U.S. patent application number 10/809062 was filed with the patent office on 2004-09-30 for protection-of telomere-1 (pot-1) protein and encoding polynucleotides.
This patent application is currently assigned to The Regents of the University of Colorado. Invention is credited to Baumann, Peter, Cech, Thomas R..
Application Number | 20040191820 10/809062 |
Document ID | / |
Family ID | 25220077 |
Filed Date | 2004-09-30 |
United States Patent
Application |
20040191820 |
Kind Code |
A1 |
Baumann, Peter ; et
al. |
September 30, 2004 |
Protection-of telomere-1 (POT-1) protein and encoding
polynucleotides
Abstract
A protein identified in humans and Schizosaccharomyces pombe,
Pot1p, binds single-stranded telomeric DNA and both stabilizes
chromosome ends and regulates telomerase activity. Compounds that
stabilize or disrupt the Pot1p-DNA interaction will be useful in
regulating the telomere length of a cell. Because telomere length
is involved in the regulation of cellular life-span, the life-span
of useful cell populations may be prolonged or undesirable cells
may be caused to cease proliferation. The identification of a Pot1
protein and its encoding DNA provides methods of screening useful
compounds or diagnosing illnesses that involve altered expression
or structure of a Pot1 protein or gene.
Inventors: |
Baumann, Peter; (Boulder,
CO) ; Cech, Thomas R.; (Potomac, MD) |
Correspondence
Address: |
SHERIDAN ROSS PC
1560 BROADWAY
SUITE 1200
DENVER
CO
80202
|
Assignee: |
The Regents of the University of
Colorado
|
Family ID: |
25220077 |
Appl. No.: |
10/809062 |
Filed: |
March 24, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10809062 |
Mar 24, 2004 |
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09816248 |
Mar 26, 2001 |
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6753411 |
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Current U.S.
Class: |
435/6.18 |
Current CPC
Class: |
C12N 9/1241 20130101;
A61K 48/00 20130101; C07K 14/39 20130101; C07K 14/4703 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method of identifying a compound that regulates the binding of
Pot1 to telomeric DNA, comprising detecting whether a candidate
compound regulates the binding of a Pot1 polypeptide to a
single-stranded telomeric DNA.
2. The method of claim 1, wherein the candidate compound is exposed
to a Pot1 polypeptide-telomeric DNA complex.
3. The method of claim 1, wherein the candidate compound is exposed
to the Pot1 polypeptide prior to exposure to the telomeric DNA.
4. The method of claim 1, wherein the step of detecting comprises
detecting whether the candidate compound strengthens the
interaction between the Pot1 polypeptide and the telomeric DNA.
5. The method of claim 1, wherein the step of detecting comprises
detecting whether the candidate compound stabilizes the interaction
between the Pot1 polypeptide and the telomeric DNA.
6. The method of claim 1, wherein the step of detecting comprises
detecting whether the candidate compound weakens the interaction
between the Pot1 polypeptide and the telomeric DNA.
7. The method of claim 1, wherein the step of detecting comprises
detecting whether the candidate compound disrupts the interaction
between the Pot1 polypeptide and the telomeric DNA.
8. The method of claim 1, wherein the step of detecting comprises
detecting whether the candidate compound interacts with the Pot1
polypeptide or a complex between the Pot1 polypeptide and the
telomeric DNA to change the binding constant of the complex between
the Pot1 polypeptide and the telomeric DNA.
9. The method of claim 1, wherein the step of detecting is
performed by detecting the ability of the candidate compound to
change the amount of a labeled probe comprising a fragment of
single-stranded telomeric DNA that interacts with the Pot1
polypeptide.
10. The method of claim 1, wherein the step of detecting is
performed using an electrophoretic mobility shift assay.
11. The method of claim 1, wherein the step of detecting is
performed using a high throughput assay for screening candidate
compounds simultaneously.
12. The method of claim 1, wherein the step of detecting is
performed using an isolated cell that recombinantly expresses the
Pot1 polypeptide.
13. The method of claim 1, further comprising testing candidate
compounds that regulate the binding of a Pot1 polypeptide to
single-stranded telomeric DNA to determine whether the candidate
compounds regulate telomere length or integrity throughout repeated
divisions in a cell culture system.
14. The method of claim 1, wherein the Pot1 polypeptide is selected
from the group consisting of: a) a Pot1 polypeptide comprising an
amino acid sequence selected from the group consisting of: SEQ ID
NO:5, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17. b) a Pot1
polypeptide comprising an amino acid sequence that is at least
about 85% identical to an amino acid sequence selected from the
group consisting of: SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:15 and
SEQ ID NO:17, wherein the polypeptide binds single-stranded
telomeric DNA; and c) a fragment of a Pot1 polypeptide as set forth
in (a) or (b), wherein the fragment binds single-stranded telomeric
DNA.
15. The method of claim 1, wherein the Pot1 polypeptide comprises
an amino acid sequence that is at least about 90% identical to an
amino acid sequence selected from the group consisting of: SEQ ID
NO:5, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17, wherein the
polypeptide binds single-stranded telomeric DNA.
16. The method of claim 1, wherein the Pot1 polypeptide is a
fragment of an amino acid sequence selected from the group
consisting of: SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID
NO:17, wherein the fragment binds single-stranded telomeric
DNA.
17. The method of claim 1, wherein the Pot1 polypeptide comprises
an amino acid sequence selected from the group consisting of: SEQ
ID NO:5, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17.
18. The method of claim 1, wherein the Pot1 polypeptide comprises
SEQ ID NO:5.
19. The method of claim 1, wherein the single-stranded telomeric
DNA is G-rich.
20. The method of claim 1, wherein the single-stranded telomeric
DNA comprises TTAGGG (positions 1-6 of SEQ ID NO:20) repeats.
21. The method of claim 1, wherein the single-stranded telomeric
DNA comprises a nucleic acid sequence selected from the group
consisting of any one of SEQ ID NOs:36-38.
22. The method of claim 1, wherein the candidate compound is
selected from the group consisting of: a small organic molecule, an
oligonucleotide, and a non-hydrolyzable DNA analogue.
23. A method of identifying a compound that interferes with the
binding of a Pot1 polypeptide to a single-stranded telomeric DNA,
comprising determining whether the candidate compound decreases the
binding of the Pot1 polypeptide to a single-stranded telomeric DNA
molecule in a mixture comprising the single-stranded telomeric DNA
molecule, the polypeptide, and the candidate compound.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/816,248, filed Mar. 26, 2001, and entitled
"Protection-of-Telomere- -1 (POT-1) Protein and Encoding
Polynucleotides." The entire disclosure of U.S. patent application
Ser. No. 09/816,248 is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Telomeres are the protein-DNA complexes that protect the
ends of linear eukaryotic chromosomes from degradation, prevent
end-to-end fusions and partake in chromosome localization and
segregation (Cooper, Curr Opin Genet Dev 10: 169-77, 2000;
McEachern et al., Annu Rev Genet 34: 331-358, 2000; Price, Curr
Opin Genet Dev 9: 218-24, 1999). Telomere length, 15-20 kb in human
embryonic or germ line cells, is maintained in part by the enzyme
telomerase. In the absence of telomerase activity, about 50-200
bases of DNA are not replicated with each round of cell division,
resulting in the eventual diminution in telomere size to typically
5-7 kb. At that length, cells enter a state of arrested growth
called replicative senescence. The maintenance of telomere length
thus is believed to play a key role in the ability of cells to
avoid replicative senescence and to propagate indefinitely, as is
the case with stem cells. Likewise, aberrant maintenance of
telomere length is believed to underlie indefinite cellular
proliferation characteristic of cancer cells (Bodnar et al.,
Science 279: 349-352, 1998; Bryan et al., 1997; McEachern et al.,
2000).
[0003] Telomeres consist of repeating units of GC-rich DNA and
terminate in a single stranded extension of the 3' strand.
Oxytricha nova telomeres, for example, consist of tandem repeats of
(TTTTGGGG) and end with a 16 nucleotide overhang of the G-rich
strand. By contrast, human telomeres have a repeating sequence
(TTAGGG)n and end with a 50-100 nucleotide overhang of the G-rich
strand. McEachern et al., 2000.
[0004] A number of proteins have been identified that specifically
interact with the double-stranded portion of the telomere or the
single-stranded 3' extension at its very end. Among the most well
characterized are the telomere end-binding proteins from
hypotrichous ciliated protozoa (Gottschling et al., Cell 47:
195-205, 1986; Price et al., Genes Dev 1: 783-93, 1987). The
.alpha. and .beta. subunit of the O. nova Telomere End-Binding
Protein (TEBP) bind specifically to the 16 nucleotide
single-stranded extension at the ends of macronuclear chromosomes
(Gray et al., Cell 67: 807-14, 1991) and form a ternary complex
whose structure has been determined using X-ray crystallography
(Horvath et al., Cell 95: 963-974, 1998). Although both protein
subunits directly interact with DNA in the ternary complex, only a
binds telomeric DNA by itself (Fang et al., Genes Dev 7: 870-82,
1993). The DNA binding domain in the .alpha. subunit has been
mapped to the N-terminal two-thirds of the polypeptide (Fang et
al., 1993) and is comprised of two "OB folds" (Horvath et al.,
1998). In vitro reconstituted .alpha.-DNA complexes are substrates
for telomerase, whereas .alpha.-.beta.-DNA complexes are not; an
observation which may indicate a function in the regulation of
telomere length (Froelich-Ammon et al., Genes Dev 12: 1504-14,
1998).
[0005] The protrusion of the G-rich strand as a single-stranded
overhang is conserved between ciliates (Klobutcher et al., Proc
Natl Acad Sci USA 78: 3015-19, 1981), yeast (Wellinger et al., Cell
72: 51-60, 1993) and mammalian cells (Makarov et al., Cell 88:
657-66, 1997; McElligott et al., Embo J 16: 3705-14, 1997; Wright
et al., Genes Dev 11: 2801-09, 1997), suggesting the existence of
similar functional mechanisms in telomere maintenance. However,
proteins sharing sequence homology with ciliate TEBPs were not
identified in the complete S. cerevisiae genome or among the
proteins that bind single-stranded telomeric DNA in vitro.
Similarly, the S. cerevisiae single-stranded telomeric DNA-binding
protein cdc13p has not been proposed to be homologous to the
ciliate TEBPs, nor have cdc13p homologues been identified in
distantly related species. (Ishikawa et al., Mol Cell Biol 13:
4301-10, 1993; Lin et al., Proc Natl Acad Sci USA 93: 13760-65,
1996; McKay et al., Nucleic Acids Res 20: 6461-64, 1992; Nugent et
al., Science 274: 249-52, 1996; Virta-Pearlman et al., Genes Dev
10: 3094-104, 1996).
[0006] The apparent absence of specific end-capping proteins in
some eukaryotes has been explained by the adoption of a telomere
structure distinct from that found in the macronuclei of
hypotrichous ciliates. This telomere structure, found at the ends
of mammalian and O. fallax chromosomes, is a large duplex loop, or
"t loop," created by the sequestration of the single-strand
overhang within the double-stranded portion of the telomeric tract
(Griffith et al., Cell 97: 503-14, 1999; Murti et al., Proc Natl
Acad Sci USA 96: 14436-39, 1999). In mammals, this architecture is
believed to be maintained by a number of proteins, including the
TTAGGG-binding factors, TRF1 and TRF2. TRF2 is believed to catalyze
the sequestration of the single-stranded DNA into the duplex region
of the DNA. Consistent with this notion is the observation that
TRF2 can cause telomeric DNA to form t loops in vitro (Griffith et
al., 1999). Other proteins have been implicated in telomere
architecture and regulation, including TIN2, which was identified
by its ability to interact with TRF1 (Kim et al., 1999).
[0007] The ability to manipulate telomere structure and metabolism
depends on the identification of those components required for the
regulation of telomere structure. Evidence has accumulated that
telomerase activity itself is not determinative of telomere
elongation or replication. For example, some cancer cell lines
maintain telomeres in the absence of telomerase activity (Bryan et
al., 1997). There is thus a pressing need in the art to identify
the functional components that regulate telomere metabolism, to
identify compounds that can be used to control the entry,
avoidance, or exit of a cell from a state of replicative
senescence. Such compounds may be useful alternatively in allowing
the indefinite propagation of useful cell lines or in halting the
growth of cancer cells in vivo for therapeutic purposes.
SUMMARY OF THE INVENTION
[0008] The present invention addresses this need by providing a
protein that caps the very ends of human chromosomes, and a related
protein that caps the ends of chromosomes in fission yeast
(Schizosaccharomyces pombe). The protein of the invention is termed
"Protection of Telomere-1," or "Pot1p," or "Pot1 protein." Specific
embodiments of these proteins are those isolated from humans and
fission yeast, hPot1p and SpPot1p, respectively. Polynucleotides
encoding a Pot1 protein are also provided.
[0009] The inventors have found that Pot1p binds single-stranded
telomeric DNA, which is a unforeseen finding, given the apparent
absence of end-capping proteins in some eukaryotes. Pot1p both
stabilizes chromosome ends and regulates telomerase activity.
Accordingly, compounds that stabilize or disrupt the Pot1p-DNA
interaction will be useful in regulating the telomere length of a
target cell or cell population. The invention thus provides a means
of altering cellular life-span, for the purpose of either
prolonging the life-span of useful cell populations or making
cancer cells enter replicative quiescence. Useful compounds with
these properties can be identified through screening methods made
possible by the discovery that a Pot1 protein binds single-stranded
telomeric DNA. The identification of a Pot1 protein and its
encoding DNA also provides a means of developing tools to diagnose
illnesses such as cancer that may involve altered expression or
structure of a Pot1 protein or gene. Such tools include
polynucleotide hybridization probes and antibodies specific for a
Pot1 protein.
[0010] Accordingly, the invention provides isolated Pot1 proteins
having the sequence set forth in SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:9, or SEQ ID NO:11. Variants of these proteins are
capable of binding single-stranded telomeric DNA and have at least
85% sequence identity with, or differ by no more than about 20
single amino acid substitutions, deletions or insertions from, a
sequence set forth in SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ
ID NO:9, or SEQ ID NO:11. The invention also provides an isolated,
naturally occurring, variant of a protein having the sequence set
forth in SEQ ID NO:13 or in SEQ ID NO:9, which may be a splicing
variant. Fragments of the Pot1 proteins of the invention are
capable of binding single-stranded telomeric DNA, and comprise the
polypeptide having the sequence set forth in SEQ ID NO:5 or SEQ ID
NO:6.
[0011] The invention further provides an isolated non-genomic
polynucleotide encoding one of the aforementioned proteins. A
vector comprising such a polynucleotide and a host cell comprising
the vector also are provided. The polynucleotide may be included in
a pharmaceutical composition, along a pharmacologically acceptable
excipient, diluent, or carrier. A method of detecting or measuring
the presence of a POT1 polynucleotide comprises contacting the a
POT1 polynucleotide, or its complement, with a biological sample
from an individual.
[0012] An antibody, or a fragment or variant thereof, is provided,
which is capable of binding a Pot1 protein. A method of raising the
antibody comprises isolating the antibody from an animal or
isolating an antibody-producing cell from an animal, following
administration of a Pot1 protein, or an antigenic fragment thereof,
to the animal. An antibody of the invention may be useful in
detecting or measuring the presence of a Pot1 polypeptide in an
individual, by contacting the antibody with a biological sample
from an individual.
[0013] The invention provides a method of increasing the life-span
of a cell, by inserting a vector comprising a POT1 polynucleotide
into the cell, where the POT1 polynucleotide is operably linked to
a promoter that allows the polynucleotide to be transcribed. The
vector comprising a POT1 polynucleotide may be administered to an
individual in a pharmaceutical composition, comprising the
polynucleotide and a pharmacologically acceptable excipient,
diluent, or carrier. In one embodiment, the carrier is capable of
preferentially delivering the polynucleotide to a specific cell
population. In another embodiment, the vector comprising the POT1
polynucleotide is inserted into the cell in vitro, which then may
be subsequently administered to an individual. The target cell may
express a second polynucleotide that encodes an exogenous protein,
such as a therapeutically useful protein.
[0014] A method of identifying a compound that interferes with the
binding of a Pot1 polypeptide to single-stranded telomeric DNA
comprises determining whether the candidate compound decreases the
binding of the Pot1 polypeptide to a single-stranded telomeric DNA
molecule in a mixture of the single-stranded telomeric DNA
molecule, the polypeptide, and the candidate compound. The compound
identified by this method may be formulated in a pharmaceutical
composition.
[0015] A method of decreasing the life-span of a cell comprises
reducing the level of Pot1p activity in a cell. The cell may be an
immortal cell line, such as a cancer cell. In one embodiment, the
method comprises delivering one of the compounds that interferes
with the binding of a Pot1 polypeptide to single-stranded telomeric
DNA.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1A: Multiple sequence alignments of the N-terminal
regions of yeast and human Pot1p and the .alpha. subunits of
ciliate TEBPs (Ec, Euplotes crassus [SEQ ID NO:1]; Sm, Stylonychia
mytilis [SEQ ID NO:2]; Ot, Oxytricha trifallax [SEQ ID NO:3]; On,
Oxytricha nova [SEQ ID NO:4]; Hs, Homo sapiens [SEQ ID NO:5]; Sp,
Schizosaccharomyces pombe [SEQ ID NO:6]). The numbers of the first
and last amino acid shown are depicted at the beginning and end of
each sequence. Sequences were aligned in ClustalW using the
Blosum35 score table followed by manual adjustment. Shaded amino
acids are conserved in 4 or more sequences.
[0017] FIG. 1B: Domain structure of the O. nova TEBP and yeast and
human Pot1p. Position of OB folds (Horvath et al., Cell 95: 963-74,
1998) and functional domains (Fang et al., Genes Dev 7: 870-82,
1993) are depicted for the O. nova TEBP. The position of the
regions aligned in FIG. 1A are indicated by open boxes.
[0018] FIG. 1C: Morphological phenotype associated with deletion of
pot1.sup.+. Colony morphology of pot1.sup.+, pot1.sup.-, trt1.sup.+
and trt1.sup.- following tetrad dissection and germination.
[0019] FIG. 1D: Phase contrast micrographs of pot1.sup.+and
pot1.sup.- cells 5 to 10 generations after germination.
[0020] FIG. 1E: Cells as in FIG. 1D but stained with DAPI to reveal
chromosome segregation defect in pot1.sup.-.
[0021] FIG. 2A: Telomere phenotype in pot1.sup.- strains. Genomic
DNA from the indicated diploid and haploid strains was digested
with Eco RI, which cleaves S. pombe DNA about 1.0-1.2 kb from the
chromosome ends, and then fractionated by 1.1% agarose gel
electrophoresis, transferred to a nylon membrane and hybridized to
a telomeric probe. A probe against the single-copy pol.alpha. gene
was used as a loading control.
[0022] FIG. 2B: Genomic DNA was digested with NsiI, fractionated by
0.8% agarose gel electrophoresis, transferred to a nylon membrane
and hybridized to a probe against Telomere Associated Sequences
internal to the telomere itself (TAS2 sequences).
[0023] FIG. 2C: The blot shown in FIG. 2B was stripped and
hybridized to a probe against Telomere Associated Sequences that
are internal to TAS2 (TAS3 sequences).
[0024] FIG. 3A: DNA-binding specificity of S. pombe Pot1p, using
conditions described in the Examples. SpPot1p was incubated with
the indicated DNA substrates. Complexes were analyzed by
nondenaturing gel electrophoresis. The SpPot1p-DNA complex is
indicated by an open arrow.
[0025] FIG. 3B: Same as FIG. 3A except that the added protein
contained truncated Pot1p as well as full length protein. Truncated
Pot1p-DNA complex is indicated by a closed arrow.
[0026] FIG. 4A: Expression of hPOT1 and DNA-binding. RT-PCR
amplification of GAPDH and hPOT1 mRNA in various human tissues.
[0027] FIG. 4B: Binding of hPot1p to human C-strand (SEQ ID NO: 19)
(CCCTAA).sub.5, G-strand (SEQ ID NO: 20) (TTAGGG).sub.5 and duplex
(SEQ ID NO: 21) (CCCTAA).sub.5.circle-solid.(TTAGGG).sub.5. Binding
conditions and analysis were as described in FIG. 3.
[0028] FIG. 5A: Substrate specificity of S. pombe and human Pot1p.
Binding of SpPot1p to S. pombe and human G-strand DNAs.
[0029] FIG. 5B: Binding of SpPot1p (50 ng) to radiolabeled S. pombe
G-strand (1.5 fmol, or 1 ng) in the presence of 10-, 100-, and
1000-fold excess of unlabeled competitor S. pombe, human or O. nova
G-strand DNAs.
[0030] FIG. 5C: Binding of hPot1p to S. pombe and human G-strand
DNAs.
[0031] FIG. 5D: Binding of hPot1p to human G-strand DNAs under same
conditions as in FIG. 5B.
[0032] FIG. 6: Inhibition of telomerase activity by Pot1p.
Telomerase activity is assayed with telomeric primer PBoli82 (SEQ
ID NO: 22) (TGTGGTGTGTGGGTGTGC) as described in Haering et al.,
Proc. Nat'l Acad. Sci. USA 97: 6367-72, 2000. Unlabeled nucleotides
are added to a concentration of 100 .mu.M as follows: lanes a and
b, dATP, dCTP and dTTP; lanes c and d, ddATP, dCTP and dTTP; lanes
e and f, dATP, dCTP and ddTTP. For lanes b, d, and f the
oligonucleotide was preincubated with a SpPot1p preparation
containing full length protein and the N-terminal 22 kDa fragment
(100 ng/.mu.l). The Pot1 protein inhibits primer extension by
telomerase.
[0033] FIG. 7: S. pombe POT1 genomic DNA. The sequence shown (SEQ
ID NO:7) is published by the Sanger Centre as part of cosmid c26H5,
having accession number SPAC26H5. The sequence contains an upstream
promoter sequence, a coding sequence, which includes two introns, 1
and 2, and a downstream terminator sequence.
[0034] FIG. 8A: A S. pombe POT1 cDNA sequence (SEQ ID NO:8), in
which both introns 1 and 2 have been spliced out.
[0035] FIG. 8B: A SpPot1 protein (SEQ ID NO:9) encoded by the DNA
sequence of SEQ ID NO:8.
[0036] FIG. 8C: A splicing variant of the S. pombe POT1 cDNA
sequence of SEQ ID NO: 8, in which intron 2 has not been spliced
out (SEQ ID NO:10).
[0037] FIG. 8D: The SpPot1 polypeptide (SEQ ID NO: 11) encoded by
the splicing variant of SEQ ID NO:10.
[0038] FIG. 9A: A full-length hPOT1 cDNA (SEQ ID NO:12).
[0039] FIG. 9B: The hPot1p splicing variant (SEQ ID NO:13) encoded
by the polynucleotide of SEQ ID NO:12.
[0040] FIG. 9C: Another splicing variant of hPOT1 cDNA (SEQ ID
NO:14), having an inserted exon indicated by the underlined
residues.
[0041] FIG. 9D: The hPot1p splicing variant (SEQ ID NO:15) encoded
by the polynucleotide of SEQ ID NO:14. The alternatively spliced
exon gives rise to a protein that is about 50% shorter than
full-length hPOT1p and has an alternative C-terminus.
[0042] FIG. 9E: A splicing variant of hPOT1 cDNA (SEQ ID NO:16). An
exon is skipped, giving raise to a hPot1p with an alternate
C-terminus.
[0043] FIG. 9F: The hPot1p splicing variant (SEQ ID NO:17) encoded
by SEQ ID NO:16.
[0044] FIG. 10A-F: A partial genomic clone of hPOT1 (AC004925; SEQ
ID NO:18). Exons are in capital letters.
[0045] FIG. 10G: A scale diagram of SEQ ID NO:18, showing the
relative position of exons. Exons are numbered arbitrarily, because
the clone does not extend to the 5' end of the gene. The exons
present in the splicing variants of FIG. 9 are indicated. "Spice
variant #1" corresponds to SEQ DI NO:13, "Splice variant #3"
corresponds to SEQ ID NO:15, and "Splice variant #3" is SEQ ID
NO:17.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0046] The inventors provide a method to control the life-span of a
cell. The life-span of a cell depends in part on the ability of a
cell to replicate its telomeres with each round of cell division. A
Pot1 protein stabilizes chromosomes by binding the single-strand
G-rich 3' extension in the telomere, thereby avoiding loss of
telomeric DNA and concomitant chromosome fusion or degradation. In
the presence of a telomere replication mechanism, such as
telomerase or telomeric recombination, Pot1p allows cells to
undergo repeated division without reduction in the length of the
telomere and attainment of replicative senescence. The isolation of
a Pot1 protein and its encoding polynucleotide allows a method of
screening for compounds that affect the interaction between Pot1p
and telomeric DNA. These compounds will be useful in prolonging or
reducing the life span of a cell or population of cells.
