U.S. patent number RE40,948 [Application Number 09/983,543] was granted by the patent office on 2009-10-27 for apc antibodies.
This patent grant is currently assigned to Astrazeneca United Kingdom, Ltd., Cancer Institute, Japanese, Foundation for Cancer Research, The Johns Hopkins University, The University of Utah. Invention is credited to Hans Albertsen, Rakesh Anand, Mary Carlson, Joanna Groden, Philip Hedge, Geoff Joslyn, Kenneth W. Kinzler, Alexander Fred Markham, Yusuke Nakumura, Andrew Thliveris, Bert Vogelstein, Raymond White.
United States Patent |
RE40,948 |
Vogelstein , et al. |
October 27, 2009 |
APC antibodies
Abstract
A human gene termed APC is disclosed. Methods and kits are
provided for assessing mutations of the APC gene in human tissues
and body samples. APC mutations are found in familial adenomatous
polyposis patients as well as in sporadic colorectal cancer
patients. APC is expressed in most normal tissues. These results
suggest that APC is a tumor suppressor.
Inventors: |
Vogelstein; Bert (Baltimore,
MD), Kinzler; Kenneth W. (Bel Air, MD), Albertsen;
Hans (Cupertino, CA), Anand; Rakesh (Sandbach,
GB), Carlson; Mary (Salt Lake City, UT), Groden;
Joanna (Salt Lake City, UT), Hedge; Philip (Winsford,
GB), Joslyn; Geoff (Salt Lake City, UT), Markham;
Alexander Fred (Crewe, GB), Nakumura; Yusuke
(Tokyo, JP), Thliveris; Andrew (Salt Lake City,
UT), White; Raymond (Salt Lake City, UT) |
Assignee: |
The Johns Hopkins University
(Baltimore, unknown)
Astrazeneca United Kingdom, Ltd. (London, GB)
Cancer Institute, Japanese, Foundation for Cancer Research
(Tokyo, JP)
The University of Utah (Salt Lake City, UT)
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Family
ID: |
27450606 |
Appl.
No.: |
09/983,543 |
Filed: |
October 24, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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09442489 |
Nov 18, 1999 |
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08289548 |
Aug 12, 1994 |
5648212 |
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07741940 |
Aug 8, 1991 |
5352775 |
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Reissue of: |
08452654 |
May 25, 1995 |
05691454 |
Nov 25, 1997 |
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Foreign Application Priority Data
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Jan 16, 1991 [GB] |
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9100962.1 |
Jan 16, 1991 [GB] |
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9100963.9 |
Jan 16, 1991 [GB] |
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9100974.6 |
Jan 16, 1991 [GB] |
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9100975.3 |
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Current U.S.
Class: |
530/387.9;
530/828; 530/806; 530/387.7; 530/387.1; 424/174.1; 424/139.1;
424/138.1; 424/130.1 |
Current CPC
Class: |
C12Q
1/68 (20130101); C07K 14/47 (20130101); C07K
14/82 (20130101); C12Q 1/6886 (20130101); C12Q
1/6827 (20130101); A01K 2217/05 (20130101); Y10S
530/828 (20130101); C12Q 2600/172 (20130101); C12Q
2600/158 (20130101); C12Q 2600/112 (20130101); C12Q
2600/156 (20130101) |
Current International
Class: |
C07K
16/30 (20060101); A61K 39/395 (20060101) |
Field of
Search: |
;530/388.2,387.9,387.7,388.8,389.7,806,827,843,844,387.1 ;536/23.5
;424/130.1,138.1,141.1,174.1 ;435/240.27 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Kinzler et al., "Identification of FAP Locus Genes from Chromosome
5q21", Science 253: 661-665, 1991. cited by other .
Groden et al., "Identification and Characterization of the familial
Adenomatous Polyposis Coli Gene", Cell 66:589-600. cited by other
.
Joslyn et al. "Identification of Delection Mutations and Three New
Genes at the Familial Polyposis Locus" Cell, 66:601-613 (1991).
cited by other .
Nishisho et al., "Mutations of Chromosome 5q21 Genes in FAP and
Colorectal Cancer Patients" Science, 253:665-669 (1991). cited by
other .
Orita et al., Genomics, vol. 5, pp. 874-879, 1989. cited by other
.
Stanbridge et al., "Identifying Tumor Suppressor Genes in Human
Colorectal Cancer", Science 247:12-13 (1990). cited by other .
Fearon et al., "Identification of a Chromosome 18q Gene that is
Altered in Colorectal Cancer", Science 247:49-56 (1990). cited by
other .
Baker et al., "Chromosome 17 Deletions and p53 Gene Mutations in
Colorectal Carcinomas", Science, 244:217-221 (1989). cited by other
.
Bodmer et al. "Localization of the Gene for familial Adenomatous
Polyposis of Chromosome 5" Nature 328:614-616 (1987). cited by
other .
Kinzler et al, Science 253: 661-665, 1991. cited by
examiner.
|
Primary Examiner: O'Hara; Eileen B
Assistant Examiner: VanderVegt; F. Pierre
Attorney, Agent or Firm: Banner & Witcoff, LTD
Government Interests
The U.S. Government has a paid-up license in this invention and the
right in limited circumstances to require the patent owner to
license others on reasonable terms as provided for by the terms of
grants awarded by the National Institutes or Health.
Parent Case Text
.Iadd.Notice: More than one reissue application has been filed for
the reissue of Pat. No. 5,691,454. The reissue applications are
Ser. Nos. 09/983,543 (the present application), which is a
continuation reissue of Pat. No. 5,691,454, and
09/442,489..Iaddend.
This application .Iadd.is a continuation of application of Ser. No.
09/442,489, filed Nov. 18, 1999, which issued as RE38916 and which
is a Reissue Application of application Ser. No. 08/452,654, filed
May 25, 1995, which issued as Pat. No. 5,691,454, which .Iaddend.is
a division, of application Ser. No. 08/289,548, filed Aug. 12,
1994, .Iadd.which issued as U.S. Pat. No. 5,648,212, .Iaddend.which
is a division of application Ser. No. 07/741,940 filed Aug. 8, 1991
(issued as U.S. Pat. No. 5,352,775).
Claims
We claim:
.[.1. A preparation of antibodies which specifically binds to a
human APC (adenomatous polyposis coil) protein having an amino acid
sequence as shown in SEQ ID NO:1, 2, or 7, and does not
specifically bind to other human proteins..].
2. A preparation of antibodies which specifically binds to a human
APC protein which is the product of a mutant allele found in a
tumor, wherein the antibodies do not specifically bind to other
human proteins, and wherein the human APC protein is a mutant form
of the amino acid sequence shown in .[.SEQ ID NOS:2 and.].
.Iadd.SEQ ID NO: .Iaddend.7.[., and the mutant allele is a mutant
form of the nucleotide sequence shown in SEQ ID NO:1.].
.Iadd.having the sequence of SEQ ID NO: 7 but for a substitution of
Arg.fwdarw.Cys at residue 414.Iaddend..
.[.3. The preparation of claim 2 wherein the mutant allele contains
a mutation selected from the group consisting of mutations at
codons 243, 279, 288, 301, 331, 413, 437, 456, 500, 712, and
1338..].
.[.4. The preparation of claim 2 wherein the mutant allele contains
a premature stop codon..].
.[.5. The preparation of claim 2 wherein the mutant allele contains
a missense mutation..].
.[.6. The preparation of claim 2 wherein the mutant allele contains
a frameshift mutation..].
.[.7. The preparation of claim 2 wherein the mutant allele contains
a splice junction mutation..].
.[.8. The preparation of claim 2 wherein the mutant allele contains
an insertion mutation..].
Description
TECHNICAL AREA OF THE INVENTION
The invention relates to the area of cancer diagnostics and
therapeutics. More particularly, the invention relates to detection
of the germline and somatic alterations of wild-type APC genes. In
addition, it relates to therapeutic intervention to restore the
function of APC gene product.
BACKGROUND OF THE INVENTION
According to the model of Knudson for tumorigenesis (Cancer
Research, Vol. 45, p. 1482, 1985), there are tumor suppressor genes
in all normal cells which, when they become non-functional due to
mutation, cause neoplastic development. Evidence for this model has
been found in the cases of retinoblastoma and colorectal tumors.
The implicated suppressor genes in those tumors, RB, p53, DCC and
MCC, were found to be deleted or altered in many cases of the
tumors studied. (Hansen and Cavenee, Cancer Research, Vol. 47, pp:
5518-5527 (1987); Baker et al., Science, Vol. 244, p. 217 (1989);
Fearon et al., Science, Vol. 247, p. 49 (1990); Kinzler et al.
Science Vol. 251. p. 1366 (1991).)
In order to fully understand the pathogenesis of tumors, it will be
necessary to identify the other suppressor genes that play a role
in the tumorigenesis process. Prominent among these is the one(s)
presumptively located at 5q21. Cytogenetic (Herrera et al., Am J.
Med. Genet., Vol. 25, p. 473 (1986) and linkage (Leppert et al.,
Science, Vol. 238, p. 1411 (1987); Bodmer et al., Nature, Vol. 328,
p. 614 (1987)) studies have shown that this chromosome region
harbors the gene responsible for familial adenomatous polyposis
(FAP) and Gardner's Syndrome (GS). FAP is an autosomal-dominant,
inherited disease in which affected individuals develop hundreds to
thousands of adenomatous polyps, some of which progress to
malignancy. GS is a variant of FAP in which desmold tumors,
osteomas and other soft tissue tumors occur together with multiple
adenomas of the colon and rectum. A less severe form of polyposis
has been identified in which only a few (2-40) polyps develop. This
condition also is familial and is linked to the same chromosomal
markers as FAP and GS (Leppert et al., New England Journal of
Medicine, Vol. 322, pp. 904-908, 1990.) Additionally, this
chromosomal region is often deleted from the adenomas (Vogelstein
et al., N. Engl. J. Med., Vol. 319, p. 525 (1988)) and carcinomas
(Vogelstein et al., N. Engl. J. Med., Vol. 319, p. 525 (1988);
Solomon et al., Nature, Vol. 328, p. 616 (1987); Sasaki et al.,
Cancer Research, Vol. 49, p. 4402 (1989); Delattre et al., Lancet,
Vol. 2, p. 353 (1989); and Ashton-Rickardt et al., Oncogene, Vol.
4, p. 1169 (1989)) of patients without FAP (sporadic tumors). Thus,
a putative suppressor gene on chromosome 5q21 appears to play a
role in the early stages of colorectal neoplasia in beth sporadic
and familial tumors.
Although the MCC gene has been identified on 5q21 as a candidate
suppressor gene, it does not appear to be altered in FAP or GS
patients. Thus there is a need in the art for investigations of
this chromosomal region to identify genes and to determine if any
of such genes are associated with FAP and/or GS and the process of
tumorigenesis.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for
diagnosing and prognosing a neoplastic tissue of a human.
It is another object of the invention to provide a method of
detecting genetic predisposition to cancer.
It is another object of the invention to provide a method of
supplying wild-type APC gene function to a cell which has lest said
gene function.
It is yet another object of the invention to provide a kit for
determination of the nucleotide sequence of APC alleles by the
polymerase chain reaction.
It is still another object of the invention to provide nucleic acid
probes for detection of mutations in the human APC gene.
It is still another object of the invention to provide a cDNA
molecule encoding the APC gene product.
It is yet another object of the invention to provide a preparation
of the human APC protein.
It is another object of the invention to provide a method of
screening for genetic prodisposition to cancer.
It is an object of the invention to provide methods of testing
therapeutic agents for the ability to suppress neoplasia.
It is still another object of the invention to provide animals
carrying mutant APC alleles.
These and other objects of the invention are provided by one or
more of the embodiments which are described below. In one
embodiment of the present invention a method of diagnosing or
prognosing a neoplastic tissue of a human is provided comprising:
detecting somatic alteration of wild-type APC genes of their
expression products in a sporadic colorectal cancer tissue, said
alteration indicating neoplasia of the tissue.
In yet another embodiment a method is provided of detecting genetic
predisposition to cancer in a human including familial adenomatous
polyposis (FAP) and Gardner's Syndrome (GS), comprising: isolating
a human sample selected from the group consisting of blood and
fetal tissue; detecting alteration of wild-type APC gene coding
sequences or their expression products from the sample, said
alteration indicating genetic predisposition to cancer.
In another embodiment of the present invention a method is provided
for supplying wild-type APC gene function to a cell which has lost
said gene function by virtue of a mutation in the APC gene,
comprising: introducing a wild-type APC gene into a cell which has
lost said gene function such that said wild-type gene is expressed
in the cell.
In another embodiment a method of supplying wild-type APC gene
function to a cell is provided comprising: introducing a portion of
a wild-type APC gene into a cell which has lost said gene function
such that said portion is expressed in the cell, said portion
encoding a part of the APC protein which is required for
non-neoplastic growth of said cell. APC protein can also be applied
to cells or administered to animals to remediate for mutant APC
genes. Synthetic peptides or drugs can also be used to mimic APC
function in cells which have altered APC expression.
In yet another embodiment a pair of single stranded primers is
provided for determination of the nucleotide sequence of the APC
gene by polymerase chain reaction. The sequence of said pair of
single stranded DNA primers is derived from chromosome 5q band 21,
said pair of primers allowing synthesis of APC gene coding
sequences.
In still another embodiment of the invention a nucleic acid probe
is provided which is complementary to human wild-type APC gene
ceding sequences and which can form mismatches with mutant APC
genes, thereby allowing their detection by enzymatic or chemical
cleavage or by shifts in electrophoretic mobility.
In another embodiment of the invention a method is provided for
detecting the presence of a neoplastic tissue in a human. The
method comprises isolating a body sample from a human; detecting in
said sample alteration of a wild-type APC gene sequence or
wild-type APC expression product, said alteration indicating the
presence of a neoplastic tissue in the human.
In still another embodiment a cDNA molecule is provided which
comprises the coding sequence of the APC gene.
In even another embodiment a preparation of the human APC protein
is provided which is substantially free of other human proteins.
The amino acid sequence of the protein is shown in FIG. 3 (SEQ ID
NOS. 7 and 2).
In yet another embodiment of the invention a method is provided for
screening for genetic predisposition to cancer, including familial
adenomatous polyposis (FAP) and Gardner's Syndrome (GS), in a
human. The method comprises: detecting among kindred persons the
presence of a DNA polymorphism which is linked to a mutant APC
allele in an individual having a genetic predisposition to cancer,
said kindred being genetically related to the individual, the
presence of said polymorphism suggesting a predisposition to
cancer.
In another embodiment of the invention a method of testing
therapeutic agents for the ability to suppress a neoplastically
transformed phenotype is provided. The method comprises: applying a
test substance to a cultured epithelial cell which carries a
mutation in an APC allele; and determining whether said test
substance suppresses the neoplastically transformed phenotype of
the cell.
In another embodiment of the invention a method of testing
therapeutic agents for the ability to suppress a neoplastically
transformed phenotype is provided. The method comprises:
administering a test substance to an animal which carries a mutant
APC allele; and determining whether said test substance prevents or
suppresses the growth of tumors.
In still other embodiments of the invention transgenic animals are
provided. The animals carry a mutant APC allele from a second
animal species or have been genetically engineered to contain an
insertion mutation which disrupts an APC allele.
The present invention provides the art with the information that
the APC gene, a heretofore unknown gene is, in fact, a target of
mutational alterations or chromosome 5q21 and that these
alterations are associated with the process of tumorigenesis. This
information allows highly specific assays to be performed to assess
the neoplastic status of a particular tissue or the predisposition
to cancer of an individual. This invention has applicability to
Familial Adenomatous Polyposis, sporadic colorectal cancers,
Gardner's Syndrome, as well as the less severe familial polyposis
discusses above.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows an overview of yeast artificial chromosome (YAC)
contigs. Genetic distances between selected RFLP markers from
within the contigs are shown in centi-Morgans.
FIGS. 1B-1, 1B-2 and IB-3 show a detailed map of the three central
contigs. The position of the six identified genes from within the
FAP region is shown; the 5' and 3' ends of the transcripts from
these genes have in general not yet been isolated, as indicated by
the string of dots surrounding the bars denoting the genes'
positions. Selected restriction endonuclease recognition sites are
indicated. B, BssH2; S, SstII; M, MluI; N, NruI.
FIGS. 2A and 2B show the sequence of TB1 (FIG. 2A.Iadd., SEQ ID
NO:5.Iaddend.) and TB2 (FIG. 2B.Iadd., SEQ ID NO:6.Iaddend.)
.[.genes.]. .Iadd.proteins.Iaddend.. The cDNA sequence of the TB1
gene was determined from the analysis of 11 cDNA clones derived
from normal colon and liver, as described in the text. A total of
2314 bp were contained within the overlapping cDNA clones, defining
an ORF of 424 amino acids beginning at nucleotide 1. Only the
predicted amino acids from the ORF are shown. The carboxy-terminal
end of the ORF has apparently been identified, but the 5' end of
the TB1 transcript has not yet been precisely determined.
The cDNA sequence of the TB2 gene was determined from the YS-39
clone derived as described in the text. This clone consisted of
2300 bp and defined an ORF of 185 amino acids beginning at
nucleotide 1. Only the predicted amino acids are shown. The carboxy
terminal end of the ORF has apparently been identified, but the 5'
end of the TB2 transcript has not been precisely determined.
FIGS. 3A-3F show the sequence of the APC gene product (SEQ ID
NO:7). The cDNA sequence was determined through the analysis of 87
cDNA clones derived from normal colon, liver, and brain. A total of
8973 bp were contained within overlapping cDNA clones, defining an
ORF of 2842 amino acids. In frame stop codons surrounded this ORF,
as described in the text, suggesting that the entire APC gene
product was represented in the ORF illustrated. Only the predicted
amino acids are shown.
FIGS. 4A and 4B show the local similarity between human APC (SEQ ID
NO:2) and ral2 (SEQ ID NO:8) of yeast. FIG. 4A shows amino acids
203 to 233 of APC, and FIG. 4B shows amino acids 453 to 481 of APC.
Local similarity among the APC (SEQ ID NO:2) and MCC genes (SEQ ID
NO:10) genes and the m3 muscarinic acetylcholine receptor (SEQ ID
NO:9) is shown. The region of the mAChR shown corresponds to that
responsible for coupling the receptor to G protein. The connecting
lines indicate identities; dots indicate related amino acids
residues.
FIG. 5 shows the genomic map of the 1200 kb NotI fragment at the
FAP locus. The NotI fragment is shown as a bold line. Relevant
parts of the deletion chromosomes from patients 3214 and 3824 are
shown as stippled lines. Probes used to characterize the NotI
fragment and the deletions, and three YACs from which subclones
were obtained, are shown below the restriction map. The chimeric
end of YAC 183H12 is indicated by a dotted line. The orientation
and approximate position of MCC are indicated above the map.
FIG. 6A-6D show the DNA sequence (SEQ ID NO:3) and predicted amino
acid sequence of DP1 (TB2) (SEQ ID NO:4). The nucleotide numbering
begins at the most 5' nucleotide isolated. A proposed initiation
methionine (base 77) is indicated in bold type. The entire coding
sequence is presented.
FIG. 7A, FIG. 7B-1, and FIG. 7B-2 show the arrangement of exons in
DP2.5 (APC). (A) Exon 9 corresponds to nucleotides 933-1312; exon
9a corresponds to nucleotides 1236-1312. The stop codon in the cDNA
is at nucleotide 8535. (B) Partial intronic sequence surrounding
each exon is shown (SEQ ID NO:11-38). 5' intron sequences of exons
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 are shown in SEQ
ID NOS: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,
respectively. 3' intron sequences of exons 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, and 14 are shown in SEQ ID NOS: 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, respectively.
DETAILED DESCRIPTION
It is a discovery of the present invention that mutational events
associated with tumorigenesis occur in a previously unknown gene on
chromosome 5q named here the APC (Adenomatous Polyposis Coil) gene.
Although it was previously known that deletion of alleles on
chromosome 5q were common in certain types of cancers, it was not
known that a target gene of these deletions was the APC gene.
Further it was not known that other types of mutational events in
the APC gene are also associated with cancers. The mutations of the
APC gene can involve gross rearrangements, such as insertions and
deletions. Point mutations have also been observed.
According to the diagnostic and prognostic method of the present
invention, alteration of the wild-type APC gene is detected.
"Alteration of a wild-type gene" according to the present invention
encompasses all forms of mutations--including deletions. The
alteration may be due to either rearrangements such as insertions,
inversions, and deletions, or to point mutations. Deletions may be
of the entire gene or only a portion of the gene. Somatic mutations
are those which occur only in certain tissues, e.g., in the tumor
tissue, and are not inherited in the germline. Germline mutations
can be found in any of a body's tissues. If only a single allele is
somatically mutated, an early neoplastic state is indicated.
However, if both alleles are mutated then a late neoplastic state
is indicated. The finding of APC mutations thus provides both
diagnostic and prognostic information. An APC allele which is not
deleted (e.g., that on the sister chromosome to a chromosome
carrying an APC deletion) can be screened for other mutations, such
as insertions, small deletions, and point mutations. It is believed
that many mutations found in tumor tissues will be those leading to
decreased expression of the APC gene product. However, mutations
leading to non-functional gene products would also lead to a
cancerous state. Point mutational events may occur in regulatory
regions, such as in the promoter of the gene, leading to loss or
diminution of expression of the mRNA. Point mutations may also
abolish proper RNA processing, leading to loss of expression of the
APC gene product.
In order to detect the alteration of the wild-type APC gene in a
tissue, it is helpful to isolate the tissue free from surrounding
normal tissues. Means for enriching a tissue preparation for tumor
cells are known in the art. For example, the tissue may be isolated
from paraffin or cryostat sections. Cancer cells may also be
separated from normal cells by flow cytometry. These as well as
other techniques for separating tumor from normal cells are well
known in the art. If the tumor tissue is highly contaminated with
normal cells, detection of mutations is more difficult.
Detection of point mutations may be accomplished by molecular
cloning of the APC allele (or alleles) and sequencing that
allele(s) using techniques well known in the art. Alternatively,
the polymerase chain reaction (PCR) can be used to simplify gene
sequences directly from a genomic DNA preparation from the tumor
tissue. The DNA sequence of the amplified sequences can then be
determined. The polymerase chain reaction itself is well known in
the art. See, e.g., Saiki et al., Science, Vol. 239, p. 487, 1988;
U.S. Pat. No. 4,683,203; and U.S. Pat. No. 4,683,195. Specific
primers which can be used in order to amplify the gene will be
discussed in more detail below. The ligase chain reaction, which is
known in the art, can also be used to amplify APC sequences. See Wu
et al., Genomics, Vol. 4, pp. 560-569 (1989). In addition, a
technique known as allele specific PCR can be used. (See Ruano and
Kidd, Nucleic Acids Research, Vol. 17, p. 8392, 1989.) According to
this technique, primers are used which hybridize at their 3' ends
to a particular APC mutation. If the particular APC mutation is not
present, an amplification product is not observed. Amplification
Refractory Mutation System (ARMS) can also be used as disclosed in
European Patent Application Publication No. 0332435 and in Newton
et al., Nucleic Acids Research, Vol. 17, p.7, 1989. Insertions and
deletions of genes can also be detected by cloning, sequencing and
amplification. In addition, restriction fragment length
polymorphism (RFLP) probes for the gene or surrounding marker genes
can be used to score alteration of an allele or an insertion in a
polymorphic fragment. Such a method is particularly useful for
screening among kindred persons of an affected individual for the
presence of the APC mutation found in that individual. Single
stranded conformation polymorphism (SSCP) analysis can also be used
to detect base change variants of an allele. (Orita et al., Proc.
Natl. Acad. Sci. USA Vol. 86, pp. 2766-2770, 1989, and Genomics,
Vol. 5, pp. 874-879, 1989.) Other techniques for detecting
insertions and deletions as are known in the art can be used.
Alterations of wild-type genes can also be detected on the basis of
the alteration of a wild-type expression product of the gene. Such
expression products include both the APC mRNA as well as the APC
protein product. The sequences of these products are shown in FIG.
3. Point mutations may be detected by amplifying and sequencing the
mRNA or via molecular cloning of cDNA made from the mRNA. The
sequence of the cloned cDNA can be determined using DNA sequencing
techniques which are well known in the art. The cDNA can also be
sequenced via the polymerase chain reaction (PCR) which will be
discussed in more detail below.
Mismatches, according to the present invention are hybridized
nucleic acid duplexes which are not 100% homologous. The lack of
total homology may be due to deletions, insertions, inversions,
substitutions or frameshift mutations. Mismatch detection can be
used to detect point mutations in the gene or its mRNA product.
While these techniques are less sensitive than sequencing, they are
simpler to perform on a large number of tumor samples. An example
of a mismatch cleavage technique is the RNase protection method,
which is described in detail in Winter et al., Proc. Natl. Acad.
Sci. USA, Vol. 82, p. 7575, 1985 and Meyers et al., Science, Vol.
230, p. 1242, 1985. In the practice of the present invention the
method involves the use of a labeled riboprobe which is
complementary to the human wild-type APC gene coding sequence. The
riboprobe and either mRNA or DNA isolated from the tumor tissue are
annealed (hybridized) together and subsequently digested with the
enzyme RNase A which is able to detect some mismatches in a duplex
RNA structure. If a mismatch is detected by RNase A, it cleaves at
the site of the mismatch. Thus, when the annealed RNA preparation
is separated on an electrophoretic gel matrix, if a mismatch has
been detected and cleaved by RNase A, an RNA product will be seen
which is smaller than the full-length duplex RNA for the riboprobe
and the mRNA or DNA. The riboprobe need not be the full length of
the APC mRNA or gene but can be a segment of either. II the
riboprobe comprises only a segment of the APC mRNA or gene it will
be desirable to use a number of these probes to screen the whole
mRNA sequence for mismatches.
In similar fashion, DNA probes can be used to detect mismatches,
through enzymatic or chemical cleavage. See, e.g., Cotton et al.,
Proc. Natl. Acad. Sci. USA, Vol. 85, 4397 1988; and Shenk et al.,
Proc. Natl. Acad. Sci. USA, Vol. 72, p. 989, 1975. Alternatively,
mismatches can be detected by shifts in the electrophoretic
mobility of mismatched duplexes relative to matched duplexes. See,
e.g., Cariello, Human Genetics, Vol. 42, p. 726, 1988. With either
riboprobes or DNA probes, the cellular mRNA or DNA which might
contain a mutation can be amplified using PCR (see below) before
hybridization. Changes in DNA of the APC gene can also be detected
using Southern hybridization, especially if the changes are gross
rearrangements, such as deletions and insertions.
DNA sequences of the APC gene which have been amplified by use of
polymerase chain reaction may also be screened using
allele-specific probes. These probes are nucleic acid oligomers,
each of which contains a region of the APC gene sequence harboring
a known mutation. For example, one oligomer may be about 30
nucleotides in length, corresponding to a portion of the A PC gene
sequence. By use of a battery of such allele-specific probes, PCR
amplification products can be screened to identify the presence of
a previously identified mutation in the APC gene. Hybridization of
allele-specific probes with amplified APC sequences can be
performed, for example, on a nylon filter. Hybridization to a
particular probe under stringent hybridization conditions indicates
the presence of the same mutation in the tumor tissue as in the
allele-specific probe.
Alterations of APC mRNA expression can be detected by any technique
known in the art. These include Northern blot analysis, PCR
amplification and RNase protection. Diminished mRNA expression
indicates an alteration of the wild-type APC gene. Alteration of
wild-type APC genes can also be detected by screening for
alteration of wild-type APC protein. For example, monoclonal
antibodies immunoreactive with APC can be used to screen a tissue.
Lack of cognate antigen would indicate an APC mutation. Antibodies
specific for products of mutant alleles could also be used to
detect mutant APC gene product. Such immunological assays can be
done in any convenient format known in the art. These include
Western blots, immunohistochemical assays and ELISA assays. Any
means for detecting an altered APC protein can be used to detect
alteration of wild-type APC genes. Functional assays can be used,
such as protein binding determinations. For example, it is believed
that APC protein oligomerizes to itself and/or MCC protein or binds
to a G protein. Thus, an assay for the ability to bind to wild type
APC or MCC protein or that G protein can be employed. In addition,
assays can be used which detect APC biochemical function. It is
believed that APC is involved in phospholipid metabolism. Thus,
assaying the enzymatic products of the involved phospholipid
metabolic pathway can be used to determine APC activity. Finding a
mutant APC gene product indicates alteration of a wild-type APC
gene.
Mutant APC genes or gene products can also be detected in other
human body samples, such as, serum, stool, urine and sputum. The
same techniques discussed above for detection of mutant APC genes
or gene products in tissues can be applied to other body samples.
Cancer cells are sloughed off from tumors and appear in such body
samples. In addition, the APC gene product itself may be secreted
into the extracellular space and found in these body samples even
in the absence of cancer cells. By screening such body samples, a
simple early diagnosis can be achieved for many types of cancers.
In addition, the progress of chemotherapy or radiotherapy can be
monitored more easily by testing such body samples for mutant APC
genes or gene products.
The methods of diagnosis of the present invention are applicable to
any tumor in which APC has a role in tumorigenesis. Deletions of
chromosome arm 5q have been observed in tumors of lung, breast,
colon, rectum, bladder, liver, sarcomas, stomach and prostate, as
well as in leukemias and lymphomas. Thus these are likely to be
tumors in which APC has a role. The diagnostic method of the
present invention is useful for clinicians so that they can decide
upon an appropriate course of treatment. For example, a tumor
displaying alteration of both APC alleles might suggest a more
aggressive therapeutic regimen than a tumor displaying alteration
of only one APC allele.
The primer pairs of the present invention are useful for
determination of the nucleotide sequence of a particular APC allele
using the polymerase chain reaction. The pairs of single stranded
DNA primers can be annealed to sequences within or surrounding the
APC gene on chromosome 5q in order to prime amplifying DNA
synthesis of the APC gene itself. A complete set of these primers
allows synthesis of all of the nucleotides of the APC gene coding
sequences, i.e., the exons. The set of primers preferably allows
synthesis of both intron and exon sequences. Allele specific
primers can also be used. Such primers anneal only to particular
APC mutant alleles, and thus will only amplify a product in the
presence of the mutant allele as a template.
In order to facilitate subsequent cloning of amplified sequences,
primers may have restriction enzyme site sequences appended to
their 5' ends. Thus, all nucleotides of the primers are derived
from APC sequences or sequences adjacent to APC except the few
nucleotides necessary to form a restriction enzyme site. Such
enzymes and sites are well known in the art. The primers themselves
can be synthesized using techniques which are well known in the
art. Generally, the primers can be made using oligonucleotide
synthesizing machines which are commercially available. Given the
sequence of the APC open reading frame shown in FIG. 3 (SEQ ID
NO:1), design or particular primers is well within the skill of the
art.
