U.S. patent number 6,897,018 [Application Number 09/644,947] was granted by the patent office on 2005-05-24 for dlc-1 gene deleted in cancers.
This patent grant is currently assigned to The United States of America as represented by the Department of Health and Human Services, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Nicholas Popescu, Snorri S. Thorgeirsson, Bao-Zhu Yuan.
United States Patent |
6,897,018 |
Yuan , et al. |
May 24, 2005 |
DLC-1 gene deleted in cancers
Abstract
A cDNA molecule corresponding to a newly discovered human gene
is disclosed. The new gene, which is frequently deleted in liver
cancer cells and cell lines, is called the DLC-1 gene. Because the
gene is frequently deleted in liver cancer cells, but present in
normal cells, it is thought to act as a tumor suppressor. This gene
is also frequently deleted in breast and colon cancers, and its
expression is decreased or undetectable in many prostate and colon
cancers. Also disclosed is the amino acid sequence of the protein
encoded by the DLC-1 gene. Methods of using these biological
materials in the diagnosis and treatment of hepatocellular cancer,
breast cancer, colon cancer, prostate cancer, and adenocarcinomas
are presented.
Inventors: |
Yuan; Bao-Zhu (Columbia,
MD), Thorgeirsson; Snorri S. (Bethesda, MD), Popescu;
Nicholas (Bethesda, MD) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
|
Family
ID: |
34576144 |
Appl.
No.: |
09/644,947 |
Filed: |
August 23, 2000 |
Current U.S.
Class: |
435/6.14;
435/91.1; 435/91.2; 435/94; 536/23.1; 536/24.3; 536/24.31 |
Current CPC
Class: |
C07K
14/4703 (20130101); A61K 48/00 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/47 (20060101); A61K
48/00 (20060101); C12Q 001/68 () |
Field of
Search: |
;435/6,91.1,91.2,94
;536/23.1,24.3,24.31 |
References Cited
[Referenced By]
U.S. Patent Documents
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5330892 |
July 1994 |
Vogelstein et al. |
5571905 |
November 1996 |
Vogelstein et al. |
5576422 |
November 1996 |
Vogelstein et al. |
5624819 |
April 1997 |
Skolnick et al. |
5654155 |
August 1997 |
Murphy et al. |
5693473 |
December 1997 |
Shattuck-Eidens et al. |
5693536 |
December 1997 |
Vogelstein et al. |
5709999 |
January 1998 |
Shattuck-Eidens et al. |
5710001 |
January 1998 |
Skolnick et al. |
5912143 |
June 1999 |
Bandman et al. |
|
Other References
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Friend et al., New England J. of Medicine 338(2):125-126 (1998).
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Chinen et al., "Isolation of 45 exon-like fragments from
8p22.fwdarw.p21.3, a region that is commonly deleted in
hepatocellular, colorectal, and non-small cell lung carcinomas,"
Cytogenet Cell Genet, vol. 75, pp. 190-196 (1996). .
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vol. 21, pp. 178-181 (1996). .
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Ortholog of the Rat Dual Regulator p122RhoGAP," EMBL Database Entry
AF026219 (Abstract, 1977). .
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2196-2199 (1998)..
|
Primary Examiner: Ungar; Susan
Assistant Examiner: Davis; Minh-Tam
Attorney, Agent or Firm: Klarquist Sparkman, LLP
Parent Case Text
This application claims priority under 35 U.S.C. 120 from
PCT/US99/04164, filed Feb. 25, 1999, and claims benefit of U.S.
Provisional Patent Application No. 60/075,952, filed Feb. 25, 1998,
which are herein incorporated by reference.
Claims
We claim:
1. A method of diagnosing liver cancer in a subject, comprising:
detecting a deletion comprising exon 2 of a nucleic acid encoding
SEQ ID NO: 2 in a sample from the subject, wherein detection of the
deletion comprising exon 2 of a nucleic acid encoding SEQ ID NO: 2
is indicative of liver cancer.
2. The method of claim 1, wherein the sample is a peripheral blood,
a urine, a saliva, a tissue biopsy, a surgical specimen, or an
autopsy sample.
3. The method of claim 1, wherein the detection is by an
amplification reaction, a hybridization reaction, or a change in
electrophoretic mobility.
4. The method of claim 1, wherein the detection is by amplification
reaction, and the amplification reaction is polymerase chain
reaction.
5. The method of claim 1, wherein the sample is a tissue biopsy, a
surgical specimen, or an autopsy sample.
Description
FIELD OF THE INVENTION
The present invention relates to the cloning and sequencing of the
human cDNA molecule corresponding to a newly discovered gene,
called DLC-1, which is frequently deleted in liver, breast and
colon cancer cells. In addition, lower DLC-1 expression is
frequently observed in liver, colon, and prostate cancer cells,
compared to normal tissue. The present invention also relates to
methods for screening and diagnosis of a genetic predisposition to
liver cancer and other cancer types, and methods of gene therapy
utilizing recombinant DNA technologies.
BACKGROUND OF THE INVENTION
The isolation of genes involved in human cancer development is
critical for uncovering the molecular basis of cancer. One theory
of cancer development holds that there are tumor suppressor genes
in all normal cells which, when they become non-functional due to
mutations, cause neoplastic development (Knudsen et al., Cancer
Res. 45:1482, 1985). Evidence to support this theory has been found
in the cases of human retinoblastoma and colorectal tumors (see
U.S. Pat. No. 5,330,892 and references cited therein), as well as
in connection with breast and ovarian cancers (see U.S. Pat. Nos.
5,693,473 and references cited therein).
More particularly, recurrent deletions on the short arm of human
chromosome 8 in cases of liver, breast, lung and prostate cancers
have raised the possibility of the presence of tumor suppressor
genes in that location. For example, loss on the short arm of
chromosome 8 in prostrate cancer (PC) cells was described in
Brothman (Cancer Genet. Cytogenet. 95:116-21, 1997). Similar
deletions on the short arm of chromosome 8 also have been detected
in primary hepatocellular cancer (HCC), non-small cell lung
carcinoma (NSCLC) and node-negative breast carcinomas (Isola, Am.
J. Pathol. 147:905-11, 1995; and Marchio, et al., Genes Chromo.
Canc. 18:59-65, 1997).
While recurrent chromosome 8 deletions in malignant tumors support
the relevance of this lesion in carcinogenesis, scientists
previously have been unable to identify the tumor suppressor genes
involved in such deletions. This lack of knowledge concerning the
molecular genetic basis of HCC, and other cancers associated with
chromosome 8 deletions, has hampered efforts to diagnose the
predisposition to such diseases and to develop more effective
treatments aimed at curing genetic deficiencies.
Therefore, it is an object of the present invention to provide a
human cDNA molecule corresponding to a previously unknown gene
located on the short arm of chromosome 8, the deletion of which
appears to be closely associated with the development of HCC and
other cancers. The cloning and sequencing of such a cDNA molecule
enables new and improved methods of diagnosis and treatment of such
diseases.
SUMMARY OF THE INVENTION
The present invention discloses the discovery of new human gene
involved in the pathogenesis of hepatocellular cancer (HCC), the
most common primary liver cancer, and one of the most common
cancers in the world, with 251,000 new cases reported each year.
(Simonetti et al., Dig. Dis. Sci. 36:962-72, 1991; Harris et al.,
Cancer Cells 2:146-8, 1990; Marchio, et al., Genes Chromo. Cancer
18:59-65, 1997). More specifically, the present invention discloses
the isolation of the full length cDNA and the chromosomal
localization of a new gene which is frequently deleted in liver
cancer, and hence is named the DLC-1 gene.
The full-length cDNA for DLC-1 is 3850 bp long (Seq. I.D. No. 1),
encodes a protein of 1091 amino acids (Seq. I.D. No. 2), and was
localized by fluorescence in situ hybridization to chromosome 8 at
bands p210.3-22. Because the DLC-1 gene is deleted from a
significant percentage of primary HCC tumor cells and cell lines,
primary breast cancers (BC), and colorectal cancer (CRC) cell
lines, and its expression is decreased or not observed in a
significant percentage of HCC cell lines, CRC cell lines and
prostate cancer (PC) cell lines, the DLC-1 gene appears to operate
as a tumor suppressor in liver cancer and other cancers including
PC, CRC and BC.
The object of identifying the hitherto unknown DLC-1 gene has been
achieved by providing an isolated human cDNA molecule which is able
specifically to correct the cellular defects characteristic of
cells from patients with a deleted or mutated DLC-1 gene.
Specifically, the invention provides, for the first time, an
isolated cDNA molecule which, when transfected into cells derived
from a patient with a deleted or mutated DLC-1 gene, can produce
the DLC-1 protein believed to be active in suppressing HCC
pathogenesis and other cancers, such as breast, colorectal, and
prostate cancers. The invention encompasses the DLC-1 cDNA molecule
(derived from normal human liver cells), the nucleotide sequence of
this cDNA, and the putative amino acid sequence of the DLC-1
protein encoded by this cDNA.
Having herein provided the nucleotide sequence of the DLC-1 cDNA,
correspondingly provided are the complementary DNA strands of the
cDNA molecule and DNA molecules which hybridize under stringent
conditions to the DLC-1 cDNA molecule or its complementary strand.
Such hybridizing molecules include DNA molecules differing only by
minor sequence changes, including nucleotide substitutions,
deletions and additions. Also comprehended by this invention are
isolated oligonucleotides comprising at least a segment of the cDNA
molecule or its complementary strand, such as oligonucleotides
which may be employed as effective DNA hybridization probes or
primers useful in the polymerase chain reaction or as hybridization
probes. Such probes and primers are particularly useful in the
screening and diagnosis of persons genetically predisposed to HCC,
and other cancers, as the result of DLC-1 gene deletions.
Hybridizing DNA molecules and variants on the DLC-1 cDNA may
readily be created by standard molecular biology techniques.
Through the manipulation of the nucleotide sequence of the human
cDNA provided by this invention by standard molecular biology
techniques, variants of the DLC-1 protein may be made which differ
in precise amino acid sequence from the disclosed protein yet which
maintain the essential characteristics of the DLC-1 protein or
which are selected to differ in one or more characteristics from
this protein. Such variants are another aspect of the present
invention.
Also provided by the present invention are recombinant DNA vectors
comprising the disclosed DNA molecules, and transgenic host cells
containing such recombinant vectors.
Having isolated the human DLC-1 cDNA sequence, the genomic sequence
for the gene was determined according to the following method: A
human genomic library constructed using the P1 vector, pAD10SacBII,
was transferred from its original E coli host into a second E. coli
host, strain N3516, following procedures well-known in the art. A
positive P1 clone containing the DLC-1 gene was then obtained by
performing a protocol of PCR-based P1 library screening (Sheperd,
Proc. Nail. Acad. Sci. USA 91:2629-33, 1994; Neuhausen, Hum. Mol.
Genet. 3:1919-26, 1994). The PCR primers used in this screening,
designed from a genomic fragment isolated through Representational
Difference Analysis (described more fully below), are listed below:
PL7-3F 5' GACACCACCATCTCTGTGCTC 3' (Seq. I.D. No. 7) PL7-3R 5'
GCAGACTGTCCTTCGTAGTTG 3' (Seq. I.D. No. 8)
An isolated and purified biological sample of this genomic DLC-1
gene was deposited with the American Type Culture Collection (ATCC)
in Manassas, Va., on Feb. 25, 1998, under accession number 98676.
The present invention also provides for the use of the DLC-1 cDNA,
the corresponding genomic gene and of the DLC-1 protein, and
derivatives thereof, in aspects of diagnosis and treatment of HCC,
and other cancers including, but not limited to PC, BC and CRC,
resulting from DLC-1 deletion or mutation.
An embodiment of the present invention is a method for screening a
subject to determine if the subject carries a mutant DLC-1 gene, or
if the gene has been partially or completely deleted, as is thought
to occur in many HCC cases. The method comprises the steps of:
providing a biological sample obtained from the subject, which
sample includes DNA or RNA, and providing an assay for detecting in
the biological sample the presence of a mutant DLC-1 gene, a mutant
DLC-RNA, or the absence, through deletion, of the DLC-1 gene and
corresponding RNA.
The foregoing assay may be assembled in the form of a diagnostic
kit and preferably comprises either: hybridization with
oligonucleotides; PCR amplification of the DLC-1 gene or a part
thereof using oligonucleotide primers; RT-PCR amplification of the
DLC-1 RNA or a part thereof using oligonucleotide primers; or
direct sequencing of the DLC-1 gene of the subject's genome using
oligonucleotide primers. The efficiency of these molecular genetic
methods should permit a rapid classification of patients affected
by deletions or mutations of the DLC-1 gene.
A further aspect of the present invention is a method for screening
a subject to assay for the presence of a mutant or deleted DLC-1
gene, comprising the steps of: providing a biological sample of the
subject which sample contains cellular proteins, and providing an
immunoassay for quantitating the level of DLC-1 protein in the
biological sample. Diagnostic methods for the detection of mutant
or deleted DLC-1 genes made possible by this invention will provide
an enhanced ability to diagnose susceptibility to HCC and other
cancers such as PC, BC and CRC.
Another aspect of the present invention is an antibody preparation
comprising antibodies that specifically detect the DLC-1 protein,
wherein the antibodies are selected from the group consisting of
monoclonal antibodies and polyclonal antibodies.
Those skilled in the art will appreciate the utility of this
invention is not limited to the specific experimental modes and
materials described herein.
The foregoing and other features and advantages of the invention
will become more apparent from the following detailed description
and accompanying drawings.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a digital image of a Southern blot which compares primary
HCC tumor cells (T) with healthy normal liver cells (N), and
demonstrates a genomic deletion of the L7-3 clone in the HCC cells.
Primary tumors 94-25T, 95-03T and 95-06T showed 50% decrease of DNA
intensity as compared with normal liver tissues.
FIG. 2 is a digital image of a Southern blot which compares
representative HCC cell lines with healthy liver cells (NL-DNA),
and demonstrates a genomic deletion of the L7-3 clone in 9 of 11
HCC cell lines. Cell lines Sk-Hep-1, PLC/PRF/5, WRL, Focus, HLF,
Hep3B, Huh-7, Huh-6, Chang showed reduction of DNA intensity
compared with human normal liver genomic DNA.
FIG. 3 is a digital image of a Southern blot which compares
representative primary human breast cancers (T) with healthy normal
blood cells (N) from the same patient, and demonstrates a genomic
deletion of the DLC-1 gene in 7 of 15 primary breast cancers. A
representative 10 of the 15 primary tumors are shown. DNA was
digested with either (a) BglII or (b) BamHI. Cell lines IC11T,
IC12T, IC13T, IC2T, IC6T, and IC7T showed reduction of DNA
intensity compared with normal DNA.
FIG. 4 is a digital image of a Southern blot which compares
representative human colon cancer cell lines with normal DNA (lane
1), and demonstrates a genomic deletion of the DLC-1 gene in two
out of five colon cancer cell lines. Cell lines SW 1116 and SW403
(lanes 5 and 6) showed reduction of DNA intensity compared with
normal DNA (lane 1).
FIG. 5 is a digital image of a Northern blot showing the mRNA
expression of the DLC-1 gene in normal human tissues. The DLC-1
gene is expressed in all normal tissues tested as a 7.5 kb major
transcript and a 4.5 kb minor transcript.
FIG. 6 is a digital image of a Northern blot comparing the mRNA
expression of DLC-1 gene in normal human tissues NL-RNA) and HCC
cell lines. DLC-1 mRNA expression was decreased or not detected in
the WRL, 7703, Chang and Focus HCC cell lines.
FIG. 7 is a digital image of a Northern blot comparing the mRNA
expression of DLC-1 in normal human tissues (CDD33C0) and human
colon cancer cell lines. DLC-1 mRNA was expression was decreased or
not detected in HCT-15, LS147T, DLD-1, HD29, SW1116, T84, SW1417,
SW403, SW948, LS180, and SW48 cell lines.
FIG. 8 is a digital image of a Northern blot showing the mRNA
expression or DLC-1 gene in three human prostate cancer cell lines.
DLC-1 mRNA was not detected in the LN-Cap and SP3504 cell
lines.
FIG. 9 is a schematic drawing of the human DLC-1 gene. Exons 1-14
are represented boxes, with introns represented by the lines
connecting the boxes.
FIG. 10 is a schematic drawing of how the mouse DLC-1 gene was
targeted using homologous recombination. The resulting construct
can be used to generate DLC-1 homozygous knock-out mice.
SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying
sequence listing are shown using standard letter abbreviations for
nucleotide bases, and three letter code for amino acids. Only one
strand of each nucleic acid sequence is shown, but the
complementary strand is understood as included by any reference to
the displayed strand.
Seq. I.D. No. 1 is the nucleotide sequence of the human DLC-1
cDNA.
Seq. I.D. No. 2 is the amino acid sequence of the human DLC-1
protein.
Seq. I.D. Nos. 3-4 are oligonucleotide sequences of PCR primers
which can be used to amplify the entire DLC-1 cDNA molecule.
Seq. I.D. Nos. 5-6 are oligonucleotide sequences of PCR primers
which can be used to amplify the open reading frame of the DLC-1
cDNA molecule.
Seq. I.D. Nos. 7-8 are the oligonucleotide sequences of PCR primers
used to screen a human genomic library.
Seq. I.D. Nos. 9-11 are the oligonucleotide sequences of the
primers used for 5' and 3' RACE.
Seq. I.D. No. 12 is the nucleotide sequence for the L7-3 probe.
Seq. I.D. No. 13 is the nucleotide sequence for the P-35 probe.
Seq. I.D. No. 14 is the nucleotide sequence for part of the human
genomic DLC-1 sequence.
Seq. I.D. No. 15 is the nucleotide sequence for part of the human
genomic DLC-1 sequence.
Seq. I.D. No. 16 is the nucleotide sequence for part of the human
genomic DLC-1 sequence.
Seq. I.D. No. 17 is the nucleotide sequence for part of the human
genomic DLC-1 sequence.
Seq. I.D. No. 18 is the nucleotide sequence for part of the human
genomic DLC-1 sequence.
Seq. I.D. No. 19 is the nucleotide sequence for part of the human
genomic DLC-1 sequence.
Seq. I.D. No. 20 is the nucleotide sequence for part of the mouse
genomic DLC-1 sequence.
Seq. I.D. No. 21 is the nucleotide sequence for part of the mouse
genomic DLC-1 sequence.
Seq. I.D. No. 22 is the nucleotide sequence for part of the mouse
genomic DLC-1 sequence.
Seq. I.D. No. 23 is the nucleotide sequence for part of the mouse
genomic DLC-1 sequence.
Seq. I.D. No. 24 is the nucleotide sequence for part of the mouse
genomic DLC-1 sequence.
Seq. I.D. No. 25 is the nucleotide sequence for part of the mouse
genomic DLC-1 sequence.
Seq. I.D. No. 26 is the nucleotide sequence for a cDNA fragment of
the mouse DLC-1 sequence.
Seq. I.D. No. 27 is the nucleotide sequence for a cDNA fragment of
the mouse DLC-1 sequence.
Seq. I.D. No. 28 is the nucleotide sequence for a cDNA fragment of
the mouse DLC-1 sequence.
Seq. I.D. No. 29 is the nucleotide sequence for a cDNA fragment of
the mouse DLC-1 sequence.
Seq. I.D. No. 30 is the nucleotide sequence for a cDNA fragment of
the mouse DLC-1 sequence.
Seq. I.D. No. 31 is the nucleotide sequence for a cDNA fragment of
the mouse DLC-1 sequence.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses the isolation of the full length
cDNA and the chromosomal localization of a new gene, called the
DLC-1 gene. As discussed in Examples 1-3 below, deletion of the
DLC-1 gene has been detected in about half of the primary HCC tumor
cells and in a majority of the HCC cell lines which were studied.
In addition, studies of other cancers revealed that DLC-1 was also
deleted in 7 of 15 primary breast cancers and in 2 of 5 CRC cell
lines. Moreover, the DLC-1 gene was not expressed in 29% of HCC
cell lines, 64% of CRC cell lines and 67% of PC cell lines. These
frequent deletions suggest that the DLC-1 gene is a tumor
suppressor gene for HCC as well as PC, BC and CRC.
The full-length cDNA for DLC-1 is 3850 bp long (Seq. I.D. No. 1)
and encodes a protein of 1091 amino acids (Seq. I.D. No. 2).
Fluorescent in situ hybridization has generally localized the gene
on the short arm of chromosome 8 at bands p21.3-22.