[0047] The existence of end-capping proteins in humans and S. pombe
was unforeseen, given the previous inability to find such proteins.
The inventors found that S. pombe open reading frame SPAC26H5.06
contains a region of modest sequence similarity to the a subunits
of TEBP from Oxytricha nova and other ciliates (FIG. 1A).
Conservation is most apparent over a 95 amino acid stretch near the
N-termini of the proteins where the S. pombe and O. nova sequences
share 19% identity and 40% similarity. This region coincides with
the most highly conserved domain within the ciliate sequences (42%
amino acid identity [61% similarity] between O. nova and E.
crassus). Sequence alignments of hPot1p with the S. pombe protein
reveals the highest conservation near the N-terminus where the S.
pombe and human proteins share 48% similarity (26% identity) (FIG.
1A). Over the same region, the similarity of the human sequence
with the O. nova protein is 39% (23% identity). Such levels of
similarity and identity are often found between functionally
unrelated proteins, so they are insufficient to indicate homology;
therefore, tests of function were performed. No obvious sequence
similarity by primary sequence alignment is noted between hPot1p or
SpPot1p and cdc13p, the single-stranded telomeric DNA-binding
protein of S. cerevisiae.
[0048] Pot1 Proteins Prevent Chromosomal Instability
[0049] The inventors demonstrate by gene knock-out a role of the S.
pombe gene, pot1.sup.+, in telomere maintenance. A heterozygous
diploid pot.sup.+/pot1.sup.- S. pombe was constructed by the method
described in Baumann and Cech, Mol Biol Cell 11: 3265-75, 2000.
Tetrad dissections revealed that the pot1.sup.- daughters formed
only very small colonies compared to their pot1.sup.+ sisters (FIG.
1C). This immediate phenotype is in stark contrast to the
observations made with strains lacking the catalytic subunit of
telomerase (trt1.sup.-), which form wild-type sized colonies upon
sporulation (FIG. 1C) and only begin to show a growth defect on the
third re-streak, when telomeres have shortened considerably
(Nakamura et al., Science 282: 493-96, 1998). For approximately 10
generations after sporulation, pot1.sup.- colonies contained a
large number of elongated cells, most of which failed to undergo
further divisions (FIG. 1D). DAPI staining revealed a high
incidence of chromosome missegregation, often leading to daughter
cells without any chromosomal DNA (FIG. 1E).
[0050] By deleting the S. pombe pot1.sup.+ gene, the inventors have
shown that a Pot1 protein plays a pivotal role in preventing
instability of chromosome ends in vivo. Biochemical and structural
data have suggested a role for the Euplotes and Oxytricha TEBPs in
protecting the very ends of chromosomes; however, because these
organisms are not amenable to genetic studies, proof of such a
capping function in vivo has been lacking. This proof is now
provided by deletion of the pot1.sup.+ gene, which leads to
immediate chromosome instability (FIG. 2). Telomeres could not be
detected by Southern blotting of genomic DNA from pot1.sup.-
strains (FIG. 2A). Using three DNA probes that recognize distinct
subregions of the telomere associated sequence (TAS), hybridization
signals were only observed with the telomere distal TAS3 probe
(FIG. 2C), but not with TAS1 or TAS2 (FIG. 2B and data not shown).
These results indicate that around 5 kb of terminal sequence had
been lost within .about.30 generations after loss of
pot1.sup.+.
[0051] In contrast to the immediate chromosome instability caused
by an absence of functional SpPot1p, the absence of functional
telomerase causes gradual telomere shortening over many generations
without an immediate effect on chromosome stability and cell
viability (Nakamura et al., 1998). Thus, at least in S. pombe,
Pot1p apparently is more important for telomere maintenance in the
short term than telomerase.
[0052] Pot1 Proteins Specifically Bind Single-Stranded Telomeric
DNA
[0053] Pot1 proteins bind directly to single-stranded telomeric
DNA. The SpPot1 protein was expressed and purified from E. coli,
using methodology described below, and the ability of the expressed
protein to bind DNA was assayed using an electrophoretic mobility
shift assay. SpPot1p interacts specifically with the G-rich strand
of S. pombe telomeric DNA, but not with the complementary C-rich
strand or double-stranded telomeric DNA (FIG. 3A).
[0054] N-terminal fragments of the SpPot1 protein maintain the
ability to bind single-stranded telomeric DNA. Several truncated
forms co-eluted with the full length protein from the Ni-NTA column
used to purify the expressed SpPot1 protein. These polypeptides
retain the N-terminal His.sub.6 tag and thus are believed to arise
either from premature termination or from proteolytic degradation
of SpPot1p. These truncated proteins had a higher affinity for DNA
while retaining the same specificity as displayed by the full
length protein (FIG. 3B). Titration experiments indicated that the
apparent K.sub.d for binding of a predominant N-terminal fragment
of Pot1p to the G-rich oligo is approximately 10 fold higher than
for the full length protein (10 nM versus 100 nM). Further
purification and analysis by mass spectroscopy showed that the
strong shift (indicated by a closed arrow in FIG. 3B, lane d) is
attributable to the binding of a 22 kDa N-terminal fragment of
SpPot1p. Increased DNA binding likewise has been observed with
N-terminal fragments of the .alpha. subunit of TEBP from Oxytricha
nova (Fang et al., 1993).
[0055] hPot1p N-terminal fragments show the same behavior as
SpPot1p fragments. hPot1p, like SpPot1p, often lacks C-terminal
sequences due to degradation or pre-mature termination. These
truncated forms of hPot1p also show the same DNA binding
specificity as full length hPot1p obtained from in vitro
translation reactions. In gel shift assays, hPot1p binds G-rich
strands of human telomeric DNA (FIG. 4B). As with SpPot1p, binding
was not observed with the complementary C-rich strand or with
double-stranded telomeric DNA.
[0056] SpPot1p and hPot1p both bind specifically to telomeric DNA.
That is, binding of both SpPot1p and hPot1p was unaffected by the
presence of a 60-fold excess of herring sperm DNA and 2000-fold
excess of an oligonucleotide of non-telomeric sequence. To further
investigate the sequence specificity, G-rich strands of telomeric
DNA from different species were tested as substrates in DNA-binding
assays. In a side-by-side comparison, SpPot1p bound the human
telomeric sequence (GGGTTA repeat) with a lower affinity than the
S. pombe telomeric sequence (repeating units of the consensus
sequence GGTTACA) (FIG. 5A). In competition experiments, a
1000-fold excess of unlabeled S. pombe sequence abolished binding
to the radiolabeled substrate, whereas the human and O. nova DNA
competitors reduced binding by only .about.50% and <2%,
respectively (FIG. 5B). Similarly, hPot1p showed only weak binding
to the S. pombe sequence (FIG. 5C), which also was not an efficient
competitor (FIG. 5D). In contrast, the presence of a 1000-fold
excess of the O. nova sequence reduced binding to less than 25%.
Accordingly, both SpPot1p and hPot1p specifically bind telomeric
DNA, and each shows a higher affinity for telomeric DNA from their
own species.
[0057] Pot1p binds a variety of related telomeric DNA sequences.
Oligonucleotides that form a DNA-Pot1p complex, as determined by an
electrophoretic mobility shift assay, are shown in Table I, below.
The affinity between Pot1p and the oligonucleotide varies with the
particular sequence (data not shown).
1TABLE I SpPot1p-binding oligonucleotides: (SEQ ID NOS: 23-35,
respectively, in order of appearance) PBoli52 GGT TAC GGT TAC AGG
TTA CA PBoli53 CGG TTA CAC GGT TAC AGG T PBoli54 GTT ACA GGT TAC
GGT TAC GG PBoli86 TGT GGT GTG TGG GTG TGC GGT T PBoli110 GGT TAC
ACG GTT ACA GGT TAC AGG TTA CAG PBoli112 GGT TAC ACG GTT ACA GGT
TAC AGG TTA CAG GGT TAC GGT TAC G PBoli183 CTG TAA GCA TAT CAT CAT
TCG A GGT TAC PBoli184 GGT TAC GCA TAT CAT CAT TCG A ATC TCG
PBoli185 CTG TAA GCA TAT CAT CGG TTA CGG TTA C PBoli186 GGT TAC GGT
TAC CAT CAT TCG A ATC TCG PBoli187 CTG TAA GCA TAT GGT TAC TCG A
ATC TCG PBoli188 CTG TAA GC GGT TAC GGT TAC GA ATC TCG PT1 GGT TAC
AGG TTA CAG GTT AC hPot1p-binding oligonucleotides: (SEQ ID NOS:
36-38, respectively, in order of appearance) PBoli177 TTA GGG TTA
GGG TTA GGG TT PBoli178 GG TTA GGG TTA GGG TTA GGG PBoli179 TTA GGG
TTA GGG TTA GGG TTA GGG TTA GGG
[0058] hPOT1 mRNA is detected in all tissues examined, although a
high steady-state level of hPOT1 mRNA is observed in testis and
lower levels are observed in colon, skeletal muscle, and peripheral
blood lymphocytes (FIG. 5A and data not shown). In contrast with
mRNA levels of human TERT, which correlate with cellular
immortality and proliferative activity, the presence of hPOT1 mRNA
in all tissues examined is consistent with hPOT1 being a house
keeping gene, required to ensure the integrity of chromosome ends
independently of the proliferative state of cells.
[0059] Screening Methods to Identify Useful Compounds that Affect
the Interaction of Pot1p with Single-Stranded Telomeric DNA
[0060] The use of routine screens to find inhibitors and activators
of Pot1p is facilitated by providing a polynucleotide that encodes
a Pot1 protein, which allows Pot1p to be expressed recombinantly.
Pot1p thus may be expressed in vitro or in a host cell, such as E.
coli, yeast, or bacullovirus-infected insect cells, and tested
against candidate compounds. Useful compounds will be those that
affect the binding between a Pot1 polypeptide and telomeric DNA,
especially the G-rich single-stranded component.
[0061] The interaction between Pot1p and telomeric DNA is readily
assayed in vitro, by a number of routine methods that are well
known to the artisan. In vitro assays can be configured as high
throughput assays, to test candidate molecules simultaneously. In
one embodiment, such assays can be designed around the
electrophoretic mobility shift assays described in the
examples.
[0062] Candidate molecules that will be useful for the invention
generally will include small organic compounds that interact with a
Pot1 protein or a Pot1 protein-DNA complex to change the binding
constant. In one embodiment, candidate molecules are rapidly
identified by their ability to change the amount of labeled probe
that interacts with a Pot1 protein in vitro. Candidates with
possible activity are then further analyzed to determine an
apparent binding constant, which is compared to that of the control
reaction lacking a candidate molecule, to determine whether the
particular compound strengthens or weakens the interaction between
Pot1p and the telomere. Promising candidates may be subsequently
analyzed in a cell culture system, to analyze the effect of the
candidate molecule on telomere length or integrity throughout
repeated cell divisions. The examples describe a number of tests
that can be used to assay the role of Pot1p on telomere
structure.
[0063] Likely candidate compounds that will inhibit the interaction
between a Pot1 polypeptide include compounds that can act as a
substrate analogue. Since the substrate for a Pot1 protein is
telomeric DNA, such compounds include single-stranded DNA
comprising TTAGGG repeats, when used to inhibit a hPot1 protein or
single-stranded DNA comprising GGTTACA repeats, when used to
inhibit a SpPot1 protein. FIG. 5B, lanes d-e, and FIG. 5D, lanes
g-h and k provide in vitro proof of principle of the efficacy of
such inhibitors. The oligonucleotides listed in TABLE I represent a
variety of useful compounds with a known ability to act as
substrate analogues. Thus, these oligonucleotides themselves, or
analogues of these oligonucleotides with advantageous
pharmacological properties, will be useful compounds for the
inhibition of Pot1p activity.
[0064] Preferred analogues of these oligonucleotides are
non-hydrolyzable DNA analogues that have increased pharmacological
longevity and efficacy. One DNA analogue with enhanced stability
relative to DNA is a peptide nucleic acid (PNA) molecule that
comprises a Pot1 protein binding site. Such molecules, along with
methods of their formulation and delivery, are generally described
in U.S. Pat. No. 6,046,307.
[0065] Candidate molecules that will be useful for the invention
may also include small organic compounds that modulate telomerase
activity. These compounds may be administered in combination with
compounds that regulate Pot1p activity. Alternatively, these
compounds themselves are candidates for regulators of Pot1p
activity, and their possible effect on Pot1p activity can be
determined by the screening methods of the invention. These
compounds are described in U.S. Pat. Nos. 6,194,206, 6,156,763,
6,110,955, or 6,054,442, for example.
[0066] Methods to Extend the Life-Span of Cells
[0067] The inventors have shown that chromosome of cells lacking
Pot1p activity are susceptible to rapid disorganization and
destabilization. Pot1p thus maintains telomere structure and
function, which provides a means of therapeutic intervention in
cases where it is desirable to alter telomere structure and
function. Methods are provided alternatively to stabilize or to
destabilize telomere structure, depending on the desirability of
prolonging the proliferative capacity, or life-span, of the cell in
question. "Proliferative capacity" and "life-span" both are used in
this context in terms of how many times a cell can divide before it
enters replicative senescence.
[0068] Enhancing the activity of a Pot1 protein in a cell
advantageously can stabilize telomeres and thereby prolong the
life-span of the cell. Examples of suitable target cells include
those that are genetically engineered to produce a desired protein
or those that produce useful antibodies. Other desirable target
cell types include isolated stem cells, especially where disease
otherwise would deplete various stem cell populations. Additional
advantageous target cells include cells that proliferate in
response to repeated tissue injury, such as endothelial cells, or
cells whose functions are susceptible to aging or disease, such as
CD4+ cells, connective tissue fibroblasts, or cells affected by
age-related macular degeneration.
[0069] Pot1p activity can be increased in a number of ways in these
desired target cells. In one method, Pot1p activity is increased by
transfecting the cell with an expression construct that encodes a
Pot1 protein. In this embodiment, the "effector compound" is an
expression vector that directs high level or regulated expression
of a Pot1 polypeptide. The expression causes higher levels of Pot1p
to accumulate in the target cell, thereby increasing the overall
level of Pot1p activity or replacing Pot1p lost through genetic
mutation. In another method, the cell is treated with a small
effector compound that stabilizes the interaction between Pot1p and
telomeric DNA. In either case, the effector compound may be added
to a cell ex vivo to affect Pot1p expression, followed by
administration of the cell to the individual undergoing treatment.
Alternatively, the effector compound may be administered to the
cell in vivo. In this case a preferable means of administration
directs or targets the effector compound to the desired cell.
Suitable means of cell targeting are known in the art, and include
liposome encapsulation and antibody-directed targeting, or
combinations of these two.
[0070] In some instances, it may be desirable to increase Pot1p
expression temporarily. When an effector compound is administered
in vivo, this control typically can be achieved simply by
discontinuing administration. Where Pot1p expression is increased
through recombinant engineering, on the other hand, it may be
desirable to control Pot1p expression with an inducible or
regulated promoter. Expression then can be induced for as long as
desired by administering the appropriate inducer or regulatory
compound.
[0071] By contrast, an inhibitor of Pot1 protein function will be
useful in shortening the life-span of cells, whose presence is
undesirable, through the destabilization of telomere structure and
function. Such cells include those that are immortalized by
aberrant expression of telomerase, as in many cancer cell lines.
Inhibitors may be delivered to the entire body, as is currently
common in chemotherapeutic methods. Because Pot1p is expressed in a
variety of cell types in humans, and may be expressed ubiquitously,
the amount of administered inhibitor must be carefully monitored to
prevent adverse side-effects to other non-targeted cell types that
express Pot1p. As an alternative or supplement to whole-body
delivery, localized delivery may be employed. For example,
inhibitors can be formulated as a depot for internal delivery to
the site of a tumor. In another embodiment, inhibitors may be
targeted to a specific population of cells by one of the many
available means of cell targeting, such as immunotargeting.
[0072] Parasitic or pathogenic cells, e.g. yeast, whose
proliferation or life-span may be controlled by regulating telomere
length, also are desirable targets for Pot1p inhibitors.
Accordingly, one embodiment of the invention is a method of
controlling yeast infection through administration of a
therapeutically effective amount of a Pot1p inhibitor.
[0073] FIG. 6 demonstrates the ability of Pot1p to inhibit
telomerase action. Pot1p is believed to inhibit telomerase activity
through the formation of a Pot1p-telomeric DNA complex. Compounds
which strengthen or weaken this complex thus are expected to affect
the level of telomerase activity in a cell. In one embodiment of
the invention, a method in which Pot1p activity is increased in a
cell, such as by recombinant expression of a POT1 polynucleotide,
is combined with the administration of a compound that inhibits
telomerase activity. A variety of telomerase inhibitors are known
in the art, as described in U.S. Pat. No. 6,156,763, for
example.
[0074] Pot1 Polypeptides
[0075] The skilled artisan will appreciate that useful variants of
a Pot1 protein include those that maintain the capability of
binding single-stranded telomeric DNA. These variants will be
useful, for example, in methods of screening for compounds that
affect the ability of a Pot1 protein to interact with
single-stranded DNA. Other useful protein variants may not exhibit
DNA-binding activity, but may be useful for other purposes. Such
purposes include raising antibodies that specifically bind a Pot1
protein, such as a non-functional, naturally occurring mutation of
Pot1p. Such purposes also include the identification of dominant
negative inhibitors that bind other cellular proteins that normally
interact with Pot1p. Variants may occur naturally or may be created
by modifying the primary sequence of the protein through
manipulation of a polynucleotide encoding a Pot1 protein. "Protein"
and "polypeptide" are used interchangeably throughout.
[0076] "Variants" of an hPot1 and SpPot1 protein include naturally
occurring allelic variations of hPot1p and SpPot1 proteins, a
fragment of a Pot1 protein that binds single-stranded telomeric
DNA, or a fragment thereof that elicits an antigenic response when
administered to a host animal. Variants also include polypeptides
that have a modified amino acid sequence from the aforementioned
polypeptides. Because protein function depends on three-dimension
structure, skilled artisan will recognize that variants bearing the
closest structural relationship to hPot1p and SpPot1p are most
likely to preserve biological function. Sequence modifications
include amino acid substitutions, insertions, and deletions. Amino
acid insertions and deletions may be made in the interior of the
protein sequence, as well as at the amino and carboxyl termini.
Guidance in determining which and how many such sequence
modifications may be made without abolishing biological or
antigenic activity may be found using computer programs well known
in the art, for example, DNAStar software.
[0077] The sequence of variants preferably will have an 80%
identity to the full-length hPot1p and SpPot1 proteins. More
preferably, variants will have at least about 85% identity to the
full-length sequences. Even more preferably, the percent identity
will be at least about 90%, and most preferably, the percent
identity will be at least about 95%, or even 98%. Likewise,
variants of fragments of hPot1p and SpPot1 proteins will be useful
for the invention, for instance, as antigenic fragments. Such
variants will have at least about 85% identity to fragments of the
hPot1p and SpPot1 proteins. Even more preferably, the percent
identity will be at least about 90%, and most preferably, the
percent identity will be at least about 95%, or even 98%.
Preferably, antigenic fragments will be 5, 10, 15, 20, or 30 amino
acids in length. A preferred biologically active Pot1p fragment
folds into DNA-binding domain. Biologically active fragments
include the N-terminal fragments of Pot1p identified by gel shift
assays, including the 22 kDa fragment of SpPot1p.
[0078] Variants may also include "splicing variants." It is
well-known that, within a given eukaryotic gene, sequences that
encode the polypeptide gene product are non-contiguous. The protein
coding sequences, or exons, are divided by intervening non-coding
sequences, known as introns. These introns are transcribed but then
spliced out during maturation of the mRNA. Exons often correspond
to functional domains of the protein product. Go, Nature 291:90-92
(1981); Branden et al., EMBO J. 3:1307-10 (1984).
[0079] Exons themselves may be spliced out during the maturation of
the mRNA. In some cases, two exons may be mutually exclusive in the
mature mRNA. Deletion or swapping of exons is known as alternative
splicing. Andreadis et al., Ann. Rev. Cell Biol. 3:207-42 (1987).
The family of proteins produced by alternatively spliced mRNAs
exhibit different functional properties, depending on which exons
are present in the mature mRNA. Typically, alternative splicing is
regulated in a tissue-specific manner and involves only one or a
few exons within a gene.
[0080] Thus, the polynucleotides of the invention encompass
variants that differ by the addition, deletion or alternative
splicing of exons. In general, exons alternatively added to the 5'
or 3' termini of the open reading frame are encompassed by
"addition" variants, whereas alternatively spliced exons that
contribute additional coding sequences within the open reading
frame are encompassed by "insertion" variants.
[0081] Specific splicing variants encompassed by the invention are
shown in the Figures. The SpPOT1 gene, for example, has two
introns, which normally are spliced from the mature transcript.
However, in one splicing variant, intron 2 may not be spliced, so
that it is included in the mature transcript (SEQ ID NO:10).
Because the intron does not contain a stop codon, the splicing
variant mRNA gives rise to a somewhat larger polypeptide (compare
SEQ ID NO:9 and 11). When intron 1 is not spliced out, however, the
resulting protein is truncated as a result of a stop codon within
intron 1. The resulting peptide has the sequence: (SEQ ID NO:39) M
G E D V I D S L Q L N E L L N A G E Y K I G V R Y Q W I Y I C F A N
N E K G T Y I S V H. Alternatively, translational frame shifting
may lead to a significantly larger protein product. Translational
frame shifting has been observed in a number of proteins involved
in telomere metabolism. Aigner et al., EMBO J. 19: 6230-39, 2000.
Polypeptides resulting from translational frame shifting also are
considered "splicing variants" for the purposes of the
invention.
[0082] A more complex pattern of splicing variants is observed in
hPOT1 polynucleotides. In one splice variant, exon 5 is not
incorporated into the mature transcript (see FIG. 10G for
nomenclature). The resulting polypeptide is 72 kDa in size and is
shown in FIG. 9B (SEQ ID NO:13). When exon 5 is included in the
mature transcript, the resulting protein is an N-terminal fragment
that is 38 kDa in size, because of the presence of a stop codon
within exon 5 (SEQ ID NO:15). When the mature transcript lacks
exons 5 and 10, it gives rise to another N-terminal fragment 58 kDa
in size. Additional variants may arise from translational frame
shifting, as well.
[0083] Additional polypeptide sequences or other moieties, such as
covalently attached detectable tags, may be added to the proteins
of the invention. Additional polypeptide sequences may fused to
either the amino or carboxyl termini of the polypeptides of the
invention, and they may be useful, for example, in assisting the
expression, purification, and/or detection proteins of the
invention. For example, these various sequences include those well
known in the art that are useful in purification of recombinantly
expressed proteins. A preferred fusion protein, which the inventors
have reduced to practice, comprises a "His.sub.6 tag" sequence,
which facilitates purification of the recombinantly expressed
protein. A preferred purification system is the TALON.TM.
nondenaturing protein purification kit for purifying
His.sub.6-tagged proteins under native conditions (CLONTECH, Palo
Alto, Calif.).
[0084] "Isolated" polypeptides of the invention have been purified
to remove at least some portion of cellular or non-cellular
molecules with which the proteins are associated naturally.
Isolated proteins include those that are partially purified or
enriched, as well as those purified to homogeneity. Isolated
proteins also include those produced artificially, such as by
recombinant expression or by in vitro translation. The isolated
protein may be included in compositions containing other
polypeptides for specific purposes, for example, as
stabilizers.
[0085] "Substitutions, insertions, additions and deletions" refer
to changes in a particular polypeptide sequence, or any one its
naturally occurring splicing variants. "Substitutions" generally
refer to alterations in the amino acid sequence that do not change
the overall length of the polypeptide, but only alter one or more
amino acid residues, substituting one for another in the common
sense of the word. Generally speaking, the number of amino acid
substitutions for any given variant will not be more than about 20,
10, 5, or 3, such as 1-20 or any range or value therein.
