U.S. patent application number 10/164278 was filed with the patent office on 2003-01-23 for prmts as modifiers of the p53 pathway and methods of use.
Invention is credited to Belvin, Marcia, Francis-Lang, Helen, Friedman, Lori, Funke, Roel P., Li, Danxi, Plowman, Gregory D..
Application Number | 20030017489 10/164278 |
Document ID | / |
Family ID | 34865578 |
Filed Date | 2003-01-23 |
United States Patent
Application |
20030017489 |
Kind Code |
A1 |
Friedman, Lori ; et
al. |
January 23, 2003 |
PRMTs as modifiers of the p53 pathway and methods of use
Abstract
Human PRMT genes are identified as modulators of the p53
pathway, and thus are therapeutic targets for disorders associated
with defective p53 function. Methods for identifying modulators of
p53, comprising screening for agents that modulate the activity of
PRMT are provided.
Inventors: |
Friedman, Lori; (San
Francisco, CA) ; Plowman, Gregory D.; (San Carlos,
CA) ; Belvin, Marcia; (Albany, CA) ;
Francis-Lang, Helen; (San Francisco, CA) ; Li,
Danxi; (San Francisco, CA) ; Funke, Roel P.;
(South San Francisco, CA) |
Correspondence
Address: |
JAN P. BRUNELLE
EXELIXIS, INC.
170 HARBOR WAY
P.O. BOX 511
SOUTH SAN FRANCISCO
CA
94083-0511
US
|
Family ID: |
34865578 |
Appl. No.: |
10/164278 |
Filed: |
June 5, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60296076 |
Jun 5, 2001 |
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60328605 |
Oct 10, 2001 |
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60338733 |
Oct 22, 2001 |
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60357253 |
Feb 15, 2002 |
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60357600 |
Feb 15, 2002 |
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Current U.S.
Class: |
435/6.14 ;
435/15; 435/7.23; 514/44A |
Current CPC
Class: |
C12Q 2600/136 20130101;
G01N 2333/4739 20130101; C12Q 1/6886 20130101; G01N 33/574
20130101; C12Q 2600/158 20130101; G01N 2500/10 20130101 |
Class at
Publication: |
435/6 ;
435/15 |
International
Class: |
C12Q 001/68; C12Q
001/48 |
Claims
What is claimed is:
1. A method of identifying a PRMT-modulating agent, said method
comprising the steps of: (a) providing an assay system comprising a
purified PRMT polypeptide or nucleic acid or a functionally active
fragment or derivative thereof; (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 PRMT-modulating
agent.
2. The method of claim 1 wherein the PRMT polypeptide or nucleic
acid is PRMT1 (CARM1).
3. The method of claim 1 wherein the assay system comprises
cultured cells that express the PRMT polypeptide.
4. The method of claim 3 wherein the cultured cells additionally
have defective p53 function.
5. The method of claim 1 wherein the assay system includes a
screening assay comprising a PRMT polypeptide, and the candidate
test agent is a small molecule modulator.
6. The method of claim 5 wherein the assay is a transferase
assay.
7. 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.
8. The method of claim 1 wherein the assay system includes a
binding assay comprising a PRMT polypeptide and the candidate test
agent is an antibody.
9. The method of claim 1 wherein the assay system includes an
expression assay comprising a PRMT nucleic acid and the candidate
test agent is a nucleic acid modulator.
10. The method of claim 9 wherein the nucleic acid modulator is an
antisense oligomer.
11. The method of claim 9 wherein the nucleic acid modulator is a
PMO.
12. The method of claim 1 additionally comprising: (d)
administering the PRMT-modulating agent identified in (c) to a
model system comprising cells defective in p53 function and,
detecting a phenotypic change in the model system that indicates
that the p53 function is restored, wherein restoration of p53
function identifies the PRMT-modulating agent as a p53 modulating
agent.
13. The method of claim 12 wherein the model system is a mouse
model with defective p53 function.
14. A method for modulating PRMT function in a mammalian cell
comprising contacting the cell with a PRMT modulating agent.
15. The method of claim 14 wherein the PRMT modulating agent
modulates a CARM1 polypeptide or nucleic acid.
16. The method of claim 14 wherein said cell has defective p53
function, and said PRMT modulating agent restores p53 function.
17. The method of claim 14 wherein the PRMT modulating agent
specifically modulates a PRMT polypeptide comprising an amino acid
sequence selected from group consisting of SEQ ID NOs:8, 9, 10, 11,
12, and 15.
18. The method of claim 14 wherein the PRMT-modulating agent is
administered to a vertebrate animal predetermined to have a disease
or disorder resulting from a defect in p53 function.
19. The method of claim 13 wherein the PRMT-modulating agent is
selected from the group consisting of an antibody and a small
molecule.
20. The method of claim 1, comprising the additional steps of: (d)
providing a secondary assay system that measures changes in p53
function, sherein said secondary assay system comprises cultured
cells or a non-human animal expressing PRMT, (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 indicative of p53 function; 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 secondary assay system identifies the test agent or
agent derived therefrom as a candidate p53 pathway modulating
agent.
21. The method of claim 20 wherein the secondary assay system
comprises cultured cells.
22. The method of claim 20 wherein the secondary assay system
comprises a non-human animal.
23. The method of claim 22 wherein the non-human animal
mis-expresses a p53 pathway gene.
24. A method of modulating p53 pathway in a mammalian cell
comprising contacting the cell with a PRMT-modulating agent that
modulates the p53 pathway.
25. The method of claim 24 wherein the agent is administered to a
mammalian animal predetermined to have a pathology associated with
the p53 pathway.
26. The method of claim 24 wherein the agent is selected from the
group consisting of a small molecule modulator, a nucleic acid
modulator, and an antibody modulator.
27. A method for diagnosing a disease or disorder associated with
alterations in PRMT expression comprising: (a) obtaining a
biological sample from a patient; (b) contacting the sample with a
probe for PRMT expression; (c) comparing results from step (b) with
a control; (d) determining whether step (c) indicates a likelihood
of the disease or disorder.
28. The method of claim 27 wherein said disease is cancer.
29. The method according to claim 28, wherein said cancer is
selected from the group consisting of colon cancer, lung cancer,
breast cancer, and ovarian cancer.
30. The method of claim 27 wherein the probe is specific for CARM1
expression.
31. A method for treating a disorder associated with impaired PRMT
function that comprises administering a therapeutically effective
amount of a PRMT modulating agent, whereby PRMT function is
restored.
32. The method of claim 31 wherein the impaired PRMT function is
attributable to an overexpression of PRMT.
33. The method of claim 31 wherein the impaired PRMT function is
attributable to an underexpression of PRMT.
34. The method of claim 31 wherein the impaired PRMT function is
attributable to impaired CARM1.
35. A method for treating a disorder associated with impaired p53
function that comprises administering a therapeutically effective
amount of a PRMT modulating agent, whereby p53 function is
restored.
36. The method of claim 35 wherein the impaired p53 function is
attributable to an overexpression of p53.
37. The method of claim 35 wherein the impaired p53 function is
attributable to an underexpression of p53.
38. The method of claim 35 wherein the PRMT modulating agent
specifically modulates CARM1.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional patent
applications No. 60/296,076 filed Jun. 5, 2001, No. 60/328,605
filed Oct. 10, 2001, No. 60/338,733 filed Oct. 22, 2001, No.
60/357,253 filed Feb. 15, 2002, and No. 60/357,600 filed Feb. 15,
2002. The contents of the prior applications are hereby
incorporated in their entirety.
BACKGROUND OF THE INVENTION
[0002] The p53 gene is mutated in over 50 different types of human
cancers, including familial and spontaneous cancers, and is
believed to be the most commonly mutated gene in human cancer
(Zambetti and Levine, FASEB (1993) 7:855-865; Hollstein, et al.,
Nucleic Acids Res. (1994) 22:3551-3555). Greater than 90% of
mutations in the p53 gene are missense mutations that alter a
single amino acid that inactivates p53 function. Aberrant forms of
human p53 are associated with poor prognosis, more aggressive
tumors, metastasis, and short survival rates (Mitsudomi et al.,
Clin Cancer Res 2000 October; 6(10):4055-63; Koshland, Science
(1993) 262:1953).
[0003] The human p53 protein normally functions as a central
integrator of signals including DNA damage, hypoxia, nucleotide
deprivation, and oncogene activation (Prives, Cell (1998) 95:5-8).
In response to these signals, p53 protein levels are greatly
increased with the result that the accumulated p53 activates cell
cycle arrest or apoptosis depending on the nature and strength of
these signals. Indeed, multiple lines of experimental evidence have
pointed to a key role for p53 as a tumor suppressor (Levine, Cell
(1997) 88:323-331). For example, homozygous p53 "knockout" mice are
developmentally normal but exhibit nearly 100% incidence of
neoplasia in the first year of life (Donehower et al., Nature
(1992) 356:215-221).
[0004] The biochemical mechanisms and pathways through which p53
functions in normal and cancerous cells are not fully understood,
but one clearly important aspect of p53 function is its activity as
a gene-specific transcriptional activator. Among the genes with
known p53-response elements are several with well-characterized
roles in either regulation of the cell cycle or apoptosis,
including GADD45, p21/Waf1/Cip1, cyclin G, Bax, IGF-BP3, and MDM2
(Levine, Cell (1997) 88:323-331).
[0005] The family of protein arginine N-methyltransferases (PRMTs)
catalyze the sequential transfer of a methyl group from
S-adenosylmethionene to the side chain nitrogens of arginine
residues within proteins to form methylated arginine derivatives
and S-adenosyl-L-homocysteine. The methylation of arginine residues
has been implicated in the regulation of signal transduction
(Altschuler L et al. (1999) J. Interferon Cytokine Res. 19:189-195;
Tang J et al. (2000) J. Biol. Chem. 275:19866-19876; Bedford M. T
et al. (2000) J. Biol. Chem. 275:16030-16036), transcription (Chen
D et al. (1999) Science 284:2174-2177), RNA transport (McBride A E
et al. (2000) J. Biol. Chem. 275:3128-3136; Yun C et al. (2000) J.
Cell Biol. 150:707-718), and possibly splicing (Friesen W J et al.,
(2001) Mol. Cell 7:1111-1117). PRMTs are conserved in evolution
(Zhang X et al. (2000) EMBO J. 19:3509-3519; Weiss V H et al.
(2000) Nat. Struct. Biol. 7:1165-1171).
[0006] Coactivator associated arginine Methyltransferase 1
(CARM1/PRMT4) functions in a dual role as a protein
methyltransferase and a transcriptional coactivator. CARM1
interacts with the p160 coactivators to enhance nuclear receptor
transcription, enhances transcription activation by the estrogen
receptor, and methylates histone H3 (Chen D et al., supra). PRMT6
is the only PRMT capable of automethylation. Of the known PRMTs,
CARM1 and PRMT6 localize to the nucleus (Frankel A et al. (2002) J
Biol Chem. 277:3537-3543).
[0007] 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, has
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 DA, 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 p53, modifier genes can be identified that may be
attractive candidate targets for novel therapeutics.
[0008] All references cited herein, including sequence information
in referenced Genbank identifier numbers and website references,
are incorporated herein in their entireties.
SUMMARY OF THE INVENTION
[0009] We have discovered genes that modify the p53 pathway in
Drosophila, and identified their human orthologs, hereinafter
referred to as PRMT. The invention provides methods for utilizing
these p53 modifier genes and polypeptides to identify
PRMT-modulating agents that are candidate therapeutic agents that
can be used in the treatment of disorders associated with defective
or impaired p53 function and/or PRMT function. p53 function.
Preferred PRMT-modulating agents specifically bind to PRMT
polypeptides and restore p53 function. Other preferred
PRMT-modulating agents are nucleic acid modulators such as
antisense oligomers and RNAi that repress PRMT gene expression or
product activity by, for example, binding to and inhibiting the
respective nucleic acid (i.e. DNA or mRNA).
[0010] PRMT-modulating agents may be evaluated by any convenient in
vitro or in vivo assay for molecular interaction with a PRMT
polypeptide or nucleic acid. In one embodiment, candidate
PRMT-modulating agents are tested with an assay system comprising a
PRMT polypeptide or nucleic acid. In one preferred embodiment, the
PRMT polypeptide or nucleic acid is PRMT1 (also referred to as
"CARM1"). Agents that produce a change in the activity of the assay
system relative to controls are identified as candidate p53
modulating agents. The assay system may be cell-based or cell-free.
PRMT-modulating agents include, but are not limited to, PRMT
related proteins (e.g. dominant negative mutants, and
biotherapeutics); PRMT-specific antibodies; PRMT-specific antisense
oligomers and other nucleic acid modulators; and chemical agents
that specifically bind to or interact with PRMT (e.g. by binding to
a PRMT binding partner). In one specific embodiment, a small
molecule modulator is identified using a transferase assay. In
specific embodiments, the screening assay system is selected from
an apoptosis assay, a cell proliferation assay, an angiogenesis
assay, and a hypoxic induction assay.
[0011] In another embodiment, candidate p53 pathway modulating
agents are further tested using a second assay system that detects
changes in the p53 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 p53 pathway, such as an angiogenic,
apoptotic, or cell proliferation disorder (e.g. cancer).
[0012] The invention further provides methods for modulating PRMT
function and/or the p53 pathway in a mammalian cell by contacting
the mammalian cell with an agent that specifically binds a PRMT
polypeptide or nucleic acid. In a preferred embodiment, the PRMT
polypeptide or nucleic acid is CARM1. 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 the p53 pathway.