The nucleic acid probes provided by the present invention are
useful for a number of purposes. They can be used in Southern
hybridization to genomic DNA and in the RNase protection method for
detecting point mutations already discussed above. The probes can
be used to detect PCR amplification products. They may also be used
to detect mismatches with the APC gene or mRNA using other
techniques. Mismatches can be detected using either enzymes (e.g.,
S1 nuclease), chemicals (e.g., hydroxylamine or osmium tetroxide
and piperidine), or changes in electrophoretic mobility of
mismatched hybrids as compared to totally matched hybrids. These
techniques are known in the art. See, Cotton, supra, Shenk, supra,
Myers, supra, Winter, supra, and Novack et al., Proc. Natl. Acad.
Sci. USA, Vol. 83, p. 586, 1986. Generally, the probes are
complementary to APC gene coding sequences, although probes to
certain introns are also contemplated. An entire battery of nucleic
acid probes is used to compose a kit for detecting alteration of
wild-type APC genes. The kit allows for hybridization to the entire
APC gene. The probes may overlap with each other or be
contiguous.
If a riboprobe is used to detect mismatches with mRNA, it is
complementary to the mRNA of the human wild-type APC gene. The
riboprobe thus is an anti-sense probe in that it does not code for
the APC protein because it is of the opposite polarity to the sense
strand. The riboprobe generally will be labeled with a radioactive,
colorimetric, or fluorometric material, which can be accomplished
by any means known in the art. If the riboprobe is used to detect
mismatches with DNA it can be of either polarity, sense or
anti-sense. Similarly, DNA probes also may be used to detect
mismatches.
Nucleic acid probes may also be complementary to mutant alleles of
the APC gene. These are useful to detect similar mutations in other
patients on the basis of hybridization rather than mismatches.
These are discussed above and referred to as allele-specific
probes. As mentioned above, the A PC probes can also be used in
Southern hybridizations to genomic DNA to detect gross chromosomal
changes such as deletions and insertions. The probes can also be
used to select cDNA clones of APC genes from tumor and normal
tissues. In addition, the probes can be used to detect APC mRNA in
tissues to determine if expression is diminished as a result of
alteration of wild-type APC genes.
According to the present invention a method is also provided of
supplying wild-type APC function to a cell which carries mutant APC
alleles. Supplying such function should suppress neoplastic growth
of the recipient cells. The wild-type APC gene or a part of the
gene may be introduced into the cell in a vector such that the gene
remains extra-chromosomal. In such a situation the gene will be
expressed by the cell from the extrachromosomal location. If a gene
portion is introduced and expressed in a cell carrying a mutant APC
allele, the gene portion should encode a part of the APC protein
which is required for non-neoplastic growth of the cell. More
preferred is the situation where the wild-type APC gene or a part
of it is introduced into the mutant cell in such a way that it
recombines with the endogenous mutant APC gene present in the cell.
Such recombination requires a double recombination event which
results in the correction of the APC gene mutation. Vectors for
introduction of genes beth for recombination and for
extrachromosomal maintenance are known in the art and any suitable
vector may be used. Methods for introducing DNA into cells such as
electroporation, calcium phosphate co-precipitation and viral
transduction are known in the art and the choice of method is
within the competence of the routineer. Cells transformed with the
wild-type A PC gene can be used as model systems to study cancer
remission and drug treatments which promote such remission.
Similarly, cells and animals which carry a mutant APC allele can be
used as model systems to study and test for substances which have
potential as therapeutic agents. The cells are typically cultured
epithelial cells. These may be isolated from individuals with APC
mutations, either somatic or germline. Alternatively, the cell line
can be engineered to carry the mutation in the APC allele. After a
test substance is applied to the cells, the neoplastically
transformed pheno-type of the cell will be determined. Any trait of
neoplastically transformed cells can be assessed, including
anchorage-independent growth, tumorigenicity in nude mice,
invasiveness of cells, and growth factor dependence. Assays for
each of these traits are known in the art.
Animals for testing therapeutic agents can be selected after
mutagenesis of whole animals or after treatment of germline cells
or zygotes. Such treatments include insertion of mutant A PC
alleles, usually from a second animal species, as well as insertion
of disrupted homologous genes. Alternatively, the endogenous APC
gene(s) of the animals may be disrupted by insertion or deletion
mutation. After test substances have been administered to the
animals, the growth of tumors must be assessed. If the test
substance prevents or suppresses the growth of tumors, then the
test substance is a candidate therapeutic agent for the treatment
of FAP and/or sporadic cancers.
Polypeptides which have APC activity can be supplied to cells which
carry mutant or missing APC alleles. The sequence of the APC
protein is disclosed in FIG. 3 (SEQ ID NO:7). These two sequences
differ slightly and appear to be indicate the existence of two
different forms of the APC protein. Protein can be produced by
expression of the cDNA sequence in bacteria, for example, using
known expression vectors. Alternatively, APC can be extracted from
APC-producing mammalian cells such as brain cells. In addition, the
techniques of synthetic chemistry can be employed to synthesize APC
protein. Any of such techniques can provide the preparation of the
present invention which comprises the APC protein. The preparation
is substantially free of other human proteins. This is most readily
accomplished by synthesis in a microorganism or in vitro.
Active APC molecules can be introduced into cells by microinjection
or by use of liposomes, for example. Alternatively, some such
active molecules may be taken up by cells, actively or by
diffusion. Extracellular application of APC gene product may be
sufficient to affect tumor growth. Supply of molecules with APC
activity should lead to a partial reversal of the neoplastic state.
Other molecules with APC activity may also be used to effect such a
reversal, for example peptides, drugs, or organic compounds.
The present invention also provides a preparation of antibodies
immunoreactive with a human APC protein. The antibodies may be
polyclonal or monoclonal and may be raised against native APC
protein, APC fusion proteins, or mutant APC proteins. The
antibodies should be immunoreactive with APC epitopes, preferably
epitopes not present on other human proteins. In a preferred
embodiment of the invention the antibodies will immunoprecipitate
APC proteins from solution as well as react with APC protein on
Western or immunoblots of polyacrylamide gels. In another preferred
embodiment, the antibodies will detect APC proteins in paraffin or
frozen tissue sections, using immunocytochemical techniques.
Techniques for raising and purifying antibodies are well known in
the art and any such techniques may be chosen to achieve the
preparation of the invention.
Predisposition to cancers as in FAP and GS can be ascertained by
testing any tissue of a human for mutations of the APC gene. For
example, a person who has inherited a germline APC mutation would
be prone to develop cancers. This can be determined by testing DNA
from any tissue of the person's body. Most simply, blood can be
drawn and DNA extracted from the cells of the blood. In addition,
prenatal diagnosis can be accomplished by testing fetal cells,
placental cells, or amniotic fluid for mutations of the APC gene.
Alteration of a wild-type APC allele, whether for example, by point
mutation or by deletion, can be detected by any of the means
discussed above.
Molecules of cDNA according to the present invention are
intron-free, APC gene ceding molecules. They can be made by reverse
transcriptase using the APC mRNA as a template. These molecules can
be propagated in vectors and cell lines as is known in the art.
Such molecules have the sequence shown in SEQ ID NO:3. The cDNA can
also be made using the techniques of synthetic chemistry given the
sequence disclosed herein.
A short region of homology has been identified between APC and the
human m3 muscarinic acetylcholine receptor (mAChR). This homology
was largely confined to 29 residues in which 6 out of 7 amino acids
(EL(GorA)GLQA) were identified (see FIG. 4 (SEQ ID NO:9)).
Initially, it was not known whether this homology was significant,
because many other proteins had higher levels of global homology
(though few had six out of seven contiguous amino acids in common).
However, a study on the sequence elements controlling G protein
activation by mAChR subtypes (Lechleiter et al., EMBO J., p. 4381
(1990)) has shown that a 21 amino acid region from the m3 mAChR
completely mediated G protein specificity when substituted for the
21 amino acids of m2 mA ChR at the analogous protein position.
These 21 residues overlap the 19 amino acid homology between APC
and m3 mA ChR.
This connection between APC and the G protein activating region of
mAChR is intriguing in light of previous investigations relating G
proteins to cancer. For example, the RAS oncogenes, which are often
mutated in colorectal cancers (Vogelstein, et al., N. Engl. J.
Med., Vol. 319, p. 525 (1988); Bos et al., Nature Vol. 327, p. 293
(1987)), are members of the (1 protein family (Bourne, et al,
Nature, Vol. 348, p. 125 (1990)) as is an in vitro transformation
supressor (Noda et al., Proc. Natl. Acad. Sci. USA, Vol. 86, p. 162
(1989)) and genes mutated in hormone producing tumors (Candis et
al., Nature, Vol. 340, p. 692 (1989); Lyons et al., Science, Vol.
249, p. 655 (1990)). Additionally, the gene responsible for
neurofibromatosis (presumably a tumor suppressor gene) has been
shown to activate the GTPase activity of RAS (Xu et al., Cell, Vol.
63, p. 835 (1990); Martin et al., Cell, Vol. 63, p. 843 (1990);
Ballester et al., Cell, Vol. 63, p. 851 (1990)). Another
interesting link between G proteins and colon cancer involves the
drug sulindac. This agent has been shown to inhibit the growth of
benign colon tumors in patients with FAP, presumably by virtue of
its activity as a cyclooxygenase inhibitor (Waddell et al., J.
Surg. Oncology 24(1), 83 (1983); Wadell, et al., Am. J. Surg.,
157(1), 175 (1989); Charneau et al., Gastroenterologie Clinique at
Biologique 14(2), 153 (1990)). Cyclooxygenase is required to
convert arachidonic acid to prostaglandins and other biologically
active molecules. G proteins are known to regulate phospholipase A2
activity, which generates arachidonic acid from phospholipids (Role
et al., Proc. Natl. Acad. Sci. USA, Vol. 84, p. 3623 (1987);
Kurachi et al., Nature, Vol. 337, 12 555 (1989)). Therefore we
propose that wild-type APC protein functions by interacting with a
G protein and is involved in phospholipid metabolism.
The following are provided for exemplification purposes only and
are not intended to limit the scope of the invention which has been
described in broad terms above.
EXAMPLE 1
This example demonstrates the isolation of a 5.5 Mb region of human
DNA linked to the FAP locus. Six genes are identified in this
region, all of which are expressed in normal colon cells and in
colorectal, lung, ad bladder tumors.
The cosmid markers YN5.64 and YN5.48 have previously been shown to
delimit an 8 cM region containing the locus for FAP (Nakamura et
al., Am. J. Hum. Genet. Vol. 43, p. 638 (1988)). Further linkage
and pulse-field gel electrophoresis (PFGE) analysis with additional
markers has shown that the FAP locus is contained within a 4 cM
region bordered by cosmids EF5.44 and L5.99. In order to isolate
clones representing a significant portion of this locus, a yeast
artificial chromosome (YAC) library was screened with various 5p21
markers. Twenty-one YAC clones, distributed within six contigs and
including 5.5 Mb from the region between YN5.64 and YN5.48, were
obtained (FIG. 1A).
Three contigs encompassing approximately 4 Mb were contained within
the central portion of this region. The YAC's constituting these
contigs, together with the markers used for their isolation and
orientations, are shown in FIG. 1. These YAC contigs were obtained
in the following way. To initiate each contig, the sequence of a
genomic marker cloned from chromosome 5q21 was determined and used
to design primers for PCR. PCR was then carried out on pools of YAC
clones distributed in microtiter trays as previously described
(Anand et al., Nucleic Acids Research, Vol. 18, p. 1951 (1980)).
Individual YAC clones from the positive pools were identified by
further PCR or hybridization based assays, and the YAC sizes were
determined by PFGE.
To extend the areas covered by the original YAC clones,
"chromosomal walking" was performed. For this purpose, YAC termini
were isolated by a PCR based method and sequenced (Riley et al.,
Nucleic Acids Research, Vol. 18, p. 2887 (1990)). PCR primers based
on these sequences were then used to rescreen the YAC library. For
example, the sequence from an intron of the FER gene (Hao et al.,
Mol. Cell. Biol., Vol. 9, p. 1587 (1989)) was used to design PCR
primers for isolation of the 28EC1 and 5EH8 YACs. The termini of
the 28EC1 YAC were sequenced to derive markers RHE28 and LHE28,
respectively. The sequences of these two markers were then used to
isolate YAC clones 15CH12 (from RHE28) and 40CF1 and 29EF1 (from
LHE28). These five YAC's formed a contig encompassing 1200 kb
(contig 1, FIG. 1B).
Similarly, contig 2 was initiated using cosmid N5.66 sequences, and
contig 3 was initiated using sequences both from the MCC gene and
from cosmid EF5.44. A walk in the telomeric direction from YAC
14FH1 and a walk in the opposite direction from YAC 39GG3 allowed
connection of the initial contig 3 clones through YAC 37HG4 (FIG.
1B). YAC37HG4 was deposited at the National Collection of
Industrial and Marine Bacteria (NCIMB), P.O. Box 31, 23 St. Machar
Drive, Aberdeen AB2 1RY, Scotland, under Accession No. 40353 on
Dec. 17, 1990.
Multipoint linkage analysis with the various markers used to define
the contigs, combined with PFGE analysis, showed that contigs 1 and
2 were centromeric to contig 3. These contigs were used as tools to
orient and/or identify genes which might be responsible for FAP.
Six genes were found to lie within this cluster of YAC's, as
follows:
Contig #1: FER--The FER gene was discovered through its homology to
the viral oncogene ABL (Hao et al., supra). It has an intrinsic
tyrosine kinase activity, and in situ hybridization with an FER
probe showed that the gene was located at 5q11-23 (Morris et al.,
Cytogenet. Cell. Genet., Vol. 53, p. 4, (1990)). Because of the
potential role of this oncogene-related gene in neoplasia, we
decided to evaluate it further with regards to the FAP locus. A
human genomic clone from FER was isolated (MF 2.3) and used to
define a restriction fragment length polymorphism (RFLP), and the
RFLP in turn used to map FER by linkage analysis using a panel of
three generation families. This showed that FER was very tightly
linked to previously defined polymorphic markers for the FAP locus.
The genetic mapping of FER was complemented by physical mapping
using the YAC clones derived from FER sequences (FIG. 1B). Analysis
of YAC contig 1 showed that FER was within 600 kb of cosmid marker
M5.28, which maps to within 1.5 Mb of cosmid L5.99 by PFGE of human
genomic DNA. Thus, the YAC mapping results were consistent with the
FER linkage data and PFGE analyses.
Contig 2: TB1--TB1 was identified through a cross-hybridization
approach. Exons of genes are often evolutionarily conserved while
introns and intergenie regions are much less conserved. Thus, it a
human probe cross-hybridizes strongly to the DNA from non-primate
species, there is a reasonable chance that it contains exon
sequences. Subclones of the cosmids shown in FIG. 1 were used to
screen Southern blots containing rodent DNA samples. A subclone of
cosmid N5.66 (p 5.66--4) was shown to strongly hybridize to rodent
DNA, and this clone was used to screen cDNA libraries derived from
normal adult colon and fetal liver. The ends of the initial cDNA
clones obtained in this screen were then used to extend the cDNA
sequence. Eventually, 11 cDNA clones were isolated, covering 2314
bp. The gene detected by these clones was named TB1. Sequence
analysis of the overlapping clones revealed an open reading frame
(ORF) that extended for 1302 bp starting from the most 5' sequence
data obtained (FIG. 2A). If this entire open reading frame were
translated, it would encode 434 amino acids (SEQ ID NO:5). The
product of this gene was not globally homologous to any other
sequence in the current database but showed two significant local
similarities to a family of ADP, ATP carrier/translocator proteins
and mitochondrial brown fat uncoupling proteins which are widely
distributed from yeast to mammals. These conserved regions of TB1
(underlined in FIG. 2A) may define a predictive motif for this
sequence family. In addition, TB1 appeared to contain a signal
peptide (or mitochondrial targeting sequence) as well as at least 7
transmembrane domains.
Contig 3: MCC, TB2, SRP and APC--The MCC gene was also discovered
through a cross-hybridization approach, as described previously
(Kinzler et al., Science Vol. 251, p. 1366 (1991)). The MCC gene
was considered a candidate for causing FAP by virtue of its tight
genetic linkage to FAP susceptibility and its somatic mutation in
sporadic colorectal carcinomas. However, mapping experiments
suggested that the ceding region of MCC was approximately 50 kb
proximal to the centromeric end of a 200 kb deletion found in an
FAP patient. MCC cDNA probes detected a 10 kb mRNA transcript on
Northern blot analysis of which 4151 bp, including the entire open
reading frame, have been cloned. Although the 3' non-translated
portion or an alternatively spliced form of MCC might have extended
into this deletion, it was possible that the deletion did not
affect the MCC gene product. We therefore used MCC sequences to
initiate a YAC contig, and subsequently used the YAC clones to
identify genes 50 to 250 kb distal to MCC that might be contained
within the deletion.
In a first approach, the insert from YAC24ED6 (FIG. 1B) was
radiolabelled and hybridized to a cDNA library from normal colon.
One of the cDNA clones (YS39) identified in this manner detected a
3.1 kb mRNA transcript when used as a probe for Northern blot
hybridization. Sequence analysis of the YS39 clone revealed that it
encompassed 2283 nucleotides and contained an ORF that extended for
555 bp from the most 5' sequence data obtained. If all of this ORF
were translated, it would encode 185 amino acids (SEQ ID NO:6)
(FIG. 2B). The gene detected by YS39 was named TB2. Searches of
nucleotide and protein databases revealed that the TB2 gene was not
identical to any previously reported sequences nor were there any
striking similarities.
Another clone (YS11) identified through the YAC 24ED6 screen
appeared to contain portions of two distinct genes. Sequences from
one end of YS11 were identical to at least 180 bp of the signal
recognition particle protein SRP19 (Lingelbach et al. Nucleic Acids
Research, Vol. 16, p. 9431 (1988). A second ORF, from the opposite
end of clone YS11, proved to be identical to 78 bp of a novel gene
which was independently identified through a second YAC-based
approach. For the latter, DNA from yeast cells containing YAC 14FH1
(FIG. 1B) was digested with EcoRI and sub-cloned into a plasmid
vector. Plasmids that contained human DNA fragments were selected
by colony hybridization using total human DNA as a probe. These
clones were then used to search for cross-hybridizing sequences as
described above for TB1, and the cross-hybridizing clones were
subsequently used to screen cDNA libraries. One of the cDNA clones
discovered in this way (FH38) contained a long ORF (2496 bp), 78 bp
of which were identical to the above-noted sequences in YS11. The
ends of the FH38 cDNA clone were then used to initiate cDNA walking
to extend the sequence. Eventually, 85 cDNA clones were isolated
from normal colon, brain and liver cDNA libraries and found to
encompass 8973 nucleotides of contiguous transcript. The gene
corresponding to this transcript was named APC. When used as probes
for Northern blot analysis, APC cDNA clones hybridized to a single
transcript of approximately 9.5 kb, suggesting that the great
majority of the gene product was represented in the cDNA clones
obtained. Sequences from the 5' end of the APC gene were found in
YAC 37HG4 but not in YAC 14FH1. However, the 3' end of the APC gene
was found in 14FH1 as well as 37HG4. Analogously, the 5' end of the
MCC ceding region was found in YAC clones 19AA9 and 266C3 but not
24ED6 or 14FH1, while the 3' end displayed the opposite pattern.
Thus, MCC and APC transcription units pointed in opposite
directions, with the direction of transcription going from
centromeric to telomeric in the case of MCC, and telomeric to
centromeric in the case of APC. PFGE analysis of YAC DNA digested
with various restriction endonucleases showed that TB2 and SRP were
between MCC and APC, and that the 3' ends of the ceding regions of
MCC and APC were separated by approximately 150 kb (FIG. 1B).
Sequence analysis of the APC cDNA clones revealed an open reading
frame of 8,535 nucleotides. The 5' end of the ORF contained a
methionine codon (codon 1) that was preceded by an in-frame stop
codon 9 bp upstream, and the 3' end was followed by several
in-frame stop codons. The protein produced by initiation at codon 1
would contain 2,842 amino acids (SEQ ID NO:7) (FIG. 3). The results
of database searching with the APC gene product were quite complex
due to the presence of large segments with locally biased amino
acid compositions. In spite of this, APC could be roughly divided
into two domains. The N-terminal 25% of the protein had a high
content of leucine residues (12%) and showed local sequence
similarities to myosins, various intermediate filament proteins
(e.g., desmin, vimentin, neurofilaments) and Drosophila
armadillo/human plakoglobin. The latter protein is a component of
adhesive junctions (desmosomes) joining epithelial cells (Franke et
al., Proc. Natl. Acad. Sci. U.S.A., Vol. 86, p. 4027 (1989); Perfer
et al., Cell, Vol. 63, p. 1167 (1990)) The C-terminal 75% of APC
(residues 731-2832) is 17% serine by composition with serine
residues more or less uniformly distributed. This large domain also
contains local concentrations of charged (mostly acidic) and
proline residues. There was no indication of potential signal
peptides, transmembrane regions, or nuclear targeting signals in
APC, suggesting a cytoplasmic localization.
To detect short similarities to APC, a database search was
performed using the PAM-40 matrix (Altschul. J. Mol. Bio., Vol.
219, p. 555 (1991). Potentially interesting matches to several
proteins were found. The most suggestive of these involved the ral2
gene product of yeast, which is implicated in the regulation of ras
activity (Fukul et al., Mol. Cell. Biol., Vol. 9, p. 5617 (1989)).
Little is known about how ral2 might interact with ras but it is
interesting to note the positively-charged character of this region
in the context of the negatively-charged GAP interaction region of
ras. A specific electrostatic interaction between ras and
GAP-related proteins has been proposed.
Because of the proximity of the MCC and APC genes, and the fact
that both .[.am.]. .Iadd.are .Iaddend.implicated in colorectal
tumorigenesis, we searched for similarities between the two
predicted proteins. Bourne has previously noted that MCC has the
potential to form alpha helical coiled coils (Nature, Vol. 351, p.
188 (1991). Lupas and colleagues have recently developed a program
for predicting coiled coil potential from primary sequence data
(Science, Vol. 252, p. 1162 (1991) and we have used their program
to analyze both MCC and APC. Analysis of MCC indicated a
discontinuous pattern of coiled-coil domains separated by putative
"hinge" or .[."sparer".]. .Iadd."spacer" .Iaddend.regions similar
to those seen in laminin and other intermediate filament proteins.
Analysis of the APC sequence revealed two regions in the N-terminal
domain which had strong coiled coil-forming potential, and these
regions corresponded to those that showed local similarities with
myosia and IF proteins on database searching. In addition, one
other putative coiled coil region was identified in the central
region of APC. The potential for both APC and MCC to form coiled
coils is interesting in that such structures often mediate homo-
and hetero-oligomerization.
Finally, it had previously been noted that MCC shared a short
similarity with the region of the m3 muscarinic acetylcholine
receptor (mAChR) known to regulate specificity of G-protein
coupling. The APC gene also contained a local similarity to the
region of the m3 mAChR (SEQ ID NO:9) that overlapped with the MCC
similarity (SEQ ID NO:10) (FIG. 4B). Although the similarities to
ral2 (SEQ ID NO:8) (FIG. 4A) and m3 mAChR (SEQ ID NO:9) (FIG. 4B)
were not statistically significant, they were intriguing in light
of previous observations relating G-proteins to neoplasia.
Each of the six genes described above was expressed in normal colon
mucosa, as indicated by their representation in colon cDNA
libraries. To study expression of the genes in neoplastic
colorectal epithelium, we employed reverse transcription-polymerase
chain reaction (PCR) assays. Primers based on the sequences of FER,
TB1, TB2, MCC, and APC were each used to design primers for PCR
performed with cDNA templates. Each of these genes was found to be
expressed in normal colon, in each of ten cell lines derived from
colorectal cancers, and in tumor cell lines derived from lung and
bladder tumors. The ten colorectal cancer cell lines included eight
from patients with sporadic CRC and two from patients with FAP.
EXAMPLE 2
This example demonstrates a genetic analysis of the role of the FER
gene in FAP and sporadic colorectal cancers.
We considered FER as a candidate because of its proximity to the
FAP locus as judged by physical and genetic criteria (see Example
1), and its homology to known tyrosine kinases with oncogenic
potential. Primers were designed to PCR-amplify the complete coding
sequence of FER from the RNA of two colorectal cancer cell lines
derived from FAP patients, cDNA was generated from RNA and used as
a template for PCR. The primers used were
5'-AGAAGGATCCCTTGTGCAGTGTGGA-3' (SEQ ID NO:95) and
5'-GACAGGATCCTGAAGCTGAGTTTG-3' (SEQ ID NO:96). The underlined
nucleotides were altered from the true FER sequence to create BamHI
sites. The cell lines used were JW and Difi, both derived from
colorectal cancers of FAP patients. (C. Paraskeva, B. G. Buckle, D.
Sheer, C. B. Wigley, Int. J. Cancer 34, 49 (1984); M. E. Gross et
al., Cancer Res. 51, 1452 (1991). The resultant 2554 basepair
fragments were cloned and sequenced in their entirety. The PCR
products were cloned in the BamHI site of Bluescript SK
(Stratagene) and pools of at least 50 clones were sequenced en
masse using T7 polymerase, as described in Nigro et al., Nature
342, 705 (1989).
Only a single conservative amino acid change (GTG.fwdarw.CTG,
creating a val to leu substitution at codon 439) was observed. The
region surrounding this codon was then amplified from the DNA of
individuals without FAP and this substitution was found to be a
common polymorphism, not specifically associated with FAP. Based on
these results, we considered it unlikely (though still possible)
the FER gene was responsible for FAP. To amplify the regions
surrounding codon 439, the following primers were used:
5'-TCAGAAAGTGCTGAAGAG-3' (SEQ ID NO:97) and
5'-GGAATAATTAGGTCTCCAA-3' (SEQ ID NO:98). PCR products were
digested with PstI, which yields a 50 bp fragment if codon 439 is
leucine, but 26 and 24 bp fragments if it is valine. The primers
used for sequencing were chosen from the FER cDNA sequence in Hao
et al., supra.
EXAMPLE 3
This example demonstrates the genetic analysis of MCC, TB2, SRP and
APC in FAP and sporadic colorectal tumors. Each of these genes is
linked and encompassed by contig 3 (see FIG. 1).
Several lines of evidence suggested that this contig was of
particular interest. First, at least three of the four genes in
this contig were within the deleted region identified in two FAP
patients. (See Example 5 infra). Second, allelic deletions of
chromosome 5q21 in sporadic cancers appeared to be centered in this
region. (Ashton-Rickardt et al., Oncogene, in press; and Miki et
al., Japn. J. Cancer Res., in press.) Some tumors exhibited loss of
proximal RFLP markers (up to and potentially including the 5' end
of MCC), but no loss of markers distal to MCC. Other tumors
exhibited loss of markers distal to and perhaps including the 3'
end of MCC, but no loss of sequences proximal to MCC. This
suggested either that different ends of MCC were affected by loss
in all such cases, or alternatively, that two genes (one proximal
to and perhaps including MCC, the other distal to MCC) were
separate targets of deletion. Third, clones from each of the six
FAP region genes were used as probes on Southern blots containing
tumor DNA from patients with Sporadic CRC. Only two examples of
somatic changes were observed in over 200 tumors studied: a
rearrangement/deletion whose centrometric end was located within
the MCC gene (Kinzler et al., supra) and an 800 bp insertion within
the APC gene between nucleotides 4424 and 5584. Fourth, point
mutations of MCC were observed in two tumors (Kinzler et al.) supra
strongly suggesting that MCC was a target of mutation in at least
some sporadic colorectal cancers.
Based on these results, we attempted to search for subtle
alterations of contig 3 genes in patients with FAP. We chose to
examine MCC and APC, rather than TB2 or SRP, because of the somatic
mutations in MCC and APC noted above. To facilitate the
identification of subtle alterations, the genomic sequences of MCC
and APC exons were determined (see Table I, SEQ ID NO:24-38).
TABLE-US-00001 TABLE I APC EXONS EXON NUCLEOTIDES.sup.1 EXON
BOUNDARY SEQUENCE.sup.2 822 to 930
catgatgttatctgtatttacctatagtctaaattataccatctataatgtgcttaattttta-
g/GGTTCA. . . (SEQ ID NO: 24) . .
.ACCAAG/gtaacagaagattacaaaccctggtcactaatgccatgactactttgctaag (SEQ
ID NO: 25) 931 to 1039
ggatattaaagtcgtaattttgtttctaaactcatttggcccacag/GTGGAA. . . (SEQ ID
NO: 26) . . .ATCCAA/gtatgttctctatagtgtacatcgtagtgcatg (SEQ ID NO:
27) 1310 to 1405
catcattgctcttcaaataacaaagcattatggtttatgttgattttatttttcag/TGCC- AG.
. . (SEQ ID NO: 28) . .
.AACTAG/gtaagacaaaaatgttttttaatgacatagacaattactggtg (SEQ ID NO: 29)
1406 to 1545 tagatgattgtctttttcctcttgccctttttaaattag/GGGGAC. . .
(SEQ ID NO: 30) . .
.AACAAG/gtatgtttttataacatgtatttcttaaggatagctcaggtctga (SEQ ID NO:
31) 1546 to 1623
gcttggcttcaagttgtctttttaatgatcctctattctgtatttaatttacag/GCTACG- . .
. (SEQ ID NO: 32) . .
.CAGCAG/gtactatttagaatttcacctgtttttcttttttctctttttctttgaggcagggtctcac-
tctg (SEQ ID NO: 33) 1624 to 1740
gcaactagtatgattttatgtataaattaatctaaaattgattaatttgacag/GTTATT. . .
(SEQ ID NO: 34) . .
.AAAAAG/gtacctttgaaaacatttagtactataatatgaatttcatgt (SEQ ID NO: 35)
1741 to 1955 caactctaattagatgacccatattcagaaacttactag/GAATCA. . .
(SEQ ID NO: 36) . .