Substitutions preferably are conservative, such that one amino acid
is replaced with one of similar shape and charge. Conservative
substitutions are well known in the art and include, for example,
the changes of: alanine to serine; arginine to lysine; asparigine
to glutamine or histidine; aspartate to glutamate; cysteine to
serine; glutamine to asparigine; glutamate to aspartate; glycine to
proline; histidine to asparigine or glutamine; isoleucine to
leucine or valine; leucine to valine or isoleucine; lysine to
arginine, glutamine, or glutamate; methionine to leucine or
isoleucine; phenylalanine to tyrosine, leucine or methionine;
serine to threonine; threonine to serine; tryptophan to tyrosine;
tyrosine to tryptophan or phenylalanine; and valine to isoleucine
or leucine.
[0086] "Insertions" add extra amino acids to the interior (not the
amino- or carboxyl-terminal ends) of the subject polypeptide.
Insertions include amino acids encoded by exons that are
alternatively spliced into a polypeptide, such as the splicing
variants shown in FIGS. 8 and 9. "Deletions" diminish the overall
size of the polypeptide by removal of amino acids from the interior
or either end of the polypeptide. In one embodiment, deletions
remove less than about 30% of the size of the subject molecule.
Other preferred deletions include naturally occurring splicing
variants of a Pot1 protein, such as those described above. These
variants may be fragments of the size the full-length protein,
which may be considerably smaller than 30% the size of the
full-length protein.
[0087] "Additions," like insertions, also add to the overall size
of the protein; however, instead of being made within the molecule,
they are made on the N- or C-terminus of the encoded protein.
Unlike deletions, additions may be of virtually any size; however,
preferred additions do not exceed about 100% of the size of the
native molecule. "Additions" also to encompass adducts to the amino
acids of the native molecule.
[0088] In general, both the DNA and protein molecules of the
invention can be defined with reference to "sequence identity." As
used herein, "sequence identity" refers to a comparison made
between two molecules using standard algorithms well-known in the
art. Although any sequence algorithm can be used to define
"sequence identity," for clarity, the present invention defines
identity with reference to the Smith-Waterman algorithm, where the
open reading frame generally is used as the reference sequence to
define the percentage identity of polynucleotide homologues over
its length. When "sequence identity" is used with reference to a
polypeptide, the designated polypeptide is used as a reference
sequence over its length.
[0089] The choice of parameter values for matches, mismatches, and
inserts or deletions is arbitrary, although some parameter values
have been found to yield more biologically realistic results than
others. One preferred set of parameter values for the
Smith-Waterman algorithm is set forth in the "maximum similarity
segments" approach, which uses values of 1 for a matched residue
and for a mismatched residue (a residue being either a single
nucleotide or a single amino acid). Insertions and deletions
("indels"), x, are weighted as:
x.sub.k=1+k/3,
[0090] where k is the number of residues in a given insert or
deletion (Waterman, Bulletin of Mathematical Biology 46:473-500
(1984)).
[0091] Polynucleotides of the Invention
[0092] Polynucleotides of the invention are those that encode Pot1
proteins or their fragments and derivatives. These polynucleotides
include those that encode SpPot1 polypeptides. An S. pombe genomic
DNA sequence is described by the Sanger Centre as part of cosmid
clone c26H5, having accession number SPAC26H5 (SEQ ID NO:7). This
sequence contains an upstream promoter region, a coding region with
two introns, and a downstream region that contains a terminator.
Both upstream and downstream regions may play a role in the
regulation of SpPot1p expression. The introns can be alternatively
spliced, as described above (SEQ ID NOS:8 and 10). Preferred
polynucleotides are non-genomic; i.e., they correspond to
transcripts from genomic DNA. An example of non-genomic DNA is a
mRNA or cDNA encoding the polypeptides of SEQ ID NO: 9 or SEQ ID
NO:11.
[0093] The polynucleotides of the invention also include those that
encode a hPot1p and its variants and fragments. A partial genomic
clone is described for human POT1, having accession number AC004925
(SEQ ID NO:18). This partial genomic clone contains nine exons,
shown diagrammatically in FIG. 10G. Of these exons, at least exons
5 and 10 can be alternatively spliced (compare SEQ ID NOS:12, 14,
and 16). Various cDNA sequences encoding full-length hPot1p have
been described: FLJ10368 (submitted 22 Feb. 2000), FLJ11073
(submitted 22 Feb. 2000), FLJ12518 (submitted 29 Sept. 2000),
BC002923 (submitted 5 Feb. 2001), and NM.sub.--015450 (submitted 26
Feb. 2001). Various other partial cDNA sequences and ESTs that
encode portions of hPot1 protein also have been described: FLJ22851
(submitted 29 Sept. 2000), AL050120 (submitted 18 Feb. 2000). Of
the hPOT1 polynucleotides presently described, only the hPOT1 cDNA
of SEQ ID NO:12 closely resembles the sequences described in
FLJ10368, FLJ11073, and FLJ12518.
[0094] The invention also provides a nucleic acid molecule having a
sequence complementary to one of the above sequences. Such isolated
nucleic acid molecules are useful as probes for gene mapping by in
situ hybridization with chromosomes. They are particularly useful
for detecting transcription of a POT1 gene in human tissue, or
transcripts of naturally occurring homologues that may themselves
be therapeutically useful.
[0095] The polynucleotides of the invention may also be useful for
detecting transcripts of naturally occurring POT1 variants
occurring in disease states. The present polynucleotides thus may
have diagnostic application in differentiating normal and abnormal
genes, based on differential hybridization, as discussed in more
detail below. Alternatively, a diagnostic application may include
differentiating abnormally high or low levels of expression of a
normal gene.
[0096] Isolated nucleic acid molecules of the present invention
include nucleic acid molecules comprising the coding sequence for a
Pot1 protein, and nucleic acid molecules which comprise a
nucleotide sequence substantially different from those described
above but which, due to the degeneracy of the genetic code, still
encode at least one Pot1 protein as described and enabled herein.
Of course, the genetic code is well-known in the art. Thus, it
would be routine for one skilled in the art to generate such
degenerate nucleic acid variants that code for specific Pot1
proteins of the present invention. See, e.g., Ausubel, et al.
[0097] The term "hybridization" refers to formation of double
stranded polynucleotides through complementary nucleotide base
pairing. High stringency hybridization occurs at a temperature
between about 65.degree. C. and 70.degree. C. in a hybridization
solution of 6.times.SSC, 0.5% SDS, 5.times. Denhardt's solution and
100 .mu.g of non-specific carrier DNA. The preferred probe is 100
bases selected from contiguous bases of the polynucleotide sequence
set forth in SEQ ID NO:1. A high stringency wash solution contains
the equivalent in ionic strength of less than about 0.2.times.SSC
and 0.1% SDS, with a preferred stringent solution containing about
0.1.times.SSC and 0.1% SDS. High stringency washing conditions
comprise washing with 2.times.SSC with 0.05% SDS five times at room
temperature, then washing with 0.1.times.SSC with 0.1% SDS at
68.degree. C. for 1 h. Blots containing the hybridized, labeled
probe are exposed to film for one to three days.
[0098] "Isolated" nucleic acid molecules are removed from their
native or naturally occurring environment. For example, recombinant
nucleic acid molecules in a vector and/or a host cell are
considered isolated for the purposes of the present invention.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the nucleic acid molecules of the present invention. Isolated
nucleic acid molecules according to the present invention further
include such molecules produced synthetically or purified from
cells containing such nucleic acids, where the nucleic acid is in
other than a naturally occurring form. Isolated nucleic acid
molecules include genomic DNA that has been removed from the
chromosome in which it occurs naturally.
[0099] Vectors of the Invention
[0100] The term "vector" refers to a nucleic acid compound used for
introducing exogenous nucleic acid into host cells. A vector
comprises a nucleotide sequence which may encode one or more
polypeptide molecules. Plasmids, cosmids, viruses, and
bacteriophages, in a natural state or which have undergone
recombinant engineering, are non-limiting examples of commonly used
vectors to provide recombinant vectors comprising at least one
desired isolated nucleic acid molecule.
[0101] The term "promoter" refers to a nucleic acid sequence that
directs the initiation of transcription. An inducible promoter is
one that is regulated by environmental signals, such as carbon
source, heat, or metal ions.
[0102] "Host cell" refers to any eukaryotic, prokaryotic, or other
cell that is suitable for propagating and/or expressing an isolated
nucleic acid that is introduced into the host cell by any suitable
means known in the art. The cell can be part of a tissue or
organism, isolated in culture or in any other suitable form.
[0103] The present invention further provides recombinant
expression cassettes comprising a nucleic acid of the present
invention, and operably linked to transcriptional initiation
regulatory sequences that will direct the transcription of the
polynucleotide in the intended host cell. Both heterologous and
endogenous promoters can be employed to direct expression. These
promoters can also be used, for example, in recombinant expression
cassettes to drive expression of antisense nucleic acids to reduce
Pot1p content in a desired tissue.
[0104] In some embodiments, isolated nucleic acids which serve as
promoter or enhancer elements can be introduced in the appropriate
position (generally upstream) of a non-heterologous form of a
polynucleotide of the present invention so as to up or down
regulate expression of a polynucleotide of the present invention.
For example, endogenous promoters can be altered in vivo by
mutation, deletion, and/or substitution. Suitable promoters include
the phage lambda PL promoter, the E. coli lac, trp and tac
promoters, the SV40 early and late promoters and promoters of
retroviral LTRs, to name a few. Other suitable promoters will be
known to the skilled artisan. The expression constructs will
further contain sites for transcription initiation, termination
and, in the transcribed region, a ribosome binding site for
translation. The coding portion of the mature transcripts expressed
by the constructs will preferably include a translation initiation
codon at the beginning and a termination codon (UAA, UGA or UAG)
appropriately positioned at the end of the polypeptide to be
translated.
[0105] The polynucleotides can optionally be joined to a vector
containing a selectable marker for propagation in a host. Such
markers include, e.g., dihydrofolate reductase or neomycin
resistance for eukaryotic cell culture and tetracycline or
ampicillin resistance genes for culturing in E. coli and other
bacteria. Representative examples of appropriate hosts include, but
are not limited to, bacterial cells, such as E. coli, Streptomyces
and Salmonella typhimurium cells; fungal cells, such as yeast
cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells;
animal cells such as CHO, COS and Bowes melanoma cells; and plant
cells. Appropriate culture mediums and conditions for the
above-described host cells are known in the art. Among vectors
preferred for use in bacteria include pQE70, pQE60 and pQE-9,
available from Qiagen; pBS vectors, Phagescript vectors, Bluescript
vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene;
and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from
Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,
pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,
pMSG and pSVL available from Pharmacia. Other suitable vectors will
be readily apparent to the skilled artisan. Introduction of the
construct into the host cell can be effected by calcium phosphate
transfection, DEAE-dextran mediated transfection, cationic
lipid-mediated transfection, electroporation, transduction,
infection or other methods. Such methods are described in many
standard laboratory manuals, such as Sambrook, supra, Chapters 1-4
and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.
[0106] Recombinant Protein Expression
[0107] The polypeptide can be expressed in a modified form, such as
a fusion protein, and can include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
can be added to the N-terminus of a polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties can
be added to a polypeptide to facilitate purification. Such regions
can be removed prior to final preparation of a polypeptide. The
addition of peptide moieties to polypeptides to engender secretion
or excretion, to improve stability and to facilitate purification,
among others, are familiar and routine techniques in the art. Such
methods are described in many standard laboratory manuals, such as
Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel,
supra, Chapters 16, 17 and 18.
[0108] A Pot1 polypeptide can be recovered and purified from
recombinant cell cultures by well known methods. Polypeptides of
the present invention include naturally purified products, products
of chemical synthetic procedures, and products produced by
recombinant techniques from a prokaryotic or eukaryotic host,
including, for example, bacterial, yeast, higher plant, insect and
mammalian cells. Polypeptides of the invention can also include an
initial modified methionine residue, in some cases as a result of
host-mediated processes. The monitoring of the purification process
can be accomplished by DNA-binding activity assays, Western blot
techniques, radioimmunoassay, or other standard immunoassay
techniques. These methods are described in many standard laboratory
manuals, such as Sambrook, supra, Chapters 17.37-17.42; Ausubel,
supra, Chapters 10, 12, 13, 16, 18 and 20.
[0109] Antibodies of the Invention
[0110] Antibodies raised against the proteins and protein fragments
of the invention also are contemplated by the invention. In
particular, the invention contemplates antibodies raised against
Pot1p, and variants thereof. Described below are antibody products
and methods for producing antibodies capable of specifically
recognizing one or more epitopes of the presently described
proteins and their derivatives. Antibodies include, but are not
limited to polyclonal antibodies, monoclonal antibodies (mAbs),
humanized or chimeric antibodies, single chain antibodies including
single chain Fv (scFv) fragments, Fab fragments, F(ab').sub.2
fragments, fragments produced by a Fab expression library,
anti-idiotypic (anti-Id) antibodies, epitope-binding fragments, and
humanized forms of any of the above.
[0111] As known to one in the art, these antibodies may be used,
for example, in the detection of a target protein in a biological
sample. They also may be utilized as part of treatment methods,
and/or may be used as part of diagnostic techniques whereby
patients may be tested for abnormal levels or preferably for the
presence of abnormal forms of the proteins.
[0112] In general, techniques for preparing polyclonal and
monoclonal antibodies as well as hybridomas capable of producing
the desired antibody are well known in the art (Campbell, A. M.,
Monoclonal Antibody Technology: Laboratory Techniques in
Biochemistry and Molecular Biology, Elsevier Science Publishers,
Amsterdam, The Netherlands (1984); St. Groth et al., J. Immunol.
Methods 35:1-21 (1980); Kohler and Milstein, Nature 256:495-497
(1975)), the trioma technique, the human B-cell hybridoma technique
(Kozbor et al., Immunology Today 4:72 (1983); Cole et al., in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.
(1985), pp. 77-96).
[0113] i) Polyclonal Antibodies.
[0114] Polyclonal antibodies are heterogeneous populations of
antibody molecules derived from the sera of animals immunized with
an antigen, such as an inventive protein or an antigenic derivative
thereof. Polyclonal antiserum, containing antibodies to
heterogeneous epitopes of a single protein, can be prepared by
immunizing suitable animals with the expressed protein described
above, which can be unmodified or modified, as known in the art, to
enhance immunogenicity. Immunization methods include subcutaneous
or intraperitoneal injection of the polypeptide.
[0115] Effective polyclonal antibody production is affected by many
factors related both to the antigen and to the host species. For
example, small molecules tend to be less immunogenic than others
and may require the use of carriers and/or adjuvant. In addition,
host animal response may vary with site of inoculation. Both
inadequate or excessive doses of antigen may result in low titer
antisera. In general, however, small doses (high ng to low .mu.g
levels) of antigen administered at multiple intradermal sites
appears to be most reliable. Host animals may include but are not
limited to rabbits, mice, and rats, to name but a few. An effective
immunization protocol for rabbits can be found in Vaitukaitis, J.
et al., J. Clin. Endocrinol. Metab. 33:988-991 (1971).
[0116] The protein immunogen may be modified or administered in an
adjuvant in order to increase the protein's antigenicity. Methods
of increasing the antigenicity of a protein are well known in the
art and include, but are not limited to coupling the antigen with a
heterologous protein or through the inclusion of an adjuvant during
immunization.
[0117] Booster injections can be given at regular intervals, with
at least one usually being required for optimal antibody
production. The antiserum may be harvested when the antibody titer
begins to fall. Titer may be determined semi-quantitatively, for
example, by double immunodiffusion in agar against known
concentrations of the antigen. See, for example, Ouchterlony et
al., Chap. 19 in: Handbook of Experimental Immunology, Wier, ed,
Blackwell (1973). Plateau concentration of antibody is usually in
the range of 0.1 to 0.2 mg/ml of serum (about 12 .mu.M). The
antiserum may be purified by affinity chromatography using the
immobilized immunogen carried on a solid support. Such methods of
affinity chromatography are well known in the art.
[0118] Affinity of the antisera for the antigen may be determined
by preparing competitive binding curves, as described, for example,
by Fisher, Chap. 42 in: Manual of Clinical Immunology, second
edition, Rose and Friedman, eds., Amer. Soc. For Microbiology,
Washington, D. C. (1980).
[0119] ii) Monoclonal Antibodies.
[0120] Monoclonal antibodies (MAbs), are homogeneous populations of
antibodies to a particular antigen. They may be obtained by any
technique that provides for the production of antibody molecules by
continuous cell lines in culture or in vivo. MAbs may be produced
by making hybridomas, which are immortalized cells capable of
secreting a specific monoclonal antibody.
[0121] Monoclonal antibodies to any of the proteins, peptides and
epitopes thereof described herein can be prepared from murine
hybridomas according to the classical method of Kohler, G. and
Milstein, C., Nature 256:495-497 (1975) (and U.S. Pat. No.
4,376,110) or modifications of the methods thereof, such as the
human B-cell hybridoma technique (Kosbor et al., 1983, Immunology
Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:
2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.
77-96).
[0122] In one method a mouse is repetitively inoculated with a few
micrograms of the selected protein over a period of a few weeks.
The mouse is then sacrificed, and the antibody producing cells of
the spleen are isolated. The spleen cells are fused, typically
using polyethylene glycol, with mouse myeloma cells, such as
SP2/0-Ag14 myeloma cells. The excess, unfused cells are destroyed
by growth of the system on selective media comprising aminopterin
(HAT media). The successfully fused cells are diluted, and aliquots
are plated to microliter plates where growth is continued.
Antibody-producing clones (hybridomas) are identified by detection
of antibody in the supernatant fluid of the wells by immunoassay
procedures. These include ELISA, as originally described by
Engvall, Meth. Enzymol. 70:419 (1980), western blot analysis,
radioimmunoassay (Lutz et al., Exp. Cell Res. 175:109-124 (1988))
and modified methods thereof.
[0123] Selected positive clones can be expanded and their
monoclonal antibody product harvested for use. Detailed procedures
for monoclonal antibody production are described in Davis, L. et
al. BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier, New York. Section
21-2 (1989). The hybridoma clones may be cultivated in vitro or in
vivo, for instance as ascites. Production of high titers of mAbs in
vivo makes this the presently preferred method of production.
Alternatively, hybridoma culture in hollow fiber bioreactors
provides a continuous high yield source of monoclonal
antibodies.
[0124] The antibody class and subclass may be determined using
procedures known in the art (Campbell, A. M., Monoclonal Antibody
Technology: Laboratory Techniques in Biochemistry and Molecular
Biology, Elsevier Science Publishers, Amsterdam, The Netherlands
(1984)). MAbs may be of any immunoglobulin class including IgG,
IgM, IgE, IgA, IgD and any subclass thereof. Methods of purifying
monoclonal antibodies are well known in the art.
[0125] iii) Antibody Derivatives and Fragments.
[0126] Fragments or derivatives of antibodies include any portion
of the antibody which is capable of binding the target antigen, or
a specific portion thereof. Antibody fragments specifically include
F(ab').sub.2, Fab, Fab' and Fv fragments. These can be generated
from any class of antibody, but typically are made from IgG or IgM.
They may be made by conventional recombinant DNA techniques or,
using the classical method, by proteolytic digestion with papain or
pepsin. See CURRENT PROTOCOLS IN IMMUNOLOGY, chapter 2, Coligan et
al., eds., (John Wiley & Sons 1991-92).
[0127] F(ab').sub.2 fragments are typically about 110 kDa (IgG) or
about 150 kDa (IgM) and contain two antigen-binding regions, joined
at the hinge by disulfide bond(s). Virtually all, if not all, of
the Fc is absent in these fragments. Fab' fragments are typically
about 55 kDa (IgG) or about 75 kDa (IgM) and can be formed, for
example, by reducing the disulfide bond(s) of an F(ab').sub.2
fragment. The resulting free sulfhydryl group(s) may be used to
conveniently conjugate Fab' fragments to other molecules, such as
detection reagents (e.g., enzymes).
[0128] Fab fragments are monovalent and usually are about 50 kDa
(from any source). Fab fragments include the light (L) and heavy
(H) chain, variable (V.sub.L and V.sub.H, respectively) and
constant (C.sub.L and C.sub.H, respectively) regions of the
antigen-binding portion of the antibody. The H and L portions are
linked by an intramolecular disulfide bridge.
[0129] Fv fragments are typically about 25 kDa (regardless of
source) and contain the variable regions of both the light and
heavy chains (V.sub.L and V.sub.H, respectively). Usually, the
V.sub.L and V.sub.H chains are held together only by non-covalent
interacts and, thus, they readily dissociate; however, they have
the advantage of small size and they retain the same binding
properties of the larger Fab fragments. Accordingly, methods have
been developed to crosslink the V.sub.L and V.sub.H chains, using,
for example, glutaraldehyde (or other chemical crosslinkers),
intermolecular disulfide bonds (by incorporation of cysteines) and
peptide linkers.
[0130] Other antibody derivatives include single chain antibodies
(U.S. Pat. No. 4,946,778; Bird, Science 242:423-426 (1988); Huston
et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et
al., Nature 334:544-546 (1989)). Single chain antibodies are formed
by linking the heavy and light chain fragments of the Fv region via
an amino acid bridge, resulting in a single chain Fv (SCFv).
[0131] One preferred method involves the generation of scFvs by
recombinant methods, which allows the generation of Fvs with new
specificities by mixing and matching variable chains from different
antibody sources. In a typical method, a recombinant vector would
be provided which comprises the appropriate regulatory elements
driving expression of a cassette region. The cassette region would
contain a DNA encoding a peptide linker, with convenient sites at
both the 5' and 3' ends of the linker for generating fusion
proteins. The DNA encoding a variable region(s) of interest may be
cloned in the vector to form fusion proteins with the linker, thus
generating a scFv.
[0132] In an exemplary alternative approach, DNAs encoding two Fvs
may be ligated to the DNA encoding the linker, and the resulting
tripartite fusion may be ligated directly into a conventional
expression vector. The scFv DNAs generated any of these methods may
be expressed in prokaryotic or eukaryotic cells, depending on the
vector chosen.
[0133] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, such fragments include
but are not limited to: the F(ab').sub.2 fragments which can be
produced by pepsin digestion of the antibody molecule and the Fab
fragments which can be generated by reducing the disulfide bridges
of the F(ab).sub.2 fragments. Alternatively, Fab expression
libraries may be constructed (Huse et al., 1989, Science,
246:1275-1281) to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity.
[0134] Derivatives also include "chimeric antibodies" (Morrison et
al., Proc. Natl. Acad. Sci., 81:6851-6855 (1984); Neuberger et al.,
Nature, 312:604-608 (1984); Takeda et al., Nature, 314:452-454
(1985)). These chimeras are made by splicing the DNA encoding a
mouse antibody molecule of appropriate specificity with, for
instance, DNA encoding a human antibody molecule of appropriate
specificity. Thus, a chimeric antibody is a molecule in which
different portions are derived from different animal species, such
as those having a variable region derived from a murine mAb and a
human immunoglobulin constant region. These are also known
sometimes as "humanized" antibodies and they offer the added
advantage of at least partial shielding from the human immune
system. They are, therefore, particularly useful in therapeutic in
vivo applications.
[0135] iv) Labeled Antibodies.
[0136] The present invention further provides the above-described
antibodies in detectably labeled form. Antibodies can be detectably
labeled through the use of radioisotopes, affinity labels (such as
biotin, avidin, etc.), enzymatic labels (such as horseradish
peroxidase, alkaline phosphatase, etc.), fluorescent labels (such
as FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures
for accomplishing such labeling are well-known in the art, for
example see (Sternberger et al., J. Histochem. Cytochem. 18:315
(1970); Bayer et al., Meth. Enzym. 62:308 (1979); Engval et al.,
Immunol. 109:129 (1972); Goding, J. Immunol. Meth. 13:215 (1976)).
The labeled antibodies of the present invention can be used for in
vitro, in vivo, and in situ diagnostic assays.
[0137] v) Immobilized Antibodies.
[0138] The foregoing antibodies also may be immobilized on a solid
support. Examples of such solid supports include plastics such as
polycarbonate, complex carbohydrates such as agarose and sepharose,
acrylic resins and such as polyacrylamide and latex beads.
Techniques for coupling antibodies to such solid supports are well
known in the art (Weir et al., "Handbook of Experimental
Immunology" 4th Ed., Blackwell Scientific Publications, Oxford,
England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic
Press, N.Y. (1974)). The immobilized antibodies of the present
invention can be used for in vitro, in vivo, and in situ assays as
well as for immunoaffinity purification of the proteins of the
present invention.