Further evidence that the DLC-1 gene acts as a tumor suppressor is
found in its 86% homology with the rat p122 RhoGAP gene (Homma and
Emori, EMBO. J. 14:286-91, 1995). The rat p122 RhoGAP gene encodes
a GTPase activating protein that catalyzes the conversion of the
active GTP-bound Rho complex to an inactive GDP-bound one. The Rho
family proteins, a subfamily of the Ras small GTP binding
superfamily, function as important regulators in the organization
of actin cytoskeleton (Nobes, et al., Cell 81:53-62, 1995). Rho
proteins are also involved in Ras-mediated oncogenic transformation
(Khosravi-Far, et al., Adv. Cancer Res. 69:59-105, 1997). GAP genes
may function as tumor suppressors by down-regulating oncogenic Rho
proteins (Quilliam, et al. Bioessays 17:395-404, 1995; Wang, et
al., Cancer Res. 57:2478-84, 1997). Based on its substantial
homology with the rat p122 RhoGAP gene, it appears likely the DLC-1
gene is a human RhoGAP gene involved in the suppression of HCC
tumors.
Definitions
In order to facilitate review of the various embodiments of the
invention, the following definition of terms is provided:
Breast Carcinoma (BC): breast cancer thought to result, in some
instances, from the deletion or mutation of the DLC-1 tumor
suppressor gene.
cDNA (complementary DNA): a piece of DNA lacking internal,
non-coding segments (introns) and regulatory sequences which
determine transcription. cDNA is synthesized in the laboratory by
reverse transcription from messenger RNA extracted from cells.
Colorectal Carcinoma (CRC): colorectal cancer (such as
adenocarcinoma) thought to result, in some instances, from the
deletion or mutation of the DLC-1 tumor suppressor gene.
Deletion: the removal of a sequence of DNA, the regions on either
side being joined together.
DLC-1 gene: a gene, the mutation of which is associated with
hepatocellular, breast, colon and prostate carcinomas, and
particularly adenocarcinomas of those organs A mutation of the
DLC-1 gene may include nucleotide sequence changes: additions or
deletions, including deletion of large portions or all of the DLC-1
gene. The term "DLC-1 gene" is understood to include the various
sequence polymorphisms and allelic variations that exist within the
population. This term relates primarily to an isolated coding
sequence, but can also include some or all of the flanking
regulatory elements and/or intron sequences.
DLC-1 cDNA: a mammalian cDNA molecule which, when transfected into
DLC-1 cells, expresses the DLC-1 protein. The DLC-1 cDNA can be
derived by reverse transcription from the mRNA encoded by the DLC-1
gene and lacks internal non-coding segments and transcription
regulatory sequences present in the DLC-1 gene.
DLC-1 protein: the protein encoded by the DLC-1 cDNA, the altered
expression or mutation of which can predispose to the development
of certain cancers, such as hepatocellular carcinoma. This
definition is understood to include the various sequence
polymorphisms that exist, wherein amino acid substitutions in the
protein sequence do not affect the essential functions of the
protein.
DNA: deoxyribonucleic acid. DNA is a long chain polymer which
comprises the genetic material of most living organisms (some
viruses have genes comprising ribonucleic acid (RNA)). The
repeating units in DNA polymers are four different nucleotides,
each of which comprises one of the four bases, adenine, guanine,
cytosine and thymine bound to a deoxyribose sugar to which a
phosphate group is attached. Triplets of nucleotides, referred to
as codons, in DNA molecules code for amino acid in a polypeptide.
The term codon is also used for the corresponding (and
complementary) sequences of three nucleotides in the mRNA into
which the DNA sequence is transcribed.
Hepatocellular carcinoma (HCC): liver cancer thought to result, in
some instances, from the deletion or mutation of the DLC-1 tumor
suppressor gene.
Isolated: requires that the material be removed from its original
environment. For example, a naturally occurring DNA molecule
present in a living animal is not isolated, but the same DNA
molecule, separated from some or all of the coexisting materials in
the natural system, is isolated.
Mutant DLC-1 gene: a mutant form of the DLC-1 gene which in some
embodiments is associated with hepatocellular, breast, colon and/or
prostate carcinoma.
Mutant DLC-1 RNA: the RNA transcribed from a mutant DLC-1 gene.
Mutant DLC-1 protein: the protein encoded by a mutant DLC-1
gene.
Oligonucleotide: A linear polynucleotide sequence of up to about
200 nucleotide bases in length, for example a polynucleotide (such
as DNA or RNA) which is at least 6 nucleotides, for example at
least 15, 50, 100 or even 200 nucleotides long.
ORF: open reading frame. Contains a series of nucleotide triplets
(codons) coding for amino acids without any termination codons.
These sequences are usually translatable into protein.
PCR: polymerase chain reaction. Describes a technique in which
cycles of denaturation, annealing with primer, and then extension
with DNA polymerase are used to amplify the number of copies of a
target DNA sequence.
Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in this invention are conventional.
Remington's Pharmaceutical Sciences, by E. W. Martin, Mack
Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of the fusion proteins herein disclosed.
In general, the nature of the carrier will depend on the particular
mode of administration being employed. For instance, parenteral
formulations usually comprise injectable fluids that include
pharmaceutically and physiologically acceptable fluids such as
water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
Probes and primers: Nucleic acid probes and primers may readily be
prepared based on the nucleic acids provided by this invention. A
probe comprises an isolated nucleic acid attached to a detectable
label or reporter molecule. Typical labels include radioactive
isotopes, ligands, chemiluminescent agents, and enzymes. Methods
for labeling and guidance in the choice of labels appropriate for
various purposes are discussed, e.g., in Sambrook et al. (Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) and
Ausubel et al. (Current Protocols in Molecular Biology, Greene
Publishing Associates and Wiley-Intersciences, 1987).
Primers are short nucleic acids, for example DNA oligonucleotides
15 nucleotides or more in length. Primers may be annealed to a
complementary target DNA strand by nucleic acid hybridization to
form a hybrid between the primer and the target DNA strand, and
then extended along the target DNA strand by a DNA polymerase
enzyme. Primer pairs can be used for amplification of a nucleic
acid sequence, e.g., by the polymerase chain reaction (PCR) or
other nucleic-acid amplification methods known in the art.
Methods for preparing and using probes and primers are described,
for example, in Sambrook et al. (Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y., 1989), Ausubel et al (Current
Protocols in Molecular Biology, Greene Publishing Associates and
Wiley-Intersciences, 1987), and Innis et al., (PCR Protocols, A
Guide to Methods and Applications, Innis et al. (eds.), Academic
Press, Inc., San Diego, Calif., 1990). PCR primer pairs can be
derived from a known sequence, for example, by using computer
programs intended for that purpose such as Primer (Version 0.5,
.COPYRGT. 1991, Whitehead Institute for Biomedical Research,
Cambridge, Mass.).
Prostate Carcinoma (PC): prostate cancer (such as prostatic
adenocarcinoma) thought to result, in some instances, from the
deletion or mutation of the DLC-1 tumor suppressor gene.
Protein: a biological molecule expressed by a gene and comprised of
amino acids.
Purified: the term "purified" does not require absolute purity;
rather, it is intended as a relative term. Thus, for example, a
purified protein preparation is one in which the protein referred
to is more pure than the protein in its natural environment within
a cell.
Recombinant: A recombinant nucleic acid is one that has a sequence
that is not naturally occurring or has a sequence that is made by
an artificial combination of two otherwise separated segments of
sequence. This artificial combination is often accomplished by
chemical synthesis or, more commonly, by the artificial
manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques.
Representational Difference Analysis (RDA): a PCR-based subtractive
hybridization technique used to identify differences in the mRNA
transcripts present in closely related cell lines.
Sequence identity: the similarity between two nucleic acid
sequences, or two amino acid sequences, is expressed in terms of
the similarity between the sequences, otherwise referred to as
sequence identity. Sequence identity is frequently measured in
terms of percentage identity (or similarity or homology); the
higher the percentage, the more similar are the two sequences.
Methods of alignment of sequences for comparison are well-known in
the art. Various programs and alignment algorithms are described
in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and
Wunsch, J. Mol. Bio. 48:443, 1970. Pearson and Lipman, Methods in
Mol. Biol. 24: 307-31, 1988; Higgins and Sharp, Gene 73:237-44,
1988; Higgins and Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc.
Acids Res. 16:10881-90, 1988; Huang et al., Comp. Appl. BioSci.
8:155-65, 1992; and Pearson et al., Meth. Mol. Biol. 24:307-31,
1994.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et
al., J. Mol. Biol. 215:403-10, 1990) is available from several
sources, including the National Center for Biological Information
(NBCl, Bethesda, Md.) and on the Internet, for use in connection
with the sequence analysis programs blastp, blasm, blastx, tblastn
and tblastx. It can be accessed at
http://www.ncbi.nlm.nih.gov/BLAST/. A description of how to
determine sequence identity using this program is available at
http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
Homologs of the DLC-1 protein are typically characterized by
possession of at least 70% sequence identity counted over the full
length alignment with the disclosed amino acid sequence using the
NCBI Blast 2.0, gapped blastp set to default parameters. Such
homologous peptides will more preferably possess at least 75%, more
preferably at least 80% and still more preferably at least 90% or
95% sequence identity determined by this method. When less than the
entire sequence is being compared for sequence identity, homologs
will possess at least 75% and more preferably at least 85% and more
preferably still at least 90% or 95% sequence identity over short
windows of 10-20 amino acids. Methods for determining sequence
identity over such short windows are described at
http://www.ncbi.nlm.nih.gov/BLAST/blast_FAQs.html. One of skill in
the art will appreciate that these sequence identity ranges are
provided for guidance only; it is entirely possible that strongly
significant homologs or other variants could be obtained that fall
outside of the ranges provided.
The present invention provides not only the peptide homologs that
are described above, but also nucleic acid molecules that encode
such homologs.
Transformed: A transformed cell is a cell into which has been
introduced a nucleic acid molecule by molecular biology techniques.
As used herein, the term transformation encompasses all techniques
by which a nucleic acid molecule might be introduced into such a
cell, including transfection with viral vectors, transformation
with plasmid vectors, and introduction of naked DNA by
electroporation, lipofection, and particle gun acceleration.
Vector. A nucleic acid molecule as introduced into a host cell,
thereby producing a transformed host cell. A vector may include
nucleic acid sequences that permit it to replicate in a host cell,
such as an origin of replication. A vector may also include one or
more selectable marker genes and other genetic elements known in
the art.
VNTR probes: Variable Number of Tandem Repeat probes. These are
highly polymorphic DNA markers for human chromosomes. The
polymorphism is due to variation in the number of tandem repeats of
a short DNA sequence. Use of these probes enables the DNA of an
individual to be distinguished from that derived from another
individual.
Tumor: a neoplasm.
Neoplasm: abnormal growth of cells.
Cancer: malignant neoplasm that has undergone characteristic
anaplasia with loss of differentiation, increased rate of growth,
invasion of surrounding tissue, and is capable of metastasis.
Malignant: cells which have the properties of anaplasia invasion
and metastasis.
Normal cells: Non-tumor, non-malignant cells.
Mammal: This term includes both human and non-human mammals.
Similarly, the term "patient" includes both human and veterinary
subjects.
Animal: Living multicellular vertebrate organisms, a category which
includes, for example, mammals and birds.
Transgenic Cell: transformed cells which contain foreign,
non-native DNA.
Additional definitions of common terms in molecular biology may be
found in Lewin, B. "Genes V" published by Oxford University
Press.
Materials and Methods
Primary HCC Samples and HCC Cell Lines
All of the primary liver tumor DNAs were obtained from surgical
resection of HCC tissues from patients in Qidong, China. Each tumor
sample was matched with its surrounding non-cancerous liver tissue.
DNAs were extracted after diagnosis of HCC with or without
cirrhosis. The tumors were Hepatitis B virus (HBV) positive for
HBVsAg and/or PCR detection of HBVx gene. HCC cell lines were
obtained from ATCC (Manassas, Va.), Qidong Liver Cancer Institute,
China, and Dr. Curtis C. Harris (Laboratory of Human
Carcinogenesis, Division of Basic Sciences, National Cancer
Institute) (Wang, et at., Chin. J. Oncol. 3:241-4, 1981).
Breast, Prostate and Colorectal Carcinomas
All normal and CRC (adenocarcinomas) cell lines were purchased from
ATCC (Manassas, Va.). The PC cell lines (also adenocarcinomas) were
obtained from The University of Texas M.D. Anderson Cancer Center
(Houston, TX). The DNA from primary breast carcinomas and blood
cells were obtained from patients in Iceland.
Manipulation of Genetic Material
Unless otherwise specified, manipulation of genetic material was
performed according to standard laboratory procedures, such as
those described in Sambrook et al. (Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y., 1989) and Ausubel et al. (Current
Protocols in Molecular Biology, Greene Publishing Associates and
Wiley-Intersciences, 1987).
Representational Difference Analysis (RDA)
One primary HCC, having a homozygous point mutation of the p53
gene, but not in its surrounding, non-cancerous liver tissue, was
selected for analysis. RDA was performed as originally described in
Lisitsyn et al. (Proc. Natl. Acad. Sci. USA 92:151-5, 1995), with
tumor DNA as tester and normal liver DNA as driver. BgIII (Promega,
Madison, Wis.) was chosen as the restriction enzyme and its
adaptors were used for direct preparation of amplicons and
PCR-based subtractive hybridization. The final difference products
showing distinct bands in agarose gel were recovered after BgIII
digestion and ligated into the BgIII site of dephosphorylated pSP72
vector (Promega). The recombinant difference products were then
transfected into E. coli DH10B.
Characterization of RDA Probes
Plasmids with distinct DNA inserts were selected for further
analysis. DNA sequencing was performed using the Dye Terminator
Cycle DNA Sequencing kit (Perkin Elmer, Rockville, Md.). Sequencing
reaction products were purified by spin columns (Princeton
Separations, Adelphia, N.J.), and run on a 377 DNA Sequencer
(Perkin Elmer/Applied Biosystems, Foster City, Calif.). The
homology analysis was carried out by BLAST search of the GenBank
DNA databases (Altschul, et al., J. Mol. Biol. 215:403-10, 1990).
The RDA products that elicited significant homology or appeared in
multiple clones, were selected for further Southern blot and/or
Northern blot analysis.
Conditions for Southern Analysis
Genomic DNA was isolated from tumor and non-tumor cell lysates and
digested with restriction enzymes. The digested DNA was separated
by electrophoresis in a 1% agarose gel and transferred to nylon
membrane for hybridization. 50 ng of DNA probe was radio-labeled
(Prime-It RmT, Stratagene) as per the manufacturers instructions
and used for hybridization. A probe for beta-actin was used as a
standard to control for the amount of DNA loaded. Hybridization was
performed at 68.degree. C. for 24 hours using Quickhybrid solution
(Stratagene). Following hybridization, the membranes were washed
three times at 37.degree. C. for 10 min in 1.times.SSC solution
containing 0.1.times.SDS. This was followed by a single wash at
62.degree. C. for 30 min in 0.1.times.SSC solution containing
0.1.times.SDS. Blots were exposed to a Phospholmager, and analyzed
using Software ImageQuant Version 3.3 (Molecular Dynamics,
Sunnyvale, Calif.) for quantitative analysis.
Conditions for Northern Analysis
Total RNA was extracted from cell lysates using TRIzol solution
(Gibco-BRL), which was then separated in a 1% agarose gel and
transferred to nylon membrane for hybridization. 50 ng of DNA probe
was radio-labeled (Prime-It RmT, Stratagene) as per the
manufacturers instructions and used for hybridization. A probe for
GAPDH or beta-actin was used as a control for the amount of RNA
loaded. Hybridization, washing, and analysis was performed as
described above for Southern Hybridization.
5' and 3' RACE and cDNA Library Screening for cDNA Cloning
5' and 3' RACE (Rapid Amplification of cDNA Ends) were started from
a deleted fragment detected with RDA, and performed using human
placenta Marathon.TM. cDNA as template (Clontech, Inc., Palo Alto,
Calif.). The primers used for RACE, generated from the L7-3
sequence (Seq. I.D. No. 12), are as follows:
PrRACE5: 5' CACTCCGGTCCTTGTAGTCTGGAACC 3' (Seq. I.D. No. 9) was
used for the first round of PCR for 5' RACE.
PrRACE5N: 5' ATCCTCTTCATGAACTCGGGCACGG 3' (Seq. I.D. No. 10) was
used as the nested primer in the second round of 5' RACE.
PrRACE3: 5' GATCAAGGTTCTAGACTACAAGGACCG 3' (Seq. I.D. No. 11) was
used for 3' RACE.
The final 5' RACE product, exhibiting the same band pattern as the
deleted fragment in Northern blot hybridization, was labeled with
.alpha.-[.sup.32 P]-dCTP to screen a 5' Strech cDNA library
constructed from human lung tissue (Clontech, Inc.). The lambda DNA
of positive clones was converted into plasmid DNA by transfecting
lambda DNA into AM1 bacterial cells. The full-length cDNA
sequencing of positive clones was completed by primer walking and
assembled by Sequencher.TM. 3.1 program.
Fluorescence in situ Hybridization (FISH) Gene Mapping and
Comparative Genomic Hybridization (CGH)
A genomic probe isolated from human P1 library was labeled with
biotin and used for FISH chromosomal localization and CGH analysis.
For both analyses, chromosomes prepared from
methotrexate-synchronized normal peripheral lymphocyte cultures
were used. The original CGH protocol, described in Kallioniemi et
al. (Science 258:818-21, 1992), was employed with minor
modifications. The conditions of hybridization, the detection of
hybridization signals, digital-image acquisition, processing and
analysis, and direct fluorescent signal localization on banded
chromosomes were performed as previously described in Zimonjic et
al. (Cancer Genet. Cytogenet. 80:100-2, 1995).
The following examples are illustrative of the scope of the present
invention.
EXAMPLE 1
Detection or DLC-1 Deletion in Liver Cancer Cells by RDA
Primary HCC tumor samples, matched with surrounding non-cancerous
liver tissue, were obtained as described above and analyzed by RDA.
Several RDA difference products were observed after the third round
of hybridization/selection as distinct bands in agarose gel. Twenty
individual fragments were isolated and analyzed by Southern blot
hybridization for deletions. One clone, L7-3, of 600 bp (Seq. I.D.
No. 12), showed loss of heterozygosity (LOH) in the primary tumor
(FIG. 1). BLAST search revealed that the L7-3 clone had homology to
rat p122 RhoGAP cDNA (Homma and Emori, EMBO. J. 14:28691,
1995).
EXAMPLE 2
Southern Analysis
HCC Cell Lines
To determine if the L7-3 clone is represented in a region
recurrently deleted in HCC, 15 primary HCC tumors and 11
HCC-derived cell lines were examined using Southern analysis as
described above. The DNA was digested with BglII, and probed with
L7-3 (Seq. I.D. No. 12). Seven of the fifteen primary HCC tumors
(representatives are shown in FIG. 1) and 9 of the 11 HCC cell
lines (FIG. 2) hid a genomic deletion of thee L7-3 clone compared
to no deletions in the normal liver cells.
Primary Breast Carcinomas
Using Southern analysis as described above, primary human breast
cancer and corresponding patient blood cell DNA was digested with
BglII (FIG. 3a) or BamHI (FIG. 3b) and probed with full-length
DLC-1 cDNA (Seq. I.D. No. 1). Genomic deletions of DLC-1 gene were
detected in 7 of 15 human primary breast cancers (representatives
are shown in FIG. 3). Deletions were noted if the DNA intensity of
the tumor tissues exhibited at least half the intensity when
compared with their normal tissue DNA. Samples IC11T, IC12T, IC13T,
IC2T, IC6T, IC7Tare representative for the genomic deletions in
these experiments.
Southern analysis of these cells resulted in several bands. As a
control for DNA loading, the bands that remained unchanged in the
tumor cells were used.
Colon Carcinoma Cell Lines
Using Southern analysis as described above, normal genomic DNA
(Promega) and the DNA from five CRC cell lines were digested with
EcoRI, and probed with a mixture of L7-3 and P-(Seq. I.D. Nos. 12
and 13) which correspond to exon 2 and exon 7 of the human DLC-1
gene (see FIG. 9), respectively. Genomic deletions of DLC-1 gene
were detected in two of five human CRC cell lines (FIG. 4). Cell
lines SW403 and SW1116 showed half of the DNA intensity for probe
P-35 when compared with normal genomic DNA (compare lanes 5 and 6
with lane 1). Interestingly, the signal was unaltered when the L7-3
probe was used, indicating that this region (exon 2) is not
responsible for the development of CRC in these cell lines.
