U.S. patent application number 13/862082 was filed with the patent office on 2013-08-15 for pfks as modifiers of the igfr pathway and methods of use.
This patent application is currently assigned to EXELIXIS, INC.. The applicant listed for this patent is Exelixis, Inc.. Invention is credited to Lynn Margaret Bjerke, Helen Francis-Lang, Lori S. Friedman, Timothy S. Heuer, Annette L. Parks, Kenneth James Shaw.
Application Number | 20130212716 13/862082 |
Document ID | / |
Family ID | 35785569 |
Filed Date | 2013-08-15 |
United States Patent
Application |
20130212716 |
Kind Code |
A1 |
Friedman; Lori S. ; et
al. |
August 15, 2013 |
PFKs as Modifiers of the IGFR Pathway and Methods of Use
Abstract
Human PFK genes are identified as modulators of the IGFR
pathway, and thus are therapeutic targets for disorders associated
with defective IGFR function. Methods for identifying modulators of
IGFR, comprising screening for agents that modulate the activity of
PFK are provided.
Inventors: |
Friedman; Lori S.; (San
Carlos, CA) ; Francis-Lang; Helen; (San Francisco,
CA) ; Parks; Annette L.; (Newton, MA) ; Shaw;
Kenneth James; (Brisbane, CA) ; Bjerke; Lynn
Margaret; (Sutton, GB) ; Heuer; Timothy S.;
(El Granada, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Exelixis, Inc.; |
|
|
US |
|
|
Assignee: |
EXELIXIS, INC.
South San Francisco
CA
|
Family ID: |
35785569 |
Appl. No.: |
13/862082 |
Filed: |
April 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11628557 |
Sep 16, 2008 |
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PCT/US2005/021614 |
Jun 20, 2005 |
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13862082 |
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60581696 |
Jun 21, 2004 |
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Current U.S.
Class: |
800/3 ;
424/158.1; 424/9.2; 435/15; 435/375; 435/6.11; 435/6.13; 435/7.21;
506/11; 506/9 |
Current CPC
Class: |
A61P 35/00 20180101;
C12Q 1/68 20130101; C12N 9/0071 20130101; G01N 2500/00 20130101;
C12Q 1/26 20130101; C12Y 114/11002 20130101; G01N 33/74 20130101;
C12Q 1/6886 20130101; A61K 49/0008 20130101; Y10T 436/143333
20150115; A61P 43/00 20180101; G01N 33/574 20130101; A61P 31/00
20180101; C12Q 1/485 20130101 |
Class at
Publication: |
800/3 ;
424/158.1; 424/9.2; 435/15; 435/7.21; 435/6.13; 435/375; 435/6.11;
506/9; 506/11 |
International
Class: |
C12Q 1/48 20060101
C12Q001/48; G01N 33/74 20060101 G01N033/74; C12Q 1/68 20060101
C12Q001/68; A61K 49/00 20060101 A61K049/00 |
Claims
1. A method of identifying a candidate IGFR pathway modulating
agent, said method comprising the steps of: (a) providing an assay
system comprising a PFK polypeptide or nucleic acid; (b) contacting
the assay system with a test agent under conditions whereby, but
for the presence of the test agent, the system provides a reference
activity; and (c) detecting a test agent-biased activity of the
assay system, wherein a difference between the test agent-biased
activity and the reference activity identifies the test agent as a
candidate IGFR pathway modulating agent.
2. The method of claim 1 wherein the assay system comprises
cultured cells that express the PFK polypeptide.
3. The method of claim 2 wherein the cultured cells additionally
have defective IGFR function.
4. The method of claim 1 wherein the assay system includes a
screening assay comprising a PFK polypeptide, and the candidate
test agent is a small molecule modulator.
5. The method of claim 4 wherein the assay is a kinase assay.
6. The method of claim 1 wherein the assay system is selected from
the group consisting of an apoptosis assay system, a cell
proliferation assay system, an angiogenesis assay system, and a
hypoxic induction assay system.
7. The method of claim 1 wherein the assay system includes a
binding assay comprising a PFK polypeptide and the candidate test
agent is an antibody.
8. The method of claim 1 wherein the assay system includes an
expression assay comprising a PFK nucleic acid and the candidate
test agent is a nucleic acid modulator.
9. The method of claim 8 wherein the nucleic acid modulator is an
antisense oligomer.
10. The method of claim 8 wherein the nucleic acid modulator is a
PMO.
11. The method of claim 1 additionally comprising: (d)
administering the candidate IGFR pathway modulating agent
identified in (c) to a model system comprising cells defective in
IGFR function and, detecting a phenotypic change in the model
system that indicates that the IGFR function is restored.
12. The method of claim 11 wherein the model system is a mouse
model with defective IGFR function.
13. A method for modulating a IGFR pathway of a cell comprising
contacting a cell defective in IGFR function with a candidate
modulator that specifically binds to a PFK polypeptide, whereby
IGFR function is restored.
14. The method of claim 13 wherein the candidate modulator is
administered to a vertebrate animal predetermined to have a disease
or disorder resulting from a defect in IGFR function.
15. The method of claim 13 wherein the candidate modulator is
selected from the group consisting of an antibody and a small
molecule.
16. The method of claim 1, comprising the additional steps of: (d)
providing a secondary assay system comprising cultured cells or a
non-human animal expressing PFK, (e) contacting the secondary assay
system with the test agent of (b) or an agent derived therefrom
under conditions whereby, but for the presence of the test agent or
agent derived therefrom, the system provides a reference activity;
and (f) detecting an agent-biased activity of the second assay
system, wherein a difference between the agent-biased activity and
the reference activity of the second assay system confirms the test
agent or agent derived therefrom as a candidate IGFR pathway
modulating agent, and wherein the second assay detects an
agent-biased change in the IGFR pathway.
17. The method of claim 16 wherein the secondary assay system
comprises cultured cells.
18. The method of claim 16 wherein the secondary assay system
comprises a non-human animal.
19. The method of claim 18 wherein the non-human animal
mis-expresses a IGFR pathway gene.
20. A method of modulating IGFR pathway in a mammalian cell
comprising contacting the cell with an agent that specifically
binds a PFK polypeptide or nucleic acid.
21. The method of claim 20 wherein the agent is administered to a
mammalian animal predetermined to have a pathology associated with
the IGFR pathway.
22. The method of claim 20 wherein the agent is a small molecule
modulator, a nucleic acid modulator, or an antibody.
23. A method for diagnosing a disease in a patient comprising:
obtaining a biological sample from the patient; contacting the
sample with a probe for PFK expression; comparing results from step
(b) with a control; determining whether step (c) indicates a
likelihood of disease.
24. The method of claim 23 wherein said disease is cancer.
25. The method according to claim 24, wherein said cancer is a
cancer as shown in Table 1 as having >25% expression level.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional patent
application 60/581,696 filed Jun. 21, 2004. The contents of the
prior application are hereby incorporated in their entirety.
BACKGROUND OF THE INVENTION
[0002] Somatic mutations in the PTEN (Phosphatase and Tensin
homolog deleted on chromosome 10) gene are known to cause tumors in
a variety of human tissues. In addition, germline mutations in PTEN
are the cause of human diseases (Cowden disease and Bannayan-Zonana
syndrome) associated with increased risk of breast and thyroid
cancer (Nelen M R et al. (1997) Hum Mol Genet, 8:1383-1387; Liaw D
et al. (1997) Nat Genet, 1:64-67; Marsh D J et al. (1998) Hum Mol
Genet, 3:507-515). PTEN is thought to act as a tumor suppressor by
regulating several signaling pathways through the second messenger
phosphatidylinositol 3,4,5 triphosphate (PIP3). PTEN
dephosphorylates the D3 position of PIP3 and downregulates
signaling events dependent on PIP3 levels (Maehama T and Dixon J E
(1998) J Biol Chem, 22, 13375-8). In particular, pro-survival
pathways downstream of the insulin-like growth factor (IGF) pathway
are regulated by PTEN activity. Stimulation of the IGF pathway, or
loss of PTEN function, elevates PIP3 levels and activates
pro-survival pathways associated with tumorigenesis (Stambolic V et
al. (1998) Cell, 95:29-39). Consistent with this model, elevated
levels of insulin-like growth factors I and II correlate with
increased risk of cancer (Yu H et al (1999) J Natl Cancer Inst
91:151-156) and poor prognosis (Takanami I et al, 1996, J Surg
Oncol 61(3):205-8). In addition, increased levels or activity of
positive effectors of the IGF pathway, such as Akt and PI(3)
kinase, have been implicated in several types of human cancer
(Nicholson K M and Anderson N G (2002) Cellular Signalling,
14:381-395).
[0003] In Drosophila melanogaster, as in vertebrates, the Insulin
Growth Factor Receptor (IGFR) pathway includes the positive
effectors PI(3) kinase, Akt, and PDK and the inhibitor, PTEN. These
proteins have been implicated in multiple processes, including the
regulation of cell growth and size as well as cell division and
survival (Oldham S and Hafen E. (2003) Trends Cell Biol. 13:79-85;
Garafolo R S. (2002) Trends Endocr. Metab. 13:156-162; Backman S A
et al. (2002) Curr. Op. Neurobio. 12:1-7; Tapon N et al. (2001)
Curr Op. Cell Biol. 13:731-737). Activation of the pathway in
Drosophila can result in increases in cell size, cell number and
organ size (Oldham S et al. (2002) Dev. 129:4103-4109; Prober D A
and Edgar B A. (2002) Genes & Dev. 16:2286-2299; Potter C J et
al. (2001) Cell 105:357-368; Verdu J et al. (1999) Cell Biol.
1:500-506).
[0004] Phosphofructokinase (PFK) is a tetrameric enzyme that
catalyzes a key step in glycolysis, namely the conversion of
D-fructose 6-phosphate to D-fructose 1,6-bisphosphate. Separate
genes encode a muscle subunit (M) and a liver subunit (L). Muscle
Phosphofructokinase (PFKM) is a homotetramer of M subunits, liver
type Phosphofructokinase (PFKL) is a homotetramer of L-subunits,
while platelet type Phosphofructokinase (PFKP) can be composed of
any tetrameric combination of M and L subunits.
[0005] The ability, to manipulate the genomes of model organisms
such as Drosophila provides a powerful means to analyze biochemical
processes that, due to significant evolutionary conservation, have
direct relevance to more complex vertebrate organisms. Due to a
high level of gene and pathway conservation, the strong similarity
of cellular processes, and the functional conservation of genes
between these model organisms and mammals, identification of the
involvement of novel genes in particular pathways and their
functions in such model organisms can directly contribute to the
understanding of the correlative pathways and methods of modulating
them in mammals (see, for example, Mechler B M et al., 1985 EMBO J
4:1551-1557; Gateff E. 1982 Adv. Cancer Res. 37: 33-74; Watson K
L., et al., 1994 J Cell Sci. 18: 19-33; Miklos G L, and Rubin G M.
1996 Cell 86:521-529; Wassarman D A, et al., 1995 Curr Opin Gen Dev
5: 44-50; and Booth D R. 1999 Cancer Metastasis Rev. 18: 261-284).
For example, a genetic screen can be carried out in an invertebrate
model organism having underexpression (e.g. knockout) or
overexpression of a gene (referred to as a "genetic entry point")
that yields a visible phenotype. Additional genes are mutated in a
random or targeted manner. When a gene mutation changes the
original phenotype caused by the mutation in the genetic entry
point, the gene is identified as a "modifier" involved in the same
or overlapping pathway as the genetic entry point. When the genetic
entry point is an ortholog of a human gene implicated in a disease
pathway, such as IGFR, modifier genes can be identified that may be
attractive candidate targets for novel therapeutics.
[0006] All references cited herein, including patents, patent
applications, publications, and sequence information in referenced
Genbank identifier numbers, are incorporated herein in their
entireties.
SUMMARY OF THE INVENTION
[0007] We have discovered genes that modify the IGFR pathway in
Drosophila, and identified their human orthologs, hereinafter
referred to as Phosphofructokinase (PFK). The invention provides
methods for utilizing these IGFR modifier genes and polypeptides to
identify PFK-modulating agents that are candidate therapeutic
agents that can be used in the treatment of disorders associated
with defective or impaired IGFR function and/or PFK function.
Preferred PFK-modulating agents specifically bind to PFK
polypeptides and restore IGFR function. Other preferred
PFK-modulating agents are nucleic acid modulators such as antisense
oligomers and RNAi that repress PFK gene expression or product
activity by, for example, binding to and inhibiting the respective
nucleic acid (i.e. DNA or mRNA).
[0008] PFK modulating agents may be evaluated by any convenient in
vitro or in vivo assay for molecular interaction with a PFK
polypeptide or nucleic acid. In one embodiment, candidate PFK
modulating agents are tested with an assay system comprising a PFK
polypeptide or nucleic acid. Agents that produce a change in the
activity of the assay system relative to controls are identified as
candidate IGFR modulating agents. The assay system may be
cell-based or cell-free. PFK-modulating agents include PFK related
proteins (e.g. dominant negative mutants, and biotherapeutics);
PFK-specific antibodies; PFK-specific antisense oligomers and other
nucleic acid modulators; and chemical agents that specifically bind
to or interact with PFK or compete with PFK binding partner (e.g.
by binding to a PFK binding partner). In one specific embodiment, a
small molecule modulator is identified using a kinase assay. In
specific embodiments, the screening assay system is selected from a
binding assay, an apoptosis assay, a cell proliferation assay, an
angiogenesis assay, and a hypoxic induction assay.
[0009] In another embodiment, candidate IGFR pathway modulating
agents are further tested using a second assay system that detects
changes in the IGFR pathway, such as angiogenic, apoptotic, or cell
proliferation changes produced by the originally identified
candidate agent or an agent derived from the original agent. The
second assay system may use cultured cells or non-human animals. In
specific embodiments, the secondary assay system uses non-human
animals, including animals predetermined to have a disease or
disorder implicating the IGFR pathway, such as an angiogenic,
apoptotic, or cell proliferation disorder (e.g. cancer).
[0010] The invention further provides methods for modulating the
PFK function and/or the IGFR pathway in a mammalian cell by
contacting the mammalian cell with an agent that specifically binds
a PFK polypeptide or nucleic acid. The agent may be a small
molecule modulator, a nucleic acid modulator, or an antibody and
may be administered to a mammalian animal predetermined to have a
pathology associated with the IGFR pathway.
DETAILED DESCRIPTION OF THE INVENTION
[0011] A dominant loss of function screen was carried out in
Drosophila to identify genes that interact with or modulate the
IGFR signaling pathway. Modifiers of the IGFR pathway and their
orthologs were identified. The DROSOPHILA PFK gene was identified
as a modifier of the IGFR pathway. Accordingly, vertebrate
orthologs of this modifier, and preferably the human orthologs, PFK
genes (i.e., nucleic acids and polypeptides) are attractive drug
targets for the treatment of pathologies associated with a
defective IGFR signaling pathway, such as cancer.
[0012] In vitro and in vivo methods of assessing PFK function are
provided herein. Modulation of the PFK or their respective binding
partners is useful for understanding the association of the IGFR
pathway and its members in normal and disease conditions and for
developing diagnostics and therapeutic modalities for IGFR related
pathologies. PFK-modulating agents that act by inhibiting or
enhancing PFK expression, directly or indirectly, for example, by
affecting a PFK function such as enzymatic (e.g., catalytic) or
binding activity, can be identified using methods provided herein.
PFK modulating agents are useful in diagnosis, therapy and
pharmaceutical development.
[0013] Nucleic Acids and Polypeptides of the Invention
[0014] Sequences related to PFK nucleic acids and polypeptides that
can be used in the invention are disclosed in Genbank (referenced
by Genbank identifier (GI) number) as GI#s 50346003 (SEQ ID NO:1),
20560995 (SEQ ID NO:2), 21361069 (SEQ ID NO:3), 14602831 (SEQ ID
NO:4), 17390696 (SEQ ID NO:5), 13623608 (SEQ ID NO:6), 14043100
(SEQ ID NO:7), 35391 (SEQ ID NO:8), 35393 (SEQ ID NO:9), 35395 (SEQ
ID NO:10), 35397 (SEQ ID NO:11), 35399 (SEQ ID NO:12), 35400 (SEQ
ID NO:13), 35402 (SEQ ID NO:14), 35405 (SEQ ID NO:15), 35407 (SEQ
ID NO:16), 35409 (SEQ ID NO:17), 35411 (SEQ ID NO:18), 35413 (SEQ
ID NO:19), 35415 (SEQ ID NO:20), 35417 (SEQ ID NO:21), 35419 (SEQ
ID NO:22), 35421 (SEQ ID NO:23), 35423 (SEQ ID NO:24), 35424 (SEQ
ID NO:25), 35426 (SEQ ID NO:26), 35428 (SEQ ID NO:27), 21749869
(SEQ ID NO:28), 50346004 (SEQ ID NO:29), 39725712 (SEQ ID NO:30),
4505748 (SEQ ID NO:31), 12653524 (SEQ ID NO:32), 15342052 (SEQ ID
NO:33), 15215396 (SEQ ID NO:34), 18203736 (SEQ ID NO:35), 188633
(SEQ ID NO:36), 188634 (SEQ ID NO:37), 188635 (SEQ ID NO:38),
188636 (SEQ ID NO:39), 188637 (SEQ ID NO:40), 188638 (SEQ ID
NO:41), 188639 (SEQ ID NO:42), 188640 (SEQ ID NO:43), 188641 (SEQ
ID NO:44), 188642 (SEQ ID NO:45), 188643 (SEQ ID NO:46), 188644
(SEQ ID NO:47), 188645 (SEQ ID NO:48), 188646 (SEQ ID NO:49),
188647 (SEQ ID NO:50), 188648 (SEQ ID NO:51), 188649 (SEQ ID
NO:52), 188650 (SEQ ID NO:53), 188651 (SEQ ID NO:54), 188652 (SEQ
ID NO:55), 188653 (SEQ ID NO:56), 188654 (SEQ ID NO:57), 188655
(SEQ ID NO:58), 3964478 (SEQ ID NO:59), 41352062 (SEQ ID NO:60),
11321600 (SEQ ID NO:61), 12803424 (SEQ ID NO:62), and 20810529 (SEQ
ID NO:63) for nucleic acid, and GI#s 21361070 (SEQ ID NO:64),
4505749 (SEQ ID NO:65), and 11321601 (SEQ ID NO:66) for polypeptide
sequences.
[0015] The term "PFK polypeptide" refers to a full-length PFK
protein or a functionally active fragment or derivative thereof. A
"functionally active" PFK fragment or derivative exhibits one or
more functional activities associated with a full-length, wild-type
PFK protein, such as antigenic or immunogenic activity, enzymatic
activity, ability to bind natural cellular substrates, etc. The
functional activity of PFK proteins, derivatives and fragments can
be assayed by various methods known to one skilled in the art
(Current Protocols in Protein Science (1998) Coligan et al., eds.,
John Wiley & Sons, Inc., Somerset, N.J.) and as further
discussed below. In one embodiment, a functionally active PFK
polypeptide is a PFK derivative capable of rescuing defective
endogenous PFK activity, such as in cell based or animal assays;
the rescuing derivative may be from the same or a different
species. For purposes herein, functionally active fragments also
include those fragments that comprise one or more structural
domains of a PFK, such as a kinase domain or a binding domain.
Protein domains can be identified using the PFAM program (Bateman
A., et al., Nucleic Acids Res, 1999, 27:260-2). For example, the
Phosphofructokinase domain (PFAM 00365) of PFK from GI#21361070
(SEQ ID NO:64) is located at approximately amino acid residues 76
to 373, 448 to 735; the Phosphofructokinase domain of PFK from
GI#4505749 (SEQ ID NO:65) is located at approximately amino acid
residues 16 to 326, 401 to 689; and the Phosphofructokinase domain
of PFK from GI#11321601 (SEQ ID NO:66) is located at approximately
amino acid residues 25 to 335, 412 to 699. Methods for obtaining
PFK polypeptides are also further described below. In some
embodiments, preferred fragments are functionally active,
domain-containing fragments comprising at least 25 contiguous amino
acids, preferably at least 50, more preferably 75, and most
preferably at least 100 contiguous amino acids of a PFK. In further
preferred embodiments, the fragment comprises the entire
functionally active domain.
[0016] The term "PFK nucleic acid" refers to a DNA or RNA molecule
that encodes a PFK polypeptide. Preferably, the PFK polypeptide or
nucleic acid or fragment thereof is from a human, but can also be
an ortholog, or derivative thereof with at least 70% sequence
identity, preferably at least 80%, more preferably 85%, still more
preferably 90%, and most preferably at least 95% sequence identity
with human PFK. Methods of identifying orthlogs are known in the
art. Normally, orthologs in different species retain the same
function, due to presence of one or more protein motifs and/or
3-dimensional structures. Orthologs are generally identified by
sequence homology analysis, such as BLAST analysis, usually using
protein bait sequences. Sequences are assigned as a potential
ortholog if the best hit sequence from the forward BLAST result
retrieves the original query sequence in the reverse BLAST (Huynen
M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen M A
et al., Genome Research (2000) 10:1204-1210). Programs for multiple
sequence alignment, such as CLUSTAL (Thompson J D et al, 1994,
Nucleic Acids Res 22:4673-4680) may be used to highlight conserved
regions and/or residues of orthologous proteins and to generate
phylogenetic trees. In a phylogenetic tree representing multiple
homologous sequences from diverse species (e.g., retrieved through
BLAST analysis), orthologous sequences from two species generally
appear closest on the tree with respect to all other sequences from
these two species. Structural threading or other analysis of
protein folding (e.g., using software by ProCeryon, Biosciences,
Salzburg, Austria) may also identify potential orthologs. In
evolution, when a gene duplication event follows speciation, a
single gene in one species, such as Drosophila, may correspond to
multiple genes (paralogs) in another, such as human. As used
herein, the term "orthologs" encompasses paralogs. As used herein,
"percent (%) sequence identity" with respect to a subject sequence,
or a specified portion of a subject sequence, is defined as the
percentage of nucleotides or amino acids in the candidate
derivative sequence identical with the nucleotides or amino acids
in the subject sequence (or specified portion thereof), after
aligning the sequences and introducing gaps, if necessary to
achieve the maximum percent sequence identity, as generated by the
program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. (1997)
215:403-410) with all the search parameters set to default values.
The HSP S and HSP S2 parameters are dynamic values and are
established by the program itself depending upon the composition of
the particular sequence and composition of the particular database
against which the sequence of interest is being searched. A %
identity value is determined by the number of matching identical
nucleotides or amino acids divided by the sequence length for which
the percent identity is being reported. "Percent (%) amino acid
sequence similarity" is determined by doing the same calculation as
for determining % amino acid sequence identity, but including
conservative amino acid substitutions in addition to identical
amino acids in the computation.
[0017] A conservative amino acid substitution is one in which an
amino acid is substituted for another amino acid having similar
properties such that the folding or activity of the protein is not
significantly affected. Aromatic amino acids that can be
substituted for each other are phenylalanine, tryptophan, and
tyrosine; interchangeable hydrophobic amino acids are leucine,
isoleucine, methionine, and valine; interchangeable polar amino
acids are glutamine and asparagine; interchangeable basic amino
acids are arginine, lysine and histidine; interchangeable acidic
amino acids are aspartic acid and glutamic acid; and
interchangeable small amino acids are alanine, serine, threonine,
cysteine and glycine.
[0018] Alternatively, an alignment for nucleic acid sequences is
provided by the local homology algorithm of Smith and Waterman
(Smith and Waterman, 1981, Advances in Applied Mathematics
2:482-489; database: European Bioinformatics Institute; Smith and
Waterman, 1981, J. of Molec. Biol., 147:195-197; Nicholas et al.;
1998, "A Tutorial on Searching Sequence Databases and Sequence
Scoring Methods" (www.psc.edu) and references cited therein; W. R.
Pearson, 1991, Genomics 11:635-650). This algorithm can be applied
to amino acid sequences by using the scoring matrix developed by
Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, M. O.
Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research
Foundation, Washington, D.C., USA), and normalized by Gribskov
(Gribskov 1986 Nucl. Acids Res. 14(6):6745-6763). The
Smith-Waterman algorithm may be employed where default parameters
are used for scoring (for example, gap open penalty of 12, gap
extension penalty of two). From the data generated, the "Match"
value reflects "sequence identity."
[0019] Derivative nucleic acid molecules of the subject nucleic
acid molecules include sequences that hybridize to the nucleic acid
sequence of a PFK. The stringency of hybridization can be
controlled by temperature, ionic strength, pH, and the presence of
denaturing agents such as formamide during hybridization and
washing. Conditions routinely used are set out in readily available
procedure texts (e.g., Current Protocol in Molecular Biology, Vol.
1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook
et al., Molecular Cloning, Cold Spring Harbor (1989)). In some
embodiments, a nucleic acid molecule of the invention is capable of
hybridizing to a nucleic acid molecule containing the nucleotide
sequence of a PFK under high stringency hybridization conditions
that are: prehybridization of filters containing nucleic acid for 8
hours to overnight at 65.degree. C. in a solution comprising
6.times. single strength citrate (SSC) (1.times.SSC is 0.15 M NaCl,
0.015 M Na citrate; pH 7.0), 5.times.Denhardt's solution, 0.05%
sodium pyrophosphate and 100 .mu.g/ml herring sperm DNA;
hybridization for 18-20 hours at 65.degree. C. in a solution
containing 6.times.SSC, 1.times.Denhardt's solution, 100 .mu.g/ml
yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters
at 65.degree. C. for 1 h in a solution containing 0.1.times.SSC and
0.1% SDS (sodium dodecyl sulfate).
[0020] In other embodiments, moderately stringent hybridization
conditions are used that are: pretreatment of filters containing
nucleic acid for 6 h at 40.degree. C. in a solution containing 35%
formamide, 5.times.SSC, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, 0.1%
PVP, 0.1% Ficoll, 1% BSA, and 500 .mu.g/ml denatured salmon sperm
DNA; hybridization for 18-20 h at 40.degree. C. in a solution
containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl (pH7.5), 5 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml salmon sperm
DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice
for 1 hour at 55.degree. C. in a solution containing 2.times.SSC
and 0.1% SDS.
[0021] Alternatively, low stringency conditions can be used that
are: incubation for 8 hours to overnight at 37.degree. C. in a
solution comprising 20% formamide, 5.times.SSC, 50 mM sodium
phosphate (pH 7.6), 5.times.Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured sheared salmon sperm DNA;
hybridization in the same buffer for 18 to 20 hours; and washing of
filters in 1.times.SSC at about 37.degree. C. for 1 hour.
[0022] Isolation, Production, Expression, and Mis-Expression of PFK
Nucleic Acids and Polypeptides
[0023] PFK nucleic acids and polypeptides are useful for
identifying and testing agents that modulate PFK function and for
other applications related to the involvement of PFK in the IGFR
pathway. PFK nucleic acids and derivatives and orthologs thereof
may be obtained using any available method. For instance,
techniques for isolating cDNA or genomic DNA sequences of interest
by screening DNA libraries or by using polymerase chain reaction
(PCR) are well known in the art. In general, the particular use for
the protein will dictate the particulars of expression, production,
and purification methods. For instance, production of proteins for
use in screening for modulating agents may require methods that
preserve specific biological activities of these proteins, whereas
production of proteins for antibody generation may require
structural integrity of particular epitopes. Expression of proteins
to be purified for screening or antibody production may require the
addition of specific tags (e.g., generation of fusion proteins).
Overexpression of a PFK protein for assays used to assess PFK
function, such as involvement in cell cycle regulation or hypoxic
response, may require expression in eukaryotic cell lines capable
of these cellular activities. Techniques for the expression,
production, and purification of proteins are well known in the art;
any suitable means therefore may be used (e.g., Higgins S J and
Hames B D (eds.) Protein Expression: A Practical Approach, Oxford
University Press Inc., New York 1999; Stanbury P F et al.,
Principles of Fermentation Technology, 2.sup.nd edition, Elsevier
Science, New York, 1995; Doonan S (ed.) Protein Purification
Protocols, Humana Press, New Jersey, 1996; Coligan J E et al,
Current Protocols in Protein Science (eds.), 1999, John Wiley &
Sons, New York). In particular embodiments, recombinant PFK is
expressed in a cell line known to have defective IGFR function. The
recombinant cells are used in cell-based screening assay systems of
the invention, as described further below.
[0024] The nucleotide sequence encoding a PFK polypeptide can be
inserted into any appropriate expression vector. The necessary
transcriptional and translational signals, including
promoter/enhancer element, can derive from the native PFK gene
and/or its flanking regions or can be heterologous. A variety of
host-vector expression systems may be utilized, such as mammalian
cell systems infected with virus (e.g. vaccinia virus, adenovirus,
etc.); insect cell systems infected with virus (e.g. baculovirus);
microorganisms such as yeast containing yeast vectors, or bacteria
transformed with bacteriophage, plasmid, or cosmid DNA. An isolated
host cell strain that modulates the expression of, modifies, and/or
specifically processes the gene product may be used.
[0025] To detect expression of the PFK gene product, the expression
vector can comprise a promoter operably linked to a PFK gene
nucleic acid, one or more origins of replication, and, one or more
selectable markers (e.g. thymidine kinase activity, resistance to
antibiotics, etc.). Alternatively, recombinant expression vectors
can be identified by assaying for the expression of the PFK gene
product based on the physical or functional properties of the PFK
protein in in vitro assay systems (e.g. immunoassays).
[0026] The PFK protein, fragment, or derivative may be optionally
expressed as a fusion, or chimeric protein product (i.e. it is
joined via a peptide bond to a heterologous protein sequence of a
different protein), for example to facilitate purification or
detection. A chimeric product can be made by ligating the
appropriate nucleic acid sequences encoding the desired amino acid
sequences to each other using standard methods and expressing the
chimeric product. A chimeric product may also be made by protein
synthetic techniques, e.g. by use of a peptide synthesizer
(Hunkapiller et al., Nature (1984) 310:105-111).
[0027] Once a recombinant cell that expresses the PFK gene sequence
is identified, the gene product can be isolated and purified using
standard methods (e.g. ion exchange, affinity, and gel exclusion
chromatography; centrifugation; differential solubility;
electrophoresis). Alternatively, native PFK proteins can be
purified from natural sources, by standard methods (e.g.
immunoaffinity purification). Once a protein is obtained, it may be
quantified and its activity measured by appropriate methods, such
as immunoassay, bioassay, or other measurements of physical
properties, such as crystallography.
[0028] The methods of this invention may also use cells that have
been engineered for altered expression (mis-expression) of PFK or
other genes associated with the IGFR pathway. As used herein,
mis-expression encompasses ectopic expression, over-expression,
under-expression, and non-expression (e.g. by gene knock-out or
blocking expression that would otherwise normally occur).
[0029] Genetically Modified Animals
[0030] Animal models that have been genetically modified to alter
PFK expression may be used in in vivo assays to test for activity
of a candidate IGFR modulating agent, or to further assess the role
of PFK in a IGFR pathway process such as apoptosis or cell
proliferation. Preferably, the altered PFK expression results in a
detectable phenotype, such as decreased or increased levels of cell
proliferation, angiogenesis, or apoptosis compared to control
animals having normal PFK expression. The genetically modified
animal may additionally have altered IGFR expression (e.g. IGFR
knockout). Preferred genetically modified animals are mammals such
as primates, rodents (preferably mice or rats), among others.