DETAILED DESCRIPTION OF THE INVENTION
[0013] To identify modifiers of the p53 pathway in Drosophila, a
genetic modifier screen was carried out in which p53 was
overexpressed in the wing (Ollmann M, et al., Cell 2000 101:
91-101). The CG5358 gene was identified as a modifier of the p53
pathway. Accordingly, vertebrate orthologs of this modifier, and
preferably the human orthologs, PRMT genes (i.e., nucleic acids and
polypeptides) are attractive drug targets for the treatment of
pathologies associated with a defective p53 signaling pathway, such
as cancer.
[0014] In vitro and in vivo methods of assessing PRMT function are
provided herein. Modulation of the PRMT or their respective binding
partners is useful for understanding the association of the p53
pathway and its members in normal and disease conditions and for
developing diagnostics and therapeutic modalities for p53 related
pathologies. PRMT-modulating agents that act by inhibiting or
enhancing PRMT expression, directly or indirectly, for example, by
affecting a PRMT function such as enzymatic (e.g., catalytic) or
binding activity, can be identified using methods provided herein.
PRMT modulating agents are useful in diagnosis, therapy and
pharmaceutical development.
[0015] Nucleic Acids and Polypeptides of the Invention
[0016] Sequences related to PRMT nucleic acids and polypeptides
that can be used in the invention are disclosed in Genbank
(referenced by Genbank identifier (GI) number) as GI#s 5257220 (SEQ
ID NO:1), 18601083 (SEQ ID NO:2), 14759767 (SEQ ID NO:3), 11422727
(SEQ ID NO:4), 8922514 (SEQ ID NO:5), 17436208 (SEQ ID NO:6), and
12803778 (SEQ ID NO:7) for nucleic acid, and GI#s 5257221 (SEQ ID
NO:8), 18601084 (SEQ ID NO:9), 14759768 (SEQ ID NO:10), 11422728
(SEQ ID NO: 11), and 8922515 (SEQ ID NO:12) for polypeptides.
Additionally, nucleic acid sequences of SEQ ID NOs:13 and 14 and
amino acid sequence of SEQ ID NO:15 can also be used in the
invention.
[0017] PRMTs are transferase proteins with transferase domains. The
term "PRMT polypeptide" refers to a full-length PRMT protein or a
functionally active fragment or derivative thereof. A "functionally
active" PRMT fragment or derivative exhibits one or more functional
activities associated with a full-length, wild-type PRMT protein,
such as antigenic or immunogenic activity, enzymatic activity,
ability to bind natural cellular substrates, etc. The functional
activity of PRMT 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. For purposes herein, functionally active fragments also
include those fragments that comprise one or more structural
domains of a PRMT, such as a transferase 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;
http://pfam.wustl.edu). Methods for obtaining PRMT 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 any one of SEQ ID NOs:8, 9, 10, 11, or 12 (a PRMT).
In further preferred embodiments, the fragment comprises the entire
functionally active domain.
[0018] The term "PRMT nucleic acid" refers to a DNA or RNA molecule
that encodes a PRMT polypeptide. Preferably, the PRMT 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 PRMT. 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 JD 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; http://blast.wustl.edulblast/README.html) 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.
[0019] 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.
[0020] 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
http://www.ebi.ac.uk/MPsrch/; 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."
[0021] Derivative nucleic acid molecules of the subject nucleic
acid molecules include sequences that hybridize to the nucleic acid
sequence of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7. 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 any one
of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7 under stringent hybridization
conditions that comprise: 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 lh in a solution
containing 0.2.times. SSC and 0.1% SDS (sodium dodecyl
sulfate).
[0022] In other embodiments, moderately stringent hybridization
conditions are used that comprise: 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 (pH 7.5), SM
EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 .mu.g/ml denatured
salmon sperm DNA; hybridization for 18-20h at 40.degree. C. in a
solution containing 35% formamide, 5.times. SSC, 50 mM Tris-HCl (pH
7.5), 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.
[0023] Alternatively, low stringency conditions can be used that
comprise: 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.
[0024] Isolation, Production, Expression, and Mis-Expression of
PRMT Nucleic Acids and Polypeptides
[0025] PRMT nucleic acids and polypeptides, useful for identifying
and testing agents that modulate PRMT function and for other
applications related to the involvement of PRMT in the p53 pathway.
PRMT nucleic acids and derivatives and orthologs thereof may be
obtained using methods known to those skilled in the art. 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 PRMT protein
for assays used to assess PRMT 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 PRMT is expressed in a cell
line known to have defective p53 function (e.g. SAOS-2 osteoblasts,
H1299 lung cancer cells, C33A and HT3 cervical cancer cells, HT-29
and DLD-1 colon cancer cells, among others, available from American
Type Culture Collection (ATCC), Manassas, Va.). The recombinant
cells are used in cell-based screening assay systems of the
invention, as described further below.
[0026] The nucleotide sequence encoding a PRMT polypeptide can be
inserted into any appropriate expression vector. The necessary
transcriptional and translational signals, including
promoter/enhancer element, can derive from the native PRMT 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. A host cell
strain that modulates the expression of, modifies, and/or
specifically processes the gene product may be used.
[0027] To detect expression of the PRMT gene product, the
expression vector can comprise a promoter operably linked to a PRMT
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
PRMT gene product based on the physical or functional properties of
the PRMT protein in in vitro assay systems (e.g. immunoassays).
[0028] The PRMT 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).
[0029] Once a recombinant cell that expresses the PRMT 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, cite purification reference).
Alternatively, native PRMT 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.
[0030] The methods of this invention may also use cells that have
been engineered for altered expression (mis-expression) of PRMT or
other genes associated with the p53 pathway. As used herein,
mis-expression encompasses ectopic expression, overexpression,
under-expression, and non-expression (e.g. by gene knock-out or
blocking expression that would otherwise normally occur).
[0031] Genetically Modified Animals
[0032] Animal models that have been genetically modified to alter
PRMT expression may be used in in vivo assays to test for activity
of a candidate p53 modulating agent, or to further assess the role
of PRMT in a p53 pathway process such as apoptosis or cell
proliferation. Preferably, the altered PRMT expression results in a
detectable phenotype, such as decreased or increased levels of cell
proliferation, angiogenesis, or apoptosis compared to control
animals having normal PRMT expression. The genetically modified
animal may additionally have altered p53 expression (e.g. p53
knockout). Preferred genetically modified animals are mammals such
as primates, rodents (preferably mice), cows, horses, goats, sheep,
pigs, dogs and cats. 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.
[0033] 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.
(1197) Nature 385:810-813; and PCT International Publication Nos.
WO 97/07668 and WO 97/07669).
[0034] In one embodiment, the transgenic animal is a "knock-out"
animal having a heterozygous or homozygous alteration in the
sequence of an endogenous PRMT gene that results in a decrease of
PRMT function, preferably such that PRMT 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 PRMT gene is used to construct a
homologous recombination vector suitable for altering an endogenous
PRMT 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
[0035] 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).
[0036] 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 PRMT gene, e.g., by introduction of additional
copies of PRMT, or by operatively inserting a regulatory sequence
that provides for altered expression of an endogenous copy of the
PRMT gene. Such regulatory sequences include inducible,
tissue-specific, and constitutive promoters and enhancer elements.
The knock-in can be homozygous or heterozygous.
[0037] 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).
[0038] The genetically modified animals can be used in genetic
studies to further elucidate the p53 pathway, as animal models of
disease and disorders implicating defective p53 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 PRMT function and phenotypic changes are compared with
appropriate control animals such as genetically modified animals
that receive placebo treatment, and/or animals with unaltered PRMT
expression that receive candidate therapeutic agent.
[0039] In addition to the above-described genetically modified
animals having altered PRMT function, animal models having
defective p53 function (and otherwise normal PRMT function), can be
used in the methods of the present invention. For example, a p53
knockout mouse can be used to assess, in vivo, the activity of a
candidate p53 modulating agent identified in one of the in vitro
assays described below. p53 knockout mice are described in the
literature (Jacks et al., Nature 2001;410:1111-1116, 1043-1044;
Donehower et al., supra). Preferably, the candidate p53 modulating
agent when administered to a model system with cells defective in
p53 function, produces a detectable phenotypic change in the model
system indicating that the p53 function is restored, i.e., the
cells exhibit normal cell cycle progression.
[0040] Modulating Agents
[0041] The invention provides methods to identify agents that
interact with and/or modulate the function of PRMT and/or the p53
pathway. Modulating agents identified by these methods are also
part of the invention. Such agents are useful in a variety of
diagnostic and therapeutic applications associated with the p53
pathway, as well as in further analysis of the PRMT protein and its
contribution to the p53 pathway. Accordingly, the invention also
provides methods for modulating the p53 pathway comprising the step
of specifically modulating PRMT activity by administering a
PRMT-interacting or -modulating agent.
[0042] As used herein, a "PRMT-modulating agent" is any agent that
modulates PRMT function, for example, an agent that interacts with
PRMT to inhibit or enhance PRMT activity or otherwise affect normal
PRMT function. PRMT function can be affected at any level,
including transcription, protein expression, protein localization,
and cellular or extra-cellular activity. In a preferred embodiment,
the PRMT-modulating agent specifically modulates the function of
the PRMT. The phrases "specific modulating agent", "specifically
modulates", etc., are used herein to refer to modulating agents
that directly bind to the PRMT polypeptide or nucleic acid, and
preferably inhibit, enhance, or otherwise alter, the function of
the PRMT. These phrases also encompass modulating agents that alter
the interaction of the PRMT with a binding partner, substrate, or
cofactor (e.g. by binding to a binding partner of a PRMT, or to a
protein/binding partner complex, and altering PRMT function). In a
further preferred embodiment, the PRMT-modulating agent is a
modulator of the p53 pathway (e.g. it restores and/or up-regulates
p53 function), and thus is also a "p53 modulating agent".
[0043] Preferred PRMT-modulating agents include small molecule
compounds; PRMT-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.
[0044] Small Molecule Modulators
[0045] 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 less than 10,000, preferably
less than 5,000, more preferably less than 1,000, and most
preferably less than 500. 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 PRMT 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 PRMT-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).
[0046] 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 p53 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.
[0047] Protein Modulators
[0048] Specific PRMT-interacting proteins are useful in a variety
of diagnostic and therapeutic applications related to the p53
pathway and related disorders, as well as in validation assays for
other PRMT-modulating agents. In a preferred embodiment,
PRMT-interacting proteins affect normal PRMT function, including
transcription, protein expression, protein localization, and
cellular or extra-cellular activity. In another embodiment,
PRMT-interacting proteins are useful in detecting and providing
information about the function of PRMT proteins, as is relevant to
p53 related disorders, such as cancer (e.g., for diagnostic
means).
[0049] A PRMT-interacting protein may be endogenous, i.e. one that
naturally interacts genetically or biochemically with a PRMT, such
as a member of the PRMT pathway that modulates PRMT expression,
localization, and/or activity. PRMT-modulators include dominant
negative forms of PRMT-interacting proteins and of PRMT proteins
themselves. Yeast two-hybrid and variant screens offer preferred
methods for identifying endogenous PRMT-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
[0050] 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 JR 3.sup.rd,
Trends Genet (2000) 16:5-8).
[0051] An PRMT-interacting protein may be an exogenous protein,
such as a PRMT-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). PRMT antibodies are further discussed below.
[0052] In preferred embodiments, a PRMT-interacting protein
specifically binds a PRMT protein. In alternative preferred
embodiments, a PRMT-modulating agent binds a PRMT substrate,
binding partner, or cofactor.
[0053] Antibodies
[0054] In another embodiment, the protein modulator is a PRMT
specific antibody agonist or antagonist. The antibodies have
therapeutic and diagnostic utilities, and can be used in screening
assays to identify PRMT modulators. The antibodies can also be used
in dissecting the portions of the PRMT pathway responsible for
various cellular responses and in the general processing and
maturation of the PRMT.
[0055] Antibodies that specifically bind PRMT polypeptides can be
generated using known methods. Preferably the antibody is specific
to a mammalian ortholog of PRMT polypeptide, and more preferably,
to human PRMT. 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 PRMT
which are particularly antigenic can be selected, for example, by
routine screening of PRMT 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 shown in any of SEQ ID NOs:8, 9, 10, 11, or 12. 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 PRMT or substantially purified fragments thereof. If PRMT
fragments are used, they preferably comprise at least 10, and more
preferably, at least 20 contiguous amino acids of a PRMT protein.
In a particular embodiment, PRMT-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.
[0056] The presence of PRMT-specific antibodies is assayed by an
appropriate assay such as a solid phase enzyme-linked immunosorbant
assay (ELISA) using immobilized corresponding PRMT polypeptides.
Other assays, such as radioimmunoassays or fluorescent assays might
also be used.
[0057] Chimeric antibodies specific to PRMT 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).
[0058] PRMT-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).
[0059] 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).
[0060] 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).
[0061] 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 aboutlo mg/ml. Immunotherapeutic methods are further
described in the literature (U.S. Pat. No. 5,859,206;
WO0073469).
[0062] Nucleic Acid Modulators
[0063] Other preferred PRMT-modulating agents comprise nucleic acid
molecules, such as antisense oligomers or double stranded RNA
(dsRNA), which generally inhibit PRMT activity. Preferred nucleic
acid modulators interfere with the function of the PRMT nucleic
acid such as DNA replication, transcription, translocation of the
PRMT RNA to the site of protein translation, translation of protein
from the PRMT RNA, splicing of the PRMT RNA to yield one or more
mRNA species, or catalytic activity which may be engaged in or
facilitated by the PRMT RNA.
[0064] In one embodiment, the antisense oligomer is an
oligonucleotide that is sufficiently complementary to a PRMT mRNA
to bind to and prevent translation, preferably by binding to the 5'
untranslated region. PRMT-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.
[0065] 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).
[0066] Alternative preferred PRMT 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, 485490
(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).