.CCACAG/gtatatatagagttttatattacttttaaagtacagaattcatactctcaaaaa (SEQ
ID NO: 37) 1956 to 8973 tcttgatttttatttcag/GCAAAT. . . (SEQ ID NO:
38) . . .GGTATTTATGCAAAAAAAAATGTTTTTGT (SEQ ID NO: 1)
.sup.1Relative to predicted translation initiation site .sup.2Small
case letters represent introns, large case letters represent exons
The entire 3' end of the cloned APC cDNA (nt 1956-8973) appeared to
be encoded in this exon, as indicated by restriction endonuclease
mapping and sequencing of the cloned genomic DNA. The ORF ended at
nt 8535. The extreme 3' end of the APC transcript has not yet been
identified.
These sequences were used to design primers for PCR analysis of
constitutional DNA from FAP patients.
We first amplified eight exons and surrounding introns of the MCC
gene in affected individuals from 90 different FAP kindreds. The
PCR products were analyzed by a ribonuclease (RNase) protein assay.
In brief, the PCR products were hybridized to in vitro transcribed
RNA probes representing the normal genomic sequences. The hybrids
were digested with RNase A, which can cleave at single base pair
mismatches within DNA-RNA hybrids, and the cleavage products were
visualized following denaturing gel electrophoresis. Two separate
RNase protection analyses were performed for each exon, one with
the sense and one with the antisense strand. Under these
conditions, approximately 40% of all mismatches are detectable.
Although some amino acid variants of MCC were observed in FAP
patients, all such variants were found in a small percentage of
normal individuals. These variants were thus unlikely to be
responsible for the inheritance of FAP.
We next examined three exons of the .[.A PC.]. .Iadd.APC
.Iaddend.gene. The three exons examined included those containing
nt 822-930, 931-1309, and the first 300 nt of the most distal exon
(nt 1956-2256). PCR and RNase protection analysis were performed as
described in Kinzler et al. supra, using the primers underlined in
Table I (SEQ ID NO:24-38). The primers for nt 1956-2256 were
5'-GCAAATCCTAAGAGAGAACAA-3' (SEQ ID NO:99) and
5'-GATGGCAAGCTTGAGCCAG-3' (SEQ ID NO:100).
In 90 kindreds, the RNase protection method was used to screen for
mutations and in an additional 13 kindreds, the PCR products were
cloned and sequenced to search for mutations not detectable by
RNase protection. PCR products were cloned into a Bluescript vector
modified as described in T. A. Holton and M. W. Graham, Nucleic
Acids Res. 19, 1156 (1991). A minimum of 100 clones were pooled and
sequenced. Five variants were detected among the 103 kindreds
analyzed. Cloning and subsequent DNA sequencing of the PCR product
of patient P21 indicated a C to T transition in codon 413 that
resulted in a change from arginine to cysteine. This amino acid
variant was not observed in any of 200 DNA samples from individuals
without FAP. Cloning and sequencing of the PCR product from
patients P24 and P34, who demonstrated the same abnormal RNase
protection pattern indicated that both had a C to T transition at
codon 801 that resulted in a change from arginine (CGA) to a stop
codon (TGA). This change was not present in 200 individuals without
FAP. As this point mutation resulted in the predicted loss of the
recognition site for the enzyme Taq I, appropriate PCR products
could be digested with Taq I to detect the mutation. This allowed
us to determine that the stop codon co-segragated with disease
phenotype in members of the family of P24. The inheritance of this
change in affected members of the pedigree provides additional
evidence for the importance of the mutation.
Cloning and sequencing of the PCR product from FAP patient P93
indicated a C to G transversion at codon 279, also resulting in a
stop codon (change from TCA to TGA). This mutation was not present
in 200 individuals without FAP. Finally, one additional mutation
resulting in a serine (TCA) to stop codon (TGA) at codon 712 was
detected in a single patient with FAP (patient P60).
The five germline mutations identified are summarized in Table IIA,
as well as four others discussed in Example 9.
TABLE-US-00002 TABLE IIA Germline mutations of the APC gene in FAP
and GS Patients EXTRA- COLO- NIC AMINO PATIENT NUCLEOTIDE ACID
DISEASE CODON CHANGE CHANGE AGE 93 279 TCA->TGA Ser->Stop 39
Mandi- bular Osteoma 24 301 CGA->TGA Arg->Stop 46 None 34 301
CGA->TGA Arg->Stop 27 Des- moid Tumor 21 413 CGC->TGC
Arg->Cys 24 Mandi- bular Osteoma 60 712 TCA->TGA Ser->Stop
37 Mandi- bular Osteoma 3746 243 CAGAG->CAG splice- junction
3460 301 CGA->TGA Arg->Stop 3827 456 CTTTCA->CTTCA
frameshift 3712 500 T->G Tyr->Stop * The mutated nucleotides
are underlined.
In addition to these germline mutations, we identified several
somatic mutations of MCC and APC in sporadic CRC's. Seventeen MCC
exons were examined in 90 sporadic colorectal cancers by RNase
protection analysis. In each case where an abnormal RNase
protection pattern was observed, the corresponding PCR products
were cloned and sequenced. This led to the identification of six
point mutations (two described previously) (Kinzler et al., supra),
each of which was not found in the germline of these patients
(Table IIB).
TABLE-US-00003 TABLE IIB Somatic Mutations in Sporadic CRC Patients
NUCLEOTIDE AMINO ACID PATIENT CODON CHANGE CHANGE T35 MCC 12
GAG/gtaaga-> (Splice GAG/gtaaaa Donor) T16 MCC 145
ctcag/GGA-> (Splice atcag/GGA Acceptor) T47 MCC 267 CGG->CTG
Arg->Leu T81 MCC 490 TCG->TTG Ser->Leu T35 MCC 506
CGG->CAG Arg->Gln T91 MCC 698 GCT->GTT Ala->Val T34 APC
288 CCAGT->CCCAGCCAGT (Insertion) T27 APC 331 CGA->TGA
Arg->Stop T135 APC 437 CAA/gtaa->CAA/gcaa (Splice Donor) T20I
APC 1338 CAG->TAG Gln->Stop For splice site mutations, the
codon nearest to the mutation is listed The underlined nucleotides
were mutant; small case letters represent introns, large case
letters represent exons
Four of the mutations resulted in amino acid substitutions and two
resulted in the alteration of splice site consensus elements.
Mutations at analogous splice site positions in other genes have
been shown to alter RNA processing in vivo and in vitro.
Three exons of APC were also evaluated in sporadic tumors. Sixty
tumors were screened by RNase protection, and an additional 98
tumors were evaluated by sequencing. The exons examined included nt
822-930, 931-1309, and 1406-1545 (Table I). A total of three
mutations were identified, each of which proved to be somatic.
Tumor T27 contained a somatic mutation of CGA (arginine) to TGA
(stop codon) at codon 33. Tumor T135 contained a GT to GC change at
a splice donor site. Tumor T34 contained a 5 bp insertion (CAGCC
between codons 288 and 289) resulting in a stop at codon 291 due to
a frameshift. p We serendipitously discovered one additional
somatic mutation in a colorectal cancer. During our attempt to
define the sequences and splice patterns of the MCC and APC gene
products in colorectal epithelial cells, we cloned cDNA from the
colorectal cancer cell line SW480. The amino acid sequence of the
MCC gene from SW480 was identical to that previously found in
clones from human brain. The sequence of APC in SW480 cells,
however, differed significantly, in that a transition at codon 1338
resulted in a change from glutamine (CAG) to a stop codon (TAG). To
determine if this mutation was somatic, we recovered DNA from
archival paraffin blocks of the original surgical specimen (T201)
from which the tumor cell line was derived 28 years ago.
DNA was purified from paraffin sections as described in S. E.
Goelz, S. R. Hamilton, and B. Vogelstein. Biochem. Biophys. Res.
Comm. 130, 118 (1985). PCR was performed as described in reference
24, using the primers 5'-GTTCCAGCAGTGTCACAG-3' (SEQ ID NO:101) and
5'-GGGAGATTTCGCTCCTGA-3' (SEQ ID NO:102). A PCR product containing
codon 1338 was amplified from the archival DNA and used to show
that the stop codon represented a somatic mutation present in the
original primary tumor and in cell lines derived from the primary
and metastatic tumor sites, but not from normal tissue of the
patient.
The ten point mutations in the MCC and APC genes so far discovered
in sporadic CRCs are summarized in Table IIB. Analysis of the
number of mutant and wild-type PCR clones obtained from each of
these tumors showed that in eight of the ten cases, the wild-type
sequence was present in approximately equal proportions to the
mutant. This was confirmed by RFLP analysis using flanking markers
from chromosome 5q which demonstrated that only two of the ten
tumors (T135 and T201) exhibited an allelic deletion on chromosome
5q. These results are consistent with previous observations showing
that 20-40% of sporadic colorectal tumors had allelic deletions of
chromosome 5q. Moreover, these data suggest that mutations of 5q21
genes are not limited to those colorectal tumors which contain
allelic deletions of this chromosome.
EXAMPLE 4
This example characterizes small, nested deletions in DNA from two
unrelated FAP patients.
DNA from 40 FAP patients was screened with cosmids that has been
mapped into a region near the APC locus to identify small deletions
or rearrangements. Two of these cosmids, L5.71 =nd L5.79,
hybridized with a 1200 kb NotI fragment in DNAs from most of the
FAP patients screened.
The DNA of one FAP patient, 3214, showed only a 940 kb NotI
fragment instead of the expected 1200 kb fragment. DNA was analyzed
from four other members of the patient's immediate family; the 940
kb fragment was present in her affected mother (4711), but not in
the other, unaffected family members. The mother also carried a
normal 1200 kb Notl fragment that was transmitted to her two
unaffected offspring. These observations indicated that the mutant
polyposis allele is on the same chromosome as the 940 kb NotI
fragment. A simple interpretation is that APC patients 3214 and
4711 each carry a 260 kb deletion within the APC locus.
If a deletion were present, then other enzymes might also be
expected to produce fragments with altered mobilities.
Hybridization of L5.79 to NruI-digested DNAs from both affected
members of the family revealed a novel NruI fragment of 1300 kb, in
addition to the normal 1200 kb NruI fragment. Furthermore, Mlul
fragments in patients 3214 and 4711 also showed an increase in size
consistent with the deletion of an MluI site. The two chromosome 5
homologs of patient 3214 were segregated in somatic cell hybrid
lines; HHW1155 (deletion hybrid) carried the abnormal homolog and
HHW1159 (normal hybrid) carried the normal homolog.
Because patient 8214 showed bray a 940 kb NotI fragment, she had
not inherited the 1200 kb fragment present in the unaffected
father's DNA. This observation suggests that he must be
heterozygous for, and have transmitted, either a deletion of the
L5.79 probe region or a variant NotI fragment too large to resolve
on the gel system. As expected, the hybrid cell line HHW1159, which
carries the paternal homolog, revealed no resolved. Not fragment
when probed with L5.79. However, probing of HHW1159 DNA with L5.79
following digestion with other enzymes did reveal restriction
fragments, demonstrating the presence of DNA homologous to the
probe. The father is, therefore, interpreted as heterozygous for a
polymorphism at the NotI site, with one chromosome 5 having a 1200
kb NotI fragment and the other having a fragment too large to
resolve consistently on the gel. The latter was transmitted to
patient 3214.
When double digests were used to order restriction sites within the
1200 kb NotI fragment, L5.71 and L5.79 were beth found to lie on a
550 kb NotI-NruI fragment and, therefore, on the same side of an
NruI site in the 1200 kb NotI fragment. To obtain genomic
representation of sequences present over the entire 1200 kb Notl
fragment, we constructed a library of small-fragment inserts
enriched for sequences from this fragment. DNA from the somatic
cell hybrid HHW141, which contains about 40% of chromosome 5, was
digested with NotI and electrophoresed under pulsed-field gel (PFG)
conditions; EcoRI fragments from the 1200 kb region of this gel
were cloned into a phage vector. Probe Map30 was isolated from this
library. In normal individuals probe Map30 hybridizes to the 1200
kb NotI fragment and to a 200 kb NruI fragment. This latter
hybridization places Map30 distal, with respect to the locations of
L5.71 and L5.79, to the NruI site of the 550 kb NotI-NruI
fragment.
Because Map30 hybridized to the abnormal, 1300 kb Nrul fragment of
patient 3214, the locus defined by Map30 lies outside the
hypothesized deletion. Furthermore, in normal chromosomes Map30
identified a 200 kb NruI fragment and L5.79 identified a 1200 kb
NruI fragment; the hypothesized deletion must, therefore, be
removing an NruI site, or sites, lying between Map30 and L5.79, and
these two probes must flank the hypothesized deletion. A
restriction map of the genomic region, showing placement of these
probes, is shown in FIG. 5.
A NotI digest of DNA from another FAP patient, 3824 was probed with
L5.79. In addition to the 1200 kb normal NotI fragment, a fragment
of approximately 1100 kb was observed, consistent with the presence
of a 100 kb deletion in one chromosome 5. In this case, however,
digestion with NruI and MluI did not reveal abnormal bands,
indicating that if a deletion were present, its boundaries must lie
distal to the NruI and MluI sites of the fragments identified by
L5.79. Consistent with this expectation, hybridization of Map30 to
DNA from patient 3824 identified a 760 kb MluI fragment in addition
to the expected 860 kb fragment, supporting the interpretation of a
100 kb deletion in this patient. The two chromosome 5 homologs of
patient 3824 were segregated in somatic cell hybrid lines; HHW1291
was found to carry only the abnormal homolog and HHW1290 only the
normal homolog.
That the 860 kb Mlul fragment identified by Map30 is distinct from
the 830 kb MluI fragment identified previously by L5.79 was
demonstrated by hybridization of Map30 and L5.79 to a NotI-MluI
double digest of DNA from the hybrid cell (HHW1159) containing the
nondeleted chromosome 5 homolog of patient 3214. As previously
indicated, this hybrid is interpreted as missing one of the NotI
sites that define the 1200 kb fragment. A 620 kb NotI-MluI fragment
was seen with probe L5.79, and an 860 kb fragment was seen with
Map30. Therefore, the 830 kb MluI fragment recognized by probe
L5.79 must contain a NotI site in HHW1159 DNA; because the 860 kb
MluI fragment remains intact, it does not carry this NotI site and
must be distinct from the 830 kb Mlul fragment.
EXAMPLE 5
This example demonstrates the isolation of human sequences which
span the region deleted in the two unrelated FAP patients
characterized in Example 4.
A strong prediction of the hypothesis that patients 8214 and 3824
carry deletions is that some sequences present on normal chromosome
5 homologs would be missing from the hypothesized deletion
homologs. Therefore, to develop genomic probes that might confirm
the deletions, as well as to identify genes from the region, YAC
clones from a contig seeded by cosmid L5.79 were localized from a
library containing seven haploid human genome equivalents
(Albertsen et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 87, pp.
4256-4260 (1990)) with respect to the hypothesized deletions. Three
clones, YACs 57B8, 310D8, and 183H12, were found to overlap the
deleted region.
Importantly, one end of YAC 57B8 (clone AT57) was found to lie
within the patient 3214 deletion. Inverse polymerase chain reaction
(PCR) defined the end sequences of the insert of YAC 57B8. PCR
primers based on one of these end sequences repeatedly failed to
amplify DNA from the somatic cell hybrid (HHW1155) carrying the
deleted homolog of patient 3214, but did amplify a product of the
expected size from the somatic cell hybrid (HHW1159) carrying the
normal chromosome 5 homolog. This result supported the
interpretation that the abnormal restriction fragments found in the
DNA of patient 3214 result from a deletion.
Additional support for the hypothesis of deletion in DNA from
patient 3214 came from subcloned fragments of YAC 183H12, which
spans the region in question. Y11, an EcoRI fragment cloned from
YAC 183H12, hybridized to the normal, 1200 kb NotI fragment of
patient 4711, but failed to hybridize to the abnormal, 940 kb Notl
fragment of 4711 or to DNA from deletion cell line HHW1155. This
result confirmed the deletion in patient 3214.
Two additional EcoRl fragments from YAC 183H12, Y10 and Y14, were
localized within the patient 3214 deletion by their failure to
hybridize to DNA from HHW1155. Probe Y10 hybridizes to a 150 kb
NruI fragment in normal chromosome 5 homologs. Because the 3214
deletion creates the 1300 kb NruI fragment seen with the probes
L5.79 and Map30 that flank the deletion, these NruI sites and the
150 kb NruI fragment lying between must be deleted in patient 3214.
Furthermore, probe Y10 hybridizes to the same 620 kb Notl-MluI
fragment seen with probe L5.79 in normal DNA, indicating its
location as L5.79-proximal to the deleted MluI site and placing it
between the MluI site and the L5.79-proximal NruI site. The MluI
site must, therefore, lie between the NruI sites that define the
150 kb NruI fragment (see FIG. 5).
Probe Y11 also hybridized to the 150 kb NruI fragment in the normal
chromosome 5 homolog, but failed to hybridize to the 620 kb
Notl-MluI fragment, placing it L5.79-distal to the MluI site, but
proximal to the second NruI site. Hybridization to the same (860
kb) MluI fragment as Map30 confirmed the localization of probe Y11
L5.79-distal to the MluI site.
Probe Y14 was shown to be L5.79-distal to both deleted NruI sites
by virtue of its hybridization to the same 200 kb NruI fragment of
the normal chromosome 5 seen with Map30. Therefore, the order of
these EcoRI fragments derived from YAC 183H12 and deleted in
patient 3214, with respect to L5.79 and Map30, is
L5.79-Y10-Y11-Y14-Map30.
The 100 kb deletion of patient 3824 was confirmed by the failure of
aberrant restriction fragments in this DNA to hybridize with probe
Y11, combined with positive hybridizations to probes Y10 and/or
Y14. Y10 and Y14 each hybridized to the 1100 kb NotI fragment of
patient 3824 as well as to the normal 1200 kb NotI fragment, but
Y11 hybridized to the 1200 kb fragment only. In the Mlul digest,
probe Y14 hybridized to the 860 kb and 760 kb fragments of patient
3824 DNA, but probe Y11 hybridized only to the 860 k13 fragment. We
conclude that the basis for the alteration in fragment size in DNA
from patient 3824 is, indeed, a deletion. Furthermore, because
probes Y10 and Y14 are missing from the deleted 3214 chromosome,
but present on the deleted 3824 chromosome, and they have been
shown to flank probe Y11, the deletion in patient 3824 must be
nested within the patient 3214 deletion.
Probes Y10, Y11, Y14 and Map30 each hybridized to YAC 310D8,
indicating that this YAC spanned the patient 3824 deletion and at a
minimum, most of the 3214 deletion. The YAC characterizations,
therefore, confirmed the presence of deletions in the patients and
provided physical representation of the deleted region.
EXAMPLE 6
This example demonstrates that the MCC coding sequence maps outside
of the region deleted in the two FAP patients characterized in
Example 4.
An intriguing FAP candidate gene, MCC, recently was ascertained
with cosmid L5.71 and was shown to have undergone mutation in colon
carcinomas (Kinzler et al., supra). It was therefore of interest to
map this gene with respect to the deletions in APC patients.
Hybridization of MCC probes with an overlapping series of YAC
clones extending in either direction from L5.71 showed that the 3'
end of MCC must be oriented toward the region of the two APC
deletions.
Therefore, two 3' cDNA clones from MCC were mapped with respect, to
the deletions: clone 1CI (bp 2378-4181) and clone 7 (bp 2890-3560).
Clone 1CI contains sequences from the C-terminal end of the open
reading frame, which stops at nucleotide 2708, as well as 3'
untranslated sequence. Clone 7 contains sequence that is entirely
3' to the open reading frame. Importantly, the entire 3'
untranslated sequence contained in the cDNA clones consists of a
single 2.5 kb exon. These two clones were hybridized to DNAs from
the YACs spanning the FAP region. Clone 7 fails to hybridize to YAC
310D8, although it does hybridize to YACs 183H12 and 57B8; the same
result was obtained with the cDNA 1CI. Furthermore, these probes
did show hybridization to DNAs from both hybrid cell lines (HWW1159
and HWW1155) and the lymphoblastoid cell line from patient 3214,
confirming their locations outside the deleted region. Additional
mapping experiments suggested that the 3' end of the MCC cDNA clone
contig is likely to be located more than 45 kb from the deletion of
patient 3214 and, therefore, more than 100 kb from the deletion of
patient 3824.
EXAMPLE 7
This example identifies three genes within the deleted region of
chromosome 5 in the two unrelated FAP patients characterized in
Example 4.
Genomic clones were used to screen cDNA libraries in three separate
experiments. One screening was done with a phage clone derived from
YAC 310D8 known to span the 260 kb deletion of patient 3214. A
large-insert phage library was constructed from this YAC; screening
with Y11 identified .lamda.205, which mapped within both deletions.
When clone .lamda.205 was used to probe a random-, plus oligo(dT)-,
primed fetal brain cDNA library (approximately 300,000 phage), six
cDNA clones were isolated and each of them mapped entirely within
both deletions. Sequence analysis of these six clones formed a
single cDNA contig, but did not reveal an extended open reading
frame. One of the six cDNAs was used to isolate more cDNA clones,
some of which crossed the L5.71-proximal breakpoint of the 3824
deletion, as indicated by hybridization to both chromosome of this
patient. These clones also contained an open reading frame,
indicating a transcriptional orientation proximal to distal with
respect to L5.71. This gene was named DP1 (deleted in polyposis 1).
This gene is identical to TB2 described above.
cDNA walks yielded a cDNA contig of 3.0-3.5 kb, and included two
clones containing terminal poly(A) sequences. This size corresponds
to the 3.5 kb band seen by Northern analysis. Sequencing of the
first 3163 bp of the cDNA contig revealed an open reading frame
extending from the first base to nucleotide 631, followed by a 2.5
kb 3' untranslated region. The sequence surrounding the methionine
codon at base 77 conforms to the Kozak consensus of an initiation
methionine (Kozak, 1984). Failed attempts to walk farther, coupled
with the similarity of the lengths of isolated cDNA and mRNA,
suggested that the NH.sub.2-terminus of the DP1 protein had been
reached. Hybridization to a combination of genomic and YAC DNAs cut
with various enzymes indicated the genomic coverage of DP1 to be
approximately 30 kb.
Two additional probes for the locus, YS-11 and YS-39, which had
been ascertained by screening of a cDNA library with an independent
YAC probe identified with MCC sequences adjacent to L5.71, were
mapped into the deletion region. YS-39 was shown to be a cDNA
identical in sequence to DP1. Partial characterization of YS-11 had
shown that 200 bp of DNA sequence at one end was identical to
sequence coding for the 19 kb protein of the ribosomal signal
recognition particle, SRP19 (Lingelbach et al., supra).
Hybridization experiments mapped YS-11 within beth deletions. The
sequence of this clone, however, was found to be complex. Although
454 bp of the 1032 bp sequence of YS-11 were identical to the
GenBank entry for the SRP19 gene, another 578 bp appended 5' to the
SRP19 sequence was found to consist of previously unreported
sequence containing no extended open reading frame. This suggested
that YS-11 was either a chimeric clone containing two independent
inserts or a clone of an incompletely processed or aberrant
message. If YS-11 were a conventional chimeric clone, the
independent segments would not be expected to map to the same
physical region. The segments resulting from anomalous processing
of a continuous transcript, however, would map to a single
chromosomal region.
Inverse PCR with primers specific to the two ends of YS-11, the
SRP19 ,end and the unidentified region, verified that both
sequences map within the YAC 310D8; therefore, YS-11 is most likely
a clone of an immature or anomalous mRNA species. Subsequently,
both ends were shown to lie with the deleted region of patient
3824, and YS-11 was used to screen for additional cDNA clones.
Of the 14 cDNA clones selected from the fetal brain library, one
clone, V5, was of particular interest in that it contained an open
reading frame throughout, although it included only a short
identity to the first 78 5' bases of the YS-11 sequence. Following
the 78 bp of identical sequence, the two cDNA sequences diverged at
an AG. Furthermore, divergence from genomic sequence was also seen
after these 78 bp, suggesting the presence of a splice junction,
and supporting the view that YS-11 represents an irregular
message.
Starting with V5, successive 5' and 3' walks were performed; the
resulting cDNA contig consisted of more than 100 clones, which
defined a new transcript, DP2. Clones walking in the 5' directions
crossed the 3824 deletion breakpoint farthest from L5.71; since its
3' end is closer to this cosmid than its 5' end, the
transcriptional orientation of DP2 is opposite to that of MCC and
DP1.
The third screening approach relied on hybridization with a 120 kb
MluI fragment from YAC 57B8. This fragment hybridizes with probe
Y11 and completely spans the 100 kb deletion in patient 3824, the
fragment was purified on two preparative PFGs, labeled, and used to
screen a fetal brain cDNA library. A number of cDNA clones
previously identified in the development of the DP1 and DP2 contigs
were reascertained. However, 19 new cDNA clones mapped into the
patient 3824 deletion. Analysis indicated that these 19 formed a
new contig, DP3, containing a large open reading frame.
A clone from the 5' end of this new cDNA contig hybridized to the
same EcoRI fragment as the 3' end of DP2. Subsequently, the DP2 and
DP3 contigs were connected by a single 5' walking step from DP3, to
form the single contig DP2.5. The complete nucleotide sequence of
DP2.5 is shown in FIG. 9.
The consensus cDNA sequence of DP2.5 suggests that the entire
coding sequence of DP2.5 has been obtained and is 8532 bp long. The
most 5' ATG codon occurs two codons from an in-frame stop and
conforms to the Kozak initiation consensus (Kozak, Nucl. Acids.
Res., Vol. 12, p. 857-872 1984). The 3' open reading frame breaks
down over the final 1.8 kb, giving multiple stops in all frames. A
poly(A) sequence was found in one clone approximately 1 kb into the
3' untranslated region, associated with a polyadenylation signal 33
bp upstream (position 9530). The open reading frame is almost
identical to that identified as APC above.
An alternatively spliced exon at nucleotide 934 of the DP2.5
transcript is of potential interest, it was first discovered by
noting that two classes of cDNA had been isolated. The more
abundant cDNA class contains a 303 bp exon not included in the
other. The presence in vivo of the two transcripts was verified by
an exon connection experiment. Primers flanking the alternatively
spliced exon were used to amplify, by PCR, cDNA prepared from
various adult tissues. Two PCR products that differed in size by
approximately 300 bases were amplified from all the tissues tested;
the larger product was always more abundant than the smaller.
EXAMPLE 8
This example demonstrates the primers used to identify subtle
mutations in DP1, SRP19, and DP25.
To obtain DNA sequence adjacent to the exons of the genes DP1,
DP2.5, and SRP19, sequencing substrate was obtained by inverse PCR
amplification of DNAs from two YACs, 310D8 and 183H12, that span
the deletions. Ligation at low concentration cyclized the
restriction enzyme-digested YAC DNAs. Oligonucleotides with
sequencing tails, designed in inverse orientation at intervals
along the cDNAs, primed PCR amplification from the cyclized
templates. Comparison of these DNA sequences with the cDNA
sequences placed exon boundaries at the divergence points. SRP19
and DP1 were each shown to have five exons. DP2.5 consisted of 15
exons. The sequences of the oligonucleotides synthesized to provide
PCR amplification primers for the exons of each of these genes are
listed in Table III SEQ ID NO:39-94.
TABLE-US-00004 TABLE III Sequences of Primers Used for SSCP
Analyses Exon Primer 1 Primer 2 DP1 UP-TCCCCGCCTGCCGCTCTC
RP-GCAGCGGCGGCTCCCGTG UP-GTGAACGGCTCTCATGCTGC
RP-ACGTGCGGGGAGGAATGGA UP-ATGATATCTTACCAAATGATATAC
RP-TTATTCCTACTTCTTCTATACAG UP-TACCCATGCTGGCTCTTTTTC
RP-TGGGGCCATCTTGTTCCTGA UP-ACATTAGGCACAAAGCTTGCAA
RP-ATCAAGCTCCAGTAAGAAGGTA SRP19 UP-TGCGGCTCCTGGGTTGTTG
RP-GCCCCTTCCTTTCTGAGGAC UP-TTTTCTCCTGCCTCTTACTGC
RP-ATGACACCCCCCATTCCCTC UP-CCACTTAAAGCACATATATTTAGT
RP-GTATGGAAAATAGTGAAGAACC UP-TTCTTAAGTCCTGTTTTTCTTTTG
RP-TTTAGAACCTTTTTTGTGTTGTG UP-CTCAGATTATACACTAAGCCTAAC
RP-CATGTCTCTTACAGTAGTACCA DP2.5 UP-AGGTCCAAGGGTAGCCAAGG*
RP-TAAAAATGGATAAACTACAATTAAAAG UP-AAATACAGAATCATGTCTTGAAGT
RP-ACACCTAAAGATGACAATTTGAG UP-TAACTTAGATAGCAGTAATTTCCC*
RP-ACAATAAACTGGAGTACACAAGG UP-ATAGGTCATTGCTTCTTGCTGAT*
RP-TGAATTTTAATGGATTACCTAGGT UP-CTTTTTTTGCTTTTACTGATTAACG
RP-TGTAATTCATTTTATTCCTAATACCTC UP-GGTAGCCATAGTATGATTATTTCT
RP-CTACCTATTTTTATACCCACAAAC UP-AAGAAAGCCTACACCATTTTTGC
RP-GATCATTCTTAGAACCATCTTGC UP-ACCTATAGTCTAAATTATACCATC
RP-GTCATGGCATTACTGACCAG UP-AGTCGTAATTTTGTTTCTAAACTC
RP-TGAAGGACTCCGATTTCACCC* UP-TCATTCACTCACAGCCTGATGAC*
RP-GCTTTGAAACATGCACTACGAT UP-AAACATCATTGCTCTTCAAATAAC
RP-TACCATGATTTAAAAATCCACCAG UP-GATGATTGTCTTTTTCCTCTTTGC
RP-CTGAGCTATCTTAAGAAATCACTG UP-TTTTAAATGATCCTCTATTCTGTAT
RP-ACAGAGTCAGACCCTCCCTCAAAG UP-TTTCTATTCTTACTGCTAGCATT
RP-ATACACAGGTAAGAAATTAGGA UP-TAGATGACCCATATTCTCTTC
RP-CAATTAGGTCTTTTTGAGAGTA 3-A UP-GTTACTGCATACACATTGTGAC
RP-GCTTTTTGTTTCGTAACATGAAG* -B UP-AGTACAAGGATGCCAATATTATG*
RP-ACTTCTATCTTTTTCAGAACGAG* -C UP-ATTTGAATACTACAGTGTTACCC*
RP-CTTGTATTCTAATTTGGCATAAGG* -D UP-CTGCCCATACACATTCAAACAC*
RP-TGTTTGCGTCTTGCCCATCTT* -E UP-AGTCTTAAATATTCAGATGAGCAG*
RP-GTTTCTCTTCATTATATTTTATGCTA* -F UP-AAGCCTACCAATTATAGTGAACG*
RP-AGCTGATGACAAAGATGATAATC* -G UP-AAGAAACAATACAGACTTATTGTG*
RP-ATGAGTGGGGTCTCCTGAAC* -H UPATCTCCCTCCAAAAGTGGTGC*
RP-TCCATCTGGAGTACTTTCTGTG* -I UP-AGTAAATGCTGCAGTTCAGAGG*
RP-CCGTGGCATATCATCCCCC* -J UP-CCCAGACTGCTTCAAAATTACC*
RP-GAGCCTCATCTGTACTTCTGC* -K UP-CCCTCCAAATGAGTTAGCTGC*
RP-TTGTGGTATAGGTTTTACTGGTG* -L UP-ACCCAACAAAAATCAGTTAGATG*
RP-GTGGCTGGTAACTTTAGCCTC* -N UP-ATGATGTTGACCTTTCCAGGG*
RP-ATTGTGTAACTTTTCATCAGTTGC* -M UP-AAAGACATACCAGACAGAGGG*
RP-CTTTTTTGGCATTGCGGAGCT* -O UP-AAGATGACCTGTTGCAGGAATG*
RP-GAATCAGACCAAGCTTGTCTAGAT* -P UP-CAATAGTAAGTAGTTTACATCAAG*
RP-AAACAGGACTTGTACTGTAGGA* -Q UP-CAGCCCCTTCAAGCAAACATC*
RP-GAGGACTTATTCCATTTCTACC* -R UP-CAGTCTCCTGGCCGAAACTC*
RP-GTTGACTGGCGTACTAATACAG* -S UP-TGGTAATGGAGCCAATAAAAAGG*
RP-TGGGACTTTTCGCCATCCAC* -T UP-TGTCTCTATCCACACATTCGTC*
RP-ATGTTTTTCATCCTCACTTTTTGC* -U UP-GGAGAAGAACTGGAAGTTCATC*
RP-TTGAATCTTTAATGTTTGGATTTGC* -V UP-TCTCCCACAGGTAATACTCCC
RP-GCTACAACTGAATGGGGTACG -W UP-CAGGACAAAATAATCCTGTCCC
RP-ATTTTCTTACTTTCATTCTTCCTC All primers are read in the 5' to 3'
direction, the first primer in each pair lies 5' of the exon it
amplifies; the second primer lies 3' of the exon it amplifies.
