt taagcttatt ctttttctta 3853
aaagagagag gtgacgaagg aaggcaggaa tgaagaagca ctgcgtggcc tccggtggaa
3913 tgcacggggc acagccgcga ctctgcaggc agcttccccc ccatgcccag
ggctctgcgc 3973 cgtcatgtga gacttaaaaa aaaagttgaa tgacttcgtg
atactttgga cttctaaatt 4033 aaatttatca ggcataaatt atgtagaatt
agaggctttg aaaataatac tggtaggttg 4093 ctcaaaggtt ttgaaagaga
aatcgctagg taggttacta tctggctaat ccatttctta 4153 tccttgacaa
tttaattcat atttgggaaa cttttaggga aatgaaaaat aaaagtcact 4213
gagtctgggt gacatttttt aagaataata taaattcagt ttcaaactct tctcacatta
4273 aaattttgct gtgaactctt actaaaatga gttttaggtt ctgtaagtgg
aaaaatgtgc 4333 ttttatttta tgggccattt ttaccacaac taatcttgcc
ttggattact aagcatctcc 4393 tgcgatccca cagaggactg tggtggccac
aggagctgaa 4433 7 141 PRT Homo sapiens 7 Ile Gly Val Glu Asn Met
His Asn Tyr Cys Phe Val Phe Ala Leu Gly 1 5 10 15 Tyr Leu Thr Val
Cys Gln Val Thr Arg Val Tyr Ile Phe Asp Tyr Gly 20 25 30 Gln Tyr
Ser Ala Asp Phe Ser Gly Pro Met Met Ile Ile Thr Gln Lys 35 40 45
Ile Thr Ser Leu Ala Cys Glu Ile His Asp Gly Met Phe Arg Lys Asp 50
55 60 Glu Glu Leu Thr Ser Ser Gln Arg Asp Leu Ala Val Arg Arg Met
Pro 65 70 75 80 Ser Leu Leu Glu Tyr Leu Ser Tyr Asn Cys Asn Phe Met
Gly Ile Leu 85 90 95 Ala Gly Pro Leu Cys Ser Tyr Lys Asp Tyr Ile
Thr Phe Ile Glu Gly 100 105 110 Arg Ser Tyr His Ile Thr Gln Ser Gly
Glu Asn Gly Lys Glu Glu Thr 115 120 125 Gln Tyr Glu Arg Thr Glu Pro
Ser Pro Asn Val Arg Ser 130 135 140 8 1276 DNA Homo sapiens CDS
(1)...(1275) misc_feature (1)...(1276) n = A,T,C or G 8 ata gga gtg
gag aac atg cac aat tac tgc ttt gtg ttt gct ctg gga 48 Ile Gly Val
Glu Asn Met His Asn Tyr Cys Phe Val Phe Ala Leu Gly 1 5 10 15 tac
ctc aca gtg tgc caa gtt act cga gtc tat atc ttt gac tat gga 96 Tyr
Leu Thr Val Cys Gln Val Thr Arg Val Tyr Ile Phe Asp Tyr Gly 20 25
30 caa tat tct gct gat ttt tca ggc cca atg atg atc att act cag aag
144 Gln Tyr Ser Ala Asp Phe Ser Gly Pro Met Met Ile Ile Thr Gln Lys
35 40 45 atc act agt ttg gct tgc gaa ata cat gat ggg atg ttt cgg
aag gat 192 Ile Thr Ser Leu Ala Cys Glu Ile His Asp Gly Met Phe Arg
Lys Asp 50 55 60 gaa gaa ctg act tcc tca cag agg gat tta gct gta
agg cgc atg cca 240 Glu Glu Leu Thr Ser Ser Gln Arg Asp Leu Ala Val
Arg Arg Met Pro 65 70 75 80 agc tta ctg gag tat ttg agt tac aac tgt
aac ttc atg ggg atc ctg 288 Ser Leu Leu Glu Tyr Leu Ser Tyr Asn Cys
Asn Phe Met Gly Ile Leu 85 90 95 gca ggc cca ctt tgc tct tac aaa
gac tac att act ttc att gaa ggc 336 Ala Gly Pro Leu Cys Ser Tyr Lys
Asp Tyr Ile Thr Phe Ile Glu Gly 100 105 110 aga tca tac cat atc aca