[0067] 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 PRMT-specific nucleic acid modulator is used in an
assay to further elucidate the role of the PRMT in the p53 pathway,
and/or its relationship to other members of the pathway. In another
aspect of the invention, a PRMT-specific antisense oligomer is used
as a therapeutic agent for treatment of p53-related disease
states.
[0068] Assay Systems
[0069] The invention provides assay systems and screening methods
for identifying specific modulators of PRMT 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 PRMT nucleic acid or protein.
In general, secondary assays further assess the activity of a PRMT
modulating agent identified by a primary assay and may confirm that
the modulating agent affects PRMT in a manner relevant to the p53
pathway. In some cases, PRMT modulators will be directly tested in
a secondary assay.
[0070] In a preferred embodiment, the screening method comprises
contacting a suitable assay system comprising a PRMT polypeptide
with a candidate agent under conditions whereby, but for the
presence of the agent, the system provides a reference activity
(e.g. transferase 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
PRMT activity, and hence the p53 pathway. The PRMT polypeptide or
nucleic acid used in the assay may comprise any of the nucleic
acids or polypeptides described above (e.g. SEQ ID NOs 1-15). In
one preferred embodiment, the PRMT is a CARM1, comprising a nucleic
acid sequence selected from any one of SEQ ID NOs 1-3, 13 and 14,
or an amino acid sequence selected from any one of SEQ ID NOs 8-10,
and 15. In a further preferred embodiment, the CARM1 nucleic acid
comprises SEQ ID NO:13 or 14, and the protein comprises SEQ ID
NO:9, 10 or 15.
[0071] Primary Assays
[0072] The type of modulator tested generally determines the type
of primary assay.
[0073] Primary Assays for Small Molecule Modulators
[0074] 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,
immunogenicty and changes in cellular morphology or other cellular
characteristics. Appropriate screening assays may use a wide range
of detection methods including fluorescent, radioactive,
calorimetric, spectrophotometric, and amperometric methods, to
provide a read-out for the particular molecular event detected.
[0075] Cell-based screening assays usually require systems for
recombinant expression of PRMT 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
PRMT-interacting proteins are used in screens to identify small
molecule modulators, the binding specificity of the interacting
protein to the PRMT protein may be assayed by various known methods
such as substrate processing (e.g. ability of the candidate
PRMT-specific binding agents to function as negative effectors in
PRMT-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 PRMT 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.
[0076] The screening assay may measure a candidate agent's ability
to specifically bind to or modulate activity of a PRMT polypeptide,
a fusion protein thereof, or to cells or membranes bearing the
polypeptide or fusion protein. The PRMT polypeptide can be full
length or a fragment thereof that retains functional PRMT activity.
The PRMT polypeptide may be fused to another polypeptide, such as a
peptide tag for detection or anchoring, or to another tag. The PRMT
polypeptide is preferably human PRMT, or is an ortholog or
derivative thereof as described above. In a preferred embodiment,
the screening assay detects candidate agent-based modulation of
PRMT interaction with a binding target, such as an endogenous or
exogenous protein or other substrate that has PRMT--specific
binding activity, and can be used to assess normal PRMT gene
function.
[0077] Suitable assay formats that may be adapted to screen for
PRMT 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).
[0078] A variety of suitable assay systems may be used to identify
candidate PRMT and p53 pathway modulators (e.g. U.S. Pat. No.
6,020,135 (p53 modulation)). Specific preferred assays are
described in more detail below.
[0079] Transferase assays. Methyltransferase assays are well known
in the art, and may be performed as described (Tang J et al. (2000)
J Biol Chem. 275:7723-7730). Briefly, hypomethylated cell lysates
are produced, and the ability of endogenous methyltransferases
present in the hypomethylated cell lysate to methylate various
substrates after addition of [.sup.3H] S-adenosylmethionene is
evaluated.
[0080] Apoptosis assays. 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).
An apoptosis assay system may comprise a cell that expresses a
PRMT, and that optionally has defective p53 function (e.g. p53 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 p53 modulating agents. In some
embodiments of the invention, an apoptosis assay may be used as a
secondary assay to test a candidate p53 modulating agents that is
initially identified using a cell-free assay system. An apoptosis
assay may also be used to test whether PRMT function plays a direct
role in apoptosis. For example, an apoptosis assay may be performed
on cells that over- or under-express PRMT relative to wild type
cells. Differences in apoptotic response compared to wild type
cells suggests that the PRMT plays a direct role in the apoptotic
response. Apoptosis assays are described further in U.S. Pat. No.
6,133,437.
[0081] Cell proliferation and cell cycle assays. 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.
[0082] 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).
[0083] Cell proliferation may also be assayed by colony formation
in soft agar (Sambrook et al., Molecular Cloning, Cold Spring
Harbor (1989)). For example, cells transformed with PRMT are seeded
in soft agar plates, and colonies are measured and counted after
two weeks incubation.
[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 PRMT may be
stained with propidium iodide and evaluated in a flow cytometer
(available from Becton Dickinson).
[0085] Accordingly, a cell proliferation or cell cycle assay system
may comprise a cell that expresses a PRMT, and that optionally has
defective p53 function (e.g. p53 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 p53 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 p.sup.53 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 PRMT 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 PRMT relative to wild type cells. Differences in
proliferation or cell cycle compared to wild type cells suggests
that the PRMT plays a direct role in cell proliferation or cell
cycle.
[0086] Angiogenesis. 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
PRMT, and that optionally has defective p53 function (e.g. p53 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 p53 modulating agents. In some
embodiments of the invention, the angiogenesis assay may be used as
a secondary assay to test a candidate p53 modulating agents that is
initially identified using another assay system. An angiogenesis
assay may also be used to test whether PRMT function plays a direct
role in cell proliferation. For example, an angiogenesis assay may
be performed on cells that over- or under-express PRMT relative to
wild type cells. Differences in angiogenesis compared to wild type
cells suggests that the PRMT plays a direct role in
angiogenesis.
[0087] Hypoxic induction. 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 PRMT 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 PRMT, and that optionally has a
mutated p53 (e.g. p53 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 p53
modulating agents. In some embodiments of the invention, the
hypoxic induction assay may be used as a secondary assay to test a
candidate p53 modulating agents that is initially identified using
another assay system. A hypoxic induction assay may also be used to
test whether PRMT 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 PRMT relative to wild type
cells. Differences in hypoxic response compared to wild type cells
suggests that the PRMT plays a direct role in hypoxic
induction.
[0088] Cell adhesion. 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.
[0089] 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.
[0090] 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).
[0091] Primary Assays for Antibody Modulators
[0092] For antibody modulators, appropriate primary assays test is
a binding assay that tests the antibody's affinity to and
specificity for the PRMT 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 PRMT-specific
antibodies; others include FACS assays, radioimmunoassays, and
fluorescent assays.
[0093] Primary Assays for Nucleic Acid Modulators
[0094] For nucleic acid modulators, primary assays may test the
ability of the nucleic acid modulator to inhibit or enhance PRMT
gene expression, preferably mRNA expression. In general, expression
analysis comprises comparing PRMT expression in like populations of
cells (e.g., two pools of cells that endogenously or recombinantly
express PRMT) 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 PRMT 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 PRMT 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).
[0095] Secondary Assays
[0096] Secondary assays may be used to further assess the activity
of PRMT-modulating agent identified by any of the above methods to
confirm that the modulating agent affects PRMT in a manner relevant
to the p53 pathway. As used herein, PRMT-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 PRMT.
[0097] Secondary assays generally compare like populations of cells
or animals (e.g., two pools of cells or animals that endogenously
or recombinantly express PRMT) in the presence and absence of the
candidate modulator. In general, such assays test whether treatment
of cells or animals with a candidate PRMT-modulating agent results
in changes in the p53 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
p53 or interacting pathways.
[0098] Cell-Based Assays
[0099] Cell based assays may use a variety of mammalian cell lines
known to have defective p53 function (e.g. SAOS-2 osteoblasts,
H1299 lung cancer cells, C33A and HT3 cervical cancer cells, HT-29
and DLD-1 colon cancer cells, among others, available from American
Type Culture Collection (ATCC), Manassas, Va.). Cell based assays
may detect endogenous p53 pathway activity or may rely on
recombinant expression of p53 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.
[0100] Animal Assays
[0101] A variety of non-human animal models of normal or defective
p53 pathway may be used to test candidate PRMT modulators. Models
for defective p53 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 p53
pathway. Assays generally require systemic delivery of the
candidate modulators, such as by oral administration, injection,
etc.
[0102] In a preferred embodiment, p53 pathway activity is assessed
by monitoring neovascularization and angiogenesis. Animal models
with defective and normal p53 are used to test the candidate
modulator's affect on PRMT 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 PRMT. 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.
[0103] In another preferred embodiment, the effect of the candidate
modulator on PRMT is assessed via tumorigenicity assays. In one
example, xenograft human tumors are 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 PRMT 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, 0.1M
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.
[0104] Diagnostic and Therapeutic Uses
[0105] Specific PRMT-modulating agents are useful in a variety of
diagnostic and therapeutic applications where disease or disease
prognosis is related to defects in the p53 pathway, such as
angiogenic, apoptotic, or cell proliferation disorders.
Accordingly, t h e invention also provides methods for modulating
the p53 pathway in a cell, preferably a cell pre-determined to have
defective or impaired p53 function (e.g. due to overexpression,
underexpression, or misexpression of p53, or due to gene
mutations), comprising the step of administering an agent to the
cell that specifically modulates PRMT activity. Preferably, the
modulating agent produces a detectable phenotypic change in the
cell indicating that the p53 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 p53
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 p53 function by administering a
therapeutically effective amount of a PRMT-modulating agent that
modulates the p53 pathway. The invention further provides methods
for modulating PRMT function in a cell, preferably a cell
pre-determined to have defective or impaired PRMT function, by
administering a PRMT-modulating agent. Additionally, the invention
provides a method for treating disorders or disease associated with
impaired PRMT function by administering a therapeutically effective
amount of a PRMT-modulating agent. In certain embodiments the
impaired PRMT function is attributable to impaired CARM1.
[0106] The discovery that PRMT is implicated in p53 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 p53 pathway and for the identification of
subjects having a predisposition to such diseases and
disorders.
[0107] Various expression analysis methods can be used to diagnose
whether PRMT 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 FM 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 p53 signaling that express a PRMT, are identified as
amenable to treatment with a PRMT modulating agent. In a preferred
application, the p53 defective tissue overexpresses a PRMT 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 PRMT
cDNA sequences as probes, can determine whether particular tumors
express or overexpress PRMT. Alternatively, the TaqMan.RTM. is used
for quantitative RT-PCR analysis of PRMT expression in cell lines,
normal tissues and tumor samples (PE Applied Biosystems).
[0108] Various other diagnostic methods may be performed, for
example, utilizing reagents such as the PRMT oligonucleotides, and
antibodies directed against a PRMT, as described above for: (1) the
detection of the presence of PRMT gene mutations, or the detection
of either over- or under-expression of PRMT MRNA relative to the
non-disorder state; (2) the detection of either an over- or an
under-abundance of PRMT gene product relative to the non-disorder
state; and (3) the detection of perturbations or abnormalities in
the signal transduction pathway mediated by PRMT.
[0109] 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 PRMT expression, the method
comprising: a) obtaining a biological sample from the patient; b)
contacting the sample with a probe for PRMT 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
selected from the group consisting of colon cancer, lung cancer,
breast cancer, and ovarian cancer. The probe may be either DNA or
protein, including an antibody.
EXAMPLES
[0110] The following experimental section and examples are offered
by way of illustration and not by way of limitation.
[0111] I. Drosophila p53 screen
[0112] The Drosophila p53 gene was overexpressed specifically in
the wing using the vestigial margin quadrant enhancer. Increasing
quantities of Drosophila p53 (titrated using different strength
transgenic inserts in 1 or 2 copies) caused deterioration of normal
wing morphology from mild to strong, with phenotypes including
disruption of pattern and polarity of wing hairs, shortening and
thickening of wing veins, progressive crumpling of the wing and
appearance of dark "death" inclusions in wing blade. In a screen
designed to identify enhancers and suppressors of Drosophila p53,
homozygous females carrying two copies of p53 were crossed to 5663
males carrying random insertions of a piggyBac transposon (Fraser M
et al., Virology (1985) 145:356-361). Progeny containing insertions
were compared to non-insertion-bearing sibling progeny for
enhancement or suppression of the p53 phenotypes. Sequence
information surrounding the piggyBac insertion site was used to
identify the modifier genes. Modifiers of the wing phenotype were
identified as members of the p53 pathway. CG5358 was an enhancer of
the wing phenotype. Human orthologs of the modifiers are referred
to herein as PRMT.
[0113] BLAST analysis (Altschul et al., supra) was employed to
identify Targets from Drosophila modifiers. For example, amino acid
sequence of CG5358 from drosophila shares 59% and 38% sequence
identity with SEQ ID NOs:9 and 12, respectively.
[0114] 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; http://pfam.wustl.edu), 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, Calif.: AAAI Press, 1998), and dust (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.
[0115] II. Expression analysis
[0116] 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, U C Davis,
Clontech, Stratagene, and Ambion.
[0117] TaqMan analysis was used to assess expression levels of the
disclosed genes in various samples.
[0118] 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.,
http://www.appliedbiosystems.com/).
[0119] Primers for expression analysis using TaqMan assay (Applied
Biosystems, Foster City, Calif.) were prepared according to the
TaqMan 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.
[0120] Taqman 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).
[0121] 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)).
[0122] GI#14759767 (SEQID NO:3) was overexpressed in 8/30 matched
colon tumors, 7/13 matched lung tumors, and 3/7 matched ovarian
tumors. 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.