Primers that lie within the exons are identified by an asterisk. UP
represents the -21M13 universal primer sequence; RP represents the
M13 reverse primer sequence.
With the exception of exons 1, 3, 4, 9, and 15 of DP2.5 (see
below), the primer sequences were located in intron sequences
flanking the exons. The 5' primer of exon 1 is complementary to the
cDNA sequence, but extends just into the 5' Kozak consensus
sequence for the initiator methionine, allowing a survey of the
translated sequences. The 5' primer of exon 3 is actually in the 5'
coding sequences of this exon, as three separate intronic primers
simply would not amplify. The 5' primer of exon 4 just overlaps the
5' end of this exon, and we thus fail to survey the 19 most 5'
bases of this exon. For exon 9, two overlapping primer sets were
used, such that each had one end within the exon. For exon 15, the
large 3' exon of DP2.5, overlapping primer pairs were placed along
the length of the exon; each pair amplified a product of 250-400
bases.
EXAMPLE 9
This example demonstrates the use of single stranded conformation
polymorphism (SSCP) analysis as described by Orita et al. Proc.
Natl. Acad. Sci. U.S.A., Vol. 86, pp. 2766-70 (1989) and Genomics,
Vol. 5, pp. 874-879 (1989) as applied to DP1, SRP19 and DP2.5.
SSCP analysis identifies most single- or multiple-base changes in
DNA fragments up to 400 bases in length. Sequence alterations are
detected as shifts in electrophoretic mobility of single-stranded
DNA on nondenaturing acrylamide gels; the two complementary strands
of a DNA segment usually resolve as two SSCP conformers of distinct
mobilities. However, if the sample is from an individual
heterozygous for a base-pair variant within the amplified segment,
often three or more bands are seen. In some cases, even the sample
from a homozygous individual will show multiple bands.
Base-pair-change variants are identified by differences in pattern
among the DNAs of the sample set.
Exons of the candidate genes were amplified by PCR from the DNAs of
61 unrelated FAP patients and a control set of 12 normal
individuals. The five exons from DP1 revealed no unique conformers
in the FAP patients, although common conformers were observed with
exons 2 and 3 in some individuals of both affected and control
sets, indicating the presence of DNA sequence polymorphisms.
Likewise, none of the five exons of SRP19 revealed unique
conformers in DNA from FAP patients in the test panel.
Testing of exons 1 through 14 and primer sets A through N of exon
15, of the DP2.5 gene, however, revealed variant conformers
specific to FAP patients in exons 7, 8, 10, 11, and 15. These
variants were in the unrelated patients 3746, 3460, 3827, 3712, and
3751, respectively. The PCR-SSCP procedure was repeated for each of
these exons in the five affected individuals and in an expanded set
of 48 normal controls. The variant bands were reproducible in the
FAP patients but were not observed in any of the control DNA
samples. Additional variant conformers in exons 11 and 15 of the
DP2.5 gene were seen; however, each of these was found in both the
affected and control DNA sets. The five sets of conformers unique
to the FAP patients were sequenced to determine the nucleotide
changes responsible for their altered mobilities. The normal
conformers from the host individuals were sequenced also. Bands
were cut from the dried acrylamide gels, and the DNA was eluted.
PCR amplification of these DNAs provided template for
sequencing.
The sequences of the unique conformers from exons 7, 8, 10, and 11
of DP2.5 revealed dramatic mutations in the DP2.5 gene. The
sequence of the new mutation creating the exon 7 conformer in
patient 3746 was shown to contain a deletion of two adjacent
nucleotides, at positions 730 and 731 in the cDNA sequence (FIG. 7,
SEQ ID NO:1). The normal sequence at this splice junction is
CAGGGTCA (intronic sequence underlined), with the intron-exon
boundary between the two repetitions of AG. The mutant allele in
this patient has the sequence CAGGTCA. Although this change is at
the 5' splice site, comparison with known consensus sequences of
splice junctions would suggest that a functional splice junction is
maintained. If this new splice junction were functional, the
mutation would introduce a frameshift that creates a stop codon 15
nucleotides downstream. If the new splice junction were not
functional, messenger processing would be significantly
altered.
To confirm the 2-base deletion, the PCR product from FAP patient
3746 and a control DNA were electrophoresed on an acrylamide-urea
denaturing gel, along with the products of a sequencing reaction.
The sample from patient 3746 showed two bands differing in size by
2 nucleotides, with the larger band identical in mobility to the
control sample; this result was independent confirmation that
patient, 3746 is heterozygous for a 2 bp deletion.
The unique conformer found in exon 8 of patient 3460 was found to
carry a C-T transition, at position 904 in the cDNA sequence of
DP2.5 (shown in FIG. 7), which replaced the normal sequence of CGA
with TGA. This point mutation, when read in frame, results in a
stop codon replacing the normal arginine codon. This single-base
change had occurred within the context of a CG dimer, a potential
hot spot for mutation (Barker et al., 1984).
The conformer unique to FAP patient 3827 in exon 10 was found to
contain a deletion of one nucleotide (1367, 1368, or 1369) when
compared to the normal sequence found in the other bands on the
SSCP gel. This deletion, occurring within a set of three T's,
changed the sequence from CTTTCA to CTTCA; this 1 base frameshift
creates a downstream stop within 30 bases. The PCR product
amplified from this patient's DNA also was electrophoresed on an
acrylamide-urea denaturing gel, along with the PCR product from a
control DNA and products from a sequencing reaction. The patient's
PCR product showed two bands differing by 1 bp in length, with the
larger identical in mobility to the PCR product from the normal
DNA; this result confirmed the presence of a 1 bp deletion in
patient 3827.
Sequence analysis of the variant conformer of exon 11 from patient
3712 revealed the substitution of a T by a G at position changing
the normal tyrosine codon to a stop codon.
The pair of conformers observed in exon 15 of the DP2.5 gene for
FAP patient 3751 also was sequenced. These conformers were found to
carry a nucleotide substitution of C to G at position 5253, the
third base of a valine codon. No amino acid change resulted from
this substitution, suggesting that this conformer reflects a
genetically silent polymorphism.
The observation of distinct inactivating mutations in the DP2.5
gene in four unrelated patients strongly suggested that DP2.5 is
the gene involved in FAP. These mutations are summarized in Table
IIA.
EXAMPLE 10
This example demonstrates that the mutations identified in the
DP2.5 (APC) gene segregate with the FAP phenotype.
Patient 3746, described above as carrying an APC allele with a
frameshift mutation, is an affected offspring of two normal
parents. Colonoscopy revealed no polyps in either parent nor among
the patient's three siblings.
DNA samples from both parents, from the patient's wife, and from
their three children were examined. SSCP analysis of DNA from both
of the patient's parents displayed the normal pattern of conformers
for exon 7, as did DNA from the patient's wife and one of his
off-spring. The two other children, however, displayed the same new
conformers as their affected father. Testing of the patient and his
parents with highly polymorphic VNTR (variable number of tandem
repeat) markers showed a 99.98% likelihood that they are his
biological parents.
These observations confirmed that this novel conformer, known to
reflect a 2 bp deletion mutation in the DP2.5 gene, appeared
spontaneously with FAP in this pedigree and was transmitted to two
of the children of the affected individual.
EXAMPLE 11
This example demonstrates polymorphisms in the APC gene which
appear to be unrelated to disease (FAP).
Sequencing of variant conformers found among controls as well as
individuals with APC has revealed the following polymorphisms in
the APC gene; first, in exon 11, at position 1458, a substitution
of T to C creating an RsaI restriction site but no amino acid
change; and second, in exon 15, at positions 5037 and 5271,
substitutions of A to G and G to T, respectively, neither resulting
in amino acid substitutions. These nucleotide polymorphisms in the
APC gene sequence may be useful for diagnostic purposes.
EXAMPLE 12
This example shows the structure of the APC gene.
The structure of the APC gene is schematically shown in FIG. 8,
with flanking intron sequences indicated (SEQ ID NO:11-38).
The continuity of the very large (6.5 kb), most 3' exon in DP2.5
was shown in two ways. First, inverse PCR with primers spanning the
entire length of this exon revealed no divergence of the cDNA
sequence from the genomic sequence. Second, PCR amplification with
converging primers placed at intervals along the exon generated
products of the same size whether amplified from the originally
isolated cDNA, cDNA from various tissues, or genomic template. Two
forms of exon 9 were found in DP2.5: one is the complete exon; and
the other, labeled exon 9A, is the result of a splice into the
interior of the exon that deletes bases 934 to 1236 in the mRNA and
removes 101 amino acids from the predicted protein (see FIG. 3, SEQ
ID NO:1 and 2).
EXAMPLE 13
This example demonstrates the mapping of the FAP deletions with
respect to the APC exons.
Somatic cell hybrids carrying the segregated chromosomes 5 from the
100 kb (HHW1291) and 260 kb (HHW1155) deletion patients were used
to determine the distribution of the APC genes exons across the
deletions. DNAs from these cell lines were used as template, along
with genomic DNA from a normal control, for PCR-based amplification
of the APC exons.
PCR analysis of the hybrids from the 260 kb deletion of patient
3214 showed that all but one (exon 1) of the APC exons are removed
by this deletion. PCR analysis of the somatic cell hybrid HHW1291,
carrying the chromosome 5 homolog with the 100 kb deletion from
patient 3824, revealed that exons 1 through 9 are present but exons
10 through 15 are missing. This result placed the deletion
breakpoint either between exons 9 and 10 or within exon 10.
EXAMPLE 14
This example demonstrates the expression of alternately spliced APC
messenger in normal tissues and in cancer cell lines.
Tissues that express the APC gene were identified by PCR
amplification of cDNA made to mRNA with primers located within
adjacent APC exons. In addition, PCR primers that flank the
alternatively spliced exon 9 were chosen so that the expression
pattern of both splice forms could be assessed. All tissue types
tested (brain, lung, aorta, spleen, heart, kidney, liver, stomach,
placenta, and colonic mucosa) and cultured cell lines
(lymphoblasts, HL60, and choriocarcinoma) expressed both splice
forms of the APC gene. We note, however, that expression by
lymphocytes normally residing in some tissues, including colon,
prevents unequivocal assessment of expression. The large mRNA,
containing the complete exon 9 rather than only exon 9A, appears to
be the more abundant message.
Northern analysis of poly(A)-selected RNA from lymphoblasts
revealed a single band of approximately 10 kb, consistent with the
size of the sequenced cDNA.
EXAMPLE 15
This example discusses structural features of the APC protein
predicted from the sequence.
The cDNA consensus sequence of APC predicts that the longer, more
abundant form of the message codes for a 2842 or 2844 amino acid
peptide with a mass of 311.8 kd. This predicted APC peptide was
compared with the current data bases of protein and DNA sequences
using both Intelligenetics and GCG software packages. No genes with
a high degree of amino acid sequence similarity were found.
Although many short (approximately 20 amino acid) regions of
sequence similarity were uncovered, none was sufficiently strong to
reveal which, if any, might represent functional homology.
Interestingly, multiple similarities to myosins and keratins did
appear. The APC gene also was scanned for sequence motifs of known
function; although multiple glycosylation, phosphorylation, and
myristoylation sites were seen, their significance is
uncertain.
Analysis of the APC peptide sequence did identify features
important in considering potential protein structure. Hydropathy
plots (Kyte and Doolittle, J. Mol. Biol. Vol. 157, pp. 105-132
(1982)) indicate that the APC protein is notably hydrophilic. No
hydrophobic domains suggesting a signal peptide or a
membrane-spanning domain were found. Analysis of the first 1000
residues indicates that .alpha.-helical rods may form (Cohen and
Parry, Trends Biochem, Sci. Vol. 77, pp. 245-248 (1986); there is a
scarcity of proline residues and, there are a number of regions
containing heptad repeats (apolar-X-X-apolar-X-X-X). Interestingly,
in exon 9A, the deleted form of exon 9, two heptad repeat regions
are reconnected in the proper heptad repeat frame, deleting the
intervening peptide region. After the first 1000 residues, the high
proline content of the remainder of the peptide suggests a compact
rather than a rod-like structure.
The prominent feature of the second 1000 residues is a 20 amino
acid repeat that is iterated seven times with semiregular spacing
(Table 4). The intervening sequences between the seven repeat
regions contained 114, 116, 151, 205, 107, and 58 amino acids,
respectively. Finally, residues 2200-24000 contain a 200 amino acid
basic domain.
TABLE-US-00005 TABLE IV Seven Different Versions of the 20-Amino
Acid Repeat Consensus: F * V E * T P * C F S R * S S L S S L S (SEQ
ID NO: 147) 1262: Y C V E D T P I C F S R C S S L S S L S (SEQ ID
NO: 148) 1376: H T V Q E T P L M F S R C T S V S S L D (SEQ ID NO:
149) 1492: F A T E S T P D G F S C S S S L S A L S (SEQ ID NO: 150)
1643: Y C V E G T P I N F S T A T S L S D L T (SEQ ID NO: 151)
1848: T P I E G T P Y C F S R N D S L S S L D (SEQ ID NO: 152)
1953: F A I E N T P V C P S H N S S L S S L S (SEQ ID NO: 153)
2013: R H V E D T P V C F S R N S S L S S L S (SEQ ID NO: 154)
Numbers denote the first amino acid of each repeat. The consensus
sequence at the top reflects a majority amino acid at a given
position. .Iadd.In the consensus sequence, "*" indicates
"Xaa.".Iaddend.
SEQUENCE LISTINGS
1
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: .[.102.]. .Iadd.154.Iaddend. (2) INFORMATION FOR SEQ ID
NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9606 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (vii) IMMEDIATE SOURCE: (B) CLONE: DP2.5(APC) (ix)
FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 34..8562 (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: 1: GGACTCGGAA ATGAGGTCCA AGGGTAGCCA AGG ATG
GCT GCA G CT TCA TAT GAT 54 Met Ala Ala Ala Ser Tyr Asp 1 5 CAG TTG
TTA AAG CAA GTT GAG GCA CTG AAG A TG GAG AAC TCA AAT CTT 102 Gln
Leu Leu Lys Gln Val Glu Ala Leu Lys M et Glu Asn Ser Asn Leu 10 15
20 CGA CAA GAG CTA GAA GAT AAT TCC AAT CAT C TT ACA AAA CTG GAA ACT
150 Arg Gln Glu Leu Glu Asp Asn Ser Asn His L eu Thr Lys Leu Glu
Thr 25 30 35 GAG GCA TCT AAT ATG AAG GAA GTA CTT AAA C AA CTA CAA
GGA AGT ATT 198 Glu Ala Ser Asn Met Lys Glu Val Leu Lys G ln Leu
Gln Gly Ser Ile 40 45 50 55 GAA GAT GAA GCT ATG GCT TCT TCT GGA CAG
A TT GAT TTA TTA GAG CGT 246 Glu Asp Glu Ala Met Ala Ser Ser Gly
Gln I le Asp Leu Leu Glu Arg 60 65 70 CTT AAA GAG CTT AAC TTA GAT
AGC AGT AAT T TC CCT GGA GTA AAA CTG 294 Leu Lys Glu Leu Asn Leu
Asp Ser Ser Asn P he Pro Gly Val Lys Leu 75 80 85 CGG TCA AAA ATG
TCC CTC CGT TCT TAT GGA A GC CGG GAA GGA TCT GTA 342 Arg Ser Lys
Met Ser Leu Arg Ser Tyr Gly S er Arg Glu Gly Ser Val 90 95 100 TCA
AGC CGT TCT GGA GAG TGC AGT CCT GTT C CT ATG GGT TCA TTT CCA 390
Ser Ser Arg Ser Gly Glu Cys Ser Pro Val P ro Met Gly Ser Phe Pro
105 110 115 AGA AGA GGG TTT GTA AAT GGA AGC AGA GAA A GT ACT GGA
TAT TTA GAA 438 Arg Arg Gly Phe Val Asn Gly Ser Arg Glu S er Thr
Gly Tyr Leu Glu 120 125 130 135 GAA CTT GAG AAA GAG AGG TCA TTG CTT
CTT G CT GAT CTT GAC AAA GAA 486 Glu Leu Glu Lys Glu Arg Ser Leu
Leu Leu A la Asp Leu Asp Lys Glu 140 145 150 GAA AAG GAA AAA GAC
TGG TAT TAC GCT CAA C TT CAG AAT CTC ACT AAA 534 Glu Lys Glu Lys
Asp Trp Tyr Tyr Ala Gln L eu Gln Asn Leu Thr Lys 155 160 165 AGA
ATA GAT AGT CTT CCT TTA ACT GAA AAT T TT TCC TTA CAA ACA GAT 582
Arg Ile Asp Ser Leu Pro Leu Thr Glu Asn P he Ser Leu Gln Thr Asp
170 175 180 TTG ACC AGA AGG CAA TTG GAA TAT GAA GCA A GG CAA ATC
AGA GTT GCG 630 Leu Thr Arg Arg Gln Leu Glu Tyr Glu Ala A rg Gln
Ile Arg Val Ala 185 190 195 ATG GAA GAA CAA CTA GGT ACC TGC CAG GAT
A TG GAA AAA CGA GCA CAG 678 Met Glu Glu Gln Leu Gly Thr Cys Gln
Asp M et Glu Lys Arg Ala Gln 200 205 210 215 CGA AGA ATA GCC AGA
ATT CAG CAA ATC GAA A AG GAC ATA CTT CGT ATA 726 Arg Arg Ile Ala
Arg Ile Gln Gln Ile Glu L ys Asp Ile Leu Arg Ile 220 225 230 CGA
CAG CTT TTA CAG TCC CAA GCA ACA GAA G CA GAG AGG TCA TCT CAG 774
Arg Gln Leu Leu Gln Ser Gln Ala Thr Glu A la Glu Arg Ser Ser Gln
235 240 245 AAC AAG CAT GAA ACC GGC TCA CAT GAT GCT G AG CGG CAG
AAT GAA GGT 822 Asn Lys His Glu Thr Gly Ser His Asp Ala G lu Arg
Gln Asn Glu Gly 250 255 260 CAA GGA GTG GGA GAA ATC AAC ATG GCA ACT
T CT GGT AAT GGT CAG GGT 870 Gln Gly Val Gly Glu Ile Asn Met Ala
Thr S er Gly Asn Gly Gln Gly 265 270 275 TCA ACT ACA CGA ATG GAC
CAT GAA ACA GCC A GT GTT TTG AGT TCT AGT 918 Ser Thr Thr Arg Met
Asp His Glu Thr Ala S er Val Leu Ser Ser Ser 280 285 290 295 AGC
ACA CAC TCT GCA CCT CGA AGG CTG ACA A GT CAT CTG GGA ACC AAG 966
Ser Thr His Ser Ala Pro Arg Arg Leu Thr S er His Leu Gly Thr Lys
300 305 310 GTG GAA ATG GTG TAT TCA TTG TTG TCA ATG C TT GGT ACT
CAT GAT AAG 1014 Val Glu Met Val Tyr Ser Leu Leu Ser Met L eu Gly
Thr His Asp Lys 315 320 325 GAT GAT ATG TCG CGA ACT TTG CTA GCT ATG
T CT AGC TCC CAA GAC AGC 1062 Asp Asp Met Ser Arg Thr Leu Leu Ala
Met S er Ser Ser Gln Asp Ser 330 335 340 TGT ATA TCC ATG CGA CAG
TCT GGA TGT CTT C CT CTC CTC ATC CAG CTT 1110 Cys Ile Ser Met Arg
Gln Ser Gly Cys Leu P ro Leu Leu Ile Gln Leu 345 350 355 TTA CAT
GGC AAT GAC AAA GAC TCT GTA TTG T TG GGA AAT TCC CGG GGC 1158 Leu
His Gly Asn Asp Lys Asp Ser Val Leu L eu Gly Asn Ser Arg Gly 360
365 370 375 AGT AAA GAG GCT CGG GCC AGG GCC AGT GCA G CA CTC CAC
AAC ATC ATT 1206 Ser Lys Glu Ala Arg Ala Arg Ala Ser Ala A la Leu
His Asn Ile Ile 380 385 390 CAC TCA CAG CCT GAT GAC AAG AGA GGC AGG
C GT GAA ATC CGA GTC CTT 1254 His Ser Gln Pro Asp Asp Lys Arg Gly
Arg A rg Glu Ile Arg Val Leu 395 400 405 CAT CTT TTG GAA CAG ATA
CGC GCT TAC TGT G AA ACC TGT TGG GAG TGG 1302 His Leu Leu Glu Gln
Ile Arg Ala Tyr Cys G lu Thr Cys Trp Glu Trp 410 415 420 CAG GAA
GCT CAT GAA CCA GGC ATG GAC CAG G AC AAA AAT CCA ATG CCA 1350 Gln
Glu Ala His Glu Pro Gly Met Asp Gln A sp Lys Asn Pro Met Pro 425
430 435 GCT CCT GTT GAA CAT CAG ATC TGT CCT GCT G TG TGT GTT CTA
ATG AAA 1398 Ala Pro Val Glu His Gln Ile Cys Pro Ala V al Cys Val
Leu Met Lys 440 445 450 455 CTT TCA TTT GAT GAA GAG CAT AGA CAT GCA
A TG AAT GAA CTA GGG GGA 1446 Leu Ser Phe Asp Glu Glu His Arg His
Ala M et Asn Glu Leu Gly Gly 460 465 470 CTA CAG GCC ATT GCA GAA
TTA TTG CAA GTG G AC TGT GAA ATG TAT GGG 1494 Leu Gln Ala Ile Ala
Glu Leu Leu Gln Val A sp Cys Glu Met Tyr Gly 475 480 485 CTT ACT
AAT GAC CAC TAC AGT ATT ACA CTA A GA CGA TAT GCT GGA ATG 1542 Leu
Thr Asn Asp His Tyr Ser Ile Thr Leu A rg Arg Tyr Ala Gly Met 490
495 500 GCT TTG ACA AAC TTG ACT TTT GGA GAT GTA G CC AAC AAG GCT
ACG CTA 1590 Ala Leu Thr Asn Leu Thr Phe Gly Asp Val A la Asn Lys
Ala Thr Leu 505 510 515 TGC TCT ATG AAA GGC TGC ATG AGA GCA CTT G
TG GCC CAA CTA AAA TCT 1638 Cys Ser Met Lys Gly Cys Met Arg Ala Leu
V al Ala Gln Leu Lys Ser 520 525 530 535 GAA AGT GAA GAC TTA CAG
CAG GTT ATT GCA A GT GTT TTG AGG AAT TTG 1686 Glu Ser Glu Asp Leu
Gln Gln Val Ile Ala S er Val Leu Arg Asn Leu 540 545 550 TCT TGG
CGA GCA GAT GTA AAT AGT AAA AAG A CG TTG CGA GAA GTT GGA 1734 Ser
Trp Arg Ala Asp Val Asn Ser Lys Lys T hr Leu Arg Glu Val Gly 555
560 565 AGT GTG AAA GCA TTG ATG GAA TGT GCT TTA G AA GTT AAA AAG
GAA TCA 1782 Ser Val Lys Ala Leu Met Glu Cys Ala Leu G lu Val Lys
