U.S. patent application number 10/479871 was filed with the patent office on 2005-10-13 for map3ks 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 | 20050227228 10/479871 |
Document ID | / |
Family ID | 35060971 |
Filed Date | 2005-10-13 |
United States Patent
Application |
20050227228 |
Kind Code |
A1 |
Friedman, Lori ; et
al. |
October 13, 2005 |
Map3ks as modifiers of the p53 pathway and methods of use
Abstract
Human MAP3K 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
MAP3K are provided.
Inventors: |
Friedman, Lori; (San Carlos,
CA) ; Plowman, Gregory D.; (San Carlos, CA) ;
Belvin, Marcia; (Albany, CA) ; Francis-Lang,
Helen; (San Francisco, CA) ; Li, Danxi;
(Zionsville, IN) ; Funke, Roel P.; (Brisbane,
CA) |
Correspondence
Address: |
PATENT DEPT
EXELIXIS, INC.
170 HARBOR WAY
P.O. BOX 511
SOUTH SAN FRANCISCO
CA
94083-0511
US
|
Family ID: |
35060971 |
Appl. No.: |
10/479871 |
Filed: |
March 21, 2005 |
PCT Filed: |
June 3, 2002 |
PCT NO: |
PCT/US02/17457 |
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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60357253 |
Feb 15, 2002 |
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60361196 |
Mar 1, 2002 |
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Current U.S.
Class: |
435/6.16 ;
435/15; 435/7.23 |
Current CPC
Class: |
G01N 2333/91215
20130101; C12Q 2600/158 20130101; C12Q 2600/136 20130101; G01N
33/57407 20130101; G01N 2500/02 20130101; C12Q 1/6886 20130101;
A61P 35/00 20180101; C12Q 1/485 20130101 |
Class at
Publication: |
435/006 ;
435/007.23; 435/015 |
International
Class: |
C12Q 001/68; G01N
033/574; C12Q 001/48 |
Claims
What is claimed is:
1. A method of identifying a candidate p53 pathway modulating
agent, said method comprising the steps of: (a) providing an assay
system comprising a purified MAP3K 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
candidate p53 pathway modulating agent.
2. The method of claim 1 wherein the assay system comprises
cultured cells that express the MAP3K polypeptide.
3. The method of claim 2 wherein the cultured cells additionally
have defective p53 function.
4. The method of claim 1 wherein the assay system includes a
screening assay comprising a MAP3K polypeptide, and the candidate
test agent is a small molecule modulator.
5. The method of claim 4 wherein the assay is a kinase assay.
6. The method of claim 1 wherein the assay system is selected from
the group consisting of an apoptosis assay system, a cell
proliferation assay system, an angiogenesis assay system, and a
hypoxic induction assay system.
7. The method of claim 1 wherein the assay system includes a
binding assay comprising a MAP3K polypeptide and the candidate test
agent is an antibody.
8. The method of claim 1 wherein the assay system includes an
expression assay comprising a MAP3K nucleic acid and the candidate
test agent is a nucleic acid modulator.
9. The method of claim 8 wherein the nucleic acid modulator is an
antisense oligomer.
10. The method of claim 8 wherein the nucleic acid modulator is a
PMO.
11. The method of claim 1 additionally comprising: (d)
administering the candidate p53 pathway 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.
12. The method of claim 11 wherein the model system is a mouse
model with defective p53 function.
13. A method for modulating a p53 pathway of a cell comprising
contacting a cell defective in p53 function with a candidate
modulator that specifically binds to a MAP3K polypeptide comprising
an amino acid sequence selected from group consisting of SEQ ID
NOS: 8 and 9, whereby p53 function is restored.
14. The method of claim 13 wherein the candidate modulator is
administered to a vertebrate animal predetermined to have a disease
or disorder resulting from a defect in p53 function.
15. The method of claim 13 wherein the candidate modulator is
selected from the group consisting of an antibody and a small
molecule.
16. The method of claim 1, comprising the additional steps of: (d)
providing a secondary assay system comprising cultured cells or a
non-human animal expressing MAP3K, (e) contacting the secondary
assay system with the test agent of (b) or an agent derived
therefrom under conditions whereby, but for the presence of the
test agent or agent derived therefrom, the system provides a
reference activity; and (f) detecting an agent-biased activity of
the second assay system, wherein a difference between the
agent-biased activity and the reference activity of the second
assay system confirms the test agent or agent derived therefrom as
a candidate p53 pathway modulating agent, and wherein the second
assay detects an agent-biased change in the p53 pathway.
17. The method of claim 16 wherein the secondary assay system
comprises cultured cells.
18. The method of claim 16 wherein the secondary assay system
comprises a non-human animal.
19. The method of claim 18 wherein the non-human animal
mis-expresses a p53 pathway gene.
20. A method of modulating p53 pathway in a mammalian cell
comprising contacting the cell with an agent that specifically
binds a MAP3K polypeptide or nucleic acid.
21. The method of claim 20 wherein the agent is administered to a
mammalian animal predetermined to have a pathology associated with
the p53 pathway.
22. The method of claim 20 wherein the agent is a small molecule
modulator, a nucleic acid modulator, or an antibody.
23. A method for diagnosing a disease in a patient comprising: (a)
obtaining a biological sample from the patient; (b) contacting the
sample with a probe for MAP3K expression; (c) comparing results
from step (b) with a control; (d) determining whether step (c)
indicates a likelihood of disease.
24. The method of claim 23 wherein said disease is cancer.
25. The method according to claim 24, wherein said cancer is a
cancer as shown in Table 1 as having >25% expression level.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional patent
applications 60/296,076 filed Jun. 5, 2001, 60/328,605 filed Oct.
10, 2001, 60/357,253 filed Feb. 15, 2002, and 60/361,196 filed Mar.
1, 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 October 2000; 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] Protein kinases (PKs) play a crucial role in regulating
cellular processes, including growth factor response, cytoskeletal
changes, gene expression, and metabolism. PKs have very similar
sequences and can be grouped based on specificity for the acceptor
amino acid. Most PKs phosphorylate either serine/threonine or
tyrosine. However, some PKs, referred to as mixed-lineage kinases,
have features of both serine/threonine and tyrosine PKs. All PKs
have Src homology (SH) domains and can also be grouped as receptors
or nonreceptors. Receptor PKs have a transmembrane region, an
extracellular ligand-binding domain, and an intracellular catalytic
domain.
[0006] Mitogen activated protein kinase kinase kinase 12 (MAP3K12),
is a dual leucine zipper-bearing kinase, and a member of the mixed
lineage protein kinase (MLK) (Reddy, U. and Pleasure, D., (1994)
Biochem. Biophys. Res. Commun. 202: 613-620). MAP3K12 contains a
COOH-terminal and NH2-terminal proline-rich domains suggestive of
src homology 3 (SH3) domain binding regions, and can be
autophosphorylated on serine and threonine residues (Holzman, L. et
al., (1994) J. Biol. Chem. 269: 30808-30817). This kinase activates
the SAPK/JNK signaling pathway, and may play a role in neuronal
differentiation (Hirai, S., (1996) Oncogene 12: 641-650).
[0007] MAP3K13 protein, also called LZK (leucine zipper-bearing
kinase) contains double leucine/isoleucine zippers, has no apparent
signal sequence or transmembrane region but does contain a kinase
catalytic domain, and an acidic domain at its C-terminal end
(Sakuma, H. et al., (1997) J. Biol. Chem. 272: 28622-28629).
MAP3K13 shares 86.4% amino acid identity with MAP3K12 and like
MAP3K12 it is also a member of the mixed-lineage kinase family of
proteins which contain similarities to both serine/threonine and
tyrosine kinases (Sakuma, H. et al., (1997) J. Biol. Chem. 272:
28622-28629). These kinases activate the phosphorylation event of
c-Jun and turn on JNK-1 (Sakuma, H. et al., (1997) J. Biol. Chem.
272: 28622-28629).
[0008] MAP3K12 and MAP3K13 are both highly conserved genes that
have been found in organisms from yeast to man. MAP3K12 has been
implicated in neuronal cell death (Xu, Z. et al. (2001) Mol Cell
Biol 21:4713-24).
[0009] 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 D A, et al., 1995 Curr Opin Gen Dev
5: 44-50; and Booth D R. 1999 Cancer Metastasis Rev. 18: 261-284).
For example, a genetic screen can be carried out in an invertebrate
model organism having underexpression (e.g. knockout) or
overexpression of a gene (referred to as a "genetic entry point")
that yields a visible phenotype. Additional genes are mutated in a
random or targeted manner. When a gene mutation changes the
original phenotype caused by the mutation in the genetic entry
point, the gene is identified as a "modifier" involved in the same
or overlapping pathway as the genetic entry point. When the genetic
entry point is an ortholog of a human gene implicated in a disease
pathway, such as p53, modifier genes can be identified that may be
attractive candidate targets for novel therapeutics.
[0010] 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
[0011] We have discovered genes that modify the p53 pathway in
Drosophila, and identified their human orthologs, hereinafter
referred to as MAP3Ks. The invention provides methods for utilizing
these p53 modifier genes and polypeptides to identify candidate
therapeutic agents that can be used in the treatment of disorders
associated with defective p53 function. Preferred MAP3K-modulating
agents specifically bind to MAP3K polypeptides and restore p53
function. Other preferred MAP3K-modulating agents are nucleic acid
modulators such as antisense oligomers and RNAi that repress MAP3K
gene expression or product activity by, for example, binding to and
inhibiting the respective nucleic acid (i.e. DNA or mRNA).
[0012] MAP3K-specific modulating agents may be evaluated by any
convenient in vitro or in vivo assay for molecular interaction with
a MAP3K polypeptide or nucleic acid. In one embodiment, candidate
p53 modulating agents are tested with an assay system comprising a
MAP3K polypeptide or nucleic acid. Candidate 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. MAP3K-modulating agents include MAP3K
related proteins (e.g. dominant negative mutants, and
biotherapeutics); MAP3K-specific antibodies; MAP3K-specific
antisense oligomers and other nucleic acid modulators; and chemical
agents that specifically bind MAP3K or compete with MAP3K binding
target. In one specific embodiment, a small molecule modulator is
identified using a kinase assay. In specific embodiments, the
screening assay system is selected from a binding assay, an
apoptosis assay, a cell proliferation assay, an angiogenesis assay,
and a hypoxic induction assay.
[0013] 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).
[0014] The invention further provides methods for modulating the
p53 pathway in a mammalian cell by contacting the mammalian cell
with an agent that specifically binds a MAP3K polypeptide or
nucleic acid. The agent may be a small molecule modulator, a
nucleic acid modulator, or an antibody and may be administered to a
mammalian animal predetermined to have a pathology associated the
p53 pathway.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Genetic screens were designed to identify modifiers of the
p53 pathway in and Drosophila in which p53 was overexpressed in the
wing (Ollmann M, et al., Cell 2000 101: 91-101). The CG8789 gene
was identified as a modifier of the p53 pathway. Accordingly,
vertebrate orthologs of these modifiers, and preferably the human
orthologs, MAP3K 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.