Preferred non-mammalian species include zebrafish, C. elegans, and
Drosophila. Preferred genetically modified animals are transgenic
animals having a heterologous nucleic acid sequence present as an
extrachromosomal element in a portion of its cells, i.e. mosaic
animals (see, for example, techniques described by Jakobovits,
1994, Curr. Biol. 4:761-763.) or stably integrated into its germ
line DNA (i.e., in the genomic sequence of most or all of its
cells). Heterologous nucleic acid is introduced into the germ line
of such transgenic animals by genetic manipulation of, for example,
embryos or embryonic stem cells of the host animal.
[0031] Methods of making transgenic animals are well-known in the
art (for transgenic mice see Brinster et al., Proc. Nat. Acad. Sci.
USA 82: 4438-4442 (1985), U.S. Pat. Nos. 4,736,866 and 4,870,009,
both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al., and
Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1986); for particle
bombardment see U.S. Pat. No. 4,945,050, by Sandford et al.; for
transgenic Drosophila see Rubin and Spradling, Science (1982)
218:348-53 and U.S. Pat. No. 4,670,388; for transgenic insects see
Berghammer A. J. et al., A Universal Marker for Transgenic Insects
(1999) Nature 402:370-371; for transgenic Zebrafish see Lin S.,
Transgenic Zebrafish, Methods Mol Biol. (2000); 136:375-3830); for
microinjection procedures for fish, amphibian eggs and birds see
Houdebine and Chourrout, Experientia (1991) 47:897-905; for
transgenic rats see Hammer et al., Cell (1990) 63:1099-1112; and
for culturing of embryonic stem (ES) cells and the subsequent
production of transgenic animals by the introduction of DNA into ES
cells using methods such as electroporation, calcium phosphate/DNA
precipitation and direct injection see, e.g., Teratocarcinomas and
Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed.,
IRL Press (1987)). Clones of the nonhuman transgenic animals can be
produced according to available methods (see Wilmut, I. et al.
(1997) Nature 385:810-813; and PCT International Publication Nos.
WO 97/07668 and WO 97/07669).
[0032] In one embodiment, the transgenic animal is a "knock-out"
animal having a heterozygous or homozygous alteration in the
sequence of an endogenous PFK gene that results in a decrease of
PFK function, preferably such that PFK expression is undetectable
or insignificant. Knock-out animals are typically generated by
homologous recombination with a vector comprising a transgene
having at least a portion of the gene to be knocked out. Typically
a deletion, addition or substitution has been introduced into the
transgene to functionally disrupt it. The transgene can be a human
gene (e.g., from a human genomic clone) but more preferably is an
ortholog of the human gene derived from the transgenic host
species. For example, a mouse PFK gene is used to construct a
homologous recombination vector suitable for altering an endogenous
PFK gene in the mouse genome. Detailed methodologies for homologous
recombination in mice are available (see Capecchi, Science (1989)
244:1288-1292; Joyner et al., Nature (1989) 338:153-156).
Procedures for the production of non-rodent transgenic mammals and
other animals are also available (Houdebine and Chourrout, supra;
Pursel et al., Science (1989) 244:1281-1288; Simms et al.,
Bio/Technology (1988) 6:179-183). In a preferred embodiment,
knock-out animals, such as mice harboring a knockout of a specific
gene, may be used to produce antibodies against the human
counterpart of the gene that has been knocked out (Claesson M H et
al., (1994) Scan J Immunol 40:257-264; Declerck P J et al., (1995)
J Biol Chem. 270:8397-400).
[0033] In another embodiment, the transgenic animal is a "knock-in"
animal having an alteration in its genome that results in altered
expression (e.g., increased (including ectopic) or decreased
expression) of the PFK gene, e.g., by introduction of additional
copies of PFK, or by operatively inserting a regulatory sequence
that provides for altered expression of an endogenous copy of the
PFK gene. Such regulatory sequences include inducible,
tissue-specific, and constitutive promoters and enhancer elements.
The knock-in can be homozygous or heterozygous.
[0034] Transgenic nonhuman animals can also be produced that
contain selected systems allowing for regulated expression of the
transgene. One example of such a system that may be produced is the
cre/loxP recombinase system of bacteriophage P1 (Lakso et al., PNAS
(1992) 89:6232-6236; U.S. Pat. No. 4,959,317). If a cre/loxP
recombinase system is used to regulate expression of the transgene,
animals containing transgenes encoding both the Cre recombinase and
a selected protein are required. Such animals can be provided
through the construction of "double" transgenic animals, e.g., by
mating two transgenic animals, one containing a transgene encoding
a selected protein and the other containing a transgene encoding a
recombinase. Another example of a recombinase system is the FLP
recombinase system of Saccharomyces cerevisiae (O'Gorman et al.
(1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182). In a
preferred embodiment, both Cre-LoxP and Flp-Frt are used in the
same system to regulate expression of the transgene, and for
sequential deletion of vector sequences in the same cell (Sun X et
al (2000) Nat Genet 25:83-6).
[0035] The genetically modified animals can be used in genetic
studies to further elucidate the IGFR pathway, as animal models of
disease and disorders implicating defective IGFR function, and for
in vivo testing of candidate therapeutic agents, such as those
identified in screens described below. The candidate therapeutic
agents are administered to a genetically modified animal having
altered PFK function and phenotypic changes are compared with
appropriate control animals such as genetically modified animals
that receive placebo treatment, and/or animals with unaltered PFK
expression that receive candidate therapeutic agent.
[0036] In addition to the above-described genetically modified
animals having altered PFK function, animal models having defective
IGFR function (and otherwise normal PFK function), can be used in
the methods of the present invention. For example, a IGFR knockout
mouse can be used to assess, in vivo, the activity of a candidate
IGFR modulating agent identified in one of the in vitro assays
described below. Preferably, the candidate IGFR modulating agent
when administered to a model system with cells defective in IGFR
function, produces a detectable phenotypic change in the model
system indicating that the IGFR function is restored, i.e., the
cells exhibit normal cell cycle progression.
[0037] Modulating Agents
[0038] The invention provides methods to identify agents that
interact with and/or modulate the function of PFK and/or the IGFR
pathway. Modulating agents identified by the methods are also part
of the invention. Such agents are useful in a variety of diagnostic
and therapeutic applications associated with the IGFR pathway, as
well as in further analysis of the PFK protein and its contribution
to the IGFR pathway. Accordingly, the invention also provides
methods for modulating the IGFR pathway comprising the step of
specifically modulating PFK activity by administering a
PFK-interacting or -modulating agent.
[0039] As used herein, a "PFK-modulating agent" is any agent that
modulates PFK function, for example, an agent that interacts with
PFK to inhibit or enhance PFK activity or otherwise affect normal
PFK function. PFK function can be affected at any level, including
transcription, protein expression, protein localization, and
cellular or extra-cellular activity. In a preferred embodiment, the
PFK-modulating agent specifically modulates the function of the
PFK. The phrases "specific modulating agent", "specifically
modulates", etc., are used herein to refer to modulating agents
that directly bind to the PFK polypeptide or nucleic acid, and
preferably inhibit, enhance, or otherwise alter, the function of
the PFK. These phrases also encompass modulating agents that alter
the interaction of the PFK with a binding partner, substrate, or
cofactor (e.g. by binding to a binding partner of a PFK, or to a
protein/binding partner complex, and altering PFK function). In a
further preferred embodiment, the PFK-modulating agent is a
modulator of the IGFR pathway (e.g. it restores and/or upregulates
IGFR function) and thus is also an IGFR-modulating agent.
[0040] Preferred PFK-modulating agents include small molecule
compounds; PFK-interacting proteins, including antibodies and other
biotherapeutics; and nucleic acid modulators such as antisense and
RNA inhibitors. The modulating agents may be formulated in
pharmaceutical compositions, for example, as compositions that may
comprise other active ingredients, as in combination therapy,
and/or suitable carriers or excipients. Techniques for formulation
and administration of the compounds may be found in "Remington's
Pharmaceutical Sciences" Mack Publishing Co., Easton, Pa.,
19.sup.th edition.
[0041] Small Molecule Modulators
[0042] Small molecules are often preferred to modulate function of
proteins with enzymatic function, and/or containing protein
interaction domains. Chemical agents, referred to in the art as
"small molecule" compounds are typically organic, non-peptide
molecules, having a molecular weight up to 10,000, preferably up to
5,000, more preferably up to 1,000, and most preferably up to 500
daltons. This class of modulators includes chemically synthesized
molecules, for instance, compounds from combinatorial chemical
libraries. Synthetic compounds may be rationally designed or
identified based on known or inferred properties of the PFK protein
or may be identified by screening compound libraries. Alternative
appropriate modulators of this class are natural products,
particularly secondary metabolites from organisms such as plants or
fungi, which can also be identified by screening compound libraries
for PFK-modulating activity. Methods for generating and obtaining
compounds are well known in the art (Schreiber S L, Science (2000)
151: 1964-1969; Radmann J and Gunther J, Science (2000)
151:1947-1948).
[0043] Small molecule modulators identified from screening assays,
as described below, can be used as lead compounds from which
candidate clinical compounds may be designed, optimized, and
synthesized. Such clinical compounds may have utility in treating
pathologies associated with the IGFR pathway. The activity of
candidate small molecule modulating agents may be improved
several-fold through iterative secondary functional validation, as
further described below, structure determination, and candidate
modulator modification and testing. Additionally, candidate
clinical compounds are generated with specific regard to clinical
and pharmacological properties. For example, the reagents may be
derivatized and re-screened using in vitro and in vivo assays to
optimize activity and minimize toxicity for pharmaceutical
development.
[0044] Protein Modulators
[0045] Specific PFK-interacting proteins are useful in a variety of
diagnostic and therapeutic applications related to the IGFR pathway
and related disorders, as well as in validation assays for other
PFK-modulating agents. In a preferred embodiment, PFK-interacting
proteins affect normal PFK function, including transcription,
protein expression, protein localization, and cellular or
extra-cellular activity. In another embodiment, PFK-interacting
proteins are useful in detecting and providing information about
the function of PFK proteins, as is relevant to IGFR related,
disorders, such as cancer (e.g., for diagnostic means).
[0046] A PFK-interacting protein may be endogenous, i.e. one that
naturally interacts genetically or biochemically with a PFK, such
as a member of the PFK pathway that modulates PFK expression,
localization, and/or activity. PFK-modulators include dominant
negative forms of PFK-interacting proteins and of PFK proteins
themselves. Yeast two-hybrid and variant screens offer preferred
methods for identifying endogenous PFK-interacting proteins
(Finley, R. L. et al. (1996) in DNA Cloning-Expression Systems: A
Practical Approach, eds. Glover D. & Hames B. D (Oxford
University Press, Oxford, England), pp. 169-203; Fashema S F et
al., Gene (2000) 250:1-14; Drees B L Curr Opin Chem Biol (1999)
3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27:919-29;
and U.S. Pat. No. 5,928,868). Mass spectrometry is an alternative
preferred method for the elucidation of protein complexes (reviewed
in, e.g., Pandley A and Mann M, Nature (2000) 405:837-846; Yates J
R 3.sup.rd, Trends Genet (2000) 16:5-8).
[0047] An PFK-interacting protein may be an exogenous protein, such
as a PFK-specific antibody or a T-cell antigen receptor (see, e.g.,
Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring
Harbor Laboratory; Harlow and Lane (1999) Using antibodies: a
laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor
Laboratory Press). PFK antibodies are further discussed below.
[0048] In preferred embodiments, a PFK-interacting protein
specifically binds a PFK protein. In alternative preferred
embodiments, a PFK-modulating agent binds a PFK substrate, binding
partner, or cofactor.
[0049] Antibodies
[0050] In another embodiment, the protein modulator is a PFK
specific antibody agonist or antagonist. The antibodies have
therapeutic and diagnostic utilities, and can be used in screening
assays to identify PFK modulators. The antibodies can also be used
in dissecting the portions of the PFK pathway responsible for
various cellular responses and in the general processing and
maturation of the PFK.
[0051] Antibodies that specifically bind PFK polypeptides can be
generated using known methods. Preferably the antibody is specific
to a mammalian ortholog of PFK polypeptide, and more preferably, to
human PFK. Antibodies may be polyclonal, monoclonal (mAbs),
humanized or chimeric antibodies, single chain antibodies, Fab
fragments, F(ab').sub.2 fragments, fragments produced by a FAb
expression library, anti-idiotypic (anti-Id) antibodies, and
epitope-binding fragments of any of the above. Epitopes of PFK
which are particularly antigenic can be selected, for example, by
routine screening of PFK polypeptides for antigenicity or by
applying a theoretical method for selecting antigenic regions of a
protein (Hopp and Wood (1981), Proc. Natl. Acad. Sci. U.S.A.
78:3824-28; Hopp and Wood, (1983) Mol. Immunol. 20:483-89;
Sutcliffe et al., (1983) Science 219:660-66) to the amino acid
sequence of a PFK. Monoclonal antibodies with affinities of
10.sup.8 M.sup.-1 preferably 10.sup.9 M.sup.-1 to 10.sup.10
M.sup.-1, or stronger can be made by standard procedures as
described (Harlow and Lane, supra; Goding (1986) Monoclonal
Antibodies Principles and Practice (2d ed) Academic Press, New
York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and 4,618,577).
Antibodies may be generated against crude cell extracts of PFK or
substantially purified fragments thereof. If PFK fragments are
used, they preferably comprise at least 10, and more preferably, at
least 20 contiguous amino acids of a PFK protein. In a particular
embodiment, PFK-specific antigens and/or immunogens are coupled to
carrier proteins that stimulate the immune response. For example,
the subject polypeptides are covalently coupled to the keyhole
limpet hemocyanin (KLH) carrier, and the conjugate is emulsified in
Freund's complete adjuvant, which enhances the immune response. An
appropriate immune system such as a laboratory rabbit or mouse is
immunized according to conventional protocols.
[0052] The presence of PFK-specific antibodies is assayed by an
appropriate assay such as a solid phase enzyme-linked immunosorbant
assay (ELISA) using immobilized corresponding PFK polypeptides.
Other assays, such as radioimmunoassays or fluorescent assays might
also be used.
[0053] Chimeric antibodies specific to PFK polypeptides can be made
that contain different portions from different animal species. For
instance, a human immunoglobulin constant region may be linked to a
variable region of a murine mAb, such that the antibody derives its
biological activity from the human antibody, and its binding
specificity from the murine fragment. Chimeric antibodies are
produced by splicing together genes that encode the appropriate
regions from each species (Morrison et al., Proc. Natl. Acad. Sci.
(1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604-608;
Takeda et al., Nature (1985) 31:452-454). Humanized antibodies,
which are a form of chimeric antibodies, can be generated by
grafting complementary-determining regions (CDRs) (Carlos, T. M.,
J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies into a
background of human framework regions and constant regions by
recombinant DNA technology (Riechmann L M, et al., 1988 Nature 323:
323-327). Humanized antibodies contain .about.10% murine sequences
and .about.90% human sequences, and thus further reduce or
eliminate immunogenicity, while retaining the antibody
specificities (Co M S, and Queen C. 1991 Nature 351: 501-501;
Morrison S L. 1992 Ann. Rev. Immun. 10:239-265). Humanized
antibodies and methods of their production are well-known in the
art (U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and
6,180,370).
[0054] PFK-specific single chain antibodies which are recombinant,
single chain polypeptides formed by linking the heavy and light
chain fragments of the Fv regions via an amino acid bridge, can be
produced by methods known in the art (U.S. Pat. No. 4,946,778;
Bird, Science (1988) 242:423-426; Huston et al., Proc. Natl. Acad.
Sci. USA (1988) 85:5879-5883; and Ward et al., Nature (1989)
334:544-546).
[0055] Other suitable techniques for antibody production involve in
vitro exposure of lymphocytes to the antigenic polypeptides or
alternatively to selection of libraries of antibodies in phage or
similar vectors (Huse et al., Science (1989) 246:1275-1281). As
used herein, T-cell antigen receptors are included within the scope
of antibody modulators (Harlow and Lane, 1988, supra).
[0056] The polypeptides and antibodies of the present invention may
be used with or without modification. Frequently, antibodies will
be labeled by joining, either covalently or non-covalently, a
substance that provides for a detectable signal, or that is toxic
to cells that express the targeted protein (Menard S, et al., Int
J. Biol Markers (1989) 4:131-134). A wide variety of labels and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, fluorescent emitting lanthanide metals,
chemiluminescent moieties, bioluminescent moieties, magnetic
particles, and the like (U.S. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also,
recombinant immunoglobulins may be produced (U.S. Pat. No.
4,816,567). Antibodies to cytoplasmic polypeptides may be delivered
and reach their targets by conjugation with membrane-penetrating
toxin proteins (U.S. Pat. No. 6,086,900).
[0057] When used therapeutically in a patient, the antibodies of
the subject invention are typically administered parenterally, when
possible at the target site, or intravenously. The therapeutically
effective dose and dosage regimen is determined by clinical
studies. Typically, the amount of antibody administered is in the
range of about 0.1 mg/kg- to about 10 mg/kg of patient weight. For
parenteral administration, the antibodies are formulated in a unit
dosage injectable form (e.g., solution, suspension, emulsion) in
association with a pharmaceutically acceptable vehicle. Such
vehicles are inherently nontoxic and non-therapeutic. Examples are
water, saline, Ringer's solution, dextrose solution, and 5% human
serum albumin. Nonaqueous vehicles such as fixed oils, ethyl
oleate, or liposome carriers may also be used. The vehicle may
contain minor amounts of additives, such as buffers and
preservatives, which enhance isotonicity and chemical stability or
otherwise enhance therapeutic potential. The antibodies'
concentrations in such vehicles are typically in the range of about
1 mg/ml to about 10 mg/ml. Immunotherapeutic methods are further
described in the literature (U.S. Pat. No. 5,859,206;
WO0073469).
[0058] Nucleic Acid Modulators
[0059] Other preferred PFK-modulating agents comprise nucleic acid
molecules, such as antisense oligomers or double stranded RNA
(dsRNA), which generally inhibit PFK activity. Preferred nucleic
acid modulators interfere with the function of the PFK nucleic acid
such as DNA replication, transcription, translocation of the PFK
RNA to the site of protein translation, translation of protein from
the PFK RNA, splicing of the PFK RNA to yield one or more mRNA
species, or catalytic activity which may be engaged in or
facilitated by the PFK RNA.
[0060] In one embodiment, the antisense oligomer is an
oligonucleotide that is sufficiently complementary to a PFK mRNA to
bind to and prevent translation, preferably by binding to the 5'
untranslated region. PFK-specific antisense oligonucleotides,
preferably range from at least 6 to about 200 nucleotides. In some
embodiments the oligonucleotide is preferably at least 10, 15, or
20 nucleotides in length. In other embodiments, the oligonucleotide
is preferably less than 50, 40, or 30 nucleotides in length. The
oligonucleotide can be DNA or RNA or a chimeric mixture or
derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone. The oligonucleotide
may include other appending groups such as peptides, agents that
facilitate transport across the cell membrane,
hybridization-triggered cleavage agents, and intercalating
agents.
[0061] In another embodiment, the antisense oligomer is a
phosphothioate morpholino oligomer (PMO). PMOs are assembled from
four different morpholino subunits, each of which contain one of
four genetic bases (A, C, G, or T) linked to a six-membered
morpholine ring. Polymers of these subunits are joined by non-ionic
phosphodiamidate intersubunit linkages. Details of how to make and
use PMOs and other antisense oligomers are well known in the art
(e.g. see WO99/18193; Probst J C, Antisense Oligodeoxynucleotide
and Ribozyme Design, Methods. (2000) 22(3):271-281; Summerton J,
and Weller D. 1997 Antisense Nucleic Acid Drug Dev.: 7:187-95; U.S.
Pat. No. 5,235,033; and U.S. Pat. No. 5,378,841).
[0062] Alternative preferred PFK nucleic acid modulators are
double-stranded RNA species mediating RNA interference (RNAi). RNAi
is the process of sequence-specific, post-transcriptional gene
silencing in animals and plants, initiated by double-stranded RNA
(dsRNA) that is homologous in sequence to the silenced gene.
Methods relating to the use of RNAi to silence genes in C. elegans,
Drosophila, plants, and humans are known in the art (Fire A, et
al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363
(1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490
(2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119
(2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A.
et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature
404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000);
Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M.,
et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619;
Elbashir S M, et al., 2001 Nature 411:494-498; Novina C D and Sharp
P. 2004 Nature 430:161-164; Soutschek J et al 2004 Nature
432:173-178; Hsieh A C et al. (2004) NAR 32(3):893-901).
[0063] Nucleic acid modulators are commonly used as research
reagents, diagnostics, and therapeutics. For example, antisense
oligonucleotides, which are able to inhibit gene expression with
exquisite specificity, are often used to elucidate the function of
particular genes (see, for example, U.S. Pat. No. 6,165,790).
Nucleic acid modulators are also used, for example, to distinguish
between functions of various members of a biological pathway. For
example, antisense oligomers have been employed as therapeutic
moieties in the treatment of disease states in animals and man and
have been demonstrated in numerous clinical trials to be safe and
effective (Milligan J F, et al, Current Concepts in Antisense Drug
Design, J Med Chem. (1993) 36:1923-1937; Tonkinson J L et al.,
Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents,
Cancer Invest. (1996) 14:54-65). Accordingly, in one aspect of the
invention, a PFK-specific nucleic acid modulator is used in an
assay to further elucidate the role of the PFK in the IGFR pathway,
and/or its relationship to other members of the pathway. In another
aspect of the invention, a PFK-specific antisense oligomer is used
as a therapeutic agent for treatment of IGFR-related disease
states.
[0064] Assay Systems
[0065] The invention provides assay systems and screening methods
for identifying specific modulators of PFK activity. As used
herein, an "assay system" encompasses all the components required
for performing and analyzing results of an assay that detects
and/or measures a particular event. In general, primary assays are
used to identify or confirm a modulator's specific biochemical or
molecular effect with respect to the PFK nucleic acid or protein.
In general, secondary assays further assess the activity of a PFK
modulating agent identified by a primary assay and may confirm that
the modulating agent affects PFK in a manner relevant to the IGFR
pathway. In some cases, PFK modulators will be directly tested in a
secondary assay.
[0066] In a preferred embodiment, the screening method comprises
contacting a suitable assay system comprising a PFK polypeptide or
nucleic acid with a candidate agent under conditions whereby, but
for the presence of the agent, the system provides a reference
activity (e.g. kinase activity), which is based on the particular
molecular event the screening method detects. A statistically
significant difference between the agent-biased activity and the
reference activity indicates that the candidate agent modulates PFK
activity, and hence the IGFR pathway. The PFK polypeptide or
nucleic acid used in the assay may comprise any of the nucleic
acids or polypeptides described above.
[0067] Primary Assays
[0068] The type of modulator tested generally determines the type
of primary assay.
[0069] Primary Assays for Small Molecule Modulators
[0070] For small molecule modulators, screening assays are used to
identify candidate modulators. Screening assays may be cell-based
or may use a cell-free system that recreates or retains the
relevant biochemical reaction of the target protein (reviewed in
Sittampalam G S et al., Curr Opin Chem Biol (1997) 1:384-91 and
accompanying references). As used herein the term "cell-based"
refers to assays using live cells, dead cells, or a particular
cellular fraction, such as a membrane, endoplasmic reticulum, or
mitochondrial fraction. The term "cell free" encompasses assays
using substantially purified protein (either endogenous or
recombinantly produced), partially purified or crude cellular
extracts. Screening assays may detect a variety of molecular
events, including protein-DNA interactions, protein-protein
interactions (e.g., receptor-ligand binding), transcriptional
activity (e.g., using a reporter gene), enzymatic activity (e.g.,
via a property of the substrate), activity of second messengers,
immunogenicity and changes in cellular morphology or other cellular
characteristics. Appropriate screening assays may use a wide range
of detection methods including fluorescent, radioactive,
colorimetric, spectrophotometric, and amperometric methods, to
provide a read-out for the particular molecular event detected.
[0071] Cell-based screening assays usually require systems for
recombinant expression of PFK and any auxiliary proteins demanded
by the particular assay. Appropriate methods for generating
recombinant proteins produce sufficient quantities of proteins that
retain their relevant biological activities and are of sufficient
purity to optimize activity and assure assay reproducibility. Yeast
two-hybrid and variant screens, and mass spectrometry provide
preferred methods for determining protein-protein interactions and
elucidation of protein complexes. In certain applications, when
PFK-interacting proteins are used in screens to identify small
molecule modulators, the binding specificity of the interacting
protein to the PFK protein may be assayed by various known methods
such as substrate processing (e.g. ability of the candidate
PFK-specific binding agents to function as negative effectors in
PFK-expressing cells), binding equilibrium constants (usually at
least about 10.sup.7M.sup.-1, preferably at least about 10.sup.8
M.sup.-1, more preferably at least about 10.sup.9 M.sup.-1), and
immunogenicity (e.g. ability to elicit PFK specific antibody in a
heterologous host such as a mouse, rat, goat or rabbit). For
enzymes and receptors, binding may be assayed by, respectively,
substrate and ligand processing.
[0072] The screening assay may measure a candidate agent's ability
to specifically bind to or modulate activity of a PFK polypeptide,
a fusion protein thereof, or to cells or membranes bearing the
polypeptide or fusion protein. The PFK polypeptide can be full
length or a fragment thereof that retains functional PFK activity.
The PFK polypeptide may be fused to another polypeptide, such as a
peptide tag for detection or anchoring, or to another tag. The PFK
polypeptide is preferably human PFK, or is an ortholog or
derivative thereof as described above. In a preferred embodiment,
the screening assay detects candidate agent-based modulation of PFK
interaction with a binding target, such as an endogenous or
exogenous protein or other substrate that has PFK-specific binding
activity, and can be used to assess normal PFK gene function.
[0073] Suitable assay formats that may be adapted to screen for PFK
modulators are known in the art. Preferred screening assays are
high throughput or ultra high throughput and thus provide
automated, cost-effective means of screening compound libraries for
lead compounds (Fernandes P B, Curr Opin Chem Biol (1998)
2:597-603; Sundberg S A, Curr Opin Biotechnol 2000, 11:47-53). In
one preferred embodiment, screening assays uses fluorescence
technologies, including fluorescence polarization, time-resolved
fluorescence, and fluorescence resonance energy transfer. These
systems offer means to monitor protein-protein or DNA-protein
interactions in which the intensity of the signal emitted from
dye-labeled molecules depends upon their interactions with partner
molecules (e.g., Selvin P R, Nat Struct Biol (2000) 7:730-4;
Fernandes P B, supra; Hertzberg R P and Pope A J, Curr Opin Chem
Biol (2000) 4:445-451).
[0074] A variety of suitable assay systems may be used to identify
candidate PFK and IGFR pathway modulators (e.g. U.S. Pat. No.
6,165,992 and U.S. Pat. No. 6,720,162 (kinase assays); U.S. Pat.
Nos. 5,550,019 and 6,133,437 (apoptosis assays); and U.S. Pat. Nos.
5,976,782, 6,225,118 and 6,444,434 (angiogenesis assays), among
others). Specific preferred assays are described in more detail
below.
[0075] Kinases, key signal transduction proteins that may be either
membrane-associated or intracellular; catalyze the transfer of
gamma phosphate from adenosine triphosphate (ATP) to a serine,
threonine or tyrosine residue in a protein substrate. Radioassays,
which monitor the transfer from [gamma-.sup.32P or -.sup.33]ATP,
are frequently used to assay kinase activity. For instance, a
scintillation assay for p56 (lck) kinase activity monitors the
transfer of the gamma phosphate from [gamma-.sup.33P] ATP to a
biotinylated peptide substrate. The substrate is captured on a
streptavidin coated bead that transmits the signal (Beveridge M et
al., J Biomol Screen (2000) 5:205-212). This assay uses the
scintillation proximity assay (SPA), in which only radio-ligand
bound to receptors tethered to the surface of an SPA bead are
detected by the scintillant immobilized within it, allowing binding
to be measured without separation of bound from free ligand. Other
assays for protein kinase activity may use antibodies that
specifically recognize phosphorylated substrates. For instance, the
kinase receptor activation (KIRA) assay measures receptor tyrosine
kinase activity by ligand stimulating the intact receptor in
cultured cells, then capturing solubilized receptor with specific
antibodies and quantifying phosphorylation via phosphotyrosine
ELISA (Sadick M D, Dev Biol Stand (1999) 97:121-133). Another
example of antibody based assays for protein kinase activity is TRF
(time-resolved fluorometry). This method utilizes europium
chelate-labeled anti-phosphotyrosine antibodies to detect phosphate
transfer to a polymeric substrate coated onto microtiter plate
wells. The amount of phosphorylation is then detected using
time-resolved, dissociation-enhanced fluorescence (Braunwalder A F,
et al., Anal Biochem 1996 Jul. 1; 238(2):159-64). Yet other assays
for kinases involve uncoupled, pH sensitive assays that can be used
for high-throughput screening of potential inhibitors or for
determining substrate specificity. Since kinases catalyze the
transfer of a gamma-phosphoryl group from ATP to an appropriate
hydroxyl acceptor with the release of a proton, a pH sensitive
assay is based on the detection of this proton using an
appropriately matched buffer/indicator system (Chapman E and Wong C
H (2.002) Bioorg Med Chem. 10:551-5).
[0076] Apoptosis Assays.
[0077] Apoptosis or programmed cell death is a suicide program is
activated within the cell, leading to fragmentation of DNA,
shrinkage of the cytoplasm, membrane changes and cell death.
Apoptosis is mediated by proteolytic enzymes of the caspase family.
Many of the altering parameters of a cell are measurable during
apoptosis. Assays for apoptosis may be performed by terminal
deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end
labeling (TUNEL) assay. The TUNEL assay is used to measure nuclear
DNA fragmentation characteristic of apoptosis (Lazebnik et al.,
1994, Nature 371, 346), by following the incorporation of
fluorescein-dUTP (Yonehara et al., 1989, J. Exp. Med. 169, 1747).
Apoptosis may further be assayed by acridine orange staining of
tissue culture cells (Lucas, R., et al., 1998, Blood 15:4730-41).
Other cell-based apoptosis assays include the caspase-3/7 assay and
the cell death nucleosome ELISA assay. The caspase 3/7 assay is
based on the activation of the caspase cleavage activity as part of
a cascade of events that occur during programmed cell death in many
apoptotic pathways. In the caspase 3/7 assay (commercially
available Apo-ONE.TM. Homogeneous Caspase-3/7 assay from Promega,
cat#67790), lysis buffer and caspase substrate are mixed and added
to cells. The caspase substrate becomes fluorescent when cleaved by
active caspase 3/7. The nucleosome ELISA assay is a general cell
death assay known to those skilled in the art, and available
commercially (Roche, Cat#1774425). This assay is a quantitative
sandwich-enzyme-immunoassay which uses monoclonal antibodies
directed against DNA and histones respectively, thus specifically
determining amount of mono- and oligonucleosomes in the cytoplasmic
fraction of cell lysates. Mono and oligonucleosomes are enriched in
the cytoplasm during apoptosis due to the fact that DNA
fragmentation occurs several hours before the plasma membrane
breaks down, allowing for accumulation in the cytoplasm.
