U.S. patent application number 09/988686 was filed with the patent office on 2003-06-26 for chromosome 17p-linked prostrate cancer susceptibility gene and a paralog and orthologous genes.
This patent application is currently assigned to Myriad Genetics, Inc.. Invention is credited to Rommens, Johanna M., Simard, Jacques, Tavtigian, Sean V., Teng, David H. F..
Application Number | 20030120052 09/988686 |
Document ID | / |
Family ID | 24255961 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030120052 |
Kind Code |
A1 |
Tavtigian, Sean V. ; et
al. |
June 26, 2003 |
Chromosome 17p-linked prostrate cancer susceptibility gene and a
paralog and orthologous genes
Abstract
The present invention relates generally to the field of human
genetics. Specifically, the present invention relates to methods
and materials used to isolate and detect a human prostate cancer
predisposing gene (HPC2), some alleles of which cause
susceptibility to cancer, in particular prostate cancer. More
specifically, the present invention relates to germline mutations
in the HPC2 gene and their use in the diagnosis of predisposition
to prostate cancer. The invention also relates to presymptomatic
therapy of individuals who carry deleterious alleles of the HPC2
gene. The invention further relates to somatic mutations in the
HPC2 gene in human prostate cancer and their use in the diagnosis
and prognosis of human prostate cancer. Additionally, the invention
relates to somatic mutations in the HPC2 gene in other human
cancers and their use in the diagnosis and prognosis of human
cancers. The invention also relates to the therapy of human cancers
which have a mutation in the HPC2 gene, (including gene therapy,
protein replacement therapy, protein mimetics, and inhibitors). The
invention further relates to the screening of drugs for cancer
therapy. The invention also relates to the screening of the HPC2
gene for mutations, which are useful for diagnosing the
predisposition to prostate cancer. In addition, the invention
relates to a paralog of human HPC2, the paralog being named ELAC1,
and to orthologs of human HPC2, these being mouse Elac2, chimpanzee
Elac2 and gorilla Elac2.
Inventors: |
Tavtigian, Sean V.; (Salt
Lake City, UT) ; Teng, David H. F.; (Salt Lake City,
UT) ; Simard, Jacques; (St. Augustin de DesMaures,
CA) ; Rommens, Johanna M.; (Toronto, CA) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
Myriad Genetics, Inc.
Salt Lake City
UT
|
Family ID: |
24255961 |
Appl. No.: |
09/988686 |
Filed: |
November 20, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09988686 |
Nov 20, 2001 |
|
|
|
09564805 |
May 5, 2000 |
|
|
|
09564805 |
May 5, 2000 |
|
|
|
09434382 |
Nov 5, 1999 |
|
|
|
60107468 |
Nov 6, 1998 |
|
|
|
Current U.S.
Class: |
536/23.2 ;
435/226; 530/350 |
Current CPC
Class: |
C07K 16/3069 20130101;
C12Q 1/6886 20130101; C12Q 2600/156 20130101; C07K 14/47 20130101;
C12Q 2600/136 20130101; A61K 38/00 20130101; C07K 14/4748
20130101 |
Class at
Publication: |
536/23.2 ;
530/350; 435/226 |
International
Class: |
C07H 021/04; C12N
009/64; C07K 014/435 |
Goverment Interests
[0002] This application was made with Government support under
Grant Nos. CA62154 and CA64477 from the National Institutes of
Health. The United States Government has certain rights in this
invention.
Claims
What is claimed is:
1. An isolated nucleic acid coding for an ELAC1 polypeptide, said
polypeptide comprising the amino acid sequence set forth in SEQ ID
NO:220 or a modified form which is functionally equivalent.
2. The isolated nucleic acid of claim 1 wherein said nucleic acid
comprises a nucleotide sequence (a) set forth in SEQ ID NO:219, (b)
its complement, (c) a corresponding RNA or (d) a nucleotide
sequence which hybridizes under stringent conditions to the
nucleotide sequence of (a), (b) or (c).
3. The isolated nucleic acid of claim 1 comprising an allelic
variant of SEQ ID NO:219, I its complement or a corresponding
RNA.
4. The isolated nucleic acid of claim 1 comprising a mutation.
5. An isolated polypeptide of SEQ ID NO:220.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of Ser. No. 09/564,805,
filed May 5, 2000, which is a continuation-in-part of Ser. No.
09/434,382, filed Nov. 5, 1999 and is related to U.S. provisional
patent application Ser. No. 60/107,468, filed Nov. 6, 1998, and
priority is claimed thereto under 35 U.S.C. .sctn.119(e). Each of
these applications is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] The publications and other materials used herein to
illuminate the background of the invention, and in particular,
cases to provide additional details respecting the practice, are
incorporated herein by reference, and for convenience, are
referenced by author and date in the following text and
respectively grouped in the appended List of References.
[0004] The genetics of cancer is complicated, involving the
function of three loosely defined classes of genes: (1) dominant,
positive regulators of the transformed state (oncogenes); (2)
recessive, negative regulators of the transformed state (tumor
suppressor genes); and (3) genes that modify risk without playing a
direct role in the biology of transformed cells (risk
modifiers).
[0005] Specific germline alleles of certain oncogenes and tumor
suppressor genes are causally associated with predisposition to
cancer. This set of genes is referred to as tumor predisposition
genes. Some of the tumor predisposition genes which have been
cloned and characterized influence susceptibility to: 1)
Retinoblastoma (RB1); 2) Wilms' tumor (WT1); 3) Li-Fraumeni (TP53);
4) Familial adenomatous polyposis (APC); 5) Neurofibromatosis type
1 (NF1); 6) Neurofibromatosis type 2 (NF2); 7) von Hippel-Lindau
syndrome (VHL); 8) Multiple endocrine neoplasia type 2A (MEN2A); 9)
Melanoma (CDKN2 and CDK4); 10) Breast and ovarian cancer (BRCAI and
BRCA2); 11) Cowden disease (MMAC1); 12) Multiple endocrine
neoplasia (MEN1); 13) Nevoid basal cell carcinoma syndrome (PTC);
14) Tuberous sclerosis 2 (TSC2); 15) Xeroderma pigmentosum (genes
involved in nucleotide excision repair); 16) Hereditary
nonpolyposis colorectal cancer (genes involved in mismatch
repair).
[0006] Specific germline alleles of certain risk modifier genes are
also associated with predisposition to cancer, but the increased
risk is sometimes only clearly expressed when it is combined with
certain environmental, dietary, or other factors. Alcohol
dehydrogenase (ADH) oxidizes ethanol to acetaldehyde, a chemical
which is both mutagenic and carcinogenic in lab animals. The enzyme
encoded by the ADH3.sup.1 allele oxidizes ethanol relatively
quickly, whereas the enzyme encoded by the ADH3.sup.2 allele
oxidizes ethanol more slowly. ADH3.sup.1 homozygotes presumably
have a high capacity for synthesis of acetaldehyde; those who also
drink heavily are at increased risk for oral cavity, esophageal,
and (in women) breast cancer relative to ADH3.sup.2 homozygotes who
drink equally heavily (Harty et al., 1997; Hori et al., 1997;
Shields, 1997). The acetyltransferases encoded by
N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2)
catalyze the acetylation of numerous xenobiotics including the
aromatic amine carcinogens derived from smoking tobacco products.
Individuals who are homozygous for slow acetylating forms of NAT2
who are also heavy smokers are at greater risk for lung, bladder,
and (in females) breast cancer than individuals who smoke equally
heavily but are homozygous for fast acetylating forms of NAT2
(Shields, 1997; Bouchardy et al., 1998).
[0007] The risk of hormone related cancers such as breast and
prostate cancer may be modulated by allelic variants in enzymes
that play a role in estrogen or androgen metabolism, or variants in
proteins that mediate the biological effects of estrogens or
androgens. A polymorphic CAG repeat in the human androgen receptor
gene encodes a polymorphic polyglutamine tract near the
amino-terminus of the protein. The length of the polyglutamine
tract is inversely correlated with the transcriptional activation
activity of the androgen receptor and thus one aspect of the
biological response to androgens. Men whose androgen receptor
contains a relatively short polyglutamine tract are at higher risk
for prostate cancer, especially high stage/high histologic grade
prostate cancer, than men whose androgen receptor contains a
relatively long polyglutamine tract (Giovannucci et al., 1997).
[0008] Prostate cancer is the most common cancer in men in many
western countries, and the second leading cause of cancer deaths in
men. It accounts for more than 40,000 deaths in the US annually.
The number of deaths is likely to continue rising over the next 10
to 15 years. In the US, prostate cancer is estimated to cost $1.5
billion per year in direct medical expenses. In addition to the
burden of suffering, it is a major public-health issue. Numerous
studies have provided evidence for familial clustering of prostate
cancer, indicating that family history is a major risk factor for
this disease (Cannon et al., 1982; Steinberg et al., 1990; Carter
et al, 1993).
[0009] Prostate cancer has long been recognized to be, in part, a
familial disease with a genetic component (Woolf, 1960a; Cannon et
al., 1982; Carter et al., 1992). Numerous investigators have
examined the evidence for genetic inheritance and concluded that
the data are most consistent with dominant inheritance for a major
susceptibility locus or loci. Woolf (1960b), described a relative
risk of 3.0 of developing prostate cancer among first-degree
relatives of prostate cancer cases in Utah using death certificate
data. Relative risks ranging from 3 to 11 for first-degree
relatives of prostate cancer cases have been reported (Cannon et
al., 1982; Woolf, 1960b; Fincham et al., 1990; Meikle et al., 1985;
Krain, 1974; Morganti et al., 1956; Goldgar et al., 1994). Carter
et al. (1992) performed segregation analysis on families
ascertained through a single prostate cancer proband. The analysis
suggested Mendelian inheritance in a subset of families through
autosomal dominant inheritance of a rare (q=0.003), high-risk
allele with estimated cumulative risk of prostate cancer for
carriers of 88% by age 85. Inherited prostate cancer susceptibility
accounted for a significant proportion of early-onset disease, and
overall was responsible for 9% of prostate occurrence by age 85.
Recent results demonstrate that at least four loci exist which
convey susceptibility to prostate cancer as well as other cancers.
These loci are HPC1 on chromosome 1q24, (Smith et al., 1996), HPCX
on chromosome Xq27-28 (Xu et al., 1998), PCAP at lq42 (Berthon et
al., 1998) and CAPB at 1p36 (Gibbs et al., 1999a). All four
suggestions of linkage for prostate cancer predisposition were the
result of hints arising from genome-wide searches. Although only
the HPC1 linkage has so far been confirmed (Cooney et al., 1997;
Neuhausen et al., 1999; Xu and ICPCG, 2000), it is becoming clear
that a large number of genes contribute to familial prostate
cancer. It also seems clear, both from published hereditary
prostate cancer linkage studies and from genotyping of our family
resource at the above mentioned loci, that no single predisposition
locus mapped to date is by itself responsible for a large portion
of familial prostate cancer (Neuhausen et al., 1999; Eeles et al.,
1998; Gibbs et al., 1999b; Lange et al., 1999; Berry et al., 2000;
Suarez et al., 2000; Goode et al., 2000).
[0010] A comparison to the cloning of, and risk profile attributed
to, breast cancer susceptibility genes provides an instructive
example. The profusion of proposed prostate loci, coupled with
minimal confirmation or refined localization following initial
publication of these linkages, contrasts sharply with studies of
the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2.
Linkage to BRCA1 was first published in 1990 (Hall et al., 1990);
groups competing to identify this gene moved swiftly from
confirmatory studies through efforts to refine the localization to
the gene identification in 1994 (Miki et al., 1994). With expanded
genomics resources, the time from linkage (Wooster et al., 1994) to
complete cloning (Wooster et al., 1995; Tavtigian et al., 1996) of
BRCA2 was only slightly more than 1 year. Ongoing mutation
screening and careful modeling of age specific and familial risks
indicate that these two genes account for virtually all extended
breast and ovarian cancer families (Antoniou et al., 2000) and the
majority of breast cancer families with more than five cases,
especially those that include an early-onset component (Ford et
al., 1998).
[0011] Even so, a fraction of familial breast cancer risk is
manifest in smaller family clusters with average age at diagnosis.
While BRCA1 and BRCA2 only account for a portion of this component
of breast cancer risk (Peto et al., 1999), there are no published
and confirmed linkages based on these types of pedigrees to date.
Standard genetic analysis appears to be limited by the problems of
low penetrance and genetic complexity. It is possible that analysis
of genetic predisposition in families with excess prostate cancer
also reflects these issues. As absence of distinction by age at
diagnosis/onset would also be consistent with the influence of
multiple susceptibility genes harboring only moderate risk sequence
variants, one might therefore ask what relative contribution low
frequency high risk variants analogous to mutations in BRCA1/2,
versus higher frequency, moderate risk sequence variants, make to
the population risk of prostate cancer.
[0012] Indeed, evidence that moderate risk sequence variants in a
number of specific genes contribute to prostate cancer
susceptibility is beginning to accumulate. For example, a
polymorphic CAG repeat within the androgen receptor open reading
frame encoding a variable length polyglutamine tract shows an
inverse relationship between repeat length and the transcriptional
transactivation activity of the receptor (Chamberlain et al., 1994;
Kazemi-Esfarjani et al., 1995). Accordingly, a series of studies
show an association between shorter androgen receptor CAG repeat
length and prostate cancer risk (Giovannucci et al., 1997; Stanford
et al., 1997), although it is not entirely clear whether the
association is with diagnosis of prostate cancer or severity of
disease (Bratt et al., 1999). Second, a number of missense variants
have been observed in the steroid 5.alpha.-reductase type II gene
(SRD5A2), responsible for conversion of testosterone to the more
active androgen dihydrotestosterone in the prostate (Makridakis et
al., 1997). One of these variants, Ala 49 Thr, has been reported to
increase the catalytic activity of the enzyme, and is associated
with increased risk of advanced prostate cancer (Makridakis et al.,
1999; Jaffe et al., 2000). Finally, several groups have reported an
excess of prostate cancer in large BRCA2 pedigrees (Sigurdsson et
al., 1997; Breast Cancer Linkage Consortium, 1999), though the
relative risk that these mutations confer for prostate cancer is
considerably lower than for breast cancer. Further, these effects
may be variant specific as association has not been confirmed among
men who carry the Ashkenazi BRCA2 founder mutation 6174delT
(Wilkens et al., 1999; Nastiuk et al., 1999; Hubert et al., 1999).
If these and similar sequence variants play a role in a significant
fraction of prostate cancer, then models of the genetic component
of familial prostate cancer may need to incorporate both linkage
evidence for major susceptibility loci and association evidence for
moderate risk sequence variants.
[0013] The Utah population provides a unique resource for examining
the genetic basis of disease. Extended high risk pedigrees
containing many cases can be ascertained as units instead of by
expansion from individual probands. While these pedigrees are an
extremely powerful resource for linkage studies, they also allow
analysis of segregation of moderate risk sequence variants through
multiple generations of both cases and their unaffected
relatives.
[0014] Detection of genetic linkage for prostate cancer
susceptibility to a defined segment of a chromosome requires that
DNA sequence variants within that chromosomal segment confer the
cancer susceptibility. This is usually taken to mean that the
causal sequence variant(s) will either alter the expression of one
or more linked genes or will alter the function of one of the
linked genes. However, detection of the genetic linkage does not
necessarily provide evidence for what class of gene (i.e. tumor
suppressor, oncogene, or risk modifier) is affected by the causal
sequence variant(s).
[0015] Most strategies for proceeding from genetic linkage of
prostate cancer susceptibility to chromosome 17p to identification
of the 17p-linked prostate cancer predisposing gene (HPC2) require
precise genetic localization studies to define a discrete segment
of the chromosome within which the causal sequence variant(s) must
map. Gene identification projects based on precise genetic
localization are called positional cloning projects. The general
strategy in positional cloning is to find all of the genes located
within the genetically defined interval, identify sequence variants
in and around those genes, and then determine which of those
sequence variants either alter the expression or the function of
one (or more) of the associated genes. Segregation of such sequence
variants with the disease in the linked kindreds must also be
demonstrated. We have executed a positional cloning project in the
HPC2 region of chromosome 17p and found a gene, herein named HPC2,
germline mutations which predisposes individuals to prostate
cancer.
SUMMARY OF THE INVENTION
[0016] The present invention relates generally to the field of
human genetics. Specifically, the present invention relates to
methods and materials used to isolate and detect a human prostate
cancer predisposing gene (HPC2), some alleles of which cause
susceptibility to cancer, in particular prostate cancer. More
specifically, the present invention relates to germline mutations
in the HPC2 gene and their use in the diagnosis of predisposition
to prostate cancer. The invention also relates to presymptomatic
therapy of individuals who carry deleterious alleles of the HPC2
gene. The invention further relates to somatic mutations in the
HPC2 gene in human prostate cancer and their use in the diagnosis
and prognosis of human prostate cancer. Additionally, the invention
relates to somatic mutations in the HPC2 gene in other human
cancers and their use in the diagnosis and prognosis of human
cancers. The invention also relates to the therapy of human cancers
which have a mutation in the HPC2 gene, including gene therapy,
protein replacement therapy, protein mimetics, and inhibitors. The
invention further relates to the screening of drugs for cancer
therapy. The invention also relates to the screening of the HPC2
gene for mutations, which are useful for diagnosing the
predisposition to prostate cancer. The HPC2 gene is useful as a
marker for the HPC2 locus and as a marker for prostate cancer.
Finally, a paralog of HPC2 as well as orthologs of HPC2 in mouse,
chimpanzee and gorilla have been isolated and characterized.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a multipoint linkage analysis of 4 kindreds that
show suggestive evidence for linkage to the HPC2 prostate cancer
susceptibility locus relative to chromosome 17p markers.
[0018] FIGS. 2A-B are diagrams showing the order of genetic markers
and recombinant boundaries neighboring HPC2, a schematic map of
BACs spanning the HPC2 region, a schematic map of transcription
units within the HPC2 region, and two diagrams of the HPC2
transcription unit showing the locations of the exons of HPC2
relative to the BAC to which it maps and relative to each other.
The individual exons are numbered.
[0019] FIG. 3 shows recombinant, physical and transcript maps
centered at the human ELAC2 locus on chromosome 17p. The top
portion shows genetic markers and recombinants. Microsatellite
markers developed at Myriad Genetics, Inc. are given as 17-MYR####.
Nested within the arrows that represent meiotic recombinants are
the pedigree in which the recombinant occurred and, in parentheses,
the number of cases who carry the haplotype on which the
recombinant occurred. The second portion of the figure shows a BAC
contig tiling path across this interval. The T7 end of each BAC is
denoted with an arrowhead. The third portion of the figure shows
transcription units identified in the interval. The bottom portion
of the figure is an expanded view of a 40 kb segment at the SP6 end
of BAC 31k12 showing the relative positions of two exons of the
gene 04CG09 and all of the coding exons of ELAC2.
[0020] FIG. 4 is an alignment of the sequence of exon 1 of the
human HPC2 gene with exon 1 of the mouse HPC2 gene. The figure also
shows an alignment of the peptide sequence encoded by exon 1 of the
human HPC2 gene with the peptide sequence encoded by exon 1 of the
mouse HPC2 gene. The human DNA sequence is SEQ ID NO:210; the human
amino acid sequence is SEQ ID NO:211; the mouse DNA sequence is SEQ
ID NO:212 and the mouse amino acid sequence is SEQ ID NO:213.
[0021] FIGS. 5A-B show kindreds 4102 and 4289. The pedigrees have
been genotyped over a 20 cM interval extending from D17S786 to
D17S805. Haplotypes are represented by the bars; the dark gray
haplotype segregating in each pedigree is the mutation bearing
chromosome. The relative position of ELAC2 is denoted by * (white
on black or white on gray).
[0022] FIG. 5A shows kindred 4102. The dark bar denotes the 1641
insG bearing haplotype. Individuals 061 and 107 carry part of the
frameshift haplotype, but neither carries the frameshift due to
recombination events. There are no data to distinguish which of the
founders, individuals 050 and 051, carried the frameshift. The
second shared haplotype in kindred 4102 is denoted by a light gray
bar. Again, there are no data to distinguish which of the founders,
individuals 005 and 006, carried the haplotype.
[0023] FIG. 5B shows kindred 4289. Individuals 064, 066, 067, 068
and 072 share a recombinant chromosome that carries the His 781
missense change.
[0024] FIGS. 6A-B are a multiple protein alignment of ELAC1/2
family members. ELAC2 family members were selected from human
(HSA), mouse (MMU), C. elegans (CEL), A. thaliana (ATH) and S.
cerevisiae (SCE). The A. thaliana genome encodes more than one
family member; gi6850339 was selected because it aligned with fewer
gaps. ELAC1 family members were selected from human, E. coli
(Es_c), the blue-green algae Synechocystis (Syn) and the
archaebacterium Methanobacterium thermoautotrophicum (Me_t).
Alignments were based on BLASTp searches and then optimized by
inspection. The positions of Ser 217, Ala 541 and Arg 781 in human
ELAC2 are marked by down arrows. The sequences shown in FIGS. 6A-B
are: human ELAC2 is SEQ ID NO:2, mouse Elac2 is SEQ ID NO:222, C.
elegans CE16965 is SEQ ID NO:227, A. thaliana gi 6850339 is SEQ ID
NO:228, S. cerevisiae YKRO79C is SEQ ID NO:229, human ELAC1 is SEQ
ID NO:220, E. coli elaC is SEQ ID NO:230, Synechocystis gi2500943
is SEQ ID NO:231, and Methanobacterium thermoautotrophicum
gi2622965 is SEQ ID NO:232.
[0025] FIG. 7 shows recessive genotype frequencies by birth
cohort.
[0026] FIG. 8 shows the results of association tests.
[0027] FIG. 9 shows a multiple protein alignment demonstrating
conservation of sequence elements between ELAC2, PSO2 and CPSF73
families. The alignments shown for segments of PSO2 and CPSF73
family members were taken from more extensive alignments that
contain family members from a larger set of species, analogous to
the ELAC1/2 alignments of FIGS. 6A-B. The seven His or Cys residues
that are conserved across two or more of the gene families are
marked by down arrows. The position of Ala 541 in human ELAC2 is
also marked by a down arrow. The sequences shown in FIG. 9 are
partial sequences of the following: human CPSF73 is SEQ ID NO:233,
A. thaliana gi6751699 is SEQ ID NO:234, S. cerevisiae YSH1 is SEQ
ID NO:235, Synechocystis gi2496795 is SEQ ID NO:236,
Methanobacterium thermoautotrophicum gi2622312 is SEQ ID NO:237,
human ha3611 is SEQ ID NO:238, A. thaliana gi2979557 is SEQ ID
NO:239, S. cerevisiae PSO2 is SEQ ID NO:240, human ELAC2 is SEQ ID
NO:2, A. thaliana gi6850339 is SEQ ID NO:228, and S. cerevisiae
YKRO79C is SEQ ID NO:229.
[0028] FIG. 10 shows a similarity comparison among the ELAC2 family
members aligned in FIGS. 6A-B.
[0029] FIGS. 11A-D shows an analysis of ELAC1 expression in human
tissues. FIGS. 11 A-B show Multiple Tissue Northern (MTN) filters
(Clontech) probed with the human ELAC1 ORF. Note that a 3 kb ELAC1
transcript is detected in all tissues. FIGS. 11C-D show the same
filters probed with human .beta.-actin as a loading control.
[0030] FIG. 12 shows a multiple protein alignment demonstrating
similarity between an N-terminal segment of the ELAC2 family
members and the sequence context of the histidine motif shared by
ELAC1 and ELAC2 family members. Species abbreviations are as in
FIGS. 6A-B. The sequences shown in FIG. 12 are partial sequences of
the following: human ELAC2 is SEQ ID NO:2, mouse Elac2 is SEQ ID
NO:222, C. elegans CE16965 is SEQ ID NO:227, A. thaliana gi6850339
is SEQ ID NO:228, S. cerevisiae YKRO79C is SEQ ID NO:229, human
ELAC1 is SEQ ID NO:220, E. coli elaC is SEQ ID NO:230,
Synechocystis gi2500943 is SEQ ID NO:231, and Methanobacterium
thermoautotrophicum gi2622965 is SEQ ID NO:232.
[0031] FIG. 13 shows the relationship between ELAC1/2, PSO2 and
CPSF73 gene family members. The tree is a distance-based depiction
of pairwise sequence similarities determined from a manual
alignment of the .about.67 amino acids immediately surrounding the
histidine motif. ClustalX (Thompson et al., 1997) was used to
calculate the percent divergence of each sequence on a pairwise
basis and neighbor joining (Saitou and Nei, 1987) was applied to
the resulting distance matrix. The treefile produced from ClustalX
was visualized using TreeView (Page, 1996) and further edited in a
graphics program for aesthetics. The scale bar indicates amino acid
substitutions per residue.
BRIEF DESCRIPTION OF THE TABLES
[0032] Table 1 is a compilation of 2-point LOD scores for markers
in the HPC2 region.
[0033] Table 2A lists the family resource used to detect linkage of
HPC2 to chromosome 17p.
[0034] Table 2B lists two-point LOD scores using the Utah
age-specific model.
[0035] Table 3 is a summary of resource genotyped for the
association tests.
[0036] Table 4 is a list of the accession numbers of human EST
sequences used to assemble a tentative, partial cDNA sequence of
the human HPC2 gene.
[0037] Table 5 is a list of the primers used for obtaining 5' RACE
products that contained the start codon and part of the 5' UTR of
the human HPC2 gene, primers used to prepare a full length human
HPC2 expression construct, and primers used to check the sequence
of that construct.
[0038] Table 6 is a list of the accession numbers of mouse EST
sequences used to assemble a tentative, partial cDNA sequence of
the mouse HPC2 gene.
[0039] Table 7 is a list of the primers used for obtaining 5' RACE
products that contained the start codon and part of the 5' UTR of
the mouse HPC2 gene, primers used to prepare a full length mouse
HPC2 expression construct, and primers used to check the sequence
of that construct.
[0040] Table 8 is a list of the primers used to mutation screen the
human HPC2 gene from genomic DNA.
[0041] Table 9 is a summary of germline sequence variants of the
human HPC2 gene.
[0042] Table 10 is a list of the allele frequencies of HPC2.
SUMMARY OF SEQUENCE LISTING
[0043] SEQ ID NO: 1 is the nucleotide sequence for the human HPC2
cDNA from the start codon through the stop codon.
[0044] SEQ ID NO:2 is the amino acid sequence for the human HPC2
protein.
[0045] SEQ ID NO:3 is the nucleotide sequence for the human HPC2
cDNA from 50 base pairs before the start codon through the end of
the 3' UTR.
[0046] SEQ ID NO:4 to SEQ ID NO:27 are the sequences of exon 1 to
exon 24 of the human HPC2 gene.
[0047] SEQ ID NO:28 is the genomic sequence of the human HPC2
gene.
[0048] SEQ ID NOs:29-190 are nucleotide sequences of primers used
to identify the human and/or mouse HPC2 genes or to screen for
mutations.
[0049] SEQ ID NOs:191-209 are nucleotide sequences of the HPC2
around and including various sequence variants.
[0050] SEQ ID NO:210 is the nucleotide sequence of human HPC2 exon
1 and SEQ ID NO:211 is the corresponding amino acid sequence as
shown in FIG. 4.
[0051] SEQ ID NO:212 is nucleotide sequence of mouse HPC2 exon 1
and SEQ ID NO:213 is the corresponding amino acid sequence as shown
in FIG. 4.
[0052] SEQ ID NO:214 is a histidine containing motif found in
HPC2/ELA2 and ELAC1.
[0053] SEQ ID NO:215 is exon 1 of ELAC1.
[0054] SEQ ID NO:216 is exon 2 of ELAC1 plus surrounding genomic
sequence.
[0055] SEQ ID NO:217 is exon 3 of ELAC1 plus surrounding genomic
sequence.
[0056] SEQ ID NO:218 is exon 4 of ELAC1 plus surrounding genomic
sequence.
[0057] SEQ ID NO:219 is the cDNA for ELAC1 and SEQ ID NO:220 is the
amino acid sequence for ELAC1.
[0058] SEQ ID NO:221 is the cDNA for mouse ELAC2 and SEQ ID NO:222
is the amino acid sequence for mouse ELAC2.
[0059] SEQ ID NO:223 is the cDNA for chimpanzee ELAC2 and SEQ ID
NO:224 is the amino acid sequence for chimpanzee ELAC2.
[0060] SEQ ID NO:225 is the cDNA for gorilla ELAC2 and SEQ ID
NO:226 is the amino acid sequence for gorilla ELAC2.
[0061] SEQ ID NOs:227-229 are the amino acid sequences for ELAC2
family member proteins from C. elegans, A. thaliana and S.
cerevisiae as shown in FIG. 6A.
[0062] SEQ ID NOs:230-232 are the amino acid sequences for ELAC1
family member proteins from E. coli, Synechocystis and
Methanobacterium thermoautotrophicum as shown in FIGS. 6A-B.
[0063] SEQ ID NOs:233-240 are amino acid sequences of proteins from
CPSF73 and PSO2 families as shown in FIG. 9. These are,
respectively, human CPSF73, A. thaliana gi6751699, S. cerevisiae
YSH1, Synechocystis gi2496795, Methanobacterium thermoautotrophicum
gi2622312, human ha3611, A. thaliana gi2979557 and S. cerevisiae
PSO2. The sequences for the ELAC2 family of FIG. 9 are SEQ ID NO:2
for human, SEQ ID NO:228 for A. thaliana (as for FIGS. 6A-B) and
SEQ ID NO:229 for S. cerevisiae (as for FIGS. 6A-B). The sequence
listing shows the complete sequences of these proteins whereas FIG.
9 shows only portions of each sequence.
DETAILED DESCRIPTION OF TEE INVENTION
[0064] The present invention provides an isolated polynucleotide
comprising all, or a portion of the HPC2 locus or of a mutated HPC2
locus, preferably at least eight bases and not more than about 27
kb in length. Such polynucleotides may be antisense
polynucleotides. The present invention also provides a recombinant
construct comprising such an isolated polynucleotide, for example,
a recombinant construct suitable for expression in a transformed
host cell.
[0065] Also provided by the present invention are methods of
detecting a polynucleotide comprising a portion of the HPC2 locus
or its expression product in an analyte. Such methods .may further
comprise the step of amplifying the portion of the HPC2 locus, and
may further include a step of providing a set of polynucleotides
which are primers for amplification of said portion of the HPC2
locus. The method is useful for either diagnosis of the
predisposition to cancer or the diagnosis or prognosis of cancer.
The HPC2 gene is useful as a marker for the HPC2 locus and as a
marker for prostate cancer.
[0066] The present invention also provides isolated antibodies,
preferably monoclonal antibodies, which specifically bind to an
isolated polypeptide comprised of at least five amino acid residues
encoded by the HPC2 locus.
[0067] The present invention also provides kits for detecting in an
analyte a polynucleotide comprising a portion of the HPC2 locus,
the kits comprising a polynucleotide complementary to the portion
of the HPC2 locus packaged in a suitable container, and
instructions for its use.
[0068] The present invention further provides methods of preparing
a polynucleotide comprising polymerizing nucleotides to yield a
sequence comprised of at least eight consecutive nucleotides of the
HPC2 locus; and methods of preparing a polypeptide comprising
polymerizing amino acids to yield a sequence comprising at least
five amino acids encoded within the HPC2 locus.
[0069] The present invention further provides methods of screening
the HPC2 gene to identify mutations. Such methods may further
comprise the step of amplifying a portion of the HPC2 locus, and
may further include a step of providing a set of polynucleotides
which are primers for amplification of said portion of the HPC2
locus. Such methods may also include a step of providing the
complete set of short polynucleotides defined by the sequence of
HPC2 or discrete subsets of that sequence, all single-base
substitutions of that sequence or discrete subsets of that
sequence, all 1-, 2-, 3-, or 4-base deletions of that sequence or
discrete subsets of that sequence, and all 1-, 2-, 3-, or 4-base
insertions in that sequence or discrete subsets of that sequence.
The method is useful for identifying mutations for use in either
diagnosis of the predisposition to cancer or the diagnosis or
prognosis of cancer.
[0070] The present invention further provides methods of screening
suspected HPC2 mutant alleles to identify mutations in the HPC2
gene.
[0071] In addition, the present invention provides methods to
screen drugs for inhibition or restoration of HPC2 gene product
function as an anticancer therapy.
[0072] The present invention also provides the means necessary for
production of gene-based therapies directed at cancer cells. These
therapeutic agents may take the form of polynucleotides comprising
all or a portion of the HPC2 locus placed in appropriate vectors or
delivered to target cells in more direct ways such that the
function of the HPC2 protein is reconstituted. Therapeutic agents
may also take the form of polypeptides based on either a portion
of, or the entire protein sequence of HPC2. These may functionally
replace the activity of HPC2 in vivo.
[0073] Finally, the present invention provides the sequence of a
paralog of HPC2, herein called ELAC1, as well as the sequences of
HPC2 orthologs from mouse, chimpanzee and gorilla. These orthologs
are named ELAC2.
[0074] It is a discovery of the present invention that the HPC2
locus which predisposes individuals to prostate cancer, is a gene
encoding an HPC2 protein, which has been found to be non-identical
to publicly available protein or CDNA sequences. This gene is
termed HPC2 herein. It is a discovery of the present invention that
mutations in the HPC2 locus in the germline are indicative of a
predisposition to prostate cancer. Finally, it is a discovery of
the present invention that germline mutations in the HPC2 locus are
also associated with prostate cancer and other types of cancer. The
mutational events of the HPC2 locus can involve deletions,
insertions and nucleotide substitutions within the coding sequence
and the non-coding sequence.
[0075] Useful Diagnostic Techniques
[0076] According to the diagnostic and prognostic method of the
present invention, alteration of the wild-type HPC2 locus is
detected. In addition, the method can be performed by detecting the
wild-type HPC2 locus and confirming the lack of a predisposition to
cancer at the HPC2 locus. "Alteration of a wild-type gene"
encompasses all forms of mutations including deletions, insertions
and point mutations in the coding and noncoding regions. Deletions
may be of the entire gene or of only a portion of the gene. Point
mutations may result in stop codons, frameshift mutations or amino
acid substitutions. 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 and are inherited. If only a single allele is somatically
mutated, an early neoplastic state is indicated. However, if both
alleles are somatically mutated, then a late neoplastic state is
indicated. The finding of HPC2 mutations thus provides both
diagnostic and prognostic information. An HPC2 allele which is not
deleted (e.g., found on the sister chromosome to a chromosome
carrying an HPC2 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 HPC2 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 reduction or loss of
expression of the HPC2 gene product, expression of an altered HPC2
gene product, or to a decrease in mRNA stability or translation
efficiency.
[0077] Useful diagnostic techniques include, but are not limited to
fluorescent in situ hybridization (FISH), direct DNA sequencing,
PFGE analysis, Southern blot analysis, single stranded conformation
analysis (SSCA), RNase protection assay, allele-specific
oligonucleotide (ASO), dot blot analysis, hybridization using
nucleic acid modified with gold nanoparticles and PCR-SSCP, as
discussed in detail further below. Also useful is the recently
developed technique of DNA microchip technology.
[0078] Predisposition to cancers, such as prostate cancer, and the
other cancers identified herein, can be ascertained by testing any
tissue of a human for mutations of the HPC2 gene. For example, a
person who has inherited a germline HPC2 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 cells for mutations of the HPC2 gene. Alteration
of a wild-type HPC2 allele, whether, for example, by point mutation
or deletion, can be detected by any of the means discussed
herein.
[0079] There are several methods that can be used to detect DNA
sequence variation. Direct DNA sequencing, either manual sequencing
or automated fluorescent sequencing can detect sequence variation.
For a gene as large as HPC2, manual sequencing is very
labor-intensive, but under optimal conditions, mutations in the
coding sequence of a gene are rarely missed. Another approach is
the single-stranded conformation polymorphism assay (SSCA) (Orita
et al., 1989). This method does not detect all sequence changes,
especially if the DNA fragment size is greater than 200 bp, but can
be optimized to detect most DNA sequence variation. The reduced
detection sensitivity is a disadvantage, but the increased
throughput possible with SSCA makes it an attractive, viable
alternative to direct sequencing for mutation detection on a
research basis. The fragments which have shifted mobility on SSCA
gels are then sequenced to determine the exact nature of the DNA
sequence variation. Other approaches based on the detection of
mismatches between the two complementary DNA strands include
clamped denaturing gel electrophoresis (CDGE) (Sheffield et al.,
1991), heteroduplex analysis (HA) (White et al., 1992) and chemical
mismatch cleavage (CMC) (Grompe et al., 1989). None of the methods
described above will detect large deletions, duplications or
insertions, nor will they detect a regulatory mutation which
affects transcription or translation of the protein. Other methods
which might detect these classes of mutations such as a protein
truncation assay or the asymmetric assay, detect only specific
types of mutations and would not detect missense mutations. A
review of currently available methods of detecting DNA sequence
variation can be found in a recent review by Grompe (1993). Once a
mutation is known, an allele specific detection approach such as
allele specific oligonucleotide (ASO) hybridization can be utilized
to rapidly screen large numbers of other samples for that same
mutation. Such a technique can utilize probes which are labeled
with gold nanoparticles to yield a visual color result (Elghanian
etal., 1997).
[0080] In order to detect the alteration of the wild-type HPC2 gene
in a tissue, it is helpful to isolate the tissue free from
surrounding normal tissues. Means for enriching 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
techniques, as well as other techniques for separating tumor cells
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.
[0081] Detection of point mutations may be accomplished by
molecular cloning of the HPC2 allele(s) and sequencing the
allele(s) using techniques well known in the art. Alternatively,
the gene sequences can be amplified directly from a genomic DNA
preparation from the tumor tissue, using known techniques. The DNA
sequence of the amplified sequences can then be determined.
[0082] There are six well known methods for a more complete, yet
still indirect, test for confirming the presence of a
susceptibility allele: 1) single-stranded conformation analysis
(SSCA) (Orita et al., 1989); 2) denaturing gradient gel
electrophoresis (DGGE) (Wartell et al., 1990; Sheffield et al.,
1989); 3) RNase protection assays (Finkelstein et al., 1990;
Kinszler et al., 1991); 4) allele-specific oligonucleotides (ASOs)
(Conner et al., 1983); 5) the use of proteins which recognize
nucleotide mismatches, such as the E. coli mutS protein (Modrich,
1991); and 6) allele-specific PCR (Ruano and Kidd, 1989). For
allele-specific PCR, primers are used which hybridize at their 3'
ends to a particular HPC2 mutation. If the particular HPC2 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., 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 relatives of an
affected individual for the presence of the HPC2 mutation found in
that individual. Other techniques for detecting insertions and
deletions as known in the art can be used.
[0083] In the first three methods (SSCA, DGGE and RNase protection
assay), a new electrophoretic band appears. SSCA detects a band
which migrates differentially because the sequence change causes a
difference in single-strand, intramolecular base pairing. RNase
protection involves cleavage of the mutant polynucleotide into two
or more smaller fragments. DGGE detects differences in migration
rates of mutant sequences compared to wild-type sequences, using a
denaturing gradient gel. In an allele-specific oligonucleotide
assay, an oligonucleotide is designed which detects a specific
sequence, and the assay is performed by detecting the presence or
absence of a hybridization signal. In the mutS assay, the protein
binds only to sequences that contain a nucleotide mismatch in a
heteroduplex between mutant and wild-type sequences.
[0084] Mismatches, according to the present invention, are
hybridized nucleic acid duplexes in which the two strands are not
100% complementary. Lack of total homology may be due to deletions,
insertions, inversions or substitutions. Mismatch detection can be
used to detect point mutations in the gene or in 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. In
the practice of the present invention, the method involves the use
of a labeled riboprobe which is complementary to the human
wild-type HPC2 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 fall length duplex RNA for the riboprobe and the mRNA or
DNA. The riboprobe need not be the full length of the HPC2 MRNA or
gene but can be a segment of either. If the riboprobe comprises
only a segment of the HPC2 mRNA or gene, it will be desirable to
use a number of these probes to screen the whole mRNA sequence for
mismatches.
[0085] In similar fashion, DNA probes can be used to detect
mismatches, through enzymatic or chemical cleavage. See, e.g.,
Cotton et al., 1988; Shenk et al., 1975; Novack et al., 1986.
Alternatively, mismatches can be detected by shifts in the
electrophoretic mobility of mismatched duplexes relative to matched
duplexes. See, e.g., Cariello, 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 HPC2 gene can also be detected using Southern
hybridization, especially if the changes are gross rearrangements,
such as deletions and insertions.
[0086] DNA sequences of the HPC2 gene which have been amplified by
use of PCR may also be screened using allele-specific probes. These
probes are nucleic acid oligomers, each of which contains a region
of the HPC2 gene sequence harboring a known mutation. For example,
one oligomer may be about 30 nucleotides in length (although
shorter and longer oligomers are also usable as well recognized by
those of skill in the art), corresponding to a portion of the HPC2
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 HPC2 gene. Hybridization
of allele-specific probes with amplified HPC2 sequences can be
performed, for example, on a nylon filter. Hybridization to a
particular probe under high stringency hybridization conditions
indicates the presence of the same mutation in the tumor tissue as
in the allele-specific probe.
[0087] The newly developed technique of nucleic acid analysis via
microchip technology is also applicable to the present invention.
In this technique, literally thousands of distinct oligonucleotide
probes are built up in an array on a silicon chip. Nucleic acid to
be analyzed is fluorescently labeled and hybridized to the probes
on the chip. It is also possible to study nucleic acid-protein
interactions using these nucleic acid microchips. Using this
technique one can determine the presence of mutations or even
sequence the nucleic acid being analyzed or one can measure
expression levels of a gene of interest. The method is one of
parallel processing of many, even thousands, of probes at once and
can tremendously increase the rate of analysis. Several papers have
been published which use this technique. Some of these are Hacia et
al., 1996; Shoemaker et al., 1996; Chee et al., 1996; Lockhart et
al., 1996; DeRisi et al., 1996; Lipshutz et al., 1995. This method
has already been used to screen people for mutations in the breast
cancer gene BRCA1 (Hacia et al., 1996). This new technology has
been reviewed in a news article in Chemical and Engineering News
(Borman, 1996) and been the subject of an editorial (Nature
Genetics, 1996). Also see Fodor (1997).
[0088] The most definitive test for mutations in a candidate locus
is to directly compare genomic HPC2 sequences from cancer patients
with those from a control population. Alternatively, one could
sequence messenger RNA after amplification, e.g., by PCR, thereby
eliminating the necessity of determining the exon structure of the
candidate gene.
[0089] Mutations from cancer patients falling outside the coding
region of HPC2 can be detected by examining the non-coding regions,
such as introns and regulatory sequences near or within the HPC2
gene. An early indication that mutations in noncoding regions are
important may come from Northern blot experiments that reveal
messenger RNA molecules of abnormal size or abundance in cancer
patients as compared to control individuals.
[0090] Alteration of HPC2 MRNA expression can be detected by any
techniques known in the art. These include Northern blot analysis,
PCR amplification and RNase protection. Diminished mRNA expression
indicates an alteration of the wild-type HPC2 gene. Alteration of
wild-type HPC2 genes can also be detected by screening for
alteration of wild-type HPC2 protein. For example, monoclonal
antibodies immunoreactive with HPC2 can be used to screen a tissue.
Lack of cognate antigen would indicate an HPC2 mutation. Antibodies
specific for products of mutant alleles could also be used to
detect mutant HPC2 gene product. Such immunological assays can be
done in any convenient formats known in the art. These include
Western blots, immunohistochemical assays and ELISA assays. Any
means for detecting an altered HPC2 protein can be used to detect
alteration of wild-type HPC2 genes. Functional assays, such as
protein binding determinations, can be used. In addition, assays
can be used which detect HPC2 biochemical function. Finding a
mutant HPC2 gene product indicates alteration of a wild-type HPC2
gene.
[0091] Mutant HPC2 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 HPC2
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 HPC2 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 HPC2 genes or gene products.
[0092] The methods of diagnosis of the present invention are
applicable to any tumor in which HPC2 has a role in tumorigenesis.
The diagnostic method of the present invention is useful for
clinicians, so they can decide upon an appropriate course of
treatment.
[0093] The primer pairs of the present invention are useful for
determination of the nucleotide sequence of a particular HPC2
allele using PCR. The pairs of single-stranded DNA primers can be
annealed to sequences within or surrounding the HPC2 gene on
chromosome 17 in order to prime amplifying DNA synthesis of the
HPC2 gene itself. A complete set of these primers allows synthesis
of all of the nucleotides of the HPC2 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 HPC2 mutant alleles,
and thus will only amplify a product in the presence of the mutant
allele as a template.
[0094] 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 HPC2 sequences or sequences adjacent to HPC2, except
for 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 HPC2 open reading frame shown
in SEQ ID NOs: 1 and 3, design of particular primers is well within
the skill of the art.
[0095] 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 HPC2 gene or mRNA using other
techniques.
[0096] It has been discovered that individuals with the wild-type
HPC2 gene do not have cancer which results from the HPC2 allele.
However, mutations which interfere with the function of the HPC2
protein are involved in the pathogenesis of cancer. Thus, the
presence of an altered (or a mutant) HPC2 gene which produces a
protein having a loss of function, or altered function, directly
correlates to an increased risk of cancer. In order to detect an
HPC2 gene mutation, a biological sample is prepared and analyzed
for a difference between the sequence of the HPC2 allele being
analyzed and the sequence of the wild-type HPC2 allele. Mutant HPC2
alleles can be initially identified by any of the techniques
described above. The mutant alleles are then sequenced to identify
the specific mutation of the particular mutant allele.
Alternatively, mutant HPC2 alleles can be initially identified by
identifying mutant (altered) HPC2 proteins, using conventional
techniques. The mutant alleles are then sequenced to identify the
specific mutation for each allele. The mutations, especially those
which lead to an altered function of the HPC2 protein, are then
used for the diagnostic and prognostic methods of the present
invention.
[0097] Definitions
[0098] The present invention employs the following definitions:
[0099] "Amplification of Polynucleotides" utilizes methods such as
the polymerase chain reaction (PCR), ligation amplification (or
ligase chain reaction, LCR) and amplification methods based on the
use of Q-beta replicase. Also useful are strand displacement
amplification (SDA), thermophilic SDA, and nucleic acid sequence
based amplification (3SR or NASBA). These methods are well known
and widely practiced in the art. See, e.g., U.S. Pat. Nos.
4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu
and Wallace, 1989 (for LCR); U.S. Pat. Nos. 5,270,184 and 5,455,166
and Walker et al., 1992 (for SDA); Spargo et al., 1996 (for
thermophilic SDA) and U.S. Pat. No. 5,409,818, Fahy et al., 1991
and Compton, 1991 for 3SR and NASBA. Reagents and hardware for
conducting PCR are commercially available. Primers useful to
amplify sequences from the HPC2 region or HPC2 paralogs or
orthologs are preferably complementary to, and hybridize
specifically to sequences in the HPC2 region or paralog or ortholog
region or in regions that flank a target region therein. HPC2
sequences or paralog or ortholog sequences generated by
amplification may be sequenced directly. Alternatively, but less
desirably, the amplified sequence(s) may be cloned prior to
sequence analysis. A method for the direct cloning and sequence
analysis of enzymatically amplified genomic segments has been
described by Scharf, 1986.
[0100] "Analyte polynucleotide" and "analyte strand" refer to a
single- or double-stranded polynucleotide which is suspected of
containing a target sequence, and which may be present in a variety
of types of samples, including biological samples.
[0101] "Antibodies." The present invention also provides polyclonal
and/or monoclonal antibodies and fragments thereof, and immunologic
binding equivalents thereof, which are capable of specifically
binding to the HPC2 polypeptides or to polypeptides encoded by
paralogs or orthologs of HPC2 and fragments thereof or to
polynucleotide sequences from the HPC2 region, or to polynucleotide
sequences which are paralogs or orthologs of HPC2, particularly
from the HPC2 locus or a portion thereof. The term "antibody" is
used both to refer to a homogeneous molecular entity, or a mixture
such as a serum product made up of a plurality of different
molecular entities. Polypeptides may be prepared synthetically in a
peptide synthesizer and coupled to a carrier molecule (e.g.,
keyhole limpet hemocyanin) and injected over several months into
rabbits. Rabbit sera is tested for immunoreactivity to the HPC2
polypeptide or fragment or to polypeptides or fragments encoded by
paralogs or orthologs of HPC2. Monoclonal antibodies may be made by
injecting mice with the protein polypeptides, fusion proteins or
fragments thereof. Monoclonal antibodies will be screened by ELISA
and tested for specific immunoreactivity with HPC2 polypeptide or
fragments thereof. See, Harlow and Lane, 1988. These antibodies
will be useful in assays as well as pharmaceuticals.
[0102] Once a sufficient quantity of desired polypeptide has been
obtained, it may be used for various purposes. A typical use is the
production of antibodies specific for binding. These antibodies may
be either polyclonal or monoclonal, and may be produced by in vitro
or in vivo techniques well known in the art. For production of
polyclonal antibodies, an appropriate target immune system,
typically mouse or rabbit, is selected. Substantially purified
antigen is presented to the immune system in a fashion determined
by methods appropriate for the animal and by other parameters well
known to immunologists. Typical sites for injection are in
footpads, intramuscularly, intraperitoneally, or intradermally. Of
course, other species may be substituted for mouse or rabbit.
Polyclonal antibodies are then purified using techniques known in
the art, adjusted for the desired specificity.
[0103] An immunological response is usually assayed with an
immunoassay. Normally, such immunoassays involve some purification
of a source of antigen, for example, that produced by the same
cells and in the same fashion as the antigen. A variety of
immunoassay methods are well known in the art. See, e.g., Harlow
and Lane, 1988, or Goding, 1986.
[0104] Monoclonal antibodies with affinities of 10.sup.-8 M.sup.-1
or preferably 10.sup.-9 to 10.sup.-10 M.sup.-1 or stronger will
typically be made by standard procedures as described, e.g., in
Harlow and Lane, 1988 or Goding, 1986. Briefly, appropriate animals
will be selected and the desired immunization protocol followed.
After the appropriate period of time, the spleens of such animals
are excised and individual spleen cells fused, typically, to
immortalized myeloma cells under appropriate selection conditions.
Thereafter, the cells are clonally separated and the supernatants
of each clone tested for their production of an appropriate
antibody specific for the desired region of the antigen.
[0105] Other suitable techniques involve in vitro exposure of
lymphocytes to the antigenic polypeptides, or alternatively, to
selection of libraries of antibodies in phage or similar vectors.
See Huse et al., 1989. The polypeptides and antibodies of the
present invention may be used with or without modification.
Frequently, polypeptides and antibodies will be labeled by joining,
either covalently or non-covalently, a substance which provides for
a detectable signal. A wide variety of labels and conjugation
techniques are known and are reported extensively in both the
scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent agents, chemiluminescent agents, magnetic particles and
the like. Patents teaching the use of such labels include U.S. Pat.
Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;
4,275,149 and 4,366,241. Also, recombinant immunoglobulins may be
produced (see U.S. Pat. No. 4,816,567).
[0106] "Binding partner" refers to a molecule capable of binding a
ligand molecule with high specificity, as for example, an antigen
and an antigen-specific antibody or an enzyme and its inhibitor. In
general, the specific binding partners must bind with sufficient
affinity to immobilize the analyte copy/complementary strand duplex
(in the case of polynucleotide hybridization) under the isolation
conditions. Specific binding partners are known in the art and
include, for example, biotin and avidin or streptavidin, IgG and
protein A, the numerous, known receptor-ligand couples, and
complementary polynucleotide strands. In the case of complementary
polynucleotide binding partners, the partners are normally at least
about 15 bases in length, and may be at least 40 bases in length.
It is well recognized by those of skill in the art that lengths
shorter than 15 (e.g., 8 bases), between 15 and 40, and greater
than 40 bases may also be used. The polynucleotides may be composed
of DNA, RNA, or synthetic nucleotide analogs. Further binding
partners can be identified using, e.g., the two-hybrid yeast
screening assay as described herein.
[0107] A "biological sample" refers to a sample of tissue or fluid
suspected of containing an analyte polynucleotide or polypeptide
from an individual including, but not limited to, e.g., plasma,
serum, spinal fluid, lymph fluid, the external sections of the
skin, respiratory, intestinal, and genitourinary tracts, tears,
saliva, blood cells, tumors, organs, tissue and samples of in vitro
cell culture constituents.
[0108] As used herein, the terms "diagnosing" or "prognosing," as
used in the context of neoplasia, are used to indicate 1) the
classification of lesions as neoplasia, 2) the determination of the
severity of the neoplasia, or 3) the monitoring of the disease
progression, prior to, during and after treatment.
[0109] "Encode". A polynucleotide is said to "encode" a polypeptide
if, in its native state or when manipulated by methods well known
to those skilled in the art, it can be transcribed and/or
translated to produce the MRNA for and/or the polypeptide or a
fragment thereof. The anti-sense strand is the complement of such a
nucleic acid, and the encoding sequence can be deduced
therefrom.
[0110] "Isolated" or "substantially pure". An "isolated" or
"substantially pure" nucleic acid (e.g., an RNA, DNA or a mixed
polymer) is one which is substantially separated from other
cellular components which naturally accompany a native human
sequence or protein, e.g., ribosomes, polymerases, many other human
genome sequences and proteins. The term embraces a nucleic acid
sequence or protein which has been removed from its naturally
occurring environment, and includes recombinant or cloned DNA
isolates and chemically synthesized analogs or analogs biologically
synthesized by heterologous systems.
[0111] "HPC2 Allele" refers to normal alleles of the HPC2 locus as
well as alleles carrying variations that predispose individuals to
develop prostate cancer. Such predisposing alleles are also called
"HPC2 susceptibility alleles".
[0112] "HPC2 Locus", "HPC2 Gene", "HPC2 Nucleic Acids" or "HPC2
Polynucleotide" each refer to polynucleotides, all of which are in
the HPC2 region, that are likely to be expressed in normal tissue,
certain alleles of which predispose an individual to develop
prostate cancers. Mutations at the HPC2 locus may be involved in
the initiation and/or progression of other types of tumors. The
locus is indicated in part by mutations that predispose individuals
to develop cancer. These mutations fall within the HPC2 region
described infra. The HPC2 locus is intended to include coding
sequences, intervening sequences and regulatory elements
controlling transcription and/or translation. The HPC2 locus is
intended to include all allelic variations of the DNA sequence.
[0113] The term HPC2 is used interchangeably throughout this
disclosure with the terms ELAC2 and HPC2/ELAC2. This holds true
regardless of whether the term refers to a nucleic acid, allele,
gene, locus, protein or peptide.
[0114] These terms, when applied to a nucleic acid, refer to a
nucleic acid which encodes an HPC2 polypeptide, fragment, homolog
or variant, including, e.g., protein fusions or deletions. The
nucleic acids of the present invention will possess a sequence
which is either derived from, or substantially similar to a natural
HPC2-encoding gene or one having substantial homology with a
natural HPC2-encoding gene or a portion thereof.
[0115] The HPC2 gene or nucleic acid includes normal alleles of the
HPC2 gene, including silent alleles having no effect on the amino
acid sequence of the HPC2 polypeptide as well as alleles leading to
amino acid sequence variants of the HPC2 polypeptide that do not
substantially affect its function. These terms also include alleles
having one or more mutations which adversely affect the function of
the HPC2 polypeptide. A mutation may be a change in the HPC2
nucleic acid sequence which produces a deleterious change in the
amino acid sequence of the HPC2 polypeptide, resulting in partial
or complete loss of HPC2 function, or may be a change in the
nucleic acid sequence which results in the loss of effective HPC2
expression or the production of aberrant forms of the HPC2
polypeptide.
[0116] The HPC2 nucleic acid may be that shown in SEQ ID NOs:1, 3
or 28 or it may be an allele as described above or a variant or
derivative differing from that shown by a change which is one or
more of addition, insertion, deletion and substitution of one or
more nucleotides of the sequence shown. Changes to the nucleotide
sequence may result in an amino acid change at the protein level,
or not, as determined by the genetic code.
[0117] Thus, nucleic acid according to the present invention may
include a sequence different from the sequence shown in SEQ ID
NOs:1, 3 or 28 yet encode a polypeptide with the same amino acid
sequence as shown in SEQ ID NO: 1. That is, nucleic acids of the
present invention include sequences which are degenerate as a
result of the genetic code. On the other hand, the encoded
polypeptide may comprise an amino acid sequence which differs by
one or more amino acid residues from the amino acid sequence shown
in SEQ ID NO:2. Nucleic acid encoding a polypeptide which is an
amino acid sequence variant, derivative or allele of the amino acid
sequence shown in SEQ ID NO:2 is also provided by the present
invention.
[0118] The HPC2 gene also refers to (a) any DNA sequence that (i)
hybridizes to the complement of the DNA sequences that encode the
amino acid sequence set forth in SEQ ID NO:2 under highly stringent
conditions (Ausubel et al., 1992) and (ii) encodes a gene product
functionally equivalent to HPC2, or (b) any DNA sequence that (i)
hybridizes to the complement of the DNA sequences that encode the
amino acid sequence set forth in SEQ ID NO:2 under less stringent
conditions, such as moderately stringent conditions (Ausubel et
al., 1992) and (ii) encodes a gene product functionally equivalent
to HPC2. The invention also includes nucleic acid molecules that
are the complements of the sequences described herein.
[0119] The polynucleotide compositions of this invention include
RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both
sense and antisense strands, and may be chemically or biochemically
modified or may contain non-natural or derivatized nucleotide
bases, as will be readily appreciated by those skilled in the art.
Such modifications include, for example, labels, methylation,
substitution of one or more of the naturally occurring nucleotides
with an analog, intemucleotide modifications such as uncharged
linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.), charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), pendent moieties
(e.g., polypeptides), intercalators (e.g., acridine, psoralen,
etc.), chelators, alkylators, and modified linkages (e.g., alpha
anomeric nucleic acids, etc.). Also included are synthetic
molecules that mimic polynucleotides in their ability to bind to a
designated sequence via hydrogen bonding and other chemical
interactions. Such molecules are known in the art and include, for
example, those in which peptide linkages substitute for phosphate
linkages in the backbone of the molecule.
[0120] The present invention provides recombinant nucleic acids
comprising all or part of the HPC2 region or the HPC2 paralog
called ELAC1 or the mouse, chimpanzee or gorilla orthologs of HPC2,
herein called mouse ELAC2, chimpanzee ELAC2 or gorilla ELAC2. The
recombinant construct may be capable of replicating autonomously in
a host cell. Alternatively, the recombinant construct may become
integrated into the chromosomal DNA of the host cell. Such a
recombinant polynucleotide comprises a polynucleotide of genomic,
cDNA, semi-synthetic, or synthetic origin which, by virtue of its
origin or manipulation, 1) is not associated with all or a portion
of a polynucleotide with which it is associated in nature; 2) is
linked to a polynucleotide other than that to which it is linked in
nature; or 3) does not occur in nature. Where nucleic acid
according to the invention includes RNA, reference to the sequence
shown should be construed as reference to the RNA equivalent, with
U substituted for T.
[0121] Therefore, recombinant nucleic acids comprising sequences
otherwise not naturally occurring are provided by this invention.
Although the wild-type sequence may be employed, it will often be
altered, e.g., by deletion, substitution or insertion.
[0122] cDNA or genomic libraries of various types may be screened
as natural sources of the nucleic acids of the present invention,
or such nucleic acids may be provided by amplification of sequences
resident in genomic DNA or other natural sources, e.g., by PCR. The
choice of cDNA libraries normally corresponds to a tissue source
which is abundant in mRNA for the desired proteins. Phage libraries
are normally preferred, but other types of libraries may be used.
Clones of a library are spread onto plates, transferred to a
substrate for screening, denatured and probed for the presence of
desired sequences.
[0123] The DNA sequences, used in this invention will usually
comprise at least about five codons (15 nucleotides), more usually
at least about 7-15 codons, and most preferably, at least about 35
codons. One or more introns may also be present. This number of
nucleotides is usually about the minimal length required for a
successful probe that would hybridize specifically with an
HPC2-encoding sequence. In this context, oligomers of as low as 8
nucleotides, more generally 8-17 nucleotides, can be used for
probes, especially in connection with chip technology.