[0139] Pharmaceutical Compositions Comprising a POT1 Gene.
[0140] Pharmaceutical compositions comprising polynucleotides
encoding functional Pot1 polypeptides of the invention are those
useful for gene therapy to cause the overexpression of functional
Pot1 polypeptides in cells in which chromosome stabilization is
desired, or the overexpression of a variant Pot1 polypeptide with
dominant negative interference activity in cells in which
chromosome destabilization is desired.
[0141] Overexpression of POT1 in a cell may be accomplished by
transfecting a cell with a POT1 polynucleotide. The POT1
polynucleotide generally is a component on an expression vector of
the invention, defined above. The vector may be delivered to a cell
by transfection of a cell ex vivo, followed by selection and
cloning of transfected cells expressing the POT1 nucleotide and
then by administration of the stably transfected cells to an
individual in need of the modified cells.
[0142] Alternatively, the POT1 polynucleotide may be delivered to a
cell or a population of cells in an individual. Various methods of
introducing exogenous genes into cells in vivo are known in the
art. See Rosenberg et al., Science 242:1575-1578 (1988) and Wolff
et al., PNAS 86:9011-9014 (1989), which are incorporated herein by
reference. A listing of suitable vectors is set forth in Hodgson,
Bio/Technology 13: 222 (1995), which is incorporated by reference.
One example of a suitable vector is a cationic liposome, such as
DC-Chol/DOPE liposome, which is an appropriate vehicle to deliver
DNA to a wide range of tissues through intravenous injection of
DNA/cationic liposome complexes. See Caplen et al., Nature Med.
1:39-46 (1995) and Zhu et al., Science 261:209-211 (1993), herein
incorporated by reference.
[0143] Viral vector-mediated gene transfer is also a suitable
method for the introduction of the vector into a target cell.
Appropriate viral vectors include adenovirus vectors and
adeno-associated virus vectors, retrovirus vectors and herpesvirus
vectors. Adenoviruses are linear, double stranded DNA viruses
complexed with core proteins and surrounded by capsid proteins. The
common serotypes 2 and 5, which are not associated with any human
malignancies, are typically the base vectors. By deleting parts of
the virus genome and inserting the desired gene under the control
of a constitutive viral promoter, the virus becomes a
replication-deficient vector capable of transferring the exogenous
DNA to differentiated, non-proliferating cells. To enter cells, the
adenovirus interacts with specific receptors on the cell surface,
and the adenovirus surface proteins interact with the cell surface
integrins. The virus penton-cell integrin interaction provides the
signal that brings the exogenous gene-containing virus into a
cytoplasmic endosome. The adenovirus breaks out of the endosome and
moves to the nucleus, the viral capsid falls apart, and the
exogenous DNA enters the cell nucleus where it functions, in an
epichromosomal fashion, to express the exogenous gene. Detailed
discussions of the use of adenoviral vectors for gene therapy can
be found in Berkner, Biotechniques 6:616-629 (1988) and Trapnell,
Advanced Drug Delivery Rev. 12:185-199 (1993), which are herein
incorporated by reference. Adenovirus-derived vectors, particularly
non-replicative adenovirus vectors, are characterized by their
ability to accommodate exogenous DNA of 7.5 kB, relative stability,
wide host range, low pathogenicity in man, and high titers
(10.sup.4 to 10.sup.5 plaque forming units per cell). See
Stratford-Perricaudet et al., PNAS 89:2581 (1992).
[0144] Pharmaceutical compositions may be formulated with one or
more physiologically acceptable carriers or excipients. In one
embodiment, the composition is formulated for injection. Long
acting formulations are generally known in the art and can be
adapted to the administration of a POT1 polynucleotide. Such
compositions may be in the form of suspensions, solutions,
emulsions in vesicles, or any other form known in the art.
Additional suspending, stabilizing, or dispersing agents may be
added as necessary. Alternatively, the active ingredient may be in
the form of a powder for reconstitution prior to
administration.
[0145] Diagnostic Methods.
[0146] The present invention also contemplates methods for
diagnosis of human disease. In particular, patients can be screened
for the occurrence of cancers, or likelihood of occurrence of
cancers, associated with mutations in the Pot1 protein or with
changes in its level of expression. By examining a number of
patients in this manner, mutations in the gene that are associated
with a malignant cellular phenotype can be identified. In addition,
correlation of the nature of the observed mutations with subsequent
observed clinical outcomes allows development of prognostic model
for the predicted outcome in a particular patient.
[0147] Screening for mutations conveniently can be carried out at
the DNA level by use of PCR, although the skilled artisan will be
aware that many other well known methods are available for the
screening. PCR primers can be selected that flank known mutation
sites, and the PCR products can be sequenced to detect the
occurrence of the mutation. Alternatively, the 3' residue of one
PCR primer can be selected to be a match only for the residue found
in the unmutated gene. If the gene is mutated, there will be a
mismatch at the 3' end of the primer, and primer extension cannot
occur, and no PCR product will be obtained. Alternatively, primer
mixtures can be used where the 3' residue of one primer is any
nucleotide other than the nonmutated residue. Observation of a PCR
product then indicates that a mutation has occurred. Other methods
of using, for example, oligonucleotide probes to screen for
mutations are described, or example, in U.S. Pat. No. 4,871,838,
which is herein incorporated by reference in its entirety.
[0148] Alternatively, antibodies can be generated that selectively
bind either mutated or non-mutated Pot1 protein. The antibodies
then can be used to screen tissue samples for occurrence of
mutations in a manner analogous to the DNA-based methods described
above.
[0149] The diagnostic methods described above can be used not only
for diagnosis and for prognosis of existing disease, but may also
be used to predict the likelihood of the future occurrence of
disease. For example, clinically healthy patients can be screened
for mutations in the Pot1 protein that correlate with later disease
onset. Such mutations may be observed in the heterozygous state in
healthy individuals. In such cases a single mutation event can
effectively disable proper functioning of the gene encoding the
Pot1 protein and induce a transformed or malignant phenotype. This
screening also may be carried out prenatally or neonatally.
[0150] DNA molecules according to the invention also are well
suited for use in so-called "gene chip" diagnostic applications.
Such applications have been developed by, inter alia, Synteni and
Affymetrix. Briefly, all or part of the DNA molecules of the
invention can be used either as a probe to screen a polynucleotide
array on a "gene chip," or they may be immobilized on the chip
itself and used to identify other polynucleotides via hybridization
to the surface of the chip. In this manner, for example, related
genes can be identified, or expression patterns of the POT1 gene in
various tissues can be simultaneously studied. Such gene chips have
particular application for diagnosis of disease, or predisposition
to disease, which may be indicated by a change in the level or
tissue distribution of POT1 mRNA or by the presence of a particular
POT1 mRNA species. Suitable chip technology is described for
example, in Wodicka et al., Nature Biotechnology, 15:1359 (1997)
which is hereby incorporated by reference.
[0151] Detection of a Pot1 Polypeptide.
[0152] The presence of a Pot1 protein may be assayed in a
biological sample isolated from an individual. Pot1p may be
detected in any number of ways commonly known in the art. For
example, Pot1p may be detected by a specific interaction with a
labeled antibody of the invention. The antibody label allows rapid
detection of an immune complex by such well known methods as
Western blotting. Formation of an immune complex will be useful in
detecting Pot1 proteins with or without biological function. Thus,
an immune complex formation will be the preferred mode of detection
of a Pot1 protein in a sample from an individual, where the Pot1
protein in the sample is suspected of lacking activity through
genetic alteration. Such an assay thus will be useful in a
diagnostic method, to detect altered forms of Pot1p.
[0153] Alternatively, a Pot1 protein may be assayed by virtue of
its biological function. In one embodiment, a sample suspected of
containing a Pot1 polypeptide is exposed to isolated labeled
telomeric DNA. A Pot1 protein is then detected by its ability to
interact with the telomeric DNA. A convenient method of assaying
this interaction is with a gel shift assay, which is well known in
the art and used to form the Pot1p-DNA complexes in Example 2.
[0154] Pharmaceutical compositions comprising compounds that affect
Pot1p activity, and routes of administering the same.
[0155] Pharmaceutical compositions comprising compounds that affect
Pot1 protein activity can be formulated and administered according
to well known methods. These compounds include those small molecule
compounds that affect Pot1p binding to telomeric DNA identified by
the screening methods of the invention. These compounds may be
delivered in a pharmaceutically acceptable carrier vehicle.
Suitable vehicles and their formulation are described, for example,
in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed.,
Mack, Easton Pa. (1980)).
[0156] Pharmaceutical compositions are formulated to provide a
"therapeutically effective amount" of a compound that affects the
activity of a Pot1 protein. The amount of a compound required for
therapeutic efficacy depends on the individual or animal to be
treated, and on the precise condition involving a Pot1 protein. The
amount actually administered will be optimized to reduce
side-effects while having a maximum effect on the activity of a
Pot1 protein. Preferably, the amount delivered to the body will be
reduced by directed delivery to a population of target cells, where
possible.
[0157] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
Thus, the compounds and their physiologically acceptable salts may
be formulated for administration by a variety of routes. The
compounds may be delivered by parenteral, inhalation or
insufflation (either through the mouth or the nose), topical, oral,
or depot administration.
[0158] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection, repeated
injections, or continuous infusion. Formulations for injection may
be presented in unit dosage form, e.g., in ampules or in multi-dose
containers, with an added preservative. The compositions may take
such forms as suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain agents that aid in suspending,
stabilizing or dispersing the active compounds. Alternatively, the
active ingredient may be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
Instead of injection, the compounds may be administered as an
irrigation fluid used to wash areas or organs of the body.
[0159] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g. gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0160] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. The compounds may also be formulated in rectal
compositions such as suppositories or retention enemas, e.g.,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0161] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated or formulated for sustained release by methods well known in
the art. Liquid preparations for oral administration may take the
form of, for example, solutions, syrups or suspensions, or they
maybe presented as a dry product for constitution with water or
other suitable vehicle before use. Such liquid preparations may be
prepared by conventional means with pharmaceutically acceptable
additives such as suspending agents (e.g., sorbitol syrup,
cellulose derivatives or hydrogenated edible fats); emulsifying
agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g.,
almond oil, oily esters, ethyl alcohol or fractionated vegetable
oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates
or sorbic acid). The preparations may also contain buffer salts,
flavoring, coloring and sweetening agents as appropriate.
[0162] Preferred formulations for oral delivery are described by
U.S. Pat. Nos. 5,574,018 and 5,428,023. Biologically active
conjugates of a therapeutically useful protein are made with
vitamin B.sub.12 (VB.sub.12) by covalently binding the primary (5')
hydroxyl group of the ribose moiety of VB.sub.12 to the therapeutic
protein. When the resulting conjugate is orally delivered, it binds
intrinsic factor (IF) transporter protein in the gastrointestinal
tract and is then taken up through the epithelium into the
bloodstream, retaining the biological activity of the protein
therapeutic. The conjugates may be orally administered in the
presence of purified IF, resulting in greater absorption.
[0163] WO 93/25221 describes compositions formulated for oral
delivery, comprising therapeutic proteins contained in microspheres
made of protein and/or synthetic polymer. The microspheres protect
their protein contents against gastrointestinal proteases and
provide controlled and sustained release of their contents.
Microspheres can be designed to pass through the intestinal
epithelium into the blood or lymph, and they may be targeted to
particular cells or organs. Formulations and methodology useful for
targeting orally administered microparticles to various organs are
described in EP 531,497, for example.
[0164] The present invention, thus generally described, will be
understood more readily by reference to the following examples,
which are provided by way of illustration and are not intended to
be limiting of the present invention.
EXAMPLES
Example 1
Expression and Purification of SpPot1p
[0165] SpPot1p containing N-terminal V5 and His.sub.6-tags was
cloned into the pQE30 expression vector (Qiagen), which introduces
an additional N-terminal His.sub.6-tag, and expressed in E. coli
strain M15 (pRep4) using tryptone phosphate media. Following
induction (0.8 mM IPTG) for 6 hours at 24.degree. C. cells were
harvested, resuspended in lysis buffer at pH 8.0 (50 mM
NaH.sub.2PO.sub.4; 0.1 M NaCl; 2 mM imidazole; 10% glycerol; 0.2%
Tween20; 5 mM .beta.-mercaptoethanol, 1 mM PMSF) and lysed by the
addition of lysozyme (0.5 mg/ml). After 30 min the concentration of
NaCl was increased to 0.6 M, genomic DNA was sheared by sonication
and cell debris was removed by centrifugation at 10,000 g for 30
min. The supernatant was incubated with Ni-NTA resin (Qiagen) at
4.degree. C. for 90 min, which was then loaded onto a column and
washed sequentially with P buffer (50 mM NaH.sub.2PO.sub.4; 600 mM
NaCl; 10% glycerol; 0.2% Tween20; 5 mM .beta.-mercaptoethanol)
containing increasing concentrations of imidazole. Pot1p eluted
around 90 mM imidazole. Pot1 containing fractions were dialysed
against T buffer (50 mM Tris/HCl pH 8.0; 10% glycerol; 0.5 mM EDTA;
0.5 mM DTT) containing 0.2 M KCl and Pot1p was further purified on
a Q-sepharose column (Pharmacia) using a linear gradient of KCl
(0.2 M-1 M). Pot1p eluted around 0.5M KCl, was dialysed against T
buffer plus 0.2M KCl and stored in aliquots at -80.degree. C.
Example 2
DNA-Binding Specificity of SpPot1p
[0166] C-strand (CGTAACCGTAACCCTGTAACCTGTAACCTGTAACCGTGTAACC) (SEQ
ID NO: 40) and G-strand (GGTTACACGGTTACAGGTTACAGGT
TACAGGGTTACGGTTACG) (SEQ ID NO: 28) were 5' .sup.32P-labeled using
T4 polynucleotide kinase and .gamma.-.sup.32P-ATP. Duplex DNA was
generated by annealing equimolar amounts of radiolabeled C-strand
and unlabelled G-strand. Binding reactions (10 .mu.l) were carried
out in 25 mM HEPES (pH 7.5), 1 mM EDTA, 50 mM NaCl, 5% glycerol,
and 2.5 .mu.M PBoli109 (CCGTAAGCATTTCATTATTGGAAT- T
CGAGCTCGTTTTCGA) (SEQ ID NO:41) as non-specific competitor. Pot1p
(50 ng) was incubated with the indicated DNA substrates (1 ng) for
15 min at 20.degree. C. Complexes were analyzed by electrophoresis
at 4.degree. C. through a 4-20% TBE gel (Invitrogen) run at 150 V
for 80 min. The Pot1p-DNA complex is indicated by an open arrow in
FIG. 3A. FIG. 3B shows the same experiment except that the added
protein (100 ng) contained truncated Pot1p as well as full length
protein. Truncated Pot1p-DNA complex is indicated by a closed
arrow.
Example 3
Substrate Specificity of SpPot1p and hPot1p
[0167] FIG. 6A shows binding of SpPot1p to radiolabeled S. pombe
and human G-strand DNAs. FIG. 6B shows binding of SpPot1p (50 ng)
to radiolabeled G-strand (15 pg or 1.5 fmol) in the presence of
10-, 100-, and 1000-fold excess of unlabeled S. pombe, human or O.
nova G-strand. FIG. 6C shows binding of hPot1p to radiolabeled S.
pombe and human G-strand DNAs. FIG. 6D shows binding of hPot1p to
radiolabeled human G-strand DNA under same conditions as in FIG.