Lys Glu Ser 570 575 580 ACC CTC AAA AGC GTA TTG AGT GCC TTA TGG A
AT TTG TCA GCA CAT TGC 1830 Thr Leu Lys Ser Val Leu Ser Ala Leu Trp
A sn Leu Ser Ala His Cys 585 590 595 ACT GAG AAT AAA GCT GAT ATA
TGT GCT GTA G AT GGT GCA CTT GCA TTT 1878 Thr Glu Asn Lys Ala Asp
Ile Cys Ala Val A sp Gly Ala Leu Ala Phe 600 605 610 615 TTG GTT
GGC ACT CTT ACT TAC CGG AGC CAG A CA AAC ACT TTA GCC ATT 1926 Leu
Val Gly Thr Leu Thr Tyr Arg Ser Gln T hr Asn Thr Leu Ala Ile 620
625 630 ATT GAA AGT GGA GGT GGG ATA TTA CGG AAT G TG TCC AGC TTG
ATA GCT 1974 Ile Glu Ser Gly Gly Gly Ile Leu Arg Asn V al Ser Ser
Leu Ile Ala 635 640 645 ACA AAT GAG GAC CAC AGG CAA ATC CTA AGA G
AG AAC AAC TGT CTA CAA 2022 Thr Asn Glu Asp His Arg Gln Ile Leu Arg
G lu Asn Asn Cys Leu Gln 650 655 660 ACT TTA TTA CAA CAC TTA AAA
TCT CAT AGT T TG ACA ATA GTC AGT AAT 2070 Thr Leu Leu Gln His Leu
Lys Ser His Ser L eu Thr Ile Val Ser Asn 665 670 675 GCA TGT GGA
ACT TTG TGG AAT CTC TCA GCA A GA AAT CCT AAA GAC CAG 2118 Ala Cys
Gly Thr Leu Trp Asn Leu Ser Ala A rg Asn Pro Lys Asp Gln 680 685
690 695 GAA GCA TTA TGG GAC ATG GGG GCA GTT AGC A TG CTC AAG AAC
CTC ATT 2166 Glu Ala Leu Trp Asp Met Gly Ala Val Ser M et Leu Lys
Asn Leu Ile 700 705 710 CAT TCA AAG CAC AAA ATG ATT GCT ATG GGA A
GT GCT GCA GCT TTA AGG 2214 His Ser Lys His Lys Met Ile Ala Met Gly
S er Ala Ala Ala Leu Arg 715 720 725 AAT CTC ATG GCA AAT AGG CCT
GCG AAG TAC A AG GAT GCC AAT ATT ATG 2262 Asn Leu Met Ala Asn Arg
Pro Ala Lys Tyr L ys Asp Ala Asn Ile Met 730 735 740 TCT CCT GGC
TCA AGC TTG CCA TCT CTT CAT G TT AGG AAA CAA AAA GCC 2310 Ser Pro
Gly Ser Ser Leu Pro Ser Leu His V al Arg Lys Gln Lys Ala 745 750
755 CTA GAA GCA GAA TTA GAT GCT CAG CAC TTA T CA GAA ACT TTT GAC
AAT 2358 Leu Glu Ala Glu Leu Asp Ala Gln His Leu S er Glu Thr Phe
Asp Asn 760 765 770 775 ATA GAC AAT TTA AGT CCC AAG GCA TCT CAT C
GT AGT AAG CAG AGA CAC 2406 Ile Asp Asn Leu Ser Pro Lys Ala Ser His
A rg Ser Lys Gln Arg His 780 785 790 AAG CAA AGT CTC TAT GGT GAT
TAT GTT TTT G AC ACC AAT CGA CAT GAT 2454 Lys Gln Ser Leu Tyr Gly
Asp Tyr Val Phe A sp Thr Asn Arg His Asp 795 800 805 GAT AAT AGG
TCA GAC AAT TTT AAT ACT GGC A AC ATG ACT GTC CTT TCA 2502 Asp Asn
Arg Ser Asp Asn Phe Asn Thr Gly A sn Met Thr Val Leu Ser 810 815
820 CCA TAT TTG AAT ACT ACA GTG TTA CCC AGC T CC TCT TCA TCA AGA
GGA 2550 Pro Tyr Leu Asn Thr Thr Val Leu Pro Ser S er Ser Ser Ser
Arg Gly 825 830 835 AGC TTA GAT AGT TCT CGT TCT GAA AAA GAT A GA
AGT TTG GAG AGA GAA 2598 Ser Leu Asp Ser Ser Arg Ser Glu Lys Asp A
rg Ser Leu Glu Arg Glu 840 845 850 855 CGC GGA ATT GGT CTA GGC AAC
TAC CAT CCA G CA ACA GAA AAT CCA GGA 2646 Arg Gly Ile Gly Leu Gly
Asn Tyr His Pro A la Thr Glu Asn Pro Gly 860 865 870 ACT TCT TCA
AAG CGA GGT TTG CAG ATC TCC A CC ACT GCA GCC CAG ATT 2694 Thr Ser
Ser Lys Arg Gly Leu Gln Ile Ser T hr Thr Ala Ala Gln Ile 875 880
885 GCC AAA GTC ATG GAA GAA GTG TCA GCC ATT C AT ACC TCT CAG GAA
GAC 2742 Ala Lys Val Met Glu Glu Val Ser Ala Ile H is Thr Ser Gln
Glu Asp 890 895 900 AGA AGT TCT GGG TCT ACC ACT GAA TTA CAT T GT
GTG ACA GAT GAG AGA 2790 Arg Ser Ser Gly Ser Thr Thr Glu Leu His C
ys Val Thr Asp Glu Arg 905 910 915 AAT GCA CTT AGA AGA AGC TCT GCT
GCC CAT A CA CAT TCA AAC ACT TAC 2838 Asn Ala Leu Arg Arg Ser Ser
Ala Ala His T hr His Ser Asn Thr Tyr 920 925 930 935 AAT TTC ACT
AAG TCG GAA AAT TCA AAT AGG A CA TGT TCT ATG CCT TAT 2886 Asn Phe
Thr Lys Ser Glu Asn Ser Asn Arg T hr Cys Ser Met Pro Tyr 940 945
950 GCC AAA TTA GAA TAC AAG AGA TCT TCA AAT G AT AGT TTA AAT AGT
GTC 2934 Ala Lys Leu Glu Tyr Lys Arg Ser Ser Asn A sp Ser Leu Asn
Ser Val 955 960 965 AGT AGT AAT GAT GGT TAT GGT AAA AGA GGT C AA
ATG AAA CCC TCG ATT 2982 Ser Ser Asn Asp Gly Tyr Gly Lys Arg Gly G
ln Met Lys Pro Ser Ile 970 975 980 GAA TCC TAT TCT GAA GAT GAT GAA
AGT AAG T TT TGC AGT TAT GGT CAA 3030 Glu Ser Tyr Ser Glu Asp Asp
Glu Ser Lys P he Cys Ser Tyr Gly Gln 985 990 995 TAC CCA GCC GAC
CTA GCC CAT AAA ATA CAT A GT GCA AAT CAT ATG GAT 3078 Tyr Pro Ala
Asp Leu Ala His Lys Ile His S er Ala Asn His Met Asp 1000 100 5
1010 1015 GAT AAT GAT GGA GAA CTA GAT ACA CCA ATA A AT TAT AGT CTT
AAA TAT 3126 Asp Asn Asp Gly Glu Leu Asp Thr Pro Ile A sn Tyr Ser
Leu Lys Tyr 1020 1025 1030 TCA GAT GAG CAG TTG AAC TCT GGA AGG CAA
A GT CCT TCA CAG AAT GAA 3174 Ser Asp Glu Gln Leu Asn Ser Gly Arg
Gln S er Pro Ser Gln Asn Glu 1035 1040 1045 AGA TGG GCA AGA CCC AAA
CAC ATA ATA GAA G AT GAA ATA AAA CAA AGT 3222 Arg Trp Ala Arg Pro
Lys His Ile Ile Glu A sp Glu Ile Lys Gln Ser 1050 1055 1060 GAG CAA
AGA CAA TCA AGG AAT CAA AGT ACA A CT TAT CCT GTT TAT ACT 3270 Glu
Gln Arg Gln Ser Arg Asn Gln Ser Thr T hr Tyr Pro Val Tyr Thr 1065
1070 1075 GAG AGC ACT GAT GAT AAA CAC CTC AAG TTC C AA CCA CAT TTT
GGA CAG 3318 Glu Ser Thr Asp Asp Lys His Leu Lys Phe G ln Pro His
Phe Gly Gln 1080 108 5 1090 1095 CAG GAA TGT GTT TCT CCA TAC AGG
TCA CGG G GA GCC AAT GGT TCA GAA 3366 Gln Glu Cys Val Ser Pro Tyr
Arg Ser Arg G ly Ala Asn Gly Ser Glu 1100 1105 1110 ACA AAT CGA GTG
GGT TCT AAT CAT GGA ATT A AT CAA AAT GTA AGC CAG 3414 Thr Asn Arg
Val Gly Ser Asn His Gly Ile A sn Gln Asn Val Ser Gln 1115 1120 1125
TCT TTG TGT CAA GAA GAT GAC TAT GAA GAT G AT AAG CCT ACC AAT TAT
3462 Ser Leu Cys Gln Glu Asp Asp Tyr Glu Asp A sp Lys Pro Thr Asn
Tyr 1130 1135 1140 AGT GAA CGT TAC TCT GAA GAA GAA CAG CAT G AA GAA
GAA GAG AGA CCA 3510 Ser Glu Arg Tyr Ser Glu Glu Glu Gln His G lu
Glu Glu Glu Arg Pro 1145 1150 1155 ACA AAT TAT AGC ATA AAA TAT AAT
GAA GAG A AA CGT CAT GTG GAT CAG 3558 Thr Asn Tyr Ser Ile Lys Tyr
Asn Glu Glu L ys Arg His Val Asp Gln 1160 116 5 1170 1175 CCT ATT
GAT TAT AGT TTA AAA TAT GCC ACA G AT ATT CCT TCA TCA CAG 3606 Pro
Ile Asp Tyr Ser Leu Lys Tyr Ala Thr A sp Ile Pro Ser Ser Gln 1180
1185 1190 AAA CAG TCA TTT TCA TTC TCA AAG AGT TCA T CT GGA CAA AGC
AGT AAA 3654 Lys Gln Ser Phe Ser Phe Ser Lys Ser Ser S er Gly Gln
Ser Ser Lys 1195 1200 1205 ACC GAA CAT ATG TCT TCA AGC AGT GAG AAT
A CG TCC ACA CCT TCA TCT 3702 Thr Glu His Met Ser Ser Ser Ser Glu
Asn T hr Ser Thr Pro Ser Ser 1210 1215 1220
AAT GCC AAG AGG CAG AAT CAG CTC CAT CCA A GT TCT GCA CAG AGT AGA
3750 Asn Ala Lys Arg Gln Asn Gln Leu His Pro S er Ser Ala Gln Ser
Arg 1225 1230 1235 AGT GGT CAG CCT CAA AAG GCT GCC ACT TGC A AA GTT
TCT TCT ATT AAC 3798 Ser Gly Gln Pro Gln Lys Ala Ala Thr Cys L ys
Val Ser Ser Ile Asn 1240 124 5 1250 1255 CAA GAA ACA ATA CAG ACT
TAT TGT GTA GAA G AT ACT CCA ATA TGT TTT 3846 Gln Glu Thr Ile Gln
Thr Tyr Cys Val Glu A sp Thr Pro Ile Cys Phe 1260 1265 1270 TCA AGA
TGT AGT TCA TTA TCA TCT TTG TCA T CA GCT GAA GAT GAA ATA 3894 Ser
Arg Cys Ser Ser Leu Ser Ser Leu Ser S er Ala Glu Asp Glu Ile 1275
1280 1285 GGA TGT AAT CAG ACG ACA CAG GAA GCA GAT T CT GCT AAT ACC
CTG CAA 3942 Gly Cys Asn Gln Thr Thr Gln Glu Ala Asp S er Ala Asn
Thr Leu Gln 1290 1295 1300 ATA GCA GAA ATA AAA GGA AAG ATT GGA ACT
A GG TCA GCT GAA GAT CCT 3990 Ile Ala Glu Ile Lys Gly Lys Ile Gly
Thr A rg Ser Ala Glu Asp Pro 1305 1310 1315 GTG AGC GAA GTT CCA GCA
GTG TCA CAG CAC C CT AGA ACC AAA TCC AGC 4038 Val Ser Glu Val Pro
Ala Val Ser Gln His P ro Arg Thr Lys Ser Ser 1320 132 5 1330 1335
AGA CTG CAG GGT TCT AGT TTA TCT TCA GAA T CA GCC AGG CAC AAA GCT
4086 Arg Leu Gln Gly Ser Ser Leu Ser Ser Glu S er Ala Arg His Lys
Ala 1340 1345 1350 GTT GAA TTT CCT TCA GGA GCG AAA TCT CCC T CC AAA
AGT GGT GCT CAG 4134 Val Glu Phe Pro Ser Gly Ala Lys Ser Pro S er
Lys Ser Gly Ala Gln 1355 1360 1365 ACA CCC AAA AGT CCA CCT GAA CAC
TAT GTT C AG GAG ACC CCA CTC ATG 4182 Thr Pro Lys Ser Pro Pro Glu
His Tyr Val G ln Glu Thr Pro Leu Met 1370 1375 1380 TTT AGC AGA TGT
ACT TCT GTC AGT TCA CTT G AT AGT TTT GAG AGT CGT 4230 Phe Ser Arg
Cys Thr Ser Val Ser Ser Leu A sp Ser Phe Glu Ser Arg 1385 1390 1395
TCG ATT GCC AGC TCC GTT CAG AGT GAA CCA T GC AGT GGA ATG GTA AGT
4278 Ser Ile Ala Ser Ser Val Gln Ser Glu Pro C ys Ser Gly Met Val
Ser 1400 140 5 1410 1415 GGC ATT ATA AGC CCC AGT GAT CTT CCA GAT A
GC CCT GGA CAA ACC ATG 4326 Gly Ile Ile Ser Pro Ser Asp Leu Pro Asp
S er Pro Gly Gln Thr Met 1420 1425 1430 CCA CCA AGC AGA AGT AAA ACA
CCT CCA CCA C CT CCT CAA ACA GCT CAA 4374 Pro Pro Ser Arg Ser Lys
Thr Pro Pro Pro P ro Pro Gln Thr Ala Gln 1435 1440 1445 ACC AAG CGA
GAA GTA CCT AAA AAT AAA GCA C CT ACT GCT GAA AAG AGA 4422 Thr Lys
Arg Glu Val Pro Lys Asn Lys Ala P ro Thr Ala Glu Lys Arg 1450 1455
1460 GAG AGT GGA CCT AAG CAA GCT GCA GTA AAT G CT GCA GTT CAG AGG
GTC 4470 Glu Ser Gly Pro Lys Gln Ala Ala Val Asn A la Ala Val Gln
Arg Val 1465 1470 1475 CAG GTT CTT CCA GAT GCT GAT ACT TTA TTA C AT
TTT GCC ACA GAA AGT 4518 Gln Val Leu Pro Asp Ala Asp Thr Leu Leu H
is Phe Ala Thr Glu Ser 1480 148 5 1490 1495 ACT CCA GAT GGA TTT TCT
TGT TCA TCC AGC C TG AGT GCT CTG AGC CTC 4566 Thr Pro Asp Gly Phe
Ser Cys Ser Ser Ser L eu Ser Ala Leu Ser Leu 1500 1505 1510 GAT GAG
CCA TTT ATA CAG AAA GAT GTG GAA T TA AGA ATA ATG CCT CCA 4614 Asp
Glu Pro Phe Ile Gln Lys Asp Val Glu L eu Arg Ile Met Pro Pro 1515
1520 1525 GTT CAG GAA AAT GAC AAT GGG AAT GAA ACA G AA TCA GAG CAG
CCT AAA 4662 Val Gln Glu Asn Asp Asn Gly Asn Glu Thr G lu Ser Glu
Gln Pro Lys 1530 1535 1540 GAA TCA AAT GAA AAC CAA GAG AAA GAG GCA
G AA AAA ACT ATT GAT TCT 4710 Glu Ser Asn Glu Asn Gln Glu Lys Glu
Ala G lu Lys Thr Ile Asp Ser 1545 1550 1555 GAA AAG GAC CTA TTA GAT
GAT TCA GAT GAT G AT GAT ATT GAA ATA CTA 4758 Glu Lys Asp Leu Leu
Asp Asp Ser Asp Asp A sp Asp Ile Glu Ile Leu 1560 156 5 1570 1575
GAA GAA TGT ATT ATT TCT GCC ATG CCA ACA A AG TCA TCA CGT AAA GGC
4806 Glu Glu Cys Ile Ile Ser Ala Met Pro Thr L ys Ser Ser Arg Lys
Gly 1580 1585 1590 AAA AAG CCA GCC CAG ACT GCT TCA AAA TTA C CT CCA
CCT GTG GCA AGG 4854 Lys Lys Pro Ala Gln Thr Ala Ser Lys Leu P ro
Pro Pro Val Ala Arg 1595 1600 1605 AAA CCA AGT CAG CTG CCT GTG TAC
AAA CTT C TA CCA TCA CAA AAC AGG 4902 Lys Pro Ser Gln Leu Pro Val
Tyr Lys Leu L eu Pro Ser Gln Asn Arg 1610 1615 1620 TTG CAA CCC CAA
AAG CAT GTT AGT TTT ACA C CG GGG GAT GAT ATG CCA 4950 Leu Gln Pro
Gln Lys His Val Ser Phe Thr P ro Gly Asp Asp Met Pro 1625 1630 1635
CGG GTG TAT TGT GTT GAA GGG ACA CCT ATA A AC TTT TCC ACA GCT ACA
4998 Arg Val Tyr Cys Val Glu Gly Thr Pro Ile A sn Phe Ser Thr Ala
Thr 1640 164 5 1650 1655 TCT CTA AGT GAT CTA ACA ATC GAA TCC CCT C
CA AAT GAG TTA GCT GCT 5046 Ser Leu Ser Asp Leu Thr Ile Glu Ser Pro
P ro Asn Glu Leu Ala Ala 1660 1665 1670 GGA GAA GGA GTT AGA GGA GGA
GCA CAG TCA G GT GAA TTT GAA AAA CGA 5094 Gly Glu Gly Val Arg Gly
Gly Ala Gln Ser G ly Glu Phe Glu Lys Arg 1675 1680 1685 GAT ACC ATT
CCT ACA GAA GGC AGA AGT ACA G AT GAG GCT CAA GGA GGA 5142 Asp Thr
Ile Pro Thr Glu Gly Arg Ser Thr A sp Glu Ala Gln Gly Gly 1690 1695
1700 AAA ACC TCA TCT GTA ACC ATA CCT GAA TTG G AT GAC AAT AAA GCA
GAG 5190 Lys Thr Ser Ser Val Thr Ile Pro Glu Leu A sp Asp Asn Lys
Ala Glu 1705 1710 1715 GAA GGT GAT ATT CTT GCA GAA TGC ATT AAT T CT
GCT ATG CCC AAA GGG 5238 Glu Gly Asp Ile Leu Ala Glu Cys Ile Asn S
er Ala Met Pro Lys Gly 1720 172 5 1730 1735 AAA AGT CAC AAG CCT TTC
CGT GTG AAA AAG A TA ATG GAC CAG GTC CAG 5286 Lys Ser His Lys Pro
Phe Arg Val Lys Lys I le Met Asp Gln Val Gln 1740 1745 1750 CAA GCA
TCT GCG TCG TCT TCT GCA CCC AAC A AA AAT CAG TTA GAT GGT 5334 Gln
Ala Ser Ala Ser Ser Ser Ala Pro Asn L ys Asn Gln Leu Asp Gly 1755
1760 1765 AAG AAA AAG AAA CCA ACT TCA CCA GTA AAA C CT ATA CCA CAA
AAT ACT 5382 Lys Lys Lys Lys Pro Thr Ser Pro Val Lys P ro Ile Pro
Gln Asn Thr 1770 1775 1780 GAA TAT AGG ACA CGT GTA AGA AAA AAT GCA
G AC TCA AAA AAT AAT TTA 5430 Glu Tyr Arg Thr Arg Val Arg Lys Asn
Ala A sp Ser Lys Asn Asn Leu 1785 1790 1795 AAT GCT GAG AGA GTT TTC
TCA GAC AAC AAA G AT TCA AAG AAA CAG AAT 5478 Asn Ala Glu Arg Val
Phe Ser Asp Asn Lys A sp Ser Lys Lys Gln Asn 1800 180 5 1810 1815
TTG AAA AAT AAT TCC AAG GAC TTC AAT GAT A AG CTC CCA AAT AAT GAA
5526 Leu Lys Asn Asn Ser Lys Asp Phe Asn Asp L ys Leu Pro Asn Asn
Glu 1820 1825 1830 GAT AGA GTC AGA GGA AGT TTT GCT TTT GAT T CA CCT
CAT CAT TAC ACG 5574 Asp Arg Val Arg Gly Ser Phe Ala Phe Asp S er
Pro His His Tyr Thr 1835 1840 1845 CCT ATT GAA GGA ACT CCT TAC TGT
TTT TCA C GA AAT GAT TCT TTG AGT 5622 Pro Ile Glu Gly Thr Pro Tyr
Cys Phe Ser A rg Asn Asp Ser Leu Ser 1850 1855 1860 TCT CTA GAT TTT
GAT GAT GAT GAT GTT GAC C TT TCC AGG GAA AAG GCT 5670 Ser Leu Asp
Phe Asp Asp Asp Asp Val Asp L eu Ser Arg Glu Lys Ala 1865 1870 1875
GAA TTA AGA AAG GCA AAA GAA AAT AAG GAA T CA GAG GCT AAA GTT ACC
5718 Glu Leu Arg Lys Ala Lys Glu Asn Lys Glu S er Glu Ala Lys Val
Thr 1880 188 5 1890 1895 AGC CAC ACA GAA CTA ACC TCC AAC CAA CAA T
CA GCT AAT AAG ACA CAA 5766 Ser His Thr Glu Leu Thr Ser Asn Gln Gln
S er Ala Asn Lys Thr Gln 1900 1905 1910 GCT ATT GCA AAG CAG CCA ATA
AAT CGA GGT C AG CCT AAA CCC ATA CTT 5814 Ala Ile Ala Lys Gln Pro
Ile Asn Arg Gly G ln Pro Lys Pro Ile Leu 1915 1920 1925 CAG AAA CAA
TCC ACT TTT CCC CAG TCA TCC A AA GAC ATA CCA GAC AGA 5862 Gln Lys
Gln Ser Thr Phe Pro Gln Ser Ser L ys Asp Ile Pro Asp Arg 1930 1935
1940 GGG GCA GCA ACT GAT GAA AAG TTA CAG AAT T TT GCT ATT GAA AAT
ACT 5910 Gly Ala Ala Thr Asp Glu Lys Leu Gln Asn P he Ala Ile Glu
Asn Thr 1945 1950 1955 CCA GTT TGC TTT TCT CAT AAT TCC TCT CTG A GT
TCT CTC AGT GAC ATT 5958 Pro Val Cys Phe Ser His Asn Ser Ser Leu S
er Ser Leu Ser Asp Ile 1960 196 5 1970 1975 GAC CAA GAA AAC AAC AAT
AAA GAA AAT GAA C CT ATC AAA GAG ACT GAG 6006 Asp Gln Glu Asn Asn
Asn Lys Glu Asn Glu P ro Ile Lys Glu Thr Glu 1980 1985 1990 CCC CCT
GAC TCA CAG GGA GAA CCA AGT AAA C CT CAA GCA TCA GGC TAT 6054 Pro
Pro Asp Ser Gln Gly Glu Pro Ser Lys P ro Gln Ala Ser Gly Tyr 1995
2000 2005 GCT CCT AAA TCA TTT CAT GTT GAA GAT ACC C CA GTT TGT TTC
TCA AGA 6102 Ala Pro Lys Ser Phe His Val Glu Asp Thr P ro Val Cys
Phe Ser Arg 2010 2015 2020 AAC AGT TCT CTC AGT TCT CTT AGT ATT GAC
T CT GAA GAT GAC CTG TTG 6150 Asn Ser Ser Leu Ser Ser Leu Ser Ile
Asp S er Glu Asp Asp Leu Leu 2025 2030 2035 CAG GAA TGT ATA AGC TCC
GCA ATG CCA AAA A AG AAA AAG CCT TCA AGA 6198 Gln Glu Cys Ile Ser
Ser Ala Met Pro Lys L ys Lys Lys Pro Ser Arg 2040 204 5 2050 2055
CTC AAG GGT GAT AAT GAA AAA CAT AGT CCC A GA AAT ATG GGT GGC ATA
6246 Leu Lys Gly Asp Asn Glu Lys His Ser Pro A rg Asn Met Gly Gly
Ile 2060 2065 2070 TTA GGT GAA GAT CTG ACA CTT GAT TTG AAA G AT ATA
CAG AGA CCA GAT 6294 Leu Gly Glu Asp Leu Thr Leu Asp Leu Lys A sp
Ile Gln Arg Pro Asp 2075 2080 2085 TCA GAA CAT GGT CTA TCC CCT GAT
TCA GAA A AT TTT GAT TGG AAA GCT 6342 Ser Glu His Gly Leu Ser Pro
Asp Ser Glu A sn Phe Asp Trp Lys Ala 2090 2095 2100 ATT CAG GAA GGT
GCA AAT TCC ATA GTA AGT A GT TTA CAT CAA GCT GCT 6390 Ile Gln Glu
Gly Ala Asn Ser Ile Val Ser S er Leu His Gln Ala Ala 2105 2110 2115
GCT GCT GCA TGT TTA TCT AGA CAA GCT TCG T CT GAT TCA GAT TCC ATC
6438 Ala Ala Ala Cys Leu Ser Arg Gln Ala Ser S er Asp Ser Asp Ser
Ile 2120 212 5 2130 2135 CTT TCC CTG AAA TCA GGA ATC TCT CTG GGA T
CA CCA TTT CAT CTT ACA 6486 Leu Ser Leu Lys Ser Gly Ile Ser Leu Gly
S er Pro Phe His Leu Thr 2140 2145 2150 CCT GAT CAA GAA GAA AAA CCC
TTT ACA AGT A AT AAA GGC CCA CGA ATT 6534 Pro Asp Gln Glu Glu Lys
Pro Phe Thr Ser A sn Lys Gly Pro Arg Ile 2155 2160 2165 CTA AAA CCA
GGG GAG AAA AGT ACA TTG GAA A CT AAA AAG ATA GAA TCT 6582 Leu Lys
Pro Gly Glu Lys Ser Thr Leu Glu T hr Lys Lys Ile Glu Ser 2170 2175
2180 GAA AGT AAA GGA ATC AAA GGA GGA AAA AAA G TT TAT AAA AGT TTG
ATT 6630 Glu Ser Lys Gly Ile Lys Gly Gly Lys Lys V al Tyr Lys Ser
Leu Ile 2185 2190 2195 ACT GGA AAA GTT CGA TCT AAT TCA GAA ATT T CA
GGC CAA ATG AAA CAG 6678 Thr Gly Lys Val Arg Ser Asn Ser Glu Ile S
er Gly Gln Met Lys Gln 2200 220 5 2210 2215 CCC CTT CAA GCA AAC ATG
CCT TCA ATC TCT C GA GGC AGG ACA ATG ATT 6726 Pro Leu Gln Ala Asn
Met Pro Ser Ile Ser A rg Gly Arg Thr Met Ile 2220 2225 2230 CAT ATT
CCA GGA GTT CGA AAT AGC TCC TCA A GT ACA AGT CCT GTT TCT 6774 His
Ile Pro Gly Val Arg Asn Ser Ser Ser S er Thr Ser Pro Val Ser 2235
2240 2245 AAA AAA GGC CCA CCC CTT AAG ACT CCA GCC T CC AAA AGC CCT
AGT GAA 6822 Lys Lys Gly Pro Pro Leu Lys Thr Pro Ala S er Lys Ser
Pro Ser Glu 2250 2255 2260 GGT CAA ACA GCC ACC ACT TCT CCT AGA GGA
G CC AAG CCA TCT GTG AAA 6870 Gly Gln Thr Ala Thr Thr Ser Pro Arg
Gly A la Lys Pro Ser Val Lys 2265 2270 2275 TCA GAA TTA AGC CCT GTT
GCC AGG CAG ACA T CC CAA ATA GGT GGG TCA 6918 Ser Glu Leu Ser Pro
Val Ala Arg Gln Thr S er Gln Ile Gly Gly Ser 2280 228 5 2290 2295
AGT AAA GCA CCT TCT AGA TCA GGA TCT AGA G AT TCG ACC CCT TCA AGA
6966 Ser Lys Ala Pro Ser Arg Ser Gly Ser Arg A sp Ser Thr Pro Ser
Arg 2300 2305 2310 CCT GCC CAG CAA CCA TTA AGT AGA CCT ATA C AG TCT
CCT GGC CGA AAC 7014 Pro Ala Gln Gln Pro Leu Ser Arg Pro Ile G ln
Ser Pro Gly Arg Asn 2315 2320 2325 TCA ATT TCC CCT GGT AGA AAT GGA
ATA AGT C CT CCT AAC AAA TTA TCT 7062 Ser Ile Ser Pro Gly Arg Asn
Gly Ile Ser P ro Pro Asn Lys Leu Ser 2330 2335 2340 CAA CTT CCA AGG
ACA TCA TCC CCT AGT ACT G CT TCA ACT AAG TCC TCA 7110 Gln Leu Pro
Arg Thr Ser Ser Pro Ser Thr A la Ser Thr Lys Ser Ser 2345 2350 2355
GGT TCT GGA AAA ATG TCA TAT ACA TCT CCA G GT AGA CAG ATG AGC CAA
7158 Gly Ser Gly Lys Met Ser Tyr Thr Ser Pro G ly Arg Gln Met Ser
Gln 2360 236 5 2370 2375 CAG AAC CTT ACC AAA CAA ACA GGT TTA TCC A
AG AAT GCC AGT AGT ATT 7206 Gln Asn Leu Thr Lys Gln Thr Gly Leu Ser
L ys Asn Ala Ser Ser Ile 2380 2385 2390 CCA AGA AGT GAG TCT GCC TCC
AAA GGA CTA A AT CAG ATG AAT AAT GGT 7254 Pro Arg Ser Glu Ser Ala
Ser Lys Gly Leu A sn Gln Met Asn Asn Gly 2395 2400 2405 AAT GGA GCC
AAT AAA AAG GTA GAA CTT TCT A GA ATG TCT TCA ACT AAA 7302 Asn Gly
Ala Asn Lys Lys Val Glu Leu Ser A rg Met Ser Ser Thr Lys 2410 2415
2420 TCA AGT GGA AGT GAA TCT GAT AGA TCA GAA A GA CCT GTA TTA GTA
CGC 7350 Ser Ser Gly Ser Glu Ser Asp Arg Ser Glu A rg Pro Val Leu
Val Arg 2425 2430 2435 CAG TCA ACT TTC ATC AAA GAA GCT CCA AGC C CA
ACC TTA AGA AGA AAA 7398 Gln Ser Thr Phe Ile Lys Glu Ala Pro Ser P
ro Thr Leu Arg Arg Lys 2440 244 5 2450 2455 TTG GAG GAA TCT GCT TCA
TTT GAA TCT CTT T CT CCA TCA TCT AGA CCA 7446 Leu Glu Glu Ser Ala
Ser Phe Glu Ser Leu S er Pro Ser Ser Arg Pro 2460 2465 2470 GCT TCT
CCC ACT AGG TCC CAG GCA CAA ACT C CA GTT TTA AGT CCT TCC 7494 Ala
Ser Pro Thr Arg Ser Gln Ala Gln Thr P ro Val Leu Ser Pro Ser 2475
2480 2485 CTT CCT GAT ATG TCT CTA TCC ACA CAT TCG T CT GTT CAG GCT
GGT GGA 7542 Leu Pro Asp Met Ser Leu Ser Thr His Ser S er Val Gln
Ala Gly Gly 2490 2495 2500 TGG CGA AAA CTC CCA CCT AAT CTC AGT CCC
A CT ATA GAG TAT AAT GAT 7590 Trp Arg Lys Leu Pro Pro Asn Leu Ser
Pro T hr Ile Glu Tyr Asn Asp 2505 2510 2515 GGA AGA CCA GCA AAG CGC
CAT GAT ATT GCA C GG TCT CAT TCT GAA AGT 7638 Gly Arg Pro Ala Lys
Arg His Asp Ile Ala A rg Ser His Ser Glu Ser 2520 252 5 2530 2535
CCT TCT AGA CTT CCA ATC AAT AGG TCA GGA A CC TGG AAA CGT GAG CAC
7686 Pro Ser Arg Leu Pro Ile Asn Arg Ser Gly T hr Trp Lys Arg Glu
His 2540 2545 2550 AGC AAA CAT TCA TCA TCC CTT CCT CGA GTA A GC ACT
TGG AGA AGA ACT 7734 Ser Lys His Ser Ser Ser Leu Pro Arg Val S er
Thr Trp Arg Arg Thr
2555 2560 2565 GGA AGT TCA TCT TCA ATT CTT TCT GCT TCA T CA GAA TCC
AGT GAA AAA 7782 Gly Ser Ser Ser Ser Ile Leu Ser Ala Ser S er Glu
Ser Ser Glu Lys 2570 2575 2580 GCA AAA AGT GAG GAT GAA AAA CAT GTG
AAC T CT ATT TCA GGA ACC AAA 7830 Ala Lys Ser Glu Asp Glu Lys His
Val Asn S er Ile Ser Gly Thr Lys 2585 2590 2595 CAA AGT AAA GAA AAC
CAA GTA TCC GCA AAA G GA ACA TGG AGA AAA ATA 7878 Gln Ser Lys Glu
Asn Gln Val Ser Ala Lys G ly Thr Trp Arg Lys Ile 2600 260 5 2610
2615 AAA GAA AAT GAA TTT TCT CCC ACA AAT AGT A CT TCT CAG ACC GTT
TCC 7926 Lys Glu Asn Glu Phe Ser Pro Thr Asn Ser T hr Ser Gln Thr
Val Ser 2620 2625 2630 TCA GGT GCT ACA AAT GGT GCT GAA TCA AAG A CT
CTA ATT TAT CAA ATG 7974 Ser Gly Ala Thr Asn Gly Ala Glu Ser Lys T
hr Leu Ile Tyr Gln Met 2635 2640 2645 GCA CCT GCT GTT TCT AAA ACA
GAG GAT GTT T GG GTG AGA ATT GAG GAC 8022 Ala Pro Ala Val Ser Lys
Thr Glu Asp Val T rp Val Arg Ile Glu Asp 2650 2655 2660 TGT CCC ATT
AAC AAT CCT AGA TCT GGA AGA T CT CCC ACA GGT AAT ACT 8070 Cys Pro
Ile Asn Asn Pro Arg Ser Gly Arg S er Pro Thr Gly Asn Thr 2665 2670
2675 CCC CCG GTG ATT GAC AGT GTT TCA GAA AAG G CA AAT CCA AAC ATT
AAA 8118 Pro Pro Val Ile Asp Ser Val Ser Glu Lys A la Asn Pro Asn
Ile Lys 2680 268 5 2690 2695 GAT TCA AAA GAT AAT CAG GCA AAA CAA
AAT G TG GGT AAT GGC AGT GTT 8166 Asp Ser Lys Asp Asn Gln Ala Lys
Gln Asn V al Gly Asn Gly Ser Val 2700 2705 2710 CCC ATG CGT ACC GTG
GGT TTG GAA AAT CGC C TG ACC TCC TTT ATT CAG 8214 Pro Met Arg Thr
Val Gly Leu Glu Asn Arg L eu Thr Ser Phe Ile Gln 2715 2720 2725 GTG
GAT GCC CCT GAC CAA AAA GGA ACT GAG A TA AAA CCA GGA CAA AAT 8262