Therefore, the signal from L7-3 can be used as an internal control
for the amount of DNA loaded.
EXAMPLE 3
Northern Analysis
HCC Cell Lines
Considering the significant DNA sequence homology of the L7-3 clone
with rat RhoGAP cDNA, its mRNA expression was examined in both
normal human tissues and HCC-derived cell lines by Northern
analysis as described above. Analysis of mRNA isolated from several
normal human tissues, including liver, demonstrated that the L7-3
clone (Seq. I.D. No. 12) hybridized to a 7.5 kb (major) transcript
and a 4.5 kb (minor) transcript (FIG. 5) that were detected in all
normal tissues but not in 4(WRL, 7703, Chang and Focus) out of-14
human HCC-derived cell lines (FIG. 6).
Colorectal Carcinomas
Using Northern analysis as described above, RNA from normal and CRC
cell lines was prepared and probed with the full-length DLC-1 cDNA
(Seq. I.D. No. 1). In human CRC cell lines, II out of 17 (HCT-15,
LS147T, DLD-1, HD29, SW1116, T84, SW1417, SW403, SW948, LS180,
SW48) showed noticeably decreased or no expression of DLC-1 mRNA
(FIG. 7). In this experiment, the normal human colon fibroblast
cell line CDD33C0 was used as a normal control.
Prostate Carcinomas
Using Northern analysis as described above, RNA from PC cell lines
was prepared and probed with the full-length DLC-1 cDNA (Seq. I.D.
No. 1). Low levels or no DLC-1 gene expression was demonstrated by
in two (LN-Cap and SP3504) out of three human PC cell lines (FIG.
8).
EXAMPLE 4
Obtaining the DLC-1 cDNA
The cDNA for the clone L7-3 was obtained by 5' RACE and 3' RACE
coupled with cDNA library screening as described above. The
full-length cDNA of DLC-1 gene is 3850 bp long (Seq. I.D. No. 1)
and encodes a protein of 1091 amino acids (Seq. I.D. No. 2). The
estimated molecular weight of DLC-1 protein is 125 kD. The
untranslated regions of 5' end and 3' end of DLC-1 gene are 324 bp
and 250 bp, respectively (Seq. I.D. No. 1).
EXAMPLE 5
Chromosomal Localization of Human DLC-1
The DLC-1 gene was chromosomally localized using the materials and
methods described above. The majority of metaphases hybridized with
biotin or digoxigenin-labeled genomic probe had fluorescent signal
at identical sites on both chromatids of the short arm of
chromosome 8. The signal was analyzed in 100 metaphases with both
homologous labeled. Fifty metaphases were examined by imaging of
DAPI generated and enhanced G-like banding. The fluorescent signals
were distributed within region 8p21-22 However, over 50% of
doublets were at bands 8p21.3-22, the most likely location of the
DLC-1 gene.
To further characterize the region harboring the DLC-1 gene, the
primary tumor DNA used as tester in RDA (94-25T) was analyzed by
CGH. The fluorescence profile for chromosome 8 demonstrated DNA
loss on region of 8p23-q11.2 and gain on region of
8q21.1-q24.3.
EXAMPLE 6
Cloning and Characterization of Human DLC-1
The DLC-1 cDNA sequence (Seq. I.D. No. 1) described above does not
contain the introns, upstream transcriptional promoter or
regulatory regions or downstream transcriptional regulatory regions
of the DLC-1 gene. Its possible that some mutations in the DLC-1
gene that may lead to HCC are not included in the cDNA but rather
are located in other regions of the DLC-1 gene. Mutations located
outside of the open reading frame that encodes the DLC-1 protein
are not likely to affect the functional activity of the protein but
rather are likely to result in altered levels of the protein in the
cell. For example, mutations in the promoter region of the DLC-1
gene may prevent transcription of the gene and therefore lead to
the complete absence of the DLC-1 protein in the cell.
Additionally, mutations within intron sequences in the genomic gene
may also prevent expression of the DLC-1 protein. Following
transcription of a gene containing introns, the intron sequences
are removed from the RNA molecule in a process termed splicing
prior to translation of the RNA molecule which results in
production of the encoded protein. When the RNA molecule is spliced
to remove the introns, the cellular enzymes that perform the
splicing function recognize sequences around the intron/exon border
and in this manner recognize the appropriate splice sites. If there
is a mutation within the sequence of the intron close to the
junction of the intron with an exon, the enzymes may not recognize
the junction and may fail to remove the intron. If this occurs, the
encoded protein will likely be defective. Thus, mutations inside
the intron sequences within the DLC-1 gene (termed "splice site
mutations") may also lead to the development of HCC. However,
knowledge of the exon structure and intronic splice site sequences
of the DLC-1 gene is required to define the molecular basis of
these abnormalities. The provision herein of the DLC-1 cDNA
sequence (Seq. I.D. No. 1) enables the cloning of the entire DLC-1
gene (including the promoter and other regulatory regions and the
intron sequences) and the determination of its nucleotide sequence.
With this information in hand, diagnosis of a genetic
predisposition to HCC and other cancers based on DNA analysis will
comprehend all possible mutagenic events at the DLC-1 locus.
The ATCC deposit (98676) of the genomic DLC-1 gene may be utilized
in aspects of the present invention. Alternatively, the DLC-1 gene
may be isolated by one or more routine procedures, including
PCR-based screening of a human genomic P1 library as described
above. Alternatively, the method described in WO 93/22435 can be
utilized. For example, a YAC library of human genomic sequences
(Monaco and Lehrach, Proc. Natl. Acad. Sci. U.S.A. 88:4123-7, 1991)
is screened for the DLC-1 gene by the polymerase chain reaction
(PCR). The library is arranged in a number (e.g., 39) of primary
DNA pools, prepared from high-density grids each containing around
300-400 YAC clones. Primary pools are screened by PCR to identify a
pool which contains a positive clone. A secondary PCR screen is
then performed on the appropriate set of eight row and 12 column
pools, as described by Bentley et al. (Genomics 12:534-41, 1992).
PCR primers based on the DLC-1 cDNA sequence are used as a sequence
tagged site (STS) for the 3' region of the gene. The yeast DNA is
then amplified with these primers by PCR for 30 cycles of
94.degree. C. for 1 minute, 60.degree. C. for 1 minute and
72.degree. C. for 1 minute, with a final 5 minute extension at
72.degree. C. Confirmation that positive YAC clones contain the
majority of the coding sequence of the DLC-1 genomic gene is
obtained by amplification of an STS from the 5' end of the cDNA.
Exon boundaries of the DLC-1 gene are then characterized, e.g., by
the vectorette PCR method. This strategy has been described in
detail previously (Roberts et al., Genomics 13:942-50, 1992).
With the sequences of the DLC-1 cDNA and DLC-1 gene in hand,
primers derived from these sequences may be used in diagnostic
tests (described below) to determine the presence of mutations in
any part of the genomic DLC-1 gene of a patient. Such primers will
be oligonucleotides comprising a fragment of sequence from the
DLC-1 gene (either intron sequence, exon sequence or a sequence
spanning an intron-exon boundary) and will comprise at least 15
consecutive nucleotides of the DLC-1 cDNA or gene. It will be
appreciated that greater specificity may be achieved by using
primers of greater lenghts. Thus, in order to obtain enhanced
specificity, the primers used may comprise 20, 25, 30 or even 50
consecutive nucleotides of the DLC-1 cDNA or gene. Furthermore,
with the provision of the DLC-1 intron sequence information the
analysis of a large and as yet untapped source of patient material
for mutations will now be possible using methods such as chemical
cleavage of mismatches (Cotton et al., Proc Natl Acad Sci USA.
85:4397-401, 1988; Montandon et al., Nucleic Acids Res. 9:3347-58,
1989) and single-strand conformational polymorphism analysis (Orita
et al., Genomics 5:874-879, 1989).
Additional experiments may now be performed to identify and
characterize regulatory elements flanking the DLC-1 gene. These
regulatory elements may be characterized by standard techniques
including deletion analyses wherein successive nucleotides of a
putative regulatory region are removed and the effect of the
deletions are studied by either transient or long-term expression
analyses experiments. The identification and characterization of
regulatory elements flanking the genomic DLC-1 gene may be made by
functional experimentation (deletion analyses, etc.) in mammalian
cells by either transient or long-term expression analyses.
Having provided a genomic clone for the human DLC-1 gene (Seq. I.D.
Nos. 14-19), it will be apparent to one skilled in the art that
either the genomic clone or the cDNA or sequences derived from
these clones may be utilized in applications of this invention,
including but not limited to, studies of the expression of the
DLC-1 gene, studies of the function of the DLC-1 protein, the
generation of antibodies to the DLC-1 protein diagnosis and therapy
of DLC-1 deleted or mutated patients to prevent or treat the onset
of HCC. Descriptions of applications describing the use of DLC-1
cDNA are therefore intended to comprehend the use of the genomic
DLC-1 gene. It will also be apparent to one skilled in the art that
homologs of this gene may now be cloned from other species, such as
the rat or the mouse, by standard cloning methods. Such homologs
will be useful in the production of animal models of HCC.
To facilitate the detection of point mutations in liver and other
cancers that exhibit alteration at region 8p12-22, the human DLC-1
gene was cloned and the intron/exon sequences characterized (Seq.
I.D. Nos. 14-19 and FIG. 9).
Human DLC-1 is approximately 25 kb, and contains 14 exons. The
largest exon is exon 2, at 1.5 kb, while the remaining exons are
less than 300 bp on average (FIG. 9).
EXAMPLE 7
Cloning Mouse DLC-1
A full understanding of the function of DLC-1 and its role in
cancer development is essential. This understanding can be
facilitated by the generation of knock-out mice, which contain a
non-functional DLC-1 gene. Prior to generating knock-out mice, the
partial cDNA (Seq. I.D. Nos. 26-31) and partial genomic (Seq. I.D.
Nos. 20-25) mouse DLC-1 sequences were determined.
Mouse DLC-1 genomic DNA was cloned and localized to chromosome 8 by
FISH (see above for methods) using a mouse DLC-1 genomic DNA clone
as the probe. Mouse DLC-1 is in a syntenic region of the human
DLC-1 gene. The localization of DLC-1 gene in mice may permit
studies with in vivo models for carcinogenesis.
EXAMPLE 8
Generating Transgenic Mice
Methods for generating transgenic mice are described in Gene
Targeting, A. L. Joyuner ed., Oxford University Press, 1995 and
Watson, J. D. et al., Recombinant DNA 2.sup.nd 4 Ed., W.H. Freeman
and Co., New York, 1992, Chapter 14. To specifically generate
transgenic mice containing a functional deletion of the DLC-1 gene,
a 1.5 kb fragment in the front of exon 2 and another 5.5 kb
fragment spanning from intron 2 to intron 5 were used as short arm
and long arm, respectively. Between long arm and short arm, the neo
gene was introduced, generating the vector shown in FIG. 10,
referred to as the knock-out vector herein.
Using standard transgenic mouse technology, the vector shown in
FIG. 10 can be used to generate DLC-1 knock-out mice by homologous
recombination. The knock-out vector is introduced into embryonic
stem cells (ES cells) by standard methods which may include
transfection, retroviral infection or electroporation (also see
Example 11). The transfected ES cells expressing the knock-out
vector will grow in medium containing the antibiotic G418. The
neomycin resistant ES cells will be microinjected into mouse
embryos (blastocysts), which are implanted into the uterus of
pseudopregnant mice. The litter will be screened for chimeric mice
by observing their coat color. Chimeric mice are ones in which the
injected ES cells developed into the germ line, thereby allowing
transmission of the gene to their offspring. The resulting
heterozygotic mice will be mated to generate a homozygous line of
transgenic mice functionally deleted for DLC-1 . These homozygous
mice will then be screened phenotypically, for example, their
predisposition to developing cancer.
EXAMPLE 9
Preferred Method of Making the DLC-1 cDNA The foregoing discussion
describes the original means by which the DLC-1 cDNA was obtained
and also provides the nucleotide sequence of this clone. With the
provision of this sequence information, the polymerase chain
reaction (PCR) may now be utilized in a more direct and simple
method for producing the DLC-1 cDNA.
Essentially, total RNA is extracted from human cells by any one of
a variety of methods routinely used; Sambrook et al. (Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) and
Ausubel et al. (In Current Protocols in Molecular Biology, Greene
Publishing Associates and Wiley-Intersciences, 1987) provide
descriptions of methods for RNA isolation. Any human cell line
derived from a non-DLC-1 deleted individual would be suitable, such
as the widely used HeLa cell line, or the WI-38 human skin
fibroblast cell line available from the American Type Culture
Collection, Rockville, Md. The extracted RNA is then used as a
template for performing the reverse transcription-polymerase chain
reaction (RT-PCR) amplification of cDNA. Methods and conditions for
RT-PCR are described in Kawasaki et al. (In PCR Protocols, A Guide
to Methods and Applications, Innis et al. (eds.), pp. 21-27,
Academic Press, Inc., San Diego, Calif., 1990). The selection of
PCR primers will be made according to the portions of the cDNA
which are to be amplified. Primers may be chosen to amplify small
segments of a cDNA or the entire cDNA molecule. Variations in
amplification conditions may be required to accommodate primers of
differing lengths; such considerations are well known in the art
and are discussed in Innis et al. (PCR Protocols, A Guide to
Methods and Applications, Innis et al. (eds.), Academic Press,
Inc., San Diego, Calif., 1990). The entire DLC-1 cDNA molecule may
be amplified using the following combination of primers:
5' TAT GGG CTC GAG CGG CCG CCC 3' (Seq. I.D. No. 3)
5' CGC ACA GTC TTA CAT ATT CCA 3' (Seq. I.D. No. 4) The open
reading frame of the cDNA molecule may be amplified using the
following combination of primers:
5' ATG TGC AGA AAG AAG CCG GAC ACC 3' (Seq. I.D. No. 5)
5' CCT AGA TTT GGT GTC TTT GGT TTC 3' (Seq. I.D. No. 6)
These primers are illustrative only; it will be appreciated by one
skilled in the art that many different primers may be derived from
the provided cDNA sequence in order to amplify particular regions
of these cDNAs.
EXAMPLE 10
Sequence Variants of DLC-1
The nucleotide sequence of the DLC-1 cDNA is set forth in SEQ ID
NO: 1; the amino acid sequence of the DLC-1 protein is encoded by
that cDNA is set fourth ein SEQ ID NO: 2. Having presented the
nucleotide sequence of the DLC-1 cDNA and the amino acid sequence
of the protein, this invention now also facilitates the creation of
DNA molecules, and thereby proteins, which are derived from those
disclosed but which vary in their precise nucleotide or amino acid
sequence from those disclosed. Such variants may be obtained
through a combination of standard molecular biology laboratory
echniques and the nucleotide sequence information disclosed by this
invention.
Variant DNA molecules include those created by standard DNA
mutagenesis techniques, for example, M13 primer mutagenesis.
Details of these techniques are provided in Sambrook et al. (in
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,
1989, Ch. 15). By the use of such techniques, variants may be
created which differ in minor ways from those disclosed. DNA
molecules and nucleotide sequences which are derivatives of those
specifically disclosed herein and which differ from those disclosed
by the deletion, addition or substitution of nucleotides while
still encoding a protein which possesses the functional
characteristic of the DLC-1 protein are comprehended by this
invention. A Iso within the scope of this invention are small DNA
molecules which are derived from the disclosed DNA molecules. Such
small DNA molecules include oligonucleotides suitable for use as
hybridization probes or polymerase chain reaction (PCR) primers. As
such, these small DNA molecules will comprise at least a segment of
the DLC-1 cDNA molecule or the DLC-1 gene and, for the purposes of
PCR, will comprise at least a 15 nucleotide sequence and, more
preferably, a 20-50 nucleotide sequence of the DLC-1 cDNA (Seq.
I.D. No. 1) or the DLC-1 gene (Seq. I.D. Nos. 14-19) (i.e., at
least 20-50 consecutive nucleotides of the DLC-1 cDNA or gene
sequences). DNA molecules and nucleotide sequences which are
derived from the disclosed DNA molecules as described above may
also be defined as DNA sequences which hybridize under stringent
conditions to the DNA sequences disclosed, or fragments
thereof.
Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method of choice and the composition and length of the hybridizing
DNA used. Generally, the temperature of hybridization and the ionic
strength (especially the Na.sup.+ concentration) of the
hybridization buffer will determine the stringency of
hybridization. Calculations regarding hybridization conditions
required for attaining particular degrees of stringency are
discussed by Sambrook et al. (In Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y., 1989 ch. 9 and 11), herein
incorporated by reference. By way of illustration only, a
hybridization experiment may be performed by hybridization of a DNA
molecule (for example, a deviation of the DLC-1 cDNA) to a target
DNA molecule (for example, the DLC-1 cDNA) which has been
electrophoresed in an agarose gel and transferred to a
nitrocellulose membrane by Southern blotting (Southern, J. Mol.
Biol. 98:503, 1975), a technique well known in the art and
described in Sambrook et al. (Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y., 1989). Hybridization with a
target probe labeled with [.sup.32 P]-dCTP is generally carried out
in a solution of high ionic strength such as 6.times.SSC at a
temperature that is 20-25.degree. C. below the melting temperature,
T.sub.m, described below. For such Southern hybridization
experiments where the target DNA molecule on the Southern blot
contains to ng of DNA or more, hybridization is typically carried
out for 6-8 hours using 1-2 ng/ml radiolabeled probe (of specific
activity equal to 109 CPM/.mu.g or greater). Following
hybridization, the nitrocellulose filter is washed to remove
background hybridization. The washing conditions should be as
stringent as possible to remove background hybridization but to
retain a specific hybridization signal. The term T.sub.m represents
the temperature above which, under the prevailing ionic conditions,
the radiolabeled probe molecule will not hybridize to its target
DNA molecule. The T.sub.m of such a hybrid molecule may be
estimated from the following equation (Bolton and McCarthy, Proc.
Natl. Acad. Sc. USA 48:1390, 1962):
Where l=the length of the hybrid in base pairs.
This equation is valid for concentrations of Na.sup.+ in the range
of 0.01 M to 0.4 M, and it is less accurate for calculations of
T.sub.m in solutions of higher [Na.sup.+ ]. The equation is also
primarily valid for DNAs whose G+C content is in the range of 30%
to 75%, and it applies to hybrids greater than 100 nucleotides in
length (the behavior of oligonucleotide probes is described in
detail in Ch. II of Sambrook et al. (Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, N.Y., 1989).
Thus, by way of example, for a 150 base pair DNA probe derived from
the open reading frame of the DLC-1 cDNA (with a hypothetical %
GC=45%), a calculation of hybridization conditions required to give
particular stringencies may be made as follows:
For this example, it is assumed that the filter will be washed in
0.3.times.SSC solution following hybridization, thereby:
[Na.sup.+ ]=0.045M
% GC=45%
Formamide concentration=0
I=150 base pairs ##EQU1##
and so T.sub.m =74.4.degree. C.
The T.sub.m, of double-stranded DNA decreases by 1-1.5.degree. C.
with every 1% decrease in homology (Bonner et al., J. Mol. Biol
81:123, 1973). Therefore, for this given example, washing the
filter in 0.3.times.SSC at 59.4-64.4.degree. C. will produce a
stringency of hybridization equivalent to 90%; that is, DNA
molecules with more than 10% sequence variation relative to the
target DLC-1 cDNA will not hybridize. Alternatively, washing the
hybridized filter in 0.3.times.SSC at a temperature of
65.4-68.4.degree. C. will yield a hybridization stringency of 94%;
that is, DNA molecules with more than 6% sequence variation
relative to the target DLC-1 cDNA molecule will not hybridize. The
above example is given entirely by way of theoretical illustration.
One skilled in the art will appreciate that other hybridization
techniques may be utilized and that variations in experimental
conditions will necessitate alternative calculations for
stringency.
In particular embodiments of the present invention, stringent
conditions may be defined as those under which DNA molecules with
more than 25% sequence variation (also termed "mismatch") will not
hybridize. In a more particular embodiment, stringent conditions
are those under which DNA molecules with more than 15% mismatch
will not hybridize, and more preferably still, stringent conditions
are those under which DNA sequences with more than 10% mismatch
will not hybridize. In another embodiment, stringent conditions are
those under which DNA sequences with more than 6% mismatch will not
hybridize.