6B.
Example 4
Cloning of the hPOT1 Gene
[0168] Oligos PBoli164T (SEQ ID NO:42) (TTCAGATGTTATCTGTCAATCAG
AACCTG) and PBoli194B (GAACACTGTTTACATCCATAGTGATGTATTGTT CC) were
used to amplify a 614 bp fragment of hPOT1 from multiple tissue
cDNA panels (Clontech) with Advantage 2 Polymerase mix in the
buffer supplied by Clontech. Cycling parameters of touch-down PCR
were 94.degree. C. for 5 s, 68.degree. C. for 120 s (32 cycles).
The gene encoding glyceraldehyde phosphate dehydrogenase (GAPDH)
was used as a positive control for the integrity of the cDNA sample
and was amplified for 26 cycles with primers (SEQ ID NO:44)
TGAAGGTCGGAGTCAACGGATTTGGT and (SEQ ID NO:45)
CATGTGGGCCATGAGGTCCACCAC.
[0169] hPOT1 was PCR amplified from ovary cDNA and cloned into a
pQE30 expression vector. Recombinantly expressed hPot1p (carrying
an N-terminal His.sub.6-tag) was purified from E. coli. The protein
was purified over Ni-NTA resin under the same conditions as
SpPot1p. The human protein eluted at around 135 mM imidazole.
[0170] The description, specific examples, and data, while
indicating exemplary embodiments, are given by way of illustration
and are not intended to limit the present invention. Various
changes and modifications within the present invention will become
apparent to the skilled artisan from the disclosure, and thus are
considered part of the invention.
Sequence CWU 1
1
45 1 118 PRT Euplotes crassus 1 Gln Lys Ala Ala Lys Lys Asp His Tyr
Gln Tyr Ser Asp Leu Ser Ser 1 5 10 15 Ile Lys Lys Glu Gly Glu Glu
Asp Gln Tyr His Phe Tyr Gly Val Val 20 25 30 Ile Asp Ala Ser Phe
Pro Tyr Lys Gly Glu Lys Arg Tyr Val Val Thr 35 40 45 Cys Lys Val
Ala Asp Pro Ser Ser Val Ala Lys Gly Gly Lys Leu Asn 50 55 60 Thr
Val Asn Val Val Phe Phe Ser Gln Asn Phe Glu Asp Leu Pro Ile 65 70
75 80 Ile Gln Arg Val Gly Asp Ile Val Arg Val His Arg Ala Arg Leu
Gln 85 90 95 His Tyr Asn Asp Ala Lys Gln Leu Asn Val Asn Met Tyr
Tyr Arg Ser 100 105 110 Ser Trp Cys Leu Phe Ile 115 2 123 PRT
Stylonychia mytilis 2 Lys Lys Arg Glu Gln Ser Thr Arg Tyr Lys Tyr
Val Glu Leu Asn Lys 1 5 10 15 Ala Ser Leu Thr Ser Ala Glu Ala Gln
His Phe Tyr Gly Val Val Ile 20 25 30 Asp Ala Thr Phe Pro Tyr Lys
Thr Asn Gln Glu Arg Tyr Ile Cys Ser 35 40 45 Leu Lys Val Val Asp
Pro Ser Leu Tyr Leu Lys Ser Gln Lys Gly Thr 50 55 60 Gly Asp Ala
Ser Asp Tyr Ala Thr Leu Val Leu Tyr Ala Lys Arg Phe 65 70 75 80 Glu
Asp Leu Pro Ile Ile His Arg Ile Gly Asp Ile Ile Arg Val His 85 90
95 Arg Ala Thr Leu Arg Leu Tyr Asn Gly Gln Arg Gln Phe Asn Ala Asn
100 105 110 Val Phe Tyr Asn Ser Ser Trp Ala Leu Phe Ser 115 120 3
123 PRT Oxytricha trifallax 3 Lys Lys Ala Glu Lys Gly Ser Lys Tyr
Glu Tyr Val Glu Leu Thr Lys 1 5 10 15 Ala Gln Leu Thr Ser Val Thr
Ala Gln His Phe Tyr Ala Val Val Ile 20 25 30 Asp Ala Thr Phe Pro
Tyr Lys Thr Asn Gln Glu Arg Tyr Ile Cys Ser 35 40 45 Leu Lys Ile
Val Asp Pro Ser Leu Tyr Leu Lys Lys Glu Lys Gly Thr 50 55 60 Gly
Asp Asn Ser Asp Tyr Ala Thr Leu Val Leu Tyr Ala Lys Arg Phe 65 70
75 80 Glu Asp Leu Pro Ile Ile His Arg Leu Gly Asp Ile Ile Arg Ile
His 85 90 95 Arg Ala Thr Ile Arg Leu Tyr Asn Gly Gln Arg Gln Phe
Asn Ala Asn 100 105 110 Ile Phe Tyr Ser Ser Ser Trp Ala Leu Phe Ser
115 120 4 123 PRT Oxytricha nova 4 Lys Lys Ser Asp Lys Gly His Lys
Tyr Glu Tyr Val Glu Leu Ala Lys 1 5 10 15 Ala Ser Leu Thr Ser Ala
Gln Pro Gln His Phe Tyr Ala Val Val Ile 20 25 30 Asp Ala Thr Phe
Pro Tyr Lys Thr Asn Gln Glu Arg Tyr Ile Cys Ser 35 40 45 Leu Lys
Ile Val Asp Pro Thr Leu Tyr Leu Lys Gln Gln Lys Gly Ala 50 55 60
Gly Asp Ala Ser Asp Tyr Ala Thr Leu Val Leu Tyr Ala Lys Arg Phe 65
70 75 80 Glu Asp Leu Pro Ile Ile His Arg Ala Gly Asp Ile Ile Arg
Val His 85 90 95 Arg Ala Thr Leu Arg Leu Tyr Asn Gly Gln Arg Gln
Phe Asn Ala Asn 100 105 110 Val Phe Tyr Ser Ser Ser Trp Ala Leu Phe
Ser 115 120 5 109 PRT Homo sapiens 5 Met Ser Leu Val Pro Ala Thr
Asn Tyr Ile Tyr Thr Pro Leu Asn Gln 1 5 10 15 Leu Lys Gly Gly Thr
Ile Val Asn Val Tyr Gly Val Val Lys Phe Phe 20 25 30 Lys Pro Pro
Tyr Leu Ser Lys Gly Thr Asp Tyr Cys Ser Val Val Thr 35 40 45 Ile
Val Asp Gln Thr Asn Val Lys Leu Thr Cys Leu Leu Phe Ser Gly 50 55
60 Asn Tyr Glu Ala Leu Pro Ile Ile Tyr Lys Asn Gly Asp Ile Val Arg
65 70 75 80 Phe His Arg Leu Lys Ile Gln Val Tyr Lys Lys Glu Thr Gln
Gly Ile 85 90 95 Thr Ser Ser Gly Phe Ala Ser Leu Thr Phe Glu Gly
Thr 100 105 6 116 PRT Schizosaccharomyces pombe 6 Lys Ile Gly Glu
Leu Thr Phe Gln Ser Ile Arg Ser Ser Gln Glu Leu 1 5 10 15 Gln Lys
Lys Asn Thr Ile Val Asn Leu Phe Gly Ile Val Lys Asp Phe 20 25 30
Thr Pro Ser Arg Gln Ser Leu His Gly Thr Lys Asp Trp Val Thr Thr 35
40 45 Val Tyr Leu Trp Asp Pro Thr Cys Asp Thr Ser Ser Ile Gly Leu
Gln 50 55 60 Ile His Leu Phe Ser Lys Gln Gly Asn Asp Leu Pro Val
Ile Lys Gln 65 70 75 80 Val Gly Gln Pro Leu Leu Leu His Gln Ile Thr
Leu Arg Ser Tyr Arg 85 90 95 Asp Arg Thr Gln Gly Leu Ser Lys Asp
Gln Phe Arg Tyr Ala Leu Trp 100 105 110 Pro Asp Phe Ser 115 7 3980
DNA Schizosaccharomyces pombe 7 tatgagtgaa gttccatcca tgatgcaaaa
agccatgctg tcaaccttaa aaagtatatc 60 ggccattccc gatgatgtac
cccctcctta ttctgagttt gctgatgata cgacagcgca 120 agctggttct
agtaaaagag atagcgctat atctgaagat cccgatcatc acaaaagtgt 180
ttggtggtct ttgagatggc aatctcggct tgttggtcgt ggaaaatcta ctgctcttac
240 tcctgaagaa accagagcaa tacaggagca ggcaaagaca ctgaaaaagg
caggaatgga 300 ctttatgcta ttctctttct ggttacctgc cctacttttg
ctgagtatct ttggtcttcg 360 aagctatgct caaatgatcg ggggatattt
atatcgctgc ataattggca tttaggtttg 420 acgaacaacc atgcatgttt
ttttctttct tttagtttta ttcttttttg tagattatga 480 gcaaactact
gtcaaaactt aggtattatg acaatgaaat cgtatatatt atattcgatt 540
ggatcaattt tttattatat tgaaagtaat tgcttatttt gtaagttaaa cttacatggg
600 tttaaacgca tagagcaggt tggcgctttt aaaaccaaaa tagatcgttg
caggtttgct 660 gttctggatc gtgaatgcaa taccttagga aagtctttta
ataagctatc gctttttgca 720 ttgcattctt tttctaaact gaacgttaga
ttagctaaag taagcgtctt gagttttcga 780 gatgaaccgc atacattaaa
atttttaagt accaattggc atgaaccggt atgcgatctg 840 cttattataa
tactagtaaa tcttgatact cggcaaactc tttcaataat agcctagcag 900
aaactgggat atgtctaaag ttttacaact gcgctcagct taaggacttt acggcgatcc
960 atttaatagc tagccatgaa cactcataac ctcaagattg aggagtgggt
cattcttttg 1020 cttgataaag aaacaaattc attattggta aaataaaact
gaataaccct tagttcatcc 1080 taggaatttg aagaagggga atgatcaagc
ttgaacaagt aactctcacg cagtctattg 1140 aataatctga aggttcatca
ctttcaaggg gttgtcttgg tttaaaaagc ttttaccaat 1200 tccatttagg
tttctgagaa aggctaaaac tcatttgttg ttcttaaagg atatttggat 1260
cattcgttga tcaagcatgg gagaggacgt tattgacagt cttcagttga atgagttatt
1320 aaatgctgga gaatataaga ttggagtgag atatcaatgg atttatattt
gttttgctaa 1380 caatgaaaaa ggaacttaca tttcagtcca ttagaagctc
tcaagaatta caaaagaaga 1440 atactattgt caatttgttt ggaatagtaa
aagattttac ccctagtcgc caaagtctac 1500 atggaactaa gggtatgctt
gcttatcatg gtggaaacta tactttttat ttttccagtc 1560 aagagctaat
aatcatgttt ttagattggg taaccaccgt atatttgtgg gatccaacat 1620
gtgatacatc aagcatcgga ctacagatac acttgttcag caaacaggga aatgatttgc
1680 ctgtaatcaa gcaggtgggg caaccgcttt tgcttcatca aatcacatta
agaagttata 1740 gagacaggac tcaaggtttg tctaaggatc aatttcgata
tgcactttgg ccagactttt 1800 cttctaattc caaagatact ctctgtcctc
aaccaatgcc tcgtttaatg aaaacgggag 1860 acaaggaaga gcaattcgcc
ttgttgttaa ataaaatttg ggatgagcaa actaataaac 1920 ataaaaatgg
cgaattattg agtacctctt ctgctcgtca aaatcaaact ggattgagtt 1980
acccttctgt ctctttttct ctgctatcac aaataactcc acatcaacgt tgtagctttt
2040 acgctcaggt aattaaaact tggtacagtg ataaaaactt tactctttat
gtcactgatt 2100 atacggaaaa tgagcttttt tttccaatgt ctccgtatac
tagctcctcg agatggaggg 2160 gcccttttgg tcggttttct ataaggtgca
ttttatggga tgagcacgac ttttactgcc 2220 gcaactacat taaagaaggt
gactatgtgg ttatgaaaaa tgtgcgaacc aaaattgatc 2280 accttggtta
tctggaatgt atacttcatg gggattcagc aaaacgttat aatatgagta 2340
tagaaaaagt cgattcggaa gaacccgaac taaacgaaat taagtcacgt aaaaggcttt
2400 atgttcagaa ttgccaaaat ggtatagaag cagtaatcga gaaactcagt
caaagccaac 2460 aatcggaaaa tccttttatc gcccatgaat taaagcaaac
ttctgttaat gaaattacgg 2520 cccatgtcat aaatgaacct gctagtttaa
aattgactac tatttctacc atacttcatg 2580 cacctttgca gaatcttctc
aaaccgagga aacataggct acgcgttcag gtggtagatt 2640 tttggccaaa
gagtttgacg cagtttgctg tgctatctca accaccatct tcgtatgttt 2700
ggatgtttgc cttgctcgta agggatgtat cgaatgtgac tttaccggtc atattttttg
2760 attctgacgc tgcggaactt attaacagct caaaaatcca accttgcaat
ttagctgatc 2820 acccgcagat gactcttcag cttaaagaaa gattatttct
gatttggggg aacttggaag 2880 aacgcattca gcatcacata tcgaagggtg
aatcgccaac tctggctgct gaagatgttg 2940 aaacaccatg gtttgatata
tatgtcaaag aatacattcc tgtaattggg aacaccaaag 3000 accatcaatc
tttgactttt cttcagaagc gctggcgagg atttggcacg aaaattgttt 3060
gactattgtg atacaaaact tacaataatg aaatgcttac ggaaaagaaa cataagaaaa
3120 acaatattta aatttaagga aagctctata ttgggagaat tttataaagc
gagcgaattt 3180 gtactaagga aaaacacaga ggggaaacgt gaaatatcta
attgcttaga ctttatataa 3240 catcaacttc gaaataatct tagaaattaa
ttacaaaaat aataaggatt ggtttgatgt 3300 atggtggtta catctaagca
ggcttttgct tagaagttgc aagtgttgag gcatcatcat 3360 cactttcatc
gtcaacagcg aatagagctt gatgctcatc ggcactgcca tgaataatat 3420
gagggttggc tggagatgta ggacgctcat gatgcagatg caaactatca tttgagagag
3480 aggaagtcat ctcaaactca tctacatctt gagcaacttg ctcactcatt
gcgaaacgac 3540 ggttattctc ggtaggacgc cacaagtaca aaatggtaag
catcaagatc aaaacaagaa 3600 tatcagtgta tccgtaatta aggaaccaaa
gaagtttcca gtattttaag taatagttca 3660 tttgaccgta gataccaatc
aaaatggcat tggctgcgac aatcgaagca taagcgacaa 3720 tgccaaaaca
tataacaatc caaagacgag tatacatctg agccttaaca gtttgcttac 3780
gaatacggag atcacgaatt gtattattta aagccaatac aatccaaagg aacatagcga
3840 agagggtgat taaaaagaca ggagcggcaa acaaaatgac caaagactct
ttattagatg 3900 ggctaatgaa caaagatgac aagaaaaagc atgaagaaac
gaactgcaaa ccagcaagaa 3960 tttgacactt acgaagaaga 3980 8 2087 DNA
Schizosaccharomyces pombe 8 gattgaggag tgggtcattc ttttgcttga
taaagaaaca aattcattat tggtaaaata 60 aaactgaata acccttagtt
catcctagga atttgaagaa ggggaatgat caagcttgaa 120 caagtaactc
tcacgcagtc tattgaataa tctgaaggtt catcactttc aaggggttgt 180
cttggtttaa aaagctttta ccaattccat ttaggtttct gagaaaggct aaaactcatt
240 tgttgttctt aaaggatatt tggatcattc gttgatcaag catgggagag
gacgttattg 300 acagtcttca gttgaatgag ttattaaatg ctggagaata
taagattgga gaacttacat 360 ttcagtccat tagaagctct caagaattac
aaaagaagaa tactattgtc aatttgtttg 420 gaatagtaaa agattttacc
cctagtcgcc aaagtctaca tggaactaag gattgggtaa 480 ccaccgtata
tttgtgggat ccaacatgtg atacatcaag catcggacta cagatacact 540
tgttcagcaa acagggaaat gatttgcctg taatcaagca ggtggggcaa ccgcttttgc
600 ttcatcaaat cacattaaga agttatagag acaggactca aggtttgtct
aaggatcaat 660 ttcgatatgc actttggcca gacttttctt ctaattccaa
agatactctc tgtcctcaac 720 caatgcctcg tttaatgaaa acgggagaca
aggaagagca attcgccttg ttgttaaata 780 aaatttggga tgagcaaact
aataaacata aaaatggcga attattgagt acctcttctg 840 ctcgtcaaaa
tcaaactgga ttgagttacc cttctgtctc tttttctctg ctatcacaaa 900
taactccaca tcaacgttgt agcttttacg ctcaggtaat taaaacttgg tacagtgata
960 aaaactttac tctttatgtc actgattata cggaaaatga gctttttttt
ccaatgtctc 1020 cgtatactag ctcctcgaga tggaggggcc cttttggtcg
gttttctata aggtgcattt 1080 tatgggatga gcacgacttt tactgccgca
actacattaa agaaggtgac tatgtggtta 1140 tgaaaaatgt gcgaaccaaa
attgatcacc ttggttatct ggaatgtata cttcatgggg 1200 attcagcaaa
acgttataat atgagtatag aaaaagtcga ttcggaagaa cccgaactaa 1260
acgaaattaa gtcacgtaaa aggctttatg ttcagaattg ccaaaatggt atagaagcag
1320 taatcgagaa actcagtcaa agccaacaat cggaaaatcc ttttatcgcc
catgaattaa 1380 agcaaacttc tgttaatgaa attacggccc atgtcataaa
tgaacctgct agtttaaaat 1440 tgactactat ttctaccata cttcatgcac
ctttgcagaa tcttctcaaa ccgaggaaac 1500 ataggctacg cgttcaggtg
gtagattttt ggccaaagag tttgacgcag tttgctgtgc 1560 tatctcaacc
accatcttcg tatgtttgga tgtttgcctt gctcgtaagg gatgtatcga 1620
atgtgacttt accggtcata ttttttgatt ctgacgctgc ggaacttatt aacagctcaa
1680 aaatccaacc ttgcaattta gctgatcacc cgcagatgac tcttcagctt
aaagaaagat 1740 tatttctgat ttgggggaac ttggaagaac gcattcagca
tcacatatcg aagggtgaat 1800 cgccaactct ggctgctgaa gatgttgaaa
caccatggtt tgatatatat gtcaaagaat 1860 acattcctgt aattgggaac
accaaagacc atcaatcttt gacttttctt cagaagcgct 1920 ggcgaggatt
tggcacgaaa attgtttgac tattgtgata caaaacttac aataatgaaa 1980
tgcttacgga aaagaaacat aagaaaaaca atatttaaat ttaaggaaag ctctatattg
2040 ggagaatttt ataaagcgag cgaatttgta ctaaggaaaa acacaga 2087 9 555
PRT Schizosaccharomyces pombe 9 Met Gly Glu Asp Val Ile Asp Ser Leu
Gln Leu Asn Glu Leu Leu Asn 1 5 10 15 Ala Gly Glu Tyr Lys Ile Gly
Glu Leu Thr Phe Gln Ser Ile Arg Ser 20 25 30 Ser Gln Glu Leu Gln
Lys Lys Asn Thr Ile Val Asn Leu Phe Gly Ile 35 40 45 Val Lys Asp
Phe Thr Pro Ser Arg Gln Ser Leu His Gly Thr Lys Asp 50 55 60 Trp
Val Thr Thr Val Tyr Leu Trp Asp Pro Thr Cys Asp Thr Ser Ser 65 70
75 80 Ile Gly Leu Gln Ile His Leu Phe Ser Lys Gln Gly Asn Asp Leu
Pro 85 90 95 Val Ile Lys Gln Val Gly Gln Pro Leu Leu Leu His Gln
Ile Thr Leu 100 105 110 Arg Ser Tyr Arg Asp Arg Thr Gln Gly Leu Ser
Lys Asp Gln Phe Arg 115 120 125 Tyr Ala Leu Trp Pro Asp Phe Ser Ser
Asn Ser Lys Asp Thr Leu Cys 130 135 140 Pro Gln Pro Met Pro Arg Leu
Met Lys Thr Gly Asp Lys Glu Glu Gln 145 150 155 160 Phe Ala Leu Leu
Leu Asn Lys Ile Trp Asp Glu Gln Thr Asn Lys His 165 170 175 Lys Asn
Gly Glu Leu Leu Ser Thr Ser Ser Ala Arg Gln Asn Gln Thr 180 185 190
Gly Leu Ser Tyr Pro Ser Val Ser Phe Ser Leu Leu Ser Gln Ile Thr 195
200 205 Pro His Gln Arg Cys Ser Phe Tyr Ala Gln Val Ile Lys Thr Trp
Tyr 210 215 220 Ser Asp Lys Asn Phe Thr Leu Tyr Val Thr Asp Tyr Thr
Glu Asn Glu 225 230 235 240 Leu Phe Phe Pro Met Ser Pro Tyr Thr Ser
Ser Ser Arg Trp Arg Gly 245 250 255 Pro Phe Gly Arg Phe Ser Ile Arg
Cys Ile Leu Trp Asp Glu His Asp 260 265 270 Phe Tyr Cys Arg Asn Tyr
Ile Lys Glu Gly Asp Tyr Val Val Met Lys 275 280 285 Asn Val Arg Thr
Lys Ile Asp His Leu Gly Tyr Leu Glu Cys Ile Leu 290 295 300 His Gly
Asp Ser Ala Lys Arg Tyr Asn Met Ser Ile Glu Lys Val Asp 305 310 315
320 Ser Glu Glu Pro Glu Leu Asn Glu Ile Lys Ser Arg Lys Arg Leu Tyr
325 330 335 Val Gln Asn Cys Gln Asn Gly Ile Glu Ala Val Ile Glu Lys
Leu Ser 340 345 350 Gln Ser Gln Gln Ser Glu Asn Pro Phe Ile Ala His
Glu Leu Lys Gln 355 360 365 Thr Ser Val Asn Glu Ile Thr Ala His Val
Ile Asn Glu Pro Ala Ser 370 375 380 Leu Lys Leu Thr Thr Ile Ser Thr
Ile Leu His Ala Pro Leu Gln Asn 385 390 395 400 Leu Leu Lys Pro Arg
Lys His Arg Leu Arg Val Gln Val Val Asp Phe 405 410 415 Trp Pro Lys
Ser Leu Thr Gln Phe Ala Val Leu Ser Gln Pro Pro Ser 420 425 430 Ser
Tyr Val Trp Met Phe Ala Leu Leu Val Arg Asp Val Ser Asn Val 435 440
445 Thr Leu Pro Val Ile Phe Phe Asp Ser Asp Ala Ala Glu Leu Ile Asn
450 455 460 Ser Ser Lys Ile Gln Pro Cys Asn Leu Ala Asp His Pro Gln
Met Thr 465 470 475 480 Leu Gln Leu Lys Glu Arg Leu Phe Leu Ile Trp
Gly Asn Leu Glu Glu 485 490 495 Arg Ile Gln His His Ile Ser Lys Gly
Glu Ser Pro Thr Leu Ala Ala 500 505 510 Glu Asp Val Glu Thr Pro Trp
Phe Asp Ile Tyr Val Lys Glu Tyr Ile 515 520 525 Pro Val Ile Gly Asn
Thr Lys Asp His Gln Ser Leu Thr Phe Leu Gln 530 535 540 Lys Arg Trp
Arg Gly Phe Gly Thr Lys Ile Val 545 550 555 10 1740 DNA
Schizosaccharomyces pombe 10 atgggagagg acgttattga cagtcttcag
ttgaatgagt tattaaatgc tggagaatat 60 aagattggag aacttacatt
tcagtccatt agaagctctc aagaattaca aaagaagaat 120 actattgtca
atttgtttgg aatagtaaaa gattttaccc ctagtcgcca aagtctacat 180
ggaactaagg gtatgcttgc ttatcatggt ggaaactata ctttttattt ttccagtcaa
240 gagctaataa tcatgttttt agattgggta accaccgtat atttgtggga
tccaacatgt 300 gatacatcaa gcatcggact acagatacac ttgttcagca
aacagggaaa tgatttgcct 360 gtaatcaagc aggtggggca accgcttttg
cttcatcaaa tcacattaag aagttataga 420 gacaggactc aaggtttgtc
taaggatcaa tttcgatatg cactttggcc agacttttct 480 tctaattcca
aagatactct ctgtcctcaa ccaatgcctc gtttaatgaa aacgggagac 540
aaggaagagc aattcgcctt gttgttaaat aaaatttggg atgagcaaac taataaacat
600 aaaaatggcg aattattgag tacctcttct gctcgtcaaa atcaaactgg
attgagttac 660 ccttctgtct ctttttctct gctatcacaa ataactccac
atcaacgttg tagcttttac 720 gctcaggtaa ttaaaacttg gtacagtgat
aaaaacttta ctctttatgt cactgattat 780 acggaaaatg agcttttttt
tccaatgtct ccgtatacta gctcctcgag atggaggggc 840 ccttttggtc
ggttttctat aaggtgcatt
ttatgggatg agcacgactt ttactgccgc 900 aactacatta aagaaggtga
ctatgtggtt atgaaaaatg tgcgaaccaa aattgatcac 960 cttggttatc
tggaatgtat acttcatggg gattcagcaa aacgttataa tatgagtata 1020
gaaaaagtcg attcggaaga acccgaacta aacgaaatta agtcacgtaa aaggctttat
1080 gttcagaatt gccaaaatgg tatagaagca gtaatcgaga aactcagtca
aagccaacaa 1140 tcggaaaatc cttttatcgc ccatgaatta aagcaaactt
ctgttaatga aattacggcc 1200 catgtcataa atgaacctgc tagtttaaaa
ttgactacta tttctaccat acttcatgca 1260 cctttgcaga atcttctcaa
accgaggaaa cataggctac gcgttcaggt ggtagatttt 1320 tggccaaaga
gtttgacgca gtttgctgtg ctatctcaac caccatcttc gtatgtttgg 1380
atgtttgcct tgctcgtaag ggatgtatcg aatgtgactt taccggtcat attttttgat
1440 tctgacgctg cggaacttat taacagctca aaaatccaac cttgcaattt
agctgatcac 1500 ccgcagatga ctcttcagct taaagaaaga ttatttctga
tttgggggaa cttggaagaa 1560 cgcattcagc atcacatatc gaagggtgaa
tcgccaactc tggctgctga agatgttgaa 1620 acaccatggt ttgatatata
tgtcaaagaa tacattcctg taattgggaa caccaaagac 1680 catcaatctt
tgacttttct tcagaagcgc tggcgaggat ttggcacgaa aattgtttga 1740 11 579
PRT Schizosaccharomyces pombe 11 Met Gly Glu Asp Val Ile Asp Ser
Leu Gln Leu Asn Glu Leu Leu Asn 1 5 10 15 Ala Gly Glu Tyr Lys Ile
Gly Glu Leu Thr Phe Gln Ser Ile Arg Ser 20 25 30 Ser Gln Glu Leu
Gln Lys Lys Asn Thr Ile Val Asn Leu Phe Gly Ile 35 40 45 Val Lys
Asp Phe Thr Pro Ser Arg Gln Ser Leu His Gly Thr Lys Gly 50 55 60
Met Leu Ala Tyr His Gly Gly Asn Tyr Thr Phe Tyr Phe Ser Ser Gln 65