[0016] In vitro and in vivo methods of assessing MAP3K function are
provided herein. Modulation of the MAP3K 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. MAP3K-modulating agents that act by inhibiting
or enhancing MAP3K expression, directly or indirectly, for example,
by affecting a MAP3K function such as enzymatic (e.g., catalytic)
or binding activity, can be identified using methods provided
herein. MAP3K modulating agents are useful in diagnosis, therapy
and pharmaceutical development.
[0017] Nucleic Acids and Polypeptides of the Invention
[0018] Sequences related to MAP3K nucleic acids and polypeptides
that can be used in the invention are disclosed in Genbank
(referenced by Genbank identifier (GI) number) as GI#s 5454183 (SEQ
ID NO:1), 13645287 (SEQ ID NO:4), and 4758695 (SEQ ID NO:5) for
nucleic acid, and GI#s 5454184 (SEQ ID NO:8) and 4758696 (SEQ ID
NO:9) for polypeptides. Further, sequences of SEQ ID NOs: 2, 3, 6,
and 7 can also be used in the invention.
[0019] MAP3Ks are kinase proteins with kinase domains. The term
"MAP3K polypeptide" refers to a full-length MAP3K protein or a
functionally active fragment or derivative thereof. A "functionally
active" MAP3K fragment or derivative exhibits one or more
functional activities associated with a full-length, wild-type
MAP3K protein, such as antigenic or immunogenic activity, enzymatic
activity, ability to bind natural cellular substrates, etc. The
functional activity of MAP3K 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 MAP3K, such as a kinase domain or a binding domain.
Protein domains can be identified using the PFAM program (Bateman
A., et al., Nucleic Acids Res, 1999, 27:260-2;
http://pfam.wustl.edu). For example, the kinase domain of MAP3K
from GI# 5454184 (SEQ ID NO:8) is located at approximately amino
acid residues 125-366 (PFAM 00069). Likewise, the kinase domain of
MAP3K from GI# 4758696 (SEQ ID NO:9) is located at approximately
amino acid residues 168-409. Methods for obtaining MAP3K
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 or 9 (a MAP3K).
In further preferred embodiments, the fragment comprises the entire
kinase (functionally active) domain.
[0020] The term "MAP3K nucleic acid" refers to a DNA or RNA
molecule that encodes a MAP3K polypeptide. Preferably, the MAP3K
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 MAP3K. Normally, orthologs in different
species retain the same function, due to presence of one or more
protein motifs and/or 3-dimensional structures. Orthologs are
generally identified by sequence homology analysis, such as BLAST
analysis, usually using protein bait sequences. Sequences are
assigned as a potential ortholog if the best hit sequence from the
forward BLAST result retrieves the original query sequence in the
reverse BLAST (Huynen M A and Bork P, Proc Natl Acad Sci (1998)
95:5849-5856; Huynen M A et al., Genome Research (2000)
10:1204-1210). Programs for multiple sequence alignment, such as
CLUSTAL (Thompson J D et al, 1994, Nucleic Acids Res 22:46734680)
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 sinilarity" 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.
[0021] 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.
[0022] 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."
[0023] 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 1 h in a solution containing 0.2.times.SSC and
0.1% SDS (sodium dodecyl sulfate).
[0024] 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 (pH7.5), 5 mM
EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 .mu.g/ml denatured
salmon sperm DNA; hybridization for 18-20 h at 40.degree. C. in a
solution containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl
(pH7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml
salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by
washing twice for 1 hour at 55.degree. C. in a solution containing
2.times.SSC and 0.1% SDS.
[0025] 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.
[0026] Isolation, Production, Expression, and Mis-Expression of
MAP3K Nucleic Acids and Polypeptides
[0027] MAP3K nucleic acids and polypeptides, useful for identifying
and testing agents that modulate MAP3K function and for other
applications related to the involvement of MAP3K in the p53
pathway. MAP3K nucleic acids and derivatives and orthologs thereof
may be obtained using any available method. For instance,
techniques for isolating cDNA or genomic DNA sequences of interest
by screening DNA libraries or by using polymerase chain reaction
(PCR) are well known in the art. In general, the particular use for
the protein will dictate the particulars of expression, production,
and purification methods. For instance, production of proteins for
use in screening for modulating agents may require methods that
preserve specific biological activities of these proteins, whereas
production of proteins for antibody generation may require
structural integrity of particular epitopes. Expression of proteins
to be purified for screening or antibody production may require the
addition of specific tags (e.g., generation of fusion proteins).
Overexpression of a MAP3K protein for assays used to assess MAP3K
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 MAP3K 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.
[0028] The nucleotide sequence encoding a MAP3K polypeptide can be
inserted into any appropriate expression vector. The necessary
transcriptional and translational signals, including
promoter/enhancer element, can derive from the native MAP3K 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.
[0029] To detect expression of the MAP3K gene product, the
expression vector can comprise a promoter operably linked to a
MAP3K 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 MAP3K gene product based on the physical or functional
properties of the MAP3K protein in in vitro assay systems (e.g.
immunoassays).
[0030] The MAP3K 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).
[0031] Once a recombinant cell that expresses the MAP3K 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 MAP3K 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.
[0032] The methods of this invention may also use cells that have
been engineered for altered expression (mis-expression) of MAP3K or
other genes associated with the p53 pathway. As used herein,
mis-expression encompasses ectopic expression, over-expression,
under-expression, and non-expression (e.g. by gene knock-out or
blocking expression that would otherwise normally occur).
[0033] Genetically Modified Animals
[0034] Animal models that have been genetically modified to alter K
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
MAP3K in a p53 pathway process such as apoptosis or cell
proliferation. Preferably, the altered MAP3K expression results in
a detectable phenotype, such as decreased or increased levels of
cell proliferation, angiogenesis, or apoptosis compared to control
animals having normal MAP3K 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.
[0035] Methods of making transgenic animals are well-known in the
art (for transgenic mice see Brinster et al., Proc. Nat. Acad. Sci.
USA 82: 4438-4442 (1985), U.S. Pat. Nos. 4,736,866 and 4,870,009,
both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al., and
Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1986); for particle
bombardment see U.S. Pat. No., 4,945,050, by Sandford et al.; for
transgenic Drosophila see Rubin and Spradling, Science (1982)
218:348-53 and U.S. Pat. No. 4,670,388; for transgenic insects see
Berghammer A. J. et al., A Universal Marker for Transgenic Insects
(1999) Nature 402:370-371; for transgenic Zebrafish see Lin S.,
Transgenic Zebrafish, Methods Mol Biol. (2000);136:375-3830); for
microinjection procedures for fish, amphibian eggs and birds see
Houdebine and Chourrout, Experientia (1991) 47:897-905; for
transgenic rats see Hammer et al., Cell (1990) 63:1099-1112; and
for culturing of embryonic stem (ES) cells and the subsequent
production of transgenic animals by the introduction of DNA into ES
cells using methods such as electroporation, calcium phosphate/DNA
precipitation and direct injection see, e.g., Teratocarcinomas and
Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed.,
IRL Press (1987)). Clones of the nonhuman transgenic animals can be
produced according to available methods (see Wilmut, I. et al.
(1997) Nature 385:810-813; and PCT International Publication Nos.
WO 97/07668 and WO 97/07669).
[0036] In one embodiment, the transgenic animal is a "knock-out"
animal having a heterozygous or homozygous alteration in the
sequence of an endogenous MAP3K gene that results in a decrease of
MAP3K function, preferably such that MAP3K 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 MAP3K gene is used to construct a
homologous recombination vector suitable for altering an endogenous
MAP3K gene in the mouse genome. Detailed methodologies for
homologous recombination in mice are available (see Capecchi,
Science (1989) 244:1288-1292; Joyner et al., Nature (1989)
338:153-156). Procedures for the production of non-rodent
transgenic mammals and other animals are also available (Houdebine
and Chourrout, supra; Pursel et al., Science (1989) 244:1281-1288;
Simms et al., Bio/Technology (1988) 6:179-183). In a preferred
embodiment, knock-out animals, such as mice harboring a knockout of
a specific gene, may be used to produce antibodies against the
human counterpart of the gene that has been knocked out (Claesson M
H et al., (1994) Scan J Immunol 40:257-264; Declerck P J et al.,
(1995) J Biol Chem. 270:8397400).
[0037] 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 MAP3K gene, e.g., by introduction of additional
copies of MAP3K, or by operatively inserting a regulatory sequence
that provides for altered expression of an endogenous copy of the
MAP3K gene. Such regulatory sequences include inducible,
tissue-specific, and constitutive promoters and enhancer elements.
The knock-in can be homozygous or heterozygous.
[0038] 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).
[0039] 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 MAP3K function and phenotypic changes are compared with
appropriate control animals such as genetically modified animals
that receive placebo treatment, and/or animals with unaltered MAP3K
expression that receive candidate therapeutic agent.
[0040] In addition to the above-described genetically modified
animals having altered MAP3K function, animal models having
defective p53 function (and otherwise normal MAP3K 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. p5.sup.3 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.
[0041] Modulating Agents
[0042] The invention provides methods to identify agents that
interact with and/or modulate the function of MAP3K and/or the p53
pathway. 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 MAP3K 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 MAP3K activity by administering a MAP3K-interacting or
-modulating agent.
[0043] In a preferred embodiment, MAP3K-modulating agents inhibit
or enhance MAP3K activity or otherwise affect normal MAP3K
function, including transcription, protein expression, protein
localization, and cellular or extra-cellular activity. In a further
preferred embodiment, the candidate p53 pathway-modulating agent
specifically modulates the function of the MAP3K. The phrases
"specific modulating agent", "specifically modulates", etc., are
used herein to refer to modulating agents that directly bind to the
MAP3K polypeptide or nucleic acid, and preferably inhibit, enhance,
or otherwise alter, the function of the MAP3K. The term also
encompasses modulating agents that alter the interaction of the
MAP3K with a binding partner or substrate (e.g. by binding to a
binding partner of a MAP3K, or to a protein/binding partner
complex, and inhibiting function).
[0044] Preferred MAP3K-modulating agents include small molecule
compounds; MAP3K-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.
[0045] Small Molecule Modulators
[0046] 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 MAP3K 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 MAP3K-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).
[0047] 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.
[0048] Protein Modulators
[0049] Specific MAP3K-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 MAP3K-modulating agents. In a preferred embodiment,
MAP3K-interacting proteins affect normal MAP3K function, including
transcription, protein expression, protein localization, and
cellular or extra-cellular activity. In another embodiment,
MAP3K-interacting proteins are useful in detecting and providing
information about the function of MAP3K proteins, as is relevant to
p53 related disorders, such as cancer (e.g., for diagnostic
means).