Nucleosomes are not present in the cytoplasmic fraction of cells
that are not undergoing apoptosis. The Phospho-histone H2B assay is
another apoptosis assay, based on phosphorylation of histone H2B as
a result of apoptosis. Fluorescent dyes that are associated with
phosphohistone H2B may be used to measure the increase of
phosphohistone H2B as a result of apoptosis. Apoptosis assays that
simultaneously measure multiple parameters associated with
apoptosis have also been developed. In such assays, various
cellular parameters that can be associated with antibodies or
fluorescent dyes, and that mark various stages of apoptosis are
labeled, and the results are measured using instruments such as
Cellomics.TM. ArrayScan.RTM. HCS System. The measurable parameters
and their markers include anti-active caspase-3 antibody which
marks intermediate stage apoptosis, anti-PARP-p85 antibody (cleaved
PARP) which marks late stage apoptosis, Hoechst labels which label
the nucleus and are used to measure nuclear swelling as a measure
of early apoptosis and nuclear condensation as a measure of late
apoptosis, TOTO-3 fluorescent dye which labels DNA of dead cells
with high cell membrane permeability, and anti-alpha-tubulin or
F-actin labels, which assess cytoskeletal changes in cells and
correlate well with TOTO-3 label.
[0078] An apoptosis assay system may comprise a cell that expresses
a PFK, and that optionally has defective IGFR function (e.g. IGFR
is over-expressed or under-expressed relative to wild-type cells).
A test agent can be added to the apoptosis assay system and changes
in induction of apoptosis relative to controls where no test agent
is added, identify candidate IGFR modulating agents. In some
embodiments of the invention, an apoptosis assay may be used as a
secondary assay to test a candidate IGFR modulating agents that is
initially identified using a cell-free assay system. An apoptosis
assay may also be used to test whether PFK function plays a direct
role in apoptosis. For example, an apoptosis assay may be performed
on cells that over- or under-express PFK relative to wild type
cells. Differences in apoptotic response compared to wild type
cells suggests that the PFK plays a direct role in the apoptotic
response. Apoptosis assays are described further in U.S. Pat. No.
6,133,437.
[0079] Cell Proliferation and Cell Cycle Assays.
[0080] Cell proliferation may be assayed via bromodeoxyuridine
(BRDU) incorporation. This assay identifies a cell population
undergoing DNA synthesis by incorporation of BRDU into
newly-synthesized DNA. Newly-synthesized DNA may then be detected
using an anti-BRDU antibody (Hoshino et al., 1986, Int. J. Cancer
38, 369; Campana et al., 1988, J. Immunol. Meth. 107, 79), or by
other means.
[0081] Cell proliferation is also assayed via phospho-histone H3
staining, which identifies a cell population undergoing mitosis by
phosphorylation of histone H3. Phosphorylation of histone H3 at
serine 10 is detected using an antibody specific to the
phosphorylated form of the serine 10 residue of histone H3.
(Chadlee, D. N. 1995, J. Biol. Chem 270:20098-105). Cell
Proliferation may also be examined using [.sup.3H]-thymidine
incorporation (Chen, J., 1996, Oncogene 13:1395-403; Jeoung, J.,
1995, J. Biol. Chem. 270:18367-73). This assay allows for
quantitative characterization of S-phase DNA syntheses. In this
assay, cells synthesizing DNA will incorporate [.sup.3H]-thymidine
into newly synthesized DNA. Incorporation can then be measured by
standard techniques such as by counting of radioisotope in a
scintillation counter (e.g., Beckman LS 3800 Liquid Scintillation
Counter). Another proliferation assay uses the dye Alamar Blue
(available from Biosource International), which fluoresces when
reduced in living cells and provides an indirect measurement of
cell number (Voytik-Harbin S L et al., 1998, In Vitro Cell Dev Biol
Anim 34:239-46). Yet another proliferation assay, the MTS assay, is
based on in vitro cytotoxicity assessment of industrial chemicals,
and uses the soluble tetrazolium salt, MTS. MTS assays are
commercially available, for example, the Promega CellTiter 96.RTM.
AQueous Non-Radioactive Cell Proliferation Assay (Cat. #G5421).
[0082] Cell proliferation may also be assayed by colony formation
in soft agar, or clonogenic survival assay (Sambrook et al.,
Molecular Cloning, Cold Spring Harbor (1989)). For example, cells
transformed with PFK are seeded in soft agar plates, and colonies
are measured and counted after two weeks incubation.
[0083] Cell proliferation may also be assayed by measuring ATP
levels as indicator of metabolically active cells. Such assays are
commercially available, for example Cell Titer-Glo.TM., which is a
luminescent homogeneous assay available from Promega.
[0084] Involvement of a gene in the cell cycle may be assayed by
flow cytometry (Gray J W et al. (1986) Int J Radiat Biol Relat Stud
Phys Chem Med 49:237-55). Cells transfected with a PFK may be
stained with propidium iodide and evaluated in a flow cytometer
(available from Becton Dickinson), which indicates accumulation of
cells in different stages of the cell cycle.
[0085] Involvement of a gene in cell cycle may also be assayed by
FOXO nuclear translocation assays. The FOXO family of transcription
factors are mediators of various cellular functions including cell
cycle progression and cell death, and are negatively regulated by
activation of the PI3 kinase pathway. Akt phosphorylation of FOXO
family members leads to FOXO sequestration in the cytoplasm and
transcriptional inactivation (Medema, R. H et al (2000) Nature 404:
782-787). PTEN is a negative regulator of PI3 kinase pathway.
Activation of PTEN, or loss of PI3 kinase or AKT, prevents
phosphorylation of FOXO, leading to accumulation of FOXO in the
nucleus, transcriptional activation of FOXO regulated genes, and
apoptosis. Alternatively, loss of PTEN leads to pathway activation
and cell survival (Nakamura, N. et al (2000) Mol Cell Biol 20:
8969-8982). FOXO translocation into the cytoplasm is used in assays
and screens to identify members and/or modulators of the PTEN
pathway. FOXO translocation assays using GFP or luciferase as
detection reagents are known in the art (e.g., Zhang X et al (2002)
J Biol Chem 277:45276-45284; and Li et al (2003) Mol Cell Biol
23:104-118).
[0086] Accordingly, a cell proliferation or cell cycle assay system
may comprise a cell that expresses a PFK, and that optionally has
defective IGFR function (e.g. IGFR is over-expressed or
under-expressed relative to wild-type cells). A test agent can be
added to the assay system and changes in cell proliferation or cell
cycle relative to controls where no test agent is added, identify
candidate IGFR modulating agents. In some embodiments of the
invention, the cell proliferation or cell cycle assay may be used
as a secondary assay to test a candidate IGFR modulating agents
that is initially identified using another assay system such as a
cell-free assay system. A cell proliferation assay may also be used
to test whether PFK function plays a direct role in cell
proliferation or cell cycle. For example, a cell proliferation or
cell cycle assay may be performed on cells that over- or
under-express PFK relative to wild type cells. Differences in
proliferation or cell cycle compared to wild type cells suggests
that the PFK plays a direct role in cell proliferation or cell
cycle.
[0087] Angiogenesis.
[0088] Angiogenesis may be assayed using various human endothelial
cell systems, such as umbilical vein, coronary artery, or dermal
cells. Suitable assays include Alamar Blue based assays (available
from Biosource International) to measure proliferation; migration
assays using fluorescent molecules, such as the use of Becton
Dickinson Falcon HTS FluoroBlock cell culture inserts to measure
migration of cells through membranes in presence or absence of
angiogenesis enhancer or suppressors; and tubule formation assays
based on the formation of tubular structures by endothelial cells
on Matrigel.RTM. (Becton Dickinson). Accordingly, an angiogenesis
assay system may comprise a cell that expresses a PFK, and that
optionally has defective IGFR function (e.g. IGFR is over-expressed
or under-expressed relative to wild-type cells). A test agent can
be added to the angiogenesis assay system and changes in
angiogenesis relative to controls where no test agent is added,
identify candidate IGFR modulating agents. In some embodiments of
the invention, the angiogenesis assay may be used as a secondary
assay to test a candidate IGFR modulating agents that is initially
identified using another assay system. An angiogenesis assay may
also be used to test whether PFK function plays a direct role in
cell proliferation. For example, an angiogenesis assay may be
performed on cells that over- or under-express PFK relative to wild
type cells. Differences in angiogenesis compared to wild type cells
suggests that the PFK plays a direct role in angiogenesis. U.S.
Pat. Nos. 5,976,782, 6,225,118 and 6,444,434, among others,
describe various angiogenesis assays.
[0089] Hypoxic Induction.
[0090] The alpha subunit of the transcription factor, hypoxia
inducible factor-1 (HIF-1), is upregulated in tumor cells following
exposure to hypoxia in vitro. Under hypoxic conditions, HIF-1
stimulates the expression of genes known to be important in tumour
cell survival, such as those encoding glyolytic enzymes and VEGF.
Induction of such genes by hypoxic conditions may be assayed by
growing cells transfected with PFK in hypoxic conditions (such as
with 0.1% O2, 5% CO2, and balance N2, generated in a Napco 7001
incubator (Precision Scientific)) and normoxic conditions, followed
by assessment of gene activity or expression by Taqman.RTM.. For
example, a hypoxic induction assay system may comprise a cell that
expresses a PFK, and that optionally has defective IGFR function
(e.g. IGFR is over-expressed or under-expressed relative to
wild-type cells). A test agent can be added to the hypoxic
induction assay system and changes in hypoxic response relative to
controls where no test agent is added, identify candidate IGFR
modulating agents. In some embodiments of the invention, the
hypoxic induction assay may be used as a secondary assay to test a
candidate IGFR modulating agents that is initially identified using
another assay system. A hypoxic induction assay may also be used to
test whether PFK function plays a direct role in the hypoxic
response. For example, a hypoxic induction assay may be performed
on cells that over- or under-express PFK relative to wild type
cells. Differences in hypoxic response compared to wild type cells
suggests that the PFK plays a direct role in hypoxic induction.
[0091] Cell Adhesion.
[0092] Cell adhesion assays measure adhesion of cells to purified
adhesion proteins, or adhesion of cells to each other, in presence
or absence of candidate modulating agents. Cell-protein adhesion
assays measure the ability of agents to modulate the adhesion of
cells to purified proteins. For example, recombinant proteins are
produced, diluted to 2.5 g/mL in PBS, and used to coat the wells of
a microtiter plate. The wells used for negative control are not
coated. Coated wells are then washed, blocked with 1% BSA, and
washed again. Compounds are diluted to 2.times. final test
concentration and added to the blocked, coated wells. Cells are
then added to the wells, and the unbound cells are washed off.
Retained cells are labeled directly on the plate by adding a
membrane-permeable fluorescent dye, such as calcein-AM, and the
signal is quantified in a fluorescent microplate reader.
[0093] Cell-cell adhesion assays measure the ability of agents to
modulate binding of cell adhesion proteins with their native
ligands. These assays use cells that naturally or recombinantly
express the adhesion protein of choice. In an exemplary assay,
cells expressing the cell adhesion protein are plated in wells of a
multiwell plate. Cells expressing the ligand are labeled with a
membrane-permeable fluorescent dye, such as BCECF, and allowed to
adhere to the monolayers in the presence of candidate agents.
Unbound cells are washed off, and bound cells are detected using a
fluorescence plate reader.
[0094] High-throughput cell adhesion assays have also been
described. In one such assay, small molecule ligands and peptides
are bound to the surface of microscope slides using a microarray
spotter, intact cells are then contacted with the slides, and
unbound cells are washed off. In this assay, not only the binding
specificity of the peptides and modulators against cell lines are
determined, but also the functional cell signaling of attached
cells using immunofluorescence techniques in situ on the microchip
is measured (Falsey J R et al., Bioconjug Chem. 2001 May-June;
12(3):346-53).
[0095] Primary Assays for Antibody Modulators
[0096] For antibody modulators, appropriate primary assays test is
a binding assay that tests the antibody's affinity to and
specificity for the PFK protein. Methods for testing antibody
affinity and specificity are well known in the art (Harlow and
Lane, 1988, 1999, supra). The enzyme-linked immunosorbant assay
(ELISA) is a preferred method for detecting PFK-specific
antibodies; others include FACS assays, radioimmunoassays, and
fluorescent assays.
[0097] In some cases, screening assays described for small molecule
modulators may also be used to test antibody modulators.
[0098] Primary Assays for Nucleic Acid Modulators
[0099] For nucleic acid modulators, primary assays may test the
ability of the nucleic acid modulator to inhibit or enhance PFK
gene expression, preferably mRNA expression. In general, expression
analysis comprises comparing PFK expression in like populations of
cells (e.g., two pools of cells that endogenously or recombinantly
express PFK) in the presence and absence of the nucleic, acid
modulator. Methods for analyzing mRNA and protein expression are
well known in the art. For instance, Northern blotting, slot
blotting, ribonuclease protection, quantitative RT-PCR (e.g., using
the TaqMan.RTM., PE Applied Biosystems), or microarray analysis may
be used to confirm that PFK mRNA expression is reduced in cells
treated with the nucleic acid modulator (e.g., Current Protocols in
Molecular Biology (1994) Ausubel F M et al., eds., John Wiley &
Sons, Inc., chapter 4; Freeman W M et al., Biotechniques (1999)
26:112-125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm D H
and Guiseppi-Elie, A Curr Opin Biotechnol 2001, 12:41-47). Protein
expression may also be monitored. Proteins are most commonly
detected with specific antibodies or antisera directed against
either the PFK protein or specific peptides. A variety of means
including Western blotting, ELISA, or in situ detection, are
available (Harlow E and Lane D, 1988 and 1999, supra).
[0100] In some cases, screening assays described for small molecule
modulators, particularly in assay systems that involve PFK mRNA
expression, may also be used to test nucleic acid modulators.
[0101] Secondary Assays
[0102] Secondary assays may be used to further assess the activity
of PFK-modulating agent identified by any of the above methods to
confirm that the modulating agent affects PFK in a manner relevant
to the IGFR pathway. As used herein, PFK-modulating agents
encompass candidate clinical compounds or other agents derived from
previously identified modulating agent. Secondary assays can also
be used to test the activity of a modulating agent on a particular
genetic or biochemical pathway or to test the specificity of the
modulating agent's interaction with PFK.
[0103] Secondary assays generally compare like populations of cells
or animals (e.g., two pools of cells or animals that endogenously
or recombinantly express PFK) in the presence and absence of the
candidate modulator. In general, such assays test whether treatment
of cells or animals with a candidate PFK-modulating agent results
in changes in the IGFR pathway in comparison to untreated (or mock-
or placebo-treated) cells or animals. Certain assays use
"sensitized genetic backgrounds", which, as used herein, describe
cells or animals engineered for altered expression of genes in the
IGFR or interacting pathways.
Cell-Based Assays
[0104] Cell based assays may detect endogenous IGFR pathway
activity or may rely on recombinant expression of IGFR pathway
components. Any of the aforementioned assays may be used in this
cell-based format. Candidate modulators are typically added to the
cell media but may also be injected into cells or delivered by any
other efficacious means.
Animal Assays
[0105] A variety of non-human animal models of normal or defective
IGFR pathway may be used to test candidate PFK modulators. Models
for defective IGFR pathway typically use genetically modified
animals that have been engineered to mis-express (e.g.,
over-express or lack expression in) genes involved in the IGFR
pathway. Assays generally require systemic delivery of the
candidate modulators, such as by oral administration, injection,
etc.
[0106] In a preferred embodiment, IGFR pathway activity is assessed
by monitoring neovascularization and angiogenesis. Animal models
with defective and normal IGFR are used to test the candidate
modulator's affect on PFK in Matrigel.RTM. assays. Matrigel.RTM. is
an extract of basement membrane proteins, and is composed primarily
of laminin, collagen IV, and heparin sulfate proteoglycan. It is
provided as a sterile liquid at 4.degree. C., but rapidly forms a
solid gel at 37.degree. C. Liquid Matrigel.RTM. is mixed with
various angiogenic agents, such as bFGF and VEGF, or with human
tumor cells which over-express the PFK. The mixture is then
injected subcutaneously (SC) into female athymic nude mice
(Taconic, Germantown, N.Y.) to support an intense vascular
response. Mice with Matrigel.RTM. pellets may be dosed via oral
(PO), intraperitoneal (IP), or intravenous (IV) routes with the
candidate modulator. Mice are euthanized 5-12 days post-injection,
and the Matrigel.RTM. pellet is harvested for hemoglobin analysis
(Sigma plasma hemoglobin kit). Hemoglobin content of the gel is
found to correlate the degree of neovascularization in the gel.
[0107] In another preferred embodiment, the effect of the candidate
modulator on PFK is assessed via tumorigenicity assays. Tumor
xenograft assays are known in the art (see, e.g., Ogawa K et al.,
2000, Oncogene 19:6043-6052). Xenografts are typically implanted SC
into female athymic mice, 6-7 week old, as single cell suspensions
either from a pre-existing tumor or from in vitro culture. The
tumors which express the PFK endogenously are injected in the
flank, 1.times.10.sup.5 to 1.times.10.sup.7 cells per mouse in a
volume of 100 .mu.L using a 27 gauge needle. Mice are then ear
tagged and tumors are measured twice weekly. Candidate modulator
treatment is initiated on the day the mean tumor weight reaches 100
mg. Candidate modulator is delivered IV, SC, IP, or PO by bolus
administration. Depending upon the pharmacokinetics of each unique
candidate modulator, dosing can be performed multiple times per
day. The tumor weight is assessed by measuring perpendicular
diameters with a caliper and calculated by multiplying the
measurements of diameters in two dimensions. At the end of the
experiment, the excised tumors maybe utilized for biomarker
identification or further analyses. For immunohistochemistry
staining, xenograft tumors are fixed in 4% paraformaldehyde, 0.1 M
phosphate, pH 7.2, for 6 hours at 4.degree. C., immersed in 30%
sucrose in PBS, and rapidly frozen in isopentane cooled with liquid
nitrogen.
[0108] In another preferred embodiment, tumorogenicity is monitored
using a hollow fiber assay, which is described in U.S. Pat. No.
5,698,413. Briefly, the method comprises implanting into a
laboratory animal a biocompatible, semi-permeable encapsulation
device containing target cells, treating the laboratory animal with
a candidate modulating agent, and evaluating the target cells for
reaction to the candidate modulator. Implanted cells are generally
human cells from a pre-existing tumor or a tumor cell line. After
an appropriate period of time, generally around six days, the
implanted samples are harvested for evaluation of the candidate
modulator. Tumorogenicity and modulator efficacy may be evaluated
by assaying the quantity of viable cells present in the
macrocapsule, which can be determined by tests known in the art,
for example, MTT dye conversion assay, neutral red dye uptake,
trypan blue staining, viable cell counts, the number of colonies
formed in soft agar, the capacity of the cells to recover and
replicate in vitro, etc.
[0109] In another preferred embodiment, a tumorogenicity assay use
a transgenic animal, usually a mouse, carrying a dominant oncogene
or tumor suppressor gene knockout under the control of tissue
specific regulatory sequences; these assays are generally referred
to as transgenic tumor assays. In a preferred application, tumor
development in the transgenic model is well characterized or is
controlled. In an exemplary model, the "RIP1-Tag2" transgene,
comprising the SV40 large T-antigen oncogene under control of the
insulin gene regulatory regions is expressed in pancreatic beta
cells and results in islet cell carcinomas (Hanahan D, 1985, Nature
315:115-122; Parangi S et al, 1996, Proc Natl Acad Sci USA 93:
2002-2007; Bergers G et al, 1999, Science 284:808-812). An
"angiogenic switch," occurs at approximately five weeks, as
normally quiescent capillaries in a subset of hyperproliferative
islets become angiogenic. The RIP1-TAG2 mice die by age 14 weeks.
Candidate modulators may be administered at a variety of stages,
including just prior to the angiogenic switch (e.g., for a model of
tumor prevention), during the growth of small tumors (e.g., for a
model of intervention), or during the growth of large and/or
invasive tumors (e.g., for a model of regression). Tumorogenicity
and modulator efficacy can be evaluating life-span extension and/or
tumor characteristics, including number of tumors, tumor size,
tumor morphology, vessel density, apoptotic index, etc.
Diagnostic and Therapeutic Uses
[0110] Specific PFK-modulating agents are useful in a variety of
diagnostic and therapeutic applications where disease or disease
prognosis is related to defects in the IGFR pathway, such as
angiogenic, apoptotic, or cell proliferation disorders.
Accordingly, the invention also provides methods for modulating the
IGFR pathway in a cell, preferably a cell pre-determined to have
defective or impaired IGFR function (e.g. due to overexpression,
underexpression, or misexpression of IGFR, or due to gene
mutations), comprising the step of administering an agent to the
cell that specifically modulates PFK activity. Preferably, the
modulating agent produces a detectable phenotypic change in the
cell indicating that the IGFR function is restored. The phrase
"function is restored", and equivalents, as used herein, means that
the desired phenotype is achieved, or is brought closer to normal
compared to untreated cells. For example, with restored IGFR
function, cell proliferation and/or progression through cell cycle
may normalize, or be brought closer to normal relative to untreated
cells. The invention also provides methods for treating disorders
or disease associated with impaired IGFR function by administering
a therapeutically effective amount of a PFK-modulating agent that
modulates the IGFR pathway. The invention further provides methods
for modulating PFK function in a cell, preferably a cell
pre-determined to have defective or impaired PFK function, by
administering a PFK-modulating agent. Additionally, the invention
provides a method for treating disorders or disease associated with
impaired PFK function by administering a therapeutically effective
amount of a PFK-modulating agent.
[0111] The discovery that PFK is implicated in IGFR pathway
provides for a variety of methods that can be employed for the
diagnostic and prognostic evaluation of diseases and disorders
involving defects in the IGFR pathway and for the identification of
subjects having a predisposition to such diseases and
disorders.
[0112] Various expression analysis methods can be used to diagnose
whether PFK expression occurs in a particular sample, including
Northern blotting, slot blotting, ribonuclease protection,
quantitative RT-PCR, and microarray analysis. (e.g., Current
Protocols in Molecular Biology (1994) Ausubel F M et al., eds.,
John Wiley & Sons, Inc., chapter 4; Freeman W M et al.,
Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001,
33:142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol 2001,
12:41-47). Tissues having a disease or disorder implicating
defective IGFR signaling that express a PFK, are identified as
amenable to treatment with a PFK modulating agent. In a preferred
application, the IGFR defective tissue overexpresses a PFK relative
to normal tissue. For example, a Northern blot analysis of mRNA
from tumor and normal cell lines, or from tumor and matching normal
tissue samples from the same patient, using full or partial PFK
cDNA sequences as probes, can determine whether particular tumors
express or overexpress PFK. Alternatively, the TaqMan.RTM. is used
for quantitative RT-PCR analysis of PFK expression in cell lines,
normal tissues and tumor samples (PE Applied Biosystems).
[0113] Various other diagnostic methods may be performed, for
example, utilizing reagents such as the PFK oligonucleotides, and
antibodies directed against a PFK, as described above for: (1) the
detection of the presence of PFK gene mutations, or the detection
of either over- or under-expression of PFK mRNA relative to the
non-disorder state; (2) the detection of either an over or an
under-abundance of PFK gene product relative to the non-disorder
state; and (3) the detection of perturbations or abnormalities in
the signal transduction pathway mediated by PFK.
[0114] Kits for detecting expression of PFK in various samples,
comprising at least one antibody specific to PFK, all reagents
and/or devices suitable for the detection of antibodies, the
immobilization of antibodies, and the like, and instructions for
using such kits in diagnosis or therapy are also provided.
[0115] Thus, in a specific embodiment, the invention is drawn to a
method for diagnosing a disease or disorder in a patient that is
associated with alterations in PFK expression, the method
comprising: a) obtaining a biological sample from the patient; b)
contacting the sample with a probe for PFK expression; c) comparing
results from step (b) with a control; and d) determining whether
step (c) indicates a likelihood of the disease or disorder.
Preferably, the disease is cancer, most preferably a cancer as
shown in TABLE 1. The probe may be either DNA or protein, including
an antibody.
EXAMPLES
[0116] The following experimental section and examples are offered
by way of illustration and not by way of limitation.
I. Drosophila IGFR Screen
[0117] A dominant loss of function screen was carried out in
Drosophila to identify, genes that interact with or modulate the
IGFR signaling pathway. Activation of the pathway by overexpression
of IGFR at early stages in the developing Drosophila eye leads to
an increase in cell number which results in a larger and rougher
adult eye (Potter C J et al. (2001) Cell 105:357-368; Huang et al.,
1999. Dev. 126:5365-5372). We generated a fly stock with an
enlarged eye due to overexpression of IGFR and identified modifiers
of this phenotype. We then identified human orthologues of these
modifiers.
[0118] The screening stock carried two transgenes. The genotype is
as follows:
[0119] +; +; P {DmIGFR-pExp-UAS)} P {Gal4-pExp-1Xey}/TM6B
[0120] Screening stock females of the above genotype were crossed
to males from a collection of 3 classes of piggyBac-based
transposons. The resulting progeny, which contain both the
transgenes and the transposon, were scored for the effect of the
transposon on the eye overgrowth phenotype (either enhancement,
suppression or no effect). All data was recorded and all modifiers
were retested with a repeat of the original cross. Modifiers of the
eye phenotype were identified as members of the IGFR pathway.
DROSOPHILA PFK was a suppressor of the eye phenotype. Orthologs of
the modifiers are referred to herein as PFK.
[0121] BLAST analysis (Altschul et al., supra) was employed to
identify orthologs of Drosophila modifiers. For example,
representative sequences from PFK, GI#s 21361070 (SEQ ID NO:64),
4505749 (SEQ ID NO:65), and 11321601 (SEQ ID NO:66) share 55%, 58%,
and 57% amino acid identity, respectively, with the Drosophila
PFK.
[0122] Various domains, signals, and functional subunits in
proteins were analyzed using the PSORT (Nakai K., and Horton P.,
Trends Biochem Sci, 1999, 24:34-6; Kenta Nakai, Protein sorting
signals and prediction of subcellular localization, Adv. Protein
Chem. 54, 277-344 (2000)), PFAM (Bateman A., et al., Nucleic Acids
Res, 1999, 27:260-2), SMART (Ponting C P, et al., SMART:
identification and annotation of domains from signaling and
extracellular protein sequences. Nucleic Acids Res. 1999 Jan. 1;
27(1):229-32), TM-HMM (Erik L. L. Sonnhammer, Gunnar von Heijne,
and Anders Krogh: A hidden Markov model for predicting
transmembrane helices in protein sequences. In Proc. of Sixth Int.
Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J.
Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C.
Sensen Menlo Park, CA: AAAI Press, 1998), and clust (Remm M, and
Sonnhammer E. Classification of transmembrane protein families in
the Caenorhabditis elegans genome and identification of human
orthologs. Genome Res. 2000 November; 10(11):1679-89) programs. For
example, the Phosphofructokinase domain (PFAM 00365) of PFK from
GI#21361070 (SEQ ID NO:64) is located at approximately amino acid
residues 76 to 373, 448 to 735; the Phosphofructokinase domain of
PFK from GI#4505749 (SEQ ID NO:65) is located at approximately
amino acid residues 16 to 326, 401 to 689; and the
Phosphofructokinase domain of PFK from GI#11321601 (SEQ ID NO:66)
is located at approximately amino acid residues 25 to 335, 412 to
699.
II. High-Throughput In Vitro Fluorescence Polarization Assay
[0123] Fluorescently-labeled PFK peptide/substrate are added to
each well of a 96-well microtiter plate, along with a test agent in
a test buffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH
7.6). Changes in fluorescence polarization, determined by using a
Fluorolite FPM-2 Fluorescence Polarization Microtiter System
(Dynatech Laboratories, Inc), relative to control values indicates
the test compound is a candidate modifier of PFK activity.
III. High-Throughput In Vitro Binding Assay
[0124] .sup.33P-labeled PFK peptide is added in an assay buffer
(100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl.sub.2, 1% glycerol, 0.5%
NP-40, 50 mM beta-mercaptoethanol, 1 mg/ml BSA, cocktail of
protease inhibitors) along with a test agent to the wells of a
Neutralite-avidin coated assay plate and incubated at 25.degree. C.
for 1 hour. Biotinylated substrate is then added to each well and
incubated for 1 hour. Reactions are stopped by washing with PBS,
and counted in a scintillation counter. Test agents that cause a
difference in activity relative to control without test agent are
identified as candidate IGFR modulating agents.
IV. Immunoprecipitations and Immunoblotting
[0125] For coprecipitation of transfected proteins,
3.times.10.sup.6 appropriate recombinant cells containing the PFK
proteins are plated on 10-cm dishes and transfected on the
following day with expression constructs. The total amount of DNA
is kept constant in each transfection by adding empty vector. After
24 h, cells are collected, washed once with phosphate-buffered
saline and lysed for 20 min on ice in 1 ml of lysis buffer
containing 50 mM Hepes, pH 7.9, 250 mM NaCl, 20
mM-glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitrophenyl
phosphate, 2 mM dithiothreitol, protease inhibitors (complete,
Roche Molecular Biochemicals), and 1% Nonidet P-40. Cellular debris
is removed by centrifugation twice at 15,000.times.g for 15 min.
The cell lysate is incubated with 25 .mu.l of M2 beads (Sigma) for
2 h at 4.degree. C. with gentle rocking.
[0126] After extensive washing with lysis buffer, proteins bound to
the beads are solubilized by boiling in SDS, sample buffer,
fractionated by SDS-polyacrylamide gel electrophoresis, transferred
to polyvinylidene difluoride membrane and blotted with the
indicated antibodies. The reactive bands are visualized with
horseradish peroxidase coupled to the appropriate secondary
antibodies and the enhanced chemiluminescence (ECL) Western
blotting detection system (Amersham Pharmacia Biotech).
V. Kinase Assay
[0127] A purified or partially purified PFK is diluted in a
suitable reaction buffer, e.g., 50 mM Hepes, pH 7.5, containing
magnesium chloride or manganese chloride (1-20 mM) and a peptide or
polypeptide substrate, such as myelin basic protein or casein (1-10
.mu.g/ml). The final concentration of the kinase is 1-20 nM. The
enzyme reaction is conducted in microtiter plates to facilitate
optimization of reaction conditions by increasing assay throughput.
A 96-well microtiter plate is employed using a final volume 30-100
.mu.l. The reaction is initiated by the addition of
.sup.33P-gamma-ATP (0.5 .mu.Ci/ml) and incubated for 0.5 to 3 hours
at room temperature. Negative controls are provided by the addition
of EDTA, which chelates the divalent cation (Mg2.sup.+ or
Mn.sup.2+) required for enzymatic activity. Following the
incubation, the enzyme reaction is quenched using EDTA. Samples of
the reaction are transferred to a 96-well glass fiber filter plate
(MultiScreen, Millipore). The filters are subsequently washed with
phosphate-buffered saline, dilute phosphoric acid (0.5%) or other
suitable medium to remove excess radiolabeled ATP. Scintillation
cocktail is added to the filter plate and the incorporated
radioactivity is quantitated by scintillation counting
(Wallac/Perkin Elmer). Activity is defined by the amount of
radioactivity detected following subtraction of the negative
control reaction value (EDTA quench).