[0124] Techniques for nucleic acid manipulation are described
generally, for example, in Sambrook et al., 1989 or Ausubel et al.,
1992. Reagents useful in applying such techniques, such as
restriction enzymes and the like, are widely known in the art and
commercially available from such vendors as New England BioLabs,
Boehringer Mannheim, Amersham, Promega Biotec, U.S. Biochemicals,
New England Nuclear, and a number of other sources. The recombinant
nucleic acid sequences used to produce fusion proteins of the
present invention may be derived from natural or synthetic
sequences. Many natural gene sequences are obtainable from various
cDNA or from genomic libraries using appropriate probes. See,
GenBank, National Institutes of Health.
[0125] "HPC2 Region" refers to a portion of human chromosome 17
bounded by the markers D17S947 and D17S799. This region contains
the HPC2 locus, including the HPC2 gene.
[0126] As used herein, the terms "HPC2 locus", "HPC2 allele" and
"HPC2 region" all refer to the double-stranded DNA comprising the
locus, allele, or region, as well as either of the single-stranded
DNAs comprising the locus, allele or region.
[0127] As used herein, a "portion" of the HPC2 locus or region or
allele is defined as having a minimal size of at least about eight
nucleotides, or preferably about 15 nucleotides, or more preferably
at least about 25 nucleotides, and may have a minimal size of at
least about 40 nucleotides. This definition includes all sizes in
the range of 8-40 nucleotides as well as greater than 40
nucleotides. Thus, this definition includes nucleic acids of 8, 12,
15, 20, 25, 40, 60, 80, 100, 200, 300, 400, 500 nucleotides, or
nucleic acids having any number of nucleotides within these ranges
of values (e.g., 9, 10, 11, 16, 23, 30, 38, 50, 72, 121, etc.,
nucleotides), or nucleic acids having more than 500 nucleotides.
The present invention includes all novel nucleic acids having at
least 8 nucleotides derived from any of SEQ ID NOs:1 or 3-28, its
complement or functionally equivalent nucleic acid sequences. The
present invention does not include nucleic acids which exist in the
prior art. That is, the present invention includes all nucleic
acids having at least 8 nucleotides derived from any of SEQ ID
NOs:1 or 3-28 with the proviso that it does not include nucleic
acids existing in the prior art.
[0128] "HPC2 protein" or "HPC2 polypeptide" refers to a protein or
polypeptide encoded by the HPC2 locus, variants or fragments
thereof. The term "polypeptide" refers to a polymer of amino acids
and its equivalent and does not refer to a specific length of the
product; thus, peptides, oligopeptides and proteins are included
within the definition of a polypeptide. This term also does not
refer to, or exclude modifications of the polypeptide, for example,
glycosylations, acetylations, phosphorylations, and the like.
Included within the definition are, for example, polypeptides
containing one or more analogs of an amino acid (including, for
example, unnatural amino acids, etc.), polypeptides with
substituted linkages as well as other modifications known in the
art, both naturally and non-naturally occurring. Ordinarily, such
polypeptides will be at least about 50% homologous to the native
HPC2 sequence, preferably in excess of about 90%, and more
preferably at least about 95% homologous. Also included are
proteins encoded by DNA which hybridize under high or low
stringency conditions, to HPC2-encoding nucleic acids and closely
related polypeptides or proteins retrieved by antisera to the HPC2
protein(s).
[0129] An HPC2 polypeptide may be that derived from any of the
exons described herein which may be in isolated and/or purified
form, free or substantially free of material with which it is
naturally associated. The polypeptide may, if produced by
expression in a prokaryotic cell or produced synthetically, lack
native post-translational processing, such as glycosylation.
Alternatively, the present invention is also directed to
polypeptides which are sequence variants, alleles or derivatives of
an HPC2 polypeptide. Such polypeptides may have an amino acid
sequence which differs from that derived from any of the exons
described herein by one or more of addition, substitution, deletion
or insertion of one or more amino acids. Preferred such
polypeptides have HPC2 function.
[0130] Substitutional variants typically contain the exchange of
one amino acid for another at one or more sites within the protein,
and may be designed to modulate one or more properties of the
polypeptide, such as stability against proteolytic cleavage,
without the loss of other functions or properties. Amino acid
substitutions may be made on the basis of similarity in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the residues involved. Preferred
substitutions are ones which are conservative, that is, one amino
acid is replaced with one of similar shape and charge. Conservative
substitutions are well known in the art and typically include
substitutions within the following groups: glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid;
asparagine, glutamine; serine, threonine; lysine, arginine; and
tyrosine, phenylalanine.
[0131] Certain amino acids may be substituted for other amino acids
in a protein structure without appreciable loss of interactive
binding capacity with structures such as, for example,
anitigen-binding regions of antibodies or binding sites on
substrate molecules or binding sites on proteins interacting with
an HPC2 polypeptide. Since it is the interactive capacity and
nature of a protein which defines that protein's biological
functional activity, certain amino acid substitutions can be made
in a protein sequence, and its underlying DNA coding sequence, and
nevertheless obtain a protein with like properties. In making such
changes, the hydropathic index of amino acids may be considered.
The importance of the hydrophobic amino acid index in conferring
interactive biological function on a protein is generally
understood in the art (Kyte and Doolittle, 1982). Alternatively,
the substitution of like amino acids can be made effectively on the
basis of hydrophilicity. The importance of hydrophilicity in
conferring interactive biological function of a protein is
generally understood in the art (U.S. Pat. No. 4,554,101). The use
of the hydrophobic index or hydrophilicity in designing
polypeptides is further discussed in U.S. Pat. No. 5,691,198.
[0132] The length of polypeptide sequences compared for homology
will generally be at least about 16 amino acids, usually at least
about 20 residues, more usually at least about 24 residues,
typically at least about 28 residues, and preferably more than
about 35 residues.
[0133] "Operably linked" refers to a juxtaposition wherein the
components so described are in a relationship permitting them to
function in their intended manner. For instance, a promoter is
operably linked to a coding sequence if the promoter affects its
transcription or expression.
[0134] The term peptide mimetic or mimetic is intended to refer to
a substance which has the essential biological activity of an HPC2,
ELAC1 or ELAC2 polypeptide. A peptide mimetic may be a
peptide-containing molecule that mimics elements of protein
secondary structure (Johnson et al., 1993). The underlying
rationale behind the use of peptide mimetics is that the peptide
backbone of proteins exists chiefly to orient amino acid side
chains in such a way as to facilitate molecular interactions, such
as those of antibody and antigen, enzyme and substrate or
scaffolding proteins. A peptide mimetic is designed to permit
molecular interactions similar to the natural molecule. A mimetic
may not be a peptide at all, but it will retain the essential
biological activity of a natural HPC2, ELAC1 or ELAC2
polypeptide.
[0135] "Probes". Polynucleotide polymorphisms associated with HPC2
alleles which predispose to certain cancers or are associated with
most cancers are detected by hybridization with a polynucleotide
probe which forms a stable hybrid with that of the target sequence,
under highly stringent to moderately stringent hybridization and
wash conditions. If it is expected that the probes will be
perfectly complementary to the target sequence, high stringency
conditions will be used. Hybridization stringency may be lessened
if some mismatching is expected, for example, if variants are
expected with the result that the probe will not be completely
complementary. Conditions are chosen which rule out
nonspecific/adventitious bindings, that is, which minimize noise.
(It should be noted that throughout this disclosure, if it is
simply stated that "stringent" conditions are used that is meant to
be read as "high stringency" conditions are used.) Since such
indications identify neutral DNA polymorphisms as well as
mutations, these indications need further analysis to demonstrate
detection of an HPC2 susceptibility allele. An example of high
stringency conditions is to hybridize to filter bound DNA in 0.5 M
NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
65.degree. C. and to wash in 0.11.times. SSC/0.1% SDS at 68.degree.
C. (Ausubel et al., 1992). Less stringent conditions, such as
moderately stringent conditions, are defined as above but with the
wash step being in 0.2.times. SSC/0.1% SDS at 42.degree. C.
[0136] Probes for HPC2 alleles may be derived from the sequences of
the HPC2 region, its cDNA, functionally equivalent sequences, or
the complements thereof. The probes may be of any suitable length,
which span all or a portion of the HPC2 region, and which allow
specific hybridization to the HPC2 region. If the target sequence
contains a sequence identical to that of the probe, the probes may
be short, e.g., in the range of about 8-30 base pairs, since the
hybrid will be relatively stable under even highly stringent
conditions. If some degree of mismatch is expected with the probe,
i.e., if it is suspected that the probe will hybridize to a variant
region, a longer probe may be employed which hybridizes to the
target sequence with the requisite specificity.
[0137] The probes will include an isolated polynucleotide attached
to a label or reporter molecule and may be used to isolate other
polynucleotide sequences, having sequence similarity by standard
methods. For techniques for preparing and labeling probes see,
e.g., Sambrook et al., 1989 or Ausubel et al., 1992. Other similar
polynucleotides may be selected by using homologous
polynucleotides. Alternatively, polynucleotides encoding these or
similar polypeptides may be synthesized or selected by use of the
redundancy in the genetic code. Various codon substitutions may be
introduced, e.g., by silent changes (thereby producing various
restriction sites) or to optimize expression for a particular
system. Mutations may be introduced to modify the properties of the
polypeptide, perhaps to change ligand-binding affinities,
interchain affinities, or the polypeptide degradation or turnover
rate.
[0138] Probes comprising synthetic oligonucleotides or other
polynucleotides of the present invention may be derived from
naturally occurring or recombinant single- or double-stranded
polynucleotides, or be chemically synthesized. Probes may also be
labeled by nick translation, Kienow fill-in reaction, or other
methods known in the art.
[0139] Portions of the polynucleotide sequence having at least
about eight nucleotides, usually at least about 15 nucleotides, and
fewer than about 9 kb, usually fewer than about 1.0 kb, from a
polynucleotide sequence encoding HPC2 are preferred as probes. This
definition therefore includes probes of sizes 8 nucleotides through
9000 nucleotides. Thus, this definition includes probes of 8, 12,
15, 20, 25, 40, 60, 80, 100, 200, 300, 400 or 500 nucleotides or
probes having any number of nucleotides within these ranges of
values (e.g., 9, 10, 11, 16, 23, 30, 38, 50, 72, 121, etc.,
nucleotides), or probes having more than 500 nucleotides. The
probes may also be used to determine whether mRNA encoding HPC2 is
present in a cell or tissue. The present invention includes all
novel probes having at least 8 nucleotides derived from any of SEQ
ID NOs: 1 or 3-28 its complement or functionally equivalent nucleic
acid sequences. The present invention does not include probes which
exist in the prior art. That is, the present invention includes all
probes having at least 8 nucleotides derived from any of SEQ ID
NOs: 1 or 3-28 with the proviso that they do not include probes
existing in the prior art.
[0140] Similar considerations and nucleotide lengths are also
applicable to primers which may be used for the amplification of
all or part of the HPC2 gene. Thus, a definition for primers
includes primers of 8, 12, 15, 20, 25, 40, 60, 80, 100, 200, 300,
400, 500 nucleotides, or primers having any number of nucleotides
within these ranges of values (e.g., 9, 10, 11, 16, 23, 30, 38, 50,
72, 121, etc. nucleotides), or primers having more than 500
nucleotides, or any number of nucleotides between 500 and 9000. The
primers may also be used to determine whether MRNA encoding HPC2 is
present in a cell or tissue. The present invention includes all
novel primers having at least 8 nucleotides derived from the HPC2
locus for amplifying the HPC2 gene, its complement or functionally
equivalent nucleic acid sequences. The present invention does not
include primers which exist in the prior art. That is, the present
invention includes all primers having at least 8 nucleotides with
the proviso that it does not include primers existing in the prior
art.
[0141] "Protein modifications or fragments" are provided by the
present invention for HPC2, ELAC1 and ELAC2 polypeptides or
fragments thereof which are substantially homologous to primary
structural sequence but which include, e.g., in vivo or in vitro
chemical and biochemical modifications or which incorporate unusual
amino acids. Such modifications include, for example, acetylation,
carboxylation, phosphorylation, glycosylation, ubiquitination,
labeling, e.g., with radionuclides, and various enzymatic
modifications, as will be readily appreciated by those well skilled
in the art. A variety of methods for labeling polypeptides and of
substituents or labels useful for such purposes are well known in
the art, and include radioactive isotopes such as .sup.32P, ligands
which bind to labeled antiligands (e.g., antibodies), fluorophores,
chemiluminescent agents, enzymes, and antiligands which can serve
as specific binding pair members for a labeled ligand. The choice
of label depends on the sensitivity required, ease of conjugation
with the primer, stability requirements, and available
instrumentation. Methods of labeling polypeptides are well known in
the art. See Sambrook et al., 1989 or Ausubel et al., 1992.
[0142] Besides substantially full-length polypeptides, the present
invention provides for biologically active fragments of the
polypeptides. Significant biological activities include
ligand-binding, immunological activity and other biological
activities characteristic of HPC2, ELAC1 or ELAC2 polypeptides.
Immunological activities include both immunogenic function in a
target immune system, as well as sharing of immunological epitopes
for binding, serving as either a competitor or substitute antigen
for an epitope of the HPC2, ELAC1 or ELAC2 protein. As used herein,
"epitope" refers to an antigenic determinant of a polypeptide. An
epitope could comprise three amino acids in a spatial conformation
which is unique to the epitope. Generally, an epitope consists of
at least five such amino acids, and more usually consists of at
least 8-10 such amino acids. Methods of determining the spatial
conformation of such amino acids are known in the art.
[0143] For immunological purposes, tandem-repeat polypeptide
segments may be used as immunogens, thereby producing highly
antigenic proteins. Alternatively, such polypeptides will serve as
highly efficient competitors for specific binding. Production of
antibodies specific for HPC2, ELAC1 or ELAC2 polypeptides or
fragments thereof is described below.
[0144] The present invention also provides for fusion polypeptides,
comprising HPC2, ELAC1 or ELAC2 polypeptides and fragments.
Homologous polypeptides may be fusions between two or more HPC2,
ELAC1 or ELAC2 polypeptide sequences or between the sequences of
HPC2, ELAC1 or ELAC2 and a related protein. Likewise, heterologous
fusions may be constructed which would exhibit a combination of
properties or activities of the derivative proteins. For example,
ligand-binding or other domains may be "swapped" between different
new fusion polypeptides or fragments. Such homologous or
heterologous fusion polypeptides may display, for example, altered
strength or specificity of binding. Fusion partners include
immunoglobulins, bacterial .beta.-galactosidase, trpE, protein A,
.beta.-lactamase, alpha amylase, alcohol dehydrogenase and yeast
alpha mating factor. See Godowski et al., 1988.
[0145] Fusion proteins will typically be made by either recombinant
nucleic acid methods, as described below, or may be chemically
synthesized. Techniques for the synthesis of polypeptides are
described, for example, in Merrifield, 1963.
[0146] "Protein purification" refers to various methods for the
isolation of the HPC2, ELAC1 or ELAC2 polypeptides from other
biological material, such as from cells transformed with
recombinant nucleic acids encoding HPC2, ELAC1 or ELAC2 and are
well known in the art. For example, such polypeptides may be
purified by immunoaffinity chromatography employing, e.g., the
antibodies provided by the present invention. Various methods of
protein purification are well known in the art, and include those
described in Deutscher, 1990 and Scopes, 1982.
[0147] The terms "isolated", "substantially pure", and
"substantially homogeneous" are used interchangeably to describe a
protein or polypeptide which has been separated from components
which accompany it in its natural state. A monomeric protein is
substantially pure when at least about 60 to 75% of a sample
exhibits a single polypeptide sequence. A substantially pure
protein will typically comprise about 60 to 90% W/W of a protein
sample, more usually about 95%, and preferably will be over about
99% pure. Protein purity or homogeneity may be indicated by a
number of means well known in the art, such as polyacrylamide gel
electrophoresis of a protein sample, followed by visualizing a
single polypeptide band upon staining the gel. For certain
purposes, higher resolution may be provided by using HPLC or other
means well known in the art which are utilized for
purification.
[0148] An HPC2, ELAC1 or ELAC2 protein is substantially free of
naturally associated components when it is separated from the
native contaminants which accompany it in its natural state. Thus,
a polypeptide which is chemically synthesized or synthesized in a
cellular system different from the cell from which it naturally
originates will be substantially free from its naturally associated
components. A protein may also be rendered substantially free of
naturally associated components by isolation, using protein
purification techniques well known in the art.
[0149] A polypeptide produced as an expression product of an
isolated and manipulated genetic sequence is an "isolated
polypeptide," as used herein, even if expressed in a homologous
cell type. Synthetically made forms or molecules expressed by
heterologous cells are inherently isolated molecules.
[0150] "Recombinant nucleic acid" is a nucleic acid which is not
naturally occurring, or which is made by the artificial combination
of two otherwise separated segments of sequence. This artificial
combination is often accomplished by either chemical synthesis
means, or by the artificial manipulation of isolated segments of
nucleic acids, e.g., by genetic engineering techniques. Such is
usually done to replace a codon with a redundant codon encoding the
same or a conservative amino acid, while typically introducing or
removing a sequence recognition site. Alternatively, it is
performed to join together nucleic acid segments of desired
functions to generate a desired combination of functions.
[0151] "Regulatory sequences" refers to those sequences normally
within 100 kb of the coding region of a locus, but they may also be
more distant from the coding region, which affect the expression of
the gene (including transcription of the gene, and translation,
splicing, stability or the like of the messenger RNA).
[0152] "Substantial homology or similarity". A nucleic acid or
fragment thereof is "substantially homologous" ("or substantially
similar") to another if, when optimally aligned (with appropriate
nucleotide insertions or deletions) with the other nucleic acid (or
its complementary strand), there is nucleotide sequence identity in
at least about 60% of the nucleotide bases, usually at least about
70%, more usually at least about 80%, preferably at least about
90%, and more preferably at least about 95-98% of the nucleotide
bases.
[0153] Identity means the degree of sequence relatedness between
two polypeptide or two polynucleotides sequences as determined by
the identity of the match between two strings of such sequences.
Identity can be readily calculated. While there exist a number of
methods to measure identity between two polynucleotide or
polypeptide sequences, the term "identity" is well known to skilled
artisans (Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part I, Griffin, A. M., and
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991). Methods commonly employed
to determine identity between two sequences include, but are not
limited to those disclosed in Guide to Huge Computers, Martin J.
Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and
Lipman, D. (1988). Preferred methods to determine identity are
designed to give the largest match between the two sequences
tested. Such methods are codified in computer programs. Preferred
computer program methods to determine identity between two
sequences include, but are not limited to, GCG program package
(Devereux et al. (1984), BLASTP, BLASTN, FASTA (Altschul et al.
(1990); Altschul et al. (1997)).
[0154] Alternatively, substantial homology or similarity exists
when a nucleic acid or fragment thereof will hybridize to another
nucleic acid (or a complementary strand thereof) under selective
hybridization conditions, to a strand, or to its complement.
Selectivity of hybridization exists when hybridization which is
substantially more selective than total lack of specificity occurs.
Typically, selective hybridization will occur when there is at
least about 55% homology over a stretch of at least about 14
nucleotides, preferably at least about 65%, more preferably at
least about 75%, and most preferably at least about 90%. See,
Kanehisa, 1984. The length of homology comparison, as described,
may be over longer stretches, and in certain embodiments will often
be over a stretch of at least about nine nucleotides, usually at
least about 20 nucleotides, more usually at least about 24
nucleotides, typically at least about 28 nucleotides, more
typically at least about 32 nucleotides, and preferably at least
about 36 or more nucleotides.
[0155] Nucleic acid hybridization will be affected by such
conditions as salt concentration, temperature, or organic solvents,
in addition to the base composition, length of the complementary
strands, and the number of nucleotide base mismatches between the
hybridizing nucleic acids, as will be readily appreciated by those
skilled in the art. Stringent temperature conditions will generally
include temperatures in excess of 30.degree. C., typically in
excess of 37.degree. C., and preferably in excess of 45.degree. C.
Stringent salt conditions will ordinarily be less than 1000 mM,
typically less than 500 mM, and preferably less than 200 mM.
However, the combination of parameters is much more important than
the measure of any single parameter. See, e.g., Wetmur and
Davidson, 1968.
[0156] Probe sequences may also hybridize specifically to duplex
DNA under certain conditions to form triplex or other higher order
DNA complexes. The preparation of such probes and suitable
hybridization conditions are well known in the art.
[0157] The terms "substantial homology" or "substantial identity",
when referring to polypeptides, indicate that the polypeptide or
protein in question exhibits at least about 30% identity with an
entire naturally-occurring protein or a portion thereof, usually at
least about 70% identity, more usually at least about 80% identity,
preferably at least about 90% identity, and more preferably at
least about 95% identity.
[0158] Homology, for polypeptides, is typically measured using
sequence analysis software. See, e.g., the Sequence Analysis
Software Package of the Genetics Computer Group, University of
Wisconsin Biotechnology Center, 910 University Avenue, Madison,
Wis. 53705, as well as the software described above with reference
to nucleic acid homology. Protein analysis software matches similar
sequences using measures of homology assigned to various
substitutions, deletions and other modifications. Conservative
substitutions typically include substitutions within the following
groups: glycine, alanine; valine, isoleucine, leucine; aspartic
acid, glutamic acid; asparagine, glutamine; serine, threonine;
lysine, arginine; and phenylalanine, tyrosine.
[0159] "Substantially similar function" refers to the function of a
modified nucleic acid or a modified protein, with reference to the
wild-type HPC2, ELAC1 or ELAC2 nucleic acid or wild-type HPC2,
ELAC1 or ELAC2 polypeptide. The modified polypeptide will be
substantially homologous to the wild-type HPC2, ELAC1 or ELAC2
polypeptide and will have substantially the same function. The
modified polypeptide may have an altered amino acid sequence and/or
may contain modified amino acids. In addition to the similarity of
function, the modified polypeptide may have other useful
properties, such as a longer half-life. The similarity of function
(activity) of the modified polypeptide may be substantially the
same as the activity of the wild-type HPC2, ELAC1 or ELAC2
polypeptide. Alternatively, the similarity of function (activity)
of the modified polypeptide may be higher than the activity of the
wild-type HPC2, ELAC1 or ELAC2 polypeptide. The modified
polypeptide is synthesized using conventional techniques, or is
encoded by a modified nucleic acid and produced using conventional
techniques. The modified nucleic acid is prepared by conventional
techniques. A nucleic acid with a function substantially similar to
the wild-type HPC2, ELAC1 or ELAC2 gene function produces the
modified protein described above.
[0160] A polypeptide "fragment," "portion" or "segment" is a
stretch of amino acid residues of at least about five to seven
contiguous amino acids, often at least about seven to nine
contiguous amino acids, typically at least about nine to 13
contiguous amino acids and, most preferably, at least about 20 to
30 or more contiguous amino acids.
[0161] The polypeptides of the present invention, if soluble, may
be coupled to a solid-phase support, e.g., nitrocellulose, nylon,
column packing materials (e.g., Sepharose beads), magnetic beads,
glass wool, plastic, metal, polymer gels, cells, or other
substrates. Such supports may take the form, for example, of beads,
wells, dipsticks, or membranes.
[0162] "Target region" refers to a region of the nucleic acid which
is amplified and/or detected. The term "target sequence" refers to
a sequence with which a probe or primer will form a stable hybrid
under desired conditions.
[0163] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of chemistry,
molecular biology, microbiology, recombinant DNA, genetics, and
immunology. See, e.g., Maniatis et al., 1982; Sambrook et al.,
1989; Ausubel et aL, 1992; Glover, 1985; Anand, 1992; Guthrie and
Fink, 1991. A general discussion of techniques and materials for
human gene mapping, including mapping of human chromosome 1, is
provided, e.g., in White and Lalouel, 1988.
[0164] Preparation of Recombinant or Chemically Synthesized Nucleic
Acids; Vectors, Transformation, Host Cells
[0165] Large amounts of the polynucleotides of the present
invention may be produced by replication in a suitable host cell.
Natural or synthetic polynucleotide fragments coding for a desired
fragment will be incorporated into recombinant polynucleotide
constructs, usually DNA constructs, capable of introduction into
and replication in a prokaryotic or eukaryotic cell. Usually the
polynucleotide constructs will be suitable for replication in a
unicellular host, such as yeast or bacteria, but may also be
intended for introduction to (with and without integration within
the genome) cultured mammalian or plant or other eukaryotic cell
lines. The purification of nucleic acids produced by the methods of
the present invention is described, e.g., in Sambrook et al., 1989
or Ausubel et al., 1992.
[0166] The polynucleotides of the present invention may also be
produced by chemical synthesis, e.g., by the phosphoramidite method
described by Beaucage and Caruthers, 1981 or the triester method
according to Matteucci and Caruthers, 1981, and may be performed on
commercial, automated oligonucleotide synthesizers. A
double-stranded fragment may be obtained from the single-stranded
product of chemical synthesis either by synthesizing the
complementary strand and annealing the strands together under
appropriate conditions or by adding the complementary strand using
DNA polymerase with an appropriate primer sequence.
[0167] Polynucleotide constructs prepared for introduction into a
prokaryotic or eukaryotic host may comprise a replication system
recognized by the host, including the intended polynucleotide
fragment encoding the desired polypeptide, and will preferably also
include transcription and translational initiation regulatory
sequences operably linked to the polypeptide encoding segment.
Expression vectors may include, for example, an origin of
replication or autonomously replicating sequence (ARS) and
expression control sequences, a promoter, an enhancer and necessary
processing information sites, such as ribosome-binding sites, RNA
splice sites, polyadenylation sites, transcriptional terminator
sequences, and mRNA stabilizing sequences. Secretion signals may
also be included where appropriate, whether from a native HPC2
protein or from other receptors or from secreted polypeptides of
the same or related species, which allow the protein to cross
and/or lodge in cell membranes, and thus attain its functional
topology, or be secreted from the cell. Such vectors may be
prepared by means of standard recombinant techniques well known in
the art and discussed, for example, in Sambrook et al., 1989 or
Ausubel et al. 1992.
[0168] An appropriate promoter and other necessary vector sequences
will be selected so as to be functional in the host, and may
include, when appropriate, those naturally associated with HPC2,
ELAC1 or ELAC2 genes. Examples of workable combinations of cell
lines and expression vectors are described in Sambrook et al., 1989
or Ausubel et al., 1992; see also, e.g., Metzger et al., 1988. Many
useful vectors are known in the art and may be obtained from such
vendors as Stratagene, New England BioLabs, Promega Biotech, and
others. Promoters such as the trp, lac and phage promoters, tRNA
promoters and glycolytic enzyme promoters may be used in
prokaryotic hosts. Useful yeast promoters include promoter regions
for metallothionein, 3-phosphoglycerate kinase or other glycolytic
enzymes such as enolase or glyceraldehyde-3-phosphate
dehydrogenase, enzymes responsible for maltose and galactose
utilization, and others. Vectors and promoters suitable for use in
yeast expression are further described in Hitzeman et al., EP
73,675A. Appropriate non-native mammalian promoters might include
the early and late promoters from SV40 (Fiers et al., 1978) or
promoters derived from murine Moloney leukemia virus, mouse tumor
virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus
or polyoma. Insect promoters may be derived from baculovirus. In
addition, the construct may be joined to an amplifiable gene (e.g.,
DIFR) so that multiple copies of the gene may be made. For
appropriate enhancer and other expression control sequences, see
also Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor
Press, Cold Spring Harbor, N.Y. (1983). See also, e.g., U.S. Pat.
Nos. 5,691,198; 5,735,500; 5,747,469 and 5,436,146.
[0169] While such expression vectors may replicate autonomously,
they may also replicate by being inserted into the genome of the
host cell, by methods well known in the art.
[0170] Expression and cloning vectors will likely contain a
selectable marker, a gene encoding a protein necessary for survival
or growth of a host cell transformed with the vector. The presence
of this gene ensures growth of only those host cells which express
the inserts. Typical selection genes encode proteins that a) confer
resistance to antibiotics or other toxic substances, e.g.
ampicillin, neomycin, methotrexate, etc.; b) complement auxotrophic
deficiencies, or c) supply critical nutrients not available from
complex media, e.g., the gene encoding D-alanine racemase for
Bacilli. The choice of the proper selectable marker will depend on
the host cell, and appropriate markers for different hosts are well
known in the art.
[0171] The vectors containing the nucleic acids of interest can be
transcribed in vitro, and the resulting RNA introduced into the
host cell by well-known methods, e.g., by injection (see, Kubo et
al., 1988), or the vectors can be introduced directly into host
cells by methods well known in the art, which vary depending on the
type of cellular host, including electroporation; transfection
employing calcium chloride, rubidium chloride, calcium phosphate,
DEAE-dextran, or other substances; microprojectile bombardment;
lipofection; infection (where the vector is an infectious agent,
such as a retroviral genome); and other methods. See generally,
Sambrook et al., 1989 and Ausubel et al., 1992. The introduction of
the polynucleotides into the host cell by any method known in the
art, including, inter alia, those described above, will be
referred, to herein as "transformation." The cells into which have
been introduced nucleic acids described above are meant to also
include the progeny of such cells.
[0172] Large quantities of the nucleic acids and polypeptides of
the present invention may be prepared by expressing the HPC2, ELAC1
or ELAC2 nucleic acids or portions thereof in vectors or other
expression vehicles in compatible prokaryotic or eukaryotic host
cells. The most commonly used prokaryotic hosts are strains of
Escherichia coli, although other prokaryotes, such as Bacillus
subtilis or Pseudomonas may also be used.
[0173] Mammalian or other eukaryotic host cells, such as those of
yeast, filamentous fungi, plant, insect, or amphibian or avian
species, may also be useful for production of the proteins of the
present invention. Propagation of mammalian cells in culture is per
se well known. See, Jakoby and Pastan, 1979. Examples of commonly
used mammalian host cell lines are VERO and HeLa cells, Chinese
hamster ovary (CHO) cells, and W138, BHK, and COS cell lines,
although it will be appreciated by the skilled practitioner that
other cell lines may be appropriate, e.g., to provide higher
expression, desirable glycosylation patterns, or other features. An
example of a commonly used insect cell line is SF9.
[0174] Clones are selected by using markers depending on the mode
of the vector construction. The marker may be on the same or a
different DNA molecule, preferably the same DNA molecule. In
prokaryotic hosts, the transformant may be selected, e.g., by
resistance to ampicillin, tetracycline or other antibiotics.
Production of a particular product based on temperature sensitivity
may also serve as an appropriate marker.
[0175] Prokaryotic or eukaryotic cells transformed with the
polynucleotides of the present invention will be useful not only
for the production of the nucleic acids and polypeptides of the
present invention, but also, for example, in studying the
characteristics of HPC2, ELAC1 or ELAC2 polypeptides.
[0176] The HPC2, ELAC1 or ELAC2 gene products can also be expressed
in transgenic animals. Animals of any species, including, but not
limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs,
goats and non-human primates, e.g., baboons, monkeys and
chimpanzees, may be used to generate HPC2, ELAC1 or ELAC2
transgenic animals.
[0177] Any technique known in the art may be used to introduce the
HPC2, ELAC1 or ELAC2 gene transgene into animals to produce the
founder lines of transgenic animals. Such techniques include, but
are not limited to, pronuclear microinjection (U.S. Pat. No.
4,873,191); retrovirus mediated gene transfer into germ lines (Van
der Putten et al., 1985); gene targeting in embryonic stem cells
(Thompson et al., 1989); electroporation of embryos (Lo, 1983); and
sperm-mediated gene transfer (Lavitrano et al., 1989); etc. For a
review of such techniques, see Gordon (1989), which is incorporated
by reference herein in its entirety.
[0178] The present invention provides for transgenic animals that
carry the HPC2, ELAC1 or ELAC2 transgene in all their cells, as
well as animals which carry the transgene in some, but not all of
their cells, i.e., mosaic animals. The transgene may be integrated
as a single transgene or in concatamers, e.g., head-to-head tandems
or head-to-tail tandems. The transgene may also be selectively
introduced into and activated in a particular cell type by
following, for example, the teaching of Lasko et al. (1992). The
regulatory sequences required for such a cell-type specific
activation will depend upon the particular cell type of interest,
and will be apparent to those of skill in the art. When it is
desired that the HPC2, ELAC1 or ELAC2 gene transgene be integrated
into the chromosomal site of the endogenous HPC2, ELAC1 or ELAC2
gene, gene targeting is preferred. Briefly, when such a technique
is to be utilized, vectors containing some nucleotide sequences
homologous to the endogenous HPC2, ELAC1 or ELAC2 gene are designed
for the purpose of integrating, via homologous recombination with
chromosomal sequences, into and disrupting the function of the
nucleotide sequence of the endogenous HPC2, ELAC1 or ELAC2 gene.
The transgene may also be selectively introduced into a particular
cell type, thus inactivating the endogenous HPC2, ELAC1 or ELAC2
gene in only that cell type, by following, for example, the
teaching of Gu et al. (1994). The regulatory sequences required for
such a cell-type specific inactivation will depend upon the
particular cell type of interest, and will be apparent to those of
skill in the art.
[0179] Once transgenic animals have been generated, the expression
of the recombinant HPC2, ELAC1 or ELAC2 gene may be assayed
utilizing standard techniques. Initial screening may be
accomplished by Southern blot analysis or PCR techniques to analyze
animal tissues to assay whether integration of the transgene has
taken place. The level of mRNA expression of the transgene in the
tissues of the transgenic animals may also be assessed using
techniques which include, but are not limited to, Northern blot
analysis of tissue samples obtained from the animal, in situ
hybridization analysis, and RT-PCR. Samples of HPC2, ELAC1 or ELAC2
gene-expressing tissue, may also be evaluated immunocytochemically
using antibodies specific for the HPC2, ELAC1 or ELAC2 transgene
product.
[0180] Antisense polynucleotide sequences are useful in preventing
or diminishing the expression of the HPC2, ELAC1 or ELAC2 locus, as
will be appreciated by those skilled in the art. For example,
polynucleotide vectors containing all or a portion of the HPC2
locus or other sequences from the HPC2 region (particularly those
flanking the HPC2 locus) may be placed under the control of a
promoter in an antisense orientation and introduced into a cell.
Expression of such an antisense construct within a cell will
interfere with HPC2 transcription and/or translation and/or
replication.
[0181] The probes and primers based on the HPC2 gene sequences
disclosed herein are used to identify homologous HPC2 gene
sequences and proteins in other species. These HPC2 gene sequences
and proteins are used in the diagnostic/prognostic, therapeutic and
drug screening methods described herein for the species from which
they have been isolated.
[0182] Methods of Use: Nucleic Acid Diagnosis and Diagnostic
Kits
[0183] In order to detect the presence of an HPC2 allele
predisposing an individual to cancer, a biological sample such as
blood is prepared and analyzed for the presence or absence of
susceptibility alleles of HPC2. In order to detect the presence of
neoplasia, the progression toward malignancy of a precursor lesion,
or as a prognostic indicator, a biological sample of the lesion is
prepared and analyzed for the presence or absence of mutant alleles
of HPC2. Results of these tests and interpretive information are
returned to the health care provider for communication to the
tested individual. Such diagnoses may be performed by diagnostic
laboratories, or, alternatively, diagnostic kits are manufactured
and sold to health care providers or to private individuals for
self-diagnosis.
[0184] Initially, the screening method involves amplification of
the relevant HPC2 sequences. In another preferred embodiment of the
invention, the screening method involves a non-PCR based strategy.
Such screening methods include two-step label amplification
methodologies that are well known in the art. Both PCR and non-PCR
based screening strategies can detect target sequences with a high
level of sensitivity.
[0185] The most popular method used today is target amplification.
Here, the target nucleic acid sequence is amplified with
polymerases. One particularly preferred method using
polymerase-driven amplification is the polymerase chain reaction
(PCR). The polymerase chain reaction and other polymerase-driven
amplification assays can achieve over a million-fold increase in
copy number through the use of polymerase-driven amplification
cycles. Once amplified, the resulting nucleic acid can be sequenced
or used as a substrate for DNA probes.
[0186] When the probes are used to detect the presence of the
target sequences (for example, in screening for cancer
susceptibility), the biological sample to be analyzed, such as
blood or serum, may be treated, if desired, to extract the nucleic
acids. The sample nucleic acid may be prepared in various ways to
facilitate detection of the target sequence; e.g. denaturation,
restriction digestion, electrophoresis or dot blotting. The
targeted region of the analyte nucleic acid usually must be at
least partially single-stranded to form hybrids with the targeting
sequence of the probe. If the sequence is naturally
single-stranded, denaturation will not be required. However, if the
sequence is double-stranded, the sequence will probably need to be
denatured. Denaturation can be carried out by various techniques
known in the art.
[0187] Analyte nucleic acid and probe are incubated under
conditions which promote stable hybrid formation of the target
sequence in the probe with the putative targeted sequence in the
analyte. The region of the probes which is used to bind to the
analyte can be made completely complementary to the targeted region
of human chromosome 17. Therefore, high stringency conditions are
desirable in order to prevent false positives. However, conditions
of high stringency are used only if the probes are complementary to
regions of the chromosome which are unique in the genome. The
stringency of hybridization is determined by a number of factors
during hybridization and during the washing procedure, including
temperature, ionic strength, base composition, probe length, and
concentration of formamide. These factors are outlined in, for
example, Maniatis et al., 1982 and Sambrook et aL, 1989. Under
certain circumstances, the formation of higher order hybrids, such
as triplexes, quadraplexes, etc., may be desired to provide the
means of detecting target sequences.
[0188] Detection, if any, of the resulting hybrid is usually
accomplished by the use of labeled probes. Alternatively, the probe
may be unlabeled, but may be detectable by specific binding with a
ligand which is labeled, either directly or indirectly. Suitable
labels, and methods for labeling probes and ligands are known in
the art, and include, for example, radioactive labels which may be
incorporated by known methods (e.g., nick translation, random
priming or kinasing), biotin, fluorescent groups, chemiluminescent
groups (e.g., dioxetanes, particularly triggered dioxetanes),
enzymes, antibodies, gold nanoparticles and the like. Variations of
this basic scheme are known in the art, and include those
variations that facilitate separation of the hybrids to be detected
from extraneous materials and/or that amplify the signal from the
labeled moiety. A number of these variations are reviewed in, e.g.,
Matthews and Kricka, 1988; Landegren et al., 1988; Mifflin, 1989;
U.S. Pat. No. 4,868,105, and in EPO Publication No. 225,807.
[0189] As noted above, non-PCR based screening assays are also
contemplated in this invention. This procedure hybridizes a nucleic
acid probe (or an analog such as a methyl phosphonate backbone
replacing the normal phosphodiester), to the low level DNA target.
This probe may have an enzyme covalently linked to the probe, such
that the covalent linkage does not interfere with the specificity
of the hybridization. This enzyme-probe-conjugate-target nucleic
acid complex can then be isolated away from the free probe enzyme
conjugate and a substrate is added for enzyme detection. Enzymatic
activity is observed as a change in color development or
luminescent output resulting in a 103 106 increase in sensitivity.
For an example relating to the preparation of
oligodeoxynucleotide-alkaline phosphatase conjugates and their use
as hybridization probes see Jablonski et al., 1986.
[0190] Two-step label amplification methodologies are known in the
art. These assays work on the principle that a small ligand (such
as digoxigenin, biotin, or the like) is attached to a nucleic acid
probe capable of specifically binding HPC2. Allele specific probes
are also contemplated within the scope of this example and
exemplary allele specific probes include probes encompassing the
predisposing or potentially predisposing mutations summarized in
Table 9 of this patent application.
[0191] In one example, the small ligand attached to the nucleic
acid probe is specifically recognized by an antibody-enzyme
conjugate. In one embodiment of this example, digoxigenin is
attached to the nucleic acid probe. Hybridization is detected by an
antibody-alkaline phosphatase conjugate which turns over a
chemiluminescent substrate. For methods for labeling nucleic acid
probes according to this embodiment see Martin et al., 1990. In a
second example, the small ligand is recognized by a second
ligand-enzyme conjugate that is capable of specifically complexing
to the first ligand. A well known embodiment of this example is the
biotin-avidin type of interactions. For methods for labeling
nucleic acid probes and their use in biotin-avidin based assays see
Rigby et al., 1977 and Nguyen et al., 1992.
[0192] It is also contemplated within the scope of this invention
that the nucleic acid probe assays of this invention will employ a
cocktail of nucleic acid probes capable of detecting HPC2. Thus, in
one example to detect the presence of HPC2 in a cell sample, more
than one probe complementary to HPC2 is employed and in particular
the number of different probes is alternatively 2, 3, or 5
different nucleic acid probe sequences. In another example, to
detect the presence of mutations in the HPC2 gene sequence in a
patient, more than one probe complementary to HPC2 is employed
where the cocktail includes probes capable of binding to the
allele-specific mutations identified in populations of patients
with alterations in HPC2. In this embodiment, any number of probes
can be used, and will preferably include probes corresponding to
the major gene mutations identified as predisposing an individual
to prostate cancer.
[0193] Methods of Use: Peptide Diagnosis and Diagnostic Kits
[0194] The neoplastic condition of lesions can also be detected on
the basis of the alteration of wild-type HPC2 polypeptide. Such
alterations can be determined by sequence analysis in accordance
with conventional techniques. More preferably, antibodies
(polyclonal or monoclonal) are used to detect differences in, or
the absence of, HPC2 peptides. The antibodies may be prepared as
discussed above under the heading "Antibodies" and as further shown
in Examples 16 and 17. Other techniques for raising and purifying
antibodies are well known in the art and any such techniques may be
chosen to achieve the preparations claimed in this invention. In a
preferred embodiment of the invention, antibodies will
immunoprecipitate HPC2 proteins from solution as well as react with
HPC2 protein on Western or immunoblots of polyacrylamide gels. In
another preferred embodiment, antibodies will detect HPC2 proteins
in paraffin or frozen tissue sections, using immunocytochemical
techniques.
[0195] Preferred embodiments relating to methods for detecting HPC2
or its mutations include enzyme linked immunosorbent assays
(ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA)
and immunoenzymatic assays (IEMA), including sandwich assays using
monoclonal and/or polyclonal antibodies. Exemplary sandwich assays
are described by David et al. in U.S. Pat. Nos. 4,376,110 and
4,486,530, hereby incorporated by reference, and exemplified in
Example 19.
[0196] Methods of Use: Drug Screening
[0197] This invention is particularly useful for screening
compounds by using the HPC2, ELAC1 or ELAC2 polypeptide or binding
fragment thereof in any of a variety of drug screening
techniques.
[0198] The HPC2, ELAC1 or ELAC2 polypeptide or fragment employed in
such a test may either be free in solution, affixed to a solid
support, or borne on a cell surface. One method of drug screening
utilizes eucaryotic or procaryotic host cells which are stably
transformed with recombinant polynucleotides expressing the
polypeptide or fragment, preferably in competitive binding assays.
Such cells, either in viable or fixed form, can be used for
standard binding assays. One may measure, for example, for the
formation of complexes between an HPC2, ELAC1 or ELAC2 polypeptide
or fragment and the agent being tested, or examine the degree to
which the formation of a complex between an HPC2, ELAC1 or ELAC2
polypeptide or fragment and a known ligand is interfered with by
the agent being tested.
[0199] Thus, the present invention provides methods of screening
for drugs comprising contacting such an agent with an HPC2, ELAC1
or ELAC2 polypeptide or fragment thereof and assaying (i) for the
presence of a complex between the agent and the HPC2, ELAC1 or
ELAC2 polypeptide or fragment, or (ii) for the presence of a
complex between the HPC2, ELAC1 or ELAC2 polypeptide or fragment
and a ligand, by methods well known in the art. In such competitive
binding assays the HPC2, ELAC1 or ELAC2 polypeptide or fragment is
typically labeled. Free HPC2, ELAC1 or ELAC2 polypeptide or
fragment is separated from that present in a protein:protein
complex, and the amount of free (i.e., uncomplexed) label is a
measure of the binding of the agent being tested to HPC2, ELAC1 or
ELAC2 or its interference with HPC2:ligand, ELAC1 :ligand or
ELAC2:ligand binding, respectively. One may also measure the amount
of bound, rather than free, HPC2, ELAC1 or ELAC2. It is also
possible to label the ligand rather than the HPC2, ELAC1 or ELAC2
and to measure the amount of ligand binding to HPC2, ELAC1 or ELAC2
in the presence and in the absence of the drug being tested.
[0200] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to the HPC2, ELAC1 or ELAC2 polypeptides and is described in detail
in Geysen (published PCT WO 84/03564). Briefly stated, large
numbers of different small peptide test compounds are synthesized
on a solid substrate, such as plastic pins or some other surface.
The peptide test compounds are reacted with HPC2, ELAC1 or ELAC2
polypeptide and washed. Bound HPC2, ELAC1 or ELAC2 polypeptide is
then detected by methods well known in the art.
[0201] Purified HPC2, ELAC1 or ELAC2 can be coated directly onto
plates for use in the aforementioned drug screening techniques.
However, non-neutralizing antibodies to the polypeptide can be used
to capture antibodies to immobilize the HPC2, ELAC1 or ELAC2
polypeptide on the solid phase.
[0202] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
specifically binding the HPC2, ELAC1 or ELAC2 polypeptide compete
with a test compound for binding to the HPC2, ELAC1 or ELAC2
polypeptide or fragments thereof. In this manner, the antibodies
can be used to detect the presence of any peptide which shares one
or more antigenic determinants of the HPC2, ELAC1 or ELAC2
polypeptide.
[0203] A further technique for drug screening involves the use of
host eukaryotic cell lines or cells (such as described above) which
have a nonfunctional HPC2, ELAC1 or ELAC2 gene. These host cell
lines or cells are defective at the HPC2, ELAC1 or ELAC2
polypeptide level. The host cell lines or cells are grown in the
presence of drug compound. The rate of growth of the host cells is
measured to determine if the compound is capable of regulating the
growth of HPC2, ELAC1 or ELAC2 defective cells.
[0204] Briefly, a method of screening for a substance which
modulates activity of a polypeptide may include contacting one or
more test substances with the polypeptide in a suitable reaction
medium, testing the activity of the treated polypeptide and
comparing that activity with the activity of the polypeptide in
comparable reaction medium untreated with the test substance or
substances. A difference in activity between the treated and
untreated polypeptides is indicative of a modulating effect of the
relevant test substance or substances.
[0205] Prior to or as well as being screened for modulation of
activity, test substances may be screened for ability to interact
with the polypeptide, e.g., in a yeast two-hybrid system (e.g.,
Bartel et al., 1993; Fields and Song, 1989; Chevray and Nathans,
1992; Lee et al., 1995). This system may be used as a coarse screen
prior to testing a substance for actual ability to modulate
activity of the polypeptide. Alternatively, the screen could be
used to screen test substances for binding to an HPC2, ELAC1 or
ELAC2 specific binding partner, or to find mimetics of an HPC2,
ELAC1 or ELAC2 polypeptide.
[0206] Methods of Use: Rational Drug Design
[0207] The goal of rational drug design is to produce structural
analogs of biologically active polypeptides of interest or of small
molecules with which they interact (e.g., agonists, antagonists,
inhibitors) in order to fashion drugs which are, for example, more
active or stable forms of the polypeptide, or which, e.g., enhance
or interfere with the function of a polypeptide in vivo. See, e.g.,
Hodgson, 1991. In one approach, one first determines the
three-dimensional structure of a protein of interest (e.g., HPC2
polypeptide) or, for example, of the HPC2-receptor or ligand
complex, by x-ray crystallography, by computer modeling or most
typically, by a combination of approaches. Less often, useful
information regarding the structure of a polypeptide may be gained
by modeling based on the structure of homologous proteins. An
example of rational drug design is the development of HIV protease
inhibitors (Erickson et al., 1990). In addition, peptides (e.g.,
HPC2 polypeptide) are analyzed by an alanine scan (Wells, 1991). In
this technique, an amino acid residue is replaced by Ala, and its
effect on the peptide's activity is determined. Each of the amino
acid residues of the peptide is analyzed in this manner to
determine the important regions of the peptide.
[0208] It is also possible to isolate a target-specific antibody,
selected by a functional assay, and then to solve its crystal
structure. In principle, this approach yields a pharmacore upon
which subsequent drug design can be based. It is possible to bypass
protein crystallography altogether by generating anti-idiotypic
antibodies (anti-ids) to a functional, pharmacologically active
antibody. As a mirror image of a mirror image, the binding site of
the anti-ids would be expected to be an analog of the original
receptor. The anti-id could then be used to identify and isolate
peptides from banks of chemically or biologically produced banks of
peptides. Selected peptides would then act as the pharmacore.
[0209] Thus, one may design drugs which have, e.g., improved HPC2,
ELAC1 or ELAC2 polypeptide activity or stability or which act as
inhibitors, agonists, antagonists, etc. of HPC2, ELAC1 or ELAC2
polypeptide activity. By virtue of the availability of cloned HPC2,
ELAC1 and ELAC2 sequences, sufficient amounts of the HPC2, ELAC1 or
ELAC2 polypeptide may be made available to perform such analytical
studies as x-ray crystallography. In addition, the knowledge of the
HPC2, ELAC1 and ELAC2 protein sequences provided herein will guide
those employing computer modeling techniques in place of, or in
addition to x-ray crystallography.
[0210] Following identification of a substance which modulates or
affects polypeptide activity, the substance may be investigated
further. Furthermore, it may be manufactured and/or used in
preparation, i.e., manufacture or formulation, or a composition
such as a medicament, pharmaceutical composition or drug. These may
be administered to individuals.
[0211] Thus, the present invention extends in various aspects not
only to a substance identified using a nucleic acid molecule as a
modulator of polypeptide activity, in accordance with what is
disclosed herein, but also a pharmaceutical composition,
medicament, drug or other composition comprising such a substance,
a method comprising administration of such a composition comprising
such a substance, a method comprising administration of such a
composition to a patient, e.g., for treatment of prostate cancer,
use of such a substance in the manufacture of a composition for
administration, e.g., for treatment of prostate cancer, and a
method of making a pharmaceutical composition comprising admixing
such a substance with a pharmaceutically acceptable excipient,
vehicle or carrier, and optionally other ingredients.
[0212] A substance identified as a modulator of polypeptide
function may be peptide or non-peptide in nature. Non-peptide
"small molecules" are often preferred for many in vivo
pharmaceutical uses. Accordingly, a mimetic or mimic of the
substance (particularly if a peptide) may be designed for
pharmaceutical use.
[0213] The designing of mimetics to a known pharmaceutically active
compound is a known approach to the development of pharmaceuticals
based on a "lead" compound. This might be desirable where the
active compound is difficult or expensive to synthesize or where it
is unsuitable for a particular method of administration, e.g., pure
peptides are unsuitable active agents for oral compositions as they
tend to be quickly degraded by proteases in the alimentary canal.
Mimetic design, synthesis and testing is generally used to avoid
randomly screening large numbers of molecules for a target
property.
[0214] There are several steps commonly taken in the design of a
mimetic from a compound having a given target property. First, the
particular parts of the compound that are critical and/or important
in determining the target property are determined. In the case of a
peptide, this can be done by systematically varying the amino acid
residues in the peptide, e.g., by substituting each residue in
turn. Alanine scans of peptide are commonly used to refine such
peptide motifs. These parts or residues constituting the active
region of the compound are known as its "pharmacophore".
[0215] Once the pharmacophore has been found, its structure is
modeled according to its physical properties, e.g.,
stereochemistry, bonding, size and/or charge, using data from a
range of sources, e.g., spectroscopic techniques, x-ray diffraction
data and NMR. Computational analysis, similarity mapping (which
models the charge and/or volume of a pharmacophore, rather than the
bonding between atoms) and other techniques can be used in this
modeling process.
[0216] In a variant of this approach, the three-dimensional
structure of the ligand and its binding partner are modeled. This
can be especially useful where the ligand and/or binding partner
change conformation on binding, allowing the model to take account
of this in the design of the mimetic.
[0217] A template molecule is then selected onto which chemical
groups which mimic the pharmacophore can be grafted. The template
molecule and the chemical groups grafted onto it can conveniently
be selected so that the mimetic is easy to synthesize, is likely to
be pharmacologically acceptable, and does not degrade in vivo,
while retaining the biological activity of the lead compound.
Alternatively, where the mimetic is peptide-based, further
stability can be achieved by cyclizing the peptide, increasing its
rigidity. The mimnetic or mimetics found by this approach can then
be screened to see whether they have the target property, or to
what extent they exhibit it. Further optimization or modification
can then be carried out to arrive at one or more final mimetics for
in vivo or clinical testing.
[0218] Methods of Use: Gene Therapy
[0219] According to the present invention, a method is also
provided of supplying wild-type HPC2 function to a cell which
carries mutant HPC2 alleles. Supplying such a function should
suppress neoplastic growth of the recipient cells. The wild-type
HPC2 gene or a part of the gene may be introduced into the cell in
a vector such that the gene remains extrachromosomal. In such a
situation, the gene will be expressed by the cell from the
extrachromosomal location. If a gene fragment is introduced and
expressed in a cell carrying a mutant HPC2 allele, the gene
fragment should encode a part of the HPC2 protein which is required
for non-neoplastic growth of the cell. More preferred is the
situation where the wild-type HPC2 gene or a part thereof is
introduced into the mutant cell in such a way that it recombines
with the endogenous mutant HPC2 gene present in the cell. Such
recombination requires a double recombination event which results
in the correction of the HPC2 gene mutation. Vectors for
introduction of genes both 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 coprecipitation and viral
transduction are known in the art, and the choice of method is
within the competence of the practitioner. Cells transformed with
the wild-type HPC2 gene can be used as model systems to study
cancer remission and drug treatments which promote such
remission.
[0220] As generally discussed above, the HPC2 gene or fragment,
where applicable, may be employed in gene therapy methods in order
to increase the amount of the expression products of such genes in
cancer cells. Such gene therapy is particularly appropriate for use
in both cancerous and pre-cancerous cells, in which the level of
HPC2 polypeptide is absent or diminished compared to normal cells.
It may also be useful to increase the level of expression of a
given HPC2 gene even in those tumor cells in which the mutant gene
is expressed at a "normal" level, but the gene product is not fully
functional.
[0221] Gene therapy would be carried out according to generally
accepted methods, for example, as described by Friedman (1991) or
Culver (1996). Cells from a patient's tumor would be first analyzed
by the diagnostic methods described above, to ascertain the
production of HPC2 polypeptide in the tumor cells. A virus or
plasmid vector (see further details below), containing a copy of
the HPC2 gene linked to expression control elements and capable of
replicating inside the tumor cells, is prepared. Alternatively, the
vector may be replication deficient and is replicated in helper
cells for use in gene therapy. Suitable vectors are known, such as
disclosed in U.S. Pat. No. 5,252,479 and PCT published application
WO 93/07282 and U.S. Pat. Nos. 5,691,198; 5,747,469; 5,436,146 and
5,753,500. The vector is then injected into the patient, either
locally at the site of the tumor or systemically (in order to reach
any tumor cells that may have metastasized to other sites). If the
transfected gene is not permanently incorporated into the genome of
each of the targeted tumor cells, the treatment may have to be
repeated periodically.
[0222] Gene transfer systems known in the art may be useful in the
practice of the gene therapy methods of the present invention.
These include viral and nonviral transfer methods. A number of
viruses have been used as gene transfer vectors, including
papovaviruses, e.g., SV40 (Madzak et al., 1992), adenovirus
(Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992;
Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson and Akrigg,
1992; Stratford-Perricaudet et al., 1990; Schneider et al., 1998),
vaccinia virus (Moss, 1992; Moss, 1996), adeno-associated virus
(Muzyczka, 1992; Ohi et al., 1990; Russell and Hirata, 1998),
herpes viruses including HSV and EBV (Margolskee, 1992; Johnson et
al., 1992; Fink et al., 1992; Breakefield and Geller, 1987; Freese
et al., 1990; Fink et al., 1996), lentiviruses (Naldini et al.,
1996), Sindbis and Semliki Forest virus (Berglund et al., 1993),
and retroviruses of avian (Bandyopadhyay and Temin, 1984;
Petropoulos et al., 1992), murine (Miller, 1992; Miller et al.,
1985; Sorge et al., 1984; Mann and Baltimore, 1985; Miller et al.,
1988), and human origin (Shimada et al., 1991; Helseth et al.,
1990; Page et al., 1990; Buchschacher and Panganiban, 1992). Most
human gene therapy protocols have been based on disabled murine
retroviruses, although adenovirus and adeno-associated virus are
also being used.
[0223] Nonviral gene transfer methods known in the art include
chemical techniques such as calcium phosphate coprecipitation
(Graham and van der Eb, 1973; Pellicer et al., 1980); mechanical
techniques, for example microinjection (Anderson et al., 1980;
Gordon et al., 1980; Brinster et al., 1981; Costantini and Lacy,
1981); membrane fusion-mediated transfer via liposomes (Felgner et
al., 1987; Wang and Huang, 1989; Kaneda et al, 1989; Stewart et
al., 1992; Nabel et al., 1990; Lim et al., 1991); and direct DNA
uptake and receptor-mediated DNA transfer (Wolff et al., 1990; Wu
et al., 1991; Zenke et al., 1990; Wu et al., 1989; Wolff et al.,
1991; Wagner et al., 1990; Wagner et al., 1991; Cotten et al.,
1990; Curiel et al., 1991; Curiel et al., 1992). Viral-mediated
gene transfer can be combined with direct in vivo gene transfer
using liposome delivery, allowing one to direct the viral vectors
to the tumor cells and not into the surrounding nondividing cells.
Alternatively, the retroviral vector producer cell line can be
injected into tumors (Culver et al., 1992). Injection of producer
cells would then provide a continuous source of vector particles.
This technique has been approved for use in humans with inoperable
brain tumors.
[0224] In an approach which combines biological and physical gene
transfer methods, plasmid DNA of any size is combined with a
polylysine-conjugated antibody specific to the adenovirus hexon
protein, and the resulting complex is bound to an adenovirus
vector. The trimolecular complex is then used to infect cells. The
adenovirus vector permits efficient binding, internalization, and
degradation of the endosome before the coupled DNA is damaged. For
other techniques for the delivery of adenovirus based vectors see
Schneider et al. (1998) and U.S. Pat. Nos. 5,691,198; 5,747,469;
5,436,146 and 5,753,500.
[0225] Liposome/DNA complexes have been shown to be capable of
mediating direct in vivo gene transfer. While in standard liposome
preparations the gene transfer process is nonspecific, localized in
vivo uptake and expression have been reported in tumor deposits,
for example, following direct in situ administration (Nabel,
1992).
[0226] Expression vectors in the context of gene therapy are meant
to include those constructs containing sequences sufficient to
express a polynucleotide that has been cloned therein. In viral
expression vectors, the construct contains viral sequences
sufficient to support packaging of the construct. If the
polynucleotide encodes HPC2, expression will produce HPC2. If the
polynucleotide encodes an antisense polynucleotide or a ribozyme,
expression will produce the antisense polynucleotide or ribozyme.
Thus in this context, expression does not require that a protein
product be synthesized. In addition to the polynucleotide cloned
into the expression vector, the vector also contains a promoter
functional in eukaryotic cells. The cloned polynucleotide sequence
is under control of this promoter. Suitable eukaryotic promoters
include those described above. The expression vector may also
include sequences, such as selectable markers and other sequences
described herein.
[0227] Gene transfer techniques which target DNA directly to
prostate tissues, e.g., epithelial cells of the prostate, are
preferred. Receptor-mediated gene transfer, for example, is
accomplished by the conjugation of DNA (usually in the form of
covalently closed supercoiled plasmid) to a protein ligand via
polylysine. Ligands are chosen on the basis of the presence of the
corresponding ligand receptors on the cell surface of the target
cell/tissue type. One appropriate receptor/ligand pair may include
the estrogen receptor and its ligand, estrogen (and estrogen
analogues). These ligand-DNA conjugates can be injected directly
into the blood if desired and are directed to the target tissue
where receptor binding and internalization of the DNA-protein
complex occurs. To overcome the problem of intracellular
destruction of DNA, coinfection with adenovirus can be included to
disrupt endosome function.
[0228] The therapy involves two steps which can be performed singly
or jointly. In the first step, prepubescent females who carry an
HPC2 susceptibility allele are treated with a gene delivery vehicle
such that some or all of their mammary ductal epithelial precursor
cells receive at least one additional copy of a functional normal
HPC2 allele. In this step, the treated individuals have reduced
risk of prostate cancer to the extent that the effect of the
susceptible allele has been countered by the presence of the normal
allele. In the second step of a preventive therapy, predisposed
young females, in particular women who have received the proposed
gene therapeutic treatment, undergo hormonal therapy to mimic the
effects on the prostate of a full term pregnancy.