[0050] An MAP3K-interacting protein may be endogenous, i.e. one
that naturally interacts genetically or biochemically with a MAP3K,
such as a member of the MAP3K pathway that modulates MAP3K
expression, localization, and/or activity. MAP3K-modulators include
dominant negative forms of MAP3K-interacting proteins and of MAP3K
proteins themselves. Yeast two-hybrid and variant screens offer
preferred methods for identifying endogenous MAP3K-interacting
proteins (Finley, R. L. et al. (1996) in DNA Cloning-Expression
Systems: A Practical Approach, eds. Glover D. & Hames B. D
(Oxford University Press, Oxford, England), pp. 169-203; Fashema S
F et al., Gene (2000) 250:1-14; Drees B L Curr Opin Chem Biol
(1999) 3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999)
27:919-29; and U.S. Pat. No. 5,928,868). Mass spectrometry is an
alternative preferred method for the elucidation of protein
complexes (reviewed in, e.g., Pandley A and Mann M, Nature (2000)
405:837-846; Yates J R 3.sup.rd, Trends Genet (2000) 16:5-8).
[0051] An MAP3K-interacting protein may be an exogenous protein,
such as a MAP3K-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). MAP3K antibodies are further
discussed below.
[0052] In preferred embodiments, a MAP3K-interacting protein
specifically binds a MAP3K protein. In alternative preferred
embodiments, a MAP3K-modulating agent binds a MAP3K substrate,
binding partner, or cofactor.
[0053] Antibodies
[0054] In another embodiment, the protein modulator is a MAP3K
specific antibody agonist or antagonist. The antibodies have
therapeutic and diagnostic utilities, and can be used in screening
assays to identify MAP3K modulators. The antibodies can also be
used in dissecting the portions of the MAP3K pathway responsible
for various cellular responses and in the general processing and
maturation of the MAP3K.
[0055] Antibodies that specifically bind MAP3K polypeptides can be
generated using known methods. Preferably the antibody is specific
to a mammalian ortholog of MAP3K polypeptide, and more preferably,
to human MAP3K. 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 MAP3K
which are particularly antigenic can be selected, for example, by
routine screening of MAP3K 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 or 9. 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 MAP3K or substantially purified fragments thereof. If MAP3K
fragments are used, they preferably comprise at least 10, and more
preferably, at least 20 contiguous amino acids of a MAP3K protein.
In a particular embodiment, MAP3K-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 MAP3K-specific antibodies is assayed by an
appropriate assay such as a solid phase enzyme-linked immunosorbant
assay (ELISA) using immobilized corresponding MAP3K polypeptides.
Other assays, such as radioimmunoassays or fluorescent assays might
also be used.
[0057] Chimeric antibodies specific to MAP3K 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) 31:452454). 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 MS, 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] MAP3K-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 about 10 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 MAP3K-modulating agents comprise nucleic
acid molecules, such as antisense oligomers or double stranded RNA
(dsRNA), which generally inhibit MAP3K activity. Preferred nucleic
acid modulators interfere with the function of the MAP3K nucleic
acid such as DNA replication, transcription, translocation of the
MAP3K RNA to the site of protein translation, translation of
protein from the MAP3K RNA, splicing of the MAP3K RNA to yield one
or more mRNA species, or catalytic activity which may be engaged in
or facilitated by the MAP3K RNA.
[0064] In one embodiment, the antisense oligomer is an
oligonucleotide that is sufficiently complementary to a MAP3K mRNA
to bind to and prevent translation, preferably by binding to the 5'
untranslated region. MAP3K-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 MAP3K 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 MAP3K-specific nucleic acid modulator is used in an
assay to further elucidate the role of the MAP3K in the p53
pathway, and/or its relationship to other members of the pathway.
In another aspect of the invention, a MAP3K-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 MAP3K 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 MAP3K nucleic acid or protein.
In general, secondary assays further assess the activity of a MAP3K
modulating agent identified by a primary assay and may confirm that
the modulating agent affects MAP3K in a manner relevant to the p53
pathway. In some cases, MAP3K 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 MAP3K polypeptide
with a candidate agent under conditions whereby, but for the
presence of the agent, the system provides a reference activity
(e.g. kinase activity), which is based on the particular molecular
event the screening method detects. A statistically significant
difference between the agent-biased activity and the reference
activity indicates that the candidate agent modulates MAP3K
activity, and hence the p53 pathway.
[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 MAP3K 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
MAP3K-interacting proteins are used in screens to identify small
molecule modulators, the binding specificity of the interacting
protein to the MAP3K protein may be assayed by various known
methods such as substrate processing (e.g. ability of the candidate
MAP3K-specific binding agents to function as negative effectors in
MAP3K-expressing cells), binding equilibrium constants (usually at
least about 10.sup.7 N.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 MAP3K 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 MAP3K
polypeptide, a fusion protein thereof, or to cells or membranes
bearing the polypeptide or fusion protein. The MAP3K polypeptide
can be full length or a fragment thereof that retains functional
MAP3K activity. The MAP3K polypeptide may be fused to another
polypeptide, such as a peptide tag for detection or anchoring, or
to another tag. The MAP3K polypeptide is preferably human MAP3K, or
is an ortholog or derivative thereof as described above. In a
preferred embodiment, the screening assay detects candidate
agent-based modulation of MAP3K interaction with a binding target,
such as an endogenous or exogenous protein or other substrate that
has MAP3K -specific binding activity, and can be used to assess
normal MAP3K gene function.
[0077] Suitable assay formats that may be adapted to screen for
MAP3K 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) 7:7304;
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 MAP3K and p53 pathway modulators (e.g. U.S. Pat. No.
6,165,992 (kinase assays); U.S. Pat. Nos. 5,550,019 and 6,133,437
(apoptosis assays); and U.S. Pat. No. 6,020,135 (p53 modulation),
among others). Specific preferred assays are described in more
detail below.
[0079] Kinase assays. In some preferred embodiments the screening
assay detects the ability of the test agent to modulate the kinase
activity of a MAP3K polypeptide. In further embodiments, a
cell-free kinase assay system is used to identify a candidate p53
modulating agent, and a secondary, cell-based assay, such as an
apoptosis or hypoxic induction assay (described below), may be used
to further characterize the candidate p53 modulating agent. Many
different assays for kinases have been reported in the literature
and are well known to those skilled in the art (e.g. U.S. Pat. No.
6,165,992; Zhu et al., Nature Genetics (2000) 26:283-289; and
WO0073469). Radioassays, which monitor the transfer of a gamma
phosphate are frequently used. For instance, a scintillation assay
for p56 (1ck) kinase activity monitors the transfer of the gamma
phosphate from gamma-.sup.33P ATP to a biotinylated peptide
substrate; the substrate is captured on a streptavidin coated bead
that transmits the signal (Beveridge M et al., J Biomol Screen
(2000) 5:205-212). This assay uses the scintillation proximity
assay (SPA), in which only radio-ligand bound to receptors tethered
to the surface of an SPA bead are detected by the scintillant
immobilized within it, allowing binding to be measured without
separation of bound from free ligand.
[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 MAP3K,
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 MAP3K function plays a
direct role in apoptosis. For example, an apoptosis assay may be
performed on cells that over- or under-express MAP3K relative to
wild type cells. Differences in apoptotic response compared to wild
type cells suggests that the MAP3K 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 L S 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 MAP3K 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 MAP3K 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 MAP3K, 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 p53 modulating agents that
is initially identified using another assay system such as a
cell-free kinase assay system. A cell proliferation assay may also
be used to test whether MAP3K 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 MAP3K relative to wild type cells. Differences in
proliferation or cell cycle compared to wild type cells suggests
that the MAP3K 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
MAP3K, 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 MAP3K function plays a
direct role in cell proliferation. For example, an angiogenesis
assay may be performed on cells that over- or under-express MAP3K
relative to wild type cells. Differences in angiogenesis compared
to wild type cells suggests that the MAP3K 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 MAP3K 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 MAP3K, 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 MAP3K 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 MAP3K relative to wild type
cells. Differences in hypoxic response compared to wild type cells
suggests that the MAP3K 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. May-June 2001;
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 MAP3K 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 MAP3K-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 MAP3K
gene expression, preferably mRNA expression. In general, expression
analysis comprises comparing MAP3K expression in like populations
of cells (e.g., two pools of cells that endogenously or
recombinantly express MAP3K) 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 MAP3K 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 MAP3K 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 MAP3K-modulating agent identified by any of the above methods to
confirm that the modulating agent affects MAP3K in a manner
relevant to the p53 pathway. As used herein, MAP3K-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 MAP3K.
[0097] Secondary assays generally compare like populations of cells
or animals (e.g., two pools of cells or animals that endogenously
or recombinantly express MAP3K) in the presence and absence of the
candidate modulator. In general, such assays test whether treatment
of cells or animals with a candidate MAP3K-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 MAP3K 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 MAP3K 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 MAP3K 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 MAP3K 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 MAP3K 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 MAP3K-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, the invention also provides methods for modulating the
p53 pathway in a cell, preferably a cell pre-determined to have
defective p53 function, comprising the step of administering an
agent to the cell that specifically modulates MAP3K activity.
Preferably, the modulating agent produces a detectable phenotypic
change in the cell indicating that the p53 function is restored,
i.e., for example, the cell undergoes normal proliferation or
progression through the cell cycle.
[0106] The discovery that MAP3K 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 MAP3K expression occurs in a particular sample, including
Northern blotting, slot blotting, ribonuclease protection,
quantitative RT-PCR, and microarray analysis. (e.g., Current
Protocols in Molecular Biology (1994) Ausubel F M et al., eds.,
John Wiley & Sons, Inc., chapter 4; Freeman W M et al.,
Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001,
33:142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol 2001,
12:41-47). Tissues having a disease or disorder implicating
defective p53 signaling that express a MAP3K, are identified as
amenable to treatment with a MAP3K modulating agent. In a preferred
application, the p53 defective tissue overexpresses a MAP3K
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
MAP3K cDNA sequences as probes, can determine whether particular
tumors express or overexpress MAP3K. Alternatively, the TaqMan.RTM.
is used for quantitative RT-PCR analysis of MAP3K 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 MAP3K oligonucleotides, and
antibodies directed against a MAP3K, as described above for: (1)
the detection of the presence of MAP3K gene mutations, or the
detection of either over- or under-expression of MAP3K mRNA
relative to the non-disorder state; (2) the detection of either an
over- or an under-abundance of MAP3K gene product relative to the
non-disorder state; and (3) the detection of perturbations or
abnormalities in the signal transduction pathway mediated by
MAP3K.
[0109] Thus, in a specific embodiment, the invention is drawn to a
method for diagnosing a disease in a patient, the method
comprising: a) obtaining a biological sample from the patient; b)
contacting the sample with a probe for MAP3K expression; c)
comparing results from step (b) with a control; and d) determining
whether step (c) indicates a likelihood of disease. Preferably, the
disease is cancer, most preferably a cancer as shown in TABLE 1.
The probe may be either DNA or protein, including an antibody.
EXAMPLES
[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. CG8789 was an enhancer of
the wing phenotype. Human orthologs of the modifiers, are referred
to herein as MAP3K.