VI. Expression Analysis
[0128] All cell lines used in the following experiments are NCI
(National Cancer Institute) lines, and are available from ATCC
(American Type Culture Collection, Manassas, Va. 20110-2209).
Normal and tumor tissues were obtained from Impath, UC Davis,
Clontech, Stratagene, Ardais, Genome Collaborative, and Ambion.
[0129] TaqMan.RTM. analysis was used to assess expression levels of
the disclosed genes in various samples.
[0130] RNA was extracted from each tissue sample using Qiagen
(Valencia, Calif.) RNeasy kits, following manufacturer's protocols,
to a final concentration of 50 ng/.mu.l. Single stranded cDNA was
then synthesized by reverse transcribing the RNA samples using
random hexamers and 500 ng of total RNA per reaction, following
protocol 4304965 of Applied Biosystems (Foster City, Calif.).
[0131] Primers for expression analysis using TaqMan.RTM. assay
(Applied Biosystems, Foster City, Calif.) were prepared according
to the TaqMan.RTM. protocols, and the following criteria: a) primer
pairs were designed to span introns to eliminate genomic
contamination, and b) each primer pair produced only one product.
Expression analysis was performed using a 7900HT instrument.
[0132] TaqMan.RTM. reactions were carried out following
manufacturer's protocols, in 25 .mu.l total volume for 96-well
plates and 10 .mu.l total volume for 384-well plates, using 300 nM
primer and 250 nM probe, and approximately 25 ng of cDNA. The
standard curve for result analysis was prepared using a universal
pool of human cDNA samples, which is a mixture of cDNAs from a wide
variety of tissues so that the chance that a target will be present
in appreciable amounts is good. The raw data were normalized using
18S rRNA (universally expressed in all tissues and cells).
[0133] For each expression analysis, tumor tissue samples were
compared with matched normal tissues from the same patient. A gene
was considered overexpressed in a tumor when the level of
expression of the gene was 2 fold or higher in the tumor compared
with its matched normal sample. In cases where normal tissue was
not available, a universal pool of cDNA samples was used instead.
In these cases, a gene was considered overexpressed in a tumor
sample when the difference of expression levels between a tumor
sample and the average of all normal samples from the same tissue
type was greater than 2 times the standard deviation of all normal
samples (i.e., Tumor-average(all normal
samples)>2.times.STDEV(all normal samples)).
[0134] Results are shown in Table 1. Number of pairs of tumor
samples and matched normal tissue from the same patient are shown
for each tumor type. Percentage of the samples with at least
two-fold overexpression for each tumor type is provided. A
modulator identified by an assay described herein can be further
validated for therapeutic effect by administration to a tumor in
which the gene is overexpressed. A decrease in tumor growth
confirms therapeutic utility of the modulator. Prior to treating a
patient with the modulator, the likelihood that the patient will
respond to treatment can be diagnosed by obtaining a tumor sample
from the patient, and assaying for expression of the gene targeted
by the modulator. The expression data for the gene(s) can also be
used as a diagnostic marker for disease progression. The assay can
be performed by expression analysis as described above, by antibody
directed to the gene target, or by any other available detection
method.
TABLE-US-00001 TABLE 1 SEQ ID NO 2 31 61 Breast 19% 17% 22% # of
Pairs 36 36 36 Colon 10% 30% 18% # of Pairs 40 40 40 Head And 31%
15% 69% Neck # of Pairs 13 13 13 Liver 22% 44% 67% # of Pairs 9 9 9
Lung 15% 20% 68% # of Pairs 40 40 40 Lymphoma 0% 75% 50% # of Pairs
4 4 4 Ovary 37% 16% 58% # of Pairs 19 19 19 Pancreas 75% 67% 67% #
of Pairs 12 12 12 Prostate 8% 4% 0% # of Pairs 24 24 24 Skin 29%
71% 57% # of Pairs 7 7 7 Stomach 9% 27% 55% # of Pairs 11 11 11
Testis 12% 0% 25% # of Pairs 8 8 8 Thyroid 0% 43% 21% Gland # of
Pairs 14 14 14 Uterus 35% 0% 30% # of Pairs 23 23 23
VII. PFK Functional Assays
[0135] RNAi experiments were carried out to knock down expression
of PFK (SEQ ID. NO:2) in various cell lines using small interfering
RNAs (siRNA, Elbashir et al, supra).
[0136] Effect of PFK RNAi on Cell Proliferation and Growth.
[0137] Standard colony growth assays, as described above, were
employed to study the effects of decreased PFK expression on cell
growth. The results of this experiment indicated that RNAi of PFK
decreased proliferation in A549 lung cancer and A2780 ovarian
cancer cells.
[0138] [.sup.3H]-thymidine incorporation assay, as described above,
was also employed to study the effects of decreased PFK expression
on cell proliferation. The results of this experiment indicated
that RNAi of PFK decreased proliferation in A549 lung cancer
cells.
[0139] Effect of PFK RNAi on Apoptosis.
[0140] The Phospho-histone H2B assay, as described above, was
employed to study the effects of decreased PFK expression on
apoptosis. The results of this experiment indicated that RNAi of
PFK of SEQ ID NO:2 increased apoptosis in 231T breast cancer cells,
A549 lung cancer cells, and U87MG glioblastoma cells. Further, RNAi
of PFK of SEQ ID NO:2 also caused a decrease in cell count in 231T
breast cancer cells.
[0141] Involvement in PTEN/IGFR Pathway:
[0142] PFK FOXO nuclear translocation assays. FOXO nuclear
translocation assays, as described above, were employed to assess
involvement of PFK in the PTEN/IGF pathway. In these experiments,
cells with reduced expression of PFK by RNAi were transiently
transfected with a plasmid expressing GFP-tagged FOXO. Automated
imaging of cellular components, such as nucleus and cytoplasm were
then carried out to assess translocation of FOXO. Alternatively,
cells were co-transfected with siRNA directed to PFK along with a
plasmid containing FOXO, and a cassette containing a promoter, a
FOXO response element, and luciferase. Cells were then analyzed for
luciferase activity and compared with cells with no siRNA. Results
indicated that reduced expression of PFK led to translocation of
FOXO to the cytoplasm in PC3 prostate cancer cells, A549 lung
cancer cells, and A2780 ovarian cancer cells. These results suggest
involvement of PFK in the PTEN/IGFR pathway.
[0143] Pan-AKT Assays.
[0144] This assay was developed to detect involvement of PFK in the
PTEN/IGFR pathway. The assay detects changes in phosphorylation for
several substrates of AKT, such as PRAS40, BAD, 4EBP1, and RPS6.
For this experiment, antibodies were raised against phosphorylated
AKT substrates, including the consensus phosphorylated AKT
substrate sequence RxRxxS/T. Expression levels of phosphorylated
substrates were then quantitated at normal levels, in presence of a
negative control, a positive control (AKT), and then with PFK
knockout. For example, when AKT levels were reduced, expression of
all its substrates was also reduced. Results indicated that reduced
expression of PFK of SEQ ID NO:2 was similar to reduced AKT levels
in 231T breast cancer and A549 lung cancer cells.
[0145] We used RPS6 assay for one subset of experiments. RPS6 is an
IGF dependent substrate of AKT. IGF 1 treatment increases
cytoplasmic RPS6 levels. Alternatively, Lily compound LY294002, a
PI3K inhibitor, reduces AKT and cytoplasmic RPS6 levels. Cells were
plated in 96 well plates, transfected with RNAi for PFK, fixed,
treated with RPS6 antibody, and stained. Measurements were based on
percentage of population of cells with increased or decreased
staining compared with negative or positive control cells. Results
of this experiment showed that reduced expression of PFK caused an
alteration in the level of phospho RPS6 protein in 231T breast
cancer and PC3 prostate cancer cells, thus suggesting an
involvement in the IGFR pathway.
[0146] We used PRAS40 as the substrate for another subset of
experiments. For this substrate, pathway inhibition causes
decreased cytoplasmic staining and increased nuclear and
perinuclear staining. Cells were plated in 96 well plates,
transfected with RNAi for PFK, fixed, treated with PRAS40 antibody,
and stained. Measurements were based on percentage of population of
cells with increased or decreased nuclear/cytoplasmic staining
ratio compared with negative or positive control cells. Results of
this experiment showed that reduced expression of PFK altered the
level of phospho PRAS40 protein in 231T breast cancer cells, A549
lung cancer cells, and PC3 prostate cancer cells, thus suggesting
an involvement in the IGFR pathway.
[0147] We used BAD as the substrate for another subset of the
experiments. For this substrate, AKT pathway inhibition causes
decreased cytoplasmic staining and unchanged or increased nuclear
staining. Cells were plated in 96 well plates, transfected with
RNAi for PFK, fixed, permeabilized and stained with
anti-phospho-BAD antibody. Measurements were based on the
percentage of the population of cells with a decreased
Cytoplasmic/Nuclear staining ratio compared with negative or
positive control cells. Results of this experiment showed that
reduced expression of PFK caused a reduction in the level of
phospho-BAD protein in the cytoplasm in 231T breast cancer cells,
A549 lung cancer cells, and PC3 prostate cancer cells, thus
suggesting an involvement in the IGFR pathway. Taken together, the
results of the pan-AKT assay suggest involvement of PFK in the
PTEN/IGFR pathway.
Sequence CWU 1
1
6612924DNAHomo sapiens 1gacggcgacg cggcgcaggc ggcgggagtg cgagctgggc
ccgtgtttcg gccgccgcca 60tggccgcggt ggacctggag aagctgcggg cgtcgggcgc
gggcaaggcc atcggcgtcc 120tgaccagcgg cggcgacgcg caaggcatga
acgctgctgt ccgggctgtg acgcgcatgg 180gcatttatgt gggtgccaaa
gtcttcctca tctacgaggg ctatgagggc ctcgtggagg 240gaggtgagaa
catcaagcag gccaactggc tgagcgtctc caacatcatc cagctgggcg
300gcactatcat tggcagcgct cgctgcaagg cctttaccac cagggagggg
cgccgggcag 360cggcctacaa cctggtccag cacggcatca ccaacctgtg
cgtcatcggc ggggatggca 420gcctcacagg tgccaacatc ttccgcagcg
agtggggcag cctgctggag gagctggtgg 480cggaaggtaa gatctcagag
actacagccc ggacctactc gcacctgaac atcgcgggcc 540tagtgggctc
catcgataac gacttctgcg gcaccgacat gaccatcggc acggactcgg
600ccctccaccg catcatggag gtcatcgatg ccatcaccac cactgcccag
agccaccaga 660ggaccttcgt gctggaagtg atgggccggc actgcgggta
cctggcgctg gtatctgcac 720tggcctcagg ggccgactgg ctgttcatcc
ccgaggctcc acccgaggac ggctgggaga 780acttcatgtg tgagaggctg
ggtgagactc ggagccgtgg gtcccgactg aacatcatca 840tcatcgctga
gggtgccatt gaccgcaacg ggaagcccat ctcgtccagc tacgtgaagg
900acctggtggt tcagaggctg ggcttcgaca cccgtgtaac tgtgctgggc
cacgtgcagc 960ggggagggac gccctctgcc ttcgaccgga tcctgagcag
caagatgggc atggaggcgg 1020tgatggcgct gctggaagcc acgcctgaca
cgccggcctg cgtggtcacc ctctcgggga 1080accagtcagt gcggctgccc
ctcatggagt gcgtgcagat gaccaaggaa gtgcagaaag 1140ccatggatga
caagaggttt gacgaggcca cccagctccg tggtgggagc ttcgagaaca
1200actggaacat ttacaagctc ctcgcccacc agaagccccc caaggagaag
tctaacttct 1260ccctggccat cctgaatgtg ggggccccgg cggctggcat
gaatgcggcc gtgcgctcgg 1320cggtgcggac cggcatctcc catggacaca
cagtatacgt ggtgcacgat ggcttcgaag 1380gcctagccaa gggtcaggtg
caagaagtag gctggcacga cgtggccggc tggttggggc 1440gtggtggctc
catgctgggg accaagagga ccctgcccaa gggccagctg gagtccattg
1500tggagaacat ccgcatctat ggtattcacg ccctgctggt ggtcggtggg
tttgaggcct 1560atgaaggggt gctgcagctg gtggaggctc gcgggcgcta
cgaggagctc tgcatcgtca 1620tgtgtgtcat cccagccacc atcagcaaca
acgtccctgg caccgacttc agcctgggct 1680ccgacactgc tgtaaatgcc
gccatggaga gctgtgaccg catcaaacag tctgcctcgg 1740ggaccaagcg
ccgtgtgttc atcgtggaga ccatgggggg ttactgtggc tacctggcca
1800ccgtgactgg cattgctgtg ggggccgacg ccgcctacgt cttcgaggac
cctttcaaca 1860tccacgactt aaaggtcaac gtggagcaca tgacggagaa
gatgaagaca gacattcaga 1920ggggcctggt gctgcggaac gagaagtgcc
atgactacta caccacggag ttcctgtaca 1980acctgtactc atcagagggc
aagggcgtct tcgactgcag gaccaatgtc ctgggccacc 2040tgcagcaggg
tggcgctcca accccctttg accggaacta tgggaccaag ctgggggtga
2100aggccatgct gtggttgtcg gagaagctgc gcgaggttta ccgcaaggga
cgggtgttcg 2160ccaatgcccc agactcggcc tgcgtgatcg gcctgaagaa
gaaggcggtg gccttcagcc 2220ccgtcactga gctcaagaaa gacactgatt
tcgagcaccg catgccacgg gagcagtggt 2280ggctgagcct gcggctcatg
ctgaagatgc tggcacaata ccgcatcagt atggccgcct 2340acgtgtcagg
ggagctggag cacgtgaccc gccgcaccct gagcatggac aagggcttct
2400gaggccagcc atgcccacgc ccctccccag cccccaccca tgccagcgca
gcgccagggc 2460tcagatgggg cctgggctgt tgtgtctgga gcctgcaggc
aggtgggggc tgcgtccctg 2520ctcagcccat cccctgcctc tatccctggc
cacctgccag gcctccctcg ggctggtgtc 2580ttgagaccag cctgccaggc
cctccagcag gaggacagag tgccctgggg catccacctt 2640cctgcccagg
ggacgtggcg ctgtcggtgt ttggaggctg ctgccccctg gctttggcgc
2700cccatgggcc ctcagcgtct ccccatgctg ggctcactac atgggccagc
ccttgctcta 2760cctggccggt aggctgctgg cgcctaggtt gtgttgagag
ggggatgccc ctggccctgc 2820ctcactgtga cctgctcctg cccacgtgca
gcacctgtca ccttttctag aaataaaatc 2880accctgactg tggggtgcat
cggtctccgg agaaaacaaa aaaa 292423384DNAHomo sapiens 2gcgacgcggc
gcaggcggcg ggagtgcgag ctgggcccgt gtttcggccg ccgccatggc 60cgcggtggac
ctggagaagc tgcgggcgtc gggcgcgggc aaggccatcg gcgtcctgac
120cagcggcggc gacgcgcaag gtcccctgac aagcccacca ggccccctgc
tgagatggct 180gtgaccctgg gctgacccgc ccagtggcac attgactccg
cctggagctg gggagaccag 240agaggccctg tggttggacg gtggcctggg
tgcgctgctc ctgccctctc cttgccctgc 300ctcagctgct gcctgccaga
ggcgtggcac ctcacctcac acctgctccc tgctgctgag 360ccccacgcca
agctggagag cggatgagaa gcatgtgtaa ccagggtaga ggtcgagagt
420cctctcgtgg gggtctccat gttcaaggga gctgccgagg cttgagcagg
agcccccagc 480aggaaactgg ctttgccaag gcccccgctg ggacagactg
tttctttcac tgcagtcctg 540ggagccgagg gcaaggggac aggaaagagg
aagtgacctc agagcctggt ggcaccagca 600tcatgtccag gctggggggc
atgaacgctg ctgtccgggc tgtgacgcgc atgggcattt 660atgtgggtgc
caaagtcttc ctcatctacg agggctatga gggcctcgtg gagggaggtg
720agaacatcaa gcaggccaac tggctgagcg tctccaacat catccagctg
ggcggcacta 780tcattggcag cgctcgctgc aaggccttta ccaccaggga
ggggcgccgg gcagcggcct 840acaacctggt ccagcacggc atcaccaacc
tgtgcgtcat cggcggggat ggcagcctta 900caggtgccaa catcttccgc
agcgagtggg gcagcctgct ggaggagctg gtggcggaag 960gtaagatctc
agagactaca gcccggacct actcgcacct gaacatcgcg ggcctagtgg
1020gctccatcga taacgacttc tgcggcaccg acatgaccat cggcacggac
tcggccctcc 1080accgcatcat ggaggtcatc gatgccatca ccaccactgc
ccagagccac cagaggacct 1140tcgtgctgga agtgatgggc cggcactgcg
ggtacctggc gctggtatct gcactggcct 1200caggggccga ctggctgttc
atccccgagg ctccacccga ggacggctgg gagaacttca 1260tgtgtgagag
gctgggtgag actcggagcc gtgggtcccg actgaacatc atcatcatcg
1320ctgagggtgc cattgaccgc aacgggaagc ccatctcgtc cagctacgtg
aaggacctgg 1380tggttcagag gctgggcttc gacacccgtg taactgtgct
gggccacgtg cagcggggag 1440ggacgccctc tgccttcgac cggatcctga
gcagcaagat gggcatggag gcggtgatgg 1500cgctgctgga agccacgcct
gacacgccgg cctgcgtggt caccctctcg gggaaccagt 1560cagtgcggct
gcccctcatg gagtgcgtgc agatgaccaa ggaagtgcag aaagccatgg
1620atgacaagag gtttgacgag gccacccagc tccgtggtgg gagcttcgag
aacaactgga 1680acatttacaa gctcctcgcc caccagaagc cccccaagga
gaagtctaac ttctccctgg 1740ccatcctgaa tgtgggggcc ccggcggctg
gcatgaatgc ggccgtgcgc tcggcggtgc 1800ggaccggcat ctcccatgga
cacacagtat acgtggtgca cgatggcttc gaaggcctag 1860ccaagggtca
ggtgcaagaa gtaggctggc acgacgtggc cggctggttg gggcgtggtg
1920gctccatgct ggggaccaag aggaccctgc ccaagggcca gctggagtcc
attgtggaga 1980acatccgcat ctatggtatt cacgccctgc tggtggtcgg
tgggtttgag gcctatgaag 2040gggtgctgca gctggtggag gctcgcgggc
gctacgagga gctctgcatc gtcatgtgtg 2100tcatcccagc caccatcagc
aacaacgtcc ctggcaccga cttcagcctg ggctccgaca 2160ctgctgtaaa
tgccgccatg gagagctgtg accgcatcaa acagtctgcc tcggggacca
2220agcgccgtgt gttcatcgtg gagaccatgg ggggttactg tggctacctg
gccaccgtga 2280ctggcattgc tgtgggggcc gacgccgcct acgtcttcga
ggaccctttc aacatccacg 2340acttaaaggt caacgtggag cacatgacgg
agaagatgaa gacagacatt cagaggggcc 2400tggtgctgcg gaacgagaag
tgccatgact actacaccac ggagttcctg tacaacctgt 2460actcatcaga
gggcaagggc gtcttcgact gcaggaccaa tgtcctgggc cacctgcagc
2520agggtggcgc tccaaccccc tttgaccgga actatgggac caagctgggg
gtgaaggcca 2580tgctgtggtt gtcggagaag ctgcgcgagg tttaccgcaa
gggacgggtg ttcgccaatg 2640ccccagactc ggcctgcgtg atcggcctga
agaagaaggc ggtggccttc agccccgtca 2700ctgagctcaa gaaagacact
gatttcgagc accgcatgcc acgggagcag tggtggctga 2760gcctgcggct
catgctgaag atgctggcac aataccgcat cagtatggcc gcctacgtgt
2820caggggagct ggagcacgtg acccgccgca ccctgagcat ggacaagggc
ttctgaggcc 2880agccatgccc acgcccctcc ccagccccca cccatgccag
cgcagcgcca gggctcagat 2940ggggcctggg ctgttgtgtc tggagcctgc
aggcaggtgg gggctgcgtc cctgctcagc 3000ccatcccctg cctctatccc
tggccacctg ccaggcctcc ctcgggctgg tgtcttgaga 3060ccagcctgcc
aggccctcca gcaggaggac agagtgccct ggggcatcca ccttcctgcc
3120caggggacgt ggcgctgtcg gtgtttggag gctgctgccc cctggctttg
gcgccccatg 3180ggccctcagc gtctccccat gctgggctca ctacatgggc
cagcccttgc tctacctggc 3240cggtaggctg ctggcgccta ggttgtgttg
agagggggat gcccctggcc ctgcctcact 3300gtgacctgct cctgcccacg
tgcagcacct gtcacctttt ctagaaataa aatcaccctg 3360actgtggggt
gcatcggtct ccgg 338433385DNAHomo sapiens 3cgtgtttcgg ccgccgccat
ggccgcggtg gacctggaga agctgcgggc gtcgggcgcg 60ggcaaggcca tcggcgtcct
gaccagcggc ggcgacgcgc aaggtcccct gacaagccca 120ccaggccccc
tgctgagatg gctgtgaccc tgggctgacc cgcccagtgg cacattgact
180ccgcctggag ctggggagac cagagaggcc ctgtggttgg acggtggcct
gggtgcgctg 240ctcctgccct ctccttgccc tgcctcagct gctgcctgcc
agaggcgtgg cacctcacct 300cacacctgct ccctgctgct gagccccacg
ccaagctgga gagcggatga gaagcatgtg 360taaccagggt agaggtcgag
agtcctctcg tgggggtctc catgttcaag ggagctgccg 420aggcttgagc
aggagccccc agcaggaaac tggctttgcc aaggcccccg ctgggacaga
480ctgtttcttt cactgcagtc ctgggagccg agggcaaggg gacaggaaag
aggaagtgac 540ctcagagcct ggtggcacca gcatcatgtc caggctgggg
ggcatgaacg ctgctgtccg 600ggctgtgacg cgcatgggca tttatgtggg
tgccaaagtc ttcctcatct acgagggcta 660tgagggcctc gtggagggag
gtgagaacat caagcaggcc aactggctga gcgtctccaa 720catcatccag
ctgggcggca ctatcattgg cagcgctcgc tgcaaggcct ttaccaccag
780ggaggggcgc cgggcagcgg cctacaacct ggtccagcac ggcatcacca
acctgtgcgt 840catcggcggg gatggcagcc ttacaggtgc caacatcttc
cgcagcgagt ggggcagcct 900gctggaggag ctggtggcgg aaggtaagat
ctcagagact acagcccgga cctactcgca 960cctgaacatc gcgggcctag
tgggctccat cgataacgac ttctgcggca ccgacatgac 1020catcggcacg
gactcggccc tccaccgcat catggaggtc atcgatgcca tcaccaccac
1080tgcccagagc caccagagga ccttcgtgct ggaagtgatg ggccggcact
gcgggtacct 1140ggcgctggta tctgcactgg cctcaggggc cgactggctg
ttcatccccg aggctccacc 1200cgaggacggc tgggagaact tcatgtgtga
gaggctgggt gagactcgga gccgtgggtc 1260ccgactgaac atcatcatca
tcgctgaggg tgccattgac cgcaacggga agcccatctc 1320gtccagctac
gtgaaggacc tggtggttca gaggctgggc ttcgacaccc gtgtaactgt
1380gctgggccac gtgcagcggg gagggacgcc ctctgccttc gaccggatcc
tgagcagcaa 1440gatgggcatg gaggcggtga tggcgctgct ggaagccacg
cctgacacgc cggcctgcgt 1500ggtcaccctc tcggggaacc agtcagtgcg
gctgcccctc atggagtgcg tgcagatgac 1560caaggaagtg cagaaagcca
tggatgacaa gaggtttgac gaggccaccc agctccgtgg 1620tgggagcttc
gagaacaact ggaacattta caagctcctc gcccaccaga agccccccaa
1680ggagaagtct aacttctccc tggccatcct gaatgtgggg gccccggcgg
ctggcatgaa 1740tgcggccgtg cgctcggcgg tgcggaccgg catctcccat
ggacacacag tatacgtggt 1800gcacgatggc ttcgaaggcc tagccaaggg
tcaggtgcaa gaagtaggct ggcacgacgt 1860ggccggctgg ttggggcgtg
gtggctccat gctggggacc aagaggaccc tgcccaaggg 1920ccagctggag
tccattgtgg agaacatccg catctatggt attcacgccc tgctggtggt
1980cggtgggttt gaggcctatg aaggggtgct gcagctggtg gaggctcgcg
ggcgctacga 2040ggagctctgc atcgtcatgt gtgtcatccc agccaccatc
agcaacaacg tccctggcac 2100cgacttcagc ctgggctccg acactgctgt
aaatgccgcc atggagagct gtgaccgcat 2160caaacagtct gcctcgggga
ccaagcgccg tgtgttcatc gtggagacca tggggggtta 2220ctgtggctac
ctggccaccg tgactggcat tgctgtgggg gccgacgccg cctacgtctt
2280cgaggaccct ttcaacatcc acgacttaaa ggtcaacgtg gagcacatga
cggagaagat 2340gaagacagac attcagaggg gcctggtgct gcggaacgag
aagtgccatg actactacac 2400cacggagttc ctgtacaacc tgtactcatc
agagggcaag ggcgtcttcg actgcaggac 2460caatgtcctg ggccacctgc
agcagggtgg cgctccaacc ccctttgacc ggaactatgg 2520gaccaagctg
ggggtgaagg ccatgctgtg gttgtcggag aagctgcgcg aggtttaccg
2580caagggacgg gtgttcgcca atgccccaga ctcggcctgc gtgatcggcc
tgaagaagaa 2640ggcggtggcc ttcagccccg tcactgagct caagaaagac
actgatttcg agcaccgcat 2700gccacgggag cagtggtggc tgagcctgcg
gctcatgctg aagatgctgg cacaataccg 2760catcagtatg gccgcctacg
tgtcagggga gctggagcac gtgacccgcc gcaccctgag 2820catggacaag
ggcttctgag gccagccatg cccacgcccc tccccagccc ccacccatgc
2880cagcgcagcg ccagggctca gatggggcct gggctgttgt gtctggagcc
tgcaggcagg 2940tgggggctgc gtccctgctc agcccatccc ctgcctctat
ccctggccac ctgccaggcc 3000tccctccggc tggtgtcttg agaccagcct
