U.S. patent application number 11/929712 was filed with the patent office on 2009-11-05 for pancreatic cancer genes.
This patent application is currently assigned to Chiron Corporation, a Delaware corporation. Invention is credited to Giulia C. Kennedy.
Application Number | 20090275054 11/929712 |
Document ID | / |
Family ID | 27376554 |
Filed Date | 2009-11-05 |
United States Patent
Application |
20090275054 |
Kind Code |
A1 |
Kennedy; Giulia C. |
November 5, 2009 |
Pancreatic Cancer Genes
Abstract
The present invention provides the art with the DNA coding
sequences of polynucleotides that are up-or-down-regulated in
cancer and dysplasia. These polynucleotides and encoded proteins or
polypeptides can be used in the diagnosis or identification of
cancer and dysplasia. Inhibitors of the up-regulated
polynucleotides and proteins can decrease the abnormality of cancer
and dysplasia. Enhancing the expression of down-regulated
polynucleotides or introducing down-regulated proteins to cells can
decrease the growth and/or abnormal characteristics of cancer and
dysplasia.
Inventors: |
Kennedy; Giulia C.; (San
Francisco, CA) |
Correspondence
Address: |
NOVARTIS VACCINES AND DIAGNOSTICS, INC.;CORPORATE INTELLECTUAL
PROPERTY-R338
P.O. BOX 8097
EMERYVILLE
CA
94662-8097
US
|
Assignee: |
Chiron Corporation, a Delaware
corporation
|
Family ID: |
27376554 |
Appl. No.: |
11/929712 |
Filed: |
October 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10351953 |
Jan 24, 2003 |
7541142 |
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11929712 |
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09773459 |
Jan 31, 2001 |
6664054 |
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10351953 |
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09337171 |
Jun 21, 1999 |
6262249 |
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09773459 |
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60118570 |
Feb 3, 1999 |
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60090391 |
Jun 23, 1998 |
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Current U.S.
Class: |
435/7.8 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 14/47 20130101 |
Class at
Publication: |
435/7.8 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 22, 1999 |
US |
PCT/US1999/014036 |
Claims
1. A method of diagnosing cancer in a patient comprising comparing
the amount of a polypeptide in a patient sample and in a normal
sample, wherein said polypeptide comprises the polypeptide encoded
by SEQ ID NO:5, or a naturally occurring variant of SEQ ID NO:5,
wherein said naturally occurring variant hybridizes to SEQ ID NO:5
under conditions including a wash in 2.times.SCC, 0.1% SDS at
50.degree. C. for 20 minutes; and wherein a patient sample
containing more of said polypeptide than the normal sample is
identified as cancerous.
2. A method of diagnosing dysplasia in a patient comprising
comparing the amount of a polypeptide in a patient sample and in a
normal sample, wherein said polypeptide comprises the polypeptide
encoded by SEQ ID NO:5, or a naturally occurring variant of SEQ ID
NO:5, wherein said naturally occurring variant hybridizes to SEQ ID
NO:5 under conditions including a wash in 2.times.SCC, 0.1% SDS at
50.degree. C. for 20 minutes; and wherein a patient sample
containing more of said polypeptide than the normal sample is
identified as dysplastic.
3. A method of diagnosing cancer in a patient comprising
determining the amount of a polypeptide in a patient sample and in
a normal sample, wherein said polypeptide is encoded by a
polynucleotide selected from the group consisting of SEQ ID NOS:1,
3-4, 6-11, and 13-14; determining the amount of a second
polypeptide in the patient sample and in the normal sample, wherein
said polypeptide is encoded by SEQ ID NO:5; and comparing the
determined amounts; wherein a patient sample which contains more of
the second polypeptide than the normal sample and which contains
substantially the same amount of the first polypeptide, as compared
to the normal sample, is identified as cancerous.
4. The method of claim 1 or 3, wherein the cancer is pancreatic
cancer.
5. The method of claim 2, wherein the dysplasia is pancreatic
dysplasia.
6. The method of any one of claims 1-3, wherein the patient sample
and the normal sample comprise pancreatic cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of (and claims the
benefit of priority under 35 U.S.C 120) U.S. patent application
Ser. No. 10/351,953, filed Jan. 24, 2003, which is a continuation
of U.S. patent application Ser. No. 09/773,459, filed Jan. 31,
2001, now U.S. Pat. No. 6,664,054, which is a divisional of U.S.
patent application Ser. No. 09/337,171, filed Jun. 21, 1999, now
U.S. Pat. No. 6,262,249, which claims priority from provisional
application Ser. No. 60/118,570, filed Feb. 3, 1999, and
provisional application Ser. No. 60/090,391, filed Jun. 23, 1998.
This application also claims the benefit of PCT Application Number
PCT/US1999/14036 filed Jun. 22, 1999. The disclosure of the prior
applications are considered part of (and is incorporated by
reference in) the disclosure of this application
TECHNICAL AREA OF THE INVENTION
[0002] The invention relates to the area of diagnosis and treatment
of pancreatic cancer and dysplasia. More specifically, it relates
to polynucleotides which are differentially regulated in pancreatic
cancer and dysplasia.
BACKGROUND OF THE INVENTION
[0003] Pancreatic cancer is the fifth leading cause of cancer death
in the United States. According to the American Cancer Society,
approximately 28,000 people will die of pancreatic cancer in the
United States in 1998. A high risk of developing pancreatic cancer,
without a corresponding increase in the risk of developing other
cancers, may be passed along in some families. Pancreatic cancer is
most likely caused by an accumulation of mutations in specific
cancer-causing genes. Pancreatic cancer is very aggressive and
chemotherapeutic agents which may be active against other
malignancies do not work effectively when used for pancreatic
cancer.
[0004] The majority of cells in the pancreas are in the exocrine
glands, which produce pancreatic enzymes, and in the ducts that
carry the pancreatic enzymes to the bile duct and to the small
intestine. Cancers of the exocrine cells of the pancreas are
usually adenocarcinomas. Pancreatic adenocarcinomas usually begin
in the ducts of the pancreas, but may sometimes develop from the
acinar cells. About 95% of cancers of the pancreas are
adenocarcinomas. Less common cancers of the exocrine pancreas
include adenosquamous carcinomas, squamous cell carcinomas, and
giant cell carcinomas.
[0005] Because pancreatic cancer is an aggressive cancer with very
high mortality, there is a need in the art for genes that are up-or
down-regulated in tumor progression Such genes are useful for
therapeutic purposes and for diagnosis of pancreatic as well as
other cancers.
SUMMARY OF THE INVENTION
[0006] The invention provides isolated polynucleotides comprising
coding regions or portions of genes whose expression is
mis-regulated in cancer and dysplasia.
[0007] The invention also provides isolated proteins and protein
fragments whose expression is mis-regulated in cancer and
dysplasia.
[0008] The invention further provides an antibody preparation which
specifically binds to a polypeptide the expression of which is
mis-regulated in cancer and dysplasia.
[0009] The invention provides a method for diagnosing cancer and
dysplasia.
[0010] The invention still further provides therapeutic
compositions useful for treating cancer and dysplasia.
[0011] These and other objects of the invention are provided by one
or more of the embodiments described below. One embodiment of the
invention provides isolated polynucleotides comprising at least
twelve contiguous nucleotides selected from the group of
polynucleotide sequences as shown in SEQ ID NOS: 1-15.
[0012] Another embodiment of the invention provides isolated
polypeptides comprising at least six contiguous amino acids encoded
by a polynucleotide selected from the group consisting of the
polynucleotide sequences as shown in SEQ ID NOS: 1-15.
[0013] Even another embodiment of the invention provides an
antibody preparation which specifically binds to a polypeptide
comprising at least six contiguous amino acids encoded by a
polynucleotide selected from the group of polynucleotide sequences
as shown in SEQ ID NOS:1-15.
[0014] Yet another embodiment of the invention provides isolated
nucleotide probes consisting of a sequence selected from the group
consisting of the polynucleotide sequences shown in SEQ ID NOS:
1-15.
[0015] Still another embodiment of the invention provides a method
of diagnosing cancer. The amount of a polypeptide expressed from a
polynucleotide having a sequence as shown in SEQ ID NO: 12 in a
test sample of tissue of a human suspected of being cancerous is
determined. The amount of said polypeptide is also determined in a
human tissue which is normal. The determined amounts are then
compared. A test sample which contains less of the polypeptide than
the normal tissue is identified as cancerous.
[0016] A further embodiment of the invention provides an additional
method of diagnosing cancer. The amount of specific mRNA molecules
in a test sample of tissue suspected of being cancerous and in a
human tissue which is normal are determined. The mRNA molecules to
be measured are complementary to the minus strand of a
double-stranded polynucleotide sequence. The double-stranded
polynucleotide sequence is shown in SEQ ID N0.12. The determined
amounts of mRNA molecules are compared. A test sample of tissue
which contains less of the mRNA molecules than the normal tissue is
identified as cancerous.
[0017] Another embodiment of the invention provides a therapeutic
composition useful for reducing the growth rate of cancer cells The
composition is comprised of a polynucleotide comprising all or a
portion of a nucleotide sequence which is operably linked to a
promoter sequence and a pharmaceutically acceptable carrier. The
polynucleotide comprising all or a portion of a nucleotide sequence
comprises at least 18 contiguous nucleotides. The nucleotide
sequence is shown in SEQ D NO: 12.
[0018] Yet another embodiment of the invention provides a
therapeutic composition useful for reducing the growth rate of
cancer cells. The composition is comprised of a polypeptide
comprising all or a portion of an amino acid sequence expressed
from a polynucleotide sequence and a pharmaceutically acceptable
carrier. The polynucleotide sequence is shown in SEQ ID NO: 12.
[0019] Another embodiment of the invention provides a method of
diagnosing dysplasia and cancer. The amount of a polypeptide
expressed from a polynucleotide having at least one of a sequence
selected from the group consisting of the polynucleotide sequences
shown in SEQ ID NOs:2, 5, and 15 in a test sample of tissue
suspected of being dysplastic or cancerous is determined. The
amount of the polypeptide is also determined in a human tissue
which is normal. The determined amounts are compared. A test sample
of human tissue which contains more of at least one polypeptide
than the normal tissue is identified as being dysplastic or
cancerous.