[0113] BLAST analysis (Altschul et al., supra) was employed to
identify Targets from Drosophila modifiers. [For example,
representative sequences from MAP3K, GI#5454184 (SEQ ID NO:8) and
GI#4758696 (SEQ ID NO:9) share 52% and 37% amino acid identity,
respectively, with the Drosophila.CG8789.
[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. Jan. 1,
1991; 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. November 2000; 10(11):1679-89) programs.
Using PFAM, the kinase domain of MAP3K from GI# 5454184 (SEQ ID
NO:8) is located at approximately amino acid residues 125-366 (PFAM
00069). Likewise, the kinase domain of MAP3K from GI# 4758696 (SEQ
ID NO:9) is located at approximately amino acid residues
168-409.
[0115] II. High-Throughput In Vitro Fluorescence Polarization
Assay
[0116] Fluorescently-labeled MAP3K peptide/substrate are added to
each well of a 96-well microliter 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 MAP3K activity.
[0117] III. High-Throughput In Vitro Binding Assay.
[0118] .sup.33P-labeled MAP3K 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.
[0119] IV. Immunoprecipitations and Immunoblotting
[0120] For coprecipitation of transfected proteins,
3.times.10.sup.6 appropriate recombinant cells containing the MAP3K
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 P40. 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.
[0121] 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).
[0122] V. Kinase Assay
[0123] A purified or partially purified MAP3K is diluted in a
suitable reaction buffer, e.g., 50 mM Hepes, pH 7.5, containing
magnesium chloride or manganese chloride (1-20 mM) and a peptide or
polypeptide substrate, such as myelin basic protein or casein (1-10
.mu.g/ml). The final concentration of the kinase is 1-20 nM. The
enzyme reaction is conducted in microtiter plates to facilitate
optimization of reaction conditions by increasing assay throughput.
A 96-well microtiter plate is employed using a final volume 30-100
.mu.l. The reaction is initiated by the addition of
.sup.33P-gamma-ATP (0.5 .mu.Ci/ml) and incubated for 0.5 to 3 hours
at room temperature. Negative controls are provided by the addition
of EDTA, which chelates the divalent cation (Mg2.sup.+ or Mn2+)
required for enzymatic activity. Following the incubation, the
enzyme reaction is quenched using EDTA. Samples of the reaction are
transferred to a 96-well glass fiber filter plate (MultiScreen,
Millipore). The filters are subsequently washed with
phosphate-buffered saline, dilute phosphoric acid (0.5%) or other
suitable medium to remove excess radiolabeled ATP. Scintillation
cocktail is added to the filter plate and the incorporated
radioactivity is quantitated by scintillation counting
(Wallac/Perkin Elmer). Activity is defined by the amount of
radioactivity detected following subtraction of the negative
control reaction value (EDTA quench).
[0124] VI. Expression Analysis
[0125] All cell lines used in the following experiments are NCI
(National Cancer Institute) lines, and are available from ATCC
(American Type Culture Collection, Manassas, Va. 20110-2209).
Normal and tumor tissues were obtained from Impath, UC Davis,
Clontech, Stratagene, and Ambion.
[0126] TaqMan analysis was used to assess expression levels of the
disclosed genes in various samples.
[0127] 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/).
[0128] 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.
[0129] 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).
[0130] 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) ).
[0131] Results are shown in Table 1. Results from various batches
of mRNA are represented for each batch. Data presented in bold
indicate that greater than 50% of tested tumor samples of the
tissue type indicated in row 1 exhibited over expression of the
gene listed in column 1, relative to normal samples. Underlined
data indicates that between 25% to 49% of tested tumor samples
exhibited over expression. 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.
1 TABLE 1 . breast . . colon . . lung . . ovary GI#5454183 (SEQ ID
NO: 1) TaqExp_100501 2 11 . 4 30 . 1 13 . 1 7 GI#13645287 (SEQ ID
NO: 4) TaqExp_100501 3 11 . 1 30 . 2 13 . 3 7
[0132]
Sequence CWU 1
1
9 1 3365 DNA Homo sapiens 1 agcatccgga gcggagctgc agcagcgccg
ccttttgtgc tgcggccgcg gagcccccga 60 gggcccagtg ttcaccatca
taccaggggc cagaggcgat ggcttgcctc catgagaccc 120 gaacaccctc
tccttccttt gggggctttg tgtctaccct aagtgaggca tccatgcgca 180
agctggaccc agacacttct gactgcactc ccgagaagga cctgacgcct acccatgtcc
240 tgcagctaca tgagcaggat gcagggggcc cagggggagc agctgggtca
cctgagagtc 300 gggcatccag agttcgagct gacgaggtgc gactgcagtg
ccagagtggc agtggcttcc 360 ttgagggcct ctttggctgc ctgcgccctg
tctggaccat gattggcaaa gcctactcca 420 ctgagcacaa gcagcagcag
gaagaccttt gggaggtccc ctttgaggaa atcctggacc 480 tgcagtgggt
gggctcaggg gcccagggtg ctgtcttcct ggggcgcttc cacggggagg 540
aggtggctgt gaagaaggtg cgagacctca aagaaaccga catcaagcac ttgcgaaagc
600 tgaagcaccc caacatcatc actttcaagg gtgtgtgcac ccaggctccc
tgctactgca 660 tcctcatgga gttctgcgcc cagggccagc tgtatgaggt
actgcgggct ggccgccctg 720 tcaccccctc cttactggtt gactggtcca
tgggcatcgc tggtggcatg aactacctgc 780 acctgcacaa gattatccac
agggatctca agtcacccaa catgctaatc acctacgacg 840 atgtggtgaa
gatctcagat tttggcactt ccaaggagct gagtgacaag agcaccaaga 900
tgtcctttgc agggacagta gcctggatgg cccctgaggt gatccgcaat gaacctgtgt
960 ctgagaaggt cgacatctgg tcctttggcg tggtgctatg ggaactgctg
actggtgaga 1020 tcccctacaa agacgtagat tcctcagcca ttatctgggg
tgtgggaagc aacagtctcc 1080 atctgcccgt gccctccagt tgcccagatg
gtttcaagat cctgcttcgc cagtgctgga 1140 atagcaaacc acgaaatcgc
ccatcattcc gacagatcct gctgcatctg gacattgcct 1200 cagctgatgt
actctccaca ccccaggaga cttactttaa gtcccaggca gagtggcggg 1260
aagaagtaaa actgcacttt gaaaagatta agtcagaagg gacctgtctg caccgcctag
1320 aagaggaact ggtgatgagg aggagggagg agctcagaca cgccctggac
atcagggagc 1380 actatgaaag gaagctggag agagccaaca acctgtatat
ggaacttaat gccctcatgt 1440 tgcagctgga actcaaggag agggagctgc
tcaggcgaga gcaagcttta gagcggaggt 1500 gcccaggcct gctgaagcca
cacccttccc ggggcctcct gcatggaaac acaatggaga 1560 agcttatcaa
gaagaggaat gtgccacaga atctgtcacc ccatagccaa aggccagata 1620
tcctcaaggc ggagtctttg ctccctaaac tagatgcagc cctgagtggg gtggggcttc
1680 ctgggtgtcc taaggccccc ccctcaccag gacggagtcg ccgtggcaag
acccgtcacc 1740 gcaaggccag cgccaagggg agctgtgggg acctgcctgg
gcttcgtaca gctgtgccac 1800 cccatgaacc tggaggacca ggaagcccag
ggggcctagg agggggaccc tcagcctggg 1860 aggcctgccc tcccgccctc
cgtgggcttc atcatgacct cctgctccgc aaaatgtctt 1920 catcgtcccc
agacctgctg tcagcagcac tagggtcccg gggccggggg gccacaggcg 1980
gagctgggga tcctggctca ccacctccgg cccggggtga caccccacca agtgagggct
2040 cagcccctgg ctccaccagc ccagattcac ctgggggagc caaaggggaa
ccacctcctc 2100 cagtagggcc tggtgaaggt gtggggcttc tgggaactgg
aagggaaggg acctcaggcc 2160 ggggaggaag ccgggctggg tcccagcact
tgaccccagc tgcactgctg tacagggctg 2220 ccgtcacccg aagtcagaaa
cgtggcatct catcggaaga ggaggaagga gaggtagaca 2280 gtgaagtaga
gctgacatca agccagaggt ggcctcagag cctgaacatg cgccagtcac 2340
tatctacctt cagctcagag aatccatcag atggggagga aggcacagct agtgaacctt
2400 cccccagtgg cacacctgaa gttggcagca ccaacactga tgagcggcca