gccaggccct ccagcaggag gacagagtgc 3060cctggggcat ccaccttcct
gcccagggga cgtggcgctg tcggtgtttg gaggctgctg 3120ccccctggct
ttggcgcccc atgggccctc agcgtctccc catgctgggc tcactacatg
3180ggccagccct tgctctacct ggccggtagg ctgctggcgc ctaggttgtg
ttgagagggg 3240gatgcccctg gccctgcctc actgtgacct gctcctgccc
acgtgcagca cctgtcacct 3300tttctagaaa taaaatcacc ctgactgtgg
ggtgcatcgg tctccggaaa aaaaaaaaaa 3360aaaaaaaaaa aaaaaaaaaa aaaaa
338542920DNAHomo sapiens 4ggcacgaggc ccgtgtttcg gccgccgcca
tggccgcggt ggacctggag aagctgcggg 60cgtcgggcgc gggcaaggcc atcggcgtcc
tgaccagcgg cggcgacgcg caaggcatga 120acgctgctgt ccgggctgtg
acgcgcatgg gcatttatgt gggtgccaaa gtcttcctca 180tctacgaggg
ctatgagggc ctcgtggagg gaggtgagaa catcaagcag gccaactggc
240tgagcgtctc caacatcatc cagctgggcg gcactatcat tggcagcgct
cgctgcaagg 300cctttaccac cagggagggg cgccgggcag cggcctacaa
cctggtccag cacggcatca 360ccaacctgtg cgtcatcggc ggggatggca
gccttacagg tgccaacatc ttccgcagcg 420agtggggcag cctgctggag
gagctggtgg cggaaggtaa gatctcagag actacagccc 480ggacctactc
gcacctgaac atcgcgggcc tagtgggctc catcgataac gacttctgcg
540gcaccgacat gaccatcggc acggactcgg ccctccaccg catcatggag
gtcatcgatg 600ccatcaccac cactgcccag agccaccaga ggaccttcgt
gctggaagtg atgggccggc 660actgcgggta cctggcgctg gtatctgcac
tggcctcagg ggccgactgg ctgttcatcc 720ccgaggctcc acccgaggac
ggctgggaga acttcatgtg tgagaggctg ggtgagactc 780ggagccgtgg
gtcccgactg aacatcatca tcatcgctga gggtgccatt gaccgcaacg
840ggaagcccat ctcgtccagc tacgtgaagg acctggtggt tcagaggctg
ggcttcgaca 900cccgtgtaac tgtgctgggc cacgtgcagc ggggagggac
gccctctgcc ttcgaccgga 960tcctgagcag caagatgggc atggaggcgg
tgatggcgct gctggaagcc acgcctgaca 1020cgccggcctg cgtggtcacc
ctctcgggga accagtcagt gcggctgccc ctcatggagt 1080gcgtgcagat
gaccaaggaa gtgcagaaag ccatggatga caagaggttt gacgaggcca
1140cccagctccg tggtgggagc ttcgagaaca actggaacat ttacaagctc
ctcgcccacc 1200agaagccccc caaggagaag tctaacttct ccctggccat
cctgaatgtg ggggccccgg 1260cggctggcat gaatgcggcc gtgcgctcgg
cggtgcggac cggcatctcc catggacaca 1320cagtatacgt ggtgcacgat
ggcttcgaag gcctagccaa gggtcaggtg caagaagtag 1380gctggcacga
cgtggccggc tggttggggc gtggtggctc catgctgggg accaagagga
1440ccctgcccaa gggccagctg gagtccattg tggagaacat ccgcatctat
ggtattcacg 1500ccctgctggt ggtcggtggg tttgaggcct atgaaggggt
gctgcagctg gtggaggctc 1560gcgggcgcta cgaggagctc tgcatcgtca
tgtgtgtcat cccagccacc atcagcaaca 1620acgtccctgg caccgacttc
agcctgggct ccgacactgc tgtaaatgcc gccatggaga 1680gctgtgaccg
catcaaacag tctgcctcgg ggaccaagcg ccgtgtgttc atcgtggaga
1740ccatgggggg ttactgtggc tacctggcca ccgtgactgg cattgctgtg
ggggccgacg 1800ccgcctacgt cttcgaggac cctttcaaca tccacgactt
aaaggtcaac gtggagcaca 1860tgacggagaa gatgaagaca gacattcaga
ggggcctggt gctgcggaac gagaagtgcc 1920atgactacta caccacggag
ttcctgtaca acctgtactc atcagagggc aacggcgtct 1980tcgactgcag
gaccaatgtc ctgggccacc tgcagcaggg tggcgctcca accccctttg
2040accggaacta tgggaccaag ctgggggtga aggccatgct gtggttgtcg
gagaagctgc 2100gcgaggttta ccgcaaggga cgggtgttcg ccaatgcccc
agactcggcc tgcgtgatcg 2160gcctgaagaa gaaggcggcg gccttcagcc
ccgtcactga gctcaagaaa gacactgatt 2220tcgagcaccg catgccacgg
gagcagtggt ggctgagcct gcggctcatg ctgaagatgc 2280tggcacaata
ccgcatcagt atggccgcct acgtgtcagg ggagctggag cacgtgaccc
2340gccgcaccct gagcatggac aagggcttct gaggccagcc atgcccacgc
ccctccccag 2400cccccaccca tgccagcgca gcgccagggc tcagatgggg
cctgggctgt tgtgtctgga 2460gcctgcaggc aggtgggggc tgcgtccctg
ctcagcccat cccctgcctc tatccctggc 2520cacctgccag gcctccctcc
ggctggtgtc ttgagaccag cctgccaggc cctccagcag 2580gaggacagag
tgccctgggg catccacctt cctgcccagg ggacgtggcg ctgtcggtgt
2640ttggaggctg ctgccccctg gctttggcgc cccatgggcc ctcagcgtct
ccccatgctg 2700ggctcactac atgggccagc ccttgctcta cctggccggt
aggctgctgg cgcctaggtt 2760gtgttgagag ggggatgccc ctggccctgc
ctcactgtga cctgctcctg cccacgtgca 2820gcacctgtca ccttttctag
aaataaaatc accctgactg tggggtgcat cggtctccgg 2880aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 292052255DNAHomo sapiens
5gcgggtacct ggcgctggta tctgcactgg cctcaggggc cgactggctg ttcatccccg
60aggctccacc cgaggacggc tgggagaact tcatgtgtga gaggctgggt gagactcgga
120gccgtgggtc ccgactgaac atcatcatca tcgctgaggg tgccattgac
cgcaacggga 180agcccatctc gtccagctac gtgaaggacc tggtggttca
gaggctgggc ttcgacaccc 240gtgtaactgt gctgggccac gtgcagcggg
gagggacgcc ctctgccttc gaccggatcc 300tgagcagcaa gatgggcatg
gaggcggtga tggcgctgct ggaagccacg cctgacacgc 360cggcctgcgt
ggtcaccctc tcggggaacc agtcagtgcg gctgcccctc atggagtgcg
420tgcagatgac caaggaagtg cagaaagcca tggatgacaa gaggtttgac
gaggccaccc 480agctccgtgg tgggagcttc gagaacaact ggaacattta
caagctcctc gcccaccaga 540agccccccaa ggagaagtct aacttctccc
tggccatcct gaatgtgggg gccccggcgg 600ctggcatgaa tgcggccgtg
cgctcggcgg tgcggaccgg catctcccat ggacacacag 660tatacgtggt
gcacgatggc ttcgaaggcc tagccaaggg tcaggtgcaa gaagtaggct
720ggcacgacgt ggccggctgg ttggggcgtg gtggctccat gctggggacc
aagaggaccc 780tgcccaaggg ccagctggag tccattgtgg agaacatccg
catctatggt attcacgccc 840tgctggtggt cggtgggttt gaggcctatg
aaggggtgct gcagctggtg gaggctcgcg 900ggcgctacga ggagctctgc
atcgtcatgt gtgtcatccc agccaccatc agcaacaacg 960tccctggcac
cgacttcagc ctgggctccg acactgctgt aaatgccgcc atggagagct
1020gtgaccgcat caaacagtct gcctcgggga ccaagcgccg tgtgttcatc
gtggagacca 1080tggggggtta ctgtggctac ctggccaccg tgactggcat
tgctgtgggg gccgacgccg 1140cctacgtctt cgaggaccct ttcaacatcc
acgacttaaa ggtcaacgtg gagcacatga 1200cggagaagat gaagacagac
attcagaggg gcctggtgct gcggaacgag aagtgccatg 1260actactacac
cacggagttc ctgtacaacc tgtactcatc agagggcaag ggcgtcttcg
1320actgcaggac caatgtcctg ggccacctgc agcagggtgg cgctccaacc
ccctttgacc 1380ggaactatgg gaccaagctg ggggtgaagg ccatgctgtg
gttgtcggag aagctgcgcg 1440aggtttaccg caagggacgg gtgttcgcca
atgccccaga ctcggcctgc gtgatcggcc 1500tgaagaagaa ggcggtggcc
ttcagccccg tcactgagct caagaaagac actgatttcg 1560agcaccgcat
gccacgggag cagtggtggc tgagcctgcg gctcatgctg aagatgctgg
1620cacaataccg catcagtatg gccgcctacg tgtcagggga gctggagcac
gtgacccgcc 1680gcaccctgag catggacaag ggcttctgag gccagccatg
cccacgcccc tccccagccc 1740ccacccatgc cagcgcagcg ccagggctca
gatggggcct gggctgttgt gtctggagcc 1800tgcaggcagg tgggggctgc
gtccctgctc agcccatccc ctgcctctat ccctggccac 1860ctgccaggcc
tccctccggc tggtgtcttg agaccagcct gccaggccct ccagcaggag
1920gacagagtgc cctggggcat ccaccttcct gcccagggga cgtggcgctg
tcggtgtttg 1980gaggctgctg ccccctggct ttggcgcccc atgggccctc
agcgtctccc catgctgggc 2040tcactacatg ggccagccct tgctctacct
ggccggtagg ctgctggcgc ctaggttgtg 2100ttgagagggg gatgcccctg
gccctgcctc actgtgacct gctcctgccc acgtgcagca 2160cctgtcacct
tttctagaaa taaaatcacc ctgactgtgg ggtgcatcgg tctccggaga
2220aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaa 225563385DNAHomo sapiens 6cgtgtttcgg ccgccgccat
ggccgcggtg gacctggaga agctgcgggc gtcgggcgcg 60ggcaaggcca tcggcgtcct
gaccagcggc ggcgacgcgc aaggtcccct gacaagccca 120ccaggccccc
tgctgagatg gctgtgaccc tgggctgacc cgcccagtgg cacattgact
180ccgcctggag ctggggagac cagagaggcc ctgtggttgg acggtggcct
gggtgcgctg 240ctcctgccct ctccttgccc tgcctcagct gctgcctgcc
agaggcgtgg cacctcacct 300cacacctgct ccctgctgct gagccccacg
ccaagctgga gagcggatga gaagcatgtg 360taaccagggt agaggtcgag
agtcctctcg tgggggtctc catgttcaag ggagctgccg 420aggcttgagc
aggagccccc agcaggaaac tggctttgcc aaggcccccg ctgggacaga
480ctgtttcttt cactgcagtc ctgggagccg agggcaaggg gacaggaaag
aggaagtgac 540ctcagagcct ggtggcacca gcatcatgtc caggctgggg
ggcatgaacg ctgctgtccg 600ggctgtgacg cgcatgggca tttatgtggg
tgccaaagtc ttcctcatct acgagggcta 660tgagggcctc gtggagggag
gtgagaacat caagcaggcc aactggctga gcgtctccaa 720catcatccag
ctgggcggca ctatcattgg cagcgctcgc tgcaaggcct ttaccaccag
780ggaggggcgc cgggcagcgg cctacaacct ggtccagcac ggcatcacca
acctgtgcgt 840catcggcggg gatggcagcc ttacaggtgc caacatcttc
cgcagcgagt ggggcagcct 900gctggaggag ctggtggcgg aaggtaagat
ctcagagact acagcccgga cctactcgca 960cctgaacatc gcgggcctag
tgggctccat cgataacgac ttctgcggca ccgacatgac 1020catcggcacg
gactcggccc tccaccgcat catggaggtc atcgatgcca tcaccaccac
1080tgcccagagc caccagagga ccttcgtgct ggaagtgatg ggccggcact
gcgggtacct 1140ggcgctggta tctgcactgg cctcaggggc cgactggctg
ttcatccccg aggctccacc 1200cgaggacggc tgggagaact tcatgtgtga
gaggctgggt gagactcgga gccgtgggtc 1260ccgactgaac atcatcatca
tcgctgaggg tgccattgac cgcaacggga agcccatctc 1320gtccagctac
gtgaaggacc tggtggttca gaggctgggc ttcgacaccc gtgtaactgt
1380gctgggccac gtgcagcggg gagggacgcc ctctgccttc gaccggatcc
tgagcagcaa 1440gatgggcatg gaggcggtga tggcgctgct ggaagccacg
cctgacacgc cggcctgcgt 1500ggtcaccctc tcggggaacc agtcagtgcg
gctgcccctc atggagtgcg tgcagatgac 1560caaggaagtg cagaaagcca
tggatgacaa gaggtttgac gaggccaccc agctccgtgg 1620tgggagcttc
gagaacaact ggaacattta caagctcctc gcccaccaga agccccccaa
1680ggagaagtct aacttctccc tggccatcct gaatgtgggg gccccggcgg
ctggcatgaa 1740tgcggccgtg cgctcggcgg tgcggaccgg catctcccat
ggacacacag tatacgtggt 1800gcacgatggc ttcgaaggcc tagccaaggg
tcaggtgcaa gaagtaggct ggcacgacgt 1860ggccggctgg ttggggcgtg
gtggctccat gctggggacc aagaggaccc tgcccaaggg 1920ccagctggag
tccattgtgg agaacatccg catctatggt attcacgccc tgctggtggt
1980cggtgggttt gaggcctatg aaggggtgct gcagctggtg gaggctcgcg
ggcgctacga 2040ggagctctgc atcgtcatgt gtgtcatccc agccaccatc
agcaacaacg tccctggcac 2100cgacttcagc ctgggctccg acactgctgt
aaatgccgcc atggagagct gtgaccgcat 2160caaacagtct gcctcgggga
ccaagcgccg tgtgttcatc gtggagacca tggggggtta 2220ctgtggctac
ctggccaccg tgactggcat tgctgtgggg gccgacgccg cctacgtctt
2280cgaggaccct ttcaacatcc acgacttaaa ggtcaacgtg gagcacatga
cggagaagat 2340gaagacagac attcagaggg gcctggtgct gcggaacgag
aagtgccatg actactacac 2400cacggagttc ctgtacaacc tgtactcatc
agagggcaag ggcgtcttcg actgcaggac 2460caatgtcctg ggccacctgc
agcagggtgg cgctccaacc ccctttgacc ggaactatgg 2520gaccaagctg
ggggtgaagg ccatgctgtg gttgtcggag aagctgcgcg aggtttaccg
2580caagggacgg gtgttcgcca atgccccaga ctcggcctgc gtgatcggcc
tgaagaagaa 2640ggcggtggcc ttcagccccg tcactgagct caagaaagac
actgatttcg agcaccgcat 2700gccacgggag cagtggtggc tgagcctgcg
gctcatgctg aagatgctgg cacaataccg 2760catcagtatg gccgcctacg
tgtcagggga gctggagcac gtgacccgcc gcaccctgag 2820catggacaag
ggcttctgag gccagccatg cccacgcccc tccccagccc ccacccatgc
2880cagcgcagcg ccagggctca gatggggcct gggctgttgt gtctggagcc
tgcaggcagg 2940tgggggctgc gtccctgctc agcccatccc ctgcctctat
ccctggccac ctgccaggcc 3000tccctccggc tggtgtcttg agaccagcct
gccaggccct ccagcaggag gacagagtgc 3060cctggggcat ccaccttcct
gcccagggga cgtggcgctg tcggtgtttg gaggctgctg 3120ccccctggct
ttggcgcccc atgggccctc agcgtctccc catgctgggc tcactacatg
3180ggccagccct tgctctacct ggccggtagg ctgctggcgc ctaggttgtg
ttgagagggg 3240gatgcccctg gccctgcctc actgtgacct gctcctgccc
acgtgcagca cctgtcacct 3300tttctagaaa taaaatcacc ctgactgtgg
ggtgcatcgg tctccggaaa aaaaaaaaaa 3360aaaaaaaaaa aaaaaaaaaa aaaaa
338572879DNAHomo sapiens 7gtttcggccg ccgccatggc cgcggtggac
ctggagaagc tgcgggcgtc gggcgcgggc 60aaggccatcg gcgtcctgac cagcggcggc
gacgcgcaag gcatgaacgc tgctgtccgg 120gctgtgacgc gcatgggcat
ttatgtgggt gccaaagtct tcctcatcta cgagggctat 180gagggcctcg
tggagggagg tgagaacatc aagcaggcca actggctgag cgtctccaac
240atcatccagc tgggcggcac tatcattggc agcgctcgct gcaaggcctt
taccaccagg 300gaggggcgcc gggcagcggc ctacaacctg gtccagcacg
gcatcaccaa cctgtgcgtc 360atcggcgggg atggcagcct tacaggtgcc
aacatcttcc gcagcgagtg gggcagcctg 420ctggaggagc tggtggcgga
aggtaagatc tcagagacta cagcccggac ctactcgcac 480ctgaacatcg
cgggcctagt gggctccatc gataacgact tctgcggcac cgacatgacc
540atcggcacgg actcggccct ccaccgcatc atggaggtca tcgatgccat
caccaccact 600gcccagagcc accagaggac cttcgtgctg gaagtgatgg
gccggcactg cgggtacctg 660gcgctggtat ctgcactggc ctcaggggcc
gactggctgt tcatccccga ggctccaccc 720gaggacggct gggagaactt
catgtgtgag aggctgggtg agactcggag ccgtgggtcc 780cgactgaaca
tcatcatcat cgctgagggt gccattgacc gcaacgggaa gcccatctcg
840tccagctacg tgaaggacct ggtggttcag aggctgggct tcgacacccg
tgtaactgtg 900ctgggccacg tgcagcgggg agggacgccc tctgccttcg
accggatcct gagcagcaag 960atgggcatgg aggcggtgat ggcgctgctg
gaagccacgc ctgacacgcc ggcctgcgtg 1020gtcaccctct cggggaacca
gtcagtgcgg ctgcccctca tggagtgcgt gcagatgacc 1080aaggaagtgc
agaaagccat ggatgacaag aggtttgacg aggccaccca gctccgtggt
1140gggagcttcg agaacaactg gaacatttac aagctcctcg cccaccagaa
gccccccaag 1200gagaagtcta acttctccct ggccatcctg aatgtggggg
ccccggcggc tggcatgaat 1260gcggccgtgc gctcggcggt gcggaccggc
atctcccatg gacacacagt atacgtggtg 1320cacgatggct tcgaaggcct
agccaagggt caggtgcaag aagtaggctg gcacgacgtg 1380gccggctggt
tggggcgtgg tggctccatg ctggggacca agaggaccct gcccaagggc
1440cagctggagt ccattgtgga gaacatccgc atctatggta ttcacgccct
gctggtggtc 1500ggtgggtttg aggcctatga aggggtgctg cagctggtgg
aggctcgcgg gcgctacgag 1560gagctctgca tcgtcatgtg tgtcatccca
gccaccatca gcaacaacgt ccctggcacc 1620gacttcagcc tgggctccga
cactgctgta aatgccgcca tggagagctg tgaccgcatc 1680aaacagtctg
cctcggggac caagcgccgt gtgttcatcg tggagaccat ggggggttac
1740tgtggctacc tggccaccgt gactggcatt gctgtggggg ccgacgccgc
ctacgtcttc 1800gaggaccctt tcaacatcca cgacttaaag gtcaacgtgg
agcacatgac ggagaagatg 1860aagacagaca ttcagagggg cctggtgctg
cggaacgaga agtgccatga ctactacacc 1920acggagttcc tgtacaacct
gtactcatca gagggcaagg gcgtcttcga ctgcaggacc 1980aatgtcctgg
gccacctgca gcagggtggc gctccaaccc cctttgaccg gaactatggg
2040accaagctgg gggtgaaggc catgctgtgg ttgtcggaga agctgcgcga
ggtttaccgc 2100aagggacggg tgttcgccaa tgccccagac tcggcctgcg
tgatcggcct gaagaagaag 2160gcggtggcct tcagccccgt cactgagctc
aagaaagaca ctgatttcga gcaccgcatg 2220ccacgggagc agtggtggct
gagcctgcgg ctcatgctga agatgctggc acaataccgc 2280atcagtatgg
ccgcctacgt gtcaggggag ctggagcacg tgacccgccg caccctgagc
2340atggacaagg gcttctgagg ccagccatgc ccacgcccct ccccagcccc
cacccatgcc 2400agcgcagcgc cagggctcag atggggcctg ggctgttgtg
tctggagcct gcaggcaggt 2460gggggctgcg tccctgctca gcccatcccc
tgcctctatc cctggccacc tgccaggcct 2520ccctccggct ggtgtcttga
gaccagcctg ccaggccctc cagcaggagg acagagtgcc 2580ctggggcatc
caccttcctg cccaggggac gtggcgctgt cggtgtttgg aggctgctgc
2640cccctggctt tggcgcccca tgggccctca gcgtctcccc atgctgggct
cactacatgg 2700gccagccctt gctctacctg gccggtaggc tgctggcgcc
taggttgtgt tgagaggggg 2760atgcccctgg ccctgcctca ctgtgacctg
ctcctgccca cgtgcagcac ctgtcacctt 2820ttctagaaat aaaatcaccc
tgactgtggg gtgcatcggt caaaaaaaaa aaaaaaaaa 28798149DNAHomo sapiens
8acgcggcgca ggcggcggga gtgcgagctg ggcccgtgtt tcggccgccg ccatggccgc
60ggtggacctg gagaagctgc gggcgtcggg cgcgggcaag gccatcggcg tcctgaccag
120cggcggcgac cggcaaggtg gggcggggg 1499177DNAHomo sapiens
9tcctctgaga tggggagggt gtcagggcct tgcttctcag cgtgggagct gacaggtttg
60ccctgacctc cacaggcatg aacgctgctg tccgggctgt gacgcgcatg ggcatttatg
120tgggtgccaa agtcttcctc atctacgagg taaggccaag gtgggctgtg tgtgtgc
17710168DNAHomo sapiens 10agggacttgc tgccggccgc catgggttcc
cctatctcat gcctgcccac tcttgattca 60gggctatgag ggcctcgtgg agggaggtga
gaacatcaag caggccaact ggctgagcgt 120atccaacatc atccagctgg
tgaggcctgg gaacgcggat gcatgttg 16811299DNAHomo sapiens 11ccagtcctgg
gtccctctgg tgatcccagg gctgtctgcc gcctgccatc tctcctgaag 60tttctggtct
cctctgtgca gggcgcgact atcattggca cggctcgctc gaaggccttt
120accaccaggg aggggcgccg ggcagcggct aacaacctgg tccagcacgg
catcaccaac 180ctgtgcgtca tcggcgggga tggcagcctt acaggtgcca
acatcttccg cagcgagtgg 240ggcagcctgc tggaggagct ggtggcggaa
ggtgggtctg tgcccggcgc actgtaggc 29912349DNAHomo sapiens
12tgccacaggg ttcccaggca ggaggaggcc tgagcctgga actcccgggc cctgccgggc
60tgcacgcctg ggatgcgggg gaaggggtgg caggagaggg gtctctggcc ctgtggtggg
120gccagtggag cctcagccag gtcctcctgc tgctcctggc ccaggtaaga
tctcagagac 180tacagcccgg acctactcgc acctgaacat cgcgggccta
gtgggctcca tcgataacga 240cttctgcggc accgacatga ccatcggcac
ggactcggcc ctccaccgca tcatggaggt 300catcgatgcc atcaccacca
ctgcccagag gtgagtgagg ctggccgcc 34913606DNAHomo sapiens
13ctagtggatc ccaaggtctg ttttagctca gagcctgggc atgagaaggg gctgtccctg
60cctgcctctc catccactgg gtcccttgag caccccgcag aatcgggctg gcagggcgtg
120tggctggcac tgatgcatcc tcctgttcca tctccacagc caccagagga
ccttcgtgct 180ggaagtgatg ggccggcact gcgggtgagg aggggcttct
ggcccgctgg gtggcccggg 240tgctgctggg gaccgcagtg acaggtgtgg
catatttatg ctagggctca gttaatgcca 300tgggtgtgag agagccgggt
gggggcctga gcaggcaggc gctcgctcct ccaggtacct 360ggcgctggta
tctgcactgg cctcaggggc cgactggctg ttcatccccg aggctccacc
420cgaggctcca cccgaggacg gctgggagaa cttcatgtgt gagaggctgg
gtgaggtggg 480tgccgtccag cctgctgggg gccgcaggtg tcctggtgca
ctgggtagcg cccctggggt 540tttgggacca gccttgacca actcatctat
cactcatggg ttcatcagca gctgcaggtg 600cccctt 606141226DNAHomo sapiens
14tcactgagag tctgtttccc cctgtaacat gcgggttccc gccttgccca ccacccaggg
60tgcactgggg cagggagggt ggcacagctc tcctgctgga cctgcagccc atcttggccc
120tgtctgacct cacctgcact ggcgccacct cctccaggta gccctccact
cccaccacac 180ccgggccctg tctgagctgc tccaaccctg tccctgtagg
aggcattgtg gcccgtggtg 240ggcaaacatg gggcttgtgg ctcagagcga
gtcccacaaa tgttggctgg gtcattgaag 300tggacggccg aaaagctctt
tcagttccca gagagctggg ccgggctggt gtgtggtgct 360gaggcagcag
cctgaggctg gcagagggga tggtgtgtcc tatgtgcacc agtgtggacc
420cacagtggct tcagggtgca gggtgtccgg ggagccctgg ctggtgccag
cgatgcgggg 480cccctggtta taaggatgag cagatggaag ctcacagggg
ccccaggtac ctggcccagg 540ctagcccggg caggcccctc tgccctgtgc
cccgtggagc tgcagccctg tgcgtcttcc 600cgcctggaag cgttcaccag
acacaagggc ccagcccagc tgtgtgtgtg ctgggcccag 660cccgagccgg
cggggttggt ggggtgctgc ccttcccagg cgggcgggca gagctcttgt
720tcaccgcctc caggagggcg gggggtccta gtgggcccag cctcatctct
gccctcgcgc 780tccaggcctg ctttcctcct gccgggtggg gattgtgccc
tggcccatgt gggttgggaa 840tggtggcccc agggagggct cctggcaccc
gggggctgtg tccggggcag gtttccctgc 900ctggcagctg agccctgtcg
tgtctttgac ccagactcgg agccgtgggt cccgactgaa 960catcatcatc
atcgctgagg gtgccattga ccgcaacggg aagcccatct cgtccagcta
1020cgtgaaggac gtgcgtgtgg gcctgggggt ggccactggg cacctgctcc
tctaggccgt 1080gtgggctggg gctcagggct ggtccttccc actgtcctgc
agctggtggt tcagaggctg 1140ggcttcgaca cccgtgtaac tgtgctgggc
cacgtgcagc ggggagggac gccctctgcc 1200ttcgaccgga tcctggtaag tggcca
122615583DNAHomo sapiensmisc_feature(18)..(18)n is a, c, g, or t
15gctctagaac tagtggancc cccgggctgc aggaattcga tatcaagctt cgtcagcgtg
60gggggctctg gaagctgggg tttgcacatc tacagaggat gggcatgtgg cttggggtag
120agggaccaag tgggtgtgcc agcctgaacc cttccccaca gagcagcaag
atgggcatgg 180aggcggtgat ggcgctgctg gaagccacgc ctgacacgcc
ggcctgcgtg gtcaccctct 240cggggaacca gtcagtgcgg ctgcccctca
tggagtgcgt gcagatggta agccctgggc 300cccccccatc agaaccgcct
ggcccctctc cccagtcccc actcacaggc cccactgctc 360tctgggggcc
cccagcactg tgagcaccgg aggcagggcc tcgtggctgg cccagggcat
420cccaggtctc cagggagggg agggatgtga gcacatccct gggtgggacg
tngggacctg 480ggacgttccc caggaggtgt gtcggagctg cagggagcct
ggtgagcatg ggaagtcaca 540ggggtccact gccactgagc ttatgtaggc
agtggtggga gtt 58316239DNAHomo sapiens 16gtttctcttc cttaagacca
aggaagtgca gaaagccatg gatgacaaga ggtttgacga 60ggccacccag ctccgtggtg
ggtaagcccc ctcatgatac ccctgcactc ttacatggat 120gggtcccggt
gccaggcagc atgtgctcga gtggcgctat gcacgcctgg cctgggtcat
180ccttctaggc accgcgtctg aagatcgagg gaggaagggg cctgcgggtg gacaggagg
23917103DNAHomo sapiensmisc_feature(6)..(6)n is a, c, g, or t
17tgtgcntggt gtgacccgaa tcccttccag gagcttcgag aacaactgga acatttacag
60gctcctcacc caccagaagc cccccaagga gaaggtgagg cag 10318231DNAHomo
sapiensmisc_feature(30)..(31)n is a, c, g, or t 18cctcctgtgc
aggttggggg tcccctcccn nggctgtgcc tcacgctnat ctccccttct 60ctctgaagtc
taacttctcc ctggccatcc tgaatgtggg ggccccggcg gctggcatga
120atgcggccgt gcgctcggcg gtgcggaccg gcatctccca tggacacaca
gtatacgtgg 180tgcacgatgg cttcgaaggc ctagccaagg gtcaggtggg
tccggccggg g 23119181DNAHomo sapiens 19cccggcaaca ggcccaaccc
tggggggaat tggccagagg ctcaggctgg cccctgaagc 60tgcatgtcct cctggcaggt
gcaagaagta ggctggcacg acgtggccgg ctggttgggg 120cgtggtggct
ccatgctggg gaccaagagg tgagctgcct gctgcgggta cctggggacc 180t
18120110DNAHomo sapiens 20tttcttccgc aggaccctgc ccaagggcca
gctggagtcc attgtggaga acatccgcat 60ctatggtatt cacgccctgc tggtggtcgg
tgggtttgag gtgagagctg 11021208DNAHomo
sapiensmisc_feature(10)..(10)n is a, c, g, or t 21tcggtgctgn
ccttgacctg ccccgtccct actgctgcag gcctatgaag gggtgctgca 60gctggtggag
gctcgcgggc gctacgagga gctctgcatc gtcatgtgtg tcatcccagc
120caccatcagc aacaacgtcc ctggcaccga cttcagcctg ggctccgaca
ctgctgtaaa 180tgccgccatg gaggtacggn ctcctgga 20822199DNAHomo
sapiens 22accccccctt gtcccccaga gctgtgaccg catcaaacag tctgcctcgg
ggaccaagcg 60ccgtgtgttc atcgtggaga ccatgggggg ttactgtggc tacctggcca
ccgtgactgg 120cattgctgtg ggggccgacg ccgcctacgt cttcgaggac
cctttcaaca tccacgactt 180aaaggtgagc ccagcccag 19923174DNAHomo
sapiens 23tgctcctgct ggccccggat cgccggtcag cctggaattc cctccccaca
gtctccggct 60catccgtgtc cgcccctccc gcaggtcaac gtggagcaca tgacggagaa
gatgaagaca 120gacattcaga ggggcctggt gctgcggtga ggctgccgtg
ggtccctggc caca 17424144DNAHomo sapiensmisc_feature(9)..(9)n is a,
c, g, or t 24gactcaggnc ctgntgcccc ctctcaggaa cgagaagtgc catgactact
acaccacgga 60gttcctgtac aacctgtact catcagaggg caagggcgtc ttcgactgca
ggaccaatgt 120cctgggccac ctgcagcagg tgtg 14425207DNAHomo sapiens
25gatccccgat cctgtctgca ctggcgttgg ccttggccag gcagcccagg ggagtccagg