caa tct ggt gaa aat gga aaa gaa gag aca 384 Arg Ser Tyr His Ile Thr
Gln Ser Gly Glu Asn Gly Lys Glu Glu Thr 115 120 125 cag tat gaa aga
aca gna gcc atc tcc aaa tgt aag gtc atg aga ttt 432 Gln Tyr Glu Arg
Thr Xaa Ala Ile Ser Lys Cys Lys Val Met Arg Phe 130 135 140 atc tgg
agc ctt tac agc atg tat tgn act gcg gkt gtt cag aag ctc 480 Ile Trp
Ser Leu Tyr Ser Met Tyr Xaa Thr Ala Xaa Val Gln Lys Leu 145 150 155
160 tta gtt tgt ggg ctg tcc ttg tta ttt cac ttg acc atc tgt aca aca
528 Leu Val Cys Gly Leu Ser Leu Leu Phe His Leu Thr Ile Cys Thr Thr
165 170 175 tta cct gtg gag tac aac att gat gag cat ttt caa gct aca
gct tcg 576 Leu Pro Val Glu Tyr Asn Ile Asp Glu His Phe Gln Ala Thr
Ala Ser 180 185 190 tgg cca aca aag att atc tat ctg tat atc tct ctt
ttg gct gcc aga 624 Trp Pro Thr Lys Ile Ile Tyr Leu Tyr Ile Ser Leu
Leu Ala Ala Arg 195 200 205 ccc aaa tac tat ttt gca tgg acg cta gct
gat gcc att aat aat gct 672 Pro Lys Tyr Tyr Phe Ala Trp Thr Leu Ala
Asp Ala Ile Asn Asn Ala 210 215 220 gca ggc ttt ggt ttc aga ggg tat
gac gaa aat gga gca gct cgc tgg 720 Ala Gly Phe Gly Phe Arg Gly Tyr
Asp Glu Asn Gly Ala Ala Arg Trp 225 230 235 240 gac tta att tcc aat
ttg aga att caa caa ata gag atg tca aca agt 768 Asp Leu Ile Ser Asn
Leu Arg Ile Gln Gln Ile Glu Met Ser Thr Ser 245 250 255 ttc aag atg
ttt ctt gat aat tgg aat att cag aca gct ctt tgg ccc 816 Phe Lys Met
Phe Leu Asp Asn Trp Asn Ile Gln Thr Ala Leu Trp Pro 260 265 270 aaa
agg gtg tgt tat gaa cga acc tcc ttc agt cca act atc cag acg 864 Lys
Arg Val Cys Tyr Glu Arg Thr Ser Phe Ser Pro Thr Ile Gln Thr 275 280
285 ttc att ctc cct gcc att ntg gca cgg ggt ata ccc agg ata tta tct
912 Phe Ile Leu Pro Ala Ile Xaa Ala Arg Gly Ile Pro Arg Ile Leu Ser
290 295 300 aac gtt tct aac agg ggt gtt aat gac att agc agc aga gct
atg aga 960 Asn Val Ser Asn Arg Gly Val Asn Asp Ile Ser Ser Arg Ala
Met Arg 305 310 315 320 aat aac ttt aga cat tat ttc att gaa cct tcc
caa ctg aaa tta ttt 1008 Asn Asn Phe Arg His Tyr Phe Ile Glu Pro
Ser Gln Leu Lys Leu Phe 325 330 335 tat gat gtt mta aca tgg ata gta
aac tca agt agc aat aag tta cac 1056 Tyr Asp Val Xaa Thr Trp Ile
Val Asn Ser Ser Ser Asn Lys Leu His 340 345 350 agk tgk gsc att tgt
gct tct ttc tat waa acc atc act cac rkt tya 1104 Xaa Xaa Xaa Ile
Cys Ala Ser Phe Tyr Xaa Thr Ile Thr His Xaa Xaa 355 360 365 cag gtc
cgg ttt att gcc gga cat act ggt tcc tcg ata atg gcg tgc 1152 Gln
Val Arg Phe Ile Ala Gly His Thr Gly Ser Ser Ile Met Ala Cys 370 375
380 cgg aca acg cgg aga aag gta ctg gaa gtt ccg ctc cac caa gtc gtg
1200 Arg Thr Thr Arg Arg Lys Val Leu Glu Val Pro Leu His Gln Val