[0123] In further expression analysis studies, human CARM1 (SEQ ID
NO: 14) message levels in a wide variety of well-characterized
tumor cell-lines were analyzed using Taqman. Results showed that
hCARM-1 was significantly upregulated in lung and colon tumor
derived cell-lines and to a lesser extent in breast and ovarian
cell lines. In another assay, CARM-1 protein (SEQ ID NO:9) levels
in multiple tumor biopsy samples from lung and colon cancer
patients and their adjacent normal tissue counterparts were stained
with an anti-CARM-1 specific antibody. The results showed elevated
CARM-1 levels in many tumor-derived tissues but not in the
corresponding normal tissue.
[0124] III. Methylation Assay
[0125] In order to evaluate whether the full-length hCARM-l had
methylating activity we performed a methylation reaction. Mouse
CARM-1 (SEQ ID NO: 8) has been previously shown to specifically
methylate Histone H3 in vitro and in vivo. We asked whether our
human homolog was also capable of exhibiting the same substrate
preference. hCARM-1 (SEQ ID NO:9) was produced in and purified from
baculovirus infected insect cells and increasing amounts of the
purified enzyme were added to reactions containing a constant
amount of recombinant Histone H3. Our experiments showed that
hCARM-1 methylates Histone H3 efficiently. Interestingly, a
previously documented general methylation inhibitor, homocysteine,
effectively inhibited hCARM-1 mediated methylation.
[0126] Methylation activity assay: Reactions were performed in IX
methylation buffer containing 20 mM Tris.HCl, pH 8.0, 200 mM NaCI
and 0.4 mM EDTA. Reactions were assembled with 2.5 .mu.g of Histone
H3 and increasing amounts of hCARM-1 (0.25 .mu.g, 0.5 .mu.g, 1 25
.mu.g, 2.5 .mu.g, 3.75 .mu.g, 5 .mu.g, or 7.5 .mu.g). A mock
reaction where hCARM-1 (SEQ ID NO:14) was omitted was used as the
negative control. Reactions were incubated at 30.degree. C. for 1
hr. prior to loading on a 10-20% gradient SDS-PAGE. The gel was
fixed, dried, and exposed to film.
[0127] IV. Cell-Based Assays
[0128] Mouse CARM-1 has been implicated as a co-activator of the
androgen and estrogen receptor mediated signaling pathways along
with the well-known steroid co-activator GRIP-1. We were therefore
interested in testing the contribution, if any, of our human clone
to these pathways. When full-length hCARM-1 (SEQ ID NO: 14) was
co-transfected with GRIP-1 and the estrogen receptor (ER) into the
breast cancer cell line T47D, we obtained a clear hCARM-1 (SEQ ID
NO: 14) concentration-dependent increase in the estradiol mediated
induction of a reporter construct containing an ER dependent
promoter in front of the luciferase gene, when compared to the
induction obtained with GRIP-1 and ER alone. Conversely,
co-transfection of antisense oligos to hCARM-1 (SEQ ID NO: 14)
effectively abrogated activation of the ER dependent reporter in
the presence of transfected hCARM-1 (SEQ ID NO:14).
[0129] Interestingly, a similar inhibitory effect on ER dependent
activation could be obtained by transfection of CARM-1 antisense
oligos even in the absence of any exogenous (transfected) proteins.
Thus, antagonizing endogenous CARM-1 is deleterious to hormone
dependent activation by endogeous ER. Similar results were obtained
upon cotransfection of hCARM-1 (SEQ ID NO:14) antisense oligos into
MDA-MB-453 breast cancer cells to assess andogen receptor (AR)
dependent signaling. Our results therefore implicate an essential
role for hCARM-1 in AR and ER mediated signaling in cells.
[0130] Transfection assays: Cells were plated in 12-well dishes and
allowed to adhere and grow overnight to 80% confluency at the time
of transfection. Tranfections were perfomed in triplicate using
Lipofectamine 2000 (Gibco) and OptiMEM media. Total amount of DNA
transfected was held constant within experiments. Six hrs. post
transfection the Lipofectamine-DNA mix was removed and replaced
with fresh media containing 10% serum. Hormone (dihydrotestosterone
or estradiol) was added at this time and reporter activation
measured after 24 hr.
[0131] V. High-Throughput In Vitro Fluorescence Polarization
Assay
[0132] Fluorescently-labeled PRMT 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 PRMT activity.
[0133] VI. High-Throughput In Vitro Binding Assay.
[0134] .sup.33P-labeled PRMT 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 p53 modulating agents.
[0135] VII. Immunoprecipitations and Immunoblotting
[0136] For coprecipitation of transfected proteins,
3.times.10.sup.6 appropriate recombinant cells containing the PRMT
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.
[0137] 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).
Sequence CWU 1
1
15 1 3124 DNA Mus musculus 1 agggggcctg gagccggacc taagatggca
gcggcggcag cgacggcggt ggggccgggt 60 gcggggagcg ctggggtggc
gggcccgggc ggcgcggggc cctgcgctac agtgtctgtg 120 ttcccgggcg
cccgcctcct cactatcggc gacgcgaacg gcgagatcca gcggcacgcg 180
gagcagcagg cgctgcgcct tgaggtgcgc gccggaccag acgcggcggg catcgccctc
240 tacagccatg aagatgtgtg tgttttcaag tgctcggtgt cccgagagac
agagtgcagt 300 cgtgtgggca gacagtcctt catcatcacc ctgggctgca
acagcgtcct catccagttt 360 gccacacccc acgatttctg ttctttctac
aacatcctga aaacctgtcg gggccacaca 420 ctggagcgct ctgtgttcag
tgagcggaca gaggaatcct cagctgtgca gtacttccag 480 ttctatggct
acctatccca gcagcagaac atgatgcagg actatgtgcg gacaggcacc 540
taccagcgtg cgatcctgca gaaccacacg gacttcaagg acaagatcgt tctagatgtg
600 ggctgtggct ctgggatcct gtcatttttt gctgctcaag caggagccag
gaaaatttat 660 gcagtggaag ccagcaccat ggctcagcat gcagaggtcc
tggtgaagag taacaatctg 720 acagaccgca tcgtggtcat ccctggcaaa
gtagaggagg tctcattgcc tgagcaagtg 780 gacattatca tctcagagcc
catgggctac atgctcttca atgaacgaat gctcgagagc 840 tacctccatg
ccaaaaagta cctgaagcct agtggaaaca tgttccccac cattggtgat 900
gtccacctcg cacccttcac tgatgaacag ctctacatgg agcagttcac caaagccaac
960 ttccggtacc agccatcctt ccatggagtg gacctgtcgg ccctcagagg
tgccgctgtg 1020 gatgagtact tccggcaacc tgtggtggac acatttgaca
tccggatcct gatggccaaa 1080 tctgtcaagt acacagtgaa cttcttagaa
gccaaagaag gcgatttgca caggatagaa 1140 atcccattca aattccacat
gctgcattca gggctagtcc atggcttggc cttctggttc 1200 gatgttgctt
tcattggctc cataatgacc gtgtggctat ccacagcccc aacagagccc 1260
ctgacccact ggtaccaggt ccggtgcctc ttccagtcac cgttgtttgc caaggccggg
1320 gacacgctct cagggacatg tctgcttatt gccaacaaaa gacagagcta
tgacatcagt 1380 attgtggcac aggtggacca gacaggctcc aagtccagta
acctgctgga tctaaagaac 1440 cccttcttca ggtacacagg tacaacccca
tcacccccac ctggctcaca ctacacgtct 1500 ccctcggaga atatgtggaa
cacaggaagc acctataatc tcagcagcgg ggtggctgtg 1560 gctggaatgc
ctactgccta cgacctgagc agtgttattg ccggcggctc cagtgtgggt 1620
cacaacaacc tgattccctt agctaacaca gggattgtca atcacaccca ctcccggatg
1680 ggctccataa tgagcacggg cattgtccaa ggctcctcag gtgcccaggg
aggcggcggt 1740 agctccagtg cccactatgc agtcaacaac cagttcacca
tgggtggccc tgccatctct 1800 atggcctcgc ccatgtccat cccgaccaac
accatgcact atgggagtta ggtgcctcca 1860 gccgcgacag cactgcgcac
tgacagcacc aggaaaccaa atcaagtcca ggcccggcac 1920 agccagtggc
tgttccccct tgttctggag aagttgttga acacccggtc acagcctcct 1980
tgctatggga acttggacaa ttttgtacac gatgtcgccg ctgccctcaa gtacccccag
2040 cccaaccttt ggtcccgagc gcgtgttgct gccatacttt acatgagatc
ctgttggggc 2100 agccctcatc ctgttctgta ctctccactc tgacctggct
ttgacatctg ctggaagagg 2160 caagtcctcc cccaaccccc acagctgcac
ctgaccaggc aggaggaggc cagcagctgc 2220 caccacagac ctggcagcac
ccaccccaca acccgtcctt gcacctcccc tcacctgggg 2280 tggcagcaca
gccagctgga cctctccttc aactaccagg ccacatggtc accatgggcg 2340
tgacatgctg ctttttttaa ttttattttt ttacgaaaag aaccagtgtc aacccacaga
2400 ccctctgaga aacccggctg gcgcgccaag ccagcagccc ctgttcctag
gcccagaggt 2460 tctaggtgag gggtggccct gtcaagcctt cagagtgggc
acagcccctc ccaccaaagg 2520 gttcacctca aacttgaatg tacaaaccac
ccagctgtcc aaaggcctag tccctacttt 2580 ctgctactgt cctgtcctga
gccctgaagg cccccctcca tcaaaagctt gaacaggcag 2640 cccagagtgt
gtcaccctgg gctactgggg cagacaagaa acctcaaaga tctgtcacac 2700
acacacaagg aaggcgtcct ctcctgatag ctgacatagg cctgtgtgtt gcgttcacat
2760 tcatgttcta cttaatcctc tcaagacagc aaccctggga aggagcctcg
cagggacctc 2820 cccagacaag aagaaaagca aacaaggaag ggtgattaat
aagcacaggc agtttcccct 2880 attcccttac cctagagtcc ccacctgaat
ggccacagcc tgccacagga accccttggc 2940 aaaggctgga gctgctctgt
gccaccctcc tgacctgtca gggaatcaca gggccctcag 3000 gcagctggga
accaggctct ctcctgtcca tcagtaatac tccttgctcg gatggccctc 3060
ccccaccttt atataaattc tctggatcac ctttgcatag aaaataaaag tgtttgcttt
3120 gtaa 3124 2 2954 DNA Homo sapiens 2 cggcggcggc ggcggcggcg
gcggcggcgg cggcggcggc ggcggcggca gcggcggcgg 60 cctgggcccg
ggcgcagcgg cggcggcggc ggggcctgga gccggatcta agatggcagc 120
ggcggcggcg gcggtggggc cgggcgcggg cggcgcgggg tcggcggtcc cgggcggcgc
180 ggggccctgc gctaccgtgt cggtgttccc cggcgcccgc ctcctcacca
tcggcgacgc 240 gaacggcgag atccagcggc acgcggagca gcaggcgctg
cgcctcgagg tgcgcgccgg 300 cccggactcg gcgggcatcg ccctctacag