60gaaccgggcc tcacctgttt ccagggtggc gctccaaccc cctttgaccg gaactatggg
120accaagctgg gggtgaaggc catgctgtgg ttgtcggaga agctgcgcga
ggtttaccgc 180aagggtaggt ggtgggtgcg acccgag 20726327DNAHomo
sapiensmisc_feature(191)..(191)n is a, c, g, or t 26cctccctggg
gcagggcctc accatggagg gctgccacgt gcctctgttt gcaggacggg 60tgttcgccaa
tgccccagac tcggcctgcg tgatcggcct gaagaagaag gcggtggcct
120tcagccccgt cactgagctc aagaaagaca ctgatttcga gtgagttcca
ccaaagcctc 180gtggaggcgg ngtggggctg aggggtggcc cagaccttcc
ctgaggccgg tgtgccagac 240ccagccccac tggcaccctg accccgcaag
cctcctggcc ccatgtccag gtccccccag 300gccgtggaga gcagggacca tgcccaa
327271080DNAHomo sapiens 27gtggctgaag agctgccctg acccctgact
ccccatcatc ctcccatccc cgtcctgcac 60aggcaccgca tgccacggga gcagtggtgg
ctgagcctgc ggctcatgct gaagatgctg 120gcacaatacc gcatcagtat
ggccgcctac gtgtcagggg agctggagca cgtgacccgc 180cgcaccctga
gcatggacaa gggcttctga ggccagccat gcccgagctg cccctcccca
240gcccccaccc atgccagcgc acgcgccagg gctcagatgg ggcctgggct
gttgtgtctg 300gagcctgcag gcaggtgggg gctgcgtccc tgctcagccc
atcccctgcc tctactccct 360ggccacctgc caggcctccc tccggctggt
gtcttgagac cagcctgcca ggcctccagc 420aggaggacag agtgccctgg
ggcatccacc ttcctgccca ggggacgtgg cgctgtcggt 480gtttggaggc
tgctgccccc tggctttggc gccccatggg ccctcagcgt ctccccatgc
540tgggctcact acatgggcca gcccttgctc tacctggccg gtaggctgct
ggcgcctagg 600ttgtgttgag agggggatgc ccctggccct gcctcactgt
gacctgctcc tgcccacgtg 660cagcacctgt caccttttct agaaataaaa
tcaccctgac tgtggggtgc catcggtctc 720cggagagcac agctgcagaa
ctcctcaccg agcggccacg ccagtggcag cagccccagg 780ggtggaggcc
ctctggccag tgcctgggac aggtcaaagg gacatgtgcc ctgagaggcc
840acaggtgctc ttcaggactc cctgggggcc accagggtga cctgagcccc
tcctggtcct 900cccctggggg cagaagggta cagcctcact cctctgtctc
ccaacctcag cctgagtggg 960ggtctccaac ctgcaggctg gtggctggct
tgagccagta tccaggagac attgatggtg 1020gtctcgcaag aggggaaaag
aaggcacggc agagctgcgc ccaccagggg ctagggctga 1080282815DNAHomo
sapiens 28gtttcggccg ccgccatggc cgcggtggac ctggagaagc tgcgggcgtc
gggcgcgggc 60aaggccatcg gcgtcctgac cagcggcggc gacgcgcaag gcatgaacgc
tgctgtccgg 120gctgtgacgc gcatgggcat ttatgtgggt gccaaagtct
tcctcatcta cgagggctat 180gagggcctcg tggagggagg tgagaacatc
aagcaggcca actggctgag cgtctccaac 240atcatccagc tgggcggcac
tatcattggc agcgctcgct gcaaggcctt taccaccaac 300ctgtgcgtca
tcggcgggga tggcagcctc acaggtgcca acatcttccg cagcgagtgg
360ggcagcctgc tggaggagct ggtggcggaa ggtaagatct cagagactac
agcccggacc 420tactcgcacc tgaacatcgc gggcctagtg ggctccatcg
ataacgactt ctgcggcacc 480gacatgacca tcggcacgga ctcggccctc
caccgcatca tggaggtcat cgatgccatc 540accaccactg cccagagcca
ccagaggacc ttcgtgctgg aagtgatggg ccggcactgc 600gggtacctgg
cgctggtatc tgcactggcc tcaggggccg actggctgtt catccccgag
660gctccacccg aggacggctg ggagaacttc atgtgtgaga ggctgggtga
gactcggagc 720cgtgggtccc gactgaacat catcatcatc gctgagggtg
ccattgaccg caacgggaag 780cccatctcgt ccagctacgt gaaggacctg
gtggttcaga
ggctgggctt cgacacccgt 840gtaactgtgc tgggccacgt gcagcgggga
gggacgccct ctgccttcga ccggatcctg 900agcagcaaga tgggcatgga
ggcggttatg gcgctgctgg aagccacgcc tgacacgccg 960gcctgcgtgg
tcaccctctc ggggaaccag tcagtgcggc tgcccctcat ggagtgcgtg
1020cagatgacca aggaagtgca gaaagccatg gatgacaaga ggtttgacga
ggccacccag 1080ctccgtggtg ggagcttcga gaacaactgg aacatttaca
agctcctcgc ccaccagaag 1140ccccccaagg agaagtctaa cttctccctg
gccatcctga atgtgggggc cccggcggct 1200ggcatgaatg cggccgtgcg
ctcggcggtg cggaccggca tctcccatgg acacacagta 1260tacgtggtgc
acgatggctt cgaaggccta gccaagggtc aggtgcaaga agtaggctgg
1320cacgacctgg ccggctggtt ggggcgtggt ggctccatgc tggggaccaa
gaggaccctg 1380cccaagggcc agctggagtc cattgtggag aacatccgca
tctatggtat tcacgccctg 1440ctggtggtcg gtgggtttga ggcctatgaa
ggggtgctgc agctggtgga ggctcgcggg 1500cgctacgagg agctctgcat
cgtcatgtgt gtcatcccag ccaccatcag caacaacgtc 1560cctggcaccg
acttcagcct gggctccgac actgctgtaa atgccgccat ggagagctgt
1620gaccgcatca aacagtctgc ctcggggacc aagcgccgtg tgttcatcgt
ggagaccatg 1680gggggttact gtggctacct ggccaccgtg actggcattg
ctgtgggggc cgacgccgcc 1740tacgtcttcg aggacccttt caacatccac
gacttaaagg tcaacgtgga gcacatgacg 1800gagaagatga agacagacat
tcagaggggc ctggtgctgc ggaacgagaa gtgccatgac 1860tactacacca
cggagttcct gtacaacctg tactcatcag agggcaaggg cgtcttcgac
1920tgcaggacca atgtcctggg ccacctgcag cagggtggcg ctccaacccc
ctttgaccgg 1980aactatggga ccaagctggg ggtggaggcc atgctgtggt
tgtcggagaa gctgcgcgag 2040gtttaccgca agggacgggt gttcgccaat
gccccagact cggcctgcgt gatcggcctg 2100aagaagaagg cggtggcctt
cagccccgtc actgagctca agaaagacac tgatttcgag 2160caccgcatgc
cacgggagca gtggtggctg agcctgcggc tcatgctgaa gatgctggca
2220caataccgca tcagtatggc cgcctacgtg tcaggggagc tggagcacgt
gacccgccgc 2280accctgagca tggacaaggg cttctgaggc cagccatgcc
cacgcccctc cccagccccc 2340acccatgcca gcgcagcgcc agggctcaga
tggggcctgg gctgttgtgt ctggagcctg 2400caggcaggtg ggggctgcgt
ccctgctcag cccatcccct gcctctatcc ctggccacct 2460gccaggcctc
cctccggctg gtgtcttgag accagcctgc caggccctcc agcaggagga
2520cagagtgccc tggggcatcc accttcctgc ccaggggacg tggcgctgtc
ggtgtttgga 2580ggctgctgcc ccctggcttt ggcgccccat gggccctcag
cgtctcccca tgctgggctc 2640actacatggg ccagcccttg ctctacctgg
ccggtaggct gctggcgcct aggttgtgtt 2700gagaggggga tgcccctggc
cctgcctcac tgtgacctgc tcctgcccac gtgcagcacc 2760tgtcaccttt
tctagaaata aaatcaccct gactgtgggg tgcatcggtc tccgg 2815293402DNAHomo
sapiens 29gacggcgacg cggcgcaggc ggcgggagtg cgagctgggc ccgtgtttcg
gccgccgcca 60tggccgcggt ggacctggag aagctgcggg cgtcgggcgc gggcaaggcc
atcggcgtcc 120tgaccagcgg cggcgacgcg caaggtcccc tgacaagccc
accaggcccc ctgctgagat 180ggctgtgacc ctgggctgac ccgcccagtg
gcacattgac tccgcctgga gctggggaga 240ccagagaggc cctgtggttg
gacggtggcc tgggtgcgct gctcctgccc tctccttgcc 300ctgcctcagc
tgctgcctgc cagaggcgtg gcacctcacc tcacacctgc tccctgctgc
360tgagccccac gccaagctgg agagcggatg agaagcatgt gtaaccaggg
tagaggtcga 420gagtcctctc gtgggggtct ccatgttcaa gggagctgcc
gaggcttgag caggagcccc 480cagcaggaaa ctggctttgc caaggccccc
gctgggacag actgtttctt tcactgcagt 540cctgggagcc gagggcaagg
ggacaggaaa gaggaagtga cctcagagcc tggtggcacc 600agcatcatgt
ccaggctggg gggcatgaac gctgctgtcc gggctgtgac gcgcatgggc
660atttatgtgg gtgccaaagt cttcctcatc tacgagggct atgagggcct
cgtggaggga 720ggtgagaaca tcaagcaggc caactggctg agcgtctcca
acatcatcca gctgggcggc 780actatcattg gcagcgctcg ctgcaaggcc
tttaccacca gggaggggcg ccgggcagcg 840gcctacaacc tggtccagca
cggcatcacc aacctgtgcg tcatcggcgg ggatggcagc 900ctcacaggtg
ccaacatctt ccgcagcgag tggggcagcc tgctggagga gctggtggcg
960gaaggtaaga tctcagagac tacagcccgg acctactcgc acctgaacat
cgcgggccta 1020gtgggctcca tcgataacga cttctgcggc accgacatga
ccatcggcac ggactcggcc 1080ctccaccgca tcatggaggt catcgatgcc
atcaccacca ctgcccagag ccaccagagg 1140accttcgtgc tggaagtgat
gggccggcac tgcgggtacc tggcgctggt atctgcactg 1200gcctcagggg
ccgactggct gttcatcccc gaggctccac ccgaggacgg ctgggagaac
1260ttcatgtgtg agaggctggg tgagactcgg agccgtgggt cccgactgaa
catcatcatc 1320atcgctgagg gtgccattga ccgcaacggg aagcccatct
cgtccagcta cgtgaaggac 1380ctggtggttc agaggctggg cttcgacacc
cgtgtaactg tgctgggcca cgtgcagcgg 1440ggagggacgc cctctgcctt
cgaccggatc ctgagcagca agatgggcat ggaggcggtg 1500atggcgctgc
tggaagccac gcctgacacg ccggcctgcg tggtcaccct ctcggggaac
1560cagtcagtgc ggctgcccct catggagtgc gtgcagatga ccaaggaagt
gcagaaagcc 1620atggatgaca agaggtttga cgaggccacc cagctccgtg
gtgggagctt cgagaacaac 1680tggaacattt acaagctcct cgcccaccag
aagcccccca aggagaagtc taacttctcc 1740ctggccatcc tgaatgtggg
ggccccggcg gctggcatga atgcggccgt gcgctcggcg 1800gtgcggaccg
gcatctccca tggacacaca gtatacgtgg tgcacgatgg cttcgaaggc
1860ctagccaagg gtcaggtgca agaagtaggc tggcacgacg tggccggctg
gttggggcgt 1920ggtggctcca tgctggggac caagaggacc ctgcccaagg
gccagctgga gtccattgtg 1980gagaacatcc gcatctatgg tattcacgcc
ctgctggtgg tcggtgggtt tgaggcctat 2040gaaggggtgc tgcagctggt
ggaggctcgc gggcgctacg aggagctctg catcgtcatg 2100tgtgtcatcc
cagccaccat cagcaacaac gtccctggca ccgacttcag cctgggctcc
2160gacactgctg taaatgccgc catggagagc tgtgaccgca tcaaacagtc
tgcctcgggg 2220accaagcgcc gtgtgttcat cgtggagacc atggggggtt
actgtggcta cctggccacc 2280gtgactggca ttgctgtggg ggccgacgcc
gcctacgtct tcgaggaccc tttcaacatc 2340cacgacttaa aggtcaacgt
ggagcacatg acggagaaga tgaagacaga cattcagagg 2400ggcctggtgc
tgcggaacga gaagtgccat gactactaca ccacggagtt cctgtacaac
2460ctgtactcat cagagggcaa gggcgtcttc gactgcagga ccaatgtcct
gggccacctg 2520cagcagggtg gcgctccaac cccctttgac cggaactatg
ggaccaagct gggggtgaag 2580gccatgctgt ggttgtcgga gaagctgcgc
gaggtttacc gcaagggacg ggtgttcgcc 2640aatgccccag actcggcctg
cgtgatcggc ctgaagaaga aggcggtggc cttcagcccc 2700gtcactgagc
tcaagaaaga cactgatttc gagcaccgca tgccacggga gcagtggtgg
2760ctgagcctgc ggctcatgct gaagatgctg gcacaatacc gcatcagtat
ggccgcctac 2820gtgtcagggg agctggagca cgtgacccgc cgcaccctga
gcatggacaa gggcttctga 2880ggccagccat gcccacgccc ctccccagcc
cccacccatg ccagcgcagc gccagggctc 2940agatggggcc tgggctgttg
tgtctggagc ctgcaggcag gtgggggctg cgtccctgct 3000cagcccatcc
cctgcctcta tccctggcca cctgccaggc ctccctcggg ctggtgtctt
3060gagaccagcc tgccaggccc tccagcagga ggacagagtg ccctggggca
tccaccttcc 3120tgcccagggg acgtggcgct gtcggtgttt ggaggctgct
gccccctggc tttggcgccc 3180catgggccct cagcgtctcc ccatgctggg
ctcactacat gggccagccc ttgctctacc 3240tggccggtag gctgctggcg
cctaggttgt gttgagaggg ggatgcccct ggccctgcct 3300cactgtgacc
tgctcctgcc cacgtgcagc acctgtcacc ttttctagaa ataaaatcac
3360cctgactgtg gggtgcatcg gtctccggag aaaacaaaaa aa
3402302812DNAHomo sapiens 30ctaaaagagt ggatcatgac ccatgaagag
caccatgcag ccaaaaccct ggggattggc 60aaagccattg ctgtcttaac ctctggtgga
gatgcccaag gtatgaatgc tgctgtcagg 120gctgtggttc gagttggtat
cttcaccggt gcccgtgtct tctttgtcca tgagggttat 180caaggcctgg
tggatggtgg agatcacatc aaggaagcca cctgggagag cgtttcgatg
240atgcttcagc tgggaggcac ggtgattgga agtgcccggt gcaaggactt
tcgggaacga 300gaaggacgac tccgagctgc ctacaacctg gtgaagcgtg
ggatcaccaa tctctgtgtc 360attgggggtg atggcagcct cactggggct
gacaccttcc gttctgagtg gagtgacttg 420ttgagtgacc tccagaaagc
aggtaagatc acagatgagg aggctacgaa gtccagctac 480ctgaacattg
tgggcctggt tgggtcaatt gacaatgact tctgtggcac cgatatgacc
540attggcactg actctgccct gcatcggatc atggaaattg tagatgccat
cactaccact 600gcccagagcc accagaggac atttgtgtta gaagtaatgg
gccgccactg tggatacctg 660gcccttgtca cctctctgtc ctgtggggcc
gactgggttt ttattcctga atgtccacca 720gatgacgact gggaggaaca
cctttgtcgc cgactcagcg agacaaggac ccgtggttct 780cgtctcaaca
tcatcattgt ggctgagggt gcaattgaca agaatggaaa accaatcacc
840tcagaagaca tcaagaatct ggtggttaag cgtctgggat atgacacccg
ggttactgtc 900ttggggcatg tgcagagggg tgggacgcca tcagcctttg
acagaattct gggcagcagg 960atgggtgtgg aagcagtgat ggcacttttg
gaggggaccc cagatacccc agcctgtgta 1020gtgagcctct ctggtaacca
ggctgtgcgc ctgcccctca tggaatgtgt ccaggtgacc 1080aaagatgtga
ccaaggccat ggatgagaag aaatttgacg aagccctgaa gctgagaggc
1140cggagcttca tgaacaactg ggaggtgtac aagcttctag ctcatgtcag
acccccggta 1200tctaagagtg gttcgcacac agtggctgtg atgaacgtgg
gggctccggc tgcaggcatg 1260aatgctgctg ttcgctccac tgtgaggatt
ggccttatcc agggcaaccg agtgctcgtt 1320gtccatgatg gtttcgaggg
cctggccaag gggcagatag aggaagctgg ctggagctat 1380gttgggggct
ggactggcca aggtggctct aaacttggga ctaaaaggac tctacccaag
1440aagagctttg aacagatcag tgccaatata actaagttta acattcaggg
ccttgtcatc 1500attgggggct ttgaggctta cacagggggc ctggaactga
tggagggcag gaagcagttt 1560gatgagctct gcatcccatt tgtggtcatt
cctgctacag tctccaacaa tgtccctggc 1620tcagacttca gcgttggggc
tgacacagca ctcaatacta tctgcacaac ctgtgaccgc 1680atcaagcagt
cagcagctgg caccaagcgt cgggtgttta tcattgagac tatgggtggc
1740tactgtggct acctggctac catggctgga ctggcagctg gggccgatgc
tgcctacatt 1800tttgaggagc ccttcaccat tcgagacctg caggcaaatg
ttgaacatct ggtgcaaaag 1860atgaaaacaa ctgtgaaaag gggcttggtg
ttaaggaatg aaaagtgcaa tgagaactat 1920accactgact tcattttcaa
cctgtactct gaggagggga agggcatctt cgacagcagg 1980aagaatgtgc
ttggtcacat gcagcagggt gggagcccaa ccccatttga taggaatttt
2040gccactaaga tgggcgccaa ggctatgaac tggatgtctg ggaaaatcaa
agagagttac 2100cgtaatgggc ggatctttgc caatactcca gattcgggct
gtgttctggg gatgcgtaag 2160agggctctgg tcttccaacc agtggctgag
ctgaaggacc agacagattt tgagcatcga 2220atccccaagg aacagtggtg
gctgaaactg aggcccatcc tcaaaatcct agccaagtac 2280gagattgact
tggacacttc agaccatgcc cacctggagc acatcacccg gaagcggtcc
2340ggggaagctg ccgtctaaac ctctctggag tgaggggaat agattacctg
atcatggtca 2400gctcacaccc taataagtcc acatcttctc agtgttttag
ctgttttttt cattaggttt 2460ccttttattc tgtaccttgc agccatgacc
agttctggcc aggagctgga ggagcaggca 2520gtgggtggga gctcctttta
ggtagaattt aacatgactt ctgccccagc tttatctgtc 2580acacaaggct
gggcacctct agtgctactg ctagatatca cttactcagt tagaattttc
2640ctaaaaataa gctttattta tttctttgtg ataacaaaga gtcttggttc
ctctactact 2700tttactacag tgacaaattg taactacact aataaatgcc
aactggtcac tgtgaaaaaa 2760aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aa 2812312759DNAHomo sapiens 31gcctgactga
gagtggatca tgacccatga agagcaccat gcagccaaaa ccctggggat 60tggcaaagcc
attgctgtct taacctctgg tggagatgcc caaggtatga atgctgctgt
120cagggctgtg gttcgagttg gtatcttcac cggtgcccgt gtcttctttg
tccatgaggg 180ttatcaaggc ctggtggatg gtggagatca catcaaggaa
gccacctggg agagcgtttc 240gatgatgctt cagctgggag gcacggtgat
tggaagtgcc cggtgcaagg actttcggga 300acgagaagga cgactccgag
ctgcctacaa cctggtgaag cgtgggatca ccaatctctg 360tgtcattggg
ggtgatggca gcctcactgg ggctgacacc ttccgttctg agtggagtga
420cttgttgagt gacctccaga aagcaggtaa gatcacagat gaggaggcta
cgaagtccag 480ctacctgaac attgtgggcc tggttgggtc aattgacaat
gacttctgtg gcactgatat 540gaccattggc actgactctg ccctgcatcg
gatcatggaa attgtagatg ccatcactac 600cactgcccag agccaccaga
ggacatttgt gttagaagta atgggccgcc actgtggata 660cctggccctt
gtcacctctc tgtcctgtgg ggccgactgg gtttttattc ctgaatgtcc
720accagatgac gactgggagg aacacctttg tcgccgactc agcgagacaa
ggacccgtgg 780ttctcgtctc aacatcatca ttgtggctga gggtgcaatt
gacaagaatg gaaaaccaat 840cacctcagaa gacatcaaga atctggtggt
taagcgtctg ggatatgaca cccgggttac 900tgtcttgggg catgtgcaga
ggggtgggac gccatcagcc tttgacagaa ttctgggcag 960caggatgggt
gtggaagcag tgatggcact tttggagggg accccagata ccccagcctg
1020tgtagtgagc ctctctggta accaggctgt gcgcctgccc ctcatggaat
gtgtccaggt 1080gaccaaagat gtgaccaagg ccatggatga gaagaaattt
gacgaagccc tgaagctgag 1140aggccggagc ttcatgaaca actgggaggt
gtacaagctt ctagctcatg tcagaccccc 1200ggtatctaag agtggttcgc
acacagtggc tgtgatgaac gtgggggctc cggctgcagg 1260catgaatgct
gctgttcgct ccactgtgag gattggcctt atccagggca accgagtgct
1320cgttgtccat gatggtttcg agggcctggc caaggggcag atagaggaag
ctggctggag 1380ctatgttggg ggctggactg gccaaggtgg ctctaaactt
gggactaaaa ggactctacc 1440caagaagagc tttgaacaga tcagtgccaa
tataactaag tttaacattc agggccttgt 1500catcattggg ggctttgagg
cttacacagg gggcctggaa ctgatggagg gcaggaagca 1560gtttgatgag
ctctgcatcc catttgtggt cattcctgct acagtctcca acaatgtccc
1620tggctcagac ttcagcgttg gggctgacac agcactcaat actatctgca
caacctgtga 1680ccgcatcaag cagtcagcag ctggcaccaa gcgtcgggtg
tttatcattg agactatggg 1740tggctactgt ggctacctgg ctaccatggc
tggactggca gctggggccg atgctgccta 1800catttttgag gagcccttca
ccattcgaga cctgcaggca aatgttgaac atctggtgca 1860aaagatgaaa
acaactgtga aaaggggctt ggtgttaagg aatgaaaagt gcaatgagaa
1920ctataccact gacttcattt tcaacctgta ctctgaggag gggaagggca
tcttcgacag 1980caggaagaat gtgcttggtc acatgcagca gggtgggagc
ccaaccccat ttgataggaa 2040ttttgccact aagatgggcg ccaaggctat
gaactggatg tctgggaaaa tcaaagagag 2100ttaccgtaat gggcggatct
ttgccaatac tccagattcg ggctgtgttc tggggatgcg 2160taagagggct
ctggtcttcc aaccagtggc tgagctgaag gaccagacag attttgagca
2220tcgaatcccc aaggaacagt ggtggctgaa actgaggccc atcctcaaaa
tcctagccaa 2280gtacgagatt gacttggaca cttcagacca tgcccacctg
gagcacatca cccggaagcg 2340gtccggggaa gctgccgtct aaacctctct
ggagtgaggg gaatagatta cctgatcatg 2400gtcagctcac accctaataa
gtccacatct tctcagtgtt ttagctgttt ttttcattag 2460gtttcctttt
attctgtacc ttgcagccat gaccagttct ggccaggagc tggaggagca
2520ggcagtgggt gggagctcct tttaggtaga atttaacatg acttctgccc
cagctttatc 2580tgtcacacaa ggctgggcac ctctagtgct actgctagat
atcacttact cagttagaat 2640tttcctaaaa ataagcttta tttatttctt
tgtgataaca aagagtcttg gttcctctac 2700tacttttact acagtgacaa
attgtaacta cactaataaa tgccaactgg tcactgtga 2759322821DNAHomo
sapiens 32ggcacgaggc taaaagagtg gatcatgacc catgaagagc accatgcagc
caaaaccctg 60gggattggca aagccattgc tgtcttaacc tctggtggag atgcccaagg
tatgaatgct 120gctgtcaggg ctgtggttcg agttggtatc ttcaccggtg
cccgtgtctt ctttgtccat 180gagggttatc aaggcctggt ggatggtgga
gatcacatca aggaagccac ctgggagagc 240gtttcgatga tgcttcagct
gggaggcacg gtgattggaa gtgcccggtg caaggacttt 300cgggaacgag
aaggacgact ccgagctgcc tacaacctgg tgaagcgtgg gatcaccaat
360ctctgtgtca ttgggggtga tggcagcctc actggggctg acaccttccg
ttctgagtgg 420agtgacttgt tgagtgacct ccagaaagca ggtaagatca
cagatgagga ggctacgaag 480tccagctacc tgaacattgt gggcctggtt
gggtcaattg acaatgactt ctgtggcacc 540gatatgacca ttggcactga
ctctgccctg catcggatca tggaaattgt agatgccatc 600actaccactg
cccagagcca ccagaggaca tttgtgttag aagtaatggg ccgccactgt
660ggatacctgg cccttgtcac ctctctgtcc tgtggggccg actgggtttt
tattcctgaa 720tgtccaccag atgacgactg ggaggaacac ctttgtcgcc
gactcagcga gacaaggacc 780cgtggttctc gtctcaacat catcattgtg
gctgagggtg caattgacaa gaatggaaaa 840ccaatcacct cagaagacat
caagaatctg gtggttaagc gtctgggata tgacacccgg 900gttactgtct
tggggcatgt gcagaggggt gggacgccat cagcctttga cagaattctg
960ggcagcagga tgggtgtgga agcagtgatg gcacttttgg aggggacccc
agatacccca 1020gcctgtgtag tgagcctctc tggtaaccag gctgtgcgcc
tgcccctcat ggaatgtgtc 1080caggtgacca aagatgtgac caaggccatg
gatgagaaga aatttgacga agccctgaag 1140ctgagaggcc ggagcttcat
gaacaactgg gaggtgtaca agcttctagc tcatgtcaga 1200cccccggtat
ctaagagtgg ttcgcacaca gtggctgtga tgaacgtggg ggctccggct
1260gcaggcatga atgctgctgt tcgctccact gtgaggattg gccttatcca
gggcaaccga 1320gtgctcgttg tccatgatgg tttcgagggc ctggccaagg
ggcagataga ggaagctggc 1380tggagctatg ttgggggctg gactggccaa
ggtggctcta aacttgggac taaaaggact 1440ctacccaaga agagctttga
acagatcagt gccaatataa ctaagtttaa cattcagggc 1500cttgtcatca
ttgggggctt tgaggcttac acagggggcc tggaactgat ggagggcagg
1560aagcagtttg atgagctctg catcccattt gtggtcattc ctgctacagt
ctccaacaat 1620gtccctggct cagacttcag cgttggggct gacacagcac
tcaatactat ctgcacaacc 1680tgtgaccgca tcaagcagtc agcagctggc
accaagcgtc gggtgtttat cattgagact 1740atgggtggct actgtggcta
cctggctacc atggctggac tggcagctgg ggccgatgct 1800gcctacattt
ttgaggagcc cttcaccatt cgagacctgc aggcaaatgt tgaacatctg
1860gtgcaaaaga tgaaaacaac tgtgaaaagg ggcttggtgt taaggaatga
aaagtgcaat 1920gagaactata ccactgactt cattttcaac ctgtactctg
aggaggggaa gggcatcttc 1980gacagcagga agaatgtgct tggtcacatg
cagcagggtg ggagcccaac cccatttgat 2040aggaattttg ccactaagat
gggcgccaag gctatgaact ggatgtctgg gaaaatcaaa 2100gagagttacc
gtaatgggcg gatctttgcc aatactccag attcgggctg tgttctgggg
2160atgcgtaaga gggctctggt cttccaacca gtggctgagc tgaaggacca
gacagatttt 2220gagcatcgaa tccccaagga acagtggtgg ctgaaactga
ggcccatcct caaaatccta 2280gccaagtacg agattgactt ggacacttca
gaccatgccc acctggagca catcacccgg 2340aagcggtccg gggaagctgc
cgtctaaacc tctctggagt gaggggaata gattacctga 2400tcatggtcag
ctcacaccct aataagtcca catcttctca gtgttttagc tgtttttttc
2460attaggtttc cttttattct gtaccttgca gccatgacca gttctggcca
ggagctggag 2520gagcaggcag tgggtgggag ctccttttag gtagaattta
acatgacttc tgccccagct 2580ttatctgtca cacaaggctg ggcacctcta
gtgctactgc tagatatcac ttactcagtt 2640agaattttcc taaaaataag
ctttatttat ttctttgtga taacaaagag tcttggttcc 2700tctactactt
ttactacagt gacaaattgt aactacacta ataaatgcca actggtcact
2760gtgaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 2820a 2821332821DNAHomo sapiens 33ggcacgaggc taaaagagtg
gatcatgacc catgaagagc accatgcagc caaaaccctg 60gggattggca aagccattgc
tgtcttaacc tctggtggag atgcccaagg tatgaatgct 120gctgtcaggg
ctgtggttcg agttggtatc ttcaccggtg cccgtgtctt ctttgtccat
180gagggttatc aaggcctggt ggatggtgga gatcacatca aggaagccac
ctgggagagc 240gtttcgatga tgcttcagct gggaggcacg gtgattggaa
gtgcccggtg caaggacttt 300cgggaacgag aaggacgact ccgagctgcc
tacaacctgg tgaagcgtgg gatcaccaat 360ctctgtgtca ttgggggtga
tggcagcctc actggggctg acaccttccg ttctgagtgg 420agtgacttgt
tgagtgacct ccagaaagca ggtaagatca cagatgagga ggctacgaag
480tccagctacc tgaacattgt gggcctggtt gggtcaattg acaatgactt
ctgtggcacc 540gatatgacca ttggcactga ctctgccctg catcggatca
tggaaattgt agatgccatc 600actaccactg cccagagcca ccagaggaca
tttgtgttag aagtaatggg ccgccactgt 660ggatacctgg cccttgtcac
ctctctgtcc tgtggggccg actgggtttt tattcctgaa 720tgtccaccag
atgacgactg ggaggaacac ctttgtcgcc gactcagcga gacaaggacc
780cgtggttctc gtctcaacat catcattgtg gctgagggtg caattgacaa
gaatggaaaa 840ccaatcacct cagaagacat caagaatctg gtggttaagc
gtctgggata tgacacccgg 900gttactgtct tggggcatgt gcagaggggt
gggacgccat cagcctttga cagaattctg 960ggcagcagga tgggtgtgga
agcagtgatg gcacttttgg aggggacccc agatacccca 1020gcctgtgtag
tgagcctctc tggtaaccag gctgtgcgcc tgcccctcat ggaatgtgtc
1080caggtgacca aagatgtgac caaggccatg gatgagaaga aatttgacga
agccctgaag 1140ctgagaggcc ggagcttcat gaacaactgg gaggtgtaca
agcttctagc tcatgtcaga 1200cccccggtat ctaagagtgg ttcgcacaca
gtggctgtga tgaacgtggg ggctccggct 1260gcaggcatga atgctgctgt