gatgagcggt 2460 ctgatgacat gtgctcccag ggctcagaaa tcccactgga
cccacctcct tcagaggtca 2520 tccctggccc tgaacccagc tccctgccca
ttccacacca ggaacttctc agagagcggg 2580 gccctcccaa ttctgaggac
tcagactgtg acagcactga attggacaac tccaacagcg 2640 ttgatgcctt
gcgcccccca gcttccctcc ctccatgaaa gccactcgta ttccttgtac 2700
atagagaaat atttatatgg attatatata tatacatata tatatatata tgcgccacat
2760 aatcaacaga aagatggggc tgtcccagcc gtaagtcagg ctcgagggag
actgatcccc 2820 tgaccaattc acctgataaa ctctagggac actggcagct
gtggaaatga atgaggcaca 2880 gccgtagagc tgtggctaag ggcaagcccc
ttcctgcccc accccattcc ttatattcag 2940 caagcaacaa ggcaatagaa
aagccagggt tgtctttata ttctttatcc ccaaataata 3000 gggggtgggg
ggaggggcgg tgggaggggc aggagagaaa accacttaga ctgcactttt 3060
ctgttccgtt tactctgttt acacattttg cacttgggag gagggaggct aaggctgggt
3120 cctcccctct gaggtttctc aggtggcaat gtaactcatt tttttgtccc
accatttatc 3180 ttctctgccc aagccctgtc ttaaggccca gggggaggtt
aggagactga tagcatgtga 3240 tggctcaggc tgaagaaccg gggttctgtt
taagtccctg cttttatcct ggtgcctgat 3300 tggggtgggg actgtcctac
tgtaacccct gtgaaaaacc ttgaaaaata acactccatg 3360 cagga 3365 2 2830
DNA Homo sapiens misc_feature (33)..(33) "n" is A, C, G, or T 2
cgagggccca gtgttcacca tcataccagg ggncagaggc gatggcttgc ctccatgaga
60 cccgaacacc ctctccttcc tttgggggct ttgtgtctac cctaagtgag
gcatccatgc 120 gcaagctgga cccagacact tctgactgca ctcccgagaa
ggacctgacg cctacccagt 180 gtgtacttcg agatgtggta ccccttggtg
ggcagggtgg gggagggccc agcccctccc 240 caggtggaga gccgccccct
gagccttttg ccaacagtgt cctgcagcta catgagcagg 300 atgcaggggg
cccaggggga gcagctgggt cacctgagag tcgggcatcc agagttcgag 360
ctgacgaggt gcgactgcag tgccagagtg gcagtggctt ccttgagggc ctctttggct
420 gcctgcgccc tgtctggacc atgattggca aagcctactc cactgagcac
aagcagcagc 480 aggaagacct ttgggaggtc ccctttgagg aaatcctgga
cctgcagtgg gtgggctcag 540 gggcccaggg tgctgtcttc ctggggcgct
tccacgggga ggaggtggct gtgaagaagg 600 tgcgagacct caaagaaacc
gacatcaagc acttgcgaaa gctgaagcac cccaacatca 660 tcactttcaa
gggtgtgtgc acccaggctc cctgctactg catcctcatg gagttctgcg 720
cccagggcca gctgtatgag gtactgcggg ctggccgccc tgtcaccccc tccttactgg
780 ttgactggtc catgggcatc gctggtggca tgaactacct gcacctgcac
aagattatcc 840 acagggatct caagtcaccc aacatgctaa tcacctacga
cgatgtggtg aagatctcag 900 attttggcac ttccaaggag ctgagtgaca
agagcaccaa gatgtccttt gcagggacag 960 tagcctggat ggcccctgag
gtgatccgca atgaacctgt gtctgagaag gtcgacatct 1020 ggtcctttgg
cgtggtgcta tgggaactgc tgactggtga gatcccctac aaagacgtag 1080
attcctcagc cattatctgg ggtgtgggaa gcaacagtct ccatctgccc gtgccctcca
1140 gttgcccaga tggtttcaag atcctgcttc gccagtgctg gaatagcaaa
ccacgaaatc 1200 gcccatcatt ccgacagatc ctgctgcatc tggacattgc
ctcagctgat gtactctcca 1260 caccccagga gacttacttt aagtcccagg
cagagtggcg ggaagaagta aaactgcact 1320 ttgaaaagat taagtcagaa
gggacctgtc tgcaccgcct agaagaggaa ctggtgatga 1380 ggaggaggga
ggagctcaga cacgccctgg acatcaggga gcactatgaa aggaagctgg 1440
agagagccaa caacctgtat atggaactta atgccctcat gttgcagctg gaactcaagg
1500 agagggagct gctcaggcga gagcaagctt tagagcggag gtgcccaggc
ctgctgaagc 1560 cacacccttc ccggggcctc ctgcatggaa acacaatgga
gaagcttatc aagaagagga 1620 atgtgccaca gaagctgtca ccccatagca
aaaggccaga tatcctcaag acggagtctt 1680 tgctccctaa actagatgca
gccctgagtg gggtggggct tcctgggtgt cctaagggcc 1740 ccccctcacc
aggacggagt cgccgtggca agacccgtca ccgcaaggcc agcgccaagg 1800
ggagctgtgg ggacctgcct gggcttcgta cagctgtgcc accccatgaa cctggaggac
1860 caggaagccc agggggccta ggagggggac cctcagcctg ggaggcctgc
cctcccgccc 1920 tccgtgggct tcatcatgac ctcctgctcc gcaaaatgtc
ttcatcgtcc ccagacctgc 1980 tgtcagcagc actagggtcc cggggccggg
gggccacagg cggagctggg gatcctggct 2040 caccacctcc ggcccggggt
gacaccccac caagtgaggg ctcagcccct ggctccacca 2100 gcccagattc
acctggggga gccaaagggg aaccacctcc tccagtaggg cctggtgaag 2160
gtgtggggct tctgggaact ggaagggaag ggacctcagg ccggggagga agccgggctg
2220 ggtcccagca cttgacccca gctgcactgc tgtacagggc tgccgtcacc
cgaagtcaga 2280 aacgtggcat ctcatcggaa gaggaggaag gagaggtaga
cagtgaagta gagctgacat 2340 caagccagag gtggcctcag agcctgaaca
tgcgccagtc actatctacc ttcagctcag 2400 agaatccatc agatggggag
gaaggcacag ctagtgaacc ttcccccagt ggcacacctg 2460 aagttggcag
caccaacact gatgagcggc cagatgagcg gtctgatgac atgtgctccc 2520
agggctcaga aatcccactg gacccacctc cttcagaggt catccctggc cctgaaccca
2580 gctccctgcc cattccacac caggaacttc tcagagagcg gggccctccc
aattctgagg 2640 actcagactg tgacagcact gaattggaca actccaacag
cgttgatgcc ttgcggcccc 2700 cagcttccct ccctccatga aagccactcg
tattccttgt acatagagaa atatttatat 2760 aaattatata tatatacata
tatatatata tatgcgccac ataatcaaca gaaagatggg 2820 gctgtcccag 2830 3
2732 DNA Homo sapiens 3 cgagggccca gtgttcacca tcataccagg ggccagaggc
gatggcttgc ctccatgaga 60 cccgaacacc ctctccttcc tttgggggct
ttgtgtctac cctaagtgag gcatccatgc 120 gcaagctgga cccagacact
tctgactgca ctcccgagaa ggacctgacg cctacccatg 180 tcctgcagct
acatgagcag gatgcagggg gcccaggggg agcagctggg tcacctgaga 240
gtcgggcatc cagagttcga gctgacgagg tgcgactgca gtgccagagt ggcagtggct
300 tccttgaggg cctctttggc tgcctgcgcc ctgtctggac catgattggc
aaagcctact 360 ccactgagca caagcagcag caggaagacc tttgggaggt
cccctttgag gaaatcctgg 420 acctgcagtg ggtgggctca ggggcccagg
gtgctgtctt cctggggcgc ttccacgggg 480 aggaggtggc tgtgaagaag
gtgcgagacc tcaaagaaac cgacatcaag cacttgcgaa 540 agctgaagca
ccccaacatc atcactttca agggtgtgtg cacccaggct ccctgctact 600
gcatcctcat ggagttctgc gcccagggcc agctgtatga ggtactgcgg gctggccgcc
660 ctgtcacccc ctccttactg gttgactggt ccatgggcat cgctggtggc
atgaactacc 720 tgcacctgca caagattatc cacagggatc tcaagtcacc
caacatgcta atcacctacg 780 acgatgtggt gaagatctca gattttggca
cttccaagga gctgagtgac aagagcacca 840 agatgtcctt tgcagggaca
gtagcctgga tggcccctga ggtgatccgc aatgaacctg 900 tgtctgagaa
ggtcgacatc tggtcctttg gcgtggtgct atgggaactg ctgactggtg 960
agatccccta caaagacgta gattcctcag ccattatctg gggtgtggga agcaacagtc
1020 tccatctgcc cgtgccctcc agttgcccag atggtttcaa gatcctgctt
cgccagtgct 1080 ggaatagcaa accacgaaat cgcccatcat tccgacagat
cctgctgcat ctggacattg 1140 cctcagctga tgtactctcc acaccccagg
agacttactt taagtcccag gcagagtggc 1200 gggaagaagt aaaactgcac
tttgaaaaga ttaagtcaga agggacctgt ctgcaccgcc 1260 tagaagagga
actggtgatg aggaggaggg aggagctcag acacgccctg gacatcaggg 1320
agcactatga aaggaagctg gagagagcca acaacctgta tatggaactt aatgccctca
1380 tgttgcagct ggaactcaag gagagggagc tgctcaggcg agagcaagct
ttagagcgga 1440 ggtgcccagg cctgctgaag ccacaccctt cccggggcct
cctgcatgga aacacaatgg 1500 agaagcttat caagaagagg aatgtgccac
agaagctgtc accccatagc aaaaggccag 1560 atatcctcaa gacggagtct
ttgctcccta aactagatgc agccctgagt ggggtggggc 1620 ttcctgggtg
tcctaagggc cccccctcac caggacggag tcgccgtggc aagacccgtc 1680
accgcaaggc cagcgccaag gggagctgtg gggacctgcc tgggcttcgt acagctgtgc
1740 caccccatga acctggagga ccaggaagcc cagggggcct aggaggggga
ccctcagcct 1800 gggaggcctg ccctcccgcc ctccgtgggc ttcatcatga
cctcctgctc cgcaaaatgt 1860 cttcatcgtc cccagacctg ctgtcagcag
cactagggtc ccggggccgg ggggccacag 1920 gcggagctgg ggatcctggc
tcaccacctc cggcccgggg tgacacccca ccaagtgagg 1980 gctcagcccc
tggctccacc agcccagatt cacctggggg agccaaaggg gaaccacctc 2040
ctccagtagg gcctggtgaa ggtgtggggc ttctgggaac tggaagggaa gggacctcag
2100 gccggggagg aagccgggct gggtcccagc acttgacccc agctgcactg
ctgtacaggg 2160 ctgccgtcac ccgaagtcag aaacgtggca tctcatcgga
agaggaggaa ggagaggtag 2220 acagtgaagt agagctgaca tcaagccaga