ccatgaagat gtgtgtgtct ttaagtgctc 360 agtgtcccga gagacagagt
gcagccgtgt gggcaagcag tccttcatca tcaccctggg 420 ctgcaacagc
gtcctcatcc agttcgccac acccaacgat ttctgttcct tctacaacat 480
cctgaaaacc tgccggggcc acaccctgga gcggtctgtg ttcagcgagc ggacggagga
540 gtcttctgcc gtgcagtact tccagtttta tggctacctg tcccagcagc
agaacatgat 600 gcaggactac gtgcggacag gcacctacca gcgcgccatc
ctgcaaaacc acaccgactt 660 caaggacaag atcgttcttg atgttggctg
tggctctggg atcctgtcgt tttttgccgc 720 ccaagctgga gcacggaaaa
tctacgcggt ggaggccagc accatggccc agcacgctga 780 ggtcttggtg
aagagtaaca acctgacgga ccgcatcgtg gtcatcccgg gcaaggtgga 840
ggaggtgtca ctccccgagc aggtggacat catcatctcg gagcccatgg gctacatgct
900 cttcaacgag cgcatgctgg agagctacct ccacgccaag aagtacctga
agcccagcgg 960 aaacatgttt cctaccattg gtgacgtcca ccttgcaccc
ttcacggatg aacagctcta 1020 catggagcag ttcaccaagg ccaacttctg
gtaccagcca tctttccatg gagtggacct 1080 gtcggccctc cgaggtgccg
cggtggatga gtatttccgg cagcctgtgg tggacacatt 1140 tgacatccgg
atcctgatgg ccaagtctgt caagtacacg gtgaacttct tagaagccaa 1200
agaaggagat ttgcacagga tagaaatccc attcaaattc cacatgctgc attcagggct
1260 ggtccacggc ctggctttct ggtttgacgt tgctttcatc ggctccataa
tgaccgtgtg 1320 gctgtccaca gccccgacag agcccctgac ccactggtac
caggtgcggt gcctgttcca 1380 gtcaccactg ttcgccaagg caggggacac
gctctcaggg acatgtctgc ttattgccaa 1440 caaaagacag agctacgaca
tcagtattgt ggcccaggtg gaccagaccg gctccaagtc 1500 cagtaacctc
ctggatctga aaaacccctt ctttagatac acgggcacaa cgccctcacc 1560
cccacccggc tcccactaca catctccctc ggaaaacatg tggaacacgg gcagcaccta
1620 caacctcagc agcgggatgg ccgtggcagg gatgccgacc gcctatgact
tgagcagtgt 1680 tattgccagt ggctccagcg tgggccacaa caacctgatt
cctttagcca acacggggat 1740 tgtcaatcac acccactccc ggatgggctc
cataatgagc acggggattg tccaagggtc 1800 ctccggcgcc cagggcagtg
gtggtggcag cacgagtgcc cactatgcag tcaacagcca 1860 gttcaccatg
ggcggccccg ccatctccat ggcgtcgccc atgtccatcc cgaccaacac 1920
catgcactac gggagctagg ggcccgcccc gcggactgac agcaccagga aaccaaatga
1980 tgtccctgcc cgccgccccc gccgggcggc tttccccctt gtactggaga
agctcgaaca 2040 cccggtcaca gctctctttg ctatgggaac tgggacactt
ttttacacga tgttgccgcc 2100 gtccccaccc taacccccac ctcccggccc
tgagcgtgtg tcgctgccat attttacaca 2160 aaatcatgtt gtgggagccc
tcgtcccccc tcctgcccgc tctaccctga cctgggcttg 2220 tcatctgctg
gaacaggcgc catggggcct gccagccctg cctgccaggt cccttagcac 2280
ctgtccccct gcctgtctcc agtgggaagg tagcctggcc aggcggggcc tccccttcga
2340 cgaccaggcc tcggtcacaa cggacgtgac atgctgcttt ttttaatttt
atttttttat 2400 gaaaagaacc agtgtcaatc cgcagaccct ctgtgaagcc
aggccggccg ggccgagcca 2460 gcagcccctc tccctagact cagaggcgcc
gcggggaggg gtggccccgc cgaggcttca 2520 ggggccccct ccccaccaaa
gggttcacct cacacttgaa tgtacaaccc accccactgt 2580 cgggaaggcc
tccgtcctcg gcccctgcct cttgctgctg tcctgtcccc gagcccctgc 2640
aggtcccccc ccgccccccc actcaagagt tagagcaggt ggctgcaggc cttgggcccg
2700 gagggaaggc cactgccggc cacttggggc agacacagac acctcaagga
tctgtcacgg 2760 aaggcgtcct ttttccttgt agctaacgtt aggcctgagt
agctcccctc catccttgta 2820 gacgctccag tccctactac tgtgacggca
tttccatccc tcccctgccc gggaagggac 2880 cttgcaggga cctctccctc
caaaaaaaga aaaaaagaaa aagaaagaaa aaataaatga 2940 ggaaacgtgt tgca
2954 3 2143 DNA Homo sapiens 3 cgcatcgtgg tcatcccggg caaggtggag
gaggtgtcac tccccgagca ggtggacatc 60 atcatctcgg agcccatggg
ctacatgctc ttcaacgagc gcatgctgga gagctacctc 120 cacgccaaga
agtacctgaa gcccagcgga aacatgtttc ctaccattgg tgacgtccac 180
cttgcaccct tcacggatga acagctctac atggagcagt tcaccaaggc caacttctgg
240 taccagccat ctttccatgg agtggacctg tcggccctcc gaggtgccgc
ggtggatgag 300 tatttccggc agcctgtggt ggacacattt gacatccgga
tcctgatggc caagtctgtc 360 aagtacacgg tgaacttctt agaagccaaa
gaaggagatt tgcacaggat agaaatccca 420 ttcaaattcc acatgctgca
ttcagggctg gtccacggcc tggctttctg gtttgacgtt 480 gctttcatcg
gctccataat gaccgtgtgg ctgtccacag ccccgacaga gcccctgacc 540
cactggtacc aggtgcggtg cctgttccag tcaccactgt tcgccaaggc aggggacacg
600 ctctcaggga catgtctgct tattgccaac aaaagacaga gctacgacat
cagtattgtg 660 gcccaggtgg accagaccgg ctccaagtcc agtaacctcc
tggatctgaa aaaccccttc 720 tttagataca cgggcacaac gccctcaccc
ccacccggct cccactacac atctccctcg 780 gaaaacatgt ggaacacggg
cagcacctac aacctcagca gcgggatggc cgtggcaggg 840 atgccgaccg
cctatgactt gagcagtgtt attgccagtg gctccagcgt gggccacaac 900
aacctgattc ctttagccaa cacggggatt gtcaatcaca cccactcccg gatgggctcc
960 ataatgagca cggggattgt ccaaggggtc ctccggcgcc cagggcagtg
gtggtggcag 1020 cacgagtgcc cactatgcag tcaacagcca gttcaccatg
ggcggccccg ccatctccat 1080 ggcgtcgccc atgtccatcc cgaccaacac
catgcactac gggagctagg ggcccgcccc 1140 gcggactgac agcaccagga
aaccaaatga tgtccctgcc cgccgccccc gccgggcggc 1200 tttccccctt
gtactggaga agctcgaaca cccggtcaca gctctctttg ctatgggaac 1260
tgggacactt ttttacacga tgttgccgcc gtccccaccc taacccccac ctcccggccc
1320 tgagcgtgtg tcgctgccat attttacaca aaatcatgtt gtgggagccc
tcgtcccccc 1380 tcctgcccgc tctaccctga cctgggcttg tcatctgctg
gaacaggcgc catggggcct 1440 gccagccctg cctgccaggt cccttagcac
ctgtccccct gcctgtctcc agtgggaagg 1500 tagcctggcc aggcggggcc
tccccttcga cgaccaggcc tcggtcacaa cggacgtgac 1560 atgctgcttt
ttttaatttt atttttttat gaaaagaacc agtgtcaatc cgcagaccct 1620
ctgtgaagcc aggccggccg ggccgagcca gcagcccctc tccctagact cagaggcgcc
1680 gcggggaggg gtggccccgc cgaggcttca ggggccccct ccccaccaaa
gggttcacct 1740 cacacttgaa tgtacaaccc accccactgt cgggaaggcc
tccgtcctcg gcccctgcct 1800 cttgctgctg tcctgtcccc gagcccctgc
aggtcccccc ccgccccccc actcaagagt 1860 tagagcaggt ggctgcaggc
cttgggcccg gagggaaggc cactgccggc cacttggggc 1920 agacacagac
acctcaagga tctgtcacgg aaggcgtcct ttttccttgt agctaacgtt 1980
aggcctgagt agctcccctc catccttgta gacgctccag tccctactac tgtgacggca
2040 tttccatccc tcccctgccc gggaagggac cttgcaggga cctctccctc
caaaaaaaga 2100 aaaaaagaaa aagaaagaaa aaataaatga ggaaacgtgt tgc
2143 4 2506 DNA Homo sapiens 4 agatggcgcg gagcgggagg cggccctgga
gcgaccccgg aggactaagc gggaacggga 60 ccagctgtac tacgagtgct
actcggacgt ttcggtccac gaggagatga tcgcggaccg 120 cgtccgcacc
gatgcctacc gcctgggtat ccttcggaac tgggcagcac tgcgaggcaa 180
gacggtactg gacgtgggcg cgggcaccgg cattctgagc atcttctgtg cccaggccgg
240 ggcccggcgc gtgtacgcgg tagaggccag cgccatctgg caacaggccc
gggaggtggt 300 gcggttcaac gggctggagg accgggtgca cgtcctgccg
ggaccagtgg agactgtaga 360 gttgccggaa caggtggatg ccatcgtgag
cgagtggatg ggctacggac tcctgcacga 420 gtccatgctg agctccgtcc
tccacgcgcg aaccaagtgg ctgaaggagg gcggtcttct 480 cctgccggcc
tccgccgagc tcttcatagc ccccatcagc gaccagatgc tggaatggcg 540
cctgggcttc tggagccagg tgaagcagca ctatggtgtg gacatgagct gcctggaggg
600 cttcgccacg cgctgtctca tgggccactc ggagatcgtt gtgcagggat
tgtccggcga 660 ggacgtgctg gcccggccgc agcgctttgc tcagctagag
ctctcccgcg ccggcttgga 720 gcaggagctg gaggccggag tgggcgggcg
cttccgctgc agctgctatg gctcggcgcc 780 catgcatggc tttgccatct
ggttccaggt gaccttccct ggaggggagt cggagaaacc 840 cctggtgctg
tccacctcgc cttttcaccc ggccactcac tggaaacagg cgctcctcta 900
cctgaacgag ccggtgcaag tggagcaaga cacggacgtt tcaggagaga tcacgctgct
960 gccctcccgg gacaaccccc gtcgcctgcg cgtgctgctg cgctacaaag
tgggagacca 1020 ggaggagaag accaaagact ttgccatgga ggactgagcg
ttgccttttc tcccagctac 1080 ctcccaaagc agcctgacct gcgtgggaga
ggcgtagcga ggtcggaggg gaaagggaga 1140 tcccacgtgc aagtaggggg
aatatctccc ccttttccct catagcctct agggagggag 1200 agtgacttca
ttctccattt gaagagattc ttctggtgat gtttacttaa aaagtgatcc 1260
ccctcaacaa cggatacagc gtgcttatta ttgggcattt agcctcaaaa gcatgtagta
1320 ccaagcactt gtatttccgt atattttgtt tcgcggggga gtgaggggga
agaacacgga 1380 tgaaaatgtc agtttttgaa gggtccatgc acatccctga
cacctcacac cttatctaag 1440 tctgaagctg gggagaaagg ggttcattta
gacttcatac atttccagta cgactttagt 1500 atctctccag agccatattt
tctcagtccg aattaattcc ccctccctag gtgcctgtag 1560 gctatggtac
ttcttcctca ttgttttcta ggtaaacttc actactggta attaagggga 1620
aggatatgag gaagcagttt aaatagccct gttctcatta ctctgaccac atacatcata
1680 gggtgctaaa gttgatgaac acattaatcc gttaagtaaa atggactttg
taattgtaca 1740 gcatacctaa gaaactcaga aggtgcattt aagagagaga
cctgaaagaa atagtatgga 1800 tttttaaaaa ttcttgtctc tactattata
accaaaaaat atttcttgta tgtcccataa 1860 aaatatttgt gtaattctta
tgaaacaggc tggtagagga ggtttctgag cctagcccaa 1920 gggcttattc
atcaccatgg gtaaattatt taaactcact taattaagga aaatattttc 1980
ccagctagaa aagtatactc attctcattt aaactctctc atttggaggg atcatgtgag
2040 ttggcctact tacaagtagt gaaagttccc ttttcagttt tgttttgttt
tgttttgttt 2100 ttctctttca ctcagccaaa tgtgaaagtt gtgaatttag
gaaaatcact tgtaatgaag 2160 tgtgaatctt gttatcaaat ttatttctct
gatgtttcct tccttatcct tgtagccaat 2220 aaaacattga cattctcacg
ttttatagat gaggtaaaaa gtcttgtgtg ctgtgagtta 2280 taatgctttt
gcctttttaa tattattagt tcttaagtgt tacagcccct tcagaatata 2340
acttcaggac aattcaaact atgcttaatg tatgattttc gagcttctgt atgctaagaa
2400 aataggtgtg aaaaactggt gttctgaaat agcctaacat ttattgtaat
tctgaatttt 2460 ctgccctttt attcattgca tattaaagta ttagagtata aaaact
2506 5 2506 DNA Homo sapiens 5 agatggcgcg gagcgggagg cggccctgga
gcgaccccgg aggactaagc gggaacggga 60 ccagctgtac tacgagtgct
actcggacgt ttcggtccac gaggagatga tcgcggaccg 120 cgtccgcacc