tcgctccact gtgaggattg gccttatcca gggcaaccga 1320gtgctcgttg
tccatgatgg tttcgagggc ctggccaagg ggcagataga ggaagctggc
1380tggagctatg ttgggggctg gactggccaa ggtggctcta aacttgggac
taaaaggact 1440ctacccaaga agagctttga acagatcagt gccaatataa
ctaagtttaa cattcagggc 1500cttgtcatca ttgggggctt tgaggcttac
acagggggcc tggaactgat ggagggcagg 1560aagcagtttg atgagctctg
catcccattt gtggtcattc ctgctacagt ctccaacaat 1620gtccctggct
cagacttcag cgttggggct gacacagcac tcaatactat ctgcacaacc
1680tgtgaccgca tcaagcagtc agcagctggc accaagcgtc gggtgtttat
cattgagact 1740atgggtggct actgtggcta cctggctacc atggctggac
tggcagctgg ggccgatgct 1800gcctacattt ttgaggagcc cttcaccatt
cgagacctgc aggcaaatgt tgaacatctg 1860gtgcaaaaga tgaaaacaac
tgtgaaaagg ggcttggtgt taaggaatga aaagtgcaat 1920gagaactata
ccactgactt cattttcaac ctgtactctg aggaggggaa gggcatcttc
1980gacagcagga agaatgtgct tggtcacatg cagcagggtg ggagcccaac
cccatttgat 2040aggaattttg ccactaagat gggcgccaag gctatgaact
ggatgtctgg gaaaatcaaa 2100gagagttacc gtaatgggcg gatctttgcc
aatactccag attcgggctg tgttctgggg 2160atgcgtaaga gggctctggt
cttccaacca gtggctgagc tgaaggacca gacagatttt 2220gagcatcgaa
tccccaagga acagtggtgg ctgaaactga ggcccatcct caaaatccta
2280gccaagtacg agattgactt ggacacttca gaccatgccc acctggagca
catcacccgg 2340aagcggtccg gggaagctgc cgtctaaacc tctctggagt
gaggggaata gattacctga 2400tcatggtcag ctcacaccct aataagtcca
catcttctca gtgttttagc tgtttttttc 2460attaggtttc cttttattct
gtaccttgca gccatgacca gttctggcca ggagctggag 2520gagcaggcag
tgggtgggag ctccttttag gtagaattta acatgacttc tgccccagct
2580ttatctgtca cacaaggctg ggcacctcta gtgctactgc tagatatcac
ttactcagtt 2640agaattttcc taaaaataag ctttatttat ttctttgtga
taacaaagag tcttggttcc 2700tctactactt ttactacagt gacaaattgt
aactacacta ataaatgcca actggtcact 2760gtgaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2820a 2821342821DNAHomo
sapiens 34ggcacgaggc taaaagagtg gatcatgacc catgaagagc accatgcagc
caaaaccctg 60gggattggca aagccattgc tgtcttaacc tctggtggag atgcccaagg
tatgaatgct 120gctgtcaggg ctgtggttcg agttggtatc ttcaccggtg
cccgtgtctt ctttgtccat 180gagggttatc aaggcctggt ggatggtgga
gatcacatca aggaagccac ctgggagagc 240gtttcgatga tgcttcagct
gggaggcacg gtgattggaa gtgcccggtg caaggacttt 300cgggaacgag
aaggacgact ccgagctgcc tacaacctgg tgaagcgtgg gatcaccaat
360ctctgtgtca ttgggggtga tggcagcctc actggggctg acaccttccg
ttctgagtgg 420agtgacttgt tgagtgacct ccagaaagca ggtaagatca
cagatgagga ggctacgaag 480tccagctacc tgaacattgt gggcctggtt
gggtcaattg acaatgactt ctgtggcacc 540gatatgacca ttggcactga
ctctgccctg catcggatca tggaaattgt agatgccatc 600actaccactg
cccagagcca ccagaggaca tttgtgttag aagtaatggg ccgccactgt
660ggatacctgg cccttgtcac ctctctgtcc tgtggggccg actgggtttt
tattcctgaa 720tgtccaccag atgacgactg ggaggaacac ctttgtcgcc
gactcagcga gacaaggacc 780cgtggttctc gtctcaacat catcattgtg
gctgagggtg caattgacaa gaatggaaaa 840ccaatcacct cagaagacat
caagaatctg gtggttaagc gtctgggata tgacacccgg 900gttactgtct
tggggcatgt gcagaggggt gggacgccat cagcctttga cagaattctg
960ggcagcagga tgggtgtgga agcagtgatg gcacttttgg aggggacccc
agatacccca 1020gcctgtgtag tgagcctctc tggtaaccag gctgtgcgcc
tgcccctcat ggaatgtgtc 1080caggtgacca aagatgtgac caaggccatg
gatgagaaga aatttgacga agccctgaag 1140ctgagaggcc ggagcttcat
gaacaactgg gaggtgtaca agcttctagc tcatgtcaga 1200cccccggtat
ctaagagtgg ttcgcacaca gtggctgtga tgaacgtggg ggctccggct
1260gcaggcatga atgctgctgt tcgctccact gtgaggattg gccttatcca
gggcaaccga 1320gtgctcgttg tccatgatgg tttcgagggc ctggccaagg
ggcagataga ggaagctggc 1380tggagctatg ttgggggctg gactggccaa
ggtggctcta aacttgggac taaaaggact 1440ctacccaaga agagctttga
acagatcagt gccaatataa ctaagtttaa cattcagggc 1500cttgtcatca
ttgggggctt tgaggcttac acagggggcc tggaactgat ggagggcagg
1560aagcagtttg atgagctctg catcccattt gtggtcattc ctgctacagt
ctccaacaat 1620gtccctggct cagacttcag cgttggggct gacacagcac
tcaatactat ctgcacaacc 1680tgtgaccgca tcaagcagtc agcagctggc
accaagcgtc gggtgtttat cattgagact 1740atgggtggct actgtggcta
cctggctacc atggctggac tggcagctgg ggccgatgct 1800gcctacattt
ttgaggagcc cttcaccatt cgagacctgc aggcaaatgt tgaacatctg
1860gtgcaaaaga tgaaaacaac tgtgaaaagg ggcttggtgt taaggaatga
aaagtgcaat 1920gagaactata ccactgactt cattttcaac ctgtactctg
aggaggggaa gggcatcttc 1980gacagcagga agaatgtgct tggtcacatg
cagcagggtg ggagcccaac cccatttgat 2040aggaattttg ccactaagat
gggcgccaag gctatgaact ggatgtctgg gaaaatcaaa 2100gagagttacc
gtaatgggcg gatctttgcc aatactccag attcgggctg tgttctgggg
2160atgcgtaaga gggctctggt cttccaacca gtggctgagc tgaaggacca
gacagatttt 2220gagcatcgaa tccccaagga acagtggtgg ctgaaactga
ggcccatcct caaaatccta 2280gccaagtacg agattgactt ggacacttca
gaccatgccc acctggagca catcacccgg 2340aagcggtccg gggaagctgc
cgtctaaacc tctctggagt gaggggaata gattacctga 2400tcatggtcag
ctcacaccct aataagtcca catcttctca gtgttttagc tgtttttttc
2460attaggtttc cttttattct gtaccttgca gccatgacca gttctggcca
ggagctggag 2520gagcaggcag tgggtgggag ctccttttag gtagaattta
acatgacttc tgccccagct 2580ttatctgtca cacaaggctg ggcacctcta
gtgctactgc tagatatcac ttactcagtt 2640agaattttcc taaaaataag
ctttatttat ttctttgtga taacaaagag tcttggttcc 2700tctactactt
ttactacagt gacaaattgt aactacacta ataaatgcca actggtcact
2760gtgaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 2820a 2821352819DNAHomo sapiens 35ggcacgaggc agcggcggag
gagagctaag actaaaagag tggatcatga cccatgaaga 60gcaccatgca gccaaaaccc
tggggattgg caaagccatt gctgtcttaa cctctggtgg 120agatgcccaa
ggtatgaatg ctgctgtcag ggctgtggtt cgagttggta tcttcaccgg
180tgcccgtgtc ttctttgtcc atgagggtta tcaaggcctg gtggatggtg
gagatcacat 240caaggaagcc acctgggaga gcgtttcgat gatgcttcag
ctgggaggca cggtgattgg 300aagtgcccgg tgcaaggact ttcgggaacg
agaaggacga ctccgagctg cctacaacct 360ggtgaagcgt gggatcacca
atctctgtgt cattgggggt gatggcagcc tcactggggc 420tgacaccttc
cgttctgagt ggagtgactt gttgagtgac ctccagaaag caggtaagat
480cacagatgag gaggctacga agtccagcta cctgaacatt gtgggcctgg
ttgggtcaat 540tgacaatgac ttctgtggca ccgatatgac cattggcact
gactctgccc tgcatcggat 600catggaaatt gtagatgcca tcactaccac
tgcccagagc caccagagga catttgtgtt 660agaagtaatg ggccgccact
gtggatacct ggcccttgtc acctctctgt cctgtggggc 720cgactgggtt
tttattcctg aatgtccacc agatgacgac tgggaggaac acctttgtcg
780ccgactcagc gagacaagga cccgtggttc tcgtctcaac atcatcattg
tggctgaggg 840tgcaattgac aagaatggaa aaccaatcac ctcagaagac
atcaagaatc tggtggttaa 900gcgtctggga tatgacaccc gggttactgt
cttggggcat gtgcagaggg gtgggacgcc 960atcagccttt gacagaattc
tgggcagcag gatgggtgtg gaagcagtga tggcactttt 1020ggaggggacc
ccagataccc cagcctgtgt agtgagcctc tctggtaacc aggctgtgcg
1080cctgcccctc atggaatgtg tccaggtgac caaagatgtg accaaggcca
tggatgagaa 1140gaaatttgac gaagccctga agctgagagg ccggagcttc
atgaacaact gggaggtgta 1200caagcttcta gctcatgtca gacccccggt
atctaagagt ggttcgcaca cagtggctgt 1260gatgaacgtg ggggctccgg
ctgcaggcat gaatgctgct gttcgctcca ctgtgaggat 1320tggccttatc
cagggcaacc gagtgctcgt tgtccatgat ggtttcgagg gcctggccaa
1380ggggcagata gaggaagctg gctggagcta tgttgggggc tggactggcc
aaggtggctc 1440taaacttggg actaaaagga ctctacccaa gaagagcttt
gaacagatca gtgccaatat 1500aactaagttt aacattcagg gccttgtcat
cattgggggc tttgaggctt acacaggggg 1560cctggaactg atggagggca
ggaagcagtt tgatgagctc tgcatcccat ttgtggtcat 1620tcctgctaca
gtctccaaca atgtccctgg ctcagacttc agcgttgggg ctgacacagc
1680actcaatact atctgcacaa cctgtgaccg catcaagcag tcagcagctg
gcaccaagcg 1740tcgggtgttt atcattgaga ctatgggtgg ctactgtggc
tacctggcta ccatggctgg 1800actggcagct ggggccgatg ctgcctacat
ttttgaggag cccttcacca ttcgagacct 1860gcaggcaaat gttgaacatc
tggtgcaaaa gatgaaaaca actgtgaaaa ggggcttggt 1920gttaaggaat
gaaaagtgca atgagaacta taccactgac ttcattttca acctgtactc
1980tgaggagggg aagggcatct tcgacagcag gaagaatgtg cttggtcaca
tgcagcaggg 2040tgggagccca accccatttg ataggaattt tgccactaag
atgggcgcca aggctatgaa 2100ctggatgtct gggaaaatca aagagagtta
ccgtaatggg cggatctttg ccaatactcc 2160agattcgggc tgtgttctgg
ggatgcgtaa gagggctctg gtcttccaac cagtggctga 2220gctgaaggac
cagacagatt ttgagcatcg aatccccaag gaacagtggt ggctgaaact
2280gaggcccatc ctcaaaatcc tagccaagta cgagattgac ttggacactt
cagaccatgc 2340ccacctggag cacatcaccc ggaagcggtc cggggaagcg
gccgtctaaa cctctctgga 2400gtgaggggaa tagattacct gatcatggtc
agctcacacc ctaataagtc cacatcttct 2460cagtgtttta gctgtttttt
tcattaggtt tccttttatt ctgtaccttg cagccatgac 2520cagttctggc
caggagctgg aggagcaggc agtgggtggg agctcctttt aggtagaatt
2580taacatgact tctgccccag ctttatctgt cacacaaggc tgggcacctc
tagtgctact 2640gctagatatc acttactcag ttagaatttt cctaaaaata
agctttattt atttctttgt 2700gataacaaag agtcttggtt cctctactac
ttttactaca gtgacaaatt gtaactacac 2760taataaatgc caactggtca
ctgtgaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 281936751DNAHomo sapiens
36ttcactgcca gaatcccgcc cccatccctc cccactgcag gcttgagaag tgggcctcac
60agacacgacc cttacctctt gacactttca ttccaaatag ctagcttttc tgtactttct
120ctgagaaagg taatgtaaat gtaaaagaag tttttgaggc tattctatag
aagatgtcat 180aattcaatgt tcctcaaagg cagggactat atttattttt
ttaaacctct cggcacctaa 240tgcaaaatct agcacacaga ctcccaagaa
atgtttgctt aaagacggat agagggccac 300aggggcaggt tttattagca
gttgctgaag atgttggaaa tgcaaggtgt ctcaggcagt 360ctggactagg
aaaaagttct gtgttgaaaa caagagaagc aacgaaataa tttattattc
420ccattaaata aagatgctga tggctctcga gatagatatg aggaccccag
gactctgggg 480agtgaaggaa ctaagccgct gccatatctg agctgagtac
ttagggggag gaggaagagg 540aggagaaagg caagcaggag gaggcggact
tcttgtcagc atctgttagt ggaggttggg 600aagcctctcc tccttccccc
tccctctttg cctccacctg gctcctcccc atgttcgtcc 660atcacccctc
ccccctttcc caaggacaat ctgcaagaaa gcagcggcgg aggagagcta
720agactaaaag gcaagagggg ccattgagtg a 75137435DNAHomo sapiens
37ctttttctcc tccctttctc atttctattc agtcaacttc tcttttccct gaccttagtt
60tattccccaa ataggttcca atcggggtgg gagggatcag ggaggaatta ggaccttagt
120agtcttgggc ttgattacat gacatttcag cttgtcagtc tacaagggtg
tggctttcct 180ctggaagaag tccaaagctc tcaggctgca aagctcagac
ttggtatagt gggagagcct 240gactgaggtg gctctagcca gtctaattgc
cgttccttta gctagtggca tcttgattcc 300tgctgtgtct taactgacca
ttgtcttaaa ttctagagtg gatcatgacc catgaagagc 360accatgcagc
caaaaccctg gggattggca aagccattgc tgtcttaacc tctggtggag
420atgcccaagg taagg 4353895DNAHomo sapiens 38tgtccctcct ttcaggtatg
aatgctgctg tcagggctgt ggttcgagtt ggtatcttca 60ccggtgcccg tgtcttcttt
gtccatgagg ttggt 953999DNAHomo sapiens 39taatgtgtca cacagggtta
tcaaggcctg gtggatggtg gagatcacat caaggaagcc 60acctgggaga gcgtttcgat
gatgcttcag ctggtatgt 9940211DNAHomo sapiens 40ctacctcttc caaagggagg
cacggtgatt ggaagtgccc ggtgcaagga ctttcgggaa 60cgagaaggac gactccgagc
tgcctacaac ctggtgaagc gtgggatcac caatctctgt 120gtcattgggg
gtgatggcag cctcactggg gctgacacct tccgttctga gtggagtgac
180ttgttgagtg acctccagaa agcaggtaag a 21141187DNAHomo sapiens
41gcttctcatt gtcaggtaag atcacagatg aggaggctac gaagtccagc tacctgaaca
60ttgtgggcct ggttgggtca attgacaatg acttctgtgg cactgatatg accattggca
120ctgactctgc cctgcatcgg atcatggaaa ttgtagatgc catcactacc
actgcccaga 180ggtaagg 1874266DNAHomo sapiens 42gtctatctct
tgcagccacc agaggacatt tgtgttagaa gtaatgggcc gccactgtgg 60gtaaga
6643129DNAHomo sapiens 43gactctcatc tcagatacct ggcccttgtc
acctctctgt cctgtggggc cgactgggtt 60tttattcctg aatgtccacc agatgacgac
tgggaggaac acctttgtcg ccgactcagc 120gaggtactt 12944117DNAHomo
sapiens 44tttgttctca accagacaag gacccgtggt tctcgtctca acatcatcat
tgtggctgag 60ggtgcaattg acaagaatgg aaaaccaatc acctcagaag acatcaagaa
tgttcgt 11745114DNAHomo sapiens 45tgttggtccc ttcagctggt ggttaagcgt
ctgggatatg acacccgggt tactgtcttg 60gggcatgtgc agaggggtgg gacgccatca
gcctttgaca gaattctggt aagt 11446147DNAHomo sapiens 46tctgggctcc
tgcagggcag caggatgggt gtggaagcag tgatggcact tttggagggg 60accccagata
ccccagcctg tgtagtgagc ctctctggta accaggctgt gcgcctgccc
120ctcatggaat gtgtccaggt ggtaagt 1474786DNAHomo sapiens
47gtgctccccc ctcagaccaa agatgtgacc aaggccatgg atgagaagaa atttgacgaa
60gccctgaagc tgagaggccg gtgagg 864885DNAHomo sapiens 48ttgtacttcc
tacaggagct tcatgaacaa ctgggaggtg tacaagcttc tagctcatgt 60cagacccccg
gtatctaagg tactg 8549171DNAHomo sapiens 49cttcctcctg tatagagtgg
ttcgcacaca gtggctgtga tgaacgtggg ggctccggct 60gcaggcatga atgctgctgt
tcgctccact gtgaggattg gccttatcca gggcaaccga 120gtgctcgttg
tccatgatgg tttcgagggc ctggccaagg ggcaggtatg g 1715092DNAHomo
sapiens 50ggcttatccc cacagataga ggaagctggc tggagctatg ttgggggctg
gactggccaa 60ggtggctcta aacttgggac taaaaggtaa gt 9251109DNAHomo
sapiens 51tcactgatca actaggactc tacccaagaa gagctttgaa cagatcagtg
ccaatataac 60taagtttaac attcagggcc ttgtcatcat tgggggcttt gaggtgagt
10952174DNAHomo sapiens 52ctctcttctt cttaggctta cacagggggc
ctggaactga tggagggcag gaagcagttt 60gatgagctct gcatcccatt tgtggtcatt
cctgctacag tctccaacaa tgtccctggc 120tcagacttca gcgttggggc
tgacacagca ctcaatacta tctgcacagt gaga 17453186DNAHomo sapiens
53ccattgtcct tgcagacctg tgaccgcatc aagcagtcag cagctggcac caagcgtcgg
60gtgtttatca ttgagactat gggtggctac tgtggctacc tggctaccat ggctggactg
120gcagctgggg ccgatgctgc ctacattttt gaggagccct tcaccattcg
agacctgcag 180gtagct 1865483DNAHomo sapiens 54ctctttcatt ttcaggcaaa
tgttgaacat ctggtgcaaa agatgaaaac aactgtgaaa 60aggggcttgg tgttaaggta
cct 8355133DNAHomo sapiens 55ttctccacct ggcaggaatg aaaagtgcaa
tgagaactat accactgact tcattttcaa 60cctgtactct gaggagggga agggcatctt
cgacagcagg aagaatgtgc ttggtcacat 120gcagcaggta ggg 13356121DNAHomo
sapiens 56tgtttctttc tccagggtgg gagcccaacc ccatttgata ggaattttgc
cactaagatg 60ggcgccaagg ctatgaactg gatgtctggg aaaatcaaag agagttaccg
taatggtagg 120t 12157127DNAHomo sapiens 57catccctcat tgcagggcgg
atctttgcca atactccaga ttcgggctgt gttctgggga 60tgcgtaagag ggctctggtc
ttccaaccag tggctgagct gaaggaccag acagattttg 120agtgagt
12758556DNAHomo sapiens 58ctcattcctc tgtaggcatc gaatccccaa
ggaacagtgg tggctgaaac tgaggcccat 60cctcaaaatc ctagccaagt acgagattga
cttggacact tcagaccatg cccacctgga 120gcacatcacc cggaagcggt
ccggggaagc tgccgtctaa acctctctgg agtgagggga 180atagattacc
tgatcatggt cagctcacac cctaataagt ccacatcttc tcagtgtttt
240agctgttttt ttcattaggt ttccttttat tctgtacctt gcagccatga
ccagttctgg 300ccaggagctg gaggagcagg cagtgggtgg gagctccttt
taggtagaat ttaacatgac 360ttctgcccca gctttatctg tcacacaagg
ctgggcacct ctagtgctac tgctagatat 420cacttactca gttagaattt
tcctaaaaat aagctttatt tatttctttg tgataacaaa 480gagtcttggt
tcctctacta cttttactac agtgacaaat tgtaactaca ctaataaatg
540ccaactggtc actgtg 556592661DNAHomo sapiens 59ccatgaagag
caccatgcag ccaaaaccct ggggattggc aaagccattg ctgtcttaac 60ctctggtgga
gatgcccaag gtatgaatgc tgctgtcagg gctgtggttc gagttggtat
120cttcaccggt gcccgtgtct tctttgtcca tgagggttat caaggcctgg
tggatggtgg 180agatcacatc aaggaagcca cctgggagag cgtttcgatg
atgcttcagc tgggaggcac 240ggtgattgga agtgcccggt gcaaggactt
tcgggaacga gaaggacgac tccgagctgc 300ctacaacctg gtgaagcgtg
ggatcaccaa tctctgtgtc attgggggtg atggcagcct 360cactggggct
gacaccttcc gttctgagtg gagtgacttg ttgagtgacc tccagaaagc
420aggtaagatc acagatgagg aggctacgaa gtccagctac ctgaacattg
tgggcctggt 480tgggtcaatt gacaatgact tctgtggcac cgatatgacc
attggcactg actctgccct 540gcatcggatc atggaaattg tagatgccat
cactaccact gcccagagcc accagaggac 600atttgtgtta gaagtaatgg
gccgccactg tggatacctg gcccttgtca cctctctgtc 660ctgtggggcc
gactgggttt ttattcctga atgtccacca gatgacgact gggaggaaca
720cctttgtcgc cgactcagcg agacaaggac ccgtggttct cgtctcaaca
tcatcattgt 780ggctgagggt gcaattgaca agaatggaaa accaatcacc
tcagaagaca tcaagaatgg 840cagcaggatg ggtgtggaag cagtgatggc
acttttggag gggaccccag ataccccagc 900ctgtgtagtg agcctctctg
gtaaccaggc tgtgcgcctg cccctcatgg aatgtgtcca 960ggtgaccaaa
gatgtgacca aggccatgga tgagaagaaa tttgacgaag ccctgaagct
1020gagaggccgg agcttcatga acaactggga ggtgtacaag cttctagctc
atgtcagacc 1080cccggtatct aagagtggtt cgcacacagt ggctgtgatg
aacgtggggg ctccggctgc 1140aggcatgaat gctgctgttc gctccactgt
gaggattggc cttatccagg gcaaccgagt 1200gctcgttgtc catgatggtt
tcgagggcct ggccaagggg cagatagagg aagctggctg 1260gagctatgtt
gggggctgga ctggccaagg tggctctaaa cttgggacta aaaggactct
1320acccaagaag agctttgaac agatcagtgc caatataact aagtttaaca
ttcagggcct 1380tgtcatcatt gggggctttg aggcttacac agggggcctg
gaactgatgg agggcaggaa 1440gcagtttgat gagctctgca tcccatttgt
ggtcattcct gctacagtct ccaacaatgt 1500ccctggctca gacttcagcg
ttggggctga cacagcactc aatactatct gcacaacctg 1560tgaccgcatc
aagcagtcag cagctggcac caagcgtcgg gtgtttatca ttgagactat
1620gggtggctac tgtggctacc tggctaccat ggctggactg gcagctgggg
ccgatgctgc 1680ctacattttt gaggagccct tcaccattcg agacctgcag
gcaaatgttg aacatctggt 1740gcaaaagatg aaaacaactg tgaaaagggg
cttggtgtta aggaatgaaa agtgcaatga 1800gaactatacc actgacttca
ttttcaacct gtactctgag gaggggaagg gcatcttcga 1860cagcaggaag
aatgtgcttg gtcacatgca gcagggtggg agcccaaccc catttgatag
1920gaattttgcc actaagatgg gcgccaaggc tatgaactgg atgtctggga
aaatcaaaga 1980gagttaccgt aatgggcgga
tctttgccaa tactccagat tcgggctgtg ttctggggat 2040gcgtaagagg
gctctggtct tccaaccagt ggctgagctg aaggaccaga cagattttga
2100gcatcgaatc cccaaggaac agtggtggct gaaactgagg cccatcctca
aaatcctagc 2160caagtacgag attgacttgg acacttcaga ccatgcccac
ctggagcaca tcacccggaa 2220gcggtccggg gaagcggccg tctaaacctc
tctggagtga ggggaataga ttacctgatc 2280atggtcagct cacaccctaa
taagtccaca tcttctcagt gttttagctg tttttttcat 2340taggtttcct
tttattctgt accttgcagc catgaccagt tctggccagg agctggagga
2400gcaggcagtg ggtgggagct ccttttaggt agaatttaac atgacttctg
ccccagcttt 2460atctgtcaca caaggctggg cacctctagt gctactgcta
gatatcactt actcagttag 2520aattttccta aaaataagct ttatttattt
ctttgtgata acaaagagtc ttggttcctc 2580tactactttt actacagtga
caaattgtaa ctacactaat aaatgccaac tggtcactgt 2640gaaaaaaaaa
aaaaaaaaaa a 2661602628DNAHomo sapiens 60gcacccggac gtgcggctcc
cctcggcctc ctcgccatgg acgcggacga ctcccgggcc 60cccaagggct ccttgcggaa
gttcctggag cacctctccg gggccggcaa ggccatcggc 120gtgctgacca
gcggcgggga tgctcaaggt atgaacgctg ccgtccgtgc cgtggtgcgc
180atgggtatct acgtgggggc caaggtgtac ttcatctacg agggctacca
gggcatggtg 240gacggaggct caaacatcgc agaggccgac tgggagagtg
tctccagcat cctgcaagtg 300ggcgggacga tcattggcag tgcgcggtgc
caggccttcc gcacgcggga aggccgcctg 360aaggctgctt gcaacctgct
gcagcgcggc atcaccaacc tgtgtgtgat cggcggggac 420gggagcctca
ccggggccaa cctcttccgg aaggagtgga gtgggctgct ggaggagctg
480gccaggaacg gccagatcga taaggaggcc gtgcagaagt acgcctacct
caacgtggtg 540ggcatggtgg gctccatcga caatgatttc tgcggcaccg
acatgaccat cggcacggac 600tccgccctgc acaggatcat cgaggtcgtc
gacgccatca tgaccacggc ccagagccac 660cagaggacct tcgttctgga
ggtgatggga cgacactgtg ggtacctggc cctggtgagt 720gccttggcct
gcggtgcgga ctgggtgttc cttccagaat ctccaccaga ggaaggctgg
780gaggagcaga tgtgtgtcaa actctcggag aaccgtgccc ggaaaaaaag
gctgaatatt 840attattgtgg ctgaaggagc aattgatacc caaaataaac
ccatcacctc tgagaaaatc 900aaagagcttg tcgtcacgca gctgggctat
gacacacgtg tgaccatcct cgggcacgtg 960cagagaggag ggaccccttc
ggcattcgac aggatcttgg ccagccgcat gggagtggag 1020gcagtcatcg
ccttgctaga ggccaccccg gacaccccag cttgcgtcgt gtcactgaac
1080gggaaccacg ccgtgcgcct gccgctgatg gagtgcgtgc agatgactca
ggatgtgcag 1140aaggcgatgg acgagaggag atttcaagat gcggttcgac
tccgagggag gagctttgcg 1200ggcaacctga acacctacaa gcgacttgcc
atcaagctgc cggatgatca gatcccaaag 1260accaattgca acgtagctgt
catcaacgtg ggggcacccg cggctgggat gaacgcagcc 1320gtacgctcag
ctgtgcgcgt gggcattgcc gacggccaca ggatgctcgc catctatgat
1380ggctttgacg gcttcgccaa gggccagatc aaagaaatcg gctggacaga
tgtcgggggc 1440tggaccggcc aaggaggctc cattcttggg acaaaacgcg
ttctcccggg gaagtacttg 1500gaagagatcg ccacacagat gcgcacgcac
agcatcaacg cgctgctgat catcggtgga 1560ttcgaggcct acctgggact
cctggagctg tcagccgccc gggagaagca cgaggagttc 1620tgtgtcccca
tggtcatggt tcccgctact gtgtccaaca atgtgccggg ttccgatttc
1680agcatcgggg cagacaccgc cctgaacact atcaccgaca cctgcgaccg
catcaagcag 1740tccgccagcg gaaccaagcg gcgcgtgttc atcatcgaga
ccatgggcgg ctactgtggc 1800tacctggcca acatgggggg gctcgcggcc
ggagctgatg ccgcatacat tttcgaagag 1860cccttcgaca tcagggatct
gcagtccaac gtggagcacc tgacggagaa aatgaagacc 1920accatccaga
gaggccttgt gctcagaaat gagagctgca gtgaaaacta caccaccgac
1980ttcatttacc agctgtattc agaagagggc aaaggcgtgt ttgactgcag
gaagaacgtg 2040ctgggtcaca tgcagcaggg tggggcaccc tctccatttg
atagaaactt tggaaccaaa 2100atctctgcca gagctatgga gtggatcact
gcaaaactca aggaggcccg gggcagagga 2160aaaaaattta ccaccgatga
ttccatttgt gtgctgggaa taagcaaaag aaacgttatt 2220tttcaacctg
tggcagagct gaagaagcaa acggattttg agcacaggat tcccaaagaa
2280cagtggtggc tcaagctacg gcccctcatg aaaatcctgg ccaagtacaa
ggccagctat 2340gacgtgtcgg actcaggcca gctggaacat gtgcagccct
ggagtgtctg acccagtccc 2400gcctgcatgt gcctgcagcc accgtggact
gtctgttttt gtaacactta agttatttta 2460tcagcacttt atgcacgtat
tattgacatt aatacctaat cggcgagtgc ccatctgccc 2520cacctgctcc
agtgcgtgct gtctgtggag tgtgtctcat gctttcagat gtgcatatga
2580gcagaattaa ttaaacattt gcctatgact ccaacaaaaa aaaaaaaa
2628612591DNAHomo sapiens 61cccggacgtg cggctcccct cggcctcctc
gccatggacg cggacgactc ccgggccccc 60aagggctcct tgcggaagtt cctggagcac
ctctccgggg ccggcaaggc catcggcgtg 120ctgaccagcg gcggggatgc
tcaaggtatg aacgctgccg tccgtgccgt ggtgcgcatg 180ggtatctacg
tgggggccaa ggtgtacttc atctacgagg gctaccaggg catggtggac
240ggaggctcaa acatcgcaga ggccgactgg gagagtgtct ccagcatcct