[0229] Methods of Use: Peptide Therapy
[0230] Peptides which have HPC2, ELAC1 or ELAC2 activity can be
supplied to cells which carry mutant or missing HPC2, ELAC1 or
ELAC2 alleles. Protein can be produced by expression of the cDNA
sequence in bacteria, for example, using known expression vectors.
Alternatively, HPC2, ELAC1 or ELAC2 polypeptide can be extracted
from HPC2-, ELAC1- or ELAC2-producing mammalian cells. In addition,
the techniques of synthetic chemistry can be employed to synthesize
HPC2, ELAC1 or ELAC2 protein. Any of such techniques can provide
the preparation of the present invention which comprises the HPC2,
ELAC1 or ELAC2 protein. Preparation is substantially free of other
human proteins. This is most readily accomplished by synthesis in a
microorganism or in vitro.
[0231] Active HPC2, ELAC1 or ELAC2 molecules can be introduced into
cells by microinjection or by use of liposomes, for example.
Alternatively, some active molecules may be taken up by cells,
actively or by diffusion. Extracellular application of the HPC2,
ELAC1 or ELAC2 gene product may be sufficient to affect tumor
growth. Supply of molecules with HPC2 activity should lead to
partial reversal of the neoplastic state. Other molecules with HPC2
activity (for example, peptides, drugs or organic compounds) may
also be used to effect such a reversal. Modified polypeptides
having substantially similar function are also used for peptide
therapy.
[0232] Methods of Use: Transformed Hosts
[0233] Similarly, cells and animals which carry a mutant HPC2,
ELAC1 or ELAC2 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 HPC2, ELAC1 or ELAC2 mutations,
either somatic or germline. Alternatively, the cell line can be
engineered to carry the mutation in the HPC2, ELAC1 or ELAC2
allele, as described above. After a test substance is applied to
the cells, the neoplastically transformed phenotype of the cell is
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.
[0234] 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 HPC2, ELAC1
or ELAC2 alleles, usually from a second animal species, as well as
insertion of disrupted homologous genes. Alternatively, the
endogenous HPC2, ELAC1 or ELAC2 gene(s) of the animals may be
disrupted by insertion or deletion mutation or other genetic
alterations using conventional techniques (Capecchi, 1989;
Valancius and Smithies, 1991; Hasty et al., 1991; Shinkai et al.,
1992; Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et
al., 1992; Donehower et al., 1992) to produce knockout or
transplacement animals. A transplacement is similar to a knockout
because the endogenous gene is replaced, but in the case of a
transplacement the replacement is by another version of the same
gene. 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 the
cancers identified herein. These animal models provide an extremely
important testing vehicle for potential therapeutic products.
[0235] In one embodiment of the invention, transgenic animals are
produced which contain a functional transgene encoding a functional
HPC2, ELAC1 or ELAC2 polypeptide or variants thereof. Transgenic
animals expressing HPC2, ELAC1 or ELAC2 transgenes, recombinant
cell lines derived from such animals and transgenic embryos may be
useful in methods for screening for and identifying agents that
induce or repress function of HPC2, ELAC1 or ELAC2. Transgenic
animals of the present invention also can be used as models for
studying indications such as disease.
[0236] In one embodiment of the invention, an HPC2, ELAC1 or ELAC2
transgene is introduced into a non-human host to produce a
transgenic animal expressing a human or murine HPC2, ELAC1 or ELAC2
gene. The transgenic animal is produced by the integration of the
transgene into the genome in a manner that permits the expression
of the transgene. Methods for producing transgenic animals are
generally described by Wagner and Hoppe (U.S. Pat. No. 4,873,191;
which is incorporated herein by reference), Brinster et al. 1985;
which is incorporated herein by reference in its entirety) and in
"Manipulating the Mouse Embryo; A Laboratory Manual" 2nd edition
(eds., Hogan, Beddington, Costantini and Long, Cold Spring Harbor
Laboratory Press, 1994; which is incorporated herein by reference
in its entirety).
[0237] It may be desirable to replace the endogenous HPC2, ELAC1 or
ELAC2 by homologous recombination between the transgene and the
endogenous gene; or the endogenous gene may be eliminated by
deletion as in the preparation of "knock-out" animals. Typically,
an HPC2, ELAC1 or ELAC2 gene flanked by genomic sequences is
transferred by microinjection into a fertilized egg. The
microinjected eggs are implanted into a host female, and the
progeny are screened for the expression of the transgene.
Transgenic animals may be produced from the fertilized eggs from a
number of animals including, but not limited to reptiles,
amphibians, birds, mammals, and fish. Within a particularly
preferred embodiment, transgenic mice are generated which
overexpress HPC2 or express a mutant form of the polypeptide.
Alternatively, the absence of an HPC2, ELAC1 or ELAC2 in
"knock-out" mice permits the study of the effects that loss of
HPC2, ELAC1 or ELAC2 protein has on a cell in vivo. Knock-out mice
also provide a model for the development of HPC2-related
cancers.
[0238] Methods for producing knockout animals are generally
described by Shastry (1995, 1998) and Osterrieder and Wolf (1998).
The production of conditional knockout animals, in which the gene
is active until knocked out at the desired time is generally
described by Feil et al. (1996), Gagneten et al. (1997) and Lobe
and Nagy (1998). Each of these references is incorporated herein by
reference.
[0239] As noted above, transgenic animals and cell lines derived
from such animals may find use in certain testing experiments. In
this regard, transgenic animals and cell lines capable of
expressing wild-type or mutant HPC2, ELAC1 or ELAC2 may be exposed
to test substances. These test substances can be screened for the
ability to reduce overexpression of wild-type HPC2, ELAC1 or ELAC2
or impair the expression or function of mutant HPC2, ELAC1 or
ELAC2.
[0240] Pharmaceutical Compositions and Routes of Administration
[0241] The HPC2, ELAC1 or ELAC2 polypeptides, antibodies, peptides
and nucleic acids of the present invention can be formulated in
pharmaceutical compositions, which are prepared according to
conventional pharmaceutical compounding techniques. See, for
example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack
Publishing Co., Easton, Pa.). The composition may contain the
active agent or pharmaceutically acceptable salts of the active
agent. These compositions may comprise, in addition to one of the
active substances, a pharmaceutically acceptable excipient,
carrier, buffer, stabilizer or other materials well known in the
art. Such materials should be non-toxic and should not interfere
with the efficacy of the active ingredient. The carrier may take a
wide variety of forms depending on the form of preparation desired
for administration, e.g., intravenous, oral, intrathecal, epineural
or parenteral.
[0242] For oral administration, the compounds can be formulated
into solid or liquid preparations such as capsules, pills, tablets,
lozenges, melts, powders, suspensions or emulsions. In preparing
the compositions in oral dosage form, any of the usual
pharmaceutical media may be employed, such as, for example, water,
glycols, oils, alcohols, flavoring agents, preservatives, coloring
agents, suspending agents, and the like in the case of oral liquid
preparations (such as, for example, suspensions, elixirs and
solutions); or carriers such as starches, sugars, diluents,
granulating agents, lubricants, binders, disintegrating agents and
the like in the case of oral solid preparations (such as, for
example, powders, capsules and tablets). Because of their ease in
administration, tablets and capsules represent the most
advantageous oral dosage unit form, in which case solid
pharmaceutical carriers are obviously employed. If desired, tablets
may be sugar-coated or enteric-coated by standard techniques. The
active agent can be encapsulated to make it stable to passage
through the gastrointestinal tract while at the same time allowing
for passage across the blood brain barrier. See for example, WO
96/11698.
[0243] For parenteral administration, the compound may be dissolved
in a pharmaceutical carrier and administered as either a solution
or a suspension. Illustrative of suitable carriers are water,
saline, dextrose solutions, fructose solutions, ethanol, or oils of
animal, vegetative or synthetic origin. The carrier may also
contain other ingredients, for example, preservatives, suspending
agents, solubilizing agents, buffers and the like. When the
compounds are being administered intrathecally, they may also be
dissolved in cerebrospinal fluid.
[0244] The active agent is preferably administered in a
therapeutically effective amount. The actual amount administered,
and the rate and time-course of administration, will depend on the
nature and severity of the condition being treated. Prescription of
treatment, e.g. decisions on dosage, timing, etc., is within the
responsibility of general practitioners or specialists, and
typically takes account of the disorder to be treated, the
condition of the individual patient, the site of delivery, the
method of administration and other factors known to practitioners.
Examples of techniques and protocols can be found in Remington 's
Pharmaceutical Sciences.
[0245] Alternatively, targeting therapies may be used to deliver
the active agent more specifically to certain types of cell, by the
use of targeting systems such as antibodies or cell specific
ligands. Targeting may be desirable for a variety of reasons, e.g.
if the agent is unacceptably toxic, or if it would otherwise
require too high a dosage, or if it would not otherwise be able to
enter the target cells.
[0246] Instead of administering these agents directly, they could
be produced in the target cell, e.g. in a viral vector such as
described above or in a cell based delivery system such as
described in U.S. Pat. No. 5,550,050 and published PCT application
Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO
96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635,
designed for implantation in a patient. The vector could be
targeted to the specific cells to be treated, or it could contain
regulatory elements which are more tissue specific to the target
cells. The cell based delivery system is designed to be implanted
in a patient's body at the desired target site and contains a
coding sequence for the active agent. Alternatively, the agent
could be administered in a precursor form for conversion to the
active form by an activating agent produced in, or targeted to, the
cells to be treated. See for example, EP 425,731A and WO
90/07936.
[0247] As disclosed in the following Examples, on the basis of
segregating mutations of HPC2 in kindreds 4102 and 4289, plus
association between carriage of the common missense changes Leu 217
and Thr 541 with a diagnosis of prostate cancer, we conclude that
HPC2 is a prostate cancer susceptibility gene.
[0248] While a 1641 insG frameshift found in kindred 4102 will
clearly disrupt protein function, this is not obviously the case
for the His 781 missense change in kindred 4289. Interestingly,
this missense change occurred on a chromosome that also carries Leu
217 and Thr 541. Thus one might entertain an additive hypothesis to
explain the relative strength of the three missense bearing alleles
that we have observed. Substitution of Leu for Ser 217 may change
the character of a normally hydrophilic segment of the protein; the
phenotype conferred is sufficiently modest that it is only detected
when the variant is homozygous. Ala 541 is immediately adjacent to
the histidine motif. At the position corresponding to Ala 541 in
the ELAC1/2, CPSF73 and PSO2 gene families, the most common residue
is alanine; when not alanine, the residue is hydrophobic, amide, or
basic (FIGS. 6A-B, 9 and 12). Although threonine is observed at
this position in other histidine motif containing gene families, it
is rare or absent in these three closely related gene families.
Thus, from sequence conservation considerations, it is quite
reasonable that the Leu 217+ Thr 541 allele should be more
deleterious than Leu 217 alone, apparently sufficiently deleterious
to be detected in a co-dominant to dominant association test. The
kindred 4289 allele carries all three missense changes, Leu 217,
Thr 541 and His 781. Examination of the pedigree suggests that the
allele is dominant and sufficiently deleterious to demonstrate
visible segregation with prostate cancer in an extended pedigree.
Interestingly, the youngest affected carrier of this variant,
4289.003, is homozygous for Leu 217 and Thr 541. Thus his mother,
the second ovarian cancer case in the pedigree, is an obligate
carrier of a Leu 217+Thr 541 allele. The observation of two ovarian
cancer cases in this pedigree, both of whom carry deleterious
alleles of ELAC2, is consistent with the possibility that the
phenotype conferred by deleterious variants in this gene is not
restricted to prostate cancer susceptibility.
[0249] The potential contributions of the androgen receptor CAG
repeat and SRD5A2 Ala 49 Thr missense change to prostate cancer
risk were first detected in association studies using sporadic
cases and unaffected controls. However, straightforward deduction
from the considerable literature on sib pair analyses would predict
that such sequence variants should be enriched among affected sibs
versus isolated cases, and it follows that such sequence variants
should contribute to a larger fraction of familial than truly
sporadic prostate cancer cases. Thus one might expect genotypes at
moderate risk susceptibility genes such as the androgen receptor,
SRD5A2, and the common missense changes in HPC2/ELAC2, to confound
linkage studies aimed at detecting and localizing lower prevalence,
higher risk susceptibility genes. However, inclusion of genotype
information from pedigree members at multiple moderate risk loci
may allow refined definition of the liability classes used by
multipoint linkage software, thereby increasing the power of the
analysis. Stratification of cases by genotype would also facilitate
positional cloning projects by providing another criterion by which
to distinguish between true recombinant carriers and confounding
sporadic cases.
[0250] The genetic data presented demonstrate that there are
deleterious sequence variants in HPC2/ELAC2 that contribute to
prostate cancer risk. Elucidating the functional alteration by
which a moderate risk sequence variant such as Leu 217 contributes
to a late onset pathology could prove difficult because its
manifestation could be quite subtle. However, a mutation as
dramatic as a frameshift leading to protein truncation within the
likely active site of an enzyme should have a more easily detected
effect on cell physiology. Conservation of the C-terminal domain of
the gene through the eubacteria and archaebacteria, combined with
the observation that the S. cerevisiae ortholog YRK079C is
essential, emphasize that the function of the ELAC1/2 gene family
is of fundamental biological interest.
[0251] The identification of the association between the HPC2 gene
mutations and prostate cancer permits the early presymptomatic
screening of individuals to identify those at risk for developing
prostate cancer. To identify such individuals, HPC2 alleles are
screened for mutations either directly or after cloning the
alleles. The alleles are tested for the presence of nucleic acid
sequence differences from the normal allele using any suitable
technique, including but not limited to, one of the following
methods: fluorescent in situ hybridization (FISH), direct DNA
sequencing, PFGE analysis, Southern blot analysis, single stranded
conformation analysis (SSCP), linkage analysis, RNase protection
assay, allele specific oligonucleotide (ASO), dot blot analysis and
PCR-SSCP analysis. Also useful is the recently developed technique
of DNA microchip technology. For example, either (1) the nucleotide
sequence of both the cloned alleles and normal HPC2 gene or
appropriate fragment (coding sequence or genomic sequence) are
determined and then compared, or (2) the RNA transcripts of the
HPC2 gene or gene fragment are hybridized to single stranded whole
genomic DNA from an individual to be tested, and the resulting
heteroduplex is treated with Ribonuclease A (RNase A) and run on a
denaturing gel to detect the location of any mismatches. Two of
these methods can be carried out according to the following
procedures.
[0252] The alleles of the HPC2 gene in an individual to be tested
are cloned using conventional techniques. For example, a blood
sample is obtained from the individual. The genomic DNA isolated
from the cells in this sample is partially digested to an average
fragment size of approximately 20 kb. Fragments in the range from
18-21 kb are isolated. The resulting fragments are ligated into an
appropriate vector. The sequences of the clones are then determined
and compared to the normal HPC2 gene.
[0253] Alternatively, polymerase chain reactions (PCRs) are
performed with primer pairs for the 5 region or the exons of the
HPC2 gene. PCRs can also be performed with primer pairs based on
any sequence of the normal HPC2 gene. For example, primer pairs for
one of the introns can be prepared and utilized. Finally, RT-PCR
can also be performed on the mRNA. The amplified products are then
analyzed by single stranded conformation polymorphisms (SSCP) using
conventional techniques to identify any differences and these are
then sequenced and compared to the normal gene sequence.
[0254] Individuals can be quickly screened for common HPC2 gene
variants by amplifying the individual s DNA using suitable primer
pairs and analyzing the amplified product, e.g., by dot-blot
hybridization using allele-specific oligonucleotide probes.
[0255] The second method employs RNase A to assist in the detection
of differences between the normal HPC2 gene and defective genes.
This comparison is performed in steps using small (.about.500 bp)
restriction fragments of the HPC2 gene as the probe. First, the
HPC2 gene is digested with a restriction enzyme(s) that cuts the
gene sequence into fragments of approximately 500 bp. These
fragments are separated on an electrophoresis gel, purified from
the gel and cloned individually, in both orientations, into an SP6
vector (e.g., pSP64 or pSP65). The SP6-based plasmids containing
inserts of the HPC2 gene fragments are transcribed in vitro using
the SP6 transcription system, well known in the art, in the
presence of [.alpha.-.sup.32P]GTP, generating radiolabeled RNA
transcripts of both strands of the gene.
[0256] Individually, these RNA transcripts are used to form
heteroduplexes with the allelic DNA using conventional techniques.
Mismatches that occur in the RNA:DNA heteroduplex, owing to
sequence differences between the HPC2 fragment and the HPC2 allele
subdlone from the individual, result in cleavage in the RNA strand
when treated with RNase A. Such mismatches can be the result of
point mutations or small deletions in the individual's allele.
Cleavage of the RNA strand yields two or more small RNA fragments,
which run faster on the denaturing gel than the RNA probe
itself.
[0257] Any differences which are found, will identify an individual
as having a molecular variant of the HPC2. These variants can take
a number of forms. The most severe forms would be frame shift
mutations or large deletions which would cause the gene to code for
an abnormal protein or one which would significantly alter protein
expression. Less severe disruptive mutations would include small
in-frame deletions and nonconservative base pair substitutions
which would have a significant effect on the protein produced, such
as changes to or from a cysteine residue, from a basic to an acidic
amino acid or vice versa, from a hydrophobic to hydrophilic amino
acid or vice versa, or other mutations which would affect secondary
or tertiary protein structure. Silent mutations or those resulting
in conservative amino acid substitutions would not generally be
expected to disrupt protein function.
[0258] Genetic testing will enable practitioners to identify
individuals at risk prostate cancer, at, or even before, birth.
Presymptomatic diagnosis of these epilepsies will enable prevention
of these disorders.
EXAMPLES
[0259] The present invention is further detailed in the following
Examples, which are offered by way of illustration and are not
intended to limit the invention in any manner. Standard techniques
well known in the art or the techniques specifically described
below are utilized.
Example 1
Linkage Analysis
[0260] All participants signed informed consent documents. This
research project has the approval of the University of Utah School
of Medicine Institutional Review Board. Ninety-seven percent of
cancer cases have been confirmed through medical records (and/or
through the Utah Cancer Registry for prostate cancer cases
diagnosed in Utah). Two-point linkage analysis was performed with
the package LINKAGE (Lathrop et al., 1984) using the FASTLINK
implementation (Cottingham et al., 1993; Schaffer et al., 1994).
The statistical analysis for the inheritance of susceptibility to
prostate cancer used a model that assumes age-specific incidence
rates from the Utah Cancer Registry, and a relative risk of 2.5 for
first-degree relatives. Susceptibility to prostate cancer was
assumed due to a dominant allele with a population frequency of
0.003. The details of the model are more thoroughly defined in
Neuhausen et al. (1999). Marker allele frequencies were estimated
from unrelated individuals present in the pedigrees. Linkage in the
presence of heterogeneity was assessed by the admixture test
(A-test) of Ott (1986), using HOMOG, which postulates two family
types, linked and unlinked. Three-point linkage analysis was
performed using VITESSE (O'Connell and Weeks, 1995).
Example 2
Physical Mapping
[0261] BAC DNA was purified and directly sequenced as previously
described (Couch et al., 1996). DNA sequences at the SP6 and T7
ends of isolated BAC clones were used to develop STSs that were
used for mapping and contig extension. Greater than 95% sequence
coverage of the FIG. 1 BAC tiling path was obtained by sequencing
plasmid sublibraries generated from these clones. The sequence data
obtained were assembled into contigs using Acembly, version 4.3 (U.
Sauvage, D. Thierry-Mieg and J. Thierry-Mieg; Centre National de la
Recherche Scientifique, France). Subsequently, a complete sequence
of this interval was released by the MIT genome center.
Example 3
Genetic Localization of HPC2
[0262] A. Early Studies
[0263] A set of high risk prostate cancer kindreds has been
collected in Utah since 1990 for the purpose of localization of
prostate cancer susceptibility loci. In February 1996, linkage
analysis of data from a genome scan performed on a subset of the
families noted evidence for linkage with markers on chromosome 17p.
Subsequent analysis of more markers in this region of chromosome
17p in a larger set of families has led to strong linkage evidence
for a susceptibility gene.
1TABLE 1 Chromosome 17p Two-point Linkage Evidence Marker 17p map
position Heterogeneity Lod Score D17S786 20.0 4.21 Myr 0022 25.5
3.99 Myr 0088 27.0 3.46 D17S947 31.6 2.32 Myr 0084 31.9 3.02 Myr
0079 32.0 0.99 D17S805 43.6 2.25
[0264] The study of specific kindreds with strong evidence of
linkage to chromosome 17p allows the definition of a most likely
region for the susceptibility locus by identifying the smallest
inherited piece of chromosome 17p shared by the prostate cancer
cases in the kindred. The minimal genetically defined region is
based on a telomeric recombinant in kindred 4325 and a centromeric
recombinant in kindred 4320. Kindred 4325 was ascertained from a
sibship of early onset prostate cancer cases. There are 6 affected
brothers in this family, one of whom also has an affected son. Five
of the 6 affected brothers, and the affected son, all share the
same piece of chromosome 17p from somewhere below marker myrOO65
down to and including marker D17S805. Kindred 4320 was also
ascertained from a sibship of early onset prostate cancer cases. In
this kindred 3 affected brothers and an affected nephew share a
piece of chromosome 17p from D17S786 down to and including myr0084.
Together, the kindred 4325 and kindred 4320 recombinants define a
minimal region of about 1 megabase (FIG. 2A); this localization is
well supported by a larger set of recombinants in both
directions.
[0265] B. Recent Studies
[0266] We originally performed a genome-wide search for prostate
cancer predisposition loci using a small set of Utah high risk
prostate cancer pedigrees and a set of 300 polymorphic markers. The
pedigrees were not selected for early age of cancer onset, but were
a subset of families ascertained using the Utah Population
Database. The first eight pedigrees analyzed gave suggestive
evidence of linkage on chromosome 17p near marker D17S520, although
significance was not established. We increased the density of
markers in the region and expanded the analysis to 33 pedigrees
(Table 2A). Analysis of the additional data, using a dominant model
integrated with Utah age-specific incidence, yielded the two-point
linkage evidence shown in Table 2B. A maximum two-point LOD score
of 4.5 was observed at marker D17S1289, theta=0.07, and a maximum
three-point LOD score of 4.3 was observed using the markers
D17S1289 and D17S921. Based on these data, we initiated a
positional cloning project, focusing on the interval between
D17S1289 and D17S921.
2TABLE 2A Family Resource Used to Detect Linkage to 17p Number of
pedigrees 3.3 Total number of cases 338 Total number of typed cases
188 Mean number of cases/pedigree (range) 10.2 (2-29) Mean number
of typed cases/pedigree (range) 5.7 (1-16) Mean age of typed cases
at diagnosis (range) 68.3 (35-88)
[0267]
3TABLE 2B Two-point LOD Scores Using Utah Age-specific Model
distance Heterogeneity Marker (cM).dagger. Max LOD.Yen. (theta) LOD
(alpha, theta) D17S796 -- 0.11 (.37) 0.10 (1.00, 0.4) D17S952 10.2
0.90 (.17) 0.87 (1.00, 0.2) D17S786 10.4 0.00 (.50) 0.95 (0.20,
0.0) D17S945 12.7 0.38 (.28) 1.41 (0.25, 0.0) D17S520 15.0 0.69
(.26) 0.64 (1.00, 0.3) D17S974 15.1 1.01 (.20) 1.20 (0.40, 0.01)
D17S1289 15.2 4.53 (.07) 4.43 (1.00, 0.1) D17S1159 15.4 0.50 (.27)
1.38 (0.25, 0.0) GATA134G03 15.7 0.48 (.20) 0.78 (0.75, 0.2)
D17S954 16.2 0.00 (.50) 0.11 (0.40, 0.2) D17S969 18.2 0.54 (.21)
0.55 (0.85, 0.2) D17S799 22.0 0.30 (.26) 0.44 (0.70, 0.2) D17S921
25.2 1.41 (.10) 1.42 (0.95, 0.1) D17S953 29.2 1.04 (.25) 0.94
(1.00, 0.3) D17S925 31.2 0.02 (.45) 0.00 (1.00, 0.0) D17S798 36.2
0.02 (.43) 0.02 (1.00, 0.4) .dagger.Distances estimated from data
using CRIMAP (Lander and Green, 1987). .Yen.Maximum LOD scores
interpolated using the standard quadratic function.
[0268] In order to refine the localization of the implied
susceptibility gene, we expanded to the set of 127 families (Table
3) which have now been typed at both this locus and the HPC1 locus.
Although the overall data set neither provides significant LOD
score evidence for linkage on chromosome 17 nor provides sufficient
evidence for de novo identification of the HPC1 locus (Neuhausen et
al., 1999), complete haplotyping of the pedigree resource revealed
a similar number of prostate cancer-associated haplotypes at each
locus.
4TABLE 3 Summary of Resource Genotyped for the Association Tests
Number of pedigrees 127 Total number of cases 2,402 Total number of
typed cases 700 Total number of typed pedigree unaffecteds 3,295
Total number of typed divergent controls 243 Mean number of
cases/pedigree (range) 18.3 (3-74) Mean number of typed
cases/pedigree (range) 5.5 (1-34) Mean age of typed cases at
diagnosis (range) 66.5 (39-88)
[0269] Early in our analysis, we observed that at both 17p and HPC1
many of our pedigrees segregate haplotypes that are shared by four
or more cases, but also contain enough noncarrying cases with
respect to either locus to eliminate any linkage evidence within
the pedigree, as estimated by LOD score. For instance, 12 affected
individuals from kindred 4333 share an HPC1 haplotype and 9
affecteds in kindred 4344 share a 17p haplotype, but neither
pedigree shows LOD score evidence for linkage at either locus.
While we recognize that this phenomenon may be due simply to lack
of linkage, we hypothesized that the underlying cause is actually
genetic complexity that is greater than the linkage models can
accommodate. We subsequently used multipoint haplotyping software
(Thomas et al., 2000) to define segregating haplotypes, and then
classified those haplotypes into three groups, depending on
strength of evidence: group 1 haplotypes, used for both
localization and mutation screening, were defined as haplotypes
shared by 4 or more cases and giving a LOD score .gtoreq.1.0 in the
pedigree where they were identified, or haplotypes shared by 6 or
more cases irrespective of LOD score; group 2 haplotypes, used for
mutation screening only, were defined as haplotypes shared by 4
cases with 0.5<LOD<1.0 in the pedigree where they were
identified, or haplotypes shared by 5 cases with LOD<1.0; and
finally, haplotypes that failed to meet either of the above
criteria.
[0270] Considering group 1 and 2 haplotypes together, evidence at
HPC1 and 17p is quite similar: 43 haplotypes at HPC1 versus 42 at
17p and 258 affected haplotype carriers at HPC1 versus 232 at 17p.
Focusing on the group 1 haplotypes, evidence at HPC1 is relatively
stronger: 26 group 1 haplotypes at HPC1 versus 18 at 17p and an
average of 7.2 affected carriers per group 1 haplotype at HPC1
versus 6.6 at 17p. However, there is one other critical difference
between the linkage evidence for the two regions. At HPC1, meiotic
recombinant mapping using the group 1 haplotypes has thus far
failed to define a consistent region. This is also reflected in the
ICPCG HPC1 study (Xu, 2000); in this work, most of the evidence for
linkage comes from a combination of the Utah and Hopkins data sets,
but the locations with the best evidence for linkage in each of the
individual sets map approximately 15 cM apart. In contrast,
recombinant mapping in affected carriers of 17p group 1 haplotypes
defined a consistent region (FIG. 3). As a result, we were able to
focus our contig assembly, transcript map development, and mutation
screening efforts on an approximately 1 MB interval centered on
D17S947 (FIG. 3).
[0271] One of the genes mapping near D17S947 shares amino acid
sequence similarity with members of the NCB1 Cluster of Orthologous
Groups (Tatusov et al., 1997) COG1234, typified by the
uncharacterized E. coli ORF elaC and the uncharacterized S.
cerevisiae ORF YKRO79C. On mutation screening this candidate gene
from the genomic DNA of prostate cancer cases carrying 17p group 1
haplotypes, a germline frameshift mutation, 1641 insG, was found in
a carrier from kindred 4102. Following detection of this
frameshift, the gene, which we shall refer to as ELAC2 because it
is the larger of two human genes that we have found that are
homologs of E. coli elaC, was subjected to careful sequence and
intense genetic analyses.
Example 4
[0272] Contig Assembly and Genomic Sequencing in the Minimal
Genetically Defined HPC2 Region
[0273] Contig Assembly.
[0274] Given a genetically defined interval flanked by meiotic
recombinants, one needs to generate a contig of genomic clones that
spans that interval. Publicly available resources, such as the
Whitehead integrated maps of the human genome (e.g., the WICGR Chr
17 map) provide aligned chromosome maps of genetic markers, other
sequence tagged sites (STSs), radiation hybrid map data, and CEPH
yeast artificial chromosome (YAC) clones.
[0275] Oligonucleotide primer pairs for the markers located in the
interval were synthesized and used to screen libraries of bacterial
artificial chromosomes (BACs) to identify BACs in the region. The
initial set of markers used was D17S969, WI-2437, WI-2335, D17S947,
and D17S799 (FIG. 2A). BACs identified with these markers were
end-sequenced. PCR primers designed from those end sequences were
used as markers to arrange the initial BACs into contigs. The
outermost marker from each contig was used in successive rounds of
BAC library screening, eventually enabling the completion of a BAC
clone contig that spanned the genetically defined interval. A set
of overlapping but non-redundant BAC clones that spanned this
interval (FIG. 2A) was then selected for use in subsequent
molecular cloning protocols such as genomic sequencing.
[0276] Genomic Sequencing.
[0277] Given a tiling path of BAC clones across a defined interval,
one useful gene finding strategy is to generate an almost complete
genomic sequence of that interval. Two types of random genomic
clone sublibraries were prepared from each BAC on the tiling path;
these were Sau 3A partial digest libraries with inserts in the 5 to
8 kb size range, and random shear libraries with inserts in the 1.0
to 1.5 kb size range. Plasmid DNA from individual clones from the
Sau 3A sublibraries sufficient in number to generate an, on
average, 1.times. redundant sequence of each BAC was prepared using
an Autogen robotic plasmid preparation machine (Integrated
Separation Systems). Insert DNA from individual clones from the
random shear sublibraries sufficient in number to generate an, on
average, 5.times. redundant sequence of each BAC, was prepared by
PCR with vector primers directly from aliquots of bacterial
cultures of each individual clone. The resulting DNA templates were
subjected to DNA sequencing from both ends with Ml 3 forward or
reverse fluorescent dye-labeled primers on ABI 377 sequencers.
[0278] These sequences were assembled into sequence contigs using
the program Acem.bly (Thierry-Mieg et al., 1995; Durbin and
Thierry-Mieg, 1991). The genomic sequence contigs were placed in a
Genetic Data Environment (GDE) (Smith et al., 1994) local database
for -'subsequent similarity searches. Similarities among genomic
DNA sequences and GenBank entries--both DNA and protein--were
identified using BLAST (Altschul et al., 1990). The DNA sequences
were also characterized with respect to short period repeats, CpG
content, and long open reading frames.
Example 5
Sequence Assembly of the Human HPC2 Gene
[0279] A BLASTn (Altschul et al., 1990) search of genomic sequences
from BAC 31k12 against dbEST identified two independent sets of
human ESTs that, when parsed across the BAC 31 k112 genomic
sequences, revealed the presence of two independent multi-exon
candidate genes, 04CG09 and the HPC2 gene (FIG. 2B). A subset of
the EST sequences assigned to HPC2 (Table 4) was assembled to
produce a tentative partial cDNA sequence for the gene.
5TABLE 4 Human ESTs Used to Assemble a Tentative Partial Human HPC2
cDNA Sequence EST Accession # Exon Span AA679618 1.fwdarw.6 Z17886
4.fwdarw.8 W37591 7.fwdarw.12 AA310236 12.fwdarw.16 R55841
15.fwdarw.19 T34216 18.fwdarw.21 AA634909 20.fwdarw.24 AA504412
23.fwdarw.24 R42795 24.fwdarw.polyA
[0280] The individual exons of the human HPC2 gene were identified
by parsing that tentative cDNA sequence across the BAC 31k12
genomic sequence (see schematics in FIG. 2B). After we had
identified the HPC2 gene, the MIT genome sequencing completely
sequenced another BAC, 597 m12, that also contains all of the exons
of HPC2 (GenBank accession # AC005277) The sequence of the human
HPC2 gene was corrected both by comparison of the sequences of the
individual exons from the tentative cDNA assembly to the
corresponding genomic sequences of BACs 31k12 and 597 m12, and by
mutation screening the gene from a set of human genomic DNAs (see
Example 8).
[0281] The original tentative human HPC2 cDNA sequence contained
neither the start codon nor any of the 5' UTR. These were obtained
by biotin capture 5' RACE (Tavtigian et al., 1996). Briefly, a
biotinylated reverse primer, CA4cg07.BR2, was designed from the
sequence of the third exon of the human HPC2 gene and used, along
with the anchor primer SampA, for a first round of PCR
amplification from human fetal liver cDNA that had been prepared
such that the 5' ends of cDNA molecules are anchored with the
sequence 5tag1. The resulting PCR products were captured on
streptavidin paramagnetic particles (Dynal), washed, and used as
template in a second round PCR amplification. A phosphorylated
reverse primer, CA4cg07.PR2, was designed from the sequence of the
second exon of the human HPC2 sequence and used, along with the
nested phosphorylated anchor primer 5ampB, for the second round PCR
amplification. The resulting 5' RACE products were gel purified and
sequenced with the primer CA4cg07.PR2 using dye-terminator
chemistry and ABI 377 sequencers. Analysis of the sequences of
these 5' RACE products yielded both the start codon and part of the
5' UTR including an in-frame stop codon (FIG. 4). Sequences of the
human primers used for 5' RACE are given in Table 5.
[0282] A full length human HPC2 cDNA was amplified from human head
and neck cDNA using the primers CA4cg7.ATG and CA4cg7.TGA. The cDNA
was ligated into the vector pGEM-T Easy (Promega) and transformed
into E. coli. The sequence of the cDNA clone was confirmed by dye
terminator sequencing on ABI 377 sequencers. Sequences of primers
used to amplify the cDNA construct and confirm the sequence of the
cDNA clone are also given in Table 5.
6TABLE 5 Primers Used in 5' RACE, cDNA Cloning and Sequence
Confirmation of a Full-length Human HPC2 cDNA Sequence (SEQ ID NO:)
5'RACE PRIMERS Stag1 CAG GAA TTC AGC ACA TAC TCA TTG TTC Agn n (29)
5AmpA CAG GAA TTC AGC ACA TAC TCA (30) 5AmpB (P)TT CAG CAC ATA CTC
ATT GTT CA (31) CA4cg07.BR2 (B)TG AAC GCC TTC TCC ACA GT (32)
CA4cg07.PR2 (P)GT ACC CGC TGC CAC CAC (33) EXPRESSION CONSTRUCT
PRIMERS CA4cg7.ATG GCT AGG ATC CGC CAC CAT GTG GGC GCT TTG CTC (34)
CA4cg7.TGA GCT ACT CGA GTC ACT GGG CTC TGA CCT TC (35) SEQUENCING
PRIMERS M13F20 GTA AAA CGA CGG CCA GT (36) M13R20 GGA AAC AGC TAT
GAC CAT G (37) CA4cg7F1 TGC GCA CGC GAG AGA AG (38) CA4cg7RI CGC
TTC TCT CGC GTG CG (39) CA4cg7F2 TCT AAT GTT GGG GGC TTA (40)
CA4cg7R2 TAA GCC CCC AAC ATT AGA (41) CA4cg7F3 TGA AAA TGA GCC ACA
CCT (42) CA4cg7R3 AGG TGT GGC TCA TTT TCA (43) CA4cg7F4 CAT TCA ACC
CAT CTG TGA (44) CA4cg7R4 TCA CAG ATG GGT TGA ATG (45) CA4cg7F5 TGA
ATG CCT CCT CAA GTA (46) CA4cg7R5 TAC TTG AGG AGG CAT TCA (47)
CA4cg7F6 GCT ACT GGA CTG TGG TGA (48) CA4cg7R6 TCA CCA CAG TCC AGT
AGC (49) CA4cg7F7 TGG AAG AGT TTC AGA CCT G (50) CA4cg7R7 CAG GTC
TGA AAC TCT TCC A (51) CA4cg7F8 CGC AGG GAC GCA CCA TA (52)
CA4cg7R8 GGT TGA ACT CGG AGA AGA (53) CA4cg7F9 CAA CTG GAA AAA TAC
CTC G (54) CA4cg7F10 GCA GAG TCC AGA AAG GC (55) CA4Cg7F11 AGA GGA
AAC TTC TTG GTG C (56) CA4cg7F12 ACC AAG GAA AGG CAG ATG (57)
CA4cg7F13 GTC AAC ATA AGC CCC GAC (58) CA4cg7F14 GGC TGC TGT GTT
TGT GTC (59) CA4cg7R14 GAA GGC ATT TGG CAG GA (60) CA4cg7F15 TAT
GAT TCC TGC CAA ATG (61) CA4cg7R15 TCC AGC GAG AGG TGT GC (62)
CA4cg7F16 TGC GAG GCT CTG GTC CG (63) CA4cg7R16 GGG CAT TGT TGG AAA
GTC (64) CA4cg7F17 TGT TTG CTG GCG ACA TC (65) nn--the last 2
nucleotides of the anchor sequence 5tag1 are specific for each cDNA
prep. (P) indicates phosphate at the 5' end of the oligo (B)
indicates biotin at the 5' end of the oligo
Example 6
Sequence Assembly of the Mouse HPC2 Gene
[0283] A BLAST search of the assembled HPC2 cDNA sequence against
dbEST identified 5 mouse ESTs that derived from a very similar
gene, the mouse ortholog of HPC2, Mm.HPC2; their accession numbers
are listed in Table 6.
7TABLE 6 Mouse ESTs Used to Assemble a Tentative Partial Mm.HPC2
cDNA Sequence EST Accession # Exon Span AA563096 1.fwdarw.5
AA518169 8.fwdarw.14 AI132016 16.fwdarw.17 AA184645 19.fwdarw.24
AA174437 24.fwdarw.24
[0284] The original partial Mm.HPC2 cDNA sequence contained the
start codon but little of the 5' UTR. More extensive 5' UTR
sequence was obtained by 5' RACE. Briefly, a biotinylated reverse
primer, m04cg07BR1, was designed from the sequence of the fourth
exon of the mouse HPC2 gene and used, along with the anchor primer
5ampA, for a first round of PCR amplification from mouse embryo
cDNA that had been prepared such that the 5' ends of cDNA molecules
are anchored with the sequence 5tag1. The resulting PCR products
were captured on streptavidin paramagnetic particles (Dynal),
washed, and used as template in a second round PCR amplification. A
phosphorylated reverse primer, m04cg07PR1, was designed from the
sequence of the third exon of the mouse HPC2 sequence and used,
along with the nested phosphorylated anchor primer 5ampB, for the
second round PCR amplification. The resulting 5' RACE products were
gel purified and sequenced with the primers m04cg07PR1 and m04cg07
exon2 rev using dye-terminator chemistry and ABI 377 sequencers.
Analysis of the sequences of these 5' RACE products yielded both
the start codon and part of the 5' UTR including an in-frame stop
codon (FIG. 4). Sequences of the primers used for 5' RACE are given
in Table 7.
[0285] More extensive 5' UTR sequence, sequence that may be from
the promoter, and the sequences of intron 1 and intron 2 of the
mouse HPC2 gene were obtained by genomic sequencing. BAC 428n12 was
obtained from a mouse genomic library by screening the library by
PCR with a pair of primers (04CG7.m11f1 and 04CG7. m11r1, Table 7)
derived from exon 11 of the mouse HPC2 cDNA sequence. A primer pair
derived from the SP6 end sequence of BAC 428n12 (428n12.S6.F1 and
428n12.S6.F1, Table 7) was used to screen the mouse BAC library by
PCR; several overlapping BACs, including BAC 199n11, were
identified. BACs 428n12 and 199n11 were sequenced with a series of
13 sequencing primers (mcg7f1 to mcg7r7, Table 7) derived from
mouse HPC2 cDNA dye-terminator chemistry and ABI 377 sequencers. A
subset of these sequences were assembled into a genomic sequence
contig extending from 280 bp upstream of the ATG start codon of
exon 1 into exon 3.
[0286] A full length mouse HPC2 cDNA is amplified from mouse
embryo, placenta, or fetal brain cDNA using the primers msCA4cg7.f
out and msCA4cg7.r out The cDNA is reamplified with the primers
msCA4cg7.ATG and msCA4cg7.TGA. The resulting PCR products are gel
purified, ligated into the vector pGEM-T Easy (Promega), and
transformed into E. coli. The sequence of the cDNA clone are
confirmed dye terminator sequencing on ABI 377 sequencers.
Sequences of primers in use to amplify the cDNA construct are also
given in Table 7.
8TABLE 7 Primers Used in 5' RACE and cDNA Cloning of a Full-length
Mouse HPC2 cDNA Sequence (SEQ ID NO:) 5'RACE PRIMERS 5tag1 CAG GAA
TTC AGC ACA TAC TCA TTG TTC Agn n (66) 5AmpA CAG GAA TTG AGC ACA
TAC TCA (67) 5AmpB (P)TT CAG CAC ATA CTC ATT GTT CA (68) m04cg07BR1
(B)CA GAA CAC ATT TGG GAA GC (69) m04cg07PR1 (P)GA TGT TGT CCA AGC
GAG C (70) BAC library screening primers 04CG7.m11f1 TGA CAC ACA
GCA CCT GA (71) 04CG7.m11r1 GAA GAT GTC AGG GTG GA (72)
428n12.S6.FI CAG GCA TAC CAC TAC AGA (73) 428n12.S6.R1 TAT CAA CTT
CTA GGC AAG TG (74) Genomic sequencing primers mcg7f1 GGA GGA TGT
CGC AGG GTT C (75) mcg7r1 GAA CCC TGC GAC ATG GTG C (76) mcg7f2 TCG
GAG GGT TCG GCT CGT C (77) mcg7r2 AAC CCT GCG ACA TGG TGC G (78)
mcg7f3 AAA GAC CCA CTG CGA CAC C (79) mcg7r3 GCA GGT GTC GCA GTG
GGT C (80) mcg7f4 GCG AAC ACC GTG TAC CTG CA (81) mcg7r4 CAG GTA
CAC GGT GTT CGG G (82) mcg7f5 GTC TTC TCG GAA TAC AAC AGG (83)
mcg7r5 CTG TTG TAT TCC GAG AAG AC (84) mcg7f6 AAG GCG TCC AAC GAC
TTA TG (85) mcg7r6 AGT CGT TGG ACG CCT TCT CC (86) mcg7r7 TCC GAG
TCA GAA AGA TGT TG (87) EXPRESSION CONSTRUCT PRIMERS PRIMARY PGR
msCA4cg7.f out GCC TTG TCA GCC TGG TG (88) msCA4cg7.r out AGG AAG
TGA GCA GAG CG (89) SECONDARY PCR msCA4cg7.ATG GGT AAA GCT TGC CAC
CAT GTG GGC GCT CCG CTC (90) msCA4cg7.TGA GCT ACT CGA GTC ACA CTC
GCG CTC CTA (91) SEQUENCING PRIMERS m04cg07 exon2 rev GCC TTC TCC
GCA GTT A (92) nn--the last 2 nucleotides of the anchor sequence
5tag1 are specific for each cDNA prep. (P) indicates phosphate at
the 5' end of the oligo (B) indicates biotin at the 5' end of the
oligo
Example 7
Northern Blots
[0287] Prehybridization and hybridization were performed at
42.degree. C. in 50% formamide, 5.times. SSPE, 1.0% SDS, 5.times.
Denhardt's mixture, 0.2 mg/mL denatured salmon sperm DNA, and 2
gg/mL poly(A). Dextran sulfate (4% v/v) was included in the
hybridization solution only. The membranes were washed twice in
2.times. SSC/0.1% SDS at 20.degree. C. for 30 minutes, followed by
a stringency wash in 0.1.times. SSC/0.1% SDS at 50.degree. C. for
30 minutes.
Example 8
Mutation Screening of the Human HPC2 Gene
[0288] Using genomic DNAs from prostate kindred members, prostate
cancer affecteds, and tumor cell lines as templates, nested PCR
amplifications were performed to generate PCR products to screen
for mutations in the HPC2 gene. The primers listed in Table 8 were
used to amplify segments of the HPC2 gene. Using the outer primer
pair for each amplicon (1A-1P, i.e., forward A and reverse P of
amplicon 1), 10-20 ng of genomic DNA were subjected to a 25 cycle
pnmary amplification, after which the PCR products were diluted
45-fold and reamplified using nested M13-tailed primers (1B-1Q,
1C-1R i.e., nested forward B and nested reverse Q of amplicon 1 or
nested forward C and nested reverse R of amplicon 1) for another 23
cycles. In general, samples were amplified with Taq Platinum (Life
Technologies) DNA polymerase; cycling parameters included an
initial denaturation step at 95.degree. C. for 3 min, followed by
cycles of denaturation at 96.degree. C. (12 s), annealing at
55.degree. C. (15 s) and extension at 72.degree. C. (30-60 s).
After the PCR reactions, excess primers and deoxynucleotide
triphosphates were digested with exonuclease I (United States
Biochemicals) and shrimp alkaline phosphatase (Amersham). PCR
products were sequenced with M13 forward or reverse fluorescent
(Big Dye, ABI) dye-labeled primers on ABI 377 sequencers.
Chromatograms were analyzed for the presence of polymorphisms or
sequence aberrations in either the Macintosh program Sequencher
(Gene Codes) or the Java program Mutscreen. We obtained more than
95% double strand sequence coverage for the entire open reading
frame of all samples screened.
9TABLE 8 Primers Used to Mutation Screen the HPC2 Gene from Genomic
DNA Exon/Primer name Sequence (SEQ ID NO:) HPC2 exon 1
ca4cg7.m1Anew CCG CTT GAG ACG CTC TAG TAT (93) ca4cg7.m1P GCT CCG
AAA GTG CTG ACA G (94) ca4cg7.m1Bnew GTT TTC CCA GTC ACG ACG TTT
CTA TTG GAT GAG CAG CCT (95) ca4cg7.m1Qnew AGG AAA CAG CTA TGA CCA
TGC CTG CGA TAT GGT GCG TC (96) ca4cg7.m1C GTT TTC CCA GTC ACG ACG
CTC AGT TTT GGT GGA GAC G (97) ca4cg7.m1Rnew AGG AAA CAG CTA TGA
CCA TGT GCC CCG ATG CTC AGA G (98) HPC2 exons 2 & 3 (primary)
ca4cg7.m2&23 A2 AAT GGT GTC AGA GAG TTT ACA G (99)
ca4cg7.m2&23P GCT ATT TGG GAG GCT GAG G (100) HPC2 exon 2
(nested) ca4cg7.m2B GTT TTC CCA GTC ACG ACG AAT GGT GTC AGA GAG TTT
ACA G (101) ca4cg7.m2Q AGG AAA CAG CTA TGA CCA TGA ACA AGG ACC ACT
TTT GCT AT (102) HPC2 exon 3 (nested) ca4cg7.m23B GTT TTC CCA GTC
ACG ACG TTT ATA GCA AAA GTG GTC CU G (103) ca4cg7.m23Q AGG AAA CAG
CTA TGA CCA TGA GAC TTC CCA CCA GCC TC (104) HPC2 exon 4
ca4.cg07.m24A CCT TGC TGC TTC ACC CTA G (105) ca4.cg07.m24P TGC TTT
ATA TGT GCT GCT ACG (106) ca4.cg07.m24B GTT TTC CCA GTC ACG ACG CAT
CTT CCC TGG TTG TAC TTC (107) ca4.cg07.m24Q AGG AAA CAG CTA TGA CCA
TCT GGA GGG CAG AAG ACT GAT (108) HPC2 exon 5 ca4cg7.m3A CTA CAT
TTG TTC AAC CAT AAC TG (109) ca4cg7.m3P GAT TTT GAG GTT TGA TGT TGA
TG (110) ca4cg7.m3B GTT TTC CCA GTC ACG ACG CAT TTG TTC AAC CAT AAC
TGC (111) ca4cg7.m3Q AGG AAA CAG CTA TGA CCA TAT TTG AGA GGT CAG
GGC ATA (112) HPC2 exon 6 ca4cg7.m4A TCG TGT CAG ATT CCC ACC ATA
(113) ca4cg7.m4P AGG CAT AAG TCA GAC ATC CGT (114) ca4cg7.m4B GTT
TTC CCA GTC ACG ACG GTT ACT CTT CCC ACA CAT CTT C (115) ca4cg7.m4Q
AGG AAA CAG CTA TGA CCA TCA CAG CAA GTG TTC AGT TTC TA (116) HPC2
exon 7 ca4cg7.m5A CAT TCC CAT GTA TGA ACG TCT (117) ca4cg7.m5P ATA
GTA AGC CCA GGA AGA AGGA (118) ca4cg7.m5B GTT TTC CCA GTC ACG ACG
CAT TCC CAT GTA TGA ACG TC T (119) ca4cg7.m5Q AGG AAA CAG CTA TGA
CCA TCT ACA AGC ATT ACA AGG CAG AG (120) HPC2 exon 8 ca4cg7.m6A AGT
GTC TTC AGC CTT TGT ATT G (121) ca4cg7.m6P ATC TGC TAT CTC TTC TTG
TCT CA (122) ca4cg7.m6B GTT TTC CCA GTC ACG ACG ATC GGG TCA TAA TCA
GTC TGT G (123) ca4cg7.m6Q AGG AAA CAG CTA TGA CCA TAT CTC TTC TTG
TCT CAG GTA ACA (124) HPC2 exons 9 & 10 (primary)
ca4cg7.m7&8A CTT CTG AAA GCA ATA AAC GCA T (125)
ca4cg7.m7&8P GAT GTC CAA ACT GTT CCA CG (126) HPC2 exon 9
(nested) ca4cg7.m7B GTT TTC CCA GTC ACG ACG TAA AAC CAA CCT TCT TCA
TTA G (127) ca4cg7.m7Q AGG AAA CAG CTA TGA CCA TAG CAA TGA TGG GAG
CGA TG (128) HPC2 exon 10 (nested) ca4cg7.m8B GTT TTC CCA GTC ACG
ACG GGC TTC TGG GGA CTC ACT G (129) ca4cg7.m8Q AGG AAA CAG CTA TGA
CCA TCC TTC AAA AGT GGT GTC TGT AG (130) HPC2 exon 11 ca4.cg07.m9A
GTA TCC ACA AAG AGA CCA GAA G (131) ca4.cgO0.m9P CAC CAA CTA CCA
ACA GTG ACT TA (132) ca4.cg07.m9B GTT TTC CCA GTC ACG ACG GCT CAC
TGG ATA GGA TAT GTC AT (133) ca4.cg07.m9Q AGG AAA CAG CTA TGA CCA
TCC AGA AAC ACA GCT CTT GCC (134) HPC2 exon 12 ca4.cg07.m10A GCT
TGC CAG ATA CAG GAA TC (135) ca4.cg07.m10P ACA GAA AGT TTA GGC AGG
TG (136) ca4.cg07.m10B GTT TTC CCA GTC ACG ACG ACG ATA CCC CTC CCT
GGC T (137) ca4.cg07.m10Q AGG AAA CAG CTA TGA CCA TAC AGA AAG TTT
AGG CAG GTG (138) HPC2 exons 13 & 14 (primary)
ca4.cg07.m11&12A CCT CTC ACT CTT CCC AGC AC (139)
ca4.cg07.m11&12P GGA GTA GGC TGC TTT TCT AAA T (140) HPC2 exon
13 (nested) ca4.cg07.m11B GTT TTC CCA GTC ACG ACG GAA CAC CTC ATC
CTC ATT ACC A (141) ca4.cg07.m11Q AGG AAA CAG CTA TGA CCA TAA GAG
ACA AAA CAC ATT CAT GG (142) HPC2 exon 14 (nested) ca4.cg07.m12B
GTT TTC CCA GTC ACG ACG GTT TCC GCT GTA AGG TAG TGT (143)
ca4.cg07.m12Q AGG AAA CAG CTA TGA CCA TCT GGA ACA TTT ACT ATG TGG
CTA (144) HPC2 exon 15 ca4.cg07.m13A TGC TAG TGG GTA GAG GTC AG
(145) ca4.cg07.m13P ACT GAA AGC CAG GTT AGA ATG (146) ca4.cg07.m13B
GTT TTC CCA GTC ACG ACG ACC CTG TCC GTC ACC TGA G (147)
ca4.cg07.m13Q AGG AAA CAG CTA TGA CCA TCC CAC CAG CAC TCC ACT TA
(148) HPC2 exon 16 ca4cg07.m14A TGT GAA GAC GGG ATA ACC TGA (149)
ca4cg07.m14P GAC AGG GCT TGA TAC CGC A (150) ca4cg07.m14B GTT TTC
CCA GTC ACG ACG ATG CTG GCT CAC TTT TGA CC (151) ca4cg07.m14Q AGG
AAA CAG CTA TGA CCA TGAC TGG TGA GTA CAG CAG GA (152) HPC2 exon 17
ca4.cg07.m15A CCA GCC TTT GTG TAA GTC TAC (153) ca4.cg07.m15P TCT
GGG CAA GTT TGG AAG C (154) ca4.cg07.m15B GTT TTC CCA GTC ACG ACG
TCC AAA GCA GAC ATC AGC CTC (155) ca4.cg07.m15Q AGG AAA CAG CTA TGA
CCA TGG AGG AAA AGA CGC AGC CA (156) HPC2 exon 18 ca4.cg07.m16A CGC
TTT CTG CCT GTG ACA T (157) ca4.cg07.m16P TTC TGT CCT TCA GCC AAT
GC (158) ca4.cg07.m16B GTT TTC CCA GTC ACG ACG TTA GAG GCT GGT GGG
TGA C (159) ca4.cg07.m16Q AGG AAA CAG CTA TGA CCA TCA TCT CAA TAA
AAA CTG GAG TGC (160) HPC2 exon 19 ca4.cg07.m17A CAC TTG ATG GGC
GTT CTG AG (161) ca4.cg07.m17P TTC TGT CCT TCA GCC AAT GC (162)
ca4.cg07.m17B GTT TTC CCA GTC ACG ACG TTC CAG CGG TTT ACA CAT CA
(163) ca4.cg07.m17Q AGG AAA CAG CTA TGA CCA TTA CCC CAG TGT CCA CCT
TG (164) HPC2 exons 20 & 21 (primary) CA4CG7.m18&22A GGG
TTC TCC AGC CAA AGA CT (165) CA4CG7.m18&22P CTG AGT CTC CTG CCT
CTG C (166) HPC2 exon 20 (nested) ca4.cg07.m18B GTT TTC CCA GTC ACG
ACG GGG TTC TCC AGC CAA AGA CT (167) ca4.cg07.m18Q AGG AAA CAG CTA
TGA CCA TGT GGG GCT GGA AGG CTC TG (168) HPC2 exon 21 (nested)
ca4.cg07.m22B GTT TTC CCA GTC ACG ACG AAG AGG TAA GGG GCA CAG C
(169) ca4.cg07.m22Q AGG AAA CAG CTA TGA CCA TCT GAG TCT CCT GCC TCT
GC (170) HPC2 exon 22 ca4.cg07.m19A GCT GAG TGT TGA GAC CAG GA
(171) ca4.cg07.m19P AGA CAA ACG ACG GCT GCT C (172) ca4.cg07.m19B
GTT TTC CCA GTC ACG ACG TTG AGA CCA GGA AAC AGC AC (173)
ca4.cg07.m19Q AGG AAA CAG CTA TGA CCA TGA GAG GAT GTG GGC GAC AA
(174) HPC2 exon 23 ca4.cg07.m20A GGG AGA TGG TGC TGG CTA C (175)
ca4.cg07.m20P CCT GGT TAG TGA TGG GTA GAT (176) ca4.cg07.m20B GTT
TTC CCA GTC ACG ACG CAG GGT CTG TGC CAC TGT C (177) ca4.cg07.m20Q
AGG AAA CAG CTA TGA CCA TCT CAG TGT GTA GAG TCC TGT C (178) HPC2
exon 24 splice acceptor and open reading frame ca4.cg07.m21A TTG
ATT TTG AGA GCA TCT GGA C (179) ca4.cg07.m21P CTC GGA CAC TTA GAC
CCA CTG (180) ca4.cg07.m21B1 GTT TTC CCA GTC ACG ACG TGC ATC CCT
TCC AGC TCC T (181) ca4.cg07.m21Q AGG AAA CAG CTA TGA CCA TGA CAC
ACA GCC TTC TGA GTT CA (182) ca4.cg07.m21C GTT TTC CCA GTC ACG ACG
CCA CAC AGA GGA GCC ACA G (183) ca4.cg07.m21R AGG AAA CAG CTA TGA
CCA TAC CAG TCC TAA GAG GCA TCT ATA (184) HPC2 exon 24 3'
untranslated region ca4.cg07.m21.3'UTR A CCA CAC AGA GGA GCC ACA G
(185) ca4.cg07.m21.3'UTR P CCA GAG GTG CTC ACT ACG AC (186)
ca4.cg07.m21.3'UTR B GTT TTC CCA GTC ACG ACG AGG TCA GAG CCC AGT
GAA GAT (187) ca4.cg07.m21.3'UTR Q AGG AAA CAG CTA TGA CCA TCA TCT
GCT TGC TTC CGT GTG (188) ca4.cg07.m21.3'UTR C GTT TTC CCA GTC ACG
ACG TCA GGA TAG GTG GTA TGG AGC (189) ca4.cg07.m21.3'UTR R AGG AAA
CAG CTA TGA CCA TCG GAC ACT TAG ACC CAC TGA T (190)
[0289]
10TABLE 9 Sequence Variants Variant name Sequence (SEQ ID NO:)
Coding effect* C650T AGACTCCGAGTYGAATGAAAATG (191) Ser217Leu A1560G
GGTGAGGGCACRTTTGGGCAGCT (192) Thr520Thr G1621A
GCACCCTGGCTRCTGTGTTTGTG (193) Ala541Thr 1641 insG (normal)
GTGTCCCACCTG-CACGCAGATCA (194) (with insertion of G)
GTGTCCCACCTGGCACGCAGATCA (195) frameshift C1722T
AAGCCGCTTCAYCCTTTGCTGGT (196) His574His A1893G
GCTGTTGCGAACRTGTGATTTGGA (197) Thr631Thr C2632G
GAGGCTTGGGSTCCCACATAAG (198) C2687T CCTGGCACAGCYGCGGGCCAGGA (199)
G2801A AATCCAGCAAARTGATTCCCTGC (200) IVS2 T-11C
Taaatgttttytcattcttag (201) IVS5 T-14C Ttgctgttgtgyggttttcttgt
(202) IVS10 23insGAT (normal) ggttttcttgat-tcagcagttaca (203) (with
insertion of GAT) Ggttttcttgatgattcagcagttaca (204) IVS13 C15T
Ggtctcagacyggccccttgtc (205) IVS14 A17T Tgccatcttgawctaatggaatc
(206) IVS14 T-8C Cttctctctctycctgcagggat (207) IVS16 C41T
Catcaagggcaygtttacttttt (208) IVS19 C26G Cagccttgcccsctgggctgttg
(209) *based on conceptual translation of the HPC2 ORF for each
allele of the sequence variant.
[0290] Kindred 4102 was ascertained as a high risk cluster with
eight prostate cancer cases in a three generation pedigree.
Genotyping revealed that six of the eight cases shared a chromosome
17p haplotype. The youngest (age at diagnosis of 46) affected
carrier of this shared haplotype, 4102.013 (i.e., kindred #4102,
individual #013; FIG. 5A), was selected for mutation screening. On
mutations screening lymphocyte DNA from 4102.013, we detected a
frameshift, 1641 insG, in ELAC2. A test for segregation revealed
that the frameshift was not on the father's chromosome, but rather
was inherited through the carrier's mother, 4102.002. Her affected
uncle 4102.053 was diagnosed with and died of prostate cancer at
age 76 in the 1960s. Genotyping of his children demonstrated that
he was an obligate frameshift carrier. In all, there are five male
frameshift carriers over age 45 in the pedigree. Of these, three
have prostate cancer, the fourth has a PSA of 5.7 at age 71, and
the fifth has a PSA of 4.2 at age 74 (FIG. 5A). The frameshift
occurs at His 548, within the histidine motif (FIGS. 6A-B) and is
predicted to be quite disruptive to the protein.