Val Asp Ala Pro Asp Gln Lys Gly Thr Glu I le Lys Pro Gly Gln Asn
2730 2735 2740 AAT CCT GTC CCT GTA TCA GAG ACT AAT GAA A GT CCT ATA
GTG GAA CGT 8310 Asn Pro Val Pro Val Ser Glu Thr Asn Glu S er Pro
Ile Val Glu Arg 2745 2750 2755 ACC CCA TTC AGT TCT AGC AGC TCA AGC
AAA C AC AGT TCA CCT AGT GGG 8358 Thr Pro Phe Ser Ser Ser Ser Ser
Ser Lys H is Ser Ser Pro Ser Gly 2760 276 5 2770 2775 ACT GTT GCT
GCC AGA GTG ACT CCT TTT AAT T AC AAC CCA AGC CCT AGG 8406 Thr Val
Ala Ala Arg Val Thr Pro Phe Asn T yr Asn Pro Ser Pro Arg 2780 2785
2790 AAA AGC AGC GCA GAT AGC ACT TCA GCT CGG C CA TCT CAG ATC CCA
ACT 8454 Lys Ser Ser Ala Asp Ser Thr Ser Ala Arg P ro Ser Gln Ile
Pro Thr 2795 2800 2805 CCA GTG AAT AAC AAC ACA AAG AAG CGA GAT T CC
AAA ACT GAC AGC ACA 8502 Pro Val Asn Asn Asn Thr Lys Lys Arg Asp S
er Lys Thr Asp Ser Thr 2810 2815 2820 GAA TCC AGT GGA ACC CAA AGT
CCT AAG CGC C AT TCT GGG TCT TAC CTT 8550 Glu Ser Ser Gly Thr Gln
Ser Pro Lys Arg H is Ser Gly Ser Tyr Leu 2825 2830 2835 GTG ACA TCT
GTT TAAAAGAGAG GAAGAATGAA ACTAAGAAAA T TCTATGTTA 8602 Val Thr Ser
Val 2840 ATTACAACTG CTATATAGAC ATTTTGTTTC AAATGAAACT TTAAAAGACT G
AAAAATTTT 8662 GTAAATAGGT TTGATTCTTG TTAGAGGGTT TTTGTTCTGG
AAGCCATATT T GATAGTATA 8722 CTTTGTCTTC ACTGGTCTTA TTTTGGGAGG
CACTCTTGAT GGTTAGGAAA A AATAGAAAG 8782 CCAAGTATGT TTGTACAGTA
TGTTTTACAT GTATTTAAAG TAGCATCCCA T CCCAACTTC 8842 CTTAATTATT
GCTTGTCTAA AATAATGAAC ACTACAGATA GGAAATATGA T ATATTGCTG 8902
TTATCAATCA TTTCTAGATT ATAAACTGAC TAAACTTACA TCAGGGGAAA A TTGGTATTT
8962 ATGCAAAAAA AAAATGTTTT TGTCCTTGTG AGTCCATCTA ACATCATAAT T
AATCATGTG 9022 GCTGTGAAAT TCACAGTAAT ATGGTTCCCG ATGAACAAGT
TTACCCAGCC T GCTTTGCTT 9082 ACTGCATGAA TGAAACTGAT GGTTCAATTT
CAGAAGTAAT GATTAACAGT T ATGTGGTCA 9142 CATGATGTGC ATAGAGATAG
CTACAGTGTA ATAATTTACA CTATTTTGTG C TCCAAACAA 9202 AACAAAAATC
TGTGTAACTG TAAAACATTG AATGAAACTA TTTTACCTGA A CTAGATTTT 9262
ATCTGAAAGT AGGTAGAATT TTTGCTATGC TGTAATTTGT TGTATATTCT G GTATTTGAG
9322 GTGAGATGGC TGCTCTTTAT TAATGAGACA TGAATTGTGT CTCAACAGAA A
CTAAATGAA 9382 CATTTCAGAA TAAATTATTG CTGTATGTAA ACTGTTACTG
AAATTGGTAT T TGTTTGAAG 9442 GGTTTGTTTC ACATTTGTAT TAATTAATTG
TTTAAAATGC CTCTTTTAAA A GCTTATATA 9502 AATTTTTTCT TCAGCTTCTA
TGCATTAAGA GTAAAATTCC TCTTACTGTA A TAAAAACAT 9562 TGAAGAAGAC
TGTTGCCACT TAACCATTCC ATGCGTTGGC ACTT 960 6 (2) INFORMATION FOR SEQ
ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2843 amino
acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Ala Ala Ala
Ser Tyr Asp Gln Leu Leu L ys Gln Val Glu Ala Leu 1 5 10 15 Lys Met
Glu Asn Ser Asn Leu Arg Gln Glu L eu Glu Asp Asn Ser Asn 20 25 30
His Leu Thr Lys Leu Glu Thr Glu Ala Ser A sn Met Lys Glu Val Leu 35
40 45 Lys Gln Leu Gln Gly Ser Ile Glu Asp Glu A la Met Ala Ser Ser
Gly 50 55 60 Gln Ile Asp Leu Leu Glu Arg Leu Lys Glu L eu Asn Leu
Asp Ser Ser 65 70 75 80 Asn Phe Pro Gly Val Lys Leu Arg Ser Lys M
et Ser Leu Arg Ser Tyr 85 90 95 Gly Ser Arg Glu Gly Ser Val Ser Ser
Arg S er Gly Glu Cys Ser Pro 100 105 110 Val Pro Met Gly Ser Phe
Pro Arg Arg Gly P he Val Asn Gly Ser Arg 115 120 125 Glu Ser Thr
Gly Tyr Leu Glu Glu Leu Glu L ys Glu Arg Ser Leu Leu 130 135 140
Leu Ala Asp Leu Asp Lys Glu Glu Lys Glu L ys Asp Trp Tyr Tyr Ala
145 150 155 160 Gln Leu Gln Asn Leu Thr Lys Arg Ile Asp S er Leu
Pro Leu Thr Glu 165 170 175 Asn Phe Ser Leu Gln Thr Asp Leu Thr Arg
A rg Gln Leu Glu Tyr Glu 180 185 190 Ala Arg Gln Ile Arg Val Ala
Met Glu Glu G ln Leu Gly Thr Cys Gln 195 200 205 Asp Met Glu Lys
Arg Ala Gln Arg Arg Ile A la Arg Ile Gln Gln Ile 210 215 220 Glu
Lys Asp Ile Leu Arg Ile Arg Gln Leu L eu Gln Ser Gln Ala Thr 225
230 235 240 Glu Ala Glu Arg Ser Ser Gln Asn Lys His G lu Thr Gly
Ser His Asp 245 250 255 Ala Glu Arg Gln Asn Glu Gly Gln Gly Val G
ly Glu Ile Asn Met Ala 260 265 270 Thr Ser Gly Asn Gly Gln Gly Ser
Thr Thr A rg Met Asp His Glu Thr 275 280 285 Ala Ser Val Leu Ser
Ser Ser Ser Thr His S er Ala Pro Arg Arg Leu 290 295 300 Thr Ser
His Leu Gly Thr Lys Val Glu Met V al Tyr Ser Leu Leu Ser 305 310
315 320 Met Leu Gly Thr His Asp Lys Asp Asp Met S er Arg Thr Leu
Leu Ala 325 330 335 Met Ser Ser Ser Gln Asp Ser Cys Ile Ser M et
Arg Gln Ser Gly Cys 340 345 350 Leu Pro Leu Leu Ile Gln Leu Leu His
Gly A sn Asp Lys Asp Ser Val 355 360 365 Leu Leu Gly Asn Ser Arg
Gly Ser Lys Glu A la Arg Ala Arg Ala Ser 370 375 380 Ala Ala Leu
His Asn Ile Ile His Ser Gln P ro Asp Asp Lys Arg Gly 385 390 395
400 Arg Arg Glu Ile Arg Val Leu His Leu Leu G lu Gln Ile Arg Ala
Tyr 405 410 415 Cys Glu Thr Cys Trp Glu Trp Gln Glu Ala H is Glu
Pro Gly Met Asp 420 425 430 Gln Asp Lys Asn Pro Met Pro Ala Pro Val
G lu His Gln Ile Cys Pro 435 440 445 Ala Val Cys Val Leu Met Lys
Leu Ser Phe A sp Glu Glu His Arg His 450 455 460 Ala Met Asn Glu
Leu Gly Gly Leu Gln Ala I le Ala Glu Leu Leu Gln 465 470 475 480
Val Asp Cys Glu Met Tyr Gly Leu Thr Asn A sp His Tyr Ser Ile Thr
485 490 495 Leu Arg Arg Tyr Ala Gly Met Ala Leu Thr A sn Leu Thr
Phe Gly Asp 500 505 510 Val Ala Asn Lys Ala Thr Leu Cys Ser Met L
ys Gly Cys Met Arg Ala 515 520 525 Leu Val Ala Gln Leu Lys Ser Glu
Ser Glu A sp Leu Gln Gln Val Ile 530 535 540 Ala Ser Val Leu Arg
Asn Leu Ser Trp Arg A la Asp Val Asn Ser Lys 545 550 555 560 Lys
Thr Leu Arg Glu Val Gly Ser Val Lys A la Leu Met Glu Cys Ala 565
570 575 Leu Glu Val Lys Lys Glu Ser Thr Leu Lys S er Val Leu Ser
Ala Leu 580 585 590 Trp Asn Leu Ser Ala His Cys Thr Glu Asn L ys
Ala Asp Ile Cys Ala 595 600 605 Val Asp Gly Ala Leu Ala Phe Leu Val
Gly T hr Leu Thr Tyr Arg Ser 610 615 620 Gln Thr Asn Thr Leu Ala
Ile Ile Glu Ser G ly Gly Gly Ile Leu Arg 625 630 635 640 Asn Val
Ser Ser Leu Ile Ala Thr Asn Glu A sp His Arg Gln Ile Leu 645 650
655 Arg Glu Asn Asn Cys Leu Gln Thr Leu Leu G ln His Leu Lys Ser
His 660 665 670 Ser Leu Thr Ile Val Ser Asn Ala Cys Gly T hr Leu
Trp Asn Leu Ser 675 680 685 Ala Arg Asn Pro Lys Asp Gln Glu Ala Leu
T rp Asp Met Gly Ala Val 690 695 700 Ser Met Leu Lys Asn Leu Ile
His Ser Lys H is Lys Met Ile Ala Met 705 710 715 720 Gly Ser Ala
Ala Ala Leu Arg Asn Leu Met A la Asn Arg Pro Ala Lys 725 730 735
Tyr Lys Asp Ala Asn Ile Met Ser Pro Gly S er Ser Leu Pro Ser Leu
740 745 750 His Val Arg Lys Gln Lys Ala Leu Glu Ala G lu Leu Asp
Ala Gln His 755 760 765 Leu Ser Glu Thr Phe Asp Asn Ile Asp Asn L
eu Ser Pro Lys Ala Ser 770 775 780 His Arg Ser Lys Gln Arg His Lys
Gln Ser L eu Tyr Gly Asp Tyr Val 785 790 795 800 Phe Asp Thr Asn
Arg His Asp Asp Asn Arg S er Asp Asn Phe Asn Thr 805 810 815 Gly
Asn Met Thr Val Leu Ser Pro Tyr Leu A sn Thr Thr Val Leu Pro 820
825 830 Ser Ser Ser Ser Ser Arg Gly Ser Leu Asp S er Ser Arg Ser
Glu Lys 835 840 845 Asp Arg Ser Leu Glu Arg Glu Arg Gly Ile G ly
Leu Gly Asn Tyr His 850 855 860 Pro Ala Thr Glu Asn Pro Gly Thr Ser
Ser L ys Arg Gly Leu Gln Ile 865 870 875 880 Ser Thr Thr Ala Ala
Gln Ile Ala Lys Val M et Glu Glu Val Ser Ala 885 890 895 Ile His
Thr Ser Gln Glu Asp Arg Ser Ser G ly Ser Thr Thr Glu Leu 900 905
910 His Cys Val Thr Asp Glu Arg Asn Ala Leu A rg Arg Ser Ser Ala
Ala 915 920 925 His Thr His Ser Asn Thr Tyr Asn Phe Thr L ys Ser
Glu Asn Ser Asn 930 935 940 Arg Thr Cys Ser Met Pro Tyr Ala Lys Leu
G lu Tyr Lys Arg Ser Ser 945 950 955 960 Asn Asp Ser Leu Asn Ser
Val Ser Ser Asn A sp Gly Tyr Gly Lys Arg 965 970 975 Gly Gln Met
Lys Pro Ser Ile Glu Ser Tyr S er Glu Asp Asp Glu Ser 980 985 990
Lys Phe Cys Ser Tyr Gly Gln Tyr Pro Ala A sp Leu Ala His Lys Ile
995 1000 1005 His Ser Ala Asn His Met Asp Asp Asn Asp G ly Glu Leu
Asp Thr Pro 1010 1015 1020 Ile Asn Tyr Ser Leu Lys Tyr Ser Asp Glu
G ln Leu Asn Ser Gly Arg 1025 103 0 1035 1040 Gln Ser Pro Ser Gln
Asn Glu Arg Trp Ala A rg Pro Lys His Ile Ile 1045 1050 1055 Glu Asp
Glu Ile Lys Gln Ser Glu Gln Arg G ln Ser Arg Asn Gln Ser 1060 1065
1070 Thr Thr Tyr Pro Val Tyr Thr Glu Ser Thr A sp Asp Lys His Leu
Lys 1075 1080 1085 Phe Gln Pro His Phe Gly Gln Gln Glu Cys V al Ser
Pro Tyr Arg Ser 1090 1095 1100 Arg Gly Ala Asn Gly Ser Glu Thr Asn
Arg V al Gly Ser Asn His Gly 1105 111 0 1115 1120 Ile Asn Gln Asn
Val Ser Gln Ser Leu Cys G ln Glu Asp Asp Tyr Glu 1125 1130 1135 Asp
Asp Lys Pro Thr Asn Tyr Ser Glu Arg T yr Ser Glu Glu Glu Gln 1140
1145 1150 His Glu Glu Glu Glu Arg Pro Thr Asn Tyr S er Ile Lys Tyr
Asn Glu 1155 1160 1165 Glu Lys Arg His Val Asp Gln Pro Ile Asp T yr
Ser Leu Lys Tyr Ala 1170 1175 1180 Thr Asp Ile Pro Ser Ser Gln Lys
Gln Ser P he Ser Phe Ser Lys Ser 1185 119 0 1195 1200 Ser Ser Gly
Gln Ser Ser Lys Thr Glu His M et Ser Ser Ser Ser Glu 1205 1210 1215
Asn Thr Ser Thr Pro Ser Ser Asn Ala Lys A rg Gln Asn Gln Leu His
1220 1225 1230 Pro Ser Ser Ala Gln Ser Arg Ser Gly Gln P ro Gln Lys
Ala Ala Thr 1235 1240 1245 Cys Lys Val Ser Ser Ile Asn Gln Glu Thr
I le Gln Thr Tyr Cys Val 1250 1255 1260 Glu Asp Thr Pro Ile Cys Phe
Ser Arg Cys S er Ser Leu Ser Ser Leu 1265 127 0 1275 1280 Ser Ser
Ala Glu Asp Glu Ile Gly Cys Asn G ln Thr Thr Gln Glu Ala 1285 1290
1295 Asp Ser Ala Asn Thr Leu Gln Ile Ala Glu I le Lys Gly Lys Ile
Gly 1300 1305 1310 Thr Arg Ser Ala Glu Asp Pro Val Ser Glu V al Pro
Ala Val Ser Gln 1315 1320 1325 His Pro Arg Thr Lys Ser Ser Arg Leu
Gln G ly Ser Ser Leu Ser Ser 1330 1335 1340 Glu Ser Ala Arg His Lys
Ala Val Glu Phe P ro Ser Gly Ala Lys Ser 1345 135 0 1355 1360 Pro
Ser Lys Ser Gly Ala Gln Thr Pro Lys S er Pro Pro Glu His Tyr
1365 1370 1375 Val Gln Glu Thr Pro Leu Met Phe Ser Arg C ys Thr Ser
Val Ser Ser 1380 1385 1390 Leu Asp Ser Phe Glu Ser Arg Ser Ile Ala
S er Ser Val Gln Ser Glu 1395 1400 1405 Pro Cys Ser Gly Met Val Ser
Gly Ile Ile S er Pro Ser Asp Leu Pro 1410 1415 1420 Asp Ser Pro Gly
Gln Thr Met Pro Pro Ser A rg Ser Lys Thr Pro Pro 1425 143 0 1435
1440 Pro Pro Pro Gln Thr Ala Gln Thr Lys Arg G lu Val Pro Lys Asn
Lys 1445 1450 1455 Ala Pro Thr Ala Glu Lys Arg Glu Ser Gly P ro Lys
Gln Ala Ala Val 1460 1465 1470 Asn Ala Ala Val Gln Arg Val Gln Val
Leu P ro Asp Ala Asp Thr Leu 1475 1480 1485 Leu His Phe Ala Thr Glu
Ser Thr Pro Asp G ly Phe Ser Cys Ser Ser 1490 1495 1500 Ser Leu Ser
Ala Leu Ser Leu Asp Glu Pro P he Ile Gln Lys Asp Val 1505 151 0
1515 1520 Glu Leu Arg Ile Met Pro Pro Val Gln Glu A sn Asp Asn Gly
Asn Glu 1525 1530 1535 Thr Glu Ser Glu Gln Pro Lys Glu Ser Asn G lu
Asn Gln Glu Lys Glu 1540 1545 1550 Ala Glu Lys Thr Ile Asp Ser Glu
Lys Asp L eu Leu Asp Asp Ser Asp 1555 1560 1565 Asp Asp Asp Ile Glu
Ile Leu Glu Glu Cys I le Ile Ser Ala Met Pro 1570 1575 1580 Thr Lys
Ser Ser Arg Lys Gly Lys Lys Pro A la Gln Thr Ala Ser Lys 1585 159 0
1595 1600 Leu Pro Pro Pro Val Ala Arg Lys Pro Ser G ln Leu Pro Val
Tyr Lys 1605 1610 1615 Leu Leu Pro Ser Gln Asn Arg Leu Gln Pro G ln
Lys His Val Ser Phe 1620 1625 1630 Thr Pro Gly Asp Asp Met Pro Arg
Val Tyr C ys Val Glu Gly Thr Pro 1635 1640 1645 Ile Asn Phe Ser Thr
Ala Thr Ser Leu Ser A sp Leu Thr Ile Glu Ser 1650 1655 1660 Pro Pro
Asn Glu Leu Ala Ala Gly Glu Gly V al Arg Gly Gly Ala Gln 1665 167 0
1675 1680 Ser Gly Glu Phe Glu Lys Arg Asp Thr Ile P ro Thr Glu Gly
Arg Ser 1685 1690 1695 Thr Asp Glu Ala Gln Gly Gly Lys Thr Ser S er
Val Thr Ile Pro Glu 1700 1705 1710 Leu Asp Asp Asn Lys Ala Glu Glu
Gly Asp I le Leu Ala Glu Cys Ile 1715 1720 1725 Asn Ser Ala Met Pro
Lys Gly Lys Ser His L ys Pro Phe Arg Val Lys 1730 1735 1740 Lys Ile
Met Asp Gln Val Gln Gln Ala Ser A la Ser Ser Ser Ala Pro 1745 175 0
1755 1760 Asn Lys Asn Gln Leu Asp Gly Lys Lys Lys L ys Pro Thr Ser
Pro Val 1765 1770 1775 Lys Pro Ile Pro Gln Asn Thr Glu Tyr Arg T hr
Arg Val Arg Lys Asn 1780 1785 1790 Ala Asp Ser Lys Asn Asn Leu Asn
Ala Glu A rg Val Phe Ser Asp Asn 1795 1800 1805 Lys Asp Ser Lys Lys
Gln Asn Leu Lys Asn A sn Ser Lys Asp Phe Asn 1810 1815 1820 Asp Lys
Leu Pro Asn Asn Glu Asp Arg Val A rg Gly Ser Phe Ala Phe 1825 183 0
1835 1840 Asp Ser Pro His His Tyr Thr Pro Ile Glu G ly Thr Pro Tyr
Cys Phe 1845 1850 1855 Ser Arg Asn Asp Ser Leu Ser Ser Leu Asp P he
Asp Asp Asp Asp Val 1860 1865 1870 Asp Leu Ser Arg Glu Lys Ala Glu
Leu Arg L ys Ala Lys Glu Asn Lys 1875 1880 1885 Glu Ser Glu Ala Lys
Val Thr Ser His Thr G lu Leu Thr Ser Asn Gln 1890 1895 1900 Gln Ser
Ala Asn Lys Thr Gln Ala Ile Ala L ys Gln Pro Ile Asn Arg 1905 191 0
1915 1920 Gly Gln Pro Lys Pro Ile Leu Gln Lys Gln S er Thr Phe Pro
Gln Ser 1925 1930 1935 Ser Lys Asp Ile Pro Asp Arg Gly Ala Ala T hr
Asp Glu Lys Leu Gln 1940 1945 1950 Asn Phe Ala Ile Glu Asn Thr Pro
Val Cys P he Ser His Asn Ser Ser 1955 1960 1965 Leu Ser Ser Leu Ser
Asp Ile Asp Gln Glu A sn Asn Asn Lys Glu Asn 1970 1975 1980 Glu Pro
Ile Lys Glu Thr Glu Pro Pro Asp S er Gln Gly Glu Pro Ser 1985 199 0
1995 2000 Lys Pro Gln Ala Ser Gly Tyr Ala Pro Lys S er Phe His Val
Glu Asp 2005 2010 2015 Thr Pro Val Cys Phe Ser Arg Asn Ser Ser L eu
Ser Ser Leu Ser Ile 2020 2025 2030 Asp Ser Glu Asp Asp Leu Leu Gln
Glu Cys I le Ser Ser Ala Met Pro 2035 2040 2045 Lys Lys Lys Lys Pro
Ser Arg Leu Lys Gly A sp Asn Glu Lys His Ser 2050 2055 2060 Pro Arg
Asn Met Gly Gly Ile Leu Gly Glu A sp Leu Thr Leu Asp Leu 2065 207 0
2075 2080 Lys Asp Ile Gln Arg Pro Asp Ser Glu His G ly Leu Ser Pro
Asp Ser 2085 2090 2095 Glu Asn Phe Asp Trp Lys Ala Ile Gln Glu G ly
Ala Asn Ser Ile Val 2100 2105 2110 Ser Ser Leu His Gln Ala Ala Ala
Ala Ala C ys Leu Ser Arg Gln Ala 2115 2120 2125 Ser Ser Asp Ser Asp
Ser Ile Leu Ser Leu L ys Ser Gly Ile Ser Leu 2130 2135 2140 Gly Ser
Pro Phe His Leu Thr Pro Asp Gln G lu Glu Lys Pro Phe Thr 2145 215 0
2155 2160 Ser Asn Lys Gly Pro Arg Ile Leu Lys Pro G ly Glu Lys Ser
Thr Leu 2165 2170 2175 Glu Thr Lys Lys Ile Glu Ser Glu Ser Lys G ly
Ile Lys Gly Gly Lys 2180 2185 2190 Lys Val Tyr Lys Ser Leu Ile Thr
Gly Lys V al Arg Ser Asn Ser Glu 2195 2200 2205 Ile Ser Gly Gln Met
Lys Gln Pro Leu Gln A la Asn Met Pro Ser Ile 2210 2215 2220 Ser Arg
Gly Arg Thr Met Ile His Ile Pro G ly Val Arg Asn Ser Ser 2225 223 0
2235 2240 Ser Ser Thr Ser Pro Val Ser Lys Lys Gly P ro Pro Leu Lys
Thr Pro 2245 2250 2255 Ala Ser Lys Ser Pro Ser Glu Gly Gln Thr A la
Thr Thr Ser Pro Arg 2260 2265 2270 Gly Ala Lys Pro Ser Val Lys Ser
Glu Leu S er Pro Val Ala Arg Gln 2275 2280 2285 Thr Ser Gln Ile Gly
Gly Ser Ser Lys Ala P ro Ser Arg Ser Gly Ser 2290 2295 2300 Arg Asp
Ser Thr Pro Ser Arg Pro Ala Gln G ln Pro Leu Ser Arg Pro 2305 231 0
2315 2320 Ile Gln Ser Pro Gly Arg Asn Ser Ile Ser P ro Gly Arg Asn
Gly Ile 2325 2330 2335 Ser Pro Pro Asn Lys Leu Ser Gln Leu Pro A rg
Thr Ser Ser Pro Ser 2340 2345 2350 Thr Ala Ser Thr Lys Ser Ser Gly
Ser Gly L ys Met Ser Tyr Thr Ser 2355 2360 2365 Pro Gly Arg Gln Met
Ser Gln Gln Asn Leu T hr Lys Gln Thr Gly Leu 2370 2375 2380 Ser Lys
Asn Ala Ser Ser Ile Pro Arg Ser G lu Ser Ala Ser Lys Gly 2385 239 0
2395 2400 Leu Asn Gln Met Asn Asn Gly Asn Gly Ala A sn Lys Lys Val
Glu Leu 2405 2410 2415 Ser Arg Met Ser Ser Thr Lys Ser Ser Gly S er
Glu Ser Asp Arg Ser 2420 2425 2430 Glu Arg Pro Val Leu Val Arg Gln
Ser Thr P he Ile Lys Glu Ala Pro 2435 2440 2445 Ser Pro Thr Leu Arg
Arg Lys Leu Glu Glu S er Ala Ser Phe Glu Ser 2450 2455 2460 Leu Ser
Pro Ser Ser Arg Pro Ala Ser Pro T hr Arg Ser Gln Ala Gln 2465 247 0
2475 2480 Thr Pro Val Leu Ser Pro Ser Leu Pro Asp M et Ser Leu Ser
Thr His 2485 2490 2495 Ser Ser Val Gln Ala Gly Gly Trp Arg Lys L eu
Pro Pro Asn Leu Ser 2500 2505 2510 Pro Thr Ile Glu Tyr Asn Asp Gly
Arg Pro A la Lys Arg His Asp Ile 2515 2520 2525 Ala Arg Ser His Ser
Glu Ser Pro Ser Arg L eu Pro Ile Asn Arg Ser 2530 2535 2540 Gly Thr
Trp Lys Arg Glu His Ser Lys His S er Ser Ser Leu Pro Arg 2545 255 0
2555 2560 Val Ser Thr Trp Arg Arg Thr Gly Ser Ser S er Ser Ile Leu
Ser Ala 2565 2570 2575 Ser Ser Glu Ser Ser Glu Lys Ala Lys Ser G lu
Asp Glu Lys His Val 2580 2585 2590 Asn Ser Ile Ser Gly Thr Lys Gln
Ser Lys G lu Asn Gln Val Ser Ala 2595 2600 2605 Lys Gly Thr Trp Arg
Lys Ile Lys Glu Asn G lu Phe Ser Pro Thr Asn 2610 2615 2620 Ser Thr
Ser Gln Thr Val Ser Ser Gly Ala T hr Asn Gly Ala Glu Ser 2625 263 0
2635 2640 Lys Thr Leu Ile Tyr Gln Met Ala Pro Ala V al Ser Lys Thr
Glu Asp 2645 2650 2655 Val Trp Val Arg Ile Glu Asp Cys Pro Ile A sn
Asn Pro Arg Ser Gly 2660 2665 2670 Arg Ser Pro Thr Gly Asn Thr Pro
Pro Val I le Asp Ser Val Ser Glu 2675 2680 2685 Lys Ala Asn Pro Asn
Ile Lys Asp Ser Lys A sp Asn Gln Ala Lys Gln 2690 2695 2700 Asn Val
Gly Asn Gly Ser Val Pro Met Arg T hr Val Gly Leu Glu Asn 2705 271 0
2715 2720 Arg Leu Thr Ser Phe Ile Gln Val Asp Ala P ro Asp Gln Lys
Gly Thr 2725 2730 2735 Glu Ile Lys Pro Gly Gln Asn Asn Pro Val P ro
Val Ser Glu Thr Asn 2740 2745 2750 Glu Ser Pro Ile Val Glu Arg Thr
Pro Phe S er Ser Ser Ser Ser Ser 2755 2760 2765 Lys His Ser Ser Pro
Ser Gly Thr Val Ala A la Arg Val Thr Pro Phe 2770 2775 2780 Asn Tyr
Asn Pro Ser Pro Arg Lys Ser Ser A la Asp Ser Thr Ser Ala 2785 279 0
2795 2800 Arg Pro Ser Gln Ile Pro Thr Pro Val Asn A sn Asn Thr Lys
Lys Arg 2805 2810 2815 Asp Ser Lys Thr Asp Ser Thr Glu Ser Ser G ly
Thr Gln Ser Pro Lys 2820 2825 2830 Arg His Ser Gly Ser Tyr Leu Val
Thr Ser V al 2835 2840 (2) INFORMATION FOR SEQ ID NO: 3: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 3172 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap
iens (vii) IMMEDIATE SOURCE: (B) CLONE: DP1(TB2) (ix) FEATURE: (A)
NAME/KEY: CDS (B) LOCATION: 1..630 (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 3: GCA GTC GCC GCT CCA GTC TAT CCG GCA CTA G GA ACA GCC CCG
GGN GGC 48 Ala Val Ala Ala Pro Val Tyr Pro Ala Leu G ly Thr Ala Pro
Gly Gly 1 5 10 15 GAG ACG GTC CCC GCC ATG TCT GCG GCC ATG A GG GAG
AGG TTC GAC CGG 96 Glu Thr Val Pro Ala Met Ser Ala Ala Met A rg Glu
Arg Phe Asp Arg 20 25 30 TTC CTG CAC GAG AAG AAC TGC ATG ACT GAC C
TT CTG GCC AAG CTC GAG 144 Phe Leu His Glu Lys Asn Cys Met Thr Asp
L eu Leu Ala Lys Leu Glu 35 40 45 GCC AAA ACC GGC GTG AAC AGG AGC
TTC ATC G CT CTT GGT GTC ATC GGA 192 Ala Lys Thr Gly Val Asn Arg
Ser Phe Ile A la Leu Gly Val Ile Gly 50 55 60 CTG GTG GCC TTG TAC
CTG GTG TTC GGT TAT G GA GCC TCT CTC CTC TGC 240 Leu Val Ala Leu
Tyr Leu Val Phe Gly Tyr G ly Ala Ser Leu Leu Cys 65 70 75 80 AAC
CTG ATA GGA TTT GGC TAC CCA GCC TAC A TC TCA ATT AAA GCT ATA 288
Asn Leu Ile Gly Phe Gly Tyr Pro Ala Tyr I le Ser Ile Lys Ala Ile 85
90 95 GAG AGT CCC AAC AAA GAA GAT GAT ACC CAG T GG CTG ACC TAC TGG
GTA 336 Glu Ser Pro Asn Lys Glu Asp Asp Thr Gln T rp Leu Thr Tyr
Trp Val 100 105 110 GTG TAT GGT GTG TTC AGC ATT GCT GAA TTC T TC
TCT GAT ATC TTC CTG 384 Val Tyr Gly Val Phe Ser Ile Ala Glu Phe P
he Ser Asp Ile Phe Leu 115 120 125 TCA TGG TTC CCC TTC TAC TAC ATG
CTG AAG T GT GGC TTC CTG TTG TGG 432 Ser Trp Phe Pro Phe Tyr Tyr
Met Leu Lys C ys Gly Phe Leu Leu Trp 130 135 140 TGC ATG GCC CCG
AGC CCT TCT AAT GGG GCT G AA CTG CTC TAC AAG CGC 480 Cys Met Ala
Pro Ser Pro Ser Asn Gly Ala G lu Leu Leu Tyr Lys Arg 145 150 155
160 ATC ATC CGT CCT TTC TTC CTG AAG CAC GAG T CC CAG ATG GAC AGT
GTG 528 Ile Ile Arg Pro Phe Phe Leu Lys His Glu S er Gln Met Asp
Ser Val 165 170 175 GTC AAG GAC CTT AAA GAC AAG TCC AAA GAG A CT
GCA GAT GCC ATC ACT 576 Val Lys Asp Leu Lys Asp Lys Ser Lys Glu T
hr Ala Asp Ala Ile Thr 180 185 190 AAA GAA GCG AAG AAA GCT ACC GTG
AAT TTA C TG GGT GAA GAA AAG AAG 624 Lys Glu Ala Lys Lys Ala Thr
Val Asn Leu L eu Gly Glu Glu Lys Lys 195 200 205 AGC ACC TAAACCAGAC
TAAACCAGAC TGGATGGAAA CTTCCTGCCC T CTCTGTACC 680 Ser Thr 210
TTCCTACTGG AGCTTGATGT TATATTAGGG ACTGTGGTAT AATTATTTTA A TAATGTTGC
740 CTTGGAAACA TTTTTGAGAT ATTAAAGATT GGAATGTGTT GTAAGTTTCT T
TGCTTACTT 800 TTACTGTCTA TATATATAGG GAGCACTTTA AACTTAATGC
AGTGGGCAGT G TCCACGTTT 860 TTGGAAAATG TATTTTGCCT CTGGGTAGGA
AAAGATGTAT GTTGCTATCC T GCAGGAAAT 920 ATAAACTTAA AATAAAATTA
TATACCCCAC AGGCTGTGTA CTTTACTGGG C TCTCCCTGC 980 ACGSATTTTC
TCTGTAGTTA CATTTAGGRT AATCTTTATG GTTCTACTTC C TRTAATGTA 1040
CAATTTTATA TAATTCNGRA ATGTTTTTAA TGTATTTGTG CACATGTACA T ATGGAAATG
1100 TTACTGTCTG ACTACANCAT GCATCATGCT CATGGGGAGG GAGCAGGGGA A
GGTTGTATG 1160 TGTCATTTAT AACTTCTGTA CAGTAAGACC ACCTGCCAAA
AGCTGGAGGA A CCATTGTGC 1220
TGGTGTGGTC TACTAAATAA TACTTTAGGA AATACGTGAT TAATATGCAA G TGAACAAAG
1280 TGAGAAATGA AATCGAATGG AGATTGGCCT GGTTGTTTCC GTAGTATATG G
CATATGAAT 1340 ACCAGGATAG CTTTATAAAG CAGTTAGTTA GTTAGTTACT
CACTCTAGTG A TAAATCGGG 1400 AAATTTACAC ACACACACAC ACACACACAC
ACACACACAC ACACACACAC A CACACACAG 1460 AGTACCCTGT AACTCTCAAT
TCCCTGAAAA ACTAGTAATA CTGTCTTATC T GCTATAAAC 1520 TTTACATATT
TGTCTATTGT CAAGATGCTA CANTGGAMNC CATTTCTGGT T TTATCTTCA 1580
NAGSGGAGAN ACATGTTGAT TTAGTCTTCT TTCCCAATCT TCTTTTTTAA M CCAGTTTNA
1640 GGMNCTTCTG RAGATTTGYC CACCTCTGAT TACATGTATG TTCTYGTTTG T
ATCATKAGC 1700 AACAACATGC TAATGRCGAC ACCTAGCTCT RAGMGCAATT
CTGGGAGANT G ARAGGNWGT 1760 ATARAGTMNC CCATAATCTG CTTGGCAATA