[0020] A further embodiment of the invention provides another
method of diagnosing dysplasia. The amount of a polypeptide
expressed from a polynucleotide having a sequence selected from the
group consisting of the polynucleotide sequences shown in SEQ ID
NOS: 1, 3-4, 6-11, and 13-14 is determined in a test sample of
tissue suspected of being dysplastic. The amount of said
polypeptide is also determined in a human tissue which is normal
The two amounts are then compared. A test sample of human tissue
which contains more of said polypeptide than the normal tissue is
identified as being dysplastic.
[0021] Another embodiment of the invention provides an additional
method of diagnosing cancer.
[0022] The amount of a polypeptide expressed from a polynucleotide
having a sequence selected from the group consisting of the
polynucleotide sequences shown in SEQ ID NOs: 2, 5, and 15, is
determined in a test sample of tissue suspected of containing
cancer, and in a human tissue which is normal. The amount of a
polypeptide expressed from a polynucleotide having a sequence
selected from the group consisting of the polynucleotide sequences
shown in SEQ ID NOs: 1, 3-4, 6-11, and 13-14 is also determined in
the test sample, and in the normal tissue The determined amounts of
said polypeptides are then compared. A test sample of tissue which
contains more of the polypeptide expressed from a polynucleotide
having a sequence selected from the group consisting of the
polynucleotide sequences shown in SEQ ID NOs:2, 5, and 15, as
compared to the normal tissue, and which contains substantially the
same amount of a polypeptide expressed from a polynucleotide
selected from the group as shown in SEQ ID NOs:1, 3-4, 6-11, and
13-14, as compared to the normal tissue, is identified as
cancerous.
[0023] Even another embodiment of the invention provides a method
of diagnosing dysplasia and cancer The amount of specific mRNA
molecules is determined in a test sample of tissue suspected of
being dysplastic or cancerous and in a human tissue which is normal
The mRNA molecules measured are complementary to the minus strand
of a double-stranded polynucleotide sequence The double-stranded
polynucleotide sequence is selected from the group of
polynucleotides as shown in SEQ ID NOs. 2, 5, and 15 The determined
amounts of mRNA molecules are compared. A test sample of human
tissue which contains more of the mRNA molecules than the normal
tissue is identified as being dysplastic or cancerous.
[0024] Yet another embodiment of the invention provides a method of
diagnosing dysplasia. The amounts of specific mRNA molecules in a
test sample of human tissue suspected of being dysplastic and in a
human tissue which is normal are determined. The mRNA molecules are
complementary to the minus strand of a double-stranded
polynucleotide sequence. The double-stranded polynucleotide
sequence is selected from the group of polynucleotides as shown in
SEQ ID NOS:1, 3-4, 6-11, and 13-14. The determined amounts of mRNA
molecules are then compared. A test sample of human tissue which
contains more of the mRNA molecules than the normal tissue is
identified as being dysplastic.
[0025] Still another embodiment of the invention provides a method
of diagnosing cancer. The amounts of a first set of specific mRNA
molecules in a test sample of tissue of a human suspected of being
cancerous and in a human tissue which is normal are determined. The
mRNA molecules are complementary to the minus strand of a
double-stranded polynucleotide sequence. The double-stranded
polynucleotide sequence is selected from the group of
polynucleotide sequences as shown in SEQ ID NOs: 1, 3-4, 6-11, and
13-14. In addition, the amounts of a second set of specific mRNA
molecules in a test sample of tissue of a human suspected of being
cancerous and in a human tissue which is normal are determined. The
mRNA molecules are complementary to the minus strand of a
double-stranded polynucleotide sequence. The double-stranded
polynucleotide sequence is selected from the group of
polynucleotide sequences as shown in SEQ ID NOs: 2, 5, and 15. The
determined amounts of the first and second sets of mRNA molecules
are compared. A test sample of human tissue which contains more of
the second set of mRNA molecules than the normal tissue, and which
contains substantially the same amount of the first set of mRNA
molecules, as compared to the normal tissue, is identified as
cancerous.
[0026] Yet another embodiment of the invention provides a
therapeutic composition useful for decreasing the amount of
translation of an mRNA molecule in a cell. The composition
comprises an antisense polynucleotide complementary to the plus
strand of a double-stranded polynucleotide. The double-stranded
polynucleotide is selected from the group consisting of
polynucleotides comprising a nucleotide sequence as shown in SEQ ID
NOs:1-11, and 13-15, wherein said antisense polynucleotide binds to
an mRNA molecule. The composition also includes a pharmaceutically
acceptable carrier.
[0027] A further embodiment of the invention provides a therapeutic
composition useful for reducing the expression of a polypeptide.
The composition comprises an antibody which specifically binds to a
polypeptide expressed from a polynucleotide selected from the group
consisting of polynucleotides comprising a nucleotide sequence as
shown in SEQ ID NOs:1-11 and 13-15. The composition also includes a
pharmaceutically acceptable carrier.
[0028] Another embodiment of the invention provides a therapeutic
composition useful for reducing the translation from an mRNA
molecule The composition comprises a ribozyme which binds to an
mRNA molecule, wherein a portion of said ribozyme is complementary
to the plus strand of a double-stranded polynucleotide. The
polynucleotide is selected from the group consisting of the
polynucleotides comprising a sequence as shown in SEQ ID NOs: 1-11,
and 13-15. The composition also comprises a pharmaceutically
acceptable carrier.
[0029] The present invention provides the art with useful
polynucleotides which represent expressed sequences of genes.
Expression of the genes is mis-regulated in cancer. The invention
also provides the art with diagnostic methods based on the over-
and under-expression of the genes and the polypeptides encoded by
the genes in cancer and dysplastic cells Inhibitors of the
over-expressed polynucleotides and polypeptides can be used to
reduce the growth of cancer cells and dysplastic cells. The
polynucleotides and polypeptides which are under-expressed in
cancer and dysplasia can be delivered therapeutically to reduce the
abnormal characteristics of cancer cells and dysplastic cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0030] Polynucleotides that are mis-regulated in cancer and
dysplasia are disclosed. The mis-regulated polynucleotide sequences
are shown in SEQ D NOS: 1-15. The polynucleotides are mis-regulated
as follows:
[0031] SEQ ID NO: 12 is down-regulated in cancer;
[0032] SEQ ID NOs:2, 5, and 15 are up-regulated in cancer and
dysplasia; and
[0033] SEQ ID NOS:1, 3-4, 6-11, and 13-14 are up-regulated in
dysplasia only.
[0034] Polynucleotides that are differentially regulated in cancer
or dysplasia or both can be useful in the diagnosis and treatment
of these diseases. Dysplasia is an atypical proliferation of
epithelial or mesenchymal cells that may represent an early stage
of cancer; however, dysplasia does not necessarily progress to
cancer. Epithelial dysplasia results in the loss of normal
orientation of one epithelial cell to another, accompanied by
alterations in cellular and nuclear size and shape. Cancer is a
proliferation of malignant cells that are no longer under normal
physiologic control.
[0035] The subgenomic polynucleotides of the invention contain less
than, a whole chromosome and are preferably intron-free. The
subgenomic polynucleotides of the invention can be isolated and
purified free from other nucleotide sequences by standard nucleic
acid purification techniques, for example, using PCR, cloning,
and/or restriction enzymes and probes to isolate fragments
comprising the encoding sequences. Subgenomic polynucleotides of
the invention can include all or a contiguous portion of a gene
coding region. In one embodiment, an isolated and purified
subgenomic polynucleotide of the invention comprises at least 10,
11, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 74, 80, 90,
100, 125, 150, 154, 175, 200, 250, 300, or 350 contiguous
nucleotides selected from the polynucleotide sequences as shown in
SEQ ID NOs:1-15. In a preferred embodiment, the polynucleotide
molecules comprise a contiguous sequence of at least twelve
nucleotides selected from the group consisting of the
polynucleotides shown in SEQ ID NOs:1-15.
[0036] An open reading frame is a region of DNA that consists
exclusively of triplets that represent amino acids The open reading
frame of the polynucleotide sequences of the invention can be
determined by examining all three possible reading frames in both
directions. If a reading frame contains termination codons it
cannot be read into protein and is not considered an open reading
frame. Usually, no more than one of the six possible frames is open
in any single stretch of DNA. An extensive open reading frame is
unlikely to exist by chance because of the lack of selective
pressure to prevent the accumulation of nonsense codons. Therefore,
the identification of a lengthy open reading frame is taken to be
prima facie evidence that the sequence is translated into protein
in that frame. Lewin, ed,. 1990, Genes IV, Cell Press, Cambridge,
Mass.
[0037] Subgenomic polynucleotides of the invention can be used,
inter alia, to produce proteins or polypeptides, as probes for the
detection of mRNA of the invention in samples or extracts of human
cells, to generate additional copies of the polynucleotides, and to
generate ribozymes or antisense oligonucleotides. The subgenomic
polynucleotides can also be used as single stranded DNA probes or
as triple strand forming oligonucleotides The probes can be used to
determine the presence or absence of the polynucleotide sequences
as shown in SEQ ID NOs:1-15 or variants thereof in a sample.
[0038] The sequence of a nucleic acid comprising at least 15
contiguous nucleotides of at least any one of SEQ ID NO:1-15,
preferably the entire sequence of at least any one of SEQ ID NO:
1-15, is not limited and can be any sequence of A, T, G, and/or C
(for DNA) and A, U, G, and/or C (for RNA) or modified bases
thereof, including inosine and pseudouridine. The choice of
sequence will depend on the desired function and can be dictated by
coding regions desired, the intron-like regions desired, and the
regulatory regions desired.
[0039] Where the entire sequence of any one of SEQ ID NO:1-15 is
within the nucleic acid, the nucleic acid obtained is referred to
herein as a polynucleotide comprising the sequence of any one of
SEQ ID NO: 1-15.
[0040] Both secreted and membrane-bound polypeptides of the present
invention are of interest. For example, levels of secreted
polypeptides can be assayed conveniently in body fluids, such as
blood and urine. Membrane-bound polypeptides are useful for
constructing vaccine antigens or inducing an immune response. Such
antigens would comprise all or part of the extracellular region of
the membrane-bound polypeptides.