ggtggcctca gagcctgaac atgcgccagt 2280 cactatctac cttcagctca
gagaatccat cagatgggga ggaaggcaca gctagtgaac 2340 cttcccccag
tggcacacct gaagttggca gcaccaacac tgatgagcgg ccagatgagc 2400
ggtctgatga catgtgctcc cagggctcag aaatcccact ggacccacct ccttcagagg
2460 tcatccctgg ccctgaaccc agctccctgc ccattccaca ccaggaactt
ctcagagagc 2520 ggggccctcc caattctgag gactcagact gtgacagcac
tgaattggac aactccaaca 2580 gcgttgatgc cttgcggccc ccagcttccc
tccctccatg aaagccactc gtattccttg 2640 tacatagaga aatatttata
taaattatat atatatacat atatatatat atatgcgcca 2700 cataatcaac
agaaagatgg ggctgtccag cc 2732 4 3378 DNA Homo sapiens 4 gttttggagc
cctctcttaa gtcagaactc tgtcccaaaa atcttctgag tgtcatctca 60
ggactttggt tatactcatg gcacgatggc caactttcag gagcacctga gctgctcctc
120 ttctccacac ttacccttca gtgaaagcaa aaccttcaat ggactacaag
atgagctcac 180 agctatgggg aaccaccctt ctcccaagct gctcgaggac
cagcaggaaa aggggatggt 240 acgaacagag ctaatcgaga gcgtgcacag
ccccgtcacc acaacagtgt tgacgagcgt 300 aagtgaggat tccagggacc
agtttgagaa cagcgttctt cagctaaggg aacacgatga 360 atcagagacg
gcggtgtctc aggggaacag caacacggtg gacggagaga gcacaagcgg 420
aactgaagac ataaagattc agttcagcag gtcaggcagt ggcagtggtg ggtttcttga
480 aggactattt ggatgcttaa ggcctgtatg gaatatcatt gggaaggcat
attccactga 540 ttacaaattg cagcagcaag atacttggga agtgccattt
gaggagatct cagagctgca 600 gtggctgggt agtggagccc aaggagcggt
cttcttgggc aagttccggg cggaagaggt 660 ggccatcaag aaagtgagag
aacagaatga gacggatatc aagcatttga ggaagttgaa 720 gcaccctaac
atcatcgcat tcaagggtgt ttgtactcag gccccatgtt attgtattat 780
catggaatac tgtgcccatg gacaactcta cgaggtctta cgagctggca ggaagatcac
840 acctcgattg ctagtagact ggtccacagg aattgcaagt ggaatgaatt
atttgcacct 900 ccataaaatt attcatcgtg atctcaaatc acctaatgtt
ttagtgaccc acacagatgc 960 ggtaaaaatt tcagattttg gtacatctaa
ggaactcagt gacaaaagta ccaagatgtc 1020 atttgctggc acggtcgcat
ggatggcgcc agaggtgata cggaatgaac ctgtctctga 1080 aaaagttgat
atatggtctt ttggagtggt gctttgggag ctgctgacag gagagatccc 1140
ttacaaagat gtagattctt cagccattat ctggggtgtt ggaagcaaca gcctccacct
1200 tccagttcct tccacttgcc ctgatggatt caaaatcctt atgaaacaga
cgtggcagag 1260 taaacctcga aaccgacctt cttttcggca gacactcatg
catttagaca ttgcctctgc 1320 agatgtactt gccaccccac aagaaactta
cttcaagtct caggctgaat ggagagaaga 1380 agtgaaaaaa cattttgaga
agatcaaaag tgaaggaact tgtatacacc ggttagatga 1440 agaactgatt
cgaaggcgca gagaagagct caggcatgcg ctggatattc gtgaacacta 1500
tgagcggaag cttgagcggg cgaataattt atacatggaa ttgagtgcca tcatgctgca
1560 gctagaaatg cgggagaagg agctcattaa gcgtgagcaa gcagtggaaa
agaagtatcc 1620 tgggacctac aaacgacacc ctgttcgtcc tatcatccat
cccaatgcca tggagaaact 1680 catgaaaagg aaaggagtgc ctcacaaatc
tgggatgcag accaaacggc cagacttgtt 1740 gagatcagaa gggatcccca
ccacagaagt ggctcccact gcatcccctt tgtccggaag 1800 tcccaaaatg
tccacttcta gcagcaagag ccgatatcga agcaaaccac gccaccgccg 1860
agggaatagc agaggcagcc atagtgactt tgccgcaatc ttgaaaaacc agccagccca
1920 ggaaaattca ccccatccca cttacctgca ccaagctcaa tcccaatacc
cttctcttca 1980 tcaccataat tctctgcagc agcaatacca gcagccccct
cctgccatgt cccagagtca 2040 ccatcccaga ctcaatatgc acggacagga
catagcaacc tgcgccaaca acctgaggta 2100 tttcggccca gcagcagccc
tgcggagccc actcagcaac catgctcaga gacagctgcc 2160 cggctcgagc
cctgacctca tctccacagc catggctgca gactgctgga gaagttctga 2220
gcctgacaag ggccaagctg gtccctgggg ctgttgccag gctgacgctt atgacccctg
2280 ccttcagtgc aggccagaac agtatgggtc cttagacata ccctctgctg
agccagtggg 2340 gaggagccct gacctttcca agtcaccagc acataatcct
ctcttggaaa acgcccagag 2400 ttctgagaaa acggaagaaa atgaattcag
cggctgtagg tctgagtcat ccctcggcac 2460 ctctcatctc ggcacccctc
cagcgctacc tcgaaaaaca aggcctctgc agaagagtgg 2520 agatgactcc
tcagaagagg aagaagggga agtagatagt gaagttgaat ttccacgaag 2580
acagaggccc catcgctgta tcagcagctg ccagtcatat tcaaccttta gctctgagaa
2640 tttctctgtg tctgatggag aagagggaaa taccagtgac cactcaaaca
gtcctgatga 2700 gttagctgat aaacttgaag accgcttggc agagaagcta
gacgacctgc tgtcccagac 2760 gccagagatt cccattgaca tatcctcaca
ctcggatggg ctctctgaca aggagtgtgc 2820 cgtgcgccgt gtgaagactc
agatgtctct gggcaagctg tgtgtggagg aacgtggcta 2880 tgagaacccc
atgcagtttg aagaatcgga ctgtgactct tcagatgggg agtgttctga 2940
tgccacagtt aggaccaata aacactacag ctctgctacc tggtaatgaa ggaatacaca
3000 tcctgaagat ctcgtgacta tactggcatt tcagatccac cccaccccca
gactcatccc 3060 actctctccc agcattttgt ctgggaagag agactacccc
atctttacca ccccctagaa 3120 atgagctgca ataacaggaa catgagactt
cgcaaatctc tggaaaataa tatccaaatg 3180 aaattaagtc tcactgaaca
tttcaatcaa gaatggcagg gatctatttt attgaatatt 3240 ctagctactg
taacattgat atttattttt gtttgacatt ttaacacttt gtactgcaaa 3300
gagtgaacta tatatgagat agagagacaa taatttcttg caaaaaaaaa aagagataaa
3360 agaaagaaca gaaaaaaa 3378 5 3569 DNA Homo sapiens misc_feature
(3283)..(3283) "n" is A, C, G, or T 5 aatttacatc cattcatgaa
tctgtgacgt cagcaagcct ttgggctcct ttgcggtggg 60 ctggaggatt
gtgtgggtgg aatccccctc ccctttattt ttccaattct gcaaggcttt 120
taaaattcac cttacatctt ttcaaagcaa gaaaatggaa cagcatgtgt aggaattctt
180 cgttgttgtt ttggagccct ctcttaagtc agaactctgt cccaaaaatc
ttctgagtgt 240 catctcagga ctttggttat actcatggca cgatggccaa
ctttcaggag cacctgagct 300 gctcctcttc tccacactta cccttcagtg
aaagcaaaac cttcaatgga ctacaagatg 360 agctcacagc tatggggaac
cacccttctc ccaagctgct cgaggaccag caggaaaagg 420 ggatggtacg
aacagagcta atcgagagcg tgcacagccc cgtcaccaca acagtgttga 480
cgagcgtaag tgaggattcc agggaccagt ttgagaacag cgttcttcag ctaagggaac
540 acgatgaatc agagacggcg gtgtctcagg ggaacagcaa cacggtggac
ggagagagca 600 caagcggaac tgaagacata aagattcagt tcagcaggtc
aggcagtggc agtggtgggt 660 ttcttgaagg actatttgga tgcttaaggc
ctgtatggaa tatcattggg aaggcatatt 720 ccactgatta caaattgcag
cagcaagata cttgggaagt gccatttgag gagatctcag 780 agctgcagtg
gctgggtagt ggagcccaag gagcggtctt cttgggcaag ttccgggcgg 840
aagaggtggc catcaagaaa gtgagagaac agaatgagac ggatatcaag catttgagga
900 agttgaagca ccctaacatc atcgcattca agggtgtttg tactcaggcc
ccatgttatt 960 gtattatcat ggaatactgt gcccatggac aactctacga
ggtcttacga gctggcagga 1020 agatcacacc tcgattgcta gtagactggt
ccacaggaat tgcaagtgga atgaattatt 1080 tgcacctcca taaaattatt
catcgtgatc tcaaatcacc taatgtttta gtgacccaca 1140 cagatgcggt
aaaaatttca gattttggta catctaagga actcagtgac aaaagtacca 1200
agatgtcatt tgctggcacg gtcgcatgga tggcgccaga ggtgatacgg aatgaacctg
1260 tctctgaaaa agttgatata tggtcttttg gagtggtgct ttgggagctg
ctgacaggag 1320 agatccctta caaagatgta gattcttcag ccattatctg
gggtgttgga agcaacagcc 1380 tccaccttcc agttccttcc acttgccctg
atggattcaa aatccttatg aaacagacgt 1440 ggcagagtaa acctcgaaac
cgaccttctt ttcggcagac actcatgcat ttagacattg 1500 cctctgcaga
tgtacttgcc accccacaag aaacttactt caagtctcag gctgaatgga 1560
gagaagaagt gaaaaaacat tttgagaaga tcaaaagtga aggaacttgt atacaccggt
1620 tagatgaaga actgattcga aggcgcagag aagagctcag gcatgcgctg
gatattcgtg 1680 aacactatga gcggaagctt gagcgggcga ataatttata
catggaattg agtgccatca 1740 tgctgcagct agaaatgcgg gagaaggagc
tcattaagcg tgagcaagca gtggaaaaga 1800 agtatcctgg gacctacaaa
cgacaccctg ttcgtcctat catccatccc aatgccatgg 1860 agaaactcat
gaaaaggaaa ggagtgcctc acaaatctgg gatgcagacc aaacggccag 1920
acttgttgag atcagaaggg atccccacca cagaagtggc tcccactgca tcccctttgt
1980 ccggaagtcc caaaatgtcc acttctagca gcaagagccg atatcgaagc
aaaccacgcc 2040 accgccgagg gaatagcaga ggcagccata gtgactttgc
cgcaatcttg aaaaaccagc 2100 cagcccagga aaattcaccc catcccactt
acctgcacca agctcaatcc caataccctt 2160 ctcttcatca ccataattct