70 75 80 Glu Leu Ile Ile Met Phe Leu Asp Trp Val Thr Thr Val Tyr
Leu Trp 85 90 95 Asp Pro Thr Cys Asp Thr Ser Ser Ile Gly Leu Gln
Ile His Leu Phe 100 105 110 Ser Lys Gln Gly Asn Asp Leu Pro Val Ile
Lys Gln Val Gly Gln Pro 115 120 125 Leu Leu Leu His Gln Ile Thr Leu
Arg Ser Tyr Arg Asp Arg Thr Gln 130 135 140 Gly Leu Ser Lys Asp Gln
Phe Arg Tyr Ala Leu Trp Pro Asp Phe Ser 145 150 155 160 Ser Asn Ser
Lys Asp Thr Leu Cys Pro Gln Pro Met Pro Arg Leu Met 165 170 175 Lys
Thr Gly Asp Lys Glu Glu Gln Phe Ala Leu Leu Leu Asn Lys Ile 180 185
190 Trp Asp Glu Gln Thr Asn Lys His Lys Asn Gly Glu Leu Leu Ser Thr
195 200 205 Ser Ser Ala Arg Gln Asn Gln Thr Gly Leu Ser Tyr Pro Ser
Val Ser 210 215 220 Phe Ser Leu Leu Ser Gln Ile Thr Pro His Gln Arg
Cys Ser Phe Tyr 225 230 235 240 Ala Gln Val Ile Lys Thr Trp Tyr Ser
Asp Lys Asn Phe Thr Leu Tyr 245 250 255 Val Thr Asp Tyr Thr Glu Asn
Glu Leu Phe Phe Pro Met Ser Pro Tyr 260 265 270 Thr Ser Ser Ser Arg
Trp Arg Gly Pro Phe Gly Arg Phe Ser Ile Arg 275 280 285 Cys Ile Leu
Trp Asp Glu His Asp Phe Tyr Cys Arg Asn Tyr Ile Lys 290 295 300 Glu
Gly Asp Tyr Val Val Met Lys Asn Val Arg Thr Lys Ile Asp His 305 310
315 320 Leu Gly Tyr Leu Glu Cys Ile Leu His Gly Asp Ser Ala Lys Arg
Tyr 325 330 335 Asn Met Ser Ile Glu Lys Val Asp Ser Glu Glu Pro Glu
Leu Asn Glu 340 345 350 Ile Lys Ser Arg Lys Arg Leu Tyr Val Gln Asn
Cys Gln Asn Gly Ile 355 360 365 Glu Ala Val Ile Glu Lys Leu Ser Gln
Ser Gln Gln Ser Glu Asn Pro 370 375 380 Phe Ile Ala His Glu Leu Lys
Gln Thr Ser Val Asn Glu Ile Thr Ala 385 390 395 400 His Val Ile Asn
Glu Pro Ala Ser Leu Lys Leu Thr Thr Ile Ser Thr 405 410 415 Ile Leu
His Ala Pro Leu Gln Asn Leu Leu Lys Pro Arg Lys His Arg 420 425 430
Leu Arg Val Gln Val Val Asp Phe Trp Pro Lys Ser Leu Thr Gln Phe 435
440 445 Ala Val Leu Ser Gln Pro Pro Ser Ser Tyr Val Trp Met Phe Ala
Leu 450 455 460 Leu Val Arg Asp Val Ser Asn Val Thr Leu Pro Val Ile
Phe Phe Asp 465 470 475 480 Ser Asp Ala Ala Glu Leu Ile Asn Ser Ser
Lys Ile Gln Pro Cys Asn 485 490 495 Leu Ala Asp His Pro Gln Met Thr
Leu Gln Leu Lys Glu Arg Leu Phe 500 505 510 Leu Ile Trp Gly Asn Leu
Glu Glu Arg Ile Gln His His Ile Ser Lys 515 520 525 Gly Glu Ser Pro
Thr Leu Ala Ala Glu Asp Val Glu Thr Pro Trp Phe 530 535 540 Asp Ile
Tyr Val Lys Glu Tyr Ile Pro Val Ile Gly Asn Thr Lys Asp 545 550 555
560 His Gln Ser Leu Thr Phe Leu Gln Lys Arg Trp Arg Gly Phe Gly Thr
565 570 575 Lys Ile Val 12 1905 DNA Homo sapiens 12 atgtctttgg
ttccagcaac aaattatata tatacacccc tgaatcaact taagggtggt 60
acaattgtca atgtctatgg tgttgtgaag ttctttaagc ccccatatct aagcaaagga
120 actgattatt gctcagttgt aactattgtg gaccagacaa atgtaaaact
aacttgcctg 180 ctctttagtg gaaactatga agcccttcca ataatttata
aaaatggaga tattgttcgc 240 tttcacaggc tgaagattca agtatataaa
aaggagactc agggtatcac cagctctggc 300 tttgcatctt tgacgtttga
gggaactttg ggagccccta tcatacctcg cacttcaagc 360 aagtatttta
acttcactac tgaggaccac aaaatggtag aagccttacg tgtttgggca 420
tctactcata tgtcaccgtc ttggacatta ctaaaattgt gtgatgttca gccaatgcag
480 tattttgacc tgacttgtca gctcttgggc aaagcagaag tggacggagc
atcatttctt 540 ctaaaggtat gggatggcac caggacacca tttccatctt
ggagagtctt aatacaagac 600 cttgttcttg aaggtgattt aagtcacatc
catcggctac aaaatctgac aatagacatt 660 ttagtctacg ataaccatgt
tcatgtggca agatctctga aggttggaag ctttcttaga 720 atctatagcc
ttcataccaa acttcaatca atgaattcag agaatcagac aatgttaagt 780
ttagagtttc atcttcatgg aggtaccagt tacggtcggg gaatcagggt cttgccagaa
840 agtaactctg atgtggatca actgaaaaag gatttagaat ctgcaaattt
gacagccaat 900 cagcattcag atgttatctg tcaatcagaa cctgacgaca
gctttccaag ctctggatca 960 gtatcattat acgaggtaga aagatgtcaa
cagctatctg ctacaatact tacagatcat 1020 cagtatttgg agaggacacc
actatgtgcc attttgaaac aaaaagctcc tcaacaatac 1080 cgcatccgag
caaaattgag gtcatataag cccagaagac tatttcagtc tgttaaactt 1140
cattgcccta aatgtcattt gctgcaagaa gttccacatg agggcgattt ggatataatt
1200 tttcaggatg gtgcaactaa aaccccagtt gtcaagttac aaaatacatc
attatatgat 1260 tcaaaaatct ggaccactaa aaatcaaaaa ggacgaaaag
tagcagttca ttttgtgaaa 1320 aataatggta ttctcccgct ttcaaatgaa
tgtctacttt tgatagaagg aggtacactc 1380 agtgaaattt gcaaactctc
gaacaagttt aatagtgtaa ttcctgtgag atctggccac 1440 gaagacctgg
aacttttgga cctttcagca ccatttctta tacaaggaac aatacatcac 1500
tatggatgta aacagtgttc tagtttgaga tccatacaaa atctaaattc cctggttgat
1560 aaaacatcgt ggattccttc ttctgtggca gaagcactgg gtattgtacc
cctccaatat 1620 gtgtttgtta tgacctttac acttgatgat ggaacaggag
tactagaagc ctatctcatg 1680 gattctgaca aattcttcca gattccagca
tcagaagttc tgatggatga tgaccttcag 1740 aaaagtgtgg atatgatcat
ggatatgttt tgtcctccag gaataaaaat tgatgcatat 1800 ccgtggttgg
aatgcttcat caagtcatac aatgtcacaa atggaacaga taatcaaatt 1860
tgctatcaga tttttgacac cacagttgca gaagatgtaa tctaa 1905 13 634 PRT
Homo sapiens 13 Met Ser Leu Val Pro Ala Thr Asn Tyr Ile Tyr Thr Pro
Leu Asn Gln 1 5 10 15 Leu Lys Gly Gly Thr Ile Val Asn Val Tyr Gly
Val Val Lys Phe Phe 20 25 30 Lys Pro Pro Tyr Leu Ser Lys Gly Thr
Asp Tyr Cys Ser Val Val Thr 35 40 45 Ile Val Asp Gln Thr Asn Val
Lys Leu Thr Cys Leu Leu Phe Ser Gly 50 55 60 Asn Tyr Glu Ala Leu
Pro Ile Ile Tyr Lys Asn Gly Asp Ile Val Arg 65 70 75 80 Phe His Arg
Leu Lys Ile Gln Val Tyr Lys Lys Glu Thr Gln Gly Ile 85 90 95 Thr
Ser Ser Gly Phe Ala Ser Leu Thr Phe Glu Gly Thr Leu Gly Ala 100 105
110 Pro Ile Ile Pro Arg Thr Ser Ser Lys Tyr Phe Asn Phe Thr Thr Glu
115 120 125 Asp His Lys Met Val Glu Ala Leu Arg Val Trp Ala Ser Thr
His Met 130 135 140 Ser Pro Ser Trp Thr Leu Leu Lys Leu Cys Asp Val
Gln Pro Met Gln 145 150 155 160 Tyr Phe Asp Leu Thr Cys Gln Leu Leu
Gly Lys Ala Glu Val Asp Gly 165 170 175 Ala Ser Phe Leu Leu Lys Val
Trp Asp Gly Thr Arg Thr Pro Phe Pro 180 185 190 Ser Trp Arg Val Leu
Ile Gln Asp Leu Val Leu Glu Gly Asp Leu Ser 195 200 205 His Ile His
Arg Leu Gln Asn Leu Thr Ile Asp Ile Leu Val Tyr Asp 210 215 220 Asn
His Val His Val Ala Arg Ser Leu Lys Val Gly Ser Phe Leu Arg 225 230
235 240 Ile Tyr Ser Leu His Thr Lys Leu Gln Ser Met Asn Ser Glu Asn
Gln 245 250 255 Thr Met Leu Ser Leu Glu Phe His Leu His Gly Gly Thr
Ser Tyr Gly 260 265 270 Arg Gly Ile Arg Val Leu Pro Glu Ser Asn Ser
Asp Val Asp Gln Leu 275 280 285 Lys Lys Asp Leu Glu Ser Ala Asn Leu
Thr Ala Asn Gln His Ser Asp 290 295 300 Val Ile Cys Gln Ser Glu Pro
Asp Asp Ser Phe Pro Ser Ser Gly Ser 305 310 315 320 Val Ser Leu Tyr
Glu Val Glu Arg Cys Gln Gln Leu Ser Ala Thr Ile 325 330 335 Leu Thr
Asp His Gln Tyr Leu Glu Arg Thr Pro Leu Cys Ala Ile Leu 340 345 350
Lys Gln Lys Ala Pro Gln Gln Tyr Arg Ile Arg Ala Lys Leu Arg Ser 355
360 365 Tyr Lys Pro Arg Arg Leu Phe Gln Ser Val Lys Leu His Cys Pro
Lys 370 375 380 Cys His Leu Leu Gln Glu Val Pro His Glu Gly Asp Leu
Asp Ile Ile 385 390 395 400 Phe Gln Asp Gly Ala Thr Lys Thr Pro Val
Val Lys Leu Gln Asn Thr 405 410 415 Ser Leu Tyr Asp Ser Lys Ile Trp
Thr Thr Lys Asn Gln Lys Gly Arg 420 425 430 Lys Val Ala Val His Phe
Val Lys Asn Asn Gly Ile Leu Pro Leu Ser 435 440 445 Asn Glu Cys Leu
Leu Leu Ile Glu Gly Gly Thr Leu Ser Glu Ile Cys 450 455 460 Lys Leu
Ser Asn Lys Phe Asn Ser Val Ile Pro Val Arg Ser Gly His 465 470 475
480 Glu Asp Leu Glu Leu Leu Asp Leu Ser Ala Pro Phe Leu Ile Gln Gly
485 490 495 Thr Ile His His Tyr Gly Cys Lys Gln Cys Ser Ser Leu Arg
Ser Ile 500 505 510 Gln Asn Leu Asn Ser Leu Val Asp Lys Thr Ser Trp
Ile Pro Ser Ser 515 520 525 Val Ala Glu Ala Leu Gly Ile Val Pro Leu
Gln Tyr Val Phe Val Met 530 535 540 Thr Phe Thr Leu Asp Asp Gly Thr
Gly Val Leu Glu Ala Tyr Leu Met 545 550 555 560 Asp Ser Asp Lys Phe
Phe Gln Ile Pro Ala Ser Glu Val Leu Met Asp 565 570 575 Asp Asp Leu
Gln Lys Ser Val Asp Met Ile Met Asp Met Phe Cys Pro 580 585 590 Pro
Gly Ile Lys Ile Asp Ala Tyr Pro Trp Leu Glu Cys Phe Ile Lys 595 600
605 Ser Tyr Asn Val Thr Asn Gly Thr Asp Asn Gln Ile Cys Tyr Gln Ile
610 615 620 Phe Asp Thr Thr Val Ala Glu Asp Val Ile 625 630 14 1298
DNA Homo sapiens 14 atgtctttgg ttccagcaac aaattatata tatacacccc
tgaatcaact taagggtggt 60 acaattgtca atgtctatgg tgttgtgaag
ttctttaagc ccccatatct aagcaaagga 120 actgattatt gctcagttgt
aactattgtg gaccagacaa atgtaaaact aacttgcctg 180 ctctttagtg
gaaactatga agcccttcca ataatttata aaaatggaga tattgttcgc 240
tttcacaggc tgaagattca agtatataaa aaggagactc agggtatcac cagctctggc
300 tttgcatctt tgacgtttga gggaactttg ggagccccta tcatacctcg
cacttcaagc 360 aagtatttta acttcactac tgaggaccac aaaatggtag
aagccttacg tgtttgggca 420 tctactcata tgtcaccgtc ttggacatta
ctaaaattgt gtgatgttca gccaatgcag 480 tattttgacc tgacttgtca
gctcttgggc aaagcagaag tggacggagc atcatttctt 540 ctaaaggtat
gggatggcac caggacacca tttccatctt ggagagtctt aatacaagac 600
cttgttcttg aaggtgattt aagtcacatc catcggctac aaaatctgac aatagacatt
660 ttagtctacg ataaccatgt tcatgtggca agatctctga aggttggaag
ctttcttaga 720 atctatagcc ttcataccaa acttcaatca atgaattcag
agaatcagac aatgttaagt 780 ttagagtttc atcttcatgg aggtaccagt
tacggtcggg gaatcagggt cttgccagaa 840 agtaactctg atgtggatca
actgaaaaag gatttagaat ctgcaaattt gacagccaat 900 cagcattcag
atgttatctg tcaatcagaa cctgacgaca gctttccaaa tggagtctcg 960
cttcgtcctc caggctggag ttcagtggca cggtctcggc tcattgcagc ctccacctcc
1020 tgagttcaag cttctcctgc ctcagcctcc caagtagctg ggattacagg
ctctggatca 1080 gtatcattat acgaggtaga aagatgtcaa cagctatctg
ctacaatact tacagatcat 1140 cagtatttgg agaggacacc actatgtgcc
attttgaaac aaaaagctcc tcaacaatac 1200 cgcatccgag caaaattgag
gtcatataag cccagaagac tatttcagtc tgttaaactt 1260 cattgcccta
aatgtcattt gctgcaagaa gttccaca 1298 15 340 PRT Homo sapiens 15 Met
Ser Leu Val Pro Ala Thr Asn Tyr Ile Tyr Thr Pro Leu Asn Gln 1 5 10
15 Leu Lys Gly Gly Thr Ile Val Asn Val Tyr Gly Val Val Lys Phe Phe
20 25 30 Lys Pro Pro Tyr Leu Ser Lys Gly Thr Asp Tyr Cys Ser Val
Val Thr 35 40 45 Ile Val Asp Gln Thr Asn Val Lys Leu Thr Cys Leu
Leu Phe Ser Gly 50 55 60 Asn Tyr Glu Ala Leu Pro Ile Ile Tyr Lys
Asn Gly Asp Ile Val Arg 65 70 75 80 Phe His Arg Leu Lys Ile Gln Val
Tyr Lys Lys Glu Thr Gln Gly Ile 85 90 95 Thr Ser Ser Gly Phe Ala
Ser Leu Thr Phe Glu Gly Thr Leu Gly Ala 100 105 110 Pro Ile Ile Pro
Arg Thr Ser Ser Lys Tyr Phe Asn Phe Thr Thr Glu 115 120 125 Asp His
Lys Met Val Glu Ala Leu Arg Val Trp Ala Ser Thr His Met 130 135 140
Ser Pro Ser Trp Thr Leu Leu Lys Leu Cys Asp Val Gln Pro Met Gln 145
150 155 160 Tyr Phe Asp Leu Thr Cys Gln Leu Leu Gly Lys Ala Glu Val
Asp Gly 165 170 175 Ala Ser Phe Leu Leu Lys Val Trp Asp Gly Thr Arg
Thr Pro Phe Pro 180 185 190 Ser Trp Arg Val Leu Ile Gln Asp Leu Val
Leu Glu Gly Asp Leu Ser 195 200 205 His Ile His Arg Leu Gln Asn Leu
Thr Ile Asp Ile Leu Val Tyr Asp 210 215 220 Asn His Val His Val Ala
Arg Ser Leu Lys Val Gly Ser Phe Leu Arg 225 230 235 240 Ile Tyr Ser
Leu His Thr Lys Leu Gln Ser Met Asn Ser Glu Asn Gln 245 250 255 Thr
Met Leu Ser Leu Glu Phe His Leu His Gly Gly Thr Ser Tyr Gly 260 265
270 Arg Gly Ile Arg Val Leu Pro Glu Ser Asn Ser Asp Val Asp Gln Leu
275 280 285 Lys Lys Asp Leu Glu Ser Ala Asn Leu Thr Ala Asn Gln His
Ser Asp 290 295 300 Val Ile Cys Gln Ser Glu Pro Asp Asp Ser Phe Pro
Asn Gly Val Ser 305 310 315 320 Leu Arg Pro Pro Gly Trp Ser Ser Val
Ala Arg Ser Arg Leu Ile Ala 325 330 335 Ala Ser Thr Ser 340 16 1816
DNA Homo sapiens 16 atgtctttgg ttccagcaac aaattatata tatacacccc
tgaatcaact taagggtggt 60 acaattgtca atgtctatgg tgttgtgaag
ttctttaagc ccccatatct aagcaaagga 120 actgattatt gctcagttgt
aactattgtg gaccagacaa atgtaaaact aacttgcctg 180 ctctttagtg
gaaactatga agcccttcca ataatttata aaaatggaga tattgttcgc 240
tttcacaggc tgaagattca agtatataaa aaggagactc agggtatcac cagctctggc
300 tttgcatctt tgacgtttga gggaactttg ggagccccta tcatacctcg
cacttcaagc 360 aagtatttta acttcactac tgaggaccac aaaatggtag
aagccttacg tgtttgggca 420 tctactcata tgtcaccgtc ttggacatta
ctaaaattgt gtgatgttca gccaatgcag 480 tattttgacc tgacttgtca
gctcttgggc aaagcagaag tggacggagc atcatttctt 540 ctaaaggtat
gggatggcac caggacacca tttccatctt ggagagtctt aatacaagac 600
cttgttcttg aaggtgattt aagtcacatc catcggctac aaaatctgac aatagacatt
660 ttagtctacg ataaccatgt tcatgtggca agatctctga aggttggaag
ctttcttaga 720 atctatagcc ttcataccaa acttcaatca atgaattcag
agaatcagac aatgttaagt 780 ttagagtttc atcttcatgg aggtaccagt
tacggtcggg gaatcagggt cttgccagaa 840 agtaactctg atgtggatca
actgaaaaag gatttagaat ctgcaaattt gacagccaat 900 cagcattcag
atgttatctg tcaatcagaa cctgacgaca gctttccaag ctctggatca 960
gtatcattat acgaggtaga aagatgtcaa cagctatctg ctacaatact tacagatcat
1020 cagtatttgg agaggacacc actatgtgcc attttgaaac aaaaagctcc
tcaacaatac 1080 cgcatccgag caaaattgag gtcatataag cccagaagac
tatttcagtc tgttaaactt 1140 cattgcccta aatgtcattt gctgcaagaa
gttccacatg agggcgattt ggatataatt 1200 tttcaggatg gtgcaactaa
aaccccagat gtcaagctac aaaatacatc attatatgat 1260
tcaaaaatct ggaccactaa aaatcaaaaa ggacgaaaag tagcagttca ttttgtgaaa
1320 aataatggta ttctcccgct ttcaaatgaa tgtctacttt tgatagaagg
aggtacactc 1380 agtgaaattt gcaaactctc gaacaagttt aatagtgtaa
ttcctgtgag atctggccac 1440 gaagacctgg aacttttgga cctttcagca
ccatttctta tacaaggaac aatacatcac 1500 tatggcactg ggtattgtac
ccctccaata tgtgtttgtt atgaccttta cacttgatga 1560 tggaacagga
gtactagaag cctatctcat ggattctgac aaattcttcc agattccagc 1620
atcagaagtt ctgatggatg atgaccttca gaaaagtgtg gatatgatca tggatatgtt
1680 ttgtcctcca ggaataaaaa ttgatgcata tccgtggttg gaatgcttca
tcaagtcata 1740 caatgtcaca aatggaacag ataatcaaat ttgctatcag
atttttgaca ccacagttgc 1800 agaagatgta atctaa 1816 17 518 PRT Homo
sapiens 17 Met Ser Leu Val Pro Ala Thr Asn Tyr Ile Tyr Thr Pro Leu
Asn Gln 1 5 10 15 Leu Lys Gly Gly Thr Ile Val Asn Val Tyr Gly Val
Val Lys Phe Phe 20 25 30 Lys Pro Pro Tyr Leu Ser Lys Gly Thr Asp
Tyr Cys Ser Val Val Thr 35 40 45 Ile Val Asp Gln Thr Asn Val Lys
Leu Thr Cys Leu Leu Phe Ser Gly 50 55 60 Asn Tyr Glu Ala Leu Pro
Ile Ile Tyr Lys Asn Gly Asp Ile Val Arg 65 70 75 80 Phe His Arg Leu
Lys Ile Gln Val Tyr Lys Lys Glu Thr Gln Gly Ile 85 90 95 Thr Ser
Ser Gly Phe Ala Ser Leu Thr Phe Glu Gly Thr Leu Gly Ala 100 105 110
Pro Ile Ile Pro Arg Thr Ser Ser Lys Tyr Phe Asn Phe Thr Thr Glu 115
120 125 Asp His Lys Met Val Glu Ala Leu Arg Val Trp Ala Ser Thr His
Met 130 135 140 Ser Pro Ser Trp Thr Leu Leu Lys Leu Cys Asp Val Gln
Pro Met Gln 145 150 155 160 Tyr Phe Asp Leu Thr Cys Gln Leu Leu Gly
Lys Ala Glu Val Asp Gly 165 170 175 Ala Ser Phe Leu Leu Lys Val Trp
Asp Gly Thr Arg Thr Pro Phe Pro 180 185 190 Ser Trp Arg Val Leu Ile
Gln Asp Leu Val Leu Glu Gly Asp Leu Ser 195 200 205 His Ile His Arg
Leu Gln Asn Leu Thr Ile Asp Ile Leu Val Tyr Asp 210 215 220 Asn His
Val His Val Ala Arg Ser Leu Lys Val Gly Ser Phe Leu Arg 225 230 235
240 Ile Tyr Ser Leu His Thr Lys Leu Gln Ser Met Asn Ser Glu Asn Gln
245 250 255 Thr Met Leu Ser Leu Glu Phe His Leu His Gly Gly Thr Ser
Tyr Gly 260 265 270 Arg Gly Ile Arg Val Leu Pro Glu Ser Asn Ser Asp
Val Asp Gln Leu 275 280 285 Lys Lys Asp Leu Glu Ser Ala Asn Leu Thr
Ala Asn Gln His Ser Asp 290 295 300 Val Ile Cys Gln Ser Glu Pro Asp
Asp Ser Phe Pro Ser Ser Gly Ser 305 310 315 320 Val Ser Leu Tyr Glu
Val Glu Arg Cys Gln Gln Leu Ser Ala Thr Ile 325 330 335 Leu Thr Asp
His Gln Tyr Leu Glu Arg Thr Pro Leu Cys Ala Ile Leu 340 345 350 Lys
Gln Lys Ala Pro Gln Gln Tyr Arg Ile Arg Ala Lys Leu Arg Ser 355 360
365 Tyr Lys Pro Arg Arg Leu Phe Gln Ser Val Lys Leu His Cys Pro Lys
370 375 380 Cys His Leu Leu Gln Glu Val Pro His Glu Gly Asp Leu Asp
Ile Ile 385 390 395 400 Phe Gln Asp Gly Ala Thr Lys Thr Pro Asp Val
Lys Leu Gln Asn Thr 405 410 415 Ser Leu Tyr Asp Ser Lys Ile Trp Thr
Thr Lys Asn Gln Lys Gly Arg 420 425 430 Lys Val Ala Val His Phe Val
Lys Asn Asn Gly Ile Leu Pro Leu Ser 435 440 445 Asn Glu Cys Leu Leu
Leu Ile Glu Gly Gly Thr Leu Ser Glu Ile Cys 450 455 460 Lys Leu Ser
Asn Lys Phe Asn Ser Val Ile Pro Val Arg Ser Gly His 465 470 475 480
Glu Asp Leu Glu Leu Leu Asp Leu Ser Ala Pro Phe Leu Ile Gln Gly 485
490 495 Thr Ile His His Tyr Gly Thr Gly Tyr Cys Thr Pro Pro Ile Cys
Val 500 505 510 Cys Tyr Asp Leu Tyr Thr 515 18 27377 DNA Homo
sapiens 18 gatctttttt tctgggctaa ttcatatgac tcaaattcat tatagttgca
taataataat 60 gttatgcttt tttcattttt catttaatag atgttgagat
cgttaccagt tttttgctct 120 tacaaataat actttaataa acatccttga
atatatgtac ttccatgttt ttacttctcc 180 acaataaact aaaagtgagg
tcgatgtatc taaggttatg cacatttttt aatagatgct 240 gccagattat
ttaccaaagg tcatagaaat ttatatccaa atagcagtgt aggagaatat 300
actttactca caccttcaca gtattggaag ttaacactat atgtaatttt tgacagttaa
360 gcaggtgaaa ggtgttttct tacttaattt tcctggctac ttggaaactt
gaaaatctta 420 ctatatattt acaaacgttt ttaattccct cttcctcaga
ttttctgctc ttactcttta 480 tctgattttc tgttgaatta tatttttgtc
agtttgtggg caaccatgta tgttttacac 540 attttcttat ttgactactt
ttatggtttc tgccattatt tccatctcat gttgtaatgg 600 ccaatattaa
ttactaaatt agatttattg aaattatacc atgccagctt gagatgtcca 660
ttcaagtcct cttgacttgg atttttatac cacttattag caatattgag gatatgtttg
720 tgtatgatgc tttataaaat aaattataaa aacataatgt actgttatgt
ataatagaat 780 gtaagctaaa gtgattacaa aatacacatt tttaaagtct
taagttcttc tttttagaaa 840 gcattttgta accttagtgc tatgactact
acttttgctt tcttgttaga gtaaaatcct 900 atttttgatg ttcatttggt
cattctatta aatttcataa gtttactatt ttatccatct 960 ccgcttttat
ttcctctaca ctgtattttt tcaacatgat aaaaactttc atacatggta 1020
gaattaaaac agttgtacaa tgaatactca aataactacc agctagactc tccaataact
1080 attttacttt gtgtgctctg tcacgtgtat ttatttctac atatctcttt
tttttttttt 1140 ttttcttttg agatggagtc tcgcttcgtc ctccaggctg
gagttcagtg gcacggtctc 1200 ggctcattgc agcctccacc tcctgagttc
aagcttctcc tgcctcagcc tcccaagtag 1260 ctgggattac aggtgcccac
caccacgccc agctaatttt tgtattttta gtagagacac 1320 agtttcacca
tgttggccag gctggtctcg aactcctgac cttagataat ctgcccgcct 1380
cggcctccta aagtgctggg attacaggtg caagccaccg tgcctggcct atgtgcctct