gcaagtgggc 300gggacgatca ttggcagtgc gcggtgccag gccttccgca
cgcgggaagg ccgcctgaag 360gctgcttgca acctgctgca gcgcggcatc
accaacctgt gtgtgatcgg cggggacggg 420agcctcaccg gggccaacct
cttccggaag gagtggagtg ggctgctgga ggagctggcc 480aggaacggcc
agatcgataa ggaggccgtg cagaagtacg cctacctcaa cgtggtgggc
540atggtgggct ccatcgacaa tgatttctgc ggcaccgaca tgaccatcgg
cacggactcc 600gccctgcaca ggatcatcga ggtcgtcgac gccatcatga
ccacggccca gagccaccag 660aggaccttcg ttctggaggt gatgggacga
cactgtgggt acctggccct ggtgagtgcc 720ttggcctgcg gtgcggactg
ggtgttcctt ccagaatctc caccagagga aggctgggag 780gagcagatgt
gtgtcaaact ctcggagaac cgtgcccgga aaaaaaggct gaatattatt
840attgtggctg aaggagcaat tgatacccaa aataaaccca tcacctctga
gaaaatcaaa 900gagcttgtcg tcacgcagct gggctatgac acacgtgtga
ccatcctcgg gcacgtgcag 960agaggaggga ccccttcggc attcgacagg
atcttggcca gccgcatggg agtggaggca 1020gtcatcgcct tgctagaggc
caccccggac accccagctt gcgtcgtgtc actgaacggg 1080aaccacgccg
tgcgcctgcc gctgatggag tgcgtgcaga tgactcagga tgtgcagaag
1140gcgatggacg agaggagatt tcaagatgcg gttcgactcc gagggaggag
ctttgcgggc 1200aacctgaaca cctacaagcg acttgccatc aagctgccgg
atgatcagat cccaaagacc 1260aattgcaacg tagctgtcat caacgtgggg
gcacccgcgg ctgggatgaa cgcggccgta 1320cgctcagctg tgcgcgtggg
cattgccgac ggccacagga tgctcgccat ctatgatggc 1380tttgacggct
tcgccaaggg ccagatcaaa gaaatcggct ggacagatgt cgggggctgg
1440accggccaag gaggctccat tcttgggaca aaacgcgttc tcccggggaa
gtacttggaa 1500gagatcgcca cacagatgcg cacgcacagc atcaacgcgc
tgctgatcat cggtggattc 1560gaggcctacc tgggactcct ggagctgtca
gccgcccggg agaagcacga ggagttctgt 1620gtccccatgg tcatggttcc
cgctactgtg tccaacaatg tgccgggttc cgatttcagc 1680atcggggcag
acaccgccct gaacactatc accgacacct gcgaccgcat caagcagtcc
1740gccagcggaa ccaagcggcg cgtgttcatc atcgagacca tgggcggcta
ctgtggctac 1800ctggccaaca tgggggggct cgcggccgga gctgatgccg
catacatttt cgaagagccc 1860ttcgacatca gggatctgca gtccaacgtg
gagcacctga cggagaaaat gaagaccacc 1920atccagagag gccttgtgct
cagaaatgag agctgcagtg aaaactacac caccgacttc 1980atttaccagc
tgtattcaga agagggcaaa ggcgtgtttg actgcaggaa gaacgtgctg
2040ggtcacatgc agcagggtgg ggcaccctct ccatttgata gaaactttgg
aaccaaaatc 2100tctgccagag ctatggagtg gatcactgca aaactcaagg
aggcccgggg cagaggaaaa 2160aaatttacca ccgatgattc catttgtgtg
ctgggaataa gcaaaagaaa cgttattttt 2220caacctgtgg cagagctgaa
gaagcaaacg gattttgagc acaggattcc caaagaacag 2280tggtggctca
agctacggcc cctcatgaaa atcctggcca agtacaaggc cagctatgac
2340gtgtcggact caggccagct ggaacatgtg cagccctgga gtgtctgacc
cagtcccgcc 2400tgcatgtgcc tgcagccacc gtggactgtc tgtttttgta
acacttaagt tattttatca 2460gcactttatg cacgtattat tgacattaat
acctaatcgg cgagtgccca tctgccccac 2520cagctccagt gcgtgctgtc
tgtggagtgt gtctcatgct ttcagatgtg catatgagca 2580gaattaatta a
2591622636DNAHomo sapiens 62gcacccggac gtgcggctcc cctcggcctc
ctcgccatgg acgcggacga ctcccgggcc 60cccaagggct ccttgcggaa gttcctggag
cacctctccg gggccggcaa ggccatcggc 120gtgctgacca gcggcgggga
tgctcaaggt atgaacgctg ccgtccgtgc cgtggtgcgc 180atgggtatct
acgtgggggc caaggtgtac ttcatctacg agggctacca gggcatggtg
240gacggaggct caaacatcgc agaggccgac tgggagagtg tctccagcat
cctgcaagtg 300ggcgggacga tcattggcag tgcgcggtgc caggccttcc
gcacgcggga aggccgcctg 360aaggctgctt gcaacctgct gcagcgcggc
atcaccaacc tgtgtgtgat cggcggggac 420gggagcctca ccggggccaa
cctcttccgg aaggagtgga gtgggctgct ggaggagctg 480gccaggaacg
gccagatcga taaggaggcc gtgcagaagt acgcctacct caacgtggtg
540ggcatggtgg gctccatcga caatgatttc tgcggcaccg acatgaccat
cggcacggac 600tccgccctgc acaggatcat cgaggtcgtc gacgccatca
tgaccacggc ccagagccac 660cagaggacct tcgttctgga ggtgatggga
cgacactgtg ggtacctggc cctggtgagt 720gccttggcct gcggtgcgga
ctgggtgttc cttccagaat ctccaccaga ggaaggctgg 780gaggagcaga
tgtgtgtcaa actctcggag aaccgtgccc ggaaaaaaag gctgaatatt
840attattgtgg ctgaaggagc aattgatacc caaaataaac ccatcacctc
tgagaaaatc 900aaagagcttg tcgtcacgca gctgggctat gacacacgtg
tgaccatcct cgggcacgtg 960cagagaggag ggaccccttc ggcattcgac
aggatcttgg ccagccgcat gggagtggag 1020gcagtcatcg ccttgctaga
ggccaccccg gacaccccag cttgcgtcgt gtcactgaac 1080gggaaccacg
ccgtgcgcct gccgctgatg gagtgcgtgc agatgactca ggatgtgcag
1140aaggcgatgg acgagaggag atttcaagat gcggttcgac tccgagggag
gagctttgcg 1200ggcaacctga acacctacaa gcgacttgcc atcaagctgc
cggatgatca gatcccaaag 1260accaattgca acgtagctgt catcaacgtg
ggggcacccg cggctgggat gaacgcggcc 1320gtacgctcag ctgtgcgcgt
gggcattgcc gacggccaca ggatgctcgc catctatgat 1380ggctttgacg
gcttcgccaa gggccagatc aaagaaatcg gctggacaga tgtcgggggc
1440tggaccggcc aaggaggctc cattcttggg acaaaacgcg ttctcccggg
gaagtacttg 1500gaagagatcg ccacacagat gcgcacgcac agcatcaacg
cgctgctgat catcggtgga 1560ttcgaggcct acctgggact cctggagctg
tcagccgccc gggagaagca cgaggagttc 1620tgtgtcccca tggtcatggt
tcccgctact gtgtccaaca atgtgccggg ttccgatttc 1680agcatcgggg
cagacaccgc cctgaacact atcaccgaca cctgcgaccg catcaagcag
1740tccgccagcg gaaccaagcg gcgcgtgttc atcatcgaga ccatgggcgg
ctactgtggc 1800tacctggcca acatgggggg gctcgcggcc ggagctgatg
ccgcatacat tttcgaagag 1860cccttcgaca tcagggatct gcagtccaac
gtggagcacc tgacggagaa aatgaagacc 1920accatccaga gaggccttgt
gctcagaaat gagagctgca gtgaaaacta caccaccgac 1980ttcatttacc
agctgtattc agaagagggc aaaggcgtgt ttgactgcag gaagaacgtg
2040ctgggtcaca tgcagcaggg tggggcaccc tctccatttg atagaaactt
tggaaccaaa 2100atctctgcca gagctatgga gtggatcact gcaaaactca
aggaggcccg gggcagagga 2160aaaaaattta ccaccgatga ttccatttgt
gtgctgggaa taagcaaaag aaacgttatt 2220tttcaacctg tggcagagct
gaagaagcaa acggattttg agcacaggat tcccaaagaa 2280cagtggtggc
tcaagctacg gcccctcatg aaaatcctgg ccaagtacaa ggccagctat
2340gacgtgtcgg actcaggcca gctggaacat gtgcagccct ggagtgtctg
acccagtccc 2400gcctgcatgt gcctgcagcc accgtggact gtctgttttt
gtaacactta agttatttta 2460tcagcacttt atgcacgtat tattgacatt
aatacctaat cggcgagtgc ccatctgccc 2520caccagctcc agtgcgtgct
gtctgtggag tgtgtctcat gctttcagat gtgcatatga 2580gcagaattaa
ttaaacattt gcctatgact ccaaaaaaaa aaaaaaaaaa aaaaaa
2636632671DNAHomo sapiens 63gagtcaggcg cgcgcgggca gggtccccat
tgcctgctgc gcacccggac gtgcggctcc 60cctcggcctc ctcgccatgg acgcggacga
ctcccgggcc cccaagggct ccttgcggaa 120gttcctggag cacctctccg
gggccggcaa ggccatcggc gtgctgacca gcggcgggga 180tgctcaaggt
atgaacgctg ccgtccgtgc cgtggtgcgc atgggtatct acgtgggggc
240caaggtgtac ttcatctacg agggctacca gggcatggtg gacggaggct
caaacatcgc 300agaggccgac tgggagagtg tctccagcat cctgcaagtg
ggcgggacga tcattggcag 360tgcgcggtgc caggccttcc gcacgcggga
aggccgcctg aaggctgctt gcaacctgct 420gcagcgcggc atcaccaacc
tgtgtgtgat cggcggggac gggagcctca ccggggccaa 480cctcttccgg
aaggagtgga gtgggctgct ggaggagctg gccaggaacg gccagatcga
540taaggaggcc gtgcagaaat acgcctacct caacgtggtg ggcatggtgg
gctccatcga 600caatgatttc tgcggcaccg acatgaccat cggcacggac
tccgccctgc acaggatcat 660cgaggtcgtc gacgccatca tgaccacggc
ccagagccac cagaggacct tcgttctgga 720ggtgatggga cgacactgtg
ggtacctggc cctggtgagt gccttggcct gcggtgcgga 780ctgggtgttc
cttccagaat ctccaccaga ggaaggctgg gaggagcaga tgtgtgtcaa
840actctcggag aaccgtgccc ggaaaaaaag gctgaatatt attattgtgg
ctgaaggagc 900aattgatacc caaaataaac ccatcacctc tgagaaaatc
aaagagcttg tcgtcacgca 960gctgggctat gacacacgtg tgaccatcct
cgggcacgtg cagagaggag ggaccccttc 1020ggcattcgac aggatcttgg
ccagccgcat gggagtggag gcagtcatcg ccttgctaga 1080ggccaccccg
gacaccccag cttgcgtcgt gtcactgaac gggaaccacg ccgtgcgcct
1140gccgctgatg gagtgcgtgc agatgactca ggatgtgcag aaggcgatgg
acgagaggag 1200atttcaagat gcggttcgac tccgagggag gagctttgcg
ggcaacctga acacctacaa 1260gcgacttgcc atcaagctgc cggatgatca
gatcccaaag accaattgca acgtagctgt 1320catcaacgtg ggggcacccg
cggctgggat gaacgcggcc gtacgctcag ctgtgcgcgt 1380gggcattgcc
gacggccaca ggatgctcgc catctatgat ggctttgacg gcttcgccaa
1440gggccagatc aaagaaatcg gctggacaga tgtcgggggc tggaccggcc
aaggaggctc 1500cattcttggg acaaaacgcg ttctcccggg gaagtacttg
gaagagatcg ccacacagat 1560gcgcacgcac agcatcaacg cgctgctgat
catcggtgga ttcgaggcct acctgggact 1620cctggagctg tcagccgccc
gggagaagca cgaggagttc tgtgtcccca tggtcatggt 1680tcccgctact
gtgtccaaca atgtgccggg ttccgatttc agcatcgggg cagacaccgc
1740cctgaacact atcaccgaca cctgcgaccg catcaagcag tccgccagcg
gaaccaagcg 1800gcgcgtgttc atcatcgaga ccatgggcgg ctactgtggc
tacctggcca acatgggggg 1860gctcgcggct ggagctgatg ccgcatacat
tttcgaagag cccttcgaca tcagggatct 1920gcagtccaac gtggagcacc
tgacggagaa aatgaagacc accatccaga gaggccttgt 1980gctcagaaat
gagagctgca gtgaaaacta caccaccgac ttcatttacc agctgtattc
2040agaagagggc aaaggcgtgt ttgactgcag gaagaacgtg ctgggtcaca
tgcagcaggg 2100tggggcaccc tctccatttg atagaaactt tggaaccaaa
atctctgcca gagctatgga 2160gtggatcact gcaaaactca aggaggcccg
gggcagagga aaaaaattta ccaccgatga 2220ttccatttgt gtgctgggaa
taagcaaaag aaacgttatt tttcaacctg tggcagagct 2280gaagaagcaa
acggattttg agcacaggat tcccaaagaa cagtggtggc tcaagctacg
2340gcccctcatg aaaatcctgg ccaagtacaa ggccagctat gacgtgtcgg
actcaggcca 2400gctggaacat gtgcagccct ggagtgtctg acccagtccc
gcctgcatgt gcctgcagcc 2460accgtggact gtctcttttt gtaacactta
agttatttta tcagcacttt atgcacgtat 2520tattgacatt aatacctaat
cggcgagtgc ccatctgccc caccagcccc agtgcgtgct 2580gtctgtggag
tgtgtctcat gctttcagat gtgcatatga gcagaattaa ttaaacattt
2640gcctacaaaa aaaaaaaaaa aaaaaaaaaa a 267164827PRTHomo sapiens
64Met Cys Asn Gln Gly Arg Gly Arg Glu Ser Ser Arg Gly Gly Leu His 1
5 10 15 Val Gln Gly Ser Cys Arg Gly Leu Ser Arg Ser Pro Gln Gln Glu
Thr 20 25 30 Gly Phe Ala Lys Ala Pro Ala Gly Thr Asp Cys Phe Phe
His Cys Ser 35 40 45 Pro Gly Ser Arg Gly Gln Gly Asp Arg Lys Glu
Glu Val Thr Ser Glu 50 55 60 Pro Gly Gly Thr Ser Ile Met Ser Arg
Leu Gly Gly Met Asn Ala Ala 65 70 75 80 Val Arg Ala Val Thr Arg Met
Gly Ile Tyr Val Gly Ala Lys Val Phe 85 90 95 Leu Ile Tyr Glu Gly
Tyr Glu Gly Leu Val Glu Gly Gly Glu Asn Ile 100 105 110 Lys Gln Ala
Asn Trp Leu Ser Val Ser Asn Ile Ile Gln Leu Gly Gly 115 120 125 Thr
Ile Ile Gly Ser Ala Arg Cys Lys Ala Phe Thr Thr Arg Glu Gly 130 135
140 Arg Arg Ala Ala Ala Tyr Asn Leu Val Gln His Gly Ile Thr Asn Leu
145 150 155 160 Cys Val Ile Gly Gly Asp Gly Ser Leu Thr Gly Ala Asn
Ile Phe Arg 165 170 175 Ser Glu Trp Gly Ser Leu Leu Glu Glu Leu Val
Ala Glu Gly Lys Ile 180 185 190 Ser Glu Thr Thr Ala Arg Thr Tyr Ser
His Leu Asn Ile Ala Gly Leu 195 200 205 Val Gly Ser Ile Asp Asn Asp
Phe Cys Gly Thr Asp Met Thr Ile Gly 210 215 220 Thr Asp Ser Ala Leu
His Arg Ile Met Glu Val Ile Asp Ala Ile Thr 225 230 235 240 Thr Thr
Ala Gln Ser His Gln Arg Thr Phe Val Leu Glu Val Met Gly 245 250 255
Arg His Cys Gly Tyr Leu Ala Leu Val Ser Ala Leu Ala Ser Gly Ala 260
265 270 Asp Trp Leu Phe Ile Pro Glu Ala Pro Pro Glu Asp Gly Trp Glu
Asn 275 280 285 Phe Met Cys Glu Arg Leu Gly Glu Thr Arg Ser Arg Gly
Ser Arg Leu 290 295 300 Asn Ile Ile Ile Ile Ala Glu Gly Ala Ile Asp
Arg Asn Gly Lys Pro 305 310 315 320 Ile Ser Ser Ser Tyr Val Lys Asp
Leu Val Val Gln Arg Leu Gly Phe 325 330 335 Asp Thr Arg Val Thr Val
Leu Gly His Val Gln Arg Gly Gly Thr Pro 340 345 350 Ser Ala Phe Asp
Arg Ile Leu Ser Ser Lys Met Gly Met Glu Ala Val 355 360 365 Met Ala
Leu Leu Glu Ala Thr Pro Asp Thr Pro Ala Cys Val Val Thr 370 375 380
Leu Ser Gly Asn Gln Ser Val Arg Leu Pro Leu Met Glu Cys Val Gln 385
390 395 400 Met Thr Lys Glu Val Gln Lys Ala Met Asp Asp Lys Arg Phe
Asp Glu 405 410 415 Ala Thr Gln Leu Arg Gly Gly Ser Phe Glu Asn Asn
Trp Asn Ile Tyr 420 425 430 Lys Leu Leu Ala His Gln Lys Pro Pro Lys
Glu Lys Ser Asn Phe Ser 435 440 445 Leu Ala Ile Leu Asn Val Gly Ala
Pro Ala Ala Gly Met Asn Ala Ala 450 455 460 Val Arg Ser Ala Val Arg
Thr Gly Ile Ser His Gly His Thr Val Tyr 465 470 475 480 Val Val His
Asp Gly Phe Glu Gly Leu Ala Lys Gly Gln Val Gln Glu 485 490 495 Val
Gly Trp His Asp Val Ala Gly Trp Leu Gly Arg Gly Gly Ser Met 500 505
510 Leu Gly Thr Lys Arg Thr Leu Pro Lys Gly Gln Leu Glu Ser Ile Val
515 520 525 Glu Asn Ile Arg Ile Tyr Gly Ile His Ala Leu Leu Val Val
Gly Gly 530 535 540 Phe Glu Ala Tyr Glu Gly Val Leu Gln Leu Val Glu
Ala Arg Gly Arg 545 550
555 560 Tyr Glu Glu Leu Cys Ile Val Met Cys Val Ile Pro Ala Thr Ile
Ser 565 570 575 Asn Asn Val Pro Gly Thr Asp Phe Ser Leu Gly Ser Asp
Thr Ala Val 580 585 590 Asn Ala Ala Met Glu Ser Cys Asp Arg Ile Lys
Gln Ser Ala Ser Gly 595 600 605 Thr Lys Arg Arg Val Phe Ile Val Glu
Thr Met Gly Gly Tyr Cys Gly 610 615 620 Tyr Leu Ala Thr Val Thr Gly
Ile Ala Val Gly Ala Asp Ala Ala Tyr 625 630 635 640 Val Phe Glu Asp
Pro Phe Asn Ile His Asp Leu Lys Val Asn Val Glu 645 650 655 His Met
Thr Glu Lys Met Lys Thr Asp Ile Gln Arg Gly Leu Val Leu 660 665 670
Arg Asn Glu Lys Cys His Asp Tyr Tyr Thr Thr Glu Phe Leu Tyr Asn 675
680 685 Leu Tyr Ser Ser Glu Gly Lys Gly Val Phe Asp Cys Arg Thr Asn
Val 690 695 700 Leu Gly His Leu Gln Gln Gly Gly Ala Pro Thr Pro Phe
Asp Arg Asn 705 710 715 720 Tyr Gly Thr Lys Leu Gly Val Lys Ala Met
Leu Trp Leu Ser Glu Lys 725 730 735 Leu Arg Glu Val Tyr Arg Lys Gly
Arg Val Phe Ala Asn Ala Pro Asp 740 745 750 Ser Ala Cys Val Ile Gly
Leu Lys Lys Lys Ala Val Ala Phe Ser Pro 755 760 765 Val Thr Glu Leu
Lys Lys Asp Thr Asp Phe Glu His Arg Met Pro Arg 770 775 780 Glu Gln
Trp Trp Leu Ser Leu Arg Leu Met Leu Lys Met Leu Ala Gln 785 790 795
800 Tyr Arg Ile Ser Met Ala Ala Tyr Val Ser Gly Glu Leu Glu His Val
805 810 815 Thr Arg Arg Thr Leu Ser Met Asp Lys Gly Phe 820 825
65780PRTHomo sapiens 65Met Thr His Glu Glu His His Ala Ala Lys Thr
Leu Gly Ile Gly Lys 1 5 10 15 Ala Ile Ala Val Leu Thr Ser Gly Gly
Asp Ala Gln Gly Met Asn Ala 20 25 30 Ala Val Arg Ala Val Val Arg
Val Gly Ile Phe Thr Gly Ala Arg Val 35 40 45 Phe Phe Val His Glu
Gly Tyr Gln Gly Leu Val Asp Gly Gly Asp His 50 55 60 Ile Lys Glu
Ala Thr Trp Glu Ser Val Ser Met Met Leu Gln Leu Gly 65 70 75 80 Gly
Thr Val Ile Gly Ser Ala Arg Cys Lys Asp Phe Arg Glu Arg Glu 85 90
95 Gly Arg Leu Arg Ala Ala Tyr Asn Leu Val Lys Arg Gly Ile Thr Asn
100 105 110 Leu Cys Val Ile Gly Gly Asp Gly Ser Leu Thr Gly Ala Asp
Thr Phe 115 120 125 Arg Ser Glu Trp Ser Asp Leu Leu Ser Asp Leu Gln
Lys Ala Gly Lys 130 135 140 Ile Thr Asp Glu Glu Ala Thr Lys Ser Ser
Tyr Leu Asn Ile Val Gly 145 150 155 160 Leu Val Gly Ser Ile Asp Asn
Asp Phe Cys Gly Thr Asp Met Thr Ile 165 170 175 Gly Thr Asp Ser Ala
Leu His Arg Ile Met Glu Ile Val Asp Ala Ile 180 185 190 Thr Thr Thr
Ala Gln Ser His Gln Arg Thr Phe Val Leu Glu Val Met 195 200 205 Gly
Arg His Cys Gly Tyr Leu Ala Leu Val Thr Ser Leu Ser Cys Gly 210 215
220 Ala Asp Trp Val Phe Ile Pro Glu Cys Pro Pro Asp Asp Asp Trp Glu
225 230 235 240 Glu His Leu Cys Arg Arg Leu Ser Glu Thr Arg Thr Arg
Gly Ser Arg 245 250 255 Leu Asn Ile Ile Ile Val Ala Glu Gly Ala Ile
Asp Lys Asn Gly Lys 260 265 270 Pro Ile Thr Ser Glu Asp Ile Lys Asn
Leu Val Val Lys Arg Leu Gly 275 280 285 Tyr Asp Thr Arg Val Thr Val
Leu Gly His Val Gln Arg Gly Gly Thr 290 295 300 Pro Ser Ala Phe Asp
Arg Ile Leu Gly Ser Arg Met Gly Val Glu Ala 305 310 315 320 Val Met
Ala Leu Leu Glu Gly Thr Pro Asp Thr Pro Ala Cys Val Val 325 330 335
Ser Leu Ser Gly Asn Gln Ala Val Arg Leu Pro Leu Met Glu Cys Val 340
345 350 Gln Val Thr Lys Asp Val Thr Lys Ala Met Asp Glu Lys Lys Phe
Asp 355 360 365 Glu Ala Leu Lys Leu Arg Gly Arg Ser Phe Met Asn Asn
Trp Glu Val 370 375 380 Tyr Lys Leu Leu Ala His Val Arg Pro Pro Val
Ser Lys Ser Gly Ser 385 390 395 400 His Thr Val Ala Val Met Asn Val
Gly Ala Pro Ala Ala Gly Met Asn 405 410 415 Ala Ala Val Arg Ser Thr
Val Arg Ile Gly Leu Ile Gln Gly Asn Arg 420 425 430 Val Leu Val Val
His Asp Gly Phe Glu Gly Leu Ala Lys Gly Gln Ile 435 440 445 Glu Glu
Ala Gly Trp Ser Tyr Val Gly Gly Trp Thr Gly Gln Gly Gly 450 455 460
Ser Lys Leu Gly Thr Lys Arg Thr Leu Pro Lys Lys Ser Phe Glu Gln 465
470 475 480 Ile Ser Ala Asn Ile Thr Lys Phe Asn Ile Gln Gly Leu Val
Ile Ile 485 490 495 Gly Gly Phe Glu Ala Tyr Thr Gly Gly Leu Glu Leu
Met Glu Gly Arg 500 505 510 Lys Gln Phe Asp Glu Leu Cys Ile Pro Phe
Val Val Ile Pro Ala Thr 515 520 525 Val Ser Asn Asn Val Pro Gly Ser
Asp Phe Ser Val Gly Ala Asp Thr 530 535 540 Ala Leu Asn Thr Ile Cys
Thr Thr Cys Asp Arg Ile Lys Gln Ser Ala 545 550 555 560 Ala Gly Thr
Lys Arg Arg Val Phe Ile Ile Glu Thr Met Gly Gly Tyr 565 570 575 Cys
Gly Tyr Leu Ala Thr Met Ala Gly Leu Ala Ala Gly Ala Asp Ala 580 585
590 Ala Tyr Ile Phe Glu Glu Pro Phe Thr Ile Arg Asp Leu Gln Ala Asn
595 600 605 Val Glu His Leu Val Gln Lys Met Lys Thr Thr Val Lys Arg
Gly Leu 610 615 620 Val Leu Arg Asn Glu Lys Cys Asn Glu Asn Tyr Thr
Thr Asp Phe Ile 625 630 635 640 Phe Asn Leu Tyr Ser Glu Glu Gly Lys
Gly Ile Phe Asp Ser Arg Lys 645 650 655 Asn Val Leu Gly His Met Gln
Gln Gly Gly Ser Pro Thr Pro Phe Asp 660 665 670 Arg Asn Phe Ala Thr
Lys Met Gly Ala Lys Ala Met Asn Trp Met Ser 675 680 685 Gly Lys Ile
Lys Glu Ser Tyr Arg Asn Gly Arg Ile Phe Ala Asn Thr 690 695 700 Pro
Asp Ser Gly Cys Val Leu Gly Met Arg Lys Arg Ala Leu Val Phe 705 710
715 720 Gln Pro Val Ala Glu Leu Lys Asp Gln Thr Asp Phe Glu His Arg
Ile 725 730 735 Pro Lys Glu Gln Trp Trp Leu Lys Leu Arg Pro Ile Leu
Lys Ile Leu 740 745 750 Ala Lys Tyr Glu Ile Asp Leu Asp Thr Ser Asp
His Ala His Leu Glu 755 760 765 His Ile Thr Arg Lys Arg Ser Gly Glu
Ala Ala Val 770 775 780 66784PRTHomo sapiens 66Met Asp Ala Asp Asp
Ser Arg Ala Pro Lys Gly Ser Leu Arg Lys Phe 1 5 10 15 Leu Glu His
Leu Ser Gly Ala Gly Lys Ala Ile Gly Val Leu Thr Ser 20 25 30 Gly
Gly Asp Ala Gln Gly Met Asn Ala Ala Val Arg Ala Val Val Arg 35 40
45 Met Gly Ile Tyr Val Gly Ala Lys Val Tyr Phe Ile Tyr Glu Gly Tyr
50 55 60 Gln Gly Met Val Asp Gly Gly Ser Asn Ile Ala Glu Ala Asp
Trp Glu 65 70 75 80 Ser Val Ser Ser Ile Leu Gln Val Gly Gly Thr Ile
Ile Gly Ser Ala 85 90 95 Arg Cys Gln Ala Phe Arg Thr Arg Glu Gly
Arg Leu Lys Ala Ala Cys 100 105 110 Asn Leu Leu Gln Arg Gly Ile Thr
Asn Leu Cys Val Ile Gly Gly Asp 115 120 125 Gly Ser Leu Thr Gly Ala
Asn Leu Phe Arg Lys Glu Trp Ser Gly Leu 130 135 140 Leu Glu Glu Leu
Ala Arg Asn Gly Gln Ile Asp Lys Glu Ala Val Gln 145 150 155 160 Lys
Tyr Ala Tyr Leu Asn Val Val Gly Met Val Gly Ser Ile Asp Asn 165 170
175 Asp Phe Cys Gly Thr Asp Met Thr Ile Gly Thr Asp Ser Ala Leu His
180 185 190 Arg Ile Ile Glu Val Val Asp Ala Ile Met Thr Thr Ala Gln
Ser His 195 200 205 Gln Arg Thr Phe Val Leu Glu Val Met Gly Arg His
Cys Gly Tyr Leu 210 215 220 Ala Leu Val Ser Ala Leu Ala Cys Gly Ala
Asp Trp Val Phe Leu Pro 225 230 235 240 Glu Ser Pro Pro Glu Glu Gly
Trp Glu Glu Gln Met Cys Val Lys Leu 245 250 255 Ser Glu Asn Arg Ala
Arg Lys Lys Arg Leu Asn Ile Ile Ile Val Ala 260 265 270 Glu Gly Ala
Ile Asp Thr Gln Asn Lys Pro Ile Thr Ser Glu Lys Ile 275 280 285 Lys
Glu Leu Val Val Thr Gln Leu Gly Tyr Asp Thr Arg Val Thr Ile 290 295
300 Leu Gly His Val Gln Arg Gly Gly Thr Pro Ser Ala Phe Asp Arg Ile
305 310 315 320 Leu Ala Ser Arg Met Gly Val Glu Ala Val Ile Ala Leu
Leu Glu Ala 325 330 335 Thr Pro Asp Thr Pro Ala Cys Val Val Ser Leu
Asn Gly Asn His Ala 340 345 350 Val Arg Leu Pro Leu Met Glu Cys Val
Gln Met Thr Gln Asp Val Gln 355 360 365 Lys Ala Met Asp Glu Arg Arg
Phe Gln Asp Ala Val Arg Leu Arg Gly 370 375 380 Arg Ser Phe Ala Gly
Asn Leu Asn Thr Tyr Lys Arg Leu Ala Ile Lys 385 390 395 400 Leu Pro
Asp Asp Gln Ile Pro Lys Thr Asn Cys Asn Val Ala Val Ile 405 410 415
Asn Val Gly Ala Pro Ala Ala Gly Met Asn Ala Ala Val Arg Ser Ala 420
425 430 Val Arg Val Gly Ile Ala Asp Gly His Arg Met Leu Ala Ile Tyr
Asp 435 440 445 Gly Phe Asp Gly Phe Ala Lys Gly Gln Ile Lys Glu Ile
Gly Trp Thr 450 455 460 Asp Val Gly Gly Trp Thr Gly Gln Gly Gly Ser
Ile Leu Gly Thr Lys 465 470 475 480 Arg Val Leu Pro Gly Lys Tyr Leu
Glu Glu Ile Ala Thr Gln Met Arg 485 490 495 Thr His Ser Ile Asn Ala
Leu Leu Ile Ile Gly Gly Phe Glu Ala Tyr 500 505 510 Leu Gly Leu Leu
Glu Leu Ser Ala Ala Arg Glu Lys His Glu Glu Phe 515 520 525 Cys Val
Pro Met Val Met Val Pro Ala Thr Val Ser Asn Asn Val Pro 530 535 540
Gly Ser Asp Phe Ser Ile Gly Ala Asp Thr Ala Leu Asn Thr Ile Thr 545
550 555 560 Asp Thr Cys Asp Arg Ile Lys Gln Ser Ala Ser Gly Thr Lys
Arg Arg 565 570 575 Val Phe Ile Ile Glu Thr Met Gly Gly Tyr Cys Gly
Tyr Leu Ala Asn 580 585 590 Met Gly Gly Leu Ala Ala Gly Ala Asp Ala
Ala Tyr Ile Phe Glu Glu 595 600 605 Pro Phe Asp Ile Arg Asp Leu Gln
Ser Asn Val Glu His Leu Thr Glu 610 615 620 Lys Met Lys Thr Thr Ile
Gln Arg Gly Leu Val Leu Arg Asn Glu Ser 625 630 635 640 Cys Ser Glu
Asn Tyr Thr Thr Asp Phe Ile Tyr Gln Leu Tyr Ser Glu 645 650 655 Glu
Gly Lys Gly Val Phe Asp Cys Arg Lys Asn Val Leu Gly His Met 660 665
670 Gln Gln Gly Gly Ala Pro Ser Pro Phe Asp Arg Asn Phe Gly Thr Lys
675 680 685 Ile Ser Ala Arg Ala Met Glu Trp Ile Thr Ala Lys Leu Lys
Glu Ala 690 695 700 Arg Gly Arg Gly Lys Lys Phe Thr Thr Asp Asp Ser
Ile Cys Val Leu 705 710 715 720 Gly Ile Ser Lys Arg Asn Val Ile Phe
Gln Pro Val Ala Glu Leu Lys 725 730 735 Lys Gln Thr Asp Phe Glu His
Arg Ile Pro Lys Glu Gln Trp Trp Leu 740 745 750 Lys Leu Arg Pro Leu
Met Lys Ile Leu Ala Lys Tyr Lys Ala Ser Tyr 755 760 765 Asp Val Ser
Asp Ser Gly Gln Leu Glu His Val Gln Pro Trp Ser Val 770 775 780
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