gatgcctacc gcctgggtat ccttcggaac tgggcagcac tgcgaggcaa 180
gacggtactg gacgtgggcg cgggcaccgg cattctgagc atcttctgtg cccaggccgg
240 ggcccggcgc gtgtacgcgg tagaggccag cgccatctgg caacaggccc
gggaggtggt 300 gcggttcaac gggctggagg accgggtgca cgtcctgccg
ggaccagtgg agactgtaga 360 gttgccggaa caggtggatg ccatcgtgag
cgagtggatg ggctacggac tcctgcacga 420 gtccatgctg agctccgtcc
tccacgcgcg aaccaagtgg ctgaaggagg gcggtcttct 480 cctgccggcc
tccgccgagc tcttcatagc ccccatcagc gaccagatgc tggaatggcg 540
cctgggcttc tggagccagg tgaagcagca ctatggtgtg gacatgagct gcctggaggg
600 cttcgccacg cgctgtctca tgggccactc ggagatcgtt gtgcagggat
tgtccggcga 660 ggacgtgctg gcccggccgc agcgctttgc tcagctagag
ctctcccgcg ccggcttgga 720 gcaggagctg gaggccggag tgggcgggcg
cttccgctgc agctgctatg gctcggcgcc 780 catgcatggc tttgccatct
ggttccaggt gaccttccct ggaggggagt cggagaaacc 840 cctggtgctg
tccacctcgc cttttcaccc ggccactcac tggaaacagg cgctcctcta 900
cctgaacgag ccggtgcaag tggagcaaga cacggacgtt tcaggagaga tcacgctgct
960 gccctcccgg gacaaccccc gtcgcctgcg cgtgctgctg cgctacaaag
tgggagacca 1020 ggaggagaag accaaagact ttgccatgga ggactgagcg
ttgccttttc tcccagctac 1080 ctcccaaagc agcctgacct gcgtgggaga
ggcgtagcga ggtcggaggg gaaagggaga 1140 tcccacgtgc aagtaggggg
aatatctccc ccttttccct catagcctct agggagggag 1200 agtgacttca
ttctccattt gaagagattc ttctggtgat gtttacttaa aaagtgatcc 1260
ccctcaacaa cggatacagc gtgcttatta ttgggcattt agcctcaaaa gcatgtagta
1320 ccaagcactt gtatttccgt atattttgtt tcgcggggga gtgaggggga
agaacacgga 1380 tgaaaatgtc agtttttgaa gggtccatgc acatccctga
cacctcacac cttatctaag 1440 tctgaagctg gggagaaagg ggttcattta
gacttcatac atttccagta cgactttagt 1500 atctctccag agccatattt
tctcagtccg aattaattcc ccctccctag gtgcctgtag 1560 gctatggtac
ttcttcctca ttgttttcta ggtaaacttc actactggta attaagggga 1620
aggatatgag gaagcagttt aaatagccct gttctcatta ctctgaccac atacatcata
1680 gggtgctaaa gttgatgaac acattaatcc gttaagtaaa atggactttg
taattgtaca 1740 gcatacctaa gaaactcaga aggtgcattt aagagagaga
cctgaaagaa atagtatgga 1800 tttttaaaaa ttcttgtctc tactattata
accaaaaaat atttcttgta tgtcccataa 1860 aaatatttgt gtaattctta
tgaaacaggc tggtagagga ggtttctgag cctagcccaa 1920 gggcttattc
atcaccatgg gtaaattatt taaactcact taattaagga aaatattttc 1980
ccagctagaa aagtatactc attctcattt aaactctctc atttggaggg atcatgtgag
2040 ttggcctact tacaagtagt gaaagttccc ttttcagttt tgttttgttt
tgttttgttt 2100 ttctctttca ctcagccaaa tgtgaaagtt gtgaatttag
gaaaatcact tgtaatgaag 2160 tgtgaatctt gttatcaaat ttatttctct
gatgtttcct tccttatcct tgtagccaat 2220 aaaacattga cattctcacg
ttttatagat gaggtaaaaa gtcttgtgtg ctgtgagtta 2280 taatgctttt
gcctttttaa tattattagt tcttaagtgt tacagcccct tcagaatata 2340
acttcaggac aattcaaact atgcttaatg tatgattttc gagcttctgt atgctaagaa
2400 aataggtgtg aaaaactggt gttctgaaat agcctaacat ttattgtaat
tctgaatttt 2460 ctgccctttt attcattgca tattaaagta ttagagtata aaaact
2506 6 2577 DNA Homo sapiens 6 atgtcgcagc ccaagaaaag aaagcttgag
tcggggggcg gcggcgaagg aggggaggga 60 actgaagagg aagatggcgc
ggagcgggag gcggccctgg agcgaccccg gaggactaag 120 cgggaacggg
accagctgta ctacgagtgc tactcggacg tttcggtcca cgaggagatg 180
atcgcggacc gcgtccgcac cgatgcctac cgcctgggta tccttcggaa ctgggcagca
240 ctgcgaggca agacggtact ggacgtgggc gcgggcaccg gcattctgag
catcttctgt 300 gcccaggccg gggcccggcg cgtgtacgcg gtagaggcca
gcgccatctg gcaacaggcc 360 cgggaggtgg tgcggttcaa cgggctggag
gaccgggtgc acgtcctgcc gggaccagtg 420 gagactgtag agttgccgga
acaggtggat gccatcgtga gcgagtggat gggctacgga 480 ctcctgcacg
agtccatgct gagctccgtc ctccacgcgc gaaccaagtg gctgaaggag 540
ggcggtcttc tcctgccggc ctccgccgag ctcttcatag cccccatcag cgaccagatg
600 ctggaatggc gcctgggctt ctggagccag gtgaagcagc actatggtgt
ggacatgagc 660 tgcctggagg gcttcgccac gcgctgtctc atgggccact
cggagatcgt tgtgcaggga 720 ttgtccggcg aggacgtgct ggcccggccg
cagcgctttg ctcagctaga gctctcccgc 780 gccggcttgg agcaggagct
ggaggccgga gtgggcgggc gcttccgctg cagctgctat 840 ggctcggcgc
ccatgcatgg ctttgccatc tggttccagg tgaccttccc tggaggggag 900
tcggagaaac ccctggtgct gtccacctcg ccttttcacc cggccactca ctggaaacag
960 gcgctcctct acctgaacga gccggtgcaa gtggagcaag acacggacgt
ttcaggagag 1020 atcacgctgc tgccctcccg ggacaacccc cgtcgcctgc
gcgtgctgct gcgctacaaa 1080 gtgggagacc aggaggagaa gaccaaagac
tttgccatgg aggactgagc gttgcctttt 1140 ctcccagcta cctcccaaag
cagcctgacc tgcgtgggag aggcgtagcg aggtcggagg 1200 ggaaagggag
atcccacgtg caagtagggg gaatatctcc cccttttccc tcatagcctc 1260
tagggaggga gagtgacttc attctccatt tgaagagatt cttctggtga tgtttactta
1320 aaaagtgatc cccctcaaca acggatacag cgtgcttatt attgggcatt
tagcctcaaa 1380 agcatgtagt accaagcact tgtatttccg tatattttgt
ttcgcggggg agtgaggggg 1440 aagaacacgg atgaaaatgt cagtttttga
agggtccatg cacatccctg acacctcaca 1500 ccttatctaa gtctgaagct
ggggagaaag gggttcattt agacttcata catttccagt 1560 acgactttag
tatctctcca gagccatatt ttctcagtcc gaattaattc cccctcccta 1620
ggtgcctgta ggctatggta cttcttcctc attgttttct aggtaaactt cactactggt
1680 aattaagggg aaggatatga ggaagcagtt taaatagccc tgttctcatt
actctgacca 1740 catacatcat agggtgctaa agttgatgaa cacattaatc
cgttaagtaa aatggacttt 1800 gtaattgtac agcataccta agaaactcag
aaggtgcatt taagagagag acctgaaaga 1860 aatagtatgg atttttaaaa
attcttgtct ctactattat aaccaaaaaa tatttcttgt 1920 atgtcccata
aaaatatttg tgtaattctt atgaaacagg ctggtagagg aggtttctga 1980
gcctagccca agggcttatt catcaccatg ggtaaattat ttaaactcac ttaattaagg
2040 aaaatatttt cccagctaga aaagtatact cattctcatt taaactctct
catttggagg 2100 gatcatgtga gttggcctac ttacaagtag tgaaagttcc
cttttcagtt ttgttttgtt 2160 ttgttttgtt tttctctttc actcagccaa
atgtgaaagt tgtgaattta ggaaaatcac 2220 ttgtaatgaa gtgtgaatct
tgttatcaaa tttatttctc tgatgtttcc ttccttatcc 2280 ttgtagccaa
taaaacattg acattctcac gttttataga tgaggtaaaa agtcttgtgt 2340
gctgtgagtt ataatgcttt tgccttttta atattattag ttcttaagtg ttacagcccc
2400 ttcagaatat aacttcagga caattcaaac tatgcttaat gtatgatttt
cgagcttctg 2460 tatgctaaga aaataggtgt gaaaaactgg tgttctgaaa
tagcctaaca tttattgtaa 2520 ttctgaattt tctgcccttt tattcattgc
atattaaagt attagagtat aaaaact 2577 7 2234 DNA Homo sapiens 7
ggcacgaggc ggaggactaa gcgggaacgg gaccagctgt actacgagtg ctactcggac
60 gtttcggtcc acgaggagat gatcgcggac cgcgtccgca ccgatgccta
ccgcctgggt 120 atccttcgga actgggcagc actgcgaggc aagacggtac
tggacgtggg cgcgggcacc 180 ggcattctga gcatcttctg tgcccaggcc
ggggcccggc gcgtgtacgc ggtagaggcc 240 agcgccatct ggcaacaggc
ccgggaggtg gtgcggttca acgggctgga ggaccgggtg 300 cacgtcctgc
cgggaccagt ggagactgta gagttgccgg aacaggtgga tgccatcgtg 360
agcgagtgga tgggctacgg actcctgcac gagtccatgc tgagctccgt cctccacgcg
420 cgaaccaagt ggctgaagga gggcggtctt ctcctgccgg cctccgccga
gctcttcata 480 gcccccatca gcgaccagat gctggaatgg cgcctgggct
tctggagcca ggtgaagcag 540 cactatggtg tggacatgag ctgcctggag
ggcttcgcca cgcgctgtct catgggccac 600 tcggagatcg ttgtgcaggg
attgtccggc gaggacgtgc tggcccggcc gcagcgcttt 660 gctcagctag
agctctcccg cgccggcttg gagcaggagc tggaggccgg agtgggcggg 720
cgcttccgct gcagctgcta tggctcggcg cccatgcatg gctttgccat ctggttccag
780 gtgaccttcc ctggagggga gtcggagaaa cccctggtgc tgtccacctc
gccttttcac 840 ccggccactc actggaaaca ggcgctcctc tacctgaacg
agccggtgca agtggagcaa 900 gacacggacg tttcaggaga gatcacgctg
ctgccctccc gggacaaccc ccgtcgcctg 960 cgcgtgctgc tgcgctacaa
agtgggagac caggaggaga agaccaaaga ctttgccatg 1020 gaggactgag
cgttgccttt tctcccagct acctcccaaa gcagcctgac ctgcgtggga 1080
gaggcgtagc gaggtcggag gggaaaggga gatcccacgt gcaagtaggg ggaatatctc
1140 cctcttttcc ctcatagcct ctagggaggg agagtgactt cattctccat
ttgaagagat 1200 tcttctggtg atgtttactt aaaaagtgat ccccctcaac
aacggataca gcgtgcttat 1260 tattgggcat ttagcctcaa aagcatgtag
taccaagcac ttgtatttcc gtatattttg 1320 tttcgcgggg gagtgagggg
gaagaacacg gatgaaaatg tcagtttttg aagggtccat 1380 gcacatccct
gacacctcac accttatcta agtctgaagc tggggagaaa ggggttcatt 1440
tagacttcat acatttccag tacgacttta gtatctctcc agagccatat tttctcagtc
1500 cgaattaatt ccccctccct aggtgcctgt aggctatggt acttcttcct
cattgttttc 1560 taggtaaact tcactactgg taattaaggg gaaggatatg
aggaagcagt ttaaatagcc 1620 ctgttctcat tactctgacc acatacatca
tagggtgcta aagttgatga acacattaat 1680 ccgttaagta aaatggactt
tgtaattgta cagcatacct aagaaactca gaaggtgcat 1740 ttaagagaga
gacctgaaag aaatagtatg gatttttaaa aattcttgtc tctactatta 1800
taaccaaaaa atatttcttg tatgtcccat aaaaatattt gtgtaattct tatgaaacag
1860 gctggtagag gaggtttctg agcctagccc aagggcttat tcatcaccat
gggtaaatta 1920 tttaaactca cttaattaag gaaaatattt tcccagctag
aaaagtatac tcattctcat 1980 ttaaactctc tcatttggag ggatcatgtg
agttggccta cttacaagta gtgaaagttc 2040 ccttttcagt tttgttttgt
tttgttttgt ttttctcttt cactcagcca aatgtgaaag 2100 ttgtgaattt
aggaaaatca cttgtaatga agtgtgaatc ttgttatcaa atttatttct 2160
ctgatgtttc cttccttatc cttgtagcca ataaaacatt gacattctca cgttttaaaa
2220 aaaaaaaaaa aaaa 2234 8 608 PRT Mus musculus 8 Met Ala Ala Ala
Ala Ala Thr Ala Val Gly Pro Gly Ala Gly Ser Ala 1 5 10 15 Gly Val
Ala Gly Pro Gly Gly Ala Gly Pro Cys Ala Thr Val Ser Val 20 25 30
Phe Pro Gly Ala Arg Leu Leu Thr Ile Gly Asp Ala Asn Gly Glu Ile 35
40 45 Gln Arg His Ala Glu Gln Gln Ala Leu Arg Leu Glu Val Arg Ala