The degeneracy of the genetic code further widens the scope of the
present invention as it enables major variations in the nucleotide
sequence of a DNA molecule while maintaining the amino acid
sequence of the encoded protein. For example, the sixteenth amino
acid residue of the DLC-1 protein is alanine. This is encoded in
the DLC-1 cDNA by the nucleotide codon triplet GCC. Because of the
degeneracy of the genetic code, three other nucleotide codon
triplets, GCT, GCG and GCA, also code for alanine. Thus, the
nucleotide sequence of the DLC-1 cDNA could be changed at this
position to any of these three codons without affecting the amino
acid composition of the encoded protein or the characteristics of
the protein. The genetic code and variations in nucleotide codons
for particular amino acids is presented in Tables 1 and 2. Based
upon the degeneracy of the genetic code, variant DNA molecules may
be derived from the cDNA molecules disclosed herein using standard
DNA mutagenesis techniques as described above, or by synthesis of
DNA sequences. DNA sequences which do not hybridize under stringent
conditions to the cDNA sequences disclosed by virtue of sequence
variation based on the degeneracy of the genetic code are herein
also comprehended by this invention.
The invention also includes DNA sequences that are substantially
identical to any of the DNA sequences disclosed herein, where
substantially identical means a sequence that has identical
nucleotides in at least 75% of the aligned nucleotides, for example
80%, 85%, 90%, 95% or 98% identity of the aligned sequences.
TABLE I The Genetic Code First Position Second Position (3' end)
Third (5' end) T C A G Position T Phe Ser Tyr Cys T Phe Ser Tyr Cys
C Leu Ser Stop (och) Stop A Leu Ser Stop (amb) Trp G C Leu Pro His
Arg T Leu Pro His Arg C Leu Pro Gln Arg A Leu Pro Gln Arg G A Ile
Thr Asn Ser T Ile Thr Asn Ser C Ile Thr Lys Arg A Met Thr Lys Arg G
G Val Ala Asp Gly T Val Ala Asp Gly C Val Ala Glu Gly A Val (Met)
Ala Glu Gly G "Stop (och)" stands for the ochre termination
triplet, and "Stop (amb)" for the amber. ATG is the most common
initiator codon; GTG usually codes for valine, but it can also code
for methionine to initiate an mRNA chain.
TABLE 2 The Degeneracy of the Genetic Code Number of Total Number
of Synonymous Codons Amino Acid Codons 6 Leu, Ser, Arg 18 4 Gly,
Pro, Ala, Val, Thr 20 3 Ile 3 2 Phe, Tyr, Cys, His, Gln, 18 Glu,
Asn, Asp, Lys 1 Met, Trp 2 Total number of codons for amino acids
61 Number of codons for termination 3 Total number of codons in
genetic code 64
One skilled in the art will recognize that the DNA mutagenesis
techniques described above may be used not only to produce variant
DNA molecules, but will also facilitate the production of proteins
which differ in certain structural aspects from the DLC-1 protein,
yet which proteins are clearly derivative of this protein and which
maintain the essential characteristics of the DLC-1 protein. Newly
derived proteins may also be selected in order to obtain variations
on the characteristic of the DLC-1 protein, as will be more fully
described below. Such derivatives include those with variations in
amino acid sequence including minor deletions, additions and
substitutions.
While the site for introducing an amino acid sequence variation is
predetermined, the mutation per se need not be predetermined. For
example, in order to optimize the performance of a mutation at a
given site, random mutagenesis may be conducted at the target codon
or region and the expressed protein variants screened for the
optimal combination of desired activity. Techniques for making
substitution mutations at predetermined sites in DNA having a known
sequence as described above are well known.
Amino acid substitutions are typically of single residues;
insertions usually will be on the order of about from 1 to 10 amino
acid residues; and deletions will range about from 1 to 30
residues. Deletions or insertions preferably are made in adjacent
pairs, i.e., a deletion of 2 residues or insertion of 2 residues.
Substitutions, deletions, insertions or any combination thereof may
be combined to arrive at a final construct. Obviously, the
mutations that are made in the DNA encoding the protein must not
place the sequence out of reading frame and preferably will not
create complementary regions that could produce secondary mRNA
structure.
Substitutional variants are those in which at least one residue in
the amino acid sequence has been removed and a different residue
inserted in its place. Such substitutions generally are made in
accordance with the following Table 3 when it is desired to finely
modulate the characteristics of the protein. Table 3 shows amino
acids which may be substituted for an original amino acid in a
protein and which are regarded as conservative substitutions.
TABLE 3 Original Residue Conservative Substitutions Ala Ser Arg Lys
Asn gln, his Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His asn; gln
Ile leu, val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met;
leu; tyr Ser Thr Thr Ser Trp Tyr Tyr trp; phe Val ile; leu
Substantial changes in function or immunological identity are made
by selecting substitutions that are less conservative than those in
Table 3, i.e., selecting residues that differ more significantly in
their effect on maintaining (a) the structure of the polypeptide
backbone in the area of the substitution, for example, as a sheet
or helical conformation, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the bulk of the side chain. The
substitutions which in general are expected to produce the greatest
changes in protein properties will be those in which (a) a
hydrophilic residue, e.g., seryl or threonyl, is substituted for
(or by) a hydrophobic residue, e.g., leucyl, isoleucyl,
phenylalanyl, valyl or alanyl; (b) a cysteine or proline is
substituted for (or by) any other residue; (c) a residue having an
electropositive side chain, e.g., lysyl, arginyl, or histadyl, is
substituted for (or by) an electronegative residue, e.g., glutamyl
or aspartyl; or (d) a residue having a bulky side chain, e.g.,
phenylalanine, is substituted for (or by) one not having a side
chain, e.g., glycine.
The effects of these amino acid substitutions or deletions or
additions may be assessed for derivatives of the DLC-1 protein by
assays in which DNA molecules encoding the derivative proteins are
transfected into DLC-1 cells using routine procedures.
The DLC-1 gene, DLC-1 cDNA, DNA molecules derived therefrom and the
protein encoded by the cDNA and derivatives thereof may be utilized
in aspects of both the study of HCC and for diagnostic and
therapeutic applications related to HCC. Utilities of the present
invention include, but are not limited to, those utilities
described in the examples presented herein. Those skilled in the
art will recognize that the utilities herein described are not
limited to the specific experimental modes and materials presented
and will appreciate the wider potential utility of this
invention.
EXAMPLE 11
Expression of DLC-1 cDNA Sequences
With the provision of the DLC-1 cDNA (Seq. I.D. No. I), the
expression and purification of the DLC-1 protein by standard
laboratory techniques is now enabled. The purified protein may be
used for functional analyses, antibody production, diagnostics and
patient therapy. Furthermore, the DNA sequence of the DLC-1 cDNA
can be manipulated in studies to understand the expression of the
gene and the function of its product. Mutant forms of the DLC-1 may
be isolated based upon information contained herein, and may be
studied in order to detect alteration in expression patterns in
terms of relative quantities, tissue specificity and functional
properties of the encoded mutant DLC-1 protein. Partial or
full-length cDNA sequences, which encode for the subject protein,
may be ligated into bacterial expression vectors. Methods for
expressing large amounts of protein from a cloned gene introduced
into Escherichia coli (E. coli) may be utilized for the
purification, localization and functional analysis of proteins. For
example, fusion proteins consisting of amino terminal peptides
encoded by a portion of the E. coli lacZ or trpE gene linked to
DLC-1 proteins may be used to prepare polyclonal and monoclonal
antibodies against these proteins. Thereafter, these antibodies may
be used to purify proteins by immunoaffinity chromatography, in
diagnostic assays to quantitate the levels of protein and to
localize proteins in tissues and individual cells by
immunofluorescence.
Intact native protein may also be produced in E. coli in large
amounts for functional studies. Methods and plasmid vectors for
producing fusion proteins and intact native proteins in bacteria
are described in Sambrook et al. (In Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, N.Y., 1989, ch. 17) herein
incorporated by reference. Such fusion proteins may be made in
large amounts, are easy to purify, and can be used to elicit
antibody response. Native proteins can be produced in bacteria by
placing a strong, regulated promoter and an efficient ribosome
binding site upstream of the cloned gene. If low levels of protein
are produced, additional steps may be taken to increase protein
production; if high levels of protein are produced, purification is
relatively easy. Suitable methods are presented in Sambrook et al.
(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,
1989) and are well known in the art. Often, proteins expressed at
high levels are found in insoluble inclusion bodies. Methods for
extracting proteins from these aggregates are described by Sambrook
et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor, N.Y., 1989, ch. 17). Vector systems suitable for the
expression of lacZ fusion genes include the pUR series of vectors
(Ruther and Muller-Hill, EMBO J. 2:1791, 1983), pEX 1-3 (Stanley
and Luzio, EMBO J. 3:1429, 1984) and pMR100 (Gray et al., Proc.
Natl. Acad. Sci. USA 79:6598, 1982). Vectors suitable for the
production of intact native proteins include pKC30 (Shimatake and
Rosenberg, Nature 292:128, 1981), pKK177-3 (Amann and Brosius, Gene
40:183, 1985) and pET-3 (Studiar and Moffatt, J. Mol. Biol.
189:113, 1986). DLC-1 fusion proteins may be isolated from protein
gels, lyophilized, ground into a powder and used as an antigen. The
DNA sequence can also be transferred from its existing context in
pREP4 to other cloning vehicles, such as other plasmids,
bacteriophages, cosmids, animal viruses and yeast artificial
chromosomes (YACs) (Burkeet al., Science 236:806-12, 1987). These
vectors may then be introduced into a variety of hosts including
somatic cells, and simple or complex organisms, such as bacteria,
fungi (Timberlake and Marshall, Science 244:1313-7, 1989),
invertebrates, plants (Gasser and Fraley, Science 244:1293, 1989),
and pigs (Pursel et al., Science 244:1281-8, 1989), which cell or
organisms are rendered transgenic by the introduction of the
heterologous DLC-1 cDNA.
For expression in mammalian cells, the cDNA sequence may be ligated
to heterologous promoters, such as the simian virus (SV) 40,
promoter in the pSV2 vector (Mulligan and Berg, Proc. Natl. Acad.
Sci. USA 78:2072-6, 1981), and introduced into cells, such as
monkey COS-1 cells (Gluzman, Cell 23:175-182, 1981), to achieve
transient or long-term expression. The stable integration of the
chimeric gene construct may be maintained in mammalian cells by
biochemical selection, such as neomycin (Southern and Berg, J. Mot.
Appl. Genet. 1:32741, 1982) and mycophenolic acid (Mulligan and
Berg, Proc. Natl. Acad. Sci. USA 78:2072-6, 1981).
DNA sequences can be manipulated with standard procedures such as
restriction enzyme digestion, fill-in with DNA polymerase, deletion
by exonuclease, extension by terminal deoxynucleotide transferase,
ligation of synthetic or cloned DNA sequences, site-directed
sequence-alteration via single-stranded bacteriophage intermediate
or with the use of specific oligonucleotides in combination with
PCR.
The cDNA sequence (or portions derived from it) or a mini gene (a
cDNA with an intron and its own promoter) may be introduced into
eukaryotic expression vectors by conventional techniques. These
vectors are designed to permit the transcription of the cDNA in
eukaryotic cells by providing regulatory sequences that initiate
and enhance the transcription of the cDNA and ensure its proper
splicing and polyadenylation. Vectors containing the promoter and
enhancer regions of the SV40 or long terminal repeat (LTR) of the
Rous Sarcoma virus and polyadenylation and splicing signal from
SV40 are readily available (Mulligan and Berg, Proc. Natl. Acad.
Sci. USA 78:2072-6, 1981; Gorman et al., Proc. Natl. Acad Sci USA
78:6777-6781, 1982). The level of expression of the cDNA can be
manipulated with this type of vector, either by using promoters
that have different activities (for example, the baculovirus pAC373
can express cDNAs at high levels in S. frugiperda cells (Summers
and Smith, In: Genetically Altered Viruses and the Environment,
Fields et al. (Eds.) 22:319-328, Cold Spring Harbor Laboratory
Press. Cold Spring Harbor, New York, 1985) or by using vectors that
contain promoters amenable to modulation, for example, the
glucocorticoid-responsive promoter from the mouse mammary tumor
virus (Lee et al., Nature 294:228, 1982). The expression of the
cDNA can be monitored in the recipient cells 24 to 72 hours after
introduction (transient expression).
In addition, some vectors contain selectable markers such as the
gpt (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-6, 1981)
or neo (Southern and Berg, J. Mot Appl. Genet. 1:327-41, 1982)
bacterial genes. These selectable markers permit selection of
transfected cells that exhibit stable, long-term expression of the
vectors (and therefore the cDNA). The vectors can be maintained in
the cells as episomal, freely replicating entities by using
regulatory elements of viruses such as papilloma (Sarver et al.,
Mol. Cell Biol. 1:486, 1981) or Epstein-Barr (Sugden et al., Mol
Cell Biol. 5:410, 1985). Alternatively, one can also produce cell
lines that have integrated the vector into genomic DNA. Both of
these types of cell lines produce the gene product on a continuous
basis. One can also produce cell lines that have amplified the
number of copies of the vector (and therefore of the cDNA as well)
to create cell lines that can produce high levels of the gene
product (Alt et al., J. Biol. Chem. 253:1357, 1978).
The transfer of DNA into eukaryotic, in particular human or other
mammalian cells, is now a conventional technique. The vectors are
introduced into the recipient cells as pure DNA (transfection) by,
for example, precipitation with calcium phosphate (Graham and
vander Eb, Virology 52:466, 1973) or strontium phosphate (Brash et
al., Mol. Cell Biol 7:2013, 1987), electroporation (Neumann et al.,
EMBO J. 1:841, 1982), lipofection (Felgner et al., Proc. Natl. Acad
Sci USA 84:7413, 1987), DEAE dextran (McCuthan et al., J. Natl.
Cancer Inst. 41:351, 1968), microinjection (Mueller et al., Cell
15:579, 1978), protoplast fusion (Schafner, Proc. Natl. Acad. Sci.
USA 77:2163-7, 1980), or pellet guns (Klein et al., Nature 327:70,
1987). Alternatively, the cDNA can be introduced by infection with
virus vectors. Systems are developed that use, for example,
retroviruses (Bernstein et al., Gen. Engrg. 7:235, 1985),
adenoviruses (Ahmad et al., J. Virol. 57:267, 1996), or Herpes
virus (Spaete et al, Cell 30:295, 1982).
These eukaryotic expression systems can be used for studies of the
DLC-1 gene and mutant forms of this gene, the DLC-1 protein and
mutant forms of this protein. Such uses include, for example, the
identification of regulatory elements located in the 5' region of
the DLC-1 gene on genomic clones that can be isolated from human
genomic DNA libraries using the information contained in the
present invention. The eukaryotic expression systems may also be
used to study the function of the normal complete protein, specific
portions of the protein, or of naturally occurring or artificially
produced mutant proteins.
Using the above techniques, the expression vectors containing the
DLC-1 gene sequence or fragments or variants or mutants thereof can
be introduced into human cells, mammalian cells from other species
or non-mammalian cells as desired. The choice of cell is determined
by the purpose of the treatment. For example, monkey COS cells
(Gluzman. Cell 23:175-182, 1981) that produce high levels of the
SV40 T antigen and permit the replication of vectors containing the
SV40 origin of replication may be used. Similarly, Chinese hamster
ovary (CHO), mouse NIH 3T3 fibroblasts or human fibroblasts or
lymphoblasts (as described herein) may be used.
The following is provided as one exemplary method to express DLC-1
polypeptide from the cloned DLC-1 cDNA sequences in mammalian
cells. Cloning vector pXTI, commercially available from Stratagene,
contains the Long Terminal Repeats (LTRs) and a portion of the GAG
gene from Moloney Murine Leukemia Virus. The position of the viral
LTRs allows highly efficient, stable transfection of the region
within the LTRs. The vector also contains the Herpes Simplex
Thymidine Kinase promoter (TK), active in embryonal cells and in a
wide variety of tissues in mice, and a selectable neomycin gene
conferring G418 resistance. Two unique restriction sites Bg/II and
XhoI are directly downstream from the TK promoter. DLC-1 cDNA,
including the entire open reading frame for the DLC-1 protein and
the 3' untranslated region of the cDNA is cloned into one of the
two unique restriction sites downstream from the promoter.
The ligated product is transfected into mouse NIH 3T3 cells using
Lipofectin (Life Technologies, Inc.) under conditions outlined in
the product specification. Positive transfectants are selected
after growing the transfected cells in 600 .mu.g/ml G418 (Sigma,
St. Louis, Mo.). The protein is released into the supernatant and
may be purified by standard immunoaffinity chromatography
techniques using antibodies raised against the DLC-1 protein, as
described below.
Expression of the DLC-1 protein in eukaryotic cells may also be
used as a source of proteins to raise antibodies. The DLC-1 protein
may be extracted following release of the protein into the
supernatant as described above, or, the cDNA sequence may be
incorporated into a eukaryotic expression vector and expressed as a
chimeric protein with, for example, .beta.-globin. Antibody to
.beta.-globin is thereafter used to purify the chimeric protein.
Corresponding protease cleavage sites engineered between the
.beta.-globin gene and the cDNA are then used to separate the two
polypeptide fragments from one another after translation. One
useful expression vector for generating .beta.-globin chimeric
proteins is pSG5 (Stratagene). This vector encodes rabbit
.beta.-globin.
The present invention thus encompasses recombinant vectors which
comprise all or part of the DLC-1 gene or cDNA sequences, for
expression in a suitable host. The DLC-1 DNA is operatively linked
in the vector to an expression control sequence in the recombinant
DNA molecule so that the DLC-1 polypeptide can be expressed. The
expression control sequence may be selected from the group
consisting of sequences that control the expression of genes of
prokaryotic or eukaryotic cells and their viruses and combinations
thereof. The expression control sequence may be specifically
selected from the group consisting of the lac system, the trp
system, the tac system, the trc system, major operator and promoter
regions of phage lambda, the control region of fd coat protein, the
early and late promoters of SV40, promoters derived from polyoma,
adenovirus, retrovirus, baculovirus and simian virus, the promoter
for 3-phosphoglycerate kinase, the promoters of yeast acid
phosphatase, the promoter of the yeast alpha-mating factors and
combinations thereof.
The host cell, which may be transfected with the vector of this
invention, may be selected from the group consisting of E-coli,
Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus or
other bacilli; other bacteria; yeast; fungi; insect; mouse or other
animal; or plant hosts; or human tissue cells.
It is appreciated that for mutant or variant DLC-1 DNA sequences,
similar systems are employed to express and produce the mutant
product.
EXAMPLE 12
Production of an Antibody to DLC-1 Protein
Monoclonal or polyclonal antibodies may be produced to either the
normal DLC-1 protein or mutant forms of this protein. Optimally,
antibodies raised against the DLC-1 protein would specifically
detect the DLC-1 protein. That is, such antibodies would recognize
and bind the DLC-1 protein and would not substantially recognize or
bind to other proteins found in human cells. The determination that
an antibody specifically detects the DLC-1 protein is made by any
one of a number of standard immunoassay methods; for instance, the
Western blotting technique (Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, N.Y., 1989). To determine
that a given antibody preparation (such as one produced in a mouse)
specifically detects the DLC-1 protein by Western blotting, total
cellular protein is extracted from human cells (for example,
lymphocytes) and electrophoresed on a sodium dodecyl
sulfate-polyacrylamide gel. Tte proteins are then transferred to a
membrane (for example, nitrocellulose) by Western blotting, and the
antibody preparation is incubated with the membrane. After washing
the membrane to remove non-specifically bound antibodies, the
presence of specifically bound antibodies is detected by the use of
an anti-mouse antibody conjugated to an enzyme such as alkaline
phosphatase; application of the substrate
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results
in the production 0.25 of a dense blue compound by immuno-localized
alkaline phosphatase. Antibodies which specifically detect the
DLC-1 protein will, by this technique, be shown to bind to the
DLC-1 protein band (which will be localized at a given position on
the gel determined by its molecular weight). Non-specific binding
of the antibody to other proteins may occur and may be detectable
as a weak signal on the Western blot. The non-specific nature of
this binding will be recognized by one skilled in the art by the
weak signal obtained on the Western blot relative to the strong
primary signal arising from the specific antibody-DLC-1 protein
binding.