[0041] Because both secreted and membrane-bound polypeptides
comprise a fragment of contiguous hydrophobic amino acids,
hydrophobicity predicting algorithms can be used to identify such
polypeptides.
[0042] A signal sequence is usually encoded by both secreted and
membrane-bound polypeptide genes to direct a polypeptide to the
surface of the cell. The signal sequence usually comprises a
stretch of hydrophobic residues. Such signal sequences can fold
into helical structures.
[0043] Membrane-bound polypeptides typically comprise at least one
transmembrane region that possesses a stretch of hydrophobic amino
acids that can transverse the membrane. Some transmembrane regions
also exhibit a helical structure.
[0044] Hydrophobic fragments within a polypeptide can be identified
by using computer algorithms. Such algorithms include Hopp &
Woods, Proc. Natl. Acad. Sci. USA 78:3824-3828 (1981); Kyte &
Doolittle, J. Mol. Biol. 157:105-132 (1982); and RAOAR algorithm,
Degli Esposti et al., Eur. J. Biochem. 190:207-219 (1990).
[0045] Another method of identifying secreted and membrane-bound
polypeptides is to translate the present polynucleotides, SEQ ID
NO: 1-15, in all six frames and determine if at least 8 contiguous
hydrophobic amino acids are present. Those translated polypeptides
with at least 8; more typically, 10; even more typically, 12
contiguous hydrophobic amino acids are considered to be either a
putative secreted or membrane bound polypeptide. Hydrophobic amino
acids include alanine, glycine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, proline, threonine, tryptophan,
tyrosine, and valine.
[0046] The polypeptides of the invention include those encoded by
the disclosed polynucleotides. These polypeptides can also be
encoded by nucleic acids that, by virtue of the degeneracy of the
genetic code, are not identical in sequence to the disclosed
polynucleotides. Thus, the invention includes within its scope
nucleic acids comprising polynucleotides encoding a protein or
polypeptide expressed by a polynucleotide having the sequence of
any one of SEQ ID NO: 1-15. Also within the scope of the invention
are variants; variants of polypeptides include mutants, fragments,
and fusions. Mutants can include amino acid substitutions.
additions or deletions. The amino acid substitutions can be
conservative amino acid substitutions or substitutions to eliminate
non-essential amino acids, such as to alter a glycosylation site, a
phosphorylation site or an acetylation site, or to minimize
misfolding by substitution or deletion of one or more cysteine
residues that are not necessary for function. Conservative amino
acid substitutions are those that preserve the general charge,
hydrophobicity/hydrophilicity, and/or steric bulk of the amino acid
substituted For example, substitutions between the following groups
are conservative: Gly/Ala, VaVIleLeu, AspIGlu, Lys/Arg, Asn/Gln,
Ser/Cys, Thr, and Phe/Trp/Tyr.
[0047] Cysteine-depleted muteins are variants within the scope of
the invention. These variants can be constructed according to
methods disclosed in U.S. Pat. No. 4,959,314, "Cysteine-Depleted
Muteins of Biologically Active Proteins." The patent discloses how
to substitute other amino acids for cysteines, and how to determine
biological activity and effect of the substitution. Such methods
are suitable for proteins according to this invention that have
cysteine residues suitable for such substitutions, for example to
eliminate disulfide bond formation.
[0048] The protein variants described herein are encoded by
polynucleotides that are within the scope of the invention. The
genetic code can be used to select the appropriate codons to
construct the corresponding variants.
[0049] The invention encompasses polynucleotide sequences having at
least 65% sequence identity to any one of SEQ ID NOS: 1-15 as
determined by the Smith-Waterman homology search algorithm as
implemented in MSPRCH program (Oxford Molecular) using an affine
gap search with the following search parameters gap open penalty of
12, and gap extension penalty of 1.
[0050] Polynucleotide probes comprising at least 12 contiguous
nucleotides selected from the nucleotide sequence of a
polynucleotide of SEQ ID NO:1-15 are used for a variety of
purposes, including identification of human chromosomes and
determining transcription levels.
[0051] The nucleotide probes are labeled, for example, with a
radioactive, fluorescent, biotinylated, or chemiluminescent label,
and detected by well known methods appropriate for the particular
label selected Protocols for hybridizing nucleotide probes to
preparations of metaphase chromosomes are also well known in the
art A nucleotide probe will hybridize specifically to nucleotide
sequences in the chromosome preparations which are complementary to
the nucleotide sequence of the probe. A probe that hybridizes
specifically to a polynucleotide should provide a detection signal
at least 5-. lo-, or 20-fold higher than the background
hybridization provided with other unrelated sequences.
[0052] Polynucleotides of the present invention are used to
identify a chromosome on which the corresponding gene resides.
Using fluorescence in situ `hybridization (FISH) on normal
metaphase spreads, comparative genomic hybridization allows total
genome assessment of changes in relative copy number of DNA
sequences. See Schwartz and Samad, Current Opinions in
Biotechnology (1994) 8:70-74; Kallioniemi et al., Seminars in
Cancer Biology (1993) 4:41-46; Valdes and Tagle, Methods in
Molecular Biology (1997) 68:1, Boultwood, ed., Human Press, Totowa,
N.J.
[0053] Preparations of human metaphase chromosomes are prepared
using standard cytogenetic techniques from human primary tissues or
cell lines. Nucleotide probes comprising at least 12 contiguous
nucleotides selected from the nucleotide sequence of SEQ ID NOS:
1-15 are used to identify the corresponding chromosome. The
nucleotide probes are labeled, for example, with a radioactive,
fluorescent, biotinylated, or chemiluminescent label, and detected
by well known methods appropriate for the particular label
selected. Protocols for hybridizing nucleotide probes to
preparations of metaphase chromosomes are also well known in the
art. A nucleotide probe will hybridize specifically to nucleotide
sequences in the chromosome preparations that are complementary to
the nucleotide sequence of the probe A probe that hybridizes
specifically to a polynucleotide-related gene provides a detection
signal at least 5-, 10-, 25 or 20-fold higher than the background
hybridization provided with non-polynucleotide coding
sequences.
[0054] Polynucleotides are mapped to particular chromosomes using,
for example, radiation hybrids or chromosome-specific hybrid
panels. See Leach et al., Advances in, Genetics, (1995) 33:63-99,
Walter et al., Nature Genetics (1994) 7:22-28; Walter and
Goodfellow, Trends in Genetics. (1992) 9:352. Such mapping can be
useful in identifying the function of the polynucleotide-related
gene by its proximity to other genes with known function. Function
can also be assigned to the related gene when particular syndromes
or diseases map to the same chromosome.
[0055] A polynucleotide will be useful in forensics, genetic
analysis, mapping, and diagnostic applications if the corresponding
region of a gene is polymorphic in the human population. A
particular polymorphic form of the polynucleotide may be used to
either identify a sample as deriving from a suspect or rule out the
possibility that the sample derives from the suspect. Any means for
detecting a polymorphism in a gene are used, including but not
limited to electrophoresis of protein polymorphic variants,
differential sensitivity to restriction enzyme cleavage, and
hybridization to an allele-specific probe.
[0056] Any naturally occurring variants of the nucleotide sequences
which encode variants thereof are within the scope of this
invention. Allelic variants of subgenomic polynucleotides of the
invention can occur and can be identified by hybridization of
putative allelic variants with nucleotide sequences disclosed
herein under stringent conditions. For example, by using the
following wash conditions--2.times.SCC, 0.1% SDS, room temperature
twice, 30 minutes each, then 2.times.SCC, 0.1% SDS, 50.degree. C.
once, 30 minutes; then 2.times.SCC, room temperature twice, 10
minutes each--allelic variants of the polynucleotides of the
invention can be identified which contain at most about 25-30% base
pair mismatches. More preferably, allelic variants contain 15-25%
base pair mismatches, even more preferably 5-15%, or 2-5%, or 1-2%
base pair mismatches.
[0057] Amplification by the polymerase chain reaction (PCR) can be
used to obtain the polynucleotides of the invention, using either
genomic DNA or cDNA as a template. The polynucleotides of the
invention may also be obtained using reverse transcriptase and mRNA
molecules that are complementary to the minus strand of a
double-stranded sequence wherein said double-stranded sequence is
selected from the group of polynucleotides comprising a sequence as
shown in SEQ ID NOS: 1-15. Using the polynucleotide sequences
disclosed herein, subgenomic polynucleotide molecules of the
invention can also be made using the techniques of synthetic
chemistry.
[0058] Probes specific to the polynucleotides of the invention may
be generated using the polynucleotide sequences disclosed in SEQ ID
NOS: 1-15. The probes are preferably at least 12, 14, 16, 18, 20,
22, 24, or 25 nucleotides in length and can be less than 2, 1, 0.5,
0.1, or 0.05 kb in length. The probes can be synthesized chemically
or can be generated from longer polynucleotides using restriction
enzymes. The probes can be labeled, for example, with a
radioactive, biotinylated, or fluorescent tag.
[0059] Subgenomic polynucleotides of the invention can be
propagated in vectors and cell lines using techniques well known in
the art. Expression systems in bacteria include those described in
Chang et al., Nature (1978) 275: 615; Goeddel et al., Nature (1979)
281: 544; Goeddel et al., Nucleic Acids Res. (1980) 8: 4057; EP
36,776; U.S. Pat. No. 4,551,433; deBoer et al., Proc. Natl. Acad.
Sci. USA (1983) 80: 21-25; and Siebenlist et al., Cell (1980) 20:
269.
[0060] Expression systems in yeast include those described in
Hinnen et al., Proc. Natl. Acad. Sci. USA (1978) 75: 1929; Ito et
al., J. Bacteriol. (1983) 153: 163; Kurtz et al., Mol. Cell. Biol.
(1986) 6:142, Kunze et al. J. Basic Microbiol. (1985) 25:141;
Gleeson et al., J. Gen. Microbiol. (1986) 132: 3459, Roggenkamp et
al., Mol. Gen. Genet. (1986) 202: 302; Das et al., J. Bacteriol.
(1984) 158: 1165; De Louvencourt et. al., J. Bacteriol. (1983)
154:737, Van den Berg et al., Bio Technology (1990) .delta.: 135;
Kunze et al., J. Basic Microbiol. (1985) 25:141; Cregg et al., Mol.