[0291] As the frameshift 1641 insG was found in an individual with
early onset prostate cancer, we screened an additional 45 prostate
cancer cases with early age at diagnosis (Dx.ltoreq.55 years),
irrespective of evidence of linkage to any locus, for mutations in
ELAC2. An alteration, Arg 781 His, was identified in individual
4289.003, diagnosed with prostate cancer at age 50. Upon expansion
of his pedigree, the mutation was traced back four generations to
4289.006, who had affected descendants from five known wives.
Prostate cancer cases who carry the missense change have been found
among the descendants from three of these five marriages. Of
thirteen prostate cancer cases in the pedigree, six carry the
missense change, three are unknown, and four are non-carriers. In
addition, a female carrier of this missense change, 4289.183, was
diagnosed with ovarian cancer at age 43 (FIG. 5B). Within the
generations with phenotype information, there are only two
unaffected male mutation carriers over age 45; 4289.068 (PSA of 0.6
at age 60) and 4289.063, who died of a heart attack at age 62. We
have no additional information on 4289.063; however, two of
4289.063's sons and a grandson are carriers who have been diagnosed
with prostate cancer. The missense change occurs in a very highly
charged stretch of amino acid residues near the C-terminus of the
protein. Arg 781 is conserved in mouse (FIGS. 6A-B), and the charge
character of the sequence segment is conserved in C. elegans. While
one cannot definitively predict that this missense change will
affect protein function, expansion from a single affected mutation
carrier to a pedigree with a LOD score of 1.3 provides good
evidence that the mutation is in fact deleterious.
[0292] The identification of two mutations provides strong evidence
that ELAC2 is a prostate cancer susceptibility gene. However, after
screening 42 haplotypes with evidence for linkage at 17p, we have
found only these two high-risk mutations. Thus it seems that only a
small fraction of prostate cancer pedigrees segregate obvious
mutations in the ELAC2 coding sequence. We do not yet know what
fraction of the pedigrees harbor subtle gene rearrangements or
regulatory mutations.
[0293] Taken together, the observation that the frameshift HPC2
1641insG segregates with prostate cancer across three generations
of kindred 4102, and the inference from shared sequence similarity
that the frameshift HPC2 1641insG must be deleterious to the
function of the HPC2 protein, establish that deleterious germline
mutations in the HPC2 gene confer susceptibility to prostate
cancer.
Example 9
Common Missense Changes in HPC2
[0294] When our original set of linked pedigrees was screened for
mutations in ELAC2, we observed several occurrences of the
non-conservative missense change Ser 217 Leu. This missense change
is embedded in an extremely hydrophilic segment of the protein
sequence. Like the common human allele, the mouse and C. elegans
residues at this position are also serine. Although the sequence of
this segment is not well conserved, its hydrophilic character is
(FIGS. 6A-B); thus substitution of a bulky hydrophobic residue for
Ser 217 could result in structural consequences to the protein.
[0295] We analyzed this sequence variant in our pedigree cases,
unaffected pedigree members, and an unrelated set of males who have
no diagnosis of cancer (divergent controls). The total number of
individuals typed exceeded 4,000 (Table 3), with an overall allele
frequency of 30% for Leu 217. A logistic regression was performed
for disease status to delineate effects of genotypes at Ser 217 Leu
versus birth year (a demographic datum collected on all
participants). We observed a significant interaction between
genotype and birth year (p=0.027), indicating that association
tests should be performed which appropriately considered birth
cohort. FIG. 7 illustrates this birth effect, showing that genotype
frequencies differ across birth cohorts for cases, but appear more
uniform for the unaffected controls. We subsequently chose to
analyze the effect of genotype in individuals born after 1919,
since the data suggest that a different risk pattern may exist for
individuals born before this date.
[0296] Association tests are consistent with the hypothesis that
the Leu 217 variant is deleterious or in disequilibrium with
another deleterious variant. Prostate cancer patients born between
1920 and 1959 have a significantly higher proportion of Leu 217
homozygotes than either the divergent controls (57/429 vs. 9/148,
p-value=0.026) or the unaffected pedigree members (57/429 vs.
220/2371, p-value=0.013) (FIGS. 7 and 8). That Leu 217 is so common
could be explained by the allele contributing to a common disease
in a recessive manner.
[0297] Upon mutation screening ELAC2 in the set of early onset
prostate cancer cases, we also observed several occurrences of a
second non-conservative missense change, Ala 541 Thr. This missense
change occurs at the border of the histidine motif (FIGS. 6A-B and
9) and thus may well affect the protein's function. This variant
has been examined in the same set of cases and controls, where it
has an overall allele frequency of 4%. Thr 541 is in strong
disequilibrium with Leu 217; in fact, we have yet to observe a
chromosome that carries Thr 541 that does not also carry Leu 217.
Another logistic regression was performed to investigate effects of
genotypes at Ala 541 Thr. Again, a significant interaction between
genotype and birth year was found (p=0.003), along with evidence
for an effect of genotype at Ala 541 Thr on disease status. Table
10 shows the allele frequencies.
11TABLE 10 Allele Definitions Allele Defining Sequence Variant(s)
Note 0 wt Matches mouse at polymorphic positions 1 Leu 217 Allele
frequency = 26.0% 2 Leu 217 + Thr 541 Allele frequency = 3.9%
[0298] The carrier frequency of Thr 541 is significantly higher in
prostate cancer cases than divergent controls such that the variant
appears to be dominant and deleterious (carrier frequency of 42/429
vs. 5/148, p-value=0.022) (FIG. 8). In contrast, the Thr 541
carrier frequency is not significantly higher in the cases than the
unaffected pedigree members. However, in the comparison between
cases and pedigree unaffecteds, when Leu 217 homozygotes are
subdivided into Thr 541 carriers and non-carriers, the presence of
Thr 541 is associated with a higher odds ratio (2.0 vs. 1.4) and
the model remains statistically significant (p-value=0.017, trend
test p-value 0.004) (FIG. 8). Thus both comparisons support the
hypothesis that the allele bearing both Thr 541 and Leu 217 is more
deleterious than the allele bearing just Leu 217.
Example 10
Identification of HPC2-interacting Proteins by Two-hybrid
Analysis
[0299] DNA fragments encoding all or portions of HPC2 are ligated
to a two-hybrid DNA-binding domain vector such as pGBT.C such that
the coding sequence of HPC2 is in-frame with coding sequence for
the Gal4p DNA-binding domain. A plasmid that encodes a DNA-binding
domain fusion to a fragment of HPC2 is introduced into the yeast
reporter strain (such as J692) along with a library of cDNAs fused
to an activation domain. Transformants are spread onto 20-150 mm
plates of selective media, such as yeast minimal media lacking
leucine, tryptophan, and histidine, and containing 25 mM
3-amino-1,2,4-triazole. After one week incubation at 30.degree. C.,
yeast colonies are assayed for expression of the lacZ reporter gene
by .beta.-galactosidase filter assay. Colonies that both grow in
the absence of histidine and are positive for production of
.beta.-galactosidase are chosen for further characterization.
[0300] The activation domain plasmid is purified from positive
colonies by the smash-and-grab technique. These plasmids are
introduced into E. coli (e.g., DH10B (Gibco BRL) by electroporation
and purified from E. coli by the alkaline lysis method. To test for
the specificity of the interaction, specific activation domain
plasmids are cotransformed into strain J692 with plasmids encoding
various DNA-binding domain fusion proteins, including fusions to
segments of HPC2 and human lamin C. Transformants from these
experiments are assayed for expression of the HIS3 and lacZ
reporter genes. Positives that express reporter genes with Hs.HPC2
constructs and not with lamin C constructs encode bona fide
HPC2-interacting proteins. These proteins are identified and
characterized by sequence analysis of the insert of the appropriate
activation domain plasmid.
[0301] This procedure is repeated with mutant forms of the HPC2
gene, to identify proteins that interact with only the mutant
protein or to determine whether a mutant form of the HPC2 protein
can or cannot interact with a protein known to interact with
wild-type HPC2.
Example 11
[0302] Identification and Sequencing of Orthologs and a Paralog of
the Human HPC2 Gene
[0303] All species living on the Earth now are thought to have
evolved from a single common ancestor that lived in the distant
past, perhaps 3.5 to 4 billion years ago. This means that any pair
of species must share a common ancestor species that lived at some
time in the past. Admittedly, this view is a bit simplistic
because, for instance, the nuclear genomes and mitochondrial
genomes of eukaryotes are thought to have independent prokaryotic
ancestries. During the evolution of an ancestral species into two
or more extant daughter species, the genes present in the genome of
the ancestral species evolve into the genes present in the genomes
of the daughter species. The evolutionary history of the genes
present in the daughter species can be quite complex because the
individual genes can evolve through a diverse set of processes
including nucleotide substitution, insertion, deletion, gene
duplication, gene conversion, lateral transfer, etc. Even so, the
evolutionary history of related genes in related organisms can
often be sorted out, especially if the pair/set of species share a
relatively recent common ancestor or if the genes being analyzed
evolved primarily through nucleotide substitutions and/or small
insertions and/or small deletions, but not gene duplications or
gene conversions. When, upon analysis, it appears that a single
gene in one species and a single gene in another species have
evolved from a single gene in a common ancestor species, those
genes are termed orthologs.
[0304] Knowledge of the identity of genes orthologous to
disease-related human genes can often be quite useful.
[0305] The human HPC2 cDNA sequence was assembled from a
combination of ESTs, hybrid selected clones, and 5' RACE (Rapid
Amplification of cDNA Ends) products; the orthologous mouse Elac2
cDNA sequence was assembled from ESTs and 5' RACE products.
Conceptual translation of the human cDNA sequence yielded a protein
of 826 amino acids; parsing the cDNA sequence across the
corresponding genomic sequence revealed 24 coding exons (FIG. 3).
Mouse Elac2 encodes a protein of 831 residues in 25 exons. BLAST
(Altschul et al., 1990) searches of the ELAC2 sequence against
GenBank readily revealed a single ortholog in S. cerevisiae
(YKR079C) and a single ortholog in C. elegans (CE16965,
CELE04A4.4), but two related sequences in S. pombe and A. thaliana.
Alignment of representative family members revealed a block of good
conservation near the N-termini and a series of blocks of high
similarity across the C-terminal half of the proteins (FIGS. 6A-B
and 10).
[0306] Hybridization of RNA blots to labeled fragments of human
ELAC2 cDNA revealed a single transcript of approximately 3 kb
(FIGS. 11A-D), in agreement with our full-length cDNA assembly of
2,970 bp. The transcript was detected in all tissues surveyed and,
like BRCA1 and BRCA2, was most abundant in testis. The apparent
size of the transcript agrees well with our full length cDNA
assembly, 2970 bp. There was no evidence from RNA blots, EST
sequences, or RT-PCR experiments of significant alternative
splicing of the transcript.
[0307] In the course of surveying ESTs derived from this gene, we
identified a small number of human and rabbit ESTs derived from a
second, related gene. The human cDNA sequence of this related gene
was assembled from a combination of ESTs and 5' RACE products.
Conceptual translation revealed that the transcript encodes a
protein of 363 residues. Radiation hybrid mapping placed the gene
at approximately 365 cR on chromosome 18. When this sequence, along
with representative sequences from a eubacterium (E. coli elaC), a
cyanobacterium (Synechocystis sp. gi2500943/SLR0050) and an
archaebacterium (M. thermoautotrophicum gi2622965) was added into
the multiprotein alignment (FIGS. 6A-B), it became apparent that
two distinct groups of proteins were represented; a group of larger
proteins (800-900 aa) restricted to the eukaryotes, and a group of
smaller proteins (300 to 400 aa) that align with the C-terminal
half of the former group and includes sequences from the
eukaryotes, eubacteria, and archaebacteria. As the 363 residue
human protein falls into this second group and is more similar to
E. coli elaC than is ELAC2, we will refer to it as ELAC1.
[0308] The alignment revealed a striking histidine containing
motif, .phi..phi..phi.[S/T]H.times.H.times.DH.times..times.G (SEQ
ID NO:214), where .phi. can be any large hydrophobic residue, near
the N-terminus of the ELAC1 group, and in the C-terminal portion of
the ELAC2 group. This motif is reminiscent of the histidine motif
found in the metallo-.beta.-lactamases (Melino et al., 1998) and
suggests, in accord with the annotation for COG1234
(www.ncbi.nlm.nih.gov/COG/index.html), that the proteins are
metal-dependent hydrolases. While assembling the multiple
alignment, we observed that the sequence within which the histidine
motif is embedded also aligns with the ELAC2 N-terminal conserved
block (FIG. 12), leading us to predict that some structural feature
of the protein is repeated. Even so, the N-terminal copy of the
repeated sequence would not necessarily retain metal-dependent
hydrolase activity, as the histidine motif itself is not
conserved.
[0309] Thorough BLAST searches of GenBank using sequences
containing this histidine motif, combined with iterative motif
searches (Nevill-Manning et al., 1998) using the eMOTIF SCAN
website (http://dna.stanford.edu/scan- ), revealed two other
families of proteins that share extended amino acid sequence
similarity with members of COG1234. The similarity includes 4 to 6
shared motifs distributed across the ELAC1 domain (FIG. 9). One
such family is the PSO2 (or SNM1) family of DNA inter-strand
crosslink repair proteins (Haase et al., 1989; Meniel et al., 1995;
Niegemann and Brendel, 1994), present only in eukaryotes. The
second family encodes the 73 kDa subunit of the mRNA cleavage and
polyadenylation specificity factor (CPSF73) (Chanfreau et al.,
1996; Jenny et al., 1994; Jenny et al., 1996). Surprisingly,
members of this latter gene family are present in both eukaryotes
and archaebacteria, as well as a cyanobacterium. These three gene
families, ELAC1/2, PSO2 and CPSF73, are equally similar to each
other (FIGS. 9 and 13); indeed they were originally placed in a
single COG (Tatusov et al., 1997). While PSo2 is required for
repair of DNA inter-strand crosslinks following treatment of cells
with, for instance, 8-methoxypsoralen plus UV-irradiation (Menial
et al., 1995), the actual substrate for the protein's presumptive
metal-dependent hydrolase activity has not been defined. Similarly,
although CPSF73 is a component of the mRNA 3' end cleavage and
polyadenylation specificity factor, it has neither the 3' end
cleavage nor the polyadenylation activity, and the substrate for
its presumptive metal-dependent hydrolase activity is unknown.
While the S. cerevisiae CPSF73 ortholog YSH1 (BRR5) is an essential
gene, PSo2 is not. Given the phylogenetic conservation of the ELAC1
domain and the observation that S. cerevisiae encodes only a single
member of this gene family, YKR079C, we asked whether it is an
essential gene. To answer this question, we performed one-step gene
disruption of YKR079C using URA3 as a selectable marker in yeast
diploid cells. Two heterozygote knockout strains were sporulated
and tetrads were dissected. Each tetrad yielded 1 or 2 viable
haploid colonies; these were all URA.sup.- and YKR079C wt. Thus we
concluded that, like YSH1, YKR079C is an essential gene.
[0310] In addition to the histidine motif and the local sequence
context in which it is embedded, ELAC1/2, PSo2 and CPSF73 proteins
share a series of sequence features, some shared pairwise between
the gene families and others by all three. Strikingly, all three
families have three or four conserved histidine or cysteine
positions, past the histidine motif, that lie within these shared
regions and can be aligned across the gene families (FIG. 9). The
arrangement is reminiscent of the binuclear zinc binding active
site of some metallo-.beta. lactamases (Carfi et al., 1998; Fabiane
et al., 1998) and the shared similarity between the metallo-.beta.
lactamases and glyoxalase II (Melino et al., 1998). This series of
sequence similarities leads to three predictions. First, the
extended similarity between the ELAC1/2, PSO2 and CPSF73 protein
families suggests that they share a domain of approximately 300
residues, and this domain constitutes a metal-dependent hydrolase
that coordinates two-divalent cations in its active site. Second,
the overall fold of this domain is likely to be similar to that of
the metallo-, lactamases. Third, similarity between the region
surrounding the ELAC1/2 histidine motif and the N-terminus of the
ELAC2 proteins suggests that these proteins are comprised of two
structurally similar domains and arose from a direct
repeat/duplication of an ancestral ELAC1-type gene.
[0311] A number of members of the ELAC1/2 family are annotated in
GenBank as sulfatases or sulfatase homologs. The annotation appears
to be assigned through sequence similarity to the atsA gene of
Alteromonas carrageenovora. The atsA protein contains a histidine
motif and has been demonstrated to have aryl sulfatase activity in
vitro (Barbeyron et al., 1995), though its sequence does not
contain any of the typical sulfatase motifs listed by PROSITE. No
other experimentally verified aryl sulfatase contains the histidine
motif. As the E. coli protein most similar to A. carrageenovora
atsA is elaC, atsA may well be a diverged member of the ELAC1 gene
family (BLASTp and alignment not shown). Accordingly, ELAC1 family
members should be tested for aryl sulfatase activity; however, it
is not apparent whether ELAC1 and ELAC2 family members have the
same substrate.
[0312] In addition to the paralog and the mouse ortholog mmELAC2
(for Mus musculus ELAC2), orthologs of HPC2 have been identified in
chimpanzee and gorilla. These are ptELAC2 (Pan troglodytes ELAC2)
and ggELAC2 (Gorilla gorilla ELAC2).
Example 12
Multiple Protein Sequence Alignments
[0313] For the alignment of FIGS. 6A-B, shading criteria were
identity (white on black) or conservative substitution (white on
gray) for all ELAC2 sequences with a residue at that position, with
four of the five sequences actually having to have a residue at
that position. Shaded positions in the ELAC2 sequences were
propagated into the ELAC1 sequences. For the alignment of FIG. 12,
two shading criteria were used: (1) Identity or conservative
substitution across the ELAC2 N-terminal alignment and identity or
conservative substitution across either the ELAC1 or ELAC2 His
motif. (2) Identity or conservative substitution across both the
ELAC1 and ELAC2 His motif, with some conservation across the ELAC2
N-terminal alignment. For the alignment of FIG. 9, shading criteria
were identity or conservative substitution across two out of the
three (CPSF73, PSO2, ELAC2) protein families represented.
Example 13
Analysis of the HPC2 Gene
[0314] The structure and function of HPC2 gene are determined
according to the following methods.
[0315] Biological Studies.
[0316] Mammalian expression vectors containing HPC2 cDNA are
constructed and transfected into appropriate prostate carcinoma
cells with lesions in the gene. Wild-type HPC2 cDNA as well as
altered HPC2 cDNA are utilized. The altered HPC2 cDNA can be
obtained from altered HPC2 alleles or produced as described below.
Phenotypic reversion in cultures (e.g., cell morphology, doubling
time, anchorage-independent growth) and in animals (e.g.,
tumorigenicity) is examined. The studies will employ both wild-type
and mutant forms of the gene.
[0317] Molecular Genetics Studies.
[0318] In vitro mutagenesis is performed to construct deletion
mutants and missense mutants (by single base-pair substitutions in
individual codons and alanine scanning mutagenesis). The mutants
are used in biological, biochemical and biophysical studies.
[0319] Mechanism Studies. The ability of HPC2 protein to bind to
known and unknown DNA sequences is examined. Its ability to
transactivate promoters is analyzed by transient reporter
expression systems in mammalian cells. Conventional procedures such
as particle-capture and yeast two-hybrid system are used to
discover and identify any functional partners. The nature and
functions of the partners are characterized. These partners in turn
are targets for drug discovery.
[0320] Structural Studies.
[0321] Recombinant proteins are produced in E. coli, yeast, insect
and/or mammalian cells and are used in crystallographic and NMR
studies. Molecular modeling of the proteins is also employed. These
studies facilitate structure-driven drug design.
Example 14
S. cerevisiae Gene Knockout
[0322] The URA3 gene was PCR amplified with tailed primers
resulting in a product flanked by 42 bp of YKRO79C coding sequences
(amino acids 3-16 and 818-831). The resulting PCR product was
transformed into yeast diploid strain YPH501 (Stratagene);
URA.sup.+ clones were screened for disruption by the presence of a
shorter PCR product at the YKR079C locus. The knock-out clones were
further confirmed by sequencing the shorter PCR product for the
presence of URA3 sequences. Two heterozygote knockout strains were
sporulated and tetrads dissected. Each tetrad yielded 1 or 2 viable
colonies. These were genotyped at YKRO79C and tested for growth on
URA.sup.- plates.
Example 15
Association Tests
[0323] STSs for Ser 217 Leu and Ala 541 Thr were amplified by
allele specific PCR using fluorescently labeled oligos. Allele
calls were made with our automated genotyping system. Genotype
calls required good allele calls at both markers. Logistic
regression analyses were performed using the SPSS statistical
software package. The chi-squared statistics for the 2.times.2
contingency tables were calculated with the Yates correction. The
trend statistic for the 3.times.2 contingency table was calculated
with the Cochran-Armitage trend test (Cochran, 1954; Armitage,
1955) using a simple linear trend (0,1,2) for the row scores.
Example 16
Generation of Polyclonal Antibody against HPC2
[0324] Segments of HPC2 coding sequence are expressed as fusion
protein in E. coli. The overexpressed proteins are purified by gel
elution and used to immunize rabbits and mice using a procedure
similar to the one described by Harlow and Lane, 1988. This
procedure has been shown to generate Abs against various other
proteins (for example, see Kraemer, et al., 1993).
[0325] Briefly, a stretch of HPC2 coding sequence was cloned as a
fusion protein in plasmid PET5A (Novagen, Inc., Madison, Wis.). The
HPC2 incorporated sequences might include SEQ ID NOs:1, 3 or 28 or
portions thereof. After induction with IPTG, the overexpression of
a fusion protein with the expected molecular weight is verified by
SDS/PAGE. Fusion proteins are purified from the gel by
electroelution. The identification of the protein as the HPC2
fusion product is verified by protein sequencing at the N-terminus.
Next, the purified protein is used as immunogen in rabbits. Rabbits
are immunized with 100 .mu.g of the protein in complete Freund's
adjuvant and boosted twice in 3 week intervals, first with 100
.mu.g of immunogen in incomplete Freund's adjuvant followed by 100
.mu.g of immunogen in PBS. Antibody containing serum is collected
two weeks thereafter.
[0326] This procedure can be repeated to generate antibodies
against mutant forms of the HPC2 protein. These antibodies, in
conjunction with antibodies to wild type HPC2, are used to detect
the presence and the relative level of the mutant forms in various
tissues and biological fluids.
Example 17
[0327] Generation of Monoclonal Antibodies Specific for HPC2
[0328] Monoclonal antibodies are generated according to the
following protocol. Mice are immunized with immunogen comprising
intact HPC2 or HPC2 peptides (wild type or mutant) conjugated to
keyhole limpet hemocyanin using glutaraldehyde or EDC as is well
known.
[0329] The immunogen is mixed with an adjuvant. Each mouse receives
four injections of 10 to 100 .mu.g of immunogen and after the
fourth injection blood samples are taken from the mice to determine
if the serum contains antibody to the immunogen. Serum titer is
determined by ELISA or RIA. Mice with sera indicating the presence
of antibody to the immunogen are selected for hybridoma
production.
[0330] Spleens are removed from immune mice and a single cell
suspension is prepared (see Harlow and Lane, 1988). Cell fusions
are performed essentially as described by Kohler and Milstein,
1975. Briefly, P3.65.3 myeloma cells (American Type Culture
Collection, Rockville, Md.) are fused with immune spleen cells
using polyethylene glycol as described by Harlow and Lane, 1988.
Cells are plated at a density of 2.times.10.sup.5 cells/well in 96
well tissue culture plates. Individual wells are examined for
growth and the supernatants of wells with growth are tested for the
presence of HPC2 specific antibodies by ELISA or RIA using wild
type or mutant HPC2 target protein. Cells in positive wells are
expanded and subcloned to establish and confirm monoclonality.
[0331] Clones with the desired specificities are expanded and grown
as ascites in mice or in a hollow fiber system to produce
sufficient quantities of antibody for characterization and assay
development.
Example 18
[0332] Isolation of HPC2 Binding Peptides
[0333] Peptides that bind to the HPC2 gene product are isolated
from both chemical and phage-displayed random peptide libraries as
follows.
[0334] Fragments of the HPC2 gene product are expressed as GST and
His-tag fusion proteins in both E. coli and SF9 cells. The fusion
protein is isolated using either a glutathione matrix (for GST
fusions proteins) or nickel chelation matrix (for His-tag fusion
proteins). This target fusion protein preparation is either
screened directly as described below, or eluted with glutathione or
imidizole. The target protein is immobilized to either a surface
such as polystyrene; or a resin such as agarose; or solid supports
using either direct absorption, covalent linkage reagents such as
glutaraldehyde, or linkage agents such as biotin-avidin.
[0335] Two types of random peptide libraries of varying lengths are
generated: synthetic peptide libraries that may contain derivatized
residues, for example by phosphorylation or myristylation, and
phage-displayed peptide libraries which may be phosphorylated.
These libraries are incubated with immobilized HPC1 gene product in
a variety of physiological buffers. Next, unbound peptides are
removed by repeated washes, and bound peptides recovered by a
variety of elution reagents such as low or high pH, strong
denaturants, glutathione, or imidizole. Recovered synthetic peptide
mixtures are sent to commercial services for peptide
micro-sequencing to identify enriched residues. Recovered phage are
amplified, rescreened, plaque purified, and then sequenced to
determined the identity of the displayed peptides.
[0336] Use of HPC1 Binding Peptides.
[0337] Peptides identified from the above screens are synthesized
in larger quantities as biotin conjugates by commercial services.
These peptides are used in both solid and solution phase
competition assays with HPC 1 and its interacting partners
identified in yeast 2-hybrid screens. Versions of these peptides
that are fused to membrane-permeable motifs (Lin et al., 1995;
Rojas et al., 1996) will be chemically synthesized, added to
cultured cells and the effects on growth, apoptosis,
differentiation, cofactor response, and internal changes will be
assayed.
Example 19
Sandwich Assay for HPC2
[0338] Monoclonal antibody is attached to a solid surface such as a
plate, tube, bead, or particle. Preferably, the antibody is
attached to the well surface of a 96-well ELISA plate. 100 .mu.l
sample (e.g., serum, urine, tissue cytosol) containing the HPC2
peptide/protein (wild-type or mutant) is added to the solid phase
antibody. The sample is incubated for 2 hrs at room temperature.
Next the sample fluid is decanted, and the solid phase is washed
with buffer to remove unbound material. 100 .mu.L of a second
monoclonal antibody (to a different determinant on the HPC2
peptide/protein) is added to the solid phase. This antibody is
labeled with a detector molecule (e.g., 125-I, enzyme, fluorophore,
or a chromophore) and the solid phase with the second antibody is
incubated for two hrs at room temperature. The second antibody is
decanted and the solid phase is washed with buffer to remove
unbound material.
[0339] The amount of bound label, which is proportional to the
amount of HPC2 peptide/protein present in the sample, is
quantified. Separate assays are performed using monoclonal
antibodies which are specific for the wild-type HPC2 as well as
monoclonal antibodies specific for each of the mutations identified
in HPC2.
[0340] While the invention has been disclosed in this patent
application by reference to the details of preferred embodiments of
the invention, it is to be understood that the disclosure is
intended in an illustrative rather than in a limiting sense, as it
is contemplated that modifications will readily occur to those
skilled in the art, within the spirit of the invention and the
scope of the appended claims.
LIST OF REFERENCES
[0341] Altschul S F, et al. (1990). J. Mol. Biol. 215: 403-410.
[0342] Altschul S F, et al. (1997). Nucl. Acids Res.
25:3389-3402.
[0343] Anand R (1992). Techniques for the Analysis of Complex
Genomes, (Academic Press).
[0344] Anderson W F, et al. (1980). Proc. Natl. Acad. Sci. USA
77:5399-5403.
[0345] Antoniou A C, et al. (2000). Genet. Epidemiol.
18:173-190.
[0346] Armitage P (1955). Biometrics 11:375-386.
[0347] Ausubel F M, et al. (1992). Current Protocols in Molecular
Biology, (J. Wiley and Sons, NY).
[0348] Bandyopadhyay P K and Temin H M (1984). Mol. Cell. Biol.
4:749-754.
[0349] Barbeyron T, et al. (1995). Microbiology 141:2897-2904.
[0350] Bartel P L, et al. (1993). "Using the 2-hybrid system to
detect protein-protein interactions." In: Cellular Interactions in
Development: A Practical Approach, Oxford University Press, pp.
153-179.
[0351] Beaucage S L and Caruthers M H (1981). Tetra. Letts.
22:1859-1862.
[0352] Berglund P, et al. (1993). Biotechnology 11:916-920.
[0353] Berkner K L (1992). Curr. Top. Microbiol. Immunol.
158:39-66.
[0354] Berkner K L, et al. (1988). BioTechniques 6:616-629.
[0355] Berry R, et al. (2000). Am. J. Hum. Genet. 66:539-546.
[0356] Berthon P, et al. (1998). Am. J. Hum. Genet.
62:1416-1424.
[0357] Borman S (1996). Chemical & Engineering News, December 9
issue, pp.42-43.
[0358] Bouchardy C, et al. (1998). Pharmacogenetics 8:291-298.
[0359] Bratt O, et al. (1999). Br. J. Cancer 81:672-676.
[0360] Breakefield X O and Geller A I (1987). Mol. Neurobiol.
1:337-371.
[0361] Breast Cancer Linkage Consortium (1999). J. Natl. Cancer
Inst. 91:1310-1316.
[0362] Brinster R L, et al. (1981). Cell 27:223-231.
[0363] Buchschacher G L and Panganiban A T (1992). J. Virol.
66:2731-2739.
[0364] Cannon L, et al. (1982). Cancer Surveys 1:47-69.
[0365] Capecchi M R (1989). Science 244:1288-1292.
[0366] Carfi A, et al. (1998). Acta Crystallogr. D Biol.
Crystallogr. 54:45-57.
[0367] Cariello N F (1988). Am. J. Human Genetics 42:726-734.
[0368] Carter B S, et al. (1992). Proc. Natl. Acad. Sci. USA
89:3367-3371.
[0369] Carter B S, et al. (1993). J. Urol. 150:797-802.
[0370] Chamberlain N L, et al. (1994). Nucl. Acids Res.
22:3181-3186.
[0371] Chanfreau G, et al. (1996). Science 274:1511-1514.
[0372] Chee M, et al. (1996). Science 274:610-614.
[0373] Chevray P M and Nathans D N (1992). Proc. Natl. Acad. Sci.
USA 89:5789-5793.
[0374] Cochran W G (1954). Biometrics 10:417-451.
[0375] Compton J (1991). Nature 350:91-92.
[0376] Conner B J, et al. (1983). Proc. Natl. Acad. Sci. USA
80:278-282.
[0377] Cooney K A, et al. (1997). J. Natl. Cancer Inst.
89:955-959.
[0378] Costantini F and Lacy E (1981). Nature 294:92-94.
[0379] Cotten M, et al. (1990). Proc. Natl. Acad. Sci. USA
87:4033-4037.
[0380] Cottingham R W, et al. (1993). Am. J. Hum. Genet.
53:252-263.
[0381] Cotton R G, et al. (1988). Proc. Natl. Acad. Sci. USA
85:4397-4401.
[0382] Couch F J, et al. (1996). Genomics 36:86-99.
[0383] Culver K W, et al. (1992). Science 256:1550-1552.
[0384] Curiel D T, et al. (1991). Proc. Natl. Acad. Sci. USA
88:8850-8854.
[0385] Curiel D T, et al. (1992). Hum. Gene Ther. 3:147-154.
[0386] DeRisi J, et al. (1996). Nature Genetics 14:457-460.
[0387] Deutscher, M (1990). Meth. Enzymology 182:83-89 (Academic
Press, San Diego, Calif.).
[0388] Donehower L A, et al. (1992). Nature 356:215-221.
[0389] Durbin R and Thierry-Mieg J (1991). A C. elegans Database.
Documentation, code and data available from anonymous FTP servers
at lirmm.lirmm.fr, cele.mrc-lmb.cam.ac.uk and ncbi.nlm.nih.gov.
[0390] Editorial (1996). Nature Genetics 14:367-370.
[0391] Eeles R A, et al. (1998). Am. J. Hum. Genet. 62:653-658.
[0392] Elghanian R, et al. (1997). Science 277:1078-1081.
[0393] Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor
Press, Cold Spring Harbor, N.Y. (1983).
[0394] Erickson J, et al. (1990). Science 249:527-533.
[0395] Fabiane S M, et al. (1998). Biochemistry 37:12404-12411.
[0396] Fahy E, et al. (1991). PCR Methods Appl. 1:25-33.
[0397] Feil R, et al., (1996). Proc. Natl. Acad. Sci. USA
93:10887-10890.
[0398] Felgner P L, et al. (1987). Proc. Natl. Acad. Sci. USA
84:7413-7417.
[0399] Fields S and Song O -K (1989). Nature 340:245-246.
[0400] Fiers W, et al. (1978). Nature 273:113-120.
[0401] Fincham S M, et al. (1990). The Prostate 17:189-206.
[0402] Fink D J, et al. (1992). Hum. Gene Ther. 3:11-19.
[0403] Fink D J, et al. (1996). Ann. Rev. Neurosci. 19:265-287.
[0404] Finkelstein J, et al. (1990). Genomics 7:167-172.
[0405] Fodor S P A (1997). Science 277:393-395.
[0406] Ford D, et al. (1998). Am. J. Hum. Genet. 62:676-689.
[0407] Freese A, et al. (1990). Biochem. Pharmacol.
40:2189-2199.
[0408] Friedman T (1991). In: Therapy for Genetic Diseases, T.
Friedman, ed., Oxford University Press, pp. 105-121.
[0409] Gagneten S, et al. (1997). Nucl. Acids Res.
25:3326-3331.
[0410] Gibbs M, et al. (1999a). Am. J. Hum. Genet. 64:776-787.
[0411] Gibbs M, et al. (1999b). Am. J. Hum. Genet.
64:1087-1095.
[0412] Giovannucci E, et al. (1997). Proc. Natl. Acad. Sci. USA
94:3320-3323.
[0413] Glover D (1985). DNA Cloning, I and II (Oxford Press).
[0414] Goding (1986). Monoclonal Antibodies: Principles and
Practice, 2d ed. (Academic Press, NY).
[0415] Godowski P J, et al. (1988). Science 241:812-816.
[0416] Goldgar D E, et al. (1994). J. Natl. Can. Inst.
86:3:200-209.
[0417] Goode E L, et al. (2000). Genet. Epidemiol. 18:251-275.
[0418] Gordon J W, et al. (1980). Proc. Natl. Acad. Sci. USA
77:7380-7384.
[0419] Gordon J W (1989). Intl. Rev. Cytol. 115:171-229.
[0420] Gorziglia M and Kapikian A Z (1992). J. Virol.
66:4407-4412.
[0421] Graham F L and van der Eb A J (1973). Virology
52:456-467.
[0422] Grompe M (1993). Nature Genetics 5:111-117.
[0423] Grompe M, et al. (1989). Proc. Natl. Acad. Sci. USA
86:5855-5892.
[0424] Gu H, et al. (1994). Science 265:103-106.
[0425] Guthrie G and Fink G R (1991). Guide to Yeast Genetics and
Molecular Biology (Academic Press).
[0426] Haase E, et al. (1989). Mol. Gen. Genet. 218:64-71.
[0427] Hacia J G, et al. (1996). Nature Genetics 14:441-447.
[0428] Hall J M, et al. (1990). Science 250:1684-1689.
[0429] Harlow E and Lane D (1988). Antibodies: A Laboratory Manual
(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
[0430] Harty L C, et al. (1997). J. Natl. Cancer Inst.
89:1698-1705.
[0431] Hasty P. et al. (1991). Nature 350:243-246.
[0432] Helseth E, et al. (1990). J. Virol. 64:2416-2420.
[0433] Hodgson J (1991). Bio/Technology 9:19-21.
[0434] Hori H, et al. (1997). J. Clin. Gastroenterol.
25:568-575.
[0435] Hubert A, et al. (1999). Am. J. Hum. Genet. 65:921-924.
[0436] Huse W D, et al. (1989). Science 246:1275-1281.
[0437] Innis M A, et al. (1990). PCR Protocols: A Guide to Methods
and Applications (Academic Press, San Diego, Calif.).
[0438] Jablonski E, et al. (1986). Nucl. Acids Res.
14:6115-6128.
[0439] Jaffe J M, et al. (2000). Cancer Res. 60:1626-1630.
[0440] Jakoby W B and Pastan I H (eds.) (1979). Cell Culture.
Methods in Enzymology, Vol. 58 (Academic Press, Inc., Harcourt
Brace Jovanovich (NY)).
[0441] Jenny A, et al. (1994). Mol. Cell. Biol. 14:8183-8190.
[0442] Jenny A, et al. (1996). Science 274:1514-1517.
[0443] Johnson P A, et al. (1992). J. Virol. 66:2952-2965.
[0444] Johnson, et al. (1993). "Peptide Turn Mimetics" In:
Biotechnology and Pharmacy, Pezzuto et al., eds., Chapman and Hall,
NY.
[0445] Kaneda Y, et al. (1989). J. Biol. Chem. 264:12126-12129.
[0446] Kanehisa M (1984). Nucl. Acids Res. 12:203-213.
[0447] Kazemi-Esfarjani P, et al. (1995). Hum. Mol. Genet.
4:523-527.
[0448] Kinszler K W, et al. (1991). Science 251:1366-1370.
[0449] Kohler G and Milstein C (1975). Nature 256:495-497.
[0450] Krain L S (1974). Preventive Medicine 3:154-159.
[0451] Kubo T, et al. (1988). FEBS Lett. 241:119-125.
[0452] Kyte J and Doolittle R F (1982). J. Mol. Biol.
157:105-132.
[0453] Landegren U, et al. (1988). Science 242:229-237.
[0454] Lander E S and Green P (1987). Proc. Natl. Acad. Sci. USA
84:2363-2367.
[0455] Lange E M, et al. (1999). Clin. Cancer Res. 5:4013-4020.
[0456] Lasko M, et al. (1992). Proc. Natl. Acad. Sci. USA
89:6232-6236.
[0457] Lathrop G M (1984). Proc. Natl. Acad. Sci. USA
81:3443-3446.
[0458] Lavitrano M, et al. (1989). Cell 57:717-723.
[0459] Lee J E, et al. (1995). Science 268:836-844.
[0460] Lim C S, et al. (1991). Circulation 83:2007-2011.
[0461] Lin Y Z, et al. (1995). J. Biol. Chem. 270:14255-14258.
[0462] Lipshutz R J, et al. (1995). BioTechniques 19:442-447.
[0463] Lo C W (1983). Mol. Cell. Biol. 3:1803-1814.
[0464] Lobe C G and Nagy A (1998). Bioessays 20:200-208.
[0465] Lockhart D J, et al. (1996). Nature Biotechnology
14:1675-1680.
[0466] Madzak C, et al. (1992). J. Gen. Virol. 73:1533-1536.
[0467] Makridakis N, et al. (1997). Cancer Res. 57:1020-1022.
[0468] Makridakis N M, et al. (1999). Lancet 354:975-978.
[0469] Maniatis T, et al. (1982). Molecular Cloning: A Laboratory
Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.).
[0470] Mann R and Baltimore D (1985). J. Virol. 54:401-407.
[0471] Margolskee R F (1992). Curr. Top. Microbiol. Immunol.
158:67-95.
[0472] Martin R, et al. (1990). BioTechniques 9:762-768.
[0473] Matteucci M D and Caruthers M H (1981). J. Am. Chem. Soc.
103:3185.
[0474] Matthews J A and Kricka L J (1988). Anal. Biochem.
169:1.
[0475] Meikle A W, et al. (1985). Prostate 6:121-128.
[0476] Melino S, et al. (1998). TIBS 23:381-382.
[0477] Meniel V, et al. (1995). Mutagenesis 10:543-548.
[0478] Merrifield B (1963). J. Am. Chem. Soc. 85:2149-2156.
[0479] Metzger D, et al. (1988). Nature 334:31-36.
[0480] Mifflin T E (1989). Clinical Chem. 35:1819-1825.
[0481] Miki Y, et al. (1994). Science 266:66-71.
[0482] Miller A D (1992). Curr. Top. Microbiol. Immunol.
158:1-24.
[0483] Miller A D, et al. (1985). Mol. Cell. Biol. 5:431-437.
[0484] Miller A D, et al. (1988). J. Virol. 62:4337-4345.
[0485] Modrich P (1991). Ann. Rev. Genet. 25:229-253.
[0486] Mombaerts P, et al. (1992). Cell 68:869-877.
[0487] Morganti G, et al. (1956). Acta Geneticae Medicae et
Gemellogogiae 6:304-305.
[0488] Moss B (1992). Curr. Top. Microbiol. Immunol 158:25-38.
[0489] Moss B (1996). Proc. Natl. Acad. Sci. USA
93:11341-11348.
[0490] Muzyczka N (1992). Curr. Top. Microbiol. Immunol.
158:97-129.
[0491] Nabel (1992). Hum. Gene Ther. 3:399-410.
[0492] Nabel E G, et al. (1990). Science 249:1285-1288.
[0493] Naldini L, et al. (1996). Science 272:263-267.
[0494] Nastiuk K L, et al. (1999). Prostate 40:172-177.
[0495] Nevill-Manning C G, et al. (1998). Proc. Natl. Acad. Sci.
USA 95:5865-5871.
[0496] Neuhausen S L, et al. (1999). Hum. Mol. Genet.
8:2437-2442.
[0497] Newton C R, et al. (1989). Nucl. Acids Res.
17:2503-2516.
[0498] Nguyen Q, et al. (1992). BioTechniques 13:116-123.
[0499] Niegemann E and Brendel M (1994). Mutat. Res.
315:275-279.
[0500] Novack D F, et al. (1986). Proc. Natl. Acad. Sci. USA
83:586-590.
[0501] O'Connell J R and Weeks D E (1995). Nat. Genet.
11:402-408.
[0502] Ohi S, et al. (1990). Gene 89:279-282.
[0503] Orita M, et al. (1989). Proc. Natl. Acad. Sci. USA
86:2776-2770.
[0504] Osterrieder N and Wolf E (1998). Rev. Sci. Tech.
17:351-364.
[0505] Ott J (1986). Genet. Epidemiol. Suppl. 1:251-257.
[0506] Page K A, et al. (1990). J. Virol. 64:5270-5276.
[0507] Page R D M (1996). Computer Applications in the Biosciences
12:357-358.
[0508] Pellicer A, et al. (1980). Science 209:1414-1422.
[0509] Peto J, et al. (1999). J. Natl. Cancer Inst. 91:943-949.
[0510] Petropoulos C J, et al. (1992). J. Virol. 66:3391-3397.
[0511] Philpott K L, et al. (1992). Science 256:1448-1452.
[0512] Quantin B, et al. (1992). Proc. Natl. Acad. Sci. USA
89:2581-2584.
[0513] Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack
Publishing Co., Easton, Pa.).
[0514] Rigby P W J, et al. (1977). J. Mol. Biol. 113:237-251.
[0515] Rojas M, et al. (1996). J. Biol. Chem. 271:27456-27461.
[0516] Rosenfeld M A, et al. (1992). Cell 68:143-155.
[0517] Ruano G and Kidd K K (1989). Nucl. Acids Res. 17:8392.
[0518] Russell D and Hirata R (1998). Nature Genetics
18:323-328.
[0519] Saitou N and Nei M (1987). Mol. Biol. Evol. 4:406-425.
[0520] Sambrook J, et al. (1989). Molecular Cloning: A Laboratory
Manual, 2nd Ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.).
[0521] Schaffer A A, et al. (1994). Hum. Hered. 44:225-237.
[0522] Scharf S J (1986). Science 233:1076-1078.
[0523] Schneider G. et al. (1998). Nature Genetics 18:180-183.
[0524] Scopes R (1982). Protein Purification: Principles and
Practice, (Springer-Verlag, NY).
[0525] Shastry B S (1995). Experientia 51:1028-1039.
[0526] Shastry B S (1998). Mol. Cell. Biochem. 181:163-179.
[0527] Sheffield V C, et al. (1989). Proc. Natl. Acad. Sci. USA
86:232-236.
[0528] Sheffield V C, et al. (1991). Am. J. Hum. Genet.
49:699-706.
[0529] Shenk T E, et al. (1975). Proc. Natl. Acad. Sci. USA
72:989-993.
[0530] Shields P B (1997). Proc. Dept. Defense BCRP Era of Hope
meeting, Vol. 1 ("Frontiers in Prevention and Detection"),
pp.9-10.
[0531] Shimada T, et al. (1991). J. Clin. Invest. 88:1043-1047.
[0532] Shinkai Y, et al. (1992). Cell 68:855-867.
[0533] Shoemaker D D, et al. (1996). Nature Genetics
14:450-456.
[0534] Sigurdsson S, et al. (1997). J. Mol. Med. 75:758-761.
[0535] Smith J R, et al. (1996). Science 274:1371-1374.
[0536] Smith S W, et al. (1994). CABIOS 10:671-675.
[0537] Snouwaert J N, et al. (1992). Science 257:1083-1088.
[0538] Sorge J, et al. (1984). Mol. Cell. Biol. 4:1730-1737.
[0539] Spargo C A, et al. (1996). Mol. Cell. Probes 10:247-256.
[0540] Stanford J L, et al. (1997). Cancer Res. 57:1194-1198.
[0541] Steinberg G D, et al. (1990). Prostate 17:337-347.
[0542] Stewart M J, et al. (1992). Hum. Gene Ther. 3:267-275.
[0543] Stratford-Perricaudet L D, et al. (1990). Hum. Gene Ther.
1:241-256.
[0544] Suarez B K, et al. (2000). Am. J. Hum. Genet.
66:933-944.
[0545] Tatusov R L, et al. (1997). Science 278:631-637.
[0546] Tavtigian S V, et al. (1996). Nat. Genet. 12:333-337.
[0547] Thierry-Mieg D, et al. (1995). Ace.mbly. A graphic
interactive program to support shotgun and directed sequencing
projects.
[0548] Thomas A, et al. (2000). Statistics and Computing In
press.
[0549] Thompson J D, et al. (1997). Nucl. Acids Res.
25:4876-4882.
[0550] Thompson S, et al. (1989). Cell 56:313-321.
[0551] Valancius V and Smithies O (1991). Mol. Cell Biol. 11:
1402-1408.
[0552] Van der Putten H, et al. (1985). Proc. Natl. Acad. Sci. USA
82:6148-6152.
[0553] Wagner E, et al. (1990). Proc. Natl. Acad. Sci. USA
87:3410-3414.
[0554] Wagner E, et al. (1991). Proc. Natl. Acad. Sci. USA
88:4255-4259.
[0555] Walker G T, et al. (1992). Nucl. Acids Res.
20:1691-1696.
[0556] Wang C Y and Huang L (1989). Biochemistry 28:9508-9514.
[0557] Wartell R M, et al. (1990). Nucl. Acids Res.
18:2699-2705.
[0558] Wells J A (1991). Methods in Enzymol. 202:390-411.
[0559] Wetmur J G and Davidson N (1968). J. Mol. Biol.
31:349-370.
[0560] White M B, et al. (1992). Genomics 12:301-306.
[0561] White R and Lalouel J M (1988). Annu. Rev. Genet.
22:259-279.
[0562] Wilkens E P, et al. (1999). Prostate 39:280-284.
[0563] Wilkinson G W and Akrigg A (1992). Nucleic Acids Res.
20:2233-2239.
[0564] Wolff J A, et al. (1990). Science 247:1465-1468.
[0565] Wolff J A, et al. (1991). BioTechniques 11:474-485.
[0566] Woolf C M (1960a). Cancer 13:361-364.
[0567] Woolf C M (1960b). Cancer 13:739-744.
[0568] Wooster R, et al. (1994). Science 265:2088-2090.
[0569] Wooster R, et al. (1995). Nature 378:789-792.
[0570] Wu D Y and Wallace R B (1989). Genomics 4:560-569.
[0571] Wu C H, et al. (1989). J. Biol. Chem. 264:16985-16987.
[0572] Wu G Y, et al. (1991). J. Biol. Chem. 266:14338-14342.
[0573] Xu J, et al. (1998). Nat. Genet. 20:175-179.
[0574] Xu J (2000). Am. J. Hum. Genet. 66:945-957.
[0575] Zenke M, et al. (1990). Proc. Natl. Acad. Sci. USA
87:3655-3659.