ctgcagcagc aataccagca gccccctcct gccatgtccc 2220 agagtcacca
tcccagactc aatatgcacg gacaggacat agcaacctgc gccaacaacc 2280
tgaggtattt cggcccagca gcagccctgc ggagcccact cagcaaccat gctcagagac
2340 agctgcccgg ctcgagccct gacctcatct
ccacagccat ggctgcagac tgctggagaa 2400 gttctgagcc tgacaagggc
caagctggtc cctggggctg ttgccaggct gacgcttatg 2460 acccctgcct
tcagtgcagg ccagaacagt atgggtcctt agacataccc tctgctgagc 2520
cagtggggag gagccctgac ctttccaagt caccagcaca taatcctctc ttggaaaacg
2580 cccagagttc tgagaaaacg gaagaaaatg aattcagcgg ctgtaggtct
gagtcatccc 2640 tcggcacctc tcatctcggc acccctccag cgctacctcg
aaaaacaagg cctctgcaga 2700 agagtggaga tgactcctca gaagaggaag
aaggggaagt agatagtgaa gttgaatttc 2760 cacgaagaca gaggccccat
cgctgtatca gcagctgcca gtcatattca acctttagct 2820 ctgagaattt
ctctgtgtct gatggagaag agggaaatac cagtgaccac tcaaacagtc 2880
ctgatgagtt agctgataaa cttgaagacc gcttggcaga gaagctagac gacctgctgt
2940 cccagacgcc agagattccc attgacatat cctcacactc ggatgggctc
tctgacaagg 3000 agtgtgccgt gcgccgtgtg aagactcaga tgtctctggg
caagctgtgt gtggaggaac 3060 gtggctatga gaaccccatg cagtttgaag
aatcggactg tgactcttca gatggggagt 3120 gttctgatgc cacagttagg
accaataaac actacagctc tgctacctgg taatgaagga 3180 atacacatcc
tgaagatctc gtgactatac tggcatttca gatccacccc acccccagac 3240
tcatcccact ctctcccagc attttgtctg ggaagagaga ctnacccatc tttacccacc
3300 ccctagaaat gagctgcaat aacaggaaca tgagacttcg caaatctctg
gaaaataata 3360 tccaaatgaa attaagtctc actgaacatt tcaatcaaga
atggcaggga tctattttat 3420 tgaatattct agctactgta acattgatat
ttatttttgt ttgacatttt aacactttgt 3480 actgcaaaga gtgaactata
tatgagatag agagacaata atttcttgca aaaaaaaaaa 3540 gagataaaag
aaagaacaaa aaaaaaaaa 3569 6 2910 DNA Homo sapiens 6 acgatggcca
actttcagga gcacctgagc tgctcctctt ctccacactt acccttcagt 60
gaaagcaaaa ccttcaatgg actacaagat gagctcacag ctatggggaa ccacccttct
120 cccaagctgc tcgaggacca gcaggaaaag gggatggtac gaacagagct
aatcgagagc 180 gtgcacagcc ccgtcaccac aacagtgttg acgagcgtaa
gtgaggattc cagggaccag 240 tttgagaaca gcgttcttca gctaagggaa
cacgatgaat cagagacggc ggtgtctcag 300 gggaacagca acacggtgga
cggagagagc acaagtggaa ctgaagacat aaagattcag 360 ttcagcaggt
caggcagtgg cagtggtggg tttcttgaag gactatttgg atgcttaagg 420
cctgtatgga atatcattgg gaaggcatat tccactgatt acaaattgca gcagcaagat
480 acttgggaag tgccatttga ggagatctca gagctgcagt ggctgggtag
tggagcccaa 540 ggagcggtct tcttgggcaa gttccgggcg gaagaggtgg
ccatcaagaa agtgagagaa 600 cagaatgaga cggatatcaa gcatttgagg
aagttgaagc accctaacat catcgcattc 660 aagggtgttt gtactcaggc
cccatgttat tgtattatca tggaatactg tgcccatgga 720 caactctacg
aggtcttacg agctggcagg aagatcacac ctcgattgct agtagactgg 780
tccacaggaa ttgcaagtgg aatgaattat ttgcacctcc ataaaattat tcatcgtgat
840 ctcaaatcac ctaatgtttt agtgacccac acagatgcgg taaaaatttc
agattttggt 900 acatctaagg aactcagtga caaaagtacc aagatgtcat
ttgctggcac ggtcgcatgg 960 atggcgccag aggtgatacg gaatgaacct
gtctctgaaa aagttgatat atggtctttt 1020 ggagtggtgc tttgggagct
gctgacagga gagatccctt acaaagatgt agattcttca 1080 gccattatct
ggggtgttgg aagcaacagc ctccaccttc cagttccttc cacttgccct 1140
gatggattca aaatccttat gaaacagacg tggcagagta aacctcgaaa ccgaccttct
1200 tttcggcaga cactcatgca tttagacatt gcctctgcag atgtacttgc
caccccacaa 1260 gaaacttact tcaagtctca ggctgaatgg agagaagaag
tgaaaaaaca ttttgagaag 1320 atcaaaagtg aaggaacttg tatacaccgg
ttagatgaag aactgattcg aaggcgcaga 1380 gaagagctca ggcatgcgct
ggatattcgt gaacactatg agcggaagct tgagcgggcg 1440 aataatttat
acatggaatt gagtgccatc atgctgcagc tagaaatgcg ggagaaggag 1500
ctcattaagc gtgagcaagc agtggaaaag aagtatcctg ggacctacaa acgacaccct
1560 gttcgtccta tcatccatcc caatgccatg gagaaactca tgaaaaggaa
aggagtgcct 1620 cacaaatctg ggatgcagac caaacggcca gacttgttga
gatcagaagg gatccccacc 1680 acagaagtgg ctcccactgc atcccctttg
tccggaagtc ccaaaatgtc cacttctagc 1740 agcaagagcc gatatcgaag
caaaccacgc caccgccgag ggaatagcag aggcagccat 1800 agtgactttg
ccgcaatctt gaaaaaccag ccagcccagg aaaattcacc ccatcccact 1860
tacctgcacc aagctcaatc ccaataccct tctcttcatc accataattc tctgcagcag
1920 caataccagc agccccctcc tgccatgtcc cagagtcacc atcccagact
caatatgcac 1980 ggacaggaca tagcaacctg cgccaacaac ctgaggtatt
tcggcccagc agcagccctg 2040 cggagcccac tcagcaacca tgctcagaga
cagctgcccg gctcgagccc tgacctcatc 2100 tccacagcca tggctgcaga
ctgctggaga agttctgagc ctgacaaggg ccaagctggt 2160 ccctggggct
gttgccaggc tgacgcttat gacccctgcc ttcagtgcag gccagaacag 2220
tatgggtcct tagacatacc ctctgctgag ccagtgggga ggagccctga cctttccaag
2280 tcaccagcac ataatcctct cttggaaaac gcccagagtt ctgagaaaac
ggaagaaaat 2340 gaattcagcg gctgtaggtc tgagtcatcc ctcggcacct
ctcatctcgg cacccctcca 2400 gcgctacctc gaaaaacaag gcctctgcag
aagagtggag atgactcctc agaagaggaa 2460 gaaggggaag tagatagtga
agttgaattt ccacgaagac agaggcccca tcgctgtatc 2520 agcagctgcc
agtcatattc aacctttagc tctgagaatt tctctgtgtc tgatggagaa 2580
gagggaaata ccagtgacca ctcaaacagt cctgatgagt tagctgataa acttgaagac
2640 cgcttggcag agaagctaga cgacctgctg tcccagacgc cagagattcc
cattgacata 2700 tcctcacact cggatgggct ctctgacaag gagtgtgccg
tgcgccgtgt gaagactcag 2760 atgtctctgg gcaagctgtg tgtggaggaa
cgtggctatg agaaccccat gcagtttgaa 2820 gaatcggact gtgactcttc
agatggggag tgttctgatg ccacagttag gaccaataaa 2880 cactacagct
ctgctacctg gtaatgaagg 2910 7 333 DNA Homo sapiens 7 tacctataca
tggagtattg tgcccatgga caactctacg aggtcttacg agctggcagg 60
aagatcacac ctcgattgct agtagactgg tccacaggaa ttgcaagtgg aatgaattat
120 ttgcacctcc ataaaattat tcatcgtgat ctcaaatcac ctaatgtttt
agtgacccac 180 acagatgcgg taaaaatttc agattttggt acatctaagg
aactcagtga caaaagtacc 240 aagatgtcat ttgctggcac ggtcgcatgg
atggcgccag aggtgatacg gaatgaacct 300 gtctctgaaa aagttgatat
ctggtctatg gta 333 8 859 PRT Homo sapiens 8 Met Ala Cys Leu His Glu
Thr Arg Thr Pro Ser Pro Ser Phe Gly Gly 1 5 10 15 Phe Val Ser Thr
Leu Ser Glu Ala Ser Met Arg Lys Leu Asp Pro Asp 20 25 30 Thr Ser
Asp Cys Thr Pro Glu Lys Asp Leu Thr Pro Thr His Val Leu 35 40 45
Gln Leu His Glu Gln Asp Ala Gly Gly Pro Gly Gly Ala Ala Gly Ser 50
55 60 Pro Glu Ser Arg Ala Ser Arg Val Arg Ala Asp Glu Val Arg Leu
Gln 65 70 75 80 Cys Gln Ser Gly Ser Gly Phe Leu Glu Gly Leu Phe Gly
Cys Leu Arg 85 90 95 Pro Val Trp Thr Met Ile Gly Lys Ala Tyr Ser
Thr Glu His Lys Gln 100 105 110 Gln Gln Glu Asp Leu Trp Glu Val Pro
Phe Glu Glu Ile Leu Asp Leu 115 120 125 Gln Trp Val Gly Ser Gly Ala
Gln Gly Ala Val Phe Leu Gly Arg Phe 130 135 140 His Gly Glu Glu Val
Ala Val Lys Lys Val Arg Asp Leu Lys Glu Thr 145 150 155 160 Asp Ile
Lys His Leu Arg Lys Leu Lys His Pro Asn Ile Ile Thr Phe 165 170 175
Lys Gly Val Cys Thr Gln Ala Pro Cys Tyr Cys Ile Leu Met Glu Phe 180
185 190 Cys Ala Gln Gly Gln Leu Tyr Glu Val Leu Arg Ala Gly Arg Pro
Val 195 200 205 Thr Pro Ser Leu Leu Val Asp Trp Ser Met Gly Ile Ala
Gly Gly Met 210 215 220 Asn Tyr Leu His Leu His Lys Ile Ile His Arg
Asp Leu Lys Ser Pro 225 230 235 240 Asn Met Leu Ile Thr Tyr Asp Asp
Val Val Lys Ile Ser Asp Phe Gly 245 250 255 Thr Ser Lys Glu Leu Ser
Asp Lys Ser Thr Lys Met Ser Phe Ala Gly 260 265 270 Thr Val Ala Trp
Met Ala Pro Glu Val Ile Arg Asn Glu Pro Val Ser 275 280 285 Glu Lys
Val Asp Ile Trp Ser Phe Gly Val Val Leu Trp Glu Leu Leu 290 295 300
Thr Gly Glu Ile Pro Tyr Lys Asp Val Asp Ser Ser Ala Ile Ile Trp 305
310 315 320 Gly Val Gly Ser Asn Ser Leu His Leu Pro Val Pro Ser Ser