GTTAAGTCAA TCTATCTTCA G TTTTTCTCT 1820 GGCCTTTAAG GTCAAACACA
AGAGGCTTCC CTAGTTTACA AGTCAGAGTC A CTTGTAGTC 1880 CATTTAAATG
CCCTCATCCG TATTCTTTGT GTTGATAAGC TGCACAKGAC T ACATAGTAA 1940
GTACAGANCA GTAAAGTTAA NNCGGATGTC TCCATTGATC TGCCAANTCG N TATAGAGAG
2000 CAATTTGTCT GGACTAGAAA ATCTGAGTTT TACACCATAC TGTTAAGAGT C
CTTTTGAAT 2060 TAAACTAGAC TAAAACAAGT GTATAACTAA ACTAACAAGA
TTAAATATCC A GCCAGTACA 2120 GTATTTTTTA AGGCAAATAA AGATGATTAG
CTCACCTTGA GNTAACAATC A GGTAAGATC 2180 ATNACAATGT CTCATGATGT
NAANAATATT AAAGATATCA ATACTAAGTG A CAGTATCAC 2240 NNCTAATATA
ATATGGATCA GAGCATTTAT TTTGGGGAGG AAAACAGTGG T GATTACCGG 2300
CATTTTATTA AACTTAAAAC TTTGTAGAAA GCAAACAAAA TTGTTCTTGG G AGAAAATCA
2360 ACTTTTAGAT TAAAAAAATT TTAAGTAWCT AGGAGTATTT AAATCCTTTT C
CCATAAATA 2420 AAAGTACAGT TTTCTTGGTG GCAGAATGAA AATCAGCAAC
NTCTAGCATA T AGACTATAT 2480 AATCAGATTG ACAGCATATA GAATATATTA
TCAGACAAGA TGAGGAGGTA C AAAAGTTAC 2540 TATTGCTCAT AATGACTTAC
AGGCTAAAAN TAGNTNTAAA ATACTATATT A AATTCTGAA 2600 TGCAATTTTT
TTTTGTTCCC TTGAGACCAA AATTTAAGTT AACTGTTGCT G GCAGTCTAA 2660
GTGTAAATGT TAACAGCAGG AGAAGTTAAG AATTGAGCAG TTCTGTTGCA T GATTTCCCA
2720 AATGAAATAC TGCCTTGGCT AGAGTTTGAA AAACTAATTG AGCCTGTGCC T
GGCTAGAAA 2780 ACAAGCGTTT ATTTGAATGT GAATAGTGTT TCAAAGGTAT
GTAGTTACAG A ATTCCTACC 2840 AAACAGCTTA AATTCTTCAA GAAAGAATTC
CTGCAGCAGT TATTCCCTTA C CTGAAGGCT 2900 TCAATCATTT GGATCAACAA
CTGCTACTCT CGGGAAGACT CCTCTACTCA C AGCTGAAGA 2960 AAATGAGCAC
ACCCTTCACA CTGTTATCAC CTATCCTGAA GATGTGATAC A CTGAATGGA 3020
AATAAATAGA TGTAAATAAA ATTGAGWTCT CATTTAAAAA AAACCATGTG C CCAATGGGA
3080 AAATGACCTC ATGTTGTGGT TTAAACAGCA ACTGCACCCA CTAGCACAGC C
CATTGAGCT 3140 ANCCTATATA TACATCTCTG TCAGTGCCCC TC 3172 (2)
INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 210 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
4: Ala Val Ala Ala Pro Val Tyr Pro Ala Leu G ly Thr Ala Pro Gly Gly
1 5 10 15 Glu Thr Val Pro Ala Met Ser Ala Ala Met A rg Glu Arg Phe
Asp Arg 20 25 30 Phe Leu His Glu Lys Asn Cys Met Thr Asp L eu Leu
Ala Lys Leu Glu 35 40 45 Ala Lys Thr Gly Val Asn Arg Ser Phe Ile A
la Leu Gly Val Ile Gly 50 55 60 Leu Val Ala Leu Tyr Leu Val Phe Gly
Tyr G ly Ala Ser Leu Leu Cys 65 70 75 80 Asn Leu Ile Gly Phe Gly
Tyr Pro Ala Tyr I le Ser Ile Lys Ala Ile 85 90 95 Glu Ser Pro Asn
Lys Glu Asp Asp Thr Gln T rp Leu Thr Tyr Trp Val 100 105 110 Val
Tyr Gly Val Phe Ser Ile Ala Glu Phe P he Ser Asp Ile Phe Leu 115
120 125 Ser Trp Phe Pro Phe Tyr Tyr Met Leu Lys C ys Gly Phe Leu
Leu Trp 130 135 140 Cys Met Ala Pro Ser Pro Ser Asn Gly Ala G lu
Leu Leu Tyr Lys Arg 145 150 155 160 Ile Ile Arg Pro Phe Phe Leu Lys
His Glu S er Gln Met Asp Ser Val 165 170 175 Val Lys Asp Leu Lys
Asp Lys Ser Lys Glu T hr Ala Asp Ala Ile Thr 180 185 190 Lys Glu
Ala Lys Lys Ala Thr Val Asn Leu L eu Gly Glu Glu Lys Lys 195 200
205 Ser Thr 210 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 434 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (vii)
IMMEDIATE SOURCE: (B) CLONE: TB1 (xi) SEQUENCE DESCRIPTION: SEQ ID
NO: 5: Val Ala Pro Val Val Val Gly Ser Gly Arg A la Pro Arg His Pro
Ala 1 5 10 15 Pro Ala Ala Met His Pro Arg Arg Pro Asp G ly Phe Asp
Gly Leu Gly 20 25 30 Tyr Arg Gly Gly Ala Arg Asp Glu Gln Gly P he
Gly Gly Ala Phe Pro 35 40 45 Ala Arg Ser Phe Ser Thr Gly Ser Asp
Leu G ly His Trp Val Thr Thr 50 55 60 Pro Pro Asp Ile Pro Gly Ser
Arg Asn Leu H is Trp Gly Glu Lys Ser 65 70 75 80 Pro Pro Tyr Gly
Val Pro Thr Thr Ser Thr P ro Tyr Glu Gly Pro Thr 85 90 95 Glu Glu
Pro Phe Ser Ser Gly Gly Gly Gly S er Val Gln Gly Gln Ser 100 105
110 Ser Glu Gln Leu Asn Arg Phe Ala Gly Phe G ly Ile Gly Leu Ala
Ser 115 120 125 Leu Phe Thr Glu Asn Val Leu Ala His Pro C ys Ile
Val Leu Arg Arg 130 135 140 Gln Cys Gln Val Asn Tyr His Ala Gln His
T yr His Leu Thr Pro Phe 145 150 155 160 Thr Val Ile Asn Ile Met
Tyr Ser Phe Asn L ys Thr Gln Gly Pro Arg 165 170 175 Ala Leu Trp
Lys Gly Met Gly Ser Thr Phe I le Val Gln Gly Val Thr 180 185 190
Leu Gly Ala Glu Gly Ile Ile Ser Glu Phe T hr Pro Leu Pro Arg Glu
195 200 205 Val Leu His Lys Trp Ser Pro Lys Gln Ile G ly Glu His
Leu Leu Leu 210 215 220 Lys Ser Leu Thr Tyr Val Val Ala Met Pro P
he Tyr Ser Ala Ser Leu 225 230 235 240 Ile Glu Thr Val Gln Ser Glu
Ile Ile Arg A sp Asn Thr Gly Ile Leu 245 250 255 Glu Cys Val Lys
Glu Gly Ile Gly Arg Val I le Gly Met Gly Val Pro 260 265 270 His
Ser Lys Arg Leu Leu Pro Leu Leu Ser L eu Ile Phe Pro Thr Val 275
280 285 Leu His Gly Val Leu His Tyr Ile Ile Ser S er Val Ile Gln
Lys Phe 290 295 300 Val Leu Leu Ile Leu Lys Arg Lys Thr Tyr A sn
Ser His Leu Ala Glu 305 310 315 320 Ser Thr Ser Pro Val Gln Ser Met
Leu Asp A la Tyr Phe Pro Glu Leu 325 330 335 Ile Ala Asn Phe Ala
Ala Ser Leu Cys Ser A sp Val Ile Leu Tyr Pro 340 345 350 Leu Glu
Thr Val Leu His Arg Leu His Ile G ln Gly Thr Arg Thr Ile 355 360
365 Ile Asp Asn Thr Asp Leu Gly Tyr Glu Val L eu Pro Ile Asn Thr
Gln 370 375 380 Tyr Glu Gly Met Arg Asp Cys Ile Asn Thr I le Arg
Gln Glu Glu Gly 385 390 395 400 Val Phe Gly Phe Tyr Lys Gly Phe Gly
Ala V al Ile Ile Gln Tyr Thr 405 410 415 Leu His Ala Ala Val Leu
Gln Ile Thr Lys I le Ile Tyr Ser Thr Leu 420 425 430 Leu Gln (2)
INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 185 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi)
ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (vii) IMMEDIATE
SOURCE: (B) CLONE: YS-39(TB2) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
6: Glu Leu Arg Arg Phe Asp Arg Phe Leu His G lu Lys Asn Cys Met Thr
1 5 10 15 Asp Leu Leu Ala Lys Leu Glu Ala Lys Thr G ly Val Asn Arg
Ser Phe 20 25 30 Ile Ala Leu Gly Val Ile Gly Leu Val Ala L eu Tyr
Leu Val Phe Gly 35 40 45 Tyr Gly Ala Ser Leu Leu Cys Asn Leu Ile G
ly Phe Gly Tyr Pro Ala 50 55 60 Tyr Ile Ser Ile Lys Ala Ile Glu Ser
Pro A sn Lys Glu Asp Asp Thr 65 70 75 80 Gln Trp Leu Thr Tyr Trp
Val Val Tyr Gly V al Phe Ser Ile Ala Glu 85 90 95 Phe Phe Ser Asp
Ile Phe Leu Ser Trp Phe P ro Phe Tyr Tyr Ile Leu 100 105 110 Lys
Cys Gly Phe Leu Leu Trp Cys Met Ala P ro Ser Pro Ser Asn Gly 115
120 125 Ala Glu Leu Leu Tyr Lys Arg Ile Ile Arg P ro Phe Phe Leu
Lys His 130 135 140 Glu Ser Gln Met Asp Ser Val Val Lys Asp L eu
Lys Asp Lys Ala Lys 145 150 155 160 Glu Thr Ala Asp Ala Ile Thr Lys
Glu Ala L ys Lys Ala Thr Val Asn 165 170 175 Leu Leu Gly Glu Glu
Lys Lys Ser Thr 180 185 (2) INFORMATION FOR SEQ ID NO: 7: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: .[.2842.].
.Iadd.2843.Iaddend. amino acids (B) TYPE: amino acid (C)
STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (vii)
IMMEDIATE SOURCE: (B) CLONE: APC (xi) SEQUENCE DESCRIPTION: SEQ ID
NO: 7: Met Ala Ala Ala Ser Tyr Asp Gln Leu Leu L ys Gln Val Glu Ala
Leu 1 5 10 15 Lys Met Glu Asn Ser Asn Leu Arg Gln Glu L eu Glu Asp
Asn Ser Asn 20 25 30 His Leu Thr Lys Leu Glu Thr Glu Ala Ser A sn
Met Lys Glu Val Leu 35 40 45 Lys Gln Leu Gln Gly Ser Ile Glu Asp
Glu A la Met Ala Ser Ser Gly 50 55 60 Gln Ile Asp Leu Leu Glu Arg
Leu Lys Glu L eu Asn Leu Asp Ser Ser 65 70 75 80 Asn Phe Pro Gly
Val Lys Leu Arg Ser Lys M et Ser Leu Arg Ser Tyr 85 90 95 Gly Ser
Arg Glu Gly Ser Val Ser Ser Arg S er Gly Glu Cys Ser Pro 100 105
110 Val Pro Met Gly Ser Phe Pro Arg Arg Gly P he Val Asn Gly Ser
Arg 115 120 125 Glu Ser Thr Gly Tyr Leu Glu Glu Leu Glu L ys Glu
Arg Ser Leu Leu 130 135 140 Leu Ala Asp Leu Asp Lys Glu Glu Lys Glu
L ys Asp Trp Tyr Tyr Ala 145 150 155 160 Gln Leu Gln Asn Leu Thr
Lys Arg Ile Asp S er Leu .Iadd.Pro.Iaddend. Leu Thr Glu 165 170 175
Asn Phe Ser Leu Gln Thr Asp Met Thr Arg A rg Gln Leu Glu Tyr Glu
180 185 190 Ala Arg Gln Ile Arg Val Ala Met Glu Glu G ln Leu Gly
Thr Cys Gln 195 200 205 Asp Met Glu Lys Arg Ala Gln Arg Arg Ile A
la Arg Ile Gln Gln Ile 210 215 220 Glu Lys Asp Ile Leu Arg Ile Arg
Gln Leu L eu Gln Ser Gln Ala Thr 225 230 235 240 Glu Ala Glu Arg
Ser Ser Gln Asn Lys His G lu Thr Gly Ser His Asp 245 250 255 Ala
Glu Arg Gln Asn Glu Gly Gln Gly Val G ly Glu Ile Asn Met Ala 260
265 270 Thr Ser Gly Asn Gly Gln Gly Ser Thr Thr A rg Met Asp His
Glu Thr 275 280 285 Ala Ser Val Leu Ser Ser Ser Ser Thr His S er
Ala Pro Arg Arg Leu 290 295 300 Thr Ser His Leu Gly Thr Lys Val Glu
Met V al Tyr Ser Leu Leu Ser 305 310 315 320 Met Leu Gly Thr His
Asp Lys Asp Asp Met S er Arg Thr Leu Leu Ala 325 330 335 Met Ser
Ser Ser Gln Asp Ser Cys Ile Ser M et Arg Gln Ser Gly Cys 340 345
350 Leu Pro Leu Leu Ile Gln Leu Leu His Gly A sn Asp Lys Asp Ser
Val 355 360 365 Leu Leu Gly Asn Ser Arg Gly Ser Lys Glu A la Arg
Ala Arg Ala Ser 370 375 380 Ala Ala Leu His Asn Ile Ile His Ser Gln
P ro Asp Asp Lys Arg Gly 385 390 395 400 Arg Arg Glu Ile Arg Val
Leu His Leu Leu G lu Gln Ile Arg Ala Tyr 405 410 415 Cys Glu Thr
Cys Trp Glu Trp Gln Glu Ala H is Glu Pro Gly Met Asp 420 425 430
Gln Asp Lys Asn Pro Met Pro Ala Pro Val G lu His Gln Ile Cys Pro
435 440 445 Ala Val Cys Val Leu Met Lys Leu Ser Phe A sp Glu Glu
His Arg His 450 455 460 Ala Met Asn Glu Leu Gly Gly Leu Gln Ala I
le Ala Glu Leu Leu Gln 465 470 475 480 Val Asp Cys Glu Met Tyr Gly
Leu Thr Asn A sp His Tyr Ser Ile Thr 485 490 495 Leu Arg Arg Tyr
Ala Gly Met Ala Leu Thr A sn Leu Thr Phe Gly Asp 500 505 510 Val
Ala Asn Lys Ala Thr Leu Cys Ser Met L ys Gly Cys Met Arg Ala 515
520 525
Leu Val Ala Gln Leu Lys Ser Glu Ser Glu A sp Leu Gln Gln Val Ile
530 535 540 Ala Ser Val Leu Arg Asn Leu Ser Trp Arg A la Asp Val
Asn Ser Lys 545 550 555 560 Lys Thr Leu Arg Glu Val Gly Ser Val Lys
A la Leu Met Glu Cys Ala 565 570 575 Leu Glu Val Lys Lys Glu Ser
Thr Leu Lys S er Val Leu Ser Ala Leu 580 585 590 Trp Asn Leu Ser
Ala His Cys Thr Glu Asn L ys Ala Asp Ile Cys Ala 595 600 605 Val
Asp Gly Ala Leu Ala Phe Leu Val Gly T hr Leu Thr Tyr Arg Ser 610
615 620 Gln Thr Asn Thr Leu Ala Ile Ile Glu Ser G ly Gly Gly Ile
Leu Arg 625 630 635 640 Asn Val Ser Ser Leu Ile Ala Thr Asn Glu A
sp His Arg Gln Ile Leu 645 650 655 Arg Glu Asn Asn Cys Leu Gln Thr
Leu Leu G ln His Leu Lys Ser His 660 665 670 Ser Leu Thr Ile Val
Ser Asn Ala Cys Gly T hr Leu Trp Asn Leu Ser 675 680 685 Ala Arg
Asn Pro Lys Asp Gln Glu Ala Leu T rp Asp Met Gly Ala Val 690 695
700 Ser Met Leu Lys Asn Leu Ile His Ser Lys H is Lys Met Ile Ala
Met 705 710 715 720 Gly Ser Ala Ala Ala Leu Arg Asn Leu Met A la
Asn Arg Pro Ala Lys 725 730 735 Tyr Lys Asp Ala Asn Ile Met Ser Pro
Gly S er Ser Leu Pro Ser Leu 740 745 750 His Val Arg Lys Gln Lys
Ala Leu Glu Ala G lu Leu Asp Ala Gln His 755 760 765 Leu Ser Glu
Thr Phe Asp Asn Ile Asp Asn L eu Ser Pro Lys Ala Ser 770 775 780
His Arg Ser Lys Gln Arg His Lys Gln Ser L eu Tyr Gly Asp Tyr Val
785 790 795 800 Phe Asp Thr Asn Arg His Asp Asp Asn Arg S er Asp
Asn Phe Asn Thr 805 810 815 Gly Asn Met Thr Val Leu Ser Pro Tyr Leu
A sn Thr Thr Val Leu Pro 820 825 830 Ser Ser Ser Ser Ser Arg Gly
Ser Leu Asp S er Ser Arg Ser Glu Lys 835 840 845 Asp Arg Ser Leu
Glu Arg Glu Arg Gly Ile G ly Leu Gly Asn Tyr His 850 855 860 Pro
Ala Thr Glu Asn Pro Gly Thr Ser Ser L ys Arg Gly Leu Gln Ile 865
870 875 880 Ser Thr Thr Ala Ala Gln Ile Ala Lys Val M et Glu Glu
Val Ser Ala 885 890 895 Ile His Thr Ser Gln Glu Asp Arg Ser Ser G
ly Ser Thr Thr Glu Leu 900 905 910 His Cys Val Thr Asp Glu Arg Asn
Ala Leu A rg Arg Ser Ser Ala Ala 915 920 925 His Thr His Ser Asn
Thr Tyr Asn Phe Thr L ys Ser Glu Asn Ser Asn 930 935 940 Arg Thr
Cys Ser Met Pro Tyr Ala Lys Leu G lu Tyr Lys Arg Ser Ser 945 950
955 960 Asn Asp Ser Leu Asn Ser Val Ser Ser Ser A sp Gly Tyr Gly
Lys Arg 965 970 975 Gly Gln Met Lys Pro Ser Ile Glu Ser Tyr S er
Glu Asp Asp Glu Ser 980 985 990 Lys Phe Cys Ser Tyr Gly Gln Tyr Pro
Ala A sp Leu Ala His Lys Ile 995 1000 1005 His Ser Ala Asn His Met
Asp Asp Asn Asp G ly Glu Leu Asp Thr Pro 1010 1015 1020 Ile Asn Tyr
Ser Leu Lys Tyr Ser Asp Glu G ln Leu Asn Ser Gly Arg 1025 103 0
1035 1040 Gln Ser Pro Ser Gln Asn Glu Arg Trp Ala A rg Pro Lys His
Ile Ile 1045 1050 1055 Glu Asp Glu Ile Lys Gln Ser Glu Gln Arg G ln
Ser Arg Asn Gln Ser 1060 1065 1070 Thr Thr Tyr Pro Val Tyr Thr Glu
Ser Thr A sp Asp Lys His Leu Lys 1075 1080 1085 Phe Gln Pro His Phe
Gly Gln Gln Glu Cys V al Ser Pro Tyr Arg Ser 1090 1095 1100 Arg Gly
Ala Asn Gly Ser Glu Thr Asn Arg V al Gly Ser Asn His Gly 1105 111 0
1115 1120 Ile Asn Gln Asn Val Ser Gln Ser Leu Cys G ln Glu Asp Asp
Tyr Glu 1125 1130 1135 Asp Asp Lys Pro Thr Asn Tyr Ser Glu Arg T yr
Ser Glu Glu Glu Gln 1140 1145 1150 His Glu Glu Glu Glu Arg Pro Thr
Asn Tyr S er Ile Lys Tyr Asn Glu 1155 1160 1165 Glu Lys Arg His Val
Asp Gln Pro Ile Asp T yr Ser Leu Lys Tyr Ala 1170 1175 1180 Thr Asp
Ile Pro Ser Ser Gln Lys Gln Ser P he Ser Phe Ser Lys Ser 1185 119 0
1195 1200 Ser Ser Gly Gln Ser Ser Lys Thr Glu His M et Ser Ser Ser
Ser Glu 1205 1210 1215 Asn Thr Ser Thr Pro Ser Ser Asn Ala Lys A rg
Gln Asn Gln Leu His 1220 1225 1230 Pro Ser Ser Ala Gln Ser Arg Ser
Gly Gln P ro Gln Lys Ala Ala Thr 1235 1240 1245 Cys Lys Val Ser Ser
Ile Asn Gln Glu Thr I le Gln Thr Tyr Cys Val 1250 1255 1260 Glu Asp
Thr Pro Ile Cys Phe Ser Arg Cys S er Ser Leu Ser Ser Leu 1265 127 0
1275 1280 Ser Ser Ala Glu Asp Glu Ile Gly Cys Asn G ln Thr Thr Gln
Glu Ala 1285 1290 1295 Asp Ser Ala Asn Thr Leu Gln Ile Ala Glu I le
Lys Glu Lys Ile Gly 1300 1305 1310 Thr Arg Ser Ala Glu Asp Pro Val
Ser Glu V al Pro Ala Val Ser Gln 1315 1320 1325 His Pro Arg Thr Lys
Ser Ser Arg Leu Gln G ly Ser Ser Leu Ser Ser 1330 1335 1340 Glu Ser
Ala Arg His Lys Ala Val Glu Phe S er Ser Gly Ala Lys Ser 1345 135 0
1355 1360 Pro Ser Lys Ser Gly Ala Gln Thr Pro Lys S er Pro Pro Glu
His Tyr 1365 1370 1375 Val Gln Glu Thr Pro Leu Met Phe Ser Arg C ys
Thr Ser Val Ser Ser 1380 1385 1390 Leu Asp Ser Phe Glu Ser Arg Ser
Ile Ala S er Ser Val Gln Ser Glu 1395 1400 1405 Pro Cys Ser Gly Met
Val Ser Gly Ile Ile S er Pro Ser Asp Leu Pro 1410 1415 1420 Asp Ser
Pro Gly Gln Thr Met Pro Pro Ser A rg Ser Lys Thr Pro Pro 1425 143 0
1435 1440 Pro Pro Pro Gln Thr Ala Gln Thr Lys Arg G lu Val Pro Lys
Asn Lys 1445 1450 1455 Ala Pro Thr Ala Glu Lys Arg Glu Ser Gly P ro
Lys Gln Ala Ala Val 1460 1465 1470 Asn Ala Ala Val Gln Arg Val Gln
Val Leu P ro Asp Ala Asp Thr Leu 1475 1480 1485 Leu His Phe Ala Thr
Glu Ser Thr Pro Asp G ly Phe Ser Cys Ser Ser 1490 1495 1500 Ser Leu
Ser Ala Leu Ser Leu Asp Glu Pro P he Ile Gln Lys Asp Val 1505 151 0
1515 1520 Glu Leu Arg Ile Met Pro Pro Val Gln Glu A sn Asp Asn Gly
Asn Glu 1525 1530 1535 Thr Glu Ser Glu Gln Pro Lys Glu Ser Asn G lu
Asn Gln Glu Lys Glu 1540 1545 1550 Ala Glu Lys Thr Ile Asp Ser Glu
Lys Asp L eu Leu Asp Asp Ser Asp 1555 1560 1565 Asp Asp Asp Ile Glu
Ile Leu Glu Glu Cys I le Ile Ser Ala Met Pro 1570 1575 1580 Thr Lys
Ser Ser Arg Lys Ala Lys Lys Pro A la Gln Thr Ala Ser Lys 1585 159 0
1595 1600 Leu Pro Pro Pro Val Ala Arg Lys Pro Ser G ln Leu Pro Val
Tyr Lys 1605 1610 1615 Leu Leu Pro Ser Gln Asn Arg Leu Gln Pro G ln
Lys His Val Ser Phe 1620 1625 1630 Thr Pro Gly Asp Asp Met Pro Arg
Val Tyr C ys Val Glu Gly Thr Pro 1635 1640 1645 Ile Asn Phe Ser Thr
Ala Thr Ser Leu Ser A sp Leu Thr Ile Glu Ser 1650 1655 1660 Pro Pro
Asn Glu Leu Ala Ala Gly Glu Gly V al Arg Gly Gly Ala Gln 1665 167 0
1675 1680 Ser Gly Glu Phe Glu Lys Arg Asp Thr Ile P ro Thr Glu Gly
Arg Ser 1685 1690 1695 Thr Asp Glu Ala Gln Gly Gly Lys Thr Ser S er
Val Thr Ile Pro Glu 1700 1705 1710 Leu Asp Asp Asn Lys Ala Glu Glu
Gly Asp I le Leu Ala Glu Cys Ile 1715 1720 1725 Asn Ser Ala Met Pro
Lys Gly Lys Ser His L ys Pro Phe Arg Val Lys 1730 1735 1740 Lys Ile
Met Asp Gln Val Gln Gln Ala Ser A la Ser Ser Ser Ala Pro 1745 175 0
1755 1760 Asn Lys Asn Gln Leu Asp Gly Lys Lys Lys L ys Pro Thr Ser
Pro Val 1765 1770 1775 Lys Pro Ile Pro Gln Asn Thr Glu Tyr Arg T hr
Arg Val Arg Lys Asn 1780 1785 1790 Ala Asp Ser Lys Asn Asn Leu Asn
Ala Glu A rg Val Phe Ser Asp Asn 1795 1800 1805 Lys Asp Ser Lys Lys
Gln Asn Leu Lys Asn A sn Ser Lys Asp Phe Asn 1810 1815 1820 Asp Lys
Leu Pro Asn Asn Glu Asp Arg Val A rg Gly Ser Phe Ala Phe 1825 183 0
1835 1840 Asp Ser Pro His His Tyr Thr Pro Ile Glu G ly Thr Pro Tyr
Cys Phe 1845 1850 1855 Ser Arg Asn Asp Ser Leu Ser Ser Leu Asp P he
Asp Asp Asp Asp Val 1860 1865 1870 Asp Leu Ser Arg Glu Lys Ala Glu
Leu Arg L ys Ala Lys Glu Asn Lys 1875 1880 1885 Glu Ser Glu Ala Lys
Val Thr Ser His Thr G lu Leu Thr Ser Asn Gln 1890 1895 1900 Gln Ser
Ala Asn Lys Thr Gln Ala Ile Ala L ys Gln Pro Ile Asn Arg 1905 191 0
1915 1920 Gly Gln Pro Lys Pro Ile Leu Gln Lys Gln S er Thr Phe Pro
Gln Ser 1925 1930 1935 Ser Lys Asp Ile Pro Asp Arg Gly Ala Ala T hr
Asp Glu Lys Leu Gln 1940 1945 1950 Asn Phe Ala Ile Glu Asn Thr Pro
Val Cys P he Ser His Asn Ser Ser 1955 1960 1965 Leu Ser Ser Leu Ser
Asp Ile Asp Gln Glu A sn Asn Asn Lys Glu Asn 1970 1975 1980 Glu Pro
Ile Lys Glu Thr Glu Pro Pro Asp S er Gln Gly Glu Pro Ser 1985 199 0
1995 2000 Lys Pro Gln Ala Ser Gly Tyr Ala Pro Lys S er Phe His Val
Glu Asp 2005 2010 2015 Thr Pro Val Cys Phe Ser Arg Asn Ser Ser L eu
Ser Ser Leu Ser Ile 2020 2025 2030 Asp Ser Glu Asp Asp Leu Leu Gln
Glu Cys I le Ser Ser Ala Met Pro 2035 2040 2045 Lys Lys Lys Lys Pro
Ser Arg Leu Lys Gly A sp Asn Glu Lys His Ser 2050 2055 2060 Pro Arg
Asn Met Gly Gly Ile Leu Gly Glu A sp Leu Thr Leu Asp Leu 2065 207 0
2075 2080 Lys Asp Ile Gln Arg Pro Asp Ser Glu His G ly Leu Ser Pro
Asp Ser 2085 2090 2095 Glu Asn Phe Asp Trp Lys Ala Ile Gln Glu G ly
Ala Asn Ser Ile Val 2100 2105 2110 Ser Ser Leu His Gln Ala Ala Ala
Ala Ala C ys Leu Ser Arg Gln Ala 2115 2120 2125 Ser Ser Asp Ser Asp
Ser Ile Leu Ser Leu L ys Ser Gly Ile Ser Leu 2130 2135 2140 Gly Ser
Pro Phe His Leu Thr Pro Asp Gln G lu Glu Lys Pro Phe Thr 2145 215 0
2155 2160 Ser Asn Lys Gly Pro Arg Ile Leu Lys Pro G ly Glu Lys Ser
Thr Leu 2165 2170 2175 Glu Thr Lys Lys Ile Glu Ser Glu Ser Lys G ly
Ile Lys Gly Gly Lys 2180 2185 2190 Lys Val Tyr Lys Ser Leu Ile Thr
Gly Lys V al Arg Ser Asn Ser Glu 2195 2200 2205 Ile Ser Gly Gln Met
Lys Gln Pro Leu Gln A la Asn Met Pro Ser Ile 2210 2215 2220 Ser Arg
Gly Arg Thr Met Ile His Ile Pro G ly Val Arg Asn Ser Ser 2225 223 0
2235 2240 Ser Ser Thr Ser Pro Val Ser Lys Lys Gly P ro Pro Leu Lys
Thr Pro 2245 2250 2255 Ala Ser Lys Ser Pro Ser Glu Gly Gln Thr A la
Thr Thr Ser Pro Arg 2260 2265 2270 Gly Ala Lys Pro Ser Val Lys Ser
Glu Leu S er Pro Val Ala Arg Gln 2275 2280 2285 Thr Ser Gln Ile Gly
Gly Ser Ser Lys Ala P ro Ser Arg Ser Gly Ser 2290 2295 2300 Arg Asp
Ser Thr Pro Ser Arg Pro Ala Gln G ln Pro Leu Ser Arg Pro 2305 231 0
2315 2320 Ile Gln Ser Pro Gly Arg Asn Ser Ile Ser P ro Gly Arg Asn
Gly Ile 2325 2330 2335 Ser Pro Pro Asn Lys Leu Ser Gln Leu Pro A rg
Thr Ser Ser Pro Ser 2340 2345 2350 Thr Ala Ser Thr Lys Ser Ser Gly
Ser Gly L ys Met Ser Tyr Thr Ser 2355 2360 2365 Pro Gly Arg Gln Met
Ser Gln Gln Asn Leu T hr Lys Gln Thr Gly Leu 2370 2375 2380 Ser Lys
Asn Ala Ser Ser Ile Pro Arg Ser G lu Ser Ala Ser Lys Gly 2385 239 0
2395 2400 Leu Asn Gln Met Asn Asn Gly Asn Gly Ala A sn Lys Lys Val
Glu Leu 2405 2410 2415 Ser Arg Met Ser Ser Thr Lys Ser Ser Gly S er
Glu Ser Asp Arg Ser 2420 2425 2430 Glu Arg Pro Val Leu Val Arg Gln
Ser Thr P he Ile Lys Glu Ala Pro 2435 2440 2445 Ser Pro Thr Leu Arg
Arg Lys Leu Glu Glu S er Ala Ser Phe Glu Ser 2450 2455 2460 Leu Ser
Pro Ser Ser Arg Pro Ala Ser Pro T hr Arg Ser Gln Ala Gln 2465 247 0
2475 2480 Thr Pro Val Leu Ser Pro Ser Leu Pro Asp M et Ser Leu Ser
Thr His 2485 2490 2495 Ser Ser Val Gln Ala Gly Gly Trp Arg Lys L eu
Pro Pro Asn Leu Ser 2500 2505 2510 Pro Thr Ile Glu Tyr Asn Asp Gly
Arg Pro A la Lys Arg His Asp Ile 2515 2520 2525 Ala Arg Ser His Ser
Glu Ser Pro Ser Arg L eu Pro Ile Asn Arg Ser
2530 2535 2540 Gly Thr Trp Lys Arg Glu His Ser Lys His S er Ser Ser
Leu Pro Arg 2545 255 0 2555 2560 Val Ser Thr Trp Arg Arg Thr Gly
Ser Ser S er Ser Ile Leu Ser Ala 2565 2570 2575 Ser Ser Glu Ser Ser
Glu Lys Ala Lys Ser G lu Asp Glu Lys His Val 2580 2585 2590 Asn Ser
Ile Ser Gly Thr Lys Gln Ser Lys G lu Asn Gln Val Ser Ala 2595 2600
2605 Lys Gly Thr Trp Arg Lys Ile Lys Glu Asn G lu Phe Ser Pro Thr
Asn 2610 2615 2620 Ser Thr Ser Gln Thr Val Ser Ser Gly Ala T hr Asn
Gly Ala Glu Ser 2625 263 0 2635 2640 Lys Thr Leu Ile Tyr Gln Met
Ala Pro Ala V al Ser Lys Thr Glu Asp 2645 2650 2655 Val Trp Val Arg
Ile Glu Asp Cys Pro Ile A sn Asn Pro Arg Ser Gly 2660 2665 2670 Arg
Ser Pro Thr Gly Asn Thr Pro Pro Val I le Asp Ser Val Ser Glu 2675
2680 2685 Lys Ala Asn Pro Asn Ile Lys Asp Ser Lys A sp Asn Gln Ala
Lys Gln 2690 2695 2700 Asn Val Gly Asn Gly Ser Val Pro Met Arg T hr
Val Gly Leu Glu Asn 2705 271 0 2715 2720 Arg Leu Asn Ser Phe Ile
Gln Val Asp Ala P ro Asp Gln Lys Gly Thr 2725 2730 2735 Glu Ile Lys
Pro Gly Gln Asn Asn Pro Val P ro Val Ser Glu Thr Asn 2740 2745 2750