Cell. Biol. (1985) 5:3376; U.S. Pat. No. 4,837,148, U.S. Pat. No.
4,929,555; Beach and Nurse, Nature (1981) 300: 706; Davidow et al.,
Curr. Genet. (1985) 10: 380; Gaillardin et al., Curr. Genet. (1985)
10: 49; Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:
284-289; Tilburn et al., Gene (1983) 26: 205-221, Yelton et al.,
Proc. Natl. Acad. Sci. USA (1984) 81: 1470-1474; Kelly and Hynes.
EMBO J. (1985) 4: 475-479; EP 244,234; and WO 91/00357.
[0061] Expression of the subgenomic polynucleotides of the
invention in insects can be accomplished as described in U.S. Pat.
No. 4,745,051, Friesen et al. (1986) "The Regulation of Baculovirus
Gene Expression" in: THE MOLECULAR BIOLOGY OF BACULOVIRUSES (W.
Doerfler, ed.); EP 127,339; EP 155,476; Vlak et al., J. Gen. Virol.
(1988) 69: 765-776; Miller et al., Am. Rev. Microbiol. (1988) 42:
177; Carbonell et al., Gene (1988) 73: 409; Maeda et al., Nature
(1985) 315: 592-594; Lebacq-Verheyden et al., Mol. Cell. Biol.
(1988) .delta.: 3129, Smith et al., Proc. Natl. Acad. Sci.
USA(1985) 82:8404; Miyajima et al., Gene (1987) 58: 273; and Martin
et al., DNA (1988) 7:99. Numerous baculoviral strains and variants
and corresponding permissive insect host cells from hosts are
described in Luckow et al., Bio/Technology (1988) .delta.: 47-55;
Miller et al., in GENETIC ENGINEERING (Setlow, J. K. et al. eds.),
Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al.,
Nature, (1985) 315: 592-594.
[0062] Mammalian expression of the subgenomic polynucleotides of
the invention can be accomplished as described in Dijkema et al.,
EMBO J (1985) 4: 76; Gorman et al., Proc. Natl. Acad. Sci. USA
(1982) 79:6777; Boshart et al., Cell (1985) 41: 521; and U.S. Pat.
No. 4,399,216. Other features of mammalian expression can be
facilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:
44; Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat. No.
4,767,704; U.S. Pat. No. 4,657,866; U.S. Pat. No. 4,927,762; U.S.
Pat. No. 4,560,655; WO 901103430; WO 87/00195, and U.S. Pat. No. RE
30,985.
[0063] The subgenomic polynucleotides of the invention can be on
linear or circular molecules. They can be on autonomously
replicating molecules (vectors) or on molecules without replication
sequences. They can be regulated by their own or by other
regulatory sequences, as is known in the art The subgenomic
polynucleotides of the invention can be introduced into suitable
host cells using a variety of techniques which are available in the
art, such as transferrin-polycation-mediated DNA transfer,
transfection with naked or encapsulated nucleic acids,
liposome-mediated DNA transfer, intracellular transportation of
DNA-coated latex beads, protoplast fusion, viral infection,
electroporation, gene gun, and calcium phosphate-mediated
transfection.
[0064] The invention provides a method of detecting expression of a
polynucleotide in, for example. a biological sample, which can be
useful, inter alia, for diagnosing cancer or dysplasia. The basis
for this method is the discovery that the polynucleotide
sequence(s) as shown in:
[0065] SEQ ID NO: 12 is down-regulated in cancer;
[0066] SEQ ID NOs: 2. 5, and 15 are up-regulated in cancer and
dysplasia; and
[0067] SEQ ID NOS: 1. 3-4, 6-11, and 13-14 are up-regulated in
dysplasia only.
[0068] In patients who have been diagnosed with pancreatic
dysplasia or cancer, the detection of levels of the expression
products of the polynucleotide sequences of the invention, either
mRNA or protein, can be used to diagnose or prognose a disorder, to
monitor treatment of the disorder, or to screen agents which affect
the disorder.
[0069] The expression products of the polynucleotide sequences of
the invention, either mRNA or proteins, can be detected in a body
sample for diagnosis or prognosis. The body sample can be, for
example, a solid tissue or a fluid sample. The patient from whom
the body sample is obtained can be healthy or can already be
identified as having a condition in which altered expression of a
protein of the invention is implicated.
[0070] In one embodiment, the body sample is assayed for the level
of a protein expressed from a polynucleotide sequence of the
invention. The protein could be detected by, for example,
antibodies to the proteins. The antibodies can be labeled, for
example, with a radioactive, fluorescent, biotinylated, or
enzymatic tag and detected directly, or can be detected using
indirect immunochemical methods, using a labeled secondary
antibody. The presence of the protein can be assayed, for example,
in tissue sections by immunocytochemistry, or in lysates, using
Western blotting, as is known in the art.
[0071] The level of the protein in a tissue sample suspected of
being cancerous or dysplastic is compared with the level of the
protein in a normal tissue. A higher level of the polypeptides
expressed from polynucleotide sequences as shown in SEQ ID NOS: 1,
3-4, 6-11, and 13-14 in the suspect tissue, as compared to the
normal tissue, indicates the presence of dysplastic cells in the
suspect tissue. A higher level of the polypeptides expressed from
polynucleotide sequences as shown in SEQ ID NOs: 2, 5, and 15 in
the suspect tissue, as compared to the normal tissue, indicates the
presence dysplastic cells or cancerous cells or both in the suspect
tissue. A lower level of the polypeptide expressed from the
polynucleotide sequence as shown in SEQ ID NO: 12 in the suspect
tissue, as compared to the normal tissue, indicates the presence of
cancerous cells in the suspect tissue.
[0072] Additionally, a differentiation between cancer or dysplasia
in a patient's diagnosis can be made. The expression of a
polynucleotide sequence of the invention that is up-regulated in
dysplastic cells only (i.e. SEQ ID NOS: 1, 3-4, 6-11, and 13-14)
and the expression of a polynucleotide that is up-regulated in both
dysplastic cells and cancerous cells (i.e., SEQ ID NOS:2, 5, and
15) can be used to screen a patient's tissues. If examination of a
patient's tissues reveals that there is no up-regulation of a
polynucleotide sequence that is up-regulated in dysplastic cells
only (i.e., SEQ ID NOS:1, 3-4, 6-11, and 13-14), and that there is
up-regulation of a polynucleotide sequence that is up-regulated in
both cancerous cells and dysplastic cells (i.e. SEQ ID NOs:2, 5,
and 15), then the patient is diagnosed with cancer.
[0073] Alternatively, the presence of mRNA expressed from the
polynucleotide sequences of the invention in two tissues can be
compared. mRNA can be detected, for example, by in situ
hybridization in tissue sections, by reverse transcriptase-PCR, or
in Northern blots containing poly A+ mRNA. One of skill in the art
can readily determine differences in the size or amount of mRNA
transcripts between two tissues, using Northern blots and
nucleotide probes. For example, the level of mRNA of the invention
in a tissue sample suspected of being cancerous or dysplastic is
compared with the expression of the mRNA in a normal tissue. Any
methods known in the art for determining the amounts of specific
mRNAs can be used.
[0074] A higher level of mRNA expressed from polynucleotide
sequences as shown in SEQ ID NOS: 1, 3-4, 6-11, and 13-14 in the
suspect tissue, as compared to the normal tissue, indicates the
presence dysplastic cells in the suspect tissue. A higher level of
mRNA expressed from the polynucleotide sequences as shown in SEQ ID
NOs:2, 5, and 15 in the suspect tissue, as compared to the normal
tissue, indicates the presence dysplastic cells or cancerous cells
or both in the suspect tissue. A lower level of the mRNA expressed
from the polynucleotide sequence as shown in SEQ ID NO: 12 in the
suspect tissue, as compared to the, normal tissue, indicates the
presence of cancerous cells in the suspect tissue. Any combinations
of these sequences can be used to determine a diagnosis.
[0075] Optionally, the level of a particular expression product of
a polynucleotide sequence of the invention in a body sample can be
quantitated. Quantitation can be accomplished, for example, by
comparing the level of expression product detected in the body
sample with the amounts of product present in a standard curve. A
comparison can be made visually or using a technique such as
densitometry, with or without computerized assistance Alternative
methods can be used, for example ELISA, western blot,
immunoprecipitation, radioimmunoassay, etc. Any method known in the
art for detecting and quantitating a particular protein can be
used.
[0076] Reagents specific for the polynucleotides and polypeptides
of the invention, such as antibodies and nucleotide probes, can be
supplied in a kit for detecting the presence of an expression
product in a biological sample. The kit can also contain buffers or
labeling components, as well as instructions for using the reagents
to detect and quantify expression products in the biological
sample.
[0077] Polynucleotide expression in a cell can be increased or
decreased, as desired. Polynucleotide expression can be altered for
therapeutic purposes, as described below, or can be used to
identify and study the role of therapeutic agents in cancer and
other diseases.
[0078] Decreasing the expression of genes containing sequences
selected from the group consisting of the sequences as shown in SEQ
ID NOs:1, 3-4, 6-11, and 13-14 is useful, for example, as a
therapeutic for altering the abnormal characteristics of dysplastic
cells. Decreasing the expression of polynucleotide sequences
selected from the group consisting of the sequences as shown in SEQ
ID NOs: 2, 5, and 15 is useful, for example, as a therapeutic agent
for decreasing the growth rate of dysplastic and cancer cells.
[0079] Expression of the polynucleotide sequences of the invention
can be altered using an antisense oligonucleotide sequence
Therapeutic compositions for decreasing gene expression comprise an
expression construct containing polynucleotides encoding all or a
portion of a polynucleotide sequence selected from the group
consisting of SEQ ID NOS:1-11, and 13-15 Within the expression
construct, the polynucleotide segment is orientated in the
antisense direction and is located downstream from a promoter.
Transcription of the polynucleotide segment initiates at the
promoter.
[0080] Preferably, the antisense oligonucleotide sequence is at
least ten nucleotides in length, but longer sequences of at least
11, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 74, 80, 90, 100,
125, 150, 162, 175, 200, 250, 300, or 350 contiguous nucleic acids
can also be used. Antisense oligonucleotide molecules can be
provided in a DNA construct and introduced into cells whose
division is to be decreased, as described above. A more complete
description of gene transfer vectors, especially retroviral vectors
is contained in U S. Ser. No. 08/869,309, which is incorporated
herein by reference.