Gly 50 55 60 Pro Asp Ala Ala Gly Ile Ala Leu Tyr Ser His Glu Asp
Val Cys Val 65 70 75 80 Phe Lys Cys Ser Val Ser Arg Glu Thr Glu Cys
Ser Arg Val Gly Arg 85 90 95 Gln Ser Phe Ile Ile Thr Leu Gly Cys
Asn Ser Val Leu Ile Gln Phe 100 105 110 Ala Thr Pro His Asp Phe Cys
Ser Phe Tyr Asn Ile Leu Lys Thr Cys 115 120 125 Arg Gly His Thr Leu
Glu Arg Ser Val Phe Ser Glu Arg Thr Glu Glu 130 135 140 Ser Ser Ala
Val Gln Tyr Phe Gln Phe Tyr Gly Tyr Leu Ser Gln Gln 145 150 155 160
Gln Asn Met Met Gln Asp Tyr Val Arg Thr Gly Thr Tyr Gln Arg Ala 165
170 175 Ile Leu Gln Asn His Thr Asp Phe Lys Asp Lys Ile Val Leu Asp
Val 180 185 190 Gly Cys Gly Ser Gly Ile Leu Ser Phe Phe Ala Ala Gln
Ala Gly Ala 195 200 205 Arg Lys Ile Tyr Ala Val Glu Ala Ser Thr Met
Ala Gln His Ala Glu 210 215 220 Val Leu Val Lys Ser Asn Asn Leu Thr
Asp Arg Ile Val Val Ile Pro 225 230 235 240 Gly Lys Val Glu Glu Val
Ser Leu Pro Glu Gln Val Asp Ile Ile Ile 245 250 255 Ser Glu Pro Met
Gly Tyr Met Leu Phe Asn Glu Arg Met Leu Glu Ser 260 265 270 Tyr Leu
His Ala Lys Lys Tyr Leu Lys Pro Ser Gly Asn Met Phe Pro 275 280 285
Thr Ile Gly Asp Val His Leu Ala Pro Phe Thr Asp Glu Gln Leu Tyr 290
295 300 Met Glu Gln Phe Thr Lys Ala Asn Phe Arg Tyr Gln Pro Ser Phe
His 305 310 315 320 Gly Val Asp Leu Ser Ala Leu Arg Gly Ala Ala Val
Asp Glu Tyr Phe 325 330 335 Arg Gln Pro Val Val Asp Thr Phe Asp Ile
Arg Ile Leu Met Ala Lys 340 345 350 Ser Val Lys Tyr Thr Val Asn Phe
Leu Glu Ala Lys Glu Gly Asp Leu 355 360 365 His Arg Ile Glu Ile Pro
Phe Lys Phe His Met Leu His Ser Gly Leu 370 375 380 Val His Gly Leu
Ala Phe Trp Phe Asp Val Ala Phe Ile Gly Ser Ile 385 390 395 400 Met
Thr Val Trp Leu Ser Thr Ala Pro Thr Glu Pro Leu Thr His Trp 405 410
415 Tyr Gln Val Arg Cys Leu Phe Gln Ser Pro Leu Phe Ala Lys Ala Gly
420 425 430 Asp Thr Leu Ser Gly Thr Cys Leu Leu Ile Ala Asn Lys Arg
Gln Ser 435 440 445 Tyr Asp Ile Ser Ile Val Ala Gln Val Asp Gln Thr
Gly Ser Lys Ser 450 455 460 Ser Asn Leu Leu Asp Leu Lys Asn Pro Phe
Phe Arg Tyr Thr Gly Thr 465 470 475 480 Thr Pro Ser Pro Pro Pro Gly
Ser His Tyr Thr Ser Pro Ser Glu Asn 485 490 495 Met Trp Asn Thr Gly
Ser Thr Tyr Asn Leu Ser Ser Gly Val Ala Val 500 505 510 Ala Gly Met
Pro Thr Ala Tyr Asp Leu Ser Ser Val Ile Ala Gly Gly 515 520 525 Ser
Ser Val Gly His Asn Asn Leu Ile Pro Leu Ala Asn Thr Gly Ile 530 535
540 Val Asn His Thr His Ser Arg Met Gly Ser Ile Met Ser Thr Gly Ile
545 550 555 560 Val Gln Gly Ser Ser Gly Ala Gln Gly Gly Gly Gly Ser
Ser Ser Ala 565 570 575 His Tyr Ala Val Asn Asn Gln Phe Thr Met Gly
Gly Pro Ala Ile Ser 580 585 590 Met Ala Ser Pro Met Ser Ile Pro Thr
Asn Thr Met His Tyr Gly Ser 595 600 605 9 608 PRT Homo sapiens 9
Met Ala Ala Ala Ala Ala Ala Val Gly Pro Gly Ala Gly Gly Ala Gly 1 5
10 15 Ser Ala Val Pro Gly Gly Ala Gly Pro Cys Ala Thr Val Ser Val
Phe 20 25 30 Pro Gly Ala Arg Leu Leu Thr Ile Gly Asp Ala Asn Gly
Glu Ile Gln 35 40 45 Arg His Ala Glu Gln Gln Ala Leu Arg Leu Glu
Val Arg Ala Gly Pro 50 55 60 Asp Ser Ala Gly Ile Ala Leu Tyr Ser
His Glu Asp Val Cys Val Phe 65 70 75 80 Lys Cys Ser Val Ser Arg Glu
Thr Glu Cys Ser Arg Val Gly Lys Gln 85 90 95 Ser Phe Ile Ile Thr
Leu Gly Cys Asn Ser Val Leu Ile Gln Phe Ala 100 105 110 Thr Pro Asn
Asp Phe Cys Ser Phe Tyr Asn Ile Leu Lys Thr Cys Arg 115 120 125 Gly
His Thr Leu Glu Arg Ser Val Phe Ser Glu Arg Thr Glu Glu Ser 130 135
140 Ser Ala Val Gln Tyr Phe Gln Phe Tyr Gly Tyr Leu Ser Gln Gln Gln
145 150 155 160 Asn Met Met Gln Asp Tyr Val Arg Thr Gly Thr Tyr Gln
Arg Ala Ile 165 170 175 Leu Gln Asn His Thr Asp Phe Lys Asp Lys Ile
Val Leu Asp Val Gly 180 185 190 Cys Gly Ser Gly Ile Leu Ser Phe Phe
Ala Ala Gln Ala Gly Ala Arg 195 200 205 Lys Ile Tyr Ala Val Glu Ala
Ser Thr Met Ala Gln His Ala Glu Val 210 215 220 Leu Val Lys Ser Asn
Asn Leu Thr Asp Arg Ile Val Val Ile Pro Gly 225 230 235 240 Lys Val
Glu Glu Val Ser Leu Pro Glu Gln Val Asp Ile Ile Ile Ser 245 250 255
Glu Pro Met Gly Tyr Met Leu Phe Asn Glu Arg Met Leu Glu Ser Tyr 260
265 270 Leu His Ala Lys Lys Tyr Leu Lys Pro Ser Gly Asn Met Phe Pro
Thr 275 280 285 Ile Gly Asp Val His Leu Ala Pro Phe Thr Asp Glu Gln
Leu Tyr Met 290 295 300 Glu Gln Phe Thr Lys Ala Asn Phe Trp Tyr Gln
Pro Ser Phe His Gly 305 310 315 320 Val Asp Leu Ser Ala Leu Arg Gly
Ala Ala Val Asp Glu Tyr Phe Arg 325 330 335 Gln Pro Val Val Asp Thr
Phe Asp Ile Arg Ile Leu Met Ala Lys Ser 340 345 350 Val Lys Tyr Thr
Val Asn Phe Leu Glu Ala Lys Glu Gly Asp Leu His 355 360 365 Arg Ile
Glu Ile Pro Phe Lys Phe His Met Leu His Ser Gly Leu Val 370 375 380
His Gly Leu Ala Phe Trp Phe Asp Val Ala Phe Ile Gly Ser Ile Met 385
390 395 400 Thr Val Trp Leu Ser Thr Ala Pro Thr Glu Pro Leu Thr His
Trp Tyr 405 410 415 Gln Val Arg Cys Leu Phe Gln Ser Pro Leu Phe Ala
Lys Ala Gly Asp 420 425 430 Thr Leu Ser Gly Thr Cys Leu Leu Ile Ala
Asn Lys Arg Gln Ser Tyr 435 440 445 Asp Ile Ser Ile Val Ala Gln Val
Asp Gln Thr Gly Ser Lys Ser Ser 450 455 460 Asn Leu Leu Asp Leu Lys
Asn Pro Phe Phe Arg Tyr Thr Gly Thr Thr 465 470 475 480 Pro Ser Pro
Pro Pro Gly Ser His Tyr Thr Ser Pro Ser Glu Asn Met 485 490 495 Trp
Asn Thr Gly Ser Thr Tyr Asn Leu Ser Ser Gly Met Ala Val Ala 500 505
510 Gly Met Pro Thr Ala Tyr Asp Leu Ser Ser Val Ile Ala Ser Gly Ser
515 520 525 Ser Val Gly His Asn Asn Leu Ile Pro Leu Ala Asn Thr Gly
Ile Val 530 535 540 Asn His Thr His Ser Arg Met Gly Ser Ile Met Ser
Thr Gly Ile Val 545 550 555 560 Gln Gly Ser Ser Gly Ala Gln Gly Ser
Gly Gly Gly Ser Thr Ser Ala 565 570 575 His Tyr Ala Val Asn Ser Gln
Phe Thr Met Gly Gly Pro Ala Ile Ser 580 585 590 Met Ala Ser Pro Met
Ser Ile Pro Thr Asn Thr Met His Tyr Gly Ser 595 600 605 10 357 PRT
Homo sapiens 10 Met Gly Tyr Met Leu Phe Asn Glu Arg Met Leu Glu Ser
Tyr Leu His 1 5 10 15 Ala Lys Lys Tyr Leu Lys Pro Ser Gly Asn Met
Phe Pro Thr Ile Gly 20 25 30 Asp Val His Leu Ala Pro Phe Thr Asp
Glu Gln Leu Tyr Met Glu Gln 35 40 45 Phe Thr Lys Ala Asn Phe Trp
Tyr Gln Pro Ser Phe His Gly Val Asp 50 55 60 Leu Ser Ala Leu Arg
Gly Ala Ala Val Asp Glu Tyr Phe Arg Gln Pro 65 70 75 80 Val Val Asp
Thr Phe Asp Ile Arg Ile Leu Met Ala Lys Ser Val Lys 85 90 95 Tyr
Thr Val Asn Phe Leu Glu Ala Lys Glu Gly Asp Leu His Arg Ile 100 105
110 Glu Ile Pro Phe Lys Phe His Met Leu His Ser Gly Leu Val His Gly
115 120 125 Leu Ala Phe Trp Phe Asp Val Ala Phe Ile Gly Ser Ile Met
Thr Val 130 135 140 Trp Leu Ser Thr Ala Pro Thr Glu Pro Leu Thr His
Trp Tyr Gln Val 145 150 155 160 Arg Cys Leu Phe Gln Ser Pro Leu Phe
Ala Lys Ala Gly Asp Thr Leu 165 170 175 Ser Gly Thr Cys Leu Leu Ile
Ala Asn Lys Arg Gln Ser Tyr Asp Ile 180 185 190 Ser Ile Val Ala Gln
Val Asp Gln Thr Gly Ser Lys Ser Ser Asn Leu 195 200 205 Leu Asp Leu
Lys Asn Pro Phe Phe Arg Tyr Thr Gly Thr Thr Pro Ser 210 215 220 Pro
Pro Pro Gly Ser His Tyr Thr Ser Pro Ser Glu Asn Met Trp Asn 225 230
235 240 Thr Gly Ser Thr Tyr Asn Leu Ser Ser Gly Met Ala Val Ala Gly
Met 245 250 255 Pro Thr Ala Tyr Asp Leu Ser Ser Val Ile Ala Ser Gly
Ser Ser Val 260 265 270 Gly His Asn Asn Leu Ile Pro Leu Ala Asn Thr
Gly Ile Val Asn His 275 280 285 Thr His Ser Arg Met Gly Ser Ile Met
Ser Thr Gly Ile Val Gln Gly 290 295 300 Val Leu Arg Arg Pro Gly Gln
Trp Trp Trp Gln His Glu Cys Pro Leu 305 310 315 320 Cys Ser Gln Gln
Pro Val His His Gly Arg Pro Arg His Leu His Gly 325 330 335 Val Ala
His Val His Pro Asp Gln His His Ala Leu Arg Glu Leu Gly 340 345 350
Ala Arg Pro Ala Asp 355 11 351 PRT Homo sapiens 11 Asp Gly Ala Glu
Arg Glu Ala Ala Leu Glu Arg Pro Arg Arg Thr Lys 1 5 10 15 Arg Glu
Arg Asp Gln Leu Tyr Tyr Glu Cys Tyr Ser Asp Val Ser Val 20 25 30
His Glu Glu Met Ile Ala Asp Arg Val Arg Thr Asp Ala Tyr Arg Leu 35
40 45 Gly Ile Leu Arg Asn Trp Ala Ala Leu Arg Gly Lys Thr Val Leu
Asp 50 55 60 Val Gly Ala Gly Thr Gly Ile Leu Ser Ile Phe Cys Ala
Gln Ala Gly 65 70 75 80 Ala Arg Arg Val Tyr Ala Val Glu Ala Ser Ala
Ile Trp Gln Gln Ala 85 90 95 Arg Glu Val Val Arg Phe Asn Gly Leu
Glu Asp Arg Val His Val Leu 100 105 110 Pro Gly Pro Val Glu Thr Val
Glu Leu Pro Glu Gln Val Asp Ala Ile 115 120 125 Val Ser Glu Trp Met
Gly Tyr Gly Leu Leu His Glu Ser Met Leu Ser 130 135 140 Ser Val Leu
His Ala Arg Thr Lys Trp Leu Lys Glu Gly Gly Leu Leu 145 150 155 160
Leu Pro Ala Ser Ala Glu Leu Phe Ile Ala Pro Ile Ser Asp Gln Met 165
170 175 Leu Glu Trp Arg Leu Gly Phe Trp Ser Gln Val Lys Gln His Tyr
Gly 180 185 190 Val Asp Met Ser Cys Leu Glu Gly Phe Ala Thr Arg Cys
Leu Met Gly 195 200 205 His Ser Glu Ile Val Val Gln Gly Leu Ser Gly
Glu Asp Val Leu Ala 210 215 220 Arg Pro Gln Arg Phe Ala Gln Leu Glu
Leu Ser Arg Ala Gly Leu Glu 225 230 235 240 Gln Glu Leu Glu Ala Gly
Val Gly Gly Arg Phe Arg Cys Ser Cys Tyr 245 250 255 Gly Ser Ala Pro
Met His Gly Phe Ala Ile Trp Phe Gln Val Thr Phe 260 265 270 Pro Gly
Gly Glu Ser Glu Lys Pro Leu Val Leu Ser Thr Ser Pro Phe 275 280 285
His Pro Ala Thr His Trp Lys Gln Ala Leu Leu Tyr Leu Asn Glu Pro 290
295 300 Val Gln Val Glu Gln Asp Thr Asp Val Ser Gly Glu Ile Thr Leu
Leu 305 310