1440 tcattcatta atttatattt tttatacatt tcaaagtaag ttgcagacat
aagtacattt 1500 tctaaacact gtggtatgaa cataattagc tagagtttag
tagttattta gagtttttta 1560 tttttgaggt aaaattagca gtgaaatgga
caactttcca ttttatgaac cactccatga 1620 gttttgacta atacataaac
gtgtaaccca aatccctcta gatttgctgt tctagaactt 1680 tgaaaaaatt
gaatcatatg tactcttttt gtatatacta tatgtttttg agagttaatc 1740
acattgttgc atatatcatt agtttgtttc ctttttaatg cctagtcaca tgatatgcgg
1800 tagacatttt ttctttagat aggaatttct agttgttatg acatcatttg
tttccttttt 1860 cctattagat ggcttcaatg tctttgtcaa aaatcaagcg
agtataaatg tgggcttatg 1920 tctaggcttc ccattcaatg cttactagta
tagtgtgaag tatgcatttt cctcacacta 1980 aattttcagt tattgcagca
ccatttgcat tctccttgca ttgctttgct gctttagtaa 2040 aaaatcaaaa
tacaatgtaa atgtgggttt atttccaggc tctctattta atttaattca 2100
gttgatctat ttttcaatcc tgatgccagt accgtgttgt cttaaattac tgtaagttta
2160 tagtaagtct tgaagtcatg tacatggttc tccaactttg ttatttttta
aaatgttatt 2220 taatattcta gattttctgc acttccacat aagtgatagc
atctgctttg caatctctac 2280 aataaagcct ctgctatttg tttgtttgtt
gttgttttga ggcagagtct cattctgttg 2340 cccaggctgg agtgcaatgg
cacaatctca gctcactgca gcctccacct cctgggttca 2400 agtgattctc
atgcctcagc ctgctgagta gctgggatta caggcatctg caccacactt 2460
ggctaatttt tgtatttgta gtagagatgg ggtttcacca ttttggccag gctggtctct
2520 aactcctgat ctcaagtgat ctgcccacct cagtcctccg aagtgttggg
attataggcg 2580 tgagccactg tgcccacccc agcctctgct attttcgaag
gattatgctg aatttacaga 2640 ttaatttgga gagaattgat atcttaacaa
tattgagcct tctaaatcat gaatgtggca 2700 tatctcacca tttatttata
ttttcttcag tttctctcag caacgctcca ttgttttcag 2760 ttctacaatg
aagttgtaat ggacttaatt tttttgcctt ttccttttta taggctctgg 2820
atcagtatca ttatacgagg tagaaagatg tcaacagcta tctgctacaa gtaagactat
2880 gtatcatttt tgagatgggc acagtaatga gcataataaa gtctgcctct
acacttacca 2940 gctaatccat ttctttctaa tagtagaaca catatccttt
aaagctaaaa tatgtccata 3000 tttaactttc ttcttctacc gtgtcttgtt
ggcataaaat ggaacccata aagataacgt 3060 gtctttacat tgcatatttt
aagtcatcta tctctaacag acttaatgtt taaaacagat 3120 atgttttaaa
cattaaatac atgatgtatt tgaagtcatg tatctctgtt agagttacat 3180
gacttaaaat gtgcaatgta aagacacata tctttaaact attacatgaa gagttatcct
3240 gtcacatgat gcatttaaca gtgtaccata aaggagctcc ttgcaatatg
cctcaaaatt 3300 ttaatttaat gttagtaatg atagtgtgtc tatcaagtac
cctccttctg ctacatcagc 3360 taagattaaa aaaaaatttt cagaaaaata
tttttaacca caaatttatt aaatgtgcta 3420 ttgtaaaaat tttaatttct
caaattggag aaggaagata acaaatgtga atggaagaag 3480 gattgatgaa
atcttttaat gttgtgttgt aattggaggt accattatgt actcatgttt 3540
tctaggtaaa tacagaagtc gatgtagctg tgtgtatgta tgatacgcat atattcacac
3600 gtgtacacat ttgtttatat tataggggtg tgtgtgtgtg tgtgtgtgtc
agtatgaatg 3660 tgtgttcata tgtaccctat ctctctctcc atgaaaaagc
atagaggcag cagcactcca 3720 gttgccataa gcacacctgg tgctcagatc
ttggtttata aataatattt ctctctaaag 3780 gaatcagagc tccttggtga
aacagcagat ttctgaacta gaacaaggga attacaagat 3840 tagtatggag
taaccttgta ctagaaagta agggggttct cagttaatga tgaaactcgt 3900
caaatggctt aggatagaac atgtctagga acatttgagc atcaaaacaa ataatactaa
3960 ttgagtaaag caggaatgca tgagcccatg ttgatgatga taaaggaaaa
ataaaatata 4020 tggggttaag tggaaatatc tttcttaaag taaaataaca
aatataaaag ggataatgaa 4080 attagaaaaa aaaaagctac cattttgtaa
ccatgatagt cattgttgag ttagttgtga 4140 atctgtggat tctaaactat
caggatattt gatgaaaaat aagatattta cattttctct 4200 agtatattct
tgttaaatac aagggggaaa cagtaagttt ttagtagaga agtgattgga 4260
cactaccttt accagctgaa taaagtttag gtctacagta atagaaacac tcactttgta
4320 tgccccttga tgtgatgcac tgagaagcat acagtatcac ttacgcatta
ttcctgccaa 4380 aaatgcataa gctaaatctg agcctgagga ataaccagac
aacacccaaa ttggtgttta 4440 ttctacagaa taaatggctg tactcttcaa
atatatcagt gttgtgaaag ataaagaaaa 4500 gccgaggact tattttacat
taaagaagtc taaagagaca tgagaattaa atgtgataca 4560 tggtccagaa
ttggatctta gacttgaaaa taaaatgaat gctaagaaga acattttgag 4620
gacaattgta gaaatttgag taatgtttgt taattaattc gattatagta ataaatcagt
4680 taaatgttct aatgttgaaa attgcctgta attatgtcaa taaaatgtct
tcttttgaaa 4740 tacatactgg aggatttaga ggaaaggagg cataatgtct
ggtagttatt ctcaaatgat 4800 tcaataatat ttatgtggtg agagacagat
aaagacaggc acagtgacaa tgataaatgt 4860 gcaaaaatgt taacaattgg
tgaatcttgg tgaatattat acagaaggtc tttgtattgt 4920 ttttgcaatt
ttccttaagt ttgaaagcat tttaaaatga aaagttaaaa actttaggtt 4980
aaaatatgag tttgaagcaa ttgctcttat cactgtgtag caatgtacac taaattgatc
5040 aggtctgcca atggcctttt tttttttttt tttttttttg aggcggagtc
tcgctgtcgc 5100 ccaggctgga gtgcagtggc actatcttgg ctcactgcaa
gctctgcctt ccgggttcac 5160 gccattctcc tgcctcagcc tcccgagtag
ctgggactac aggtgcccgc caccacaccg 5220 gctaattttt tgtattttta
gtagagacgg ggtttcaccg tgttagccag gatggtctcg 5280 ctctcttgac
ctcgtgatct acccgcctcg gcctcccaaa gtgctgggat tacaggcgtg 5340
agccaccgcg cccggtgcca atggcctttt taaaagcatc accagctggg tgcagtggct
5400 cacgcccgta atcccagcac tttgggaggc cgaggcgggc agatcacctg
aggacgggag 5460 ttcgaagcca gcctgaccaa catggagaaa ccccgtttct
actagaagta caaaaattag 5520 ctgggcgtgg tggtgcatgc ctgtaatccc
agctacttag gaggctgagg caggagaatc 5580 gcttgaacct gggaggtaga
ggttgtggtg agcagagatc gcaccattgc actccagcct 5640 gggcaacaag
agggaaactc cgtctccgaa aaaaaaaaaa aaaaaccaca atcgccacca 5700
caacaaaatg ttccactgta ataaatgttc cactctgatg taataaatgt tccactctga
5760 taaaggcaag tgagaaataa taaatgatga atatatttgg gcagactcat
ttgtcacaga 5820 agtatcttaa atataaactt tattaactga aatatttgaa
aagaggtgta attacttgaa 5880 atatctaatt aagtgataca gagagccttg
ttggtaaact tctgtccttc ttggccattt 5940 gctccttgaa ggaaaactaa
ttcaacaaga atttcattgg attaaagctc agtactgaaa 6000 ggaattgtct
tcgccattga ggttaataag atttgtacat catttccctt ttctaaaaca 6060
catgaaagtg ttaagctaga atgtatagca agctgttgcc ttaagctaag ggtcaccagc
6120 aattttatac tttttcccag taaaaactga tcactacaat cccaggccat
ctttccacaa 6180 gtagctgagg agacctattg tacctatttc ccaggcaatt
gctcctaatg cttttgtctg 6240 agtttttttt ccagtttgac tcaacttcct
cttatttttc ctctccctcc tcctccactc 6300 cctccttcca actccccaaa
cttcctcttc tccactacta caccactcct gtgacagtta 6360 gatcaccctt
aatgtccctt cctattctta atctgatttt ataatgatgg ttctgtaaaa 6420
agtaactgat ttgaaacatc caagagcctg caaataatat ttgcaaataa tattttacaa
6480 gtgtgttttg ttacattctt ttgtggcaga caccagttag aacttaaacg
gttgcctagc 6540 gtaatatttt cttagctaaa taaaccttgc ttttttgaat
gcttactagg cagttaagtt 6600 acttatttct tcccccaaat tatccagcgt
ttatttagta cacatttgtt gagtacctac 6660 tgtgcctggc actatgctag
tgggccttgg gtatacatca gggaataaag acataaccct 6720 tcctttcatg
gagtgacact taatagagct taaattaatt agattttata gtatatattt 6780
ggttcaggag gatgcatgtc ataaatatga ttcttgttat tctgattgaa tataaaaatt
6840 ctttacagta cttacagatc atcagtattt ggagaggaca ccactatgtg
ccattttgaa 6900 acaaaaagct cctcaacaat accgcatccg agcaaaattg
aggtcatata agcccagaag 6960 actatttcag tctgttaaac ttcattgccc
taaatgtcat ttgctgtgag tattttccat 7020 aataaaacaa acgttttcat
attatttgtg tgtatatgta cacatatgta taattttgtg 7080 tcttaggaat
aagtaaattg ttaatatata tattatattt tgcaagaatg gtaaattttt 7140
taggtaaagt gctaaattct tagagaataa attattctga tagtaataaa agtgggtgct
7200 attttcagat ctaaaattca gcttagtcac tctgataaag gcaaatgaga
aataataaat 7260 gatgaatata tttgggcaga ctcatttgtc acagaagtat
cttctgaaat ataaaccttt 7320 attaactgaa atttttgaaa ggagttgtaa
ttacttgaaa tatctaatta agtgataaag 7380 agagccttgt tggtaaactt
ctgtcctgct taataactag aatataataa atataattta 7440 aattttcttt
agtaattgag aatttctcag tgcctttact ctgaacatca gtgattatat 7500
aaatatgtaa taaatgtata taactgtttt gtaatccttt tactacataa tcggctcaag
7560 acatattctg aaaatcattt ttaaaagctc ctcatctttt tgcaatttgc
ctacttttcc 7620 tctgaatatc taaaatgatg ttttggaaaa tgtagataat
tgatggttat atgcatttgg 7680 atgccctaaa ttgagtcttc actaaaatgt
gctacaatgt gtaaatatct atgtacatcg 7740 ccatgtattt gtgtgcttat
aaattgtgag tatctgtgtt cattaatata catatatttt 7800 ccaatccaaa
atttgggttt gtttgaagaa attttttatt ttaaaatctc tttaaataaa 7860
atgtgaggga actgttttta cccatttgag cttgaaatgg tggttgggat taaaatgtat
7920 atataaggat tttagataat tcttcaaata ttatcaaact ttggtttatt
gaattttgta 7980 aaatcataca gctttgtaaa ataaaaccac tctccgcgat
cattttttaa acaaataagg 8040 atattatctc agaaattaac ggaaactgtc
taaagttaca cagttaactg gcaacagaac 8100 cagaagaaag ccatacacct
tttgattcca aatgatgcca tttctgctac atggtaccta 8160 accatatgac
ttcttaaaat tattaattat taaacagaat tggaaatatt attagtttag 8220
aagtgccctt ctccctaagt gtggtaagtg gatatttaac tggagtgaag acggggccac
8280 tgcatttttt tctcctactg ggaaatttag cattctttac agaggagaaa
aaaattgatg 8340 ctagaaataa ttatgagtaa ctttgtatca caaaaccagg
catagaaatc actggtagtt 8400 aatgtaaata tgatttggat atacttaccc
acaaaatatc aaataattat ctattgaaaa 8460 aaagttattt gttctgcaaa
gtgaattatc tccataattt acataattta agaaaaagta 8520 actgactcat
ctacatgtaa gaatgatact ttttaatttg ataacttgtt aaatggaaat 8580
cttcacgctt acaccaaaat cgatttctat catttcattg ccaataattt taggcaagaa
8640 gttccacatg agggcgattt ggatataatt tttcaggatg gtgcaactaa
aaccccagat 8700 gtcaagctac aaaatacatc attatatgat tcaaaaatct
ggaccactaa aaatcaaaaa 8760 ggacgaaaag tagcagttca ttttgtgaaa
aataatggta ttctcccgct ttcaaatgaa 8820 tgtctacttt tgatagaagg
taagatattt aagtcactgt tttgttagaa tactcctttt 8880 gcatattttt
cctaattaat tattgtttaa tacattttac agacaaccta gtacatataa 8940
agtaaaaata gtatttaaat ttaacaaaat tgaatatata tgttaactag gttcaaatat
9000 atataagcac acgttcataa atttatctta attacatttg aaattgtact
tcagactcaa 9060 gtgttaacat ttaactatat tgttggattg cattttattt
tgtcaatgct aagctgattg 9120 tctagttaag taataataaa agaggctgat
tgcttatgta ccattgctgt tttcttggcc 9180 tctggatgtc actgttgttt
catagaaata gggtgaaagt catctattgt atcaaaatca 9240 aagaagagac
cattgaaaca agtaaagata acttgacaag ttttaaatga aatttatcat 9300
gtttggtttt tcattttctt ttcattttca tctaattttt atctcattta tctaaaatat
9360 gtactgtgaa ttttttttca tggcaaattt agagtttttc ttaaggcttc
tcttcccttg 9420 taaccttttc attgtttttc ttaaggcttt ccttcccttg
aaaccttttc attgtttttc 9480 ttaaggcttt ccttcccttg aaaccttttc
attgtttttc tgaaggcttt tcttcccttg 9540 aaaccttttg taatagaaga
aaaatacctt ctttaatttg ccttagagta atatttaact 9600 ttatttttaa
taaatgaggg aattctatgt aaattataga ctttgggtga ttatgtgtca 9660
gtataggttc atttttaaca aatgtaccac gctggtagag gatgttgata ctggaggagg
9720 ctagcatgta tggtagaagg ggatacggaa aatctctgta ccttcctctt
aattttgctg 9780 tgaacctaaa actgctcctt aaaaaaaaaa aaaatgaagt
cttaaaaaga aaacatagaa 9840 tgtacaacac tgagagtaaa ccctaatata
gactggactt tgagtgataa tggtttgtta 9900 gtaatgtaaa gtgtggactt
tgagtgataa tggtttgtta ctaatgtaaa ctgtggactt 9960 tgagtgataa
tggtttttta aaataggttt cttgattgac taaatttacc actctggtgc 10020
aagatgttga taatggggaa gaggctaggg gacataggga aactttgtac cttttgctta
10080 attttgcagt gaacctaaaa ctgcttttta aaaaaggctt atttaaaaaa
ataatgagaa 10140 tgtatgtaaa agcactttga aatgtaaaag gaatataaga
aatgtgagct atttttattt 10200 tatgtttcta agtattataa cctggaccaa
gggctaggat cttactgcag tatggcactg 10260 ctctggttag gaagtaacaa
aatcaaaaac tgacctggac ttagagatga accaaagaaa 10320 acgatataaa
tacaaagtca ttcttagact ttaaggacct gcagcagtat tcactgatat 10380
tcatgccaag ttaatgcagt tgacactatt ttattgtgac catagtttac attagggttc
10440 actcattctg ctttacagtt ctttatgttt tgacaaatgc agaataccat
gtacccacca 10500 ttagagtctc atataaaaca gtatcactta atttctgtaa
aagctctaag atctgtgtcc 10560 agattttttt ttgcatgcag atgtccagtt
ttccagtacc atttcttaaa aagactgttc 10620 cttctccatt gaattgcctt
tgcttctttg tcaaaccagt ttgtgtgaat ttgcttctgt 10680 gttctctatt
ctgttttaat ctgtctgtta ttttcctaat atcacaccat ccttatttct 10740
aaagctatat agtaattctt gaaattgtgt agtgtttgtc ctgcaacttt cttctttttc
10800 ttgagtattg tgttggctat tgtaaatctt ttgcatttcc atgtaaactt
tataatcagt 10860 ttgtcaatat ccaaaaataa cttgctggga tttttattaa
gattgccagc tgggcgcagt 10920 ggctcactct ggtaatctta gcactttggg
aggccgaggc aggcagatca cctgaggtcg 10980 ggagttcgag accagcctga
ccaacatgaa gaaaccctgt ctctactaaa aatacaaaat 11040 tagccaggca
tcatggtgca tacctgtaat cccaactact cgggaggctg aggcagtaga 11100
atggcttgaa cccgggaggc ggaggttgcg gtgagccgag atcgcgccat tgcactccag
11160 cctgggtaac aagagcgaaa cttcatctca aaaaaaaaaa agattgccat
aatctataag 11220
tcacggtgga gacagagaac taacaacttg atgttattga cgatgaacat ggactatctt
11280 tctatgtaga tcttcttaga tccctttaac tagggtttta tagttttact
cagataaacc 11340 ttataaatcc aacaaaatat agatcacatt ttgttagctt
tatatctaag tattttcttt 11400 tttggtgcta attatttaat gttaaattca
aactttgatt atttattgct tatgtatagg 11460 gaagcaattg attttttttt
taattaacct tgtatcctct accgttgcta taattgcttg 11520 ttatttcagg
aatttttttg ttgtgatttc ctgtaaacaa agacagctta tttcttcctt 11580
cctaatatgt ataccttttg tttccttttc ttactgcatt agatagggct tccagtacaa
11640 tattgaatag gagcaatgag agggaatgtt cttgctttta tcccagtctt
aggtggaaag 11700 tgtcaccatt aaatgtaatt ttagctgtgg ctattttatc
gatgttcttt atcaagttga 11760 agaagttccc caatattcct agtttgctga
gaatttttat tattaatgat gttggatttt 11820 atcaaatgct ttttctattg
catctattaa tatgatcata caatttttct tctttagcct 11880 attaatgtga
taaattacat taattgattt tgaggtgttt aaccagcctt gcctacctaa 11940
aataaatctc atttggtcat ggtgaataat tattttcttt tttgattcaa tttttaaata
12000 ctttctgagt atttttttat gtgttttctt aagagaagtt gatcaatagg
tcttcattct 12060 tgtaatgtat ttggttatgt attagaatat tgctggcctc
ataagagtta ggaaacattc 12120 cctctacttc cattttctgg aatacatagt
agagaattag tgtcatttca gtgtttgggt 12180 agacttagct attgaaacaa
tctgagcctg gtgacttttt tcaagattat tattattgat 12240 ttaatttctc
tatagacata gacctattca gattatctgt ttctccttgt gtgagttttg 12300
atagattatg cctttcaaga aatggaacca ttttatctaa ggtgtcaaac ttgtgggttc
12360 gaattgttta taatatttat ttattattaa cactatattt taaactgcat
aacatttaac 12420 ttcctctgaa acattttgta ttgtttccaa ttgaattgaa
tccaatttgt atggaactct 12480 aatgtcactg aatcatttta tcataatatt
tattattaat acctataatt tactgaatag 12540 actatgtgtc aggcactgta
ctagtttagt attttatctt taactctcat aacagttctt 12600 ctgtaagctg
gatatatccc ctttgtaaac agaagaggaa actgagacca agagaaaatg 12660
gtgaagtact caaggttaaa gacttaataa atgtcagaaa aaaattcaaa cttaggcctt
12720 tctgtctcca tagtccatgt taaatatttc tactgattgc aaataaattg
ctctcagtta 12780 ggatgtctcc agatacaaac cttgagaaat gtagtatgca
catatataca tgtaaatgtc 12840 tttctttgtt cttattcatt tgtttagcac
atgtttattg aatgcctact atgtgccaga 12900 cactgattta ggcattagtg
gcaatgtagc aaacacaaca aagttcttcc tttcatggac 12960 tttacattaa
gaggaaatca ctaaaatatt gatagtaata gtcactcatg gctctaagtg 13020
ctttacaaat attaactcat ttaatcttta taatgatctt acagagtaac attattctca
13080 gttttgcaaa tggggaaact gttataccag agtttaagta acttgaccaa
ggttgtccag 13140 cttatgtgcc agagccaaac tcgtgtgact ggccagtgtg
aatgactaga tgagctctca 13200 ccagattctt tgaaatagtg tttttgggga
ggaactcata gagaaaagag ttagtgaatg 13260 gtcacctatt gcagttttga
acagtaggca ggagtctctt cagcagggct aggtatcagt 13320 ctccaaaaga
tagactaact tttgggctgt gaaactttta agtagcatgc ttagggaata 13380
ttgttttgag tttttaagca tgcataatga gagtttctat ctagctgcaa tatgatatag
13440 cagaactctg gcttccagta acaaagagct tgggggaagg aggatgggaa
cagggcaagt 13500 taaaatgcca cagagctcac cgttcttgcc aaaattcagc
cctttttctg gagcaaacac 13560 tccttggatt gttgaaggcc tctggtaatt
tccagaattc taaaaaaggt tttacagttt 13620 ttgccaatat tcttactgct
gttatagtca agtgtgtctt tggatgtcct cactctgcta 13680 taccagaagt
gcttctcctt tataattgaa tgttgacatt acaaattcta cccaaatttt 13740
aggaaataca cagaggtatt ttttaaatcc ttttcatttt gcctggagag aggaagcatt
13800 attagctaag taaaaaggac actgccttct aataatggat gccattggac
aatacttctc 13860 agccagcctg gtcatttgaa tgcttactct gtcatagaat
taactgtgat aattttccca 13920 ggaaaaatga acaaatttta tatgtgaatt
catattacat gaactactca tatctatatt 13980 taaatgaaat attgacctga
aaattgagat ttaaactcta aatttgccca gatattaatt 14040 agtatatagc
aaattagtga gaatctgatc ataacttagc ttttaattta tattccctct 14100
tttggttatt tgaaccaaag tgttcctgaa ataaagagca atttgtttaa atttaagaag
14160 ttggttaaaa tttcacaagc tttatatttt accaaagtct cagcattttt
gtgcattgat 14220 ttttttaatc aatgtatagg attgtacatt tacaaattaa
tattttttac atacattcat 14280 tgtctttttc tgtcaattcc tttagtcttt
tattatacct cacacgttat ttaataggac 14340 tgtacttgtc tacattttat
ttgcactact tgaaggattt atttattctc ttaacaggag 14400 gtacactcag
tgaaatttgc aaactctcga acaagtttaa tagtgtaatt cctgtgagat 14460
ctggccacga agacctggaa cttttggacc tttcagcacc atttcttata caaggaacaa
14520 tacatcacta tgggtatttt gttttgtttt gttttgtttt gttttgttta
ttatactttt 14580 aagttctggg gtcatgtgct gaacatggag gtttgttacg
taggtataca cgtgctattg 14640 tggtttgctg cacccatcaa cccgtcacct
gcattaggca tttctcctaa tgctgtcctt 14700 cccctagcct cccaccccct
gacaggccct ggtgtgtgat gttcccctcc ctgtctccat 14760 gtgttctcat
tgttcaactc ccacttatga gtgagaacat gcagtgtttg gttttctgtt 14820
ctggtgttag tttgctgaga atgatggttt ccggctttat ccatatgcct ggcaaggaca
14880 tgaactcatc ctttttttgg ctgcatagta ttccatggtg cgtatgtgcc
acattttctt 14940 aatccagtct atcactgatg gacatttggt atagttccag
gtctttgcta ttgtgaatag 15000 tgctgcaata aacgtacatg tgcatgtgtc
tttatagcag aatgatttat aatcctttgg 15060 gtatataccc agtaatggga
ttgctggatc aaatggtatt tctagttcta gatccttgag 15120 gagttgccat
accgtgttcc acaaagattg aactaattta cactcccacc aacagtgtaa 15180
aagcattcct gtttctccac attgtctcaa gcatctgttg tttcctgact ttttaatgat
15240 cgccattcta agtggcgtga gatggtatct cattgtggtt ttgatttgca
tttctctaat 15300 gatcagtgac attgagcttt ctttcatatg tttgttggct
gtgtaaatgt ctccttttaa 15360 gaactgtctg ttcatatcct tcacccactt
tttgatgggg ttgttttttt cttttaaatt 15420 taagttcttt gtagagtcta
gatattagcc ctttgtcaga tggattgcaa aaatttcctc 15480 ccattctgta
ggttgcctgt ttactctgat gatagtttct tttgccgtgc agaagctctt 15540
tagtttaatt aggtcccatt tgtcaatttt ggcttttatt gcctttgctt ttggtgtttt
15600 agacatgaag tctttgccca tgcctatgtc ctgaatggta ttgcccaggt
ttccttctag 15660 gatttttatg gttttaggtc ttacatttaa gtctttaatc
catcttgagt tgatttttgt 15720 ataaggtgta aggggatcca gtttcagttt
tctgcatatg gctagccagt tttcccaaca 15780 tttattaaat agggaatcct
ttccccattg cttgtttttg tcaggtttgt caaagatcag 15840 atggttgcag
atgtgtggtg gtgttttcaa ctgagaaaac ttttggaatt aaaaactgtt 15900