[0081] The antisense oligonucleotides can be composed of
deoxyribonucleotides, ribonucleotides, or a combination of both.
Oligonucleotides can be synthesized manually or by an automated
synthesizer, by covalently linking the 5' end of one nucleotide
with the 3' end of another nucleotide with phosphodiester or
non-phosphodiester internucleotide linkages such as
alkylphosphonates, phosphorothioates, phosphorodithioates,
alkylphosphonothioates, alkylphosphonates, phosphoramidates,
phosphate esters, carbamates, acetamidate, carboxymethyl esters,
carbonates, and phosphate triesters. See Brown, 1994, Meth. Mol.
Biol. 20:1-8; Sonveaux, 1994, Meth. Mol. Biol. 26:1-72; Uhlmann et
al., 1990, Chem. Rev. 90:543-583.
[0082] Although precise complementarity is not required for
successful duplex formation between an antisense molecule and the
complementary coding sequence of a gene, antisense molecules with
no more than one mismatch are preferred. One skilled in the art can
easily use the calculated melting point of an antisense-sense pair
to determine the degree of mismatch which will be tolerated between
a particular antisense oligonucleotide and a particular coding
sequence of the selected gene.
[0083] The antisense oligonucleotides of the invention can be
modified without affecting their ability to hybridize to a
polynucleotide coding sequence of the present invention. These
modifications can be internal or at one or both ends of the
antisense molecule. For example, internucleoside phosphate linkages
can be modified by adding cholesteryl or diamine moieties with
varying numbers of carbon residues between the amino groups and
terminal ribose. Modified bases or sugars or both, such as
arabinose instead of ribose, or a 3',5'-substituted oligonucleotide
in which the 3' hydroxyl group or the 5' phosphate group are
substituted, can also be employed in a modified antisense
oligonucleotide These modified oligonucleotides can be prepared by
methods well known in the art. Agrawal et al., 1992, Trends
Biotechnol. 10: 152-158; Uhlmann et al., 1990, Chem. Rev. 90:
543-584, Uhlmann et al., 1987, Tetrahedron. Lett. 215:
3539-3542.
[0084] Expression of the polynucleotides of the invention can also
be decreased by delivering polyclonal, monoclonal, or single chain
antibodies that specifically bind to polypeptides expressed from
the polynucleotide sequences as shown in SEQ ID NOS:1-11 and 13-15.
Antibodies specific to these proteins bind to the protein and
prevent the protein from functioning in the cell. Blocking protein
expression or function is useful for preventing, reducing the
effects of, or curing cancer and dysplasia.
[0085] In one embodiment of the invention, expression of the
polynucleotides selected from the group consisting of the
polynucleotide sequences shown in SEQ ID 15 NOS. 1-11, and 13-15
are decreased using a ribozyme, an RNA molecule with catalytic
activity. See, e.g., Cech, 1987, Science 236: 1532-1539, Cech,
1990, Ann. Rev. Biochem. 59:543-568, Cech, 1992, Curr. Opinion
Struct. Biol. 2: 605-609; Couture and Stinchcomb, 1996, Trends
Genet. 12:510-515. Ribozymes an be used to inhibit gene function by
cleaving an RNA sequence, as is known in the art (e.g., Haseloff et
al., U.S. Pat. No. 5,641,673).
[0086] The coding sequence of a polynucleotide of the invention can
be used to generate a ribozyme which will specifically bind to RNA
transcribed from said polynucleotide. Methods of designing and
constructing ribozymes which can cleave other RNA molecules in
trans a highly sequence specific manner have been developed and
described in the art (see Haseloff, J. et al. (1988), Nature
334:585-591). For example, the cleavage activity of ribozymes can
be targeted to specific RNAs by engineering a discrete
"hybridization" region into the ribozyme. The hybridization region
contains a sequence complementary to the target RNA and thus
specifically hybridizes with the target (see, for example, Gerlach,
W. L. et al., EP 321,201). Longer complementary sequences can be
used to increase the affinity of the hybridization sequence for the
target. The hybridizing and cleavage regions of the ribozyme of the
invention can be integrally related; thus, upon hybridizing to the
target RNA through the complementary regions, the catalytic region
of the ribozyme can cleave the target.
[0087] Ribozymes of the invention can be introduced into cells as
part of a DNA construct, as is known in the art. The DNA construct
can also include transcriptional regulatory elements, such as a
promoter element, an enhancer or UAS element, and a transcriptional
terminator signal, for controlling the transcription of the
ribozyme in the cells.
[0088] Mechanical methods, such, as microinjection,
liposome-mediated transfection, electroporation, gene gun, or
calcium phosphate precipitation, can be used to introduce the
ribozyme-containing DNA construct into cells whose division it is
desired to decrease, as described above. Alternatively. if it is
desired that the DNA construct be stably retained by the cells, the
DNA construct can be supplied on a plasmid and maintained as a
separate element or integrated into the genome of the cells, as is
known in the art.
[0089] As taught in Haseloff et al., U.S. Pat. No. 5,641,673, the
ribozymes of the invention can be engineered so that their
expression will occur in response to factors which induce
expression of a polynucleotides of the invention. The ribozyme can
also be engineered to provide an additional level of regulation, so
that destruction of RNA occurs only when both the ribozyme and the
corresponding gene are induced in the cells.
[0090] Preferably, the mechanism used to decrease expression of the
polynucleotides of the invention, whether antisense nucleotide
sequence, antibody, or ribozyme decreases expression of the
polynucleotide by 50%, 60%, 70%, or 80%. Most preferably,
expression of the polynucleotide is decreased by 90%, 95%, 99%, or
100%. The effectiveness of the mechanism chosen to alter expression
of the polynucleotide can be assessed using methods well known in
the art, such as hybridization of nucleotide probes to mRNA of the
polynucleotide, quantitative RT-PCR, or detection of a protein
using specific antibodies of the invention.
[0091] Increased expression of a polynucleotide is useful to
decrease the growth rate of cancer cells where the particular
polynucleotide is down-regulated in cancer cells, such as the
polynucleotide sequence as shown in SEQ ID NO: 12. Therapeutic
compositions for increasing polynucleotide expression comprise an
expression construct containing all or a portion of the
polynucleotide sequence as shown in SEQ ID NO: 12. Within an
expression construct, the polynucleotide segment is oriented in the
sense direction and is located downstream from the promoter.
Transcription of the polynucleotide segment initiates at the
promoter. The expression construct can be introduced into cells
along with a pharmaceutically acceptable carrier to decrease the
growth rate of cancer cells or ameliorate other abnormal
characteristics. Expression of the polynucleotide sequence can be
monitored by detecting production of mRNA which hybridizes to the
delivered polynucleotide or by detecting protein encoded by the
delivered polynucleotide.
[0092] Proteins that are expressed from the polynucleotide
sequences of the invention can be produced recombinantly in
prokaryotic or eukaryotic host cells, such as bacteria, yeast,
insect, or mammalian cells, using expression vectors known in the
art. Enzymes can be used to generate less than full length
polypeptides by enzymatic proteolysis of full-length proteins of
the invention. Alternatively, synthetic chemistry methods, such as
solid-phase peptide synthesis, can be used to synthesize the
proteins and polypeptides.
[0093] Species homologs of human subgenomic polynucleotides or the
encoded polypeptides can be identified by making suitable probes or
primers and screening cDNA expression libraries from other species,
such as mice, monkeys, yeast, or bacteria. Mammalian homologs are
preferred, however.
[0094] Proteins or polypeptides expressed from the polynucleotide
sequences as shown in SEQ ID NO: 1-15 can be isolated and purified
from human cells that express the proteins. The proteins can be
obtained substantially free from other human proteins by standard
protein purification methods, such as size exclusion
chromatography, ion exchange chromatography, ammonium sulfate
fractionation, affinity chromatography, or preparative gel
electrophoresis.
[0095] Proteins or polypeptides expressed from the polynucleotides
of the invention can also be used in a fusion protein, for example,
as an immunogen. The fusion protein comprises two protein segments.
The first protein segment consists of at least six, eight, ten,
twelve, fifteen, twenty or thirty contiguous amino acids of a
polypeptide sequence expressed from a polynucleotide sequence as
shown in SEQ ID NOS:1-15. The first protein segment is fused to a
second protein segment by means of a peptide bond. The second
protein segment can be a full-length protein or a fragment of a
protein. Techniques for making fusion proteins, either
recombinantly or by covalently linking two protein segments, are
well known in the art.
[0096] The second protein or protein fragment of a fusion protein
can be derived from another type of protein or a similar protein.
The second protein or protein fragment can be labeled with a
detectable marker, such as a radioactive or fluorescent tag, or can
be an enzyme that will generate a detectable product Enzymes
suitable for this purpose, such as p-galactosidase, are well-known
in the art. A fusion protein can be used, for example, to target
the proteins of the invention or polypeptides to a particular
location in a cell or tissue. in various assays, such as the yeast
two-hybrid technique, or as an immunogen.
[0097] The proteins or polypeptides expressed from the
polynucleotides of the invention can be used for generating
antibodies The antibodies can be used, inter alia, to detect and
quantitate expression of the cognate protein. Proteins or
polypeptides expressed from the polynucleotides of the invention
comprising at least six, eight, ten, twelve, fifteen, twenty or
thirty consecutive amino acids can be used as immunogens. The
proteins or polypeptides can be used to obtain a preparation of
antibodies which specifically bind to a protein or polypeptide of
the invention. The antibodies can be polyclonal or monoclonal
Techniques for raising both polyclonal and monoclonal antibodies
are well known in the art.
[0098] Single chain antibodies can also be constructed. Single
chain antibodies which specifically bind to a protein or
polypeptide expressed from the polynucleotides of the invention can
be isolated, for example, from single-chain immunoglobulin display
libraries, as are known in the art. The library is "panned" against
a protein or polypeptide, and a number of single chain antibodies
which bind different epitopes of the polypeptide with high-affinity
can be isolated. Hayashi et al., 1995, Gene 160: 129-30. Such
libraries are known and available to those in the art. The
antibodies can also be constructed using the polymerase chain
reaction (PCR), using hybridoma cDNA as a template. Thirion et al.,
1996, Eur. J. Cancer Prev. 5: 507-11.