Cys Pro 325 330 335 Asp Gly Phe Lys Ile Leu Leu Arg Gln Cys Trp Asn
Ser Lys Pro Arg 340 345 350 Asn Arg Pro Ser Phe Arg Gln Ile Leu Leu
His Leu Asp Ile Ala Ser 355 360 365 Ala Asp Val Leu Ser Thr Pro Gln
Glu Thr Tyr Phe Lys Ser Gln Ala 370 375 380 Glu Trp Arg Glu Glu Val
Lys Leu His Phe Glu Lys Ile Lys Ser Glu 385 390 395 400 Gly Thr Cys
Leu His Arg Leu Glu Glu Glu Leu Val Met Arg Arg Arg 405 410 415 Glu
Glu Leu Arg His Ala Leu Asp Ile Arg Glu His Tyr Glu Arg Lys 420 425
430 Leu Glu Arg Ala Asn Asn Leu Tyr Met Glu Leu Asn Ala Leu Met Leu
435 440 445 Gln Leu Glu Leu Lys Glu Arg Glu Leu Leu Arg Arg Glu Gln
Ala Leu 450 455 460 Glu Arg Arg Cys Pro Gly Leu Leu Lys Pro His Pro
Ser Arg Gly Leu 465 470 475 480 Leu His Gly Asn Thr Met Glu Lys Leu
Ile Lys Lys Arg Asn Val Pro 485 490 495 Gln Asn Leu Ser Pro His Ser
Gln Arg Pro Asp Ile Leu Lys Ala Glu 500 505 510 Ser Leu Leu Pro Lys
Leu Asp Ala Ala Leu Ser Gly Val Gly Leu Pro 515 520 525 Gly Cys Pro
Lys Ala Pro Pro Ser Pro Gly Arg Ser Arg Arg Gly Lys 530 535 540 Thr
Arg His Arg Lys Ala Ser Ala Lys Gly Ser Cys Gly Asp Leu Pro 545 550
555 560 Gly Leu Arg Thr Ala Val Pro Pro His Glu Pro Gly Gly Pro Gly
Ser 565 570 575 Pro Gly Gly Leu Gly Gly Gly Pro Ser Ala Trp Glu Ala
Cys Pro Pro 580 585 590 Ala Leu Arg Gly Leu His His Asp Leu Leu Leu
Arg Lys Met Ser Ser 595 600 605 Ser Ser Pro Asp Leu Leu Ser Ala Ala
Leu Gly Ser Arg Gly Arg Gly 610 615 620 Ala Thr Gly Gly Ala Gly Asp
Pro Gly Ser Pro Pro Pro Ala Arg Gly 625 630 635 640 Asp Thr Pro Pro
Ser Glu Gly Ser Ala Pro Gly Ser Thr Ser Pro Asp 645 650 655 Ser Pro
Gly Gly Ala Lys Gly Glu Pro Pro Pro Pro Val Gly Pro Gly 660 665 670
Glu Gly Val Gly Leu Leu Gly Thr Gly Arg Glu Gly Thr Ser Gly Arg 675
680 685 Gly Gly Ser Arg Ala Gly Ser Gln His Leu Thr Pro Ala Ala Leu
Leu 690 695 700 Tyr Arg Ala Ala Val Thr Arg Ser Gln Lys Arg Gly Ile
Ser Ser Glu 705 710 715 720 Glu Glu Glu Gly Glu Val Asp Ser Glu Val
Glu Leu Thr Ser Ser Gln 725 730 735 Arg Trp Pro Gln Ser Leu Asn Met
Arg Gln Ser Leu Ser Thr Phe Ser 740 745 750 Ser Glu Asn Pro Ser Asp
Gly Glu Glu Gly Thr Ala Ser Glu Pro Ser 755 760 765 Pro Ser Gly Thr
Pro Glu Val Gly Ser Thr Asn Thr Asp Glu Arg Pro 770 775 780 Asp Glu
Arg Ser Asp Asp Met Cys Ser Gln Gly Ser Glu Ile Pro Leu 785 790 795
800 Asp Pro Pro Pro Ser Glu Val Ile Pro Gly Pro Glu Pro Ser Ser Leu
805 810 815 Pro Ile Pro His Gln Glu Leu Leu Arg Glu Arg Gly Pro Pro
Asn Ser 820 825 830 Glu Asp Ser Asp Cys Asp Ser Thr Glu Leu Asp Asn
Ser Asn Ser Val 835 840 845 Asp Ala Leu Arg Pro Pro Ala Ser Leu Pro
Pro 850 855 9 966 PRT Homo sapiens 9 Met Ala Asn Phe Gln Glu His
Leu Ser Cys Ser Ser Ser Pro His Leu 1 5 10 15 Pro Phe Ser Glu Ser
Lys Thr Phe Asn Gly Leu Gln Asp Glu Leu Thr 20 25 30 Ala Met Gly
Asn His Pro Ser Pro Lys Leu Leu Glu Asp Gln Gln Glu 35 40 45 Lys
Gly Met Val Arg Thr Glu Leu Ile Glu Ser Val His Ser Pro Val 50 55
60 Thr Thr Thr Val Leu Thr Ser Val Ser Glu Asp Ser Arg Asp Gln Phe
65 70 75 80 Glu Asn Ser Val Leu Gln Leu Arg Glu His Asp Glu Ser Glu
Thr Ala 85 90 95 Val Ser Gln Gly Asn Ser Asn Thr Val Asp Gly Glu
Ser Thr Ser Gly 100 105 110 Thr Glu Asp Ile Lys Ile Gln Phe Ser Arg
Ser Gly Ser Gly Ser Gly 115 120 125 Gly Phe Leu Glu Gly Leu Phe Gly
Cys Leu Arg Pro Val Trp Asn Ile 130 135 140 Ile Gly Lys Ala Tyr Ser
Thr Asp Tyr Lys Leu Gln Gln Gln Asp Thr 145 150 155 160 Trp Glu Val
Pro Phe Glu Glu Ile Ser Glu Leu Gln Trp Leu Gly Ser 165 170 175 Gly
Ala Gln Gly Ala Val Phe Leu Gly Lys Phe Arg Ala Glu Glu Val 180 185
190 Ala Ile Lys Lys Val Arg Glu Gln Asn Glu Thr Asp Ile Lys His Leu
195 200 205 Arg Lys Leu Lys His Pro Asn Ile Ile Ala Phe Lys Gly Val
Cys Thr 210 215 220 Gln Ala Pro Cys Tyr Cys Ile Ile Met Glu Tyr Cys
Ala His Gly Gln 225 230 235 240 Leu Tyr Glu Val Leu Arg Ala Gly Arg
Lys Ile Thr Pro Arg Leu Leu 245 250 255 Val Asp Trp Ser Thr Gly Ile
Ala Ser Gly Met Asn Tyr Leu His Leu 260 265 270 His Lys Ile Ile His
Arg Asp Leu Lys Ser Pro Asn Val Leu Val Thr 275 280 285 His Thr Asp
Ala Val Lys Ile Ser Asp Phe Gly Thr Ser Lys Glu Leu 290 295 300 Ser
Asp Lys Ser Thr Lys Met Ser Phe Ala Gly Thr Val Ala Trp Met 305 310
315 320 Ala Pro Glu Val Ile Arg Asn Glu Pro Val Ser Glu Lys Val Asp
Ile 325 330 335 Trp Ser Phe Gly Val Val Leu Trp Glu Leu Leu Thr Gly
Glu Ile Pro 340 345 350 Tyr Lys Asp Val Asp Ser Ser Ala Ile Ile Trp
Gly Val Gly Ser Asn 355 360 365 Ser Leu His Leu Pro Val Pro Ser Thr
Cys Pro Asp Gly Phe Lys Ile 370 375 380 Leu Met Lys Gln Thr Trp Gln
Ser Lys Pro Arg Asn Arg Pro Ser Phe 385 390 395 400 Arg Gln Thr Leu
Met His Leu Asp Ile Ala Ser Ala Asp Val Leu Ala 405 410 415 Thr Pro
Gln Glu Thr Tyr Phe Lys Ser Gln Ala Glu Trp Arg Glu Glu 420 425 430
Val Lys Lys His Phe Glu Lys Ile Lys Ser Glu Gly Thr Cys Ile His 435
440 445 Arg Leu Asp Glu Glu Leu Ile Arg Arg Arg Arg Glu Glu Leu Arg
His 450 455 460 Ala Leu Asp Ile Arg Glu His Tyr Glu Arg Lys Leu Glu
Arg Ala Asn 465 470 475 480 Asn Leu Tyr Met Glu Leu Ser Ala Ile Met
Leu Gln Leu Glu Met Arg 485 490 495 Glu Lys Glu Leu Ile Lys Arg Glu
Gln Ala Val Glu Lys Lys Tyr Pro 500 505 510 Gly Thr Tyr Lys Arg His
Pro Val Arg Pro Ile Ile His Pro Asn Ala 515 520 525 Met Glu Lys Leu
Met Lys Arg Lys Gly Val Pro His Lys Ser Gly Met 530 535 540 Gln Thr
Lys Arg Pro Asp Leu Leu Arg Ser Glu Gly Ile Pro Thr Thr 545 550 555
560 Glu Val Ala Pro Thr Ala Ser Pro Leu Ser Gly Ser Pro Lys Met Ser
565 570 575 Thr Ser Ser Ser Lys Ser Arg Tyr Arg Ser Lys Pro Arg His
Arg Arg 580 585 590 Gly Asn Ser Arg Gly Ser His Ser Asp Phe Ala Ala
Ile Leu Lys Asn 595 600 605 Gln Pro Ala Gln Glu Asn Ser Pro His Pro
Thr Tyr Leu His Gln Ala 610 615 620 Gln Ser Gln Tyr Pro Ser Leu His
His His Asn Ser Leu Gln Gln Gln 625 630 635 640 Tyr Gln Gln Pro Pro
Pro Ala Met Ser Gln Ser His His Pro Arg Leu 645 650 655 Asn Met His
Gly Gln Asp Ile Ala Thr Cys Ala Asn Asn Leu Arg Tyr 660 665 670 Phe
Gly Pro Ala Ala Ala Leu Arg Ser Pro Leu Ser Asn His Ala Gln 675 680
685 Arg Gln Leu Pro Gly Ser Ser Pro Asp Leu Ile Ser Thr Ala Met Ala
690 695 700 Ala Asp Cys Trp Arg Ser Ser Glu Pro Asp Lys Gly Gln Ala
Gly Pro 705 710 715 720 Trp Gly Cys Cys Gln Ala Asp Ala Tyr Asp Pro
Cys Leu Gln Cys Arg 725 730 735 Pro Glu Gln Tyr Gly Ser Leu Asp Ile
Pro Ser Ala Glu Pro Val Gly 740 745 750 Arg Ser Pro Asp Leu Ser Lys
Ser Pro Ala His Asn Pro Leu Leu Glu 755 760 765 Asn Ala Gln Ser Ser
Glu Lys Thr Glu Glu Asn Glu Phe Ser Gly Cys 770 775 780 Arg Ser Glu
Ser Ser Leu Gly Thr Ser His Leu Gly Thr Pro Pro Ala 785 790 795 800
Leu Pro Arg Lys Thr Arg Pro Leu Gln Lys Ser Gly Asp Asp Ser Ser 805
810 815 Glu Glu Glu Glu Gly Glu Val Asp Ser Glu Val Glu Phe Pro Arg
Arg 820 825 830 Gln Arg Pro His Arg Cys Ile Ser Ser Cys Gln Ser Tyr
Ser Thr Phe
835 840 845 Ser Ser Glu Asn Phe Ser Val Ser Asp Gly Glu Glu Gly Asn
Thr Ser 850 855 860 Asp His Ser Asn Ser Pro Asp Glu Leu Ala Asp Lys
Leu Glu Asp Arg 865 870 875 880 Leu Ala Glu Lys Leu Asp Asp Leu Leu
Ser Gln Thr Pro Glu Ile Pro 885 890 895 Ile Asp Ile Ser Ser His Ser
Asp Gly Leu Ser Asp Lys Glu Cys Ala 900 905 910 Val Arg Arg Val Lys
Thr Gln Met Ser Leu Gly Lys Leu Cys Val Glu 915 920 925 Glu Arg Gly
Tyr Glu Asn Pro Met Gln Phe Glu Glu Ser Asp Cys Asp 930 935 940 Ser
Ser Asp Gly Glu Cys Ser Asp Ala Thr Val Arg Thr Asn Lys His 945 950
955 960 Tyr Ser Ser Ala Thr Trp 965
* * * * *
References