[0576] U.S. Pat. No. 3,817,837
[0577] U.S. Pat. No. 3,850,752
[0578] U.S. Pat. No. 3,939,350
[0579] U.S. Pat. No. 3,996,345
[0580] U.S. Pat. No. 4,275,149
[0581] U.S. Pat. No. 4,277,437
[0582] U.S. Pat. No. 4,366,241
[0583] U.S. Pat. No. 4,376,110
[0584] U.S. Pat. No. 4,486,530
[0585] U.S. Pat. No. 4,554,101
[0586] U.S. Pat. No. 4,683,195
[0587] U.S. Pat. No. 4,683,202
[0588] U.S. Pat. No. 4,816,567
[0589] U.S. Pat. No. 4,868,105
[0590] U.S. Pat. No. 4,873,191
[0591] U.S. Pat. No. 5,252,479
[0592] U.S. Pat. No. 5,270,184
[0593] U.S. Pat. No. 5,409,818
[0594] U.S. Pat. No. 5,436,146
[0595] U.S. Pat. No. 5,455,166
[0596] U.S. Pat. No. 5,550,050
[0597] U.S. Pat. No. 5,691,198
[0598] U.S. Pat. No. 5,735,500
[0599] U.S. Pat. No. 5,747,469
[0600] Hitzeman et al., EP 73,675A
[0601] EPO Publication No. 225,807
[0602] EP 425,731A
[0603] European Patent Application Publication No. 0332435
[0604] WO 84/03564
[0605] WO 90/07936
[0606] WO 92/19195
[0607] WO 93/07282
[0608] WO 94/25503
[0609] WO 95/01203
[0610] WO 95/05452
[0611] WO 96/02286
[0612] WO 96/02646
[0613] WO 96/11698
[0614] WO 96/40871
[0615] WO 96/40959
[0616] WO 97/12635
Sequence CWU 1
1
240 1 2481 DNA Homo sapiens CDS (1)..(2478) 1 atg tgg gcg ctt tgc
tcg ctg ctg cgg tcc gcg gcc gga cgc acc atg 48 Met Trp Ala Leu Cys
Ser Leu Leu Arg Ser Ala Ala Gly Arg Thr Met 1 5 10 15 tcg cag gga
cgc acc ata tcg cag gca ccc gcc cgc cgc gag cgg ccg 96 Ser Gln Gly
Arg Thr Ile Ser Gln Ala Pro Ala Arg Arg Glu Arg Pro 20 25 30 cgc
aag gac ccg ctg cgg cac ctg cgc acg cga gag aag cgc gga ccg 144 Arg
Lys Asp Pro Leu Arg His Leu Arg Thr Arg Glu Lys Arg Gly Pro 35 40
45 tcg ggg tgc tcc ggc ggc cca aac acc gtg tac ctg cag gtg gtg gca
192 Ser Gly Cys Ser Gly Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala
50 55 60 gcg ggt agc cgg gac tcg ggc gcc gcg ctc tac gtc ttc tcc
gag ttc 240 Ala Gly Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser
Glu Phe 65 70 75 80 aac cgg tat ctc ttc aac tgt gga gaa ggc gtt cag
aga ctc atg cag 288 Asn Arg Tyr Leu Phe Asn Cys Gly Glu Gly Val Gln
Arg Leu Met Gln 85 90 95 gag cac aag tta aag gtt gct cgc ctg gac
aac ata ttc ctg aca cga 336 Glu His Lys Leu Lys Val Ala Arg Leu Asp
Asn Ile Phe Leu Thr Arg 100 105 110 atg cac tgg tct aat gtt ggg ggc
tta agt gga atg att ctt act tta 384 Met His Trp Ser Asn Val Gly Gly
Leu Ser Gly Met Ile Leu Thr Leu 115 120 125 aag gaa acc ggg ctt cca
aag tgt gta ctt tct gga cct cca caa ctg 432 Lys Glu Thr Gly Leu Pro
Lys Cys Val Leu Ser Gly Pro Pro Gln Leu 130 135 140 gaa aaa tac ctc
gaa gca atc aaa ata ttt tct ggt cca ttg aaa gga 480 Glu Lys Tyr Leu
Glu Ala Ile Lys Ile Phe Ser Gly Pro Leu Lys Gly 145 150 155 160 ata
gaa ctg gct gtg cgg ccc cac tct gcc cca gaa tac gag gat gaa 528 Ile
Glu Leu Ala Val Arg Pro His Ser Ala Pro Glu Tyr Glu Asp Glu 165 170
175 acc atg aca gtt tac cag atc ccc ata cac agt gaa cag agg agg gga
576 Thr Met Thr Val Tyr Gln Ile Pro Ile His Ser Glu Gln Arg Arg Gly
180 185 190 aag cac caa cca tgg cag agt cca gaa agg cct ctc agc agg
ctc agt 624 Lys His Gln Pro Trp Gln Ser Pro Glu Arg Pro Leu Ser Arg
Leu Ser 195 200 205 cca gag cga tct tca gac tcc gag tcg aat gaa aat
gag cca cac ctt 672 Pro Glu Arg Ser Ser Asp Ser Glu Ser Asn Glu Asn
Glu Pro His Leu 210 215 220 cca cat ggt gtt agc cag aga aga ggg gtc
agg gac tct tcc ctg gtc 720 Pro His Gly Val Ser Gln Arg Arg Gly Val
Arg Asp Ser Ser Leu Val 225 230 235 240 gta gct ttc atc tgt aag ctt
cac tta aag aga gga aac ttc ttg gtg 768 Val Ala Phe Ile Cys Lys Leu
His Leu Lys Arg Gly Asn Phe Leu Val 245 250 255 ctc aaa gca aag gag
atg ggc ctc cca gtt ggg aca gct gcc atc gct 816 Leu Lys Ala Lys Glu
Met Gly Leu Pro Val Gly Thr Ala Ala Ile Ala 260 265 270 ccc atc att
gct gct gtc aag gac ggg aaa agc atc act cat gaa gga 864 Pro Ile Ile
Ala Ala Val Lys Asp Gly Lys Ser Ile Thr His Glu Gly 275 280 285 aga
gag att ttg gct gaa gag ctg tgt act cct cca gat cct ggt gct 912 Arg
Glu Ile Leu Ala Glu Glu Leu Cys Thr Pro Pro Asp Pro Gly Ala 290 295
300 gct ttt gtg gtg gta gaa tgt cca gat gaa agc ttc att caa ccc atc
960 Ala Phe Val Val Val Glu Cys Pro Asp Glu Ser Phe Ile Gln Pro Ile
305 310 315 320 tgt gag aat gcc acc ttt cag agg tac caa gga aag gca
gat gcc ccc 1008 Cys Glu Asn Ala Thr Phe Gln Arg Tyr Gln Gly Lys
Ala Asp Ala Pro 325 330 335 gtg gcc ttg gtg gtt cac atg gcc cca gca
tct gtg ctt gtg gac agc 1056 Val Ala Leu Val Val His Met Ala Pro
Ala Ser Val Leu Val Asp Ser 340 345 350 agg tac cag cag tgg atg gag
agg ttt ggg cct gac acc cag cac ttg 1104 Arg Tyr Gln Gln Trp Met
Glu Arg Phe Gly Pro Asp Thr Gln His Leu 355 360 365 gtc ctg aat gag
aac tgt gcc tca gtt cac aac ctt cgc agc cac aag 1152 Val Leu Asn
Glu Asn Cys Ala Ser Val His Asn Leu Arg Ser His Lys 370 375 380 att
caa acc cag ctc aac ctc atc cac ccg gac atc ttc ccc ctg ctc 1200
Ile Gln Thr Gln Leu Asn Leu Ile His Pro Asp Ile Phe Pro Leu Leu 385
390 395 400 acc agt ttc cgc tgt aag aag gag ggc ccc acc ctc agt gtg
ccc atg 1248 Thr Ser Phe Arg Cys Lys Lys Glu Gly Pro Thr Leu Ser
Val Pro Met 405 410 415 gtt cag ggt gaa tgc ctc ctc aag tac cag ctc
cgt ccc agg agg gag 1296 Val Gln Gly Glu Cys Leu Leu Lys Tyr Gln
Leu Arg Pro Arg Arg Glu 420 425 430 tgg cag agg gat gcc att att act
tgc aat cct gag gaa ttc ata gtt 1344 Trp Gln Arg Asp Ala Ile Ile
Thr Cys Asn Pro Glu Glu Phe Ile Val 435 440 445 gag gcg ctg cag ctt
ccc aac ttc cag cag agc gtg cag gag tac agg 1392 Glu Ala Leu Gln
Leu Pro Asn Phe Gln Gln Ser Val Gln Glu Tyr Arg 450 455 460 agg agt
gcg cag gac ggc cca gcc cca gca gag aaa aga agt cag tac 1440 Arg
Ser Ala Gln Asp Gly Pro Ala Pro Ala Glu Lys Arg Ser Gln Tyr 465 470
475 480 cca gaa atc atc ttc ctt gga aca ggg tct gcc atc ccg atg aag
att 1488 Pro Glu Ile Ile Phe Leu Gly Thr Gly Ser Ala Ile Pro Met
Lys Ile 485 490 495 cga aat gtc agt gcc aca ctt gtc aac ata agc ccc
gac acg tct ctg 1536 Arg Asn Val Ser Ala Thr Leu Val Asn Ile Ser
Pro Asp Thr Ser Leu 500 505 510 cta ctg gac tgt ggt gag ggc aca ttt
ggg cag ctg tgc cgt cat tac 1584 Leu Leu Asp Cys Gly Glu Gly Thr
Phe Gly Gln Leu Cys Arg His Tyr 515 520 525 gga gac cag gtg gac agg
gtc ctg ggc acc ctg gct gct gtg ttt gtg 1632 Gly Asp Gln Val Asp
Arg Val Leu Gly Thr Leu Ala Ala Val Phe Val 530 535 540 tcc cac ctg
cac gca gat cac cac acg ggc ttg cca agt atc ttg ctg 1680 Ser His
Leu His Ala Asp His His Thr Gly Leu Pro Ser Ile Leu Leu 545 550 555
560 cag aga gaa cgc gcc ttg gca tct ttg gga aag ccg ctt cac cct ttg
1728 Gln Arg Glu Arg Ala Leu Ala Ser Leu Gly Lys Pro Leu His Pro
Leu 565 570 575 ctg gtg gtt gcc ccc aac cag ctc aaa gcc tgg ctc cag
cag tac cac 1776 Leu Val Val Ala Pro Asn Gln Leu Lys Ala Trp Leu
Gln Gln Tyr His 580 585 590 aac cag tgc cag gag gtc ctg cac cac atc
agt atg att cct gcc aaa 1824 Asn Gln Cys Gln Glu Val Leu His His
Ile Ser Met Ile Pro Ala Lys 595 600 605 tgc ctt cag gaa ggg gct gag
atc tcc agt cct gca gtg gaa aga ttg 1872 Cys Leu Gln Glu Gly Ala
Glu Ile Ser Ser Pro Ala Val Glu Arg Leu 610 615 620 atc agt tcg ctg
ttg cga aca tgt gat ttg gaa gag ttt cag acc tgt 1920 Ile Ser Ser
Leu Leu Arg Thr Cys Asp Leu Glu Glu Phe Gln Thr Cys 625 630 635 640
ctg gtg cgg cac tgc aag cat gcg ttt ggc tgt gcg ctg gtg cac acc
1968 Leu Val Arg His Cys Lys His Ala Phe Gly Cys Ala Leu Val His
Thr 645 650 655 tct ggc tgg aaa gtg gtc tat tcc ggg gac acc atg ccc
tgc gag gct 2016 Ser Gly Trp Lys Val Val Tyr Ser Gly Asp Thr Met
Pro Cys Glu Ala 660 665 670 ctg gtc cgg atg ggg aaa gat gcc acc ctc
ctg ata cat gaa gcc acc 2064 Leu Val Arg Met Gly Lys Asp Ala Thr
Leu Leu Ile His Glu Ala Thr 675 680 685 ctg gaa gat ggt ttg gaa gag
gaa gca gtg gaa aag aca cac agc aca 2112 Leu Glu Asp Gly Leu Glu
Glu Glu Ala Val Glu Lys Thr His Ser Thr 690 695 700 acg tcc caa gcc
atc agc gtg ggg atg cgg atg aac gcg gag ttc att 2160 Thr Ser Gln
Ala Ile Ser Val Gly Met Arg Met Asn Ala Glu Phe Ile 705 710 715 720
atg ctg aac cac ttc agc cag cgc tat gcc aag gtc ccc ctc ttc agc
2208 Met Leu Asn His Phe Ser Gln Arg Tyr Ala Lys Val Pro Leu Phe
Ser 725 730 735 ccc aac ttc agc gag aaa gtg gga gtt gcc ttt gac cac
atg aag gtc 2256 Pro Asn Phe Ser Glu Lys Val Gly Val Ala Phe Asp
His Met Lys Val 740 745 750 tgc ttt gga gac ttt cca aca atg ccc aag
ctg att ccc cca ctg aaa 2304 Cys Phe Gly Asp Phe Pro Thr Met Pro
Lys Leu Ile Pro Pro Leu Lys 755 760 765 gcc ctg ttt gct ggc gac atc
gag gag atg gag gag cgc agg gag aag 2352 Ala Leu Phe Ala Gly Asp
Ile Glu Glu Met Glu Glu Arg Arg Glu Lys 770 775 780 cgg gag ctg cgg
cag gtg cgg gcg gcc ctc ctg tcc agg gag ctg gca 2400 Arg Glu Leu
Arg Gln Val Arg Ala Ala Leu Leu Ser Arg Glu Leu Ala 785 790 795 800
ggc ggc ctg gag gat ggg gag cct cag cag aag cgg gcc cac aca gag
2448 Gly Gly Leu Glu Asp Gly Glu Pro Gln Gln Lys Arg Ala His Thr
Glu 805 810 815 gag cca cag gcc aag aag gtc aga gcc cag tga 2481
Glu Pro Gln Ala Lys Lys Val Arg Ala Gln 820 825 2 826 PRT Homo
sapiens 2 Met Trp Ala Leu Cys Ser Leu Leu Arg Ser Ala Ala Gly Arg
Thr Met 1 5 10 15 Ser Gln Gly Arg Thr Ile Ser Gln Ala Pro Ala Arg
Arg Glu Arg Pro 20 25 30 Arg Lys Asp Pro Leu Arg His Leu Arg Thr
Arg Glu Lys Arg Gly Pro 35 40 45 Ser Gly Cys Ser Gly Gly Pro Asn
Thr Val Tyr Leu Gln Val Val Ala 50 55 60 Ala Gly Ser Arg Asp Ser
Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe 65 70 75 80 Asn Arg Tyr Leu
Phe Asn Cys Gly Glu Gly Val Gln Arg Leu Met Gln 85 90 95 Glu His
Lys Leu Lys Val Ala Arg Leu Asp Asn Ile Phe Leu Thr Arg 100 105 110
Met His Trp Ser Asn Val Gly Gly Leu Ser Gly Met Ile Leu Thr Leu 115
120 125 Lys Glu Thr Gly Leu Pro Lys Cys Val Leu Ser Gly Pro Pro Gln
Leu 130 135 140 Glu Lys Tyr Leu Glu Ala Ile Lys Ile Phe Ser Gly Pro
Leu Lys Gly 145 150 155 160 Ile Glu Leu Ala Val Arg Pro His Ser Ala
Pro Glu Tyr Glu Asp Glu 165 170 175 Thr Met Thr Val Tyr Gln Ile Pro
Ile His Ser Glu Gln Arg Arg Gly 180 185 190 Lys His Gln Pro Trp Gln
Ser Pro Glu Arg Pro Leu Ser Arg Leu Ser 195 200 205 Pro Glu Arg Ser
Ser Asp Ser Glu Ser Asn Glu Asn Glu Pro His Leu 210 215 220 Pro His
Gly Val Ser Gln Arg Arg Gly Val Arg Asp Ser Ser Leu Val 225 230 235
240 Val Ala Phe Ile Cys Lys Leu His Leu Lys Arg Gly Asn Phe Leu Val
245 250 255 Leu Lys Ala Lys Glu Met Gly Leu Pro Val Gly Thr Ala Ala
Ile Ala 260 265 270 Pro Ile Ile Ala Ala Val Lys Asp Gly Lys Ser Ile
Thr His Glu Gly 275 280 285 Arg Glu Ile Leu Ala Glu Glu Leu Cys Thr
Pro Pro Asp Pro Gly Ala 290 295 300 Ala Phe Val Val Val Glu Cys Pro
Asp Glu Ser Phe Ile Gln Pro Ile 305 310 315 320 Cys Glu Asn Ala Thr
Phe Gln Arg Tyr Gln Gly Lys Ala Asp Ala Pro 325 330 335 Val Ala Leu
Val Val His Met Ala Pro Ala Ser Val Leu Val Asp Ser 340 345 350 Arg
Tyr Gln Gln Trp Met Glu Arg Phe Gly Pro Asp Thr Gln His Leu 355 360
365 Val Leu Asn Glu Asn Cys Ala Ser Val His Asn Leu Arg Ser His Lys
370 375 380 Ile Gln Thr Gln Leu Asn Leu Ile His Pro Asp Ile Phe Pro
Leu Leu 385 390 395 400 Thr Ser Phe Arg Cys Lys Lys Glu Gly Pro Thr
Leu Ser Val Pro Met 405 410 415 Val Gln Gly Glu Cys Leu Leu Lys Tyr
Gln Leu Arg Pro Arg Arg Glu 420 425 430 Trp Gln Arg Asp Ala Ile Ile
Thr Cys Asn Pro Glu Glu Phe Ile Val 435 440 445 Glu Ala Leu Gln Leu
Pro Asn Phe Gln Gln Ser Val Gln Glu Tyr Arg 450 455 460 Arg Ser Ala
Gln Asp Gly Pro Ala Pro Ala Glu Lys Arg Ser Gln Tyr 465 470 475 480
Pro Glu Ile Ile Phe Leu Gly Thr Gly Ser Ala Ile Pro Met Lys Ile 485
490 495 Arg Asn Val Ser Ala Thr Leu Val Asn Ile Ser Pro Asp Thr Ser
Leu 500 505 510 Leu Leu Asp Cys Gly Glu Gly Thr Phe Gly Gln Leu Cys
Arg His Tyr 515 520 525 Gly Asp Gln Val Asp Arg Val Leu Gly Thr Leu
Ala Ala Val Phe Val 530 535 540 Ser His Leu His Ala Asp His His Thr
Gly Leu Pro Ser Ile Leu Leu 545 550 555 560 Gln Arg Glu Arg Ala Leu
Ala Ser Leu Gly Lys Pro Leu His Pro Leu 565 570 575 Leu Val Val Ala
Pro Asn Gln Leu Lys Ala Trp Leu Gln Gln Tyr His 580 585 590 Asn Gln
Cys Gln Glu Val Leu His His Ile Ser Met Ile Pro Ala Lys 595 600 605
Cys Leu Gln Glu Gly Ala Glu Ile Ser Ser Pro Ala Val Glu Arg Leu 610
615 620 Ile Ser Ser Leu Leu Arg Thr Cys Asp Leu Glu Glu Phe Gln Thr
Cys 625 630 635 640 Leu Val Arg His Cys Lys His Ala Phe Gly Cys Ala
Leu Val His Thr 645 650 655 Ser Gly Trp Lys Val Val Tyr Ser Gly Asp
Thr Met Pro Cys Glu Ala 660 665 670 Leu Val Arg Met Gly Lys Asp Ala
Thr Leu Leu Ile His Glu Ala Thr 675 680 685 Leu Glu Asp Gly Leu Glu
Glu Glu Ala Val Glu Lys Thr His Ser Thr 690 695 700 Thr Ser Gln Ala
Ile Ser Val Gly Met Arg Met Asn Ala Glu Phe Ile 705 710 715 720 Met
Leu Asn His Phe Ser Gln Arg Tyr Ala Lys Val Pro Leu Phe Ser 725 730
735 Pro Asn Phe Ser Glu Lys Val Gly Val Ala Phe Asp His Met Lys Val
740 745 750 Cys Phe Gly Asp Phe Pro Thr Met Pro Lys Leu Ile Pro Pro
Leu Lys 755 760 765 Ala Leu Phe Ala Gly Asp Ile Glu Glu Met Glu Glu
Arg Arg Glu Lys 770 775 780 Arg Glu Leu Arg Gln Val Arg Ala Ala Leu
Leu Ser Arg Glu Leu Ala 785 790 795 800 Gly Gly Leu Glu Asp Gly Glu
Pro Gln Gln Lys Arg Ala His Thr Glu 805 810 815 Glu Pro Gln Ala Lys
Lys Val Arg Ala Gln 820 825 3 2958 DNA Homo sapiens misc_feature
(51)..(2531) coding sequence as in SEQ ID NO1 3 cgcgggcgta
ggtgaccggc ggctttctca gttttggtgg agacgggcgc atgtgggcgc 60
tttgctcgct gctgcggtcc gcggccggac gcaccatgtc gcagggacgc accatatcgc
120 aggcacccgc ccgccgcgag cggccgcgca aggacccgct gcggcacctg
cgcacgcgag 180 agaagcgcgg accgtcgggg tgctccggcg gcccaaacac
cgtgtacctg caggtggtgg 240 cagcgggtag ccgggactcg ggcgccgcgc
tctacgtctt ctccgagttc aaccggtatc 300 tcttcaactg tggagaaggc
gttcagagac tcatgcagga gcacaagtta aaggttgctc 360 gcctggacaa
catattcctg acacgaatgc actggtctaa tgttgggggc ttaagtggaa 420
tgattcttac tttaaaggaa accgggcttc caaagtgtgt actttctgga cctccacaac
480 tggaaaaata cctcgaagca atcaaaatat tttctggtcc attgaaagga
atagaactgg 540 ctgtgcggcc ccactctgcc ccagaatacg aggatgaaac
catgacagtt taccagatcc 600 ccatacacag tgaacagagg aggggaaagc
accaaccatg gcagagtcca gaaaggcctc 660 tcagcaggct cagtccagag
cgatcttcag actccgagtc gaatgaaaat gagccacacc 720 ttccacatgg
tgttagccag agaagagggg tcagggactc ttccctggtc gtagctttca 780
tctgtaagct tcacttaaag agaggaaact tcttggtgct caaagcaaag gagatgggcc
840 tcccagttgg gacagctgcc atcgctccca tcattgctgc tgtcaaggac
gggaaaagca 900 tcactcatga aggaagagag attttggctg aagagctgtg
tactcctcca gatcctggtg 960 ctgcttttgt ggtggtagaa tgtccagatg
aaagcttcat tcaacccatc tgtgagaatg 1020 ccacctttca gaggtaccaa
ggaaaggcag atgcccccgt ggccttggtg gttcacatgg 1080 ccccagcatc
tgtgcttgtg gacagcaggt accagcagtg gatggagagg tttgggcctg 1140
acacccagca cttggtcctg aatgagaact gtgcctcagt tcacaacctt cgcagccaca
1200 agattcaaac ccagctcaac ctcatccacc cggacatctt ccccctgctc
accagtttcc 1260 gctgtaagaa ggagggcccc accctcagtg tgcccatggt
tcagggtgaa tgcctcctca 1320 agtaccagct ccgtcccagg agggagtggc
agagggatgc cattattact tgcaatcctg 1380 aggaattcat agttgaggcg
ctgcagcttc ccaacttcca gcagagcgtg caggagtaca 1440 ggaggagtgc
gcaggacggc ccagccccag cagagaaaag aagtcagtac ccagaaatca 1500
tcttccttgg aacagggtct gccatcccga tgaagattcg aaatgtcagt gccacacttg
1560 tcaacataag ccccgacacg tctctgctac tggactgtgg tgagggcaca
tttgggcagc 1620 tgtgccgtca ttacggagac
caggtggaca gggtcctggg caccctggct gctgtgtttg 1680 tgtcccacct
gcacgcagat caccacacgg gcttgccaag tatcttgctg cagagagaac 1740
gcgccttggc atctttggga aagccgcttc accctttgct ggtggttgcc cccaaccagc
1800 tcaaagcctg gctccagcag taccacaacc agtgccagga ggtcctgcac
cacatcagta 1860 tgattcctgc caaatgcctt caggaagggg ctgagatctc
cagtcctgca gtggaaagat 1920 tgatcagttc gctgttgcga acatgtgatt
tggaagagtt tcagacctgt ctggtgcggc 1980 actgcaagca tgcgtttggc
tgtgcgctgg tgcacacctc tggctggaaa gtggtctatt 2040 ccggggacac
catgccctgc gaggctctgg tccggatggg gaaagatgcc accctcctga 2100
tacatgaagc caccctggaa gatggtttgg aagaggaagc agtggaaaag acacacagca
2160 caacgtccca agccatcagc gtggggatgc ggatgaacgc ggagttcatt
atgctgaacc 2220 acttcagcca gcgctatgcc aaggtccccc tcttcagccc
caacttcagc gagaaagtgg 2280 gagttgcctt tgaccacatg aaggtctgct
ttggagactt tccaacaatg cccaagctga 2340 ttcccccact gaaagccctg
tttgctggcg acatcgagga gatggaggag cgcagggaga 2400 agcgggagct
gcggcaggtg cgggcggccc tcctgtccag ggagctggca ggcggcctgg 2460
aggatgggga gcctcagcag aagcgggccc acacagagga gccacaggcc aagaaggtca
2520 gagcccagtg aagatctggg agaccctgaa ctcagaaggc tgtgtgtctt
ctgccccacg 2580 cacgcacccg tatctgccct ccttgctggt agaagctgaa
gagcacggtc ccccaggagg 2640 cagctcagga taggtggtat ggagctgtgc
cgaggcttgg gctcccacat aagcactagt 2700 ctatagatgc ctcttaggac
tggtgcctgg cacagccgcg ggccaggagg ctgccacacg 2760 gaagcaagca
gatgaactaa tttcatttca aggcagtttt taaagaagtc ttggaaacag 2820
acggcggcac ctttcctcta atccagcaaa gtgattccct gcacaccaga gacaagcaga
2880 gtaacaggat cagtgggtct aagtgtccga gacttaacga aaatagtatt
tcagctgcaa 2940 taaagattga gtttgcaa 2958 4 295 DNA Homo sapiens
misc_feature (51)..(295) exon 1 4 cgcgggcgta ggtgaccggc ggctttctca
gttttggtgg agacgggcgc atgtgggcgc 60 tttgctcgct gctgcggtcc
gcggccggac gcaccatgtc gcagggacgc accatatcgc 120 aggcacccgc
ccgccgcgag cggccgcgca aggacccgct gcggcacctg cgcacgcgag 180
agaagcgcgg accgtcgggg tgctccggcg gcccaaacac cgtgtacctg caggtggtgg
240 cagcgggtag ccgggactcg ggcgccgcgc tctacgtctt ctccgagttc aaccg
295 5 51 DNA Homo sapiens misc_feature (1)..(51) exon 2 5
gtatctcttc aactgtggag aaggcgttca gagactcatg caggagcaca a 51 6 71
DNA Homo sapiens misc_feature (1)..(71) exon 3 6 gttaaaggtt
gctcgcctgg acaacatatt cctgacacga atgcactggt ctaatgttgg 60
gggcttaagt g 71 7 65 DNA Homo sapiens misc_feature (1)..(65) exon 4
7 gaatgattct tactttaaag gaaaccgggc ttccaaagtg tgtactttct ggacctccac
60 aactg 65 8 58 DNA Homo sapiens misc_feature (1)..(58) exon 5 8
gaaaaatacc tcgaagcaat caaaatattt tctggtccat tgaaaggaat agaactgg 58
9 69 DNA Homo sapiens misc_feature (1)..(69) exon 6 9 ctgtgcggcc
ccactctgcc ccagaatacg aggatgaaac catgacagtt taccagatcc 60 ccatacaca
69 10 120 DNA Homo sapiens misc_feature (1)..(120) exon 7 10
gtgaacagag gaggggaaag caccaaccat ggcagagtcc agaaaggcct ctcagcaggc
60 tcagtccaga gcgatcttca gactccgagt cgaatgaaaa tgagccacac
cttccacatg 120 11 59 DNA Homo sapiens misc_feature (1)..(59) exon 8
11 gtgttagcca gagaagaggg gtcagggact cttccctggt cgtagctttc atctgtaag
59 12 59 DNA Homo sapiens misc_feature (1)..(59) exon 9 12
cttcacttaa agagaggaaa cttcttggtg ctcaaagcaa aggagatggg cctcccagt 59
13 73 DNA Homo sapiens misc_feature (1)..(73) exon 10 13 tgggacagct
gccatcgctc ccatcattgc tgctgtcaag gacgggaaaa gcatcactca 60
tgaaggaaga gag 73 14 113 DNA Homo sapiens misc_feature (1)..(113)
exon 11 14 attttggctg aagagctgtg tactcctcca gatcctggtg ctgcttttgt
ggtggtagaa 60 tgtccagatg aaagcttcat tcaacccatc tgtgagaatg
ccacctttca gag 113 15 96 DNA Homo sapiens misc_feature (1)..(96)
exon 12 15 gtaccaagga aaggcagatg cccccgtggc cttggtggtt cacatggccc
cagcatctgt 60 gcttgtggac agcaggtacc agcagtggat ggagag 96 16 139 DNA
Homo sapiens misc_feature (1)..(139) exon 13 16 gtttgggcct
gacacccagc acttggtcct gaatgagaac tgtgcctcag ttcacaacct 60
tcgcagccac aagattcaaa cccagctcaa cctcatccac ccggacatct tccccctgct
120 caccagtttc cgctgtaag 139 17 86 DNA Homo sapiens misc_feature
(1)..(86) exon 14 17 aaggagggcc ccaccctcag tgtgcccatg gttcagggtg
aatgcctcct caagtaccag 60 ctccgtccca ggagggagtg gcagag 86 18 119 DNA
Homo sapiens misc_feature (1)..(119) exon 15 18 ggatgccatt
attacttgca atcctgagga attcatagtt gaggcgctgc agcttcccaa 60
cttccagcag agcgtgcagg agtacaggag gagtgcgcag gacggcccag ccccagcag
119 19 97 DNA Homo sapiens misc_feature (1)..(97) exon 16 19
agaaaagaag tcagtaccca gaaatcatct tccttggaac agggtctgcc atcccgatga
60 agattcgaaa tgtcagtgcc acacttgtca acataag 97 20 139 DNA Homo
sapiens misc_feature (1)..(139) exon 17 20 ccccgacacg tctctgctac
tggactgtgg tgagggcaca tttgggcagc tgtgccgtca 60 ttacggagac
caggtggaca gggtcctggg caccctggct gctgtgtttg tgtcccacct 120
gcacgcagat caccacacg 139 21 39 DNA Homo sapiens misc_feature
(1)..(39) exon 18 21 ggcttgccaa gtatcttgct gcagagagaa cgcgccttg 39
22 110 DNA Homo sapiens misc_feature (1)..(110) exon 19 22
gcatctttgg gaaagccgct tcaccctttg ctggtggttg cccccaacca gctcaaagcc
60 tggctccagc agtaccacaa ccagtgccag gaggtcctgc accacatcag 110 23
100 DNA Homo sapiens misc_feature (1)..(100) exon 20 23 tatgattcct
gccaaatgcc ttcaggaagg ggctgagatc tccagtcctg cagtggaaag 60
attgatcagt tcgctgttgc gaacatgtga tttggaagag 100 24 121 DNA Homo
sapiens misc_feature (1)..(121) exon 21 24 tttcagacct gtctggtgcg
gcactgcaag catgcgtttg gctgtgcgct ggtgcacacc 60 tctggctgga
aagtggtcta ttccggggac accatgccct gcgaggctct ggtccggatg 120 g 121 25
79 DNA Homo sapiens misc_feature (1)..(79) exon 22 25 ggaaagatgc
caccctcctg atacatgaag ccaccctgga agatggtttg gaagaggaag 60
cagtggaaaa gacacacag 79 26 145 DNA Homo sapiens misc_feature
(1)..(145) exon 23 26 cacaacgtcc caagccatca gcgtggggat gcggatgaac
gcggagttca ttatgctgaa 60 ccacttcagc cagcgctatg ccaaggtccc
cctcttcagc cccaacttca gcgagaaagt 120 gggagttgcc tttgaccaca tgaag
145 27 655 DNA Homo sapiens misc_feature (1)..(228) exon 24 27
gtctgctttg gagactttcc aacaatgccc aagctgattc ccccactgaa agccctgttt
60 gctggcgaca tcgaggagat ggaggagcgc agggagaagc gggagctgcg
gcaggtgcgg 120 gcggccctcc tgtccaggga gctggcaggc ggcctggagg
atggggagcc tcagcagaag 180 cgggcccaca cagaggagcc acaggccaag
aaggtcagag cccagtgaag atctgggaga 240 ccctgaactc agaaggctgt
gtgtcttctg ccccacgcac gcacccgtat ctgccctcct 300 tgctggtaga
agctgaagag cacggtcccc caggaggcag ctcaggatag gtggtatgga 360
gctgtgccga ggcttgggct cccacataag cactagtcta tagatgcctc ttaggactgg
420 tgcctggcac agccgcgggc caggaggctg ccacacggaa gcaagcagat
gaactaattt 480 catttcaagg cagtttttaa agaagtcttg gaaacagacg
gcggcacctt tcctctaatc 540 cagcaaagtg attccctgca caccagagac
aagcagagta acaggatcag tgggtctaag 600 tgtccgagac ttaacgaaaa
tagtatttca gctgcaataa agattgagtt tgcaa 655 28 26664 DNA Homo
sapiens misc_feature (910)..(13104) exon 1 910-1154; exon 2
1736-1786; exon 3 1925-1995; exon 4 3025-3089; exon 5 4361-4418;
exon 6 5582- 5650; exon 7 7075-7194; exon 8 8186-8244; exon 9
12878-12936; exon 10 13032-13104; 28 tatcaggtga ctgaattcta
tattctgaag taggagatac tgttattgct gttattacat 60 tttacacata
agaaagctga ggctctgaga ggtcaagatc acgcagctaa caaatgagcc 120
aagactcttg ctttagagct tgtcctctat tcttgctttt ctttccaaaa aacactacaa
180 tttttgtttt gttttgtttt gttttgagac agggtctcga ggtgtcaccc
aggctggagt 240 gcagtggcgc gatttcgact caccgcaacc tccgcctccg
cgcttaagcg attctcctgc 300 ctcagcctcc caagtagctg ggactacaag
ctcgggacac cacgtaaaaa tgatcaagtt 360 ctaacatgta tgcatacgaa
ttacaatgga aataaaatta gcaaagcgct tatgctaatg 420 ctcaatacaa
ttgatttcct cacatttaat cctcacaacc actacaacca cctctaactc 480
aagctctgag ggactgacgt gcccggagga cacagctctt atctggtgag aacaggagcg
540 ttttagcgaa actccaaact cctaggtccc gccttcccca ggaaggcttt
tcctggcact 600 gtgcttccgg aagtcccgcc ccaggagaaa aacagcttcc
ggaaaaaatt gcggccggca 660 aaccggaaca gaactagggg cggggccgct
tgagacgctc tagtattcct ctactctatg 720 gccactgtca attgacaagt
cccgagcggt aaagctcctt tctattggat gagcagcctc 780 gcgtaggcgg
gaagctcggt gcacggcgcg ctgattggct ggatcsgcca tgcggagcgg 840
ctaggtggtg cacgggaaac gcgggcgtag gtgaccggcg gctttctcag ttttggtgga
900 gacgggcgca tgtgggcgct ttgctcgctg ctgcggtccg cggccggacg
caccatgtcg 960 cagggacgca ccatatcgca ggcacccgcc cgccgcgagc
ggccgcgcaa ggacccgctg 1020 cggcacctgc gcacgcgaga gaagcgcgga
ccgtcggggt gctccggcgg cccaaacacc 1080 gtgtacctgc aggtggtggc
agcgggtagc cgggactcgg gcgccgcgct ctacgtcttc 1140 tccgagttca
accggtcagt caacgagcca cgccccgtcc cgctgggccc tcagtgcggc 1200
gcagcctctg agcatcgggg cacctcccag ggcttcggct tccctgcttc acacatgtgg
1260 ttcactgttg cgggggttcg tggagttatg gtgggtggga aatccgagat
tctttgcatc 1320 catgtgattt ctgcggatct gtgaagaact tcaggcctgg
gtctgagcgt ccttttccca 1380 acccttgggc cccggcctgg ctgtcagcac
tttcggagct ccaccctctt ccgtgcaccc 1440 caaggccagt gtgtcgttgt
tagcgtgtgg ggtggacaga tctggtgtgt agccggtggt 1500 ggagaaagga
ctcattttgt cctagcaccc acacacacag gcccccactc ctctccacct 1560
ctgctaagga gggctcaaaa cccaccagca taaatgtggc tcggtagtcc aacgtggact
1620 tttaattttt ttttcttttt tttttttcca gagtctacaa taaaacatct
aattggtgtc 1680 agagagttta cagaataaaa ccttctgaat gtcttgtgta
atgtttgtct tgtaggtatc 1740 tcttcaactg tggagaaggc gttcagagac
tcatgcagga gcacaagtga gtcagtctct 1800 tgctttcgga gggggagttg
attacggggc ttgaaagccg aaatgagagg ccagttgttt 1860 tttatagcaa
aagtggtcct tgttctgttc atgttatcct gtttaaatgt tttytcattc 1920
ttaggttaaa ggttgctcgc ctggacaaca tattcctgac acgaatgcac tggtctaatg
1980 ttgggggctt aagtggtgag tatattcttt gcagtgtcag aggctggtgg
gaagtctctg 2040 ggattttaac cggctttacc atttttccaa gtctggggtg
ggcagctact tttttttttt 2100 tttttttttt tgtcagtggc gtgatcttgg
ctcactgcaa cctttgcctt ctgggctcag 2160 gtgatcccct cacctcagcc
tcccaaatag ctgggaccac acgtgtgccc catcacacct 2220 ggctaatttt
ttttgtatgt tttgtagcga cggggttttg ctatgttgcc caggctggtc 2280
tcaaacttct gcgatcctcc tgtctcggcc tcccagagtg ctgggattac aggcatgagc
2340 caccgcacct ggcctggaat tctttttata ccagcccagt cagcagcagc
acagagcatt 2400 aaaagctgtg actcaggaga acagatttta atatggatac
cacctcttaa gtgttaccat 2460 ccacttagtt tcttgcgttg cggggacaga
gatttgtggc agtaaactgg agagtctagc 2520 agtggtgatt acagttaata
tgtttaccgc agacgccatt ggcacattgg cagccacaca 2580 catacccact
gtccagatta ccctgtcatt tatgtctatc aaccggaagg tcaggattgt 2640
gttgcagcca aattgtgtgg gcttggtggc atggaccgga aggagtgaag tgttagacca
2700 gtctcccttc tcagggctga gactagggtg aggcacttag ggtgccagcc
cttcacttgc 2760 atgattcctt acattttgca cactgggtgc cttgctgctt
caccctagtg acagctcagc 2820 ccattctaga ggcatttaaa gaatatttgg
tgtctgttac acctctagct ggcatcactt 2880 ctgctctgta catcttccct
ggttgtactt ccaaagctgg aaggtggaga tgtagataaa 2940 tagttggatt
agtacggggt gctcctcctg ttagtgacga caggtcaaat tgatgagaga 3000
tctgatttta tgcatccttt ttaggaatga ttcttacttt aaaggaaacc gggcttccaa
3060 agtgtgtact ttctggacct ccacaactgg tgagtctttc ctgacacatc
tttcaaaagc 3120 aatctttcct tttgtaatat cagtaacaag aattttcctt
tttgcaaatc agtcttctgc 3180 cctccagaga tacctggtcg ttgaaacgct
tcccctttca agttaaaaag acttgagttc 3240 tgattaacta tgtgaccttg
atcaagttac tttacctttc tgagctttag tttattcatc 3300 tataagatga
ctatcacgtt tcatagagtt gttaaagatt aaatgacgta gcagcacata 3360
taaagcacta aatcacttta ttagatatat gtttggcacc aagtaggcac acaagaaagg
3420 gcagcttttg tttttattca ataaatttct gacatcttct tacctttcag
tccagcttat 3480 tacactcttg agaaggcgtg tgtgtgttgt tgaatataac
agttcatttt ccagtcctta 3540 agaagaaagt caccaagacc tgttaagtct
ttccccaaaa taacgtttga aatccatcca 3600 tttgtctctt attgaggcct
tccttatttc tgttttctat gcctgtaaac tacaatagcc 3660 tcccatattc
attctcgcct tcctgtaatc catctgccac acagcagcca gagaggtcac 3720
ttcaagacag aaaagtagtg tgtcacttgc caccctaaag cccttcatgg gctccccatt
3780 gcaatacaat caaaacacct tgatatggcc tacaagtcct gtaggccccg
gccgctaccc 3840 acacttccat ctgtacccat cgctgaactg cagctgcatg
ggctgactct tatgtccctc 3900 taactccctg gccacttcag gactttcgcc
cttccgcggg ttccctctgc ctcttctaat 3960 tgctgcctat attgttactg
aaccttcagg gctcagctag agggtcattt actccagaac 4020 tgcctcttct
tctctagaca agttggatcc cagccttctg tatttttcat tttccttgca 4080
gagcacttag cataatgcca ctaagctgtt tctgttatcg tgtttccttt tgtctcctcc
4140 actggcctga ttagagcaag gcctccatct ctttttcctg ctatatcctt
ggcatctgat 4200 ataatggata ctcagtaaat atttgtaata aatgatgttc
aaaatattta ctaagctttg 4260 ttttatgttg atacctattg gtaacctttt
aaatacttga atagttgctg tgttctacat 4320 ttgttcaacc ataactgctc
atttctttgt ttttcattag gaaaaatacc tcgaagcaat 4380 caaaatattt
tctggtccat tgaaaggaat agaactgggt acgtctttgt ctgtgactca 4440
tcctctgcta tttctaactt atatatgccc tgacctctca aattagaatc cattaaaaac
4500 atcaacatca aacctcaaaa tcaaatgctt catcaccacg agattttttt
tttttttttt 4560 ttttttggat agagtcttgc tttcttacca ggctggagtg
cagtggcatg atctcggctc 4620 actgcaacct ccacctcctg ggttcaagcc
attctcccac ctcagcctcc tgagtagcta 4680 ggactacagg cgcatgccat
cacgctcagc taattttttg tatttttagt agagacgggg 4740 tttcaccatg
ttggccagga tagtctcgat ctcttgacct tgtgatctgc ccgcctcagc 4800
ctcccaaaat gagctaccat gtggctggag atgggatttc taaatagtga cattttctgt
4860 gttcccacct catgctgtaa aaataggggc caggtcggca ggagtgattg
aacagctgat 4920 gcctgcctgt gtacatgctg tgtggcattc tccatccaga
cggcagggct cctgcctcag 4980 ttccagaggt gcttctcgtc gttgagttgc
tttgagttgg gggcgggggt gacaagggtt 5040 ccctagaggt tttgtggcca
actttgtaca ttgaaacgca gctccagctg cgcagggggg 5100 cttacagcct
cttgatggga agaggcctca ctgaggatgc tagtagggct cttgtcctgg 5160
cactggtgtg tatctgtggc ttgttaatac tcctctttta tagaaacact aatactttgt
5220 ttcaaaatat acatcagctc ttctggtttg cgatgatagg ttccctggct
tcactattct 5280 gtttgttaac ttgggtctct gaaagttgag tactagtttc
ttgtttttca atttttaacg 5340 gatagtcacc aaagattata atgtcttttc
atctggctgt agtaaatata aatggctgac 5400 caaaatacac ttttatttat
ttcctaaaaa tggtaatctc cttagaaagt ctggttttcg 5460 tgtcagattc
ccaccataat tctgaggcaa ttcagttgct cgtggttggt gatcctgaag 5520
ttactcttcc cacacatctt cactaatgca atcactttgc tgttgtgygg ttttcttgta
5580 gctgtgcggc cccactctgc cccagaatac gaggatgaaa ccatgacagt
ttaccagatc 5640 cccatacaca gtgagtatga aagccaggtt tcccaggagg
agggtgtacg tcctgagtaa 5700 agaaaacatg gatgaaaata gaaactgaac
acttgctgtg ggcaccctgt tttgtgttct 5760 gagcatgatt agaaaattta
gttgaggaat gaagatatgg ctcctgccct ggcttataaa 5820 cttacggatg
tctgacttat gcctaatgat agtgattatg ctttggaata ttagataatc 5880
aagcactgtt ggtaaataga ttgcattcaa gtttgcacat tcattgcttg gaggtttttt
5940 cccacaggcg taataccctc ttttgatcag acgatcatga agaggtttgc
acagatagat 6000 ttttttaaat aaataatgat tacagcaacc taaaagaagt
gttgttgggg gttagaagct 6060 cctgcaaatt ccgaagtatc agggccagat
gatgtggtct tagcttagga aaagagttag 6120 tcttgtcctt gaacttggct
aaagacattc atgtctggtt ttacttacat gtgaagagag 6180 taccaagcag
taggggtatt tccttgttag tactaactaa tgtgatgctt actaagtagt 6240
gctgatgggt gacagaccag agcacccagc aaaggccaga gaagtccaga acctggcgag
6300 gagatgaggc ttacactgac tgaaggcaga aggcagcagg gaggagagga
atgtgccgga 6360 gcaatggcac aagtgctcct aggccagtgc tgtgatgagc
tgatcagcac tcccattgcc 6420 tggcttgctc ctcctgctca gatgccttct
ctcacctgac ccctgctgta gccaccccca 6480 gcctgagttg catccacctg
tttgttgtcc atttccagca ccctgttctt cgctccatgg 6540 catgtgacag
ttaactttca tatgtgattt gcgtgatcga tgttaacatg ctcagttttg 6600
ccgatcactg ttttttcagt gtccagcggc cctcagtgag tgaacttacg ttcattctcg
6660 ttgcagctgt gctttagctt cttagagcag cgaatttttt tcccttgatc
ttgagcctta 6720 actaaatgta aaatgaggct ccttcttgag ataggtaccc
tttgggtcta tgtgttttag 6780 cgggagtgat gataataaat aagcatgtct
acaacccaca tgctgtttag ataacacgtt 6840 gttgagttgg tactgtggcc
gaggctgtga gctaagcaga aacataaaca ttaataggac 6900 ataggtgcag
cccagaaacc aggtaggaag ttaactaact agttatttcc tactgtatag 6960
taaaaggtgt gctgatttaa ttggcgttct ggcattccca tgtatgaacg tctgggcctt
7020 ggctgtcagc tcaccttgtg cagtgtgtaa tttggtggta tctgtactga
ccaggtgaac 7080 agaggagggg aaagcaccaa ccatggcaga gtccagaaag
gcctctcagc aggctcagtc 7140 cagagcgatc ttcagactcc gagtygaatg
aaaatgagcc acaccttcca catggtaata 7200 gtataaacaa aacagagcag
cagaaaggct tgcgttttct taattctctg ccttgtaatg 7260 cttgtagaga
gtcattattg taagaaagcc aggtgtgtaa acagatcctt cttcctgggc 7320
ttactataac ttggcccgtt gggggaatga gaagggttgt tgtaaaggtg gcagcctgca
7380 actttaataa tgaccagtcc acagttttgg ccacccaggg tctgggtagg
cccaaaactg 7440 tgttctgttt tcccagagga gaacagggcc tgacaaacgg
attcattttg tatttttcat 7500 taatgtaaca tttatgcaaa ttttccatta
atgtggaaac tataactgct aagccaatga 7560 gacagtcaaa tcagtgagag
gctctgcacg tcttccagaa tgacagccca ctgggaaacg 7620 gagttaaaag
tccaagatga gatgtagctc aggagtcagg ccgcttcggg agtttgttgt 7680
ccttaacaga aggtcagcgt tggcaaagct cggcagctcc tctttctgtc ctgaggtctt
7740 gtctagtgac tgagaacagg ctgaccccta tgtgctgtcc ttgtttggat
ggcaccgggt 7800 aaagactgac accagcattt tctctgcagg cctttgaact
tttgtgttat ttcatatatt 7860 atatgtgtta taaagcacat tacaatatat
ttttctctgt cttctccagt cctaggtgaa 7920 atgtgtcatt taaaaaaaat
ttcacttgcc attctaaagt ttttctggtg agagttttgt 7980 gtttttcatt
tacgcaaaca catctccaca taagtaggga aaaaaagtct tcttgagtat 8040
attagtgtct tcagcctttg tattgggaca gtagcgtcca ttaattttta tgtgaagtga
8100 aattaggtat cgggtcataa tcagtctgtg atgtcttcac agctttcaca
tttaccttgt 8160 gataatcaag tgtgtttttc ctcaggtgtt agccagagaa
gaggggtcag ggactcttcc 8220 ctggtcgtag ctttcatctg taaggtaagg
aagactttcc ggagggctgt acatgactgg 8280 ggtcttggtc agcgacctct
ggtttgcact ttttcattaa tttgagggta ggcactcctg 8340 ttacctgaga
caagaagaga tagcagatct tcagaaaagc tgatggaagg ccgggtgcag 8400
tggctcacgc ctgtaatccc agcactttgg gagtccaagg caggtggatc acgaggtcag
8460 gagtttgaga acagcctgac caacgtggtg aaaccctgtc tgtactaaaa
atacaaaaat 8520 tagctgggtg tggtggcgca tgcctgtaat cccagctact
tgagaggcca
aggcaagaga 8580 atcgcttgaa cacaggaggc ggaggttgca gtgagttgag
attgcaccat tgcactccag 8640 cctgggtgac agagcaagac tctctcaaag
aaaaaaaaaa ttcgatagaa atgacactgg 8700 caatgagcct gcaacaagta
ttactactga cctttcataa ttgtcatcac ttgtaggttt 8760 cagagtttag
atgctctgtt tctcaaaata accccatact tttatttcct tttaaatttt 8820
tttccagtgc cctgtcagcc tccgtacatt tttttttttt ttttttgaga ccatgtctgt
8880 ctccatcgcc taggctggag tgtgcagtgg cacaatctcg gctcactgca
gcctccacct 8940 cccaggttca agtgattctc ctgcctcagc ctcccaagta
gctaggatta taggtgcgcg 9000 ccaccacacc cagttaattt ttgtattttt
agtagagatg gggtttcacc atgttggcca 9060 ggctggtttc actcctgacc
tcaggtgatc cacccacctt ggcctcccaa aatgctggga 9120 ttacaggcgt
gaagcactgt gcctggtcca tattctttta tatttgccaa tgattggtcc 9180
ttttagaatt cagaaattat tgaaggcagc tgtgtttgtt ttccttcaac tccatcaggc
9240 ctttattcaa agtcttttaa ctctgtttta ctttatttca ttcccctgca
atagctaagg 9300 tctaacacca gattaattgg aatattagct agcattcaca
aaggcctaga tctgtaactc 9360 tgaaattggt caaattccat taaaaatttt
tgttacaata agctgtttgt aagatctgac 9420 tagtggctta tttttaatag
aattttgcat taaaatttta tcaatacaat ttgcaacaaa 9480 tttgtctaaa
tatgtgaaaa gatttcattg cctttttgtg ggcttagatt attttttaat 9540
gttgattttg aaatatattt ggaattgtta tctaaattct aaaagctaca agtgaaaata
9600 ataatgaaag taagtagtta atattagtgg caagatcatt gccagtatca
tttctatcga 9660 tttatttgaa taatgtgatt ttcataaaag ttaagtacta
ctgttaacag gcttattact 9720 tgtatgtttc tgagttttag atagcaaaat
cattttttaa agttttaaaa atattttatt 9780 tttgataatc tatatttata
ttgtctgatt tttaaactgt tttctatggt aatctttaaa 9840 tcgtattcct
gctttccgga ataggtaaca gtgagcatga tgaaaagtga caagctcact 9900
tttacacact cgggcagttg ccctattatc aggcagccgt tcctgggggc tgccagctgc
9960 ctgccctggc ttttccatct ccttccttgc tgtcttctgc ggctccttct
gagggctgct 10020 gtcactggat tagcctataa cgcctttccc ctcttctaat
taatttgctg ctctcaggtg 10080 aggttttgga aagcaataaa gctgagctag
gtcaagttcc aggagtctct tggcatgagg 10140 acctgaaaaa ctcatctgtt
ggaagacctc ggctttgggc agctggtgca ctgttggggc 10200 gttattggct
gcgttctggc tctcatcagt cttccagata ctctgcattc ctcagagagg 10260
aacatatctc catgggttga gttcagctcc cagggagatg ggtttccctg ccttaagtcg
10320 gcaagtacct ttttttttct ttttttgaga cagagtctcg ctctgtcacc
aggctggagt 10380 gcagtggtgc gatcttggct cactgcaacc tctgcctccc
agggtcaagc agttctcctg 10440 cctcagcctc ccgagtagct gggactacag
gagcgcacca ccatgcccag ctaatttttg 10500 tattttttta gtagagacgg
ggtttcacca tgttggccag gatggtctgg atctcttgat 10560 ttcctgatcc
gcctgccttg gcctcccaaa gtgctgggat tacaggcgtg agccatcatg 10620
accagccttt atgtttcttt gtttgttttg tttttctgag atggagtctc gctctgttgc
10680 ccaggctgga gtgcagtgtt gccatctcga cttactgcaa cctctgcctt
ccaggttcaa 10740 gtgattcctt gcctcagcct cccgtgtagc tgggatcaca
ggtgcctgcc accatgcccg 10800 gctaattttt gtattgttag tagacacagg
gtttcgccat gttggccagg ctagtctcga 10860 actcctgacc tcaagtgatc
tgccttcctc agcctcctaa agtgctgggg ttacaggagt 10920 gaaccaccat
gcccagcctt caattacctt ttatttattt tatttattta tttatttttg 10980
agacggagtc tttctgtgtt gcccaggctg gagtgcggtg gcgcaatctt agctcactgc
11040 aacctcctcc tcccaggctc aagtgattct catgcatcag cttcccgagt
agctgggact 11100 tcaggtgccc gccaccacac ttggctaatt tttgtgtttt
tagtagagac ggggtttcac 11160 catgttggcc aggctggtct tgaatttctg
acctcaaatg atcctcctgc ttcagcctcc 11220 caaagtgctg ggattacagg
cgtgagccac tgcccccaac agcaagtacc ttttaaacat 11280 tagagacatt
tagttgccat cctcaaaccc gtttgggtgt gtggagagaa tgttgggtcg 11340
tgacatggtt gttagttatc taaagatgtc agccatcaat catcactgtg tgatgtgcac
11400 actgaagctg taatccttca tctaggatga tattttttaa gatggaaaat
tctacaaccc 11460 tgagaataag gatttcagat ccaaatttga gactcagccc
tacgagtaac tctttaactt 11520 cagagagtta aaagaagatg cacagttgat
gaagatttaa aggagaaaat ggaaatcaaa 11580 tgtcatttag cactcaaagg
cctacatgtc atttctgaca tttttctgtt tgtgtgaaat 11640 tttttttttc
ctataaaatg attgtgaagt tttctggtag aattattgtt tgcctttcta 11700
atgtaatagc atattagggt tttttttttt ttctttttct ttttttgaga cagagtctca
11760 ctctgtcgcc caggctggag tgcagtggca cgatctcggg tcactgcaat
cttccgcctc 11820 ctgggttcct gcctcagcct cccgagtagc tgggactaca
ggcgcacgtc accacacccg 11880 gctaattttt tgtattttta gtagtgacag
ggattcaccg tcttagccag ggtggtcttg 11940 atctcctgac ctcatgatct
acccgcctcg gcttcccaaa gtgctgggat tacaggcatg 12000 agccgctgtg
cctggctatt agagattttt tattataatt tatctccaag ataaaagcag 12060
tgacattata ttgccacata attgaaaaat acaagagaaa taaaaatcat ccatgctttt
12120 gttagcctat cactgtcatt gaaatattat gttacatggc agtttgcttg
ctggttgctc 12180 tgttaggcaa cgctctggtg acattccttt agctattaat
tgaggaatgt agaatgacag 12240 aacagtgttt ctcctcaatg atacttgaag
gatatttatt aactttcata ttgaattaca 12300 ttttattaaa tttataatga
gttaatgctg ggaaataaaa cactgattta agtcattttg 12360 gcttttagta
ctaaagcatt tgacaataaa tgacttcttc agaatatggt ataccttctg 12420
aaagcaataa acgcatttta atgaattgta aggaaacaac atcattttat tttttatttt
12480 tttttttgag acagactttc gcttttgttg cctaggctgg agtgcaatgg
cgcgatctcg 12540 gctcactgca acatccgcct ctgggttcaa gcgattctcc
tgcctcagct tcctgagttg 12600 ctgggattac aggcacgtgc caccacgcct
ggccaatttt gtatttttag tagagacggg 12660 gtttctccac gttggtcagg
ctggtctcaa actcctgacc tcaggtgatc tgcccgcctc 12720 agcctccgaa
agtactggaa ttacaggcgt gagccaccgt gcctggccaa cattattatt 12780
tttttttaat ctagaaaaat acacttctaa gaaaattgat taaaaccaac cttcttcatt
12840 agcccctaag atcacatcta tgttctcttt gttgcagctt cacttaaaga
gaggaaactt 12900 cttggtgctc aaagcaaagg agatgggcct cccagtgtga
gtgtgggggg taaggcttct 12960 ggggactcac tgggtacacc tgtccactta
aggaaatcac atttcacaga ggccttgcct 13020 cttcatttca gtgggacagc
tgccatcgct cccatcattg ctgctgtcaa ggacgggaaa 13080 agcatcactc
atgaaggaag agaggtgaga tgcctggttt tcttgatnca gcagttacag 13140
gtagggtctg aaatgctggg cagagtctgt cttcttcagg ccctacagac accacttttg
13200 aaggacgtgg aacagtttgg acatcactca gctaagtgat aaaatggcct
cttttatctg 13260 tgtttgtccc gcatgtcaac acggctgcat tcgagcattt
ttgtagattg tccatttagg 13320 atctagtcac cgtcctcctt aaagggtgca
tgctttcctt ggtacttgag ctcaggacag 13380 tgtctaacaa cagaccccat
atggatgggc ctggggttta tggtccagag gaatgccaca 13440 gtattctatg
tcaagatatt tcctctgact tctgaggaca ttaggaccag tggccacaga 13500
ctgaagaaaa ccttaatgcc aagcctcctt tcctggccag tgtaggcctg aagtgcctca
13560 acctgacagt tacctgttta ggtatccaca aagagaccag aagggtgttg
atggtgatgt 13620 gtaaagttgg ttttgtgctt tgtttacctc tcagctcact
ggataggata tgtcatgtta 13680 gcagttgcct tgaaggcagt tcagtttggt
ggctgagctg tgacccccag tgggcgggct 13740 tatttggttt tgcagatttt
ggctgaagag ctgtgtactc ctccagatcc tggtgctgct 13800 tttgtggtgg
tagaatgtcc agatgaaagc ttcattcaac ccatctgtga gaatgccacc 13860
tttcagaggt aatgaggggt ctctagggtg ggagaagtga gagctgaaac ccagcccagc
13920 atcgacatgg gcatcttgtg gcaagagctg tgtttctggg aagaccacta
tctgggttta 13980 cagttcagag gccggcactc ctgccttaag tcactgttgg
tagttggtgg gctccggtgt 14040 acacagcctc aaagtgaaat tagaaaagat
tgaaaactag aaacaactga ggactagaaa 14100 ttcaactaga actcttacag
ctcttatacc agaagaaatt ctagaacttt tttgaattct 14160 aactaatgcc
ccagattatc atttggatta ttttgaactg aattaatttt cttccattac 14220
ctgcattgaa acaaatgagg tgggtcagag tgtgtgagac tgtcgtggtc aagagtccgt
14280 gttatgggat ggactcacag ctggggaatg tcttttgggc taactgccac
tctgttgttg 14340 tcctctatcg aagttaacca gttttgcggt tcagctttca
ttccagatgg aatcatcttt 14400 gacccaccta tctgagtttg aatcttttcc
cccactctta atggtttacc tgtatttttc 14460 ctgttcctag tttgtatcta
tctgtatttt ttcacttgtt tttttctact taccacaaca 14520 aatccttttg
ggctgctgta ccccttccga gtcagagcgt taggagttgt ttcatggtct 14580
gctttattct ctgtgggtga atttggatgc gctggtagcc ccggctttgt attttaatcc
14640 agttttgggc agcaaaacct cttcaatgaa tcaggtgtca tttgagagcc
atgtgtggat 14700 gtgtgatgat gctgggatag ataaaaatag ctactgtgta
tatttctttt taaagggaac 14760 tggagggaaa cacatcagca tgttagtaag
tggtctgttg tccaggtggt gaaatttcag 14820 atgattttca tttctcgtgc
ctgtgtctca ggtcctctgg aaggcagaca ccagggtggc 14880 attggaggtg
caggaggttt attcgaggaa atttgactgt gagagaggaa ggagagaggg 14940
agcaggagga ggcagggaga gcctgggtct ggctttgcag gttggacccg tatgagtgga
15000 gagggtagga aggaagtgca gtgctgagaa aggatcagcc aggcctactg