[0099] The single chain antibody can be mono- or bi-specific, and
can be bivalent or tetravalent. Construction of tetravalent
bispecific single chain antibodies is taught in Coloma and
Morrison, 1997, Nat. Biotechnol. 15: 159-63 Construction of
bivalent bispecific single chain antibodies is taught in Mallender
and Voss, 1994, J. Biol. Chem. 269: 199-206.
[0100] A nucleotide sequence encoding the single chain antibody can
then be constructed using manual or automated nucleotide synthesis,
cloned into DNA expression vectors using standard recombinant DNA
methodologies, and introduced into cells which express the selected
gene, as described below. Alternatively, the antibodies can be
produced directly using filamentous phage technology Verhaar et
al., 1995, Int. J. Cancer 61:497-501; Nicholls et al., 1993. J.
Immunol. Meth. 165:81-91.
[0101] The antibodies bind specifically to the epitopes of the
proteins or polypeptides expressed from the polynucleotides of the
invention. In a preferred embodiment, the epitopes are not present
on other human proteins. Typically a minimum number of contiguous
amino acids to encode an epitope is 6, 8, or 10. However, more can
be used, for example, at least 15, 25, or 50, especially to form
epitopes which involve non-contiguous residues or particular
conformations.
[0102] Antibodies that bind specifically to the proteins or
polypeptides include those that bind to full-length proteins or
polypeptides Specific binding antibodies do not detect other
proteins on Western blots of human cells, or provide a signal at
least ten-fold lower than the signal provided by the target protein
of the invention. Antibodies which have such specificity can be
obtained by routine screening. In a preferred embodiment of the
invention, the antibodies immunoprecipitate the proteins or
polypeptides expressed from the polynucleotides the invention from
cell extracts or solution. Additionally, the antibodies can react
with proteins or polypeptides expressed from the polynucleotides of
the invention in tissue sections or on Western blots of
polyacrylamide gels. Preferably the antibodies do not exhibit
nonspecific cross-reactivity with other human proteins on Western
blots or in immunocytochemical assays.
[0103] Techniques for purifying antibodies to the proteins or
polypeptides expressed from the polynucleotides of the invention
are available in the art. In a preferred embodiment, the antibodies
are passed over a column to which a particular protein or
polypeptide expressed from the polynucleotides of the invention is
bound. The bound antibodies are then eluted, for example, with a
buffer having a high salt concentration.
[0104] Therapeutic compositions of the invention also comprise a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers are well known to those in the art. Such carriers include,
but are not limited to, large, slowly metabolized macromolecule,
such as proteins, polysaccharides, polylactic acids, polyglycolic
acids, polymeric amino acids, amino acid copolymers, and inactive
virus particles. Pharmaceutically acceptable salts can also be used
in the composition, for example, mineral salts such as
hydrochlorides, hydrobromides, phosphates, or sulfates, as well as
the salts of organic acids such as acetates, proprionates,
malonates, or benzoates.
[0105] Therapeutic compositions can also contain liquids, such as
water, saline, glycerol, and ethanol, as well as substances such as
wetting agents, emulsifying agents, or pH buffering agents.
Liposomes, such as those described in U.S. Pat. No. 5,422,120, WO
95/13796, WO 91/14445, or EP 524,968 B1, can also be used as a
carrier for the therapeutic composition.
[0106] Typically, a therapeutic composition is prepared as an
injectable, either as a liquid solution or suspension, however,
solid forms suitable for solution in, or suspension in, liquid
vehicles prior to injection can also be prepared. A composition can
also be formulated into an enteric coated tablet or gel capsule
according to known methods in the art, such as those described in
U.S. Pat. No. 4,853,230, EP 225,189, AU 9,224,296, and AU
9,230,801.
[0107] Administration of the therapeutic agents of the invention
can include local or systemic administration, including injection,
oral administration, particle gun, or catheterized administration,
and topical administration. Various methods can be used to
administer a therapeutic composition directly to a specific site in
the body.
[0108] For treatment of tumors, for example, a small tumor or
metastatic lesion can be located and a therapeutic composition
injected several times in several different locations within the
body of the tumor. Alternatively, arteries which serve a tumor can
be identified, and a therapeutic composition injected into such an
artery, in order to deliver the composition directly into the
tumor.
[0109] A tumor which has a necrotic center can be aspirated and the
composition injected directly into the now empty center of the
tumor. A therapeutic composition can be directly administered to
the surface of a tumor, for example, by topical application of the
composition. X-ray imaging can be used to assist in certain of the
above delivery methods. Combination therapeutic agents, including a
protein or polypeptide or a subgenomic polynucleotide and other
therapeutic agents, can be administered simultaneously or
sequentially.
[0110] Receptor-mediated targeted delivery can be used to deliver
therapeutic compositions containing subgenomic polynucleotides,
proteins, or reagents such as antibodies, ribozymes, or antisense
oligonucleotides of the invention to specific tissues.
Receptor-mediated delivery techniques are described in, for
example, Findeis et al. (1993), Trends in Biotechnol. 11, 202-05;
Chiou et al. (1994), GENE THERAPEUTICS. METHODS AND APPLICATIONS OF
DIRECT GENE TRANSFER (J. A. Wolff, ed.); Wu & Wu (1988), J.
Biol. Chem. 263, 621-24, Wu et al. (1994), J. Biol. Chem. 269,
542-46; Zenke et al (1990), Proc. Natl. Acad. Sci. U.S.A. 87:
3655-59; Wu et al. (1991), J. Biol. Chem. 266, 338-42.
[0111] Alternatively, therapeutic compositions can be introduced
into human cells ex vivo and the cells then replaced into the
human. Cells can be removed from a variety of locations including,
for example, from a selected tumor or from an affected organ. In
addition, a therapeutic composition can be inserted into
non-affected, for example, dermal fibroblasts or peripheral blood
leukocytes. If desired, particular fractions of cells such as a T
cell subset or stem cells can also be specifically removed from the
blood (see, for example, PCT WO 91/16116). The removed cells can
then be contacted with a therapeutic composition utilizing any of
the above-described techniques, followed by the return of the cells
to the human, preferably to or within the vicinity of a tumor or
other site to be treated. The methods described above can
additionally comprise the steps of depleting fibroblasts or other
non-contaminating tumor cells subsequent to removing tumor cells
from a human, and/or the step of inactivating the cells, for
example, by irradiation.
[0112] Both the dose of a composition and the means of
administration can be determined based on the specific qualities of
the therapeutic composition, the condition, age, and weight of the
patient, the progression of the disease, and other relevant
factors. Preferably, a therapeutic composition of the invention
increases or decreases expression of a polynucleotide by 50%, 60%,
70%, or 80%. Most preferably, expression of the polynucleotide is
increased or decreased by 90%, 95%, 99%, or 100%. The effectiveness
of the mechanism chosen to alter expression of the polynucleotide
can be assessed using methods well known in the art, such as
hybridization of nucleotide probes to mRNA of the polynucleotide,
quantitative RT-PCR, or detection of a protein or polypeptide using
specific antibodies.
[0113] If the composition contains protein, polypeptide, or
antibody, effective dosages of the composition are in the range of
about 5 .mu.g to about 50 .mu.g/kg of patient body weight, about 50
.mu.g to about 5 mg/kg, about 100 .mu.g to about 500 .mu.g/kg of
patient body weight, and about 200 to about 250 .mu.kg.
[0114] Therapeutic compositions containing subgenomic
polynucleotides can be administered--in a range of about 100 ng to
about 200 mg of DNA for local administration in a gene therapy
protocol. Concentration ranges of about 500 ng to about 50 mg,
about 1 .mu.g to about 2 mg, about 5 .mu.g to about 500 .mu.g, and
about 20 .mu.g to about 100 .mu.g of DNA can also be used during a
gene therapy protocol. Factors such as method of action and
efficacy of transformation and expression are considerations that
will effect the dosage required for ultimate efficacy of the
subgenomic polynucleotides. Where greater expression is desired
over a larger area of tissue, larger amounts of subgenomic
polynucleotides or the same amounts re-administered in a successive
protocol of administrations, or several administrations to
different adjacent or close tissue portions of, for example, a
tumor site, may be required to effect a positive therapeutic
outcome. In all cases, routine experimentation in clinical trials
will determine specific ranges for optimal therapeutic effect.
[0115] The therapeutic compositions are useful in treating
pancreatic cancer and pancreatic dysplasia, as well as other types
of cancers such as: bone cancer; brain tumors; breast cancer;
endocrine system cancers, such as cancers of the thyroid,
pituitary, and adrenal glands and the pancreatic islets;
gastrointestinal cancers, such as cancer of the anus, colon,
esophagus, gallbladder, stomach, liver, and rectum; genitourinary
cancers such as cancer of the penis, prostate and testes;
gynecological cancers, such as cancer of the ovaries, cervix,
endometrium, uterus, fallopian tubes, vagina, and vulva; head and
neck cancers, such as hypopharyngeal, laryngeal, oropharyngeal
cancers, lip, mouth and oral cancers, cancer of the salivary gland,
cancer of the aerodigestive tract and sinus cancer, leukemia;
lymphomas including Hodgkin's and non-Hodgkin's lymphoma,
metastatic cancer; myelomas; sarcomas; skin cancer; urinary tract
cancers including bladder, kidney and urethral cancers; and
pediatric cancers, such as pediatric brain tumors, leukemia,
lymphomas, sarcomas, liver cancer and neuroblastoma and
retinoblastoma.
[0116] The following example provides data and experimental
procedures. However, the invention is not limited to the example.
The invention is defined in the specification as a whole which
includes the claims.
Example 1
[0117] A family was identified that had several members who had
been diagnosed with pancreatic cancer. The pathological features of
disease in the family included progression from normal to
metaplasia to dysplasia to cancer. Tissues were obtained from a
member of the family diagnosed with pancreatic cancer, from a
member of the family diagnosed with dysplasia of pancreatic cells,
from a person unrelated to the family diagnosed with pancreatitis,
and from a person unrelated to the family with a normal
pancreas.