Substantially pure DLC-1 protein suitable for use as an immunogen
is isolated from transfected or transformed cells. Concentration of
protein in the final preparation is adjusted, for example, by
concentration on an Amicon filter device, to the level of a few
micrograms per milliliter. Monoclonal or polyclonal antibody to the
protein can then be prepared as follows:
Monoclonal Antibody Production by Hybridoma Fusion
Monoclonal antibody to epitopes of the DLC-1 protein identified and
isolated as described can be prepared from murine hybridomas
according to the classical method of Kohler and Milstein (Nature
256:495, 1975) or derivative methods thereof. Briefly, a mouse is
repetitively inoculated with a few micrograms of the selected
protein over a period of a few weeks. The mouse is then sacrificed,
and the antibody-producing cells of the spleen isolated. The spleen
cells are fused by means of polyethylene glycol with mouse myeloma
cells, and the excess unfused cells destroyed by growth of the
system on selective media comprising aminopterin (HAT media). The
successfully fused cells are diluted and aliquots of the dilution
placed in wells of a microtiter plate where growth of the culture
is continued. Antibody-producing clones are identified by detection
of antibody in the supernatant fluid of the wells by immunoassay
procedures, such as ELISA, as originally described by Engvall
(Enzymol. 70:419, 1980), and derivative methods thereof. Selected
positive clones can be expanded and their monoclonal antibody
product harvested for use. Detailed procedures for monoclonal
antibody production are described in Harlow and Lane (Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, New York,
1988).
Polyclonal Antibody Production by Immunization
Polyclonal antiserum containing antibodies to heterogenous epitopes
of a single protein can be prepared by immunizing suitable animals
with the expressed protein, which can be unmodified or modified to
enhance immunogenicity. Effective polyclonal antibody production is
affected by many factors related both to the antigen and the host
species. For example, small molecules tend to be less immunogenic
than others and may require the use of carriers and adjuvant. Also,
host animals vary in response to site of inoculations and dose,
with both inadequate or excessive doses of antigen resulting in low
titer antisera. Small doses (ng level) of antigen administered at
multiple intradermal sites appears to be most reliable. An
effective immunization protocol for rabbits can be found in
Vaitukaitis et al. (J. Clin. Endocrinol. Metab. 33:988-91,
1971).
Booster injections can be given at regular intervals, and antiserum
harvested when antibody titer thereof, as determined
semi-quantitatively, for example, by double immunodiffusion in,
agar against known concentrations of the antigen, begins to fall.
See, for example, Ouchterlony et al. (In Handbook of Experimental
Immunology, Wier, D. (ed.) chapter 19, Blackwell, 1973). Plateau
concentration of antibody is usually in the range of 0.1 to 0.2
mg/ml of serum (about 12 .mu.M). Affinity of the antisera for the
antigen is determined by preparing competitive binding curves, as
described, for example, by Fisher (Manual of Clinical Immunology,
Ch. 42, 1980).
Antibodies Raised against Synthetic Peptides
A third approach to raising antibodies against the DLC-1 protein is
to use synthetic peptides synthesized on a commercially available
peptide synthesizer based upon the predicted amino acid sequence of
the DLC-1 protein.
Antibodies Raised by Injection of DLC-1 Gene
Antibodies may be raised against the DLC-1 protein by subcutaneous
injection of a DNA vector which expresses the DLC-1 protein into
laboratory animals, such as mice. Delivery of the recombinant
vector into the animals may be achieved using a hand-held form of
the Biolistic system (Sanford et al., Particulate Sci. Technol.
5:27-37, 1987) as described by Tang et al. (Nature 356:152-4,
1992). Expression vectors suitable for this purpose may include
those which express the DLC-1 gene under the transcriptional
control of either the human .beta.-actin promoter or the
cytomegalovirus (CMV) promoter.
Antibody preparations prepared according to these protocols are
useful in quantitative immunoassays which determine concentrations
of antigen-bearing substances in biological samples; they are also
used semi-quantitatively or qualitatively to identify the presence
of antigen in a biological sample.
EXAMPLE 13
DNA-Based Diagnosis
One major application of the DLC-1 sequence information presented
herein is in the area of genetic testing for predisposition to HCC,
BC, PC and/or CRC owing to DLC-1 deletion or mutation. The gene
sequence of the DLC-1 gene, including intron-exon boundaries is
also useful in such diagnostic methods. Individuals carrying
mutations in the DLC-1 gene, or having heterozygous or homozygous
deletions of the DLC-1 gene, may be detected at the DNA level with
the use of a variety of techniques. For such a diagnostic
procedure, a biological sample of the subject, which biological
sample contains either DNA or RNA derived from the subject, is
assayed for a mutated or deleted DLC-1 gene. Suitable biological
samples include samples containing genomic DNA or RNA obtained from
body cells, such as those present in peripheral blood, urine,
saliva, tissue biopsy, surgical specimen, amniocentesis samples and
autopsy material. The detection in the biological sample of either
a mutant DLC-1 gene, a mutant DLC-1 RNA, or a homozygously or
heterozygously deleted DLC-1 gene, may be performed by a number of
methodologies, as outlined below.
A preferred embodiment of such detection techniques is the
polymerase chain reaction amplification of reverse transcribed RNA
(RT-PCR) of RNA isolated from lymphocytes followed by direct DNA
sequence determination of the products. The presence of one or more
nucleotide differences between the obtained sequence and the cDNA
sequences, and especially, differences in the ORF portion of the
nucleotide sequence are taken as indicative of a potential DLC-1
gene mutation.
Alternatively, DNA extracted from lymphocytes or other cells may be
used directly for amplification. The direct amplification from
genomic DNA would be appropriate for analysis of the entire DLC-1
gene including regulatory sequences located upstream and downstream
from the open reading frame. Recent reviews of direct DNA diagnosis
have been presented by Caskey (Science 236:1223-8, 1989) and by
Landegren et al. (Science 242:229-37, 1989).
Further studies of DLC-1 genes isolated from DLC-1 patients may
reveal particular mutations, or deletions, which occur at a high
frequency within this population of individuals. In this case,
rather than sequencing the entire DLC-1 gene, it may be possible to
design DNA diagnostic methods to specifically detect the most
common DLC-1 mutations or deletions.
The detection of specific DNA mutations may be achieved by methods
such as hybridization using specific oligonucleotides (Wallace et
al., Cold Spring Harbor Symp. Quant. Biol. 51:257-61, 1986), direct
DNA sequencing (Church and Gilbert, Proc. Natl. Acad Sci USA
81:1991-5, 1988), the use of restriction enzymes (Flavell et al.,
Cell 15:25, 1978; Geever et al., Proc. Natl. Acad Sci USA 78:5081,
1981), discrimination on the basis of electrophoretic mobility in
gels with denaturing reagent (Myers and Maniatis, Cold Spring
Harbor Symp. Quant. Biol. 51:275-84, 1986), RNase protection (Myers
et al., Science 230:1242, 1985), chemical cleavage (Cotton et al.,
Proc. Natl. Acad. Sci. USA 85:4397401, 1988), and the
ligase-mediated detection procedure (Landegren et al., Science
241:1077, 1988).
Oligonucleotides specific to normal or mutant sequences are
chemically synthesized using commercially available machines,
labeled radioactively with isotopes (such as .sup.32 P) or
non-radioactively, with tags such as biotin (Ward and Langer et
al., Proc. Natl. Acad. Sci. USA 78:6633-57, 1981), and hybridized
to individual DNA samples immobilized on membranes or other solid
supports by dot-blot or transfer from gels after electrophoresis.
The presence of these specific sequences are visualized by methods
such as autoradiography or fluorometric (Landegren, et al., Science
242:229-37, 1989) or calorimetric reactions (Gebeyehu et al.,
Nucleic Acids Res. 15:4513-34, 1987). The absence of hybridization
would indicate a mutation in the particular region of the gene, or
deleted DLC-1 gene.
Sequence differences between normal and mutant forms of the DLC-1
gene may also be revealed by the direct DNA sequencing method of
Church and Gilbert (Proc. Natl. Acad. Sci. USA 81: 1991-5, 1988).
Cloned DNA segments may be used as probes to detect specific DNA
segments. The sensitivity of this method is greatly enhanced when
combined with PCR (Wrichnik et al., Nucleic Acids Res. 15:529-42,
1987; Wong et al., Nature 330:384-386, 1987; Stoflet et al.,
Science 239:491-4, 1988). In this approach, a sequencing primer
which lies within the amplified sequence is used with
double-stranded PCR product or single-stranded template generated
by a modified PCR. The sequence determination is performed by
conventional procedures with radiolabeled nucleotides or by
automatic sequencing procedures with fluorescent tags.
Sequence alterations may occasionally generate fortuitous
restriction enzyme recognition sites or may eliminate existing
restriction sites. Changes in restriction sites are revealed by the
use of appropriate enzyme digestion followed by conventional
gel-blot hybridization (Southern, J. Mol. Biol. 98:503, 1975). DNA
fragments carrying the site (either normal or mutant) are detected
by their reduction in size or increase of corresponding restriction
fragment numbers. Genomic DNA samples may also be amplified by PCR
prior to treatment with the appropriate restriction enzyme;
fragments of different sizes are then visualized under UV light in
the presence of ethidium bromide after gel electrophoresis.
Genetic testing based on DNA sequence differences may be achieved
by detection of alteration in electrophoretic mobility of DNA
fragments in gels with or without denaturing reagent. Small
sequence deletions and insertions can be visualized by
high-resolution gel electrophoresis. For example, a PCR product
with small deletions is clearly distinguishable from a normal
sequence on an 8% non-denaturing polyacrylamide gel (WO 91/10734;
Nagamine et al., Am. J. Hum. Genet. 45:337-9, 1989). DNA fragments
of different sequence compositions may be distinguished on
denaturing formamide gradient gels in which the mobilities of
different DNA fragments are retarded in the gel at different
positions according to their specific "partial-melting"
temperatures (Myers et al., Science -230:1242, 1985).
Alternatively, a method of detecting a mutation comprising a single
base substitution or other small change could be based on
differential primer length in a PCR. For example, an invariant
primer could be used in addition to a primer specific for a
mutation. The PCR products of the normal and mutant genes can then
be differentially detected in acrylamide gels.
In addition to conventional gel-electrophoresis and
blot-hybridization methods, DNA fragments may also be visualized by
methods where the individual DNA samples are not immobilized on
membranes. The probe and target sequences may be both in solution,
or the probe sequence may be immobilized (Saiki et al., Proc. Nat.
Acad. Sci. USA 86:6230-4, 1989). A variety of detection methods,
such as autoradiography involving radioisotopes, direct detection
of radioactive decay (in the presence or absence of scintillant),
spectrophotometry involving calorigenic reactions and fluorometry
involved fluorogenic reactions, may be used to identify specific
individual genotypes.
If more than one mutation is frequently encountered in the DLC-1
gene, a system capable of detecting such multiple mutations would
be desirable. For example, a PCR with multiple, specific
oligonucleotide primers and hybridization probes may be used to
identify all possible mutations at the same time (Chamberlain et
al. Nucl. Acids Res. 16:1141-55, 1988). The procedure may involve
immobilized sequence-specific oligonucleotides probes (Saiki et
al., Proc. Nat. Acad. Sci. USA 86:6230-4, 1989).
The following Example describes one method by which deletions of
the DLC-1 gene may be detected.
EXAMPLE 14
Two Step Assay to Detect the Presence of DLC-1 Gene in a Sample
Patient liver, breast, prostate and/or colorectal tissue sample is
processed according to the method disclosed by Antonarakis, et al.
(New Eng. J. Med. 313:842-848, 1985), separated through a 1%
agarose gel and transferred to a nylon membrane for Southern blot
analysis. Membranes are UV cross linked at 150 ml using a GS Gene
Linker (Bio-Rad). A DLC-1 probe is subcloned into pTZ18U. The
phagemids are transformed into E. coli MV 1190 infected with M13KO7
helper phage (Bio-Rad, Richmond, Calif.). Single stranded DNA is
isolated according to standard procedures (see Sambrook, et al.
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,
1989).
Blots are prehybridized for 15-30 min. at 65.degree. C. in 7%
sodium dodecyl sulfate (SDS) in 0.5M NaPO.sub.4. The methods follow
those described by Nguyen, et al. (BioTechniques 13:116-123, 1992).
The blots are hybridized overnight at 65.degree. C. in 7% SDS, 0.5M
NaPO.sub.4 with 25-50 ng/ml single stranded probe DNA.
Post-hybridization washes consist of two 30 min. washes in 5% SDS,
40 mM NaPO.sub.4 at 65.degree. C., followed by two 30-min washes in
1% SDS, 40 mM NaPO.sub.4 at 65.degree. C.
Next the blots are rinsed with phosphate buffered saline (pH 6.8)
for 5 min at room temperature and incubated with 0.2% casein in PBS
for 5 min. The blots are then preincubated for 5-10 minutes in a
shaking water bath at 45.degree. C. with hybridization buffer
consisting or 6M urea, 0.3M NaCl, and 5.times. Denhardt's solution
(see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y., 1989). The buffer is removed and replaced with
50-75 .mu.Vcm.sup.2 fresh hybridization buffer plus 2.5 nM of the
covalently cross-linked oligonucleotide sequence complementary to
the universal primer site (UP-AP, Bio-Rad). The blots are
hybridized for 20-30 min at 45.degree. C. and post hybridization
washes are incubated at 45.degree. C. as two 10 min washes in 6 M
urea, 1.times.standard saline citrate (SSC), 0.1% SDS and one 10
min wash in 1.times.SSC, 0.1% Triton.RTM. X-100. The blots are
rinsed for 10 min at room temperature with 1XSSC.
Blots are incubated for 10 min at room temperature with shaking in
the substrate buffer consisting of 0.1M diethanolamine, 1 mM
MgCl.sub.2, 0.02% sodium azide, pH 10.0. Individual blots are
placed in heat sealable bags with substrate buffer and 0.2 mM AMPPD
(3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane
, disodium salt, Bio-Rad). After a 20 min incubation at room
temperature with shaking, the excess AMPPD solution is removed. The
blot is exposed to X-ray film overnight. Positive bands indicate
the presence of the DLC-1 gene. Patient samples which show no
hybridizing bands lack the DLC-1 gene, indicating the possibility
of ongoing cancer, or an enhanced susceptibility to developing
cancer in the future.
EXAMPLE 15
Quantitation of DLC-1 Protein
An alternative method of diagnosing DLC-1 gene deletion or mutation
is to quantitate the level of DLC-1 protein in the cells of an
individual. This diagnostic tool would be useful for detecting
reduced levels of the DLC-1 protein which result from, for example,
mutations in the promoter regions of the DLC-1 gene or mutations
within the coding region of the gene which produced truncated,
non-functional polypeptides, as well as from deletions of the
entire DLC-1 gene. The determination of reduced DLC-1 protein
levels would be an alternative or supplemental approach to the
direct determination of DLC-1 gene deletion or mutation status by
the methods outlined above. The availability of antibodies specific
to the DLC-1 protein will facilitate the quantitation of cellular
DLC-1 protein by one of a number of immunoassay methods which are
well known in the art and are presented in Harlow and Lane
(Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory,
New York, 1988).
For the purposes of quantitating the DLC-1 protein, a biological
sample of the subject, which sample includes cellular proteins, is
required. Such a biological sample may be obtained from body cells,
such as those present in peripheral blood, urine, saliva, tissue
biopsy, amniocentesis samples, surgical specimens and autopsy
material, particularly liver cells. Quantitation of DLC-1 protein
is achieved by immunoassay and compared to levels of the protein
found in healthy cells. A significant (e.g., 50% or greater)
reduction in the amount of DLC-1 protein in the cells of a subject
compared to the amount of DLC-1 protein found in normal human cells
would be taken as an indication that the subject may have deletions
or mutations in the DLC-1 gene locus.
EXAMPLE 16
Gene Therapy
A new gene therapy approach for DLC-1 patients is now made possible
by the present invention. Essentially, liver cells may be removed
from a patient having deletions or mutations of the DLC-1 gene, and
then transfected with an expression vector containing the DLC-1
cDNA. These transfected liver cells will thereby produce functional
DLC-1 protein and can be reintroduced into the patient. In addition
to liver cells, breast, colorectal, prostate, or other cells may be
used, depending on the cancer of interest.
The scientific and medical procedures required for human cell
transfection are now routine procedures. The provision herein or
DLC-1 cDNAs now allows the development of human gene therapy based
upon these procedures. Immunotherapy of melanoma patients using
genetically engineered tumor-infiltrating lymphocytes (TILs) has
been reported by Rosenberg et al. (N. Engl. J. Med. 323:570-8,
1990). In that study, a retrovirus vector was used to introduce a
gene for neomycin resistance into TILs. A similar approach may be
used to introduce the DLC-1 cDNA into patients affected by DLC-1
deletions or mutations.
Retroviruses have been considered the preferred vector for
experiments in gene therapy, with a high efficiency of infection
and stable integration and expression (Orkin et al., Prog. Med.
Genet. 7:130, 1988). The full length DLC-1 gene or cDNA can be
cloned into a retroviral vector and driven from either its
endogenous promoter or from the retroviral LTR (long terminal
repeat). Other viral transfection systems may also be utilized for
this type of approach, including Adeno-Associated virus (AAV)
(McLaughlin et al., J. Virol. 62:1963, 1988), Vaccinia virus (Moss
et al., Annu. Rev. Immunol. 5:305, 1987), Bovine Papilloma virus
(Rasmussen et al., Methods Enzymol. 139:642, 1987) or members of
the herpesvirus group such as Epstein-Barr virus (Margolskee et
al., Mol. Cell. Biol. 8:283747, 1988). Recent developments in gene
therapy techniques include the use of RNA-DNA hybrid
oligonucleotides, as described by Cole-Strauss, et al. (Science
273:1386-9, 1996). This technique may allow for site-specific
integration of cloned sequences, permitting accurately targeted
gene replacement.
Having illustrated and described the principles of isolating the
human DLC-1 cDNA and its corresponding genomic genes, the protein
and modes of use of these biological molecules, it should be
apparent to one skilled in the art that the invention can be
modified in arrangement and detail without departing from such
principles. We claim all modifications coming within the spirit and
scope of the claims presented herein.
SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
NUMBER OF SEQ ID NOS: 31 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1 <211> LENGTH: 3850 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (325)..(3600)
<400> SEQUENCE: 1 tatgggctcg agcggccgcc cgggcaggtg cccgagcgag
ggcgcttcgc tcccagccag 60 gacatggccg cacctctccg catcaggagc
gccggctcac ggacttctcg cccaactccc 120 tgagcgctcc ctcgtttcga
tctttagaaa accccgcttt ctttctgggg ccgtgacgag 180 gggcagggag
cggcgagcaa ggatgcgttg aggaccgcga gggcgcgcgt ctcgggtgcc 240
gccgtgggtc ccgacgcgga agccgagccg cctccgcctg cctcgacttc cccacagcgc
300 ttccgccgcc gcctgccgtg cttg atg tgc aga aag aag ccg gac acc atg
351 Met Cys Arg Lys Lys Pro Asp Thr Met 1 5 atc cta aca caa att gaa
gcc aag gaa gct tgt gat tgg cta cgg gca 399 Ile Leu Thr Gln Ile Glu
Ala Lys Glu Ala Cys Asp Trp Leu Arg Ala 10 15 20 25 act ggt ttc ccc
cag tat gca cag ctt tat gaa gat ttc ctg ttc ccc 447 Thr Gly Phe Pro
Gln Tyr Ala Gln Leu Tyr Glu Asp Phe Leu Phe Pro 30 35 40 atc gat
att tcc ttg gtc aag aga gag cat gat ttt ttg gac aga gat 495 Ile Asp
Ile Ser Leu Val Lys Arg Glu His Asp Phe Leu Asp Arg Asp 45 50 55
gcc att gag gct cta tgc agg cgt cta aat act tta aac aaa tgt gcg 543
Ala Ile Glu Ala Leu Cys Arg Arg Leu Asn Thr Leu Asn Lys Cys Ala 60
65 70 gtg atg aag cta gaa att agt cct cat cgg aaa cga agt gac gat
tca 591 Val Met Lys Leu Glu Ile Ser Pro His Arg Lys Arg Ser Asp Asp
Ser 75 80 85 gac gag gat gag cct tgt gcc atc agt ggc aaa tgg act
ttc caa agg 639 Asp Glu Asp Glu Pro Cys Ala Ile Ser Gly Lys Trp Thr
Phe Gln Arg 90 95 100 105 gac agc aag agg tgg tcc cgg ctt gaa gag
ttt gat gtc ttt tct cca 687 Asp Ser Lys Arg Trp Ser Arg Leu Glu Glu
Phe Asp Val Phe Ser Pro 110 115 120 aaa caa gac ctg gtc cct ggg tcc
cca gac gac tcc cac ccg aag gac 735 Lys Gln Asp Leu Val Pro Gly Ser
Pro Asp Asp Ser His Pro Lys Asp 125 130 135 ggc ccc agc ccc gga ggc
acg ctg atg gac ctc agc gag cgc cag gag 783 Gly Pro Ser Pro Gly Gly
Thr Leu Met Asp Leu Ser Glu Arg Gln Glu 140 145 150 gtg tct tcc gtc
cgc agc ctc agc agc act ggc agc ctc ccc agc cac 831 Val Ser Ser Val
Arg Ser Leu Ser Ser Thr Gly Ser Leu Pro Ser His 155 160 165 gcg ccc
ccc agc gag gat gct gcc acc ccc cgg act aac tcc gtc atc 879 Ala Pro
Pro Ser Glu Asp Ala Ala Thr Pro Arg Thr Asn Ser Val Ile 170 175 180
185 agc gtt tgc tcc tcc agc aac ttg gca ggc aat gac gac tct ttc ggc
927 Ser Val Cys Ser Ser Ser Asn Leu Ala Gly Asn Asp Asp Ser Phe Gly
190 195 200 agc ctg ccc tct ccc aag gaa ctg tcc agc ttc agc ttc agc
atg aaa 975 Ser Leu Pro Ser Pro Lys Glu Leu Ser Ser Phe Ser Phe Ser
Met Lys 205 210 215 ggc cac gaa aaa act gcc aag tcc aag acg cgc agt
ctg ctg aaa cgg 1023 Gly His Glu Lys Thr Ala Lys Ser Lys Thr Arg
Ser Leu Leu Lys Arg 220 225 230 atg gag agc ctg aag ctc aag agc tcc
cat cac agc aag cac aaa gcg 1071 Met Glu Ser Leu Lys Leu Lys Ser
Ser His His Ser Lys His Lys Ala 235 240 245 ccc tca aag ctg ggg ttg
atc atc agc ggg ccc atc ttg caa gag ggg 1119 Pro Ser Lys Leu Gly
Leu Ile Ile Ser Gly Pro Ile Leu Gln Glu Gly 250 255 260 265 atg gat
gag gag aag ctg aag cag ctc agc tgc gtg gag atc tcc gcc 1167 Met
Asp Glu Glu Lys Leu Lys Gln Leu Ser Cys Val Glu Ile Ser Ala 270 275
280 ctc aat ggc aac cgc atc aac gtc ccc atg gta cga aag agg agc gtt
1215 Leu Asn Gly Asn Arg Ile Asn Val Pro Met Val Arg Lys Arg Ser
Val 285 290 295 tcc aac tcc acg cag acc agc agc agc agc agc cag tcg
gag acc agc 1263 Ser Asn Ser Thr Gln Thr Ser Ser Ser Ser Ser Gln
Ser Glu Thr Ser 300 305 310 agc gcg gtc agc acg ccc agc cct gtt acg
agg acc cgg agc ctc agt 1311 Ser Ala Val Ser Thr Pro Ser Pro Val
Thr Arg Thr Arg Ser Leu Ser 315 320 325 gcg tgc aac aag cgg gtg ggc
atg tac tta gag ggc ttc gat cct ttc 1359 Ala Cys Asn Lys Arg Val
Gly Met Tyr Leu Glu Gly Phe Asp Pro Phe 330 335 340 345 aat cag tca
aca ttt aac aac gtg gtg gag cag aac ttt aag aac cgc 1407 Asn Gln
Ser Thr Phe Asn Asn Val Val Glu Gln Asn Phe Lys Asn Arg 350 355 360
gag agc tac cca gag gac acg gtg ttc tac atc cct gaa gat cac aag
1455 Glu Ser Tyr Pro Glu Asp Thr Val Phe Tyr Ile Pro Glu Asp His
Lys 365 370 375 cct ggc act ttc ccc aaa gct ctc acc aat ggc agt ttc
tcc ccc tcg 1503 Pro Gly Thr Phe Pro Lys Ala Leu Thr Asn Gly Ser
Phe Ser Pro Ser 380 385 390 ggg aat aac ggc tct gtg aac tgg agg acg
gga agc ttc cac ggc cct 1551 Gly Asn Asn Gly Ser Val Asn Trp Arg
Thr Gly Ser Phe His Gly Pro 395 400 405 ggc cac atc agc ctc agg agg
gaa aac agt agc gac agc ccc aag gaa 1599 Gly His Ile Ser Leu Arg
Arg Glu Asn Ser Ser Asp Ser Pro Lys Glu 410 415 420 425 ctg aag aga
cgc aat tct tcc agc tcc atg agc agc cgc ctg agc atc 1647 Leu Lys
Arg Arg Asn Ser Ser Ser Ser Met Ser Ser Arg Leu Ser Ile 430 435 440
tac gac aac gtg ccg ggc tcc atc ctc tac tcc agt tca ggg gac ctg
1695 Tyr Asp Asn Val Pro Gly Ser Ile Leu Tyr Ser Ser Ser Gly Asp
Leu 445 450 455 gcg gat ctg gag aac gag gac atc ttc ccc gag ctg gac
gac atc ctc 1743 Ala Asp Leu Glu Asn Glu Asp Ile Phe Pro Glu Leu
Asp Asp Ile Leu 460 465 470 tac cac gtg aag ggg atg cag cgg ata gtc
aat cag tgg tcg gag aag 1791 Tyr His Val Lys Gly Met Gln Arg Ile
Val Asn Gln Trp Ser Glu Lys 475 480 485 ttt tct gat gag gga gat tcg
gac tca gcc ctg gac tcg gtc tct ccc 1839 Phe Ser Asp Glu Gly Asp
Ser Asp Ser Ala Leu Asp Ser Val Ser Pro 490 495 500 505 tgc ccg tcc
tct cca aaa cag ata cac ctg gat gtg gac aac gac cga 1887 Cys Pro
Ser Ser Pro Lys Gln Ile His Leu Asp Val Asp Asn Asp Arg 510 515 520
acc aca ccc agc gac ctg gac agc aca ggc aac tcc ctg aat gaa ccg
1935 Thr Thr Pro Ser Asp Leu Asp Ser Thr Gly Asn Ser Leu Asn Glu
Pro 525 530 535 gaa gag ccc tcc gag atc ccg gaa aga agg gat tct ggg
gtt ggg gct 1983 Glu Glu Pro Ser Glu Ile Pro Glu Arg Arg Asp Ser
Gly Val Gly Ala 540 545 550 tcc cta acc agg tcc aac agg cac cga ctg
aga tgg cac agt ttc cag 2031 Ser Leu Thr Arg Ser Asn Arg His Arg
Leu Arg Trp His Ser Phe Gln 555 560 565 agc tca cat cgg cca agc ctc
aac tct gta tca cta cag att aac tgc 2079 Ser Ser His Arg Pro Ser
Leu Asn Ser Val Ser Leu Gln Ile Asn Cys 570 575 580 585 cag tct gtg
gcc cag atg aac ctg ctg cag aaa tac tca ctc cta aag 2127 Gln Ser
Val Ala Gln Met Asn Leu Leu Gln Lys Tyr Ser Leu Leu Lys 590 595 600
cta acg gcc ctg ctg gag aaa tac aca cct tct aac aag cat ggt ttt
2175 Leu Thr Ala Leu Leu Glu Lys Tyr Thr Pro Ser Asn Lys His Gly
Phe 605 610 615 agc tgg gcc gtg ccc aag ttc atg aag agg atc aag gtt
cca gac tac 2223 Ser Trp Ala Val Pro Lys Phe Met Lys Arg Ile Lys
Val Pro Asp Tyr 620 625 630 aag gac cgg agt gtg ttt ggg gtc cca ctg
acg gtc aac gtg cag cgc 2271 Lys Asp Arg Ser Val Phe Gly Val Pro
Leu Thr Val Asn Val Gln Arg 635 640 645 aca gga caa ccg ttg cct cag
agc atc cag cag gcc atg cga tac ctc 2319 Thr Gly Gln Pro Leu Pro
Gln Ser Ile Gln Gln Ala Met Arg Tyr Leu 650 655 660 665 cgg aac cat
tgt ttg gat cag gtt ggg ctc ttc aaa aaa tcg ggg gtc 2367 Arg Asn
His Cys Leu Asp Gln Val Gly Leu Phe Lys Lys Ser Gly Val 670 675 680
aag tcc cgg att cag gct ctg cgc cag atg aat gaa ggt gcc ata gac
2415 Lys Ser Arg Ile Gln Ala Leu Arg Gln Met Asn Glu Gly Ala Ile
Asp 685 690 695 tgt gtc aac tac gaa gga cag tct gct tat gac gtg gca
gac atg ctg 2463 Cys Val Asn Tyr Glu Gly Gln Ser Ala Tyr Asp Val
Ala Asp Met Leu 700 705 710 aag cag tat ttt cga gat ctt cct gag cca
cta atg acg aac aaa ctc 2511 Lys Gln Tyr Phe Arg Asp Leu Pro Glu
Pro Leu Met Thr Asn Lys Leu 715 720 725 tcg gaa acc ttt cta cag atc
tac caa tat gtg ccc aag gac cag cgc 2559 Ser Glu Thr Phe Leu Gln
Ile Tyr Gln Tyr Val Pro Lys Asp Gln Arg 730 735 740 745 ctg cag gcc
atc aag gct gcc atc atg ctg ctg cct gac gag aac cgg 2607 Leu Gln
Ala Ile Lys Ala Ala Ile Met Leu Leu Pro Asp Glu Asn Arg 750 755 760
gtg gtt ctg cag acc ctg ctt tat ttc ctg tgc gat gtc aca gca gcc
2655 Val Val Leu Gln Thr Leu Leu Tyr Phe Leu Cys Asp Val Thr Ala
Ala 765 770 775 gta aaa gaa aac cag atg acc cca acc aac ctg gcc gtg
tgc tta gcg 2703 Val Lys Glu Asn Gln Met Thr Pro Thr Asn Leu Ala
Val Cys Leu Ala 780 785 790 cct tcc ctc ttc cat ctc aac acc ctg aag
aga gag aat tcc tct ccc 2751 Pro Ser Leu Phe His Leu Asn Thr Leu
Lys Arg Glu Asn Ser Ser Pro 795 800 805 agg gta atg caa aga aaa caa
agt ttg ggc aaa cca gat cag aaa gat 2799 Arg Val Met Gln Arg Lys
Gln Ser Leu Gly Lys Pro Asp Gln Lys Asp 810 815 820 825 ttg aat gaa
aac cta gct gcc act caa ggg ctg gcc cat atg atc gcc 2847 Leu Asn
Glu Asn Leu Ala Ala Thr Gln Gly Leu Ala His Met Ile Ala 830 835 840
gag tgc aag aag ctt ttc cag gtt ccc gag gaa atg agc cga tgt cgt
2895 Glu Cys Lys Lys Leu Phe Gln Val Pro Glu Glu Met Ser Arg Cys
Arg 845 850 855 aat tcc tat acc gaa caa gag ctg aag ccc ctc act ctg
gaa gca ctc 2943 Asn Ser Tyr Thr Glu Gln Glu Leu Lys Pro Leu Thr
Leu Glu Ala Leu 860 865 870 ggg cac ctg ggt aat gat gac tca gct gac
tac caa cac ttc ctc cag 2991 Gly His Leu Gly Asn Asp Asp Ser Ala
Asp Tyr Gln His Phe Leu Gln 875 880 885 gac tgt gtg gat ggc ctg ttt
aaa gaa gtc aaa gag aag ttt aaa ggc 3039 Asp Cys Val Asp Gly Leu
Phe Lys Glu Val Lys Glu Lys Phe Lys Gly 890 895 900 905 tgg gtc agc
tac tcc act tcg gag cag gct gag ctg tcc tat aag aag 3087 Trp Val
Ser Tyr Ser Thr Ser Glu Gln Ala Glu Leu Ser Tyr Lys Lys 910 915 920
gtg agc gaa gga ccc cgt ctg agg ctt tgg agg tca gtc att gaa gtc
3135 Val Ser Glu Gly Pro Arg Leu Arg Leu Trp Arg Ser Val Ile Glu
Val 925 930 935 cct gct gtg cca gag gaa atc tta aag cgc cta ctt aaa
gaa cag cac 3183 Pro Ala Val Pro Glu Glu Ile Leu Lys Arg Leu Leu
Lys Glu Gln His 940 945 950 ctc tgg gat gta gac ctg ttg gat tca aaa
gtg atc gaa att ctg gac 3231 Leu Trp Asp Val Asp Leu Leu Asp Ser
Lys Val Ile Glu Ile Leu Asp 955 960 965 agc caa act gaa att tac cag
tat gtc caa aac agt atg gca cct cat 3279 Ser Gln Thr Glu Ile Tyr
Gln Tyr Val Gln Asn Ser Met Ala Pro His 970 975 980 985 cct gct cga
gac tac gtt gtt tta aga acc tgg agg act aat tta ccc 3327 Pro Ala
Arg Asp Tyr Val Val Leu Arg Thr Trp Arg Thr Asn Leu Pro 990 995
1000 aaa gga gcc tgt gcc ctt tta cta acc tct gtg gat cac gat cgc
gca 3375 Lys Gly Ala Cys Ala Leu Leu Leu Thr Ser Val Asp His Asp
Arg Ala 1005 1010 1015 cct gtg gtg ggt gtg agg gtt aat gtg ctc ttg
tcc agg tat ttg att 3423 Pro Val Val Gly Val Arg Val Asn Val Leu
Leu Ser Arg Tyr Leu Ile 1020 1025 1030 gaa ccc tgt ggg cca gga aaa
tcc aaa ctc acc tac atg tgc aga gtt 3471 Glu Pro Cys Gly Pro Gly
Lys Ser Lys Leu Thr Tyr Met Cys Arg Val 1035 1040 1045 gac tta agg
ggc cac atg cca gaa tgg tac aca aaa tct ttt gga cat 3519 Asp Leu
Arg Gly His Met Pro Glu Trp Tyr Thr Lys Ser Phe Gly His 1050 1055
1060 1065 ttg tgt gca gct gaa gtt gta aag atc cgg gat tcc ttc agt
aac cag 3567 Leu Cys Ala Ala Glu Val Val Lys Ile Arg Asp Ser Phe
Ser Asn Gln 1070 1075 1080 aac act gaa acc aaa gac acc aaa tct agg
tga tcactgaagc aacgcaaccg 3620 Asn Thr Glu Thr Lys Asp Thr Lys Ser
Arg 1085 1090 cttccaccac catggtgttt gtttttagaa gttttgccag
tccttgaaga atgggttctg 3680 tgtgtaatcc tgaaacaaag aaaactacaa
gctggagtgt aggaattgac tatagcaatt 3740 tgatacattt ttaaagctgc
ttcctgtttg ttgagggtct gtattcatag accttgactg 3800 gaatatgtaa
gactgtgcga aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3850 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2 <211>
LENGTH: 1091 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 2 Met Cys Arg Lys Lys Pro Asp Thr Met
Ile Leu Thr Gln Ile Glu Ala 1 5 10 15 Lys Glu Ala Cys Asp Trp Leu
Arg Ala Thr Gly Phe Pro Gln Tyr Ala 20 25 30 Gln Leu Tyr Glu Asp
Phe Leu Phe Pro Ile Asp Ile Ser Leu Val Lys 35 40 45 Arg Glu His
Asp Phe Leu Asp Arg Asp Ala Ile Glu Ala Leu Cys Arg 50 55 60 Arg
Leu Asn Thr Leu Asn Lys Cys Ala Val Met Lys Leu Glu Ile Ser 65 70
75 80 Pro His Arg Lys Arg Ser Asp Asp Ser Asp Glu Asp Glu Pro Cys
Ala 85 90 95 Ile Ser Gly Lys Trp Thr Phe Gln Arg Asp Ser Lys Arg
Trp Ser Arg 100 105 110 Leu Glu Glu Phe Asp Val Phe Ser Pro Lys Gln
Asp Leu Val Pro Gly 115 120 125 Ser Pro Asp Asp Ser His Pro Lys Asp
Gly Pro Ser Pro Gly Gly Thr
130 135 140 Leu Met Asp Leu Ser Glu Arg Gln Glu Val Ser Ser Val Arg
Ser Leu 145 150 155 160 Ser Ser Thr Gly Ser Leu Pro Ser His Ala Pro
Pro Ser Glu Asp Ala 165 170 175 Ala Thr Pro Arg Thr Asn Ser Val Ile
Ser Val Cys Ser Ser Ser Asn 180 185 190 Leu Ala Gly Asn Asp Asp Ser
Phe Gly Ser Leu Pro Ser Pro Lys Glu 195 200 205 Leu Ser Ser Phe Ser
Phe Ser Met Lys Gly His Glu Lys Thr Ala Lys 210 215 220 Ser Lys Thr
Arg Ser Leu Leu Lys Arg Met Glu Ser Leu Lys Leu Lys 225 230 235 240
Ser Ser His His Ser Lys His Lys Ala Pro Ser Lys Leu Gly Leu Ile 245
250 255 Ile Ser Gly Pro Ile Leu Gln Glu Gly Met Asp Glu Glu Lys Leu
Lys 260 265 270 Gln Leu Ser Cys Val Glu Ile Ser Ala Leu Asn Gly Asn
Arg Ile Asn 275 280 285 Val Pro Met Val Arg Lys Arg Ser Val Ser Asn
Ser Thr Gln Thr Ser 290 295 300 Ser Ser Ser Ser Gln Ser Glu Thr Ser
Ser Ala Val Ser Thr Pro Ser 305 310 315 320 Pro Val Thr Arg Thr Arg
Ser Leu Ser Ala Cys Asn Lys Arg Val Gly 325 330 335 Met Tyr Leu Glu
Gly Phe Asp Pro Phe Asn Gln Ser Thr Phe Asn Asn 340 345 350 Val Val
Glu Gln Asn Phe Lys Asn Arg Glu Ser Tyr Pro Glu Asp Thr 355 360 365
Val Phe Tyr Ile Pro Glu Asp His Lys Pro Gly Thr Phe Pro Lys Ala 370
375 380 Leu Thr Asn Gly Ser Phe Ser Pro Ser Gly Asn Asn Gly Ser Val
Asn 385 390 395 400 Trp Arg Thr Gly Ser Phe His Gly Pro Gly His Ile
Ser Leu Arg Arg 405 410 415 Glu Asn Ser Ser Asp Ser Pro Lys Glu Leu
Lys Arg Arg Asn Ser Ser 420 425 430 Ser Ser Met Ser Ser Arg Leu Ser
Ile Tyr Asp Asn Val Pro Gly Ser 435 440 445 Ile Leu Tyr Ser Ser Ser
Gly Asp Leu Ala Asp Leu Glu Asn Glu Asp 450 455 460 Ile Phe Pro Glu
Leu Asp Asp Ile Leu Tyr His Val Lys Gly Met Gln 465 470 475 480 Arg
Ile Val Asn Gln Trp Ser Glu Lys Phe Ser Asp Glu Gly Asp Ser 485 490
495 Asp Ser Ala Leu Asp Ser Val Ser Pro Cys Pro Ser Ser Pro Lys Gln
500 505 510 Ile His Leu Asp Val Asp Asn Asp Arg Thr Thr Pro Ser Asp
Leu Asp 515 520 525 Ser Thr Gly Asn Ser Leu Asn Glu Pro Glu Glu Pro
Ser Glu Ile Pro 530 535 540 Glu Arg Arg Asp Ser Gly Val Gly Ala Ser
Leu Thr Arg Ser Asn Arg 545 550 555 560 His Arg Leu Arg Trp His Ser
Phe Gln Ser Ser His Arg Pro Ser Leu 565 570 575 Asn Ser Val Ser Leu
Gln Ile Asn Cys Gln Ser Val Ala Gln Met Asn 580 585 590 Leu Leu Gln
Lys Tyr Ser Leu Leu Lys Leu Thr Ala Leu Leu Glu Lys 595 600 605 Tyr
Thr Pro Ser Asn Lys His Gly Phe Ser Trp Ala Val Pro Lys Phe 610 615
620 Met Lys Arg Ile Lys Val Pro Asp Tyr Lys Asp Arg Ser Val Phe Gly
625 630 635 640 Val Pro Leu Thr Val Asn Val Gln Arg Thr Gly Gln Pro
Leu Pro Gln 645 650 655 Ser Ile Gln Gln Ala Met Arg Tyr Leu Arg Asn
His Cys Leu Asp Gln 660 665 670 Val Gly Leu Phe Lys Lys Ser Gly Val
Lys Ser Arg Ile Gln Ala Leu 675 680 685 Arg Gln Met Asn Glu Gly Ala
Ile Asp Cys Val Asn Tyr Glu Gly Gln 690 695 700 Ser Ala Tyr Asp Val