gaaagcccag 15060 agcagagctt gccagataca ggaatcccac gtccattgga
aatggcccag caccggggtc 15120 tgccgtgagc agcctgctgt gagagcatgg
cctgggcgtg gaggctgtca gctcactgca 15180 gtgctgcaga gggccgcacg
atacccctcc ctggctgcgt ggtccctgtc ttggtgtgtc 15240 ctgagtctgc
atcactttgt aaagccccac tcttctgccc aggtaccaag gaaaggcaga 15300
tgcccccgtg gccttggtgg ttcacatggc cccagcatct gtgcttgtgg acagcaggta
15360 ccagcagtgg atggagaggt atggagccca gcccagcggc acttggggta
actcttctgg 15420 gcagtggtgg attccccttt cctcccctcg tgctctttcc
agcgctacct acccttctgc 15480 acctgcctaa actttctgtg ggattcctgc
cttcccagaa ttctaggctt cccagatctg 15540 tgctacactc gtgaagaaaa
tgcaccgcta ggtggcgcag tgtccacacg attccattta 15600 ttttacaccc
tccacactct tcagggtgtc tgaacaaata ctgccgtttg gttgaggatt 15660
ccataagtga attccaaaga agagattgca gctataaaat gatagcttcc atttactgaa
15720 tgcccacttt gtgggaggca gtgtgtgaaa tacccttcat ttcacttcat
ttcctctagg 15780 gtcgtcgcca gcagccctgg gaggtagatg tttagtcact
ggaaggcatc tttttcctcg 15840 gggcatcgct ggccagggcc aggtggagga
gtatgagttg agctcgggtg cggggtgacc 15900 ttgggctgct ttttggcccc
tgcccgtatc tccccacatg gcccgtttac ctgcccctca 15960 ctccatggcc
tgctctcctg ctgtctcttt cattcctcag ggtttgggtc ccctatttgt 16020
atgccctgga catcttcttt ttcttgtttt tcctctcact cttcccagca cacctgaaag
16080 gcagctgagc tagggaacac cgggctttga gacagcagga gtgggaccat
gtttggccat 16140 gtagtaacac tgcttggggc aagtcactga actgtttgaa
cacctcatcc tcattaccac 16200 tcctgagctc agcaccactc ctcaggggga
gctgcctcct aacagacgct gcaaatgccg 16260 ggtctgtttc ttcacaggtt
tgggcctgac acccagcact tggtcctgaa tgagaactgt 16320 gcctcagttc
acaaccttcg cagccacaag attcaaaccc agctcaacct catccacccg 16380
gacatcttcc ccctgctcac cagtttccgc tgtaaggtag tgtctcagac yggccccttg
16440 tcggcccagc tctcgtcccc tctctttctc tccatgaatg tgttttgtct
ctttcagaag 16500 gagggcccca ccctcagtgt gcccatggtt cagggtgaat
gcctcctcaa gtaccagctc 16560 cgtcccagga gggagtggca gaggtctgtg
ccatcttgaa ctaatggaat cgtctcagtc 16620 gagttgggaa acatttctgt
aaatagccac atagtaaatg ttccaggagg ctctccagac 16680 catatggtct
ctgttgtaac tattcaactc cgctttgagc acaaaagaaa cacggacaat 16740
aagctaatga atgggcttgg ctgtgtgcca gcgtgaattt atttagaaaa gcagcctact
16800 ccaggctggg ttgaggtggg cggattgggg ccagtagttc tccttttcca
aaattgcctt 16860 gcatgggaat agcagtgata gagctcgtgt gtttcacagt
atagaaaata ggaaatgtgt 16920 gatgaacaaa gtcacccata atcctgttgc
ccagagataa tgattgataa cattttgtgt 16980 ttcttgattt gtgtatgtgg
gtttatattg tcagtctttt cctgtatcac taaacagtct 17040 taagtaacaa
gatttttatt ggtattccaa atagggatgt ttactcattt gggatgtttc 17100
caattttttg ttgtttttaa tgaatgaaac aataaatgtc ttatatataa atctttgatg
17160 ggaactctgt tcccttcaag tcattcctaa atgtgggatt actggcccag
agtgtgagac 17220 ttgttaaggt acttgataaa tgtaagatgc catcttgaaa
gcctcttcca gtacaatcca 17280 accaggaaag tgaacagcct tactgcccca
catctttatt ttaattaatt aatttattta 17340 ttttatttat ttatttattt
ttttgagacg gagtttcact cttgttgccc aggctggagt 17400 gcaatggcgt
gatctcagct cactgcaacc tccgcctccc gggttcaagc atttctcctg 17460
ccccagcctc ccgaatagct ggaattacag gcgcctgcta ccacgcccgg ctaatttttt
17520 gtaattttag cagagacggg tttcaccatg ttggcaggct ggtctcgaac
tcctgacctc 17580 aggtgatcca cccacctcag cctcccaaag tgttgggatt
acaggcgtga gccgtgcccg 17640 gcctgtttta atttttaagg atctgaacct
tgattttaag tttcctgccc actccacagt 17700 atttgtatta gaatagagca
tgtgctggat tatgactgga tgctgtgtgc tgttgaggtt 17760 gggtagttgg
ggccctttaa gagactatac tagcaagact cgggcccaca ggcaacatca 17820
cggggttgaa gaacctggtg tccctttgtt ggcatctgcg caggctctta acacacagca
17880 gcgatacaca gccctagccg acattcagat ttaccttgtg cttgtgaaaa
atattgcaca 17940 gggcctgccc tagacctagt gaattagaat cttgagagtt
aggcttggga ctcacaagct 18000 cccagatgat tttaatgctc agcgaggttg
aagagccgcc tgtccaagga gttgccactc 18060 cgtgtgatct ggggcttgct
aggaaagtgg gatctcaggc ctcactgcag agctgccgaa 18120 ctggcttctg
cgttttgcca aggttcctgg gtgtgaacat gagtttcaga gtcactcctc 18180
tagggcccct gcttctcagc tcggaccatt gacccctcag aggacatttg gcaacatctg
18240 gaaacgttct tggttgtcac agcctaggag gtgggtagtg gtgctgctag
tgggtagagg 18300 tcaggggtac tgcaccagga cagcagcact ggccacagaa
aaaaactgtc ttgccctgag 18360 catcagtagt tccccgttga ctggccctga
ggcagagcga tgcagcatcc aaaaggcggt 18420 ggagcagacc tgccccagat
cctagtcact taaccttcag tgttgatctg aaggaacttc 18480 ctgcagattg
tccccctgaa tttattctgg acatccccaa tggggtctgc tgaggccata 18540
taccctgtcc gtcacctgag atgcttctct ctctycctgc agggatgcca ttattacttg
18600 caatcctgag gaattcatag ttgaggcgct gcagcttccc aacttccagc
agagcgtgca 18660 ggagtacagg aggagtgcgc aggacggccc agccccagca
ggtgagtggg agcccacaga 18720 gcagcctttc tttcctgggc tctgcccctg
ctgctgtttt cctagcatta agtggagtgc 18780 tggtggggcg cattctaacc
tggcttttca gtctaatcca gggcttctct actcagctct 18840 acattagaat
tatagtcatt ggaggagggg gctttgggga gtttaagaat cccaattcct 18900
ggctgggcgc ggtggctcac acctgtaatc ccagcacttc gggaggccga ggcaggtgga
18960 tcgcgaggtc gggagatcga gaccatcctg gctaacatga tgaaaccccg
tctctactaa 19020 aaatacaaaa attagctggg cgtggcggcg ggcgcctgta
gtcccagcta ctcgggagac 19080 tgaggcagga gaatggcgag aacccgggag
gcggagcttg cagtgagcca aggtcgtgcc 19140 actgcgctcc agcctggacg
acagagtgag actccgtctc aaaaaaaaaa atcccaattc 19200 ctgtgcccca
tcccacccaa tcagagcatt tggcgatggc acccaggcat tcttggcaag 19260
gcacgcactg agtgaaacgt tttagtgaac acctgtggaa agagctctga gcagggactt
19320 ggctggcaga gatctagtcc tggctttgcg gatgcaaatc catggaggat
cttggccacg 19380 tcactcaact gaggctgagg gccgggcaca ggctttggaa
ccatcgggtc tccctggatt 19440 tgaatcctga ccctgcctct taccatcttc
actggagacc tgggcgtctg agcctgtttc 19500 ccccttggga agcagagcat
ttcctacctg gtagggctgg gaggatgcga ccgaagtgca 19560 tggtcttgca
gtgagagctg gatgcaaggc acacactgtt ctcttgaaat aaatgacagt 19620
tcccagcata aagaaatgtc attttttaaa tgtaaaagaa ttacagcaat tcttttgaag
19680 aaaggactgg agaatttatt tgttcttctt agccttttgg tgacagatag
cctgtgggtc 19740 ccacactggt gcgaagtcct ttgtttcaga gcggttgcca
ggggcctgcc agtccccctc 19800 ctgggaagct ggatagaact atgttgctta
cccatctgtc ttagtctgtg ttttgttatt 19860 ataaaagaat atgtgagact
gggtaattta tcaagaaaag aggtgtattt agttcacggt 19920 tctgcgggct
gagaattgaa ggtcacggcc ctagcttcca gtgaaggctt ccatgctgca 19980
tcataacgtg gcagaggagc gcaagtagga agtggacgct tgtgaagacg ggataacctg
20040 agctgcactc tggctttata acaacccccc tctcctggga acaaatccat
tcccttgaga 20100 agtaatgcag tctcctgaga gccagtactt actactgcag
ctccaagcca ctcaggaggg 20160 tccgtccctg tagcccaaac gccttccact
aggccccgcc tcccaaaacc gccatactag 20220 ggagcacgtt tccacatgag
gtctggggac aaaccaatga cactcaaacc attgcacctt 20280 ctcatggctg
catgctggct cacttttgac ccaaaggaat ggattgtttc acatggattt 20340
tttcacagag aaaagaagtc agtacccaga aatcatcttc cttggaacag ggtctgccat
20400 cccgatgaag attcgaaatg tcagtgccac acttgtcaac ataaggtatg
ctgctttccc 20460 aggaagcatc cttccatcaa gggcaygttt actttttaaa
caaaagtcct gctgtactca 20520 ccagtcgatt tgaaatgcgg tatcaagccc
tgtcacttgt catgtcgact ggagtgtcca 20580 ggagaggagc gtggccttac
tgcattttat agcctcagta gcaaacttta ccctgggaat 20640 caccaaaatt
catcccatga tgtcttttaa taaacagctg attttactgt gggcagtaca 20700
cctagctaag aaattagctc ctttaatttt tacattaatc ctatgaagtg gtgaataact
20760 acccattttg ttgatgagtg acctgatatt cagagaggtg acttgctatg
gttcctacag 20820 ctggtaagtg gggcatctga agtttgagcg gggacttggg
gtcttgattg ctacatggta 20880 ttgtccccca gccatttgtt ggtagtatgt
taaaaagctt tagggttttg cacatttgtg 20940 ttcagaacct ttattggatt
ccccttgaca tgttttttag ttgattctct tgggtttgcc 21000 tggggtcatc
agcagagaga ttagtcaaat gcgttgtgac atgtacacgt tatctctaca 21060
gatagtatgt gaagaaaata agattgtgaa ttaccaggtt tgttttaaat tttgctctgc
21120 catcttacat gctagtggtg gatgataaac aaccaaatag tgcattaaat
atatacagca 21180 gtgacgagat gtgccctgac atcagaaata tacaatctgg
ggtgtgtttc tctgtggatg 21240 aggacatgca ataaagcagc ttggagtgag
ccggcctctc ccgggggctg agatcctggg 21300 ggaagaaggg ctttttgagt
ttgacctgac accctgcgag cagcttttga accagctgaa 21360 gctaatggga
aggtgctatt gccaccttgc ctccgcctcc cgactccttt ttcccccaga 21420
aggtaatgtc ttagcaccgg ggcttctctc tgcaaaatgg gtgcagccct ctcagtgttc
21480 gtggctcctc ccagagaatg aaggaggcca gagcgggtca gcactctctc
tgccttggag 21540 cagagcttct gaaatggact gcacagcaga atagcccaag
aagtttgtca gaatccagac 21600 ttccagagcc ctgcctaaaa ccaagtcaga
aaccccgagt gacacctggg agtctgcgtt 21660 aactggctcc ctgaatgaag
cacctgcagc ccgccctgca ccaggtgtct ttgaggacat 21720 gagctgagga
aaccccgacc acttgcaaag ggggaaaagt ccgatggcag ctggacctag 21780
aaagagtctc atatggccca gtgcctgtcc tggtattttc aacagaggct gtggccacag
21840 tcaatctgca tggtcagatt cattgttagg actaaatgct ttaagcctcc
tataaacttt 21900 tttttttttt ttttgatgcc cagcctttgt gtaagtctac
ttgaaagggt ttcagggttc 21960 catggatact tctttgctat aaagaggatg
acacatgtaa aatcaccttt atggttaaat 22020 taattggctt ttatattagc
tcctcaaagc aaagcaggag agacagaaat ttctgcagtt 22080 gcttcttggt
cctgtccaaa gcagacatca gcctctgaac catcagcagt cttcctagtg 22140
gcagtgactc tcttcctctt ctcttctgca gccccgacac gtctctgcta ctggactgtg
22200 gtgagggcac rtttgggcag ctgtgccgtc attacggaga ccaggtggac
agggtcctgg 22260 gcaccctggc tgctgtgttt gtgtcccacc tgcacgcaga
tcaccacacg gtgagtgttg 22320 ggctggacca caaagctgga gcctggagga
ggcactgcca cgttgagttg gccctttggc 22380 tgcgtctttt cctccgcttc
caaacttgcc cagagctttt gttactcatc tctggctagg 22440 aaatggtttt
ttgcaaaact caacatagtc cttctgcgcc acaagaatgt cttctcttcc 22500
tgttcagttc ctttcctgca gcaggacagg tttgagttta cccagccttc cttgagtctt
22560 gaatctcaca cggcctgctc agcggaagct ttgaccggat gcaggaggtg
tggctatgag 22620 accctcacct tggtctcctg gggtgccggg ccctgggccg
ttgccctctt cccagcacgg 22680 gtcgtgtcgc tttctgcctg tgacatttca
gggccatggc gcagggggct cggcctgtgc 22740 cacccccact gcggctgtgt
tagaggctgg tgggtgacgt cgggctggca actcctgcaa 22800 gagagagggc
tgcagaccct aacccggagg ggatggccct ggggcctggc tgacgcatgt 22860
ctcctgtttc cttgccaggg cttgccaagt atcttgctgc agagagaacg cgccttggta
22920 agtgtggcac ttgatgggcg ttctgagttt cagcggttta cacatcatcc
gccatgcctc 22980 ttggcactcc agtttttatt gagatgttct gtcgtcgagt
cggcacttgc attttttgtt 23040 ccaggcatct ttgggaaagc cgcttcaccc
tttgctggtg gttgccccca accagctcaa 23100 agcctggctc cagcagtacc
acaaccagtg ccaggaggtc ctgcaccaca tcaggtgagc 23160 atccagggca
gcctggcccg stgggctgtt gcttgctgcc gtctccttca gaagctcaag 23220
gtggacactg gggtagttac caatatcccc cagcagcctt gcccttgaca tggtcccaga
23280 tggcagaagc aggggagaag tgcattggct gaaggacaga aaccattaga
tagttcccat 23340 gtaatgctta ttttcttaga agcatttctt cccagtcctc
atttgagttc tgagctgctt 23400 tctaaacttc gagcagcttt tcttgatgag
acagttccag agccaagcac ccaaatagtg 23460 gctagcacag agaatgtcca
tagcaggtgt gtggctagct ggcaggtggc accatcctca 23520 ccccaagggg
aaggagtccc ctctgctgga gccatccgtg gcccgtgctg cctgagccgg 23580
aggcagcatt cacctgctgg gtttctccca gtggcctaga ggctttggtt
tggctcttta 23640 tatttgactg ctgtttcctc atcatagtga ctatgattta
actcatgttt tctcctaaga 23700 atgattttgg ggttctccag ccaaagactt
aaactttggt tccagatgtc caagaaacgt 23760 ttattatcat tttaaatgtt
ttgtcttttt acagtatgat tcctgccaaa tgccttcagg 23820 aaggggctga
gatctccagt cctgcagtgg aaagattgat cagttcgctg ttgcgaacrt 23880
gtgatttgga agaggtaagg ggcacagccg caggcatcat gggggcgagg tggggagcag
23940 agctgcagag ccctccagcc ccaccctttc agtttcagac ctgtctggtg
cggcactgca 24000 agcatgcgtt tggctgtgcg ctggtgcaca cctctggctg
gaaagtggtc tattccgggg 24060 acaccatgcc ctgcgaggct ctggtccgga
tgggtgagta gaggaagaag caagccaccc 24120 tgaggttgct ctggggtttg
tgtagctgga ggtgaatgca ggtgggcttg cagggaaacg 24180 tcagcagagg
caggagactc aggtccccac cctcagagtc tctggttgtc atcctagtag 24240
gcagacccag ggccagggga gctgagtgtt gagaccagga aacagcacgt gactgaggcc
24300 tgtgtgccgc tctcgcagag aactctgccc tgatccttgt gctgcttctc
cagggaaaga 24360 tgccaccctc ctgatacatg aagccaccct ggaagatggt
ttggaagagg aagcagtgga 24420 aaagacacac aggtagcaaa ggccggtcag
tccttgtcgc ccacatcctc tccctccccc 24480 actacgtgac actgagcagc
cgtcgtttgt ctccactgat gtggggctgc cctgcttcct 24540 atcaagggct
atgggggctt ccttgacctg tggcagtgct cacaggctct tggcctttat 24600
ttttgcagaa ttttctaagc aagattctag agtgaggcac agttttttga aagcatctag
24660 aaatcggctg aataaactat aagccatgtc agggaattgc caggggaagg
cgggggctgg 24720 gggactgaat ttttggctgc taatttcaac gaaagagtgc
attaccccag gtgggccctg 24780 tggtttctct tgggtgccct catggacaga
tttggcagcc agcacagagg gtgggcttca 24840 tccaggggtg tgtgcgaagg
ctctggccct caggggagat tgtgctggct acggaggtgc 24900 ccgttaagaa
aacccaccag cttccccggg tgccctggca gttgatggcc agggtctgtg 24960
ccactgtctg ctttgcagtc ttgcagttga gttcagcttc agtctgctct gtccttcacc
25020 tgcagcacaa cgtcccaagc catcagcgtg gggatgcgga tgaacgcgga
gttcattatg 25080 ctgaaccact tcagccagcg ctatgccaag gtccccctct
tcagccccaa cttcagcgag 25140 aaagtgggag ttgcctttga ccacatgaag
gtctgtatgt cacacggaca gcacagggcg 25200 gggacggggc agggagacag
gactctacac actgagtagg acggtcagct ggagtttgct 25260 ttcttatttg
gggccaccgt gggaaaaggt tatctaccca tcactaacca ggtcgaacca 25320
ccctgggttt gctggtgaga cccacctcct gcaggggcca actagtcttc agtctcagtt
25380 cactggaaat ttctgagaat ccttttaggc ctggactgct cacacagtca
tggcatttga 25440 gcctcagcac agacctgtga gacaggtggt tgcctcttgt
gagtgggaaa gccaggcctg 25500 acccttggcc ttccggaatg aaggggcaga
gccggagcca ggcctcgttt ttcaggagct 25560 tgattttgag agcatctgga
ctgctctccc ttccctctcc ggaggccctt agccaggcct 25620 ggggagcctc
tgccccttta gagggttccc tccatgccat tcttttttcc atttcagctg 25680
tggcctgttg gcttgtgcca aggaaggggc gttggcgctg ctgtgtgagc acatgactgc
25740 atcccttcca gctcctgtcc cccacccctg cccctctgag acatgtcctt
gtcttctatt 25800 gtgtcttcta ggtctgcttt ggagactttc caacaatgcc
caagctgatt cccccactga 25860 aagccctgtt tgctggcgac atcgaggaga
tggaggagcg cagggagaag cgggagctgc 25920 ggcaggtgcg ggcggccctc
ctgtccaggg agctggcagg cggcctggag gatggggagc 25980 ctcagcagaa
gcgggcccac acagaggagc cacaggccaa gaaggtcaga gcccagtgaa 26040
gatctgggag accctgaact cagaaggctg tgtgtcttct gccccacgca cgcacccgta
26100 tctgccctcc ttgctggtag aagctgaaga gcacggtccc ccaggaggca
gctcaggata 26160 ggtggtatgg agctgtgccg aggcttgggc tcccacataa
gcactagtct atagatgcct 26220 cttaggactg gtgcctggca cagccgcggg
ccaggaggct gccacacgga agcaagcaga 26280 tgaactaatt tcatttcaag
gcagttttta aagaagtctt ggaaacagac ggcggcacct 26340 ttcctctaat
ccagcaaagt gattccctgc acaccagaga caagcagagt aacaggatca 26400
gtgggtctaa gtgtccgaga cttaacgaaa atagtatttc agctgcaata aagattgagt
26460 ttgcaattgt gagttctttt gcttcctcct gctgctgcta cagagcaggg
tctgctgtgc 26520 accaccttgg agaaggctct ctgtgctgta gtgtggcagc
tgcctggtac ccgggtggct 26580 tggaagaagt cagctcccgt cgtagtgagc
acctctggaa cctgtcctca gagagccacc 26640 cttattcgcc aagtcttttt gaca
26664 29 31 DNA Homo sapiens 29 caggaattca gcacatactc attgttcagn n
31 30 21 DNA Homo sapiens 30 caggaattca gcacatactc a 21 31 22 DNA
Homo sapiens 31 ttcagcacat actcattgtt ca 22 32 19 DNA Homo sapiens
32 tgaacgcctt ctccacagt 19 33 17 DNA Homo sapiens 33 gtacccgctg
ccaccac 17 34 33 DNA Homo sapiens 34 gctaggatcc gccaccatgt
gggcgctttg ctc 33 35 29 DNA Homo sapiens 35 gctactcgag tcactgggct
ctgaccttc 29 36 17 DNA Homo sapiens 36 gtaaaacgac ggccagt 17 37 19
DNA Homo sapiens 37 ggaaacagct atgaccatg 19 38 17 DNA Homo sapiens
38 tgcgcacgcg agagaag 17 39 17 DNA Homo sapiens 39 cgcttctctc
gcgtgcg 17 40 18 DNA Homo sapiens 40 tctaatgttg ggggctta 18 41 18
DNA Homo sapiens 41 taagccccca acattaga 18 42 18 DNA Homo sapiens
42 tgaaaatgag ccacacct 18 43 18 DNA Homo sapiens 43 aggtgtggct
cattttca 18 44 18 DNA Homo sapiens 44 cattcaaccc atctgtga 18 45 18
DNA Homo sapiens 45 tcacagatgg gttgaatg 18 46 18 DNA Homo sapiens
46 tgaatgcctc ctcaagta 18 47 18 DNA Homo sapiens 47 tacttgagga
ggcattca 18 48 18 DNA Homo sapiens 48 gctactggac tgtggtga 18 49 18
DNA Homo sapiens 49 tcaccacagt ccagtagc 18 50 19 DNA Homo sapiens
50 tggaagagtt tcagacctg 19 51 19 DNA Homo sapiens 51 caggtctgaa
actcttcca 19 52 17 DNA Homo sapiens 52 cgcagggacg caccata 17 53 18
DNA Homo sapiens 53 ggttgaactc ggagaaga 18 54 19 DNA Homo sapiens
54 caactggaaa aatacctcg 19 55 17 DNA Homo sapiens 55 gcagagtcca
gaaaggc 17 56 19 DNA Homo sapiens 56 agaggaaact tcttggtgc 19 57 18
DNA Homo sapiens 57 accaaggaaa ggcagatg 18 58 18 DNA Homo sapiens
58 gtcaacataa gccccgac 18 59 18 DNA Homo sapiens 59 ggctgctgtg
tttgtgtc 18 60 17 DNA Homo sapiens 60 gaaggcattt ggcagga 17 61 18
DNA Homo sapiens 61 tatgattcct gccaaatg 18 62 17 DNA Homo sapiens
62 tccagccaga ggtgtgc 17 63 17 DNA Homo sapiens 63 tgcgaggctc
tggtccg 17 64 18 DNA Homo sapiens 64 gggcattgtt ggaaagtc 18 65 17
DNA Homo sapiens 65 tgtttgctgg cgacatc 17 66 31 DNA Homo sapiens 66
caggaattca gcacatactc attgttcagn n 31 67 21 DNA Homo sapiens 67
caggaattca gcacatactc a 21 68 22 DNA Homo sapiens 68 ttcagcacat
actcattgtt ca 22 69 19 DNA Homo sapiens 69 cagaacacat ttgggaagc 19
70 18 DNA Homo sapiens 70 gatgttgtcc aagcgagc 18 71 17 DNA Homo
sapiens 71 tgacacacag cacctga 17 72 17 DNA Homo sapiens 72
gaagatgtca gggtgga 17 73 18 DNA Homo sapiens 73 caggcatacc actacaga
18 74 20 DNA Homo sapiens 74 tatcaacttc taggcaagtg 20 75 19 DNA
Homo sapiens 75 gcaccatgtc gcagggttc 19 76 19 DNA Homo sapiens 76
gaaccctgcg acatggtgc 19 77 19 DNA Homo sapiens 77 tcgcagggtt
cggctcgtc 19 78 19 DNA Homo sapiens 78 aaccctgcga catggtgcg 19 79
19 DNA Homo sapiens 79 aaagacccac tgcgacacc 19 80 19 DNA Homo
sapiens 80 gcaggtgtcg cagtgggtc 19 81 20 DNA Homo sapiens 81
ccgaacaccg tgtacctgca 20 82 19 DNA Homo sapiens 82 caggtacacg
gtgttcggg 19 83 21 DNA Homo sapiens 83 gtcttctcgg aatacaacag g 21
84 20 DNA Homo sapiens 84 ctgttgtatt ccgagaagac 20 85 20 DNA Homo
sapiens 85 aaggcgtcca acgacttatg 20 86 20 DNA Homo sapiens 86
agtcgttgga cgccttctcc 20 87 20 DNA Homo sapiens 87 tccgagtcag
aaagatgttg 20 88 17 DNA Homo sapiens 88 gccttgtcag cctggtg 17 89 17
DNA Homo sapiens 89 aggaagtgag cagagcg 17 90 33 DNA Homo sapiens 90
gctaaagctt gccaccatgt gggcgctccg ctc 33 91 27 DNA Homo sapiens 91
gctactcgag tcacactcgc gctccta 27 92 16 DNA Homo sapiens 92
gccttctccg cagtta 16 93 21 DNA Homo sapiens 93 ccgcctgaga
cgctctagta t 21 94 19 DNA Homo sapiens 94 gctccgaaag tgctgacag 19
95 39 DNA Homo sapiens 95 gttttcccag tcacgacgtt tctattggat
gagcagcct 39 96 38 DNA Homo sapiens 96 aggaaacagc tatgaccatg
cctgcgatat ggtgcgtc 38 97 37 DNA Homo sapiens 97 gttttcccag
tcacgacgct cagttttggt ggagacg 37 98 37 DNA Homo sapiens 98
aggaaacagc tatgaccatg tgccccgatg ctcagag 37 99 22 DNA Homo sapiens
99 aatggtgtca gagagtttac ag 22 100 19 DNA Homo sapiens 100
gctatttggg aggctgagg 19 101 40 DNA Homo sapiens 101 gttttcccag
tcacgacgaa tggtgtcaga gagtttacag 40 102 41 DNA Homo sapiens 102
aggaaacagc tatgaccatg aacaaggacc acttttgcta t 41 103 40 DNA Homo
sapiens 103 gttttcccag tcacgacgtt tatagcaaaa gtggtccttg 40 104 38
DNA Homo sapiens 104 aggaaacagc tatgaccatg agacttccca ccagcctc 38
105 19 DNA Homo sapiens 105 ccttgctgct tcaccctag 19 106 21 DNA Homo
sapiens 106 tgctttatat gtgctgctac g 21 107 39 DNA Homo sapiens 107
gttttcccag tcacgacgca tcttccctgg ttgtacttc 39 108 39 DNA Homo
sapiens 108 aggaaacagc tatgaccatc tggagggcag aagactgat 39 109 23
DNA Homo sapiens 109 ctacatttgt tcaaccataa ctg 23 110 23 DNA Homo
sapiens 110 gattttgagg tttgatgttg atg 23 111 39 DNA Homo sapiens
111 gttttcccag tcacgacgca tttgttcaac cataactgc 39 112 39 DNA Homo
sapiens 112 aggaaacagc tatgaccata tttgagaggt cagggcata 39 113 21
DNA Homo sapiens 113 tcgtgtcaga ttcccaccat a 21 114 21 DNA Homo
sapiens 114 aggcataagt cagacatccg t 21 115 40 DNA Homo sapiens 115
gttttcccag tcacgacggt tactcttccc acacatcttc 40 116 41 DNA Homo
sapiens 116 aggaaacagc tatgaccatc acagcaagtg ttcagtttct a 41 117 21
DNA Homo sapiens 117 cattcccatg tatgaacgtc t 21 118 22 DNA Homo
sapiens 118 atagtaagcc caggaagaag ga 22 119 39 DNA Homo sapiens 119
gttttcccag tcacgacgca ttcccatgta tgaacgtct 39 120 41 DNA Homo
sapiens 120 aggaaacagc tatgaccatc tacaagcatt acaaggcaga g 41 121 22
DNA Homo sapiens 121 agtgtcttca gcctttgtat tg 22 122 23 DNA Homo
sapiens 122 atctgctatc tcttcttgtc tca 23 123 40 DNA Homo sapiens
123 gttttcccag tcacgacgat cgggtcataa tcagtctgtg 40 124 42 DNA Homo
sapiens 124 aggaaacagc tatgaccata tctcttcttg tctcaggtaa ca 42 125
22 DNA Homo sapiens 125 cttctgaaag caataaacgc at 22 126 20 DNA Homo
sapiens 126 gatgtccaaa ctgttccacg 20 127 40 DNA Homo sapiens 127
gttttcccag tcacgacgta aaaccaacct tcttcattag 40 128 38 DNA Homo
sapiens 128 aggaaacagc tatgaccata gcaatgatgg gagcgatg 38 129 37 DNA
Homo sapiens 129 gttttcccag tcacgacggg cttctgggga ctcactg 37 130 41
DNA Homo sapiens 130 aggaaacagc tatgaccatc cttcaaaagt ggtgtctgta g
41 131 22 DNA Homo sapiens 131 gtatccacaa agagaccaga ag 22 132 23
DNA Homo sapiens 132 caccaactac caacagtgac tta 23 133 41 DNA Homo
sapiens 133 gttttcccag tcacgacggc tcactggata ggatatgtca t 41 134 39
DNA Homo sapiens 134 aggaaacagc tatgaccatc cagaaacaca gctcttgcc 39
135 20 DNA Homo sapiens 135 gcttgccaga tacaggaatc 20 136 20 DNA
Homo sapiens 136 acagaaagtt taggcaggtg 20 137 37 DNA Homo sapiens
137 gttttcccag tcacgacgac gatacccctc cctggct 37 138 39 DNA Homo
sapiens 138 aggaaacagc tatgaccata cagaaagttt aggcaggtg 39 139 20
DNA Homo sapiens 139 cctctcactc ttcccagcac 20 140 22 DNA Homo
sapiens 140 ggagtaggct gcttttctaa at 22 141 40 DNA Homo sapiens 141
gttttcccag tcacgacgga acacctcatc ctcattacca 40 142 41 DNA Homo
sapiens 142 aggaaacagc tatgaccata agagacaaaa cacattcatg g 41 143 39
DNA Homo sapiens 143 gttttcccag tcacgacggt ttccgctgta aggtagtgt 39
144 42 DNA Homo sapiens 144 aggaaacagc tatgaccatc tggaacattt
actatgtggc ta 42 145 20 DNA Homo sapiens 145 tgctagtggg tagaggtcag
20 146 21 DNA Homo sapiens 146 actgaaagcc aggttagaat g 21 147 37
DNA Homo sapiens 147 gttttcccag tcacgacgac cctgtccgtc acctgag 37
148 38 DNA Homo sapiens 148 aggaaacagc tatgaccatc ccaccagcac
tccactta 38 149 21 DNA Homo sapiens 149 tgtgaagacg ggataacctg a 21
150 19 DNA Homo sapiens 150 gacagggctt gataccgca 19 151 38 DNA Homo
sapiens 151 gttttcccag tcacgacgat gctggctcac ttttgacc 38 152 39 DNA
Homo sapiens 152 aggaaacagc tatgaccatg actggtgagt acagcagga 39 153
21 DNA Homo sapiens 153 ccagcctttg tgtaagtcta c 21 154 19 DNA Homo
sapiens 154 tctgggcaag tttggaagc 19 155 39 DNA Homo sapiens 155
gttttcccag tcacgacgtc caaagcagac atcagcctc 39 156 38 DNA Homo
sapiens 156 aggaaacagc tatgaccatg gaggaaaaga cgcagcca 38 157 19 DNA
Homo sapiens 157 cgctttctgc ctgtgacat 19 158 20 DNA Homo sapiens
158 ttctgtcctt cagccaatgc 20 159 37 DNA Homo sapiens 159 gttttcccag
tcacgacgtt agaggctggt gggtgac 37 160 42 DNA Homo sapiens 160
aggaaacagc tatgaccatc atctcaataa aaactggagt gc 42 161 20 DNA Homo
sapiens 161 cacttgatgg gcgttctgag 20 162 20 DNA Homo sapiens 162
ttctgtcctt cagccaatgc 20 163 38 DNA Homo sapiens 163 gttttcccag
tcacgacgtt ccagcggttt acacatca 38 164 38 DNA Homo sapiens 164
aggaaacagc tatgaccatt accccagtgt ccaccttg 38 165 20 DNA Homo
sapiens 165 gggttctcca gccaaagact 20 166 19 DNA Homo sapiens 166
ctgagtctcc tgcctctgc 19 167 38 DNA Homo sapiens 167 gttttcccag
tcacgacggg gttctccagc caaagact 38 168 38 DNA Homo sapiens 168
aggaaacagc tatgaccatg tggggctgga aggctctg 38 169 37 DNA Homo
sapiens 169 gttttcccag tcacgacgaa gaggtaaggg gcacagc 37 170 38 DNA
Homo sapiens 170 aggaaacagc tatgaccatc
tgagtctcct gcctctgc 38 171 20 DNA Homo sapiens 171 gctgagtgtt
gagaccagga 20 172 19 DNA Homo sapiens 172 agacaaacga cggctgctc 19
173 38 DNA Homo sapiens 173 gttttcccag tcacgacgtt gagaccagga
aacagcac 38 174 38 DNA Homo sapiens 174 aggaaacagc tatgaccatg
agaggatgtg ggcgacaa 38 175 19 DNA Homo sapiens 175 gggagatggt
gctggctac 19 176 21 DNA Homo sapiens 176 cctggttagt gatgggtaga t 21
177 37 DNA Homo sapiens 177 gttttcccag tcacgacgca gggtctgtgc
cactgtc 37 178 40 DNA Homo sapiens 178 aggaaacagc tatgaccatc
tcagtgtgta gagtcctctc 40 179 22 DNA Homo sapiens 179 ttgattttga
gagcatctgg ac 22 180 21 DNA Homo sapiens 180 ctcggacact tagacccact
g 21 181 37 DNA Homo sapiens 181 gttttcccag tcacgacgtg catcccttcc
agctcct 37 182 41 DNA Homo sapiens 182 aggaaacagc tatgaccatg
acacacagcc ttctgagttc a 41 183 37 DNA Homo sapiens 183 gttttcccag
tcacgacgcc acacagagga gccacag 37 184 42 DNA Homo sapiens 184
aggaaacagc tatgaccata ccagtcctaa gaggcatcta ta 42 185 19 DNA Homo
sapiens 185 ccacacagag gagccacag 19 186 20 DNA Homo sapiens 186
ccagaggtgc tcactacgac 20 187 39 DNA Homo sapiens 187 gttttcccag
tcacgacgag gtcagagccc agtgaagat 39 188 39 DNA Homo sapiens 188
aggaaacagc tatgaccatc atctgcttgc ttccgtgtg 39 189 39 DNA Homo
sapiens 189 gttttcccag tcacgacgtc aggataggtg gtatggagc 39 190 40
DNA Homo sapiens 190 aggaaacagc tatgaccatc ggacacttag acccactgat 40
191 23 DNA Homo sapiens 191 agactccgag tygaatgaaa atg 23 192 23 DNA
Homo sapiens 192 ggtgagggca crtttgggca gct 23 193 23 DNA Homo
sapiens 193 gcaccctggc trctgtgttt gtg 23 194 23 DNA Homo sapiens
194 gtgtcccacc tgcacgcaga tca 23 195 24 DNA Homo sapiens 195
gtgtcccacc tggcacgcag atca 24 196 23 DNA Homo sapiens 196
aagccgcttc aycctttgct ggt 23 197 24 DNA Homo sapiens 197 gctgttgcga
acrtgtgatt tgga 24 198 22 DNA Homo sapiens 198 gaggcttggg
stcccacata ag 22 199 23 DNA Homo sapiens 199 cctggcacag cygcgggcca
gga 23 200 23 DNA Homo sapiens 200 aatccagcaa artgattccc tgc 23 201
21 DNA Homo sapiens 201 taaatgtttt ytcattctta g 21 202 23 DNA Homo
sapiens 202 ttgctgttgt gyggttttct tgt 23 203 24 DNA Homo sapiens
203 ggttttcttg attcagcagt taca 24 204 27 DNA Homo sapiens 204
ggttttcttg atgattcagc agttaca 27 205 23 DNA Homo sapiens 205
gtgtctcaga cyggcccctt gtc 23 206 23 DNA Homo sapiens 206 tgccatcttg
awctaatgga atc 23 207 23 DNA Homo sapiens 207 cttctctctc tycctgcagg
gat 23 208 23 DNA Homo sapiens 208 catcaagggc aygtttactt ttt 23 209
23 DNA Homo sapiens 209 cagccttgcc csctgggctg ttg 23 210 350 DNA
Homo sapiens CDS (51)..(293) 210 cgcgggcgta ggtgaccggc ggctttctca
gttttggtgg agacgggcgc atg tgg 56 Met Trp 1 gcg ctt tgc tcg ctg ctg
cgg tcc gcg gcc gga cgc acc atg tcg cag 104 Ala Leu Cys Ser Leu Leu
Arg Ser Ala Ala Gly Arg Thr Met Ser Gln 5 10 15 gga cgc acc ata tcg
cag gca ccc gcc cgc cgc gag cgg ccg cgc aag 152 Gly Arg Thr Ile Ser
Gln Ala Pro Ala Arg Arg Glu Arg Pro Arg Lys 20 25 30 gac ccg ctg
cgg cac ctg cgc acg cga gag aag cgc gga ccg tcg ggg 200 Asp Pro Leu
Arg His Leu Arg Thr Arg Glu Lys Arg Gly Pro Ser Gly 35 40 45 50 tgc
tcc ggc ggc cca aac acc gtg tac ctg cag gtg gtg gca gcg ggt 248 Cys
Ser Gly Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala Ala Gly 55 60
65 agc cgg gac tcg ggc gcc gcg ctc tac gtc ttc tcc gag ttc aac 293
Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe Asn 70 75
80 cggtcagtca acgagccacg ccccgtcccg ctgggccctc agtgcggcgc agcctct
350 211 81 PRT Homo sapiens 211 Met Trp Ala Leu Cys Ser Leu Leu Arg
Ser Ala Ala Gly Arg Thr Met 1 5 10 15 Ser Gln Gly Arg Thr Ile Ser
Gln Ala Pro Ala Arg Arg Glu Arg Pro 20 25 30 Arg Lys Asp Pro Leu
Arg His Leu Arg Thr Arg Glu Lys Arg Gly Pro 35 40 45 Ser Gly Cys
Ser Gly Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala 50 55 60 Ala
Gly Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe 65 70
75 80 Asn 212 326 DNA Mus musculus CDS (51)..(269) 212 tggcggcgtg
aggggtctgg ctgccttgtc agcctggtgt ggtcgggtgc atg tgg 56 Met Trp 1
gcg ctc cgc tca ctg ttg cgt ccc ctt ggc ctg cgc acc atg tcg cag 104
Ala Leu Arg Ser Leu Leu Arg Pro Leu Gly Leu Arg Thr Met Ser Gln 5
10 15 ggt tcg gct cgt cgg ccg cgg cca ccc aag gac cca ctg cga cac
ctg 152 Gly Ser Ala Arg Arg Pro Arg Pro Pro Lys Asp Pro Leu Arg His
Leu 20 25 30 cgt acg cgg gag aag cgc ggc ccg ggt ccc ggg ggc ccg
aac acc gtg 200 Arg Thr Arg Glu Lys Arg Gly Pro Gly Pro Gly Gly Pro
Asn Thr Val 35 40 45 50 tac ctg cag gtg gtg gcg gcg ggc ggc cgg gac
gcg ggg gct gct ctc 248 Tyr Leu Gln Val Val Ala Ala Gly Gly Arg Asp
Ala Gly Ala Ala Leu 55 60 65 tat gtc ttc tcg gaa tac aac aggtcagagt
gggccgacag ccctggggga 299 Tyr Val Phe Ser Glu Tyr Asn 70 ttggccccag
cgccacgtgc tcgggag 326 213 73 PRT Mus musculus 213 Met Trp Ala Leu
Arg Ser Leu Leu Arg Pro Leu Gly Leu Arg Thr Met 1 5 10 15 Ser Gln
Gly Ser Ala Arg Arg Pro Arg Pro Pro Lys Asp Pro Leu Arg 20 25 30
His Leu Arg Thr Arg Glu Lys Arg Gly Pro Gly Pro Gly Gly Pro Asn 35
40 45 Thr Val Tyr Leu Gln Val Val Ala Ala Gly Gly Arg Asp Ala Gly
Ala 50 55 60 Ala Leu Tyr Val Phe Ser Glu Tyr Asn 65 70 214 13 PRT
Artificial Sequence Description of Artificial SequenceHistidine
containing motif. 214 Xaa Xaa Xaa Xaa His Xaa His Xaa Asp His Xaa
Xaa Gly 1 5 10 215 127 DNA Homo sapiens misc_feature (1)..(127)
Exon 1. 215 tttaatacga ctcactatag ggaatttggc cctcgagnng aattcggcac
gagggtagcc 60 ccgcgacagc tgggccgagg gtgcgggcct gcgctccctc
ggctcctggc gcgggctcgg 120 ggagagg 127 216 983 DNA Homo sapiens
intron (1)..(300) Upstream intron of exon 2. 216 gtctccatag
ttttgccttt ttgagaacat catatagtta gaattcagct atagttttta 60
attgcctggg tttggttatt tttgtttgtt tgggtgtgtg aacaattata caagatttgt
120 taacttgtag ttttagccaa gttattaaaa ccttactgtg gatatgtgtg
gaatactatg 180 agagaccaag aatccagact gttctaaata accaaaaagt
aataatagag ataaatatta 240 caggaatatg tttttggtcc agtgatatga
aataatcccc agatgatctt tctgttgcag 300 ggtggaagat gtctatggat
gtgacattcc tggggacggg tgcagcatac ccatctccaa 360 cccggggtgc
ctctgctgtg gtccttcggt gtgaaggcga gtgctggctc tttgactgtg 420
gggagggaac acagacacag cttatgaaaa gccaacttaa agcaggttag tgtgccttca
480 gctatctcat taagaatttt ttgttgttct gcttcatttt cttggctctc
cttggacatt 540 ttgtttagaa acagccctga tggttgcatc ccacttcagt
gctacaccct ggtgagactt 600 ggaaggcctg caggcatctg gccacgtcca
ctgaacttca tttacttatt tacttgcttt 660 tcatttatcc tgtagatgct
gaaagcaagg attcatgtag gcttggggtt tgggaaatgt 720 cgtgggatac
accaggcata ttagatgaac actgccttag caaggaagca gtgtacatac 780
ttacctccac caggagatag ttttcatgag aggatgcaaa gggtaggaaa tgtttggagg
840 aggagatgtt gttttcctct tggggttatc aggtaaactt ctcagagaag
ttgacctgtg 900 gattgtcaaa gagagagatt tcaggctgag agaagaaggc
atttcatcag gggatggagt 960 gagcagagcc acacctggga gat 983 217 1287
DNA Homo sapiens intron (1)..(300) Intron upstream of exon 3. 217
gtgagctatg atcacaccac tgcactccag cctggatgac agagcaagac ctgtctcttt
60 aaaaaaaaaa aaactattaa aaacaaacaa acaaaaaacc acctggtgaa
ataaagcctg 120 tcttcttgtt tttggaatca tgtagcaaaa tgtaaatgaa
taagtttatg atgataagta 180 gaacttttaa attcaattta ctatttttaa
tgtaaattgt taggcttgtt tcaaatagct 240 ttgtatgggt ttttagttaa
tgaaaaattt ccaaacgtat ttctctatct caatcaaaag 300 ggagaattac
caagatcttc atcacacacc ttcatggaga ccatttcttt ggccttcctg 360
ggctcctctg cacaatcagc ctgcagagtg gctccatggt gtccaaacag cctattgaaa
420 tctatggccc tgtagggctt cgggacttta tctggcgaac catggaactc
tctcacacgg 480 agctggtctt ccattatgtg gttcatgaac tggttcctac
agcagatcaa tgtcctgcag 540 aagaactaaa agaatttgcg catgtgaata
gagcagacag tcctcccaaa gaggaacaag 600 gaagaactat cctgttagac
tcagaagaaa actcatacct tctgtttgat gatgaacaat 660 ttgttgtaaa
agcatttcgc ctctttcaca gaattccctc atttgggttt tcagtcgtgg 720
aaaagaaacg cccaggtaaa ctcaatgcac agaaacttaa agaccttggt aagtgttttt
780 ttgttttttg ttttttcccg ccttctcatc aatagggctc ctgttgactg
aagctataag 840 aaatgtcata gtaaggccag gagttgtggc tcacgcctgt
aatcctagca ctttgggagg 900 ccgaggtggg aggatcactt gagttcggga
gttcaagacc agcctgggca acatggcgaa 960 accccatctc tactaaaaat
acaaaaagta actgggtgtg gtgtcatgtg cctgtagtcc 1020 cagctacttg
gggggctgag gcaggaggat cacttgaacc tgggaggtca aggctgcagt 1080
aagccaagat agtgttacta tactccagct tgggtgacaa agcgaaactc tgtctcaaaa
1140 aaaaaaaagt gtcatagtaa gcttccactc ctctatccca ggcctgaaac
tgacaatttc 1200 tcacttagtc ctttgtccaa agttgcttat taagaaatcc
atggggccaa aaaaatgcta 1260 tttagagcaa acccagtata catttga 1287 218
1378 DNA Homo sapiens intron (1)..(300) Intron upstream of exon 4.
218 tgtgcacagc agagaatcaa gaatgttaca gtgactacaa taaggtccta
gtgatactta 60 ggagactaaa acttgtctga catgtatgca tgggaaatgt
ttcaagtact aaggcattgc 120 taatatcaat caacactgaa attttaaaaa
tgtataaatc cagttttcca caagtagtaa 180 aacatttata acaattatgg
atgccttttc cattagctat ttgcaatgct gttaaaatag 240 actcttgaaa
agtcataaat tccattccta tgatgtaatg ttatctgcct tcatcattag 300
gtgttccacc aggtcctgcc tatgggaagc tgaaaaatgg aatttctgtt gttctggaaa
360 atggggttac aatttctccc caagatgtct taaaaaagcc tattgttgga
agaaaaatct 420 gcatattggg tgactgctct ggggttgtgg gtgatggagg
agtaaaactg tgctttgaag 480 cagacctgtt gatccacgaa gcaaccctgg
atgatgccca gatggacaaa gcaaaggagc 540 atggccacag cacaccacag
atggcagcaa catttgcaaa gttgtgccgt gcaaagaggc 600 tggttctgac
tcacttcagt cagaggtaca aaccagttgc cttggccaga gaaggagaaa 660
cagatggcat tgcagaacta aaaaagcaag ctgaatcagt gttagatctc caagaagtga
720 ctctagcaga agattttatg gtgataagca ttccaatcaa gaaatgaaac
cagtgttcct 780 gagtgcacac tgacatgtct gtgaatatgt tactgaacct
atagtccagt ttttttattt 840 cttgttttag tctgaaatta tttgggccct
aataatccta aaaagaatgg agctgcattg 900 atgaattggc tcagtattta
aagggagcaa actttttgat aataaatctt tttaagagaa 960 aaaaaaccca
gcatcctttt tgaagtccag atttgtcaaa atgatagact attcagttat 1020
acatcttatt ttgtgctact accacagata gccaatattc catgcagtcc tgggcttagc
1080 ttctgcccag ctttattgct gctattggca aagagcacag gactcagccc
tcgtggctaa 1140 aaatggtatt ttggcagttt gtattgaatc tgtttgtgtt
attaacagaa gagggagaaa 1200 tgtcatgaga cgttggacag gcaggattga
tgatagcatg accatagctt tgctggaata 1260 ctgaatgcag ggtttggcta
ggtgtttatt ttaacatttt attaaacttt ctatttgggt 1320 cttaacccat
ggttctcaac tggggtgaca ctgctcctct agaacaggtt gaaatatg 1378 219 1462
DNA Homo sapiens CDS (136)..(1224) 219 tttaatacga ctcactatag
ggaatttggc cctcgagnng aattcggcac gagggtagcc 60 ccgcgacagc
tgggccgagg gtgcgggcct gcgctccctc ggctcctggc gcgggctcgg 120
ggagaggggt ggaag atg tct atg gat gtg aca ttc ctg ggg acg ggt gca
171 Met Ser Met Asp Val Thr Phe Leu Gly Thr Gly Ala 1 5 10 gca tac
cca tct cca acc cgg ggt gcc tct gct gtg gtc ctt cgg tgt 219 Ala Tyr
Pro Ser Pro Thr Arg Gly Ala Ser Ala Val Val Leu Arg Cys 15 20 25
gaa ggc gag tgc tgg ctc ttt gac tgt ggg gag gga aca cag aca cag 267
Glu Gly Glu Cys Trp Leu Phe Asp Cys Gly Glu Gly Thr Gln Thr Gln 30
35 40 ctt atg aaa agc caa ctt aaa gca ggg aga att acc aag atc ttc
atc 315 Leu Met Lys Ser Gln Leu Lys Ala Gly Arg Ile Thr Lys Ile Phe
Ile 45 50 55 60 aca cac ctt cat gga gac cat ttc ttt ggc ctt cct ggg
ctc ctc tgc 363 Thr His Leu His Gly Asp His Phe Phe Gly Leu Pro Gly
Leu Leu Cys 65 70 75 aca atc agc ctg cag agt ggc tcc atg gtg tcc
aaa cag cct att gaa 411 Thr Ile Ser Leu Gln Ser Gly Ser Met Val Ser
Lys Gln Pro Ile Glu 80 85 90 atc tat ggc cct gta ggg ctt cgg gac
ttt atc tgg cga acc atg gaa 459 Ile Tyr Gly Pro Val Gly Leu Arg Asp
Phe Ile Trp Arg Thr Met Glu 95 100 105 ctc tct cac acg gag ctg gtc
ttc cat tat gtg gtt cat gaa ctg gtt 507 Leu Ser His Thr Glu Leu Val
Phe His Tyr Val Val His Glu Leu Val 110 115 120 cct aca gca gat caa
tgt cct gca gaa gaa cta aaa gaa ttt gcg cat 555 Pro Thr Ala Asp Gln
Cys Pro Ala Glu Glu Leu Lys Glu Phe Ala His 125 130 135 140 gtg aat
aga gca gac agt cct ccc aaa gag gaa caa gga aga act atc 603 Val Asn
Arg Ala Asp Ser Pro Pro Lys Glu Glu Gln Gly Arg Thr Ile 145 150 155
ctg tta gac tca gaa gaa aac tca tac ctt ctg ttt gat gat gaa caa 651
Leu Leu Asp Ser Glu Glu Asn Ser Tyr Leu Leu Phe Asp Asp Glu Gln 160
165 170 ttt gtt gta aaa gca ttt cgc ctc ttt cac aga att ccc tca ttt
ggg 699 Phe Val Val Lys Ala Phe Arg Leu Phe His Arg Ile Pro Ser Phe
Gly 175 180 185 ttt tca gtc gtg gaa aag aaa cgc cca ggt aaa ctc aat
gca cag aaa 747 Phe Ser Val Val Glu Lys Lys Arg Pro Gly Lys Leu Asn
Ala Gln Lys 190 195 200 ctt aaa gac ctt ggt gtt cca cca ggt cct gcc
tat ggg aag ctg aaa 795 Leu Lys Asp Leu Gly Val Pro Pro Gly Pro Ala
Tyr Gly Lys Leu Lys 205 210 215 220 aat gga att tct gtt gtt ctg gaa
aat ggg gtt aca att tct ccc caa 843 Asn Gly Ile Ser Val Val Leu Glu
Asn Gly Val Thr Ile Ser Pro Gln 225 230 235 gat gtc tta aaa aag cct
att gtt gga aga aaa atc tgc ata ttg ggt 891 Asp Val Leu Lys Lys Pro
Ile Val Gly Arg Lys Ile Cys Ile Leu Gly 240 245 250 gac tgc tct ggg
gtt gtg ggt gat gga gga gta aaa ctg tgc ttt gaa 939 Asp Cys Ser Gly
Val Val Gly Asp Gly Gly Val Lys Leu Cys Phe Glu 255 260 265 gca gac
ctg ttg atc cac gaa gca acc ctg gat gat gcc cag atg gac 987 Ala Asp
Leu Leu Ile His Glu Ala Thr Leu Asp Asp Ala Gln Met Asp 270 275 280
aaa gca aag gag cat ggc cac agc aca cca cag atg gca gca aca ttt
1035 Lys Ala Lys Glu His Gly His Ser Thr Pro Gln Met Ala Ala Thr
Phe 285 290 295 300 gca aag ttg tgc cgt gca aag agg ctg gtt ctg act
cac ttc agt cag 1083 Ala Lys Leu Cys Arg Ala Lys Arg Leu Val Leu
Thr His Phe Ser Gln 305 310 315 agg tac aaa cca gtt gcc ttg gcc aga
gaa gga gaa aca gat ggc att 1131 Arg Tyr Lys Pro Val Ala Leu Ala
Arg Glu Gly Glu Thr Asp Gly Ile 320 325 330 gca gaa cta aaa aag caa
gct gaa tca gtg tta gat ctc caa gaa gtg 1179 Ala Glu Leu Lys Lys
Gln Ala Glu Ser Val Leu Asp Leu Gln Glu Val 335 340 345 act cta gca
gaa gat ttt atg gtg ata agc att cca atc aag aaa 1224 Thr Leu Ala
Glu Asp Phe Met Val Ile Ser Ile Pro Ile Lys Lys 350 355 360
tgaaaccagt gttcctgagt gcacactgac atgtctgtga atatgttact gaacctatag
1284 tccagttttt ttatttcttg ttttagtctg aaattatttg ggccctaata
atcctaaaaa 1344 gaatggagct gcattgatga attggctcag tatttaaagg
gagcaaactt tttgataata 1404 aatcttttta agagaaaaaa aaaaaaaaga
aaaaagatct ataattaagc aggggcat 1462 220 363 PRT Homo sapiens 220
Met Ser Met Asp Val Thr Phe Leu Gly Thr Gly Ala Ala Tyr Pro Ser 1 5
10 15 Pro Thr Arg Gly Ala Ser Ala Val Val Leu Arg Cys Glu Gly Glu
Cys 20 25 30 Trp Leu Phe Asp Cys Gly Glu Gly Thr Gln Thr Gln Leu
Met Lys Ser 35 40 45 Gln Leu Lys Ala Gly
Arg Ile Thr Lys Ile Phe Ile Thr His Leu His 50 55 60 Gly Asp His
Phe Phe Gly Leu Pro Gly Leu Leu Cys Thr Ile Ser Leu 65 70 75 80 Gln
Ser Gly Ser Met Val Ser Lys Gln Pro Ile Glu Ile Tyr Gly Pro 85 90
95 Val Gly Leu Arg Asp Phe Ile Trp Arg Thr Met Glu Leu Ser His Thr
100 105 110 Glu Leu Val Phe His Tyr Val Val His Glu Leu Val Pro Thr
Ala Asp 115 120 125 Gln Cys Pro Ala Glu Glu Leu Lys Glu Phe Ala His
Val Asn Arg Ala 130 135 140 Asp Ser Pro Pro Lys Glu Glu Gln Gly Arg
Thr Ile Leu Leu Asp Ser 145 150 155 160 Glu Glu Asn Ser Tyr Leu Leu
Phe Asp Asp Glu Gln Phe Val Val Lys 165 170 175 Ala Phe Arg Leu Phe
His Arg Ile Pro Ser Phe Gly Phe Ser Val Val 180 185 190 Glu Lys Lys
Arg Pro Gly Lys Leu Asn Ala Gln Lys Leu Lys Asp Leu 195 200 205 Gly
Val Pro Pro Gly Pro Ala Tyr Gly Lys Leu Lys Asn Gly Ile Ser 210 215
220 Val Val Leu Glu Asn Gly Val Thr Ile Ser Pro Gln Asp Val Leu Lys
225 230 235 240 Lys Pro Ile Val Gly Arg Lys Ile Cys Ile Leu Gly Asp
Cys Ser Gly 245 250 255 Val Val Gly Asp Gly Gly Val Lys Leu Cys Phe
Glu Ala Asp Leu Leu 260 265 270 Ile His Glu Ala Thr Leu Asp Asp Ala
Gln Met Asp Lys Ala Lys Glu 275 280 285 His Gly His Ser Thr Pro Gln
Met Ala Ala Thr Phe Ala Lys Leu Cys 290 295 300 Arg Ala Lys Arg Leu
Val Leu Thr His Phe Ser Gln Arg Tyr Lys Pro 305 310 315 320 Val Ala
Leu Ala Arg Glu Gly Glu Thr Asp Gly Ile Ala Glu Leu Lys 325 330 335
Lys Gln Ala Glu Ser Val Leu Asp Leu Gln Glu Val Thr Leu Ala Glu 340
345 350 Asp Phe Met Val Ile Ser Ile Pro Ile Lys Lys 355 360 221
2470 DNA Mus musculus CDS (1)..(2466) 221 atg tgg gcg ctc cgc tca
ctg ttg cgt ccc ctt ggc ctg cgc acc atg 48 Met Trp Ala Leu Arg Ser
Leu Leu Arg Pro Leu Gly Leu Arg Thr Met 1 5 10 15 tcg cag ggt tcg
gct cgt cgg ccg cgg cca ccc aaa gac cca ctg cga 96 Ser Gln Gly Ser
Ala Arg Arg Pro Arg Pro Pro Lys Asp Pro Leu Arg 20 25 30 cac ctg
cgt acg cgg gag aag cgc ggc ccg ggt ccc ggg ggc ccg aac 144 His Leu
Arg Thr Arg Glu Lys Arg Gly Pro Gly Pro Gly Gly Pro Asn 35 40 45
acc gtg tac ctg cag gtg gtg gcg gcg ggc ggc cgg gac gcg ggg gct 192
Thr Val Tyr Leu Gln Val Val Ala Ala Gly Gly Arg Asp Ala Gly Ala 50
55 60 gct ctc tat gtc ttc tcg gaa tac aac agg tac ctt ttt aac tgc
gga 240 Ala Leu Tyr Val Phe Ser Glu Tyr Asn Arg Tyr Leu Phe Asn Cys
Gly 65 70 75 80 gaa ggc gtc caa cga ctt atg cag gaa cac aag act gaa
agt cgc tcg 288 Glu Gly Val Gln Arg Leu Met Gln Glu His Lys Thr Glu
Ser Arg Ser 85 90 95 ctt gac aac atc ttt ctg act cgg atg cat tgg
tca aat gtt ggg ggg 336 Leu Asp Asn Ile Phe Leu Thr Arg Met His Trp
Ser Asn Val Gly Gly 100 105 110 ttg tgt gga atg att tta act tta aag
gaa acc ggg ctt ccc aaa tgt 384 Leu Cys Gly Met Ile Leu Thr Leu Lys
Glu Thr Gly Leu Pro Lys Cys 115 120 125 gtt ctg tct gga cca cca cag
ctg gag aaa tat cta gaa gca atc aaa 432 Val Leu Ser Gly Pro Pro Gln
Leu Glu Lys Tyr Leu Glu Ala Ile Lys 130 135 140 ata ttt tct ggt cca
ttg aaa gga ata gaa ctg gcc gtg cgg cct cac 480 Ile Phe Ser Gly Pro
Leu Lys Gly Ile Glu Leu Ala Val Arg Pro His 145 150 155 160 tct gca
cca gaa tac aag gat gag acc atg act gtt tac cag gtc cct 528 Ser Ala
Pro Glu Tyr Lys Asp Glu Thr Met Thr Val Tyr Gln Val Pro 165 170 175
atc cac agt gaa cgg agg tgt gga aag caa cag cca tcc cag agc ccc 576