Val 385 390 395 400 ggg gac act tgg gac agc tct tcc aca agc gcg ccg
aag ccg gac aca 1248 Gly Asp Thr Trp Asp Ser Ser Ser Thr Ser Ala
Pro Lys Pro Asp Thr 405 410 415 acg acg ggg cgg ggg ggt ggg gca acc
c 1276 Thr Thr Gly Arg Gly Gly Gly Ala Thr 420 425 9 425 PRT Homo
sapiens VARIANT (1)...(425) Xaa = Any Amino Acid 9 Ile Gly Val Glu
Asn Met His Asn Tyr Cys Phe Val Phe Ala Leu Gly 1 5 10 15 Tyr Leu
Thr Val Cys Gln Val Thr Arg Val Tyr Ile Phe Asp Tyr Gly 20 25 30
Gln Tyr Ser Ala Asp Phe Ser Gly Pro Met Met Ile Ile Thr Gln Lys 35
40 45 Ile Thr Ser Leu Ala Cys Glu Ile His Asp Gly Met Phe Arg Lys
Asp 50 55 60 Glu Glu Leu Thr Ser Ser Gln Arg Asp Leu Ala Val Arg
Arg Met Pro 65 70 75 80 Ser Leu Leu Glu Tyr Leu Ser Tyr Asn Cys Asn
Phe Met Gly Ile Leu 85 90 95 Ala Gly Pro Leu Cys Ser Tyr Lys Asp
Tyr Ile Thr Phe Ile Glu Gly 100 105 110 Arg Ser Tyr His Ile Thr Gln
Ser Gly Glu Asn Gly Lys Glu Glu Thr 115 120 125 Gln Tyr Glu Arg Thr
Xaa Ala Ile Ser Lys Cys Lys Val Met Arg Phe 130 135 140 Ile Trp Ser
Leu Tyr Ser Met Tyr Xaa Thr Ala Xaa Val Gln Lys Leu 145 150 155
160 Leu Val Cys Gly Leu Ser Leu Leu Phe His Leu Thr Ile Cys Thr Thr
165 170 175 Leu Pro Val Glu Tyr Asn Ile Asp Glu His Phe Gln Ala Thr
Ala Ser 180 185 190 Trp Pro Thr Lys Ile Ile Tyr Leu Tyr Ile Ser Leu
Leu Ala Ala Arg 195 200 205 Pro Lys Tyr Tyr Phe Ala Trp Thr Leu Ala
Asp Ala Ile Asn Asn Ala 210 215 220 Ala Gly Phe Gly Phe Arg Gly Tyr
Asp Glu Asn Gly Ala Ala Arg Trp 225 230 235 240 Asp Leu Ile Ser Asn
Leu Arg Ile Gln Gln Ile Glu Met Ser Thr Ser 245 250 255 Phe Lys Met
Phe Leu Asp Asn Trp Asn Ile Gln Thr Ala Leu Trp Pro 260 265 270 Lys
Arg Val Cys Tyr Glu Arg Thr Ser Phe Ser Pro Thr Ile Gln Thr 275 280
285 Phe Ile Leu Pro Ala Ile Xaa Ala Arg Gly Ile Pro Arg Ile Leu Ser
290 295 300 Asn Val Ser Asn Arg Gly Val Asn Asp Ile Ser Ser Arg Ala
Met Arg 305 310 315 320 Asn Asn Phe Arg His Tyr Phe Ile Glu Pro Ser
Gln Leu Lys Leu Phe 325 330 335 Tyr Asp Val Xaa Thr Trp Ile Val Asn
Ser Ser Ser Asn Lys Leu His 340 345 350 Xaa Xaa Xaa Ile Cys Ala Ser
Phe Tyr Xaa Thr Ile Thr His Xaa Xaa 355 360 365 Gln Val Arg Phe Ile
Ala Gly His Thr Gly Ser Ser Ile Met Ala Cys 370 375 380 Arg Thr Thr
Arg Arg Lys Val Leu Glu Val Pro Leu His Gln Val Val 385 390 395 400
Gly Asp Thr Trp Asp Ser Ser Ser Thr Ser Ala Pro Lys Pro Asp Thr 405
410 415 Thr Thr Gly Arg Gly Gly Gly Ala Thr 420 425 10 29 DNA Homo
sapiens 10 tgaggtatcc cagagcaaac acaaagcag 29 11 28 DNA Homo
sapiens 11 tcagttcttc atccttccga aacatccc 28
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References