Ala Asp Met Leu Lys Gln Tyr Phe Arg Asp Leu 705 710 715 720 Pro Glu
Pro Leu Met Thr Asn Lys Leu Ser Glu Thr Phe Leu Gln Ile 725 730 735
Tyr Gln Tyr Val Pro Lys Asp Gln Arg Leu Gln Ala Ile Lys Ala Ala 740
745 750 Ile Met Leu Leu Pro Asp Glu Asn Arg Val Val Leu Gln Thr Leu
Leu 755 760 765 Tyr Phe Leu Cys Asp Val Thr Ala Ala Val Lys Glu Asn
Gln Met Thr 770 775 780 Pro Thr Asn Leu Ala Val Cys Leu Ala Pro Ser
Leu Phe His Leu Asn 785 790 795 800 Thr Leu Lys Arg Glu Asn Ser Ser
Pro Arg Val Met Gln Arg Lys Gln 805 810 815 Ser Leu Gly Lys Pro Asp
Gln Lys Asp Leu Asn Glu Asn Leu Ala Ala 820 825 830 Thr Gln Gly Leu
Ala His Met Ile Ala Glu Cys Lys Lys Leu Phe Gln 835 840 845 Val Pro
Glu Glu Met Ser Arg Cys Arg Asn Ser Tyr Thr Glu Gln Glu 850 855 860
Leu Lys Pro Leu Thr Leu Glu Ala Leu Gly His Leu Gly Asn Asp Asp 865
870 875 880 Ser Ala Asp Tyr Gln His Phe Leu Gln Asp Cys Val Asp Gly
Leu Phe 885 890 895 Lys Glu Val Lys Glu Lys Phe Lys Gly Trp Val Ser
Tyr Ser Thr Ser 900 905 910 Glu Gln Ala Glu Leu Ser Tyr Lys Lys Val
Ser Glu Gly Pro Arg Leu 915 920 925 Arg Leu Trp Arg Ser Val Ile Glu
Val Pro Ala Val Pro Glu Glu Ile 930 935 940 Leu Lys Arg Leu Leu Lys
Glu Gln His Leu Trp Asp Val Asp Leu Leu 945 950 955 960 Asp Ser Lys
Val Ile Glu Ile Leu Asp Ser Gln Thr Glu Ile Tyr Gln 965 970 975 Tyr
Val Gln Asn Ser Met Ala Pro His Pro Ala Arg Asp Tyr Val Val 980 985
990 Leu Arg Thr Trp Arg Thr Asn Leu Pro Lys Gly Ala Cys Ala Leu Leu
995 1000 1005 Leu Thr Ser Val Asp His Asp Arg Ala Pro Val Val Gly
Val Arg Val 1010 1015 1020 Asn Val Leu Leu Ser Arg Tyr Leu Ile Glu
Pro Cys Gly Pro Gly Lys 1025 1030 1035 1040 Ser Lys Leu Thr Tyr Met
Cys Arg Val Asp Leu Arg Gly His Met Pro 1045 1050 1055 Glu Trp Tyr
Thr Lys Ser Phe Gly His Leu Cys Ala Ala Glu Val Val 1060 1065 1070
Lys Ile Arg Asp Ser Phe Ser Asn Gln Asn Thr Glu Thr Lys Asp Thr
1075 1080 1085 Lys Ser Arg 1090 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence PCR primer <400> SEQUENCE: 3 tatgggctcg
agcggccgcc c 21 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 4 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence
PCR primer <400> SEQUENCE: 4 cgcacagtct tacatattcc a 21
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence PCR primer
<400> SEQUENCE: 5 atgtgcagaa agaagccgga cacc 24 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence PCR primer <400> SEQUENCE:
6 cctagatttg gtgtctttgg tttc 24 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 7 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence PCR primer <400> SEQUENCE: 7 gacaccacca
tctctgtgct c 21 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 8 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence
PCR primer <400> SEQUENCE: 8 gcagactgtc cttcgtagtt g 21
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 9
<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence primer <400>
SEQUENCE: 9 cactccggtc cttgtagtct ggaacc 26 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 10 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence primer <400> SEQUENCE: 10 atcctcttca
tgaactcggg cacgg 25 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 11 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence
primer <400> SEQUENCE: 11 gatcaaggtt ctagactaca aggaccg 27
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 12
<211> LENGTH: 691 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence probe <400>
SEQUENCE: 12 ccngcaganc tcgaaaatat gcttcggcat gtctgccacg tcataagcag
actgtccttc 60 gtagttgaca cagtctatgg caccctcatt catctggcgc
agagcctgaa tccgggactt 120 gacccccgat tttctgaaga gcccaacctg
tcggaagagc aacactaagt gtggggtaca 180 ttcacgtgga cgcagtgttt
acaccacaca actagaagaa gctgcatgta atccgagctc 240 ccctgagtac
gtgaacccgc aggcagcgct ctcacctgat ccaaacaatg gttccggggg 300
tatcgcacgg cctgctggat gctctcaggc aacggttgtc ctgtgcgctg cacgttgacc
360 gtcaggggac cccaaacaca ctccggtcct tgtagtctgg aaccttgatc
ctcttcatga 420 actcgggcac ggccctgtta aagagcacag agatggtggt
gtcggcggan acatgctcac 480 ttgtctgtct acacttgtcc aattctgcag
gcaaaccctg tgggctccag attctgatat 540 catccaatga attcgagctc
gtaccgggga tcctctaaaa tccaacttgc aggcattcca 600 gcttcagctg
ctccaatttc tatatgttcc cctaaatcgt atttttttga aacataaggt 660
tattttttta attgtaccnc gttcctaacn a 691 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 13 <211> LENGTH: 301
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence probe <400> SEQUENCE: 13 gaggctctat
gcaggcgtct aaatacttta aacaaatgtg cggtgatgaa gctagaaatt 60
agtcctcatc ggaaacgaag tgacgattca gacgaggatg agccttgtgc catcagtggc
120 aaatggactt tccaaaggga cagcaagagg tggtcccggc ttgaagagtt
tgatgtcttt 180 tctccaaaac aagacctggt ccctgggtcc ccagacgact
cccacccgaa ggacggcccc 240 agccccggag gcacgctgat ggacctcagc
gagcgccagg aggtgtcttc cgtccgcagc 300 c 301 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 14 <211> LENGTH: 3006
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(3006) <223> OTHER INFORMATION: n represents a
or g or c or t/u
<400> SEQUENCE: 14 cnggcagatc tcgaanatac tgcttcggca
tgtctgccac gtcataagca gactgtcctt 60 cgtagttgac acagtctatg
gcaccytcat tcatctggcg cagagcctga atccgggact 120 tgacccccga
ttttctgaag agcccaacct gtcggaagag caacactaag tgtggggtac 180
attcacgtgg acgcagtgtt tacaccacac aactagaaga agctgcatgt aatccgagct
240 cccctgagta cgtggacccg caggcagcgc tctcacctga tccaaacaat
ggttccggrg 300 gtatcgcayg gcctgctgga tgctctgagg caacggttgt
cctgtgcgct gcacgttgac 360 cgtcagtggg accccaaaca cactccggtc
cttgtagtct ggaaccttga tcctcttcat 420 gaacttgggc acggcctgtt
aaagaacaca gagatggtgg tgttggcgga gacatgctca 480 cttgtctgtc
tacacttgtc caattctgca ggcaaaccct gtgggctcca gatctgtgct 540
aatacggtgg ctacttaaat ttaaattaaa caaaatgaca aattcagttc cccagtggta
600 ctggccacac ttcaggtgct ccttcatctt ttgtgctcag tacctactgt
attggcctgt 660 gcagataaag aacattccta tcatccagac agttctcctg
gacagtgctg ttctagatct 720 tctaagagtg ggggttgaca ggtccgtttc
ctcagttagg agcgtccttc caccttgaac 780 ctggagaatt ggggtctaca
gtcttaagga agctgatgga tttccttaca gaatggcggt 840 ataggatgga
acaagcagaa aacaacatgt aataccctaa ttaggtgcat ctgatagagt 900
gtgaaaaaca aggtcccttt tgtcttgaaa aaagggtaag aatcacttct gagttcttga
960 tgagatcgaa agcatttagg gtcaaaaggc gcagataaca catgatggga
aaacagcaat 1020 gagagcctaa cacaatggga gccaactcca gagctcaaca
gtgaatgacc tgaagtcaaa 1080 ataaaatctg ctgctgatga cccggagaac
attacatctt taggtttcta aaggaagatg 1140 gaaaaggaac aatgggggtt
ttgtgagccg accccaggct ccctggtgtc ctgaaaccag 1200 gtccacccca
gcactatatg caacagcagg aaacccatgt catgcatttc aggctgtcaa 1260
gcagaaattc cagctctcca aatgacctct ctgaacagga cccgaaaggg caaggccaaa
1320 caggaaaaga accttgtgta ggattcctcc ctgctccaca gatcccacca
tgtgaggctt 1380 ttacagttgg ttttgagtca ctggaaacac tgaccagaac
acaagaagta ttatggactt 1440 tcagattctt gagggtttgg tggggatggg
ggtgggccac tccgaaatga gaatctaaaa 1500 tatgcagttt taaatagcca
gcagggaaaa cattactcta agcacagagg aactccagag 1560 aagacagact
gctttgcctt ttgaatgctc accagcagcc atggcatgtt actgtttata 1620
gctccaggaa aggtaaaacg aaagagcaaa gttaagtttg tatttccata cagttaagtg
1680 tgtggtatca tggctataag tgtgcataat actcgctttg tcgggggaga
aaagcccgac 1740 ggcggaatgt gaaaagaaca cattacgatc cccaccgaga
atctgaagca tgtgaggata 1800 aaccggtcaa tacttatttc tgtcattcag
aacaaacaac ttctgtattt agcaaggctc 1860 acataataac agcctttgaa
cgggagtgct ttgatgctga agttaaatct gctatgatcc 1920 taaggagagg
aggagctgga gacaaaaaga acagtttcct tgctttgccg actttctcaa 1980
gcaacttggg tttgctacag agtgctacta atgaaatggg cggcttctcc atttttatca
2040 aatatggtag tgtgcgactg gataataaac actcagatta ctgaaaagac
ttaaggattc 2100 ccagatgaca ctgaaaaatg cactgagatg tcaatctaga
aacatttctc tgcttggcac 2160 tgatagcaga aaaattaaga tgtacccaga
ttaggtgata tccatgaccc atctagcctt 2220 acagcctacc cctcacattc
tatatactaa ggagctatat ttttcaaagt aattatgaac 2280 aatttgtaca
atgcatttca tctctacatt tgagtctata atatgttaga gtagtgaatt 2340
ccttaaaata attattcact gttagacagt ctttgctaga aaaaaagtaa cctgaattct
2400 ttagcacagg tggatgctac aaatayctgc mcrkscrrmy kywykakymy
tattattatt 2460 attattattt tttgagatag agtcttactc tgtcacccag
gctggagtgc agtagcctta 2520 tcttggggct cactgcaacc tccatcttct
gggctcaagg gattctcatg cctcagcttc 2580 ctgagtagat gggattacag
gtgcatgcca ccacactcag ctaatttttg tatttttagt 2640 agagaatggg
gttcgccaat gttggccagc tggtctcaaa ctcctggcgt catgtgatcc 2700
acctatgtca gattcccaag atgctgggat acaggcatga gccacacacc cgccccaaga
2760 tgatttctaa aaacaggcat gaatacggta taagaacagg twctgtaant
caagnaattc 2820 caaganggtc tcaywawatc twatkgttgt ccttctcctc
cayccagaaa tacratctgm 2880 tactgtgcat acattwactg awagtggawk
atyctawtat tattgggaan gancccctat 2940 caccacntga ccctaagagt
attgnatttt caccccntca tctggcgata tgacntgccc 3000 gngggg 3006
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 15
<211> LENGTH: 305 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 15 tcaaaggcat gggaaatgat
agattttatg catttgaact agcaaacaga tgtttctcat 60 tttatttcca
tgctttctaa cttaaataat tcatcagctt ttctttcttt tctctgatag 120
gggccacatg ccagaatggt acacaaaatc ttttggacat ttgtgtgcag ctgaagttgt
180 aaagatccgg gattccttca gtaaccagaa cactgaaacc aaagacacca
aatctaggtg 240 atcactgaag caacgcaacc gcttccacca ccatggtgtt
tgtttctaga acttttgcca 300 gtcct 305 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 16 <211> LENGTH: 466
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(466) <223> OTHER INFORMATION: n represents a
or g or c or t/u <400> SEQUENCE: 16 tggattnccn tgncactgaa
aaatacatcc tctttccagg tgagcgaagg accccctctg 60 aggctttgga
ggtcagtcat tgaagtccct gctgtgccag aggaaatctt aaagcgccta 120
cttaaagaac agcacctctg ggatgtagac ctgttggatt caaaagtgat cgaaattctg
180 gacagccaaa ctgaaattta ccagtatgtc caaaacagta tggcacctca
tcctgctcga 240 gactacgttg ttttaaggtg agcgcttccc agttgttttt
ttgtgacaag gatgactcca 300 tatatgaacc aagcctatat gtcactgatc
ttacaagatg gtataattat ttaaagtaga 360 ggccgggcat atggtggctc
acacctgtaa tcccagcact ctgggaggcc aaggtgggag 420 gatcacttga
ggccagcagt tcaagaccag cctggntaat atagca 466 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 17 <211> LENGTH: 692
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(692) <223> OTHER INFORMATION: n represents a
or g or c or t/u <400> SEQUENCE: 17 ccngcaganc tcgaaaatat
gcttcggcat gtctgccacg tcataagcag actgtccttc 60 gtagttgaca
cagtctatgg caccctcatt catctggcgc agancctgaa tccgggactt 120
gacccccgat tttctgaaga gcccaacctg tcggaagagc aacactaagt gtggggtaca
180 ttcacgtgga cgcagtgttt acaccacaca actagaagaa gctgcatgta
atccgagctc 240 ccctgagtac gtgaacccgc aggcagcgct ctcacctgat
ccaaacaatg gttccggggg 300 tatcgcacgg cctgctggat gctctgaggc
aacggttgtc ctgtgcgctg cacgttgacc 360 gtcaggggac cccaaacaca
ctccggtcct tgtagtctgg aaccttgatc ctcttcatga 420 actcgggcac
ggccctgtta aagagcacag agatggtggt gtcggcggan acatgctcac 480
ttgtctgtct acacttgtcc aattctgcag gcaaaccctg tgggctccag attctgatat
540 catccaatga attcnanctc ngtaccgggg atcctctaaa atccaacttg
caggcattcc 600 agcttcagct gctccaattt ctatatgttc ccctaaatcn
tatttttttg aaacataagg 660 ttattttttt aattgtaccn cgttcctaac na 692
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 18
<211> LENGTH: 315 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (314) <223> OTHER
INFORMATION: n represents a or g or c or t/u <400> SEQUENCE:
18 tttcgtgtga ggggcttagc tcttgttcgg tataggaatt acgacatcgg
ctcatttcct 60 cgggaacctg tgcggaacat gacagacaga aaggaggtga
gtccacctgt actcaatctc 120 aatgcccatc agtggaaaag actgggtagg
aacaatggcc tggtccttaa agcagtgcag 180 gcatcttccc gccggaggtg
ggctatcatg ctgaccgcac gtgttatcac gaggatatga 240 acagatcacc
tccataaatg tatctgaaat cttatttcca tgtaaggtct ttggaaagtt 300
agagtagggg gagnc 315 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 19 <211> LENGTH: 281 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(281)
<223> OTHER INFORMATION: n represents a or g or c or t/u
<400> SEQUENCE: 19 ctcnngactg tgtggatggc ctgtttaaag
aagtcaaaga gaagtttaaa ggctgggtca 60 ngctactcca cttcggagca
ggctgagctg tcctataaga aggtaaggct tcaccctgtt 120 gtcggctagt
tgagtccagg agtcgaagct tgggtccatc agagataaca cgcttttgcc 180
aactaatctg tctggggatc tgtagcccac aacctccctt gtagagctgg gcaccggggt
240 gagtaagatc cccgtggtga gagtggaaac cgnncaaagc a 281 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 20 <211>
LENGTH: 1713 <212> TYPE: DNA <213> ORGANISM: Mus
musculus <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1713) <223> OTHER INFORMATION: n
represents a or g or c or t/u <400> SEQUENCE: 20 ttgaacgctt
gggtaccggg ccccccctcg aggtcgacgg tatcgataag cttgatatcg 60
aattcctgca gcccggggga tccaatccct gggtccccag acaactctcg tttgcaaagc
120 gccacaagcc acgaaagcat gctgacagac ctcagcgagc accaggaggt
ggcctctgtc 180 cgaagcctca gcagcaccag cagcagcgtc cccacccacg
cagcccacag tggagatgcc 240 actacgcccc gaaccaattc cgtcatcagc
gtctgctcct ccggacactt tgtaggcaac 300 gatgactctt tttccagcct
gccgtctccc aaggaactgt ccagcttcag ttttagcatg 360 aaaggccacc
acgagaagaa caccaagtcg aagacgcgga gcctgctcaa acgcatggag 420
agcctgaagc tcaagggctc ccaccacagc aagcacaagg cgccttccaa gctggggttg
480 atcatcagtg ctcccattct gcaggagggt atggatgagg cgaagctgaa
gcagctgaac 540 tgtgtggaga tctcagccct caatggcaac cacatcaacg
tgcccatggt accggaaaag 600 gagccgtgtc taacttcacc cagaccagca
gcaagcagca gccaatcaga gaccagcagc 660 gcggtcagca cacccagccc
ggtcaccagg acccggagcc tcagcacctg taacaagcgg 720 gtgggcatgt
atctagaggg cttcgaccca ttcagtcagt ccaccttgaa caacgtgacg 780
gagcagaact ataaaaaccg tgagagctac ccagaggaca cggtgttcta cattcccgaa
840 gatcacaagc ccggcacctt ccctaaggcc ctctcccatg gcagtttctg
tccctcggga 900 aacagttctg tgaactggag gaccggaagc ttccatggcc
ccggccatct cagcctacgg 960 agagaaaaca gccatgacag tcctaaggag
ctgaagagac gcaattcttc cagctctctg 1020 agcagccgcc tgagcatcta
tgataacgta ccgggttcta tcctgtactc cagctcggga 1080 gaactggccg
acctggagaa tgaggacatc ttccctgagc tggatgacat tctctaccac 1140
gtgaagggga tgcagcggat agtcaaccag tggtccgaga agttttccga cgagggagac
1200 tcggactcag ccctggactc tgtctctcct tgcccgtcat cttcaaaaca
gattcacctg 1260 gatgtggacc atgaccgaag gacacccagt gacctggaca
gcacaggcaa ctccttcaat 1320 gagcccgaag agcccactga tatccggaaa
gaagagactt ccggggtggg ggctttcctt 1380 gaccagtgca ataggtaagg
gaaaggcgtt gctttctcgg atgcattcca aaaggtgggg 1440 gaaattcaaa
gaaaggggtc ttgctttggg tggggattgg agttctngat anttttgcca 1500
agttccttgg aaaattcctt aggggaattg gatncccaac cngggaagaa cccccaaaca
1560 aatccccnaa cngggaaaaa ggnggttttt attnaaaacc tggggtnntt
gaaacccttt 1620 gggccattca aangggattn ccntacccag gtggggancc
cttggaaana aangggtggg 1680 tggttttgga aacnaatttt tagtcccngg gcc
1713 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 21
<211> LENGTH: 4767 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(4767) <223> OTHER
INFORMATION: n represents a or g or c or t/u <400> SEQUENCE:
21 cataccaagt gaggtgtaat tgtttaaacc aaaaagtttg aaggatatgg