Ile His Ser Glu Arg Arg Cys Gly Lys Gln Gln Pro Ser Gln Ser Pro 180
185 190 aga aca tct ccc aac agg ctc agt ccc aaa cag tca tcg gac tct
gga 624 Arg Thr Ser Pro Asn Arg Leu Ser Pro Lys Gln Ser Ser Asp Ser
Gly 195 200 205 tca gct gaa aat ggg cag tgc caa cag gaa agc atg ggg
cag gga ccc 672 Ser Ala Glu Asn Gly Gln Cys Gln Gln Glu Ser Met Gly
Gln Gly Pro 210 215 220 tcc tta gtg gta gct ttt gtc tgc aag ctt cac
ttg agg aaa gga aac 720 Ser Leu Val Val Ala Phe Val Cys Lys Leu His
Leu Arg Lys Gly Asn 225 230 235 240 ttc ttg gtg ctt aaa gca aag gag
ctg ggc ctt cct gtt ggg acg gcc 768 Phe Leu Val Leu Lys Ala Lys Glu
Leu Gly Leu Pro Val Gly Thr Ala 245 250 255 gcc att gca ccc atc att
gct gct gtc aag gac ggg aag agt atc act 816 Ala Ile Ala Pro Ile Ile
Ala Ala Val Lys Asp Gly Lys Ser Ile Thr 260 265 270 tac gaa gga aga
gag att gct gct gaa gag ctt tgt aca ccc cca gat 864 Tyr Glu Gly Arg
Glu Ile Ala Ala Glu Glu Leu Cys Thr Pro Pro Asp 275 280 285 cct ggt
ctt gta ttc atc gtg gta gag tgt cct gat gaa gga ttc atc 912 Pro Gly
Leu Val Phe Ile Val Val Glu Cys Pro Asp Glu Gly Phe Ile 290 295 300
ctg ccc atc tgt gag aac gac acc ttt aaa agg tac cag gca gag gct 960
Leu Pro Ile Cys Glu Asn Asp Thr Phe Lys Arg Tyr Gln Ala Glu Ala 305
310 315 320 gat gca cct gtg gcg ctg gtg gtc cac ata gcc cca gaa tct
gta ctc 1008 Asp Ala Pro Val Ala Leu Val Val His Ile Ala Pro Glu
Ser Val Leu 325 330 335 atc gac agc aga tac cag cag tgg atg gag agg
ttc ggg cct gac aca 1056 Ile Asp Ser Arg Tyr Gln Gln Trp Met Glu
Arg Phe Gly Pro Asp Thr 340 345 350 cag cac ctg att ctg aat gag aat
tgc ccc tcg gtc cac aac ctg cgc 1104 Gln His Leu Ile Leu Asn Glu
Asn Cys Pro Ser Val His Asn Leu Arg 355 360 365 agc cac aag att cag
acc cag ctc agc ctc atc cac cct gac atc ttc 1152 Ser His Lys Ile
Gln Thr Gln Leu Ser Leu Ile His Pro Asp Ile Phe 370 375 380 ccc cag
ctt acc agc ttc tat agt aag gag gaa ggg tcc acc ctc agc 1200 Pro
Gln Leu Thr Ser Phe Tyr Ser Lys Glu Glu Gly Ser Thr Leu Ser 385 390
395 400 gtg cca aca gtt cgg ggt gaa tgc ctc ctc aag tat tca gtc cgc
ccc 1248 Val Pro Thr Val Arg Gly Glu Cys Leu Leu Lys Tyr Ser Val
Arg Pro 405 410 415 aag aga gag tgg cag agg gat acc aca ctc gac tgc
aat act gat gaa 1296 Lys Arg Glu Trp Gln Arg Asp Thr Thr Leu Asp
Cys Asn Thr Asp Glu 420 425 430 ttc ata gct gag gcc ttg gag ctc ccc
agt ttc cag gag agt gtg gag 1344 Phe Ile Ala Glu Ala Leu Glu Leu
Pro Ser Phe Gln Glu Ser Val Glu 435 440 445 gag tat cgg aag aac gtg
cag gaa aac cca gcc cca gca gag aaa aga 1392 Glu Tyr Arg Lys Asn
Val Gln Glu Asn Pro Ala Pro Ala Glu Lys Arg 450 455 460 agc cag tat
cct gaa att gtc ttc ctg ggt acg ggg tct gcc atc cca 1440 Ser Gln
Tyr Pro Glu Ile Val Phe Leu Gly Thr Gly Ser Ala Ile Pro 465 470 475
480 atg gag atc cga aat gtc agt tcc aca ctc gtc aac cta agc cct gac
1488 Met Glu Ile Arg Asn Val Ser Ser Thr Leu Val Asn Leu Ser Pro
Asp 485 490 495 aag tca gtg ctc ctg gat tgt gga gaa ggc act ttt ggg
cag ttg tgc 1536 Lys Ser Val Leu Leu Asp Cys Gly Glu Gly Thr Phe
Gly Gln Leu Cys 500 505 510 cgt cat tac gga cag caa ata gac cga gtc
tta tgc agc ctc acg gct 1584 Arg His Tyr Gly Gln Gln Ile Asp Arg
Val Leu Cys Ser Leu Thr Ala 515 520 525 gtg ttt gtg tcc cac ctg cac
gcc gac cac cac acg ggc ttg ctg aat 1632 Val Phe Val Ser His Leu
His Ala Asp His His Thr Gly Leu Leu Asn 530 535 540 atc ttg ctg cag
aga gag cat gcg ttg gca tct ctg ggg aaa ccc ttc 1680 Ile Leu Leu
Gln Arg Glu His Ala Leu Ala Ser Leu Gly Lys Pro Phe 545 550 555 560
cag ccc ttg ctt gtg gtg gct cct acc cag ctc agg gcc tgg ctg cag
1728 Gln Pro Leu Leu Val Val Ala Pro Thr Gln Leu Arg Ala Trp Leu
Gln 565 570 575 cag tat cac aac cac tgc cag gag att ctg cac cac gtc
agt atg att 1776 Gln Tyr His Asn His Cys Gln Glu Ile Leu His His
Val Ser Met Ile 580 585 590 cct gcc aaa tgc ctt cag aaa ggg gca gag
gtc tcc aat act aca ttg 1824 Pro Ala Lys Cys Leu Gln Lys Gly Ala
Glu Val Ser Asn Thr Thr Leu 595 600 605 gaa agg ctg ata agc ttg ctg
ttg gaa aca tgt gac tta gaa gaa ttt 1872 Glu Arg Leu Ile Ser Leu
Leu Leu Glu Thr Cys Asp Leu Glu Glu Phe 610 615 620 cag acc tgc ctg
gta cgg cac tgc aag cat gct ttt ggc tgt gca ctg 1920 Gln Thr Cys
Leu Val Arg His Cys Lys His Ala Phe Gly Cys Ala Leu 625 630 635 640
gta cat tca tct ggc tgg aaa gtc gtc tac tcg ggg gat acc atg ccc
1968 Val His Ser Ser Gly Trp Lys Val Val Tyr Ser Gly Asp Thr Met
Pro 645 650 655 tgt gag gct ctg gtc cag atg ggg aaa gat gcc acc ctc
ctg ata cat 2016 Cys Glu Ala Leu Val Gln Met Gly Lys Asp Ala Thr
Leu Leu Ile His 660 665 670 gaa gcc act ctg gag gat cnc ttg gaa gag
gaa gca gta gag agg aca 2064 Glu Ala Thr Leu Glu Asp Xaa Leu Glu
Glu Glu Ala Val Glu Arg Thr 675 680 685 cac agc acc acc tcc cag gct
att aat gtg ggg atg cgg atg aat gcg 2112 His Ser Thr Thr Ser Gln
Ala Ile Asn Val Gly Met Arg Met Asn Ala 690 695 700 gag ttc atc atg
ctg aac cac ttc agt cag cgg tac gcn aag atc ccc 2160 Glu Phe Ile
Met Leu Asn His Phe Ser Gln Arg Tyr Xaa Lys Ile Pro 705 710 715 720
ctt ttc agc cct gac ttc aac gag aaa gtt ggc atc gcc ttt gac cac
2208 Leu Phe Ser Pro Asp Phe Asn Glu Lys Val Gly Ile Ala Phe Asp
His 725 730 735 atg aag gtc tgn ttt gga gac ttc ccg aca gtg ccc aag
ctg att ccc 2256 Met Lys Val Xaa Phe Gly Asp Phe Pro Thr Val Pro
Lys Leu Ile Pro 740 745 750 cca ctg aag gcc ctg ttt gca ggt gac att
gaa gag atg gtg gaa cgc 2304 Pro Leu Lys Ala Leu Phe Ala Gly Asp
Ile Glu Glu Met Val Glu Arg 755 760 765 agg gag aag agg gag cta cgg
ctg gtg cga gca gcc ctc ctg acc cag 2352 Arg Glu Lys Arg Glu Leu
Arg Leu Val Arg Ala Ala Leu Leu Thr Gln 770 775 780 cag gca gac agc
cca gag gac aga gaa ccc caa cag aag cgg gcc cac 2400 Gln Ala Asp
Ser Pro Glu Asp Arg Glu Pro Gln Gln Lys Arg Ala His 785 790 795 800
aca gat gaa cca cac agc cca cag agc aag aag gag agc gtg gca aac
2448 Thr Asp Glu Pro His Ser Pro Gln Ser Lys Lys Glu Ser Val Ala
Asn 805 810 815 act tta gga gcg cga gtg tgag 2470 Thr Leu Gly Ala
Arg Val 820 222 822 PRT Mus musculus 222 Met Trp Ala Leu Arg Ser
Leu Leu Arg Pro Leu Gly Leu Arg Thr Met 1 5 10 15 Ser Gln Gly Ser
Ala Arg Arg Pro Arg Pro Pro Lys Asp Pro Leu Arg 20 25 30 His Leu
Arg Thr Arg Glu Lys Arg Gly Pro Gly Pro Gly Gly Pro Asn 35 40 45
Thr Val Tyr Leu Gln Val Val Ala Ala Gly Gly Arg Asp Ala Gly Ala 50
55 60 Ala Leu Tyr Val Phe Ser Glu Tyr Asn Arg Tyr Leu Phe Asn Cys
Gly 65 70 75 80 Glu Gly Val Gln Arg Leu Met Gln Glu His Lys Thr Glu
Ser Arg Ser 85 90 95 Leu Asp Asn Ile Phe Leu Thr Arg Met His Trp
Ser Asn Val Gly Gly 100 105 110 Leu Cys Gly Met Ile Leu Thr Leu Lys
Glu Thr Gly Leu Pro Lys Cys 115 120 125 Val Leu Ser Gly Pro Pro Gln
Leu Glu Lys Tyr Leu Glu Ala Ile Lys 130 135 140 Ile Phe Ser Gly Pro
Leu Lys Gly Ile Glu Leu Ala Val Arg Pro His 145 150 155 160 Ser Ala
Pro Glu Tyr Lys Asp Glu Thr Met Thr Val Tyr Gln Val Pro 165 170 175
Ile His Ser Glu Arg Arg Cys Gly Lys Gln Gln Pro Ser Gln Ser Pro 180
185 190 Arg Thr Ser Pro Asn Arg Leu Ser Pro Lys Gln Ser Ser Asp Ser
Gly 195 200 205 Ser Ala Glu Asn Gly Gln Cys Gln Gln Glu Ser Met Gly
Gln Gly Pro 210 215 220 Ser Leu Val Val Ala Phe Val Cys Lys Leu His
Leu Arg Lys Gly Asn 225 230 235 240 Phe Leu Val Leu Lys Ala Lys Glu
Leu Gly Leu Pro Val Gly Thr Ala 245 250 255 Ala Ile Ala Pro Ile Ile
Ala Ala Val Lys Asp Gly Lys Ser Ile Thr 260 265 270 Tyr Glu Gly Arg
Glu Ile Ala Ala Glu Glu Leu Cys Thr Pro Pro Asp 275 280 285 Pro Gly
Leu Val Phe Ile Val Val Glu Cys Pro Asp Glu Gly Phe Ile 290 295 300
Leu Pro Ile Cys Glu Asn Asp Thr Phe Lys Arg Tyr Gln Ala Glu Ala 305
310 315 320 Asp Ala Pro Val Ala Leu Val Val His Ile Ala Pro Glu Ser
Val Leu 325 330 335 Ile Asp Ser Arg Tyr Gln Gln Trp Met Glu Arg Phe
Gly Pro Asp Thr 340 345 350 Gln His Leu Ile Leu Asn Glu Asn Cys Pro
Ser Val His Asn Leu Arg 355 360 365 Ser His Lys Ile Gln Thr Gln Leu
Ser Leu Ile His Pro Asp Ile Phe 370 375 380 Pro Gln Leu Thr Ser Phe
Tyr Ser Lys Glu Glu Gly Ser Thr Leu Ser 385 390 395 400 Val Pro Thr
Val Arg Gly Glu Cys Leu Leu Lys Tyr Ser Val Arg Pro 405 410 415 Lys
Arg Glu Trp Gln Arg Asp Thr Thr Leu Asp Cys Asn Thr Asp Glu 420 425
430 Phe Ile Ala Glu Ala Leu Glu Leu Pro Ser Phe Gln Glu Ser Val Glu
435 440 445 Glu Tyr Arg Lys Asn Val Gln Glu Asn Pro Ala Pro Ala Glu
Lys Arg 450 455 460 Ser Gln Tyr Pro Glu Ile Val Phe Leu Gly Thr Gly
Ser Ala Ile Pro 465 470 475 480 Met Glu Ile Arg Asn Val Ser Ser Thr
Leu Val Asn Leu Ser Pro Asp 485 490 495 Lys Ser Val Leu Leu Asp Cys
Gly Glu Gly Thr Phe Gly Gln Leu Cys 500 505 510 Arg His Tyr Gly Gln
Gln Ile Asp Arg Val Leu Cys Ser Leu Thr Ala 515 520 525 Val Phe Val
Ser His Leu His Ala Asp His His Thr Gly Leu Leu Asn 530 535 540 Ile
Leu Leu Gln Arg Glu His Ala Leu Ala Ser Leu Gly Lys Pro Phe 545 550
555 560 Gln Pro Leu Leu Val Val Ala Pro Thr Gln Leu Arg Ala Trp Leu
Gln 565 570 575 Gln Tyr His Asn His Cys Gln Glu Ile Leu His His Val
Ser Met Ile 580 585 590 Pro Ala Lys Cys Leu Gln Lys Gly Ala Glu Val
Ser Asn Thr Thr Leu 595 600 605 Glu Arg Leu Ile Ser Leu Leu Leu Glu
Thr Cys Asp Leu Glu Glu Phe 610 615 620 Gln Thr Cys Leu Val Arg His
Cys Lys His Ala Phe Gly Cys Ala Leu 625 630 635 640 Val His Ser Ser
Gly Trp Lys Val Val Tyr Ser Gly Asp Thr Met Pro 645 650 655 Cys Glu
Ala Leu Val Gln Met Gly Lys Asp Ala Thr Leu Leu Ile His 660 665 670
Glu Ala Thr Leu Glu Asp Xaa Leu Glu Glu Glu Ala Val Glu Arg Thr 675
680 685 His Ser Thr Thr Ser Gln Ala Ile Asn Val Gly Met Arg Met Asn
Ala 690 695 700 Glu Phe Ile Met Leu Asn His Phe Ser Gln Arg Tyr Xaa
Lys Ile Pro 705 710 715 720 Leu Phe Ser Pro Asp Phe Asn Glu Lys Val
Gly Ile Ala Phe Asp His 725 730 735 Met Lys Val Xaa Phe Gly Asp Phe
Pro Thr Val Pro Lys Leu Ile Pro 740 745 750 Pro Leu Lys Ala Leu Phe
Ala Gly Asp Ile Glu Glu Met Val Glu Arg 755 760 765 Arg Glu Lys Arg
Glu Leu Arg Leu Val Arg Ala Ala Leu Leu Thr Gln 770 775 780 Gln Ala
Asp Ser Pro Glu Asp Arg Glu Pro Gln Gln Lys Arg Ala His 785 790
795
800 Thr Asp Glu Pro His Ser Pro Gln Ser Lys Lys Glu Ser Val Ala Asn
805 810 815 Thr Leu Gly Ala Arg Val 820 223 2908 DNA Pan
troglodytes CDS (1)..(2478) 223 atg tgg gcg ctt tgc tcg ctg ctg cgg
tcc gcg gcc gga cgc acc atg 48 Met Trp Ala Leu Cys Ser Leu Leu Arg
Ser Ala Ala Gly Arg Thr Met 1 5 10 15 tcg cag gga cgc acc ata tcg
cag gca ccc gcc cgc cgc gag cgg ccg 96 Ser Gln Gly Arg Thr Ile Ser
Gln Ala Pro Ala Arg Arg Glu Arg Pro 20 25 30 cgc aag gac ccg ctg
cgg cac ctg cgc acg cga gag aag cgc gga ccg 144 Arg Lys Asp Pro Leu
Arg His Leu Arg Thr Arg Glu Lys Arg Gly Pro 35 40 45 tcg ggg tgc
tcc ggc ggc cca aac acc gtg tac ctg cag gtg gtg gca 192 Ser Gly Cys
Ser Gly Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala 50 55 60 gcg
ggt agc cgg gac tcg ggc gcc gcg ctc tac gtc ttc tcc gag ttc 240 Ala
Gly Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe 65 70
75 80 aac cgg tat ctc ttc aac tgt gga gaa ggc att cag aga ctc atg
cag 288 Asn Arg Tyr Leu Phe Asn Cys Gly Glu Gly Ile Gln Arg Leu Met
Gln 85 90 95 gag cac aag tta aag gtt gct cgc ctg gac aac ata ttc
ctg aca cga 336 Glu His Lys Leu Lys Val Ala Arg Leu Asp Asn Ile Phe
Leu Thr Arg 100 105 110 atg cac tgg tct aat gtt ggg ggc tta agt gga
atg att ctt act tta 384 Met His Trp Ser Asn Val Gly Gly Leu Ser Gly
Met Ile Leu Thr Leu 115 120 125 aag gaa acc ggg ctt cca aag tgt gta
ctt tct gga cct cca caa ctg 432 Lys Glu Thr Gly Leu Pro Lys Cys Val
Leu Ser Gly Pro Pro Gln Leu 130 135 140 gaa aaa tac ctc gaa gca atc
aaa ata ttt tct ggt cca ttg aaa gga 480 Glu Lys Tyr Leu Glu Ala Ile
Lys Ile Phe Ser Gly Pro Leu Lys Gly 145 150 155 160 ata gaa ctg gct
gtg cgg ccc cac tct gcc cca gaa tac gag gat gaa 528 Ile Glu Leu Ala
Val Arg Pro His Ser Ala Pro Glu Tyr Glu Asp Glu 165 170 175 acc atg
aca gtt tac cag atc ccc ata cac agt gaa cag agg agg gga 576 Thr Met
Thr Val Tyr Gln Ile Pro Ile His Ser Glu Gln Arg Arg Gly 180 185 190
aag cac caa cca tgg cag agt cca gaa agg cct ctc agc agg ctc agt 624
Lys His Gln Pro Trp Gln Ser Pro Glu Arg Pro Leu Ser Arg Leu Ser 195
200 205 cca gag cga tct tca gac tcc gag tca aat gaa aat gag cca cac
ctt 672 Pro Glu Arg Ser Ser Asp Ser Glu Ser Asn Glu Asn Glu Pro His
Leu 210 215 220 cca cat ggt gtt agc cag aga aga ggg gtc agg gac tct
tcc ctg gtc 720 Pro His Gly Val Ser Gln Arg Arg Gly Val Arg Asp Ser
Ser Leu Val 225 230 235 240 gta gct ttc atc tgt aag ctt cac tta aag
aga gga aac ttc ttg gtg 768 Val Ala Phe Ile Cys Lys Leu His Leu Lys
Arg Gly Asn Phe Leu Val 245 250 255 ctc aaa gca aag gag atg ggc ctc
cca gtt ggg aca gct gcc atc gct 816 Leu Lys Ala Lys Glu Met Gly Leu
Pro Val Gly Thr Ala Ala Ile Ala 260 265 270 ccc atc att gct gct gtc
aag gac ggg aaa agc atc act cat gaa gga 864 Pro Ile Ile Ala Ala Val
Lys Asp Gly Lys Ser Ile Thr His Glu Gly 275 280 285 aga gag att ttg
gct gaa gag ctg tgt act cct cca gat cct ggt gct 912 Arg Glu Ile Leu
Ala Glu Glu Leu Cys Thr Pro Pro Asp Pro Gly Ala 290 295 300 gct ttt
gtg gtg gta gaa tgt cca gat gaa agc ttc att caa ccc atc 960 Ala Phe
Val Val Val Glu Cys Pro Asp Glu Ser Phe Ile Gln Pro Ile 305 310 315
320 tgt gag aat gcc acc ttt cag agg tac caa gga aag gca gat gcc ccc
1008 Cys Glu Asn Ala Thr Phe Gln Arg Tyr Gln Gly Lys Ala Asp Ala
Pro 325 330 335 gtg gcc ttg gtg gtt cac atg gcc cca gaa tct gtg ctt
gtg gac agc 1056 Val Ala Leu Val Val His Met Ala Pro Glu Ser Val
Leu Val Asp Ser 340 345 350 agg tac cag cag tgg atg gag agg ttt ggg
cct gac acc cag cac ttg 1104 Arg Tyr Gln Gln Trp Met Glu Arg Phe
Gly Pro Asp Thr Gln His Leu 355 360 365 gtc ctg aat gag aac tgt gcc
tca gtt cac aac ctt cgc agc cac aag 1152 Val Leu Asn Glu Asn Cys
Ala Ser Val His Asn Leu Arg Ser His Lys 370 375 380 att caa acc cag
ctc aac ctc atc cac ccg gac atc ttc ccc ctg ctc 1200 Ile Gln Thr
Gln Leu Asn Leu Ile His Pro Asp Ile Phe Pro Leu Leu 385 390 395 400
acc agt ttc ccc tgt aag aag gag ggc ccc acc ctc agt gtg ccc atg
1248 Thr Ser Phe Pro Cys Lys Lys Glu Gly Pro Thr Leu Ser Val Pro
Met 405 410 415 gtt cag ggt gaa tgc ctc ctc aag tac cag ctc cgt ccc
agg agg gag 1296 Val Gln Gly Glu Cys Leu Leu Lys Tyr Gln Leu Arg
Pro Arg Arg Glu 420 425 430 tgg cag agg gat gcc att att act tgc aat
cct gag gaa ttc ata att 1344 Trp Gln Arg Asp Ala Ile Ile Thr Cys
Asn Pro Glu Glu Phe Ile Ile 435 440 445 gag gcg ctg cag ctt ccc aac
ttc cag cag agt gtg cag gag tac agg 1392 Glu Ala Leu Gln Leu Pro
Asn Phe Gln Gln Ser Val Gln Glu Tyr Arg 450 455 460 agg agt gcg cag
gac ggc cca gcc cca gca gag aaa aga agt cag tac 1440 Arg Ser Ala
Gln Asp Gly Pro Ala Pro Ala Glu Lys Arg Ser Gln Tyr 465 470 475 480
cca gaa atc atc ttc ctt gga aca ggg tct gcc atc ccg atg aag att
1488 Pro Glu Ile Ile Phe Leu Gly Thr Gly Ser Ala Ile Pro Met Lys
Ile 485 490 495 cga aat gtc agt gcc aca ctt gtc aac ata agc ccc gac
acg tct ctg 1536 Arg Asn Val Ser Ala Thr Leu Val Asn Ile Ser Pro
Asp Thr Ser Leu 500 505 510 cta ctg gac tgt ggt gag ggc acg ttt ggg
cag ctg tgc cgt cat tac 1584 Leu Leu Asp Cys Gly Glu Gly Thr Phe
Gly Gln Leu Cys Arg His Tyr 515 520 525 gga gac cag gtg gac agg gtc
ctg ggc acc ctg gct gct gtg ttt gtg 1632 Gly Asp Gln Val Asp Arg
Val Leu Gly Thr Leu Ala Ala Val Phe Val 530 535 540 tcc cac ctg cac
gca gat cac cac acg ggc ttg cta aat atc ttg ctg 1680 Ser His Leu
His Ala Asp His His Thr Gly Leu Leu Asn Ile Leu Leu 545 550 555 560
cag aga gaa cga gcc ttg gca tct ttg gga aag ccc ttt cac cct ttg
1728 Gln Arg Glu Arg Ala Leu Ala Ser Leu Gly Lys Pro Phe His Pro
Leu 565 570 575 ctg gtg gtt gcc ccc aac cag ctc aaa gcc tgg ctc cag
cag tac cac 1776 Leu Val Val Ala Pro Asn Gln Leu Lys Ala Trp Leu
Gln Gln Tyr His 580 585 590 aac cag tgc cag gag gtc ctg cac cac atc
agt atg att cct gcc aaa 1824 Asn Gln Cys Gln Glu Val Leu His His
Ile Ser Met Ile Pro Ala Lys 595 600 605 tgc ctt cag gaa ggg gct gag
atc tcc agt cct gca gtg gaa aga ttg 1872 Cys Leu Gln Glu Gly Ala
Glu Ile Ser Ser Pro Ala Val Glu Arg Leu 610 615 620 atc agt tcg ctg
ttg cga aca tgt gat ttg gaa gag ttt cag acc tgt 1920 Ile Ser Ser
Leu Leu Arg Thr Cys Asp Leu Glu Glu Phe Gln Thr Cys 625 630 635 640
ctg gtg cgg cac tgc aag cat gcg ttt ggc tgt gcg ctg gtg cac acc
1968 Leu Val Arg His Cys Lys His Ala Phe Gly Cys Ala Leu Val His
Thr 645 650 655 tct ggc tgg aaa gtg gtc tat tcc ggg gac acc atg ccc
tgc gag gct 2016 Ser Gly Trp Lys Val Val Tyr Ser Gly Asp Thr Met
Pro Cys Glu Ala 660 665 670 ctg gtc cgg atg ggg aaa gat gcc acc ctc
ctg ata cat gaa gcc acc 2064 Leu Val Arg Met Gly Lys Asp Ala Thr
Leu Leu Ile His Glu Ala Thr 675 680 685 ctg gaa gac ggt ttg gaa gag
gaa gca gtg gaa aag aca cac agc aca 2112 Leu Glu Asp Gly Leu Glu
Glu Glu Ala Val Glu Lys Thr His Ser Thr 690 695 700 acg tcc caa gcc
atc agc gtg ggg atg cgg atg aac gcg gag ttc att 2160 Thr Ser Gln
Ala Ile Ser Val Gly Met Arg Met Asn Ala Glu Phe Ile 705 710 715 720
atg ctg aac cac ttc agc cag cgc tat gcc aag gtc ccc ctc ttc agc
2208 Met Leu Asn His Phe Ser Gln Arg Tyr Ala Lys Val Pro Leu Phe
Ser 725 730 735 ccc aac ttc aac gag aaa gtg gga gtt gcc ttt gac cac
atg aag gtc 2256 Pro Asn Phe Asn Glu Lys Val Gly Val Ala Phe Asp
His Met Lys Val 740 745 750 tgc ttt gga gac ttt gca aca atg ccc aag
ctg att ccc cca ctg aaa 2304 Cys Phe Gly Asp Phe Ala Thr Met Pro
Lys Leu Ile Pro Pro Leu Lys 755 760 765 gcc ctg ttt gct ggc gac atc
gag gag atg gag gag cgc agg gag aag 2352 Ala Leu Phe Ala Gly Asp
Ile Glu Glu Met Glu Glu Arg Arg Glu Lys 770 775 780 cgg gag ctg cgg
cag gtg cgg gcg gcc ctc ctg tcc agg gag ctg gca 2400 Arg Glu Leu
Arg Gln Val Arg Ala Ala Leu Leu Ser Arg Glu Leu Ala 785 790 795 800
ggc ggc ctg gag gat ggg gag cct cag cag aaa cgg gcc cac aca gag
2448 Gly Gly Leu Glu Asp Gly Glu Pro Gln Gln Lys Arg Ala His Thr
Glu 805 810 815 gag cca cag gcc aag aag gtc aga gcc cag tgaagatctg
ggagaccctg 2498 Glu Pro Gln Ala Lys Lys Val Arg Ala Gln 820 825
aattcagaag gctgtgtgtc ttctgcccca cgcacgcacc cgtatctgcc ctccttgctg
2558 gtagaagctg aagagcacgg tcccccagga ggcagctcag gataggtggt
atggagctgt 2618 gccaaggctt gggctcccac ataagcacta gtctatagat
gcctcttagg actggtgcct 2678 ggcacagccg cgggacagga ggctgccaca
cggaagcaag cagatgaact aatttcattt 2738 caaggcagtt tttaaagaag
gcttggaaac agacggcagc acctttcctc taatccagca 2798 aagtgattcc
ctgcacacca gagacaagca gagtaacagg atcagtgggt ctaagtgtcc 2858
gagacttaac gaaaatagta tttcagctgc aataaagatt gagtttgcaa 2908 224 826
PRT Pan troglodytes 224 Met Trp Ala Leu Cys Ser Leu Leu Arg Ser Ala
Ala Gly Arg Thr Met 1 5 10 15 Ser Gln Gly Arg Thr Ile Ser Gln Ala
Pro Ala Arg Arg Glu Arg Pro 20 25 30 Arg Lys Asp Pro Leu Arg His
Leu Arg Thr Arg Glu Lys Arg Gly Pro 35 40 45 Ser Gly Cys Ser Gly
Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala 50 55 60 Ala Gly Ser
Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe 65 70 75 80 Asn
Arg Tyr Leu Phe Asn Cys Gly Glu Gly Ile Gln Arg Leu Met Gln 85 90
95 Glu His Lys Leu Lys Val Ala Arg Leu Asp Asn Ile Phe Leu Thr Arg
100 105 110 Met His Trp Ser Asn Val Gly Gly Leu Ser Gly Met Ile Leu
Thr Leu 115 120 125 Lys Glu Thr Gly Leu Pro Lys Cys Val Leu Ser Gly
Pro Pro Gln Leu 130 135 140 Glu Lys Tyr Leu Glu Ala Ile Lys Ile Phe
Ser Gly Pro Leu Lys Gly 145 150 155 160 Ile Glu Leu Ala Val Arg Pro
His Ser Ala Pro Glu Tyr Glu Asp Glu 165 170 175 Thr Met Thr Val Tyr
Gln Ile Pro Ile His Ser Glu Gln Arg Arg Gly 180 185 190 Lys His Gln
Pro Trp Gln Ser Pro Glu Arg Pro Leu Ser Arg Leu Ser 195 200 205 Pro
Glu Arg Ser Ser Asp Ser Glu Ser Asn Glu Asn Glu Pro His Leu 210 215
220 Pro His Gly Val Ser Gln Arg Arg Gly Val Arg Asp Ser Ser Leu Val
225 230 235 240 Val Ala Phe Ile Cys Lys Leu His Leu Lys Arg Gly Asn
Phe Leu Val 245 250 255 Leu Lys Ala Lys Glu Met Gly Leu Pro Val Gly
Thr Ala Ala Ile Ala 260 265 270 Pro Ile Ile Ala Ala Val Lys Asp Gly
Lys Ser Ile Thr His Glu Gly 275 280 285 Arg Glu Ile Leu Ala Glu Glu
Leu Cys Thr Pro Pro Asp Pro Gly Ala 290 295 300 Ala Phe Val Val Val
Glu Cys Pro Asp Glu Ser Phe Ile Gln Pro Ile 305 310 315 320 Cys Glu
Asn Ala Thr Phe Gln Arg Tyr Gln Gly Lys Ala Asp Ala Pro 325 330 335
Val Ala Leu Val Val His Met Ala Pro Glu Ser Val Leu Val Asp Ser 340
345 350 Arg Tyr Gln Gln Trp Met Glu Arg Phe Gly Pro Asp Thr Gln His
Leu 355 360 365 Val Leu Asn Glu Asn Cys Ala Ser Val His Asn Leu Arg
Ser His Lys 370 375 380 Ile Gln Thr Gln Leu Asn Leu Ile His Pro Asp
Ile Phe Pro Leu Leu 385 390 395 400 Thr Ser Phe Pro Cys Lys Lys Glu
Gly Pro Thr Leu Ser Val Pro Met 405 410 415 Val Gln Gly Glu Cys Leu
Leu Lys Tyr Gln Leu Arg Pro Arg Arg Glu 420 425 430 Trp Gln Arg Asp
Ala Ile Ile Thr Cys Asn Pro Glu Glu Phe Ile Ile 435 440 445 Glu Ala
Leu Gln Leu Pro Asn Phe Gln Gln Ser Val Gln Glu Tyr Arg 450 455 460
Arg Ser Ala Gln Asp Gly Pro Ala Pro Ala Glu Lys Arg Ser Gln Tyr 465
470 475 480 Pro Glu Ile Ile Phe Leu Gly Thr Gly Ser Ala Ile Pro Met
Lys Ile 485 490 495 Arg Asn Val Ser Ala Thr Leu Val Asn Ile Ser Pro
Asp Thr Ser Leu 500 505 510 Leu Leu Asp Cys Gly Glu Gly Thr Phe Gly
Gln Leu Cys Arg His Tyr 515 520 525 Gly Asp Gln Val Asp Arg Val Leu
Gly Thr Leu Ala Ala Val Phe Val 530 535 540 Ser His Leu His Ala Asp
His His Thr Gly Leu Leu Asn Ile Leu Leu 545 550 555 560 Gln Arg Glu
Arg Ala Leu Ala Ser Leu Gly Lys Pro Phe His Pro Leu 565 570 575 Leu
Val Val Ala Pro Asn Gln Leu Lys Ala Trp Leu Gln Gln Tyr His 580 585
590 Asn Gln Cys Gln Glu Val Leu His His Ile Ser Met Ile Pro Ala Lys
595 600 605 Cys Leu Gln Glu Gly Ala Glu Ile Ser Ser Pro Ala Val Glu
Arg Leu 610 615 620 Ile Ser Ser Leu Leu Arg Thr Cys Asp Leu Glu Glu
Phe Gln Thr Cys 625 630 635 640 Leu Val Arg His Cys Lys His Ala Phe
Gly Cys Ala Leu Val His Thr 645 650 655 Ser Gly Trp Lys Val Val Tyr
Ser Gly Asp Thr Met Pro Cys Glu Ala 660 665 670 Leu Val Arg Met Gly
Lys Asp Ala Thr Leu Leu Ile His Glu Ala Thr 675 680 685 Leu Glu Asp
Gly Leu Glu Glu Glu Ala Val Glu Lys Thr His Ser Thr 690 695 700 Thr
Ser Gln Ala Ile Ser Val Gly Met Arg Met Asn Ala Glu Phe Ile 705 710
715 720 Met Leu Asn His Phe Ser Gln Arg Tyr Ala Lys Val Pro Leu Phe
Ser 725 730 735 Pro Asn Phe Asn Glu Lys Val Gly Val Ala Phe Asp His
Met Lys Val 740 745 750 Cys Phe Gly Asp Phe Ala Thr Met Pro Lys Leu
Ile Pro Pro Leu Lys 755 760 765 Ala Leu Phe Ala Gly Asp Ile Glu Glu
Met Glu Glu Arg Arg Glu Lys 770 775 780 Arg Glu Leu Arg Gln Val Arg
Ala Ala Leu Leu Ser Arg Glu Leu Ala 785 790 795 800 Gly Gly Leu Glu
Asp Gly Glu Pro Gln Gln Lys Arg Ala His Thr Glu 805 810 815 Glu Pro
Gln Ala Lys Lys Val Arg Ala Gln 820 825 225 2892 DNA Gorilla
gorilla CDS (1)..(2478) 225 atg tgg gcg ctt tgc tcg ctg ctg cgg tcc
gcg gcc gga cgc acc atg 48 Met Trp Ala Leu Cys Ser Leu Leu Arg Ser
Ala Ala Gly Arg Thr Met 1 5 10 15 tcg cag gga cgc acc ata tcg cag
gca ccc gcc cgc cgc gag cgg ccg 96 Ser Gln Gly Arg Thr Ile Ser Gln
Ala Pro Ala Arg Arg Glu Arg Pro 20 25 30 cgc aag gac ccg ctg cgg
cac ctg cgc acg cga gag aag cgc gga ccg 144 Arg Lys Asp Pro Leu Arg
His Leu Arg Thr Arg Glu Lys Arg Gly Pro 35 40 45 tcg ggg tgc tcc
ggg ggc cca aac acc gtg tac ctg cag gtg gtg gca 192 Ser Gly Cys Ser
Gly Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala 50 55 60 gcg ggt
agc cgg gac tcg ggc gcc gcg ctc tac gtc ttc tcc gag ttc 240 Ala Gly
Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe 65 70 75 80
aac cgg tat ctc ttc aac tgt gga gaa ggc gtt cag aga ctc atg cag 288
Asn Arg Tyr Leu Phe Asn Cys Gly Glu Gly Val Gln Arg Leu Met Gln 85
90 95 gag cac aag tta aag gtt gtt cgc ctg gac aac ata ttc ctg aca
cga 336 Glu His Lys Leu Lys Val Val Arg Leu Asp Asn Ile Phe Leu Thr
Arg 100 105
110 atg cac tgg tct aat gtt ggg ggc tta agt gga atg att ctt act tta
384 Met His Trp Ser Asn Val Gly Gly Leu Ser Gly Met Ile Leu Thr Leu
115 120 125 aag gaa acc ggg ctt cca aag tgt gta ctt tct gga cct cca
cag ctg 432 Lys Glu Thr Gly Leu Pro Lys Cys Val Leu Ser Gly Pro Pro
Gln Leu 130 135 140 gaa aaa tac ctc gaa gca atc aaa ata ttt tct ggt
cca ttg aaa gga 480 Glu Lys Tyr Leu Glu Ala Ile Lys Ile Phe Ser Gly
Pro Leu Lys Gly 145 150 155 160 ata gaa ctg gct gtg cgg ccc cac tct
gcc cca gaa tac gag gat gaa 528 Ile Glu Leu Ala Val Arg Pro His Ser
Ala Pro Glu Tyr Glu Asp Glu 165 170 175 acc atg aca gtt tac cag atc
ccc ata cac agt gaa cag agg agg gga 576 Thr Met Thr Val Tyr Gln Ile
Pro Ile His Ser Glu Gln Arg Arg Gly 180 185 190 agg cac caa cca tgg
cag agt cca gaa agg cct ctc agc agg ctc agt 624 Arg His Gln Pro Trp
Gln Ser Pro Glu Arg Pro Leu Ser Arg Leu Ser 195 200 205 cca gag cga
tct tca gac tcc gag tcg aat gaa aat gag cca cac ctt 672 Pro Glu Arg
Ser Ser Asp Ser Glu Ser Asn Glu Asn Glu Pro His Leu 210 215 220 cca
cat ggt gtt agc cag aga aga ggg gtc agg gac tct tcc ctg gtc 720 Pro
His Gly Val Ser Gln Arg Arg Gly Val Arg Asp Ser Ser Leu Val 225 230
235 240 gta gct ttc atc tgt aag ctt cac tta aag aga gga aac ttc ttg
gtg 768 Val Ala Phe Ile Cys Lys Leu His Leu Lys Arg Gly Asn Phe Leu
Val 245 250 255 ctc aaa gca aag gag atg ggc ctc cca gtt ggg aca gct
gcc atc gct 816 Leu Lys Ala Lys Glu Met Gly Leu Pro Val Gly Thr Ala
Ala Ile Ala 260 265 270 ccc atc att gct gct gtc aag gac ggg aaa agc
atc act cat gaa gga 864 Pro Ile Ile Ala Ala Val Lys Asp Gly Lys Ser
Ile Thr His Glu Gly 275 280 285 aga gag att ttg gct gaa gag ctg tgt
act cct cca gat cct ggt gct 912 Arg Glu Ile Leu Ala Glu Glu Leu Cys
Thr Pro Pro Asp Pro Gly Ala 290 295 300 gct ttt gtg gtg gta gaa tgt
cca gat gaa agc ttc att caa ccc atc 960 Ala Phe Val Val Val Glu Cys
Pro Asp Glu Ser Phe Ile Gln Pro Ile 305 310 315 320 tgt gag aat gcc
acc ttt cag agg tac caa gga aag gca gat gcc ccc 1008 Cys Glu Asn
Ala Thr Phe Gln Arg Tyr Gln Gly Lys Ala Asp Ala Pro 325 330 335 gtg
gcc ttg gtg gtt cac atg gcc cca gaa tct gtg ctt gtg gac agc 1056
Val Ala Leu Val Val His Met Ala Pro Glu Ser Val Leu Val Asp Ser 340
345 350 agg tac cag cag tgg atg gag agg ttt ggg cct gac acc cag cac
ttg 1104 Arg Tyr Gln Gln Trp Met Glu Arg Phe Gly Pro Asp Thr Gln
His Leu 355 360 365 gtc ctg aat gag aac tgt gcc tca gtt cac aac ctt
cgc agc cac aag 1152 Val Leu Asn Glu Asn Cys Ala Ser Val His Asn
Leu Arg Ser His Lys 370 375 380 att caa acc cag ctc aac ctc atc cac
ccg gac atc ttc ccc ctg ctc 1200 Ile Gln Thr Gln Leu Asn Leu Ile
His Pro Asp Ile Phe Pro Leu Leu 385 390 395 400 acc agt ttc ccc tgt
aag aag gag ggc ccc acc ctc agt gtg ccc atg 1248 Thr Ser Phe Pro
Cys Lys Lys Glu Gly Pro Thr Leu Ser Val Pro Met 405 410 415 gtt cag
ggt gaa tgc ctc ctc aag tac cag ctc cgt ccc agg agg gaa 1296 Val
Gln Gly Glu Cys Leu Leu Lys Tyr Gln Leu Arg Pro Arg Arg Glu 420 425
430 tgg cag agg gat gcc att atc act tgc aat cct gag gaa ttc ata gtt
1344 Trp Gln Arg Asp Ala Ile Ile Thr Cys Asn Pro Glu Glu Phe Ile
Val 435 440 445 gag gcg ctg cag ctt ccc aac ttc cag cag agt gtg cag
gag tac agg 1392 Glu Ala Leu Gln Leu Pro Asn Phe Gln Gln Ser Val
Gln Glu Tyr Arg 450 455 460 agg agt gtg cag gac gtc cca gcc cca gca
gag aaa aga agt cag tac 1440 Arg Ser Val Gln Asp Val Pro Ala Pro
Ala Glu Lys Arg Ser Gln Tyr 465 470 475 480 cca gaa atc atc ttc ctt
gga aca ggg tct gcc atc ccc atg aag att 1488 Pro Glu Ile Ile Phe
Leu Gly Thr Gly Ser Ala Ile Pro Met Lys Ile 485 490 495 cga aat gtc
agt gcc aca ctt gtc aac ata agc ccc gac acg tct ctg 1536 Arg Asn
Val Ser Ala Thr Leu Val Asn Ile Ser Pro Asp Thr Ser Leu 500 505 510
cta ctg gac tgt ggt gag ggc acg ttt ggg cag ctg tgc cgt cat tac
1584 Leu Leu Asp Cys Gly Glu Gly Thr Phe Gly Gln Leu Cys Arg His
Tyr 515 520 525 gga gac cag gtg gac agg gtc ctg ggc acc ctg gct gct
gtg ttt gtg 1632 Gly Asp Gln Val Asp Arg Val Leu Gly Thr Leu Ala
Ala Val Phe Val 530 535 540 tcc cac ctg cac gca gat cac cac acg ggc
ttg cta aat atc ttg ctg 1680 Ser His Leu His Ala Asp His His Thr
Gly Leu Leu Asn Ile Leu Leu 545 550 555 560 cag aga gaa caa gcc ttg
gca tct ttg gga aag ccc ctt cac cct ttg 1728 Gln Arg Glu Gln Ala
Leu Ala Ser Leu Gly Lys Pro Leu His Pro Leu 565 570 575 ctg gtg gtt
gcc ccc agc cag ctc aaa gcc tgg ctc cag cag tac cac 1776 Leu Val
Val Ala Pro Ser Gln Leu Lys Ala Trp Leu Gln Gln Tyr His 580 585 590
aac cag tgc cag gag gtc ctg cac cac atc agt atg att cct gcc aaa
1824 Asn Gln Cys Gln Glu Val Leu His His Ile Ser Met Ile Pro Ala
Lys 595 600 605 tgc ctt cag gaa ggg gct gag atc tcc agt cct gca gtg
gaa aga ttg 1872 Cys Leu Gln Glu Gly Ala Glu Ile Ser Ser Pro Ala
Val Glu Arg Leu 610 615 620 atc agt tcg ctg ttg cga aca tgt gat ttg
gaa gag ttt cag acc tgt 1920 Ile Ser Ser Leu Leu Arg Thr Cys Asp
Leu Glu Glu Phe Gln Thr Cys 625 630 635 640 ctg gtg cgg cac tgc aag
cat gcg ttt ggc tgt gcg ctg gtg cac acc 1968 Leu Val Arg His Cys
Lys His Ala Phe Gly Cys Ala Leu Val His Thr 645 650 655 tct ggc tgg
aaa gtg gtc tat tcc ggg gac acc atg ccc tgc gag gct 2016 Ser Gly
Trp Lys Val Val Tyr Ser Gly Asp Thr Met Pro Cys Glu Ala 660 665 670
ctg gtc cgc atg ggg aaa gat gcc acc ctc ctg ata cat gaa gcc acc
2064 Leu Val Arg Met Gly Lys Asp Ala Thr Leu Leu Ile His Glu Ala
Thr 675 680 685 ctg gaa gat ggt ttg gaa gag gaa gca gtg gaa aag aca
cac agc aca 2112 Leu Glu Asp Gly Leu Glu Glu Glu Ala Val Glu Lys
Thr His Ser Thr 690 695 700 acg tcc caa gcc atc agc gtg ggg atg cgg
atg aac gcg gag ttc att 2160 Thr Ser Gln Ala Ile Ser Val Gly Met
Arg Met Asn Ala Glu Phe Ile 705 710 715 720 atg ctg aac cac ttc agc
cag cgc tat gcc aag gtc ccc ctc ttc agc 2208 Met Leu Asn His Phe
Ser Gln Arg Tyr Ala Lys Val Pro Leu Phe Ser 725 730 735 ccc aac ttc
aac gag aaa gtg gga gtt gcc ttt gac cac atg aag gtc 2256 Pro Asn
Phe Asn Glu Lys Val Gly Val Ala Phe Asp His Met Lys Val 740 745 750
tgc ttt gga gac ttt cca aca atg ccc aag ctg att ccc cca ctg aaa
2304 Cys Phe Gly Asp Phe Pro Thr Met Pro Lys Leu Ile Pro Pro Leu
Lys 755 760 765 gcc ctg ttt gcc ggc gac atc gag gag atg gag gag cgc
agg gag aag 2352 Ala Leu Phe Ala Gly Asp Ile Glu Glu Met Glu Glu
Arg Arg Glu Lys 770 775 780 cgg gag ctg cgg cag gtg cgg gcg gcc ctc
ctg tcc ggg gag ctg gca 2400 Arg Glu Leu Arg Gln Val Arg Ala Ala
Leu Leu Ser Gly Glu Leu Ala 785 790 795 800 ggc ggc ctg gag gat ggg
gag cct cag cag aaa cgg gcc cac aca gag 2448 Gly Gly Leu Glu Asp
Gly Glu Pro Gln Gln Lys Arg Ala His Thr Glu 805 810 815 gag cca cag
gcc aag aag gtc aga gcc cag tgaagatctg ggagaccctg 2498 Glu Pro Gln
Ala Lys Lys Val Arg Ala Gln 820 825 aattcagaag gctgtgtgtc
ttctgcccca cgcacgcacc cgtatctgcc ctccttgctg 2558 gtagaagctg
aagagcacgg tcccccagga ggcagctcag gataggtggt atggagctgt 2618
gccgaggctt aggctcccac ataagcacta gtctataggt gcctggcaca gccgcgggac
2678 aggaggctgc cacacggaag caagcagatg aactaatttc atttcaaggc
agtttttaaa 2738 gaagtcttgg aaacagacgg cagcaccttt cctctaatcc
agcaaagtga ttccctgcac 2798 accagagaca agcagagtaa caggatcact
gggtctaagt gtccgagact taacgaaaat 2858 agtatttcag ctgcaataaa
gattgagttt gcaa 2892 226 826 PRT Gorilla gorilla 226 Met Trp Ala
Leu Cys Ser Leu Leu Arg Ser Ala Ala Gly Arg Thr Met 1 5 10 15 Ser
Gln Gly Arg Thr Ile Ser Gln Ala Pro Ala Arg Arg Glu Arg Pro 20 25
30 Arg Lys Asp Pro Leu Arg His Leu Arg Thr Arg Glu Lys Arg Gly Pro
35 40 45 Ser Gly Cys Ser Gly Gly Pro Asn Thr Val Tyr Leu Gln Val
Val Ala 50 55 60 Ala Gly Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val
Phe Ser Glu Phe 65 70 75 80 Asn Arg Tyr Leu Phe Asn Cys Gly Glu Gly
Val Gln Arg Leu Met Gln 85 90 95 Glu His Lys Leu Lys Val Val Arg
Leu Asp Asn Ile Phe Leu Thr Arg 100 105 110 Met His Trp Ser Asn Val
Gly Gly Leu Ser Gly Met Ile Leu Thr Leu 115 120 125 Lys Glu Thr Gly
Leu Pro Lys Cys Val Leu Ser Gly Pro Pro Gln Leu 130 135 140 Glu Lys
Tyr Leu Glu Ala Ile Lys Ile Phe Ser Gly Pro Leu Lys Gly 145 150 155
160 Ile Glu Leu Ala Val Arg Pro His Ser Ala Pro Glu Tyr Glu Asp Glu
165 170 175 Thr Met Thr Val Tyr Gln Ile Pro Ile His Ser Glu Gln Arg
Arg Gly 180 185 190 Arg His Gln Pro Trp Gln Ser Pro Glu Arg Pro Leu
Ser Arg Leu Ser 195 200 205 Pro Glu Arg Ser Ser Asp Ser Glu Ser Asn
Glu Asn Glu Pro His Leu 210 215 220 Pro His Gly Val Ser Gln Arg Arg
Gly Val Arg Asp Ser Ser Leu Val 225 230 235 240 Val Ala Phe Ile Cys
Lys Leu His Leu Lys Arg Gly Asn Phe Leu Val 245 250 255 Leu Lys Ala
Lys Glu Met Gly Leu Pro Val Gly Thr Ala Ala Ile Ala 260 265 270 Pro
Ile Ile Ala Ala Val Lys Asp Gly Lys Ser Ile Thr His Glu Gly 275 280
285 Arg Glu Ile Leu Ala Glu Glu Leu Cys Thr Pro Pro Asp Pro Gly Ala
290 295 300 Ala Phe Val Val Val Glu Cys Pro Asp Glu Ser Phe Ile Gln
Pro Ile 305 310 315 320 Cys Glu Asn Ala Thr Phe Gln Arg Tyr Gln Gly
Lys Ala Asp Ala Pro 325 330 335 Val Ala Leu Val Val His Met Ala Pro
Glu Ser Val Leu Val Asp Ser 340 345 350 Arg Tyr Gln Gln Trp Met Glu
Arg Phe Gly Pro Asp Thr Gln His Leu 355 360 365 Val Leu Asn Glu Asn
Cys Ala Ser Val His Asn Leu Arg Ser His Lys 370 375 380 Ile Gln Thr
Gln Leu Asn Leu Ile His Pro Asp Ile Phe Pro Leu Leu 385 390 395 400
Thr Ser Phe Pro Cys Lys Lys Glu Gly Pro Thr Leu Ser Val Pro Met 405
410 415 Val Gln Gly Glu Cys Leu Leu Lys Tyr Gln Leu Arg Pro Arg Arg
Glu 420 425 430 Trp Gln Arg Asp Ala Ile Ile Thr Cys Asn Pro Glu Glu
Phe Ile Val 435 440 445 Glu Ala Leu Gln Leu Pro Asn Phe Gln Gln Ser
Val Gln Glu Tyr Arg 450 455 460 Arg Ser Val Gln Asp Val Pro Ala Pro
Ala Glu Lys Arg Ser Gln Tyr 465 470 475 480 Pro Glu Ile Ile Phe Leu
Gly Thr Gly Ser Ala Ile Pro Met Lys Ile 485 490 495 Arg Asn Val Ser
Ala Thr Leu Val Asn Ile Ser Pro Asp Thr Ser Leu 500 505 510 Leu Leu
Asp Cys Gly Glu Gly Thr Phe Gly Gln Leu Cys Arg His Tyr 515 520 525
Gly Asp Gln Val Asp Arg Val Leu Gly Thr Leu Ala Ala Val Phe Val 530
535 540 Ser His Leu His Ala Asp His His Thr Gly Leu Leu Asn Ile Leu
Leu 545 550 555 560 Gln Arg Glu Gln Ala Leu Ala Ser Leu Gly Lys Pro
Leu His Pro Leu 565 570 575 Leu Val Val Ala Pro Ser Gln Leu Lys Ala
Trp Leu Gln Gln Tyr His 580 585 590 Asn Gln Cys Gln Glu Val Leu His
His Ile Ser Met Ile Pro Ala Lys 595 600 605 Cys Leu Gln Glu Gly Ala
Glu Ile Ser Ser Pro Ala Val Glu Arg Leu 610 615 620 Ile Ser Ser Leu
Leu Arg Thr Cys Asp Leu Glu Glu Phe Gln Thr Cys 625 630 635 640 Leu
Val Arg His Cys Lys His Ala Phe Gly Cys Ala Leu Val His Thr 645 650
655 Ser Gly Trp Lys Val Val Tyr Ser Gly Asp Thr Met Pro Cys Glu Ala
660 665 670 Leu Val Arg Met Gly Lys Asp Ala Thr Leu Leu Ile His Glu
Ala Thr 675 680 685 Leu Glu Asp Gly Leu Glu Glu Glu Ala Val Glu Lys
Thr His Ser Thr 690 695 700 Thr Ser Gln Ala Ile Ser Val Gly Met Arg
Met Asn Ala Glu Phe Ile 705 710 715 720 Met Leu Asn His Phe Ser Gln
Arg Tyr Ala Lys Val Pro Leu Phe Ser 725 730 735 Pro Asn Phe Asn Glu
Lys Val Gly Val Ala Phe Asp His Met Lys Val 740 745 750 Cys Phe Gly
Asp Phe Pro Thr Met Pro Lys Leu Ile Pro Pro Leu Lys 755 760 765 Ala
Leu Phe Ala Gly Asp Ile Glu Glu Met Glu Glu Arg Arg Glu Lys 770 775
780 Arg Glu Leu Arg Gln Val Arg Ala Ala Leu Leu Ser Gly Glu Leu Ala
785 790 795 800 Gly Gly Leu Glu Asp Gly Glu Pro Gln Gln Lys Arg Ala
His Thr Glu 805 810 815 Glu Pro Gln Ala Lys Lys Val Arg Ala Gln 820
825 227 844 PRT Caenorhabditis elegans 227 Met Lys Met Leu Phe Phe
Gly Ile Lys Val Ser Arg His Leu Ile Ser 1 5 10 15 Ser Thr Ser Cys
Leu Phe Lys Asp Asn Asn Glu Glu Leu Leu Glu Ser 20 25 30 Ile Lys
Glu Arg Ile Ala Arg Asn Arg Arg Ile Leu Gln Lys His Ser 35 40 45
Ser Ser His Leu Lys Ala Arg Glu Val Asn Ala Ser Ile Ser Asn Leu 50
55 60 Arg Gln Ser Met Ala Ala Val Gln Lys Lys Gln Lys Ala Ala His
Glu 65 70 75 80 Pro Pro Ala Asn Ser Ile Val Asn Ile Pro Ser Gln Val
Ser Ile Glu 85 90 95 Val Leu Gly Asn Gly Thr Gly Leu Leu Arg Ala
Cys Phe Ile Leu Arg 100 105 110 Thr Pro Leu Lys Thr Tyr Met Phe Asn
Cys Pro Glu Asn Ala Cys Arg 115 120 125 Phe Leu Trp Gln Leu Arg Ile
Arg Ser Ser Ser Val Val Asp Leu Phe 130 135 140 Ile Thr Ser Ala Asn
Trp Asp Asn Ile Ala Gly Ile Ser Ser Ile Leu 145 150 155 160 Leu Ser
Lys Glu Ser Asn Ala Leu Ser Thr Arg Leu His Gly Ala Met 165 170 175
Asn Ile Lys His Phe Leu Glu Cys Ile Arg Pro Phe Gln Asp Ser Asp 180
185 190 Tyr Gly Ser Cys Lys Tyr Pro Ser Gln Val Glu Glu Arg Pro Tyr
Thr 195 200 205 Met Glu Asn Tyr Glu Asp Ala Gly Leu Lys Val Thr Tyr
Ile Pro Leu 210 215 220 Ser Pro Pro Leu Asn Ile Gly Ser Asn Asn Glu
Lys Ser Lys Asn Val 225 230 235 240 Lys Val Asn Asn Val Asp Ile Ala
Phe Leu Ile Glu Met Lys Glu Ala 245 250 255 Ala Arg Arg Ile Asp Thr
Met Lys Leu Met Glu Leu Lys Val Pro Lys 260 265 270 Gly Pro Leu Ile
Gly Lys Leu Lys Ser Gly Glu Ala Val Thr Leu Pro 275 280 285 Asp Gly
Arg Thr Ile Gln Pro Asp Gln Val Phe Ser Ser Asp Lys Val 290 295 300
Glu Gly Asp Lys Pro Leu Leu Leu Val Thr Glu Cys Thr Thr Glu Asp 305
310 315 320 His Val Lys Ala Leu Ile Asp Ser Ser Ser Leu Gln Pro Phe
Leu Asn 325 330 335 Gly Glu Lys Gln Leu Asp Tyr Met Val His Ile Ser
Asp Asp Ala Val 340 345 350 Ile Asn Thr Pro Thr Tyr Arg His Leu Met
Glu Lys Leu Asn Asn Pro 355 360 365 Ser Ile Thr His Leu Leu Ile Asn
Gly Gly Asn Pro Val Ile Pro Ala 370 375 380 Val Glu Ser Val Tyr
Lys His Thr Arg Leu Leu Arg Ser Ile Ala Pro 385 390 395 400 Ser Leu
Phe Pro Ala Leu His Pro Ile Asp Trp Ser Gly Ile Ile Thr 405 410 415
Gln Asn Glu Glu Leu Ser Gln Arg Gln Asp Gln Phe Ile Arg Val Ala 420
425 430 Pro Met Gln Arg Tyr Trp Met Arg Arg Gly Ala Ser Phe Asn Glu
Glu 435 440 445 Pro Ile Val Asn Asn Leu Leu Ala Ala Glu Pro Glu Leu
Ser Asp Lys 450 455 460 Ala Lys Glu Leu Ile Lys Glu Tyr Gln Lys Leu
Glu Lys Glu Asn Lys 465 470 475 480 Met Asp Cys Glu Phe Pro Lys Leu
Thr Phe Phe Gly Thr Ser Ser Ala 485 490 495 Val Pro Ser Lys Tyr Arg
Asn Val Thr Gly Tyr Leu Val Glu Ala Ser 500 505 510 Glu Asn Ser Ala
Ile Leu Ile Asp Val Gly Glu Gly Thr Tyr Gly Gln 515 520 525 Met Arg
Ala Val Phe Gly Glu Asp Gly Cys Lys Gln Leu Leu Val Asn 530 535 540
Leu Asn Cys Val Leu Ile Thr His Ala His Gln Asp His Met Asn Gly 545
550 555 560 Leu Tyr Thr Ile Ile Ala Arg Arg Lys Glu Ala Phe Glu Ser
Leu Gly 565 570 575 Ala Pro Tyr Arg Pro Leu Val Leu Val Cys Asn Arg
Asn Val Leu Lys 580 585 590 Pro Met Lys Thr Tyr Ser Ile Cys Phe Glu
Asn Ile Glu His Leu Leu 595 600 605 Glu Ile Val Asp Ile Ser Arg Tyr
Pro Leu Thr Pro Pro Gly Ser Pro 610 615 620 Gly Gly Pro Pro Gly Lys
Arg Pro Arg Leu Pro Ser Pro His Leu Pro 625 630 635 640 Pro Ser Arg
Asp Val Leu Gln Asp Met Ser Ser Ser Phe Asp Lys Lys 645 650 655 Ala
Trp Lys Leu Asp Glu Leu Lys Ala Val Gln Val His His Thr Arg 660 665
670 Met Ala Asn Gly Phe Val Met Arg Val Ala Gly Lys Arg Ile Val Phe
675 680 685 Ser Gly Asp Thr Lys Pro Cys Asp Leu Leu Val Glu Glu Gly
Lys Asp 690 695 700 Ala Asp Val Leu Val His Glu Ser Thr Phe Glu Asp
Gly His Glu Val 705 710 715 720 Asp Met Thr Pro Lys Pro Pro Lys Lys
Leu Ala Lys Ile Ser Ser Leu 725 730 735 Ala Asp Ala Met Arg Lys Arg
His Ser Thr Met Gly Gln Ala Val Asp 740 745 750 Val Gly Lys Arg Met
Asn Ala Lys His Ile Ile Leu Thr His Phe Ser 755 760 765 Ala Arg Tyr
Pro Lys Val Pro Val Leu Pro Glu Tyr Leu Asp Lys Glu 770 775 780 Asn
Ile Gly Val Ala Met Asp Met Leu Arg Val Arg Phe Asp His Leu 785 790
795 800 Pro Leu Val Ser Lys Leu Leu Pro Ile Phe Arg Glu Val Phe Val
Ala 805 810 815 Glu Leu Phe Glu Leu Thr Ile Lys Lys Glu Gln Arg Val
Leu Lys Asp 820 825 830 Lys Glu Leu Ser Glu Lys Arg Gly Gln Leu Lys
Ala 835 840 228 837 PRT Arabidopsis thaliana 228 Met Glu Asn Asn
Glu Ala Thr Asn Gly Ser Lys Ser Ser Ser Asn Ser 1 5 10 15 Phe Val
Phe Asn Lys Arg Arg Ala Glu Gly Phe Asp Ile Thr Asp Lys 20 25 30
Lys Lys Arg Asn Leu Glu Arg Lys Ser Gln Lys Leu Asn Pro Thr Asn 35
40 45 Thr Ile Ala Tyr Ala Gln Ile Leu Gly Thr Gly Met Asp Thr Gln
Asp 50 55 60 Thr Ser Ser Ser Val Leu Leu Phe Phe Asp Lys Gln Arg
Phe Ile Phe 65 70 75 80 Asn Ala Gly Glu Gly Leu Gln Arg Phe Cys Thr
Glu His Lys Ile Lys 85 90 95 Leu Ser Lys Ile Asp His Val Phe Leu
Ser Arg Val Cys Ser Glu Thr 100 105 110 Ala Gly Gly Leu Pro Gly Leu
Leu Leu Thr Leu Ala Gly Ile Gly Glu 115 120 125 Glu Gly Leu Ser Val
Asn Val Trp Gly Pro Ser Asp Leu Asn Tyr Leu 130 135 140 Val Asp Ala
Met Lys Ser Phe Ile Pro Arg Ala Ala Met Val His Thr 145 150 155 160
Arg Ser Phe Gly Pro Ser Ser Thr Pro Asp Pro Ile Val Leu Val Asn 165
170 175 Asp Glu Val Val Lys Ile Ser Ala Ile Ile Leu Lys Pro Cys His
Ser 180 185 190 Glu Glu Asp Ser Gly Asn Lys Ser Gly Asp Leu Ser Val
Val Tyr Val 195 200 205 Cys Glu Leu Pro Glu Ile Leu Gly Lys Phe Asp
Leu Glu Lys Ala Lys 210 215 220 Lys Val Phe Gly Val Lys Pro Gly Pro
Lys Tyr Ser Arg Leu Gln Ser 225 230 235 240 Gly Glu Ser Val Lys Ser
Asp Glu Arg Asp Ile Thr Val His Pro Ser 245 250 255 Asp Val Met Gly
Pro Ser Leu Pro Gly Pro Ile Val Leu Leu Val Asp 260 265 270 Cys Pro