315 320 Pro Ser Arg Asp Asn Pro Arg Arg Leu Arg Val Leu Leu Arg Tyr
Lys 325 330 335 Val Gly Asp Gln Glu Glu Lys Thr Lys Asp Phe Ala Met
Glu Asp 340 345 350 12 316 PRT Homo sapiens 12 Met Ile Ala Asp Arg
Val Arg Thr Asp Ala Tyr Arg Leu Gly Ile Leu 1 5 10 15 Arg Asn Trp
Ala Ala Leu Arg Gly Lys Thr Val Leu Asp Val Gly Ala 20 25 30 Gly
Thr Gly Ile Leu Ser Ile Phe Cys Ala Gln Ala Gly Ala Arg Arg 35 40
45 Val Tyr Ala Val Glu Ala Ser Ala Ile Trp Gln Gln Ala Arg Glu Val
50 55 60 Val Arg Phe Asn Gly Leu Glu Asp Arg Val His Val Leu Pro
Gly Pro 65 70 75 80 Val Glu Thr Val Glu Leu Pro Glu Gln Val Asp Ala
Ile Val Ser Glu 85 90 95 Trp Met Gly Tyr Gly Leu Leu His Glu Ser
Met Leu Ser Ser Val Leu 100 105 110 His Ala Arg Thr Lys Trp Leu Lys
Glu Gly Gly Leu Leu Leu Pro Ala 115 120 125 Ser Ala Glu Leu Phe Ile
Ala Pro Ile Ser Asp Gln Met Leu Glu Trp 130 135 140 Arg Leu Gly Phe
Trp Ser Gln Val Lys Gln His Tyr Gly Val Asp Met 145 150 155 160 Ser
Cys Leu Glu Gly Phe Ala Thr Arg Cys Leu Met Gly His Ser Glu 165 170
175 Ile Val Val Gln Gly Leu Ser Gly Glu Asp Val Leu Ala Arg Pro Gln
180 185 190 Arg Phe Ala Gln Leu Glu Leu Ser Arg Ala Gly Leu Glu Gln
Glu Leu 195 200 205 Glu Ala Gly Val Gly Gly Arg Phe Arg Cys Ser Cys
Tyr Gly Ser Ala 210 215 220 Pro Met His Gly Phe Ala Ile Trp Phe Gln
Val Thr Phe Pro Gly Gly 225 230 235 240 Glu Ser Glu Lys Pro Leu Val
Leu Ser Thr Ser Pro Phe His Pro Ala 245 250 255 Thr His Trp Lys Gln
Ala Leu Leu Tyr Leu Asn Glu Pro Val Gln Val 260 265 270 Glu Gln Asp
Thr Asp Val Ser Gly Glu Ile Thr Leu Leu Pro Ser Arg 275 280 285 Asp
Asn Pro Arg Arg Leu Arg Val Leu Leu Arg Tyr Lys Val Gly Asp 290 295
300 Gln Glu Glu Lys Thr Lys Asp Phe Ala Met Glu Asp 305 310 315 13
1780 DNA Homo sapiens 13 caccgaattc gccggatcta agatggcagc
ggcggcggcg gcggtggggc cgggcgcggg 60 cggcgcgggg tcggcggtcc
cgggcggcgc ggggccctgc gctaccgtgt cggtgttccc 120 cggcgcccgc
ctcctcacca tcggcgacgc gaacggcgag atccagcggc acgcggagca 180
gcaggcgctg cgcctcgagg tgcgcgccgg cccggactcg gcgggcatcg ccctctacag
240 ccatgaagat gtgtgtgtct ttaagtgctc agtgtcccga gagacagagt
gcagccgtgt 300 gggcaagcag tccttcatca tcaccctggg ctgcaacagc
gtcctcatcc agttcgccac 360 acccaacgat ttctgttcct tctacaacat
cctgaaaacc tgccggggcc acaccctgga 420 gcggtctgtg ttcagcgagc
ggacggagga gtcttctgcc gtgcagtact tccagtttta 480 tggctacctg
tcccagcagc agaacatgat gcaggactac gtgcggacag gcacctacca 540
gcgcgccatc ctgcaaaacc acaccgactt caaggacaag atcgttcttg atgttggctg
600 tggctctggg atcctgtcgt tttttgccgc ccaagctgga gcacggaaaa
tctacgcggt 660 ggaggccagc accatggccc agcacgctga ggtcttggtg
aagagtaaca acctgacgga 720 ccgcatcgtg gtcatcccgg gcaaggtgga
ggaggtgtca ctccccgagc aggtggacat 780 catcatctcg gagcccatgg
gctacatgct cttcaacgag cgcatgctgg agagctacct 840 ccacgccaag
aagtacctga agcccagcgg aaacatgttt cctaccattg gtgacgtcca 900
ccttgcaccc ttcacggatg aacagctcta catggagcag ttcaccaagg ccaacttctg
960 gtaccagcca tctttccatg gagtggacct gtcggccctc cgaggtgccg
cggtggatga 1020 gtatttccgg cagcctgtgg tggacacatt tgacatccgg
atcctgatgg ccaagtctgt 1080 caagtacacg gtgaacttct tagaagccaa
agaaggagat ttgcacagga tagaaatccc 1140 attcaaattc cacatgctgc
attcagggct ggtccacggc ctggctttct ggtttgacgt 1200 tgctttcatc
ggctccataa tgaccgtgtg gctgtccaca gccccgacag agcccctgac 1260
ccactggtac caggtgcggt gcctgttcca gtcaccactg ttcgccaagg caggggacac
1320 gctctcaggg acatgtctgc ttattgccaa caaaagacag agctacgaca
tcagtattgt 1380 ggcccaggtg gaccagaccg gctccaagtc cagtaacctc
ctggatctga aaaacccctt 1440 ctttagatac acgggcacaa cgccctcacc
cccacccggc tcccactaca catctccctc 1500 ggaaaacatg tggaacacgg
gcagcaccta caacctcagc agcgggatgg ccgtggcagg 1560 gatgccgacc
gcctatgact tgagcagtgt tattgccagt ggctccagcg tgggccacaa 1620
caacctgatt cctttagggt cctccggcgc ccagggcagt ggtggtggca gcacgagtgc
1680 ccactatgca gtcaacagcc agttcaccat gggcggcccc gccatctcca
tggcgtcgcc 1740 catgtccatc ccgaccaaca ccatgcacta cgggagctag 1780 14
1849 DNA Homo sapiens 14 caccgaattc gccggatcta agatggcagc
ggcggcggcg gcggtggggc cgggcgcggg 60 cggcgcgggg tcggcggtcc
cgggcggcgc ggggccctgc gctaccgtgt cggtgttccc 120 cggcgcccgc
ctcctcacca tcggcgacgc gaacggcgag atccagcggc acgcggagca 180
gcaggcgctg cgcctcgagg tgcgcgccgg cccggactcg gcgggcatcg ccctctacag
240 ccatgaagat gtgtgtgtct ttaagtgctc agtgtcccga gagacagagt
gcagccgtgt 300 gggcaagcag tccttcatca tcaccctggg ctgcaacagc
gtcctcatcc agttcgccac 360 acccaacgat ttctgttcct tctacaacat
cctgaaaacc tgccggggcc acaccctgga 420 gcggtctgtg ttcagcgagc
cgacggagga gtcttctgcc gtgcagtact tccagtttta 480 tggctacctg
tcccagcagc agaacatgat gcaggactac gtgcggacag gcacctacca 540
gcgcgccatc ctgcaaaacc acaccgactt caaggacaag atcgttcttg atgttggctg
600 tggctctggg atcctgtcgt tttttgccgc ccaagctgga gcacggaaaa
tctacgcggt 660 ggaggccagc accatggccc agcacgctga ggtcttggtg
aagagtaaca acctgacgga 720 ccgcatcgtg gtcatcccgg gcaaggtgga
ggaggtgtca ctccccgagc aggtggacat 780 catcatctcg gagcccatgg
gctacatgct cttcaacgag cgcatgctgg agagctacct 840 ccacgccaag
aagtacctga agcccagcgg aaacatgttt cctaccattg gtgacgtcca 900
ccttgcaccc ttcacggatg aacagctcta catggagcag ttcaccaagg ccaacttctg
960 gtaccagcca tctttccatg gagtggacct gtcggccctc cgaggtgccg
cggtggatga 1020 gtatttccgg cagcctgtgg tggacacatt tgacatccgg
atcctgatgg ccaagtctgt 1080 caagtacacg gtgaacttct tagaagccaa
agaaggagat ttgcacagga tagaaatccc 1140 attcaaattc cacatgctgc
attcagggct ggtccacggc ctggctttct ggtttgacgt 1200 tgctttcatc
ggctccataa tgaccgtgtg gctgtccaca gccccgacag agcccctgac 1260
ccactggtac caggtgcggt gcctgttcca gtcaccactg ttcgccaagg caggggacac
1320 gctctcaggg acatgtctgc ttattgccaa caaaagacag agctacgaca
tcagtattgt 1380 ggcccaggtg gaccagaccg gctccaagtc cagtaacctc
ctggatctga aaaacccctt 1440 ctttagatac acgggcacaa cgccctcacc
cccacccggc tcccactaca catctccctc 1500 ggaaaacatg tggaacacgg
gcagcaccta caacctcagc agcgggatgg ccgtggcagg 1560 gatgccgacc
gcctatgact tgagcagtgt tattgccagt ggctccagcg tgggccacaa 1620
caacctgatt cctttagcca acacggggat tgtcaatcac acccactccc ggatgggctc
1680 cataatgagc acggggattg tccaagggtc ctccggcgcc cagggcagtg
gtggtggcag 1740 cacgagtgcc cactatgcag tcaacagcca gttcaccatg
ggcggccccg ccatctccat 1800 ggcgtcgccc atgtccatcc cgaccaacac
catgcactac gggagctag 1849 15 585 PRT Homo sapiens 15 Met Ala Ala
Ala Ala Ala Ala Val Gly Pro Gly Ala Gly Gly Ala Gly 1 5 10 15 Ser
Ala Val Pro Gly Gly Ala Gly Pro Cys Ala Thr Val Ser Val Phe 20 25
30 Pro Gly Ala Arg Leu Leu Thr Ile Gly Asp Ala Asn Gly Glu Ile Gln
35 40 45 Arg His Ala Glu Gln Gln Ala Leu Arg Leu Glu Val Arg Ala
Gly Pro 50 55 60 Asp Ser Ala Gly Ile Ala Leu Tyr Ser His Glu Asp
Val Cys Val Phe 65 70 75 80 Lys Cys Ser Val Ser Arg Glu Thr Glu Cys
Ser Arg Val Gly Lys Gln 85 90 95 Ser Phe Ile Ile Thr Leu Gly Cys
Asn Ser Val Leu Ile Gln Phe Ala 100 105 110 Thr Pro Asn Asp Phe Cys
Ser Phe Tyr Asn Ile Leu Lys Thr Cys Arg 115 120 125 Gly His Thr Leu
Glu Arg Ser Val Phe Ser Glu Arg Thr Glu Glu Ser 130 135 140 Ser Ala
Val Gln Tyr Phe Gln Phe Tyr Gly Tyr Leu Ser Gln Gln Gln 145 150 155
160 Asn Met Met Gln Asp Tyr Val Arg Thr Gly Thr Tyr Gln Arg Ala Ile
165 170 175 Leu Gln Asn His Thr Asp Phe Lys Asp Lys Ile Val Leu Asp
Val Gly 180 185 190 Cys Gly Ser Gly Ile Leu Ser Phe Phe Ala Ala Gln
Ala Gly Ala Arg 195 200 205 Lys Ile Tyr Ala Val Glu Ala Ser Thr Met
Ala Gln His Ala Glu Val 210 215 220 Leu Val Lys Ser Asn Asn Leu Thr
Asp Arg Ile Val Val Ile Pro Gly 225 230 235 240 Lys Val Glu Glu Val
Ser Leu Pro Glu Gln Val Asp Ile Ile Ile Ser 245 250 255 Glu Pro Met
Gly Tyr Met Leu Phe Asn Glu Arg Met Leu Glu Ser Tyr 260 265 270 Leu
His Ala Lys Lys Tyr Leu Lys Pro Ser Gly Asn Met Phe Pro Thr 275 280
285 Ile Gly Asp Val His Leu Ala Pro Phe Thr Asp Glu Gln Leu Tyr Met
290 295 300 Glu Gln Phe Thr Lys Ala Asn Phe Trp Tyr Gln Pro Ser Phe
His Gly 305 310 315 320 Val Asp Leu Ser Ala Leu Arg Gly Ala Ala Val
Asp Glu Tyr Phe Arg 325 330 335 Gln Pro Val Val Asp Thr Phe Asp Ile
Arg Ile Leu Met Ala Lys Ser 340 345 350 Val Lys Tyr Thr Val Asn Phe
Leu Glu Ala Lys Glu Gly Asp Leu His 355 360 365 Arg Ile Glu Ile Pro
Phe Lys Phe His Met Leu His Ser Gly Leu Val 370 375 380 His Gly Leu
Ala Phe Trp Phe Asp Val Ala Phe Ile Gly Ser Ile Met 385 390 395 400
Thr Val Trp Leu Ser Thr Ala Pro Thr Glu Pro Leu Thr His Trp Tyr 405
410 415 Gln Val Arg Cys Leu Phe Gln Ser Pro Leu Phe Ala Lys Ala Gly
Asp 420 425 430 Thr Leu Ser Gly Thr Cys Leu Leu Ile Ala Asn Lys Arg
Gln Ser Tyr 435 440 445 Asp Ile Ser Ile Val Ala Gln Val Asp Gln Thr
Gly Ser Lys Ser Ser 450 455 460 Asn Leu Leu Asp Leu Lys Asn Pro Phe
Phe Arg Tyr Thr Gly Thr Thr 465 470 475 480 Pro Ser Pro Pro Pro Gly
Ser His Tyr Thr Ser Pro Ser Glu Asn Met 485 490 495 Trp Asn Thr Gly
Ser Thr Tyr Asn Leu Ser Ser Gly Met Ala Val Ala 500 505 510 Gly Met
Pro Thr Ala Tyr Asp Leu Ser Ser Val Ile Ala Ser Gly Ser 515 520 525
Ser Val Gly His Asn Asn Leu Ile Pro Leu Gly Ser Ser Gly Ala Gln 530
535 540 Gly Ser Gly Gly Gly Ser Thr Ser Ala His Tyr Ala Val Asn Ser
Gln 545 550 555 560 Phe Thr Met Gly Gly Pro Ala Ile Ser Met Ala Ser
Pro Met Ser Ile 565 570 575 Pro Thr Asn Thr Met His Tyr Gly Ser 580
585
* * * * *
References