[0118] Ductal cells from the tissues of each of these subjects were
cultured and mRNA was isolated from the cultures. The mRNA was
subjected to reverse transcriptase polymerase chain reaction using
200 primer pairs (10 anchored and 20 arbitrary primers). The
resulting cDNA was subjected to a differential display in which the
cDNA from each of the 4 samples were compared on a gel. Bands of
cDNA that appeared to be up-or down-regulated in the dysplastic or
pancreatic cancer samples, as compared to the normal and
pancreatitis samples, were cut from the gel, amplified, cloned, and
sequenced.
[0119] The following polynucleotides sequences, as shown in SEQ ID
NOS: 1-5, were identified as being mis-regulated in pancreatic
cancer or dysplasia or both:
TABLE-US-00001 TABLE 1 Up-Regulated and Down-Regulated
Polynucleotides in Pancreatic Cancer and Dysplasia SEQ ID NO
Regulation Status SEQ ID NO: 1 Up in dysplasia only SEQ ID NO: 2 Up
in dysplasia and cancer SEQ ID NO: 3 Up in dysplasia only SEQ ID
NO: 4 Up in dysplasia only SEQ ID NO: 5 Up in dysplasia and cancer
SEQ ID NO: 6 Up in dysplasia only SEQ ID NO: 7 Up in dysplasia only
SEQ ID NO: 8 Up in dysplasia only SEQ ID NO: 9 Up in dysplasia only
SEQ ID NO: 10 Up in dysplasia only SEQ ID NO: 11 Up in dysplasia
only SEQ ID NO: 12 Down in cancer only SEQ ID NO: 13 Up in
dysplasia only SEQ ID NO: 14 Up in dysplasia only SEQ ID NO: 15 Up
in dysplasia and cancer
[0120] All patents, published patent applications and publications
cited herein are incorporated by reference as if set forth fully
herein.
[0121] Although certain preferred embodiments have been described
herein, it is not intended that such embodiments be construed as
limitations on the scope of the invention except as set forth in
the following claims.
Sequence CWU 1
1
151492DNAHomo sapien 1atgaactcgg tttaagacag ggcttcttca ccattgcgag
agcgttcacc gggacgagtg 60gcaagagtct tggcttggat agcatgaaga gccccagtac
aaggaagaat actggaaatg 120ctcaattcgt ggagcgcgtt taaacgacga
tttatttggt tttcaatgac cgaggactta 180tgacaggatg attacatttg
accttgggac atgaacgctt ggactgctga cttgtgtgta 240aagctgtttt
gtttgtttgt gtcttgcttg acagtggttc tcgatcatgg tgatacctga
300tgctttggac atgtccactt actcctctat tattcgttgg atcattgttt
attctgatag 360atagtgactt atgttcggat gtcgatcaca ggattgtgat
tgttagtcca ctgtatctct 420gatcgaatag gtctatatat tattatttag
atagaaaaag tagcaatcca cttaggagat 480ttattgatct gc 4922545DNAHomo
sapien 2tcaggtttga ggctggaaaa agaatcatcc cttcctttcg agttgagatt
gtttctcatt 60ttataagtag cttttatttt atttgaaatt tgaatttctc ttaaaatggt
agagtatacc 120aactttacag aaaggggaaa aaagtcacct actgactgaa
cacagctttt accaatttga 180gcgtctcctt gcagtctttt gaaatacgta
tatgggttac accattgtaa acatgtgttc 240agagcttgca attcataaat
atgtttatgt ccgttatcta atgtgagctc aaaacacaat 300aagagggtca
gggttgtgaa gaaggcagga caggaattat ttaacccatt tttcaaatga
360gaaaactgtg gcccagatac agaatgtcac ttgctaaaat cacatacatt
gaaaccagtt 420ctctccagca tgtcacagtg cttctgtgtt agagcccaag
ttacaaacca aagtgtacaa 480gggcacagat tattagcaat ttacatttaa
aaatttttat atttcctaac tgatacatat 540taatt 5453978DNAHomo sapien
3gtaggtgttg tatttctact ttacaggtag gaaaatggag gctaagaaaa gttaatttgt
60ccgagggccc tctgatgata gtgaaactgg gatggaacct ctgcctgctt gcttctgagg
120tctgggctcc taactactgc tctactgcct cgagccaaga gatttacgcc
ctattaagca 180atttgttgtg cgataaattg gaagacacag cagataagca
aacaactcaa gcaaccaggt 240cggttcctgg agtttctgaa ttgttgggac
caaggggccg tgcagaggta accacagctg 300gcgtagtgtg gttgaggtag
ccctattagc cttttagttg ctgttactaa tttatttctc 360agtggtcaat
gaaccaattg gccatcaatc agctttgtgt ataggtcatg ctcccatggc
420tctgacccag gttgctgctc agagttggca tcgtggctaa aatattacta
gaggtcaaag 480atatgtgtgt gtttgtggtt gatttagtcg agtgatctag
aggaatctga accttagaga 540ctgaagaaga accagcattt ctgggcaata
atacttgagt taaggagagt gtagcaaaac 600tctaggttag cattggcagt
ccctaggatt cagactgtag gcctaaatga ccctcagtcc 660agagctgtac
ctaatgagga caatacattt taatgtgagt ccattcttaa cagcaaaatt
720tcctctttgc ttgtcaccag ggaaaaatgg gtttgcatag aaaaggtgga
gattgagggg 780gaagcagaat ggacaaggag taaagaggga atccaactac
ttagatttga gctttcgttc 840ttctttggta gttgtagagg tgagcttacc
aaagcataga tgacaggcaa tgtggtatac 900aagttactac actccaaaag
tctggggttc ttacttattt tgtgcatgac atccaaagta 960gcctaataaa atcttttc
9784978DNAHomo sapien 4gtagatgttg tatttctact ttacaggtag gaaaatggag
gctaagaaaa gttaatttgt 60ccgagggccc tctgatgata gtgaaactgg gatggaacct
ctgcctgctt gcttctgagg 120tctgggctcc taactactgc tctactgcct
cgagccaaga gatttacgcc ctattaagca 180atttgttgtg cgataaattg
gaagacacag cagataagca aacaactcaa gcaaccaggt 240cagttcctgg
agtttctgaa ttgttgggac caaggggccg tgcagaggta accacagctg
300gcgtagtgtg gttgaggtag ccctattagc cttttagttg ctgttactaa
tttatttctc 360agtggtcaat gaaccaattg gccatcaatc agctttgtgt
ataggtcatg ttcccatggc 420tctgacccag gttgctgctc agagttggca
tcgtggctaa aatattacta gaggtcaaag 480atatgtgtgt gtttgtggtt
gatttagtcg agtgatctag aggaatctga accttagaga 540ctgaagaaga
accagcattt ctgggcaata atacttgagt taaggagagt gtagcaaaac
600tctaggttag cattggcagt ccctaggatt cagactgtag gcctaaatga
ccctcagtcc 660agagctgtac ctaatgagga caatacattt taatgtgagt
ccattcttaa cagcaaaatt 720tcctctttgc ttgtcaccag ggaaaaatgg
gtttgcatag aaaaggtgga gattgagggg 780gaagcagaat ggacaaggag
taaagaggga atccaactac ttagatttga gctttcgttc 840ttctttggta
gttgtagagg tgagcttacc aaagcataga tgacaggcaa tgtggtatac
900aagttactac actccaaaag tctggggttc ttacttattt tgtgcatgac
atccaaagta 960gcctaataaa atcttttc 9785539DNAHomo sapien 5aatagacatt
atactttcta tgtgtggaaa agagtttttc aaagatatga aactgtaaaa 60tatttgttag
ttccagccta tatatttgct ggttggagta tagctgactc attgaaatca
120aagtcaattt tttggaattt aatgtttttc atatgcttgt tcactgttat
agttcctcag 180aaactgctgg aatttcgtta cttcatttta ccttatgtca
tttataggct taacatacct 240ctgcctccca catccagact catttgtgaa
ctgagctgct atgcagttgt taatttcata 300acttttttca tctttctgaa
caagactttt cagtggccaa atagtcagga cattcaaagg 360tttatgtggt
aatatcagtg atatttcgaa ctgtgaaaat ggacttaata attagaccat
420ttctacaaag aacaactgaa taggtggaaa acatggaatt tcttttaggt
gcagtggtgg 480tcttcaaatt acattagttt tttttatata tattttaaac
atatgtaaga aattaagtg 5396491DNAHomo sapien 6atgaactcgg tttaagacag
ggcttcttca ccattgcgag aacgttcacc gggacgagtg 60gcaagagtct tggcttggat
agcatgaaga gccccagtac aaggaagaat actggaaatg 120ctcaattcgt
ggagcgcgtt taaacgacga tttatttggt tttcaatgac cgaggactta
180tgacaggatg attacatttg accttgggac atgaacgctt ggactgctga
cttgtgtgta 240aagctgtttg tttgtttgtg tcttgcttga cagtggttct
cgatcatgat gatacctgat 300gctttggaca tgtccactta ctcctctatt
attcgttgga tcattgttta ttctgataga 360tagtgactta tgttcggatg
tcgatcacag gattgtgatt gttagtccac tgtatctctg 420atcgaatagg
tctatatatt attatttaga tagaaaaagt agcaatccac ttaggagatt
480tattgatctg c 4917491DNAHomo sapien 7atgaactcgg tttaagacag
ggcttcttca ccattgcgag aacgttcacc gggacgagtg 60gcaagagtct tggcttggat
agcatgaaga gccccagtac aaggaagaat actggaaatg 120ctcaattcgt
ggagcgcgtt taaacgacga tttatttggt tttcaatgac cgagacttat
180gacaggatga ttacatttga ccttgggaca tgaacgcttg gactgctgac