caaaagccag 60 acttaaattt ccatttttcc tttttttttt tttttttaag
ggaaattctt attcaatgtg 120 taagtgctca ctatcatctc tggggaggca
gagggagaaa aaaaatacct ggtaattcaa 180 agccagtctg ggctacacag
caagatcgtc cctcaaaaaa gtacttttta attaaaagag 240 agaaattatt
ccgaatccat agaaatagtc gttggagtat tgggaggtgg gaagcccaag 300
gcccttgtcc atgtagtcac acataatggc agtggcttgg gctttcatag aagggcacac
360 gtggggacct tcccttgtgg gctttctgac tcttcactta ctgcatatgc
ctactgcaga 420 gatttcctct ggactggagc actgggactt tctttctaaa
aatataaagt tcagtaatga 480 ccaacaatta tgattaggct agtaggcttt
tgttcatttt taaaaattgt atgtgtgtga 540 gtattttacc tcacgcatag
tgtatgtacc gtgcctctga agacggaaga aaacattagc 600 ttcccctgga
actggagtta cagatggttg taagccacca tgtaagtgct gggatttgaa 660
ctcaggtcca tctggaagaa cagccagtgc ggtacccact gagccatctc tccagccccc
720 tgcccagtgt tcttaaagtg ttagtctacg gtagcagatg atttggtccc
ttgaagaaat 780 tctttcccct caatcttgct agcttgactg ataacctaaa
cccattgagg aagctctgat 840 cacgagcaag ctctactccg gactggaaga
gtgttcagtg tgtctcaaag cacgtacttg 900 tggtgttgta aaccgtgagc
catgctgaga cgcctcttgt gaaatgtctt cccgtggctt 960 caggaacatt
tcagaccgct gttttccttt ggagttaaaa ctgactcctt ctaccaacac 1020
gtggaaagaa ttgtgaacat cagctggtag ttgtcatatg aaaaaacaaa acaaaacaaa
1080 acaaaaaact atgttgtctg tcactgtcat cttcagtatg tactttgtcc
ccaaatcacc 1140 atgacatgcc aaagccgtgt caagcattgc agagacattc
taaccttgtt gctcttacta 1200 ttcagtttaa aaagaagcaa gtaattgtgg
gaaggtaggg gatgcttgga agaggacttt 1260 gctatgtaga ccaaactggg
ctagaactca acaatcctcc tgcctcagcc tcccatgtgc 1320 tacatgcaac
aaacaaggag cttaaacatt tttttttttt atgaatgcca ggaaaaccta 1380
caggaatttg aagaattttt gtgggagcct ctgttttctt atttcttctt ctgtcttatt
1440 ttaaatgcaa gaaggggcag acctccacct gctctccttt tatctgtgcg
cctccagccc 1500 tagccccaac cttgtgctgc aaagctcttg aagcttcgac
attgcacctt tggctccatc 1560 tgtcttgaaa aacggaccca aggcacccaa
gagataagac ctgcacattc ctgctgggcc 1620 cttgccttgg gtggcggcgg
ggtcagaatg cccaaggcca cagatggtta ctgatagcgc 1680 tatctcggcc
acctacttga acgatcctac ttcaggtcct cttggctggc ttttctatat 1740
tttcttttct tttctgccat tgttaatact tgtttcacaa ccaactgtag aggttgctgt
1800 ctttgggcac cagagccact gtgctttaat cctgggttct taggcaagat
tcctaagctc 1860 tctaagcccc gcccccatcc cctttcgtcc cttataaaat
aaagataaat catagtatct 1920 gtggcagaag gttgtcagag gactgaagac
gagccagtgc agtgtcaccc aaagacagtg 1980 gcagttcacc tagttagaac
catattttaa ttcttggttg acagagcacg actgtatgta 2040 tctatgtggt
agcaagtgat gtttcaatgt ttgtgtgtaa ggtgaatgag tgaattatgg 2100
gggttaacat atccgatagc ttataggttt atcatcttgt ctggagtata tgcaaattgg
2160 ctattttaaa atataaaatt aatattaact atagtcaccc tggtatgcca
agtcgccctg 2220 cacttgctgc ctgcctcttt gcgactccct gtcccttccc
aacttctggt gaccatcctt 2280 ctgttccctt ctatgaaatg agtttcttct
ctggtcagaa ctactatctt atgtccctag 2340 tacccctccg gaaatctgag
ggtcctgctc tttggagatc ctagagcatg cggatgggtg 2400 aggggaaatc
attgaaaaac cacagaaacc cagagaggaa gcggcacgcc cctagtctgg 2460
tgccaccagc ataaaaagtt aaagttgact tttctcaaac caacctcctg ggtcttttgt
2520 tgtttgactt aaactggcgt gtgtgaagtt actccacctc cccaagcccc
ataggcctcc 2580 atgcctagta aatttggtta taaacaccac tcagccatta
aagccccaat gcagtccagt 2640 ggagatttga ttacgggttc gattaatgaa
tcccagacct aagactaact taaccattgc 2700 tcactcttaa agccttgaaa
aaaactgggg gagtgaaaca ttacatttgg ttgtgtcctt 2760 taactgagac
ccctcagcaa gggaccctac acccttctga gcctccagtg tctctcaact 2820
gttcctcctg ccctycccca ctcctccagt gtctctcaac tgttcctcct gccctccccc
2880 actcctttcc catgcaagga gaggtttttc tgaaagagtt ggtgttctgt
tttatctcag 2940 tttattattc tataaacagg cttccacata atctatagaa
tcaaaggcag gcttctcagg 3000 ctgcagagat actacctatc ctggtgcatc
caagttgtca gagcaggacc cgggagataa 3060 agcccagcag ggtacaagat
cagttccaag tggagggaat taagcggctc ttattccatg 3120 gaaaaaaaaa
agcaaggttg caataattcg ggaaagaaat aaaagactga tgggtgtgtg 3180
tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgta agcttatgag gcaacaagca
3240 gacgcattta aaaaggaaga ctttggtgat gatcatctgg aagattctag
aaagaactga 3300 ggcccaggga cctgtcactc acactttgca tactaggtag
cgagtagata acgggtgcta 3360 ctgttgtttt ttgttttttt tttttctcct
atgactttta atgaagctga ttgattgatg 3420 actgattgat cgattgattg
attgatggtt gattgatcga ctgattgatt tccattgtgc 3480 taaggattga
acttgaagcc ttgtgtgtcc ttcgcaagta cctgatcact gaactactct 3540
gccgtccccc tttctctaat gtggctaaac cgatatcatt ggcgatgggg gcaactcgtt
3600 caaagctgca gtttgactcc catctcagcg gggactgtgt tctaagggcc
tgtttgtgct 3660 cagtgagatt tttaaaataa tcatttgtgc agttgctgtc
gatactgaaa acagtctctc 3720 ctgataggac tgagtaataa agaggcctgg
aacttcgcct ctgtataata aattcaagca 3780 ataaaagtca ccttctgaca
tggacatttc tgaggcccat tgtccttctt aattattact 3840 tgagtgagaa
gggtgcactg agcactttgc ctgcaacctt ccccagttcc tactgctggc 3900
ctgttgccct tgaagtgggc ctgccattga tgctgtagca tgccgtctaa caagaaatag
3960 aatggcactt ttgtgttaga caagcttttt tttttttttt tgagaataga
actcactagc 4020
tagaccaggc tggcctccaa ctcacagaga cctacttgcc tctgccttct gggtattaag
4080 attaaagacg tgcactacca tcctgggact ccattacccg ctatgtaatt
gaagtgtagc 4140 atacctgccg aaactagaaa tgagttccga gaagctcata
ttgtatgggt cagttgttca 4200 gtttgattgc ccattcgtgg ttcctttctc
tgctcacggc ctttctctgc tctgcaggcg 4260 cttaaatact ctaaacaagt
gtgcagtcat gaagctggag attagtcctc accggaagcg 4320 agtgagtacc
aaaattacat gggggggggg ggcagggaca gcaggcacac taaccaagac 4380
aggacttgta tctacactct gtaaaaggcc ctgtttgtcc attcctcaac atgttaaaac
4440 ccctatttgg agacagtagt ggatggtggc atctactgct ctggacttga
agaaatctgt 4500 tacttttccc agtgaactcc atggctacca tgtgattcaa
agcatgaagc ctattgaatc 4560 tccagaggaa tttcacattg ctccctagag
gaaataaagc taacattctg taggacctct 4620 tcctgtttcc tggatggaac
agtagctcca tctcgaagct gtcaagatga aaggggaagg 4680 ctggcttggg
ggatactgta ggagatgtgg atcgtggggg gtggggagga agacgccgga 4740
gcaggaaatc ccatacactc tgtggna 4767 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 22 <211> LENGTH: 1072
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (6) <223> OTHER INFORMATION: n represents a or g or
c or t/u <400> SEQUENCE: 22 ttgaanccca agctggagct ccccgcggtg
gcggccgctc tagaactagt ggatccagat 60 acagttcttg tctttaaact
ctgactatgg acaggaatta tatcctgccc acgacccatc 120 cagcctgact
gtccacatct tacactctac actcaaggct gaggattcta gattatgaag 180
agttagacat ctaatacatt tctattttaa aaatatagtt gctctgtggt ggggcatggt
240 ggcacatggc tttaatccta gcacccagag gaggtagagg caggtgaatc
tctgagttca 300 aggccagcct ggtatatata gcactgactg ctctcccaga
ggtcctgagt tcaatttcca 360 gcaaccatat ggtggctcac aaccatctgg
aatggaatcc gatgccctct tctggtgtgt 420 ctgaagacag ctacagtgta
ctcatacata caataaataa ttcttaaaaa aaaaaacaaa 480 aacaaaaaca
aaaactcaaa cacaaacaaa cagtatatat gtaagatatt atagctaacc 540
acttaagttt attattctct gagcattttt gccagaaagg tctgcttcta aataaacaac
600 aaagcaaaaa caccccaaag tccaaacaaa aaccccaaac tttttagcac
aggtagattt 660 ctcaggttat gctcaaaacc ttcattcaaa actgaccgac
agcgtgatgg agtgtgggct 720 cagcatgaac aagggcctga acgcatctca
ggcaaccacg tgatggctga aaacccaacc 780 aaccagtcct gcagttaact
ccctgaggct ccaggagttt gagcagcatg gagaacatag 840 cctggaggat
gtggagacca cctgcttaaa ggttgatgga ctggtgacat tgacagagga 900
cagaacggtc ctaagctgag tgctggggac aacctcaggg agcatgatgg catcccccca
960 gggccattgc tcactgctca ctacgagctg gctctcttac cagctgaagc
cgtgcttgtt 1020 ggaggcgtgt cttttccagc agggccgtca atttcaggag
ccagtttttc tt 1072 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 23 <211> LENGTH: 1104 <212> TYPE:
DNA <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1104) <223> OTHER INFORMATION: n represents a or g or c
or t/u <400> SEQUENCE: 23 ttggamracy sggtaccggg ccccccctcg
cggtcgacgg tatcgataag cttgatatcg 60 aattcctgca gcccggggga
tcctgctttg ggaaaaagac gtcaaactct tcaaggcggg 120 accaccgctt
gctgtccctc tggaaagtcc acttgccgct tatggcgcaa ggctcatctt 180
catccgaatc ctcactctga aaacacagaa tgaagccatt tatgtactgg gccaagcagg
240 gggcagaagg cagaacacag gttaagggcc aggccacagc ccaaaggata
ttcccagtgt 300 ccattgctca gttctcttat gtaacaaaga tggatttaaa
gacattatta ttgggctgga 360 gggatggctc agccgttaag aacactgacc
gcttttccag aggtcctgag ttcaaatccc 420 agcaaccaca aggtggctca
acaaccatct gtaatgagat ctgatgccct cttccggtgt 480 gtctgaagac
agccatagtg tacttatata taatataaat aaatccttaa aaaaagagac 540
attattatta ctttatttta tttagagaat gtatttgcat gtatgtgtat atgtatggat
600 gtatatgaat gttcacaccg tgttcagacg actaccagtc agtgtgagtt
ttctccttca 660 gtcatataga actgggtcgt caggcttggc aacaggccga
ctgtcattta accagcccag 720 atgtaaagac tttaacagaa gtctgaccaa
gtgttgccag ctaaacaagt cattttattg 780 aaaccctggc tcgttgggcc
attcactaat cgctcacaaa ggggacctct gagatgggcc 840 gaaaattcaa
gcatgcaaaa tattctgaac tggaatcaga gtcaacagtc gtgggactcc 900
ctctggattg cctccagttt aactgcgtgt tgacagagtg tgtttatata ctcgtgtgca
960 attaaaaaaa aaaaaaagct attttcaaac agcagaatgg cagctgagga
ctctaggtcc 1020 aaagagaaaa gacanggnat ttcttttaaa agaactgaag
accatttaan cgagccatct 1080 gtggcagaaa aggnaaaata gant 1104
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 24
<211> LENGTH: 725 <212> TYPE: DNA <213> ORGANISM:
Mus musculus <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(725) <223> OTHER
INFORMATION: n represents a or g or c or t/u <400> SEQUENCE:
24 aannccctga tatcccggaa agaagagact ccggggtggg ggcttccctg
accaggtgca 60 ataggtaagg aagggcgttg cttctcgatg catccagagg
tggggaatca agaagggtct 120 gcttggtggg attgagtctg atatttgcag
tcctgcaaat tcctagggac tgcatccaac 180 caggagaccc caacaatccc
aacgggaaag gagtattata aactgggtat gaacctttgg 240 tcatcaagga
tgcagacagt ggaccctgga agatggtggt gtttgaacaa tatagtcagg 300
ccttatccac cgtggggtgt acttagacgt gcttaaagtg cttgcatctt gattctcctg
360 cagttccaaa tcttcggttt cagccaggca cagatgagaa ctactcaggg
gagaaactgt 420 cttctccgtc attataccct gggtaataga gtgtgaccgt
gaactactag caggttgtta 480 tagcaatctg gcttataaac ttacattaaa
tggggagggt gctcccgatg tgcgtagaca 540 ctatccatct tctataagag
gcctgagtgt actgagtcca catatctgct atgtctggaa 600 ccaaccttca
ggggttacaa agacagtggg ggtggggggg aggcagggaa aggaagatcg 660
atgctcttgg ttcctgatga tcagaagatt ggtcccagct tactcctttc cgcctgttct
720 ttttg 725 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 25 <211> LENGTH: 528 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(528) <223> OTHER
INFORMATION: n represents a or g or c or t/u <400> SEQUENCE:
25 agacgnggtc ccactgactg tgaacgtgca gcgctcagga cagcccctgc
cccagagcat 60 ccagcaggcc atgcgctacc tccgtaacca ctgtctggac
caggtgagta cagctgcctg 120 tggatcccac tcgtgggagc ggagctttgg
gctgcatgtt tttttttcta gtttcgtggg 180 gaagggtcct gcttccacac
ccatccctgc tgttctcctt ccaaaaggtc gggctcttca 240 ggaagtcagg
tgtcaaatcc cggatccagg ctctacgcca gatgaatgaa agcgctgaag 300
ataatgtcaa ctatgaaggc cagtctgctt atgatgtggc agacatgtta aagcaatatt
360 ttcgagatct tcctgagccc ctcatgacga acaaactntn cgaaaccttn
ctgcagatct 420 accagtgtaa gcgttctttg gtcttcttaa gnaactgatg
tcgggttcat gggaccaact 480 gagcacacaa gcctttttna tgccatcctt
ttgaaanaaa aacttnat 528 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 26 <211> LENGTH: 393 <212> TYPE:
DNA <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(393)
<223> OTHER INFORMATION: n represents a or g or c or t/u
<400> SEQUENCE: 26 aacanaanat tccggatttc ctcagggacc
tggaaanaat tcttgcattc agcaatcatg 60 tgggccagcc cttggantcg
ccgctaggtt ttcattcagg tctttctggt ctggtttgcc 120 caaactctgt
tttctttgca ttacccttgg anaaaaattc tctcncttca gggtgttgag 180
gtggaanang gacggagcta ggcacacagc caggttggtg ggagtcatct ggttttcttt
240 cacagccgct gtgacatcgc tcaggaaata aaaaantgtc tgcanaacct
cccggttctc 300 ntcgggcagg ancataatgg ccgccttgat ggcttggang
cgctggtcct tgggcacata 360 ctggtatatc tgcanggaan gttcggaaaa ttt 393
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 27
<211> LENGTH: 601 <212> TYPE: DNA <213> ORGANISM:
Mus musculus <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(601) <223> OTHER
INFORMATION: n represents a or g or c or t/u <400> SEQUENCE:
27 ccaagctgaa ttccgggcgc cttgtgcttg ctgtggtggg agcccttgaa
gcttcaggct 60 ctccatgcgt ttgagcaggc tccgcgtctt cgacttggtg
ttcttctcgt ggtggccttt 120 catgctaaaa ctgaagctgg acagttcctt
gggagacggc aggctggaaa aagagtcatc 180 gttgcctaca aagtgtccgg
aggagcagac gctgatgacg gaattggttc ggggcgtant 240 ggcatctcca
ctgtgggctg cgtgggtggg gacgctgctg ctggtgctgc tgangcttcg 300
gacagangcc acctcctggt gctcgctgag gtctgtcagc atgctttcgt ggcttgtggc
360 gctttgcaaa cgaaanttgt ctggggaccc agggattgga tcctgctttg
ggaaaaagac 420 tcaaactctt caaggcggga ccaccgcttg ctgtccctct
ggaaagtcca cttgccgctt 480 atggcgcaag gctcatcttc atccgaatcc
tcacttcgct tccggtgaag actaatctcc 540 acttcntgaa tgcacacttg
tttanaatat ttaacncctg canaaaacct ccatggcgtc 600 t 601 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 28 <211>
LENGTH: 260 <212> TYPE: DNA <213> ORGANISM: Mus
musculus <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(260) <223> OTHER INFORMATION: n
represents a or g or c or t/u <400> SEQUENCE: 28 ggcttangga
agtgccgggc ttgtgatctt cgggaatgta gaacaccgtg tcctctgggt 60
agctctcacg gtttttatag ttctgctccg tcncnttgtt caaggtggac tgactgaatg
120 ggtccaancc ctctaaatac atgcccaccc gcttgttaca ggtgctgagg
ctccgggtcc 180 tggtgaccgg gctgggtgtg ctgaccgcnc tgctggtctc
tgattggctg ctgctgctgc 240 tggtctgggt ggaattagac 260 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 29 <211>
LENGTH: 358 <212> TYPE: DNA <213> ORGANISM: Mus
musculus <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(358) <223> OTHER INFORMATION: n
represents a or g or c or t/u <400> SEQUENCE: 29 ctgattccgg
gttgacatta tcttcagcgc tttcattcat ctggcgtaga gcctggatcc 60
gggatttgac acctgacttc ctgaagancc cgacctggtc cagacagtgg ttacggaggt
120 agcgcatggc ctgctggatg ctctggggca ggggctgtcc tgagcgctgc
acgttcacag 180 tcagtgggac cccaaacaca ctccggtcct tgtagtctgg
aaccttgatc cttttcatga 240 acttgggcac agcccagctg aanccgtgct
tgttggangg cgtgtncttt tccagcaggg 300 ccgtcaattt caggagcgag
tntttctgca gcaggttcat ctgggccaca gactggca 358 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 30 <211> LENGTH: 154
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(154) <223> OTHER INFORMATION: n represents a
or g or c or t/u <400> SEQUENCE: 30 aattccgggc gatgtcacag
cggctgtgaa agaaaaccag atgactccca ccaacctggc 60 tgtgtgccta
gctccgtccc tcttccacct caacaccctg aancnataga attcttctcc 120
aagggtaatg canatgaaaa cagagtttgg gcaa 154 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 31 <211> LENGTH: 294
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(294) <223> OTHER INFORMATION: n represents a
or g or c or t/u <400> SEQUENCE: 31 aagctggaat ccggtgcgct
ccagccttga gccatggctg tgcgtcctcg ctgttggagc 60 cacggctccc
cagctccgtg ccccgctccc tgagagtgct cccttcgcgg tggcaatcta 120
aaacccacga ttttgcccga gctggggcga agcgtaagga agctgcgaac cangatgtgc
180 tgacgaccgc gaggggctcg cgtcccggct gccaccgtgg gtcccgacgt
gggatcccga 240 tnacttctgg cngcctcgac tttcccagtg cgctcccgtc
gncctgcgcc gacc 294
* * * * *
References