gaagagtaat ttttattagt ttatttcatt ggttactata tgttcagcat gaacttacag
15960 tgtatcaact tatatgtact aggtttttct ggcatatatc tgttcttttg
ataagcatat 16020 atagtgagag tacacgcaat gtgtgaggca taaggctgct
gtcttttgat tcctcagcca 16080 gaggctggta ctcacttgtt ttctttaaca
gtgaggattt agattccagt tacagagaaa 16140 aattcagagc tgcaaaccta
gtaaaaatta agtgattcaa tttcagaatt tctgagccac 16200 taaattacaa
atttgctgcc actgaaaatt ggaatataaa agaattcatt aggagctata 16260
aacagatttc tacatttaga aggagggggt agggataaaa tctcctctac tgcttgatga
16320 aacaatcacc ctggacacat tctgatttga gaaaccttgg attataacat
atgttttatc 16380 atcctattcc tctttctttc cgacttctac atttgtagca
attagtagtc attgtcataa 16440 tgtgtaaatc ctgattgaaa aattatatac
tggttgaaaa atattatacg gtaagcatga 16500 tacctcccta attgtgtggt
aaagtcactg ttaggcattg ccctctgtcc ttccaacata 16560 tcataaaatt
ttagccataa agcgaaagtg tatgccactg acttaaatct ctgtgttata 16620
gctgttttta ctgatatact cagtgtctaa ttctccctct cattagactc atgatctgag
16680 agtccatctt ttttgaaaat aaaatgattt ttaattaagc caattaatta
aaaaattaaa 16740 actcataaaa ttcagttttt cttgtataat aagtcactga
gctttctctt tttgcatgct 16800 catcctcgct cacttgcttt tgttctttcc
cctttctctc tattttgcct tgccagtact 16860 gggcaccgtg acgcgtctaa
accaggaaag gaaatattca tattcatttt aaactctgaa 16920 atactactac
ttcttttact agaagtctca aaaaaattac cttaaggacc ccattttttt 16980
tttttttttt gagatgaagt cttgctctat tgcccagata ggagtgcagt ggcatgatct
17040 cagctcactg caacctctgc ctccccggtt caagcgattc tcctgtctca
acccccccgc 17100 cgagtagctg ggactacagg catgcaccac taacacccgg
ctgattgttt cgtattgtta 17160 ttagaaacga ggtttcacca tgttggccag
gctggttttg acctcctgac cttaggtgat 17220 ctgcccacct cggcctccca
aagtgctggg attacaggtg tgagccactg tgcccaacca 17280 aggctgttga
ctttttactg gttgcttcaa aactaaggca aatgctgttc acactccaga 17340
ttttaagaca tttttacatt ttttattact tgagtttcat catcaaaagc cagtatatct
17400 tttaattgat tcttcttttt atttttgggt tatgaaataa ttttaactta
tagaaaaatt 17460 aaaaaagtaa catcacaaca attacgtatc caccatttag
atttaacaaa tcgtaacgtt 17520 ttgacattat ttcagacttt tttttttttt
tttttttttg gagacagtgt cattctgata 17580 cccaggctga agtggcatga
tttcagctca ttgtagcctt gacatcctgg gctcaagcaa 17640 tcctactatc
tcagcctccc aactagctgg gactacaggt gcacaccacc acacctggct 17700
aatttttgta gggatggggt tttgccatgt tgcccaggct gttcttgaac tctggagttc
17760 aagcaatctg cctaccttgg cctccaaact tttttttttt tttttttttt
ttatttttaa 17820 gaaattaaat gttacagaga agtagtataa tgccatatca
atcccttctc taactctttt 17880 ttctcagagg tagctacttt tccaaacttg
gattaaatcc ttctcatcaa tgtttttatg 17940 ccttcattat atgtgtgaac
tcttaagcag tatggcatat ttttcatttt ttaaatttat 18000 ataaactgtt
tcgtactatg ccaagccttt tgcagcttgc tttttttgat tcattaaaat 18060
tttcaagatt taccactatt gacgcatgta gatttagatt atttaacatc tttggagtat
18120 gttatgaaat atcagaattt attagcctat tttcctatta atggatatgt
gttatttttt 18180 gtttcattta cagaccataa tgaagtcacg ttatatgttt
tcttgtctat ttcccttgtc 18240 ataaaatgag ttcagtgggt cataaacagt
ttttttttaa attatatgat gtggttgtag 18300 taaaaaatgg aatgagaggg
aatggataat agagaacatt ttacacagta agggtcagtg 18360 ttgtttccta
aactttcatt tcaattgtat gtgtatgtat gtattactaa gatatgatat 18420
taaatgaatt tcttactgtg agtccttaac aaaaatgttt gaaagttact cctaaggtgt
18480 ttacctgaaa ttagaattac tggattataa ggtgtatata agttttgctt
tatgggaaga 18540 aataccaaat tgttcttccc atggttttaa caatatatgg
tcccatcagt aatgtataaa 18600 attttagttt ctaccaagtt cactccaaca
cttggtatta gtctatttct gtctgatact 18660 tggcattaat tttgtaattt
tgtcaggcca gcgagcatca gatggtatcc ataatgtttt 18720 tatttgtatt
tcctagatgt ctagtgtgtt taagcagccc ccgtgtttat cagctacata 18780
ggtttcctac tctatgaatt ccatgttcac atcttttgcc tgtttttcta tgtggttact
18840 gatttctttg ttggttcatg tgtgagcgca catacatgta attgattgta
aggtttcttt 18900 ccgtgttaga gatactaatc tttgtcagtt tcatccatac
ttctagtgta ttccatgcct 18960 ttttaacttt atggtttctt gtgttttata
ggttttttta aaatttttgt ttggtaattg 19020 ctttataggt tactctcatc
cctttgcttt caagtttctg gcattctaat ttgtatgtca 19080 ctcataaata
aaagcttatg gctaaatttt agttttaata gtggagttta aatatgttct 19140
taagttattg atatatttag tttatgtttc taattttttc tgtttcccct ttcactgctt
19200 tggaagtaag tagttctgta tttaattttg acttaatatc cttaattttt
aatttttata 19260 ctaactttaa taatgtctaa tgctaatcaa tatcgtagtc
tttttcttag gcaataatat 19320 tcttttgtta aattgacatc ttttattaga
aaagaaacac ctatatattt aataaataga 19380 agggtataag atgtaatgtg
gttaccctct tgttttcctc aaagtgcaaa tgaaaacaaa 19440 ttgcatggac
ctttcgaact tttattttta ttcaagtata tcttttcaag tatattttct 19500
tatcaacatc tcataaacat tatgatgatg cataataaaa aataaattac tcatagttaa
19560 aatatgttgg tattcaagta aagcaaaata actgtactac acaatgcaca
actttagtgt 19620 attgtgtagt cttagattta tatacatttc aaaagttaac
tatggaatta ggcatcataa 19680 actacaaacc tctggatatg tgcttactaa
aaatattaat tatctagaat cttgcatgtt 19740 gtgactgttt agtaattttt
ctctattggc catatttatt aacactttga atttattaag 19800 atattactta
cagaggccag gtatggtggc tcacacctgt aatcccagta ctttgggagg 19860
ccaaggcagg cagatggctt gagctcagga gttgagacca gcctgggcat tgtggcaaga
19920 ccctgtctct ataaaattac aaaaatcacc caggcatggt ggtgtgcaac
tgtggttcta 19980 gctacttgga aggctgaggt gggaggctca cttgagccca
ggaggcagag gtgacagtgc 20040 ctgggtgaca gagtgagacc ttgtcttaaa
aaatatatat atatagatat agatatagat 20100 atagatatag atcatagaat
cagagaattc ttagagatga tcattttctt caacttttca 20160 ttttaacaaa
taaggaaatt gagagcaaaa ttaattaatg atttggacct ggaaccgagc 20220
accctgttct caatttagag ttgtttattc tgaatcttat actgtctttt ttattgccct
20280 tatgtaataa gcttactctt tcataattct cttgtgaaac aaacaagcac
attacaatat 20340 aggggatgca gtattcttct gtttaataat ttatatttta
aaactacaca tgtttgagca 20400 gtaaaaagtt ataacaaaca agctaaatta
tttttaaata tttatggttc tttcttttat 20460 aaatttcaga tgtaaacagt
gttctagttt gagatccata caaaatctaa attccctggt 20520 tgataaaaca
tcgtggattc cttcttctgt ggcagaaggt tagctaaatt tccatgccct 20580
gcaattttaa ctgtttgttt acaaggttat ttcacctact tatatttcag tatacctgaa
20640 agtatacctg ttccttcttt gtatacttat tccttcctct gtaagataaa
cagactttgt 20700 aaatttaaag atatctgcca agccttcctt tagtctgtat
ttcttcaagc aggcaccgtc 20760 acatactttc ccctatgcct tactattttg
tttttcctcc tcagtaagca ttccacttta 20820 ccagtgcttt tctcagaatt
tggcattcag agctggacat tgtgctgcag atgttgtttg 20880 gccaattcag
aatagagtga aattattatt tacctgaaac tggacactca gcttctacta 20940
gcctgaaatg tcattgtata gctatttatt tgtacacttg gttttgtttt ctttcctttt
21000 tgatacagcc atctcatgtt ttatttgtgg tccagtgaaa tcctagggtc
ctgtcacatg 21060 aacttcttga acttggtctt ctcattctat tcttaatgta
attttttttt ctgtcacatg 21120 aacttcttga acttggtctc ttcgttctat
tcttaatgta atatctttgt ttttatggtt 21180 cctgggagta ggtgctaagt
tcatctttct tagttttagt tcacagtttt aacctattga 21240 gaccttttga
agcctaaaat tcagttcccc tgtattaatg tctgttgtat gccctagttc 21300
atgtctgtat gtcctaattt attcttactt tccctgttaa ttagttatac tgtttaaata
21360 tgggttccac agataaaagc taataaaaca ttctataaat tgagtatctt
ccatttccaa 21420 acaagaagat atttatctta acctgtgaat tttcatttta
cccagtatgt ctaatttctt 21480 atttcttcct tatcttacca aattattaaa
tctcagattc tgacattctt gtccattcaa 21540 ccagatgata tccctttttt
cttttttaaa gttataaatt attcccctag cttataatag 21600 aaaggagaga
ggcatgctaa aacggtattt aactgcatgc tattttttag aatattctgt 21660
attttaattt tatctttcat aaaactaaca tgcaatgagt tacatttcat gaatcacttt
21720 ttgtggtttc tatggaggct atcaactgtt ttttttattt atttattttt
atttattttg 21780 agacagagcc ttactctgtc gcccaggctg gagtgcagtg
gtgcaatctc gactcactgc 21840 aacctctgcc tcccaggttc aagcaattct
catgcctcag cctccagagg agctggaatt 21900 acaggtgtat gttaccaagc
ctagctattt tttttggtat ttttagtaga gacagggttt 21960 catcatgttg
gccaggctgg tcttgaactc ctcaagatcc gcccaggtga tctgcccacc 22020
tcagcctccc aaagtgctga gaatacaggt gtgagggtgt caacttattt taaatacgtt
22080 aatatttaat caaaaagatt aaattgctta tcataagata ttctccctat
gtaggtatag 22140 tgaaatattc caaaatgaat ctgctaaatg agcttaatta
taggttgagt atctgtggag 22200 ttaaaaacac aaactgtcct ctgctctgcc
accacagcaa tcagcgcaga agacttatgt 22260 gaccaaatgc ataggggttt
tcacccacac accaagcagg caatccctca gcagacgcca 22320 gctgggtgtc
ctccagttca attctgacac tatctacctg gagataatgc caagtttttc 22380
tttgtatctt gagttatttt agtaaataaa atttacaggt ctatactatc ataaaacaat
22440 tttaacttta ccttgataat aaggaatagc agactcatat ggtttgatct
ttttttcctt 22500 cactagcact gggtattgta cccctccaat atgtgtttgt
tatgaccttt acacttgatg 22560 atggaacagg agtactagaa gcctatctca
tggattctgt aagtatcaga ggtaataaag 22620 atatttttaa ttaaaaaata
atatttaaaa aattgaatac atttattcat acctgctttg 22680 ttcctaaaag
gacttaaggc accttaaaaa tataagtaaa atatgagcac ataaatcttg 22740
aatcatctgt gtatgtatct ctttttttat ttgacactaa atcttaacat ttgaatagtg
22800 aaaaattaag gaacagggat ttaaagagtc attccctata ccatggccaa
aatgcagaga 22860 tacggccaca ctatggaagc attatttgta gtcaacattt
tatcgtactt ttgtttgttt 22920 gtttgtttgt ttgtttgttt tttgagatgg
agtcttgctc tgttgcccag gctggagtgc 22980 agtggcacga tctcagctca
ctgcaacctc cgcctcccgg gttcaagcag ttctctgcct 23040 cagcctccca
agtagctggg attgcaggta tgcaccacca cgcccagcta atttttgtat 23100
ttttgtagag acagggtttc accatcttgg ccaggctggt cttgaactcc tgacctcatg
23160 atccacccac ccttggcctc ccaaagtgct gagattacag gcgtgagcca
ccgtgcccag 23220 ccttgatcat actttttaaa cctccacatt tcatattaga
ggaatgaagt tactttaaca 23280 gggaagatag atattattgt ataaagtttt
gaggcagtct acaaaacctt cctcatttct 23340 gacactaatt gcaattggaa
gtcctcaagg ccactcttag atttgataat tcacaagact 23400 cctagaactc
actgaaaact gttatactga cagttacaga ttattacagc taaaggatgt 23460
acattaaaat cagataatga aagagatgta taggacagag tccaggaaag ttccagacat
23520 ggaacttata gttgtcctct ccccatagag ttgtggactg ttactttccc
tgcaacagtg 23580 tgtagcagta tacataatat attgccagat agggaagctc
tgctaaaaga ttttagtggg 23640 actctatcac gtaggtatgg ttgactgccc
atatggctga tcatagtctt cagcccctct 23700 tgagatcaag ctgataccac
atgctccaaa ctttccaccc tacatcatat tgttaaacta 23760 ttcatagtga
cccagggctt ccaggcaaaa atacttctat caagtgtgac atagaaaggg 23820
cttagagatt acgttccaca agctaaggtc aaagcccaga cctctcttag ggtaaagtta
23880 aaatgtttac tacatggatt ggaaaagatc tgagttatag ttgagaggag
aatttttctc 23940 ccacctacac aattcattta acctttcatt aaatatttaa
tgagcacctg ctatgtacta 24000 ggtactatcc tatgtgatgg agacacagcg
gtgaacaaag taaacaaaat tccttccttc 24060 ttgaaactta taacatagta
gggaagagaa aaattaaata actatataat acatatactg 24120 tatgttatat
tcatttaagc ttagcacaag attttttttt ctatgcacaa agagaatagt 24180
cagcctcatt gtttttaaat cattattacc atcatcatta ttaaatcaga gcaatttact
24240 tgattacgtg tatctcaaag ctattttaag attaaagagt aaataagatt
ttggagttga 24300 gaccagcatt ctagtttatg aattctacaa tcttgataga
gggaaactgt ctagattatc 24360 ttttaattgg acaatattga aatatgtgtt
aataataaca ttaaaaagga ttaatattat 24420 ttcctttttt tttctctcat
gaaacatttt taaggacaaa ttcttccaga ttccagcatc 24480 agaagttctg
atggatgatg accttcagaa aagtgtggat atgatcatgg atatgttttg 24540
tcctccagga ataaaaattg gtaggcaaga atattttaac aatcccacac ttcttttact
24600 tgagatagca ctaacatata tgtactctgt ggacttttag aagtctgaaa
gctttgcttc 24660 caaatgattt actaagtagt gagtgattac tctatgatca
acctttgatg aagagagtgg 24720 cagggataaa atagttatga atcataattc
ctgcagtcaa aagattttta aaatattttt 24780 aaatatagga aagggagata
gttttgatca caagcacatt tgacattgtc atgctacaag 24840 cattttagtt
gaaattagac caaaagtgat gaattgttgg cagtaaacat tttctgtaac 24900
aaactccaat tatccaattt aattcatgga ttaatttttt tatttattgt taactagttt
24960 cagattttac aagcttttgt tttaccaatt ttttgtgagc tttgttttct
gcataaacat 25020 ttgattaata aaccagatct tcctcatttc aaattgtact
gcttatacct gctgccactg 25080 aattttcctt ctgtgactat atttgtactt
atgttgaaac ttgcagatct aagtcatatt 25140 aagacaattt tgatttttct
aacaattttt tatcgtagga aattttacca gctgcagatt 25200 tagcagctgg
tttaattttt atatactatt tttaatcagg ctttactctc cctggtcaat 25260
ctttgcatct tataatagtt acataatgat aggaatttgt gttgatctct aaccaagttt
25320 aacttgaata cctttatttg ttgtcagttt taatttgtgt taactgtttg
gattcttttg 25380 gatagatttc tagaagtaag tctttatatc caaaagcatg
ggcctggtag acccattgta 25440 accactattt tagattttta aaatatatac
caaccatttt gaaacccaag atgtactcac 25500 tgttacctgc ttgtggcaaa
aaattcaaat tagtcacaat tgctccaaaa caataacatg 25560 aatctagtat
gtattttgaa gagagaataa tgttaaattt ggaagggacg tttacttact 25620
tttcaagcca aaataaatgt taatttttct agctcagtgg taagcttagg tacctatttc
25680 agagttattt attttgtttt aatggttaaa tcgctttttt tgtttttgtt
tttgtagatg 25740 catatccgtg gttggaatgc ttcatcaagt catacaatgt
cacaaatgga acagataatc 25800 aaatttgcta tcagattttt gacaccacag
ttgcagaaga tgtaatctaa tattgccatc 25860 caatttagca tacataaaat
gttgccactc accttccctg tttgagcttc ttttcctgac 25920 ctgagttttg
tatcagcaat gttgatgatg ttagcatggg tatgggatta gaaaatgtcc 25980
ttaccttaaa tctcttggct tttactgggt gcaaggtaaa taatggctat ggattttgtt
26040 ttgctttctg ttttgctttt gtacaaagag acctgcttaa acaagtactg
ctgagataag 26100 tgtctgatca agctacagtg tactttaagt agaaatggca
aagttgcttt gttggggtgc 26160 tgatactgat gattttagga taaattcatt
tctttaaact tgtaatacat ggttttattg 26220 cttgtttctc tccaggatag
tagagatttc tctatttcac ctcaacctaa taaaagtggt 26280
cagatttata atgttaatga cttaatatta tccttttcta atagtctcat gtaaaatatg
26340 ccgctattac aacttacaac taattgaatg agatgttaac ttagtaaaat
agtttgattt 26400 ttacctgaca gtgtttgtca aatttaaaat catgaatatt
caattttata caaacattta 26460 tatatatata tatagatttg tgtatgttat
ttgccaaaga cagatataaa ttacctggtt 26520 taatattagt gaagaataaa
taagtgcaca catttcaact gtttcattta tttgccctaa 26580 gttgagctga
aaaatgatat gaggcaaaga atcgaaatag gtgtggcaat gcagcagatg 26640
tttagggctg tctacatccc aggtactgtg ctaagcacta aacatgtatt tgatcctcac
26700 agcaacctat ttttccgata agaaatctga ggcttgattg ataagctgac
ttgactaagt 26760 tcacacagtt tgtaaaagct agagtctgtg ccttaattca
cataatctct attcagagcc 26820 tgtactgtta accactcaag gattctggaa
cagaagctaa cagttttctg caacgagtct 26880 ttgacttaaa catctgaaat
aacattggaa atagattata agaggagtca gtgtgttttt 26940 ctatagtttc
aaaatacttt taacatctta ttgtcaaaaa gattggataa ctgactttct 27000
ttgctcataa taactctaaa ttctagttcc tgagtacatt aacacatctt ctttacctaa
27060 ctaccaatgt cccccatcat cgacttatca gcttgtttga gacaatgaga
aagactgatt 27120 ttattttcaa gaatatagac tcttggttca aaacattttc
aggaaaaata ttttaaaacc 27180 ctacagttga acaggtgtgt ttccgtgttg
atgatgtgct caggatacaa aggtgaaata 27240 aacatttttt ctgccttcag
gaagccctca atctagaaga gtagaggtcc aaaggtgcca 27300 tatgttcaca
ctgtgagcct gcaagatctc cacgttaaca aaggaaaact cttcctatga 27360
atcttcatga tgatagg 27377 19 30 DNA Homo sapiens 19 ccctaaccct
aaccctaacc ctaaccctaa 30 20 30 DNA Homo sapiens 20 ttagggttag
ggttagggtt agggttaggg 30 21 60 DNA Homo sapiens 21 ccctaaccct
aaccctaacc ctaaccctaa ttagggttag ggttagggtt agggttaggg 60 22 18 DNA
Artificial Sequence Description of Artificial Sequence Telomeric
primer PBoli82 22 tgtggtgtgt gggtgtgc 18 23 20 DNA Artificial
Sequence Description of Artificial Sequence SpPot1p-binding
oligonucleotide 23 ggttacggtt acaggttaca 20 24 19 DNA Artificial
Sequence Description of Artificial Sequence SpPot1p-binding
oligonucleotide 24 cggttacacg gttacaggt 19 25 20 DNA Artificial
Sequence Description of Artificial Sequence SpPot1p-binding
oligonucleotide 25 gttacaggtt acggttacgg 20 26 22 DNA Artificial
Sequence Description of Artificial Sequence SpPot1p-binding
oligonucleotide 26 tgtggtgtgt gggtgtgcgg tt 22 27 30 DNA Artificial
Sequence Description of Artificial Sequence SpPot1p-binding
oligonucleotide 27 ggttacacgg ttacaggtta caggttacag 30 28 43 DNA
Artificial Sequence Description of Artificial Sequence
SpPot1p-binding oligonucleotide 28 ggttacacgg ttacaggtta caggttacag
ggttacggtt acg 43 29 28 DNA Artificial Sequence Description of
Artificial Sequence SpPot1p-binding oligonucleotide 29 ctgtaagcat
atcatcattc gaggttac 28 30 28 DNA Artificial Sequence Description of
Artificial Sequence SpPot1p-binding oligonucleotide 30 ggttacgcat
atcatcattc gaatctcg 28 31 28 DNA Artificial Sequence Description of
Artificial Sequence SpPot1p-binding oligonucleotide 31 ctgtaagcat
atcatcggtt acggttac 28 32 28 DNA Artificial Sequence Description of
Artificial Sequence SpPot1p-binding oligonucleotide 32 ggttacggtt
accatcattc gaatctcg 28 33 28 DNA Artificial Sequence Description of
Artificial Sequence SpPot1p-binding oligonucleotide 33 ctgtaagcat
atggttactc gaatctcg 28 34 28 DNA Artificial Sequence Description of
Artificial Sequence SpPot1p-binding oligonucleotide 34 ctgtaagcgg
ttacggttac gaatctcg 28 35 20 DNA Artificial Sequence Description of
Artificial Sequence SpPot1p-binding oligonucleotide 35 ggttacaggt
tacaggttac 20 36 20 DNA Artificial Sequence Description of
Artificial Sequence hPot1p-binding oligonucleotide 36 ttagggttag
ggttagggtt 20 37 20 DNA Artificial Sequence Description of
Artificial Sequence hPot1p-binding oligonucleotide 37 ggttagggtt
agggttaggg 20 38 30 DNA Artificial Sequence Description of
Artificial Sequence hPot1p-binding oligonucleotide 38 ttagggttag
ggttagggtt agggttaggg 30 39 45 PRT Schizosaccharomyces pombe 39 Met
Gly Glu Asp Val Ile Asp Ser Leu Gln Leu Asn Glu Leu Leu Asn 1 5 10
15 Ala Gly Glu Tyr Lys Ile Gly Val Arg Tyr Gln Trp Ile Tyr Ile Cys
20 25 30 Phe Ala Asn Asn Glu Lys Gly Thr Tyr Ile Ser Val His 35 40
45 40 43 DNA Artificial Sequence Description of Artificial Sequence
C-strand binding specificity of SpPot1p 40 cgtaaccgta accctgtaac
ctgtaacctg taaccgtgta acc 43 41 40 DNA Artificial Sequence
Description of Artificial Sequence PBoli109 oligonucleotide 41
ccgtaagcat ttcattattg gaattcgagc tcgttttcga 40 42 29 DNA Artificial
Sequence Description of Artificial Sequence PBoli164T
oligonucleotide 42 ttcagatgtt atctgtcaat cagaacctg 29 43 35 DNA
Artificial Sequence Description of Artificial Sequence PBoli194B
oligonucleotide 43 gaacactgtt tacatccata gtgatgtatt gttcc 35 44 26
DNA Artificial Sequence Description of Artificial Sequence Primer
44 tgaaggtcgg agtcaacgga tttggt 26 45 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 45 catgtgggcc atgaggtcca
ccac 24
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