ttgtgtgtaa 240agctgttttg tttgtttgtg tcttgcttga cagtggttct
cgatcatgat gatacctgat 300gctttggaca tgtccactta ctcctctatt
attcgttgga tcattgttta ttctgataga 360tagtgactta tgttcggatg
tcgatcacag gattgtgatt gttagtccac tgtatctctg 420atcgaatagg
tctatatatt attatttaga tagaaaaagt agcaatccac ttaggagatt
480tattgatctg c 4918492DNAHomo sapien 8gcagatcaat aaatctccta
agtggattgc tactttttct atctaaataa taatatatag 60acctattcga tcagagatac
agtggactaa caatcacaat cctgtgatcg acatccgaac 120ataagtcact
atctatcaga ataaacaatg atccaacgaa taatagagga gtaagtggac
180atgtccaaag catcaggtat catcatgatc gagaaccact gtcaagcaag
acacaaacaa 240acaaaacagc tttacacaca agtcagcagt ccaagcgttc
atgtcccaag gtcaaatgta 300atcatcctgt cataagtcct cggtcattga
aaaccaaata aatcgtcgtt taaacgcgct 360ccacgaattg agcatttcca
gtattcttcc ttgtactggg gctcttcatg ctatccaagc 420caagactctt
gccactcgtc ccggtgaacg ttctcgcaat ggtgaagaag ccctgtctta
480aaccgagttc at 4929492DNAHomo sapien 9atgaactcgg tttaagacag
ggcttcttca ccattgcgag aacgttcacc gggacgagtg 60gcaagagtct tggcttggat
agcatgaaga gccccagtac aaggaagaat actggaaatg 120ctcaattcgt
ggagcgcgtt taaacgacga tttatttggt tttcaatgac cgaggactta
180tgacaggatg attacatttg accttgggac atgaacgctt ggactgctga
cttgtgtgta 240aagctgtttt gtttgtttgt gtcttgcttg acagtggttc
tcgatcatga tgatacctga 300tgctttggac atgtccactt actcctctat
tattcgttgg atcattgttt attctgatag 360atagtgactt atgttcggat
gtcgatcaca gggttgtgat tgttagtcca ctgtatctct 420gatcgaatag
gtctatatat tattatttag atagaaaaag tagcaatcca cttaggagat
480ttattgatct gc 49210492DNAHomo sapien 10atgaactcgg tttaagacag
ggcttcttca ccattgcgag aacgttcacc gggacgagtg 60gcaagagtct tggcttggat
agcatgaaga gccccagtac aaggaagaat actggaaatg 120ctcaattcgt
ggagcgcgtt taaacgacga tttatttggt tttcaatgac cgaggactta
180tgacaggatg attacatttg accttgggac atgaacgctt ggactgctga
cttgtgtgta 240aagctgtttt gtttgtttgt gtcttgcttg acagtggttc
tcgatcatga tgatacctga 300tgctttggac atgtccactt actcctctat
tattcgttgg atcattgttt attctgatag 360atagtgactt atgttcggat
gtcgatcaca ggattgtgat tgttagtcca ctgtatctct 420gatcgaatag
gtctatatat tattatttag atagaaaaag tagcaatcca cttaggagat
480ttattgatct gc 49211492DNAHomo sapien 11atgaactcgg tttaagacag
ggcttcttca ccattgcgag aacgttcacc gggacgagtg 60gcaagagtct tggcttggat
agcatgaaga gccccagtac aaggaagaat actggagatg 120ctcaattcgt
ggagcgcgtt taaacgacga tttatttggt tttcaatgac cgaggactta
180tgacaggatg attacatttg accttgggac atgaacgctt ggactgctga
cttgtgtgta 240aagctgtttt gtttgtttgt gtcttgcttg acagtggttc
tcgatcatga tgatacctga 300tgctttggac atgtccactt actcccctat
tattcgttgg atcattgttt attctgatag 360atagtgactt atgttcggat
gtcgatcaca ggattgtgat tgttagtcca ctgtatctct 420gatcgaatag
gtctatatat tattatttag atagaaaaag tagcaatcca cttaggagat
480ttattgatct gc 49212826DNAHomo sapien 12tttaggcttc tgcaggggac
tctgtacatg tgcgttggca ttatggatcg atttttacag 60gttcagccag tttcccggaa
gaagcttcaa ttagttggga ttactgctct gctcttggcc 120tccaagtatg
aggagatgtt ttctccaaat attgaagact ttgtttacat cacagacaat
180gcttatacca gttcccaaat ccgagaaatg gaaactctaa ttttgaaaga
atcgaaattt 240gagttgggtc gacccttgcc actacacttc ttaaggcgag
catcaaaagc cggggaggtt 300gatgttgaac agcacacttt agccaagtat
ttgatggagc tgactctcat cgactatgat 360atggtgcatt atcatccttc
taaggtagca gcagctgctt cctgcttgtc tcagaaggtt 420ctaggacaag
gaaaatggaa cttaaagcag cagtattaca caggatacac agagaatgaa
480gtattggaag tcatgcagca catggccaag aatgtggtga aagtaaatga
aaacttaact 540aaattcatcg ccatcaagaa taagtatgca agcagcaaac
tcctgaagat cagcatgatc 600cctcagctga actcaaaagc cgtcaaagac
cttgcctctc cactgatagg aaggtcctag 660gctgccgtgg cccctgggga
tgtgtgcttc attgtgccct ttttcttatt ggtttagaac 720tcttgatttt
gtacatagtc ctctggtcta tctcatgaaa cctcttctca gaccagtttt
780ctaaacatat attgaggaaa aataaagcga ttggtttttc ttaagg
82613819DNAHomo sapien 13ctggagagaa aacccataaa tgccccgaat
gtgggagagc ctttttttat cagtcattcc 60ttatgagaca tatgaaaatt cacactggag
agaaaccgta tgaatgtggg aaatgtggga 120aagcctttag atattcctta
caccttaata aacatttaag aaagcatgtt gtgcagaaga 180agccctacga
atgtgaagaa tgtgggaaag tcattcggga gtcctcaaaa tatacacata
240taaggagcca cactggagag aaaccctata aatgtaagac atgtggaaaa
gactttgcaa 300agtcgccagg acttaaaaaa catcttaaga ctcacaaaga
tgagaagccc tgtgaatgaa 360aggaaggtgg aaaatttttc attaattttc
tgactgtacc aaacatgtga ggaggacata 420ttggaaggga gctcaagggg
ttagcatgag tgagaacatc ttccctgaac tctcgtatct 480tacagaaatg
tgaaaaaaaa ccctgtgaag gtaaagtcta cagaaagcct ttcatcttca
540ttcatcttga gtagacattt gttctcaccc tggagagaaa ctgcgaatct
aaaaggaata 600tgacaaagcc ttcagcgtgg tctcaaattc atggttcata
caagaactca cactgcagag 660actccttacg gaaataaaaa atgtaggaaa
gacctgccgg ccgcggtggc tcatgcctgt 720aatcccagca ctttgggagg
ccgaggcggg cggatcacga ggtcaggaga tcaagaccat 780cctggctaac
acggtgatac cccgtctcta ctaaaaata 819141386DNAHomo sapien
14gtagatgttg tatttctact ttacaggtag gaaaatggag gctaagaaaa gttaatttgt
60ccgagggccc tctgatgata gtgaaactgg gatggaacct ctgcctgctt gcttctgagg
120tctgggctcc taactactgc tctactgcct cgagccaaga gatctacgcc
ctattaagca 180atttgttgtg cgataaattg gaagacacag cagataagca
aacaactcaa gcaaccaggt 240cagttcctgg agtttctgaa ttgttgggac
caaggggccg tgcagaggta accacagctg 300gcgtagtgtg gttgaggtag
ccctattagc cttttagttg ctgttactaa tttatttctc 360agtggtcaat
gaaccaattg gccatcaatc agctttgtgt ataggtcatg ttcccatggc
420tctgacccag gttgctgctc agagttggca tcgtggctaa aatattacta
gaggtcaaag 480atatgtgtgt gtttgtggtt gatttagtcg agtgatctag
aggaatctga accttagaga 540ctgaagaaga accagcattt ctgggcaata
atacttgagt taaggagagt gtagcaaaac 600tctaggttag cattggcagt
ccctagaatt cagactgtag gcctaaatga ccctcagtcc 660agagctgtac
ctaatgagga caatacattt taatgtgagt ccattcttaa cagcaaaatt
720tcctctttgc ttgtcaccag ggaaaaatgg gtttgcatag aaaaggtgga
gattgagggg 780gaagcagaat ggacaaggag taaagaggga atccaactac
ttagatttga gctttcgttc 840ttctttggta gttgtagagg tgagcttacc
aaagcataga tgacaggcaa tgtggtatac 900aagttactac actccaaaag
tctggggttc ttacttattt tgtgcatgac atccaaagta 960gcctaataaa
atcttttcac agaaaaaaaa gctttacttt cctttgccaa atttttaact
1020ttttattctg aaataatttc agaattattg aaaaatttag agactaggac
aacccagatt 1080cctcaaatat taacacttta ccacatctgc cttctcattc
ctctctatat acataggtgc 1140atgtgtggtt ttaatgttta tttatataca
tatcattatt attttcttaa ctgtttgaga 1200gtaagttgaa gacatgatgc
tccttactct ttaaatactt cagtgtgtat ttcctaaaaa 1260gcaggccatg
ttctacatca tcacagtata attatcaaaa ttgggaaatt aatattaatg
1320caatactatt tatcaaattt taagatctta ttcaaatttc acttgctggc
ctaataatgt 1380tctttc 138615547DNAHomo sapien 15aaaagtctgc
tttgaggcaa aggtaaccca gaatctccca atgaaagaag gctgcacaga 60ggtctctctc
cttcgagttg ggtggtctgt tgatttttcc cgtccacagc ttggtgaaga
120tgaattctct tacggtttcg atggacgagg actcaaggca gaaaatggac
aatttgagga 180atttggccag acttttgggg agaatgatgt tattggctgc
tttgctaatt ttgagactga 240agaagtagaa ctttccttct ccaagaatgg
agaagaccta ggtgtggcat tctggatcag 300caaggattcc ctggcagacc
gggcccttct accccatgtc ctctgcaaaa attgtgttgt 360agaattaaac
ttcggtcaga aggaggagcc cttcttccca ccaccagaag agtttgtgtt
420cattcatgct gtgcctgttg aggagcgtgt acgcactgca gtccctccca
agaccacaga 480ggaatgtgag gtgattctga tggtgggact acccggatct
ggaaagaccc agtgggcact 540gaaatat 547
* * * * *