Glu Ser Ser Ile Val Glu Arg Thr Pro Phe S er Ser Ser Ser Ser Ser
2755 2760 2765 Lys His Ser Ser Pro Ser Gly Thr Val Ala A la Arg Val
Thr Pro Phe 2770 2775 2780 Asn Tyr Asn Pro Ser Pro Arg Lys Ser Ser
A la Asp Ser Thr Ser Ala 2785 279 0 2795 2800 Arg Pro Ser Gln Ile
Pro Thr Pro Val Asn A sn Asn Thr Lys Lys Arg 2805 2810 2815 Asp Ser
Lys Thr Asp Ser Thr Glu Ser Ser G ly Thr Gln Ser Pro Lys 2820 2825
2830 Arg His Ser Gly Ser Tyr Leu Val Thr Ser V al 2835 2840 (2)
INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (vii) IMMEDIATE SOURCE: (B) CLONE:
ral2(yeast) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Leu Thr Gly
Ala Lys Gly Leu Gln Leu Arg A la Leu Arg Arg Ile Ala 1 5 10 15 Arg
Ile Glu Gln Gly Gly Thr Ala Ile Ser P ro Thr Ser Pro Leu 20 25 30
(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 29 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (vii) IMMEDIATE SOURCE: (B) CLONE: m3(mAChR) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 9: Leu Tyr Trp Arg Ile Tyr Lys Glu
Thr Glu L ys Arg Thr Lys Glu Leu 1 5 10 15 Ala Gly Leu Gln Ala Ser
Gly Thr Glu Ala G lu Thr Glu 20 25 (2) INFORMATION FOR SEQ ID NO:
10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 amino acids (B)
TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (vii) IMMEDIATE
SOURCE: (B) CLONE: MCC (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Leu Tyr Pro Asn Leu Ala Glu Glu Arg Ser A rg Trp Glu Lys Glu Leu 1
5 10 15 Ala Gly Leu Arg Glu Glu Asn Glu Ser Leu T hr Ala Met 20 25
(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 11: GTATCAAGAC TGTGACTTTT AATTGTAGTT TATCCATTTT 40 (2)
INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 12: TTTAGAATTT CATGTTAATA TATTGTGTTC TTTTTAACAG 40 (2)
INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 13: GTAGATTTTA AAAAGGTGTT TTAAAATAAT TTTTTAAGCT 40 (2)
INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 14: AAGCAATTGT TGTATAAAAA CTTGTTTCTA TTTTATTTAG 40 (2)
INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 15: GTAACTTTTC TTCATATAGT AAACATTGCC TTGTGTACTC 40 (2)
INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 16: NNNNNNNNNN NNNGTCCCTT TTTTTAAAAA AAAAAAATAG 40 (2)
INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 17: GTAAGTAACT TGGCAGTACA ACTTATTTGA AACTTTAATA 40 (2)
INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 18: ATACAAGATA TTGATACTTT TTTATTATTT GTGGTTTTAG 40 (2)
INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 19: GTAAGTTACT TGTTTCTAAG TGATAAAACA GYGAAGAGCT 40 (2)
INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 20: AATAAAAACA TAACTAATTA GGTTTCTTGT TTTATTTTAG 40 (2)
INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 21: GTTAGTAAAT TSCCTTTTTT GTTTGTGGGT ATAAAAATAG 40 (2)
INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 22: ACCATTTTTG CATGTACTGA TGTTAACTCC ATCTTAACAG 40 (2)
INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 23: GTAAATAAAT TATTTTATCA TATTTTTTAA AATTATTTAA 40 (2)
INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 64 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 24: CATGATGTTA TCTGTATTTA CCTATAGTCT AAATTATACC ATCTATAATG T
GCTTAATTT 60 TTAG 64
(2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 25: GTAACAGAAG ATTACAAACC CTGGTCACTA ATGCCATGAC TACTTTGCTA A
G 52 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 26: GGATATTAAA GTCGTAATTT
TGTTTCTAAA CTCATTTGGC CCACAG 46 (2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap
iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GTATGTTCTC
TATAGTGTAC ATCGTAGTGC ATGTTTCAAA 40 (2) INFORMATION FOR SEQ ID NO:
28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 56 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: CATCATTGCT
CTTCAAATAA CAAAGCATTA TGGTTTATGT TGATTTTATT T TTCAG 56 (2)
INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 29: GTAAGACAAA AATGTTTTTT AATGACATAG ACAATTACTG GTG 43 (2)
INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 30: TTAGATGATT GTCTTTTTCC TCTTGCCCTT TTTAAATTAG 40 (2)
INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 31: GTATGTTTTT ATAACATGTA TTTCTTAAGA TAGCTCAGGT ATGA 44 (2)
INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 32: GCTTGGCTTC AAGTTGNCTT TTTAATGATC CTCTATTCTG TATTTAATTT A
CAG 54 (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 65 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 33: GTACTATTTA GAATTTCACC
TGTTTTTCTT TTTTCTCTTT TTCTTTGAGG C AGGGTCTCA 60 CTCTG 65 (2)
INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 52 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 34: GCAACTAGTA TGATTTTATG TATAAATTAA TCTAAAATTG ATTAATTTCC A
G 52 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 35: GTACCTTTGA AAACATTTAG
TACTATAATA TGAATTTCAT GT 42 (2) INFORMATION FOR SEQ ID NO: 36: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap
iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: CCAACTCNAA
TTAGATGACC CATATTCAGA AACTTACTAG 40 (2) INFORMATION FOR SEQ ID NO:
37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: GTATATATAG
AGTTTTATAT TACTTTTAAA GTACAGAATT CATACTCTCA A AAA 54 (2)
INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 38: ATTGTGACCT TAATTTTGTG ATCTCTTGAT TTTTATTTCA G 41 (2)
INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 39: TCCCCGCCTG CCGCTCTC 18 (2) INFORMATION FOR SEQ ID NO:
40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: GCAGCGGCGG
CTCCCGTG 18 (2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 41: GTGAACGGCT CTCATGCTGC 20 (2)
INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 42: ACGTGCGGGG AGGAATGGA 19 (2) INFORMATION FOR SEQ ID NO:
43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: ATGATATCTT
ACCAAATGAT ATAC 24 (2) INFORMATION FOR SEQ ID NO: 44: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 44: TTATTCCTAC TTCTTCTATA CAG 23
(2) INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 45: TACCCATGCT GGCTCTTTTT C 21 (2) INFORMATION FOR SEQ ID
NO: 46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B)
TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 46: TGGGGCCATC TTGTTCCTGA 20 (2)
INFORMATION FOR SEQ ID NO: 47: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 47: ACATTAGGCA CAAAGCTTGC AA 22 (2) INFORMATION FOR SEQ ID
NO: 48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: ATCAAGCTCC
AGTAAGAAGG TA 22 (2) INFORMATION FOR SEQ ID NO: 49: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 49: TGCGGCTCCT GGGTTGTTG 19 (2)
INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 50: GCCCCTTCCT TTCTGAGGAC 20 (2) INFORMATION FOR SEQ ID NO:
51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: TTTTCTCCTG
CCTCTTACTG C 21 (2) INFORMATION FOR SEQ ID NO: 52: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 52: ATGACACCCC CCATTCCCTC 20 (2)
INFORMATION FOR SEQ ID NO: 53: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 53: CCACTTAAAG CACATATATT TAGT 24 (2) INFORMATION FOR SEQ ID
NO: 54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: GTATGGAAAA
TAGTGAAGAA CC 22 (2) INFORMATION FOR SEQ ID NO: 55: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 55: TTCTTAAGTC CTGTTTTTCT TTTG 24
(2) INFORMATION FOR SEQ ID NO: 56: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 56: TTTAGAACCT TTTTTGTGTT GTG 23 (2) INFORMATION FOR SEQ ID
NO: 57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: CTCAGATTAT
ACACTAAGCC TAAC 24 (2) INFORMATION FOR SEQ ID NO: 58: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 58: CATGTCTCTT ACAGTAGTAC CA 22
(2) INFORMATION FOR SEQ ID NO: 59: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 59: AGGTCCAAGG GTAGCCAAGG 20 (2) INFORMATION FOR SEQ ID NO:
60: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: TAAAAATGGA
TAAACTACAA TTAAAAG 27 (2) INFORMATION FOR SEQ ID NO: 61: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap
iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: AAATACAGAA
TCATGTCTTG AAGT 24 (2) INFORMATION FOR SEQ ID NO: 62: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 62: ACACCTAAAG ATGACAATTT GAG 23
(2) INFORMATION FOR SEQ ID NO: 63: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 63: TAACTTAGAT AGCAGTAATT TCCC 24 (2) INFORMATION FOR SEQ ID
NO: 64: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: ACAATAAACT
GGAGTACACA AGG 23 (2) INFORMATION FOR SEQ ID NO: 65: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 65: ATAGGTCATT GCTTCTTGCT GAT 23
(2) INFORMATION FOR SEQ ID NO: 66: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 66: TGAATTTTAA TGGATTACCT AGGT 24 (2) INFORMATION FOR SEQ ID
NO: 67: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: CTTTTTTTGC
TTTTACTGAT TAACG 25 (2) INFORMATION FOR SEQ ID NO: 68: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 68: TGTAATTCAT TTTATTCCTA
ATA.[.G.]. .Iadd.C.Iaddend.CTC 27 (2) INFORMATION FOR SEQ ID NO:
69: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: GGTAGCCATA
GTATGATTAT TTCT 24 (2) INFORMATION FOR SEQ ID NO: 70: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 70: CTACCTATTT TTATACCCAC AAAC 24
(2) INFORMATION FOR SEQ ID NO: 71: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 71: AAGAAAGCCT ACACCATTTT TGC 23 (2) INFORMATION FOR SEQ ID
NO: 72: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: GATCATTCTT
AGAACCATCT TGC 23 (2) INFORMATION FOR SEQ ID NO: 73: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 73: ACCTATAGTC TAAATTATAC CATC 24
(2) INFORMATION FOR SEQ ID NO: 74: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 74: GTCATGGCAT TAGTGACCAG 20 (2) INFORMATION FOR SEQ ID NO:
75: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: AGTCGTAATT
TTGTTTCTAA ACTC 24 (2) INFORMATION FOR SEQ ID NO: 76: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 76: TGAAGGACTC GGATTTCAC.[.G.].
.Iadd.C.Iaddend. C 21 (2) INFORMATION FOR SEQ ID NO: 77: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap
iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: TCATTCACTC
ACAGCCTGAT GAC 23 (2) INFORMATION FOR SEQ ID NO: 78: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 78: GCTTTGAAAC ATGCACTACG AT 22
(2) INFORMATION FOR SEQ ID NO: 79: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 79: AAACATCATT GCTCTTCAAA TAAC 24 (2) INFORMATION FOR SEQ ID
NO: 80: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80: TACCATGATT
TAAAAATCCA CCAG 24 (2) INFORMATION FOR SEQ ID NO: 81: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 81: GATGATTGTC TTTTTCCTCT TGC 23
(2) INFORMATION FOR SEQ ID NO: 82: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 82: CTGAGCTATC TTAAGAAATA CATG 24 (2) INFORMATION FOR SEQ ID
NO: 83: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83: TTTTAAATGA
TCCTCTATTC TGTAT 25 (2) INFORMATION FOR SEQ ID NO: 84: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 84: ACAGAGTCAG ACCCTGCCTC AAAG 24
(2) INFORMATION FOR SEQ ID NO: 85: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 85: TTTCTATTCT TACTGCTAGC ATT 23 (2) INFORMATION FOR SEQ ID
NO: 86: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86: ATACACAGGT
AAGAAATTAG GA 22 (2) INFORMATION FOR SEQ ID NO: 87: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 87: TAGATGACCC ATATTCTGTT TC 22
(2) INFORMATION FOR SEQ ID NO: 88: (i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 88: CAATTAGGTC TTTTTGAGAG TA 22 (2) INFORMATION FOR SEQ ID
NO: 89: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89: GTTACTGCAT
ACACATTGTG AC 22 (2) INFORMATION FOR SEQ ID NO: 90: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 90: GCTTTTTGTT TCCTAACATG AAG 23
(2) INFORMATION FOR SEQ ID NO: 91: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 91: TCTCCCACAG GTAATACTCC C 21 (2) INFORMATION FOR SEQ ID
NO: 92: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92: GCTAGAACTG
AATGGGGTAC G 21 (2) INFORMATION FOR SEQ ID NO: 93: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 93: CAGGACAAAA TAATCCTGTC CC 22
(2) INFORMATION FOR SEQ ID NO: 94: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 94: ATTTTCTTAG TTTCATTCTT CCTC 24 (2) INFORMATION FOR SEQ ID
NO: 95: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95: AGAAGGATCC
CTTGTGCAGT GTGGA 25 (2) INFORMATION FOR SEQ ID NO: 96: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 96 GACAGGATCC TGAAGCTGAG TTTG 24
(2) INFORMATION FOR SEQ ID NO: 97: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 97: TCAGAAAGTG CTGAAGAG 18 (2) INFORMATION FOR SEQ ID NO:
98: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98: GGAATAATTA
GGTCTCCAA 19 (2) INFORMATION FOR SEQ ID NO: 99: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 99: GCAAATCCTA AGAGAGAACA A 21 (2)
INFORMATION FOR SEQ ID NO: 100: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 100: GATGGCAAGC TTGAGCCAG 19 (2) INFORMATION FOR SEQ ID NO:
101: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101: GTTCCAGCAG
TGTCACAG 18 (2) INFORMATION FOR SEQ ID NO: 102: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 102: GGGAGATTTC GCTCCTGA 18
.Iadd.(2) INFORMATION FOR SEQ ID NO: 103: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 103: AGTACAAGGA TGCCAATATT ATG 23
(2) INFORMATION FOR SEQ ID NO: 104: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 104: ACTTCTATCT TTTTCAGAAC GAG 23 (2) INFORMATION FOR SEQ ID
NO: 105: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105: ATTTGAATAC
TACAGTGTTA CCC 23 (2) INFORMATION FOR SEQ ID NO: 106: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 106: CTTGTATTCT AATTTGGCAT AAGG 24
(2) INFORMATION FOR SEQ ID NO: 107: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 107: CTGCCCATAC ACATTCAAAC AC 22 (2) INFORMATION FOR SEQ ID
NO: 108: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108: TGTTTGCGTC
TTGCCCATCT T 21 (2) INFORMATION FOR SEQ ID NO: 109:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap
iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109: AGTCTTAAAT
ATTCAGATGA GCAG 24 (2) INFORMATION FOR SEQ ID NO: 110: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 110: GTTTCTCTTC ATTATATTTT ATGCTA
26 (2) INFORMATION FOR SEQ ID NO: 111: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 111: AAGCCTACCA ATTATAGTGA ACG 23
(2) INFORMATION FOR SEQ ID NO: 112: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 112: AGCTGATGAC AAAGATGATA ATC 23 (2) INFORMATION FOR SEQ ID
NO: 113: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113: AAGAAACAAT
ACAGACTTAT TGTG 24 (2) INFORMATION FOR SEQ ID NO: 114: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 114: ATGAGTGGGG TCTCCTGAAC 20 (2)
INFORMATION FOR SEQ ID NO: 115: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 115: ATCTCCCTCC AAAAGTGGTG C 21 (2) INFORMATION FOR SEQ ID
NO: 116: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: TCCATCTGGA
GTACTTTCTG TG 22 (2) INFORMATION FOR SEQ ID NO: 117: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 117: AGTAAATGCT GCAGTTCAGA GG 22
(2) INFORMATION FOR SEQ ID NO: 118: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 118: CCGTGGCATA TCATCCCCC 19 (2) INFORMATION FOR SEQ ID NO:
119: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo
sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119: CCCAGACTGC
TTCAAAATTA CC 22 (2) INFORMATION FOR SEQ ID NO: 120: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 120: GAGCCTCATC TGTACTTCTG C 21
(2) INFORMATION FOR SEQ ID NO: 121: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 121: CCCTCCAAAT GAGTTAGCTG C 21 (2) INFORMATION FOR SEQ ID
NO: 122: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122: TTGTGGTATA
GGTTTTACTG GTG 23 (2) INFORMATION FOR SEQ ID NO: 123: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 123: ACCCAACAAA AATCAGTTAG ATG 23
(2) INFORMATION FOR SEQ ID NO: 124: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 124: GTGGCTGGTA ACTTTAGCCT C 21 (2) INFORMATION FOR SEQ ID
NO: 125: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125: ATGATGTTGA
CCTTTCCAGG G 21 (2) INFORMATION FOR SEQ ID NO: 126: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 126: ATTGTGTAAC TTTTCATCAG TTGC 24
(2) INFORMATION FOR SEQ ID NO: 127: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 127: AAAGACATAC CAGACAGAGG G 21 (2) INFORMATION FOR SEQ ID
NO: 128: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 128: CTTTTTTGGC
ATTGCGGAGC T 21 (2) INFORMATION FOR SEQ ID NO: 129: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 129: AAGATGACCT GTTGCAGGAA TG
22
(2) INFORMATION FOR SEQ ID NO: 130: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 130: GAATCAGACC AAGCTTGTCT AGAT 24 (2) INFORMATION FOR SEQ
ID NO: 131: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 131: CAATAGTAAG
TAGTTTACAT CAAG 24 (2) INFORMATION FOR SEQ ID NO: 132: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 132: AAACAGGACT TGTACTGTAG GA 22
(2) INFORMATION FOR SEQ ID NO: 133: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 133: CAGCCCCTTC AAGCAAACAT C 21 (2) INFORMATION FOR SEQ ID
NO: 134: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134: GAGGACTTAT
TCCATTTCTA CC 22 (2) INFORMATION FOR SEQ ID NO: 135: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 135: CAGTCTCCTG GCCGAAACTC 20 (2)
INFORMATION FOR SEQ ID NO: 136: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 136: GTTGACTGGC GTACTAATAC AG 22 (2) INFORMATION FOR SEQ ID
NO: 137: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137: TGGTAATGGA
GCCAATAAAA AGG 23 (2) INFORMATION FOR SEQ ID NO: 138: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 138: TGGGACTTTT CGCCATCCAC 20 (2)
INFORMATION FOR SEQ ID NO: 139: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 139: TGTCTCTATC CACACATTCG TC 22 (2) INFORMATION FOR SEQ ID
NO: 140: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140: ATGTTTTTCA
TCCTCACTTT TTGC 24 (2) INFORMATION FOR SEQ ID NO: 141: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 141: GGAGAAGAAC TGGAAGTTCA TC 22
(2) INFORMATION FOR SEQ ID NO: 142: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 142: TTGAATCTTT AATGTTTGGA TTTGC 25 (2) INFORMATION FOR SEQ
ID NO: 143: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 143: TCTCCCACAG
GTAATACTCC C 21 (2) INFORMATION FOR SEQ ID NO: 144: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 144: GCTACAACTG AATGGGGTAC G 21
(2) INFORMATION FOR SEQ ID NO: 145: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 145: CAGGACAAAA TAATCCTGTC CC 22 (2) INFORMATION FOR SEQ ID
NO: 146: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 146: ATTTTCTTAC
TTTCATTCTT CCTC 24 (2) INFORMATION FOR SEQ ID NO: 147: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL
SOURCE: (A) ORGANISM: Artificial sequence (consensus) (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: 147: Phe Xaa Val Glu Xaa Thr Pro Xaa Cys
Phe S er Arg Xaa Ser Ser Leu 1 5 10 15 Ser Ser Leu Ser 20 (2)
INFORMATION FOR SEQ ID NO: 148: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM:
Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 148: Tyr Cys
Val Glu Asp Thr Pro Ile Cys Phe S er Arg Cys Ser Ser Leu 1 5 10 15
Ser Ser Leu Ser 20 (2) INFORMATION FOR SEQ ID NO: 149: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 149: His Thr Val Gln Glu Thr Pro Leu Met Phe S er Arg Cys
Thr Ser Val 1 5 10 15 Ser Ser Leu Asp 20 (2) INFORMATION FOR SEQ ID
NO: 150: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 150: Phe Ala Thr Glu Ser Thr
Pro Asp Gly Phe S er Cys Ser Ser Ser Leu 1 5 10 15 Ser Ala Leu Ser
20 (2) INFORMATION FOR SEQ ID NO: 151: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 151: Tyr Cys Val Glu Gly Thr Pro Ile Asn Phe S er Thr Ala
Thr Ser Leu 1 5 10 15 Ser Asp Leu Thr 20 (2) INFORMATION FOR SEQ ID
NO: 152: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 152: Thr Pro Ile Glu Gly Thr Pro
Tyr Cys Phe S er Arg Asn Asp Ser Leu 1 5 10 15 Ser Ser Leu Asp 20
(2) INFORMATION FOR SEQ ID NO: 153: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A)
ORGANISM: Homo sap iens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 153:
Phe Ala Ile Glu Asn Thr Pro Val Cys Pro S er His Asn Ser Ser Leu 1
5 10 15 Ser Ser Leu Ser 20 (2) INFORMATION FOR SEQ ID NO: 154: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi)
ORIGINAL SOURCE: (A) ORGANISM: Homo sap iens (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: 154: Arg His Val Glu Asp Thr Pro Val Cys
Phe S er Arg Asn Ser Ser Leu 1 5 10 15 Ser Ser Leu Ser
20.Iaddend.
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