Thr Glu Ser His Ala Ala Glu Leu Phe Ser Leu Lys Ser Leu 275 280 285
Glu Ser Tyr Tyr Ser Ser Pro Asp Glu Gln Thr Ile Gly Ala Lys Phe 290
295 300 Val Asn Cys Ile Ile His Leu Ser Pro Ser Ser Val Thr Ser Ser
Pro 305 310 315 320 Thr Tyr Gln Ser Trp Met Lys Lys Phe His Leu Thr
Gln His Ile Leu 325 330 335 Ala Gly His Gln Arg Phe Leu Pro Leu Leu
Ile Ile Val Ser His Gln 340 345 350 Lys Thr Val Arg Lys Asn Met Ala
Phe Pro Ile Leu Lys Ala Ser Ser 355 360 365 Arg Ile Ala Ala Arg Leu
Asn Tyr Leu Cys Pro Gln Phe Phe Pro Ala 370 375 380 Pro Gly Phe Trp
Pro Ser Gln Leu Thr Asp Asn Ser Ile Ile Asp Pro 385 390 395 400 Thr
Pro Ser Asn Lys Phe Asn Leu Arg Pro Val Ala Ile Arg Gly Ile 405 410
415 Asp Arg Ser Cys Ile Pro Ala Pro Leu Thr Ser Ser Glu Val Val Asp
420 425 430 Glu Leu Leu Ser Glu Ile Pro Glu Ile Lys Asp Lys Ser Glu
Glu Ile 435 440 445 Lys Gln Phe Trp Asn Lys Gln His Asn Lys Thr Ile
Ile Glu Lys Leu 450 455 460 Trp Leu Ser Glu Cys Asn Thr Val Leu Pro
Asn Cys Leu Glu Lys Ile 465 470 475 480 Arg Arg Asp Asp Met Glu Ile
Val Ile Leu Gly Thr Gly Ser Ser Gln 485 490 495 Pro Ser Lys Tyr Arg
Asn Val Ser Ala Ile Phe Ile Asp Leu Phe Ser 500 505 510 Arg Gly Ser
Leu Leu Leu Asp Cys Gly Glu Gly Thr Leu Gly Gln Leu 515 520 525 Lys
Arg Arg Tyr Gly Leu Asp Gly Ala Asp Glu Ala Val Arg Lys Leu 530 535
540 Arg Cys Ile Trp Ile Ser His Ile His Ala Asp His His Thr Gly Leu
545 550 555 560 Ala Arg Ile Leu Ala Leu Arg Ser Lys Leu Leu Lys Gly
Val Thr His 565 570 575 Glu Pro Val Ile Val Val Gly Pro Arg Pro Leu
Lys Arg Phe Leu Asp 580 585 590 Ala Tyr Gln Arg Leu Glu Asp Leu Asp
Met Glu Phe Leu Asp Cys Arg 595 600 605 Ser Thr Thr Ala Thr Ser Trp
Ala Ser Leu Glu Ser Gly Gly Glu Ala 610 615 620 Glu Gly Ser Leu Phe
Thr Gln Gly Ser Pro Met Gln Ser Val Phe Lys 625 630 635 640 Arg Ser
Asp Ile Ser Met Asp Asn Ser Ser Val Leu Leu Cys Leu Lys 645 650 655
Asn Leu Lys Lys Val Leu Ser Glu Ile Gly Leu Asn Asp Leu Ile Ser 660
665 670 Phe Pro Val Val His Cys Pro Gln Ala Tyr Gly Val Val Ile Lys
Ala 675 680 685 Ala Glu Arg Val Asn Ser Val Gly Glu Gln Ile Leu Gly
Trp Lys Met 690 695 700 Val Tyr Ser Gly Asp Ser Arg Pro Cys Pro Glu
Thr Val Glu Ala Ser 705 710 715 720 Arg Asp Ala Thr Ile Leu Ile His
Glu Ala Thr Phe Glu Asp Ala Leu 725 730 735 Ile Glu Glu Ala Leu Ala
Lys Asn His Ser Thr Thr Lys Glu Ala Ile 740 745 750 Asp Val Gly Ser
Ala Ala Asn Val Tyr Arg Ile Val Leu Thr His Phe 755 760 765 Ser Gln
Arg Tyr Pro Lys Ile Pro Val Ile Asp Glu Ser His Met His 770 775 780
Asn Thr Cys Ile Ala Phe Asp Leu Met Ser Ile Asn Met Ala Asp Leu 785
790 795 800 His Val Leu Pro Lys Val Leu Pro Tyr Phe Lys Thr Leu Phe
Arg Asp 805 810 815 Glu Met Val Glu Asp Glu Asp Ala Asp Asp Val Ala
Met Asp Asp Leu 820 825 830 Lys Glu Glu Ala Leu 835 229 838 PRT
Saccharomyces cerevisiae 229 Met Phe Thr Phe Ile Pro Ile Thr His
Pro Thr Ser Asp Thr Lys His 1 5 10 15 Pro Leu Leu Leu Val Gln Ser
Ala His Gly Glu Lys Tyr Phe Phe Gly 20 25 30 Lys Ile Gly Glu Gly
Ser Gln Arg Ser Leu Thr Glu Asn Lys Ile Arg 35 40 45 Ile Ser Lys
Leu Lys Asp Ile Phe Leu Thr Gly Glu Leu Asn Trp Ser 50 55 60 Asp
Ile Gly Gly Leu Pro Gly Met Ile Leu Thr Ile Ala Asp Gln Gly 65 70
75 80 Lys Ser Asn Leu Val Leu His Tyr Gly Asn Asp Ile Leu Asn Tyr
Ile 85 90 95 Val Ser Thr Trp Arg Tyr Phe Val Phe Arg Phe Gly Ile
Asp Leu Asn 100 105 110 Asp His Ile Met Lys Asp Lys Glu Val Tyr Lys
Asp Lys Ile Ile Ala 115 120 125 Val Lys Ser Phe Asn Val Leu Lys Asn
Gly Gly Glu Asp Arg Leu Gly 130 135 140 Val Phe Asp Ser Phe Gln Lys
Gly Val Leu Arg Ser Ile Val Ala Lys 145 150 155 160 Met Phe Pro Lys
His Ala Pro Thr Asp Arg Tyr Asp Pro Ser Ser Asp 165 170 175 Pro His
Leu Asn Val Glu Leu Pro Asp Leu Asp Ala Lys Val Glu Val 180 185 190
Ser Thr Asn Tyr Glu Ile Ser Phe Ser Pro Val Arg Gly Lys Phe Lys 195
200 205 Val Glu Glu Ala Ile Lys Leu Gly Val Pro Lys Gly Pro Leu Phe
Ala 210 215 220 Lys Leu Thr Lys Gly Gln Thr Ile Thr Leu Asp Asn Gly
Ile Val Val 225 230 235 240 Thr Pro Glu Gln Val Leu Glu Asn Glu Arg
His Phe Ala Lys Val Leu 245 250 255 Ile Leu Asp Ile Pro Asp Asp Leu
Tyr Leu Asn Ala Phe Val Glu Lys 260 265 270 Phe Lys Asp Tyr Asp Cys
Ala Glu Leu Gly Met Val Tyr Tyr Phe Leu 275 280 285 Gly Asp Glu Val
Thr Ile Asn Asp Asn Leu Phe Ala Phe Ile Asp Ile 290 295 300 Phe Glu
Lys Asn Asn Tyr Gly Lys Val Asn His Met Ile Ser His Asn 305 310 315
320 Lys Ile Ser Pro Asn Thr Ile Ser Phe Phe Gly Ser Ala Leu Thr Thr
325 330 335 Leu Lys Leu Lys Ala Leu Gln Val Asn Asn Tyr Asn Leu Pro
Lys Thr 340 345 350 Asp Arg Val Phe Ser Lys Asp Phe Tyr Asp Arg Phe
Asp Thr Pro Leu 355 360 365 Ser Arg Gly Thr Ser Met Cys Lys Ser Gln
Glu Glu Pro Leu Asn Thr 370 375 380 Ile Ile Glu Lys Asp Asn Ile His
Ile Phe Ser Gln Asn Lys Thr Val 385 390 395 400 Thr Phe Glu Pro Phe
Arg Met Asn Glu Glu Pro Met Lys Cys Asn Ile 405 410 415 Asn Gly Glu
Val Ala Asp Phe Ser Trp Gln Glu Ile Phe Glu Glu His 420 425 430 Val
Lys Pro Leu Glu Phe Pro Leu Ala Asp Val Asp Thr Val Ile Asn 435 440
445 Asn Gln Leu His Val Asp Asn Phe Asn Asn Ser Ala Glu Lys Lys Lys
450 455 460 His Val Glu Ile Ile Thr Leu Gly Thr Gly Ser Ala Leu Pro
Ser Lys 465 470 475 480 Tyr Arg Asn Val Val Ser Thr Leu Val Lys Val
Pro Phe Thr Asp Ala 485 490 495 Asp Gly Asn Thr Ile Asn Arg Asn Ile
Met Leu Asp Ala Gly Glu Asn 500 505 510 Thr Leu Gly Thr Ile His Arg
Met Phe Ser Gln Leu Ala Val Lys Ser 515 520 525 Ile Phe Gln Asp Leu
Lys Met Ile Tyr Leu Ser His Leu His Ala Asp 530 535 540 His His Leu
Gly Ile Ile Ser Val Leu Asn Glu Trp Tyr Lys Tyr Asn 545 550 555 560
Lys Asp Asp Glu Thr Ser Tyr Ile Tyr Val Val Thr Pro Trp Gln Tyr 565
570 575 His Lys Phe Val Asn Glu Trp Leu Val Leu Glu Asn Lys Glu Ile
Leu 580 585 590 Lys Arg Ile Lys Tyr Ile Ser Cys Glu His Phe Ile Asn
Asp Ser Phe 595 600 605 Val Arg Met Gln Thr Gln Ser Val Pro Leu Ala
Glu Phe Asn Glu Ile 610 615 620 Leu Lys Glu Asn Ser Asn Gln Glu Ser
Asn Arg Lys Leu Glu Leu Asp 625 630 635 640 Arg Asp Ser Ser Tyr Arg
Asp Val Asp Leu Ile Arg Gln Met Tyr Glu 645 650 655 Asp Leu Ser Ile
Glu Tyr Phe Gln Thr Cys Arg Ala Ile His Cys Asp 660 665 670 Trp Ala
Tyr Ser Asn Ser Ile Thr Phe Arg Met Asp Glu Asn Asn Glu 675 680 685
His Asn Thr Phe Lys Val Ser Tyr Ser Gly Asp Thr Arg Pro Asn Ile 690
695 700 Glu Lys Phe Ser Leu Glu Ile Gly Tyr Asn Ser Asp Leu Leu Ile
His 705 710 715 720 Glu Ala Thr Leu Glu Asn Gln Leu Leu Glu Asp Ala
Val Lys Lys Lys 725 730 735 His Cys Thr Ile Asn Glu Ala Ile Gly Val
Ser Asn Lys Met Asn Ala 740 745 750 Arg Lys Leu Ile Leu Thr His Phe
Ser Gln Arg Tyr Pro Lys Leu Pro 755 760 765 Gln Leu Asp Asn Asn Ile
Asp Val Met Ala Arg Glu Phe Cys Phe Ala 770 775 780 Phe Asp Ser Met
Ile Val Asp Tyr Glu Lys Ile Gly Glu Gln Gln Arg 785 790 795 800 Ile
Phe Pro Leu Leu Asn Lys Ala Phe Val Glu Glu Lys Glu Glu Glu 805 810
815 Glu Asp Val Asp Asp Val Glu Ser Val Gln Asp Leu Glu Val Lys Leu
820 825 830 Lys Lys His Lys Lys Asn 835 230 311 PRT Escherichia
coli 230 Met Lys Arg Asp Glu Leu Met Glu Leu Ile Phe Leu Gly Thr
Ser Ala 1 5 10 15 Gly Val Pro Thr Arg Thr Arg Asn Val Thr Ala Ile
Leu Leu Asn Leu 20 25 30 Gln His Pro Thr Gln Ser Gly Leu Trp Leu
Phe Asp Cys Gly Glu Gly 35 40 45 Thr Gln His Gln Leu Leu His Thr
Ala Phe Asn Pro Gly Lys Leu Asp 50 55 60 Lys Ile Phe Ile Ser His
Leu His Gly Asp His Leu Phe Gly Leu Pro 65 70 75 80 Gly Leu Leu Cys
Ser Arg Ser Met Ser Gly Ile Ile Gln Pro Leu Thr 85 90 95 Ile Tyr
Gly Pro Gln Gly Ile Arg Glu Phe Val Glu Thr Ala Leu Arg 100 105 110
Ile Ser Gly Ser Trp Thr Asp Tyr Pro Leu Glu Ile Val Glu Ile Gly 115
120 125 Ala Gly Glu Ile Leu Asp Asp Gly Leu Arg Lys Val Thr Ala Tyr
Pro 130 135 140 Leu Glu His Pro Leu Glu Cys Tyr Gly Tyr Arg Ile Glu
Glu His Asp 145 150 155 160 Lys Pro Gly Ala Leu Asn Ala Gln Ala Leu
Lys Ala Ala Gly Val Pro 165 170 175 Pro Gly Pro Leu Phe Gln Glu Leu
Lys Ala Gly Lys Thr Ile Thr Leu 180 185 190 Glu Asp Gly Arg Gln Ile
Asn Gly Ala Asp Tyr Leu Ala Ala Pro Val 195 200 205 Pro Gly Lys Ala
Leu Ala Ile Phe Gly Asp Thr Gly Pro Cys Asp Ala 210 215 220 Ala Leu
Asp Leu Ala Lys Gly Val Asp Val Met Val His Glu Ala Thr 225 230 235
240 Leu Asp Ile Thr Met Glu Ala Lys Ala Asn Ser Arg Gly His Ser Ser
245 250 255 Thr Arg Gln Ala Ala Thr Leu Ala Arg Glu Ala Gly Val Gly
Lys Leu 260 265 270 Ile Ile Thr His Val Ser Ser Arg Tyr Asp Asp Lys
Gly Cys Gln His 275 280 285 Leu Leu Arg Glu Cys Arg Ser Ile Phe Pro
Ala Thr Glu Leu Ala Asn 290 295 300 Asp Phe
Thr Val Phe Asn Val 305 310 231 326 PRT Synechocystis sp. 231 Met
Glu Ile Thr Phe Leu Gly Thr Ser Ser Gly Val Pro Thr Arg Asn 1 5 10
15 Arg Asn Val Ser Ser Ile Ala Leu Arg Leu Pro Gln Arg Ala Glu Leu
20 25 30 Trp Leu Phe Asp Cys Gly Glu Gly Thr Gln His Gln Phe Leu
Arg Ser 35 40 45 Glu Val Lys Ile Ser Gln Leu Thr Arg Ile Phe Ile
Thr His Leu His 50 55 60 Gly Asp His Ile Phe Gly Leu Met Gly Leu
Leu Ala Ser Ser Gly Leu 65 70 75 80 Ala Gly Ser Gly Gln Gly Ile Glu
Ile Tyr Gly Pro Glu Gly Leu Gly 85 90 95 Asp Tyr Leu Glu Ala Cys
Cys Arg Phe Ser Ser Thr His Leu Gly Lys 100 105 110 Arg Leu Lys Val
His Thr Val Arg Glu Asn Gly Leu Ile Tyr Glu Asp 115 120 125 Lys Asp
Phe Gln Val His Cys Gly Leu Leu Lys His Arg Ile Pro Ala 130 135 140
Tyr Gly Tyr Arg Val Glu Glu Lys Gln Arg Pro Gly Arg Phe Asn Val 145
150 155 160 Glu Gln Ala Glu Ala Leu Gly Ile Pro Phe Gly Pro Ile Tyr
Gly Gln 165 170 175 Leu Lys Gln Gly Lys Thr Val Thr Leu Glu Asp Gly
Arg Arg Ile Arg 180 185 190 Gly Gln Asp Leu Cys Glu Pro Pro Glu Pro
Gly Arg Lys Phe Val Tyr 195 200 205 Cys Thr Asp Thr Val Phe Cys Glu
Glu Ala Ile Ala Leu Ala Gln Glu 210 215 220 Ala Asp Leu Leu Val His
Glu Ala Thr Phe Ala His Gln Asp Ala Gln 225 230 235 240 Leu Ala Phe
Asp Arg Leu His Ser Thr Ser Thr Met Ala Ala Gln Val 245 250 255 Ala
Leu Leu Ala Asn Val Lys Gln Leu Ile Met Thr His Phe Ser Pro 260 265
270 Arg Tyr Ala Pro Gly Asn Pro Leu Gln Leu Glu Asn Leu Leu Ala Glu
275 280 285 Ala Gln Ala Ile Phe Pro Asn Thr Arg Leu Ala Arg Asp Phe
Leu Thr 290 295 300 Val Glu Ile Pro Arg Arg Thr Ala Asp Pro Ala Ile
Ala Met Ser Thr 305 310 315 320 Pro Gln Ala Ser Pro Ala 325 232 307
PRT Methanobacterium thermoautotrophicum 232 Met Met Glu Val Thr
Phe Leu Gly Thr Ser Ser Ala Val Pro Ser Lys 1 5 10 15 Asn Arg Asn
His Thr Ser Ile Ala Leu Arg Ile Pro Gly Glu Ile Phe 20 25 30 Leu
Phe Asp Cys Gly Glu Gly Thr Gln Arg Gln Met Ala Leu Ala Gly 35 40
45 Ile Ser Pro Met Lys Val Thr Arg Ile Phe Ile Thr His Leu His Gly
50 55 60 Asp His Ile Leu Gly Ile Pro Gly Met Ile Gln Ser Met Gly
Phe Arg 65 70 75 80 Gly Arg Glu Glu Pro Leu Asp Ile Tyr Gly Pro Pro
Gly Ile His Glu 85 90 95 Leu His Glu Cys Ile Met Lys Met Gly Tyr
Phe Thr Leu Asp Phe Asp 100 105 110 Ile Asn Val His Glu Val Arg Gly
Gly Thr Val Val Glu Glu Asp Asp 115 120 125 Tyr Arg Val Thr Ser Ala
Pro Ala Ser His Ser Val Phe Asn Leu Ala 130 135 140 Tyr Cys Phe Glu
Glu Lys Lys Arg Pro Arg Phe Leu Arg Glu Lys Ala 145 150 155 160 Ile
Ala Leu Gly Leu Lys Pro Gly Pro Ala Phe Gly Lys Leu His Arg 165 170
175 Gly Ile Pro Val Arg Val Gly Asp Arg Ile Ile Met Pro Glu Glu Val
180 185 190 Leu Gly Ser Pro Arg Lys Gly Val Lys Val Cys Tyr Ser Gly
Asp Thr 195 200 205 Arg Pro Cys Glu Ser Val Ile Lys Leu Ala Glu Gly
Ala Glu Leu Leu 210 215 220 Ile His Glu Ser Thr Leu Glu Ala Gly Ser
Glu Asp Lys Ala Ala Glu 225 230 235 240 Ser Gly His Ser Thr Ala Arg
Glu Ala Ala Glu Val Ala Arg Ser Ala 245 250 255 Gly Val Lys Arg Leu
Ile Leu Thr His Leu Ser Thr Arg Tyr Lys Arg 260 265 270 Thr Glu Val
Ile Leu Glu Ala Ala Arg Gln Val Phe Pro Val Thr Asp 275 280 285 Val
Ala Asp Asp Leu Met Thr Val Glu Val Lys Ala Tyr Asp Ser Ser 290 295
300 Pro Asp Ser 305 233 684 PRT Homo sapiens 233 Met Ser Ala Ile
Pro Ala Glu Glu Ser Asp Gln Leu Leu Ile Arg Pro 1 5 10 15 Leu Gly
Ala Gly Gln Glu Val Gly Arg Ser Cys Ile Ile Leu Glu Phe 20 25 30
Lys Gly Arg Lys Ile Met Leu Asp Cys Gly Ile His Pro Gly Leu Glu 35
40 45 Gly Met Asp Ala Leu Pro Tyr Ile Asp Leu Ile Asp Pro Ala Glu
Ile 50 55 60 Asp Leu Leu Leu Ile Ser His Phe His Leu Asp His Cys
Gly Ala Leu 65 70 75 80 Pro Trp Phe Leu Gln Lys Thr Ser Phe Lys Gly
Arg Thr Phe Met Thr 85 90 95 His Ala Thr Lys Ala Ile Tyr Arg Trp
Leu Leu Ser Asp Tyr Val Lys 100 105 110 Val Ser Asn Ile Ser Ala Asp
Asp Met Leu Tyr Thr Glu Thr Asp Leu 115 120 125 Glu Glu Ser Met Asp
Lys Ile Glu Thr Ile Asn Phe His Glu Val Lys 130 135 140 Glu Val Ala
Gly Ile Lys Phe Trp Cys Tyr His Ala Gly His Val Leu 145 150 155 160
Gly Ala Ala Met Phe Met Ile Glu Ile Ala Gly Val Lys Leu Leu Tyr 165
170 175 Thr Gly Asp Phe Ser Arg Gln Glu Asp Arg His Leu Met Ala Ala
Glu 180 185 190 Ile Pro Asn Ile Lys Pro Asp Ile Leu Ile Ile Glu Ser
Thr Tyr Gly 195 200 205 Thr His Ile His Glu Lys Arg Glu Glu Arg Glu
Ala Arg Phe Cys Asn 210 215 220 Thr Val His Asp Ile Val Asn Arg Gly
Gly Arg Gly Leu Ile Pro Val 225 230 235 240 Phe Ala Leu Gly Arg Ala
Gln Glu Leu Leu Leu Ile Leu Asp Glu Tyr 245 250 255 Trp Gln Asn His
Pro Glu Leu His Asp Ile Pro Ile Tyr Tyr Ala Ser 260 265 270 Ser Leu
Ala Lys Lys Cys Met Ala Val Tyr Gln Thr Tyr Val Asn Ala 275 280 285
Met Asn Asp Lys Ile Arg Lys Gln Ile Asn Ile Asn Asn Pro Phe Val 290
295 300 Phe Lys His Ile Ser Asn Leu Lys Ser Met Asp His Phe Asp Asp
Ile 305 310 315 320 Gly Pro Ser Val Val Met Ala Ser Pro Gly Met Met
Gln Ser Gly Leu 325 330 335 Ser Arg Glu Leu Phe Glu Ser Trp Cys Thr
Asp Lys Arg Asn Gly Val 340 345 350 Ile Ile Ala Gly Tyr Cys Val Glu
Gly Thr Leu Ala Lys His Ile Met 355 360 365 Ser Glu Pro Glu Glu Ile
Thr Thr Met Ser Gly Gln Lys Leu Pro Leu 370 375 380 Lys Met Ser Val
Asp Tyr Ile Ser Phe Ser Ala His Thr Asp Tyr Gln 385 390 395 400 Gln
Thr Ser Glu Phe Ile Arg Ala Leu Lys Pro Pro His Val Ile Leu 405 410
415 Val His Gly Glu Gln Asn Glu Met Ala Arg Leu Lys Ala Ala Leu Ile
420 425 430 Arg Glu Tyr Glu Asp Asn Asp Glu Val His Ile Glu Val His
Asn Pro 435 440 445 Arg Asn Thr Glu Ala Val Thr Leu Asn Phe Arg Gly
Glu Lys Leu Ala 450 455 460 Lys Val Met Gly Phe Leu Ala Asp Lys Lys
Pro Glu Gln Gly Gln Arg 465 470 475 480 Val Ser Gly Ile Leu Val Lys
Arg Asn Phe Asn Tyr His Ile Leu Ser 485 490 495 Pro Cys Asp Leu Ser
Asn Tyr Thr Asp Leu Ala Met Ser Thr Val Lys 500 505 510 Gln Thr Gln
Ala Ile Pro Tyr Thr Gly Pro Phe Asn Leu Leu Cys Tyr 515 520 525 Gln
Leu Gln Lys Leu Thr Gly Asp Val Glu Glu Leu Glu Ile Gln Glu 530 535
540 Lys Pro Ala Leu Lys Val Phe Lys Asn Ile Thr Val Ile Gln Glu Pro
545 550 555 560 Gly Met Val Val Leu Glu Trp Leu Ala Asn Pro Ser Asn
Asp Met Tyr 565 570 575 Ala Asp Thr Val Thr Thr Val Ile Leu Glu Val
Gln Ser Asn Pro Lys 580 585 590 Ile Arg Lys Gly Ala Val Gln Lys Val
Ser Lys Lys Leu Glu Met His 595 600 605 Val Tyr Ser Lys Arg Leu Glu
Ile Met Leu Gln Asp Ile Phe Gly Glu 610 615 620 Asp Cys Val Ser Val
Lys Asp Asp Ser Ile Leu Ser Val Thr Val Asp 625 630 635 640 Gly Lys
Thr Ala Asn Leu Asn Leu Glu Thr Arg Thr Val Glu Cys Glu 645 650 655
Glu Gly Ser Glu Asp Asp Glu Ser Leu Arg Glu Met Val Glu Leu Ala 660
665 670 Ala Gln Arg Leu Tyr Glu Ala Leu Thr Pro Val His 675 680 234
693 PRT Arabidopsis thaliana 234 Met Ala Ser Ser Ser Thr Ser Leu
Lys Arg Arg Glu Gln Pro Ile Ser 1 5 10 15 Arg Asp Gly Asp Gln Leu
Ile Val Thr Pro Leu Gly Ala Gly Ser Glu 20 25 30 Val Gly Arg Ser
Cys Val Tyr Met Ser Phe Arg Gly Lys Asn Ile Leu 35 40 45 Phe Asp
Cys Gly Ile His Pro Ala Tyr Ser Gly Met Ala Ala Leu Pro 50 55 60
Tyr Phe Asp Glu Ile Asp Pro Ser Ser Ile Asp Val Leu Leu Ile Thr 65
70 75 80 His Phe His Ile Asp His Ala Ala Ser Leu Pro Tyr Phe Leu
Glu Lys 85 90 95 Thr Thr Phe Asn Gly Arg Val Phe Met Thr His Ala
Thr Lys Ala Ile 100 105 110 Tyr Lys Leu Leu Leu Thr Asp Tyr Val Lys
Val Ser Lys Val Ser Val 115 120 125 Glu Asp Met Leu Phe Asp Glu Gln
Asp Ile Asn Lys Ser Met Asp Lys 130 135 140 Ile Glu Val Ile Asp Phe
His Gln Thr Val Glu Val Asn Gly Ile Lys 145 150 155 160 Phe Trp Cys
Tyr Thr Ala Gly His Val Leu Gly Ala Ala Met Phe Met 165 170 175 Val
Asp Ile Ala Gly Val Arg Ile Leu Tyr Thr Gly Asp Tyr Ser Arg 180 185
190 Glu Glu Asp Arg His Leu Arg Ala Ala Glu Leu Pro Gln Phe Ser Pro
195 200 205 Asp Ile Cys Ile Ile Glu Ser Thr Ser Gly Val Gln Leu His
Gln Ser 210 215 220 Arg His Ile Arg Glu Lys Arg Phe Thr Asp Val Ile
His Ser Thr Val 225 230 235 240 Ala Gln Gly Gly Arg Val Leu Ile Pro
Ala Phe Ala Leu Gly Arg Ala 245 250 255 Gln Glu Leu Leu Leu Ile Leu
Asp Glu Tyr Trp Ala Asn His Pro Asp 260 265 270 Leu His Asn Ile Pro
Ile Tyr Tyr Ala Ser Pro Leu Ala Lys Lys Cys 275 280 285 Met Ala Val
Tyr Gln Thr Tyr Ile Leu Ser Met Asn Asp Arg Ile Arg 290 295 300 Asn
Gln Phe Ala Asn Ser Asn Pro Phe Val Phe Lys His Ile Ser Pro 305 310
315 320 Leu Asn Ser Ile Asp Asp Phe Asn Asp Val Gly Pro Ser Val Val
Met 325 330 335 Ala Thr Pro Gly Gly Leu Gln Ser Gly Leu Ser Arg Gln
Leu Phe Asp 340 345 350 Ser Trp Cys Ser Asp Lys Lys Asn Ala Cys Ile
Ile Pro Gly Tyr Met 355 360 365 Val Glu Gly Thr Leu Ala Lys Thr Ile
Ile Asn Glu Pro Lys Glu Val 370 375 380 Thr Leu Met Asn Gly Leu Thr
Ala Pro Leu Asn Met Gln Val His Tyr 385 390 395 400 Ile Ser Phe Ser
Ala His Ala Asp Tyr Ala Gln Thr Ser Thr Phe Leu 405 410 415 Lys Glu
Leu Met Pro Pro Asn Ile Ile Leu Val His Gly Glu Ala Asn 420 425 430
Glu Met Met Arg Leu Lys Gln Lys Leu Leu Thr Glu Phe Pro Asp Gly 435
440 445 Asn Thr Lys Ile Met Thr Pro Lys Asn Cys Glu Ser Val Glu Met
Tyr 450 455 460 Phe Asn Ser Glu Lys Leu Ala Lys Thr Ile Gly Arg Leu
Ala Glu Lys 465 470 475 480 Thr Pro Asp Val Gly Asp Thr Val Ser Gly
Ile Leu Val Lys Lys Gly 485 490 495 Phe Thr Tyr Gln Ile Met Ala Pro
Asp Glu Leu His Val Phe Ser Gln 500 505 510 Leu Ser Thr Ala Thr Val
Thr Gln Arg Ile Thr Ile Pro Phe Val Gly 515 520 525 Ala Phe Gly Val
Ile Lys His Arg Leu Glu Lys Ile Phe Glu Ser Val 530 535 540 Glu Phe
Ser Thr Asp Glu Glu Ser Gly Leu Pro Ala Leu Lys Val His 545 550 555
560 Glu Arg Val Thr Val Lys Gln Glu Ser Glu Lys His Ile Ser Leu Gln
565 570 575 Trp Ser Ser Asp Pro Ile Ser Asp Met Val Ser Asp Ser Ile
Val Ala 580 585 590 Leu Ile Leu Asn Ile Ser Arg Glu Val Pro Lys Ile
Val Met Glu Glu 595 600 605 Glu Asp Ala Val Lys Ser Glu Glu Glu Asn
Gly Lys Lys Val Glu Lys 610 615 620 Val Ile Tyr Ala Leu Leu Val Ser
Leu Phe Gly Asp Val Lys Leu Gly 625 630 635 640 Glu Asn Gly Lys Leu
Val Ile Arg Val Asp Gly Asn Val Ala Gln Leu 645 650 655 Asp Lys Glu
Ser Gly Glu Val Glu Ser Glu His Ser Gly Leu Lys Glu 660 665 670 Arg
Val Arg Val Ala Phe Glu Arg Ile Gln Ser Ala Val Lys Pro Ile 675 680
685 Pro Leu Ser Ala Ser 690 235 779 PRT Saccharomyces cerevisiae
235 Met Glu Arg Thr Asn Thr Thr Thr Phe Lys Phe Phe Ser Leu Gly Gly
1 5 10 15 Ser Asn Glu Val Gly Arg Ser Cys His Ile Leu Gln Tyr Lys
Gly Lys 20 25 30 Thr Val Met Leu Asp Ala Gly Ile His Pro Ala Tyr
Gln Gly Leu Ala 35 40 45 Ser Leu Pro Phe Tyr Asp Glu Phe Asp Leu
Ser Lys Val Asp Ile Leu 50 55 60 Leu Ile Ser His Phe His Leu Asp
His Ala Ala Ser Leu Pro Tyr Val 65 70 75 80 Met Gln Arg Thr Asn Phe
Gln Gly Arg Val Phe Met Thr His Pro Thr 85 90 95 Lys Ala Ile Tyr
Arg Trp Leu Leu Arg Asp Phe Val Arg Val Thr Ser 100 105 110 Ile Gly
Ser Ser Ser Ser Ser Met Gly Thr Lys Asp Glu Gly Leu Phe 115 120 125
Ser Asp Glu Asp Leu Val Asp Ser Phe Asp Lys Ile Glu Thr Val Asp 130
135 140 Tyr His Ser Thr Val Asp Val Asn Gly Ile Lys Phe Thr Ala Phe
His 145 150 155 160 Ala Gly His Val Leu Gly Ala Ala Met Phe Gln Ile
Glu Ile Ala Gly 165 170 175 Leu Arg Val Leu Phe Thr Gly Asp Tyr Ser
Arg Glu Val Asp Arg His 180 185 190 Leu Asn Ser Ala Glu Val Pro Pro
Leu Ser Ser Asn Val Leu Ile Val 195 200 205 Glu Ser Thr Phe Gly Thr
Ala Thr His Glu Pro Arg Leu Asn Arg Glu 210 215 220 Arg Lys Leu Thr
Gln Leu Ile His Ser Thr Val Met Arg Gly Gly Arg 225 230 235 240 Val
Leu Leu Pro Val Phe Ala Leu Gly Arg Ala Gln Glu Ile Met Leu 245 250
255 Ile Leu Asp Glu Tyr Trp Ser Gln His Ala Asp Glu Leu Gly Gly Gly
260 265 270 Gln Val Pro Ile Phe Tyr Ala Ser Asn Leu Ala Lys Lys Cys
Met Ser 275 280 285 Val Phe Gln Thr Tyr Val Asn Met Met Asn Asp Asp
Ile Arg Lys Lys 290 295 300 Phe Arg Asp Ser Gln Thr Asn Pro Phe Ile
Phe Lys Asn Ile Ser Tyr 305 310 315 320 Leu Arg Asn Leu Glu Asp Phe
Gln Asp Phe Gly Pro Ser Val Met Leu 325 330 335 Ala Ser Pro Gly Met
Leu Gln Ser Gly Leu Ser Arg Asp Leu Leu Glu 340 345 350 Arg Trp Cys
Pro Glu Asp Lys Asn Leu Val Leu Ile Thr Gly Tyr Ser 355 360 365 Ile
Glu Gly Thr Met Ala Lys Phe Ile Met Leu Glu Pro Asp Thr Ile 370 375
380 Pro Ser Ile Asn Asn Pro Glu Ile Thr Ile Pro Arg Arg Cys Gln Val
385 390 395 400 Glu Glu Ile Ser Phe Ala Ala His Val Asp Phe Gln Glu
Asn Leu Glu
405 410 415 Phe Ile Glu Lys Ile Ser Ala Pro Asn Ile Ile Leu Val His
Gly Glu 420 425 430 Ala Asn Pro Met Gly Arg Leu Lys Ser Ala Leu Leu
Ser Asn Phe Ala 435 440 445 Ser Leu Lys Gly Thr Asp Asn Glu Val His
Val Phe Asn Pro Arg Asn 450 455 460 Cys Val Glu Val Asp Leu Glu Phe
Gln Gly Val Lys Val Ala Lys Ala 465 470 475 480 Val Gly Asn Ile Val
Asn Glu Ile Tyr Lys Glu Glu Asn Val Glu Ile 485 490 495 Lys Glu Glu
Ile Ala Ala Lys Ile Glu Pro Ile Lys Glu Glu Asn Glu 500 505 510 Asp
Asn Leu Asp Ser Gln Ala Glu Lys Gly Leu Val Asp Glu Glu Glu 515 520
525 His Lys Asp Ile Val Val Ser Gly Ile Leu Val Ser Asp Asp Lys Asn
530 535 540 Phe Glu Leu Asp Phe Leu Ser Leu Ser Asp Leu Arg Glu His
His Pro 545 550 555 560 Asp Leu Ser Thr Thr Ile Leu Arg Glu Arg Gln
Ser Val Arg Val Asn 565 570 575 Cys Lys Lys Glu Leu Ile Tyr Trp His
Ile Leu Gln Met Phe Gly Glu 580 585 590 Ala Glu Val Leu Gln Asp Asp
Asp Arg Val Thr Asn Gln Glu Pro Lys 595 600 605 Val Lys Glu Glu Ser
Lys Asp Asn Leu Thr Asn Thr Gly Lys Leu Ile 610 615 620 Leu Gln Ile
Met Gly Asp Ile Lys Leu Thr Ile Val Asn Thr Leu Ala 625 630 635 640
Val Val Glu Trp Thr Gln Asp Leu Met Asn Asp Thr Val Ala Asp Ser 645
650 655 Ile Ile Ala Ile Leu Met Asn Val Asp Ser Ala Pro Ala Ser Val
Lys 660 665 670 Leu Ser Ser His Ser Cys Asp Asp His Asp His Asn Asn
Val Gln Ser 675 680 685 Asn Ala Gln Gly Lys Ile Asp Glu Val Glu Arg
Val Lys Gln Ile Ser 690 695 700 Arg Leu Phe Lys Glu Gln Phe Gly Asp
Cys Phe Thr Leu Phe Leu Asn 705 710 715 720 Lys Asp Glu Tyr Ala Ser
Asn Lys Glu Glu Thr Ile Thr Gly Val Val 725 730 735 Thr Ile Gly Lys
Ser Thr Ala Lys Ile Asp Phe Asn Asn Met Lys Ile 740 745 750 Leu Glu
Cys Asn Ser Asn Pro Leu Lys Gly Arg Val Glu Ser Leu Leu 755 760 765
Asn Ile Gly Gly Asn Leu Val Thr Pro Leu Cys 770 775 236 554 PRT
Synechocystis sp. 236 Met Thr Phe Ser Val Pro Thr Gln Gly Lys Ala
Phe Ala Asn Ile Ser 1 5 10 15 Phe Leu Pro Tyr Gly Val Gly Pro Arg
Asp Gly Gly Ile Cys Leu Glu 20 25 30 Leu His Leu Gly Pro Tyr Arg
Ile Leu Leu Asp Cys Gly Leu Glu Asp 35 40 45 Leu Thr Pro Leu Leu
Ala Ala Asp Pro Gly Thr Val Asp Leu Val Phe 50 55 60 Cys Ser His
Ala His Arg Asp His Gly Leu Gly Leu Trp Gln Phe His 65 70 75 80 Gln
Gln Phe Pro His Ile Pro Ile Leu Ala Ser Glu Val Thr Gln Arg 85 90
95 Leu Leu Pro Leu Asn Trp Pro Asp Glu Phe Val Pro Pro Phe Cys Arg
100 105 110 Val Leu Pro Trp Arg Ser Pro Gln Glu Val Leu Pro Gly Leu
Thr Val 115 120 125 Glu Leu Leu Pro Ala Gly His Leu Pro Gly Ala Ala
Leu Ile Leu Leu 130 135 140 Glu Tyr His Asn Gly Asp Arg Leu Tyr Arg
Val Ile Tyr Thr Gly Asp 145 150 155 160 Tyr Cys Leu Ser His Leu Gln
Leu Val Asp Gly Leu Ala Leu Thr Pro 165 170 175 Leu Arg Gly Leu Lys
Pro Asp Val Leu Ile Leu Glu Gly His Tyr Gly 180 185 190 Asn Arg Arg
Leu Pro His Arg Arg Gln Gln Glu Lys Gln Phe Ile Gln 195 200 205 Ala
Ile Glu Thr Val Leu Ala Lys Gly Arg Asn Ile Leu Leu Pro Val 210 215
220 Pro Pro Leu Gly Leu Ala Gln Glu Ile Leu Lys Leu Leu Arg Thr His
225 230 235 240 His Gln Phe Thr Gly Arg Gln Val Asn Leu Trp Ala Gly
Glu Ser Val 245 250 255 Ala Arg Gly Cys Asp Ala Tyr Gln Gly Ile Ile
Asp His Leu Pro Asp 260 265 270 Asn Val Arg Asn Phe Ala Gln His Gln
Pro Leu Phe Trp Asp Asp Lys 275 280 285 Val Tyr Pro His Leu Arg Pro
Leu Thr Asp Asp Gln Gly Glu Leu Ser 290 295 300 Leu Ser Ala Pro Ser
Ile Val Ile Thr Thr Thr Trp Pro Ala Phe Trp 305 310 315 320 Pro Ser
Pro Ala Ala Leu Pro Gly Leu Trp Thr Val Phe Met Pro Gln 325 330 335
Leu Leu Thr Leu Pro Ser Cys Leu Val Asn Phe Ala Trp Gln Asp Leu 340
345 350 Glu Glu Phe Pro Lys Tyr Glu Leu Glu Asp Tyr Leu Leu Ala Asp
His 355 360 365 Ser Asp Gly Arg Asn Thr Thr Gln Leu Ile His Asn Leu
Arg Pro Gln 370 375 380 His Leu Val Phe Val His Gly Gln Pro Ser Asp
Ile Glu Asp Leu Thr 385 390 395 400 Ser Leu Glu Glu Leu Gln Ser Arg
Tyr Gln Leu His Ser Pro Ala Ala 405 410 415 Gly Asn Ala Val Ala Leu
Pro Ile Gly Asp Arg Phe Val Gln Pro Thr 420 425 430 Pro Pro Pro Pro
Gln Ile Tyr Glu Gly Glu Ile His Glu Leu Glu Pro 435 440 445 Asn Lys
Gln Ile His His Leu Gly Glu Val Val Ile His Leu Asp Gly 450 455 460
Gln Ile Leu Glu Asn Ser Arg Trp Gly Lys Phe Gly Glu Thr Gly Ile 465
470 475 480 Val Gln Ala Arg Trp Gln Gly Glu Glu Leu Val Leu Arg Gly
Ile Ser 485 490 495 Gln Arg Glu Leu Leu Lys Gln Asn Gln Ser Ser Lys
Arg Pro Val Asp 500 505 510 Phe Asp Cys Cys Ala Asn Cys Arg His Phe
Gln His Tyr His Cys Arg 515 520 525 Asn Pro Val Ser Pro Leu Met Gly
Leu Glu Val Arg Ala Asp Gly His 530 535 540 Cys Pro Val Phe Glu Ser
Val Ala Ser Ser 545 550 237 636 PRT Methanobacterium
thermoautotrophicum 237 Met Val Ser Glu Met Leu Glu Glu Ile Lys Arg
Thr Ile Met Gln Arg 1 5 10 15 Leu Pro Glu Arg Val Gln Val Ala Lys
Val Glu Phe Glu Gly Pro Glu 20 25 30 Val Val Ile Tyr Thr Lys Asn
Pro Glu Ile Ile Thr Glu Asn Gly Asn 35 40 45 Leu Ile Arg Asp Ile
Ala Lys Asp Ile Arg Lys Arg Ile Ile Ile Arg 50 55 60 Ser Asp Arg
Ser Val Leu Met Asp Pro Glu Lys Ala Ile Arg Lys Ile 65 70 75 80 His
Glu Ile Val Pro Glu Glu Ala Lys Ile Thr Asn Ile Ser Phe Asp 85 90
95 Asp Val Thr Cys Glu Val Ile Ile Glu Ala Arg Lys Pro Gly Leu Val
100 105 110 Ile Gly Lys Tyr Gly Ser Thr Ser Arg Glu Ile Val Lys Asn
Thr Gly 115 120 125 Trp Ala Pro Lys Ile Leu Arg Thr Pro Pro Ile Ser
Ser Glu Ile Ile 130 135 140 Glu Arg Ile Arg Arg Thr Leu Arg Lys Asn
Ser Lys Glu Arg Lys Lys 145 150 155 160 Ile Leu Gln Gln Leu Gly Asn
Arg Ile His Gln Lys Pro Lys Tyr Asp 165 170 175 Asn Asp Trp Ala Arg
Leu Thr Ala Met Gly Gly Phe Arg Glu Val Gly 180 185 190 Arg Ser Cys
Leu Tyr Leu Gln Thr Pro Asn Ser Arg Val Leu Leu Asp 195 200 205 Cys
Gly Val Asn Val Ala Gly Gly Asp Asp Lys Asn Ser Tyr Pro Tyr 210 215
220 Leu Asn Val Pro Glu Phe Thr Leu Asp Ser Leu Asp Ala Val Ile Ile
225 230 235 240 Thr His Ala His Leu Asp His Ser Gly Phe Leu Pro Tyr
Leu Tyr His 245 250 255 Tyr Gly Tyr Asp Gly Pro Val Tyr Cys Thr Ala
Pro Thr Arg Asp Leu 260 265 270 Met Thr Leu Leu Gln Leu Asp His Ile
Asp Ile Ala His Arg Glu Asp 275 280 285 Glu Pro Leu Pro Phe Asn Val
Lys His Val Lys Lys Ser Val Lys His 290 295 300 Thr Ile Thr Leu Asp
Tyr Gly Glu Val Thr Asp Ile Ala Pro Asp Ile 305 310 315 320 Arg Leu
Thr Leu His Asn Ala Gly His Ile Leu Gly Ser Ala Met Ala 325 330 335
His Leu His Ile Gly Asp Gly Gln His Asn Met Val Tyr Thr Gly Asp 340
345 350 Phe Lys Tyr Glu Gln Ser Arg Leu Leu Glu Ala Ala Ala Asn Arg
Phe 355 360 365 Pro Arg Ile Glu Thr Leu Val Met Glu Ser Thr Tyr Gly
Gly His Glu 370 375 380 Asp Val Gln Pro Ser Arg Asn Arg Ala Glu Lys
Glu Leu Val Lys Thr 385 390 395 400 Ile Tyr Ser Thr Leu Arg Arg Gly
Gly Lys Ile Leu Ile Pro Val Phe 405 410 415 Ala Val Gly Arg Ala Gln
Glu Leu Met Ile Val Leu Glu Glu Tyr Ile 420 425 430 Arg Thr Gly Ile
Ile Asp Glu Val Pro Val Tyr Ile Asp Gly Met Ile 435 440 445 Trp Glu
Ala Asn Ala Ile His Thr Ala Arg Pro Glu Tyr Leu Ser Lys 450 455 460
Asp Leu Arg Asp Gln Ile Phe His Met Gly His Asn Pro Phe Ile Ser 465
470 475 480 Asp Ile Phe His Lys Val Asn Gly Met Asp Glu Arg Arg Glu
Ile Val 485 490 495 Glu Gly Glu Pro Ser Ile Ile Leu Ser Thr Ser Gly
Met Leu Thr Gly 500 505 510 Gly Asn Ser Leu Glu Tyr Phe Lys Trp Leu
Cys Glu Asp Pro Asp Asn 515 520 525 Ser Leu Val Phe Val Gly Tyr Gln
Ala Glu Gly Ser Leu Gly Arg Arg 530 535 540 Ile Gln Lys Gly Trp Lys
Glu Ile Pro Leu Lys Asp Glu Asp Asp Lys 545 550 555 560 Met Arg Val
Tyr Asn Val Arg Met Asn Ile Lys Thr Ile Glu Gly Phe 565 570 575 Ser
Gly His Ser Asp Arg Arg Gln Leu Met Glu Tyr Val Lys Arg Ile 580 585
590 Ser Pro Lys Pro Glu Lys Ile Leu Leu Cys His Gly Asp Asn Tyr Lys
595 600 605 Thr Leu Asp Leu Ala Ser Ser Ile Tyr Arg Thr Tyr Arg Ile
Glu Thr 610 615 620 Lys Thr Pro Leu Asn Leu Glu Thr Val Arg Ile Gln
625 630 635 238 1040 PRT Homo sapiens 238 Met Leu Glu Asp Ile Ser
Glu Glu Asp Ile Trp Glu Tyr Lys Ser Lys 1 5 10 15 Arg Lys Pro Lys
Arg Val Asp Pro Asn Asn Gly Ser Lys Asn Ile Leu 20 25 30 Lys Ser
Val Glu Lys Ala Thr Asp Gly Lys Tyr Gln Ser Lys Arg Ser 35 40 45
Arg Asn Arg Lys Arg Ala Ala Glu Ala Lys Glu Val Lys Asp His Glu 50
55 60 Val Pro Leu Gly Asn Ala Gly Cys Gln Thr Ser Val Ala Ser Ser
Gln 65 70 75 80 Asn Ser Ser Cys Gly Asp Gly Ile Gln Gln Thr Gln Asp
Lys Glu Thr 85 90 95 Thr Pro Gly Lys Leu Cys Arg Thr Gln Lys Ser
Gln His Val Ser Pro 100 105 110 Lys Ile Arg Pro Val Tyr Asp Gly Tyr
Cys Pro Asn Cys Gln Met Pro 115 120 125 Phe Ser Ser Leu Ile Gly Gln
Thr Pro Arg Trp His Val Phe Glu Cys 130 135 140 Leu Asp Ser Pro Pro
Arg Ser Glu Thr Glu Cys Pro Asp Gly Leu Leu 145 150 155 160 Cys Thr
Ser Thr Ile Pro Phe His Tyr Lys Arg Tyr Thr His Phe Leu 165 170 175
Leu Ala Gln Ser Arg Ala Gly Asp His Pro Phe Ser Ser Pro Ser Pro 180
185 190 Ala Ser Gly Gly Ser Phe Ser Glu Thr Lys Ser Gly Val Leu Cys
Ser 195 200 205 Leu Glu Glu Arg Trp Ser Ser Tyr Gln Asn Gln Thr Asp
Asn Ser Val 210 215 220 Ser Asn Asp Pro Leu Leu Met Thr Gln Tyr Phe
Lys Lys Ser Pro Ser 225 230 235 240 Leu Thr Glu Ala Ser Glu Lys Ile
Ser Thr His Ile Gln Thr Ser Gln 245 250 255 Gln Ala Leu Gln Phe Thr
Asp Phe Val Glu Asn Asp Lys Leu Val Gly 260 265 270 Val Ala Leu Arg
Leu Ala Asn Asn Ser Glu His Ile Asn Leu Pro Leu 275 280 285 Pro Glu
Asn Asp Phe Ser Asp Cys Glu Ile Ser Tyr Ser Pro Leu Gln 290 295 300
Ser Asp Glu Asp Thr His Asp Ile Asp Glu Lys Pro Asp Asp Ser Gln 305
310 315 320 Glu Gln Leu Phe Phe Thr Glu Ser Ser Lys Asp Gly Ser Leu
Glu Glu 325 330 335 Asp Asp Asp Ser Cys Gly Phe Phe Lys Lys Arg His
Gly Pro Leu Leu 340 345 350 Lys Asp Gln Asp Glu Ser Cys Pro Lys Val
Asn Ser Phe Leu Thr Arg 355 360 365 Asp Lys Tyr Asp Glu Gly Leu Tyr
Arg Phe Asn Ser Leu Asn Asp Leu 370 375 380 Ser Gln Pro Ile Ser Gln
Asn Asn Glu Ser Thr Leu Pro Tyr Asp Leu 385 390 395 400 Ala Cys Thr
Gly Gly Asp Phe Val Leu Phe Pro Pro Ala Leu Ala Gly 405 410 415 Lys
Leu Ala Ala Ser Val His Gln Ala Thr Lys Ala Lys Pro Asp Glu 420 425
430 Pro Glu Phe His Ser Ala Gln Ser Asn Lys Gln Lys Gln Val Ile Glu
435 440 445 Glu Ser Ser Val Tyr Asn Gln Val Ser Leu Pro Leu Val Lys
Ser Leu 450 455 460 Met Leu Lys Pro Phe Glu Ser Gln Val Glu Gly Tyr
Leu Ser Ser Gln 465 470 475 480 Pro Thr Gln Asn Thr Ile Arg Lys Leu
Ser Ser Glu Asn Leu Asn Ala 485 490 495 Lys Asn Asn Thr Asn Ser Ala
Cys Phe Cys Arg Lys Ala Leu Glu Gly 500 505 510 Val Pro Val Gly Lys
Ala Thr Ile Leu Asn Thr Glu Asn Leu Ser Ser 515 520 525 Thr Pro Ala
Pro Lys Tyr Leu Lys Ile Leu Pro Ser Gly Leu Lys Tyr 530 535 540 Asn
Ala Arg His Pro Ser Thr Lys Val Met Lys Gln Met Asp Ile Gly 545 550
555 560 Val Tyr Phe Gly Leu Pro Pro Lys Arg Lys Glu Glu Lys Leu Leu
Gly 565 570 575 Glu Ser Ala Leu Glu Gly Ile Asn Leu Asn Pro Val Pro
Ser Pro Asn 580 585 590 Gln Lys Arg Ser Ser Gln Cys Lys Arg Lys Ala
Glu Lys Ser Leu Ser 595 600 605 Asp Leu Glu Phe Asp Ala Ser Thr Leu
His Glu Ser Gln Leu Ser Val 610 615 620 Glu Leu Ser Ser Glu Arg Ser
Gln Arg Gln Lys Lys Arg Cys Arg Lys 625 630 635 640 Ser Asn Ser Leu
Gln Glu Gly Ala Cys Gln Lys Arg Ser Asp His Leu 645 650 655 Ile Asn
Thr Glu Ser Glu Ala Val Asn Leu Ser Lys Val Lys Val Phe 660 665 670
Thr Lys Ser Ala His Gly Gly Leu Gln Arg Gly Asn Lys Lys Ile Pro 675
680 685 Glu Ser Ser Asn Val Gly Gly Ser Arg Lys Lys Thr Cys Pro Phe
Tyr 690 695 700 Lys Lys Ile Pro Gly Thr Gly Phe Thr Val Asp Ala Phe
Gln Tyr Gly 705 710 715 720 Val Val Glu Gly Cys Thr Ala Tyr Phe Leu
Thr His Phe His Ser Asp 725 730 735 His Tyr Ala Gly Leu Ser Lys His
Phe Thr Phe Pro Val Tyr Cys Ser 740 745 750 Glu Ile Thr Gly Asn Leu
Leu Lys Asn Lys Leu His Val Gln Glu Gln 755 760 765 Tyr Ile His Pro
Leu Pro Leu Asp Thr Glu Cys Ile Val Asn Gly Val 770 775 780 Lys Val
Val Leu Leu Asp Ala Asn His Cys Pro Gly Ala Val Met Ile 785 790 795
800 Leu Phe Tyr Leu Pro Asn Gly Thr Val Ile Leu His Thr Gly Asp Phe
805 810 815 Arg Ala Asp Pro Ser Met Glu Arg Ser Leu Leu Ala Asp Gln
Lys Val 820 825 830 His Met Leu Tyr Leu Asp Thr Thr Tyr Cys Ser Pro
Glu Tyr Thr Phe 835 840 845 Pro Ser Gln Gln Glu Val Ile Arg Phe Ala
Ile Asn Thr Ala Phe Glu 850 855 860 Ala Val Thr Leu Asn Pro His Ala
Leu Val Val Cys Gly Thr Tyr Ser 865 870 875 880 Ile Gly
Lys Glu Lys Val Phe Leu Ala Ile Ala Asp Val Leu Gly Ser 885 890 895
Lys Val Gly Met Ser Gln Glu Lys Tyr Lys Thr Leu Gln Cys Leu Asn 900
905 910 Ile Pro Glu Ile Asn Ser Leu Ile Thr Thr Asp Met Cys Ser Ser
Leu 915 920 925 Val His Leu Leu Pro Met Met Gln Ile Asn Phe Lys Gly
Leu Gln Ser 930 935 940 His Leu Lys Lys Cys Gly Gly Lys Tyr Asn Gln
Ile Leu Ala Phe Arg 945 950 955 960 Pro Thr Gly Trp Thr His Ser Asn
Lys Phe Thr Arg Ile Ala Asp Val 965 970 975 Ile Pro Gln Thr Lys Gly
Asn Ile Ser Ile Tyr Gly Ile Pro Tyr Ser 980 985 990 Glu His Ser Ser
Tyr Leu Glu Met Lys Arg Phe Val Gln Trp Leu Lys 995 1000 1005 Pro
Gln Lys Ile Ile Pro Thr Val Asn Val Gly Thr Trp Lys Ser Arg 1010
1015 1020 Ser Thr Met Glu Lys Tyr Phe Arg Glu Trp Lys Leu Glu Ala
Gly Tyr 1025 1030 1035 1040 239 723 PRT Arabidopsis thaliana 239
Met Ser Asn Thr Val Glu Asp Asp Asp Asp Asp Phe Gln Ile Pro Pro 1 5
10 15 Ser Ser Gln Leu Ser Ile Arg Lys Pro Leu His Pro Thr Asn Ala
Asn 20 25 30 Asn Ile Ser His Arg Pro Pro Asn Lys Lys Pro Arg Leu
Cys Arg Tyr 35 40 45 Pro Gly Lys Glu Asn Val Thr Pro Pro Pro Ser
Pro Asp Pro Asp Leu 50 55 60 Phe Cys Ser Ser Ser Thr Pro His Cys
Ile Leu Asp Cys Ile Pro Ser 65 70 75 80 Ser Val Asp Cys Ser Leu Gly
Asp Phe Asn Gly Pro Ile Ser Ser Leu 85 90 95 Gly Glu Glu Asp Lys
Glu Asp Lys Asp Asp Cys Ile Lys Val Asn Arg 100 105 110 Glu Gly Tyr
Leu Cys Asn Ser Met Glu Ala Arg Leu Leu Lys Ser Arg 115 120 125 Ile
Cys Leu Gly Phe Asp Ser Gly Ile His Glu Asp Asp Glu Gly Phe 130 135
140 Val Glu Ser Asn Ser Glu Leu Asp Val Leu Ile Asn Leu Cys Ser Glu
145 150 155 160 Ser Glu Gly Arg Ser Gly Glu Phe Ser Leu Gly Lys Asp
Asp Ser Ile 165 170 175 Gln Cys Pro Leu Cys Ser Met Asp Ile Ser Ser
Leu Ser Glu Glu Gln 180 185 190 Arg Gln Val His Ser Asn Thr Cys Leu
Asp Lys Ser Tyr Asn Gln Pro 195 200 205 Ser Glu Gln Asp Ser Leu Arg
Lys Cys Glu Asn Leu Ser Ser Leu Ile 210 215 220 Lys Glu Ser Ile Asp
Asp Pro Val Gln Leu Pro Gln Leu Val Thr Asp 225 230 235 240 Leu Ser
Pro Val Leu Lys Trp Leu Arg Ser Leu Gly Leu Ala Lys Tyr 245 250 255
Glu Asp Val Phe Ile Arg Glu Glu Ile Asp Trp Asp Thr Leu Gln Ser 260
265 270 Leu Thr Glu Glu Asp Leu Leu Ser Ile Gly Ile Thr Ser Leu Gly
Pro 275 280 285 Arg Lys Lys Ile Val Asn Ala Leu Ser Gly Val Arg Asp
Pro Phe Ala 290 295 300 Ser Ser Ala Glu Val Gln Ala Gln Ser His Cys
Thr Ser Gly His Val 305 310 315 320 Thr Glu Arg Gln Arg Asp Lys Ser
Thr Thr Arg Lys Ala Ser Glu Pro 325 330 335 Lys Lys Pro Thr Ala Asn
Lys Leu Ile Thr Glu Phe Phe Pro Gly Gln 340 345 350 Ala Thr Glu Gly
Thr Lys Ile Arg Thr Ala Pro Lys Pro Val Ala Glu 355 360 365 Lys Ser
Pro Ser Asp Ser Ser Ser Arg Arg Ala Val Arg Arg Asn Gly 370 375 380
Asn Asn Gly Lys Ser Lys Val Ile Pro His Trp Asn Cys Ile Pro Gly 385
390 395 400 Thr Pro Phe Arg Val Asp Ala Phe Lys Tyr Leu Thr Arg Asp
Cys Cys 405 410 415 His Trp Phe Leu Thr His Phe His Leu Asp His Tyr
Gln Gly Leu Thr 420 425 430 Lys Ser Phe Ser His Gly Lys Ile Tyr Cys
Ser Leu Val Thr Ala Lys 435 440 445 Leu Val Asn Met Lys Ile Gly Ile
Pro Trp Glu Arg Leu Gln Val Leu 450 455 460 Asp Leu Gly Gln Lys Val
Asn Ile Ser Gly Ile Asp Val Thr Cys Phe 465 470 475 480 Asp Ala Asn
His Cys Pro Gly Ser Ile Met Ile Leu Phe Glu Pro Ala 485 490 495 Asn
Gly Lys Ala Val Leu His Thr Gly Asp Phe Arg Tyr Ser Glu Glu 500 505
510 Met Ser Asn Trp Leu Ile Gly Ser His Ile Ser Ser Leu Ile Leu Asp
515 520 525 Thr Thr Tyr Cys Asn Pro Gln Tyr Asp Phe Pro Lys Gln Glu
Ala Val 530 535 540 Ile Gln Phe Val Val Glu Ala Ile Gln Ala Glu Ala
Phe Asn Pro Lys 545 550 555 560 Thr Leu Phe Leu Ile Gly Ser Tyr Thr
Ile Gly Lys Glu Arg Leu Phe 565 570 575 Leu Glu Val Ala Arg Val Leu
Arg Glu Lys Ile Tyr Ile Asn Pro Ala 580 585 590 Lys Leu Lys Leu Leu
Glu Cys Leu Gly Phe Ser Lys Asp Asp Ile Gln 595 600 605 Trp Phe Thr
Val Lys Glu Glu Glu Ser His Ile His Val Val Pro Leu 610 615 620 Trp
Thr Leu Ala Ser Phe Lys Arg Leu Lys His Val Ala Asn Arg Tyr 625 630
635 640 Thr Asn Arg Tyr Ser Leu Ile Val Ala Phe Ser Pro Thr Gly Trp
Thr 645 650 655 Ser Gly Lys Thr Lys Lys Lys Ser Pro Gly Arg Arg Leu
Gln Gln Gly 660 665 670 Thr Ile Ile Arg Tyr Glu Val Pro Tyr Ser Glu
His Ser Ser Phe Thr 675 680 685 Glu Leu Lys Glu Phe Val Gln Lys Val
Ser Pro Glu Val Ile Ile Pro 690 695 700 Ser Val Asn Asn Asp Gly Pro
Asp Ser Ala Ala Ala Met Val Ser Leu 705 710 715 720 Leu Val Thr 240
661 PRT Saccharomyces cerevisiae 240 Met Ser Arg Lys Ser Ile Val
Gln Ile Arg Arg Ser Glu Val Lys Arg 1 5 10 15 Lys Arg Ser Ser Thr
Ala Ser Ser Thr Ser Glu Gly Lys Thr Leu His 20 25 30 Lys Asn Thr
His Thr Ser Ser Lys Arg Gln Arg Thr Leu Thr Glu Phe 35 40 45 Asn
Ile Pro Thr Ser Ser Asn Leu Pro Val Arg Ser Ser Ser Tyr Ser 50 55
60 Phe Ser Arg Phe Ser Cys Ser Thr Ser Asn Lys Asn Thr Glu Pro Val
65 70 75 80 Ile Ile Asn Asp Asp Asp His Asn Ser Ile Cys Leu Glu Asp
Thr Ala 85 90 95 Lys Val Glu Ile Thr Ile Asp Thr Asp Glu Glu Glu
Leu Val Ser Leu 100 105 110 His Asp Asn Glu Val Ser Ala Ile Glu Asn
Arg Thr Glu Asp Arg Ile 115 120 125 Val Thr Glu Leu Glu Glu Gln Val
Asn Val Lys Val Ser Thr Glu Val 130 135 140 Ile Gln Cys Pro Ile Cys
Leu Glu Asn Leu Ser His Leu Glu Leu Tyr 145 150 155 160 Glu Arg Glu
Thr His Cys Asp Thr Cys Ile Gly Ser Asp Pro Ser Asn 165 170 175 Met
Gly Thr Pro Lys Lys Asn Ile Arg Ser Phe Ile Ser Asn Pro Ser 180 185
190 Ser Pro Ala Lys Thr Lys Arg Asp Ile Ala Thr Ser Lys Lys Pro Thr
195 200 205 Arg Val Lys Leu Val Leu Pro Ser Phe Lys Ile Ile Lys Phe
Asn Asn 210 215 220 Gly His Glu Ile Val Val Asp Gly Phe Asn Tyr Lys
Ala Ser Glu Thr 225 230 235 240 Ile Ser Gln Tyr Phe Leu Ser His Phe
His Ser Asp His Tyr Ile Gly 245 250 255 Leu Lys Lys Ser Trp Asn Asn
Pro Asp Glu Asn Pro Ile Lys Lys Thr 260 265 270 Leu Tyr Cys Ser Lys
Ile Thr Ala Ile Leu Val Asn Leu Lys Phe Lys 275 280 285 Ile Pro Met
Asp Glu Ile Gln Ile Leu Pro Met Asn Lys Arg Phe Trp 290 295 300 Ile
Thr Asp Thr Ile Ser Val Val Thr Leu Asp Ala Asn His Cys Pro 305 310
315 320 Gly Ala Ile Ile Met Leu Phe Gln Glu Phe Leu Ala Asn Ser Tyr
Asp 325 330 335 Lys Pro Ile Arg Gln Ile Leu His Thr Gly Asp Phe Arg
Ser Asn Ala 340 345 350 Lys Met Ile Glu Thr Ile Gln Lys Trp Leu Ala
Glu Thr Ala Asn Glu 355 360 365 Thr Ile Asp Gln Val Tyr Leu Asp Thr
Thr Tyr Met Thr Met Gly Tyr 370 375 380 Asn Phe Pro Ser Gln His Ser
Val Cys Glu Thr Val Ala Asp Phe Thr 385 390 395 400 Leu Arg Leu Ile
Lys His Gly Lys Asn Lys Thr Phe Gly Asp Ser Gln 405 410 415 Arg Asn
Leu Phe His Phe Gln Arg Lys Lys Thr Leu Thr Thr His Arg 420 425 430
Tyr Arg Val Leu Phe Leu Val Gly Thr Tyr Thr Ile Gly Lys Glu Lys 435
440 445 Leu Ala Ile Lys Ile Cys Glu Phe Leu Lys Thr Lys Leu Phe Val
Met 450 455 460 Pro Asn Ser Val Lys Phe Ser Met Met Leu Thr Val Leu
Gln Asn Asn 465 470 475 480 Glu Asn Gln Asn Asp Met Trp Asp Glu Ser
Leu Leu Thr Ser Asn Leu 485 490 495 His Glu Ser Ser Val His Leu Val
Pro Ile Arg Val Leu Lys Ser Gln 500 505 510 Glu Thr Ile Glu Ala Tyr
Leu Lys Ser Leu Lys Glu Leu Glu Thr Asp 515 520 525 Tyr Val Lys Asp
Ile Glu Asp Val Val Gly Phe Ile Pro Thr Gly Trp 530 535 540 Ser His
Asn Phe Gly Leu Lys Tyr Gln Lys Lys Asn Asp Asp Asp Glu 545 550 555
560 Asn Glu Met Ser Gly Asn Thr Glu Tyr Cys Leu Glu Leu Met Lys Asn
565 570 575 Asp Arg Asp Asn Asp Asp Glu Asn Gly Phe Glu Ile Ser Ser
Ile Leu 580 585 590 Arg Gln Tyr Lys Lys Tyr Asn Lys Phe Gln Val Phe
Asn Val Pro Tyr 595 600 605 Ser Glu His Ser Ser Phe Asn Asp Leu Val
Lys Phe Gly Cys Lys Leu 610 615 620 Lys Cys Ser Glu Val Ile Pro Thr
Val Asn Leu Asn Asn Leu Trp Lys 625 630 635 640 Val Arg Tyr Met Thr
Asn Trp Phe Gln Cys Trp Glu Asn Val Arg Lys 645 650 655 Thr Arg Ala
Ala Lys 660
* * * * *
References